Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of growth and tissue remodelling during the mouse craniofacial and cardiac development Iamaroon, Anak 1996

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1996-147703.pdf [ 20.42MB ]
Metadata
JSON: 831-1.0087734.json
JSON-LD: 831-1.0087734-ld.json
RDF/XML (Pretty): 831-1.0087734-rdf.xml
RDF/JSON: 831-1.0087734-rdf.json
Turtle: 831-1.0087734-turtle.txt
N-Triples: 831-1.0087734-rdf-ntriples.txt
Original Record: 831-1.0087734-source.json
Full Text
831-1.0087734-fulltext.txt
Citation
831-1.0087734.ris

Full Text

CHARACTERIZATION OF GROWTH A N D TISSUE REMODELLING DURING THE MOUSE CRANIOFACIAL A N D CARDIAC DEVELOPMENT A N A K I A M A R O O N D.D.S., Chulalongkorn University, 1987 M.S., University of Minnesota, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES (Department of Oral Biology) W e accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1996 © Anak Iamaroon, 1906 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Orod, B'oflo The University of British Columbia Vancouver, Canada Date ^ p t g ^ b e i r \1 \0\0\Q DE-6 (2/88) 11 ABSTRACT Craniofac ia l and cardiac development share many c o m m o n basic biological processes. Remodelling of the extracellular matrix (ECM) is believed to play an important role during mammalian embryogenesis. But the role of tissue remodel l ing d u r i n g morphogenesis of the craniofacial complex and heart remains unclear. Therefore, I hypothesized that changes of basement membrane components and growth factors were associated w i t h remodelling and growth of the embryonic primary palate, the future premaxillary area, and the heart. The present investigation encompassed four projects. First was the characterization of the distribution of major basement membrane components; laminin , type IV collagen and fibronectin; by indirect immunofluorescence i n the pr imary palate as the epithelial seam is disrupted and the mesenchymal bridge forms and enlarges. The results showed that localized disrupt ion of basement membrane components occurred simultaneously w i t h mesenchymal bridge formation and enlargement during primary palate formation. The purposes of the second part were to characterize the distribution patterns of epidermal growth factor (EGF) and transforming growth factor-alpha ( T G F - a ) and their receptor, epidermal growth factor receptor (EGF-R), by immunohistochemistry and to analyze regional patterns of cell proliferation by 5-bromodeoxyuridine (BrdU) incorparation and proliferating cell nuclear antigen ( P C N A ) immunolocalization during primary palate morphogenesis. The results showed that E G F , T G F - a , and EGF-R were labelled more intensely i n the tips and peripheral regions of the facial prominences where cell proliferation was most pronounced. This suggested that E G F and T G F - a stimulate cell proliferation during outgrowth of prominences during primary palate morphogenesis. I l l The purpose of the third part was to look for the presence of enzymes invo lved i n degradation of the basement membrane of the epithelial seam dur ing primary palate morphogenesis. Protein expression of a candidate matrix metalloproteinase ( M M P ) , 72-kDa gelatinase (MMP-2) was studied by indirect immunofluorescence and zymography. The results revealed that M M P - 2 was present i n the area of fusion i n the primary palate and also i n the tips and per ipheral regions of the facial prominences. This local izat ion of M M P - 2 suggested that regional differences i n tissue remodel l ing are i n v o l v e d i n direct ional enlargement of the facial prominences. Ge la t in z y m o g r a p h y conf i rmed the presence of active and latent M M P - 2 i n the deve loping craniofacial complex. The purpose of the last part was to examine the distribution of M M P - 2 and its substrates: type IV collagen, l a m i n i n and f ibronect in d u r i n g heart morphogenesis by indirect immunohistochemistry. The results showed that the distribution patterns of M M P - 2 were highly correlated w i t h that of the E C M components from embryonic day 9-13. Collectively, these data indicate that the mechanisms of growth and tissue remodell ing dur ing craniofacial and heart development are complex and may involve multiple interactions between various molecular factors; including E C M components (type IV collagen, laminin, and fibronectin), growth factors (EGF and TGF-a) and their receptor (EGF-R), and matrix metalloproteinase (MMP-2) . i v TABLE OF CONTENTS Abstract i i Table of Contents i v List of Figures v i i List of Tables v i i i List of Diagrams ix List of Abbreviations x Preface x i i Acknowledgements x i i i Dedication x i v Chapter One: General Introduction 1 A . Review of the Literature 1 1. Morphogenesis of the primary palate and mandible 1 2. Morphogenesis of the heart 13 3. Morphogenesis of the eye 21 4. Extracellular matrix and its degradation i n craniofacial and heart development 23 4.1 L a m i n i n 25 4.2 Type IV collagen 27 4.3 Fibronectin 29 4.4 Remodelling of the E C M 30 5. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) 32 5.1 M M P s and TIMPs 32 5.2 M M P s , TIMPs and growth factors 40 V 6. Growth factors 42 6.1 Epidermal growth factor 45 6.2 Transforming growth factor-alpha 47 6.3 Epidermal growth factor receptor 49 7. C e l l proliferation i n the developing primary palate 51 B. Hypotheses and aims 54 Chapter Two: Distribution of basement membrane components i n the developing mouse primary palate 56 Introduction 56 Materials and Methods 57 Results 59 Discussion 64 Chapter Three: Ce l l proliferation and distribution of EGF, T G F - a , and EGF-receptor in the developing mouse primary palate 69 Introduction 69 Materials and Methods 70 Results 74 Discussion 83 Chapter Four: Distribution of 72-kDa gelatinase in the developing craniofacial complex of the mouse embryo 90 Introduction 90 Materials and Methods 92 Results 95 Discussion 109 v i Chapter Five: Immunolocalization of 72-kDa gelatinase and extacellular matrix components during mouse cardiac development 115 Introduction 115 Materials and Methods 118 Results 121 Discussion 132 Chapter Six: General Discussion 137 Chapter Seven: Conclusions and Future Work 144 BIBLIOGRAPHY 149 APPENDIX 1: The primary antibodies and their concentrations and sources 191 APPENDIX 2: The secondary antibodies and their concentrations and sources 193 APPENDIX 3: Positive control tissues 194 APPENDIX 4: Samples of mouse embryos used for immunohistochemistry 195 V l l LIST OF FIGURES Fig. 1 Photographs of frontal views of the human embryo 10 Fig. 2 Frontal sections of the human embryo 10 Fig. 3 Photographs of the frontal views of the mouse embryo 11 Fig. 4 Frontal sections of the mouse embryo 11 Figs. 5-8 Distribution of the basement membranes components i n the developing primary palate 61 Figs. 9-12 Distribution of EGF-R, EGF, and T G F - a i n the developing primary palate 77 Figs. 13-18 Distribution of B r d U and P C N A in the developing primary palate 79 Figs. 19-20 Immunolocalization of 72-kDa gelatinase in the developing primary palate 100 Fig. 21 Immunolocalization of 72-kDa gelatinase i n the developing mandible, zymogram 102 Figs. 22,23 Immunolocalization of 72-kDa gelatinase i n the developing eye 103 Figs. 24-26 Co-localization of 72-kDa gelatinase, EGF, and T G F - a in the developing primary palate 105 Figs. 27-30 Immunolocalization of 72-kDa gelatinase and extracellular matrix components during mouse cardiac development 124 V l l l LIST OF DIAGRAMS Diagram I Diagram II Diagram III Diagram IV Diagram V Facial development Heart development Eye development A protease cascade of activation of M M P s in vivo. Schematic model of the sequential events of molecular inductions and interactions during morphogenesis of the primary palate. 9 20 22 35 137 ix LIST OF TABLES Table I Distribution of E C M and other molecules during facial development 4 Table II Distribution of E C M and other molecules during heart development 15 Table III M M P family members 33 Table IV Growth factor families 42 Table V Distribution of 72-kDa gelatinase, EGF and TGF-cc during primary palate morphogenesis 99 X LIST OF ABBREVIATIONS A B C avidin-biotin-peroxidase complex A V C atrio-ventricular canal B M P bone morphogenetic protein B r d U 5'-bromodeoxyuridine C L / P cleft l ip w i t h or without cleft palate C R A B P cellular retinoic acid binding protein C R B P cellular retinol b inding protein CT-1 cardiotrophin-1 D A B 3,3'-diaminobenzidine hydrogen peroxide D i l 14-dioctadecyl-33/3V3'-tetramethylindocarbocyanine perchlorate EC endocardial cushion ECGF endothelial cell growth factor E C M extracellular matrix EGF epidermal growth factor E G F - R epidermal growth factor receptor E H S Engelbreth-Holm-Swarm ES embryonic stem cells F G F fibroblast growth factor F G F R fibroblast growth factor receptor FITC fluorescein isothiocyanate H B G F heparin-binding growth factor H G F / S F hepatocyte growth factor/scatter factor IGF insulin-l ike growth factor IL-1 interleukin-1 XI LLP leukemia inhibitory factor L N lateral nasal prominence LPS lipopolysaccharide M E E medial edge epithelium M M P matrix metalloproteinase M N medial nasal prominence M T - M M P membrane-type matrix metalloproteinase M X maxil lary prominence N G F nerve growth factor P A plasminogen activator PBS phosphate-buffered saline P C N A proliferating cell nuclear antigen P D G F platelet-derived growth factor R A R retinoic acid receptor R T - P C R reverse transcription polymerase chain reaction S P A R C secreted protein acidic and rich i n cysteine T G F - a transforming growth factor- alpha TGF-(3 transforming growth factor-beta T I M P tissue inhibitor of matrix metalloproteinase T N F tumor necrosis factor T P A 12-O-tetradecanoyl phorbol 13-acetate T R E TPA-responsive element T R I T C tetramethylrhodamine isothiocyanate TS tail somite V C A M - 1 vascular cell adhesion molecule-1 x i i P R E F A C E Some of the material inc luded i n this thesis has been previous ly published or has been submitted, as noted below: Iamaroon, A v and Diewert V . M . (1996): Distribution of basement membrane components i n the mouse pr imary palate. J. Craniofac. Genet. Dev. B io l . 16:48-51. Iamaroon, A . , Tait, B., and Diewert, V . M . (1996): C e l l proli feration and expression of E G F , T G F - a , and EGF-R i n the developing primary palate. J. Dent. Res. (in press). Iamaroon, A . , W a l l o n , U . M . , O v e r a l l , C M . , and Diewert , V . M . (1996): Express ion of 72-kDa gelatinase (matrix metalloproteinase 2) i n the developing craniofacial complex of the mouse embryo. A r c h . Oral Biol , (in press). Iamaroon, A . , W a l l o n , U . M . , O v e r a l l , C M . , and Diewert , V . M . (1996): I m m u n o l o c a l i z a t i o n of 72-kDa gelatinase and extracel lular matr ix components during mouse cardiac development. J. Anat. (in revision). These publications as wel l as this thesis are the principal work of the candidate, A n a k Iamaroon. However , the thesis supervisor, Dr . V . M . Diewert, offered editorial comments on the manuscripts and contributed advice and suggestions throughout the course of the experiments that comprise these publications and this thesis. Drs. C M . Overal l and U . M . Wal lon also provided comments on the manuscripts and were responsible for preparation, affinity purification, and specificity evaluation of the anti-72-kDa gelatinase antibody. B. Tait was responsible for animal preparation for B r d U injection and tissue sectioning. The candidate and thesis supervisor agree that contributions of the respective parties are as stated above. Df . A n a k Iamaroon ~ Dr. Diewert (candidate) (supervisor and co-author) X l l l ACKNOWLEDGEMENTS I w o u l d l ike to sincerely thank D r . V i r g i n i a M . Diewert for having provided direction, suggestions, and friendship for the past four years. H a v i n g been working w i t h her is enjoyable and valuable. I w o u l d like to also thank the others of m y supervisory committee: D r . D . M . Brunette, D r . C M . Overa l l , and D r . J . M . Richman for their he lpfu l suggestions and guidance. Their h igh w o r k i n g standard and knowledge i n science are worth recognizing. I am grateful to B. Tait for the technical assistance and warmest friendship. Starting out to live in Vancouver w o u l d have been greatly difficult without her. Special thanks also go to Dr . C . Tse, Dr . A . Kuttan, Dr . U . M . Wal lon , D r . H . M o o n , D . Nagy, A . Wong, J. Sarnowski, and J. Firth for their technical assistance and friendship. I am indebted w i t h a group of special friends whose fr iendship has enormously inspired me and made this thesis possible. These people include W i l l y and Carol Anne Soong and their family, Dr . C . Tantikitti , D r . R.J. G o r l i n , Pia Pibulsravudt, A p i n y a Doungchan, Dr . M . Romero, Dr . E. L u n d , Dr . B. W u , Dr . J. Uitto, Dr . L . Ratkay, Dr . P. Pan, Dr . B. Steffensen, Dr . R. Brownsey, Tony N g , and others whose names are not mentioned. This success w i l l always be theirs as w e l l . Last but not least, this thesis was made possible by financial supports from the Government of Thai land, the Univers i ty Graduate Fe l lowship , and the Medical Research Counci l grants through Dr. V . M . Diewert. x i v namo tassa bhagavato arahato sammasambuddhassa For m y father and mother whose endless support is beyond words and for all teachers w h o m I have fortunately known. 1 CHAPTER 1: GENERAL INTRODUCTION A. Review of the Literature 1. Morphogenesis of the primary palate and mandible The human primary palate starts forming at about 33 days after conception (stage 15) as the area which w i l l form the nose becomes induced and thickened to form the nasal placode (O'Rahilly and Mul ler , 1987). The nasal placode is flanked by two elevated prominences, the medial and lateral nasal prominences. A succession of different inductors i n c l u d i n g the central nervous system is believed to involve formation of the nasal organ (Jacobson, 1963a, 1963b). Previous studies i n the chick (Johnson, 1966; LeLievre and Le Douar in , 1975; Lumsden et ah, 1991) and mouse embryos (Nichols, 1986; Serbedzija et ah, 1992; Osumi-Yamashita et ah, 1994; Trainor and Tarn, 1995) have f o u n d that the mesenchyme underlying the placode and adjacent prominences originates from neural crest cells and mesodermal cells. Migrat ing neural crest cells reach the facial regions at the stages 10-14 i n chick (Lumsden et ah, 1991) and 9-to-10-day-old mouse embryos (Osumi-Yamashita et ah, 1994). The muscles of mastication and superficial muscles of the face, on the other hand, are derived from the somitomeres of the head (Noden, 1988). More recently, the spatial distribution of the mesoderm derived from somitomeres and neural crest cells dur ing mouse craniofacial morphogenesis was studied by micromanipulative cell grafting and cell labelling (Trainor and Tarn, 1995). The results revealed a distinct segregation of these two cell populations w i t h i n the first three branchial arches i n w h i c h neural crest cells are located i n the periphery of the branchial arches and envelop the somitomere-derived core tissues on the rostral, lateral, and caudal sides of the arch. However , cells from these two sources mixed extensively i n the peri-ocular, peri-otic, frontonasal, and cervical mesenchyme. 2 A t about 37 days after conception i n humans (stage 16), the medial nasal prominence starts contacting and fusing w i t h the lateral nasal and maxil lary prominences (O'Rahil ly and M u l l e r , 1987). The area of contact is called the epithelial plate or seam or nasal f in. This structure forms the continuity between the nasal cavity and the roof of the stomodeum. A t about 41 days after conception i n humans (stage 17) (Figs. 1,2), the epithel ia l seam becomes disrupted and invaded by the mesenchyme from the prominences, forming the mesenchymal bridge (Streeter, 1948; Warbr ick , 1960; Vermei j -Keers , 1972; Diewert and Shiota, 1990). Diewert and V a n der Meer (1991) analyzed the growth of the epithelial and mesenchymal components during normal pr imary palate formation i n humans. It was found that during stage 17, the mesenchymal bridge formed through the epithelial seam and the size of the mesenchymal bridge increased rapidly to occupy up to 50% the total area. By stages 18 and 19, total primary palate area increased and the mesenchymal bridge enlarged to constitute 65 to 85% of the total area. A t about 44 days after conception i n humans (stage 18), the nasal f in posterior to the area of the mesenchymal bridge persists forming an oronasal membrane which separates the nasal cavity from the stomodeal cavity. Subsequently, this epithelial membrane ruptures to form a communicat ion between the respiratory passage and the pharynx, the primitive choana (Streeter, 1948; Warbrick, 1960; Tamarin, 1982). Successful primary palate formation involves a closely t imed sequence of local cellular events w i t h spatial changes associated w i t h craniofacial growth. D i s r u p t i o n of pr imary palate formation, predisposed both to genetic and environmental factors, leads to cleft l ip malformation (see review i n Diewert and W a n g , 1992). Previous studies have shown that teratogenic agents can induce cleft l ip malformation i n animals (Sulik et ah, 1979; Trasler and Leong, 1982). More recently, genetic studies revealed that two epistatic loci are involved w i t h cleft l ip malformation i n mice (Juriloff, 1995). A multifactorial threshold m o d e l was proposed to expla in h o w genetic and environmental factors contribute to cleft l ip malformation in mice (Diewert et ah, 1993b). Mouse embryos have been used as a model for the study of the primary palate development since their morphogenesis is similar to that of the human (see review i n Diewert and Wang, 1992) (Fig. 3). The mouse primary palate starts forming at about 10 days and 18 hours (Reed, 1933; Trasler, 1968; W a n g et ah, 1995) w i t h the appearance of the lateral nasal and medial nasal prominences. Subsequently, the medial nasal prominence makes contact w i t h the lateral nasal and maxil lary prominences at the inferior part of the nasal groove forming an epithelial seam or nasal f in from posterior to anterior direction. The scanning and transmission electron microscopic studies showed that fusion between the prominences occurs by a temporal sequence of events which include the loss of microvi l l i by the epithelial cells (Mil l icovsky and Johnston, 1981). After a brief per iod of quiescence, the epithelial cells begin to f i l l the nasal groove by producing a series of surface projections that increase i n size and complexity as the process of fusion nears termination. Recently, W a n g et ah (1995) have studied growth of the internal morphological components during primary palate morphogenesis i n mice w i t h and without genetic cleft l ip l iabi l i ty. It was found that i n cleft l ip strains ( A / W y S n and C L / F r ) enlargement of the epithelial seam and replacement of epithelia by a mesenchymal bridge are delayed compared wi th noncleft l ip strains (C57BL/6J and BALB/cByJ ) . However, i n both cleft and noncleft l ip strains, the primit ive choanae open at the same stages. Therefore, cleft l ip strains have a narrower w i n d o w of primary palate formation. These may contribute to h igh frequencies of cleft l ip malformation i n cleft l ip-l iable strains (Wang, 1992; Diewert and Wang, 1992; Wang et ah, 1995). Various E C M molecules (type IV collagen, laminin, fibronectin), growth factors (fibroblast growth factor-8, bone morphogenetic proteins) and receptors (retinoic acid receptors), oncoprotein (ras), secreted protein (Wnt-5a), and transcriptional factors (AP-2, Msx-1, Msx-2, goosecoid, Pax-3, Pax-6), have been found i n the developing facial prominences and suggested to be important for the facial development (Table I). Table I: Distribution of E C M and other molecules during facial development. Molecules Localization References AP-2 BMP-2A BMP-4 c-met M X P , F N M , L N M , M N P , M D (mouse, days 10.5-12.5) ectoderm, mesenchyme M N P , M D (mouse, days 11.5,12.5) F N M , M X P , M D , L N P (chick, stages 20,24,28) ectoderm, mesenchyme F N M , M X P , M D (mouse, day 9) F N M , M X P , M D , L N P (chick, stages 20,24,28) ectoderm, mesenchyme M N P , L N P , M X P , M D (mouse, days 9-10) ectoderm Mitchel l et ah, 1991 Lyons et ah, 1990 Francis-West et ah, 1994 Jones et ah, 1991 Francis-West et ah, 1994 Andermarcher et ah, 1996 Collagen IV CRABP-I CRBP FGF-8 Fibronectin goosecoid HGF/SF Maden et al, 1991 Dolle et al, 1990 Dolle et al, 1990 M X P , roof of stomodeum X u et al, 1990 (chick, stages 22-31) basement membrane F N M , M X P , M D (chick, stage 20) F N M , M D (mouse, days 10.5,12.5) mesenchyme F N M , M D (mouse, days 10.5,12.5) mesenchyme M X P , M D , nasal placode, nasal pit epithelia (mouse, days 9.5,10.5) ectoderm M X P , roof of stomodeum, M D (chick, stages 22-31) X u et al, 1990 (mouse, days 12-18) Richman and Diewert, 1987 basement membrane, mesenchyme Crossley and Mart in , 1995 MacArthur et al, 1995 Ohuchi et al, 1994 L N P , M N P , M D (mouse, day 10.5) mesenchyme M N P , L N P , M X P , M D (mouse, days 9-10) mesenchyme Gaunt et al, 1993 Andermarcher et al, 1996 Laminin Msx-1 (Hox-7) Msx-2 (Hox-8) Pax-3 Pax-6 RARoc R A R p M X P , roof of stomodeum (chick, stages 22-31) basement membrane M X P , F N M , M D (chick, stage 25) L N P , M N P , M X P , M D (mouse, day 9.5-11.5) mesenchyme M X P , F N M , M D (chick, stage 25) L N P , M N P , M X P , M D (mouse, day 10.5) ectoderm, mesenchyme nasal prominence, M X P , M D (mouse, days 10-13) mesenchyme nasal placode (mouse, days 9.5,10.5) ectoderm L N P , M N P , M X P , M D (mouse, days 10,11) F N M , M D (mouse, day 12.5) mesenchyme L N P , M D (mouse, days 10,11) X u et al, 1990 Nish ikawa et al, 1994 Brown et al, 1993 Robert et al, 1989 Brown et al, 1993 MacKenzie et al, 1992 Nish ikawa et al, 1994 MacKenzie et al, 1992 Gould ing et al, 1991 Grindley et al, 1995 Osumi-Yamashita et al, 1990 Dolle et al, 1990 Osumi-Yamashita et al, 1990 R A R y ras onco-proteins Wnt-5a F N M , M D (mouse, day 10.5) F N M , M X P , L N P (chick, stage 20) mesenchyme L N P , M N P , M X P , M D (mouse, days 10,11) F N M , M D (mouse, days 10.5,12.5) ectoderm, mesenchyme L N P , M N P , M X P (mouse, days 11,12) ectoderm, mesenchyme L N P , M N P , M D (mouse, day 10.5) ectoderm, mesenchyme Dolle et al, 1990 Rowe et al, 1991 Osumi-Yamashita et al, 1990 Dolle et al, 1990 Wang et al, 1995 Gavin et al, 1990 B M P , bone morphogenetic protein; CRABP,cel lular retinoic acid binding protein; C R B P , cellular retinol b i n d i n g protein; F G F , fibroblast growth factor; F N M , frontonasal mass; H G F / S F , hepatocyte growth factor /scatter factor; L N P , lateral nasal prominence ; M D , m a n d i b u l a r prominence ; M N P , m e d i a l nasal prominence; M X P , maxillary prominence; R A R , retinoic acid receptor The p r i m a r y palate and the secondary palate share m a n y s imi lar developmental events inc luding fusion of the facial prominences, epithelial seam format ion and e l iminat ion. In addi t ion , the maxi l la ry prominence contributes to not only the lateral part of the primary palate, but also the whole 8 secondary palate. The secondary palate starts to form w i t h bilateral outgrowths from the maxillary prominences at embryonic day 12 i n mice, day 6 i n chickens, and day 45 i n humans (see review i n Ferguson, 1988). Initially, the maxil lary prominences grow vertically and then elevate to a horizontal position above the tongue. Subsequently, the two palatal shelves make contact and fuse to form a m i d l i n e epithelial seam, s imilar to the pr imary palate. D i s r u p t i o n of the epithelial seam in the secondary palate have been extensively studied and at least three mechanisms for elimination of the medial edge epithel ium (MEE) have been proposed. Programmed cell death or apoptosis was initially suggested as a mechanism of the seam el iminat ion based on ultrastructural evidence of autolytic epithelial cells (Hudson and Shapiro, 1973; Greene and Pratt, 1976). More recently by using in situ labelling for nuclear D N A fragmentation (TdT-mediated dUTP-biot in nick end labelling, T U N E L , method), apoptotic cells were observed along the epithelial seams of both primary (Pellier and Astic , 1994) and secondary (Mori et al., 1994) palates. Recent in vitro and in vivo studies by using vital cell labelling techniques have suggested that M E E cells undergo epithelial-mesenchymal transformation (Grif f i th and H a y , 1992; Shuler et al., 1992). Alternatively, migration of M E E cells to the adjacent oral and nasal epithelia is also indicated, based on using D i l labelling and confocal microscopy (Carette and Ferguson, 1992). The fate of the epithelial cells dur ing seam regression i n the primary palate needs to be investigated. The first branchial arch gives rise not only to the maxil lary prominence but also the mandibular prominence. The mandible starts to develop by elongation and outgrowth of the mandibular prominence. Subsequently, differentiation of tissues, including Meckel's cartilage, membranous bone, tooth germs and muscles, takes place (Frommer and M a r g o l i e s , 1971). The mesenchymal cells i n the mandibular pr imordia include both mesodermally-derived and neural crest-derived mesenchymal cells. The neural crest-derived mesenchymal cells w i l l give rise to chondrocytes and osteoblasts (Smith and H a l l , 1990), whereas the mesodermally-derived mesenchymal cells w i l l give rise to skeletal muscles (Noden, 1988). Frontonasa l p r o m i n e n c e N a s a l p lacode N a s a l P " O r a l o p e n i n g O r a l plate ( s t 0 m a d e u m ) M a x i l l a r y process M a n d i b u l a r process 2 n d visceral arch M e d i a n nasal process Lateral nasal process N a s o l a c r i m a l f u r r o w axil lary process Ma Mandible 1st visceral f u r r o w Lateral nasal process P h i l t r u m A u d i t o r y h i l l o c k E x t e r n a l ear Diagram I: Frontal views of the human embryo showing development of the face. (From Foundations of A n i m a l Development by Hopper A . F . and Hart N . H . , 1980.) 10 12 Figs. 1 A - C : Photographs of frontal views of the craniofacial morphology of the h u m a n embryos i n the Kyoto Collect ion. A t the stage (S)17 (A), the facial prominences have already made contact and formed the epithelial seam. A t stage 18 (B), the facial prominences become more enlarged and completely form the primary palate at stage 19 (C). (modified from Diewert and Shiota, 1990) Figs. 2 A - F : Frontal sections of human embryonic pr imary palate formation showing fusion between the medial nasal prominence ( M N P ) w i t h the lateral nasal (LNP) and maxil lary prominences (MXP) at early stage 17 (A). Higher magnification shows an epithelial seam (between arrows) (D). A t stage 18, the epithelial seam becomes widely disrupted and replaced by a mesenchymal bridge (B). Higher magnification shows disrupt ion of the epithelial seam w i t h a replacement of a mesenchymal bridge (between arrows) (E). A t stage 19, the mesenchymal bridge enlarges w i t h the outgrowth of the pr imary palate (C). Grooves between prominences are markedly reduced. Higher magnification reveals a midline region of the primary palate (F). (modified from Diewert and Shiota, 1990) Figs. 3 A , B : Photographs showing frontal views of the craniofacial morphology of mouse embryos d u r i n g the stages of epithelial seam formation (A) and mesenchymal bridge formation (B). Figs. 4 A - D : The scanning electron micrographs reveal the fusion between the medial nasal prominence w i t h the lateral nasal and maxi l lary prominences (between arrows) d u r i n g the stages of epithelial seam formation (A) and mesenchymal bridge formation (C). Frontal sections through the face showing an intact epithel ial seam (between arrows) (B) and at the later stage, in i t ia l disruption of the epithelial seam and mesenchymal bridge formation (between arrows) (D). (modified from the Ph.D. thesis, Wang, 1992) 13 2. Morphogenesis of the heart The heart, like the face, is formed from two types of mesenchyme: neural crest-derived ectomesenchymal and mesodermal. Crania l neural crest cells contribute not only to the mesenchyme of the branchial arches, but also to mesenchymal walls of the aortic arch arteries (Phillips et al., 1987; Fuki ishi and Morr iss -Kay, 1992) and conduction tissue cells of the heart (Gorza et al., 1988; F i logamo et al., 1990). By us ing quai l chick chimeras, neural crest cells presumptive for branchial arches 3, 4, and 6 were found distributed to the outflow tract region of the heart (Phillips et al., 1987). Similarly, neural crest cells labelled w i t h D i l between occipital somites 1 and 2 or 3 and 4 migrated wi th in and dorsal to branchial arches 3 and 4 and into the outflow tract of the heart i n rat embryos (Fukiishi and Morr iss -Kay, 1992). A b n o r m a l migration of cranial neural crest cells is believed to be the cause of DiGeorge anomaly which includes abnormal i t ies of the craniofac ia l , t h y m u s , p a r a t h y r o i d , t h y r o i d , and cardiovascular systems (Lammer and Opitz , 1986). Morphogenesis of the heart can be divided into two phases, the pretubular and tubular phases. The pretubular phase is init iated by anterior-lateral migration of the cardiogenic mesoderm cells from the primitive streak that leads to formation of the heart tube (Garcia-Martinez and Schoenwolf, 1993). The o r i g i n of the precursors of the cardiac myoblasts , however , remains controversial. In avian embryos, it was suggested that the precursors migrate into the prospective pericardial region of the intra-embryonic coelom from either side of the foregut diverticulum (Rosenquist and De Haan, 1966). In mouse embryos, it was found that the precursors develop in situ by the differentiation of coelomic mesothelial cells that line the w a l l of the prospective pericardial component of the intra-embryonic coelom (Kaufman and Navaratnam, 1981). More recently, by u t i l i z i n g m y o s i n i m m u n o s t a i n i n g , it was proposed that the early rat 14 myocardium is a single epithelial unit ly ing i n front of the prechordal plate and the heart tube is formed by cohesive movement of the myocardial epithelium (Suzuki et al, 1995). The heart p r i m o r d i u m or cardiogenic plate is in i t i a l ly formed as a crescent-shaped zone of mesoderm cephalic to the embryonic disc at about embryonic day 15 i n humans (Hopper and Hart , 1980) and embryonic day 7 i n mice (Sissman, 1970). Subsequently, the heart pr imordium becomes canalized to form the endothelial tube on either side of the open gut. A s the floor of the foregut is closed, the paired endothelial tubes are brought together and then fuse to form a single, endocardial tube ly ing i n the midl ine. The endothelial tube is enveloped by the epimyocardia l rudiments . Therefore, at this stage, the pr imi t ive heart tube is composed of two layers: an endocardium and a m y o c a r d i u m w i t h E C M , the cardiac jelly, f i l l ed i n between the two layers ( M a r k w a l d et al, 1984). A s the heart further develops, the m y o c a r d i u m , part icular ly i n the ventricles, loses its epithel ial organizat ion and forms trabeculations at about embryonic day 9 i n mice shortly after the first myocardial contractions (Sissman, 1970). Subsequently, these trabeculations contribute to the interventricular septum and papillary muscles (Pexieder and Janecek, 1984; H a y et al, 1984). In later development, the heart tube becomes regionalized, forming atria and ventricles. The endocardium i n the regions of the atrioventricular canal and outf low tract undergoes epithel ial-mesenchymal transformation (Manasek, 1976). A s a result, endocardial cushion tissues are developed and give rise to septal and valvular structures (Van Mierop et al., 1962). Formation of the endocardial cushion tissue is a biphasic process: ini t ial ly , the endothelial cells become activated and subsequently, actively invade the underlying cardiac jelly (Runyan et al, 1992). The endocardial cushion tissue w i l l give rise to septal and valvular structures (Van Mierop et al, 1962). 15 Extracellular matrix is believed to play an important role i n heart development. The functions of the extracellular matrix include precardiac cell migrat ion (Linask and Lash , 1988), epithelial-mesenchymal transformation (Icardo and Manasek, 1984; Mjaatvedt et al, 1987; L i t v i n et al, 1992), valve formation (Swiderski et al., 1994), heart septation (Ahumada et al, 1981), and epicardial formation (Giudice and Steinberg, 1981). Molecules k n o w n to be present i n the cardiac jelly include hyaluronic acid (Manasek et al., 1973), sulfated proteoglycan (Gessner et al, 1965), fibronectin, laminin, types I-VI,VIII collagens (Kitten et al, 1987; Little et al., 1989; Kosher and Solursh, 1898; Drake et al, 1990; Samuel et al, 1994; Swiderski et al, 1994), and elastin (Hurle et al, 1994) (see Table II). Changes i n the distribution of these E C M molecules occur throughout heart development. For example, dur ing heart tube formation, fibronectin is found on the basal surfaces of the myocardium and endocardium. In later development, fibronectin increases i n myocardium and endocardium at the onset of trabeculation and decreases as trabeculation is completed. These findings suggested that fibronectin is involved i n cardiac trabeculation (Icardo and Manasek, 1983). Table II: Distribution of E C M and other molecules during heart development. Molecules Localization References BMP-2A atrial myocardium, A V C myocardium (mouse, days 9.5-14.5) Lyons et al, 1990 c-met Collagen I Collagen II Collagen III Collagen IV Collagen V Collagen VI 16 myocardium, endocardium, aorta (mouse, days 8, 9.5) cardiac jelly (chick, stage 7) subepicardium (chick, day 12) between endocardium and epimyocardium (chick, stages 14-19) E C tissue, cardiac jelly, myocardium, valves (chick, stages 18-23) E C tissue, valves (chick, stages 18-23) subepicardium (chick, day 8) cardiac jelly (chick, stages 7-9) myocardium, endocardium (chick, stage 17) E C tissue, valves (chick, stages 18-23) E C tissue, epicardium, valves (chick, stages 28-40) Andermarcher et al, 1996 Drake et al, 1990 Tidball 1992 Kosher and Solursh, 1989 Swiderski et al, 1994 Swiderski et al, 1994 Tidball 1992 Drake et al, 1990 Kit ten et al, 1987 Swiderski et al, 1994 Hurle et al, 1994 Collagen VIII CRABP-I CRBP CT-1 Elastin FGF-8 FGFR-1 Fibronectin HGF/SF 17 myocardium, valves (mouse, days 11-17, chick stages 19-33) E C tissue (mouse, day 12.5) r i E C tissue, epicardium (mouse, day 12.5) myocardium (mouse, days 8.5-15.5) OFT, E C tissue (chick, stages 22-29) epicardium, valves (chick, stages 30-40) heart primordia (mouse, days 7.75, 8.0) heart primordia, myocardium (chick, stages 8-30) myocardium, endocardium (chick, stages 14-17) cardiac jelly, E C tissue (chick, stages 16-29) pericardium, myocardium, endocardium (rat, somite stages 4-16) myocardium, endocardium (mouse, day 8) Sage and Iruela-Arispe, 1990 Iruela-Arispe and Sage, 1991 Dolle et al, 1990 Dolle et al, 1990 Sheng et al, 1996 Hur le et al, 1994 Hurle et al, 1994 Crossley and Mart in , 1995 Sugi et al, 1995 Icardo and Manasek, 1983; , Kitten et al, 1987 Icardo and Manasek, 1984 Tuckett and Morriss-Kay, 1986 Andermarcher et al, 1996 18 IGFsI&II Interleukin-la (IL-la) Laminin MMP-2 Msx-1 (Hox-7) RAR-a RAR -p RAR-y Rat interstitial collagenase-3 myocardium (chick, stage28) myocardium, endocardium, E C tissue (rat, days 10.5-14.5) cardiac jelly (chick, stages 9-15) myocardium, endocardium (chick, stage 17) pericardium, myocardium, endocardium (rat, somite stages 8-16) myocardial cell l ining (mouse, day 9) E C tissue (mouse, day 10.5) myocardium, E C tissue (mouse, day 12.5) no expression i n heart E C tissue (mouse, day 12.5) myocardium, endocardium, E C tissue (rat, days 10.5-14.5) Ralphs et al, 1990 Nakagawa et al, 1992 Drake et al, 1990 Kitten et al, 1987 Tuckett and Morriss-Kay, 1986 Reponen et al, 1992 Robert et al, 1989 Dolle et al, 1990 Dolle et al, 1990 Dolle et al, 1990 Nakagawa et al, 1992 19 TGF-pl TGF-p2 TGF-p3 TIMP-3 Urokinase PA V C A M - 1 E C tissue, endocardium (mouse, days 9.5,10.5) myocardium, A V C , OFT (mouse, days 9.5-12.5) O F T (mouse, day 8.5) myocardium, trabeculae (mouse, day 12.5) E C tissue, endocardium (quail, stage 17) epicardium, myocardium, intraventricular septum (mouse, days 8.75,11.5) Akhurst et al, 1990 Dickson et al, 1993 Dickson et al, 1993 Apte et al, 1994 McGuire and O r k i n , 1992 Kwee et al, 1995 A V C , atrio-ventricular canal; B M P , bone morphogenetic protein; c-met, H G F / S F receptor; C R A B P , cellular retinoic acid b inding protein; C R B P , cellular retinol b inding protein; CT-1 , cardiotrophin-1; E C , endocardial cushion; FGF, fibroblast growth factor; F G F R , fibroblast growth factor receptor; H G F / S F , hepatocyte growth factor/scatter factor; IGF, insulin-like growth factor; OFT, outflow tract; P A , plasminogen activator; R A R , retinoic acid receptor; T G F , transforming growth factor; T I M P , tissue inhibitor of metalloproteases; V C A M - 1 , vascular cell adhesion molecule-1 20 Neural plate orsal aorta Pericardia coelom myocard ia p r i m o r d i a doco rdiac jel ly rdial primordia B Neural groove tube Dorsal aorta Amnion— (cut) Pericardia 1 coelom Endocardium Cardiac jelly E pi myocardium Diagram II: Transverse sections of human heart showing four stages i n the fusion of the paired cardiac pr imordia . (From Patten's H u m a n Embryology. Elements of Cl inical Development. Corliss, 1976) 21 3. Morphogenesis of the eye Eye morphogenesis is an important event d u r i n g the craniofacial development. Often, the eyes are affected i n craniofacial syndromes such as holoprosencephalies, fetal alcoholic syndrome, and cyclopia (see review i n Johnston and Bronsky, 1995). The embryonic eye is formed as bi lateral evaginations of the forebrain, the optic vesicles (human: 22d; mouse: 9d) (Mann, 1964; Hopper and Hart , 1980). The optic vesicles extend laterally unti l they make contact w i t h the surface ectoderm of the head. Simultaneously, the surface ectoderm at the contact area becomes elongated and forms the lens placode. Subsequently, the lens placode and optic vesicle invaginate result ing i n formation of the lens pit and double-layered optic cup. The lens pit continues to invaginate unti l it separates from the surface ectoderm and forms a hol low bal l , the lens vesicle, embedded i n the underlying mesenchyme (Fig. 4). Fo l lowing formation of the lens vesicle, the epithelial cells at the posterior aspect, the posterior lens epithelium, are elongated and f i l l up the space of the lens vesicle, forming the lens fibers and producing lens-specific proteins, crystallins. A s the optic cup forms, the inner cell layer differentiates into the neural retina, while the outer cell layer differentiates into the retinal pigment epithelium, producing melanin. The optic stalk provides a pathway for the future optic nerve (for reviews see M c A v o y , 1980; Piatigorsky, 1981; Kaufman, 1992). Invagination of the lens ectoderm is believed to involve a multistep process of tissue interactions and inductions. Init ial ly, lens induct ion was speculated to depend on specific interactions between the presumptive lens ectoderm and optic vesicle (Spemann, 1901; L e w i s , 1907). H o w e v e r , later experiments have shown that other tissues can also induce lens formation from the presumptive lens ectoderm (Karkinen-Jaaskelainen, 1978; M i z u n o , 1972). These findings suggested that the presumptive lens ectoderm is predetermined 22 and capable of differentiation into lens under the appropriate conditions (Piatigorsky, 1981). M a n y mechanisms of invagination of the lens ectoderm have been proposed. These mechanisms include intracellular forces mainly from actin microfilaments (Wrenn and Wessells, 1969), forces generated by cell population pressure (Zwaan and Hendrix , 1973), localized cell death (Silver and Hughes, 1973), asymmetric migration of cells into the placode (Bancroft and Bellairs, 1977), and a close proximity of optic-vesicle E C M constituents which provide a stimulus for the basal growth of presumptive lens ectoderm (Parmigiani and M c A v o y , 1984; Peterson et al, 1995). wall of forebroin Rathke's pocket primary optic vesicle -wal l of forebroin fr—— head ectoderm optic cup head mesenchyme 9 days sensory layer of retina . pigment layer of retina 10 days sensory layer of retina ' pigment layer of retina 11 days early eyelid choroid fissure central retinal artery 11 days pigment layer of retina ,sensory layer of retina anter ior chambe 12 days posterior chamber optic stalk lens epi thel ium early eyel id eye muscle y optic nerve I3days I4days Diagram III: Sections of the developing eye of 9-tol4-day-old mouse embryo, (from The mouse. Its Reproduction and Development. Rugh, 1968) 23 4. Extracellular matrix and its degradation i n craniofacial and heart development Tissue remodel l ing is bel ieved to be important for embryogenesis particularly dur ing cell migration, proliferation and differentiation and tissue outgrowth (Matrisian, 1992; Werb et al, 1992). M a n y organ systems show changes of E C M molecules during their development. The mechanism of remodelling of E C M is thought to be involved w i t h matrix metalloproteinases (MMPs) because M M P s are found to be physiologically and pathologically capable of degrading a w i d e variety of E C M molecules (reviewed by Birkedal-Hansen et al., 1993; O v e r a l l , 1994). M M P s have been found to be expressed i n a number of developmental events, for example blastocyst outgrowth (Behrendtsen et al., 1992), tooth morphogenesis (Overall and Limeback, 1988; Sahlberg et al, 1992; He ik inhe imo and Salo, 1995), and branching morphogenesis of the salivary gland (Nakanishi et al, 1986; Hayakawa et al, 1992). The expression of 72-kDa gelatinase, i n particular, was studied by in situ hybridization and found i n the mesenchyme of the developing branchial arches, cornea, l imb, kidney, lung, and bone (Reponen et al., 1992). These data suggest that M M P s may play a significant role i n tissue remodelling i n the embryo. The basement membrane acts as an interface between histological ly dissimilar tissues that arise from different primary germ layers. Ultrastructurally, the basement membrane can be divided into three major zones; (1) the lamina lucida, an electron lucent zone 25-50 n m beneath the plasma membrane, (2) the lamina densa, an amorphous or finely fibrillar electron-dense layer ly ing beneath the lamina luc ida and extending for a thickness of 20-50 n m , and (3) the basement membrane re t iculum, a f ibr i l lar area below the lamina densa containing bundles of microfibril-like structures, anchoring fibrils (see review in Burgeson, 1993). The major molecular components of basement membranes are type IV col lagen, l a m i n i n , entactin, and heparan sulfate proteoglycan. 