Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Epithelium is required for maintaining FGFR-2 expression levels in facial Mesenchyme of the developing… Matovinovic, Elizabeth 1997

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

Item Metadata

Download

Media
831-ubc_1997-0569.pdf [ 5.17MB ]
Metadata
JSON: 831-1.0088270.json
JSON-LD: 831-1.0088270-ld.json
RDF/XML (Pretty): 831-1.0088270-rdf.xml
RDF/JSON: 831-1.0088270-rdf.json
Turtle: 831-1.0088270-turtle.txt
N-Triples: 831-1.0088270-rdf-ntriples.txt
Original Record: 831-1.0088270-source.json
Full Text
831-1.0088270-fulltext.txt
Citation
831-1.0088270.ris

Full Text

E P I T H E L I U M IS R E Q U I R E D F O R M A I N T A I N I N G F G F R - 2 E X P R E S S I O N  LEVELS  IN F A C I A L M E S E N C H Y M E O F T H E D E V E L O P I N G C H I C K E M B R Y O . by ELIZABETH MATOVINOVIC  B . A . , The University of Manitoba,  1994  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Health Sciences)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A September  1997  © Elizabeth Mato vino vie,  1997  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  jJ f  The University of British Columbia Vancouver, Canada Da«.  Oct  ;i  i<m  ABSTRACT  In the developing chick embryo, F G F R - 2 expression patterns correlate with outgrowth of facial prominences: frontonasal mass prominences which form the prenasal cartilage and upper beak express high levels of F G F R - 2 receptor while maxillary prominences which form the flattened corners of the beak and palatal shelves express low F G F R - 2 transcript levels.  Facial epithelium is an  abundant source of F G F s and is required to support outgrowth of mesenchymal tissue including cartilage rod formation.  Since F G F R - 2 is highly expressed in  regions of facial outgrowth and epithelium is required for outgrowth of facial prominences, epithelium could be required to maintain F G F R - 2 transcripts in facial mesenchyme.  To test this hypothesis, we removed epithelium to inhibit  outgrowth of regions of the embryonic face, grafted frontonasal mass and maxillary prominences into a host limb bud and then examined changes in F G F R - 2 expression using in situ hybridization. We also hybridized adjacent sections with collagen II probe to identify regions undergoing chondrogenesis. Our results indicate that removal of epithelium from frontonasal mass lead to a decrease in F G F R - 2 and collagen II expression 24 hours after grafting to host and that neither F G F R - 2 nor collagen II expression increased to expected levels at 48 hours. These results suggest that there are signals in the epithelium required for increasing F G F R - 2 and collagen II gene transcription and the expression of these genes are linked to outgrowth of facial prominences. We localized F G F 8  transcripts in the developing chick face to determine whether F G F 8 is a putative epithelial signal required for F G F R - 2 expression. Our results indicate that F G F 8 ectodermal expression does not overlap F G F R - 2 expression i n mesenchymal tissue and therefore is not a signal required for the expression of this receptor.  iv  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  v i  List of Figures  Acknowledgement  Introduction  v u  V 1 U  1  Chapter One  The embryonic chick face  2  Epithelial-mesenchymal interactions  4  Fibroblast growth factors (FGFs)  5  Fibroblast growth factor receptors (FGFRs)  A i m of study  Chapter Two Materials and Methods  ChapterThree Results  ChapterFour Discussion....  Summary  References  1  vi  LIST O F T A B L E S Table 1  Expression scores of F G F R - 2 and Collagen II in individual frontonasal mass specimens  30  LIST O F FIGURES  Figure 1. Diagrammatic representation of F G F 4 and 8 and F G F R expression at stage 28 Figure 2. Grafting method  10 13  Figure 3. Dark field and bright field views of stage 24 and stage 28 heads  21  Figure 4. Expression of F G F R - 2 and collagen II m R N A in adjacent sections of grafts of frontonasal mass prominences Figure 5. Expression in frontonasal mass grafts  23 25  Figure 6. Expression of F G F R - 2 and collagen II m R N A in adjacent sections of grafts of maxillary prominences Figure 7. Expression in maxillary grafts  27 29  Figure 8. Dark field views of stage 20, 24, and 28 heads hybridized to antisense F G F 8 riboprobe  32  ACKNOWLEDGEMENTS  My greatest thanks goes to Dr. Joy Richman who g a v e me the opportunity to work on this project and o p e n e d the door for me to pursue research in the biological sciences. I also thank her for providing figuresl and 2 of this thesis. I thank Dr. Tim O'Connor for serving on my committee, providing guidance whenever I asked for it a n d for introducing me to various aspects of developmental biology in Anatomy 504. I also thank Dr. Virginia Diewert for serving on my committee. I thank Dr. Shen Hua for sharing her wealth of knowledge about molecular biology and her overall research experience with me. I also thank her for a wonderful friendship. I also thank Sara, Angus, Mehrnaz, A n a n d a , Kirsi, Maryam, Marni a n d Rob, a n d Chris a n d Deb for their friendship during my time in this department. Additional thanks to Rob for being my dentist. I thank Sandra Babich for her technical expertise. I thank Dr. Hannu Larjava for his helpful discussions on integrins a n d matrix proteins. I thank Dr. Chris Overall for making his journals accessible to everybody in the department including me. I thank Ingrid Ellis, Linda Gerow a n d of course Cindy Goundrey for their help with anything and everything. I thank Aida el-Husseini for giving me love and delicious suppers. I thank m a m a and tata, Alaa el-Husseini, Aref el-Husseini, Dr. Steven Vincent, Dr. Don Brunette, Dr. Stan Mendyk, Dr. Claudio Stern, Dr. Roger Tsien, and Dr. Stephen Hawking who have inspired me in different ways during this degree.  INTRODUCTION Factors involved in the development o f an organism increase with time almost exponentially, hence, developmental biologists tend to focus on a specific aspect of development which can range from studying molecular involved  in specifying  cell  fate  to  how different  tissues  determinants  may regulate  morphogenesis. M y research seeks to link gene expression with morphogenesis by addressing how tissue interactions regulate gene transcription. Generally, most vertebrates have a similar design where a midline splits the face in two symmetrical halves which results in the formation of two ears, two eyes, one nose and one mouth.  This design is so similar among vertebrates that it is very  difficult to distinguish an early chicken embryo from a mouse embryo from a human embryo. Hence, studying face development in one organism has relevance to understanding face development in many organisms. Studying  face  development  also  has clinical relevance  for humans.  Craniofacial birth defects like cleft lip/palate affects 1:1000 individuals (Ross and Johnson, 1972) and craniosynostosis is inherited in 1:2500 individuals (Cohen, 1986).  Craniofacial research  can be applied to future  treating  of facial  abnormalities like cleft lip, hemifacial microsomia and craniosynostosis  (Winter  and Baraitser, 1994). The genetic cause of some of these craniofacial defects has been determined. The main feature of Crouzon's syndrome is craniosynostosis and the genetic cause is mutations in genes that code for fibroblast growth factor receptors [FGFRs] (Yamaguchi and Rossant, 1995). This project aims to elucidate  fibroblast growth factor [FGF] and F G F R functions in craniofacial development. Hence, by identifying proteins involved in normal face development we can apply this knowledge to understanding human abnormalities. I have chosen to carry out my studies on the avian embryo because manipulations  can be carried  out in ovo, embryonic  stages are described  (Hamburger and Hamilton, 1951), easily monitored and the rate o f development can be controlled by altering ambient temperature.  It is also possible to study long  term effects of early manipulations in the avian embryo and these effects can be related to human development.  THE EMBRYONIC CHICK FACE In vertebrates, the majority of the embryonic face and head is comprised of tissue originating from cranial neural crest cells (Hall and Horstadius, 1988). The vertebrate neural crest is a population of cells which arise from the dorsal edges of the neural folds as they close to form the neural tube.  Neural crest cells migrate  extensively along well characterized pathways comprised of extracellular  matrix  molecules like fibronectin, tenascin and proteoglycans which guide these cells (Bronner-Fraser,  1996)  to populate  target sites and form  sensory  neurons,  peripheral nervous system neurons, pigment cells, catecholamine secreting cells, and craniofacial  skeletal  tissue  (Le Douarin,  1982).  