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The distribution of glycoconjugates in the basal lamina and ECM during esophageal muscle formation in… Reimer, Corinne L. 1989

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THE DISTRIBUTION OF GLYCOCONJUGATES IN THE BASAL LAMINA AND ECM DURING ESOPHAGEAL MUSCLE FORMATION IN EMBRYOS OF THE STARFISH PISASTER OCHRACEUS AS REVEALED BY LECTIN HISTOCHEMISTRY By CORINNE L REIMER B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Deptartment of Anatomy, U n i v e r s i t y of B r i t i s h Columbia We accept this , thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1989 © Corinne L Reimer, 1989 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 ANATOMY The University of British Columbia Vancouver, Canada Date AUGUST 21, 1989 DE-6 (2/88) i i ABSTRACT Morphogenetic events cons is t of complex i n t e r a c t i o n s of c e l l s and e x t r a c e l l u l a r mater ia ls r e s u l t i n g in the movement and rearrangement of groups of c e l l s and t h e i r subsequent d i f f e r e n t i a t i o n to form organs or organ systems. Although we can pred ic t these movements f o r any given event, we have l i t t l e understanding of how morphogenesis i s c o n t r o l l e d . In the s t a r f i s h P i s a s t e r ochraceus. assembly o f mesenchyme c e l l s on one p a r t i c u l a r region of the l a rva l gut, the oesophagus, and t h e i r subsequent d i f f e r e n t i a t i o n in to muscle i s an example of a simple morphogenetic event which i s r e a d i l y access ib le fo r study. In t h e i r migrat ion to the gut, the mesenchyme c e l l s t rave l through a r i c h substrate o f ECM. Upon t h e i r a r r i v a l at the presumptive esophagus, they come to s e t t l e on the BL underly ing the endodermal ep i the l ium. It i s qu i te poss ib le that i n t e r a c t i o n s between the mesenchymal c e l l s and 'the ECM/basal lamina are important in d i r e c t i n g and regu la t ing t h e i r d i f f e r e n t i o n in to muscle. The basal laminae and ECM of vertebrates and invertebrates i s r i c h in g lycoconjugates , inc lud ing g lycoprote ins , proteoglycans and glycosaminoglycans. U l t r a s t r u c t u r a l studies of embryos of the as tero id P i s a s t e r ochraceus have demonstrated that at the l a t e g a s t r u l a stage, the endodermal basal lamina i s th inner and less a l c i a n o p h i l i c in the esophageal r e g i o n . FITC and c o l l o i d a l gold l a b e l l e d l e c t i n s , which act as s p e c i f i c probes for carbohydrate mo i t i es , usua l l y those at the terminal end of o l igosacchar ide chains , have been used to l o c a l i z e these sugars at the l i g h t and e lect ron microscope l e v e l s . These studies show i i i that a heterogeneity ex i s ts with respect to terminal sugars in the basal lamina, i . e . l e c t i n binding of the basal lamina i s not uniform in a l l regions o f the embyro. S p e c i f i c a l l y , a s t a t i s t i c a l ana lys i s of l e c t i n binding determined that l a b e l l i n g with the two l e c t i n s , Au2g-Con A and A u 2 5 - L F A was s i g n i f i c a n t l y reduced in the esophageal region as compared with the other regions of the embyro, while l a b e l l i n g of the BL with AU25-DBA showed a s i m i l a r i n t e n s i t y in a l l areas of the embryo. These r e s u l t s confirmed the a l c i a n blue r e s u l t s descr ibed above and suggest that there are some sugar conta in ing molecules, perhaps s p e c i f i c g l y c o p r o t e i n s , GAGs and/or proteoglycans, which are present in reduced q u a n t i t i e s in t h i s reg ion . In a d d i t i o n , these studies show a d i s t i n c t l a b e l l i n g pattern of the ECM through which the mesenchymal c e l l s migrate on route to the esophagus. D i f f e r e n t l e c t i n s label d i f f e r e n t regions of the ECM, however i t can not yet been said whether there i s a r e g i o n a l l y d i s t i n c t pattern in the area of the migratory path of mesenchymal c e l l s to the esophagus. Proteoglycans and GAGs are involved in c e l l movement in vertebrates and s u l f a t e d glycoconjugates have been shown to be necessary fo r mesenchyme c e l l movement in ech ino ids . A decrease in proteoglycans and GAGs in the esophageal BL could therefore help to d i r e c t movement of the presumptive muscle c e l l s to the esophagus by prov id ing a "stop" s i g n a l . i v TABLE OF CONTENTS PAGE Abstract i i L i s t of Tables vi L i s t of Appendices v i i L i s t of Figures v i i i L i s t of Abbreviat ions xi Acknowledgments xiv 1. INTRODUCTION 1 1.1 General Introduct ion 1 1.2 Epithel ial-Mesenchymal Interact ions 1 1.3 Role o f the BL in Epithel ial-Mesenchymal Interact ions 4 1.4 ECM-Structure and Role in Morphogenesis 7 1.5 ECM as a D i r e c t o r in Morphgenesis 8 1.6 Techniques Used to V i s u a l i z e Glycoconjugates 09 1.7 Invertebrate ECM and Migrat ion 19 1.8 Rat ionale fo r the Current Model System 20 1.9 Development of the Esophagus in P i s a s t e r Ochraceus 21 2. MATERIALS & METHODS 25 2.1 Rearing o f P i sas te r Ochraceus embryos 25 2.2 LM & TEM Morphology of Esophageal Muscle Formation 26 2.3 Lect ins Used for t h i s Study 27 2.4 FITC-Lect in L a b e l l i n g of the BL & ECM During Esophageal 28 Muscle Formation 2.5 C o l l o i d a l Gold Lect in L a b e l l i n g of the BL & ECM During 29 Esophageal Muscle Formation V PAGE 2.6 Autoradiography of 3 H-Sugars Incorporated Into Late 32 Gastru la Embryos 3. RESULTS 44 3.1 Formation of the Esophageal Musculature 44 3.2 F i x a t i o n 53 3.3 FITC-Lect in L a b e l l i n g of the BL and ECM 59 3.4 TEM Lect in L a b e l l i n g of the BL and ECM 69 3.5 I n t r a c e l l u l a r Granules 88 3.6 Autoradiaography 91 4. CONCLUSIONS AND DISCUSSION 95 4.1 Opening Remarks 95 4.2 F ixat ions 96 4.3 Nature of Lect in-B ind ing S i tes 100 4.4 Lect in Binding to the BL and ECM of JL. Ochraceus 104 4.5 Impl icat ions of the Results as They Relate to Development 107 5. THE HYALINE LAYER: ANOTHER EXTRACELLULAR MATRIX 111 5.1 Introduct ion 111 5.2 Results 116 5.3 Discuss ion 133 6 REFERENCES 136 vi LIST OF TABLES TABLE PAGE 1 St ructure of Monosaccharides 13 2 Lect ins & The i r Propert ies 35 3 FITC-Lect ins & S p e c i f i c Inh ib i to rs Used in t h i s Study 36 4 Lect in Concentrations fo r Conjugation to Gold 36 5 The E f f e c t of Various F ixat ives on FITC-Lect in Binding S i tes 57 6 Number o f C o l l o i d a l Gold P a r t i c l e s Bound/Length of BL A f t e r 86 L a b e l l i n g with Au25-Con A 7 Number of C o l l o i d a l Gold P a r t i c l e s Bound/Length of BL A f t e r 87 L a b e l l i n g with AU25-LFA 8 Number of C o l l o i d a l Gold P a r t i c l e s Bound/Length of BL A f t e r 87 L a b e l l i n g with AU25-DBA 9 D i s t r i b u t i o n of 3 H-Glucosamine A f t e r a 6 Hr Label in the 92 Late Gast ru la Embryo of P i sas te r Ochraceus 10 Lec t in -B ind ing S p e c i f i c i t i e s fo r GAGs 101 11 Proposed Lect in Binding S i tes 103 12 Au25-Lect in L a b e l l i n g of the Hyal ine Layer: A Regional 128 Breakdown vi i LIST OF APPENDICES APPENDIX PAGE 1 F i x a t i v e s Used f o r TEM and C o l l o i d a l Go ld-Lect in L a b e l l i n g 37 2 F i x a t i v e s Used for FITC-Lect in L a b e l l i n g 38 3 Buffers 39 4 Gelvatol/DABCO "Anti fad ing" Mounting Medium for f luorescence 40 Microscopy 5 Preparat ion o f C o l l o i d a l Gold 41 6 M i c r o t i t r a t i o n Assay for Determination of Optimal [Prote in] 42 Required to S t a b i l i z e C o l l o i d a l Gold 7 Preparat ion of Lect in-Gold Conjugates 43 vi i i LIST OF FIGURES FIGURE PAGE 1 A LM saggi ta l sect ion of P. ochraceus in la te g a s t r u l a . 46 2 A TEM saggi ta l sect ion through the esophagus, ear ly g a s t r u l a . 48 3 A TEM saggi ta l sect ion through the esophagus, l a t e g a s t r u l a . 48 4 A TEM sect ion through the esophagus showing e p i t h e l i a l - 50 mesenchymal i n t e r a c t i o n , l a t e g a s t r u l a . 5a A TEM c r o s s - s e c t i o n through the esophagus, l a te b i p i n n a r i a . 52 b A TEM c r o s s - s e c t i o n of an esophageal muscle c e l l , l a t e 52 b i p i n n a r i a . 6a A LM sect ion of a g lutara ldehyde/a lc ian blue f i xed embryo, 55 l a t e g a s t r u l a , showing the ectoderm. b A LM sect ion of a paraformaldehyde/CPC-fixed embryo, l a t e 55 g a s t r u l a showing the ectoderm and ECM. 7a A saggi ta l sect ion of an embryo in l a t e g a s t r u l a , sta ined with 62 FITC-Con A. b A saggi ta l sect ion of an embyro in l a t e g a s t r u l a , stained with 62 the control sugar/conjugate s o l u t i o n , mannose/FITC-Con A. 8 A se r ies o f sect ions through an embryo in la te g a s t r u l a 64 sta ined with FITC-Con A, showing at higher magni f i cat ion the l a b e l l i n g patterns of the BL and ECM. 9a A saggi ta l sect ion of an embryo in la te g a s t r u l a , stained with 66 FITC-WGA. b A saggi ta l sect ion of an embryo in l a t e g a s t r u l a , stained with 66 the control sugar/conjugate s o l u t i o n , glcNAc/FITC-WGA. 10 A se r ies o f sect ions through an embryo in la te g a s t r u l a , 68 sta ined with FITC-WGA, showing at higher magni f i cat ion the l a b e l l i n g patterns of the BL and ECM. 11a A TEM through the stomach of an embryo in la te g a s t r u l a , f i xed 71 and embedded convent iona l l y . b A TEM through the stomach of an embyro in l a t e g a s t r u l a , 71 embedded in lowicry l K4M to enable histochemical s t a i n i n g with Au25-conjugates. ix FIGURE PAGE 12 A TEM through the stomach of an embyro in l a t e g a s t r u l a , 73 showing at higher magni f icat ion the u l t r a s t r u c t u r e of the BL and ECM as preserved with glutaradehyde/alc ian blue fol lowed by embedding in lowicry l K4M. 13 A TEM through the ectoderm stained with AU25-DBA. 77 14a A TEM through the ectoderm stained with Au25-Con A. 79 b A TEM through the ectoderm, stained with the control sugar/ 79 conjugate s o l u t i o n , mannose/Au25-Con A. 15a A TEM through the stomach region stained with AU25-LFA. 81 b A TEM through the esophagus stained with the control sugar/ 81 conjugate s o l u t i o n , s i a l i c acid/Au25-LFA. 16a A TEM through the ectoderm stained with AU25-WGA. 83 b A TEM through the ectoderm stained with the control sugar/ 83 conjugate s o l u t i o n , glcNAc/Au25-WGA. 17a A TEM through the esophagus stained with AU25-SBA. 85 b A TEM through the ectoderm stained with the control sugar/ 85 conjugate s o l u t i o n , galNAc/Au25-SBA. 18a A sagg i ta l sect ion through an embryo in l a t e g a s t r u l a , f i xed 90 with Bouin's and stained with FITC-WGA to show l a b e l l i n g of i n t r a c e l l u l a r granules . b A sagg i ta l sect ion through an embryo in la te g a s t r u l a , f i xed 90 with Bouin's and stained with FITC-Con A to show l a b e l l i n g of i n t r a c e l l u a r granules . 