24 Immunoelectron microscopic studies revealed a preferential localization of type IV collagen, l aminin , proteoglycan, and entactin i n the lamina densa w i t h occasional projections into the lamina lucida (see review i n T i m p l and Dziadek, 1986). These components are present i n vir tual ly al l basement membranes, although their proportions may vary among basement membranes (see review i n F u r t h m a y r , 1993). Other basement membrane components i n c l u d i n g fibronectin, S P A R C , amyloid P, some complement components, and molecular isoforms of l a m i n i n and type IV collagen, are more restricted i n their distribution (see reviews in T i m p l , 1989; Fitch and Linsenmayer, 1994). In development, some the major components of the basement membrane are present i n mesenchyme. Type IV collagen is present not only i n basement membranes, but also i n the mesenchyme of the l imb bud (Solursh and Jensen, 1988), tooth mesenchyme (Heikinheimo and Salo, 1995), and cardiac jelly of the heart (Little et al., 1989). Similarly, laminin was also detected i n the mesenchyme of the mandibular arch and the guidance pathway of the trigeminal axon i n chicken embryos (Riggott and M o o d y , 1987), the cranial mesenchyme (Tuckett and Morriss-Kay, 1986), and early ganglia and nerve roots (Rogers et al., 1986). Tissue interactions are defined as events i n w h i c h d iss imi lar cell populat ions act on one another to alter cell behavior i n developmental ly significant ways. It is believed that every organ i n the adult body of many species arises as a result of tissue interactions (Wessells, 1977). The basement membrane has been speculated to play a crucial role in epithelial-mesenchymal interactions during embryogenesis (Grobstein, 1954; Bernfield et al, 1984). Alterations of the basement membrane components are important for branching morphogenesis of the salivary gland (Bernfield et al, 1984) and the lung (Grant et al, 1983; Schuger et al, 1990). Degradation of the basement membrane is believed to al low the transmission of inductive signals from one tissue to another, for example i n 25 formation of kidney tubules (Wartiovaara et al, 1974; Ekblom, 1981) and tooth morphogenesis (Thesleff, 1981; Sahlberg et al., 1992). Epithel ial-mesenchymal interactions have been found to be important for facial morphogenesis in chick embryos (Wedden, 1987; Richman and Tickle, 1989,1992). The facial ectoderm, particularly, is required for the outgrowth and accompanying differentiation of cartilage to form rod-like structures wi th in the frontonasal mass and mandible i n chick embryos (Wedden, 1987; Richman and Tickle, 1989). Recombinations between epithelium and mesenchyme from different prominences showed that the epithelia are interchangeable and appear to be equivalent (Richman and Tickle , 1989). It was also found that removal of the epi thel ium from the maxi l lary prominence explant disturbed the continued growth of the explant (Saber et al., 1989). Recombination experiments from maxi l lary epi thel ium-maxi l lary mesenchyme, l imb apical ectodermal r idge-maxil lary mesenchyme, and stage 28 epithelium-stage 22 mesenchyme indicated that direct epithelial-mesenchymal contact and normal architecture of the interface between the two tissues are important for the viabi l i ty of the tissue explant. Therefore, it was suggested that the basement membrane within the tissue interface may influence epithelial-mesenchymal interactions. Each of the major extracellular molecules w i l l be discussed in the next section. 4.1 Laminin L a m i n i n is the most abundant glycoprotein i n the basement membrane composed of three genetically distinct polypeptide chains referred to as a (A) (400 kDa), (3 (Bl) (220 kDa), and y (B2) (200 kDa) chains. These three chains are connected to each other by disulfide bonds (Timpl, 1989; Burgeson et al., 1994). L a m i n i n is synthesized by different types of cells inc luding epithelial , and 26 endothelial cells, muscle cells and fibroblasts (Paulsson, 1992). Molecules k n o w n to b ind laminin include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, hepar in , cell-surface glycoproteins, sulfatides, and gangliosides (Kle inman et al, 1987). L a m i n i n interacts w i t h cells v ia the transmembrane glycoprotein, integrin. This interaction is believed to mediate the signal to the cytoskeleton and influence cell proliferation and differentiation (Paulsson, 1992). So far, eight different laminin chains; including a l , a2 (M), a3 (K), f31, f32 (S), (33, y l , and y2 (B2t); and seven different heterotrimeric assembly forms; inc luding laminins-1, -2, -3, -4, -5, -6, and -7; have been characterized (see reviews i n Burgeson et al, 1994; T i m p l and Brown, 1994). These findings suggest that the functional diversity of basement membranes arises i n part f rom the particular laminin isoforms they contain (Miner and Sanes, 1994). For example, laminin a chain polypeptide was mainly detected i n basement membranes of epithelial cells, suggesting that this chain is important for morphogenesis of the epithelial sheets (Klein et al, 1990). Renal glomerular basement membrane contains a , 02 and y chains, while basement membrane of the extrasynaptic muscle contains oc2, P and y chains (Sanes et al, 1990). Antibodies are available to various isoforms of laminin (Miner and Sanes, 1994; Durham and Snyder, 1995). L a m i n i n appears as the first E C M protein during embryogenesis as early as the two-cell stage (Dziadek and T i m p l , 1985). Only the p and y polypeptides are synthesized unti l the 8-cell stage. Then an a chain starts to appear from the 16-cell stage (Cooper and MacQueen , 1983). L a m i n i n plays a diverse role i n numerous biological activities i n c l u d i n g neurite outgrowth, cell adhesion, p r o l i f e r a t i o n , m i g r a t i o n , e p i t h e l i a l ce l l d i f f e r e n t i a t i o n , phagocytos i s , angiogenesis, and tumor metastases (see reviews i n E k b l o m , 1993; Kle inman et 27 al, 1993; Yurchenco, 1994). L a m i n i n is suggested to be important for embryonic l u n g morphogenesis since anti- laminin-treated explants showed a marked inhibi t ion of branching morphogenesis and a distortion of the bronchial tree (Schuger et al., 1990, 1991). Different temporal patterns of l a m i n i n a, P and y chains dur ing l u n g development suggested that each chain is independently regulated and may play important, but different roles i n fetal lung development (Durham and Snyder, 1995). In kidney development, w h e n the mesenchyme transforms into the epithelium and forms tubular structures, the mesenchyme expresses pr imari ly the laminin P and y chains, based on immunofluorescence, Western and Northern blot analyses (see review i n Ekblom, 1993). Later, as the epithelial cells develop, the m R N A expression of l aminin a l chain increases. These f indings were confirmed by in situ hybr idizat ion results i n w h i c h the expression of the a l chain was mainly localized to areas of developing epithelial cells (Ekblom et al, 1990). These data suggest that local cell-cell interactions enhance product ion of l aminin chains and epithelial cell polarizat ion may be associated w i t h the presence of laminin containing the a chain (Ekblom, 1993). Recently, mutations i n human y2 chain gene was found to be correlated w i t h a skin disease, epidermolysis bullosa (Pulkkinen et al, 1994). 4.2 Type IV collagen Type IV collagen is the pr inc ipal collagenous component found as a molecular network i n the basement membrane. One of the major functions of type IV collagen is to provide mechanical support for the tissues (Timpl, 1989). Type IV collagen also possesses b inding activities for E C M such as laminin , proteoglycan, entactin (nidogen), and cells (T impl , 1989). Type IV collagen 28 comprises three polypeptide chains [al(IV)2 a2(IV)]. Recently, variant type IV collagen oc3, a4, a5 and a6 chains have been identified and found i n a more restricted tissue distribution (Hudson et al, 1993). For example, the oc3 and a4(IV) collagen chains are present i n the synaptic basal lamina at the neuromuscular junction, whi le the a l and a2(IV) collagen chains are found extrasynaptically (Sanes et al, 1990). In rat kidney development, a l and a2(IV) collagen chains and the l a m i n i n B l appear to be fetal components of the glomerular basement membrane. Subsequently, there is a developmental switch to a3-a5(IV) chains and S- laminin i n mature glomerular basement membrane (Miner and Sanes, 1994). Type IV collagen is believed to be the first collagen to appear i n the embryo. It was found i n the inner cell mass of the 3- to 4-day-old blastocysts (Leivo et al., 1980) and subsequently in the basement membranes of the ectoderm and endoderm, notochord and neural tube (Hay, 1991). Immunohistochemical studies revealed that the pathways for neural crest migration not only contain fibronectin, type I collagen, laminin, heparan sulfate proteoglycan and entactin but also type IV collagen. Studies of neural crest cell migrat ion on various substrates in vitro demonstrated that type IV collagen, laminin, fibronectin and type I collagen permit migration whereas type II collagen does not (Fitch and Linsenmayer, 1994). Type IV collagen and laminin were implicated as important for endothelial cell attachment and polarization during embryonic angiogenesis (Grant et al., 1990). Mutation of type IV collagen gene, that causes impairment of triple helix stability, leads to embryonic lethality in C. elegans (Guo et ah, 1991). 29 4.3 Fibronectin Fibronectin is a glycoprotein characteristically present i n adult connective-tissue stroma and embryonic mesenchyme. The protein comprises a dimer of two similar 250-kDa subunits held together by a pair of disulfide bonds at their carboxyl termini . Fibronectin is encoded by a single gene. However , several isoforms occur as a result of an alternative spl ic ing of its precursor m R N A (Yamada, 1991). Fibronectin is not only found i n the E C M of the mesenchyme but also i n basement membranes of the embryo (Stenman and Vaher i , 1978; T i m p l and M a r t i n , 1982; Yamada, 1991). However , the or ig in of basement membrane fibronectin remains controversial. Since fibronectin is present i n plasma, therefore fibronectin can be deposited i n the basement membrane by simple infiltration. Alternatively, fibronectin can be synthesized by cells resting on the basement membrane (Liotta et al., 1986). D u r i n g mouse embryogenesis, fibronectin is first detected i n the inner cell mass during primitive endoderm formation and at the the interface between the trophectoderm and the parietal endoderm (Zetter and Mart in , 1978: Leivo et al., 1980). Subsequently, fibronectin is present i n between the mesodermal and ectodermal layers (Wartiovaara et al., 1979). Later i n development, fibronectin has been found to become associated wi th different tissues. Thus, it is speculated that fibronectin plays different roles i n different regions of the embryo at specific per iods of time (Thiery et al., 1989). These include cel l adhesion, cel l p r o l i f e r a t i o n , m i g r a t i o n of mesenchymal and neura l crest cells , ce l l dif ferentiat ion, spreading and growth of endothel ial cells and structural organization of embryonic matrix (Bronner-Fraser, 1986; Yamada, 1991; Lesot et al., 1990; Thiery et al., 1989; H a y , 1991). Recently, fibronectin n u l l homozygous mouse embryos were generated and found to implant and initiate gastrulation normally (George et al., 1993). However, i n later development various detected 30 abnormalit ies were f o u n d i n c l u d i n g deformed neura l tubes, absence of notochord and somites, deformed vessels and heart, and severe defects i n mesodermally-derived tissues all of which led to early embryonic lethality. These findings suggested that these abnormalities arise from fundamental deficits i n mesodermal migration, adhesion, proliferation or differentiation as a result of the absence of fibronectin. 4.4 Remodelling of the E C M D u r i n g embryonic development, E C M molecules, w h i c h are the major components of the tissue stroma, have to be locally and co-ordinately degraded and synthesized i n order to facilitate morphogenesis. The basement membrane is a good example for remodell ing of the E C M since it is f o u n d to be rap id ly remodelled d u r i n g embryonic development (Bernfield, 1981; T i m p l and Dziadek, 1986). There are a number of situations i n w h i c h the basement membrane is completely degraded as the organs or structures undergo regression, differentiation or invasion. For example, type IV collagen, laminin , heparan sulfate proteoglycan, and fibronectin were found to become irregular and discontinuous, as the male Mul ler ian duct regresses (Ikawa et al, 1984). These findings indicate that degradation of the E C M around the male Mul ler ian duct is a prior event to regression of the structure. D u r i n g early tooth development, the basement membrane between the enamel organ and the dental papi l la is thought to play a role i n mediating epithelio-mesenchymal interactions, resulting i n odontoblast differentiation. In the late bel l stage of tooth development, w h e n the odontoblasts secrete predentine, the dental basement membrane degrades. Subsequently, the preameloblast undergoes differentiation (Hurmerinta and Thesleff, 1981; Thesleff and Hurmerinta , 1981; Sahlberg et al., 1992). D u r i n g the i n v o l u t i o n of the m a m m a r y g land, the 31 basement membrane around the secretory alveoli becomes discontinuous and degraded as the secretory alveolar structures degenerate (Warburton et al., 1982). In early secondary palate formation, the midline epithelial seam is formed by fusion of the medial edge epithelia of the palatal shelves (Ferguson, 1988). Subsequently, the epithelial seam as wel l as the basement membrane becomes disrupted w i t h ingrowth of the mesenchymal tissue. Breakdown of the seam basement membrane was demonstrated by immunolocal izat ion of type IV collagen (Ferguson, 1988) and laminin (Shuler et al., 1992). Similar events, i n which the basement membrane is degraded, have also found in ductal growth of the submandibular gland (Bernfield et al., 1984), angiogenesis (Form et al., 1986), and tumor invasion and metastasis (Barsky et al., 1983). However, the molecular events of E C M remodelling during embryonic development are addressed i n the present work. 32 5. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) In general, degradation of the extracellular matrix involves at least four distinct pathways including plasminogen-dependent, matrix metalloproteinase, phagocytic, and osteoclastic pathways (see review i n Birkedal-Hansen et al., 1993). This review focuses on the matrix metalloproteinase pathway because it is thought to play a major role i n tissue remodel l ing d u r i n g the embryonic development. -5:i MMPs and TIMPs Matr ix metalloproteinases (MMPs) are believed to play a major role i n physiological degradation of E C M because they are found to be present in various types of both adult and embryonic cells and tissues and able to degrade a wide variety of E C M molecules i n c l u d i n g type I -V, VI I -X collagens, gelat in, f ibronectin, l a m i n i n , proteoglycans, and elastin (see Table III). M M P s are classified into the same family on the basis of extracellular substrate specificity, zinc dependence, inhibit ion by tissue inhibitors of metalloproteinases (TIMPs), secretion as a zymogen, and D N A sequence relatedness (Matr is ian, 1990; Alexander and Werb, 1991). M M P family is composed of four members; (1) the collagenases, (2) gelatinases/type IV collagenases, (3) stromelysins, and (4) membrane-type M M P s ( M T - M M P s ) (Table III). The collagenases specifically degrade native type I-III collagens to produce thermally unstable degradation fragments. U p o n collagen denaturation, gelatin and the collagenase cleavage products can be further degraded by other M M P s inc luding gelatinases and stromelysins (reviewed by Overall , 1994). The 72-kDa (MMP-2) and 92-kDa type IV gelatinases (MMP-9) also degrade native types IV ,V (Fessler et al., 1984), VII (Seltzer et al., 1989), X (Welgus et al., 1990) collagens and elastin (Senior et al., 33 1991). Al though 72-kDa gelatinase can degrade fibronectin and laminin, 92-kDa gelatinase does not (Okada et al, 1990; Nagase et al, 1991). Stromelysins have a broader substrate specificity inc luding proteoglycans, l a m i n i n , f ibronectin, gelatin, and the globular port ion of type IV collagen (Galloway et al, 1988; Nicholson et al, 1989). Recently, nucleotide and amino acid sequences of human and mouse 72-k D a gelatinases were characterized. It was found that the overall amino acid sequence s imilari ty between the mouse and human enzymes proper (active enzyme form) is 96.6%. The zinc-binding domain is 100% identical. The sequence of the mouse carboxyl terminus contains two residues more than the human enzyme (Reponen et al., 1992). H u m a n 92-kDa gelatinase exhibits 49% homology to human 72-kDa gelatinase (Wilhelm et al, 1989). Table III: M M P family members. MMPs Substrates I. Collagenases -Interstitial collagenase collagens I,II,III,VII, ( M M P - 1 , fibroblast collagenase) VIII,X, gelatin - P M N collagenase collagens I,II,III,VII, (MMP-5) VIILX, gelatin II. Gelatinases -Gelatinase A gelatin, collagens IV ,V , ( M M P - 2 , 72-kDa gelatinase, 72-kDa type IV collagenase) VII,X,XI, elastin, fibronectin, laminin 34 -Gelatinase B (MMP-9 , 92-kDa gelatinase, 92-kDa type IV collagenase) III. Stromelysins -Stromelysin 1 ( M M P - 3 , transin) -Stromelysin 2 (MMP-10, transin-2) -Stromelysin 3 (MMP-11) -Matr i lysin (MMP-7 , PUMP-1) IV. Membrane-type M M P s ( M T - M M P s ) - M T - M M P - 1 - M T - M M P - 2 gelatin, collagens IV ,V , elastin, fibronectin proteoglygans, laminin, fibronectin, gelatin, collagens IV,V, IX,X proteoglycans, laminin , fibronectin, gelatin, collagens III,IV,V,IX proteoglycans, gelatin, fibronectin, elastin, laminin, collagen IV progelatinase A progelatinase A ( M o d i f i e d f rom Matrisian,1992; Birkedal-Hansen et al.,1993; and B i r k e d a l -Hansen, 1995) Regulation of M M P activity occurs at different levels of expression. A t the transcriptional level, many growth factors, cytokines, oncogene products, tumor promoters, and hormones have been found to either stimulate or suppress transcription of M M P s (see reviews i n Woessner, 1991; Birkedal-Hansen et al., 1993). For example, 12-O-tetradecanoyl phorbol 13-acetate (TPA) can affect 35 transcription of M M P s via the A P - l / T R E binding site (Angel et al., 1987). M M P s are secreted as a latent, inactive form and can be in vitro activated by many mechanisms including treatment wi th organic mercurides, chaotropic agents, or proteases. For example, trypsin was found to be able to activate almost all M M P s (Liotta et al., 1979; Salo et al., 1983; Saari et al., 1990), whi le chymotrypsin can activate stromelysin-1 (Nagase et al., 1990) and neutrophil collagenase (Saari et al., 1990). Activation of the precursors of interstitial collagenase and stromelysin can occur v i a a p lasminogen-p lasmin-MMP cascade (Chapman et al., 1988; Thomson et al., 1989). Activated stromelysin, i n turn, can degrade collagenase into highly active collagenase (Nagase et al., 1991). Therefore, it was proposed that, in vivo, the precursors of interstitial collagenase and stromelysin are activated by a protease cascade mechanism (Diagram III) (Matrisian, 1992). plasminogen •• urokinase • p l a s m i n Transcriptional ( A P - l / P E A - 3 ) activation procollagenase • prostromelysin-••collagenase Stromelysin highly active collagenase Diagram III: A protease cascade of activation of M M P s in vivo ( Matrisian, 1992). 36 The in vivo mechanism of activation of progelatinase A is believed to be different from the other types of M M P s as shown above. Indeed, activation of progelatinase A has been found to be plasma membrane-dependent (Ward et al., 1994) and associated wi th a new member of M M P s , membrane-type M M P (MT-M M P - 1 ) (Sato et al., 1994; Strongin et al, 1993, 1995). M T - M M P - 1 is an integral plasma membrane protein w i t h the molecular weight of 66 kDa . It was found that expression of M T - M M P - 1 on the cell surface induces specific activation of progelatinase A (Sato et al, 1994). M t - M M P - 1 was also found to be expressed in the human l u n g carcinoma (Sato et al, 1994). Strongin et al. (1993,1995) have demonstrated that M T - M M P - 1 acts as a cell surface receptor for TIMP-2 . By binding its carboxyl-end domain to M T - M M P * TIMP-2 complex at the cell surface, the proenzyme of 72-kDa gelatinase becomes activated. More recently, M T - M M P -2 c D N A was isolated from a human placenta c D N A library (Takino et al., 1995). Like M T - M M P - 1 , M T - M M P - 2 can induce processing of progelatinase A into the activated forms. TIMPs are molecules found to be able to form non-covalent bimolecular complexes w i t h the active forms of M M P s and, i n some instances, w i t h latent M M P precursors. Thus, it was implicated that the local activity of M M P s is regulated by TIMPs (see review i n Matr is ian, 1992). TIMP-1 (Cawston, 1986) inhibits a l l M M P s as does TIMP-2 (Stetler-Stevenson et al, 1989). TIMP-2 , however, is 2 to 10 fold more effective than TIMP-1 at inhibiting 72-kDa and 92-k D a gelatinases; whereas interstitial collagenase is inhibited by TIMP-1 more than twofold more effectively than it is by TIMP-2 (Howard et al, 1991). Recently, the pr imary structure of mouse TIMP-2 was characterized and found to have ex t raordinar i ly h i g h homology (97%) to h u m a n T I M P - 2 , suggest ing a fundamental b iological role of the protein (Shimizu et al, 1992). Besides inhibit ion of M M P activities, both TIMP-1 and TIMP-2 have a potent growth-37 promoting activity for a wide range of cells. This activity was suggested to be a direct cellular effect mediated by a cell surface receptor (Hayakawa, 1994). TIMP-3 (ChIMP-3, chicken inhibitor of metalloproteinases) was recently characterized (Apte et al., 1994) and appeared to have similar fuctions as TIMP-1 and TIMP-2 (Hayakawa, 1994). Very recently, a novel TIMP-4 was cloned from a human heart c D N A l ibrary (E. Shi , personal communication). Its function remains to be determined. M M P s and TIMPs have been detected i n a wide variety of embryonic tissues and suggested to play an important role in tissue invasion, cell migration, proliferation, and differentiation dur ing embryogenesis. There are numerous examples of expression of E C M - d e g r a d i n g proteinases a n d / o r T I M P s i n developmental processes including ovulation (Curry et al., 1990), embryonic pre-and peri-implantations (Brenner et al., 1989; Behrendtsen et ah, 1992; Lefebvre et al, 1995; Harvey et al, 1995; Reponen et al, 1995; Canete-Soler et al, 1995), endoderm differentiation (Adler et al, 1990; Brenner et al, 1989), branching morphogenesis of salivary gland (Nakanishi et al, 1986; Hayakawa et al, 1992) and lung (Ganser et al, 1991), involution of the tadpole tail (Gross and Bruschi, 1971), bone and cartilage formations (Dean et al, 1985; Edwards et al., 1992; Flenniken and Wi l l i ams , 1990; Apte et al, 1994), neurite extension (Pittman, 1985; M u i r , 1994), tooth morphogenesis (Overall and Limeback, 1988; N o m u r a et al, 1989; Sahlberg et al, 1992), heart development (Nakagawa et al, 1992; M c G u i r e and O r k i n , 1992), retina development (Sheffield, 1992; Sheffield et al, 1994), myogenesis (Guerin and H o l l a n d , 1995), secondary palate development (Brinkley et al, 1995; D u et al, 1996; Morr i s -Wiman et al, 1996), mammary gland development (Talhouk et al, 1991, 1992; Dickson and Warburton, 1992; Sympson et al, 1994) and mandibular condyle development (Breckon et al, 1994). 38 Collectively, M M P s and TIMPs are suggested to play a significant role i n embryogenesis. However, the in vivo mechanism of tissue remodell ing remains speculative and circumstantial. Involvement of M M P s and TIMPs dur ing pre-and peri-implantation and blastocyst outgrowth of the mammalian embryo has been extensively studied. A t about 4.5 days p.c. in mice, the trophectodermal cells of the blastocyst penetrate the uterine epithelium and its basement membrane and then invade the uterine decidual stroma. Studies of embryonic implantation i n culture have been studied by placing the blastocyst on a complex E C M (Glass e t al., 1983). Fol lowing attachment of the blastocyst to the E C M , trophoblast cells migrate out and invade the E C M by degrading it. Several M M P s , inc luding collagenase, stromelysin, 72-kDa and 92-kDa gelatinases, u P A and TIMP-1,-2,-3 are secreted by trophoblast cells (Brenner et al., 1989; Behrendtsen et al., 1992; Harvey et al., 1995). Exogenous TIMP-1 was found to stimulate the migration of parietal endoderm-like cells from the blastocyst outgrowths (Werb et al., 1992). These data suggest that the ability of the embryo to invade the uterine epithel ium and stroma is, at least i n part, regulated by M M P s and T I M P s (Matrisian, 1990). By in situ hybridization, Reponen et al. (1995) and Canete-Soler et al. (1995) found that 92-kDa gelatinase but not 72-kDa gelatinase was strongly expressed i n invading trophoblasts. TIMP-3 but not TIMP-1 or TIMP-2 was intensely expressed i n maternal cells i n the area surrounding the i n v a d i n g embryonic tissue (Reponen et al., 1995). These results suggested a cooperative role between 92-kDa gelatinase and TIMP-3 i n the E C M proteolysis associated w i t h implantation of the early embryo. Dur ing pre-implantation development, it was found that gelatinases are markedly induced by T G F - a in vitro (Dardik et al., 1993). These data suggest that upon stimulation by T G F - a , gelatinases, secreted from the blastocyst, may be involved i n blastocoel E C M remodel l ing and 39 migration of the parietal endoderm cells. More recently, growth factors and cytokines, particularly E G F and leukemia inhibitory factor (LIF), were found to stimulate u P A and M M P - 9 activities i n the blastocyst cultures during the early, h ighly invasive phase of implantation (Harvey et al., 1995). In the later stages, LIF was found to decrease production of both proteinases, whi le E G F had no effect. These results suggested that LIF may also down-regulate invasiveness of the blastocyst i n later development. M M P s and T I M P s are also bel ieved to participate i n branching morphogenesis of the salivary gland and lung (Nakanishi et al.,1986; Fukuda et al., 1988; Hayakawa et al., 1992; Ganser et al., 1991), a process that requires both E C M stabilization and degradation, as wel l as cell migration and proliferation (Werb et al., 1992). U p o n st imulation by E G F and T G F - a , the gelatinases, particularly 72-kDa gelatinase, were secreted by the lung explants (Ganser et al., 1991). Exogenous collagenase can inhibit branching of the sal ivary gland (Nakanishi et aZ.,1986; Fukuda et al., 1988) and lung (Ganser et al., 1991) i n culture. T I M P , on the other hand, can stimulate supernumerary b u d d i n g of the salivary gland. These findings suggested that collagenase and T I M P regulate branch ing morphogenesis through r e m o d e l l i n g of the E C M molecules (Hayakawa et al, 1992). Tissue remodelling during secondary palate formation was also shown by i m m u n o l o c a l i z a t i o n of several E C M components ( M o r r i s s - W i m a n and Brinkley, 1992). It was found that type III collagen, fibronectin, and hyaluronate are the major components of the E C M infrastructure of the deve loping secondary palate. A s development proceeds, hyaluronate was found to be expanded, while type III collagen and fibronectin become more circumscribed. Recently, the expression of both m R N A and protein of M M P s and TIMPs have been investigated i n the developing mouse secondary palate (Brinkley et al, 40 1995; D u et al, 1996; M o r r i s - W i m a n et al., 1996). By ut i l iz ing zymography and reverse transcriptase polymerase chain reaction (RT-PCR) techniques, urokinase plasminogen activator (uPA) , M M P - 1 , -2, and -9 were identi f ied in vivo (Brinkley et al, 1995) and u P A M M P - 1 , -2, -3, -7, and -9 were detected i n secondary palate explants (Morris-Wiman et al, 1996). TIMP-1 and TIMP-2 were also found to be expressed during in vivo and in vitro palatogenesis (Du et al, 1996). These data suggest that M M P s , serine protease, and TIMPs are invo lved i n E C M remodelling during secondary palate morphogenesis. 5.2 M M P s , T I M P s and growth factors In most cell types, genes of M M P s are not constitutively expressed but rather are regulated at the transcriptional level by treatment w i t h a variety of factors i n c l u d i n g g r o w t h f a c t o r s / c y t o k i n e s , okadaic a c i d , bac ter ia l l ipopolysaccharide (LPS), hormones, oncogene products and tumor promotors (see review i n Birkedal-Hansen et al, 1993). For example, expression of some M M P s is induced by interleukin-lp, tumor necrosis factor-a (MacNaul et al, 1990), platelet-derived growth factor (PDGF) , E G F (Kerr et al, 1988), bFGF (Edwards et al, 1987) and nerve growth factor (NGF) (Machida et al, 1991). TGF-p can repress the transcription of collagenase and stromelysin-1 (Edwards et al, 1987; Kerr et al, 1990) but upregulates 72-kDa and 92-kDa gelatinases (Overall et al, 1991; Salo et al, 1991). TIMP-1 and -2 genes are differently regulated. TIMP-1 expression is stimulated.by E G F , T N F - a (Mawatari et al, 1989), TGF-p (Edwards et al, 1987), retinoids and glucocorticoids (Clark et al, 1987); whereas, TIMP-2 expression is d o w n regulated by TGF-P (Stetler-Stevenson et al, 1990). TIMP-1 , i n particular, has been localized to the same embryonic tissues where TGF-p expression has 41 been detected (Heine et al., 1987; Flenniken and Wil l iams, 1990). These findings suggested that there is a functional interaction between TIMP-1 and TGF-f3 in vivo (Flenniken and Wil l iams, 1990). Based on substrate specificity, 72-kDa gelatinase appears to be a major enzyme for basement membrane degradation since it can degrade type IV collagen, laminin, and fibronectin (see Table III). In addition, 72-kDa gelatinase has been found to be associated w i t h disruption of the basement membrane of the developing mammary gland (Dickson and Warburton , 1992) and the d e v e l o p i n g tooth (Sahlberg et al., 1992; H e i k i n h e i m o and Salo, 1995). Accordingly , i n the present study, 72-kDa gelatinase was hypothesized to be involved i n epithelial seam disruption dur ing primary palate morphogenesis and remodelling of the basement membrane during heart morphogenesis. 42 6. Growth factors G r o w t h factors are smal l polypept ides funct ioning m a i n l y i n cel l proliferation and differentiation. They have been found i n a variety of tissues particularly embryonic and w o u n d tissues. In addit ion, they have also been identified i n some neoplasia and certain diseases. Growth factors can be classified into at least f ive families based on nucleotide and amino acid sequence homology and similar receptor-binding activity. These growth factor families include (1) E G F , (2) insulin-l ike growth factor (IGF), (3) fibroblast growth factor (FGF), (4) platelet-derived growth factor (PDGF) and (5) transforming growth factor-beta (TGF-P) families (Table III). G r o w t h factors can act i n autocrine, paracrine or endocrine regulation pathways (for reviews see Mercola and Stiles, 1988; Pusztai et al, 1993). Table IV: Growth Factor Families Family Members Epidermal growth factor E G F , T G F - a , amphiregulin (AP), heparin-binding EGF-l ike growth factor (HB -EGF) , Vaccinia virus growth factor (VGF) , myxomavirus growth factor (MGF) , Shope fibroma growth factor (SFGF), Schwannoma-derived growth factor ( S D G F ) , Cripto, gp30 Fibroblast growth factor FGF-1 (acidic F G F , aFGF, endothelial cell growth factor, (Heparin-binding growth factor) ECGF) 43 Insulin-like growth factor Platelet-derived growth factor Transforming growth factor-P FGF-2 (basic FGF, bFGF) FGF-3 (int-2), FGF-4 (k-FGF) FGF-5 , FGF-6 FGF-7 (keratinocyte growth factor, K G F ) FGF-8, FGF-9 IGF-I, IGF-II (Somatomedin-C) R e l a x i n P D G F a a , P D G F p p , P D G F a P T G F - p i , TGF-p2, TGF-P3, TGF-p4, TGF-p5, Inhibin a , I n h i b i n / a c t i v i n p A , Inhib in/ac t iv inpB, Mul ler ian inhibitory substance (MIS), D P P - C (decapentaplegic gene), 60A, V g l (vegetal pole genel), Vgr-1 (vegetal-pole-gene-related-1), Vgr -2 , D V R (decapentaplegic-Vg-related)/BMP-2-4, D V R / B M P - 5 - 7 , Osteogenin, OP-1 (osteogenic protein-1), OP-2, GDF-1 (growth and differentiation factor-1), G D F - 3 , G D F - 9 , N o d a l (modified from Derynck, 1992; Heikinheimo, 1993; Pusztai et al, 1993; Mi l ler and Rizzino, 1994) 44 E G F Family G r o w t h factors p lay an important role i n w o u n d h e a l i n g and embryogenesis by s t i m u l a t i n g cel l p r o l i f e r a t i o n and e n h a n c i n g ce l l differentiation (see reviews i n Mercola and Stiles, 1988; M i l o s , 1992). The E G F family inc luding E G F and T G F - a is one of the major growth factor families detected i n embryonic tissues. Previous studies of the expression of E G F , T G F - a and their common receptor, EGF-R suggest that these growth factors participate i n growth and development of various embryonic tissues for example the blastocyst (Adamson, 1993; Chia et al, 1995), tooth (Partanen and Thesleff, 1987; Partanen, 1990; Shum et al, 1993), mandible (Kronmiller et al., 1993; Shum et al, 1993), and secondary palate (Dixon et al, 1991). However, the expression of E G F , T G F - a , and EGF-R and their relationship w i t h cell proliferation during primary palate morphogenesis were u n k n o w n . Therefore, i n the present study, the expression of these molecules i n the developing primary palate was investigated. E G F and T G F - a are the pr imary E G F family members expressed i n mammalian tissues. E G F and T G F - a are similar i n many aspects, for example they exert their effects via the same integral membrane glycoprotein receptor, EGF-receptor (Cohen et al., 1980; Todaro et al, 1980; Massague, 1983), they are mitogenic peptides that regulate cell proliferation and differentiation (de Larco and Todaro, 1978; Derynck et al, 1984), and the molecular weight of each mature peptide are approximately 6 kDa (Carpenter and Wahl , 1991; Derynck et al, 1984). T G F - a shares about 33-44% homology w i t h E G F (Heath, 1993). However , the transmembrane and cytoplasmic domains of T G F - a and E G F demonstrate no similarity (Marquardt et al, 1984). The lack of similarity in these regions suggests a difference i n the function of these two growth factors (Kumar et al, 1995). TGF-45 a does not cross-react w i t h anti-EGF antibodies suggesting structural differences between E G F and T G F - a (Schlessinger et al, 1983). E G F and T G F - a and their receptor (EGF-R) have been detected i n various embryonic and adult tissues and i n both normal and pathological conditions. 6.1 EGF E G F m R N A encodes a transmembrane precursor glycoprotein of 1207 amino acids. In mouse submandibular gland, the precursor molecule is rapidly processed to the 53-amino acid form of E G F . Both the precursor and mature forms can bind to EGF-R and activate the target cells (Carpenter and Wahl , 1991). EGF was first isolated from mouse submandibular salivary glands (Cohen, 1962). Later by amino acid analysis, mouse EGF was shown to be identical w i t h human P-urogastrone (Gregory, 1975). E G F transcripts and protein were in i t i a l ly detected i n the h u m a n preimplantation embryo (Chia et al., 1995). In later development, E G F expression has been detected i n the developing mandible (Kronmiller et al., 1991a), tooth, lung (Snead et al., 1989), pancreas, skin, k idney, and submandibular salivary gland (Miettinen, 1993). E G F protein has been identified i n various fetal tissues inc luding the kidney, gastrointestinal tract, submandibular gland, lung , l iver, bra in , placenta, and amnion (Kasselberg et al., 1985; Poulsen et al., 1986; Stahlman et al, 1989; Shigeta et al, 1993). E G F and E G F - R were also found to be coexpressed i n the mouse embryonic mandible from E10 through E15 by using R T - P C R technique (Shum et al, 1993). E G F transcripts were local ized to ectomesenchymal cells associated w i t h precartilage, cartilage, bone, and tooth-forming cells (Shum et al, 1993). More recently, E G F immunoreact ivity was studied i n human fetuses and found to be present i n the placenta, skin, distal 46 tubules of the kidney, surface epithelium of the stomach, tips of the small intestinal v i l l i , and glandular structures of the stomach, duodenum, pancreas, and trachea (Poulsen et al., 1996). Physiological roles of E G F i n development have been studied in vivo and in vitro. U p o n injection into newborn mice, E G F stimulated precocious eye opening and incisor eruption (Cohen, 1962). E G F also enhanced growth and keratinization of epithelial cells (Cohen, 1962; Cohen and Elliot, 1963). It has been proposed that E G F is necessary for the initiation of odontogenesis (Kronmiller et al., 1991b; Shum et al, 1993). However, exogenous E G F altered the pattern of the dental lamina, producing supernumeraries i n the diastema region i n organ cultures (Kronmil ler , 1995), inhibited early mouse tooth morphogenesis and cytodifferentiation in vitro (Partanen et al., 1985) and reduced tooth size in vivo (Rhodes et al., 1987). Taken together, these results suggest that E G F plays a role i n tooth morphogenesis. The location of E G F transcripts i n the mesenchyme adjacent to the mandibular epithelium suggested a paracrine mechanism i n the stimulation of epithelial proliferation i n dental lamina formation (Kronmiller , 1995). A d d i t i o n of exogenous E G F to lungs i n culture resulted i n significant st imulation of branching morphogenesis (Warburton et al., 1992). By using an antisense ol igodeoxynucleotide directed against E G F precursor m R N A i n embryonic mouse l u n g i n culture, the expression of E G F and branching morphogenesis were inhibited (Seth et al, 1993). These data suggest that E G F is important for pulmonary organogenesis. 47 6.2 TGF-alpha T G F - a m R N A encodes a transmembrane precursor glycoprotein of 160 amino acids which is processed into a 50 amino acid mature protein (Derynck, 1984). T G F - a is highly conserved between man and mouse and shares about 33-44% homology w i t h E G F (Heath, 1993). T G F - a was first isolated from culture medium of mouse sarcoma virus transformed cells (de Larco and Todaro, 1978) and later found to be highly conserved between human and mouse (Heath, 1993). L ike E G F , both precursor and mature forms of T G F - a can bind to EGF-R and activate the target cells (Wong et al., 1989). Binding of T G F - a precursor w i t h E G F - R , so-called "juxtacrine" st imulat ion, is proposed to mediate cell-cell adhesion and trigger a cascade of events leading to cell duplication (Massague, 1990). M o r e recently, a role for membrane-bound TGF-a -media ted cell-cel l interactions d u r i n g the peri - implantat ion of mammal ian development was suggested (Paria et al., 1994). By using R T - P C R technique, T G F - a transcripts are able to be detected as early as i n the unfertilized egg and during preimplantation stage. T G F - a protein was immunolocalized i n all cells of the blastocyst (Rappolee et al., 1988). In later stages, T G F - a has been localized i n embryonic tissues particularly ectodermal origin for example i n the branchial arches, oral and nasopharyngeal epithelia, otic vesicle, mesonephric tubules of the kidney (Wilcox and Derynck, 1988), pancreas and gastrointestinal tract (Hormi and Lehy, 1994). T G F - a transcripts were also identified by P C R i n the mouse mandible at days 9 and 10 (Kronmiller et al, 1993). Initially, T G F - a was considered as a fetal form of E G F . However, it has also been identified i n various types of normal adult tissues inc luding skin 48 (Coffey et al., 1987), gastrointestinal tract (Cartlidge and Elder, 1989), and ovarian tissues (Chegini and W i l l i a m s , 1992) and pathological conditions inc luding psoriatic skin (Kondo et ah, 1992) and neoplasia (Derynck et al., 1987; Barton et al., 1991). T G F - a is frequently a superagonist of E G F (Derynck, 1992). Similar to EGF, injection of exogenous T G F - a into newborn mice also activated precocious eye opening and incisor eruption (Smith et al., 1985; Tarn, 1985). It was proposed that the major role of T G F - a i n development is to drive the proliferation of various cell types, especially epithelial cells. The coexpression of T G F - a and EGF-R genes by many epithelial cells suggests that T G F - a may act i n an autocrine manner (Derynck, 1992). T G F - a was found to promote the proliferation of the medial edge epithelial cells unti l fusion of the palatal shelves occurs i n secondary palate cultures (Lee and H a h n , 1991). The in vitro study showed that T G F - a can stimulate E C M formation in the developing mouse secondary palate (Dixon and Ferguson, 1992). Overexpression of T G F - a induced epithelial hyperplasia i n the l iver, pancreas, mammary gland and skin of transgenic mice, indicat ing that T G F - a functions i n epithelial proliferation (Matsui et al., 1990; Sandgren et al., 1990; Vassar and Fuchs, 1991). More recently, mice homozygous for a disrupted T G F - a (TGF-a -/-) gene appeared to be viable and fertile, suggesting that T G F - a may not be absolutely essential for embryonic development (Mann et al, 1993; Luetteke et al., 1993). These findings, however, do not rule out a role for T G F - a under natural conditions since other growth factors such as E G F might be able to compensate the effects of T G F - a i n the knockout mice (Wiley et al., 1995). 49 6.3 EGF-Receptors E G F - R is a transmembrane glycoprotein of 1186 amino acids w i t h a molecular weight of 170 kDa (Cohen et al., 1980). EGF-R is also known as c-ErbBl since it is the proto-oncogene precursor of v-erbB f o u n d i n chicken erythroblastosis virus (Adamson, 1990). Other related members of this family are ErbBl, ErbB3, and ErbBA (see reviews i n Pusztai et al., 1993; Wi ley et al., 1995). E G F - R referred i n the present work is the prototype of EGF-R, c-ErbBl. U p o n b i n d i n g to an extracellular l igand, the monomeric E G F - R protein becomes dimerize and autophosphorylated. Subsequently, signal transduction events take place that lead to changes i n cell proliferation, cell differentiation, cell adhesion, a n d / o r cell migration (see reviews i n Hernandez-Sotomayor and Carpenter, 1992; Wiley et al., 1995). EGF-R has been detected in various embryonic and adult tissues, and found to be overexpressed i n some neoplasia (Gullick et al., 1986; Carpenter and W a h l , 1991). D u r i n g preimplantation per iod, E G F - R has been detected i n mouse (Adamson, 1990) and human (Adamson, 1993; C h i a et al., 1995) blastocysts. Therefore, it was suggested that E G F - R takes part i n growth regulation of the early embryo, particularly i n the process of implantat ion (Adamson, 1990). E G F binding studies have been used to characterize E G F - R function i n many developing organ systems: heart, kidney (Shigeta et ah, 1993), tooth (Partanen and Thesleff, 1987), k idney, l iver , l u n g (Nexo and Kryger -Baggesen, 1989; Shigeta et al., 1993) and secondary palate (Sharpe et al., 1992). E G F - R m R N A and/or protein have been found i n the developing tooth (Abbott and Pratt, 1988), pancreas (Miettinen and Heikinheimo, 1992), gastrointestinal tract (Hormi and Lehy, 1994), mandible (Shum et al., 1993) and secondary palate (Dixon et al., 1991). More recently, the knockout mice lacking E G F - R were generated and showed multiple abnormalities dependent on genetic background (Threadgill et al., 1995; Sibilia and Wagner, 1995). For example, mutant fetuses 50 died at mid-gestation due to placental defects on a 129/Sv background. O n a C D - I background, the mutants l ived up to three weeks and showed abnormalities i n skin, kidney, brain, liver and gastrointestinal tract (Threadgill et al., 1995). These results suggested that EGF-R participates i n a wide range of cellular activities particularly epithelial proliferation and differentiation. A l t h o u g h T G F - a can b ind EGF-R w i t h an affinity comparable to that of E G F , there are distinguishable differences i n their biological activities (see reviews i n Derynck, 1992; Hernandez-Sotomayor and Carpenter, 1992). For example, both T G F - a and EGF can induce neovascularization in vivo. But T G F - a is found to be more potent than E G F (Schreiber et al., 1986). A monoclonal antibody to EGF-R (13A9) can block the binding of T G F - a to human E G F - R but has no affect on the affinity of E G F for b inding to E G F - R (Winkler et al., 1898). These results indicated either that the antibody stabilizes a conformation of E G F -R which is not favorable for T G F - a binding or that it blocks a part of the surface of the receptor which is necessary for T G F - a binding but not E G F binding. 