Using  radioactive and  lipophyllic dye labeling techniques, neural crest cells were shown to reach chick facial primordia at stage 14 when the face is first recognizable (Noden, 1975;  Lumsden et al, 1991). A t stage 14 in the chick, the first branchial arch is present and will form the maxillary and mandibular prominences. B y stage 15 the nasal placodes are bilateral epithelial thickenings on the edges of the presumptive frontonasal mass. The maxillary prominences are first apparent at stage 18 as buds of tissue bordering the sides of the presumptive mouth (Yee and Abbot, 1978). Non-neural crest derived tissue which contributes to face development include paraxial mesoderm adjacent to the developing brain which goes on to form craniofacial musculature (Noden, 1975; Couly and LeDouarin, 1989). Initially, facial prominences surround the presumptive oral cavity as buds o f mesenchyme covered with epithelium. The two halves of the face consist of paired lateral nasal, maxillary and mandibular prominences with the frontonasal mass prominence formed at the center to establish the midline of the face. Lateral nasal prominences form the conchae of the nose and tissues o f the external nares (Romanoff, 1960). The maxillary prominences go on to form the palatal shelves, the pterygoid, quadratojugal, palatine, jugal and maxillary bones. In the center, the frontonasal mass forms the majority o f the upper beak including prenasal cartilage, nasal septum and primary palate.  The mandibular prominences form the entire  lower beak supported by paired rods of Meckel's cartilage. A t stage 24 (4.5 days incubation) maxillary, mandibular and the frontonasal mass prominences are similar in volume but cell differentiation and cartilage formation are beginning to occur.  A t stage 26, the proximal dorsal part o f the  lateral nasal processes begin to fuse with the maxilla and this fusion is complete by  stage 30. B y stage 30-31, distinct facial outgrowth o f the frontonasal mass and mandible are visible.  The present study seeks to elucidate signals  involved in the outgrowth of facial prominences between stage 24 when all primordia are equal in volume and stage 28 where the frontonasal mass and mandible are beginning to differentiate and form cartilage rods.  EPITHELIAL-MESENCHYMAL INTERACTIONS In  the  developing  chick  face,  epithelial  signals  are  required for  mesenchymal outgrowth and differentiation of facial prominences (Wedden, 1987; Richman and Tickle, 1989; Sabera et al, 1989). Mesenchyme is programmed to specify the structure of facial prominences and can differentiate to form bone and cartilage in the absence of epithelium (Tyler, 1978; Hall, 1980; Richman and Tickle, 1989), however, without epithelium, mesenchymal outgrowth is inhibited (Wedden,  1987;  interchangeable  Richman  and Tickle,  1989).  A l l facial  epithelia  and can support morphogenesis of beak structures;  are  whereas,  epithelium from areas outside the face such as the dorsal surface of the limb and the flank, cannot provide signals necessary for normal outgrowth o f facial prominences (Richman and Tickle, 1989).  These results suggest that facial epithelia contain  specific signals to foster outgrowth of facial mesenchyme.  FGFs  Which epithelial signals are regulating mesenchymal outgrowth in developing facial prominences? Fibroblast growth factors (FGF) have been shown to regulate many aspects of growth and patterning during development (Yamaguchi and Rossant, 1995). FGF2 (Richman et al., 1997) is homogeneously expressed throughout facial prominences in both epithelium and mesenchymal tissue (summarized in fig.IB) and ectopic applications of this factor can increase the length of cartilage rods formed by frontonasal mass and mandibular mesenchyme (Richman et al., 1997). FGF4 is expressed in mandibular epithelium (Niswander and Martin, 1992; Barlow and Francis-West, 1997) and like FGF2, can also stimulate outgrowth of both mandibular and frontonasal mass mesenchyme (Richman et al., 1997). FGF8 is expressed in the epithelium lining the nasal pits, the corners of the frontonasal mass, the caudal surface of maxillary prominences and the cranial surface of mandibular prominences (Heikinheimo et al., 1994; Ohuchi et al., 1994; Crossley and Martin, 1995; Wall and Hogan, 1996; Helms et al, 1997) but its effects on facial mesenchyme are not known. FGF 10 is expressed in developing limb mesenchyme and may activate receptors found in overlying epithelial cells (Ohuchi et al., 1997); its expression pattern in facial prominences has yet to be determined. Recently, four new members of the FGF family called FGF homologous factors (FHF) have been identified (Smallwood et al.,1996) and are expressed in the developing mouse embryo, however, it is unclear whether they  are involved in face development. In sum, F G F 2 , F G F 4 , and F G F 8 are expressed in developing facial prominences and may provide epithelial signals required for outgrowth of mesenchyme.  FGFRs Endogenous F G F signals may be mediated by high affinity fibroblast growth factor receptors (FGFRs). Four distinct F G F R genes have been identified (Miller and Rizzino, 1994; Yamaguchi and Rossant, 1995). The structure o f F G F R s consists of 3 extracellular immunoglobulin domains, a transmembrane domain, and an intracellular tyrosine kinase domain which is split into 2 parts by a sequence without  tyrosine  kinase  activity.  Alternative  splicing of the  extracellular  immunoglobulin domains (Chellaiah et al.,1994) and receptor heterodimerization (Shi et al.,1993) determine ligand binding specificity in vitro.  In general, the  expression o f alternative spliced isoforms of receptors 1-3 are tissue specific (OrrUrtreger et al., 1993; Shi et al, 1994). The b exon which codes for the carboxy portion of the third immunoglobulin domain is expressed in epithelial tissues while the c exon is found in mesenchymal and neural tissues (Orr-Urtreger et al., 1993; Shi etal., 1994). F G F R - 1 , 2 and 3 are expressed in facial prominences of the developing chick embryo (Richman and Leon Delgado, 1995; Wilke et al., 1997; summarized  in f i g . l A ) .  A t stage 28, just prior to the onset of chondrogenesis,  F G F R - 1 is expressed at higher levels in the mesenchyme lateral to the nasal pits and in the lateral edges of the frontonasal mass and may play a role in stimulating fusion between the frontonasal mass, lateral nasal prominences and maxillary primordia. F G F R - 2 is expressed at high levels in the center of the frontonasal mass and may be required for both establishing the midline of the frontonasal mass and for outgrowth of the prenasal cartilage.  Moderate levels of F G F R - 2 are found at  the lateral edges of maxillary prominences and at the caudal third o f mandibular prominences.  Later on in development, F G F R - 2 is found in precartilage cell  aggregates (Szebenyi et al., 1995; Patstone et al., 1992; Peters et al., 1992; OrrUrtreger et al, 1993). F G F R - 3 transcripts are concentrated at the posterior edge of the frontonasal mass where these receptors may be transducing signals involved in the outgrowth of this primordium. In the mandible, F G F R - 3 expression is confined to the lateral third of this prominence, an area that will later form the malleus and incus (Richman and Diewert, 1988;  Richman unpublished data).  In sum,  the  mesenchymal isoforms of F G F R - 1 , 2 and 3 have unique expression patterns in developing facial prominences which correlate with different phases of outgrowth and differentiation.  AIM OF STUDY In the present study we wish to determine which mesenchymal signals are regulated by epithelium in the developing chick face.  F G F s are expressed in the  epithelium  of  developing facial  prominences  outgrowth of facial mesenchyme.  Moreover,  and  can  stimulate  F G F R expression patterns in  mesenchymal tissue correlate with relative outgrowth of facial prominences (Richman and Leon Delgado, 1995; Wilke et al., 1997; f i g . l A ) . Based on this data we hypothesize that the truncated outgrowth which occurs after removing facial epithelium may be due to a loss of signal required for maintaining mesenchymal F G F R expression. To test this hypothesis, we removed epithelium from developing facial prominences and then probed for changes in F G F R - 2 expression.  We chose  to examine F G F R - 2 because its expression pattern is more pronounced in regions of the face involved in outgrowth than the other F G F R s (fig.l A ) and consequently changes in its expression pattern can be readily detected.  Our data indicate that  epithelium is required for the temporal up-regulation of mesenchymal F G F R - 2 and collagen II expression in the frontonasal mass.  