19a A sagg i ta l sect ion through an embryo in la te g a s t r u l a 94 processed for autoradiography fo l lowing a 4 hr incubat ion with ^-g lucosamine . b A higher magni f i cat ion of 19a through the stomach. 94 c A higher magni f i cat ion of 19a through the i n t e s t i n e . 94 d A higher magni f i cat ion of 19a through the stomodeum. 94 e A control sect ion of 19a through the stomach. 94 X FIGURE PAGE 20 A TEM through the hyal ine layer of an embryo in ear ly 115 g a s t r u l a . 21a A sagg i ta l sect ion through an embyro in l a t e g a s t r u l a , stained 119 with FITC-Con A to show l a b e l l i n g of the HL. b A s e r i a l sect ion of the above stained with the control sugar/ 119 conjugate s o l u t i o n , mannose/FITC-Con A. 22a A sagg i ta l sect ion through an embryo in l a t e g a s t r u l a , stained 121 with FITC-WGA to show l a b e l l i n g of the HL. b A s e r i a l sect ion of the above stained with the control sugar/ 121 conjugate s o l u t i o n , glcNAc/FITC-WGA. 23a A sagg i ta l sect ion through an embyro in la te g a s t r u l a , stained 124 with FITC-SBA to show l a b e l l i n g of the HL. b A s e r i a l sect ion of the above, stained with the control sugar/ 124 conjugate s o l u t i o n , galNAc/FITC-SBA. 24a A saggi ta l sect ion through an embryo in l a t e g a s t r u l a , stained 126 with FITC-RCA to show l a b e l l i n g of the HL. b A s e r i a l sect ion o f the above, stained with the control sugar/ 126 conjugate s o l u t i o n , gal/FITC-RCA. 25a A TEM of the hyal ine layer stained with AU25-SBA. 130 b A TEM of the hyal ine layer stained with the control sugar/ 130 conjugate s o l u t i o n , galNAc/Au25-SBA. 26a A TEM of the hyal ine layer stained with Au25~Con A. 130 b A TEM of the hyal ine layer stained with the control sugar/ 130 conjugate s o l u t i o n , mannose/Au25-Con A. 27a A TEM through the hyal ine layer stained with AU25-WGA. 132 b A TEM through the hyal ine layer stained with the control 132 sugar/conjugate s o l u t i o n , glcNAc/Au25-WGA. 28a A TEM through the hyal ine layer stained with AU25-PNA. 132 b A TEM through the hyal ine layer stained with the control 132 sugar/conjugate s o l u t i o n , gal/AU25-PNA. xi LIST OF ABBREVIATIONS aq aqueous A u 2 5 c o l l o i d a l gold with mean p a r t i c l e s i ze diameter, 25 nm BL basal lamina(e) BM(s) basement membrane(s) C carbon CS chondro i t in s u l f a t e CPC cety l pyr id in ium c h l o r i d e DABCO 1,4 d i a z a b i c y c l o [2 .2 .2] octane DS dermatan s u l f a t e dH 2 0 d i s t i l l e d water Ec ectoderm ECM e x t r a c e l l u l a r matrix En endoderm Es esophagus FITC f luorescene isoth iocyanate fuc L-fucose GAG(s) glycosaminoglycan(s) gal galactose galNAc N-acetyl-D-galactosami ne glcNAc N-acetyl-D-glucosami ne H hydrogen HA hyaluronic ac id HL hyal ine layer xi i HSPG heparan s u l f a t e proteoglycan 3 H t r i t i u m KS kerat in s u l f a t e L e c t i n s : Con A concanaval in A GSA-II G r i f f o n i a s i m p l i c i f o l i a a g g l u t i n i n II DBA Dol ichos b i f l o r u s a g g l u t i n i n LFA Limax f lavus a g g l u t i n i n PNA peanut a g g l u t i n i n RCA Ric inus communis a g g l u t i n i n UEA-I Ulex europaeus a g g l u t i n i n - I SBA soybean a g g l u t i n i n WGA wheat germ a g g l u t i n i n sWGA succ iny lated wheat germ a g g l u t i n i n LM l i g h t microscopy man mannose uCi microCurie mOs mi l l iosmole M molar Me mesenchyme Mu muscle NC neural c res t Neu5Ac N-acetyl-neuraminic ac id Neu5Gc N-glycolo ly-neuraminic ac id nm nanometer xi i i p i i s o e l e c t r i c point PBS phosphate buffered s a l i n e PF paraformaldehyde S stomach TBS t r i s buffered s a l i n e TEM transmission e lect ron microscopy xi v ACKNOWLEDGEMENTS I consider i t a p r i v i l e d g e to have worked in Dr. Bruce Crawford's l a b . I fee l that because of him, I have come to learn what research i s " a l l about". I t was a great benef i t to be able to r e l y on his time and expert i se whenever requ i red , and because of t h i s , I have learned a great deal of research techniques and research methodology. I would a l so l i k e to thank my parents fo r t h e i r support and constant encouragement in my academic endeavors. This work was supported by grant #A0032 from NSERC. - 1 -1. INTRODUCTION 1.1 GENERAL INTRODUCTION Morphogenesis involves the organizat ion of sub-populations of c e l l s in to s p e c i f i c arrangements, which u l t i m a t e l y r e s u l t s in the development of a mature organism. In the organ izat ion of c e l l popu lat ions , a ser ies of c o n t r o l l e d or d i rec ted events take p lace: these are c e l l d i v i s i o n , c e l l migrat ion , c e l l death, c e l l adhesion, and c e l l d i f f e r e n t i a t i o n (Edelman, 1985). Our knowledge of the molecular bases of these processes has increased g r e a t l y over the past decade, but the s t r a t e g i e s o f how the primary processes are interconnected in m u l t i c e l l u l a r organisms are s t i l l unknown. Although these developmental events are u l t i m a t e l y under genomic c o n t r o l , i t i s thought that c e l l and t i s s u e i n t e r a c t i o n s such as epithel ial-mesenchymal i n t e r a c t i o n s , may be the primary regu latory mechanisms for the assembly of c e l l s during morphogenesis ( B e r n f i e l d et a l . , 1973). These i n t e r a c t i o n s involve recogn i t ion and communication among p a r t i c i p a t i n g c e l l s and inc lude d i r e c t c e l l - c e l l i n t e r a c t i o n , d i r e c t c e l l - m a t r i x i n t e r a c t i o n s as well as i n d i r e c t c e l l communication with other c e l l s and the matrix v i a the product ion of d i f f u s i b l e substances. 1.2 EPITHELIAL-MESENCHYMAL INTERACTIONS The importance of epithel ial-mesenchymal i n t e r a c t i o n s for morphogenesis and c e l l u l a r d i f f e r e n t i a t i o n i s well es tab l i shed (Rudnick, 1933; Auerbach, 1960; Taderera, 1967). For example, during the development of normal s k i n , the epidermal c e l l layer i n t e r a c t s c l o s e l y with the underly ing mesenchyme, and appears to control the d i f f e r e n t i a t i o n , growth & spreading, and o r i e n t a t i o n of the over ly ing - 2 -epidermis (McLoughlin, 1968). A s i m i l a r s i t u a t i o n i s present during s a l i v a r y gland development, during which s a l i v a r y epi thel ium undergoes c h a r a c t e r i s t i c morphogenesis only when i t i s in the presence of ( i t s own s p e c i f i c kind of) mesenchyme (Grobste in , 1968). In some cases, the requirement appears to be less r e s t r i c t i v e , so that while e p i t h e l i a s t i l l maintain a developmental requirement fo r mesenchyme, they are able to continue t h e i r c h a r a c t e r i s t i c morphogenesis in the presence of a mesenchyme which i s not t h e i r own, ( i . e . pancreat ic ep i the l ium, Grobste in , 1968). While the interdependency of epithel ium and mesenchyme seems c e r t a i n , there has been some controversy with regards to the exact nature of t h i s i n t e r a c t i o n , which has centered around the question of whether actual d i r e c t c e l l - c e l l contact i s requ i red , or whether short-range i n t e r a c t i o n s (matrix-mediated) are s u f f i c i e n t to induce d i f f e r e n t i a t i o n . The c l a s s i c experiments of Grobstein (1956) and his assoc iates (Koch & Grobste in , 1963; Kallman & Grobste in , 1966) suggested the presence of d i f f u s i b l e molecules which lead to "embryonic induct ion" in the absence of d i r e c t epithel ial-mesenchymal contacts . However, reports by Nordl ing et a l . (1971) and Saxen (1972) have ind icated that the length of time required for induct ion to occur between i n t e r a c t i n g c e l l populat ions in a t r a n s f i l t e r s i t u a t i o n tends to ru le out d i f f u s i o n of molecules as a mechanism f o r information t r a n s f e r . They postulated that induct ive information might be transmitted by d i r e c t c e l l u l a r contact or e x t r a c e l l u l a r mater ia ls which were bound to the c e l l per iphery . Both of these hypotheses require that the plasma membranes o f c e l l s from the i n t e r a c t i n g t i ssues be brought in to r e l a t i v e l y c lose a p p o s i t i o n , and furthermore, that the basal lamina (BL) in t h i s region would be degraded or changed in some way to al low for t h i s contact . - 3 -Reports by Wartiovaara et a l . (1974), Lehtonen (1975), and Saxen et a l . (1976) i n d i c a t e that d i r e c t epithel ial-mesenchymal contacts are e s s e n t i a l f o r the induct ion of kidney tubule morphogenesis. Several t r a n s f i l t e r embryonic induct ion studies of kidney tubules using Nucleopore f i l t e r s have demonstrated that heterotypic c e l l processes approximate one another and make d i r e c t contact (Wartiovaara et a l . , 1974) , but that i n t e r a c t i o n s take place only across f i l t e r s having pores which al low the formation of cytoplasmic processes (Lehtonen et a l . , 1975) . More recent studies have added embryonic lung (Bluemink et a l . , 1976) , tooth germs (S lavk in & Br ingas, 1976), rat submandibular gland ( C u t l e r & Chaudhrey, 1973; C u t l e r , 1977) and embryonic mouse mammary gland (Heuberger et a l , 1982) to the growing l i s t of t i s sues in which t r a n s i e n t epithel ial-mesenchymal contacts are seen. In a l l cases, these contacts seem more c l o s e l y assoc iated with funct iona l d i f f e r e n t i a t i o n of c e l l s rather than morphogenesis of the s t r u c t u r e . Despite th i s evidence, there does, however, appear to be cases where d i r e c t contact between ep i the l ium and mesenchyme i s not necessary fo r induct ion of d i f f e r e n t i a t i o n . During e p i t h e l i a l i n i t i a t i o n of bone formation in