51 7. Cell proliferation in the developing primary palate. D u r i n g pr imary palate formation, morphogenetic changes occur i n the craniofacial complex. By using morphometric analysis and three-dimensional computer reconstructions i n different stages of human embryos (Diewert and Wang, 1992; Diewert and Lozanoff, 1993), it has been found that the frontonasal prominence elongates vertically (height increases by seven times) and narrows to approximately half the width . The brain and the face become vertically separated. The area of the maxillary region increases extensively, particularly i n the distal region, as the maxillary prominences grow forward to meet the lateral nasal and media l nasal prominences. The growth of the lateral nasal region increases p r i m a r i l y i n w i d t h . Whereas, the growth of the medial nasal prominences appears to increase only slightly. These normal growth patterns are believed to be important for facilitating contact between facial prominences and primary palate morphogenesis (Diewert and W a n g , 1992). Regional differences i n cel l proliferation and E C M composition i n the facial prominences are believed to contribute to these changes during primary palate morphogenesis (Diewert et al, 1993a). D u r i n g embryonic development, there is ongoing d i v i s i o n of cells, resulting i n tissue proliferation and morphogenesis. A s this occurs, the parent cell accumulates certain proteins and doubles its D N A content to provide for the progeny. C e l l proliferation and D N A synthetic activity can be studied by many approaches. 5-Bromodeoxyuridine (BrdU), an analogue of thymidine, has been ut i l ized for this purpose since it can be incorporated into D N A dur ing the S-phase of the cell cycle. Its localization in the nuclei can be detected by using the ant i -BrdU antibody (Gratzner, 1982). Proliferating cell nuclear antigen ( P C N A ) , an auxil iary protein to D N A polymerase delta, has also been proposed as a marker of replicating cells since its cell concentration is directly correlated w i t h 52 the proliferative state of the cell. P C N A was shown to increase through G l , peak at the G l / S-phase interface, decrease through G2 and reach low levels i n M-phase and interphase (Celis and Celis, 1985; K u r k i et al., 1986; Casasco et al., 1993). By using immunohistochemistry w i t h the a n t i - P C N A antibody, the proliferation index of certain tissues, including embryonic (Leibovici et al., 1992; Sanders et al., 1993) and neoplastic tissues (Hal l et al, 1990; Pendleton et al, 1993), can be characterized. The advantages of using P C N A to measure growth fractions and label l ing indices include ease of staining and el iminat ion of the need for injections of B r d U for the labelling of S-phase fractions. However, P C N A is less suitable for determination of kinetic parameters such as phase durations and cell cycle time and generally cannot be used to trace lineages and migrations of cells d u r i n g differentiation (Dolbeare, 1995). A study of cell proli feration i n the gastrulating chick embryo showed that immunolocal izat ion of P C N A gave generally similar distribution patterns to those of B r d U incorporation, although there were always more P C N A - p o s i t i v e nuclei than BrdU-pos i t ive nucle i (Sanders et al., 1993). Similarly, a comparative study of rat pancreatic growth between P C N A labell ing and [3]H-thymidine autoradiography showed that P C N A index is higher than that of [3]H-thymidine. These two studies suggest that PCNA-pos i t ive cells are found not only in S-phase cells, but also i n cells that have recently completed the cell cycle (Elsasser et al, 1994). Labelling of B r d U and and [3]H-thymidine were also compared and showed similar results i n murine tumors (Wilson et al, 1987). In addition, B r d U labelling demonstrated greater sensitivity w i t h little background interference i n transformed Syrian hamster cells (Cawood and Savage, 1983). C e l l proliferation has been studied i n the embryonic face. Analys is of labelling indices by labelling chick embryos w i t h [3]H- thymidine indicated that rates of cell proliferation varied wi th in each of the facial prominences of the 53 chick embryo. Regions where rates of proliferation were maintained at elevated levels were the boundary areas of the facial prominences particularly the anterior tip of the maxi l lary and the lateral nasal prominences and the zones of attachment between the maxillary and the lateral nasal prominences (Minkoff , 1980a). The percentage of labelled cells in all areas declines w i t h advancing developmental age (Minkoff and K u n t z , 1978; Minkof f , 1980a). A pattern of declining rates of cell proliferation w i t h advancing developmental age has also been observed i n morphogenesis of the fronto-nasal prominence (Minkoff and K u n t z , 1977), l imb (Ede et al., 1975), and secondary palate (Nanda and Romeo, 1975). Recently, cell densities and proliferation were studied i n rhesus monkey embryos labelled w i t h [3]H-thymidine during primary palate formation (Diewert et al., 1993a). The results showed that labelling indices were consistently higher i n facial prominences than i n midline tissues ventral to the brain (Diewert et al., 1993a). Similarly, cell proliferation of the facial prominences was studied by B r d U incorporation in mouse embryos (Gui et al., 1993). Ce l l proliferation rates of the mesenchyme i n the facial prominences were found to remain unchanged during the early stages of development and to decrease i n the later stages. These data suggested that a differential rate may be operative as a morphogenetic mechanism during enlargement and union of the facial prominences. It was also found that epithelial cells i n the prospective fusion area showed decreased D N A synthesis i n comparison w i t h those i n the nonfusing areas, indicat ing that epithelial cell proliferation converts to a differentiation-type pattern (Gui et al., 1993). A l t h o u g h the study of cell proliferation by G u i et al. (1993) was done during embryonic mouse day 10, regional differences i n cell proliferation i n later stages (embryonic day 11): fusion of the facial prominences and mesenchymal bridge enlargement was not studied. Therefore, in the present work, regional cell proliferation at different stages during primary palate formation was undertaken. 54 B. Hypotheses N o r m a l tissue remodel l ing involves well-balanced and coordinated synthesis and degradation of the E C M molecules. Remodell ing of the E C M is believed to play an important role during embryonic development particularly i n cell migration, proliferation, differentiation, cell-cell interactions, and tissue outgrowth and invasion (Matrisian, 1992; Werb et al., 1992). Morphogenesis of the tissues i n the craniofacial complex; especially the primary palate, mandible and eye; and heart share many common basic biological processes for example cell migration, proliferation, differentiation, and tissue interactions. However , the mechanisms of tissue remodel l ing and outgrowth d u r i n g craniofacial morphogenesis and tissue remodell ing dur ing heart morphogenesis remain unclear. I, therefore, tested the hypotheses that temporo-spatial changes of certain molecular factors take place during craniofacial and heart morphogenesis and contribute to the regulation of tissue remodel l ing and outgrowth of the craniofacial complex and heart such as; 1. basement membrane components of the epithelial seam; inc luding laminin, type IV collagen, and fibronectin; are gradually disrupted i n a sequence during primary palate morphogenesis, 2. elevated levels of expression of growth factors and their receptors, particularly EGF, T G F - a , and EGF-R, are present in rapidly growing regions of the developing primary palate and are correlated w i t h the sites of increased cell proliferation, 3. 72-kDa gelatinase is expressed at high levels in the area of the epithelial seam as the basement membrane becomes disrupted dur ing pr imary palate morphogenesis, 55 4. 72-kDa gelatinase co-localized w i t h the enzyme's substrates; type IV collagen, laminin , and fibronectin; during heart morphogenesis. General A i m The aim was to increase our understanding of the molecular mechanisms of the development of the craniofacial and heart morphogenesis i n the mouse. Specific A i m s 1. To characterize changes of the major components of the basement membrane; l a m i n i n , type IV collagen, and fibronectin; that accompany the regression of the epithelial seam during morphogenesis of the mouse primary palate, 2. To characterize the distribution of the growth factors and their receptors; particularly E G F , T G F - a , and E G F - R ; d u r i n g morphogenesis of the mouse primary palate, 3. To analyze regional proliferation of the facial prominences by B r d U incorporation and P C N A immunulocal izat ion d u r i n g morphogenesis of the mouse primary palate, 4. To characterize the distribution of 72-kDa gelatinase i n association w i t h tissue outgrowth and morphogenesis of the mouse craniofacial complex particularly the primary palate, mandible and eye, 5. To characterize the co-localization of E G F , T G F - a and 72-kDa gelatinase i n association w i t h morphogenesis of the mouse primary palate, 6. To characterize the distribution of 72-kDa gelatinase i n correlation w i t h changes of the distribution of the enzyme substrates; type IV collagen, laminin , and fibronectin; during morphogenesis of the mouse heart. 56 C H A P T E R 2: Dis tr ibut ion of Basement Membrane Components i n the Developing Mouse Primary Palate Introduction The basement membrane is an ubiquitous extracellular matrix mainly found i n epithelial, nerve, and fat tissues (Timpl, 1989). Various molecules have been identified i n the basement membrane such as laminin , type IV collagen, heparan sulfate proteoglycan, fibronectin, nidogen (entactin), S P A R C , amyloid P, and some complement components (Timpl, 1989). The primary palate is formed by fusion of the medial nasal prominence w i t h the lateral nasal and maxi l lary prominences. Ini t ia l ly , the epithel ial coverings of the prominences adhere and establish an epithelial seam w h i c h becomes disrupted and replaced by a mesenchymal bridge w h i c h enlarges and unites the prominences together as a single organ, the primary palate (Streeter, 1948; Diewert and Shiota, 1990; Diewert and Wang, 1992; Diewert and Lazanoff, 1993; Diewert et al, 1993b; Wang et al, 1995). I predicted that d u r i n g pr imary palate formation, l a m i n i n , type IV collagen, and fibronectin i n the basement membrane of the epithelial seam are disrupted and rap id ly disappear i n association w i t h mesenchymal bridge formation. The temporo-spatial distributions of laminin, type IV collagen, and fibronectin were determined by means of immunohistochemistry (Iamaroon and Diewert, 1996). 57 Materials and Methods A n i m a l Maintenance and Breeding A l l mice were housed i n facilities approved by the Canadian Counc i l of A n i m a l Care and experimental protocols used were approved by the A n i m a l Care Committee of the University of British Columbia . Mice were maintained on a diet of Purina mouse chow and filtered water, in the animal unit i n the Faculty of Dentistry at the University of British Columbia , and i n windowless rooms on a 12 hour light (7:00 am to 7:00 pm), 12 hour dark cycle. The temperature was controlled at about 22° C . Three or four adult females were caged overnight w i t h a male and were examined i n the m o r n i n g for the presence of a vaginal p l u g . Ovula t ion was assumed to occur at midnight , therefore 9 am of the day the p l u g found was designated as day 0 hour 9 of gestation (Snell et al, 1940). Mouse Stocks The strains of mice used i n the present study were C D 1 ( Charles River, Wi lmington , M A ) , B A L B / c B y (Jackson Laboratory, Bar Harbor , Maine) , C L / F r (developed i n the laboratory of D r . Fraser i n M c G i l l Univers i ty and k i n d l y provided to our laboratories by D r . Trasler i n 1985), A / W y S n (Jackson, Bar Harbor, Maine), and A / J (Jackson, Bar Harbor, Maine). Tissue Collect ion and Preparation Pregnant mice were ki l led at various times from day 10 through 11 i n a carbon dioxide chamber. Gravid uteri were removed and placed i n normal saline solution. Individual embryos were dissected from the uterine decidua and staged by counting the number of tail somites (TS) from the caudal edge of the hide 58 l imb to the end of the tail (Wang, 1992). Embryonic heads were removed from the body wi th a N o . 15 surgical blade, fixed i n 4% (w/v) paraformaldehyde for 12-24 hours, and processed for paraffin embedding. The specimens were serially sectioned coronally or horizontally at a thickness of 7 |im and placed on 3-aminopropyl-triethoxy-silane-coated slides (Sigma, St. Louis , M O ) , then dried overnight . Deparaffinized sections were incubated w i t h 0.4% (w/v) pepsin (Sigma, St. Louis , M O ) i n 0.02M HC1 for 5 min . The purpose of proteolytic digestion w i t h pepsin was to remove the cross-linking i n paraformaldehyde-fixed tissues and render tissue antigen reactive. Sections without pepsin treatment were also included and found weak staining. Sections were incubated w i t h 25% (v /v) normal goat serum for 30 m i n , washed w i t h phosphate-buffered saline (PBS), and incubated w i t h anti-laminin (Sigma, St. Louis , M O , rabbit anti-EHS mouse sarcoma, L-9393), anti-type IV collagen (Chemicon, Temecula, C A , rabbit anti-human, AB748), or anti-fibronectin (Dako, Glostrup, Denmark, rabbit anti-human, A 245) antibody wi th concentrations of 1:100, 1:25, and 1:100, respectively overnight at 4° C . Subsequently, the sections were incubated i n fluorescein isothiocyanate (FITC)-conjugated (sheep anti-rabbit, Sigma, St. Louis , M O , anti-rabbit IgG, F-7512) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated (goat anti-rabbit, Sigma, St. Louis, M O , anti-rabbit IgG, T-6778) secondary antibody w i t h the concentration of 1:100 for 1 hour at 4° C and washed. Wi th in each group of the sections, a negative control slide was incubated w i t h normal goat serum as a replacement of the primary antibody. The dilution tests were performed on the antibodies against laminin, type IV collagen, and fibronectin. The results showed that the staining intensity decreased gradually as the di lut ion of the antibodies increased, as predicted. Positive control tissues included embryonic basement 59 membranes of the surface ectoderm for the antibodies against laminin , type IV collagen, and fibronectin (Timpl, 1989). Positive immunoreactivity was found i n basement membranes of the surface ectoderm as predicted (data not shown). Results A t least 33 CD1 mouse embryos were used in these studies (see Appendix 4). Consecutive sections were not employed i n these studies but selected comparable sections were used for each antibody staining. Morphogenesis of the primary palate can be categorized into three stages; (1) epithelial seam formation (7-11 TS), (2) epithelial seam disruption (12-17 TS), and (3) mesenchymal bridge enlargement (18-20 TS). Epithelial seam formation (7-11 TS) Sections of C D 1 embryos at the 7-11 tail somite stage showed intense local izat ion of l amin in , type IV collagen, and fibronectin i n the basement membrane of the epithelial seam dur ing the pr imary palate formation (Figs. 5A,B,C) . The staining was present from the anterior to posterior of the epithelial seam and was continuous w i t h the basement membranes of the stomodeal and nasal epithel ia . The staining patterns of l a m i n i n , type IV collagen, and fibronectin were uniform throughout all basement membranes. The staining of the basement membrane components was also present around the blood vessels showing extensive vascularization of the primary palate. Fibronectin was also intensely labelled i n the mesenchyme of the facial prominences (Fig. 5C). The staining of laminin, type IV collagen, and fibronectin appeared to be more diffuse i n the tip of the maxil la than i n the other regions of the basement membrane (Figs 8A,B,C) . These findings were also observed i n later stages of the primary palate morphogenesis. 60 Epithelial seam disruption (12-17 TS) The staining of laminin, type IV collagen, and fibronectin i n the basement membrane of the epithelial seam became rapidly discontinuous and fragmented in the later stages of primary palate formation (Figs. 6A,B,C). However, basement membranes of the stomodeal and nasal epithelia remained intact. A close association between the areas of basement membrane b r e a k d o w n and mesenchymal ingrowth was evident. Disrupt ion of the basement membrane components appeared to be a prior event to disappearance of the epithelial seam. The basement membrane components were also disrupted at the nasal and oral margins where merging of facial prominences continued as the primary palate enlarged. Disrupt ion of the basement membrane appeared to progress from central to anterior of the seam. Mesenchymal bridge enlargement (18-20 TS) A t the 18-20 TS stages, the epithelial seam and its basement membrane largely disappeared simultaneously w i t h mesenchymal bridge enlargement i n the primary palate (Figs. 7A,B,C). Basement membrane components were intact around the margins of the primary palate. Grooves between the prominences were markedly reduced. Fibronectin was also intensely local ized to the mesenchyme of the peripheral regions of the facial prominences; whereas it was less pronounced i n the m i d l i n e region. In the region posterior to the mesenchymal bridge, basement membrane components of the the nasal f in remained intact where formation of the primitive choana occurred (Fig. 7D). 63 Figs. 5 A - C : Epi thel ia l seam formation: Sections show intense staining of laminin (A), type IV collagen (B), and fibronectin (C) i n basement membranes of the epithelial seam (between arrows), and nasal and stomodeal epithelia. The staining of f ibronectin was also present i n the mesenchyme of the facial prominences. L N , lateral nasal; M N , medial nasal; M X , maxillary prominences. Figs. 6 A - C : Epithel ia l seam breakdown: Sections show disrupt ion of the stainings of laminin (A), type rv collagen (B), and fibronectin (C) i n the basement membrane of the epithelial seam (between arrows). However , the staining i n basement membranes of the nasal and stomodeal epithelia remained intact. Figs. 7A -C: Mesenchymal bridge enlargement: Sections show disappearance of the epithelial seam and its basement membrane. There was a reorganization of the stainings of laminin (A), type IV collagen (B), and fibronectin (C) around the margins of the primary palate (arrows). The staining of fibronectin was intensely localized to the mesenchyme particularly i n the peripheral regions of the facial prominences. Figs. 7D-E: In the region posterior to the mesenchymal bridge, the staining of laminin remained intact i n the basement membrane of the forming primit ive choana (between arrows) (D). The negative control section, incubated w i t h normal goat serum as a replacement of the primary antibody, showed absent staining i n the tissue (E). Figs. 8A -C: The staining of laminin (A), type IV collagen (B), and fibronectin (C) appeared to be more diffuse in the basement membrane at the tips of the maxilla. 64 Discussion In this study, we characterized the presence of the major components of the basement m e m b r a n e i n the p r i m a r y palate b y means of i m m u n o h i s t o c h e m i s t r y . D u r i n g e p i t h e l i a l seam f o r m a t i o n , basement membrane components were intact. Subsequently, the basement membrane components of the epithelial seam became patchy and then entirely disrupted as the mesenchymal bridge formed and enlarged. This coincides wi th rupture of the oronasal membrane or nasal f in resulting i n formation of the primit ive choana and completion of the primary palate (Tamarin, 1982). Previous studies have implicated the basement membrane i n mediation of epithelio-mesenchymal interactions i n several developing organ systems, such as the tooth (Thesleff et al., 1991), salivary glands (Bernfield et al., 1984), l u n g (Jaskoll and Slavkin, 1984), kidney (Ekblom, 1981), and genitourinary system (Ikawa et al., 1984). Indeed, epithelial-mesenchymal interactions i n the facial prominences were suggested to be mediated by the epithelial-mesenchymal interface (Saber et ah, 1989). Dur ing primary palate formation, elevated rates of cell proliferation beneath the ectoderm of the facial prominences are believed to be maintained by these epithelial-mesenchymal interactions (Minkoff , 1980a,b; Diewert et al, 1993a; G u i et al, 1993). D i s r u p t i o n of the major basement membrane components of the epithelial seam was clearly demonstrated i n this study. This phenomenon occurs rapidly after fusion of the facial prominences and formation of the epithelial seam. Similar situations, w i t h breakdown of the basement membrane and regression of the epithelial components, have also been found i n some other organ systems, such as the Muller ian ducts of male embryos (Trelstad et al, 1982; Ikawa et al, 1984), involuted mammary glands (Warburton et al, 1982; Talhouk 65 et al, 1992), and developing secondary palate (Ferguson, 1988). The mechanism of the disruption of the basement membrane and the epithelial seam has drawn considerable interest from investigators since persistence of the epithelial seam may be the abnormality leading to cleft formation (Stark, 1954; Warbrick, 1960; Diewert and Wang, 1992). A mesenchymally-produced soluble factor, hepatocyte growth factor/scatter factor ( H G F / S F ) , was isolated (Stoker et al, 1987) and proposed to be associated w i t h the disruption of the midl ine epithelial seam i n the developing secondary palate (Ferguson, 1988). M o r e recently, the m R N A expression of H G F / S F and its receptor, c-met were studied and found i n the mesoderm and endoderm along the rostro-intermediate part of the primit ive streak (Andermarcher et al, 1996). In later development, H G F / S F and c-met were expressed i n the cardiovascular system, migrating neural crest cells, branchial arches, early l imb buds, and some visceral organs. However , their expression i n the secondary palate is unknown. M a t r i x m e t a l l o p r o t e i n a s e s ( M M P s ) , e s p e c i a l l y t y p e I V collagenases/gelatinases and stromelysins, may be good candidates for this degradative process since they have been found to be capable of degrading type IV collagen, l a m i n i n , and fibronectin (Fessler et al, 1984; G a l l o w a y et al, 1988; Okada et al, 1990; Nagase et al, 1991). Indeed, the proteins expression of 72-kDa gelatinase (MMP-2) and stromelysin (MMP-3) were found i n the rat mammary gland and implicated to be responsible for basement membrane degradation dur ing mammary gland invo lut ion (Dickson and Warburton, 1992). 72-kDa gelatinase was also found to be associated w i t h breakdown of type IV collagen i n the dental basement membrane during mouse (Sahlberg et al, 1992) and human (Heikinheimo and Salo, 1995) tooth morphogenesis. Disrupt ion of the basement membrane appears to be a common process dur ing embryogenesis since it has been found to be a key event prior to 66 migration of neural crest cells (Duband and Thiery, 1982a; Duband and Thiery, 1987), somit ic mesenchyme format ion (Solursh et al, 1979), mesoderm formation from the primitive streak (Duband and Thiery, 1982b), and ameloblast differentiation during tooth formation (see review in Thesleff and Hurmerinta , 1981). Disrupt ion of the basement membrane of the epithelial seam of the secondary palate appears to occur p r i o r to e p i t h e l i a l - m e s e n c h y m a l transformation (Griffith and H a y , 1992) a n d / o r migration of the medial edge epithelial cells to the stomodeal and nasal epithelia (Carette and Ferguson, 1992), the two mechanisms proposed for seam e l iminat ion i n the deve loping secondary palate. It was suggested that degradation of the basement membrane al lows the transmission of inductive signals from one tissue to another i n k idney tubule formation (Wartiovaara et al., 197AL; E k b l o m , 1981) and tooth morphogenesis (Sahlberg et al., 1992). This inductive transmission may be also important for the fate of the medial edge epithelial cells of the secondary palate. In the primary palate, the smaller number of cells present i n the epithelial seam makes study of their fate even more difficult than that of the secondary palate (Diewert and Wang, 1992; Diewert et al, 1993b). In this study, the presence of fibronectin i n the extracellular matrix is particularly interesting since its differential distribution was observed in the 18-20 TS facial pr imordia . Fibronectin was intensely labelled i n the peripheral regions of the facial prominences; whereas the staining was less pronounced i n the midl ine regions. The pattern of distribution of fibronectin was consistent w i t h previous studies of cell density during primary palate formation i n which the cell densities were high in the facial prominences and low i n midline tissues (Diewert et al, 1993a; M i n k o f f , 1980a,b). Changes i n cell dispers ion and extracellular matrix content have been studied and found to be involved i n growth of embryonic pr imordia (Burk, 1983). The differential distr ibution of 67 fibronectin was also observed in the developing secondary palate i n w h i c h staining was found mainly i n the mesenchyme and basement membrane but appeared to be fibronectin-free in the mesenchymal core of the palatal shelves (Morr is -Wiman and Brinkley, 1992). Recently, the knock-out mice w i t h mutant fibronectin gene were generated and found to have various severe abnormalities that lead to early embryonic lethality (George et al., 1993). These abnormalities i n mouse embryos were suggested to arise from fundamental deficiencies i n mesodermal migration, adhesion, proliferation or differentiation as a result of the absence of fibronectin. Collectively, the differential distribution of fibronectin i n the developing primary palate suggests that the mesenchymal cells i n the peripheral regions of the facial prominences may utilize fibronectin as a substrate for cell adhesion, proliferation, and differentiation. A s a result, successful outgrowth of the primary palate takes place. Distribution of type IV collagen, laminin, and fibronectin was also studied during maxillary process formation i n the chick embryo between stages 22 and 31 (Xu et al., 1990). It was found that the staining of type IV collagen was more intense i n the basement membranes l in ing the roof of the stomodeum than i n the maxil lary process at al l staged examined, whereas laminin appeared to be uniformly stained throughout the basement membrane of all regions examined. It was proposed that maxillary process outgrowth may be related directly to changes i n the distribution of type IV collagen. Laminin , on the other hand, may serve mainly as a structural support (Xu et al, 1990). In the present study, the staining of type IV collagen, laminin, and fibronectin was also found to be more diffuse particularly i n the basement membrane of the tip of the maxil la . These f indings suggest that the basement membrane becomes less organized i n the region that needs to expand and outgrow. 68 In summary, the basement membrane components; l a m i n i n , type IV collagen, and fibronectin; were found to become rap id ly disrupted i n the epithelial seam during the primary palate formation. These results indicate that d i s r u p t i o n of the basement membrane is important for epi thel ia l seam regression that leads to successful mesenchymal bridge formation and fusion of the facial prominences. 69 C H A P T E R 3: C e l l Proliferation and Distr ibution of E G F , T G F - a , and E G F -Receptor i n the Developing Mouse Primary palate Introduction Both E G F and T G F - a are members of the E G F family found in various embryonic and adult tissues. They exert their actions via the same receptor, E G F -R (Massague, 1983; Hernandez-Sotomayor and Carpenter, 1992). The expression of these three molecules has been found i n many organ development systems including the preimplantation embryo (Adamson, 1993; Chia et al, 1995), tooth (Partanen and Thesleff, 1987; Partanen, 1990; Shum et ah, 1993), mandible (Kronmil ler et al., 1993; Shum et al., 1993), secondary palate (Dixon et al., 1991), and gastrointestinal tract (Miettinen et al . , 1989; Miett inen, 1993; H o r m i and Lehy, 1994). Nonsyndromic cleft l ip w i t h or without cleft palate ( C l / P ) is one of the most common human birth defects, w i t h frequencies of 1/1000 i n Caucasians (Thompson et al., 1991), 1.7/1000 i n Asians (Kobayashi, 1958; Nee l , 1958), and 2.75/1000 i n North American Indians in British Columbia (Lowry and Renwick, 1969). The eitiology of C l / P is complex and associated wi th heterogenous factors inc luding anatomical variations, racial differences, genetic and environmental factors (see review i n Johnston and Bronsky, 1995). Recent studies have suggested that genetic factors play an important role i n C l / P malformation and may involve several susceptibility loci i n humans (Farrall and Holder , 1992; Mi tche l l and Risch, 1992) and mice (Juriloff, 1995). In human studies, T G F - a locus has been indicated to be one of the potential C l / P susceptibility loci based on population association studies (Ardinger et ah, 1989; Chenevix-Trench et al., 1991, 1992; Holder et al., 1992; Sassni et al, 1993; Feng et al, 1994). Therefore, it 70 was suggested that allelic variants of T G F - a may have differential effects on pr imary palatal epithelial cells (Feng et al, 1994). A variant molecule, i n combination w i t h other factors, may cause the epithelial cells either to proliferate excessively or to differentiate prematurely in some way that preclude successful fusion of the facial prominences. However , involvement of T G F - a d u r i n g primary palate morphogenesis remains unknown. In the present study, I, therefore, undertook a comprehensive study on the temporo-spatial distribution of T G F - a along w i t h its receptor, E G F - R , and a related growth factor, E G F during primary palate morphogenesis by means of indirect immunohistochemistry w i t h conventional a n d / o r confocal laser scan microscopes. In addit ion, the regional cell proliferation w i t h i n the developing primary palate was studied by using 5-bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen ( P C N A ) immunolocalization (Iamaroon et al, 1996a). Materials and Methods Tissue collection and preparation C D 1 mice were mated overnight and embryos were collected on days 10 and 11 (plug = day 0). A small number of C L / F r mice which showed normal development of the primary palate (20-30% of embryos have spontaneous cleft lip) were also used i n this study. The TS number from the caudal edge of the h i n d l imb was determined (Wang, 1992). The heads were fixed i n 4% ( w / v ) paraformaldehyde i n PBS for labelling of EGF, T G F - a , EGF-R and P C N A , and i n 70% ethanol for labelling of B r d U for 24 hours. The paraffinized specimens were 71 serially sectioned coronally at a thickness of 7 Lim. Information of animal breeding and maintenance and mouse stocks is shown in Chapter two. For B r d U labelling, on days 10 and 11, the pregnant mice were injected intraperitoneally w i t h 0.2 m l of 10 m g / m l B r d U (Sigma, St. Louis , M O ) and ki l led 2 hours fol lowing injection. The embryos were obtained and processed as mentioned above. Immunohistochemistry For the staining of E G F and T G F - a , an indirect immunofluorescence technique was used. Briefly, deparaffinized sections were incubated w i t h 0.4% ( w / v ) pepsin (Sigma, St. Louis , M O ) i n 0.02M HC1 for 5 min . The purpose of proteolyt ic digest ion w i t h peps in was to remove the c ross - l ink ing i n paraformaldehyde-fixed tissues and render tissue antigen reactive. Sections wi thout peps in treatment were also inc luded and f o u n d weak staining. Subsequently, the sections were incubated wi th normal goat serum for 20 m i n at room temperature and a polyclonal anti-EGF (rabbit anti-mouse, Sigma, St. Louis , M O , E-2635) or polyclonal ant i -TGF-a (sheep anti-human recombinant, Chemicon, Temecula, C A , AB1412) antibody both at the concentration of 1:50 overnight at 4° C . After washing wi th PBS, the sections were incubated w i t h the TRITC (goat anti-rabbit, Sigma, St. Louis , M O , T-6778) or FITC (donkey anti-sheep, Sigma, St. Louis , M O , F-7634) conjugated secondary antibody for 1 hour at 4° C both at the concentration of 1:200. For label l ing of E G F - R , the avidin-biot in- immunoperoxidase (ABC) technique was employed as previously described (Hsu et al., 1981). Briefly, deparaffinized sections were incubated w i t h 3% (v/v) hydrogen peroxide for 20 m i n to eliminate endogenous peroxidases, washed w i t h PBS, and then digested 72 w i t h 0.4% (w/v) pepsin (Sigma, St. Louis , M O ) i n 0.02M HC1 for 5 m i n . After washing w i t h PBS, the slides were incubated w i t h normal goat serum for 20 m i n at room temperature and then w i t h a monoclonal ant i -EGF-R (mouse anti-human, Clone F4, Sigma, St. Louis , M O , E-3138) at the concentration of 1:20 overnight at 4° C . After washing w i t h PBS, the slides were incubated w i t h a biotin-conjugated secondary antibody (goat anti-mouse, Vector, Burlingame, C A , PK-4002) and then w i t h StreptABComplex (Dakopatts, Santa Barbara, C A , K377) both at the concentration of 1:100 for 30 m i n at room temperature. Dark brown staining was developed by using 3,3'-diaminobenzidine hydrogen peroxide (DAB) as a substrate. The section were counterstained w i t h methyl green. For P C N A (monoclonal mouse anti-human P C 10 antibody, Dimension, Mississauga, O N T , Canada) labell ing, sections were treated as those of E G F - R except the absence of incubation w i t h 0.4% (w/v) pepsin i n 0.02M HC1. The slides were observed and photographed w i t h the Zeiss photomicroscope a n d / o r the Zeiss confocal laser scan microscope. For B r d U labelling, deparaffinized sections were treated w i t h 2 N HC1 for 10 m i n and neutral ized by us ing sodium tetraborate ( p H 9) for 10 m i n . Subsequently, the sections were incubated w i t h normal goat serum for 20 m i n and a monoclonal a n t i - B r d U antibody (mouse ant i -human, C lone B U - 1 , Amersham, Oakvi l le , O N T , Canada, RPN202) at 4° C overnight, fo l lowed by incubation w i t h FITC conjugated secondary antibody (goat anti-mouse, Sigma, St. Louis, M O , F-9006) at the concentration of 1:200 overnight at 4° C . The specificity of antibodies against EGF-R (Gullick et al, 1986), T G F - a (Ju et al, 1991), E G F (Beerstecher et al, 1988), B r d U (Gonchoroff et aZ.,1986), and P C N A (Waseem and Lane, 1990) have been partially characterized by others. The monoclonal antibody against E G F - R specifically recognizes the cytoplasmic 73 tyrosine kinase catalytic domain of E G F - R and cross-reacts w i t h mouse tissue (Fowler et al, 1995). This antibody recognizes c-ErbBl form of E G F - R . The specificity of anti-human T G F - a antibody was also characterized and found not to cross-react w i t h E G F or TGF- f i (Chemicon, Temecula, C A ) . Positive control tissues used for anti-EGF antibody was embryonic mouse lung (Warburton et al, 1992) ; for a n t i - T G F - a ant ibody, h u m a n oral squamous ce l l carc inoma (Christensen et al, 1993, k i n d l y provided by Dr . L . Zhang), for a n t i - P C N A antibody, human tonsil (kindly provided by Dr. L . Zhang) and mouse intestine (Hall et al, 1990; Dolbeare, 1995); and for ant i -BrdU antibody, mouse intestine (Dolbeare, 1995). A l l positive control tissues showed positive immunoreactivity as predicted. W i t h i n each group of the sections, one negative control slide was incubated w i t h normal serum as a replacement of the primary antibodies. The results showed that there was no staining on the negative control slides. The di lut ion tests were performed on the antibodies against E G F , T G F - a , EGF-R, and B r d U . The results showed that the staining intensity decreased gradually as the dilution of the antibodies increased, as predicted. A series of di lut ion test for anti-P C N A antibody (PC10) was characterized (Hal l et al, 1990) and the optimal d i lut ion for the immunostaining was used as recommended (Pendleton et al, 1993) . A n irrelevant monoclonal antibody was also used as a control (mouse anti-human epithelial keratin, A E 1 , I C N , Costa Mesa, C A , 69-140). The a n t i - A E l antibody recognizes most of the acidic (type I) keratins and cross-reacts w i t h mouse keratins. The sections of the mouse primary palate were treated similar to those of B r d U and E G F - R . The result showed intense staining of keratin particularly i n the stomodeal epithelial cells (Fig. 18D). 74 Confocal microscopy Tissues from the primary palate stained w i t h antibodies against E G F and T G F - a were examined w i t h both epifluorescence and confocal microscopy using a 20X objective on the Zeiss Confocal Laser Scan Microscope (LCM10). A n argon laser (A,max=488 nm) and a helium-neon laser (A.max=543 nm) were used for FITC-and rhodamine-labelled tissues, respectively. Tissues were sectioned at 1 or 2 Lim intervals and images were printed using a video printer (UP-5000, Sony Canada) and the Polaroid 6000 f i lm recorder system. Results E G F , T G F - a , and EGF-Receptor A t least 33 embryos were used i n these study (see A p p e n d i x 4). Consecutive sections were not employed i n these studies but selected comparable sections were used for each antibody staining. The stainings of E G F , T G F - a , and E G F - R showed to have similar distribution patterns at a l l stages examined during morphogenesis of the primary palate. A small number of C L / F r strain of mice which showed normal development of the primary palate were also used i n these studies and showed similar results to those of C D 1 strain of mice. C L / F r mouse embryos wi th cleft fomation were excluded. Epi thel ia l seam formation In the anterior region of the face, the staining of E G F , T G F - a , and EGF-R (Figs. 9A-C) was found i n epithelial and mesenchymal tissues, particularly at the tips and peripheral regions of the lateral and medial nasal prominences. U p o n 75 fusion of the medial nasal prominence w i t h the lataral nasal and maxi l lary prominences, labelling of E G F , T G F - a , and EGF-R (Figs. 10A-C) was intensely present i n the fusion and peripheral areas of the facial prominences. The expression of the three molecules decreased i n the deeper regions of the facial prominences (Figs. 11A-B). However, the staining remained pronounced i n the peripheral regions of the prominences. The brain and nasal epithel ium were minimal ly stained at all stages examined. Epi thel ia l seam disruption and mesenchymal bridge enlargement A s the epithel ial seam was disrupted w i t h an enlargement of the mesenchymal bridge, the staining of EGF, T G F - a , and EGF-R (Figs. 12A-C) was absent i n the disrupted epithelial seam and the midline tissues. However , the staining remained intense i n the epithelial and mesenchymal cells i n the peripheral regions of the maxillary and nasal prominences. B r d U and P C N A A t least twenty-two embryos were used in these studies (see Appendix 4). B r d U and P C N A were found to have similar distribution patterns at all stages examined during morphogenesis of the primary palate. Epi thel ia l seam formation In the anterior region of the face, the staining of B r d U and P C N A (Figs. 13A,B) was intensely localized i n the nuclei of the epithelial and mesenchymal cells of the lateral and medial nasal prominences. The central aspect of the medial nasal prominence appeared to have fewer labelled cells than other areas. U p o n fus ion of the media l nasal prominence w i t h the lateral nasal and 76 maxillary prominences, B r d U and P C N A (Figs. 14A,B) appeared to be intensely labelled at the peripheral regions of the facial prominences. The fusion areas showed decreased labelling. In the deeper regions, the labellings of B r d U and P C N A (Figs. 15A,B; 17A,B) became dramatically reduced in the midline regions. The peripheral regions of the maxil lary and nasal prominences were stained more intensely. The brain showed positive stained cells part icularly at its superior and inferior aspects. Label l ing indices were not calculated i n these studies. Epi thel ia l seam disruption and mesenchymal bridge enlargement A s the epithelial seam regressed and was replaced by the mesenchymal bridge, the staining of B r d U and P C N A (Figs. 16A,B) appeared to be most pronounced i n the epithelial and mesenchymal cells at the peripheral regions of the maxil lary and nasal prominences. The disrupted epithelial seam and the midline areas showed decreased labellings of B r d U and P C N A . The negative controls sections, incubated w i t h 20-25% (v/v) normal goat or donkey serum as a replacement of the primary antibodies, showed absent staining (Figs. 18A-C). A monoclonal antibody against epithelial keratin (anti-A E 1 antibody) was also used as a control and found to be stained appropriately on the surface ectoderm as predicted (Fig. 18D). Mouse lung buds were used as a positive control for anti-EGF antibody (Fig. 18E) and found positive as predicted while anti-TGF-a antibody gave a negative result (Fig. 18F). 78 Figs. 9-12: Frontal sections through the developing mouse nasal region dur ing formation of the primary palate. The sites of fusion between the prominences are shown by the arrows. BR, brain; L N , lateral nasal prominence; M N , medial nasal prominence; M X , maxillary prominence; N C , nasal cavity F i g . 