Epithelium is also required for  maintaining F G F R - 2 and collagen II expression in maxillary prominences. Another aim of this study is to map the location of transcripts for a putative ligand for F G F R - 2 . Hence we localized F G F 8 m R N A transcripts in the developing chick face and found that although its expression pattern is exclusively ectodermal, it did not overlap endogenous mesenchymal F G F R - 2 expression (fig.l).  9 Figure 1.  (A) Summary of the mesenchymal expression patterns of FGF receptors in the stage 24 chick embryo. Heavier stippling indicates higher levels of FGFR lc around the nasal pits and in the mandibular prominence. Lighter stippling indicates lower levels of FGFR lc. Heavy lines indicate the location of tissues dissected for grafting. FGFR, fibroblast growth factor receptor; FNM, frontonasal mass; LNP, lateral nasal prominence; Md, mandible; Mx, maxilla. (B) Summary of the ectodermal expression patterns of FGF4 and FGF8 transcripts at stages 24 and 28. FGF2 protein is expressed in all facial ectoderm and is not depicted in this diagram.  H  FGFR lc  ^  FGFR 2c FGFR 3c  I '  I Grafted ' region  FGF-4 FGF-8  11 MATERIALS AND METHODS C h i c k e n embryos Fertilized White Leghorn chick eggs were obtained from Coastline Chicks, Abbotsford, B . C . and incubated at 3 7 . 5 ° C with humidity. T o stage the embryos, each egg was candled with a fiber optic light and gently rolled to release the embryo from the inner shell membrane. Eggs were windowed to view the embryo by cutting a hole through the eggshell with scissors. Embryos were viewed under a dissecting microscope and staged according to the criteria in Hamburger and Hamilton (1951) based on external characteristics. A t stage 22, the limb bud is as long as it is wide and is the optimum stage for serving as a host site for the grafts (fig.2). Donor tissue was obtained from stage 24 embryos.  Grafting Facial prominences in situ are not accessible for direct manipulation since embryos develop lying on one side. Also, techniques used to strip facial epithelium from mesenchymal tissue in situ (Yang and Niswander, 1995) result in rapid epithelial regeneration by surrounding tissue making it difficult to observe isolated mesenchyme behavior (Richman unpublished; McGonnal unpublished). Hence, we grafted stage 24 facial prominences into host limb buds to maintain an in vivo environment while at the same time allowing for observations at 24 and 48 hour increments (Wedden, 1987; Richman and Tickle, 1989).  Donor embryos were removed from the egg and placed on a concave dissection slide filled with dissecting media containing I X Hanks buffered salt solution and 10% fetal calf serum (fig.2). The entire maxillary prominence and the central third of the frontonasal mass were dissected with irridectomy scissors. Epithelium was removed from mesenchyme by placing the tissue pieces in 2% crude trypsin in I X Hanks buffered salt solution at 4 ° C for 30-60 minutes until epithelium  easily peeled away  from mesenchyme  (fig.2).  The tissue was  transferred to a dish of cold serum containing media to stop the trypsin action and the epithelium was cleanly separated from each piece of mesenchyme with forceps while working on a frozen slab. Four types of grafts were made: frontonasal mass mesenchyme  with epithelium and without epithelium as well as maxillary  mesenchyme with and without epithelium.  These grafts were positioned into a  prepared site on the dorsal surface of a stage 22 chick wing bud (Wedden, 1987; Richman and Tickle, 1989).  Host embryos containing grafts were incubated a  further 24 and 48 hours, fixed in 4% paraformaldehyde/phosphate buffered saline overnight, and embedded in wax as described in Rowe et al. (1991).  Grafting Method  Stage 22 host wing bud  Stage 26 host wing bud (24 h post-grafting)  In situ hybridization F G F R - 2 antisense (gift from E . Pasquale), F G F 8 antisense, collagen II antisense and sense R N A probes were used. The F G F R - 2 sequence corresponds to nucleotides 60 to 1116, includes the three extracellular immunoglobulin domains and can therefore  recognize  both the Illb and IIIc isoforms o f the third  immunoglobulin domain. A genomic clone for chick F G F 8 was the template for the F G F riboprobe (gift from J - C . Izpisua Belmonte).  The collagen II c D N A  transcribes as a l k b R N A transcript and is discussed in Devlin et al. (1988). Transverse sections were made through the trunks o f embryos which included both wing buds.  In situ hybridization was carried out as described i n •  35  Rowe et al. (1991). Riboprobes were labeled with [ S ] U T P , partially hydrolyzed, JJ  mixed with hybridization buffer: I X salts (0.5M N a C l , 0 . 1 M Tris p H 7.5, 2 0 m M N a P 0 4 p H 6.8, 50 m M E D T A p H 7.5, 5 X Denhardts), 50% formamide, 2% dextran sulphate, 50 m M D T T , 500pg/ml yeast total R N A , and placed on pretreated tissue sections.  Sections were hybridized overnight at 5 5 ° C , treated with 40ug/ml  of R N A s e , and washed up to 0.1 X S S C (0.015M N a C l , 0.015M sodium citrate; p H 7.0) for 20 minutes at 6 5 ° C .  Slides were dipped in N T B - 2 emulsion, exposed 1-2  weeks and counter-stained with malachite green. compound subjected  microscope to  in situ  Photographs were taken with a  under darkfield illumination. hybridization were  photographed with brightfield illumination.  stained  Adjacent sections not with  toluidine blue and  RESULTS Facial prominences grafted to the dorsal limb bud surface attach to the underlying mesenchyme within a few hours. A vascular connection is established within 24 hours and the graft can be identified as a swelling on the dorsal surface of the wing bud (fig.2,4,6). B y 48 hours, frontonasal mass and maxillary grafts with epithelium are larger than respective  grafts without epithelium for  compare fig.4D with fig.4J and fig.6D with 6J. located near the future elbow region (fig.2).  example,  A t 48 hours growth the graft is  Since it takes time for the graft to  revascularize, there is some degree of developmental delay compared to in vivo growth.  Donor tissue is taken from stage 24 embryos and we estimate that after 24  hours of growth on the host limb bud, the donor mesenchyme has reached stage 26. The 48 hour grafts would be equivalent to stage 28-29 when chondrogenesis in the frontonasal mass is underway. Ectoderm expresses abundant F G F R - 2 isoform Illb transcripts.  This  epithelialization  ectodermal  occurs  over  signal isolated  is  useful  in  mesenchymal  determining grafts.  In  when  re-  general,  mesenchymal grafts partly re-epithelialize at 24 hours growth on the limb bud and are fully covered with ectoderm by 48 hours.  The F G F R - 2 riboprobe recognizes  various F G F R isoforms found in both epithelial and mesenchymal tissue, however, only changes in mesenchymal gene expression are scored in these experiments. F G F R - 2 and collagen II expression levels were scored using the following system: 0 for background expression; +++  for high expression.  + for low levels; ++ for moderate expression and  Temporal and spatial expression of FGFR-2 and collagen II in the chick face in vivo At stage 24, FGFR-2 transcripts are expressed at moderate levels in the frontonasal mass center and mandibular prominences and at background levels in maxillary prominences (fig.3A). Stage 28 marks the beginning of chondrogenesis in the frontonasal mass and FGFR-2 mRNA is expressed at high levels in the center of this prominence and in developing Meckel's cartilage of the mandibular prominences (fig.3C).  FGFR-2 is expressed at low levels in lateral maxillary  mesenchyme (fig.3C). At stage 24, prior to chondrogenesis, collagen II transcripts are expressed at background levels in the frontonasal mass and maxillary prominences and at high levels in the developing Meckel's cartilage of the mandibular prominence (fig.3B). At stage 28, collagen II expression patterns mirrored those of FGFR-2.  Here,  collagen II is highly expressed in the center of the frontonasal mass (fig.3D) and in mandibular prominences (data not shown) and at moderate levels in the medial mesenchyme of maxillary prominences that will give rise to the palatal shelves (data not shown). Sections hybridized to the sense mRNA probe for collagen II showed no signal above background (data not shown).  Epithelium is required for maintaining FGFR-2 and collagen II expression in frontonasal mass grafts A t 24 hours, half the frontonasal mass grafts with epithelium contain low F G F R - 2 signal (table l;fig.5A) while the other half express moderate to high F G F R - 2 transcripts (table l;fig.4B,5A). F G F R - 2 expression levels increased after 48 hours growth where most grafts express high F G F R - 2 levels (table 1 ;fig.4E,5A). This temporal increase in frontonasal mass F G F R - 2 expression from 24 to 48 hours is consistent with in vivo expression patterns (fig.3A and C). In most cases transcripts were localized to the center of the graft. A t 24  hours, frontonasal mass grafts without epithelium express lower  F G F R - 2 levels (table l;fig.4H,5A) than grafts with epithelium (table l;fig.4B,5A). A t 48 hours, mesenchymal grafts without epithelium express low to moderate F G F R - 2 signal (table l ; f i g . 