the mandible, Hal l (1987) has shown strong evidence to support short range matrix-mediated i n t e r a c t i o n as the means of epithel ial-mesenchymal i n d u c t i o n . The molecular basis of induct ion fo l lowing epithel ial-mesenchymal i n t e r a c t i o n s i s not known, fo r once the two t i ssues have come into contact , var ious types of i n t e r a c t i o n s can take p lace . For example, a s ignal molecule may d i r e c t l y i n t e r a c t with the nucleus or genome of the responding c e l l , or a l t e r n a t i v e l y , the s ignal may be passed v ia a cytoplasmic f a c t o r , i . e . a chemical mesenger, as occurs when s te ro id hormones act on animal c e l l s . Since these i n t e r a c t i o n s involve a loca l - 4 -s ignal t ransduct ion from the mesenchyme to the epi the l ium and v ice v e r s a , an e x t r a c e l l u l a r matrix component secreted and deposited by the mesenchyme would be a natural candidate fo r a s ignal transducer. Recent ly , the d iscovery of the e x t r a c e l l u l a r matrix prote in tenasc in , which i s expressed by mesenchyme, has provided some strong evidence to support t h i s i d e a . Tenascin i s a prote in which has an unusual ly r e s t r i c t e d expression pattern in the developing embyro (Chiquet-Ehrismann et a l . , 1986). In several organs, i t i s found in the dense mesenchyme that immediately surrounds budding and growing e p i t h e l i a , but i s absent from other parts of the mesenchyme (Chiquet-Ehrismann et a l . , 1986). In a d d i t i o n , i t has a rather dramatic temporal express ion, in that i t appears to be d i r e c t l y st imulated by epithel ial-mesenchymal i n t e r a c t i o n s ; in the developing kidney, i t appears s h o r t l y a f t e r the f i r s t epithel ial-mesenchymal i n t e r a c t i o n (Aufderheide et a l . , 1987). The d i s t r i b u t i o n of tenasc in , coupled with i t s expression in development suggests that i t i s important fo r e i t h e r mesenchymal condensation, f o r e p i t h e l i a l growth, or both. 1.3 ROLE OF THE BL IN EPITHELIAL-MESENCHYMAL INTERACTIONS Recent ly , a t tent ion has been focussed on the ro le that the BL plays in epithel ial-mesenchymal i n t e r a c t i o n s . The BL i s a s t r u c t u r a l l y ordered and chemical ly s p e c i a l i z e d region of the e x t r a c e l l u l a r matrix (ECM), u s u a l l y forming a continuous sheet c l o s e l y appl ied to the basal surfaces o f e p i t h e l i a (Kefa l ides et a l . , 1979; Madri et a l . , 1984). I t thus occupies a s t r a t e g i c a l l y important p o s i t i o n as i t separates the ep i the l ium from the underly ing mesenchyme, and would appear to act as a phys ica l b a r r i e r between the two. In add i t ion to several types of col lagens and g lycoprote ins - 5 -( f i b r o n e c t i n , lamin in , e n t a c t i n , nidogen), the BL contains a large quota of a n i o n i c a l l y charged carbohydrates in the form of glycosaminoglycans (GAGs), such as chondro i t in s u l f a t e , heparan s u l f a t e and hyaluronic a c i d . (P ra t t et a l . , 1975; Weston et a l . , 1978; & Er ickson & Weston, 1983). GAGs are the carbohydrate components of proteoglycans, and cons i s t of repeat ing d isacchar ide u n i t s . Each d isacchar ide unit cons i s ts of a hexosamine, usua l ly in i t s N-acetylated form, as well as a nonnitrogenous sugar, D-glucuronic ac id or L- iduronic ac id (Schrevel et a l . , 1981). The GAG composition and arrangement of the BL v a r i e s , however, from t i s s u e to t i s s u e . For example, the BL o f the corneal ep i the l ium contains p r i m a r i l y chondro i t in s u l f a t e , which i s organized in a regu lar array in 2 planes on e i t h e r s ide of the lamina densa (Tre l s tad et a l . , 1974). In the mouse embryo s a l i v a r y g land, the BL contains hyaluronate and chondro i t in s u l f a t e (Cohn et a l . , 1977); in the pregnant mouse mammary e p i t h e l i a , hyaluronate and heparan s u l f a t e (Gordon & B e r n f i e l d , 1980), in the chick embryo notochord & neural tube, chondro i t in s u l f a t e and heparan s u l f a t e (Hay & Meier, 1974), and in the mature ra t glomerular BL as well as chick embryo lens capsule , mainly heparan s u l f a t e (Hay & Meier, 1974; Kanwar & Farquhar, 1979). I t seems l i k e l y that during epithel ial-mesenchymal i n t e r a c t i o n s in which d i r e c t c e l l - c e l l contact i s made, the b a r r i e r between the two, i . e . the BL, must be somehow overcome. Neural c res t (NC) migration provides us with an in v ivo system in which to study the BL in morphogenetic i n t e r a c t i o n s . The migratory behavior of NC c res t c e l l s i s character i zed f i r s t by a loss of c e l l to c e l l attachments, fol lowed by movement o f c e l l s to a fenestrated BL and therea f te r penetrat ion through i t . The BL, in t h i s case, appears to form a physica l b a r r i e r to c e l l m igrat ion , and the premigratory NC c e l l populat ion w i l l not emmigrate - 6 -unless the BL perforated or absent (Martins-Green et a l . , 1986; Newgreen and Er i ckson , 1986). The epithel ial-mesenchymal i n t e r a t i o n s which occur during branching morphogenesis ( i . e . during lung, s a l i v a r y gland and kidney development), have been studied extens ive ly , and an i n t e r e s t i n g observat ion has been made in that the BL i s thinned or discontinuous at the t i p s where a c t i v e growth i s taking place ( B e r n f i e l d et a l . , 1973; C u t l e r & Chaudhry, 1973; Coughl in , 1975; Lehtonen, 1975; Gal lagher , 1986a). D i rec t c e l l to c e l l contacts between the e p i t h e l i a l and mesenchymal c e l l s appear to occur through these thinned BL. There i s a l so evidence from studies on the formation of h a i r s , p a r t i c u l a r l y the s p e c i a l i z e d whiskers o f the rodent snout, that holes appear in the s p e c i a l i z e d BL on which the s u p e r f i c i a l e p i t h e l i a l c e l l s r e s t , and that processes from mesenchymal c e l l s penetrate these holes to e s t a b l i s h d i r e c t contact with the e p i t h e l i a l c e l l s (Goldberg & Hardy, 1983). Such contact immediately precedes the induct ive i n t e r a c t i o n which occurs between epi the l ium and mesenchyme in the formation of these whiskers, a t iming which i s cons istent with c e l l - t o - c e l l communication being required to mediate the i n t e r a c t i o n . In v i t r o experiments have attempted to focus on the ro le which the BL plays in epithel ial-mesenchymal i n t e r a c t i o n s . In lung organoid c u l t u r e s , f e t a l lung e p i t h e l i a l and mesenchymal c e l l s d i f f e r e n t i a t e in to type II pneumocytes and connective t i s s u e c e l l s r e s p e c t i v e l y ; i t has been shown that c e l l contacts between these two c e l l types are necessary in order f o r d i f f e r e n t i a t i o n to occur , and that the formation o f a BL by the ep i the l ium i s not a requirement fo r pneumocyte d i f f e r e n t i a t i o n to occur (Zimmerman, 1987). These observat ions have lead to the proposal that developmentally - 7 -regulated BL degradation may be a general mechanism f o r c o n t r o l l i n g morphogenetic t i s s u e i n t e r a c t i o n s by the t iming of d i r e c t c e l l contacts . (Bluemink et a l . , 1976; Goldberg & Hardy, 1983). The BL i s therefore ascr ibed more than j u s t a passive s t r u c t u r a l r o l e in morphogenesis but rather a dynamical ly changing informational ro le which in f luences the migrat ion o f c e l l populat ions and the nature of epithel ial-mesenchymal i n t e r a c t i o n s (Sanders & Prasad, 1983). 1.4 ECM-STRUCTURE & ROLE IN MORPHOGENESIS In add i t ion to epithel ial-mesenchymal i n t e r a c t i o n s , morphogenetic processes appear to be inf luenced s t rong ly by cel l -ECM i n t e r a c t i o n s . Together with mesenchymal c e l l s , ECM comprises the connective t i s s u e of the e a r l y embryo, and i t s ro le in guiding c e l l migrat ion during morphogenesis has been studied widely in several embryonic systems i n c l u d i n g NC and pr imordia l germ c e l l migrat ion (Heasman & Wyl ie, 1981; Too le , 1981; Turner et a l , 1983; Er i ckson , 1987a). The ECM through which NC c e l l s migrate has been descr ibed u l t r a s t r u c t u r a l l y : t i s s u e f i xed in the presence of c a t i o n i c dyes or tannic ac id appears to cons is t of a sta ined granular material assoc iated with matrix f i b r i l s (Hay, 1978). The ECM has a lso been analyzed by hi stochemical and immunochemical methods and includes various co l lagen types (Von Der Mark, 1980; Wart iovarra et a l . , 1980), f i b r o n e c t i n (Mayer et a l . , 1981; Newgreen & Th iery , 1980) and various GAGs such as hyaluronic a c i d , chondro i t in s u l f a t e and heparan s u l f a t e (Kv is t & Finnegan, 1970; Pratt et a l . , 1975; Weston et a l . , 1978;). Results of studies using hyaluronidase to d igest away GAGs have lead some to speculate that the sta ined granular material i s high in hyaluronic ac id (HA) or proteoglycans (Sanders, 1979; Solursh et a l . , 1979), while the f i b r i l s - 8 -are composed of col lagens (S ingley & So lursh , 1989). During morphogenesis, the ECM i s considered to a id the organ izat ion of mesenchymal c e l l s in two ways: F i r s t l y , ECM enlarges the a v a i l a b l e migratory spaces for c e l l s to t rave l through because i t i s r i c h in HA, which, in i t s hydrated s t a t e , i s high in volume (Toole et a l , 1972; Meier & Hay, 1973; T re l s tad at a l , 1974; Prat t et a l , 1975; Derby, 1978; Toole et a l , 1984). Secondly, the ECM provides a substratum, that i s , a phys ica l support or meshwork f o r the movement and migrat ion of not only mesenchymal c e l l s , but a l so more s p e c i a l i z e d c e l l s such as pr imordia l germ c e l l s and NC c e l l s (Sanders, 1986a). 