9: D u r i n g epithelial seam formation, anteriorly, E G F - R was intensely localized to the tips and peripheral regions of the L N and M N (A). The confocal micrographs show similar patterns of labelling of EGF (B) and T G F - a (C). F i g . 10: U p o n fusion of the facial prominences, the staining of E G F - R was intensely present at the fusion (between arrows) and peripheral areas of the L N and M N (A). The confocal micrographs show similar patterns of labelling of E G F (B) and T G F - a (C). F i g . 11: In the deeper regions, the staining of E G F - R remained present at the peripheral regions of the M X , L N , and M N (A). The confocal micrographs show similar patterns of labelling of E G F (B) and T G F - a (C). The midline and nasal f in (between arrows) areas were negative for staining. F i g . 12: D u r i n g epi thel ia l seam d i s r u p t i o n and mesenchymal br idge enlargement, the stainings of EGF-R (A), E G F (B), and T G F - a (C) were absent i n the disrupted epithelial seam (between arrows) but remained intense i n the peripheral regions of the facial prominences. 79 80 Figs. 13-16: Frontal sections through the developing mouse nasal region dur ing formation of the primary palate. The sites of fusion between the prominences are shown by the arrows. BR, brain; L N , lateral nasal prominence; M N , medial nasal prominence; M X , maxillary prominence; N C , nasal cavity Fig. 13: D u r i n g epithelial seam formation, anteriorly, P C N A (A) and B r d U (B) labelled cells were present in the epithelium and mesenchyme particularly of the L N and the tip of the M N . Fig. 14: U p o n fusion of the facial prominences, the stainings of P C N A (A) and B r d U (B) were intensely present at the L N and the tip of the M N . The epithelial seam (between arrows) showed reduced staining. Fig. 15: Posteriorly, the stainings of P C N A (A) and B r d U (B) were mainly present at the peripheral regions of the facial prominences. The nasal f i n (between arrows) area showed decreased staining. Fig. 16: D u r i n g epi thel ia l seam d i s r u p t i o n and mesenchymal br idge enlargement, the stainings of P C N A (A) and B r d U (B) were generally reduced but remained pronounced i n the peripheral regions of the facial prominences. The disrupted epithelial seam (between arrows) showed markedly reduced staining. 82 Figs. 17A,B: H i g h magnifications of the developing primary palate dur ing the stage of epithelial seam formation demonstrate intense labelling of P C N A (A) and B r d U incorporation (B) particularly i n the lateral nasal and maxi l lary prominences. L N , lateral nasal prominence; M N , medial nasal prominence; M X , maxi l lary prominence; N C , nasal cavity Figs. 18A-C: The negative control sections, incubated w i t h normal serum as a replacement of the pr imary palate and biotin- (A), FITC- (B), or rhodamine-conjugated secondary antibody, showed absent staining i n the developing primary palate. F ig . 18D: A monoclonal antibody, mouse anti-human epithelial keratin antibody (AE-1), was used as a control revealing intense staining particularly i n the stomodeal epithelial cells (arrowheads). The nasal and surface epithelia showed less intense labelling. F i g . 18E,F: The epithel ial l in ings of the deve loping mouse l u n g buds (arrowheads) demonstrated pronounced labelling for E G F (E) but absent staining for T G F - a (F). 83 Discussion In the present study, the expression of E G F , T G F - a , and their receptor, E G F - R (c-erbBl) was characterized by means of indirect immunohistochemistry. Interestingly, the distribution patterns of the three molecules showed striking similarity at all stages examined during primary palate morphogenesis. These observations suggest an autocrine mode of growth regulation for these growth factors dur ing outgrowth and fusion of the facial pr imordia . Previous studies have also shown the coexpression of these growth factors and their receptor d u r i n g m a n d i b u l a r m o r p h o g e n e s i s (Shum et al., 1993), e m b r y o n i c p r e i m p l a n t a t i o n (Johnson et al., 1994; C h i a et al., 1995) and malignant transformation (Derynck et al., 1987; Christensen et al., 1993; Barton et al., 1991). Alternatively, a paracrine mode of regulation may also be operative since the strongest signals of E G F and T G F - a appeared to be at the surface ectoderm. The growth factors may diffuse into the underlying mesenchyme, b ind to E G F - R at the surfaces of the mesenchymal cells and stimulate cell proliferation. A s a result, a differential proliferation of the mesenchymal cells occurred as shown i n the studies of immunolocalization of P C N A and B r d U incorporation. EGF, T G F - a , and EGF-Rs are believed to be involved i n different stages of embryogenesis i n c l u d i n g preimplantat ion, the time d u r i n g w h i c h the cell lineages are set up, and the beginning of organogenesis (Werb, 1990; Adamson, 1993). Later during secondary palate development, the role of E G F , T G F - a , and EGF-R has also been investigated (Pratt, 1987; Dixon et al, 1991,1993). Pratt (1987) speculated that T G F - a , produced by the epithel ium of the palatal shelves, stimulates epithelial proliferation i n the early stages. U p o n fusion of the palatal shelves, the epithelial seam ceases production of T G F - a and undergoes a series of 84 events of programmed cell death. M o re recently, T G F - a and E G F - R were immunolocalized to the tips of the maxillary prominences i n the early stages of the secondary palate formation (Dixon et al., 1991). U p o n fusion of the palatal shelves, T G F - a and E G F - R were intensely labelled i n the fusion area and remained i n the disrupted epithelial seam. EGF, on the other hand, was sparsely expressed at all stages examined. EGF and T G F - a were also found to be capable of stimulating production of a variety of extracellular matrix molecules by mouse embryonic palatal mesenchymal cells in vitro (Dixon et al., 1993). Therefore, it was proposed that growth factors, especially T G F - a , and EGF-R participate i n the secondary palate formation (Dixon et al., 1991,1993). Interestingly, the expression of the homeobox-containing genes encoding transcript ional factors, Msx-1 (Hox-7) and M s x - 2 (Hox-8), was loca l ized par t icular ly i n the mesenchyme and ectoderm of the tips of the facial prominences (see Table I) (Robert et al., 1989; MacKenzie et al., 1992; N i s h i k a w a et al., 1994), the regions where E G F , T G F - a , and E G F - R were also distributed d u r i n g pr imary palate morphogenesis. M s x - 1 and Msx-2 are believed to be important for epithelial-mesenchymal interactions i n the developing organs. M s x - 1 , i n par t i cular , promotes cel l pro l i fe ra t ion and suppresses cel l differentiation i n myoblastic cell culture (Song et al., 1992). Taken together, these findings i m p l y relationships between Msx-1 and Msx-2 w i t h E G F , T G F - a , and E G F - R especially i n cell proliferation during the outgrowth of the primary palate. Recently, another growth factor member, FGF-8 , was studied by in situ h y b r i d i z a t i o n and also found to be local ized to the facial prominences (Heikinheimo et al., 1994; Ohuchi et al., 1994; Crossley and Mart in , 1995). A t day 9.5, FGF-8 was expressed i n the prospective nasal placode, the commissural plate 85 of the forebrain, and the surface ectoderm of the the maxillary and mandibular components of the first branchial arch. In later development (day 10.5), the expression of FGF-8 was found i n ectodermal cells surrounding the nasal pits and surface ectoderm of the maxilla and mandible. It was suggested that FGF-8 may be a component of the epithelium-derived signal involved i n regulating the outgrowth and patterning of the facial prominences (Heikinheimo et al., 1994; Ohuchi et al., 1994; Crossley and Mart in , 1995). More recently, the FGF-8 isoforms were also immunolocal ized to the similar regions of the m R N A expression (MacArthur et al., 1995). Collectively, the multiple expressions of several growth and regulatory factors i n the deve loping p r i m a r y palate suggest that morphogenesis of the primary palate is a complex process that needs specific interactions between tissues and molecules produced at the precise locations and times. The expression of mult iple growth factors has also been investigated dur ing secondary palate morphogenesis. For example, before elevation of the palatal shelf, P D G F - A A isoform was localized only to the palatal epithelia while the PDGFoc-receptor was present i n the palatal mesenchyme, nasal and medial edge epithelia by immunohistochemistry (Qiu and Ferguson, 1995). D u r i n g palatal midl ine epithelial seam formation and disruption, both P D G F - A A and PDGFoc-receptor were colocalized i n the nasal and midl ine seam epithelia. However , the staining of P D G F - B B / p receptor were sparse or absent at all stages examined. IGF-II transcripts and peptides were localized to the mesenchyme of the horizontal prefusion palate (Ferguson et al, 1992). Subsequently, IGF-II transcripts were found to be absent during palatal fusion. IGF-II peptides, on the other hand, were localized to the nasal and medial edge epithelia. The expression patterns of TGF-p2, i n particular, was found to be remarkably similar to that of 86 IGF-II during secondary palatal formation (Fitzpatrick et al., 1990; Pelton et al., 1990). The expression of TGF-03 was first present i n the epithelial component of the vertical palatal shelf. Subsequently, TGF-[33 was co-localized w i t h T G F - p i to the medial edge epithelium of the horizontal palatal shelf and midline epithelial seam dur ing palatal fusion (Fitzpatrick et al., 1990). Similarly, aFGF and bFGF were found to be expressed mainly i n the midline epithelial seam and remain i n the epithelium of the degenerating seam (Sharpe et al., 1993). These data indicate an interactive network between different growth factors and their receptors i n control l ing the complex and r a p i d differentiation process d u r i n g palatal adhesion, fus ion , m i d l i n e epi thel ia l seam d i s r u p t i o n and mesenchymal consolidation (Qiu and Ferguson, 1995). Depletion of TGF-(33, but not TGF(31 and T G F B 2 , was found to inhibit palatal fusion i n cultures by using antisense oligonucleotides and neutralizing antibodies (Brunet et al., 1995). This inhibition could be rescued by exogenouse TGF-|33. More recently, TGF-(33 n u l l mutant mice have been generated by gene-targeting and found to have impaired palatal fusion leading to cleft palate and delayed lung development (Proetzel et al., 1995; Kaartinen et al., 1995). Other tissues including the primary palate, craniofacial complex and heart appeared to have normal development. Indeed, the palatal shelves of TGF-P3 n u l l mutants grew and elevated normally but failed to fuse. These f indings suggested that TGF-|33 acts specifically on the medial edge epithelial cells and may mediate epithelial-mesenchymal interactions dur ing secondary palate fusion (Kaartinen et al., 1995; Proetzel et al., 1995). To determine whether there is any relationship between the expression of E G F , T G F - a , and EGF-R and the sites of cell proliferation, cell proliferation was studied by using B r d U and P C N A as the markers (Gratzner, 1982; Leibovici et al., 87 1992). The distribution patterns of B r d U and P C N A were almost identical and most pronounced in the similar regions to those of the growth factors and their receptor. These findings suggest that E G F and T G F - a may play a role in cell proliferation via EGF-R during primary palate morphogenesis. The expression of P C N A , i n particular, may have a direct association w i t h some growth factors in vivo since it was found to be inducible by EGF, P D G F , and F G F in vitro (Bravo and Macdonald-Bravo, 1984; Jaskulski et al, 1988). In the present study, the labelling of B r d U and P C N A showed differential distribution during primary palate morphogenesis i n which cells at the tips and peripheral regions of the facial prominences part icularly the lateral nasal prominence showed more intense staining than those i n the deeper regions and the nasal epithel ium. In more advanced stages, the labell ing was generally reduced but remained pronounced at the peripheral regions of the prominences. These results indicate that the rate of cell proliferation remains stable i n the peripheral regions of the facial prominences, while it is reduced i n the midline regions as the pr imary palate development progresses. These f indings are consistent w i t h previous studies (Minkoff and Mart in , 1984; Minkoff , 1991; G u i et al, 1993; Diewert et al., 1993b). It was suggested that a differential rate i n the decline of cell proliferation may be operative as a morphogenetic mechanism dur ing enlargement and union of the facial prominences (Gui et al., 1993) and may be associated w i t h expression of a differentiated phenotype such as chondrogenic differentiation i n the roof of the stomodeum (Minkoff and Mart in , 1984). Regional differences of growth rates within the mesenchyme was studied i n the maxil lary prominence of chick embryos dur ing stages 19-29 by [3]H-thymidine injection (Bailey et al., 1988). Four different regions; including medial 88 side of the maxil la , ventral tip of the maxil la , lateral side of the maxi l la , and central portion of the maxilla; were characterized. It was found that growth rate of the maxil lary mesenchyme differed based on its proximity to the overlying epithelium. The rate of cell proliferation i n the facial prominences was higher in the subepithelial zone of the mesenchyme compared to that of cell populations which were located further away from the epithelium. Differences of growth rate among the regions, on the other hand, was not detected i n the later stages (stages 28-29). These data suggested that the epithelium may exert a growth regulating effect such as growth factors on the subjacent mesenchyme and this effect may be related to the stage of development (Bailey et al., 1988). Collectively, the localization of the growth factors and their receptor at the tips of the prominences prior to fusion and at the fusion area upon fusion; and the absence of their expression in the disrupted epithelial seam implicate that E G F , T G F - a , and EGF-R may selectively induce tissue proliferation resulting i n successful fusion of the prominences. In addition, the intense localization of the growth factors and their receptor as wel l as B r d U and P C N A at the peripheral regions of the facial prominences suggests that E G F , T G F - a , and E G F - R may participate i n the outgrowth and enlargement of the primary palate. Nonsyndromic cleft l ip wi th or without cleft palate ( C L / P ) is one the most common malformations of the human structure that is believed to involve unsuccessful fusion of the facial prominences d u r i n g the p r i m a r y palate formation (Diewert and Wang, 1992). Since C L / P tends to aggregate w i t h i n families, genetic factors are believed to play an important role i n its, cause (Feng et al., 1994). Based on populat ion association, several investigations have indicated that an allele of the T G F - a locus, or a gene closely l inked to it, is a risk factor for the C L / P malformation i n human (Ardinger et al., 1989; Chenevix-89 Trench et al, 1991, 1992; Holder et al, 1992; Sassani et al, 1993; Feng et al, 1994). In the present study, T G F - a was found to be present i n the facial prominences during the time of the fusion of the facial prominences. Together, these findings suggest that T G F - a may be important for the fusion of the facial prominences. Recently, knock-out mice w i t h mutant T G F - a gene were generated and found to have normal pr imary palate development (Mann et al, 1993; Luetteke et al, 1993). However, these data cannot completely preclude the importance of T G F - a in primary palate development, since the mutant mice may obtain compensatory effects from other growth factors for example EGF which is also expressed during primary palate development i n the present study. A l t h o u g h E G F and T G F - a were found to be co-localized dur ing primary palate morphogenesis, only EGF was present i n the developing lung bud. These data suggest that some tissues may not be able to fully compensate for lack of a gene product. A future study of transgenic mice lacking E G F w o u l d provide support for the role of E G F i n lung development. In summary, the temporo-spatial expression of E G F , T G F - a , and E G F - R was present dur ing primary palate morphogenesis. The three molecules were s h o w n to have s imilar dis tr ibut ion patterns at a l l stages examined. The distribution patterns of B r d U and P C N A were similar and most pronounced at the similar regions to E G F , T G F - a , and EGF-R. These findings suggest that E G F , T G F - a , and E G F - R may stimulate cell proliferation, which i n turn contribute to the fusion of the facial prominences and outgrowth of the primary palate. 90 C H A P T E R 4: Distr ibution of 72-kDa gelatinase (MMP-2) i n the developing craniofacial complex of the mouse embryo Introduction Craniofacial development is complex and involves many basic biological processes i n c l u d i n g tissue interactions, cell migrat ion, prol i ferat ion, and differentiation. A s a result, many organ systems; for example the primary and secondary palates, mandible, brain, nose, ear, and eye; are formed. Remodell ing of the extracellular matrix (ECM) was suggested to be important for cell migration, cell-cell interactions, embryo expansion, uterine implantation, and tissue invasion during mammalian embryogenesis (Werb et ah, 1992). However, the mechanisms of tissue remodelling i n the developing craniofacial complex remain unclear. Matr ix metalloproteinases (MMPs) are believed to play an important role i n physiological matrix degradation (reviewed by Overa l l , 1994). The M M P f a m i l y is composed of four enzyme classes; the collagenases, type IV collagenases/gelatinases, stromelysins, and membrane-type M M P s ( M T - M M P s ) . Interstitial collagenase specifically degrades types I-III collagens to produce thermally unstable degradation fragments which then denature to form gelatin, which is further degraded by other M M P s including gelatinases and stromelysins (reviewed by Overall , 1994). The 72-kDa (MMP-2) and 92-kDa gelatinases ( M M P -9) also degrade native types IV, V (Fessler et al., 1984), VII (Seltzer et al, 1989), and X collagens (Welgus et al., 1990). Al though 72-kDa gelatinase can degrade fibronectin and laminin, 92-kDa gelatinase does not (Okada et al., 1990). 91 The importance of 72-kDa gelatinase i n tissue r e m o d e l l i n g and embryogenesis is indicated from studies i n which 72-kDa gelatinase has been detected i n a wide variety of embryonic tissues. During tooth morphogenesis, 72-k D a gelatinase was found to be associated w i t h enamel development (Overall and Limeback, 1988) and dental basement membrane degradation i n mice (Sahlberg et al., 1992) and humans (Heikinheimo and Salo, 1995). E G F and T G F - a can induce 72-kDa gelatinase activity dur ing branching morphogenesis of the lung (Ganser et al., 1991). 72-kDa gelatinase is also associated w i t h breakdown of the basement membrane dur ing mammary gland involut ion (Talhouk et al., 1992; Dickson and Warburton, 1992), development of the mandibular condyle (Breckon et al., 1994), neurite outgrowth (Muir , 1994), and myogenesis (Guerin and Hol land , 1995). The expression of 72-kDa gelatinase has been s tudied by in situ hybr idizat ion dur ing mouse development and found to be local ized to the mesenchyme of the first, second, and third branchial arches of 10- and 11-day-old embryos but different stages of primary palate formation were not examined (Reponen et al., 1992). 72-kDa gelatinase was also expressed i n the vitreous body and corneal stroma of the developing eye and osteogenic mesenchyme of the mandible i n the 13-day-old embryo. More recently, M M P s , inc luding 72-kDa gelatinase, were detected i n the mouse facial prominences of 10- to- l l -day-old mouse embryos by zymography (Da Silveira et al., 1995), Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses (Da Silviera et al., 1996). These results have confirmed the presence of 72-kDa gelatinase i n the d e v e l o p i n g craniofacial complex and suggested its importance d u r i n g craniofacial development. However , the temporo-spatial protein expression of 72-kDa gelatinase i n the developing craniofacial complex has not been investigated. 92 The purpose of the study was to characterize relationships between 72-kDa gelatinase and early development of the mouse craniofacial complex by gelatin zymography and indirect immunofluorescence w i t h conventional and confocal laser scan microscopy. In addit ion, the co-distribution of 72-kDa gelatinase and the g r o w t h factors, E G F and T G F - a , were also investigated by indirect immunofluorescence w i t h confocal laser scan microscopy. Materials and Methods Tissue preparation C D 1 and C L / F r mice were mated overnight and embryos were collected on days 10 and 11 (plug = day 0). The number of tail somites (TS) from the caudal edge of the hind limb was determined (Wang, 1992). The heads were fixed i n 4% ( w / v ) paraformaldehyde i n PBS for 24 hours and paraff in embedded. The specimens were serially sectioned frontally through the face as previously described (Diewert and Lozanoff, 1993) at a thickness of 7 |im. Thirty four C D 1 mouse embryos were used for the facial prominences and seventeen C D 1 mouse embryos for eye studies. A small number of C L / F r mouse embryos w h i c h showed normal development of the pr imary palate were also used i n these studies (see Appendix 4). Immunofluorescence Deparaffinized sections were incubated w i t h 0.4% (w/v) pepsin i n 0.02 M HC1 for 5 m i n and then washed w i t h PBS. N o other proteolytic treatment was performed i n these studies. Subsequently, the slides were incubated i n normal goat serum for 30 m i n at room temperature and i n an antipeptide antibody 93 against the C-terminal domain of 72-kDa gelatinase (Wal lon and O v e r a l l , manuscript i n preparation) at a concentration of 1:200 overnight at 4° C . Different concentrations were tried and found that 1:200 was the opt imum concentration. Both non- and affinity-purified anti-72-kDa gelatinase antibodies were used and showed similar results. Therefore, the results from these two antibodies were used i n the present studies. After washing with PBS, the slides were incubated i n rhodamine-conjugated secondary antibody (Sigma, St. Louis, M O , goat anti-rabbit IgG crystalline TRITC conjugate, T-6778) at a concentration of 1:200 for 1 hour at 4° C and washed. The slides were observed and photographed w i t h a Zeiss photomicroscope equipped wi th a filter for rhodamine excitation. The co-localization of 72-kDa gelatinase w i t h the growth factors, E G F and T G F - a , was determined by using the adjacent consecutive sections of the primary palate of the 10- to- l l -day-old mouse embryos under the same conditions of immunosta ining. Sections from embryonic day 12 were also inc luded. The immunofluorecence studies of E G F , T G F - a , and 72-kDa gelatinase and confocal laser scan microscopy were performed as described i n Chapters 3 and 4A. For semiquantitative study, three embryos from each stage were included (see Table V) . Intensity of the fluorescence staining was recorded under the epifluorescence microscope as the fol lowing: +++ = strongly positive staining; ++ = moderately positive staining; + = weakly positive staining; - = negative staining. The modal values were recorded and shown in Table V . The anti-72-kDa gelatinase antibody was an antipeptide antibody raised i n rabbits against peptide coupled to keyhole limpet hemocyanin. The peptide sequence was selected from an analysis of the primary sequence of the C-terminal domain of human and murine 72-kDa gelatinase which indicated it to be solvent exposed. Subsequent analysis of the 3D structure of the C-terminal domain of 94 human 72-kDa gelatinase reported by Libson et al. (1995) confirmed the solvent accessibility of the peptide sequence which was located on the outer ^-strand of the hemopexin-like module 3 of the C-terminal domain. Antipeptide antiserum was collected from whole bodybleed and then affinity purif ied against the peptide coupled to an Aff igel-10 c o l u m n (BioRad). The specificity against 72-kDa gelatinase was confirmed by Western blot analysis (Wallon and Overal l , 1996, data not shown). Sections of the developing pr imary palate were also studied w i t h a polyclonal antibody against the whole molecule of h u m a n 72-kDa gelatinase (k indly p r o v i d e d by D r . W . G . Stetler-Stevenson). The results showed immunolocal izat ion i n the similar regions to those stained w i t h the antibody against the C-terminal domain of 72-kDa gelatinase (Wallon and Overal l , 1996). However , the staining appeared to be also present i n the extracellular matrix w i t h h igh background suggesting nonspecific b inding (data not shown). A small number of frozen sections of the primary palate were also immunostained w i t h the antibody against the C-terminal domain of 72-kDa gelatinase. A l t h o u g h the staining was localized i n the lateral edges of the nasal prominences and the maxi l lary t ip , s imilar to paraformaldehyde-f ixed sections, the results were inconclusive since tissue morphology was poorly preserved (data not shown). Confocal Microscopy Tissues of the craniofac ia l complex were e x a m i n e d w i t h both epifluorescence and confocal microscopy using a 20X objective on the Confocal Laser Scan Microscope (Zeiss, Germany). A helium-neon laser (Xmax=543 nm) was used for rhodamine-labelled tissues. Tissues were sectioned at 1 or 2 |im intervals and images were printed using a video printer (UP-5000, Sony Canada). 95 Gelatin Zymography 72-kDa gelatinase activity was detected by ut i l iz ing a discontinuous-SDS-p o l y a c r y l a m i d e - g e l electrophoresis-system w i t h L - m e t h o x y - 2 , 4 - d i p h e n y l -3(2H)furanone labelled (64958; F luka, Switzerland) gelatin (G-6650, Sigma) as previously described (O'Grady et al, 1984). By the use of this method, gelatin degradation could be visual ly monitored under long-wave ultraviolet l ight. Tissues from day 11 CD1 mouse heads were dissected, snap frozen, homogenized i n SDS non-reducing electrophoresis buffer, and centrifuged. The supernatants were loaded onto a 7.5% SDS-PAGE gel co-polymerized wi th 1 m g / m l of labelled gelatin. After electrophoresis the gel was washed twice i n 50 m M Tris buffer ( p H 7.5) containing 0.02% N a N 3 and 2.5% Triton X-100. The second wash was then supplemented w i t h 5 m M C a C l 2 and 1 ( i M Z n C l 2 and after incubation for 24 hours (37° C) in a 50 m M Tris buffer containing 5 m M C a C l 2 , 200 n M N a C l , 1 | i M Z n C l 2 , and 0.02% N a N 3 ( p H 7.5), the gel was viewed by long-wave ultraviolet light, photographed, and the gel then stained wi th 0.2% Coomassie Blue R-250. Results Immunofluorescence Primary Palate Morphogenesis of the primary palate can be classified into three stages; epithelial seam formation (7-11 TS), epithelial seam disruption (12-17 TS), and mesenchymal bridge enlargement (18-20 TS). D u r i n g epithelial seam formation (7-11 TS), i n the anterior region of the developing mouse face, the staining of 72-kDa gelatinase was mainly present i n epithelial and mesenchymal tissues particularly i n the tips and peripheral 96 regions of the lateral and medial nasal prominences (Figs. 19A,B). U p o n fusion of the m e d i a l nasal prominence w i t h the lateral nasal and m a x i l l a r y prominences, the staining was intensely local ized to the fus ion and the peripheral regions of the prominences (Figs. 19C,D). In the deeper region, the staining decreased i n the nasal f in area but remained h igh i n the peripheral regions of the facial prominences and the tip of the maxilla (Figs. 19E,F). A s the epithelial seam became disrupted w i t h mesenchymal bridge formation (12-17 TS), the staining of 72-kDa gelatinase was strongly localized to the fusion and the peripheral regions of the nasal prominences (Figs. 20A,B). In the deeper region, the staining decreased to background levels i n the nasal f in area but remained intense i n the peripheral regions of the lateral nasal and maxillary prominences (Figs. 20C,D). A s the mesenchymal bridge enlarged (18-20 TS), the staining of 72-kDa gelatinase was generally reduced and mainly present in the peripheral regions of the lateral nasal and maxillary prominences (Figs. 20E,F). The midl ine region, brain, and nasal epithelium showed negative staining at all stages examined. A small number of C L / F r mice were also stuidied and showed similar results to those of C D 1 mice (data not shown). M a n d i b l e and Second Branchial A r c h The i m m u n o l o c a l i z a t i o n of 72-kDa gelatinase was s tudied i n the developing mandible and second branchial arch. A t 9 day gestation, 72-kDa gelatinase staining in the mandible was at background levels (see Chapter 5, Fig. 27A). The data revealed a similar pattern of the distribution at embryonic days 10 and 11. In the anterior region of the mandible, the staining of 72-kDa gelatinase was localized to the epithelium and mesenchyme i n the tip of the mandible (Figs. 21A,B). A s the mandibular prominences merged, the distribution of 72-kDa 97 gelatinase was intensely present i n the peripheral regions of the mandible and the superior aspect of the midline area. 72-kDa gelatinase was also labelled to the peripheral regions of the second branchial arch (Figs. 21C,D). The staining of 72-kDa gelatinase remained negative in the midline tissues at all stages examined. Eye D u r i n g formation of the lens vesicle (embryonic day 10), i n the region anterior to the lens, the staining of 72-kDa gelatinase was mainly localized to the surface ectoderm and the underlying mesenchyme (Figs. 22A,B). A t the area where the surface ectoderm invaginated to form the lens vesicle (embryonic day 10), staining i n the epithelium became negative (Figs. 22C,D). However , the surface ectoderm adjacent to the lens pit remained labelled. In the later stages (embryonic day 11), as the lens completely detached from the surface ectoderm, the staining of 72-kDa gelatinase reappeared i n the deve loping corneal epithelium and mesenchyme (Figs. 22E,F). Blood cells i n the vitreous capillaries revealed autofluorescence. The lens and the optic cup and stalk showed negative staining at all stages examined. Gelatin Zymography To confirm the expression of 72-kDa gelatinase i n the developing mouse craniofacial complex, the gelatinase activity from day 11 embryos (16-18 TS) was analyzed by gelatin zymography. A l l six samples from embryonic mouse heads dur ing 16-18 TS showed similar results. The results revealed a major gelatinase band corresponding to 72-kDa gelatinase and minor bands representing the activated form of 72-kDa gelatinase (Fig. 21E). In addit ion, a minor , higher molecular weight gelatinase band was also detected of unknown identity. 98 Co-distribution of 72-kDa gelatinase, E G F , and T G F - a Sections from twelve C D 1 mouse embryos were used i n this series of experiments. The distribution patterns of EGF, T G F - a , and 72-kDa gelatinase i n the developing pr imary palate from 10-day-old embryos through 12-day-old embryos were studied by the use of confocal laser scan microscope. The results were summarized i n Table V and described below. Epithel ia l seam formation (7-11 TS) In the anterior region of the face, the staining of E G F , T G F - a and 72-kDa gelatinase was localized to the epithelial and mesenchymal tissues, particularly at the tips and peripheral regions of the lateral and medial nasal prominences (Figs. 24A,B,C). The staining of EGF, T G F - a and 72-kDa gelatinase appeared to decrease i n the central area of the medial nasal prominence. In deeper regions, the labell ing of E G F , T G F - a and 72-kDa gelatinase was intensely present i n the peripheral regions of the facial prominences and the tip of the maxilla (data not shown). The midline tissue showed reduced staining. The nasal epithelium and brain showed minimal staining at all stages examined. Epithel ia l seam disruption (12-17 TS) A s the epithelial seam became disrupted w i t h a replacement of the mesenchymal bridge, i n the anterior region to the fusion, the staining of E G F , T G F - a and 72-kDa gelatinase was localized to the tips and peripheral regions of the nasal prominences (data not shown). U p o n fusion of the media l nasal prominence w i t h the lateral nasal and maxillary prominences, the labelling of E G F , T G F - a and 72-kDa gelatinase was pronounced i n the fusion and the 99 peripheral regions of the facial prominences and the tip of the maxil la (Figs. 25A,B,C). In the deeper region, the staining of EGF, T G F - a and 72-kDa gelatinase appeared to be generally decreased but remained i n the peripheral regions of the facial prominences (data not shown). Mesenchymal bridge enlargement (18-20 TS) A s the mesenchymal bridge rapidly enlarged w i t h the outgrowth of the pr imary palate, the staining of E G F , T G F - a and 72-kDa gelatinase became generally reduced but remained strongly distributed in the peripheral regions of the lateral nasal and maxil lary prominences (Fig. 26A,B,C). The midl ine and deeper regions of the primary palate showed absent staining for EGF, T G F - a , and 72-kDa gelatinase. Dur ing embryonic day 12, the labelling of EGF, T G F - a , and 72-kDa gelatinase appeared negative in the primary palate. Table V : Distribution of 72-kDa gelatinase, EGF and T G F - a during primary palate morphogenesis. 7-11 TS 12-17 TS 18-20 TS day 12 7 2 - k D a g e l a t i n a s e + ++ + E G F ++ +++ ++ T G F - a + ++ + +++ = strongly positive staining; ++ = moderately positive staining; + = weakly positive staining; - = negative staining 1 0 0 101 o 1 0 2 2 1 B a@@[jDDQ) 2 1 D Tail Somites r 17 18 1 21E <- minor | - pro-gelatinase * active gelatinase 45 -> 1 0 3 1 0 4 1 0 5 1 0 6 Figs. 19 A - F : Epithel ia l seam formation (embryonic day 10): The confocal micrographs of anterior frontal sections show the staining of 72-kDa gelatinase i n the tips and peripheral regions of the lateral and medial nasal prominences (19A). U p o n fusion of the facial prominences, the staining was intensely localized to the fusion (between arrowheads) and peripheral regions (19C). Posteriorly, the staining remained intense i n the peripheral regions of the facial prominences. The nasal f in area showed negative staining (arrows) (19E). The corresponding hematoxylin and eosin (H&E) sections are shown (19B,D,F). L N , lateral nasal prominence; M N , media l nasal prominence; M X , max i l l a ry prominence; E, nasal epithelium. Figs . 20 A - F : Epithel ia l seam disruption (embryonic day 11): The confocal micrographs of frontal sections show intense staining of 72-kDa gelatinase at the epithelial seam (arrows) and the peripheral regions of the facial prominences (20A). Posteriorly, the staining remained pronounced i n the peripheral regions of the facial prominences (20C). The nasal epithelium and nasal f in (arrows) were negative. Dur ing mesenchymal bridge enlargement, the staining of 72-kDa gelatinase was generally reduced but remained i n the peripheral regions of the primary palate (4E). The corresponding H & E sections are shown (20B,D,F). Figs. 21 A - E : A t embryonic day 11, the staining of 72-kDa gelatinase was intensely localized i n the tip of the mandible (arrow) and the peripheral region of the second branchial arch (21 A ) . A s the mandibular prominences merged, the staining was mainly present i n the peripheral regions of the mandible and second branchial arch and the superior aspect of the merging area of the mandible (arrow) (21C). The corresponding H & E sections are shown (21B,D). Gelatinase activity of the mouse heads was detected by gelatin zymography (21E). Two heads from each 17 and 18 tail somite stages were analyzed by a 7.5% SDS-P A G E gel impregnated w i t h gelatin labelled w i t h L-methoxy-2,4-diphenyl-1 0 7 3(2H)furanone. A l l lanes reveal similar results showing a major gelatinase band corresponding to 72-kDa gelatinase and minor bands representing activated forms of 72-kDa gelatinase. BR, brain; M D , mandible; II, second brachial arch. Figs . 22 A - F : D u r i n g lens development (embryonic day 10), the confocal micrographs show intense staining of 72-kDa gelatinase i n the surface ectoderm and mesenchyme at the anterior region to the forming lens (22A). A t the region where the surface ectoderm invaginated to form the lens vesicle, the staining became absent in the epithelium (arrow) (22C). A t later stages (embryonic day 11), the staining reappeared i n the surface ectoderm and mesenchyme as the lens completely formed (22E). The optic cup and stalk showed negative staining at all stages examined. The corresponding H & E sections are shown (22B,D,F). L , lens; O C , optic cup; OS, optic stalk. Figs. 23 A - C : Dur ing the stage of lens differentiation (embryonic day 13), 72-kDa gelatinase was mainly localized to the developing corneal epi thel ium and mesenchyme (arrowheads) (23A). The staining appeared negative i n the other structures of the developing eye. The negative control sections f rom the developing eye (23B) and primary palate (23C), incubated w i t h normal serum as a replacement of the pr imary antibody, showed absent staining. Blood cells i n capillaries revealed autofluorescence. Figs. 24-26: Frontal sections through the developing mouse nasal region during formation of the primary palate. The sites of the fusion of the facial prominences are shown by the arrows. BR, brain; L N , lateral nasal prominence; M N , medial nasal prominence; M X , maxillary prominence; N C nasal cavity F ig . 24: D u r i n g the epithelial seam formation (7-11 TS), i n the anterior region of the face, the confocal micrographs show intense staining of 72-kDa gelatinase (A), E G F (B) and T G F - a (C) i n the epithelial and mesenchymal cells at the tips and peripheral regions of the lateral and medial nasal prominences. 1 0 8 F i g . 25: A s the epithelial seam became disrupted (12-17 TS), the confocal micrographs show strong labelling of 72-kDa gelatinase (A), E G F (B) and T G F - a (C) i n the epithelial and mesenchymal cells at the peripheral regions of the nasal and maxillary prominences and the tip of the maxilla. F ig . 26: D u r i n g enlargement of the mesenchymal bridge w i t h the outgrowth of the primary palate (18-20 TS), the confocal micrographs show markedly reduced staining of 72-kDa gelatinase (A), E G F (B) and T G F - a (C) i n the facial prominences. However, the labelling of 72-kDa gelatinase (A), E G F (B) and TGF-a (C) remained pronounced in the peripheral regions of the primary palate. 109 Discussion The temporo-spatial distribution of 72-kDa gelatinase was demonstrated i n early development of the mouse craniofacial complex, part icular ly i n the p r i m a r y palate, mandible , second branchial arch, and eye, by indirect immunofluorescence w i t h conventional and confocal laser scan microscopy. Staining of 72-kDa gelatinase was found to be intensely local ized i n the peripheral regions and the tips of the primary palate, mandible, and second branchial arch. These regions of high expression, particularly of the pr imary palate, were consistent w i t h the outgrowth regions characterized i n previous studies (Diewert and Wang, 1992; Diewert and Lozanoff, 1993). It was found that the size of the maxillary region increases rapidly, particularly i n the most distal regions, as the maxil lary prominence grows forward to meet the medial nasal and lateral nasal prominences. Whereas, the size of the lateral nasal prominence increases w i t h a predominantly lateral growth pattern. These outgrowth regions of the primary palate also show intense localization of fibronectin (see Chapter 2). Thus, adaptation of existing E C M and basement membrane to the expanded form of the rapidly growing facial proprominences appears to require adaptive remodelling by 72-kDa gelatinase. It was suggested that M M P function i n tissue remodelling may be to disrupt E C M receptor-substrate contacts to mobilize cells or degrade the E C M i n preparation for a change i n composition (Werb et al., 1992). Alternatively, the binding of cells to matrix components may reduce their proliferation (Mart in and Sank, 1991). Therefore, the induct ion of M M P s by growth factors may serve to release the cell from its contacts w i t h collagen and al low expression of the growth factor receptors required for cell proliferation. More specifically, the fibronectin degrading activity of 72-kDa gelatinase may release cells from this important cell attachment protein and facilitate alterations i n cell shape and cell proliferation. Indeed, it was recently identif ied that a 1 1 0 fibronectin b inding site on 72-kDa gelatinase localized to the hemopexin-like domain of the enzyme (Wallon and Overall , submitted) which may be involved i n this process. The results here are somewhat different f rom the previous in situ hybridizat ion study of 72-kDa gelatinase expression by Reponen et al. (1992) where 72-kDa gelatinase m R N A was f o u n d m a i n l y expressed i n the mesenchyme wi th the surface ectoderm being completely negative. However , in the present study, it was clearly found that some regions of the surface ectoderm, particularly i n the peripheral areas of the facial prominences, were strongly positive for 72-kDa gelatinase. Similar f indings have also been reported for human breast (Monteagudo et al, 1990; Soini et al, 1994), colon (Levy et al, 1991), and ovarian cancers (Aut io-Harmainen et al., 1993) where 72-kDa gelatinase protein has been localized to the cancer cells by immunohistochemistry, whereas the m R N A transcripts were found i n the stromal cells adjacent to invading tumor cells of breast (Soini et al, 1994), colon (Pyke et al, 1993), and ovarian cancers (Autio-Harmainen et al., 1993) by in situ hybridization. A t these sites, it appears that 72-kDa gelatinase is synthesized by the stromal cells and exported to the E C M where b inding to the tumor cells occurs in trans. However , i n the present study, although 72-kDa gelatinase may be localized to cell surfaces on cells not expressing 72-kDa gelatinase m R N A , partly explaining the discrepant results to Reponen et al. (1992), cytoplasmic staining was also a prominent feature of the outer cell layers indicating cellular synthesis in these regions. The presence of 72-kDa gelatinase i n the craniofacial complex was confirmed by functional assays using gelatin zymography. The data revealed that 72-kDa gelatinase is the predominant gelatinase expressed dur ing craniofacial development. In addit ion, an activated form of the enzyme was also detected showing that 72-kDa gelatinase is functional during craniofacial morphogenesis. 