4 K , 5 A ) which is a slight increase in expression from levels found at 24 hours but the level of transcripts is much lower in isolated mesenchyme grafts at 48 hours than grafts with epithelium (table l;fig.4E,5A). A t 24 hours, half the frontonasal mass grafts without epithelium expressed low collagen II levels (table 1; fig. 5B) while the other half expressed moderate to high levels (tablel;fig.4I,5B).  A t 48 hours, collagen II signal is at background  levels in almost all grafts without epithelium (table 1 ;fig.4L,5B) compared to grafts with epithelium where transcripts are high at 48 hours (table l;fig.4F,5B). In  frontonasal mass grafts with epithelium, areas expressing collagen II overlap areas expressing F G F R - 2 (compare fig.4E with F).  FGFR-2 and collagen II transcript levels are strongly correlated in frontonasal mass grafts A t 24 hours growth, expression levels o f F G F R - 2 correlate with collagen II expression levels in most frontonasal mass with epithelium specimens (table 1). For example, both F G F R - 2 and collagen II expression are at background levels in specimen 2 and 5, low in specimens 1 and 8, moderate in specimens 4 and 6, and high in specimens 7 and 9 (table 1).  A t 48 hours growth, expression levels  correlate in 6 out o f 8 specimens (table 1).  This correlation is not evident in  frontonasal mass grafts without epithelium at the 48 hour time point (table 1).  Epithelium is required for maintaining FGFR-2 and collagen II expression in maxillary grafts Maxillary grafts with epithelium express low to background F G F R - 2 levels at 24 hours (fig.6B; fig.7A).  However, at 48 hours, specimens display low to  moderate F G F R - 2 expression (fig.6E; fig.7A) confined to one edge of the graft which  resembles  the  prominences (fig.3C).  in vivo lateral  expression  pattern  found  in stage  28  Tissue orientation was not monitored during the grafting  procedure hence, this peripheral expression pattern was not in the same location for each specimen with respect to the host limb bud.  Maxillary grafts without  epithelium express low to background F G F R - 2 levels throughout the mesenchyme (fig.6H and K ; fig.7A).  A l l maxillary grafts with epithelium express low or background collagen II levels at 24 hours (fig.6C; fig.7B). A t 48 hours, specimens display low to moderate collagen II transcripts concentrated at one side of the graft (fig.6F; fig.7B).  However, this concentrated region of collagen II expression did not  overlap areas expressing F G F R - 2 transcripts (compare fig.6E with 6F). maxillary  grafts with epithelium, grafts without epithelium express  background collagen II levels after 24 hours growth (fig.61; fig 7B).  Like low  to  In contrast,  maxillary grafts without epithelium had decreased levels of collagen II expression 48 hours after grafting (fig.6L) compared to respective grafts with epithelium (fig.6F; fig.7B).  Temporal and spatial expression of F G F 8 in the chick face in vivo In order to correlate the expression of a potential ligand for F G F R - 2 , we mapped the expression of F G F 8 .  A t stage 20, F G F 8 transcripts are abundant in  epithelium surrounding the nasal pits (fig.8A,B) and extend across the frontonasal mass (data not shown). A t stage 24, F G F 8 is highly expressed in epithelium surrounding the nasal pits which extends to the lateral thirds of the frontonasal mass but is down regulated in the central third. The caudal and medial surface of the maxillae, the proximal and cranial surface of the mandible and the caudal surface of the second branchial arch express high F G F 8 transcript levels (fig.8C,D).  A t stage  28, high F G F 8 expression continues in epithelium surrounding the nasal pits and in the lateral thirds of the frontonasal mass (fig.8E,F).  The caudal surface of the  maxillae continue to express high levels of F G F 8 (fig.8E,F).  F i g u r e 3. Dark field ( A , C ) and bright field (B,D) views of stage 24 ( A , B ) and stage 28 (C,D) heads (frontal sections).  Panels A and C were hybridized to antisense F G F R - 2  riboprobe. Panels B and D were hybridized to collagen II c R N A probe. Scale bar = 250pm K e y : (e)epithelium, (h)hours, (md) mandible, (mx)maxilla, (mm) frontonasal mass, (mc) Meckel's cartilage, (np)nasal pit A)  Moderate  FGFR-2  expression  across  mesenchyme o f the mandible at stage 24.  the  frontonasal  mass  and  caudal  Epithelium contains abundant F G F R - 2  transcripts. B) High collagen II expression in condensing Meckel's cartilage  and cranial  mesenchyme of the frontonasal mass at stage 24. C) H i g h F G F R - 2 expression in the frontonasal mass center (arrowheads) and in condensing Meckel's cartilage  at stage 28.  The lateral edges  prominences also express some F G F R - 2 signal (arrows with tails).  of maxillary Epithelium  contains abundant F G F R - 2 transcripts. D) High collagen II expression in the frontonasal mass center (arrowheads) and in developing Meckel's cartilage overlapping F G F R - 2 expression (fig.IB) at stage 28. Collagen II is expressed  at low levels in the medial portion of maxillary  prominences (data not shown).  21  F i g u r e 4. Expression of  F G F R - 2 ( B , E , H , K ) and collagen II (C,F,I,L) m R N A in adjacent  sections of grafts of frontonasal mass prominences.  Panels A , D , G , J are sections  stained with toluidine blue which clearly show graft position in the host limb bud. Scale bar =250 um K e y : (u)ulna, (g)graft, (h)hours, (l)limb (FNM)frontonasal mass B ) F G F R - 2 expression in frontonasal mass grafts with epithelium at 24 hours growth.  F G F R - 2 expression is abundant in epithelium (arrow with tails) while  mesenchymal expression is moderate (arrowheads). C) Collagen II expression in frontonasal mass grafts with epithelium at 24 hours growth. Transcripts are abundant in the centre of the graft (arrowheads). E) F G F R - 2 expression in frontonasal mass grafts with epithelium at 48 hours growth.  FGFR-2  expression  is high in epithelium (arrow with tails)  and  mesenchyme (arrowheads). H) Frontonasal mass grafts without epithelium at 24 hours. F G F R - 2 expression is low in mesenchymal tissue (arrowheads) and epithelium does not cover the entire graft (arrows with tails). I) Collagen II expression in frontonasal mass grafts without epithelium at 24 hours growth. Collagen II is expressed  at high levels at the  edges  of the  graft  mesenchyme (arrows with tails). K ) F G F R - 2 expression in frontonasal mass grafts without epithelium at 48 hours growth.  FGFR-2  is expressed  at high levels in the epithelium which has  regenerated over graft (arrowhead) and at background in the mesenchyme.  This  background level expression differs from the high expression levels found in grafts with epithelium at 48 hours (compare with panel E). L) Collagen II expression in frontonasal mass grafts without epithelium at 48 hours growth. Collagen II expression is at background which differs from the high expression levels found in grafts with epithelium at 48 hours (compare with panel F) .  23  F i g u r e 5.  Gene expression levels in frontonasal mass grafts. K e y : (0) background expression, (+) low expression, (++) moderate expression, (+++) high expression, (Epi) epithelium, (Mes) mesenchyme, (H) hour A ) Summary of F G F R - 2 expression levels in frontonasal mass grafts. A t 24 hours, grafts without epithelium express lower F G F R - 2 levels then grafts with epithelium. A t 48 hours, most grafts with epithelium express high F G F R - 2 levels while half the grafts without epithelium express low to background F G F R - 2 levels. B ) Summary of collagen II expression levels i n frontonasal mass grafts. A t 48 hours, most grafts with epithelium express high collagen II levels while almost all the grafts without epithelium express background collagen II levels.  25  EXPRESSION IN FRONTONASAL MASS GRAFTS 6-  FGFR2  5432C  10--  Ffl  ii 24HMes  Expression levels  24HEpi + Mes  48H Mes  48HEpi+Mes  mo E3 +  C M ©  U  s  Collagen I I  24HMes  24H Epi + Mes  48H Mes  T y p e of graft  48H Epi +Mes  •  ++  •  +++  F i g u r e 6.  