1.5 ECM AS A DIRECTOR IN MORPHOGENESIS The r o l e that ECM plays in c e l l migration has been studied widely in NC c e l l migrat ion (Duband & Th iery , 1982; Th iery et a l , 1982; Le Douarin et a l , 1984; Brauer et a l . , 1985; Bronner-Fraser , 1986; E r i ckson , 1987b). NC c e l l s fo l low a well def ined pathway during t h e i r migratory route . They a r i s e in the ectoderm, and at neurulat ion are pos i t ioned at the top of the neural f o l d s . From here, they break through t h e i r BL and migrate i n d i v i d u a l l y as mesenchymal c e l l s through an ECM-rich area , and eventua l ly d i f f e r e n t i a t e in to a large number of d e r i v a t i v e t i s s u e s (Weston, 1970, 1983; LeDouarin, 1980, 1982). Considerable e f f o r t has gone in to i d e n t i f y i n g and i n v e s t i g a t i n g the organ izat ion o f matrix substances in the ECM migratory pathways of NC c e l l s , l a r g e l y because NC c e l l s se lec t a p a r t i c u l a r pathway which var ies depending on t h e i r ax ia l ( a n t e r i o r - p o s t e r i o r ) l o c a t i o n . For example, in the c r a n i a l reg ion , NC c e l l s enter the d o r s a l - l a t e r a l pathway and avoid the ventra l pathway, whereas in the trunk reg ion , the major i ty of the NC c e l l s enter the ventral pathway ( i . e . between the somite and neural - 9 -tube) , and not the d o r s a l - l a t e r a l pathway (Anderson & Meier, 1981). I t i s be l ieved that one determining f a c t o r in d i r e c t i n g NC c e l l migrat ion i s the actual composition of the ECM in these pathways (Weston et a l . , 1978; Bolender et a l . , 1980; Newgreen & Th ie ry , 1980; Er ickson & Tur ley , 1983; Brauer et a l . , 1985). Recent studies (Brauer & Markwald, 1988) have revealed that the i n i t i a l pathway taken by NC c e l l s at e i ther ax ia l leve l i s enriched in f i b r o n e c t i n - c o n t a i n i n g p a r t i c l e s and lacks s u l f a t e d po lyanions . Because f i b r o n e c t i n p a r t i c l e s are a l so present in areas that NC c e l l s do not enter , f i b r o n e c t i n alone can not be accred i ted for d i r e c t i n g the paths of c e l l movement; add i t iona l fac tors are probably involved in t h i s process, and one such f a c t o r could be the presence o f su l fa ted proteoglycans (Kv is t & Finnegan 1970). In v i t r o studies have shown that su l fa ted proteoglycans i n h i b i t c e l l attachment and migrat ion in f i b r o n e c t i n - r i c h substrates (Rich et a l . , 1981; Newgreen et a l . , 1982). I t i s therefore poss ib le that su l fa ted components of ECM in some unknown way block or i n h i b i t the f ib ronect in-NC c e l l i n t e r a c t i o n and in doing so may d i r e c t or s e l e c t the pathway for NC c e l l migrat ion . Future advances in understanding the i n t r i c a t e ways in which the ECM in f luences c e l l s during morphogenesis w i l l come as a r e s u l t of i d e n t i f y i n g and l o c a l i z i n g candidate macromolecules in the matr ix . In add i t ion to antibody techniques, several techniques which v i s u a l i z e glycoconjugates s p e c i f i c a l l y are c u r r e n t l y being used to character i ze components of the ECM. These w i l l be discussed next. 1.6 TECHNIQUES USED TO VISUALIZE GLYCOCONJUGATES Morphological and histochemical studies of ECM have, in the past , l a r g e l y r e l i e d upon the use of c a t i o n i c dyes such as a l c i a n blue -10-incorportated in to f i x a t i v e s or used a f t e r the processing of t i s s u e to s t a i n ECM components. Cat ion ic dyes are successful because of the charged nature o f ECM, due p r i m a r i l y to GAGs. However, more r e c e n t l y , use of these low r e s o l u t i o n c a t i o n i c dyes has been replaced by techniques in which ind iv idua l g lycoprote ins and carbohydrates are mapped both s p a t i a l l y and temporal ly during morphogenetic processes. 3 One technique which i s used i s autoradiography of H-sugars. In th i s method, r a d i o a c t i v e l y l a b e l l e d sugars placed in the cu l ture medium are taken up by c e l l s and are incorporated in the var ious d i f f e r e n t carbohydrate conta in ing structures present in the organism. Fol lowing chases in "co ld" medium, the l o c a l i z a t i o n of these r a d i o a c t i v e l y l a b e l l e d sugars can be determined, as well as t h e i r rate of metabolism 3 in the organism. Autoradiographic studies using H-glucosamine have been used in the past to i d e n t i f y e p i t h e l i a l sur face-assoc iated GAGs of the s a l i v a r y g land, lung and u r e t e r i c bud ( B e r n f i e l d & Banerjee, 1972). Another technique which has been very successfu l in l o c a l i z i n g t i s s u e carbohydrates i s l e c t i n h is tochemistry . Lect ins-General Lect ins are def ined as prote ins or g lycoprote ins which bind to carbohydrates in a very s p e c i f i c manner. The major i ty of l e c t i n s are i s o l a t e d from p l a n t s , although l e c t i n s have a lso been i s o l a t e d from b a c t e r i a , sponges, s n a i l s , the sera of f i s h , and the hemolymph of l o b s t e r s . Lect ins have at least two sugar-binding s i t e s , and t h e i r s p e c i f i c i t y i s usua l l y defined in terms of monosaccharides or o l igosacchar ides that i n h i b i t the agg lut inat ion r e a c t i o n . Because of the s i m i l a r i t i e s l e c t i n s and ant ibodies share in the binding mechanisms they employ, l e c t i n s have a lso been def ined " loose ly" - l i -as ant ibodies s p e c i f i c e x c l u s i v e l y fo r carbohydrates, as apposed to being s p e c i f i c fo r a p r o t e i n . There are some fundamental d i f fe rences between l e c t i n s and ant ibodies however, which leave l e c t i n s in a catagory a l l of t h e i r own. The primary d i f fe rence i s that l e c t i n s are of non-immune o r i g i n , while ant ibodies are a d i r e c t r e s u l t of an immune r e a c t i o n . Unl ike ant ibod ies , the exact funct ion of endogenous l e c t i n s i s not c l e a r , however several ro les have been proposed. For p lant l e c t i n s , these inc lude (a) the poss ib le involvement in defense against pathogens, p a r t i c u l a r l y during ear ly seed germination, where they may act as f u n g i s t a t i c or b a c t e r i o s t a t i c agents (Mirelman et a l . , 1975); (b) a poss ib le r o l e in the packaging or m o b i l i z a t i o n of the storage mater ia ls that accumulate in seeds p r i o r to dormancy (Boyd et a l . , 1958), and (c) a poss ib le funct ion in c e l l wall e longat ion (Labav i tch , 1981). For vertebrate l e c t i n s , several ro les have a lso been proposed. Soluble l e c t i n s are thought to p a r t i c i p a t e in the organizat ion of e x t r a c e l l u l a r g lycoconjugates , f o r example, ch icken- lactose l e c t i n - I i n t e r a c t s with glycoconjugates in the ECM between c e l l s (Barondes, 1984). Membrane l e c t i n s are genera l l y bel ieved to p a r t i c i p a t e in s h u t t l i n g so luble g lycoprote ins from the outs ide to the i n t e r i o r of the c e l l and within i n t r a c e l l u l a r compartments (Ashwell & M o r e l l , 1974). Although there i s no d e f i n i t i v e proof fo r any of these proposed f u n c t i o n s , studies on the p r o p e r t i e s , d i s t r i b u t i o n and regu lat ion of l e c t i n s as well as on t h e i r endogenous receptors w i l l l i k e l y lead to an ult imate e l u c i d a t i o n of t h e i r var ious r o l e s . -12-L e c t i n s - S p e c i f i c i t v fo r Carbohydrates Although some l e c t i n s bind e x c l u s i v e l y to one carbohydrate moiety, most l e c t i n s bind to two or three d i f f e r e n t sugars which are s t r u c t u r a l l y s i m i l a r . Usua l l y , however, one sugar binds to the l e c t i n with a greater a f f i n i t y than do i t s s t r u c t u r a l l y s i m i l a r counterparts . The reason that there i s a degree of v a r i a b i l i t y among many lec t in -carbohydrate i n t e r a c t i o n s has to do with the areas of the sugar with which the l e c t i n i n t e r a c t s . For example, as a general r u l e , l e c t i n s t o l e r a t e very l i t t l e v a r i a t i o n at Carbon-3 (C-3) of the sugars they b ind , and consequently a l e c t i n which binds to a sugar with an a-OH group at C-3 (eg, D-glucose) w i l l not bind to a carbohydrate possessing a I3-0H group at C-3, such as L-fucose ( tab le 1) (Goldste in & Poretz , 1986). The C-4 hydroxyl group i s a l so c r i t i c a l l y involved in d i r e c t i n g 1 e c t i n - b i n d i n g s p e c i f i c i t y . This i s demonstrated in that l e c t i n s which bind mannose/glucose, res idues , sugars which have B-OH groups at C-4, do not i n t e r a c t with ga lactose , which has an a-OH group at C-4 ( tab le 1) . I t fo l lows that galNAc-binding l e c t i n s do not bind to glcNAc residues f o r the same reasons (A l len et a l . , 1973). V a r i a t i o n s at C-2 impart some degree of s p e c i f i c i t y to l e c t i n binding as w e l l . For example, Con A binds p r i m a r i l y mannose ( a -OH at C-2) , but i t w i l l a l so bind to glucose and glcNAc (B-OH at C-2) , although to a l e s s e r extent. L ikewise, l e c t i n s which p r e f e r e n t i a l l y bind galNAc a lso react with ga lactose . Lect ins d i f f e r markedly with respect to t h e i r anomeric s p e c i f i c i t i e s , that i s , the o r i e n t a t i o n of the C-l hydroxyl group. Some l e c t i n s , fo r example Con A, have anomeric s p e c i f i c i t y and bind only to the a-anomers (Smith & G o l d s t e i n , 1967), whereas o thers , l i k e SBA and RCA bind to both a and B-anomers (L i s et a l . , 1970). - 1 3 -TABLE 1: STRUCTURE OF MONOSACCHARIDES I D-glucose III D-mannose V N-acetylgalactosamine VII N-acetylneuraminic ac id II D-galactose IV N-acetylglucosamine VI L-fucose VIII D-xylose -14-Most l e c t i n s i n t e r a c t with the nonreducing terminal g lycosy l groups o f po lysacchar ide and g lycoprote in chain ends. One exception i s Con A, which in add i t ion to i t s i n t e r a c t i o n with a-mannopyranosyl and a -g lucopyranosy l terminal groups, binds in terna l 2-0-cc-manno-pyranosyl residues (Goldste in et a l . , 1973). Another exception i s WGA, which i n t e r a c t s with in terna l 4 -0-subst i tuted glcNAc residues (A l l en et a l . , 1973). Although some l e c t i n s appear to bind only a s ing le g lycosy l u n i t , many have been found to possess extended binding s i t e s and accommodate 2-5 sugar res idues . For example, WGA i n t e r a c t s most s t rong ly with 13(1,4)-linked glcNAc oligomers and PNA binds p r e f e r e n t i a l l y to gal(31,3galNAc units (Wu, 1984). Lect in-Carbohydrate Interact