111 These results were consistent w i t h those of previous investigations by gelatin zymography (Da Silveira et ah, 1995), Western blot and R T - P C R analyses (Da Silveira et al., 1996) in which 72-kDa gelatinase was found to be expressed i n the facial prominences during the primary palate formation. Taken together, these f indings indicate that 72-kDa gelatinase may p lay a signif icant role i n remodell ing the E C M i n the fast outgrowing regions of the pr imary palate, mandible, and second branchial arch. Dur ing eye development, the staining of 72-kDa gelatinase was found to be absent in the invaginating epithelium during formation of the lens vesicles. In the later stages, as the lens completely detached from the surface ectoderm, the staining of 72-kDa gelatinase reappeared i n the developing corneal epithelium and mesenchyme, whereas the lens and optic cup remained negative at all stages examined. These observations imply that i n order to al low invagination of the surface ectoderm to form the lens vesicle, stabilization of the E C M i n the invaginating areas may be needed. Indeed, it was found that there is a strong adhesion between the optic vesicle and the lens placode (McKeehan, 1971). Later immunohistochemical studies have also confirmed that the staining of types I and IV collagens, laminin, and fibronectin is intensely localized to the interspace between the lens and the optic vesicle throughout the invagination of the lens vesicle (Parmigiani and M c A v o y , 1984; Hilfer and Randolph, 1993; Peterson et al., 1995). Recently, Sheffield et al. (1992, 1994) have demonstrated participation of plaminogen activator and high molecular weight metalloproteinases, but not 72-k D a gelatinase, during histogenesis of the embryonic retina and suggested that these proteinases play a role i n fiber outgrowth from retinal cells. Lastly, a further indicat ion of the importance of M M P s i n the development and maintenance of the E C M of the eye has been demonstrated by the presence of specific mutations of TIMP-3 gene i n Sorsby's fundus dystrophy (Weber et al., 1 1 2 1994) and the elevated expression of TIMP-3 m R N A i n retinas affected by simplex retinitis pigmentosa (Jones et al., 1994). Other M M P s have also been implicated i n developmental process i n w h i c h epithel ial invaginat ion occurs. In branching morphogenesis of the salivary gland, collagenase inhibits cleft formation of the gland, whereas T I M P induces supernumerary clefts (Hayakawa et al., 1992). Therefore, it was proposed that collagenase and T I M P regulate cleft formation through modulat ion of the E C M . Similarly, it was found that addition of purified mammalian collagenase to lung cultures inhibited epithelial branching and produced end b u d enlargement (Ganser et al., 1991). However, a more recent study showed that stromelysin-1 can induce hyperproliferation and differentiation of mammary epithelial cells in v i r g i n female transgenic mice that express autoactivated isoforms of s t romelys ins-1 (Sympson et al., 1994). D u r i n g lactat ion, s tromelysin-1 demonstrated lytic activities, d isrupt ing basement membrane integrity and reducing mammary-specif ic function. Implantation of slow-release pellets containing T I M P - 1 , on the other hand, delayed mammary gland involut ion (Talhouk et al., 1992). These findings suggest that epithelial invagination, like lens development, is complex and requires well-balanced expression between M M P s and TIMPs. It is important that regulation of tissue remodelling be finely balanced to enable sufficient degradation of E C M to facilitate tissue proliferation whi le mainta ining tissue integrity. Indeed, regulation of M M P s takes place i n a complex but coordinated manner and at several levels. Regulation of M M P s can occur locally by TIMPs which form 1:1 complexes wi th activated M M P s to inhibit their activity (for review see Matr is ian, 1992; Overal l , 1994). G r o w t h factors, cytokines, phorbol ester tumor promoters, and oncogene products can either induce or suppress expression of specific M M P genes (for review see Matrisian, 1 1 3 1992; Overal l , 1994). For example, transforming growth factor-beta (TGF-P), a growth factor associated wi th developmental processes, can induce an increase i n expression of 72-kDa gelatinase (Overall et al, 1989, 1991). T G F - a and E G F proteins were also implicated in the induction of M M P activity, particularly 72-kDa gelatinase, during lung development (Ganser et al., 1991). Gelatinase protein was also demonstrated i n the blastocyst d u r i n g mouse pre implanta t ion development (Brenner et al., 1989) and found to be induced by T G F - a (Dardik et al., 1993). It was suggested that upon stimulation by T G F - a , gelatinases, secreted from the blastocyst, may be involved i n blastocoel E C M remodel l ing and migration of the parietal endoderm cells (Dardik et al., 1993). Indeed, we have also localized E G F and T G F - a expression w i t h i n the similar regions of 72-kDa gelatinase expression shown i n the present study dur ing the pr imary palate formation (see Chapter 3). Taken together, these data indicate that E G F and TGF-a may be involved in the regulation of M M P expression during embryogenesis. Since E G F , T G F - a , and 72-kDa gelatinase were localized to the similar regions of the developing primary palate at all stages examined i n the present study, I hypothesized that E G F and T G F - a are inducers of 72-kDa gelatinase synthesis in vivo and participate i n the regulation of cell proliferation and tissue outgrowth d u r i n g pr imary palate morphogenesis. Further investigation is needed to be performed i n order to study the induction of 72-kDa gelatinase by E G F and T G F - a . For example; E G F and T G F - a can be supplemented to organ cultures of primary palate, as was done i n lung explants (Ganser et ah, 1991). If E G F and T G F - a are inducers of 72-kDa gelatinase, an increase of 72-kDa gelatinase expression i n the primary palate w i l l be detected. 72-kDa gelatinase 1 1 4 expression i n culture can be evaluated by zymography, Western blots, and quantitative R T - P C R analyses. In conclusion, the temporo-spatial distribution of 72-kDa gelatinase during early craniofacial development was found to be associated w i t h the tips of growing facial prominences. These findings suggest that 72-kDa gelatinase may be invo lved , at least i n part, i n tissue remodell ing d u r i n g the early mouse craniofacial morphogenesis. 1 1 5 C H A P T E R 5: Immunolocalization of 72-kDa Gelatinase and Extracellular Matr ix Components during Mouse Cardiac Development Introduction The heart tube is initially formed by convergence of the two lateral cardiac pr imordia i n the ventral midl ine at embryonic day 7 i n mice (Sissman, 1970; H o p p e r and Har t , 1980). The heart tube is composed of two layers: an endothelium and a myocardium, which are separated by an acellular sleeve of extracellular matrix (ECM), the cardiac jelly or myocardial basement membrane ( M a r k w a l d et al, 1984; Kitten et al., 1987). A s the heart develops further, the myocardium, particularly i n the ventricles, loses its epithelial organization and forms trabeculations at about embryonic day 9 i n mice shortly after the first myocardia l contractions (Sissman, 1970). These trabeculations subsequently contribute to the interventricular septum and papil lary muscles (Pexieder and Janecek, 1984; H a y et al. , 1984). In later development, the endothelial cells i n the regions of the atrioventricular canal and outflow tract undergo transformation into mesenchymal cells (Manasek, 1976; Runyan and M a r k w a l d , 1983). They embed in the cardiac jelly forming endocardial cushion tissue. The ventral and dorsal endocardial cushions are first definable at embryonic day 10, unite at embryonic day 11 i n mice (Sissman, 1970), and later give rise to septal and valvular structures (Van Mierop et al, 1962; Manasek, 1976). Extracel lular matrix appears to p lay an important role i n heart development, several components of w h i c h have been studied i n c l u d i n g fibronectin, laminin , type IV collagen, and elastin. Fibronectin is believed to r e g u l a t e p r e c a r d i a c c e l l m i g r a t i o n ( L i n a s k a n d L a s h , 1988). Immunohistochemical studies on stages 8-18 of chick embryos revealed that 1 1 6 f ibronectin was first found on the basal surfaces of the m y o c a r d i u m and endocardium dur ing fusion of the the heart pr imordia . Later the intensity of f ibronectin increases at the onset of trabeculations and then decreases as trabeculations are completed (Icardo and Manasek, 1983). Similar findings were found i n rat heart embryos during 4-16 somite stages (Tuckett and Morriss-Kay, 1986) . In addition, to being important for trabecular formation, fibronectin was also found to be closely associated wi th surfaces of migrating mesenchymal cells in the cardiac jelly of the endocardial cushion tissues (Icardo and Manasek, 1984). The distribution of fibronectin was demonstrated as a gradient extending from the myocardium towards the endocardium and appeared to contact endothelial cells just prior to epithelial-mesenchymal transformation (Mjaatvedt et al., 1987). This suggests the importance of f ibronect in i n epi the l ia l -mesenchymal transformation dur ing heart development. Moreover, transgenic homozygous mouse embryos l a c k i n g f ibronect in showed var ious defects i n heart development inc luding abnormal fusion of the heart p r i m o r d i a , thickened m y o c a r d i u m , deficient cardiac jelly, and abnormal or absent endocardium (George et al., 1993). The results i n the transgenic mouse study also support the importance of fibronectin in heart development. Immunolocal ization revealed that l aminin first appears i n the cardiac jelly at stage 15 of chick embryos (Little et al., 1989). Later laminin was localized to the basal surfaces of the myocardium and endocardium at stage 17 of chick embryos (Kitten et al., 1987) and during 8-16 somite stages of rat embryos (Tuckett and Morriss-Kay, 1986). O n the other hand, type IV collagen was first detected in E C M between the splanchnic mesoderm and endoderm of pretubular-heart chick embryos (stage 9) (Drake et al., 1990) and found on the basal surfaces of the myocardium and endocardium at the stage 17 of chick embryos (Kitten et al., 1987) . More recently, elastin was immuriolocalized to the outflow tract and the 1 1 7 base of the atrioventricular cushion dur ing stages 22-29 of embryonic chick embryos (Hurle et al, 1994). In later development, elastin was found i n the d e v e l o p i n g v a l v u l a r apparatus, subendocardial space of the atria, and epicardium. Thus, the distribution of these E C M molecules changes throughout heart development . A c c o r d i n g l y , unders tanding the processes of tissue remodell ing and the proteinases involved i n the developing heart is central to the understanding of heart morphogenesis and may provide insight into the remodelling processes that occur later in life during heart diseases. Dur ing cardiac development, rat interstitial collagenase-3 (Nakagawa et al., 1992) and plasminogen activator (McGuire and O r k i n , 1992) have been localized to embryonic heart tissues and suggested to be important for heart morphogenesis. M M P s have also been found to be i n v o l v e d w i t h tissue remodel l ing i n the normal adult (Tyagi et al., 1993) and diseased hearts (Takahashi et al., 1990) and very recently a nove l tissue inhibi tor of metalloproteinases (TIMP-4) was cloned from a human heart c D N A library (Eric Shi , personal communication) indicat ing the importance of M M P s i n the development and maintenance of the myocardial structures. A l t h o u g h 92-kDa gelatinase m R N A has not been detected i n embryonic heart tissue (Reponen et al., 1994; Canete-Soler et ah, 1995), the expression of 72-kDa gelatinase was detected i n the heart tissue of 9-day-old mice by in situ h y b r i d i z a t i o n and newborn and adult mice by Northern analysis (Reponen et al., 1992). However, the temporo-spatial expression of 72-kDa gelatinase protein dur ing early heart morphogenesis has not been investigated in detail. Col lect ively , remodell ing of the E C M is a major event d u r i n g heart morphogenesis and may be associated w i t h M M P activity. In the present study, the expression of 72-kDa gelatinase protein was investigated and correlated w i t h changes i n the distr ibution of substrates of the enzyme: type IV collagen, 1 1 8 laminin, and fibronectin during early morphogenesis of the mouse heart using indirect immunohistochemistry w i t h conventional and confocal laser scan microscopy. Materials and Methods Tissue preparation C D 1 mice were mated overnight and embryos were collected on days 9-13 (plug = day 0), fixed i n 4% (w/v) paraformaldehyde i n PBS for 24 hours, and paraffin embedded. The specimens were serially sectioned sagittally (9- and 10-day-old embryos) and coronally (10- to 13-day-old embryos) at a thickness of 7 um. Sections f rom at least 60 mouse embryos were used i n these studies (see Appendix 4). Immunohistochemistry The immunolocalization of 72-kDa gelatinase and the E C M components was s tudied on the embryonic heart f rom day 9 w h e n the ventr icular trabeculation begins (Sissman, 1970) unti l day 13 when the ventricles are largely composed of trabeculae carneae ( K a u f m a n , 1992). The a v i d i n - b i o t i n -immunoperoxidase technique was used as previously described (Hsu et ah, 1981). Deparaffinized sections were incubated w i t h 3% (v/v) hydrogen peroxide for 20 m i n to eliminate endogenous peroxidases. After washing w i t h PBS, the slides were incubated w i t h normal goat serum for 20 m i n at room temperature and then w i t h an affinity pur i f ied anti-72-kDa gelatinase antibody (Wallon and 1 1 9 Overa l l , 1996) overnight at 4° C . After washing w i t h PBS, the slides were incubated w i t h biotin-conjugated secondary antibody (Sigma, St. Louis , M O , goat anti-rabbit biotin conjugate, B-8895) and then w i t h StreptABComplex (Dakopatts, Santa Barbara, C A , ¥377) both at the concentration of 1:100 for 30 m i n at room temperature . D a r k b r o w n s t a i n i n g was d e v e l o p e d by u s i n g 3,3'-diaminobenzidine as a substrate. The sections were counter stained w i t h methyl green. Heart tissues were also s tudied by indirect immunofluorescence techniques w i t h a non-affinity pur i f ied anti-72-kDa gelatinase antibody. The results showed similar patterns of labelling to those of an affinity purif ied anti-72-kDa gelatinase w i t h the A B C technique at all stages examined. To investigate the E C M components, deparaffinized sections were digested w i t h 0.4% (w/v) pepsin (Sigma, St. Louis , M O ) i n 0.02M HC1 for 15 m i n , washed w i t h PBS and preincubated w i t h normal goat serum for 30 m i n . Subsequently, sections were incubated overnight at 4° C w i t h rabbit ant i - laminin (Sigma, St. Louis, M O , rabbit anti-EHS mouse sarcoma, L-9393), rabbit anti-type IV collagen (Chemicon, Temecula, C A , rabbit anti-mouse, AB756), or rabbit anti-fibronectin (Dako, G l o s t r u p , D e n m a r k , rabbit a n t i - h u m a n , A 245) ant i serum at concentrations of 1:100, 1:50, and 1:100, respectively. The slides were incubated w i t h fluorescein-conjugated (Sigma, St. Louis , M O , sheep anti-rabbit IgG FITC conjugate, F-7512) or rhodamine-conjugated (Sigma, St. Louis , M O , goat anti-rabbit IgG crystal l ine T R I T C conjugate, T-6778) secondary ant ibody at concentrations of 1:100 and 1:200, respectively for 1 hour at 4° C and washed. The slides were observed and photographed w i t h the Zeiss photomicroscope equipped w i t h the appropriate filters for rhodamine and fluorescein excitation. 1 2 0 The specificity of the anti-f ibronectin antibody has been previously characterized (Van Helden et al, 1985). Pre-absorption of anti- laminin and anti-type IV collagen antibodies w i t h an excess of l aminin and type IV collagen, respectively, abolished immunoreact ivi ty on heart tissue sections (data not shown). Experimental slides were incubated i n both the primary and secondary antibodies. W i t h i n each group of the sections, the negative control slides were incubated w i t h normal goat serum as a replacement for the primary antibody. The results showed no staining of the negative control. The mouse mandibular bone forming tissue was used as positive control tissue for the antibody against 72-kDa gelatinase (Reponen et al, 1992) which was strongly positive as predicted. A n t i - t y p e IV collagen, ant i - laminin , and anti-f ibronectin antisera showed intense immunoreactivity at the basement membrane of the surface ectoderm of the primary palate, which was used as the positive control for type IV collagen, laminin, and fibronectin (see Chapter 2). Confocal Microscopy The 10- to - l l -day-o ld embryonic heart tissues were studied by indirect immunof luorescence technique. The endocardia l c u s h i o n tissue at the atrioventricular canal was examined w i t h both epifluorescence and confocal microscopy using a 20X objective on the Confocal Laser Scan Microscope (Zeiss, Germany). A H e l i u m - N e o n laser (Xmax=543 nm) was used for rhodamine-labelled tissues. Tissues were sectioned at 1 or 2 \im intervals and images were pr inted us ing a v ideo printer (UP-5000, Sony Canada , R i c h m o n d , Br i t i sh Columbia) . 121 Results 9- Day-Old Embryo D u r i n g this developmental stage, the staining of 72-kDa gelatinase was generally present i n the cytoplasm and on the surfaces of the myocytes and endothelial cells of the ventricular and atrial walls (Figs. 27A,B). The body (thoracic) w a l l overlying the pericardial cavity and mandible showed only weak staining of 72-kDa gelatinase. Of note, the staining of 72-kDa gelatinase i n the cardiac jelly of the heart and the neural tissues appeared negative. The immunofluorescent staining also showed similar patterns of distribution of 72-kDa gelatinase (Fig. 27C). The staining of type IV collagen (Fig. 29A), l a m i n i n (Fig. 29B), and fibronectin (Fig. 29C) was mainly localized to the basal surfaces of the endothelial cells and myocytes of the ventricular and atrial walls. Type IV collagen, laminin, and fibronectin were also intensely stained i n the basement membrane of the body wal l . 10- D a y - O l d Embryo D u r i n g this developmental stage, staining of 72-kDa gelatinase was uni formly present i n the cytoplasm and on the surfaces of the myocytes and endothelial cells of the ventricular and atrial walls by the A B C (Figs. 27D,E) and immunofluorescent techniques (Fig. 27F). 72-kDa gelatinase was also distributed on the surface ectoderm of the facial prominences. The endocardial cushion tissues appeared less intensely positive for 72-kDa gelatinase i n comparison w i t h the myocardial tissues when viewed wi th epifluorescence microscopy (Fig. 27G). However , confocal microscopy revealed that 72-kDa gelatinase was strongly expressed i n many endocardial cushion endothelial and mesenchymal cells (Fig. 1 2 2 27H). The less intense labelling observed by epifluorescence microscopy was related to the low cell density and high E C M content of endocardial cushion tissue. Type IV collagen (Fig. 29D), laminin (Fig. 29E), fibronectin (Fig. 29F) were strongly and uniformly labelled on the basal surfaces of the endothelial cells and myocytes of the ventricular and atrial walls and i n the basement membrane of the body w a l l . Fibronectin also showed intense localization i n the mesenchyme of the body w a l l and endocardial cushion tissue at the atrioventricular canal (Fig. 29F). 11- D a y - O l d Embryo D u r i n g this developmental stage, the ventricular wal ls are markedly thickened and have become trabeculated, whereas the atrial walls appear to be thinner and more expanded. 72-kDa gelatinase was found to be strongly labelled in the cytoplasm and on the surfaces of the endothelial cells and myocytes of the ventricular and atrial walls Figs. 28A-C). O n the other hand, the endocardial cushion tissue at the atrioventricular canal showed decreased labelling for 72-kDa gelatinase (Figs. 28A,C) compared to 9- and 10-day-old embryos. The staining of type IV collagen (Fig. 29G), l a m i n i n (Fig. 29H), and fibronectin (Fig. 291) was mainly localized to the basal surfaces of the endothelial cells and myocytes of the ventricular and atrial wal ls part icular ly i n the ventricular trabeculation. The distribution of type IV collagen, l aminin , and fibronectin was markedly decreased i n the interventricular septum. 12- D a y - O l d Embryo A s the ventricular walls became more thickened, the labelling of 72-kDa gelatinase remained intense i n the ventricular trabeculations (Fig. 28E) and atrial 1 2 3 walls (Fig. 28F). However, the distribution of 72-kDa gelatinase was dramatically reduced in the ventricular walls and the interventricular septum. The distribution of type IV collagen (Fig. 30A), laminin (Fig. 30B), and f ibronectin (Fig. 30C) was pronounced i n the ventricular trabeculations. H o w e v e r , the staining of type IV collagen, l a m i n i n , and f ibronectin was decreased i n the ventricular w a l l and the interventricular septum. 13-Day-Old Embryo A s the ventricular and atrial walls and the trabeculations matured, the staining of 72-kDa gelatinase dramatically decreased. However, there was strong labelling of 72-kDa gelatinase i n the ventricular trabeculations (Fig. 28G). The intensity of the staining of type IV collagen (Fig. 30D), laminin (Fig. 30E), and fibronectin (Fig. 30F) was also generally decreased in the heart tissue, particularly i n the ventricular and atrial walls and the interventricular septum. The labelling of type IV collagen, laminin, and fibronectin appeared reduced i n the ventricular trabeculations i n comparison w i t h the previous stages. 1 2 4 1 2 5 Figs. 27 A - H : Sagittal and coronal sections through the embryonic heart from days 9-10. A , atrial chamber; BR, brain, E C , endocardial cushion tissue; M N , mandible; N , neural tissues; V , ventricular chamber. A t the 9-day-old stage of heart development, the staining of 72-kDa gelatinase was mainly present i n the ventricular and atrial walls , whi le the staining i n the mandible and neural tissues was slightly above background (Fig. 27A). H i g h magnification of the ventricle reveals labelling i n the myocytes and on cell surfaces of the endothelial cells (arrows) and their cytoplasmic processes (arrowheads) (Fig. 27B). Similar patterns of immunofluorescent labelling was also found in the ventricular w a l l (Fig. 27C). A t the 10-day-old stage, the staining of 72-kDa gelatinase was now intensely and uniformly expressed i n the ventricular and atrial walls (Fig. 27D). H i g h magnification also shows the staining i n the myocytes and on cell surfaces of the endothelial cells (arrow) and their cytoplasmic processes (arrowheads) (Fig. 27E). The immunofluorescent staining revealed similar patterns of distribution i n the ventricular and atrial walls (Fig. 27F). The endocardial cushion tissue at the atrioventricular canal (inset) showed labelling of 72-kDa gelatinase (Fig. 27G). By using confocal microscopy, the endocardial cushion tissue revealed intense cytoplasmic staining of 72-kDa gelatinase particularly in the endothelial (arrow) and mesenchymal cells (Fig. 27H). 1 2 7 Figs. 28 A - H : Coronal sections through the embryonic heart from days 11-13. A , atrial chamber; V , ventricular chamber. A t the 11-day-old stage, the staining of 72-kDa gelatinase remained intense in the ventricular and atrial walls (Fig. 28A). Higher magnification shows the labelling i n the myocytes and endothelial cells i n the ventricular (Fig. 28B) and atrial walls (Fig. 28C). The endocardial cushion tissue (arrow) showed decreased staining of 72-kDa gelatinase (Figs. 28A,C). The negative control section, incubated w i t h normal serum as a replacement for the pr imary antibody, showed methyl green counterstaining and a l ight b r o w n background staining (Fig. 28D). A t the 12-day-old stage, 72-kDa gelatinase was strongly labelled i n the trabeculations of the ventricle (Fig. 28E) but its intensity appeared reduced i n the outer ventricular w a l l and interventricular septum (arrow) (Fig. 28E). The atrial walls also showed intense labelling (arrow) (Fig. 28F). A t the 13-day-old stage, the staining of 72-kDa gelatinase generally decreased but remained intense i n the ventricular trabeculations (Fig. 28G). The negative control section', incubated w i t h normal serum as a replacement for the primary antibody, showed methyl green counterstaining and a light brown background staining (Fig. 28H). 128 1 2 9 Figs. 29 A - H : Selected sagittal and coronal sections through the embryonic heart from days 9-11. A , atrial chamber; V , ventricular chamber. A t the 9-day-old stage, the staining of type IV collagen (Fig. 29A), laminin (Fig. 29B), and fibronectin (Fig. 29C) were localized to the basal surfaces of the myocytes and endothelial cells in the ventricular and atrial walls. A t the 10-day-old stage, type IV collagen (Fig. 29D), laminin (Fig. 29E), and fibronectin (Fig. 29F) were now uniformly and intensely stained i n the myocytes and endothelial cells i n the ventricular and atr ial w a l l s . F ibronect in was also label led i n the cardiac je l ly at the atrioventicular canal (Fig. 29F). A t the 11-day-old stage, the staining of type IV collagen (Fig. 29G), laminin (Fig. 29H), and fibronectin (Fig. 291) remained pronounced i n the ventricular walls but the intensity appreared reduced i n the interventricular septa (arrows). 1 3 0 131 Fig . 30 A - F : Selected coronal sections through the embryonic heart from days 12 and 13. W , ventricular w a l l . A t the 12-day-old stage, the labelling of type IV collagen (Fig. 30A), laminin (Fig. 30B) and fibronectin (Fig. 30C) was mainly local ized to the ventricular trabeculations (arrowheads) but the intensity appeared decreased i n the outer ventricular w a l l . A t the 13-day-old stage, the staining of type IV collagen (Fig. 30D), laminin (Fig. 30E) and fibronectin (Fig. 30F) appeared to generally decrease i n the heart tissue but remained i n the ventricular trabeculations (arrowheads). 1 3 2 Discussion In the present study, we have undertaken a comprehensive analysis of the temporo-spatial distribution of 72-kDa gelatinase as w e l l as type IV collagen, laminin and fibronectin, E C M molecules that are substrates of 72-kDa gelatinase. The expression and activity of 72-kDa gelatinase i n heart tissues reported here is consistent w i t h a role for the enzyme i n the remodell ing of these components. The temporo-spatial distribution of type IV collagen, laminin , and fibronectin, the major basement membrane components, was highly correlated w i t h that of 72-kDa gelatinase during days 9-13 of heart morphogenesis. Indeed, it was shown that the fibronectin type II-like repeats of 72-kDa gelatinase b i n d native and denatured type I collagen and elastin (Steffensen et al., 1995) whereas the C -terminal d o m a i n of the enzyme binds fibronectin and heparin (Wal lon and Overa l l , 1996), indicat ing a possible mechanism for tissue localization of the enzyme. Type IV collagen, laminin and fibronectin were found to be uniformly and intensely labelled on the basal surfaces of the myocytes and endothelial cells during the early stages of development. A s the heart matured, the staining of type IV collagen, l a m i n i n , and fibronectin was retained i n the ventricular trabeculation, but showed reduced intensity particularly i n the ventricular walls. Previous studies on the distribution of fibronectin (Icardo and Manasek, 1983; Tuckett and Morr iss -Kay, 1986) also showed reduced staining i n later stages of heart development, w h i c h w i t h our data indicate that i n addit ion to fibronectin, remodell ing of type IV collagen and laminin occurs throughout early heart morphogenesis. 72-kDa gelatinase was also uni formly present i n the myocytes and endothelial cells i n the ventricular and atrial walls at the early stages. A t later 1 3 3 stages, the staining of 72-kDa gelatinase was dramatically reduced i n the walls and mainly localized to the trabeculated ventricles. Taken together, these results indicate that 72-kDa gelatinase may be coexpressed wi th the E C M molecules and then participates i n the remodelling of the E C M during the rapid formation of the ventricular trabeculations. The expression of M M P s has also been observed d u r i n g organ development where fast growth occurs. For example, the distribution of 72-kDa gelatinase was found to be correlated w i t h outgrowth formation of the developing craniofacial complex at mouse embryonic days 10 and 11 (see Chapter 4). Moreover, 72-kDa gelatinase colocalized w i t h epidermal growth factor and transforming growth factor-a during the outgrowth of the developing mouse primary palate (see Chapters 3 and 4). Nerve growth factor upregulated 72-kDa gelatinase expression by chick dorsal root ganglionic neurons i n culture and was thought to play an important role i n E C M degradation during neurite outgrowth (Muir , 1994). M M P s , including 72-kDa gelatinase, have also been found to be expressed during mouse blastocyst outgrowth (Brenner et al., 1989; Behrendtsen et al., 1992; Harvey et al., 1995). Indeed, elevation and co-expression of E C M components (fibronectin, type I collagen, and S P A R C ) w i t h 72-kDa gelatinase by transforming growth factor-(31 (TGF-P 1), a potent growth factor involved i n tissue morphogenesis, has been detected i n human fibroblast cells (Overall et al, 1989; 1991). A t that time, it was proposed that 72-kDa gelatinase may not only be involved i n the remodell ing of newly deposited matrix, but may also perform a "quality control" role by degrading misfolded or denatured collagens i n the provisional matrix. In the present study, the decreased expression of 72-kDa gelatinase and the E C M components i n the interventricular septum and ventricular w a l l i n the later stages was evident. These data indicate that reduction of the 72-kDa 1 3 4 gelatinase expression i n those regions may occur possibly by a passive rundown of the i n d u c i n g signal or an active down-regulat ion by growth factors or hormones. Indeed, i n areas showing reduced 72-kDa gelatinase staining, isolated cells still showed intense 72-kDa gelatinase labelling. This indicates that although a tissue may exhibit a general down-regulation of 72-kDa gelatinase expression, i n d i v i d u a l cel l responsiveness varies. In a d d i t i o n , tissue inhibi tors of metalloproteinases (TIMPs) may also play an important role in local repression of 72-kDa gelatinase activity in those regions. Indeed, the expression of TIMP-3 was intensely localized to the developing mouse heart at day 12.5 and reduced at day 14.5 (Apte et al., 1994). However, TIMP-1 was not detected i n the developing heart (Nomura et al., 1989) and there is no available information on TIMP-2 expression d u r i n g heart development. The dis tr ibut ion of T IMP-4 i n the embryonic heart is currently under study in our laboratories. 72-kDa gelatinase may play a significant role in cell migration during an epithelial-mesenchymal transformation by degrading the basement membrane components, since the expression of 72-kDa gelatinase was found i n the endothelial and mesenchymal cells of the endocardial cushion tissue. Support for an important role of the enzyme i n cell migration and invasion also comes from the cell surface distribution of 72-kDa gelatinase reported here on the myocytes and endothel ial cells. This is consistent w i t h the cel l surface d i s t r i b u t i o n of endogenously activated enzyme u p o n C o n c a n a v a l i n - A stimulation (Overall and Sodek, 1990) that was later found to be associated w i t h the cell membrane (Ward et al., 1994). TGF-(31, a transcriptional inducer of 72-k D a gelatinase (Overall et al., 1991), was also localized to the endothelial cells i n the atrioventricular canal dur ing formation of the endocardial cushion tissue (Akhurst et al., 1990). In addit ion, TGF-f31, i n combination w i t h ventricular 1 3 5 m y o c a r d i u m , can mediate an epithel ial -mesenchymal transformation by cultured atrioventricular canal endothelial cells in vitro (Potts and R u n y a n , 1989). This mesenchyme containing invaded myocytes w i l l eventually form heart septal and valvular structures (Van Mierop et al., 1962; Manasek, 1976). Taken together, it is suggested that the endothelial cells i n the atrioventricular canal may produce 72-kDa gelatinase, upon stimulation by T G F - p i , i n order to degrade the basement membrane and other E C M molecules during invasion of the under ly ing tissue to form the heart mesenchyme. Involvement of 72-kDa gelatinase d u r i n g endothelial invasion into the u n d e r l y i n g tissue at the atrioventricular canal models a similar situation that may take place dur ing invasion of the tumor cells into stromal tissue. Indeed, 72-kDa gelatinase is found to be produced by the culture explants of a highly metastatic murine tumor and shows a strong correlation wi th the metastatic potential of cancer cells (reviewed by Liotta et al., 1980; Stetler-Stevenson et al, 1993). Various human cancers, for example breast (Monteagudo et al., 1990), colon (Levy et al., 1991), and ovarian (Autio-Harmainen et al., 1993) cancers demonstrate 72-kDa gelatinase i n the tumor cells. Collectively, tumor cells and endocardial cushion endothelial cells may share similar mechanisms for tissue invasion that ut i l ize 72-kDa gelatinase. Therefore, a better understanding of the mechanism of endothelial invasion of the underlying tissue i n the atrioventricular canal may prove useful i n understanding the mechanisms of invasion and metastasis of the tumors. Recently, M M P - 1 and TIMP-1 were found to be co-localized to the interstitial space between cardiac muscle bundles and w i t h i n the endothelium and subendocardial space of the endocardium in normal adult rat heart (Tyagi et al., 1995a). Northen blot results also showed the co-expression of M M P - 1 and TIMP-1 i n various adult tissues including the heart, skin, lung, liver, and kidney. 1 3 6 The expression of 72-kDa gelatinase, i n particular, was specifically higher i n the heart tissue (Tyagi et al., 1995a). U p o n induction by serum i n cultures, human heart fibroblast and endothelial cells increase the product ion of M M P - 1 and TIMP-1 at the m R N A and protein levels i n a dose-dependent manner (Tyagi et al., 1995b). Taken together, involvement of M M P s and T I M P s i n normal adult heart tissue indicate their role i n the integrity of cardiovascular structures. M M P s were also suggested to be associated w i t h the pathogenesis of various kinds of heart diseases inc luding aortic aneurysm, myocardial infarction, ischemia, and dilated cardiomyopathy (Cannon et al., 1983; Sato et al., 1983; Cleutjens et al., 1995). More recently, increased expression of M M P s inc luding neutrophil-type collagenase and 72-kDa and 92-kDa gelatinases was found to be associated w i t h idiopathic dilated cardiomyopathy (Gunja-Smith et al., 1996). 72-kDa gelatinase showed both latent and active forms. T I M P activity, on the other hand, was undetected. The newly-deposited collagen also showed poor cross-links. These changes are believed to contribute to weakening and dilatation of the ventricular w a l l . In summary, the temporo-spatial expression of 72-kDa gelatinase was shown to be associated w i t h type IV collagen, l aminin , and fibronectin and correlated w i t h the changes of this distr ibut ion pattern d u r i n g early heart morphogenesis, particularly during the 9-to-13-day-old stages. Thus, these results indicate that 72-kDa gelatinase may play an important role i n remodelling of the heart E C M components; type IV collagen, laminin, and fibronectin; which may be the in vivo substrates of 72-kDa gelatinase during early morphogenesis of the mouse heart. The current studies on the relationship of 72-kDa gelatinase expression i n heart w i t h TIMP-4 levels should also provide insight into the mechanisms of cardiac tissue remodelling i n development and disease. 1 3 7 C H A P T E R 6: G E N E R A L D I S C U S S I O N Collect ively, the characterization of the growth and tissue remodell ing during morphogenesis of the mouse primary palate i n the present study suggests a hypothesis of the sequential events of molecular inductions and interactions (Diagram IV). E G F + EGF-R, T G F - a + EGF-R upregulate^ M M P - 2 Expression at the Tips and Peripheral Regions at the Tips and Peripheral Regions of the Facial Prominences of the Facial Prominences stimulate enhances C e l l Proliferation facilitates ^ Remodelling of E C M ( B r d U incorporation, P C N A ) enhances enhances Outgrowth and Fusion of the Facial Prominences D i a g r a m IV : Schematic model of the sequential events of molecular inductions and interactions during morphogenesis of the primary palate. 1 3 8 Craniofacial growth is involved wi th morphological changes of the facial prominences and the brain . The data from computer reconstructions and morphometric analyses showed that the size of the maxil lary region increases rapidly as the maxillary prominence grows frontally to contribute to the primary palate (see review in Diewert and Wang, 1992; Diewert and Lozanoff, 1993). The size of the lateral nasal prominence also increases w i t h a predominantly lateral growth pattern. In contrast, the size of the medial nasal prominence appears to increase only slightly. Since the facial prominences are physically attached to the brain, changes of the brain morphology affect growth of the face. It was found that the brain and the face become vertically separated as the brain becomes more superiorly positioned relative to the developing face. Rapid growth of the facial prominences to facilitate tissue contact plays an important role i n primary palate morphogenesis. If a robust mesenchymal bridge is not well-established, failure of pr imary palate formation that leads to cleft l ip malformation may take place (Wang, 1992; Diewert and Wang, 1992; Wang et al, 1995). Based on the proposed model shown i n Diagram IV, a cascade of molecular signals w i t h the expression of E G F , T G F - a , and E G F - R and 72-kDa gelatinase is suggested. E G F , T G F - a , and EGF-R were mainly expressed by the epithelium and the adjacent underlying mesenchyme of the maxillary and nasal prominences during 10-to- l l -day-old mouse embryos. A t the early stages (7-17 TS), the expression of EGF, T G F - a and EGF-R was intensely localized to the tips and peripheral regions of the facial prominences. A t later stages (18-20 TS), the expression generally decreased but remained in peripheral regions of the primary palate. The uptake of B r d U and P C N A labelling, two cell proliferation markers, were most pronounced i n regions w i t h high levels of E G F , T G F - a , and E G F - R 1 3 9 expression. E G F and T G F - a act synergistically v ia EGF-Rs and may stimulate prol i ferat ion of epithelial and mesenchymal cells. A s a result, the facial prominences enlarged and grew out from the base of the head. The distribution of E G F and T G F - a i n both epithelial and mesenchymal cells in the study is interesting and indicates that EGF and T G F - a are not tissue or cell type specific, but rather are expressed i n temporal and spatial manners dur ing pr imary palate morphogenesis. These findings are similar to those that occur dur ing embryonic lung development i n which E G F and E G F - R were co-localized to a specific position-restricted distribution i n the epithel ium and mesenchyme of the pr imit ive airways (Warburton et ah, 1992). In the present study, an autocrine mode of regulation is speculated since their receptor, E G F - R was localized to the similar regions to E G F and T G F - a at all stages examined. Indeed, an autocrine mode of regulation of E G F and T G F - a was found to be important for embryonic development (Shum et al., 1993; Johnson et al., 1994; Chia et ah, 1995) and tumorigenesis (Derynck et ah, 1987; Ju et ah, 1991; Barton et al., 1991; Chris tensen et ah, 1993). In a d d i t i o n , transgenic mice w i t h overexpression of T G F - a developed liver (Jhappan et ah, 1990) and breast (Matsui et ah, 1990; Sandgren et ah, 1990) neoplasia. These neoplastic tissues also expressed EGF-R i n their epithelial components. Alternatively, a paracrine mode of regulation may also be operative since the strongest signals of E G F and T G F - a appeared to be at the surface ectoderm. The growth factors may diffuse into the underlying mesenchyme through the basement membrane, b ind to E G F - R at the surfaces of the mesenchymal cells and stimulate cell proliferation. A s a result, a differential proliferation of the mesenchymal cells occurred. Decreased staining of B r d U and P C N A as wel l as EGF, T G F - a and EGF-R i n the midline tissues and 1 4 0 deeper regions of the primary palate in the present study suggested that relative growth reduction takes place in certain regions of the primary palate and may contribute to further tissue differentiation and specialization i n those regions. Previous cell proliferation study by Minkof f and Mar t in (1984) showed similar results and suggested chondrogenic differentiation at the roof of the stomodeum. Regional differences of the labelling indices were also detected i n the maxi l lary prominence by using [3]H-thymidine injection i n chick embryos (Bailey et al, 1988). It was found that growth rate of the maxillary mesenchyme differed based on its proximity to the overlying epithelium. The rate of cell proliferation i n the facial prominences was higher i n the subepithelial zone of the mesenchyme compared to that of cell populations w h i c h were located further away from the epithelium. These results are consistent w i t h the present study and suggest that the epithelium may have growth regulating effect by secreting morphogens or growth factors into the subjacent mesenchyme (Bailey et al, 1988). 72-kDa gelatinase was found to co-localize w i t h E G F and T G F - a , particularly i n outgrowth regions of the pr imary palate, and i n the tips and peripheral regions of the facial prominences. A t advanced stages of pr imary palate formation, expression of 72-kDa gelatinase, E G F and T G F - a became dramatically reduced. These findings indicate that there may be an interaction between 72-kDa gelatinase and the two growth factors. Indeed, E G F and T G F - a have been found to stimulate synthesis of M M P s i n a variety of cells and tissues (Kerr et al, 1988; Shima et al, 1993; Lyons et al, 1993; Chen et al, 1993). For example, upon being activated by T G F - a and E G F , rat mucosal keratinocytes, under permanently serum-free conditions, increased the production of M M P - 1 and M M P - 9 (Lyons et al, 1993). Increased synthesis of 72-kDa gelatinase was also 141 found to be induced by EGF and T G F - a during in vitro branching morphogenesis of the lung (Ganser et ah, 1991). The protein expression of gelatinases, including 72-kDa gelatinase, was also demonstrated i n the blastocyst during mouse pre-implantat ion development (Brenner et ah, 1989) and found to be induced by T G F - a (Dardik et ah, 1993). In differentiating human epidermal raft cultures, T G F - a was found to induce keratinocytes to secrete type I collagenase and gelatinase (Turksen et ah, 1991). A s a result, invasion of basal cells into the collagen matrix took place. Taken together, E G F and T G F - a may regulate the expression of 72-kDa gelatinase during primary palate morphogenesis. 