Expression of F G F R - 2 ( B , E , H , K ) and collagen II (C,F,I,L) m R N A i n adjacent sections of grafts of maxillary prominences. Panels A , D , G , J are sections stained with toluidine blue which clearly show graft position i n the host limb bud. Scale bar =25Opm K e y : (e)epithelium, (u)ulna, (g)graft, (h)hours,(l) limb ( M X ) m a x i l l a , B ) F G F R - 2 expression in maxillary grafts with epithelium at 24 hours growth. F G F R - 2 expression is high in epithelium (arrows with tail) and low in mesenchymal tissue -confined to one edge of the graft (arrowheads). C) Collagen II expression i n maxillary grafts with epithelium at 24 hours growth. Collagen II expression is at background levels. E) F G F R - 2 expression i n maxillary grafts with epithelium after 48 hours growth. F G F R - 2 expression is high in epithelium (arrow with tail) and low i n mesenchymal tissue-confined to one edge of the graft (arrowheads). F) Collagen II expression i n maxillary grafts with epithelium at 48 hours growth. Collagen II expression is low and confined to one edge of the graft (arrowheads). H ) F G F R - 2 expression in maxillary grafts without epithelium at 24hours growth. F G F R - 2 expression is at background and epithelium does not cover the entire graft (arrowheads). I) Collagen II expression i n maxillary grafts without epithelium at 24 hours growth. Collagen II is expressed at background levels K ) F G F R - 2 expression in maxillary grafts without epithelium at 48 hours growth. Epithelium has regenerated over the graft and F G F R - 2 is expressed at high levels i n the epithelium (arrow with tail) and lower levels around the periphery of the graft (arrowheads). L ) Collagen II expression i n maxillary grafts without epithelium at 48 growth. Collagen II is expressed at background levels.  hours  F i g u r e 7. Gene expression levels in maxillary grafts. K e y : (0) background expression, (+) (+++)  low expression, (++)  moderate expression,  high expression, (Epi) epithelium, (Mes) mesenchyme, (H) hour  A ) Summary o f F G F R - 2 expression levels in maxillary grafts.  A t 48 hours,  grafts with epithelium display slightly more F G F R - 2 expression than grafts without epithelium. B ) Summary of collagen II expression levels in maxillary grafts. A t 48 hours, grafts with epithelium display low to moderate collagen II expression while grafts without epithelium are at background.  1 29  EXPRESSION IN MAXILLARY GRAFTS  Cfl  C  Expression levels  E U  a>  CA  24HMes  o  24H Epi + Mes  48H Mes  48HEpi+Mes  mo • +  £ 3  24HMes B  24H Epi + Mes  48HMes  Type of graft  48HEpi+Mes  •  ++  •  +++  Epithelium and Mesenchyme 24h  Mesenchyme 24h  specimen F G F R - 2  Collagen  specimen FGFR-2 Collage  no.  II  no.  1  +  +  1  +  2  0  0  2  0  3  + ++  +++ ++  3  + ++  4 5 6 7 8 9  4  0  0  5  ++ +++ + +++  ++ +++ + +++  6 7  t  + +  II  ++ +  0  +++ ++ ++  0  Mesenchyme 48h  Epithelium and Mesenchyme 48h specimen F G F R - 2  Collagen  specimen FGFR-2 C o l l a g  no.  II  no.  1 2 3 4 5 6 7 8  ++ + +++ + ++ +++ +++ +++ +++  ++ +++ +++ +++ +++ +++ +++ ++  II  1  0  0  2  + ++ ++ ++ +  0  3 4 5 6  0  +++  0  0  Table 1: Expression scores of FGFR-2 and Collagen II in individual frontonasal mass specimens. K E Y : (0) background expression, (+) low expression, (+ +) moderate expression, ( + + +) high expression, (h) hours, (no.) number  F i g u r e 8. Dark field views of stage 20 ( A , B ) , 24 (C,D) and stage 28 (E,F) heads hybridized to antisense F G F 8 riboprobe. A , C , E are frontal sections and B , C , E are parasagittal sections. Scale bar = 250pm Key: (20)stage 20, (24) stage 24, (28) stage 28, ( M D ) mandible, ( M X ) maxilla, (fnm) frontonasal mass, (NP) nasal pit, (T) telencephalon, (2 ) nd  second branchial  arch, (E) eye A ) Stage 20 frontal section. High F G F 8 expression in epithelium adjacent to the nasal pit, the lateral edges of the frontonasal mass, and at the second branchial arch. B) Stage 20 parasagittal section. High F G F 8 expression is found at the lateral edge of the frontonasal mass and in between the maxillary and mandibular prominence. Strong signal is also present at the isthmus of the mesencephalon and the rombencephalon. C) Stage 24 frontal section.  Abundant transcripts are concentrated in nasal pit  ectoderm and in epithelium encasing the maxillae. D) Stage 24 parasagittal section. Expression pattern is similar to C . E) Stage 28 frontal section.  F G F 8 is highly expressed in ectoderm covering the  globular processes and the developing maxillae.  F G F 8 transcripts are high in  lateral frontonasal mass epithelium but are not expressed in the centre of the prominence (arrowheads). F) Stage 28 parasagittal section. Expression is restricted to the medial edge o f the maxillae and in the cranial surface of the mandible.  32  33  DISCUSSION Frontonasal mass and maxillary prominences behaved autonomously when grafted to the host limb bud Previous morphological studies indicate that frontonasal mass grafts with epithelium grown for 7 days on a host embryo formed cartilage rods that were 80% of the average prenasal cartilage rod length in vivo (Richman and Tickle, 1989). These data show that the process of outgrowth and differentiation on the host limb bud is remarkably similar to in vivo growth. Our results indicate that after grafting facial prominences with epithelium, F G F R - 2 and collagen II transcript levels are similar to levels found in the respective prominences in vivo.  For example at 48 hours,  maxillary prominences with epithelium express low F G F R - 2 and collagen II levels as in vivo and control for possible effects that host limb tissue may be contributing to graft gene expression. In addition, F G F R - 2 and collagen II position-specific expression patterns in frontonasal mass and maxillary grafts were preserved in the ectopic location. The F G F R - 2 and collagen II expression in the centre of frontonasal mass grafts at 24 hours growth occurs in pre-cartilage cell aggregates. Previous grafting studies have not detected cartilage formation until 48 hours after grafting in alcian green-stained wholemounts (Richman and Tickle, 1989). Position-specific pattern of expression is also preserved in maxillary grafts; here both F G F R - 2 and collagen II transcripts maintained peripheral expression patterns concentrated to one edge of the graft. These  data are similar to in vivo expression patterns where F G F R - 2 and collagen II transcripts are found at the lateral and medial edges respectively. W h y is collagen II expressed in the ventro-medial regions of maxillary prominences? There are no known cartilaginous tissues derived from the ventromedial part of maxillary prominences  (D. Noden, personal communication).  Moreover, no cartilage forms when maxillary mesenchyme is grown i n micromass culture (Wedden et al, 1987; Langille et al,1989; Richman and Crosby, 1990). The expression of collagen II in the maxillae may be acting as a signalling molecule which controls when and where cyto-differentiative events take place (Thorogood et al., 1986) or the expression is occurring in non-chondrogenic tissues as it does in the basement membrane of epithelia, dorsal and lateral surface ectoderm, lateral and ventral gut endoderm and other tissues (Kosher and Solursh, 1989; W o o d et al.,1991; Cheah et al., 1991) as a transient expression not indicative of cartilage formation (Cheah et al., 1991). F G F R - 2 expression in dorso-lateral regions of the maxilla is also not correlated with any chondrogenic activity but may contribute to patterning bones derived from the maxillary prominence.  Epithelium is required to maintain mesenchymal FGFR-2 levels in the developing frontonasal mass Following 24 hours o f growth on a host limb bud, grafts of frontonasal mass mesenchyme have not been re-epithelialized and the mesenchyme exhibits reduced levels of F G F R - 2 expression when compared to respective grafts with epithelium. This  down-regulation of F G F R - 2  expression  suggests that  frontonasal  mass  mesenchyme requires signals provided by facial epithelium in order to maintain expression. Rapid down-regulation of gene expression following ectodermal removal has been demonstrated in the limb bud. Within hours of removing the FGF-rich apical ectodermal ridge down-regulation of AP-2, Cek-8 and Msx-1 is observed (Shen et al., 1997; Patel et al., 1996; Ros et al., 1992). While we have not done a detailed time course for F G F R - 2 down-regulation in grafts of frontonasal mass mesenchyme, it is likely that decrease in signal would be seen at earlier time points. After 48 hours of growth, frontonasal mass mesenchyme is covered with limb epithelium and gene expression has increased to moderate levels in 50% of the grafts. In contrast, intact frontonasal mass grafts with epithelium had high levels of F G F R - 2 expression, similar to the high levels found in vivo. There are two possible explanations for the modest up-regulation observed in frontonasal mesenchyme grafts at 48 hours. The first possibility is that the limb epithelium inhibited increased expression of F G F R - 2 . The second possibility is that the initial lack of facial epithelium over the grafts resulted in loss of signals necessary for the temporal upregulation of F G F R - 2 transcription. Our data do not distinguish between these two  possibilities.  