ions There i s a f a i r amount of controversy about the actual nature of forces respons ib le fo r lect in-carbohydrate i n t e r a c t i o n s . Consider ing the po lyhydroxy l i c and hydroph i l i c nature of the sugars, i t would seem l o g i c a l that po lar i n t e r a c t i o n s (hydrogen bonds and d ipo le i n t e r a c t i o n s ) would p lay a dominant ro le in these carbohydrate-prote in i n t e r a c t i o n s . Experimental support fo r t h i s view i s given from x-ray c r y s t a l l o g r a p h i c studies on Con A-methyl-a-mannopyranoside complexes (Becker et a l . , 1975; Hardman & Ainsworth, 1976). Further support fo r the involvement o f po lar i n t e r a c t i o n s comes from chemical m o d i f i c a t i o n , spectroscop ic , and proton t i t r a t i o n studies (Hassing et a l . , 1971; Hassing and G o l d s t e i n , 1972). For example, by studying f ree energy contr ibut ions of H-bonds between the hydroxyl groups of sugars and t h e i r s p e c i f i c l e c t i n s , the nature o f the groups o f the l e c t i n s involved in hydrogen bonding have been evaluated (Bhattacharyya & Brewer, 1988). These -15-studies have revealed that depending on the p a r t i c u l a r l e c t i n , hydroxyl groups on the carbohydrate i n t e r a c t d i f f e r e n t l y with a p a r t i c u l a r l e c t i n . In the RCA-galactose i n t e r a c t i o n , the C-2 hydroxyl group forms two weak hydrogen bonds in the capac i ty of a H-bond acceptor and a donor, the C-3 and C-4 hydroxyl groups forms at least one H-bond each with a charged residue of RCA, and both the C-l and C-6 hydroxyl groups appear not to be involved with l e c t i n b ind ing . Conversely , the hydrogen bonding between Con A and a-methyl mannoside shows a d i f f e r e n t p a t t e r n : the C-l and C-2 hydroxyl groups accept H-bonds from uncharged groups on Con A, the C-3 hydroxyl accepts a H-bond from a charged group, and both the C-4 and C-6 hydroxyl groups donate a H-bond to charged groups on Con A. Some other researchers (Lemieux and his co l leagues , 1982) be l ieve that lec t in-carbohydrate binding r e s u l t s from hydrophobic i n t e r a c t i o n s . In t h i s view, i t i s proposed that the lect in-combin ing s i t e recognizes i n t e r a c t i o n s between surfaces (topographic features) and not sugar units per se (Lemieux, 1982; Hindsgaul et a l . , 1982; Baker et a l . , 1983). A more conservat ive notion put fo r th by Roberts & Goldste in (1984) i s that both h y d r o p h i l i c and hydrophobic forces are invo lved . For example, the i n t e r a c t i o n between L-fucose and lima bean l e c t i n (LBL) has been studied extens ive ly , and t h i s i n t e r a c t i o n appears to have a hydrophobic character , as ca l cu la ted from entropy and enthalpy record ings . Go ldste in & Poretz (1986) view that polar i n t e r a c t i o n s between carbohydrate hydroxyl groups and the polar s ide chains of amino acids (along with some nonpolar contacts) with in a l e c t i n ' s hydrophobic binding s i t e would be an ideal model fo r s p e c i f i c carbohydrate-protein i n t e r a c t i o n s . For example, looking at the WGA-s ia ly lo l igosacchar ide system (Kronis & Carver, 1985), dominant forces s t a b i l i z i n g the -16-assoc iated complex appear to be intermolecular H-bonds and van der Waals f o r c e s . In the f u t u r e , high r e s o l u t i o n X-ray c r y s t a l l o g r a p h i c ana lys i s of l ec t in -carbohydrate complexes should resolve many of the present uncertai nt i es. Lect i ns-Markers A v a r i e t y of markers e x i s t fo r l i g h t microscopy (LM) and transmiss ion e lec t ron microscopy (TEM). Common markers f o r LM inc lude peroxidase and f l u o r e s c e i n . Peroxidase has been widely used f o r TEM as w e l l , however recent ly p a r t i c u l a t e markers such as c o l l o i d a l gold have become more popular . The f i r s t use of c o l l o i d a l gold as a marker for TEM was by Faulk & Taylor (1971) who absorbed a n t i s e r a to gold p a r t i c l e s to use as probes for immunocytochemistry. C o l l o i d a l gold was f i r s t used in l e c t i n binding studies by Horisberger et a l . (1975). The growing i n t e r e s t in t h i s marker system stems from the fac t that c o l l o i d a l gold markers can be reproduc ib ly and e a s i l y prepared in a range of s i z e s , making the system extremely f l e x i b l e , as well as the f a c t that gold probes are e lect ron dense, spher ica l and very easy to i d e n t i f y . In the present study, l e c t i n s conjugated to FITC were used for l i g h t microscopy, and l e c t i n s conjugated to c o l l o i d a l go ld , fo r transmission e lec t ron microscopy. Lect in Binding Studies of The Basement Membrane: A Review Lect ins have been used not only to map c e l l surface carbohydrates, but, more r e c e n t l y , to probe for i n t r a c e l l u l a r and e x t r a c e l l u l a r s i t e s using f i xed and sectioned m a t e r i a l . In t h i s respect , several l e c t i n - b i n d i n g studies have been c a r r i e d out on embryonic basement membranes (BMs), and there appears to be a great degree of v a r i a t i o n in -17-the carbohydrate composition of BMs between species as well as between organs with in the same spec ies . Hurle et a l . (1988) found that the l e c t i n s PNA, RCA, SBA, WGA, and sWGA sta ined the ectodermal BM of chick limb buds. With a l l of the l e c t i n s , the BM showed an undulating appearance and exhib i ted a rather uniform s t a i n i n g i n t e n s i t y . However, during (Hamburger & Hamilton) stages 22-25 of growth, a zone of increased s t a i n i n g was observed with PNA at the BM of the ap ica l ectodermal r i d g e , the area of ac t ive morphogenesis. Lect in binding studies on embryonic chick lung bud has a l so revealed some i n t e r e s t i n g pat terns . Ga l lagher , (1986b) has shown that the l e c t i n s WGA, SBA and RCA s t a i n the BM of ep i the l ium of embryonic chick lung. However, somewhat l e s s e r amounts of these l e c t i n s bind to the t i p s of newly formed buds. B lo t tner & Lindner (1987) have studied l e c t i n binding patterns in e a r l y odontogenic events in prenatal rats 13-20 days o l d . They found that Con A l a b e l l e d dental BM at days 13-15 and l a b e l l e d inner dental BM and predentin at days 17-19. They found WGA to be s p e c i f i c fo r the dental BM, and found PNA to s t rong ly label the inner dental BM while l a b e l l i n g the outer dental BM weakly. Upon comparing l e c t i n binding s i t e s in kidneys of several d i f f e r e n t animal spec ies , Ho l thof fer (1983) found d i f fe rences in the l e c t i n b inding propert ies of BMs among spec ies . For example, the adult GBM of the guinea pig stained with RCA, DBA, and UEA-I, while that of the r a b b i t stained only with RCA AND UEA-I; the BM of the dog stained with WGA and PNA, while that of the hen stained only with WGA; the BM of the human sta ined with PNA, RCA and Con A, while that o f the mouse, only with PNA. Further studies (Holthofer & V i r t a n e n , 1987) revealed that l e c t i n b inding patterns of human f e t a l glomerular basement membrane -18-(GBM) d i f f e r e d from those of human mature GBM: Fetal GBM stained with PSA, Con A, UFA, and RCA, whereas in the adult k idney, only RCA, Con A, and PNA were found to label the GBM. Katow and Solursh (1982), and DeSimone & Spiegel (1986) have revealed developmental stage s p e c i f i c patterns of l e c t i n binding in the e a r l y echinoid embryonic BM, which suggests that the regional and temporal expression of g lycoprote ins and or GAGs are t i g h t l y regulated by the developing embryo. They f i n d that Con A binds to the ent i re basal surface of the epithel ium u n t i l g a s t r u l a t i o n , at which time Con-A binding s i t e s disappear from the vegetal h a l f of the ectoderm, and are l o c a l i z e d to the BM of the animal pole region and to the base of the archenteron. Whether or not the disappearance of Con-A binding s i t e s from the vegetal h a l f ectoderm i s due to a transformation of binding s i t e s to a masked form i s not yet known; however, s ince t h i s surface a l t e r a t i o n a l so occurs on the migrat ing primary mesenchyme c e l l s , i t might r e f l e c t a regulatory mechanism in mesenchymal-ectodermal i n t e r a c t i o n s . Further studies explor ing the phylogenetic polymorphism of l e c t i n binding of BMs was explored by Ribera et a l . (1987), in t h e i r studies of vertebrate neuromuscular junct ions (NMJs). They found that DBA, SBA and PNA bound to synapt ic BM in a l l mammalian, r e p t i l i a n , and less i n t e n s l y in amphibians, whereas no s t a i n i n g was seen in the major i ty of avians and f i s h e s . In cont ras t , WGA and Con A bound c o n s i s t e n t l y to a l l species s t u d i e d . I t appears, from these s tud ies , that BMs from a wide range of t i s s u e s and species bind a v a r i e t y of l e c t i n s , i n c l u d i n g Con A, WGA, sWGA, PNA, RCA, DBA, UEA, and SBA. Although some hint i s given to suggest a species s p e c i f i c trend when the data on NMJs i s reviewed, in - 1 9 -g e n e r a l , there i s substant ia l overlap in the l e c t i n binding patterns of a l l BMs s tud ied . As w e l l , no h i s t o - s p e c i f i c trends are notable from the data presented. Of s i g n i f i c a n c e are c e r t a i n developmental ly re la ted patterns of l e c t i n - b i n d i n g , which are seen in branching morphogenesis, g a s t r u l a t i o n , and tooth development. 