72-kDa gelatinase, upon being locally induced by the growth factors, is probably synthesized into the E C M as a latent, inactive enzyme. The mechanism of activation of 72-kDa gelatinase in vivo was suggested to be involved w i t h M T -M M P s (Sato et ah, 1994) and take place on the cell surface (Ward et ah, 1994). These data are consistent w i t h the present study since the immunostaining of 72-k D a gelatinase was intensely localized on the cell surfaces of the myocytes and endothelial cells i n the heart tissue (see Chapters 5). More recently, M T - M M P - 2 was identifed and found to induce processing of progelatinase A into the activated forms, like M T - M M P - 1 (Takino et ah, 1995). Taken together, M T - M M P s may be in vivo activators for 72-kDa gelatinase d u r i n g p r i m a r y palate morphogenesis. Further investigation of M T - M M P expression i n the developing primary palate w i l l support this mode of activation of 72-kDa gelatinase in vivo. The active enzyme may enhance and facilitate cell proliferation by remodelling the E C M i n the tips and peripheral regions of the primary palate. Indeed, the expression of M M P s and TIMPs have been found to be associated w i t h cell proliferation i n many studies. For example, serum can induce cell proliferation and the expression of M M P - 1 and TIMP-1 i n human heart fibroblast and 1 4 2 endothelial cells i n cultures (Tyagi et al., 1995b). 72-kDa and 92-kDa gelatinases have been suggested to be i n v o l v e d i n basement membrane and matrix degradation i n balloon-injured carotid arterial w a l l during the period of smooth muscle cell proliferation and migration (Zempo et ah, 1994). E G F , P D G F , and insul in can induce cell proliferation and the production of M M P - 1 and M M P - 3 i n rheumatoid synovial fibroblasts i n cultures (Hiraoka et al., 1992). TIMP-1 and TIMP-2 can directly stimulate cell proliferation in a Burkitt lymphoma cell line (Hayakawa, 1994; Hayakawa et al., 1994). Collectively, 72-kDa gelatinase may play be involved i n primary palate morphogenesis by facilitating cell proliferation at the outgrowth regions. . 72-kDa gelatinase appears to be a common molecule for tissue remodelling d u r i n g embryonic development (Ganser et al., 1991; Reponen et al., 1992; Sahlberg et al., 1992; Heikinheimo and Salo, 1995), w o u n d healing (Salo et al., 1994), and tumor invasion and metastasis (Liotta et al., 1980; Monteagudo et ah, 1990; L e v y et al, 1991; A u t i o - H a r m a i n e n et al., 1993; Soini et al, 1994). The expression of 72-kDa gelatinase has also been detected i n a wide variety of normal and transformed cell lines inc luding fibroblast (Seltzer et al., 1981), chondrocytes (Murphy et al, 1989), keratinocytes (Salo et al, 1991), endothelial cells (Kalebic et al, 1983), and rheumatoid synovial cells (Okada et al, 1990). However , the substrates for 72-kDa gelatinase in vivo remain unclear. In the present s tudy, the association between the protein expression of 72-kDa gelatinase and the E C M components including type IV collagen, laminin and fibronectin was characterized i n the developing pr imary palate and heart. Previous in vitro studies showed that type IV collagen (Fessler et al, 1984), fibronectin and laminin (Okada et al, 1990; Nagase et al, 1991) are also substrates for 72-kDa gelatinase. Type IV collagen, i n particular, has been found to be associated w i t h 72-kDa gelatinase dur ing mouse (Sahlberg et al, 1992) and 1 4 3 human (Heikinkeimo and Salo, 1995) tooth development and mammary gland development (Dickson and Warbur ton , 1992). Taken together, the E C M components; particularly type IV collagen, laminin and fibronectin; may be substrates for 72-kDa gelatinase in vivo during embryonic development. Recently, Overa l l (1994) has proposed a model for M M P and T I M P regulation i n tissue remodelling. He proposed that gradients of growth factors or other regulatory molecules at sites of tissue remodell ing may modulate the expression of M M P s , TIMPs, and E C M proteins. N o r m a l connective tissue is characterized by low constitutive levels of TIMPs and 72-kDa gelatinase. A t a remodelling site, a net resorptive cell phenotype may be expected at the focus of degradation, characterized by increased M M P and reduced T I M P expression. The surrounding normal tissue is l ikely protected from M M P activity by a "green belt" comprising cells that exhibit an active formative phenotype induced by "formative factors" such as TGF-p. This "green belt" region is marked by an up-regulation of T I M P , 72-kDa gelatinase, and E C M protein synthesis to both protect local tissue and to buffer adjacent normal tissue from inappropriate M M P activity diffusing from the focus of degradation (Overall, 1994). The results from the present work suggest that gradients of E G F and TGF-a at the outgrowth regions, the remodell ing sites, of the developing facial prominences are present and operative by inducing the expression of 72-kDa gelatinase. Decreased levels of 72-kDa gelatinase i n the deeper and midl ine tissues also provide support for the model of tissue remodelling proposed by Overal l (1994). D u r i n g p r i m a r y palate morphogenesis, a number of growth factors, t ranscr ipt ion factors, and oncogene product are expressed i n the facial prominences (see Table I). This mult iple expression of molecular factors 1 4 4 indicates that the mechanism of primary palate morphogenesis is complex and needs interactions and inductions between molecules. For example, AP-2, a transcriptional factor, was found to be present in the ectoderm and mesenchyme of the mouse facial prominences (Mitchel l et al., 1991). Recently, transgenic mouse embryos lacking AP-2 gene function demonstrated exencephaly, midline clefts of the face, and absent or malformed sensory organs and cranial ganglia (Schorle et al., 1996; Zhang et al., 1996). These findings suggest that AP-2 is important for craniofacial development. Interestingly, it was found that an AP-2 binding site is located i n the first exon of 72-kDa gelatinase gene and suggested to be a regulatory element for gene transcription (Huhtala et al., 1990; Sato and Seiki , 1993). These data indicate that 72-kDa gelatinase may be a target gene regulated by AP-2 since 72-kDa gelatinase was also expressed i n the mouse facial prominences at similar stages (see Chapter 4). Some other transcriptional factors expressed in the facial prominences may be regulators of AP-2, particularly Msx-1, since the expression of Msx-1 overlaps wi th that of AP-2 (Mitchell et al., 1991; Robert et al., 1989; Brown et al., 1993). AP-2 mutants also showed abnormal expression of Msx-1 i n which it was reduced i n the proximal portions of the facial prominences and was laterally displaced i n the distal portions (Schorle et al., 1996). However, Pax-3 and twist, regulatory genes k n o w n to be required for cranial closure, appeared to be normally expressed i n AP-2 mutants (Schorle et al., 1996). These indicate that defects in cranial closure in AP-2 mutants are not caused by loss of Pax-3 and twist. 1 4 5 CHAPTER 7: CONCLUSIONS A N D FUTURE WORK CONCLUSIONS 1. The major basement membrane components inc luding laminin , type IV collagen and fibronectin, became disrupted and gradually disappeared i n association w i t h the regression of the epithelial seam and the mesenchymal bridge formation during morphogenesis of mouse primary palate. 2. E G F , T G F - a , and EGF-R were temporo-spatially co-distributed i n the enlarging facial prominences of the developing primary palate, suggesting their role i n outgrowth of the mouse primary palate tissues. 3. The distribution patterns of B r d U and P C N A were similar to those of E G F , T G F - a and EGF-R at similar stages of primary palate morphogenesis. These observations suggest that EGF, T G F - a and EGF-R may stimulate cell proliferation during outgrowth of the mouse primary palate. 4. Expression of 72-kDa gelatinase was associated w i t h morphogenesis of the pr imary palate, mandible, eye, and heart. Based on co-localization of E C M components and 72-kDa gelatinase, I suggest that 72-kDa gelatinase may enhance E C M remodelling of those developing organs. 5. 72-kDa gelatinase was found to be localized to the same regions as was E G F and T G F - a at al l stages examined dur ing morphogenesis of the mouse primary palate, suggesting that EGF and T G F - a may regulate morphogenesis of the pr imary palate, at least i n part, by inducing the expression of 72-kDa gelatinase. 1 4 6 F U T U R E W O R K Based on the results of the present study, 72-kDa gelatinase appears to be important for development of many organ systems including the primary palate, mandible, eye, and heart because the temporo-spatial distr ibution of 72-kDa gelatinase was found to be expressed at increased levels at critical times dur ing development of those organs. However, the mechanisms of 72-kDa gelatinase i n organ development, especially the functional aspects, are unknown. E G F , T G F - a , and EGF-R were also shown to be involved w i t h morphogenesis of the primary palate but their direct relationship wi th 72-kDa gelatinase and cell proliferation remains to be determined. Therefore, I further hypothesize that 1. 72-kDa gelatinase is a major M M P involved in degradation of basement membranes during fusion and outgrowth of the facial prominences, 2. elevated levels of expression of other M M P s and TIMPs are present i n the areas of fusion and outgrowth during primary palate morphogenesis, 3. growth factors, particularly T G F - a and EGF, are in vivo inducers for 72-k D a gelatinase expression during primary palate morphogenesis. Approaches 1. To further determine involvement of 72-kDa gelatinase at the transcription level i n tissue remodelling during primary palate morphogenesis, the m R N A expression of 72-kDa gelatinase can be s tudied by in situ hybridizat ion technique (Reponen et ah, 1992; Wilk inson, 1993). This technique w o u l d al low us to investigate w h i c h type of cells, epithelial or mesenchymal cells, can synthesize 72-kDa gelatinase. In situ hybridizat ion can be performed 1 4 7 either on tissue sections or whole mount embryos (Wilkinson, 1993). The mouse anti-sense 72-kDa gelatinase probe can be generated according to Reponen et al. (1992). The expression of 72-kDa gelatinase m R N A can also be confirmed by Northern blots and reverse transcription polymerase chain reaction (RT-PCR) analyses (Koopman, 1993). In order to quantitatively compare the amount of 72-k D a gelatinase m R N A from different stages of primary palate morphogenesis, the R N A a s e protection assay can be analyzed (Altaba, 1993). To further understand the mechanisms of 72-kDa gelatinase i n tissue remodell ing dur ing primary palate morphogenesis, functional tests for 72-kDa gelatinase can be investigated by several approaches. a) A first approach is to perform loss of function experiments. Organ cultures can be used for this purpose (Morriss-Kay, 1993). The developing primary palate is dissected as one unit and cultured. To test whether 72-kDa gelatinase is required for tissue outgrowth and fusion of the facial prominences, the cultured explants w i l l be incubated wi th a monoclonal anti-72-kDa gelatinase antibody specific for the catalytic domain of 72-kDa gelatinase at various times i n comparison w i t h the controls. If 72-kDa gelatinase is important for development of the primary palate, the cultured explants incubated w i t h a monoclonal anti-72-k D a gelatinase antibody w i l l show deficiency i n growth and unsuccessful fusion of the facial prominences. Alternatively, a monoclonal anti-72-kDa gelatinase antibody can be replaced by TIMPs and synthetic M M P inhibitors (Fisher et al., 1994). These approaches w i l l provide not only a better understanding of the functions of 72-kDa gelatinase during development of the primary palate but also the therapeutic potential of M M P inhibitors i n controlling 72-kDa gelatinase-dependent pathological processes such as malignant tumors and inflammatory diseases (Birkedal-Hansen, 1995). 1 4 8 b) A second approach is to perform whole embryo culture (Morriss-Kay, 1993). Similar to the organ cultures, the whole embryo culture w i l l a l low us to examine the effects of a monoclonal anti-72-kDa gelatinase antibody, TIMPs , synthetic M M P inhibitors or the other molecules of interest; such as E G F and T G F - a ; to the development of the whole embryo by introducing the antibody or the growth factors into the amniotic f lu id at various times. By introducing an anti-72-kDa gelatinase antibody, TIMPs, or synthetic M M P inhibitors i n the culture, growth of the primary palate may be abnormal. O n the other hand, by adding E G F or T G F - a i n the culture, growth of the pr imary palate and the expression of 72-kDa gelatinase may be stimulated. c) A third approach is to generate transgenic mice w i t h mutant or deleted 72-kDa gelatinase gene. The homozygous mouse embryos w i t h mutant or deleted 72-kDa gelatinase gene can be generated by two techniques, pronuclear injection (Schnieke et al., 1983) or gene targeting i n embryonic stem (ES) cells (Joyner, 1991; Robertson and Mar t in , 1993). These techniques w i l l a l low us to directly examine functional effects of 72-kDa gelatinase dur ing multiple organ development of the mouse embryo by comparing the homozygous embryos w i t h the heterozygous and normal embryos. 2. To determine whether the other M M P s ; such as interstitial collagenase, stromelysins, 92-kDa gelatinase, and M T - M M P s ; and TIMPs; such as TIMP-1 , -2, -3, and -4; are i n v o l v e d i n tissue remodel l ing d u r i n g p r i m a r y palate morphogenesis, co-localization of the proteins and m R N A s of M M P s and TIMPs can be studied on tissue sections or whole mount embryos by employing sequential immunohistochemistry and in situ hybridizat ion technique (Stern, 1993). In addition, the functional analyses of M M P s and TIMPs can be performed in vitro similar to the above experiments. 1 4 9 3. To test whether the growth factors; for example, E G F and T G F - a can induce the production of 72-kDa gelatinase from epithelial cells or mesenchymal cells, the epithelial cells or mesenchymal cells can be removed from the primary palate and cultured separately under appropriate conditions (Dixon et al., 1993). Subsequently, E G F or T G F - a w i l l be added to the culture m e d i u m at various times i n comparison w i t h the controls. Then the culture m e d i u m is collected and the expression of 72-kDa gelatinase is studied by using gelatin zymography. The experimental results can be semiquantitatively compared w i t h the controls by using densitometry on the zymograms. 1 5 0 B I B L I O G R A P H Y Abbott, B.D. , and Pratt, R . M . (1988): E G F receptor expression i n the developing tooth is altered by exogenous retinoic acid and EGF. Dev. Biol . 128:300-304. A d a m s o n , E .D. (1990): E G F receptor activities i n mammalian development. M o l . Reprod. Dev. 27:16-22. A d a m s o n , E . D . (1993): Act iv i t ies of growth factors i n pre implantat ion development. J. Cel l Biochem. 53:280-287. A d l e r , R.R., Brenner, C . A . , and Werb. Z . (1990): Expression of extracellular matr ix-degrading metalloproteinases and metalloproteinase inhibitors is developmentally regulated dur ing endoderm differentiation of embryonal carcinoma cells. Development 110:211-220. A h u m a d a , G .C . , Rennard, S.I., Figueroa, A . A . , and Silver, M . H . (1981): Cardiac f ibronectin: Developmental distr ibut ion and quantitative comparison of possible sites of synthesis. J. M o l . Cel l Cardiol . 13:667-678. Akhurs t , R.J., Lehnert, S.A., Faissner, A . , and Duffie, E. (1990): T G F beta i n mur ine morphogenetic processes: the early embryo and cardiogenesis. Development 108:645-656. Alexander, C M . , and Werb, Z . (1991): Extracellular matrix degradation. In: H a y E. (ed.) "Ce l l Biology of Extracellular Matr ix . " P lenum Press, N e w York. pp.255-302. Altaba, A . R . i . (1993): RNAase protection assays. In: Stern, C D . , and Hol land , P . W . H . (eds.) "Essential Developmental Biology." Oxford University Press, Oxford, pp.213-222. Andermarcher, E., Surani, M . A . , and Gherardi, E. (1996): Co-expression of the H G F / S F and c-met genes d u r i n g early mouse embryogenesis precedes reciprocal expression i n adjacent tissues during organogenesis. Dev. Genetics 18:254-266. Ange l , P., Imagawa, M . , C h i u , R., Stein, B., Imbra, R.J., Rahmsdorf, H.J . , Jonat, C , Herr l i ch , P., and K a r i n , M . (1987): Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Ce l l 49:729-739. 151 Apte , S.S., Hayashi , K . , Seldin, M.F . , Mattei, M . - G . , Hayashi , M . , and Olsen, B.R. (1994): Gene encoding a novel murine tissue inhibitor of metalloproteinases (TIMP), TIMP-3 , is expressed i n developing mouse epithelia, cartilage, and muscle, and is located on mouse chromosome 10. Dev. Dynamics 200:177-197. Ardinger , H . H . , Buetow, K . H . , Bel l , G.I. , Bordach, J., VanDemark, D.R. , and M u r r a y , J .C. (1989): Association of genetic variation of the transforming growth factor alpha gene wi th cleft l ip and palate. A m . J. H u m . Genet. 45:348-353. Autio-Harmainen, H . , Karttunen, T., Hurskainen, T., Hoyhtya , M . , Kauppi la , M . , and Tryggvason, K . (1993): Expression of 72- kilodalton type rV collagenase (gelatinase A ) i n benign and malignant ovarian tumors. Lab. Invest. 69:312-321. Bailey, L.J . , M i n k o f f , R., and K o c h , W . E . (1988): Relative growth rates of maxil lary mesenchyme i n the chick embryo. J Craniofac Genet Dev. B io l . 8:167-177. Bancroft, M . , and Bellairs, R. (1977): Placodes of the chick embryo studied by S E M . Anat. Embryol. 151:97-108. Barsky, S .H. , Siegal, G.P. , Jannotta, F., and Liotta, L . A . (1983): Loss of basement membrane components by invasive tumors but not by their benign counterparts. Lab. Invest. 49:140-147. Barton, C . M . , H a l l , P . A . , Huges, C . M . , Gul l ick , W.J., and Lemoine, N . R . (1991): Transforming growth factor alpha and epidermal growth factor i n human pancreatic cancer. J. Pathol. 163:111-116. Beerstecher, H.J . , Huiskens-Van Der Mei j , C . , and Warnaar, S.O. (1988): A n immunohistochemical study performed w i t h monoclonal and polyc lonal antibodies to mouse epidermal growth factor. J. Histochem. Cytochem. 36:1153-1160. Behrendtsen, O. , Alexander, C . M . , and Werb, Z . (1992): Metalloproteinases mediate extracellular matrix degradation by cells f rom mouse blastocyst outgrowths. Development 114:447-456. Bernfield, M . (1981): Organization and remodelling of the extracellular matrix i n morphogenesis. In: Connel ly , T .G. (ed.) "Morphogenesis and Pattern Formation." Raven Press, N e w York pp.139-162. 1 5 2 Bernfield, M . , Banerjee, S.D., Koda , J.E., and Rapraeger, A . C . (1984): Remodelling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: Trelstad R .L . (ed.) "The Role of Extracellular M a t r i x of Development." A l a n R Liss Inc., N e w York, pp.545-572. Birkedal-Hansen, H . (1995): Proteolytic remodelling of extracellular matrix. Cur . O p i n . Biol . 7:728-735. Birkedal-Hansen, H . , Moore , W.G. I . , Bodden, M . K . , Windsor , L.J . , B irkedal -Hansen, B., DeCarlo, A . , and Engler, J .A. (1993): Matrix metalloproteinases: a review. Crit . Rev. Oral Biol . M e d . 4(2):197-250. Bravo, R., and Macdonald-Bravo, H . (1984): Induction of the nuclear protein 'cycl in ' i n quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation wi th D N A synthesis. E .M.B.O. J. 3:3177-3181. Breckon, J.J., Hembry, R . M . , Reynolds, J.J., and Meikle, M . C . (1994): Regional and temporal changes i n the synthesis of matrix metalloproteinases and TIMP-1 during development of the rabbit mandibular condyle. J. Anat. 184:99-110. Brenner, C . A . , Adler , R.R., Rappolee, D . A . , Pedersen, R . A . , and Werb, Z . (1989): Genes for extracellular matr ix-degrading metalloproteinases and their inhibitor, T I M P , are expressed during early mammalian development. Genes Dev. 3:848-859. Brinkley, L . , D u , Y. , and Morris -Wiman, J. (1995): Matrix-degrading enzymes are present during palatal morphogenesis. J. Dent. Res. 74:66 Bronner-Fraser, M . (1986): A n antibody to the receptor for fibronectin and l a m i n i n perturbs cranial neural crest development in vivo. Dev . B i o l . 117:528-536. B r o w n , J . M . , Wedden, S.E., M i l l b u r n , G . H . , Robson, L . G . , H i l l , R.E. , Davidson, D.R., and Tickle, C . (1993): Experimental analysis of the control of expression of the homeobox-gene Msx-1 in the developing limb and face. Development 119:41-48. Brunet, C . L . , Sharpe, P . M . , and Ferguson, M.W.J . (1995): Inhibition of TGF-p3 (but not T G F - p l or TGF-(32) activity prevents normal mouse embryonic palate fusion. Int. J. Dev. Biol . 39:345-355. Burgeson, R.E. (1993): Dermal-epidermal adhesion i n skin. In: Rohrbach, D . H . , and T i m p l , R. (eds.) "Molecular and Cel lu lar Aspects of Basement Membrane." Academic Press, Inc., San Diego, pp.49-66. 1 5 3 Burgeson, R.E., Chiquet, M . , Deutzmann, R., Ikblom, P., Engel, J., Kle inman, H . , Mar t in , G.R., Ortonne, J.-P., Paulsson, M . , Sanes, J., T i m p l , R., Tryggvason, K . , Yamada, Y . , and Yurchenco, P .D. (1994): A new nomenclature for laminins. Matrix Biol . 14:209-211. Burk, D.T. (1993): Distribution of glycosaminoglycans i n the developing facial region of mouse embryos. J. Craniofac. Genet. Dev. Biol . 3:339-349. Canete-Soler, R., G u i , Y . - H . , L inask , K . K . , and Muscher , R.J. (1995): Developmenta l expression of M M P - 9 (gelatinase B) m R N A i n mouse embryos. Dev. Dynamics 204:30-40. Cannon, R .O. , Butany, J.W., M c M a n u s , B . M . , Speir, E., Kravi tz , A . B . , Bo l i , R., and Ferrans, V.J . (1983): Early degradation of collagen after acute myocardial infarction i n the rat. A m . J. Cardiol . 52:390-395. Carette, M . J . M . , and Ferguson, M.W.J . (1992): The fate of medial edge epithelial cells dur ing palatal fusion in vitro: an analysis by D i l labelling and confocal microscopy. Development 114:379-388. Carpenter, G . , and Wahl , M. I . (1991): The epidermal growth factor family. In: Sporn, M . B . , Roberts, A . B . (eds.) "Peptide Growth Factors and Their Receptors I." Springer Verlag, N e w York, pp.69-171. Cartl idge, S.A., and Elder, J.B. (1989): Transforming growth factor beta and epidermal growth factor levels in normal gastrointestinal mucosa. Br. J. Cancer 60:657-660. Casasco, A . , Giordano, M . , Danova, M . , Casaco, M . , Conaglia , I., Call igaro, A . (1993): PC10 monoclonal antibody to proliferating cell nuclear antigen as probe for cycling cell detection in developing tissues. Histochem. 99:191-199. C a w o o d , A . H . , and Savage, J.R. (1983): A comparison of the use of bromodeoxyuridine and [3H]thymidine i n studies of the cell cycle. Ce l l Tissue Kinet. 16:51-57. Cawston, T.E. (1986): Protein inhibitors of metalloproteinases. In: Barrett, A. J . and Salvesen, G . (eds.) "Proteinase Inhibitors." Elsevier Science, Amsterdam. pp.589-610. Celis, J.E. and Celis, A . (1985): Cel l cycle-dependent variations in the distribution of the nuclear protein cycl in proliferating cell nuclear antigen i n cultured cells: subdivision of S phase. Proc. Nat. Acad. Sci. U .S .A. 82:3262-3266. 1 5 4 Chapman, H . A . , Reilly, J.J., and Kobzik, L . (1988): Role of plasminogen activator i n degradation of extracellular matrix protein by l ive h u m a n alveolar macrophages. A m . Rev. Res. Dis. 137:412-419. Chegin i , N . and Wi l l iams , R.S. (1992): Immunocytochemical localization of transforming growth factors (TGFs) T G F - a and T G F - 0 i n h u m a n ovarian tissues. J. C l i n . Endocrinol. Metab. 74:973-980. Chen, L . L . , Narayanan, R., Hibbs, M.S. , Benn, P . A . , Clawson, M . L . , L u , G . , R h i m , J.S., Greenberg, B., and Mendelsohn, J. (1993): Altered epidermal growth factor s igna l t ransduct ion i n activated Ha-ras - t rans formed h u m a n keratinocytes. Biochem. Biophys. Res. C o m m . 193:167-174. Chenevix-Trench, G . , Jones, K. , Green, A . C . , Duffy, D .L . , and Mart in , N . G . (1992): Cleft l ip w i t h or without cleft palate: associations w i t h transforming growth factor and retinoic acid receptor loci. A m . J. H u m . Genet. 51:1377-1385. Chenevix-Trench, G . , Jones, K . , Green, A . C . , and Mar t in , N . G . (1991): Further evidence for an association between genetic variation i n transforming growth factor alpha and cleft l ip and palate. A m . J. H u m . Genet. 48:1012-1013. Chia , C M . , Winston, R . M . L . , and Handyside, A . H . (1995): E G F , T G F - a and EGFR expression i n human preimplantation embryos. Development 121:299-307. Christensen, M . E . , Therkildsen, M . H . , Poulsen, S.S., and Bretlau, P. (1993): Immunoreactive transforming growth factor alpha and epidermal growth factor i n oral squamous cell carcinomas. J. Pathol. 169:323-328. Clark , S.D., Kobayashi , D . K . , and Welgus, H . G . (1987): Regulation of the expression of tissue inhibitor of metalloproteinases and collagenases by retinoids and glucocorticoids in human fibroblasts. J. C l i n . Invest. 80:1280-1288. Cleutjens, J . P . M . , Kandala , J . C , Guafda , E., Guntaka, R .V . , and Weber, K .T . (1995): Regulation of collagen degradation i n the rat myocardium after infarction. 27:1281-1292. Coffey, R.J., Derynck, R., Wilcox, J .N. , Bringman, T.S., Goustin, A.S. , Moses, H . L . , and Pittelkow, M . R . (1987): Production and autoinduction of transforming growth factor-a i n human keratinocytes. Nature 328:817-820. Cohen, S. (1962): Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening i n the new-born animal. J. B io l . Chem. 237:1555-1562. 1 5 5 Cohen, S., Carpenter, G . , and K i n g , L.J. (1980): Epidermal growth factor-receptor-kinase interactions. Co-purif icat ion of receptor and epidermal growth factor-enhanced phosphorylation activity. J. Biol . Chem. 255:4834-4842. Cooper, A . R . , and MacQueen, H . A . (1983): Subunits of laminin are differentially synthesized i n mouse eggs and early embryos. Dev. Biol . 96:467-471. Cor l i ss , C . E . (1976): Patten's H u m a n Embryology . Elements of C l i n i c a l Development. M c G r a w - H i l l Book Company: N e w York, pp.68. Crossley, P . H . , and Mart in , G.R. (1995): The mouse Fgf8 gene encodes a family of polypeptides and is expressed i n regions that direct outgrowth and patterning i n the developing embryo. Development 121:439-451. Curry , T.E. Jr., M a n n , J.S., Estes, R.S., and Jones, P.B.C. (1990): a2-Macroglobul in and tissue inhibitor of metalloproteinases: collagenase inhibitors i n human preovulatory ovaries. Endocrinology 127:63-68. Dardik , A . , Doherty, A .S . , and Schultz, R . M . (1993): Protein secretion by the mouse blastocyst: St imulatory effect on secretion into the blastocoel by transforming growth factor-a. M o l . Reprod. Dev. 34:396-401. D a S i l v e i r a , A . , D u , Y . , and B r i n k l e y , L . (1995): Appearance of metalloproteinases and serine proteases during murine facial development. J. Dent. Res. 74:65. Da Silveira, A . , D u , Y. , and Brinkley, L . (1996): Occurrence of M M P s , TIMPs, and their m R N A s during murine facial morphogenesis. J. Dent. Res. 75:226. Dean D . D . , M u n i z , O.E. , Berman, I., Pita, J.C., Carreno, M . R . , Woessner, J.F. Jr., and H o w e l l D.S. (1985): Localization of collagenase i n the growth plate of rachitic rats. J. C l i n . Invest. 76:716-722. Derynck, R. (1992): The physiology of transforming growth factor-a. A d v . Cancer Res. 58:27-52. Derynck , R., G o e d d e l , D . V . , U l l r i c h , A . , Gutterman, J . U . , W i l l i a m s , R . D . , Bringman, T.S., and Berger, W . H . (1987): Synthesis of messenger R N A s for transforming growth factor a and (3 and the epidermal growth factor receptor by human tumors. Cancer Res. 47:707-712. Derynck, R., Roberts, A . B . , Winkler , M . E . , Chen, E.Y., and Goeddel , D . V . (1984): H u m a n transforming growth factor-alpha: Precursor structure and expression i n E. coli. Ce l l 38:287-297. 1 5 6 Dickson, M . C . , Slager, H . G . , Duffie, E., Mummery , C . L . , and Akhurst , R.J. (1993): R N A and protein localizations of TGFfi2 i n the early mouse embryo suggest an involvement i n cardiac development. Development 117:625-639. Dickson, S.R., and Warburton, M.J . (1992): Enhanced synthesis of gelatinase and stromelysin by myoepithelial cells dur ing involut ion of the rat mammary gland. J. Histochem. Cytochem. 40:697-703. Diewert, V . M . , and Lozanoff, S. (1993): G r o w t h and morphogenesis of the human embryonic midface dur ing primary palate formation analyzed i n frontal sections. J. Craniofac. Genet. Dev. Biol . 13:162-183. Diewert, V . M . , and Shiota, K . (1990): Morphological observations i n normal pr imary palate and cleft l ips embryos i n the Kyoto collection. Teratology 41:663-667. Diewert, V . M . , and V a n der Meer, D . (1991): G r o w t h of the human primary palate. J. Dent. Res. 69:156. Diewert, V . M . , and Wang, K . - Y . (1992): Recent advances i n primary palate and midface morphogenesis research. Crit . Rev. Oral Biol . M e d . 4(1):111-130. Diewert, V . M . , Wang, K . -Y . , and Tait B. (1993a): A morphometric analysis of cell densities i n facial prominences of the rhesus monkey embryo during primary palate formation. J. Craniofac. Genet. Dev. Biol . 13:236-249. Diewert, V . M . , Wang , K . - Y . , and Tait, B. (1993b): A new threshold model for cleft l ip i n mice. A n n . N . Y . Acad. Sci. 678:341-343. Dixon, M.J . , and Ferguson, M.W.J . (1992): The effects of epidermal growth factor, transforming growth factors alpha and beta and platelet-derived growth factor on murine palatal shelves i n organ culture. A r c h . Ora l Biol . 37:395-410. D i x o n , M. J . , Foreman, D . , Schor, S., and Ferguson, M . W . J . (1993): Epidermal growth factor and transforming growth factor alpha regulate extracellular matrix product ion by embryonic mouse palatal mesenchymal cells cultured on a variety of substrata. Roux's Arch . Dev. Biol . 203:140-150. D i x o n , M. J . , Garner, J., and Ferguson M . W . J . (1991): Immunolocal ization of epidermal growth factor (EGF), E G F receptor and transforming growth factor alpha (TGF-a) dur ing murine palatogenesis in vivo and in vitro. Anat . Embryol . 184:83-91. Dolbeare, F. (1995): Bromodeoxyuridine: a diagnostic tool i n biology and medicine, Part I: His tor ic perspectives, histochemical methods and cell kinetics. Histochem. J. 27:339-369. 1 5 7 Dol le , P. , Ruberte, E., Leroy, P., Morr iss -Kay, G . , and Chambon, P. (1990): Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic s tudy of their d i f ferent ia l pattern of t ranscr ipt ion d u r i n g mouse organogenesis. Development 110:1133-1151. Drake, C.J., Davis, L . A . , Walters, L . , Little, C . D . (1990): A v i a n vasculogenesis and the distribution of collagens I, IV, laminin , and fibronectin i n the heart primordia. J. Exp. Zool . 255:309-322. D u , Y . , Morr is -Wiman, J., D a Silveira, A . , and Brinkley, L . (1996): Occurrence of TIMPs dur ing palatal morphogenesis in vivo and in vitro. J. Dent. Res. 75:225. Duband, J.L., and Thiery, J.P. (1982a): Distribution of fibronectin i n the early phase of avian cephalic neural crest cell migration. Dev. Biol . 93:308-323. Duband, J.L., and Thiery, J.P. (1982b): Appearance and distribution of fibronectin during chick embryo gastrulation and neurulation. Dev. Biol . 94:337-350. D u b a n d , J.L., and Thiery, J.P. (1987): Distribution of laminin and collagens during avian neural crest development. Development 101:461-478. Durham, P.L. , and Snyder, J . M . (1995): Characterization of a l , (31, and y l laminin subunits during rabbit fetal lung development. Dev. Dynamics 203:408-421. Dziadek, M . , and T i m p l , R. (1985): Expression of nidogen and l a m i n i n i n basement membranes during mouse embryogenesis and i n teratocarcinoma cells. Dev. Biol . 111:372-382. Ede, D . A . , Flint, O.P., and Teague, P. (1975): Cel l proliferation i n the developing w i n g - b u d of normal and talpid mutant chick embryos. J. E m b r y o l . Exp. Morphol . 34(3): 587-607. Edwards, D.R. , Heath, J.K., Hogan, B . L . M . , N o m u r a , S., and Wi l l s , A . J . (1992): Expression of T I M P i n fetal and adult mouse tissues studied by in situ hybridizat ion. In: Birkedal-Hansen, H . , Werb, Z . , Welgus, H . G . , and V a n Wart, H . E . (eds.) "Matrix Metalloproteinases and Inhibitors." Matr ix . Spec. Suppl . N o l . Gustav Fischer, Stuttgart, pp.286-293. Edwards, D.R., M u r p h y , G . , Reynolds, J.J., Whitman, S.E., Docherty, A.J.P., Ange l , P., and Heath, J.K. (1987): Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. E M B O J. 6:1899-1904. 1 5 8 Ekblom, M . , K le in , G . , Mugrauer, G . , Fecker, L . , Deutzmann, R., T i m p l , R., and Ekblom, P. (1990): Transient and locally restricted expression of laminin A chain m R N A by developing epithelial cells dur ing kidney organogenesis. Cel l 60:337-346. Ekblom, P. (1981): Formation of basement membranes i n the embryonic kidney: an immunohistological study. J. Cel l Biol . 91:1-10. Ekblom, P. (1993): Basement membranes i n development. In: Rohrbach, D . H . and R. T i m p l , R. (eds.) "Molecular and Cel lular Aspects of Basement Membranes." Academic Press, Inc., San Diego, pp.359-383. Elsasser, H . P . , Biederbick, A . , and Kern, H .F . (1994): Growth of rat pancreatic acinar cells quantitated w i t h a monoclonal antibody against the proliferating cell nuclear antigen. Cel l Tissue Res. 276:603-609. Farrall, M . , and Holder, S. (1992): Familial recurrence-pattern analysis of cleft l ip w i t h or without cleft palate. A m . J. H u m . Genet. 50:270-277. Feng, H . , Sassani, R., Bartlett, S.P., Lee, A . , Hecht, J.T., Malcolm, S., Winter, R . M . , Vintiner, G . M . , Buetow, K . H . , and Gasser, D . L . (1994): Evidence from family studies, for l inkage d i s e q u i l i b r i u m between T G F A and a gene for nonsyndromic cleft l ip w i t h or without cleft palate. A m . J. H u m . Genet. 55:932-936. Ferguson, M.W.J . (1988): Palate formation. Development 103(Suppl):41-60. Ferguson, M.W.J . , Sharpe, P . M . , Thomas, B.L. , and Beck, F. (1992): Differential expression of insulin-like growth factors I and II (IGF-I and II) m R N A , peptide and b inding protein I during mouse palate development: comparison w i t h TGF-(3 peptide distribution. J. Anat. 181:219-238. Fessler, L.I . , Duncan, K . G . , Fessler, J . H . , Salo, T., and Tryggvason, K . (1984): Characterization of procollagen IV cleavage products by a specific tumor collagenase. J. Biol . Chem. 259:9783-9789. Filogamo, G . , Corvetti , G . , and Sisto Daneo, L . (1990): Differentiation of cardiac conducting cells from the neural crest cells. J. Autonomic Nervous System 30(Suppl):S55-57. Fisher, C , Gilberton-Beadling, S., Powers, E . A . , Petzold, G . , Poorman, R., and Mitchel l , M . A . (1994): Interstitial collagenase i n required for angiogenesis in vitro. Dev. Biol . 162:499-510. 1 5 9 Fitch, J . M . , and Linsenmayer, T.F. (1994): Interstitial basement membrane components i n development. In: Yurchenco, P .D. , Birk, D .E . , and Mecham, R.P. (eds.) "Extracellular Matrix Assembly and Structure." Academic Press, Inc., San Diego, pp.441-462. Fitzpatrick, D.R., Denhez, F., Kondaiah, P., and Akhurst , R.J. (1990): Differential expression of T G F beta isoforms i n murine palatogenesis. Development 109:585-595. Flenniken, A . M . , and Wil l iams, B.R.G. (1990): Developmental expression of the endogenous T I M P gene and a TIMP- lacZ fusion gene i n transgenic mice. Genes Dev. 4:1094-1106. Form, D . M . , Pratt, B . M . , and M a d r i , J .A. (1986): Endothelial cell proliferation dur ing angiogeneis. In vitro modulation by basement membrane components. Lab. Invest. 55:521-530. Fowler, K.J . , Walker, F., Alexander, W. , Hibbs, M . L . , Nice, E .C. , Bohmer, R . M . , M a n n , G.B. , T h u m w o o d , S., Magli t to , R., Danks, J .A. , Chetty, R., Burgess, A . W . , and D u n n , A . R . (1995): A mutation i n the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results i n impaired lactation. Proc. Nat l . Acad. Sci. U .S .A. 92:1465-1469. Francis-West, P . H . , Tatla, T., and Brickell, P . M . (1994): Expression patterns of the bone morphogenetic protein genes Bmp-4 and Bmp-2 i n the developing chick face suggest a role i n outgrowth of the primordia. Dev. Dynamics 201:168-178. Frommer, J., and Margolies, M . R . (1971): Contribution of Meckel's cartilage to ossification of the mandible i n mice. J. Dent. Res. 50:1260-1267. Fuki ishi , Y . , and Morriss-Kay, G . M . (1992): Migration of cranial neural crest cells to the pharyngeal arches and heart i n rat embryos. Ce l l Tissue Res. 268:1-8. Fukuda, Y. , Masuda, Y. , K ish i , J., Hashimoto, Y. , Hayakawa, T., Nogawa, H . , and Nahanishi , Y . (1988): The role of intersitial collagens i n cleft formation of mouse e m b r y o n i c s u b m a n d i b u l a r g l a n d d u r i n g i n i t i a l b r a n c h i n g . Development 103:259-267. Furthmayr, H . (1993): Basement membrane collagen: Structure, assembly, and biosynthesis. In: Zern, M . A . , and Reid, L . M . (eds.) "Extracellular Matrix ." Marcel Dekker, Inc., N e w York. pp.149-185. 1 6 0 Gal loway, W . A . , M u r p h y , G . , Sandy, J.D., Gavr i lovic , J., Cawston, T.E. , and Reynolds, J.J. (1988): Purif ication and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective-tissue components. Biochem. J. 209:741-752. Ganser, G . L . , Str ickl in, G.P . , and Matr is ian, L . M . (1991): E G F and T G F - a influence in vitro lung development by the induction of matrix-degrading metalloproteinases. Int. J. Dev. Biol . 35:1-8. Garcia-Martinez, V . , and Schoenwolf, G . C . (1993): Primitive-streak origin of the cardiovascular system i n avian embryos. Dev. Biol . 159:706-719. Gaunt, S.J., B l u m , M . , and De Robertis, E . M . (1993): Expression of the mouse goosecoid gene d u r i n g mid-embryogenesis may mark mesenchymal cell lineages i n the developing head, limbs and body wal l . Development 117:769-778. G a v i n , B.J., M c M a h o n , A.J . , and M c M a h o n , A . P . (1990): Expression of multiple novel W n t - l / i n t - l - r e l a t e d genes during fetal and adult mouse development. Genes Dev. 4:2319-2332. George, E.L. , Georges-Labouesse, E . N . , Patel-King, R.S., Rayburn, H . , and Hynes, R .O. (1993): Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119:1079-1091. Gessner, I .H . , Lorincz, A . E . , and Bostrom, H . (1965): A c i d mucopolysaccharide content of the cardiac jelly of the chick embryo. J. Exp. Zool . 160:291-298. Giudice , G.J., and Sternberg, M.S . (1981): A direct role for fibronectin i n early heart development. J. Cel l Biol . 91:151a. Glass, R . H . , Aggeler , J., Spindle, A . , Pedersen, R . A . , and Werb, Z . (1983): Degradation of extracellular matrix by mouse trophoblast outgrowths: A model for implantation. J. Cel l Biol . 96:1108-1116. Gonchoroff, N.J . , Katzmann, J .A., Currie, R . M . , Evans, E X . , Houck, D . W . , Kl ine , B . C . , G r e i p p , P.R., and Loken, M . R . (1986): S-phase detection w i t h an antibody to bromodeoxyuridine. J. Immunol. Methods 93:97-101. Gorza , L . , Schiaffino, S., and Vitadello, M . (1988): Heart conduction system: a neural crest derivative? Brain Res. 457:360-366. G o u l d i n g , M . D . , Chalepakis, G . , Deutsch, U . , Erselius, J.R., and Cruss, R. (1991): Pax-3, a novel murine D N A b i n d i n g protein expressed d u r i n g early neurogenesis. E M B O J . 10:1135-1147. 161 Grant, D.S., Kle inman, H . K . , and Mar t in , G.R. (1990): The role of basement membranes in vascular development. A n n . N . Y. Acad. Sci. 588:61-72. Grant, M . , Cutts, N .R . , and Brody, J.S. (1983): Alterations i n lung basement membrane dur ing fetal growth and type 2 cell development. Dev. B io l . 97:173-183. G r a t z n e r , H . G . (1982): M o n o c l o n a l ant ibody to 5 -Bromo- and 5-Iododeoxyuridine: A new reagent for detection of D N A replication. Science 218:474-475. Greene, R . M . , and Pratt, R . M . (1976): Developmental aspects of secondary palate formation. J Embryol Exp M o r p h o l 36:225-245. Gregory, H . (1975): Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257:325-327. Gri f f i th , C . M . , and H a y , E .D. (1992): Epithelial-mesenchymal transformation dur ing palatal fusion: carboxyfluorescein traces cells at light and electron microscopic levels. Development 116:1087-1099. Grindley, J.C., Davidson, D.R., and H i l l , R.E. (1995): The role of Pax-6 i n eye and nasal development. Development 121:1433-1442. Grobstein, C . (1954): Tissue interaction i n the morphogenesis of mouse embryonic rudiments in vitro. In: Rudnik, O . (ed.) "Aspects of Synthesis and order in Growth." Princeton University Press, Princeton, pp.233-256. Gross J., and Bruschi , A . B . (1971): The pattern of collagen degradation i n cultured tadpole tissues. Dev. Biol . 26:36-41. Guer in , C . W . , and H o l l a n d , P .C . (1995): Synthesis and secretion of matrix-degrading metalloproteinases by human skeletal muscle satellite cells. Dev. Dynamics 202:91-99. G u i , T., Osumi-Yamashita , N . , and Eto, K . (1993): Prol i ferat ion of nasal epithel ial and mesenchymal cells d u r i n g pr imary palate formation. J. Craniofac. Genet. Dev. Biol . 13:250-258. Gul l i ck , W.J. , Marsden, J.J., Whittle, N . , Ward , B., Bobrow, L . , and Waterfield, M . D . (1986): Expression of epidermal growth factor receptors on human cervical, ovarian, and vulva carcinomas. Cancer Res. 46:285-292. Gunja-Smith, Z . , Morales, A . R . , Romanell i , R., and Woessner, J.F. Jr. (1996): R e m o d e l i n g of h u m a n m y o c a r d i a l co l lagen i n i d i o p a t h i c d i l a t e d cardiomyopathy. 148:1639-1648. 1 6 2 G u o , X. , Johnson, J.J., and Kramer, J . M . (1991): Embryonic lethality caused by mutations i n basement membrane collagen of C. elegans. Nature 349:707-709. H a l l , P . A . , Levinson, D . A . , Woods, A . L . , Y u , C . C . W . , Kellock, D.B. , Watkins, J .A. , Barnes, D . M . , Gillett , C .E . , Camplejohn, R., Dover, R., Waseem, J .A. , and L a n e , D . P . (1990): P r o l i f e r a t i n g ce l l nuclear ant igen ( P C N A ) immunolocalization i n paraffin sections: and index of cell proliferation w i t h evidence of deregulated expression in some neoplasms. J. Pathol. 162:285-294. Harvey, M . B . , Leco, K . L . , Arcellana-Panlilio, M . Y . , Zhang, X. , Edwards, D.R., and Schultz, G . A . (1995): Proteinase expression i n early mouse embryos is regulated by leukemia inhibi tory factor and epidermal g r o w t h factor. Development 121:1005-1014. H a y , E.D. (1991): Collagen and other matrix glycoproteins i n embryogenesis. In: H a y , E .D. (ed.) "Cel l Biology of Extracellular Matrix." Plenum Press, N e w York. pp. 419-462. H a y , D . A . , M a r k w a l d , R.R., and Fitzharris, T.P. (1984): Selected views of early heart development by scanning electron microscopy. In: Johari, O . M . (ed.) "Scanning Electron Microscopy." S E M Inc., Illinois. V o l . IV, pp.1983-1993. H a y a k a w a , T. (1994): Tissue inhibitors of metalloproteinases and their cell growth-promoting activity. Cel l Structure Function 19:109-114. H a y a k a w a , T., K i s h i , J.