Both immediate and delayed contact of frontonasal mass  mesenchyme with limb ectoderm have been shown to inhibit outgrowth o f the mesenchyme (Richman and Tickle, 1989; Richman and Tickle 1992). It is clear from these data that dorsal limb ectoderm cannot completely replace facial ectoderm and one of the reasons why outgrowth is inhibited might be due to an inhibitory effect of the limb ectoderm on F G F R - 2 expression in the mesenchyme.  The alternative  possibility, that re-growth of facial epithelium would allow normal gene expression, is not supported by recent data from Imai et al. (1997). Grafts o f first arch mesenchyme into other regions o f the face do not form cartilage unless they are simultaneously grafted with facial ectoderm. Thus the delay in re-epithelialization in grafts of facial mesenchyme placed within the face may be enough to prevent the cascade of molecular events that normally precede chondrogenesis (including F G F R 2 expression).  FGFR-2  and collagen II expression are differentially affected by the removal of  epithelium Both F G F R - 2 transcript levels and expression domains in frontonasal mass grafts with epithelium closely overlap collagen II expression levels and domains suggesting that there may be a relationship between F G F R - 2 expression and chondrogenesis in this prominence.  However, in frontonasal mass grafts without  epithelium there is a slight increase of F G F R - 2 expression  while collagen II  transcripts are down-regulated to background levels. These results indicate that the  expression patterns of F G F R - 2 and collagen II are no longer as strongly correlated in isolated mesenchyme as with the presence of facial epithelia. The differential regulatory effects on F G F R - 2 and collagen II that are caused by removing epithelium may reflect the hierarchy of signaling involved in cyto-differentiation. F G F R - 2 activity may be further upstream from the collagen II signaling which occurs just prior to cartilage formation. F G F R - 2 is specifically expressed across the mid-line of the frontonasal mass in stage 24 embryos and is one of the earliest markers for the future chondrogenic region. In the limb bud, F G F R - 2 is also the earliest of the three F G F R s examined to be localized to cartilage condensations (Szebenyi et al., 1995) and precedes the expression of type II collagen (Devlin et al., 1988). We know that cartilage is formed in nearly all grafts of isolated frontonasal mass mesenchyme i f they continue developing in the host limb bud (Richman and Tickle, 1989; 1992) which means type II collagen m R N A will be ultimately be upregulated. This delay in collagen II expression may be linked to the formation of truncated cartilage rods.  Endogenous FGFs may be acting on facial mesenchyme F G F 2 , 4 and 8 are ectodermal signals which may activate mesenchymally expressed F G F R - 2 in the developing face.  F G F 2 is homogeneously  throughout facial prominences (Richman et al., 1997)  expressed  and has mitogenic activity  when bound to the mesenchymally expressed isoform of F G F R - 2 (Ornitz et al.,1996). F G F R - 2 is present in the frontonasal mass and mandibular prominences and ectopic  F G F 2 increases outgrowth of both frontonasal mass and mandibular mesenchyme (Richman et al., 1997) which makes F G F 2 a good candidate for activating F G F R - 2 in vivo. Dorsal wing ectoderm also contains abundant levels of F G F 2 protein (Savage et al.,1993) yet the presence of dorsal wing ectoderm cannot support outgrowth of facial mesenchyme (Richman and Tickle, 1989). Hence, other factors in addition to F G F 2 are required for outgrowth o f facial mesenchyme. F G F 4 can also promote outgrowth of frontonasal mass and mandibular mesenchyme (Richman et al., 1997) and has high mitogenic activity when bound to the mesenchymal isoform of F G F R - 2 (Ornitz et al., 1996), however, the in vivo distribution of F G F 4 transcripts in the developing chick face has only been detected at the medial-rostral surface of mandibular ectoderm (Barlow and Francis-West, 1997; Francis-West personal communication) where it may have paracrine regulatory effects on neighbouring mesenchymal F G F R - 2 expression. More work is needed to determine whether F G F 4 is expressed in the frontonasal mass. In the stage 24 face, the expression pattern of F G F - 8 is concentrated around the nasal pits (Helms et al., 1997; Richman et al., 1997) adjacent but not overlapping F G F R - 2 expression in the frontonasal mass. Moreover F G F - 8 has no mitogenic effects when bound to F G F R - 2 (IIIc isoform, Ornitz et al., 1996). Other putative F G F R - 2 activators include F G F - 6 (de Lapeyriere et al., 1993; Coulier et al., 1994; Han and Martin, 1993), F G F - 9 (Hecht et al.,1995; Tagashira et al., 1995), F G F - 1 0 (Ohuchi et a l , 1997) and FHF1-4 (Smallwood et al.,1996) all of which have not been described in the face.  The removal of epithelium from facial mesenchyme may have eliminated a ligand required to activate F G F R - 2 signalling and dismantled the positive  feedback  required for F G F R - 2 up-regulation.  A feedback  loop was  demonstrated in developing chick skin (Song et al., 1996). The application o f exogenous F G F 2 lead to up-regulation of F G F R - 1 expression.  Experiments are  underway to test for the presence of a similar feedback loop involving F G F R - 2 in facial mesenchyme.  Relevance to C r o u z o n ' s syndrome Mutations in fibroblast growth factor receptors ( F G F R ) have been identified in humans with craniofacial disorders including Crouzon, Jackson Weiss, Pfeiffer and Aperts syndromes (Reardon et a l , 1994; Jabs et al., 1994; Rutland et al., 1995; Wilkie et al., 1995; Oldridge et al., 1995; Galvin et al., 1996). The phenotype o f Crouzon's syndrome includes shallow orbits, parrot nose, mandibular prognathism and maxillary hypoplasia (Cohen, 1986). In the developing chick, abundant F G F R 2 transcripts are found in regions of the head affected in Crouzon's syndrome (Wilke et al., 1997).  We found that removing epithelia from facial mesenchyme  results in diminished F G F R - 2 expression which may cause the truncated outgrowth of cartilage rods that occurs later on in development (Richman and Tickle, 1989). Hence, our results in the chicken embryo may parallel the defects found in Crouzon's syndrome.  Other proteins involved in face development In addition to the F G F s , several other proteins have recently been shown to play very important roles in patterning various parts of the embryo. Proteins such as sonic hedgehog (Shh) and bone morphogenetic proteins (Bmp) are present at sites where epithelial-mesenchymal interactions occur and may mediate in vivo F G F functions. A vertebrate homolog of the Drosophila Hedgehog gene (Ingham, 1995), Shh, is expressed in many epithelial tissues, frequently at sites where inductive interactions between epithelial and mesenchymal cells occur (Roelink, 1996). Throughout embryogenesis, Shh is found in organizing centers like Hensen's node, the notochord, the floor plate of the neural tube, and the posterior part of the limb bud.  In developing limb buds,  Shh mediates polarizing activity (Riddle et al,  1993), and its expression can be induced and maintained by F G F s (Crossley et al, 1996). In addition, Shh can induce F G F and Bmp-2 expression in the limb (Laufer etal, 1994). Since Shh is involved i n patterning, interacts with F G F s and Bmps  -  molecules with distinctly localized transcripts in the developing face, it is necessary to  investigate  possible Shh functions in facial morphogenesis.  Throughout  craniofacial development, Shh is exclusively expressed in epithelium.  During  earlier stages (st. 16), Shh m R N A is expressed in the presumptive oral cavity and in the posterior margin of the 2nd branchial arch which goes on to form facial muscles bones and nerves (Wall and Hogan, 1995).  A t stage 25, Shh is expressed in  epithelium at the inferior borders between the frontonasal mass, lateral nasal prominences, and maxillary prominences where it may mediate fusion of these tissues (Helms et al., 1997). A t stage 29, Shh expression is highly restricted to the caudal edge o f frontonasal mass ectoderm, maxillae and in the midline o f the stomodeum (Helms et al., 1997). In sum, early Shh expression in the presumptive oral cavity may participate in putative signalling pathways delegating overall facial symmetry while Shh expression which occurs in already formed facial prominences may be involved in outgrowth and patterning. Interestingly,  Shh mouse knockouts  display a cyclops phenotype (Chiang et al., 1996) which already indicates that this protein is involved in establishing the mid-line of the face.  