1.7 INVERTEBRATE ECM AND MIGRATION In i n v e r t e b r a t e s , c e l l migrat ion during morphogenesis has a l so been studied qu i te extens ive ly (Okazaki , 1960; Gustafson & Wolpert, 1967; Schneider et a l . , 1978; H e i f i t z & Lennarz, 1979; Katow & So lursh , 1981; Spiegel & Burger, 1982; Desimone & S p i e g e l , 1986). Mesenchymal c e l l s migrat ing through the b lastocoe le of developing echinoderms has been a f a v o r i t e system to study cel l -ECM i n t e r a c t i o n s because of the transparency of the embryos, and the ease of which enzymes and i n h i b i t o r s can be placed in the cu l ture medium. An ECM that has an u l t r a s t r u c t u r a l appearance s i m i l a r to vertebrate ECM (strands of f i b e r s assoc iated with granules) has been descr ibed in the b lastocoe le of echinoids during e a r l y development (Endo & Noda, 1977; Katow & So lursh , 1979; Kawabe et a l . , 1981; Spiegel et a l . , 1983; Gale leo & M o r r i l l , 1983). I t appears to contain GAGs (Karp & So lursh , 1974), proteoglycans (Oguri & Yamagat, 1978) co l lagen (Pucc i -Minafra et a l . , 1972) and f i b r o n e c t i n and laminin (Spiegel et a l . , 1980, 1983; Katow et a l . , 1982; Wessel et a l . , 1984). In the developing sea u r c h i n , primary mesenchyme c e l l s are in c lose a s s o c i a t i o n with the ECM. However, i f the embryos are placed in s u l f a t e - f r e e sea water, the c e l l s do not migrate and the number o f granules in the ECM i s g r e a t l y reduced, suggesting that the granules may represent s u l f a t e - c o n t a i n i n g macromolecules and may be an important component of the migratory substratum (Katow & So lursh , -20-1981). In the developing a s t e r o i d , a material with s i m i l a r EM appearance has been descr ibed (Crawford & Chi a , 1981; Abed & Crawford, 1986a). Although i t i s probable that the ECM in the s t a r f i s h b lastocoe le contains s i m i l a r components found in the echinoid ECM, fur ther biochemical work i s necessary to confirm t h i s . 1.8 RATIONALE FOR THE CURRENT MODEL SYSTEM In higher organisms, i t i s d i f f i c u l t to f i n d a simple model system with which to approach the (complex) problem of morphogenesis. This has been overcome to a c e r t a i n extent by the use of var ious c e l l and t i s s u e c u l t u r e techniques, which serve to provide a simple and reproducib le i n v i t r o system to study t i s s u e and c e l l u l a r i n t e r a c t i o n s . Several "simple" inver tebrate embryo systems such as those of sea urch ins , tun icates and annel ids have a lso been used extens ive ly to i n v e s t i g a t e the phenomema of c e l l - c e l l communication during morphogenesis. These often provide a s i m p l i f i e d system in which to study development in v i v o . In a d d i t i o n , invertebrate embryos are e a s i l y maintained in the laboratory at a minimal cost , and can be grown in large numbers in synchronous c u l t u r e s . As tero id embryos have several added advantages which, when combined, make them a des i rab le system in which to study embryology. F i r s t l y , the as te ro id adults are very easy to feed and can be kept r i p e at least 6 months of the year . Secondly, one adult provides a very large number of gametes, enabling massive amounts of embryos to be cu l tured with great ease. T h i r d l y , the as te ro id embryos are t ransparent , which makes poss ib le the continual v i s u a l i z a t i o n of c e l l movement and migrat ion during growth. Fourth ly , development i s synchronous, a l lowing large numbers of embryos of the same stage to be - 2 1 -removed i f necessary. And f i f t h l y , the embryos do not develop calcium carbonate s p i c u l e s , as do ech ino ids , which makes them ideal fo r EM s t u d i e s . In add i t ion to the reasons mentioned above i t i s probable that the basic and most essent ia l mechanisms of development w i l l transcend p h y l e t i c d i f f e r e n c e s , as do the basic and essent ia l mechanisms of c e l l a c t i v i t i e s . I t therefore fol lows that so lv ing the puzzle of morphogenesis in the as tero id embryo could lead to a greater understanding of development in higher organisms. 1.9 DEVELOPMENT OF THE ESOPHAGUS IN PISASTER OCHRACEUS During the e a r l y development o f the a s t e r o i d P. ochraceus. mesenchyme c e l l s are formed. These c e l l s leave the t i p of the archenteron at the mid gas t ru la stage and migrate in to the b l a s t o c o e l e . Locomotion appears to involve extension and r e t r a c t i o n of f i l o p o d i a in a manner s i m i l a r to that seen during the migrat ion of secondary mesenchyme c e l l s of echinoids (Gustafson & Wolpert, 1967). Shor t l y a f t e r the egress of the mesenchyme c e l l s the archenteron begins to bend toward one region o f the ectoderm, and a b l i s t e r of BL extends from the t i p of the archenteron to spikes in the c e l l s o f the presumptive stomodeal ectoderm. Both endodermal and ectodermal c e l l s appear to migrate along t h i s BL template eventua l ly making contact to form a mouth. At approximately the time of mouth formation, the gut becomes segmented in to 4 reg ions , r e s u l t i n g in the formation of the stomodeum, esophagus, stomach and i n t e s t i n e . Some of the mesenchyme c e l l s f i n a l l y come to res t and attach e x c l u s i v e l y to the presumptive esophageal endoderm, where they subsequently develop processes which surround the esophagus and d i f f e r e n t i a t e in to muscle c e l l s ; the end r e s u l t i s an i n t e r l o c k i n g of muscle c e l l s forming a smooth muscle sheath around the esophageal -22-endoderm. The migrat ion of mesenchymal c e l l s during esophagus formation has been studied in the developing as ter iod P. ochraceus. using time lapse videomicrocopy (Crawford, unpublished data) : I t i s i n t e r e s t i n g to note that the mesenchymal c e l l s , much l i k e the NC c e l l s of ver tebrates , can choose more that one route to migrate. F i r s t l y , they can migrate to the esophagus where they fur ther d i f f e r e n t i a t e in to muscle. Secondly, they can migrate elsewhere in the b l a s t o c o e l e , where they do not d i f f e r e n t i a t e in to muscle, or i f so, do so at a much l a t e r t ime. I t i s therefore poss ib le that , j u s t as was seen in the ECM through which NC c e l l s migrate through, the ECM in the b lastocoe le of as tero ids has a d i f f e r e n t composition in d i f f e r e n t regions of the embyro. It i s poss ib le that t h i s plays an important ro le in d i r e c t i n g c e l l migrat ion . This u n d i f f e r e n t i a t e d mesenchymal c e l l populat ion provides a good system with which to study some of the primary processes of development ( c e l l m igrat ion , c e l l adhesion and c e l l d i f f e r e n t i a t i o n ) and how they i n t e r a c t with each other . Several questions can be asked regarding th i s system: For example, "How do the mesenchyme c e l l s "know" where to go? How do they know where and when to stop? What i s t e l l i n g them to d i f f e r e n t i a t e ? " . In looking for answers to these quest ions , one i n e v i t a b l y thinks of the ro le of the ECM as a c o n t r o l l e r of morphogenetic events. For example, i t i s poss ib le that the mesenchyme c e l l s are being to ld where to go simply by the pre-arranged pattern of matrix present in the b lastocoe le which they are able to i d e n t i f y . In ver tebrate systems, i t i s well documented that f i l o p o d i a of migrating c e l l s i n t e r a c t with ECM, which i s organized in to a three-dimensional array o f f i b r i l s and i n t e r s t i t i a l bodies (T re l s tad et a l . , 1974). In ech ino ids , studies invo lv ing i n h i b i t i o n of GAG and g lycoprote in - 2 3 -synthesis has suggested that the ECM in the b las tocoe le may be involved in c o n t r o l l i n g mesenchyme c e l l movement during morphogenesis (Katow & So lursh , 1979; Akasaka et a l . , 1980; Venkatasubramanian & So lursh , 1984). In a s t e r o i d s , SEM studies have shown that ECM in the b lastocoe le i s located as organized strands in routes of mesenchyme c e l l migrat ion between the endoderm and ectoderm. It was suggested that these form "roadways" f o r c e l l t rave l and thus form one mechanism f o r d i r e c t i n g mesenchyme c e l l movement (Crawford & Chi a, 1981). This means of control i s only r e l a t i v e as the c e l l s do not use these pathways e x c l u s i v e l y . Furthermore, one subpopulation migrates to the esophagus, and only the esophagus, stops and d i f f e r e n t i a t e s in to muscle c e l l s ; t h i s attachment i s character ized by int imate contacts with the BL. Fol lowing t h e i r a r r i v a l , contacts with the e p i t h e l i a l c e l l surface occur in some reg ions . An obvious question then r e s u l t s : "How do the mesenchymal c e l l s which are dest ined to form the muscle know 'where to g o ' ? " . Since the i n i t i a l attachment appears to be with the BL, i s there something d i f f e r e n t about the ECM and/or BL in t h i s region that al lows the presumptive muscle c e l l s to recognize i t ? Indeed i t has been observed (Crawford, 1988) that the BL in t h i s region appears to have less of the dense a l c i a n o p h i l i c material which i s normally attached to the f ib rous meshwork. One p o s s i b i l i t y i s that the BL in t h i s region i s b iochemica l ly d i f f e r e n t , thus prov id ing a recognizable d i f f e r e n c e for migrat ing mesenchymal c e l l s , and d i r e c t i n g them to t h i s region for f u r t h e r d i f f e r e n t i a t i o n . This could represent a developmental l y regulated degradation of the BL, or a l t e r n a t i v e l y , s ince the esophageal BL i s the l a s t BL to have formed in the developing gut (Abed & Crawford, 1986b), i t may simply be "incomplete" at the time of a r r i v a l of the presumptive muscle c e l l s , thus f a c i l i t a t i n g d i r e c t e p i t h e l i a l --24-mesenchymal i n t e r a c t i o n s . In the present study, two d i f f e r e n t techniques ( l e c t i n binding and autoradiography) were used to examine the macromolecular nature of both the BL and the ECM during the development of the esophagus in F\ ochraceus. The purpose o f these studies was to determine whether there were regional d i f fe rences in the carbohydrate composition of the BL and ECM, and s p e c i f i c a l l y , to v i s u a l i z e d i f fe rences between the BL under ly ing the esophagus and that underly ing epi the l ium in other areas o f the embryo. Lect ins represent ing the f i v e major saccharide binding groups were chosen, and were tagged to the markers FITC and c o l l o i d a l gold in order to enable v i s u a l i z a t i o n of the binding s i t e s with the 3 l i g h t and e lec t ron microscope. In a d d i t i o n , autoradiography with H-glucosamine, arabinose, mannose and fucose was undertaken in order to r e v e a l , at the l i g h t microscopic l e v e l , the uptake and d i s t r i b u t i o n of these monosaccharides. -25-2. MATERIALS