-I., and N a k a n i s h i , Y . (1992): Sa l ivary g land morphogenesis: possible involvement of collagenase. In: Birkedal-Hansen, H . , W e r b , Z . , Welgus , H . G . , and V a n Wart , H . E . (eds.) " M a t r i x Metalloproteinases and Inhibitors." Matr ix . Spec. Suppl . N o l . Gustav Fischer, Stuttgart, pp.344-351. Hayakawa, T., Yamashita, K . , Ohuchi , E., and Shinagawa, A . (1994): Ce l l growth-promoting activity of tissue inhibitor of metalloproteinase-2 (TIMP-2). J. C e l l Science 107:2373-2379. Heath, J.K. (1993): Growth Factors. Oxford University Press, Oxford, pp.19-22. Heik inhe imo, K . (1993): C e l l growth and differentiation of developing and neoplastic odontogenic tissues. P h . D . Dissertation, Univers i ty of T u r k u , F i n l a n d . Heikinheimo, K . , and Salo, T. (1995): Expression of basement membrane type IV collagen and type IV collagenases (MMP-2 and M M P - 9 ) in human fetal teeth. J. Dent. Res. 74:1226-1234. 1 6 3 Heikinheimo, K . , Vouti lainen, R., Happonen, R.-P., and Miett inen, P.J. (1993): E G F receptor and its ligands, E G F and T G F - a , i n developing and neoplastic human odontogenic tissues. Int. J. Dev. Biol . 37:387-396. Heikinheimo, M . , Lawshe, A . , Shackleford, G . M . , Wi lson, D.B. , and MacArthur , C . A . (1994): Fgf-8 expression i n the post-gastrulation mouse is localized to the developing face, limbs, and central nervous system. Mech. Dev. 48:129-138. Heine , U . I . , M u n o z , E.F., Flanders, K . C . , E l l ingsworth , L .R . , L a m , H . - Y . P . , Thompson, N . L . , Roberts, A . B . , and Sporn, M . B . (1987): Role of transforming growth factor i n the development of the mouse embryo. J. C e l l B i o l . 105:2861-2876. Hernandez-Sotomayor, T .S .M. , and Carpenter, G . (1992): Epidermal growth factor receptor: elements of intracellular communication. J. Membrane Biol . 128:81-89. Hi l fer , S.R., and Randolph, G.J. (1993): Immunolocalization of basal lamina components dur ing development of chick otic and optic pr imordia . Anat . Rec. 235:443-452. Hiraoka, K . , Sasaguri, Y . , Komiya , S., Inoue, A . , and Morimatsu, M . (1992): Ce l l prol i ferat ion-related p r o d u c t i o n of matrix metalloproteinase-1 (tissue collagenase) and -3 (stromelysin) by cultured human rheumatoid synovial fibroblasts. Biochem. Int. 27:1083-1091. Holder , S.E., Vintiner, G . M . , Farren, G . , Malco lm, S., and Winter, R . M . (1992): Confirmation of an association between RFLPs at the transforming growth factor-alpha locus and nonsyndromic cleft l ip and palate. J. M e d . Genet. 29:390-392. Hopper , A . F . , and Hart , N . H . (1980): Foundations of A n i m a l Development. Oxford University Press, Oxford. H o r m i , K . , and Lehy, T. (1994): Developmental expression of transforming growth factor-a and epidermal growth factor receptor proteins i n the human pancreas and digestive tract. Cel l Tissue Res. 278:439-450. H o w a r d , E.W., Bullen, E.C. , and Banda, M.J . (1991): Preferential inhibition of 72-and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2. J. Biol . Chem. 266:13070-13075. H s u , S . M . , Raine, L . , and Fanger, H . (1981): The use of avidin-biotin-peroxidase complex (ABC) i n immunoperoxidase technique: A comparison between A B C and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577-580. 1 6 4 H u d s o n , B .G . , Reeders, S.T., and Tryggvason, K . (1993): Type IV collagen: Structure, gene organization, and role i n human diseases. J. B io l . C h e m . 268:26033-26036. H u d s o n , C D . , and Shapiro B .L . (1973): A n autoradiographic s tudy of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium w i t h reference to the concept of programmed cell death. Archs. Oral Biol . 18:77-84. Huhtala, P., Chow, L.T., and Tryggvason, K . (1990): Structure of the human type IV collagenase gene. J. Biol . Chem. 265:11077-11082. Hur le , J . M . , Kitten, G.T., Sakai, L .Y . , Vo lp in , D . , and Solursh, M . (1994): Elastic extracellular matrix of the embryonic chick heart: an immunohistological study using laser confocal microscopy. Dev. Dynamics 200:321-332. H u r m e r i n t a , K . , and Thesleff, I. (1981): Ultrastructure of the epithelio-mesenchymal interface of the mouse tooth germ. J. Craniofac. Genet. Dev. Biol . 1:191-202. Iamaroon, A . , and Diewert V . M . (1996): Distribution of basement membrane components i n the mouse primary palate. J. Craniofac. Genet. Dev. B io l . 16:48-51. Iamaroon, A . , Tait, B., and Diewert , V . M . (1996a): C e l l proli feration and expression of E G F , T G F - a , and EGF-R i n the developing primary palate. J. Dent. Res. (in press). Iamaroon, A . , W a l l o n , U . M . , O v e r a l l , C M . , and Diewert , V . M . (1996b): Express ion of 72-kDa gelatinase (matrix metalloproteinase 2) i n the developing craniofacial complex of the mouse embryo. A r c h . Ora l Biol , (in press). Iamaroon, A . , W a l l o n , U . M . , O v e r a l l , C M . , and Diewert , V . M . (1996c): I m m u n o l o c a l i z a t i o n of 72-kDa gelatinase and extracel lular matr ix components during mouse cardiac development. J. Anat. (in revision). Icardo, J . M . , and Manasek, F.J. (1983): Fibronectin distribution during early chick embryo heart development. Dev. Biol . 95:19-30. Icardo, J . M . , and Manasek, F.J. (1984): A n indirect immunofluorescence study of the distribution of fibronectin dur ing the formation of the cushion tissue mesenchyme i n the embryonic heart. Dev. Biol . 101:336-345. 1 6 5 Ikawa, H . , Trelstad, R .L . , Hutson, J . M . , Manganaro, T.F., and Donahoe, P . K . (1984): Changing patterns of fibronectin, laminin, type IV collagen, and a basement membrane proteoglycan during rat Mul ler ian duct regression. Dev. Biol . 102:260-263. Iruela-Arispe, M . L . , and Sage, E . H . (1991): Expression of type VIII collagen dur ing morphogenesis of the chicken and mouse heart. Dev. Bio l . 144:107-118. Jacobson, A . G . (1963a): The determination and positioning of the nose, lens and ear. I. Interactions w i t h i n the ectoderm, and between the ectoderm and underlying tissues. J. Exp. Zool . 154:273-283. Jacobson, A . G . (1963b): The determination and positioning of the nose, lens and ear. II. The role of the endoderm. J. Exp. Zool . 154:283-291. Jaskoll, T.F., and Slavkin, H . C . (1984): Ultrastructural and immunofluorescence studies of basal-lamina alterations d u r i n g mouse l u n g morphogenesis. Differentiation 28:36-48. Jaskulski , D . , Gatt i , C , Traval i , S., Calabretta, B., and Baserga, R. (1988): Regulation of the proliferating cell nuclear antigen cycl in and thymidine kinase m R N A levels by growth factors. J. Biol . Chem. 263:10175-10179. Jhappan, C , Stahle, C , Harkins, R . N . , Fausto, N . , Smith, G . H . , and Merl ino , G.T. (1990): T G F a overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. C e l l 61:1137-1146. Johnson, S.E., Rothstein, J.L., and Knowles, B.B. (1994): Expression of epidermal growth factor family gene members i n early mouse development. Dev. Dynamics 201:216-226. Johnston, M . C . (1966): A radiographic study of the migration and fate of the craniofacial neural crest cells in the chick embryo. Anat. Rec. 156:143-155. Johnston, M . C , and Bronsky, P.T. (1995): Prenatal craniofacial development: new insights on normal and abnormal mechanisms. Cri t . Rev. Ora l B io l . M e d . 6:25-79. Jones, C M . , Lyons, K . M . , and Hogan, B . L . M . (1991): Involvement of bone morphogenetic protein-4 (BMP-4) and Vgl-1 i n morphogenes is and neurogenesis i n the mouse. Development 111:531-542. Jones, S.E., Jomary, C , and Neal , M.J . (1994): Expression of TIMP3 m R N A is elevated i n retinas affected by simplex retinitis pigmentosa. FEBS 352:171-174. 1 6 6 Joyner, A . L . (1991): Gene targeting and gene trap screens using embryonic stem cells: new approaches to mammalian development. BioAssays 13:649-656. Ju, W . D . , V e l u , T.J., Vass, W . C . , Papageorge, A . G . , and L o w y , D.R . (1991): Tumorigenic transformation of N I H 3T3 cells by the autocrine synthesis of transforming growth factor a. N e w Biologist 3:380-388. Juriloff, D . M . (1995): Genetic analysis of the construction of the AEJ . A congenic strain indicates that nonsyndromic CL(P) i n the mouse is causes by two loci w i t h epistatic interaction. J. Craniofac. Genet. Dev. Biol . 15:1-12. Kaartinen, V . , Voncken, J.W., Shuler, C , Warburton, D . , B u , D . , Heisterkamp, N . , and Groffen, J. (1995): Abnormal lung development and cleft palate i n mice lacking TGF-(33 indicates defects of epithelial-mesenchymal interaction. Nature Genetics 11:415-421. Kalebic, T., Garbisa, S., Glaser, B., and Liotta, L . A . (1983): Basement membrane collagen: degradation by migrating endothelial cells. Science 221:281-283. Karkinen-Jaaskelainen, M . (1978): Permissive and directive interactions i n lens induction. J. Embryol . Exp. Morphol . 44:167-179. Kasse lberg , A . G . , O r t h , D . , G r a y , M . E . and Stahlman, M . T . (1985): I m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n of h u m a n e p i d e r m a l g r o w t h factor/urogastrone i n several human tissues. J. Histochem. Cytochem. 33:315-322. Kaufman, M . H . (1992): The atlas of mouse development. Academic Press, Harcourt Brace Jovanovich, Publishers: San Diego, C A pp.75-158. Kaufman, M . H . , and Navaratnam, V . (1981): Early differentiation of the heart i n mouse embryos. J. Anat. 133:235-246. Kerr , L . D . , M i l l e r , D.B . , and Matr is ian, L . M . (1990): T G F - p l inhib i t ion of t rans in/s t romelys in gene expression is mediated through a fos-binding sequence. Cel l 61:267-278. Kerr, L . D . , Hol t , J.T., and Matrisian, L . M . (1988): Growth factors regulate transin gene expression by c-fos-dependent and c-fos-independent pathways. Science 242:1424-1427. Ki t ten , G.T. , M a r k w a l d , R.R., and Bolender, D . L . (1987): Dis t r ibut ion of basement membrane antigens i n cryopreserved early embryonic hearts. Anat. Rec. 217:370-390. 1 6 7 Kle in , G . , Ekblom, M . , Fecker, L . , T impl , R., and Ekblom, P. (1990): Differential expression of laminin A and B chains dur ing development of embryonic mouse organs. Development 110:823-837. Kleinman, H . K . , Graf, J., Iwamoto, Y. , Kitten, G.T., Ogle, R.C. , Sasaki, M . , Yamada, Y . , M a r t i n , G.R. , and Luckenbi l l -Edds , L . (1987): Role of basement membranes in cell differentiation. Annal . N Y . Acad. Sci. 513:134-145. Kle inman, H . K . , Weeks, B.S., Schnaper, H . W . , Kibbey, M . C , Yamamura, K . , and Grant , D.S. (1993): Tha laminins : a fami ly of basement membrane glycoproteins important in cell differentiation and tumor metastases. Vi tam. H o r m . 47:161-186. Kobayashi, Y . (1958): A genetic study of harelip and cleft palate. Jpn. J. H u m . Genet. 3:73-107. K o n d o , S., H o z u m i , Y . , Maejima, H . , and A s o , K . (1992): Organ culture of psoriatic skin: effect of T G F - a and TGF-P on epidermal structure in vitro. A r c h . Dermatol. Res. 284:150-153. K o o p m a n , P. (1993): Analysis of gene expression by reverse transcriptase-polymerase chain reaction. In: Stern, C D . , and H o l l a n d , P . W . H . (eds.) "Essential Developmental Biology." Oxford University Press, Oxford, pp.233-241. Kosher, R . A . , and Solursh, M . (1989): Widespread distribution of type II collagen during embryonic chick development. Dev. Biol . 131:558-566. K r o n m i l l e r , J.E. (1995): Spatial dis tr ibut ion of epidermal growth-factor transcripts and effects of exogenous epidermal growth factors on the pattern of the mouse dental lamina. A r c h . Oral Biol . 40:137-143. Kronmil ler , J.E., Upholt , W.B. , and Kollar , E.J. (1991a): Expression of epidermal growth factor m R N A i n the developing mouse mandibular process. Archs. Oral Biol . 36:405-410. K r o n m i l l e r , J.E., U p h o l t , W.B . , and K o l l a r , E.J. (1991b): Antisense E G F oligonucleotides inhibit odontogenesis i n embryonic mouse mandible in vitro. Dev. Biol . 147:484-488. Kronmil ler , J.E., Upholt , W.B. , and Kollar , E.J. (1993): Effects of retinol on the temporal expression of transforming growth factor-alpha m R N A i n the embryonic mouse mandible. A r c h . Oral Biol . 38:185-188. 1 6 8 K u r k i , P., Vanderlaan, M . , Dolbeare, F., Gray, J., and Tan, E . M . (1986): Expression of proliferating cell nuclear antigen ( P C N A ) / c y c l i n during the cell cycle. Exp. Cel l Res. 166:209-219. Kumar , V . , Bustin, S.A., and M c K a y , L A . (1995): Transforming growth factor alpha. Cel l Biol . Int. 19:373-388. Kwee, L . , Baldwin, H.S. , Shen, H . M . , Stewart, C .L . , Buck, C . A . , and Labow, M . A . (1995): Defective development of the embryonic and extraembryonic circulatory systems i n vascular cell adhesion ( V C A M - 1 ) deficient mice. Development 121:489-503. Lammer, E.J., and Opitz , J . M . (1986): The DiGeorge anomaly as a developmental field defect. A m . J. M e d . Genet. 2(Suppl):113-127. de Larco, J.E., and Todaro, G.J. (1978): G r o w t h factor from murine sarcoma virus-transformed cells. Proc. Nat l . Acad. Sci. U .S .A. 75:4001-4005. Lee, D . C . (1985): Cloning and sequence analysis of a c D N A for rat transforming growth factor-a. Nature 313:489-491. Lee, D . C , and H a h n , K . M . (1991): Expression of growth factors and their receptors i n development. In: Sporn, M . B . and Roberts, A . B . (eds.) "Peptide Growth Factors and Their Receptors II." Springer-Verlag, N e w York, pp.611-654. Lefebvre, O. , Regnier, C , Chenard, M . - P . , Wendling, C , Chambon, P., Basset, P., and Rio , M . - C . (1995): Developmental expression of mouse stromelysin-3 m R N A . Development 121:947-955. Le ibovic i , M . , M o n o d , G . , Geraudie, J., Bravo, R., and Mechal i , M . (1992): Nuclear distribution of P C N A dur ing embryonic development i n Xenopus laevis: a reinvestigation of early cell cycles. J. Cel l Sci. 102:63-69. Le ivo , I., Vaheri , A . , T i m p l , R., and Wartiovaara, J. (1980): Appearance and distribution of collagens and laminin i n the early mouse embryo. Dev. Biol . 76:100-114. Le Lievre, C , and Le Douarin, N . M . (1975): Mesenchymal derivatives of the neural crest: Analysis of chimeric quail and chick embryos. J. Embryol . Exp. M o r p h o l . 34:125-154. Lesot, H . , Kubler , M . - D . , Fausser, J .L., and Ruch, J .-V. (1990): A 165 k D a membrane antigen mediating fibronectin-vinculin interaction is involved i n murine odontoblast differentiation. Differentiation 44:25-35. 1 6 9 Levy, A . T . , Cioce, V . , Sobel, M . E . , Garbisa, S., Grigioni , W.F. , Liotta, L . A . , Stetler-Stevensen, W . G . (1991): Increased expression of the M r 72,000 type IV collagenase in human colonic adenocarcinoma. Can. Res. 51:439-444. Lewis , W . H . (1907): Lens formation from strange ectoderm i n Rana sylvatica. A m . J. Anat. 7:145-169. Libson, A . M . , Gittis, A . G . , Collier, I.E., Marmer, B.L., Goldberg, G.I., and Lattman, E.E. (1995): Crystal structure of the haemopexin-like C-terminal domain of gelatinase A . Nature Struct. Biol . 2:938-942. Linask, K . K . , and Lash, J.W. (1988): A role for fibronectin i n the migration of avian precardiac cells. I. Dose-dependent effects of fibronectin antibody. Dev. Biol . 129:315-323. Liotta, L . A . , Abe , S., Gehron Robey, P. , and M a r t i n , G.R. (1979): Prerential digestion of basement membrane collagen by an enzyme derived from a metastatic murine tumor. Proc. Nat l . Acad . Sci. U S A 76:2268-2272. Liotta, L . A . , Rao, C . N . , and Wewer, U . M . (1986): Biochemical interactions of tumor cells w i t h the basement membrane. A n n . Rev. Biochem. 55:1037-1057. Liotta, L . A . , Tryggvason, K . , Garbisa, S., Hart, I., Foltz, C M . , and Shafie, S. (1980): Metastatic potential correlates w i t h enzymatic degradation of basement membrane collagen. Nature 284:67-68. Little, C D . , Piquet, D . M . , Davis, L . A . , Walters, L . , and Drake, C.J. (1989): The distribution of laminin, collagen type IV, collagen type I and fibronectin i n the cardiac jelly-basement membrane. Anat. Rec. 224:417-425. L i t v i n , J., Montgomery, M . , Gonzalez-Sanchez, A . , Bisaha, J .G., and Bader, D . (1992): Commitment and differentiation of cardiac myocytes. Trends Cardiovas. M e d . 2:27-32. L o w r y , R.B., and Renwick, D . H . G . (1969): Incidence of cleft l ip and cleft palate i n British Columbia Indians. J. M e d . Genet. 6:67-69. Luetteke, N . C , Q i u , T . H . , Peiffer, R.L. , Oliver, P., Smithies, O. , and Lee, D . (1993): T G F - a deficiency results i n hair follicle and eye abnormalities i n targeted and waved-1 mice. Cel l 73:263-278. Lumsden , A . , Sprawson, N . , and Graham, A . (1991): Segmental or igin and migration of neural crest cells i n the hindbrain region of the chick embryo. Development 113:1281-1292. 1 7 0 Lyons , J .G., Birkedal-Hansen, B., Pierson, M . C . , Whitelock, J . M . , and Birkedal-Hansen, H . (1993): Interleukin-1 beta and transforming growth factor-a lpha/epidermal growth factor induce expression of M(r) 95,000 type IV collagenase/gelatinase and interstitial fibroblast-type collagenase by rat mucosal keratinocytes. J. Biol . Chem. 268:19143-19151. Lyons, K . M . , Pelton, R.W. , and Hogan, B. (1990): Organogenesis and pattern formation i n the mouse: R N A distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109:833-844. M a c A r t h u r , C . A . , Lawshe, A . , X u , J., Santos-Ocampo, S., H e i k i n h e i m o , M . , Chellaiah, A .T . , and Ornitz , D . M . (1995): FGF-8 isoforms activate receptor splice forms that are expressed i n mesenchymal regions of mouse development. Development 121:3603-3613. M a c h i d a , C M . , Scott, J .D., and Ciment , G . (1991): N G F - i n d u c t i o n of the metalloproteinase t rans in/s t romelys in i n PC12 cells: involvement of multiple protein kinases. J. Cel l Biol . 114:1037-1048. MacKenzie, A . , Ferguson, M.W.J . , and Sharpe, P.T. (1992): Expression patterns of the homeobox gene, Hox-8, i n the mouse embryo suggest a role in specifying tooth initiation and shape. Development 115:403-420. M a c N a u l , K . L . , Chartrain, N . , Lark, M . , Tocci, M.J . , and Hutchinson, N. I . (1990): Discoordinate expression of stromelysin, collagenase and tissue inhibitor of metalloproteinase-1 i n rheumatoid human synovial fibroblasts. Synergistic effects of interleukin-1 and tumor necrosis factor-a on s tromelys in expression. J. Biol . Chem. 265:17238-17245. Maden, M . , Hunt , P., Eriksson, U . , Kuro iwa , A . , Krumlauf, R., and Summerbell, D . (1991): Retinoic acid-binding protein, rhombomeres and the neural crest. Development 111:35-44. Manasek, F.J. (1976): Heart Development: Interactions involved i n cardiac morphogenesis. In: Poste, G . and Nicholson, G . L . (eds.) "The Cel l Surface i n A n i m a l Embryogenesis and Development." Elsevier, N o r t h H o l l a n d , Amsterdam, pp .545-598. Manasek, F.J., Re id , M . , V i n s o n , W . , Seyer, J., and Johnson, R. (1973): Glycosaminoglycan synthesis by the early embryonic chick. Dev. B io l . 35:332-348. M a n n , G.B. , Fowler, K.J. , Gabriel , A . , Nice, E . C , Wil l iams, R.L. , and D u n n , A.S . (1993): Mice w i t h a n u l l mutation of the T G F - a gene have abnormal skin architecture, w a v y hair, and curly whiskers and often develop corneal inflammation. Cel l 73:249-261. 171 M a n n , I. (1964): The development of the human eye. Grune & Stratton, N e w York. M a r k w a l d , R.R., K r u g , E.L. , Runyan, R.B., Kitten, G.T. (1984): Proteins i n cardiac jelly which induce mesenchyme formation. In: Ferrans, V.J . , Rosenquist, G . , Weinstein, C . (eds.) "Cardiac Morphogenesis." Elsevier, N e w York. pp.60-68. Marquardt , H . , Hunkapi l lar , M . W . , H o o d , L . E . , and Todaro, G.J. (1984): Rat transforming growth factor type 1: structure and relation to epidermal growth factor. Science 223:1079-1082. Mar t in , G.R., and Sank, A . C . (1991): Extracellular matrices, cells, and growth factors. In: Sporn, M . B . and Roberts, A . B . (eds) "Peptide G o w t h Factors and Their Receptors II." Springer-Verlag, Berlin, pp.463-477. Massague, J. (1983): Epidermal growth factor-like transforming growth factor. II. Interaction w i t h epidermal growth factor receptors i n h u m a n placenta membranes and A431 cells. J. Biol . Chem. 258:13614-13620. Massague, J. (1990): Transforming growth factor-a. J. Biol . Chem. 265:21393-21396. Matr i s ian , L . M . (1990): Metalloproteinases and their inhibitors i n matrix remodeling. Trend Genet. 6:121-125. Matr is ian, L . M . (1992): The matrix-degrading metalloproteinases. Bioassays 14:455-463. M a t s u i , Y . , Halter , S .A. , Hol t , J.T., Hogan , B . L . M . , and Coffey, R.J. (1990): Development of mammary hyperplasia and neoplasia i n M M T V - T G F a transgenic mice. Cel l 61:1147-1155. Mawatar i , M . , Kohno, K . , Mizoguchi , H . , Matsuda, T., A s o h , K . , van Damme, J., Welgus, H . G . , and Kuwano, M . (1989): Effects of tumor necrosis factor and epidermal growth factor on cell surface morphology, cell surface receptors, and the product ion of tissue inhibitor of metalloproteinases and IL-6 i n human microvascular endothelial cells. J. Immunol. 143:1619-1627. M c A v o y , J.W. (1980): Induction of the eye lens. Differentiation 17:137-149. M c G u i r e , P .G . , and O r k i n , R .W. (1992): Urokinase activity i n the developing avian heart: A spatial and temporal analysis. Dev. Biol . 193:24-33. McKeehan M.S . (1951): Cytological aspects of embryonic lens induction in the chick. J. Exp. Zool . 117:31-64. 1 7 2 M e r c o l a , M . , and Stiles, C D . (1988): G r o w t h factor superfamilies and mammalain embryogenesis. Development 102:451-460. Miettinen, P.J. (1993): Transforming growth factor-alpha and epidermal growth factor expression i n human fetal gastrointestinal tract. Ped. Res. 33:481-486. Miett inen, P.J., and Heikinheimo, K . (1992): Transforming growth factor-alpha (TGF-a) and insulin gene expression in human fetal pancreas. Development 114:833-840. Miett inen, P.J., Perheentupa, J., Otonkoski , T., Lahteenmaki, A . , and Panula, P. (1989): E G F - and TGF-a-l ike peptides in human fetal gut. Ped. Res. 26:25-30. M i l l e r , K . , and R i z z i n o , A . (1994): Developmental regulation and signal transduction pathways of fibroblast growth factors and their receptors. In: N i l s e n - H a m i l t o n , M . (ed.) "Growth Factors and Signal Transduction i n Development." Wiley-Liss, Inc., N e w York. pp.19-49. M i l l i c o v s k y , G . , and Johnston, M . C (1981): Act ive role of embryonic facial epithelium: N e w evidence of cellular events i n morphogenesis. J. Embryol . Exp. Morphol . 63:53-66. Miner , J .H . , and Sanes, J.R. (1994): Collagen IV a3, a4, and a5 chains i n rodent basal laminae: sequence, d is t r ibut ion , association w i t h l amin ins , and developmental switches. J. Cel l Biol . 127:879-891. Minkof f , R. (1980a): Regional variation of cell proliferation w i t h i n the facial processes of the chick embryo: a study of the role of 'merging' d u r i n g development. J. Embryol . Exp. Morphol . 57:37-49. Minkof f , R. (1980b): C e l l proliferation and migration dur ing primary palate development. In: Pratt, R . M . and Christainsen, R .L . (eds.) "Current Research Trends i n Prenatal Craniofacial Development." Elsevier, N e w York. pp.119-132. Minkoff , R. (1991): Ce l l proliferation during formation of the embryonic facial primordia. J. Craniofac. Genet. Dev. Biol . 11:251-261. Minkof f , R., and K u n t z , A . J . (1977): C e l l proliferation dur ing morphogenic change: analysis of frontonasal morphogenesis i n the chick embryo employing D N A labelling indices. J. Embryol. Exp. Morphol . 40:101-113. Minkoff , R., and Mart in , R.E. (1984): Cel l cycle analysis of facial mesenchyme i n the chick embryo: II. Label di lut ion studies and developmental fate of slow cycling cells. J. Embryol. Exp. Morphol . 81:61-73. 1 7 3 Mitchel l . , L .E . , and Risch, N . (1992): Mode of inheritance of nonsyndromic cleft l ip w i t h or without cleft palate: a reanalysis. A m . J. H u m . Genet. 51:323-332. Mitchel l , P.J., Timmons, P . M . , Herbert, J . M . , Rigby, P.W.J., and Tijan, R. (1991): Transcription factor A P - 2 is expressed i n neural crest cell lineages dur ing mouse embryogenesis. Genes Dev. 5:105-119. M i z u n o , T. (1972): Lens differentiation in vitro in the absence of optic vesicle i n the epiblast of chick blastoderm under the influence of sk in dermis. J. Embryol . Exp. Morphol . 28:117-132. Mjaatvedt, C . H . , Lepera, R.C. , and M a r k w a l d , R.R. (1987): Myocardial specificity for init iat ing endothelial-mesenchymal cell transition i n embryonic chick heart correlates w i t h a particulate distribution of fibronectin. Dev. B io l . 119:59-67. Monteagudo, C . , Merino, M.J . , San-Juan, J., Liotta, L . A . , and Stetler-Stevenson, W . G . (1990): Immunohistochemical distribution of type IV collagenase i n normal, benign, and malignant breast tissue. A m . J. Pathol. 136:585-592. M o r i , C . , N a k a m u r a , N . , Okamoto, Y . , Osawa, M . , and Shiota, K . (1994): Cytochemical identification of programmed cell death i n the fusing fetal mouse palate by specific labelling of D N A fragmentation. Anat . Embryol . 190:21-28. Morr iss -Kay, G . M . (1993): Postimplantation mammalian embryos. In: Stern, C . D . , and Hol land, P . W . H . (eds.) "Essential Developmental Biology." Oxford University Press, Oxford, pp.55-66. Morr i s -Wiman, J., and Brinkley, L . (1992): A n extracellular matrix infrastructure provides support for murine secondary palatal shelf remodelling. Anat. Rec. 234:575-586. M o r r i s - W i m a n , J., D u , Y. , and Brinkley, L . (1996): Matrix-degrading enzymes during in vitro palatogenesis. J. Dent. Res. 75:225. M u i r , D . (1994): Metalloproteinase-dependent neurite outgrowth w i t h i n a synthetic extracellular matrix is induced by nerve growth factor. Exp. C e l l Res. 210:243-252. M u r p h y , G . , H e m b r y , R . M . , M c G a r r i t y , A . M . , and Reynolds , J.J. (1989): Gelatinase (type IV collagenase) immunolocalization i n cells and tissues: use of an antiserum to rabbit bone gelatinase that identifies h igh and low M r forms. J. Cel l Sci. 92:487-495. 1 7 4 Nagase, H . , Enghi ld , J.J., Suzuki , K. , and Salvesen, G . (1990): Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl) mercuric acetate. Biochemistry 29:5783-5789. Nagase, H . , Ogata, Y. , Suzuki , K . , Enghild, J.J., and Salvesen, G . (1991): Substrate specificities and activation mechanisms of matrix metalloproteinases. Biochem. Soc. Trans. 19:715-718. Nakagawa, M . , Terracio, L . , Carver, W . , Birkedal-Hansen, H . , and Borg, T .K. (1992): Expression of collagenase and IL-loc i n developing rat hearts. Dev. Dynamics 195:87-99. Nakanishi , Y . , Sugiura, F., K i s h i , J.-L, and Hayakawa, T. (1986): Collagenase inhibitor stimulates cleft formation dur ing early morphogenesis of mouse salivary gland. Dev. Biol . 113:201-206. Nanda , R., and Romeo, D . (1975): Differential cell proliferation of embryonic rat palatal processes as determined by incorporation of tritiated thymidine. Cleft Palate 12:436-443. Neel , J.V. (1958): A study of major congenital defects i n Japanese infants. A m . J. H u m . Genet. 10:398-445. Nexo, E. and Kryger-Baggesen, N . (1989): The receptor for epidermal growth factor is present i n human fetal kidney, liver and lung. Regul. Pept. 26:1-8. Nichols , D . H . (1986): Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo. A m . J. Anat. 176:221-231. N i c h o l s o n , R., M u r p h y , G . , and Breathnach, R. (1989): H u m a n and rat m a l i g n a n t - t u m o r - a s s o c i a t e d m R N A s e n c o d e s t r o m e l y s i n - l i k e metalloproteinases. Biochemistry 28:5195-5203. Nish ikawa, K . , Nakanishi , T., A o k i , C , Hattori, T., Takahashi, K . , and Taniguchi, S. (1994): Differential expression of homeo-containing genes Msx-1 and M s x -2 and homeoprotein Msx-2 expression during chick craniofacial development. Biochem. M o l . Biol . Int. 32:763-771. N o d e n , D . M . (1988): Interactions and fates of avian craniofacial mesenchyme. Development 103: Suppl, 121-140. N o m u r a , S., Hogan , B . L . M . , Wi l l s , A. J . , Heath J.K., and Edwards , D.R. (1989): Developmental expression of tissue inhibitor of metalloproteinase (TIMP) R N A . Development 105:575-583. 1 7 5 O'Grady, R .L . , Nethery, A . , and Hunter, N . (1984): A fluorescent screening assay for collagenase us ing col lagen labeled w i t h L - m e t h o x y - 2 , 4 - d i p h e n y l -3(2H)furanone. A n a l . Biochem. 140:490-494. Ohuchi , H . , Yoshioka, H . , Tanaka, A . , K a w a k a m i , Y . , N o h n o , T., and N o j i , S. (1994): Involvement of androgen-induced growth factor (Fgf-8) gene i n mouse embryogenesis and morphogenesis . B iochem. B iophys . Res. C o m m u n . 204:882-888. Okada , Y . , M o r o d o m i , T., E n g h i l d , J.J., S u z u k i , K . , Yasui , A . , Nakanish i , I., Salvesen, G . , and Nagase, H . (1990): Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymatic properties. Eur. J. Biochem. 194:721-730. O'Rahil ly , R., and M u l l e r , F. (1987): Developmental Stages i n H u m a n Embryos. Carnegie Inst. Wash. Publ . 637: Carnegie Institute of Washington, pp.175-302. Osumi-Yamashi ta , N . , N i n o m i y a , Y . , D o i , H . , and Eto, K . (1994): The contribution of both forebrain and midbrain crest cells to the mesenchyme i n the frontonasal mass of mouse embryos. Dev. Biol . 164:409-419. Osumi-Yamashita , N . , N o j i , S., N o h n o , T., Koyama, E., D o i , H . , Eto, K . , and Tanigushi , S. (1990): Expression of retinoic acid receptor genes i n neural crest-derived cells during mouse facial development. FEBS 264:71-74. Overal l , C M . (1994): Regulation of tissue inhibitor of matrix metalloproteinase expression. A n n . N e w York. A c a d . Sci. 732:51-64. Overal l , C M . , and Limeback, H . (1988): Identification and characterization of enamel proteinases isolated from developing enamel. Biochem. J. 256:965-972. O v e r a l l , C M . , and Sodek, J. (1990): C o n c a n a v a l i n - A produces a matrix-degradative phenotype i n h u m a n f ibroblasts . I n d u c t i o n / e n d o g e n o u s activation of collagenase, 72 k D a gelatinase, & Pump-1 is accompanied by suppression of tissue inhibitor of matrix metalloproteinases. J. Biol . Chem. 265:21141-21151. Overa l l , C M . , W r a n a , J .L., and Sodek, J. (1989): Independent regulation of collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibi tor expression i n h u m a n fibroblasts by transforming growth factor-p. J. Biol . Chem. 264:1860-1869. 1 7 6 Overal l , C M . , Wrana, J.L., and Sodek, J. (1991): Transcriptional and post-transcriptional regulat ion of 72-kDa gelatinase/type IV collagenase by transforming growth factor-pl i n human fibroblasts. J. Biol . Chem. 266:14064-14071. Paria, B.C. , Das, S.K., Huet -Hudson, Y . M . , and Dey, S.K. (1994): Distribution of transforming growth factor alpha precursors i n the mouse uterus during the preimplantation period and after steroid hormone treatments. Biol . Reprod. 50:481-491. Parmigiani , C , and M c A v o y , J. (1984): Localization of laminin and fibronectin during rat lens morphogenesis. Differentiation 28:53-61. Partanen, A . M . , Ekblom, P., and Thesleff, I. (1985): Epidermal growth factor inhibits morphogenesis and cell differentiation i n cultured mouse embryonic teeth. Dev. Biol . 111:84-94. Partanen, A . M . (1990): Epidermal growth factor and transforming growth factor-alpha i n the development of epithelial-mesenchymal organs of the mouse. In: N i l s e n - H a m i l t o n , M . (ed.) " G r o w t h Factors and Development ." Academic Press, N e w York, pp.31-53. Partanen, A . M . , and Thesleff, I. (1987): Localization and quantitation of 1251-epidermal growth factor b i n d i n g i n mouse embryonic tooth and other embryonic tissues at different developmental stages. Dev. Biol . 120:186-197. Paulsson, M . (1992): Basement membrane proteins: structure, assembly and cellular interactions. Crit . Rev. Biochem. Molec. Biol . 27:93-127. Pellier, V . , and Astic , L . (1994): Detection of apoptosis by electron microscopy and in situ labelling i n the rat olfactory pit. NeuroReport 5:1429-1432. Pelton, R .W. , Hogan , B . L . M . , Mi l l e r , D . A . , and Moses, H . L . (1990): Differential expression of genes encoding TGFs (31, (32, and (33 dur ing murine palate formation. Dev. Biol . 141:456-460. Pendleton, N . , D i x o n , G.R., Burnett, H . E . , Occleston, N . L . , M y s k o w , M . W . , and Green, J .A. (1993): Expression of proliferating cell nuclear antigen ( P C N A ) i n dysplasia of the bronchial epithelium. J. Pathol. 170:169-172. Peterson , P . E . , P o w , C.S.T. , W i l s o n , D.B . , and H e i i d r i c k x , A . G . (1995): Loca l izat ion of glycoproteins and glycosaminoglycans d u r i n g early eye development i n the macaque. J. Anat. 186:31-42. 1 7 7 Pexieder, T., and Janecek, P. (1984): Organogenesis of the human embryonic and early fetal heart as studied by microdissection and S E M . In: N o r a , J.J. and Takao, A . (eds.) "Congenital Heart Disease: Causes and Processes." Futura Publishing Co. , N e w York, pp.401-421. Phil l ips, M . T . , K i rby , M . L . , and Forbes, G . (1987): Analysis of cranial neural crest distribution i n the developing heart using quail chick chimeras. Circulation Res. 60:27-30. Piatigorsky, J. (1981): Lens differentiation i n vertebrates. A review of cellular and molecular features. Differentiation 19:134-153. Pittman, R . N . (1985): Release of plasminogen activator and calcium-dependent metalloproteinases f rom cultured sympathetic and sensory neurons. Dev. Biol. 110:91-101. Potts, J.D. and Runyan, R.B. (1989): Epithelial-mesenchymal cell transformation i n the embryonic heart can be mediated, i n part, by transforming growth factor B. Dev. Biol. 134:392-401. Poulsen, S.S., Kryger-Baggesen, N . , and Nexo, E. (1996): Immunohistochemical local izat ion of epidermal growth factor i n the second-trimester h u m a n fetuses. Histochem. Cel l Biol . 105:111-117. Poulsen, S.S., N e x o , E. , Skov Olsen, P. , Hess, J., and Kirkegaard, P. (1986): Immunohistochemical localization of epidermal growth factor i n rat and man. Histochemistry 85:389-394. Pratt, R . M . (1987): Role of epidermal growth factor i n embryonic development. In: Sawyer, R . H . (ed.) "Current topics i n developmental biology." Academic Press, N e w York. V o l . 22, pp.175-193. Proetzel, G . , P a w l o w s k i , S.A., Wiles, M . V . , Y i n , M . , Boiv in , G .P . , Howies , P . N . , D i n g , J., and Ferguson, M . W . J . (1995): Transforming growth factor-B3 is required for secondary palate fusion. Nature Genetics 11:409-414. Pulkkinen, L . , Christiano, A . M . , Airenne, T., Haakana, H . , Tryggvason, K . , and Uitto, J. (1994): Mutations i n the y2 chain gene ( L A M C2) of k a l i n i n / l a m i n i n 5 i n the junctional forms of epidermolysis bullosa. Nature Genet 6:293-297. Pusztai , L . , Lewis , C .E . , Lorenzen, and McGee, J .O'D. (1993): G r o w t h factors: regulation of normal and neoplastic growth. J. Pathol. 169:191-201. Pyke, C , Ralfkiaer, E., Tryggvason, K . , and Dano, K . (1993): Messenger R N A for two type IV collagenases is located i n stromal cells i n human colon cancer. A m . J. Pathol. 142:359-365. 1 7 8 Q i u , C .X . , and Ferguson, M.W.J . (1995): The distribution of P D G F s and P D G F -receptors during murine secondary palate development. J. Anat . 186:17-29. Ralphs, J.R., Wyl ie , L . , and H i l l , D.J. (1990): Distribution of insulin-l ike growth factor peptides i n the developing chick embryo. Development 109:51-58. Rappolee, D . A . , Brenner, C . A . , Schultz, R., M a r k , D . , and Werb, Z . (1988): D e v e l o p m e n t a l express ion of P D G F , T G F - a , and T G F - 0 genes i n preimplantation mouse embryos. Science 241:1823-1825. Reed, S.C. (1933): A n embryological study of harelip i n mice. Anat. Rec. 56:101-110. Reponen, P. , Le ivo , I., Sahlberg, C . , Apte , S.S., Olsen, B.R., Thesleff, I., and Tryggvason, K . (1995): 92-kDa type IV collagenase and TIMP-3, but not 72-kDa type IV collagenase or TIMP-1 or TIMP-2, are highly expressed during mouse embryo implantation. Dev. Dynamics 202:388-396. Reponen, P. , Sahlberg, C . , H u h t a l a , P. , H u r s k a i n e n , T., Thesleff, I., and Tryggvason , K . (1992): Molecu lar c loning of mur ine 72-kDa type IV collagenase and its expression during mouse development. J. Biol . Chem. 267:7856-7862. Rhodes, J .A. , Fi tzgibbon, D . H . , Macchiarulo, P . A . , and M u r p h y , R . A . (1987): Epidermal growth factor-induced precocious incisor eruption is associated w i t h decreased tooth size. Dev. Biol . 121:247-252. Richman, J . M . , and Diewert, V . M . (1987): A n immunofluorescence study of chondrogenesis i n murine mandibular ectomesenchyme. C e l l Differentiation 21:161-173. Richman, J . M . , and Tickle, C . (1989): Epithelia are interchangeable between facial p r i m o r d i a of chick embryos and morphoogenesis is control led by the mesenchyme. Dev. Biol. 136:201-210. Richman, J . M . , and Tickle, C . (1992): Epithelial-mesenchymal interactions i n the outgrowth of l imb buds and facial pr imordia i n chick embryos. Dev. Biol . 154:299-308. Riggott, M.J . , and M o o d y , S.A. (1987): Distribution of laminin and fibronectin along peripheral trigeminal axon pathways i n the developing chick. J. Comp. Neurol . 258:580-596. Robert, B., Sassoon, D. , Jacq, B., Gehring, W . , and Buckingham, M . (1989): Hox-7, a mouse homeobox gene w i t h a nove l pattern of expression d u r i n g embryogenesis. E M B O J. 8:91-100. 179 Robertson, E.J., and M a r t i n , G.R. (1993): Embryonic stem cells and gene targeting. In: Stern, C D . , and H o l l a n d , P . W . H . (eds.) "Essential Developmental Biology." Oxford University Press, Oxford, pp.167-170. Rogers, S.L., Edson, K.J., Letourneau, P . C , and M c L o o n , S . C (1986): Distribution of laminin i n the developing peripheral nervous system of the chick. Dev. Biol . 113:429-435. Rosenquist, G . C , and De Haan, R.L. (1966): Migrat ion of precardiac cells i n the embryonic chick heart. Contrib. Embryol. 38:113-121. Rowe, A . , Richman, J . M . , and Brickell , P . M . (1991): Retinoic acid treatment alters the distribution of retinoic acid receptor-b transcripts i n the embryonic chick face. Development 111:1007-1016. R u g h , R. (1968): The mouse. Its reproduction and development. Burgess Publishing Company: Minneapolis, M N pp.117-167. Runyan, R.B., and M a r k w a l d , R.R. (1983): Invasion of mesenchyme into three dimensional collagen gels: A regional and temporal analysis of interaction i n embryonic heart. Dev. Biol . 95:108-114. Runyan, R.B., Potts, J .D., and Weeks, D . L . (1992): TGF-B3-mediated tissue interaction during embryonic heart development. M o l . Reprod. Dev. 32:152-159. Saari, H . , Suomalainen, K . , L i n d y , O. , Konttinen, Y.T. , and Sorsa, T. (1990): Activation of latent human nuetrophil collagenase by reactive oxygen species and serine proteases. Biochem. Biophys. Res. Commun. 3:979-987. Saber, G . M . , Parker, S.B., and Minkof f , R. (1989): Influence of epithelial-mesenchymal interaction on the viabi l i ty of facial mesenchyme in vitro. Anat. Rec. 225:56-66. Sage, E . H . , and Iruela-Arispe, M . L . (1990): Type VIII collagen i n murine development. Association wi th capillary formation in vitro. A n n . N . Y . A c a d . Sci. 580:17-31. Sahlberg, C , Reponen, P., Tryggvason, K . , and Thesleff, I. (1992): Association between the expression of murine 72 kDa type IV collagenase and basement membrane degradation during mouse tooth development. Archs. Ora l B io l . 37:1021-1030. Salo, T., Liotta, L . A . , Tryggvason, K . (1983): Purification and characterization of a m u r i n e basement membrane col lagen-degrading enzyme secreted by metastatic tumor cells. J. Biol . Chem. 258:3058-3063. 1 8 0 Salo, T., Lyons, J.G., Rahemtulla, F., Birkedal-Hansen, H . , and Larjava, H . (1991): Transforming growth factor-bl up regulates type IV collagenase expression in cultured human keratinocytes. J. Biol . Chem. 266:11436-11441. Salo, T., Makela , M . , Kylmanieme, M . , Aut io-Harmainen, H . , and Larjava, H . (1994): Expression of matrix metalloproteinas-2 and -9 during early human w o u n d healing. Lab. Invest. 70:176-182. Samuel, J.L., Farhadian, F., Sabri, A . , Marotte, F., Robert, V . , and Rappaport, L . (1994): Expression of fibronectin during rat fetal and postnatal development: an in situ hybridizat ion and immunohistochemical study. Cardiovas. Res. 28:1653-1661. Sanders, E.J., Varedi , M . , and French, A.S . (1993): C e l l proliferation i n the gastrularating chick embryo: a study using B r d U incorporation and P C N A localization. Development 118:389-399. Sanes, J.R., Engval l , E. , Butkowski , R., and Hunter , D . D . (1990): Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J. Cel l Biol . 111:1685-1699. Sangren, E.P., Luetteke, N . C . , Palmiter, R.D. , Brinster, R.L. , and Lee, D . L . (1990): Overexpress ion of T G F - a i n transgenic mice: induct ion of epithel ia l hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Ce l l 61:1121-1135. Sassani, R., Bartlett, S.P., Feng, H . , Golden-Sauve, A . , H a q , A . K . , Buetow, K . H . , and Gasser, D . L . (1993): Association between alleles of the transforming growth factor-alpha locus and the occurrence of cleft l ip . A m . J. M e d . Genet. 45:565-569. Sato, H . , and Seiki , M . (1993): Regulatory mechanism of 92-kDa type IV collagenase gene expression which is associated w i t h invasiveness of tumor cells. Oncogene 8:395-405. Sato, H . , Takino, T., Okada, Y. , Cao, J., Shinagawa, A . , Yamamoto, E., and Seiki M . (1994): A Matrix metalloproteinase expressed on the surface of the invasive tumour cells. Nature 370:61-65. Sato., S., Ashraf , M . , M i l l a r d , R .W. , Fujiwara, H . , and Schwartz, A . (1983): Connective tissue changes i n early ischemia of porcine myocardium: and ultrastructural study. J. M o l . Cel l Cardiol . 15:261-275. Schlessinger, J., Schreiber, A . B . , Levi , A . , Lax, I., Libermann, T., Yarden, Y . (1983): Regulation of cell proliferation by epidermal growth factor. C . R . C . Rev. Biochem. 14:93-111. 181 Schnieke, A . , Harbers, K . , and Jaenisch, R. (1983): Embryonic lethal mutant i n mice induced by retrovirus insertion into the alpha 1(1) collagen gene. Nature 304:315-320. Schorle, H . , Meier , P., Buchert, M . , Jaenisch, R., and M i t c h e l l , P.J. (1996): Transcriptional factor A P - 2 essential for cranial closure and craniofacial development. Nature 381:235-238. Schreiber, A . B . , Winkler , M . E . , and Derynck, R. (1986): Transforming growth factor-a is a more potent angiogenic mediator than epidermal growth factor. Science 232:1250-1253. Schuger, L . , O'Shea, S., Rheinheimer, J., Varani , J. (1990): L a m i n i n i n l u n g d e v e l o p m e n t : Effects of a n t i - l a m i n i n a n t i b o d y i n m u r i n e l u n g morphogenesis. Dev Biol 137:26-32. Schuger, L . , Skubitz, A . P . N . , O'Shea, K.S. , Chang, J.F., and Varani , J. (1991): Identification of laminin domains involved i n branching morphogenesis: Effects of anti- laminin monoclonal antibodies on mouse lung development. Dev. Biol . 146:531-541. Seltzer, J.L., Adams, S.A., Grant, G . A . , and Eisen, A . Z . (1981): Purification and properties of a gelatin-specific neutral protease from human skin. J. B io l . Chem. 256:4662-4668. Seltzer, J.L., Eisen, A . Z . , Bauer, E .A. , Morris , N . P . , Glanvil le , R.W., and Burgeson R.E. (1989): Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human sk in . J. B i o l . C h e m . 