Since patched, the  proposed receptor for Shh (Stone et al., 1996; Marigo et al., 1996), is almost exclusively expressed in epithelium, it is unlikely that the ligand-receptor model postulated for F G F signalling where epithelium provides the ligand required for the activation o f a mesenchymally expressed receptor applies to Shh mechanism of action.  H o w epithelial expression o f Shh interacts with underlying mesenchymal  tissue is an intriguing question which has yet to be addressed. Bmps are also expressed  at inductive sites o f epithelial-mesenchymal  interactions. Bmp-2 and Bmp-4 are members of the T G F p super family which are growth factors involved in various aspects o f development (Hogan, 1996). In the developing chick face, Bmp-2 and Bmp-4 m R N A expression is complex, shifting from epithelium to mesenchyme in regions involved in outgrowth (Francis-West et al., 1994).  Recently, ectopic applications of Bmp-2 and 4 have been shown to  participate  in a signaling cascade involving  F G F 4 that  controls  outgrowth and patterning of the facial primordia (Barlow and Francis-West, 1997) and it has been postulated that Bmp-2  and F G F 4 may interact to regulate  development o f the mandible (Barlow and Francis-West, 1997) as they do during limb development (Laufer et al., 1994). Signaling  cascades found at various  sites  of epithelial-mesenchymal  interactions may also be present in face development.  In Drosophila imaginal  discs, the B m p related factor 'decapentaplegic' (Dpp) is an important downstream target o f hedgehog (HH), a homologue of Shh, action. Here, Dpp appears to be involved in several organizing effects of H H (Roelink, 1996).  In the chick, Shh  induces ectopic mesenchymal B m p expression in the developing hindgut (Roberts et al., 1995), and in the developing wing bud (Laufer et al., 1994). Since Shh can regulate B m p expression at various sites of epithelial-mesenchymal interactions, perhaps B m p is a downstream target of Shh function in the developing face. Moreover, since F G F s can induce Shh expression in developing limbs, Shh may be a downstream target for F G F functions in the face. Therefore, the relationship that exists between these proteins in other regions of the developing embryo may also be present in the developing face.  SUMMARY  In sum, my thesis addresses the role o f overlying epithelium i n the regulation of mesenchymal F G F R - 2 expression during face development.  My  results suggest that there are signals i n overlying epithelium required for the maintenance of F G F R - 2 expression in the developing chick face. W e believe that the epithelium may provide a ligand which activates F G F R - 2 , triggering a positive feedback mechanism that is dismantled when mesenchyme is stripped of epithelium and consequently, F G F R - 2 receptor expression is not up-regulated to levels found i n mesenchyme with endogenous epithelium. W e postulate a putative mechanism for how epithelial and mesenchymal interactions occur; epithelium may provide the ligand necessary for activating receptors present i n underlying mesenchyme.  We  localized F G F 8 m R N A in the developing face to determine whether this protein colocalizes with F G F R - 2 and found that it does not. Hence, more work is needed to determine the endogenous ligand for this receptor which may be expressed i n ectoderm as well as to elucidate other proteins which may function upstream or downstream from F G F signalling to gain a better understanding o f how F G F s contribute to face development.  44  REFERENCES Barlowe, A . J. and Francis-West, P. H . (1997). Ectopic application of recombinant B M P - 2 and B M P - 4 can change patterning of developing chick face primordia. Development 124: 391-398. Bronner-Fraser, M . (1996). Manipulations of neural crest cells and their migratory pathways. Methods in Cell Biology 51:61-79. Brown, J . M . , Wedden, S.E., Millburn, G . H . , Robson, L . G . , Hill, 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. Chiang, C , Litingtung, Y . , Lee, E . , Young, K . E . , Corden, J.L., Westphal, H . , and Beachy, P . A . (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature, 383,407-413. Cheah, K . S . E . , Lau, E . T . , A u , P . K . C . , and Tam, P.P.L. (1991)  Expression of the  mouse alpha 1(11) collagen gene is not restricted to cartilage during development. Development 111: 945-953. Chellaiah, A . , McEwen, D . G . , Wener, S., X u , J., and Ornitz, D . M . (1994) Fibroblast growth factor receptor (FGFR) 3. Alternative splicing in irnmunoglobulinlike domain III creates a receptor highly specific for F G F / F G F - 1 . J Biol Chem 269: 11620-11627. Cohn, M . J . , Izpisua-Belmonte, J . C , Abud, H . , Heath, J.K., Tickle, C . (1995) Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell 80: 739-746. Cohen, M . M . (1986) "Craniosynostosis. Diagnosis, evaluation and management." N e w York: Raven Press. Coulier, F., Pizette, S., Ollendorff, V . , deLapeyriere, O . , and Birnbaum, D . (1994) The human and mouse fibroblast growth factor 6 (FGF-6) genes and their products: Possible implication in muscle development. Prog Growth Factor Res 5: 1-14. Couly, G . F . , Coltey, P . M . , and L e Douarin, N . M . (1993). The triple origin of the skull in higher vertebrates: a study in quail-chick chimeras. Development 106, 493509. Crossley, P . H . and Martin, G.R. (1995)  The mouse Fgf8 gene encodes a family of  polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121: 439-451.  45 Crossley, P . H . , Minowada, G . , MacArthur, C . A . , Martin, G . R . (1996)  Roles for  F G F 8 in the induction, initiation and maintenance of chick limb development.  Cell  84:127-136. Devlin, C.J., Brickell, P . M . , Taylor, E.R., Hornbruch, A . , Craig, R . K . and Wolpert, L . (1988). In situ hybridization reveals differential spatial distribution of m R N A s for type I and type II collagen in the chick limb bud. Development 103:111-118. deLapeyriere, O . , Ollendorff, V . , Planche, J., Ott, M . O . , Pizette, S., Coulier, F., and Birnbaum, D . (1993)  Expression of the F G F - 6 gene is restricted to developing  skeletal muscle in the mouse embryo. Development 118: 601-611. Francis-West,P.H. et al, (1994). Expression patterns of the bone morphogenetic protein genes B M P 4 and B M P 2 in the developing chick face suggest a role in outgrowth of the primordia. Dev. D y n . 201:18-178. Galvin, B . D . , Hart, K . C . , Meyer, A . N . , Webster, M . K . (1996) Constitutive receptor activation by Crouzon syndrome mutations i n fibroblast growth factor receptor (FGFR)2 and F G F R 2 / N e u chimeras. Pro. Nat. Acad. Sci. U S A 93: 7894-9. Hall,  B . K . (1980)  Tissue interactions  and the initiation of osteogenesis and  chondrogenesis in the neural crest-derived mandibular skeleton of the embryonic mouse as seen in isolated murine tissues and in recombinations of murine and avian tissues. J Embryol Exp Morphol 58:251-264. Hamburger, V . and Hamilton, H . L . (1951)  A series of normal stages in the  development of the chick embryo. J Morphol 88: 49-92. Han, J.K., and Martin, G.R. (1993) Embryonic expression of F G F - 6 is restricted to the skeletal muscle lineage. Dev Biol 158:549-554. Hecht, D . , Zimmerman, N . , Bedford, M . , A v i v i ,  A . , and Yayon, A . (1995)  Identification of fibroblast growth factor 9 (FGF-9) as a high affinity, heparin dependent ligand for F G F receptors 3 and 2 but not for F G F receptors 1 and 4. Growth Factors 12 (3): 223-33. Heikinheimo, M . , Lawshe, A . , Shackleford, G . M . , Wilson, D . B . and MacArthur, C A . (1994). F G F - 8 expression in the post-gastrulation mouse suggests a role in the development o f the face, limbs and central nervous system. M e c h Dev 48: 129-138. Helms, J.A., K i m , C . H . , H u , D . , Minkoff, R., Thaller, C , and Eichele, G . (1997). Sonic hedgehog participates in craniofacial morphogenesis and is downregulated by teratogenic doses of retinoic acid. Dev Biol 187: 25-35.  Hogan, B . L . M .  (1996). Bone Morphogenetic Proteins: multifunctional  regulators of vertebrate development. Genes Dev. 10, 1580-1594. Imai, H . , Osumi-Yamashita, N . , Kuritani, S. and Eto, K . (1997). The role o f Fgf-8 expressing region in the first pharyngeal arch. Dev Biol 186: 333, Abst. B133. Ingham, P . W . (1995). Signalling by hedgehog family proteins in Drosophila and vertebrate development. Curr. Op. in Gen. Dev. 5:492-498. Jabs, E . W . , L i , X . , Scott, A . F . , Meyers, G . , Chen, W., Eccles, M . , M a o , J.-L, Charnas, L . R . , Jackson, C . E . , and Jaye, M . (1994) Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nature. Genet. 