AND METHODS 2.1 REARING OF PISASTER OCHRACEUS EMBRYOS Glass and P l a s t i c Hare A l l g lass and p l a s t i c ware used in the rear ing of embryos was e x c l u s i v e l y used f o r t h i s purpose, and was not exposed to detergents or chemicals of any k ind . A f t e r use, containers were washed f i r s t with regu lar tap water and then with sea water. The sea water was c o l l e c t e d from V i c t o r i a B.C. rather than Vancouver to avoid f resh water contamination from the Fraser r i v e r . Before use, i t was f i l t e r e d through a Whatman #1 f i l t e r and aerated. P l a s t i c ware was discarded every 2 weeks to avoid contamination from res idual debr is l e f t over from the c u l t u r e s . Preparat ion of Gametes Adult s t a r f i s h were c o l l e c t e d i n t e r t i d a l l y in May and June of 1985 and 1987 at three locat ions on the west coast of B r i t i s h Columbia near Vancouver, V i c t o r i a , and Bamfield. They were placed in running sea water tanks (9-10°C) at the Department of Zoology, UBC, where they were kept under condi t ions of constant l i g h t in order to prevent spawning of gametes. Ovaries were removed from the s t a r f i s h by exc is ing 1 arm and ext rac t ing the organs with forceps . They were placed in a p l a s t i c p e t r i d i s h , then minced and placed in O.lmg/ml 1-methyl adenine in sea water fo r approximately 70-90 min. at 10.5°C, to allow breakdown of the germinal v e s i c l e s . The eggs were then washed two times by sett lement through 500 mis of sea water a f t e r which they were ready f o r f e r t i l i z a t i o n . Testes , i s o l a t e d from the s t a r f i s h in a s i m i l a r manner, were -26-placed in a p l a s t i c pe t r i d ish and kept "dry" u n t i l the eggs were ready for f e r t i l i z a t i o n . At t h i s po in t , a few drops of "dry" undi luted sperm were placed in 25 ml sea water to make a cloudy suspension, and the sperm suspension was examined to check for sperm m o t i l i t y . F e r t i 1 i z a t i o n S u f f i c i e n t washed eggs to cover 1/2-3/4 of the bottom of a 1 l i t e r p l a s t i c beaker were placed in 300-400 ml sea water; they were then f e r t i l i z e d by the add i t ion of 5-10 drops of a c t i v e sperm suspension, and the beakers were aggitated gent ly for 1 min. to ensure even d ispers ion of the sperm to the eggs. Once the f e r t i l i z e d eggs had s e t t l e d on the bottom of the beakers, the sea water was poured o f f and the eggs were resuspended in 500 ml f resh sea water. Fol lowing hatching, sea water conta in ing the embryos was poured in to new beakers to separate the now swimming embryos from the debris on the bottom of the beakers. Development of the embryos and larvae was c a r r i e d out at 10.5°C, and was monitored by microscopic observat ion , u n t i l i t was observed that mouth formation was complete, at which time the embryos were harvested f o r experimental use by low speed c e n t r i f u g a t i o n (120xg f o r 3 min. in 12 ml conica l cent r i fuge tubes) . 2.2 LM AND TEM MORPHOLOGY OF ESOPHAGEAL MUSCLE FORMATION Embryos at the appropr iate developmental stage were harvested and f i xed by immersion in IX glutaraldehyde in 80% sea water, pH 7.0, saturated with a l c i a n blue for 12 hours (see appendix 1) . Fol lowing two 5 min. r inses in 2.5% sodium bicarbonate b u f f e r , the embryos were post f i x e d in 2% 0 s 0 4 with 1.25% sodium bicarbonate, pH 7.4, fo r 1 hr . at room temperature (Wood & L u f t , 1965). This was fol lowed by s t a i n i n g en - 2 7 -bloc in 2% aqueous uranyl acetate fo r 30 min. The embryos were dehydrated through a graded ser ies of ethanols and propylene oxide, and then embedded in Epon 812 ( L u f t , 1961). For LM, 0.5ia sect ions were cut with a g lass kn i fe on a Porter-Blum MT-1 ultramicrotome, dr ied on g lass s l i d e s and post-s ta ined with Richardson's c a t i o n i c dye (0.5% methylene b lue , 0.57. Azure II and 0.5% borax in d i s t i l l e d water, Richardson et a l . , 1960). Sect ions were viewed and photographed on a Zeiss Photo-microscope III with Adox KB17 f i l m at 100 ASA, and the f i l m was developed 5 min. with Kodak D76 f u l l s t rength , r insed in tap water and f ixed for 4 min. in Kodak rapid f i x e r . For TEM, s i l v e r / g r e y sect ions were picked up on carbon/parlodian coated 100 mesh Copper g r i d s , counterstained with 2% uranyl acetate (aq) 8 min. and with Reynold's lead c i t r a t e 5 min. (Reynolds, 1963). They were then examined on a P h i l l i p s 301 EM, and photographed using Kodak Eastman f i n e gra in 5302 35 mm f i l m . The f i l m was developed for 5 min. with Kodak D19 f u l l s t rength , r insed and f i xed as above. 2.3 LECTINS USED FOR THIS STUDY The l e c t i n s used for t h i s study were representat ives of the 6 major carbohydrate-binding groups. These inc lude the mannose/glucose-binding l e c t i n s (Con A ) , the N-acetylgalactosamine-binding l e c t i n s (Dol ichos b i f l o r u s l e c t i n , Soybean a g g l u t i n i n ) the ga lactose-b ind ing l e c t i n s (Peanut l e c t i n , Ric inus communis a g g l u t i n i n ) , the N-acety l -glucosamine-binding l e c t i n s (Wheat germ a g g l u t i n i n ) , the L-fucose-binding l e c t i n s (Ulex Europeus a g g l u t i n i n ) , and the s i a l i c ac id-b ind ing l e c t i n s (Li max f lavus l e c t i n ) . These l e c t i n s , and some of t h e i r p roper t ies per t inent to t h i s study are l i s t e d in tab le 2. -28-2.4 FITC-LECTIN LABELLING OF THE BL & ECM DURING ESOPHAGEAL MUSCLE FORMATION F i x a t i o n and Embedding Animals in the la te g a s t r u l a / e a r l y b i p i n n a r i a stage, in which the mesenchyme c e l l s were j u s t a r r i v i n g at the esophagus, were harvested as above and f i x e d by immersion in a v a r i e t y of f i x a t i v e s f o r F I T C - l e c t i n l a b e l l i n g (see appendix #2). The best f i x a t i v e fo r the l a b e l l i n g studies was determined i n d i v i d u a l l y fo r each l e c t i n . Fol lowing f i x a t i o n , the embryos were dehydrated in a graded ser ies of ethanols as f o l l o w s : 5 min. in each of 30%, 50%, 70%, 2 changes of 5 min. each in 95%, and then embedded in JB4 h y d r o p h i l i c p l a s t i c r e s i n (JEM Sc iences) . l - 2 v i m sect ions were cut on a dry glass k n i f e , and were f l o a t e d on d i s t i l l e d water (dH 2 0) conta in ing 1-2 drops cone. NH40H/100 ml. The sect ions were dr ied on glass s l i d e s and placed in a 40°C oven for 30 min. P r i o r to s t a i n i n g , the sect ions were rehydrated in PBS for 5 min. FITC S ta in ing Sect ions were incubated in a dark moist chamber (para f i lm on moist f i l t e r paper in a pet r i d ish) fo r 1-8 hr . in 40 pi of l e c t i n so lut ions or l ec t in/sugar contro ls (the F I T C - l e c t i n s , t h e i r sources and the conjugate sugars used are l i s t e d in tab le 3 ) . The l e c t i n s were d i sso lved in PBS (see appendix #3) to make a f i n a l concentrat ion of 200 yig/ml PBS. The sugars used for contro ls were d isso lved in PBS to make a f i n a l concentrat ion of 1.0 M, and then added to the l e c t i n so lut ions in a 1:1 r a t i o , a f t e r which they were mixed by p ipet te fo r 30 min. p r i o r to s t a i n i n g . Fol lowing s t a i n i n g , the s l i d e s were washed 2 x 5 min. in PBS and 1 x 5 min. in d i s t i l l e d water before mounting c o v e r s l i p s with gelvatol/DABCO a n t i - f a d i n g mounting medium (Johnson et a l . , 1982; Taylor & Heimer, 1974). For r e c i p e , see appendix 4. - 2 9 -Microscopy S l i d e s were viewed with a Zeiss Photo-microscope III equipped with ep i f luorescence ; photographs were taken on I l f o r d HP5 f i l m , developed 17 min. with I l f o r d Microphen at f u l l strength to give an ASA of 3200, r insed and f i xed as above. 2.5 COLLOIDAL GOLD LECTIN LABELLING OF THE BL & ECM DURING ESOPHAGEAL MUSCLE FORMATION F i x a t i o n and Embedding Embryos at the appropriate developmental stage ( l a t e gast ru la/ ear ly b i p i n n a r i a ) were harvested and f i xed by immersion in a v a r i e t y of f i x a t i v e s (see appendix #1). Fol lowing f i x a t i o n , the embryos were dehydrated through a graded ser ies of ethanols (1 x 5 min. each in 30%, 50%, 70%, and 2 x 5 min. each in 95%, 100%) and then i n f i l t r a t e d with Lowicryl K4M. I n f i l t r a t i o n in to the Lowicryl res in was done gradual ly as fo l lows : 1 x 30 min. in 20%, 40%, 60%, 80% Lowicryl in 100% ethanol at room temperature, and then 2 x 2 hr . in pure Lowicryl at -2°C. Po lymer izt ion was performed in g e l a t i n capsules with UV r a d i a t i o n at -2°C f o r 24 hr . The polymerizat ion chamber cons isted of 1-15 watt f luorescent tube placed 20 cm from the g e l a t i n capsules in a box l ined with aluminum f o i l . Fol lowing po lymer izat ion , the capsules were U.V. cured a f u r t h e r 2 days at room temperature. S i l v e r / g r a y sect ions were picked up on carbon/parlodion coated 100 mesh copper or n ickel g r i d s . -30-Preparat ion of C o l l o i d a l Gold C o l l o i d a l gold was prepared using the c i t r a t e method character ized by Frens (1973), which involved the reduct ion o f ch lo roaur i c ac id (Sigma) with a sodium c i t r a t e (EM Sciences) s o l u t i o n to give monodisperse so ls having a mean diameter of =25nm ( A u 2 g ) . For d e t a i l s , see appendix #5. Preparat ion of Lect in-Gold Conjugates Lec t in -go ld conjugates were prepared using a method adapated from Morr is et a l . (1986). This involved an i n i t i a l assay to determine the appropr iate concentrat ion of each l e c t i n to add to the gold sol to ensure s t a b i l i z a t i o n fol lowed by the conjugation i t s e l f . A f t e r the conjugation step, each conjugate was washed twice in 5 mM phosphate buf fer and then d ia lysed to br ing the prote ins back up to t h e i r p h y s i o l o g i c a l s t rength . Deta i led procedures fo r both the assay and the conjugation steps can be found in appendices #6 and #7, and l e c t i n concentrat ions used for the conjugation are l i s t e d in tab le 4. Sugar contro ls were prepared by adding 0.1 ml of 1 M i n h i b i t o r y sugar so lut ions (see table 2) to 0.9 ml of l e c t i n - g o l d conjugate; th i s was ag i tated by p i p e t t i n g for 1 min, allowed to