1264:3822-3826. Senior, R . M . , Gri f f in , G .L . , Fliszar, C.J., Shapiro, S.D., Goldberg, G.I., and Welgus, H . G . (1991): H u m a n 92- and 72-kilodalton type IV collagenases are elastases. J. Biol . Chem. 266:7870-7875. Serbedzija, G . N . , Bronner-Fraser, M . , and Fraser, S.E. (1992): Vi ta l dye analysis of cranial neural crest cell migration i n the mouse embryo. Development 116:297-307. Seth, R., Shum, L . , W u , F., Wuenschnell , C , H a l l , F .L . , S lavkin , H . C . , and Warburton, D . (1993): Role of epidermal growth factor expression i n early mouse embryo l u n g branching morphogenesis i n culture: Antisense oligodeoxynucleotide inhibitory strategy. Dev. Biol . 158:555-559. Sharpe, P . M . , Brunet, C . L . , and Ferguson, M . W . J . (1992): M o d u l a t i o n of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors. Int. J. Dev. Biol . 36:275-282. 1 8 2 Sharpe, P . M . , Brunei , C . L . , Foreman, D . M . , and Ferguson, M . W . J . (1993): Localization of acidic and basic fibroblast growth factors during mouse palate development and their effects on mouse palate mesenchyme cells in vitro. Roux's A r c h . Dev. Biol . 202:132-143. Sheffield, J.B. (1992): Is there a role for metalloproteinases in chick neural retina development? In: Birkedal-Hansen, H . , Werb, Z . , Welgus, H . G . , and V a n Wart, H . E . (eds.) "Matrix Metalloproteinases and Inhibitors." Matr ix . Spec. Suppl. N o l . Gustav Fischer, Stuttgart, pp.391-392. Sheffield, J.B., Krasnopolsky, V . , and Dehlinger, E. (1994): Inhibitor of retinal growth cone activity by specific metalloproteinase inhibitors in vitro. Dev. Dynamics 200:79-88. Sheng, Z . , Pennica, D . , W o o d , W.I. , and Chien, K .R . (1996): Cardiotrophin-1 displays early expression i n the murine heart tube and promoters cardiac myocyte survival . Development 122:419-428. Shima, I., Sasaguri, Y . , K u s u k a w a , J., Nakano , R., Yamana, H . , Fujita, H . , K a k e g a w a , T., and M o r i m a t s u , M . (1993): P r o d u c t i o n of matr ix metalloproteinase 9 (92-kDa gelatinase) by human oesophageal squamous cell carcinoma in response to epidermal growth factor. Br. J. Cancer 67:721-727. Shigeta, H . , Taga, M . , Katoh, A . , and Minaguchi , H . (1993): Ontogenesis and dis tr ibut ion of epidermal growth factor immunoreact ivi ty and b i n d i n g activity i n the mouse fetal and neonatal tissues. Endocrine J. 40:641-647. Shimizu, S., Mal ik , K . , Sejima, H . , K i sh i , J., Hayakawa, T., and K o i w a i , O . (1992): C lon ing and sequencing of the c D N A encoding a mouse tissue inhibitor of metalloproteinases-2. Gene 114:291-292. Shuler, C .F . , Halpern , D . E . , G u o , Y . , and Sank, A . C . (1992): M e d i a l edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo. Dev. Biol . 154:318-330. Shum, L . , Sakakura, Y . , Bringas, P. Jr., Luo , W . , Snead, M . L . , M a y o , M . , Crohin , C , M i l l a r , S., Werb, Z . , Buckley, S., H a l l , F .L. , Warburton, D . , and Slavkin, H . C . (1993): E G F abrogation-induced fusi l l i - form dysmorphogenesis of Meckel 's cartilage during embryonic mouse mandibular morphogenesis in vitro. Development 118:903-917. Sibilia, M . , and Wagner, E.F. (1995): Strain-dependent epithelial defects i n mice lacking the E G F receptor. Science 269:234-237. 1 8 3 Si lver , J., and Hughes , A . F . W . (1973): The role of cel l death d u r i n g morphogenesis of the mammalain eye. J. Morphol . 140:159-170. Sissman, N . (1970): Developmental landmarks i n cardiac morphogenesis: comparative chronology. A m . J. Cardiol . 25:141-148. Smith, M . M . , and H a l l , B.K. (1990): Developmental and evolutionary origins of vertebrate skeletogenic and odontogenic tissues. Biol . Rev. 65:277-373. Smith, J . M . , Sporn, M . B . , Roberts, A . B . , Derynck, R., Winkler , M . E . , and Gregory, H . (1985): H u m a n transforming growth factor-a causes precocious eyelid opening i n newborn mice. Nature 315:515-518. Snead, M . L . , L u o , W . , Oliver , P., Nakamura, M . , Don-Wheeler, G . , Bessem, C , Bell , G.I., Rai l , L.B. , and Slavkin, H . C . (1989): Localization of growth factor precursor i n tooth and lung during embryonic mouse development. Dev. Biol . 134:420-429. Snell, G . D . , Fekete, E., H u m m e l , K . P . , and L a w , L . W . (1940): The relation of mating, ovulation and the estrus smear i n the house mouse to time of day. Anat. Rec. 76:39-54. Soini, Y . , Hurskainen, T., Hoyhtya, M , Oikarinen, A . , and Aut io-Harmainen H . (1994): 72-kDa and 92-kDa type IV collagenase, type IV collagen, and laminin m R N A s i n breast cancer: a study by in situ hybr idizat ion. J. Histochem. Cytochem. 42:945-951. Solursh, M . , Fisher, M . , Meier , S., and Singley, C.T. (1979): The role of the extracellular matrix i n the formation of the sclerotome. J. E m b r y o l . Exp. M o r p h o l . 54:75-98. Solursh, M . , and Jensen, K . L . (1988): Accumulat ion of basement membrane components during the onset of chondrogenesis and myogenesis i n the chick w i n g bud. Development 104:41-49. Song, K . , Wang, Y. , and Sassoon, D . (1992): Expression of Hox-7.1 i n myoblasts inhibits terminal differentiation and induces cell transformation. Nature 360:477-481. Spemann, H . (1901): Uber Correlationen in der Entwickelung des Auges. Verh. Anat. Ges. 15:61-79. Stahlman, M . T . , Or th , D . N . , and Gray, M . E . (1989): Immunocytochemical localization of epidermal growth factor in the developing human respiratory system and i n acute and chronic lung disease in the neonate. Lab. Invest. 60:539-547. 1 8 4 Stark, R.B. (1954): The pathogenesis of harelip and cleft palate. Plast. Reconstruct. Surg. 13:20-32. Steffensen, B., Wal lon , U . M . , and Overal l , C M . (1995): Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. H i g h affinity b inding to native type I collagen but not native type IV collagen. J. Biol . Chem. 270:11555-11566. Stenman, S., and Vaheri , A . (1978): Distribution of a major connective tissue protein, fibronectin, i n normal human tissue. J. Exp. M e d . 147:1054-1064. Stern, C D . (1993): Immunocytochemistry of embryonic material. In: Stern, C D . , and Hol land, P . W . H . (eds.) "Essential Developmental Biology." Oxford University Press, Oxford, pp.193-212. Stetler-Stevenson, W . G . , Brown, P.D. , Onisto, M . , Levy, T. and Liotta, L . A . (1990): Tissue inhibitor of metalloproteinases-2 (TIMP-2) m R N A expression i n tumor cell lines and human tumor tissues. J. Biol . Chem. 265:13933-13938. Stetler-Stevenson, W . G . , Krutzsch, H . C , and Liotta. L . A . (1989): Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J. Biol . Chem. 264:17374-17378. Stetler-Stevenson, W . G . , Liotta, L . A . , and Kleiner, D . E . Jr (1993): Extracellular matr ix 6: Role of matrix metalloproteinases i n tumor invas ion and metastasis. FASEB J. 7:1434-1441. Stoker, M . , Gherardi , E., Perryman, M . , and Gray, J. (1987): Scatter factor is a fibroblast-derived modulator of epithelial cell moti l i ty. Nature , (London) 327:239-242. Streeter, G . L . (1948): Developmental horizons i n human embryos. Description of age groups X V , X V I , XVII, and XVIII, being the third issue of a survey of the Carnegie collection. Contrib. Embryol. Carnegie Inst. 32:133-203. Strongin, A . Y . , Co l l i e r , I., Bannikov, G . , Marmer , B .L . , Grants, G . A . , and Goldberg, G.I. (1995): Mechanism of cell surface activation of 72-kDa type IV collagenase. J. Biol . Chem. 270:5331-5338. Strongin, A . Y . , Marmer, B.L. , Grant, G . A . , and Goldberg, G.I. (1993): Plasma membrane-dependent activation of the 72-kDa type IV collagenase is prevented by complex formation w i t h TIMP-2. J. B io l . Chem. 268:14033-14039. 1 8 5 Sugi, Y . , SasseJ., Barron, M . , and Lough, J. (1995): Developmental expression of fibroblast growth factor receptor-1 (cek-l;flg) during heart development. Dev. Dynamics 202:115-125. Sulik, K . K . , Johnston, M . C , Ambrose, L . J .H. , and Dorgad, D . (1979): Phenytoin (Di lant in) - induced cleft l ip and palate i n A / J mice: a scanning and transmission electron microscopic study. Anat. Rec. 195:243-256. S u z u k i , H . R . , Solursh, M . , and B a l d w i n , H.S. (1995): Relationship between fibronectin expression dur ing gastrulation and heart formation i n the rat embryo. Dev. Dynam. 204:259-277. Swiderski , R.E. , Daniels, K.J . , Jensen, K . L . , and Solursh, M . (1994): Type II collagen is transient expressed dur ing avian cardiac valve morphogenesis. Dev. Dynamics 200:294-304. Sympson, C.J., Talhouk, R.S., Alexander, C M . , C h i n , J.R., Clift , S .M. , Bissell, M.J . , and Werb, Z . (1994): Targeted expression of stromelysin-1 i n mammary gland provides evidence for a role of proteinases i n branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression. J. Cel l . Biol . 125:681-693. Takahashi, S., Barry, A . C , and Factor, S . M . (1990): Col lagen degradation i n ischemic rat hearts. Biochem. J. 265:233-241. Takino, T., Sato, H . , Shinagawa, A . , and Seiki, M . (1995): Identification of the second membrane-type matrix metalloproteinase ( M T - M M P - 2 ) gene from a human placenta c D N A library. M T - M M P s form a unique membrane-type subclass i n the M M P family. J. Biol . Chem. 270:23013-23020. Talhouk, R., C h i n , J.R., Unemor i , E . N . , Werb, Z . , and Bissell , M. J . (1991): Proteinases of the mammary gland: development regulation in vivo and vectorial secretion in culture. Development 112:439-449. Talhouk, R., Werb, Z . , and Bissell, M. J . (1992): Functional interplay between extracellular matrix and extracellular matrix-degrading proteinases i n the mammary gland: a coordinate system for regulating mammary epithelial function. In: Fleming, T.P. (ed.) "Epithelial Organization and Development." Chapman and H a l l , London, pp.329-351. Tarn, J.P. (1985): Physiological effects of transforming growth factor i n the new born mouse. Science 229:673-675. Tamarin, A . (1982): The formation of the primitive choanae and the junction of the primary and secondary palates in the mouse. A m . J. Anat. 165:319-337. 1 8 6 Thesleff, I., and H u r m e r i n t a , K . (1981): Tissue interactions i n tooth development. Differentiation 18:75-88. Thesleff, I., Partanen, A . , and V a i n i o , S. (1991): Epithel ial -mesenchymal interactions i n tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors. J. Craniofac. Genet. Dev. Biol . 11:229-237. Thiery, J.-P., Duband, J.-L., Dufour , S., Savagner, P., and Imhof, B . A . (1989): Roles of fibronectin in embryogenesis. In: Mosher, D.F. (ed.) "Fibronectin." Academic Press, N e w York. pp.181-212. Thompson, M . W . , Mclnnes , R.R., and W i l l a r d , H . F . (1991): Genetics i n medicine. Fifth edition, W.B. Saunders, Toronto. Thomson, B . M . , Atk inson, S.J., McGarr i ty , A . M . , Hembry, R . M . , Reynolds, J.J., and Meikle , M . C . (1989): Type I collagen degradation by mouse calvarial osteoblasts st imulated w i t h 1 ,25-dihydroxyvitamin D-3: evidence for a plaminogen-plasmin-metalloproteinase activation cascade. Biochem. et Biophy. Acta 1014:125-132. Threadgill , D . W . , Dlugosz, A . A . , Hansen, L . A . , Tennenbaum, T., L icht i , U . , Yee, D . , LaMantia , C , Mourton, T., Herrup, K . , Harris , R.C. , Barnard, J .A. , Yuspa, S .H. , Coffey, R.J., and Magnuson, T. (1995): Targeted disruption of mouse E G F receptor: effect of genetic background on mutant phenotype. Science 269:230-234. T i d b a l l , J .G. (1992): Dis t r ibut ion of collagens and f ibronect in i n the subepicardium during avian cardiac development. Anat . Embryol . 185:155-162. T i m p l , R. (1989): Structure and biological activity of basement membrane proteins. Eur. J. Biochem. 180:487-502. T i m p l , R., and Brown, J.C. (1994): The laminins. Matrix Biol . 14:275-281. T i m p l , R., and Dziadek, M . (1986): Structure, development, and molecular pathology of basement membranes. Int. Rev. Exp. Pathol. 29:1-112. T i m p l , R., and Mart in , G.R. (1982): Components of basement membranes. In Furthmayr, H . (ed.) "Immunohistochemistry of the Extracellular Matr ix . " C R C Press, Boca Raton, Florida. V o l II, pp.119-150. Todaro, G.J., Fryl ing, C , and DeLarco, J.E. (1980): Transforming growth factors produced by certain human tumor cells: polypeptides that interact w i t h epidermal growth factor receptors. Proc. Nat l . Acad . Sci. U .S .A. 77:5258-5262. 1 8 7 Trainor, P . A . , and Tarn, P.P. (1995): Cranial paraxial mesoderm and neural crest cells of the mouse embryos: co-distribution i n the craniofacial mesenchyme but distinct segregation i n branchial arches. Development 121:2569-2582. Trasler, D . G . (1968): Pathogenesis of cleft l ip and its relation to embryonic face shape in A / J and C57BL mice. Teratology 1:33-50. Trasler, D . G . , and Leong, S. (1982): Mitotic index i n mouse embryos w i t h 6-aminonicotinamide-induced and inherited cleft l ip . Teratology 25:259-265. Trelstad, R.L. , Hayashi, A . , Hayashi, K . , and Donahoe, P .K. (1982): The epithelial-mesenchymal interface of the male rat M u l l e r i a n duct: Loss of basement membrane integrity and ductal regression. Dev. Biol . 92:27-40. Tuckett, F., and Morr iss -Kay, G . M . (1986): The distribution of fibronectin, l aminin and entactin i n the neurulating rat embryo studied by indirect immunofluorescence. J. Embryol . Exp. Morphol . 94:95-112. Turksen, K . , C h o i , Y . , and Fuchs, E. (1991): Transforming growth factor alpha induces collagen degradation and cell migration i n differentiating human epidermal raft cultures. Cel l Regulation 2:613-625. Tyagi , S.C., Kumar , S.G., Banks, J., and Fortson, W . (1995a): Co-expression of tissue inhibitor and matrix metalloproteinase i n myocardium. J. M o l . C e l l Cardiol . 27:2177-2189. Tyagi , S.C., Kumar , S.G., and Glover, G . (1995b): Induction of tissue inhibitor and matrix metalloproteinase by serum i n human heart-derived fibroblast and endomyocardial endothelial cells. J. Cel l . Biochem. 58:360-371. T y a g i , S .C. , Ratajska, A . , and Weber, K . T . (1993): M y o c a r d i a l matrix metalloproteinase(s): local ization and activation. M o l . C e l l Biochem. 126:49-59. V a n Helden, W . C . H . , Kok-Verspuy, A . , Harff, G . A . , and V a n K a m p , G.J. (1985): Rate-nephrolometric determination of fibronectin i n plasma. C l i n . C h e m . 31:1182-1184. V a n M i e r o p , L . H . S . , A l l e y , R .D. , Kausel , H . W . , and Stranahan, A . (1962): The anatomy and embryology of endocardial cushion defects. J. Thorac. Cardiovasc. Surg. 43:71-83. Vassar, R., and Fuchs, E. (1991): Transgenic mice provide new insights into the role of T G F - a dur ing epidermal development and differentiation. Genes Dev. 5: 714-727. 1 8 8 Vermeij-Keers, C . (1972): Transformation i n the facial region of the human embryo. A d v . Anat. Embryol. Cel l Biol . 46:7-28. Wal lon U . M . , and Overall C M . (1996): The C-terminal hemopexin-like domain of human 72-kDa gelatinase requires calcium for structural integrity and for b i n d i n g to fibronectin and heparin: Analysis of the b i n d i n g properties of recombinant 72-kDa gelatinase C - d o m a i n to extracellular matr ix and basement membrane components. J. Biol . Chem. (submitted). Wang, K . - Y . (1992): Morphometric studies of normal and abnormal pr imary palate formation i n noncleft and cleft l ip strains of mice. P h . D . Thesis. Vancouver: University of British Columbia Library. Wang, K . - Y . , Chen, K . C , Chiang, C P . , and K u o , M . Y . P . (1995): Distribution of p 2 1 r a s dur ing primary palate formation of non-cleft and cleft strains of mice. J. Oral . Pathol. M e d . 24:103-108. Wang, K . - Y . , Juriloff, D . M . , and Diewert, V . M . (1995): Deficient and delayed primary palatal fusion and mesenchymal bridge formation i n cleft lip-liable strains of mice. J. Craniofac. Genet. Dev. Biol . 15:99-116. Warbrick, J . C (1960): The early development of the nasal cavity and upper l ip i n the human embryo. J. Anat. 94:351-362. W a r b u r t o n , M . J . , M i t c h e l l , D . , O r m e r o d , E.J., and R u d l a n d , P. (1982): Distr ibution of myoepithelial cells and basement membrane proteins i n the resting, pregnant, lactating, and i n v o l u t i n g rat m a m m a r y g land . J. Histochem. Cytochem. 30:667-676. Warburton, D . , Seth, R., Shum, L . , Horcher, P .G . , H a l l , F .L. , Werb, Z . , Slavkin, H . C (1992): Epigenetic role of epidermal growth factor expression and signalling i n embryonic mouse lung morphogenesis. Dev. Biol . 149:123-133. W a r d , R.V. , Atkinson, S.J., Reynolds, J.J., and M u r p h y , G . (1994): C e l l surface-mediated activation of progelatinase A : demonstration of the involvement of the C- terminal domain of progelatinase A i n cell surface b i n d i n g and activation of progelatinase A by primary fibroblasts. Biochem. J. 304:263-269. Wartiovaara, J., L e i v i , I., and Vaheri , A . (1979): Expression of the cell surface-associated glycoprotein, fibronectin, i n the early mouse embryo. Dev. Biol . 69:247-257. Wartiovaara, J., N o r d l i n g , S., Lehtonen, E., and Saxen, L . (1974): Transfilter induction of kidney tubules: Correlation w i t h cytoplasmic penetration into Nucleopore filters. J. Embryol. Exp. Morphol . 31:667-682. 1 8 9 Waseem, N . H . , and Lane, D .P . (1990): Monoclonal antibody analysis of the proliferating cell nuclear antigen ( P C N A ) . J. Cel l Sci. 96:121-129. Weber, B.H.F. , Vogt, G . , Pruett, R . C , Strohr, H . , and Felbor, U . (1994): Mutations i n the tissue inhibitor of metalloproteinases-3 (TIMP3) i n patients w i t h Sorsby's fundus dystrophy. Nature Genet. 8:352-356. Wedden, S.E. (1987): Epithelial-mesenchymal interactions i n the development of chick facial pr imordia and the target of retinoid action. Development 99:341-351. Welgus, H . G . , Fliszar, C.J., Seltzer, J.L., Schmid, T . M . , and Jeffrey, J.J. (1990): Differential susceptibility of type X collagen to cleavage by two mammarian interstitial collagenases and 72-kDa type IV collagenase. J. B io l . Chem. 265:13521-13527. Werb, Z . (1990): Expression of E G F and T G F - a genes i n early mammal ian development. Molec. Reprod. Dev. 27:10-15. Werb, Z . , Alexander, C M . , and Adler , R.R. (1992): Expression and function of matrix metalloproteinases i n development. In: Birkedal-Hansen, H . , Werb, Z . , Welgus, H . G . , and V a n Wart, H . E . (eds.). "Matrix Metalloproteinases and Inhibitors." Matrix. Spec. Suppl. N o . l . Gustav Fischer, Stuttgart, pp.337-343. Wessells, N . K . (1977): In: Wessells N . K . (ed.) "Tissue Interactions and Development." W . A . Benjamin, Inc., California, pp.3-5. Wilcox, J . N . , and Derynck, R. (1988): Developmental expression of transforming growth factor-alpha and beta i n mouse fetus. M o l . Ce l l Biol . 8:3415-3422. Wiley , L . M . , Adamson, E.D. , and Tsark, E . C (1995): Epidermal growth factor receptor function i n early mammalian development. BioEssays 17:839-846. Wi lhe lm, S .M. , Collier, I.E., Marmer, B.L. , Eisen, A . Z . , Grant, G . A . , and Goldberg, G.I. (1989): SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J. Biol . Chem. 264:17213-17221. Wi lk inson , D . G . (1993): In situ hybridization. In: Stern, C D . , and H o l l a n d , P . W . H . (eds.) "Essential Developmental Biology." Oxford University Press, Oxford, pp.257-274. W i l s o n , G . D . , Soranson, J .A. , and Lewis , A . A . (1987): C e l l kinetics of mouse k idney us ing bromodeoxyur idine incorporat ion and f low cytometry: preparation and staining. Cel l Tissue Kinet. 20:125-133. 1 9 0 Winkler , M . E . , O'Connor, L . , Winger, M . , and Fendly, B. (1989): Epidermal growth factor and transforming growth factor a b i n d differently to the epidermal growth factor receptor. Biochem. 28:6373-6378. Woessner, J.F. (1991): M a t r i x metalloproteinases and their inhibitors i n connective tissue remodelling. F.A.S.E.B. J. 5:2145-2154. W o n g , S.T., Winchel l , L .F . , M c C u n e , B.K. , Earp, H.S. , Teixido, J., Massague, J., Herman, B., and Lee, D . C . (1989): The TGF-a precursor expressed on the cell surface binds to the E G F receptor on adjacent cells, leading to signal transduction. Cel l 56:495-506. W r e n n , J.T., and Wessells, N . K . (1969): A n ultrastructural study of lens invagination in the mouse. J. Exp. Zool . 171:359-367. X u , Z . , Parker, S.B., and Minkoff , R. (1990): Distribution of type IV collagen, l a m i n i n and fibronectin dur ing maxil lary process formation i n the chick embryo. A m . J. Anat. 187:232-246. Yamada, K . M . (1991): Fibronectin and other cell interactive glycoproteins. In H a y , E .D. (ed.) "Cel l Biology of Extracellular Matrix." Plenum Press, N e w York, pp.111-146. Yurchenco, P .D. (1994): Assembly of laminin and type rv collagen into basement membrane networks. In: Yurchenco, P .D . , Birk, D . E . , and Mecham, R.P. (eds.) "Extracellular Matrix Assembly and Structure." Academic Press, Inc., San Diego, pp.351-388. Zempo, N . , Kenagy, R.D. , A u , Y.P.T. , Bendeck, M . , Clowes, M . M . , Reidy, M . A . , and Clowes, A . W . (1994): Matrix metalloproteinases of vascular wal l cells are increased i n balloon-injured rat carotid artery. J. Vascular Surgery 20:209-217. Zetter, B.R., and Mart in , G.R. (1978): Expression of a high molecular weight cell surface glycoprotein (LETS protein) by preimplantation mouse embryos and teratocarcinoma stem cells. Proc. Nat l . Acad . Sci. U .S .A. 75:2324-2328. Zhang, J., Hagopian-Donaldson, S., Serbedzija, G . , Elsemore, J., Plehn-Dujowich, D . , M c M a h o n , A . P . , Flavell , R . A . , and Wil l iams, T. Neura l tube, skeletal and body wal l defects in mice lacking transcriptional factor A P - 2 . Nature 381:238-241. Zwaan, J., and Hendrix , R .W. (1973): Changes in cell and organ shape during early development of the ocular lens. A m . J. Zool . 13:1039-1049. 191 APPENDIX 1: The primary antibodies and their concentrations and sources primary antibody host concentration source 1. polyclonal rabbit 1:50 anti-mouse type IV collagen 2. polyclonal rabbit 1:25 anti-human type IV collagen 3. polyclonal rabbit 1:100 anti-EHS mouse sarcoma laminin 4. polyclonal rabbit 1:100 anti-human fibronectin 5. polyclonal anti-mouse E G F rabbit 1:50 6. polyclonal sheep 1:50 anti-human recombinant T G F - a 7. monoclonal mouse 1:20 anti-human E G F - R C h e m i c o n , Temecula, C A (AB756) C h e m i c o n , Temecula, C A (AB748) Sigma, St. Louis, M O (L-9393) Dako, Glostrup, Denmark (A 245) Sigma, St. Louis, M O (E-2635) C h e m i c o n , Temecula, C A (AB1412) Sigma, St. Louis, M O (E-3138) 1 9 2 8. monoclonal mouse 1:20 anti-human P C N A (PC 10) 9. monoclonal mouse 1:10 anti-human B r d U 10. polyclonal rabbit 1:200 anti-human M M P - 2 (non-affinity-purified) 11. polyclonal rabbit 1:10 anti-human M M P - 2 (affinity-purified) D i m e n s i o n , Mississauga, O N T , Canada A m e r s h a m , Oakvi l le , O N T , Canada (RPN202) created & provided by U . M . Wal lon and C M . Overal l , U B C created & provided by U . M . Wal lon and C M . Overal l , U B C 1 9 3 A P P E N D I X 2: The secondary antibodies and their concentrations and sources secondary antibody host concentration source 1. anti-rabbit goat TRITC conjugate 1:200 2. anti-sheep FITC conjugate donkey 1:200 3. anti-mouse goat 1:100 biotin conjugate (Vectastain A B C Kit) 4. anti-mouse goat 1:200 FITC conjugate 5. anti-rabbit sheep 1:200 FITC conjugate 6. anti-rabbit goat 1:100 biotin conjugate Sigma, St. Louis, M O (T-6778) Sigma, St. Louis, M O (F-7634) Vector, B u r l i n g a m e , C A (PK-4002) Sigma, St. Louis, M O (F-9006) Sigma, St. louis, M O (F-7512) Sigma, St. Louis, M O (B-8895) 1 9 4 APPENDIX 3: Positive control tissues Antibodies Tissues References anti-EGF anti-TGF-a anti-MMP-2 anti-laminin anti-fib ronectin anti-type IV collagen anti-BrdU anti-PCNA embryonic mouse lung human oral squamous cell carcinoma embryonic mouse mandibular bone basement membranes mesenchymal tissue, basement membranes basement membranes mouse intestine mouse intestine h u m a n tonsil Warburton et al, 1992 Christensen et al, 1993 Reponen et al, 1992 T i m p l , 1989 T i m p l , 1989 T i m p l , 1989 Dolbeare, 1995 Dolbeare, 1995 H a l l et al, 1990 195 APPENDIX 4: Samples of mouse embryos used for immunohistochemistry. Laminin (Primary Palate) 7-11 TS 12-17 TS 18-20 TS C D 1 10.21.10 (9 TS) 4 slides, 4 sections/slide =16 sections C D 1 8.7 (13 TS) 11 slides, 4 sections/slide = 44 sections C D 1 B-4.8 (19 TS) 3 slides, 4 sections/slide =12 sections C D 1 10.21.2 (9 TS) 2 slides, 4 sections/slide =8 sections C D 1 6.1 (13 TS) 7 slides, 4 sections /s l ide =28 sections C D 1 B-4.10 (20 TS) 1 slide, 4 sections/slide =4 sections C D 1 1.3 (11 TS) 1 slide, 30 sections/slide =30 sections C D 1 10.21.6 (14 TS) 9 slides, 4 sections/slide =36 sections C L F R 7.8s (19 TS) 1 slide, 4 sections/slide =4 sections C L F R 1.7 (11 TS) 2 slides, 4 sections/slide = 8 sections C D 1 1.10 (15 TS) 8 slides, 4 sections/slide =32 sections C L F R 7.4s (19 TS) 2 slides, 4 sections/slide =8 sections C D 1 1.4 (15 TS) 1 slide, 30 sections/slide =30 sections C D 1 6.6 (17 TS) 14 slides, 4 sections/slide =56 sections C D 1 B-1.5 (16 TS) 2 slides, 4 sections/slide =8 sections C L F R 10.22 (13 TS) 11 slides, 4 sections/slide =44 sections C L F R 4.8 (14 TS) 1 slide, 30 sections/slide =30 sections C L F R 4.7 (14 TS) 1 slide, 30 sections/slide =30 sections Total 3 C D 1 embryos (50 sections) 1 C L F R embryo (8 sections) Total 7 CD1 embryos (234 sections) 3 C L F R embryos (104 sections) Total 2 C D 1 embryos (20 sections) 2 C L F R embryos (12 sections) 196 Type IV Collagen (Primary Palate) 7-11 TS 12-17 TS 18-20 TS C D 1 10.21 (9 TS) 6 slides, 4 sections/slide =24 sections CD1 3.2 (12 TS) 6 slides, 4 sections/slide =24 sections C D 1 B-4.8 (19 TS) 2 slides, 4 sections/slide =8 sections C D 1 10.18 (11 TS) 8 slides, 4 sections/slide =32 sections C D 1 10.8.7 (13 TS) 7 slides, 4 sections/slide =28 sections C D 1 7.1 (19 TS) 10 slides, 4 sections/slide =40 sections C D 1 10.19.9 (9 TS) 1 slide, 3 sections/slide =3 sections CD110.23.6 (13 TS) 1 0 slides, 4 sections/slides =40 sections C D 1 4.3 (20 TS) 1 slide, 4 sections/slide =4 sections C D 1 10.21 (14 TS) 12 slides, 4 sections/slide =48 sections C D 1 2.6 (20 TS) 2 slides, 4 sections/slide =8 sections C D 1 3.1 (17 TS) 1 slide, 4 sections/slide =4 sections C L F R 2.1 (19 TS) 2 slides, 4 sections/slide =8 sections C L F R 10.22 (13 TS) 1 1 slides, 4 sections/slide =44 sections Total 3 C D 1 embryos (59 sections) Total 5 CD1 embryos (144 sections) 1 C L F R embryo (44 sections) Total 4 C D 1 embryos (60 sections) 1 C L F R embryo (8 sections) Fibronectin (Primary Palate) 7-11 TS 12-17 TS 18-20 TS C D 1 D-4.7 (7 TS) 1 slide, 20 sections/slide =20 sections C D 1 B-1.8 (15 TS) 10 slides, 4 sections/slide =40 sections C D 1 2.7 (18 TS) 2 slides, 4 sections/slide =8 sections C D 1 10.21.2 (9 TS) 13 slides, 4 sections/slide =52 sections C D 1 1.5 (15 TS) 1 slide, 30 sections/slide =30 sections C D 1 B-4.8 (19 TS) 8 slides, 4 sections/slide =32 sections C D 1 1.11 (11 TS) 1 slide, 30 sections/slide =30 sections CD1 18.1 (17 TS) 2 slides, 5 sections/slide =10 sections C D 1 18.12 (19 TS) 1 slide, 4 sections/slide =4 sections C D 1 10.22 (15 TS) 15 slides, 4 sections/slide =60 sections C D 1 18.9 (19 TS) 1 slide, 4 sections/slide =4sections Total 3 C D 1 embryos (102 sections) Total 4 CD1 embryos (140 sections) Total 4 CD1 embryos (48 sections) 197 Epidermal Growth Factor (EGF) <7TS 7-11 TS 12-17 TS 18-20 TS dayl2 C D l 25.8 (3 TS) 2 slides 6 sections/slide =12 sections C D l A-9.5 (9 TS) 2 slides 30 sections/slide =60 sections C D l 3.2 (12 TS) 1 slide 30 sections/slide = 30 sections C D l 1.8 (18 TS) 1 slide 4 sections/slide =4 sections C D l 19.4 1 slide 20 sections/slide =20 sections C D l 25.5 (4 TS) 1 slides 5 sections/slide =5 sections C D l 10.21.6 (10 TS) 2 slides 4 sections/slide =8 sections C D l D-5.5 (15 TS) 6 slides 4 sections/slide =24 sections C D l 18.8(18 TS) 1 slide 4 sections/slide =4 sections C D l 19.9 1 slide 9 sections/slide =9 sections C D l 25.6 (5 TS) 2 slide 5 sections/slide =10 sections C D l 10.19.7 (11 TS) 3 slides 4 sections/slide =12 sections C D l 18.1 (17 TS) 3 slides 5 sections/slide =15 sections C D l 18.9 (19 TS) 1 slide 4 sections/slide =4 sections C D l 28.6 1 slide 10 sections/slide =10 sections C D l 1.5 (17 TS) 3 slides 4 sections/slide =12 sections C D l 18.12(19 TS) 1 slide 3 sections /s l ide =3 sections C D l 19.2 1 slide 10 sections/slide =11 sections C L F R A-1.3 (14 TS) 7 slides 4 sections/slide =28 sections C L F R A - l . l (15 TS) 9 slides 4 sections/slide =36 sections Total 3 C D l embryos (32 sections) Total 3 C D l embryos (38 sections) Total 4 C D l embryos (81 sections) 2 C L F R embryos (64 sections) Total 4 C D l embryos (15 sections) Total 4 C D l embryos (50 sections) 198 Transforming Growth Factor-alpha (TGF-alpha) <7TS 7-11 TS 12-17 TS 18-20 TS dayl2 C D l 25.8 (3 TS) 2 slides 6 sections/slide =12 sections C D l D-7.1 (8 TS) 2 slides 30 sections/slide =60 sections C D l D-5.6 (13 TS) 2 slides 30 sections/slide =60 sections C D l 1.8 (18 TS) 3 slides 4 sections/slide =12 sections C D l 19.4 1 slide 20 sections/slide =20 sections C D l 25.5 (4 TS) 1 slide 5 sections/slide =5 sections C D l B-1.9 (9 TS) 5 slides 4 sections/slide =20 sections C D l 10.23.8 (15 TS) 2 slides 4 sections/slide =8 sections C D l 1.11 (18 TS) 1 slide 4 sections/slide =4 sections C D l 19.9 1 slide 9 sections/slid e=9 sections C D l 25.6 (5 TS) 1 slide 5 sections/slide =5 sections C D l D-7.4 (10 TS) 1 slide 30 sections/slide =30 sections C D l 10.18.1 (15 TS) 1 slide 4 sections/slide =4 sections C D l 11.18.5 (18 TS) 2 slides 4 sections/slide =8 sections C D l 19.2 1 slide 13 sections/slide =13 sections C D l 18.1 (17 TS) 1 slide 5 sections/slide =5 sections C D l 11.11 (19 TS) 2 slides 4 sections/slide =8 sections C L F R 4.1 (12 TS) 5 slides 4 sections/slide =20 sections C L F R 6.7 (18 TS) 1 slide 4 sections/slide =4 sections C L F R 10.22.8 (14 TS) 4 slides 4 sections/slide =16 sections C L F R 2.5 (18 TS) 2 slides 30 sections/slide =60 sections C L F R 3.7 (15 TS) 5 slides 4 sections/slide =20 sections Total 3 C D l embryos (22 sections) Total 3 C D l embryos (110 sections) Total 4 C D l embryos (77 sections) 3 C L F R embryos (56 sections) Total 4 C D l embryos (32 sections) 2 C L F R embryos (64 sections) Total 3 C D l embryos (42 sections) 199 Epidermal Growth Factor Receptor (EGF-R) <7TS | 7-11 TS | 12-17 TS | 18-20 TS | dayl2 C D l 25.8 (3 TS) C D l 10.21.5 (8 TS) C D l D-7.3 (12 TS) C D l 11.18.5 (18 TS) C D l 19.4 2 slides 1 slide 2 slides 2 slides 1 slide 6 sections/slide 4 sections/slide 4 sections/slide 4 sections/slide 6 sections/slide =12 sections =4 sections =8 sections =8 sections =6 sections C D l 25.5 (4 TS) 1 slide 5 sections/slide =5 sections C D l B-1.9 (9 TS) 1 slide 4 section/slide =4 sections C D l 10.19.5 (15 TS) 2 slides 4 sections/slide =8 sections C D l 1.8 (18 TS) 1 slide 4 sections/slide =4 sections C D l 19.9 1 slide 5 sections/slide =5 sections C D l 25.6 (5 TS) 1 slide 5 sections/slide =5 sections C D l A-8.5 (11 TS) 1 slide 30 sections/slide =30 sections C D l 10.21.1 (15 TS) 2 slides 4 sections/slide =8 sections C D l 18.8 (18 TS) 1 slide 3 sections/slide =3 sections C D l 19.2 1 slide 6 sections/slide =6 sections C D l 10.21.5 (15 TS) 2 slides 4 sections/slide =8 sections C D l 18.9 (19 TS) 1 slide 4 sections/slide =4 sections C D l 10.23.8 (15 TS) 3 slides 4 sections/slide =12 sections C D l 18.12 (19 TS) 1 slide 4 sections/slide =4 sections C D l 18.1 (17 TS) 1 slide 5 sections/slide =5 sections C D l 1.6 (17 TS) 3 slides4 sections/slide =12 sections C D l 1.5 (17 TS) 5 slides 4 sections/slide =20 sections C L F R 4.1 (12 TS) 5 slides 4 sections/slide =20 sections Total 3 C D l e m b r y o (22 sections) Total 3 C D l embryos (38 sections) Total 5 C D l embryos (52 sections) 1 C L F R embryo (20 sections) Total 5 C D l embryos (23 sections) Total 3 C D l embryos (17 sections) 200 5-Bromodeoxyuridine (BrdU) Incorporation 7-11 TS 12-17 TS 18-22 TS C D l ul8.8 (10 TS) 2 slides (30 sections /slide) =60 sections C D l ul9.4 (12 TS) 1 slide (30 sections/slide) =30 sections C D l 5.7 (20 TS) 2 slides (30 sections/slide) =60 sections C D l u20.1 (10 TS) 2 slides (30 sections/slides) =60 sections C D l u4.3 (13 TS) 1 slide (30 sections/slide) =30 sections C D l u5.10 (22 TS) 1 slide (30 sections/slide) =30 sections C D l 4.8 (10 TS) 1 slide (30 sections/slide) =30 sections C D l u3.5 (14 TS) 1 slide (30 sections/slide) =30 sections C D l 6.3 (22 TS) 1 slide(30 sections/slide) =30 sections C D l ul9.10 (11 TS) 1 slide (30 sections/slide) =30 sections C D l 1.6 (15 TS) 1 slide (30 sections/slide) =30 sections Balb/c u7.8 (18 TS) 1 slide (30 sections/slide) =30 sections Balb/c 6.5 (9 TS) 1 slide (30 sections/slide) =30 sections Balb/c u5.7 (14 TS) 1 slide (30 sections/slide) =30 sections Balb/c u l l . 3 (20 TS) 1 slide (30 sections/slide) =30 sections Balb/c u l . 2 (10 TS) 1 slide (30 sections/slide) =30 sections Balb/c 1.6 (15 TS) 1 slide (30 sections/slide) =30 sections Balb/c u2.4 (16 TS) 1 slide (30 sections/slide) =30 sections T o t a l 4 C D l embryos (180 sections) 2 Balb/c embryos (60 sections) T o t a l 4 C D l embryos (120 sections) 3 Balb/c embryos (90 sections) T o t a l 3 C D l embryos (90 sections) 2 Balb/c embryos (60 sections) 201 Proliferating Cell Nuclear Antigen (PCNA) 7-11 TS 12-17 TS 18-22 TS C D l 1.1 (10 TS) 2 slides (30 sections/slide) =60 sections C D l B-1.7 (12 TS) 2 slides (4 sections/slide) =8 sections C D l 4.2 (18 TS) 2 slides (30 sections/slide) =60 sections C D l 1.11 (11 TS) 2 slides (30 sections /sl ide) =60 sections C D l B-1.6 (13 TS) 2 slides (4 sections/slide) = 8 sections C D l u7.7 (18 TS) 1 slide (30 sections/slide) =30 sections C D l ul7.4 (15 TS) 1 slide (30 sections/slide) =30 sections C D l B-4.8 (19 TS) 1 slide (4 sections/slide) =4 sections C D l 14 (16 TS) 1 slide (30 sections/slide) =30 sections C D l u5.2 (20 TS) 1 slide (30 sections/slide) =30 sections C D l B-1.5 (16 TS) 3 slides (4 sections/slide) =12 sections C D l u l l . 7 (21 TS) 2 slides (30 sections/slide) = 60 sections C D l 6.9 (17 TS) 1 slide (4 sections/slide) =4 sections Total 2 C D l embryos (120 sections) Total 6 C D l embryos (92 sections) Total 7 C D l embryos (188 sections) 202 72-kDa Gelatinase (Primary Palate and Mandible) <7 TS 7-11 TS 12-17 TS 18-20 TS dayl2 C D l 5 (day 9) 1 slide 4 sections/slide =4 sections C D l D-7.2 (7 TS) 2 slides 30 sections/slide =60 sections C D l D-5.10 (12 TS) 2 slides 30 sections/slide =60 sections C D l 1.8 (18 TS) 2 slides 4 sections/slide =8 sections C D l 28.6 2 slides 10 sections/slide =20 sections C D l 5.2.95 (day 9) 1 slide 6 sections/slide =6 sections C D l B-1.9 (9 TS) 5 slides 4 sections/slide =20 sections C D l 10.19.3 (13 TS) 2 slides 4 sections/slide =8 sections C D l 11.11 (19 TS) 2 slides 4 sections/slide =8 sections C D l 19.4 1 slide 5 sections/slide =5 sections C D l 25.3 (4 TS) 1 slide 6 sections/slide =6 sections C D l 10.21.2 (9TS) 8 slides 4 sections/slide =32 sections C D l D-5.7 (13 TS) 2 slides 30 sections/slide =60 sections C D l 1.1 (20 TS) 2 slides 30 sections/slide =60 sections C D l 19.9 1 slide 9 sections/slide =9 sections C D l 25.5(4 TS) 2 slides6 sections/slide 12 sections C D l 10.21.10 (9 TS) 4 slides 4 sections/slide =16 sections C D l 10.18.3 (13 TS) 1 slide 4 sections/slide =4 sections C D l 3.2 (20 TS) 1 slide 30 sections/slide =30 sections C L F R 6.2s 3 slides 5 sections/slide =15 sections C D l 25.6 (5 TS) 2 slides 5 sections/slide =10 sections C L F R 14 3 slide 2 sections/slide =6 sections C D l 18.2 (13 TS) 1 slide 4 sections/slide =4 section C D l 18.12 (19 TS) 1 slide 5 sections/slide =5 sections C L F R 6.3 s 1 slide 10 sections/slide =10 sections C L F R 1 (day 9) 2 slides 10 sections/slide =20 sections C D l 10.23.3 (15 TS) 2 slides 4 sections/slide =8 sections C L F R 1.4 (18 TS) 3 slides 3 sections/ slide =9 sections C L F R 14 3 slides 4 sections/ slide =12 sections C D l 18.11 4 slides 5 sections/slide =20 sections C L F R 10.22.8 (14 TS) 3 slides 4 sections/slide =12 sections C L F R 3.7 (15 TS) 5 slides 4 sections/slide =20 sections C L F R 10.21.3 (16 TS) 2 slides 4 sections /s l ide =8 sections Total 5 C D l embryos (38 sections) 1 C L F R embryo (20 sections) Total 4 C D l embryos (138 sections) 1 C L F R embryo (6 sections) Total 7 C D l embryos (148 sections) 2 C L F R embryos (36 sections) Total 5 C D l embryos (111 sections) 1 C L F R embryo (9 sections) Total 3 C D l embryo (34 sections) 3 C L F R embryos 37 sections) 203 72-kDa Gelatinase (Eye) daylO d a y l l dayl2 dayl3 dayl4 C D l D-7.2 C D l D-5.6 C D l 28.6 C D l 22.3 C D l 16.8s 1 slide 1 slide 2 slides 2 slides 3 slides 30 sections/slide 30 sections/slide 5 sections /s l ide 5 sections/slide 6 sections/slides =30 sections =30 sections =10 sections =10 sections =18 sections C D l D-7.5 C D l 10.23 C D l 19.4 C D l 22.2 C D l 16.7s 1 slide 2 slides 2 slide 2 slides 3 slides 30 sections/slide 4 sections/slide 20 sections/slide 5 sections/slide 15 sections/slide =30 sections =8 sections =40 sections =10 sections =45 sections C D l A-9.5 C D l 4.2 C D l 19.2 A / J 1 3 C D l 14.8s 1 slide 1 slide 1 slide 1 slide 1 slide 30 sections/slide 25 sections/slide 17 sections/slide 4 sections/slide 4 sections/slide =30 sections =25 sections =17 sections =4 sections =4 sections C D l 25.8 C L F R 6.3s A / J c C L F R 12.7s 2 slides 2 slides 2 slides 1 slide 6 sections/slide 10 sections/slide 3 sections/slide 4 sections/slide =12 sections =20 sections =6 sections =4 sections Total Total Total Total Total 4 C D l embryos (102 sections) 3 C D l embryos (63 sections) 3 C D l embryos (67 sections) 1 C L F R embryo (20 sections) 2 C D l embryos (20 sections) 2 A / J embryos (10 sections) 3 C D l embryos (67 sections) 1 C L F R embryo (4 sections) Co-distribution of 72-kDa Gelatinase, EGF and TGF-alpha (Primary Palate) 7-11 TS 12-17 TS 18-20 TS C D l 10.19.9 (9 TS) 1 slide, 3 sections/slide =3 sections C D l A-8.6 (12 TS) 1 slide, 3 sections/slide =3 sections C D l 18.8 (1 TS) 1 slide, 3 sections/slide =3 sections C D l 10.19.6 (10 TS) 1 slides, 3 sections/slide =3 sections C D l 10.8.8 (14 TS) 1 slide, 3 sections/slide =3 sections C D l 18.9 (19 TS) 1 slide, 3 sections/slide =3 sections C D l 10.19.10 (9 TS) 1 slide, 3 sections/slide =3 sections C D l A-8.7 (15 TS) 1 slide, 3 sections/slides =3 sections C D l 18.12 (19 TS) 1 slide, 3 sections/slide =3 sections Total 3 C D l embryos (9 sections) Total 3 C D l embryos (9 sections) Total 3 C D l embryos (9 sections) 204 72-kDa Gelatinase (Heart) day9 daylO d a y l l dayl2 day l3 C D l 5 1 slide 4 sections/slide =4 sections C D l 25.3 1 slide 6 sections/slide =6 sections C D l 18.1 4 slides 5 sections/slide =20 sections C D l 28.6 2 slides 10 sections/slide =20 sections C D l 22.3 1 slide 4 sections/slide =4 sections C D l 5.2.95 1 slide 6 sections/slide =6 sections C D l 25.5 2 slides 6 sections/slide =12 sections C D l 18.12 2 slides 5 sections/slide =10 sections C D l 19.4 2 slides 20 sections/slide =40 sections C D l 22.5 1 slide 4 sections/slide =4 sections C D l 5.3.95 1 slide 6 sections/slide =6 sections C D l 25.6 2 slides 5 sections/slide =10 sections C L F R 1.1 1 slide 10 sections/slide =10 sections C D l 19.2 1 slide 17 sections/slide =17 sections C D l 22.7 1 slide 4 sections/slide = 4 sections C L F R 1 2 slides lOsections/ slide =20 sections C D l 25.4 2 slides 10 sections/slide =10 sections C L F R 4.3 1 slide 5 sections/slide =5 sections C L F R 6.2s 3 slides 5 sections/slide =15 sections C D l 22.2 1 slide 4 sections/slide =4 sections C L F R 13.2 3 slides 2 sections/slide =6 sections C L F R 7.4s 1 slide 5 sections/slide =5 sections C L F R 6.3s 1 slide 10 sections/slide = 10 sections A / W 5.2 2 slides 10 sections/slide =20 sections C L F R 17.5 3 slides 4 sections/slide =12 sections T o t a l 3 C D l embryos (16 sections) 1 C L F R embryo (20 sections) T o t a l 4 C D l embryos (38 sections) 1 C L F R embryo (6 sections) T o t a l 2 C D l embryos (30 sections) 3 C L F R embryos (20 sections) 1 A / W embryo (20 sections) T o t a l 3 C D l embryos (77 sections) 3 C L F R embryos (37 sections) T o t a l 4 C D l embryos (16 sections) 205 Laminin (Heart) day9 daylO d a y l l dayl2 dayl3 C D l 5.2.95 C D l 25.3 C D l 18.13 C D l 19.9 C D l 22.3 1 slide 1 slide 1 slides 1 slide 1 slide 6 sections/slide 6 sections/slide 5 sections/slide 4 sections/slide 4 sections/slide =6 sections =6 sections =5 sections =4 sections =4 sections C D l 5.3.95 C D l 25.4 C D l 18.9 C D l 19.4 C D l 22.5 1 slide 1 slides 1 slide 1 slides 1 slide 6 sections/slide 6 sections/slide 4 sections/slide 20 sections/slide 4 sections/slide =6 sections =6 sections =10 sections =20 sections =4 sections C L F R 1 C D l 25.10 A / W 5.2 C D l 19.2 C D l 22.2 1 slides 1 slides 1 slides 1 slide 1 slide 10 sections/slide 5 sections/slide 10 sections/slid 17 sections/slide 4 sections/slide =10 sections =5 sections e=10 sections =17 sections =4 sections Total Total Total Total Total 2 C D l embryos (12 sections) 1 C L F R embryo (10 sections) 3 C D l embryos (17 sections) 2 C D l embryos (15 sections) 1 A / W embryo (10 sections) 3 C D l embryos (41 sections) 3 C D l embryos (12 sections) Type IV Collagen (Heart) day9 daylO d a y l l dayl2 dayl3 C D l 5.2.95 C D l 25.3 C D l 18.13 C D l 19.7 C D l 22.7 1 slide 1 slide 1 slide 1 slide 1 slide 6 sections/slide 6 sections/slide 5 sections/slide 4 sections/slide 4 sections/slide =6 sections =6 sections =5 sections =4 sections =4 sections C D l 5.3.95 C D l 25.5 C D l 18.9 C D l 19.2 C D l 22.5 1 slide 1 slide 1 slide 1 slide 1 slide 6 sections/slide 6 sections/slide 4 sections /s l ide 20 sections/slide 4 sections/slide =6 sections =6 sections =10 sections =20 sections =4 sections C L F R 1 C D l 25.7 C D l 18.12 C L F R 6.3s C D l 22.2 1 slide 1 slide 1 slide 1 slide 1 slide 10 sections/slide 5 sections/slide 5 sections/slide 4 sections/slide 4 sections/slide =10 sections =5 sections =5 sections =4 sections =4 sections Total Total Total Total Total 2 C D l embryos (12 sections) 1 C L F R embryo (10 sections) 3 C D l embryos (17 sections) 3 C D l embryos (15 sections) 2 C D l embryos (24 sections) 1 C L F R embryo (4 sections) 3 C D l embryos (12 sections) 206 Fibronectin (Heart) day9 daylO d a y l l dayl2 dayl3 C D l 5.2.95 C D l 25.6 C D l 18.13 C D l 19.7 C D l 22.7 1 slide 1 slide 1 slide 1 slide 1 slide 6 sections/slide 6 sections/slide 5 sections/slide 4 sections/slide 4 sections/slide =6 sections =6 sections =5 sections =4 sections =4 sections C D l 5.3.95 C D l 25.10 C D l 18.9 C D l 19.2 C D l 22.5 1 slide 1 slides 1 slide 1 slides 1 slide 6 sections/slide 6 sections/slide 4 sections/slide 20 sections/slide 4 sections/slide =6 sections =6 sections =10 sections =20 sections =4 sections C L F R 1 C D l 25.7 C D l 18.12 C D l 19.9 C D l 22.2 1 slideslO 1 slides 1 slides 1 slide 1 slide sections/slide 5 sections/slide 5 sections/slide 4 sections/slide 4 sections/slide =10 sections =5 sections =5 sections =4 sections =4 sections Total Total Total Total Total 2 C D l embryos (12 sections) 1 C L F R embryo (10 sections) 3 C D l embryos (17 sections) 3 C D l embryos (15 sections) 3 C D l embryos (28 sections) 3 C D l embryos (12 sections) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0087734/manifest

Comment

Related Items