8: 275-279. Johnson D . E . , Williams, L . T . (1993) Structural and functional diversity in the F G F receptor multigene family. A d v Can Res, 60: 1-41. Kosher, R . A . and Solursh, M . (1989)  Widespread distribution o f type II collagen  during embryonic chick development. Dev Biol 131(2): 558-66. Langille, R . M . , Paulsen, D . F . , and Solursh, M . . (1989) physiological concentrations  Differential effects o f  of retinoic acid in vitro on chondrogenesis and  myogenesis in chick craniofacial mesenchyme. Differentiation 40: 84-92. Laufer,E. et al, (1994). Sonic hedgehog and F G F - 4 act through a signalling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79:993-1003 MacArthur, C . A . , Lawshe, A . , X u , J., Santos-Ocampo, S., Heikinheimo, M . , Chellaiah, A . T . and Ornitz, D . M . (1995) forms  that  are expressed  F G F - 8 isoforms activate receptor splice  in mesenchymal  regions  of mouse  development.  Development 121: 3603-13. Miller, K . and Rizzino, A . (1994) Developmental regulation and signal transduction pathways of fibroblast growth factors and their receptors,  in "Modern Cell Biology:  vol.14 (ed. M . Nilsen-Hamilton), pp. 19-49. Toronto: Wiley-Liss Inc. Niswander, L . , and Martin, G . R. (1992)  Fgf-4 expression during gastrulation,  myogenesis, limb and tooth development in the mouse. Development 114: 755-768. Ohuchi, H . , Yoshioka, H . , Tanaka, A . , Kawakami, Y . , Nohno, T . , and Noji, S. (1994) Involvement  of  androgen-induced  growth  factor  (FGF8)  gene  in  mouse  embryogenesis and morphogenesis. Biochem Biophys Res Commun 2004: 882-888.  Ohuchi, H . , Nakagawa, T . , Yamamoto,  A . , Araga, A . , Ohata, T . ,  Ishimaru, Y . , et al., (1997) The mesenchymal factor, F G F 10, initiates and maintains the outgrowth of the chick limb bud through interaction with F G F 8 , an apical ectodermal factor. Development 124, 2235-2244. Ornitz, D . M . , X u , J., Colvin, J.S., McEwen, D . G . , MacArthur C . A . , Coulier F., Gao, G . and Goldfarb, M . (1996)  Receptor specificity of the fibroblast growth factor  family. J Biol Chem 271: 15292-7. Orr-Urtreger, A . , Bedford, M . . T . , Burakova, T . , Arman, E . , Zimmer Y . , Yayon, A . , Givol, D . and Lonai, P. (1993)  Developmental localization of the splicing  alternatives of fibroblast growth factor receptor-2 (FGFR-2). Dev Biol 158: 475-486. Patel, K . Nittenberg, R., D'Souza, D . , Irving, C , Burt, D . , Wilkinson, D . G . and Tickle, C . (1996). Expression and regulation of Cek-8, a cell to cell signalling receptor in developing chick limb buds. Development, 122, 1147-1155. Patstone, G . , Pasquale, E . B . , and Maher, P . A . (1993)  Different members of the  fibroblast growth factors receptor family are specific to distinct cell types in the developing chicken embryo. Dev Biol 155: 107-123. Peters, K . G . , Werner, S., Chen, G . , and Williams, L . T . (1992) Two F G F receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 114: 233-243. Peters, K . G . , Ornitz, D . , Werner, S., Williams, L . (1993) Unique expression pattern of the F G F receptor 3 gene during mouse organogenesis. Richman J. M . and Diewert, V . M . (1988)  Dev Biol, 155: 423-430.  The fate of Meckel's cartilage  chondrocytes in ocular culture. Dev. Biol. 129: 48-60. Richman, J. M . , and Tickle, C . (1989)  Epithelia is interchangeable between facial  primordia of chick embryos and morphogenesis is controlled by the mesenchyme. Dev Biol 136:201-210. Richman, J. M . , and Tickle, C . (1992)  Epithelial-mesenchymal interactions in the  outgrowth of limb buds and facial primordia in chick embryos. Dev Biol 154: 299308. Richman, J. M . and Leon Delgado, J. L . (1995) Locally released retinoic acid leads to facial clefts in the chick embryo but does not alter the expression of receptors for fibroblast growth factor. J Craniofac Genet Dev Biol 15: 190-204. Richman, J. M . , Herbert, M . , Matovinovic, E . , and Walin, J. (1997) Effect of fibroblast growth factors on outgrowth of facial mesenchyme. Dev. Biol. In press.  48  Riddle, et al, (1993). Sonic hedgehog mediates the polarizing activity of the Z P A . Cell 75:1401-1416. Roberts,D.J. et al, (1995). Sonic hedgehog is an endodermal signal inducing Bmp4 and H o x genes during induction and regionalization o f the chick hindgut. Development 121:3163-3174. Roelink, H . (1996). Tripartite signalling of pattern: interactions between Hedgehogs, B M P s and Wnts in the control of vertebrate development. Cur. O p . Neurobiology 6:33-40. Ros, M . A . , Lyons, G . L . , Kosher, R . A . , Upholt, W . B . , Coelho, C . N . D . and Fallon, J.F. (1992). Apical ridge dependent and independent mesodermal domains of GHox-7 and GHox-8 expression in chick limb buds. Development 116: 811-818. Rowe, A . , Richman, J. M . and Brickell, P. M . (1991) Retinoic acid treatment alters the distribution of retinoic acid receptor-beta transcripts in the embryonic chick face. Development 111: 1007-1016. Ross, B . R . and Johnston, M . C . (1972) Cleft lip and palate, Williams and Wilkins, Baltimore, pp. 1-67, 295-306. Rutland P., Pulleyn L.J., Reardon W . et al: Identical mutations in the F G F R - 2 gene cause both Pfeifer and Crouzon syndrome phenotypes. Nature Genet 1995, 9:173176. Saber,  G . M . , Parker,  S.B., and Minkoff, R. (1989).  Influence  of epithelial-  mesenchymal interaction on the viability of facial mesenchyme in vitro. Anat. Rec. 225, 56-66. Savage, M . P . , Hart, C . E . , Riley, B . B . , Sasse, J., Olwin, B . B . , and Fallon, J.F. (1993) Distribution o f F G F - 2 suggests it has a role in chick limb bud growth. Dev Dynam 198:159-170. Shen, H . , Wilke, T . A . , Ashique, A . , Narvey, M . , Zerucha, T . , Savino, E . , Williams, T . and J . M . Richman. (1997). Chicken transcription factor A P - 2 : cloning, expression and its role in outgrowth of facial prominences and limb buds. Dev Biol, in press. Shi, E . , Kan, M . , X u , J., Wangh, F., Hou, J. and McKeenan, W . L . (1993) Control of fibroblast growth factor receptor kinase signal transduction by heterodimerization of combinatorial splice variants. M o l Cell Biol 13: 3907-3918.  Shi, D . L . , Launay, C , Fromentoux, V . , Feige, J.J., Boucaut, J.C. (1994) Expression o f fibroblast growth factor receptor-2 splice variants is developmentally and tissue-specifically regulated in the amphibian embryo. Dev Biol 164 (1): 173-82. Smallwood, P . M . , Munoz-Sanjuan, I., Tong, P., Macke, J.P., Hendry, S . H . C . , Gilbert, D.J., Copeland, N . G . , Jenkins, N . A . , and Nathans, J. (1996) Fibroblast growth factor (FGF) homologous factors: New members of the F G F family implicated in nervous system development. Proc Natl Acad Sci 93: 9850-9857. Song, H . K . , Wang, Y . , and Goetinck, P.F. (1996) Fibroblast growth factor 2 can replace ectodermal signalling for feather development. Proc Natl Acad Sci 93:1024610249. Szebenyi, G . , Savage, M . P . , Olwin, B . B . , and Fallon, J.F. (1995) Changes in the expression  of fibroblast  growth  factor  receptors  mark  distinct  chondrogenesis in vitro and during chick limb skeletal patterning.  stages  of  Dev Dynam  204:446-456. Tagashira, S., Ozaki, K . , Ohta, M . , and Itoh, N . (1995) Localization of fibroblast growth factor-9 m R N A in the rat brain. M o l Brain Res 30:233-241. Thorogood, P., Bee, J., and von der Mark, K . (1986) Transient expression of collagen type  II  at  epithelio-mesenchymal  interfaces  during  morphogenesis  of the  cartilagenous neurocranium. Dev Biol 116 (2):497-509. Tyler, M . S . and McCobb, D.P. (1980)  The genesis of membrane bone in the  embryonic chick maxilla: epithelial-mesenchymal tissue recombination studies. J Embryol Exp Morphol 56: 269-81. Wall, N . A . and Hogan, B . L . M . (1996). Expression of bone morphogenetic protein-4 (BMP-4), bone morphogenetic protein-7 (BMP-7), fibroblast growth factor-8 ( F G F 8) and sonic hedgehog (SHH) during branchial arch development in the chick. Mech Dev 53:383-392. Wedden, S . E . (1987)  Epithelial-mesenchymal interactions in the development o f  chick facial primordia and the target of retinoid action. Development 99: 341-351. Wedden,  W . E . , Lewin-Smith, M . R . , and Tickle,  C . (1987) The patterns of  chondrogenesis of cells from facial primordia of chick embryos in micromass culture. Dev Biol 117:71-82. Wilke, T . A . , Gubbels S., Schwartz J., and Richman, J . M . (1997)  Expression of  fibroblast growth factor receptors ( F G F R 1 , F G F R 2 , F G F R 3 ) in the developing head and face. Dev Dynam in press.  Wood, A . , Ashurst, D . , Corbett, A . , and Thorogood, P. (1991) The transient expression of type II collagen at tissue interfaces  during mammalian  craniofacial development. Development 111: 955-968. Yamaguchi, T.P., and Rossant, J. (1995) Fibroblast growth factors in mammalian development. Curr Opin in Gen and Dev 5: 485-491. Yang, Y . and Niswander, L . (1995)  Interaction between the signalling molecules  W N T 7 a and S H H during vertebrate limb development: dorsal signals regulate anteroposterior patterning. Cell 80 (6): 939-47.  

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.831.1-0088270/manifest

Comment

Related Items