stand 1 h r . , and then used to s t a i n as above. Lect in-Go ld L a b e l l i n g P r i o r to actual s t a i n i n g , the gr ids were preincubated in a moist chamber on drops of t r i s bufferred s a l i n e (TBS), which was adjusted to the pH of the i s o e l e c t r i c point (p i ) of the p a r t i c u l a r l e c t i n or control sugar/conjugate so lu t ion f o r 5 min. at room temperature (see table 2) . They were then placed on drops of l e c t i n - g o l d conjugates or sugar - 3 1 -control conjugates fo r 30 min. Fol lowing t h i s , the gr ids were washed with a steady stream of d i s t i l l e d water fo r 10 sec, and counter stained with 2% uranyl acetate (aq) fo r 5 min, fol lowed by lead c i t r a t e fo r 1 mi n. Microscopy Stained gr ids were observed and recorded using a P h i l i p s 301 TEM with Kodak Eastman f i n e grain 5302 35 mm f i l m , which was processed as above. Morphometry Morphometric ana lys i s was done on p r i n t s from e lect ron micrographs, which were taken at a magni f icat ion of x2,000. For measurement of both the length of basal lamina and counting of gold p a r t i c l e s , negatives were pr inted at an enlargement of x 15,000. Counts were recorded in 4 - 8 random areas of s p e c i f i e d length; areas were considered random, as low magni f icat ion photographs in which gold p a r t i c l e s were not with in reso lv ing power were taken. The tota l l i n e a r length of BL measured for each region was « 0.5 urn. -32-2.6 AUTORADIOGRAPHY OF 3H-SUGARS INCORPORATED INTO LATE GASTRULA EMBRYOS L a b e l l i n g o f Embryos 0.4 ml of concentrated embryos were l a b e l l e d in 150 ml p l a s t i c ur ine specimen cups, in 12 ml sea water conta in ing 250>iCi of one of •3 "3 3 3 H-glucosamine, H-fucose, H-arabinose or H-mannose at 12°C. The beakers were rotated on a shaker at 12°C f o r 6 hr . The embryos were then c e n t r i f u g e d , washed 2 times in f resh f i l t e r e d sea water, resuspended in larger volumes of sea water in new p l a s t i c beakers, and rotated at 12°C as above fo r chase times of 3, 9 or 18 hr. Fol lowing t h i s , the embryos were again i s o l a t e d by c e n t r i f u g a t i o n , f i xed by immersion in 1% glutaraldehyde in 80% sea water, pH 7.0, saturated with a l c i a n blue fo r 10 hr . at room temperature, fol lowed by dehydration and embedding in JB4 or Epon 812 as above. High Speed Scintillation Autoradiography. S l i d e Preparat ion P r i o r to use, s l i d e s were cleaned in a mixture of 35 ml saturated sodium bichromate and 1 l i t e r concentrated s u l f u r i c ac id fo r 2 hr , fol lowed by an overnight wash in tap water. They were then washed twice fo r 1 hr . in d i s t i l l e d water with constant s t i r r i n g , covered, and put in a 40° oven to dry . From t h i s point on, the s l i d e s were handled with g loves . Sect ion i ng 0 . 5 - 2 . 0 jam sect ions of JB4 embedded material were prepared as above. -33-Emulsion Preparat ion/Dipping The emulsion ( I l f o r d K.2) was melted in complete darkness in a constant temperature water bath set at 40°C, which had been allowed to e q u i l i b r a t e fo r 1 hr . before us ing. Only enough emulsion was melted to make about 25 ml. During mel t ing , the emulsion was s t i r r e d gent ly with a g lass rod . A f t e r 45 min, a clean s l i d e was dipped in i t to t e s t for completeness o f melt ing and for a i r bubbles. Once the emulsion was ready, the s l i d e s were placed 2 at a time in an automatic dipping machine (V. A v a r l a i d ) , and were dipped f o r 5 s. A f t e r d i p p i n g , they were dr ied upr ight with the emulsion end fac ing up f o r 1-2 hr . at room temperature. For high speed autoradiography the dr ied emulsion coated s l i d e s were dipped in Aquasol fo r 5 min. (Baserga et a l . , 1969; Durie et a l . , 1975; Goldgefter et a l . , 1976; and Wolfe 1976) before being stored in l i g h t t i g h t boxes conta in ing a small amount of D r i e r i t e . The boxes were wrapped with aluminum f o i l and stored in a -70°C f r e e z e r , to ensure low background. Test s l i d e s were developed at 3 day i n t e r v a l s to determine the cor rec t exposure time. Developing In complete darkness, and at room temperature, s l i d e s were placed in g lass s t a i n i n g holders fo r 100 ml c o p l i n j a r s , and the s c i n t i l l a t i o n f l u o r was removed by soaking them in a decreasing ethanol ser ies (5 min. each in 95%, 80%, 70%, and 50%). The s l i d e s were then developed for 5 min. in Kodak D19 f u l l strength with occass ional a g i t a t i o n , r insed b r i e f l y in water and f ixed in Kodak Rapid F i x , d i l u t e d 1:1 with tap water, f o r 5 min. Fol lowing t h i s , the s l i d e s were r insed twice f o r 5 min. in d i s t i l l e d water, and stained immediately. -34-Sta in inq and Microscopy The s l i d e s were stained for 30 sec. with Richardson's s ta in (Richardson et a l . , 1960) at room temperature, washed for 10 min. in 2 changes of d i s t i l l e d water, and then covers l ipped . They were then viewed and photographed as above on a Zeiss Photo-microscope I I I . TABLE 2: LECTINS AND THEIR PROPERTIES LECTIN SOURCE ABBREV Concanavalin A Sigma Con A 106,000 (Canaval is ens i formis) Wheat germ (Tr i t i cum vulqare) Soybean (Glycine max) Castor bean (Ric inus communis) Horse gram (Dolichos b i f l o r u s ) Peanut (Arachis hypogaea) Slug (Limax f lavus) Gorse or Furze Seed (Ulex Europaeus-I) Sigma WGA Sigma SBA Sigma RCA Vector DBA 43,200 120,000 120,000 120,000 Vector PNA 111,000 PI 7.1 (Agrawal & Goldstei n 167) 8.5 (Monsigny et a l . ' 7 9 ) 5.8 (Lotan et al '74) 7.8 (Wei & Koh'78) 4.5 ( E t z l e r & Kabat'70) 6.7 (Metler'83) Sigma LFA 44,000 9.0-9.5(*) Sigma UEA-I 60,000-68,000 SUGARS NHICH INHIBIT AGGLUTINATION* man-ccl ,2man-al ,2man> <x-man>a-gl c glcNAcGl,4glcNAcBl,4glcNAc> glcNAcBl,4glcNAc>glcNAc a-galNAc=6-galNAc>a-gal gal>galNAc galNAc-al,3galNAc> a-gal NAc galBl,3galNAc>galNH2>gal Neu5Ac>Neu5Gc a-L-Fucose CONTROL SUGARS USED [1 M] (mg/ml) mannose 180 glcNAc 220 galNAc 220 galactose 180 galactose 180 Neu5Ac 310 L-Fucose 164 *Goldstein & Poretz , 1986 -36-TABLE 3: FITC-LECTINS AND SPECIFIC INHIBITORS USED IN THIS STUDY LECTIN SOURCE BLOCKING SUGAR FITC-WGA EY LAB N-acetyl-D-glucosamine FITC-SBA Sigma N-acetyl-D-galactosamine FITC-Con A Sigma Methyl-D-mannnoside FITC-RCA Sigma B-D-Galactose FITC-UEA-I Sigma L-fucose TABLE 4: LECTIN CONCENTRATIONS FOR CONJUGATION TO GOLD Lect in Amount of prote in needed to s t a b i l i z e 10 mis A U 2 5 (1*9) Con A 200 WGA 100 SBA* 100 PNA* 130 DBA 200 LFA 200 *Roth, 1983 - 3 7 -Appendix #1: Fixatives Used For TEM and Colloidal-Gold Labelling * A l l f i x a t i v e s should have a f i n a l pH of 7 . 0 - 7 . 5 , and a f i n a l osmolar i ty of 900-1000 mOs. 1. 1% Glutara ldehyde. a l c i a n blue in sea water. (Crawford & Abed. 1986) 257. g lutaraldehyde 0.4 ml 17. a l c i a n blue in sea water (saturated)^ 7.8 ml d i s t i11ed water 1.8 ml 1 S t i r a l c i a n blue in sea water 2-8 hr, and f i l t e r before use. 2. Dunlaps Phosphate buffered g lutara ldehyde. (Dunlap. 1965: Cloney &  F lo rev . 1968) Stock A: 257. glutaraldehyde 5 ml 0.34 M NaCl 20 ml Stock B: 0.4 M M i l l o n i g s phosphate buf fer 25 ml Mix stock A and B 1:1. 3. 27. Paraformaldehyde. 0.17. glutaraldehyde in PBS. 87. paraformaldehyde 2 257. g lutaraldehyde PBS 1 part 0.16 parts 3 parts 2 T o make up paraformaldehyde s o l u t i o n , d i s s o l v e dry paraformaldehyde in water at 60°C and add 1 N NaOH drop by drop u n t i l c l e a r . -38-Appendix #2: Fixatives Used For FITC-Lectin Labelling * A l l f i x a t i v e s should have a f i n a l pH of 7 . 0 - 7 . 5 , and a f i n a l osmolar i ty o f 900-1000 mOs. 1. Paraformaldehyde, ce ty lpvr id in ium c h l o r i d e (CPC) in sea water 8% paraformaldehyde 1 1 part 0.04% CPC 1 part sea water 2 parts TTO make up paraformaldehyde s o l u t i o n , d i s s o l v e dry paraformaldehyde in water at 60°C and add 1 N NaOH drop by drop u n t i l c l e a r . 2. Paraformaldehyde, a l c i a n blue in sea water 1% a l c i a n blue in sea water ( s a t u r a t e d ) 2 7 parts 8% paraformaldehyde 3 parts 2 S t i r a l c i a n blue in sea water 2-8 hr; f i l t e r before use. 3. Paraformaldehyde. CPC in M i l l o n i q ' s 8% paraformaldehyde 1 part 0.04% CPC 1 part M i l l o n i g ' s phosphate buf fer (0.2M) 2 parts 4. Paraformaldehyde in sea water with 2° f i x a t i o n of  paraformaldehyde/CPC in sea water 1° F i x : 8% paraformaldehyde 2 parts sea water 2 parts 2° F i x : 8% paraformaldehyde 1 part 0.04% CPC 1 part sea water 2 parts Boui n's saturated (aq) p i c r i c ac id 15 parts formal in (40% formaldehyde) 5 parts g l a c i a l a c e t i c ac id 1 part 6. 95% Ethanol - 3 9 -Appendix #3: Buffers 1. Phosphate buffered s a l i n e (Crawford. 1972) Stock S o l u t i o n s : Sa l ine G Stock V Sa l ine G Stock VI NaCl 160.0g MgS0 4 .7H 2 0 15.4'g KC1 8.0g CaCl2-2H 2 0 1.6g KH2PO4 3.0g or C a C l 2 . 6 H 2 0 2.4g Na2HP04.7H20 5.8g Disso lve in dH20 to 1000 ml Disso lve separate ly in dr^O; to ta l volume = 1000 ml. To make a working s o l u t i o n , d i s s o l v e : Sa l ine G Stock V 50 ml Sa l ine G Stock VI 50 ml in dH 20 to 1000ml; adjust pH to 7.4. 2. M i l l o n i a ' s phosphate b u f f e r . 0.2M ( M i l l o n i q . 1961) NaH 2 P04.H 2 0 11.08g NaOH 2.85g D isso lve in dr^O to make 400 ml 3. Sorensen's phosphate buffer (Humason. 1962) N a 2 H P 0 4 5.68g KH2PO4 1.35g Disso lve s a l t s separate ly ; add dH20 up to make 250 ml 4. T r i s Buffered Sa l ine (TBS) 0.5 M T r i s Stock, pH 7.6: Trizma (Sigma) 60.57g dH 20 500 ml 1 N HC1 370 ml D isso lve Trisma in dH 2 0; add HC1 to pH 7 .6 , and f i l l to 1000 ml with dH 2 0. -40-To make TBS, d i s s o l v e : NaCl KC1 Sa l ine G Stock VI (Ca, Mg) in 0.5 M T r i s stock, pH 7.6 8.0g 0.4g 50 ml 100 ml 5. Sodium Bicarbonate 2.5% (Mood & L u f t . 1963) NaHC03 dH 20 2.5g 100ml Adjust pH to 7.2 p r i o r to use. Appendix # 4: Gelvatol/DABCO "Anti fading" mounting medium for fluorescence microscopy. (Tay lor & Heimer, 1974; Johnson et a l . , 1982,) . 1. D isso lve 20g ge lvato l 20/30 (Monsanto) in 80 ml 0.2 M T r i s (2.42 g Trisma/100 ml, pH 8.6) by s t i r r i n g overn ight . 2. D isso lve 3.75 g 1,4 Diazob icyc lo [2 .2 .2] octane, DABCO, (A ld r i ch) in 40 ml g lycero l at 50-60°C. 3. Add g lycero l to ge lvato l mixture and s t i r . 4. Remove undissolved ge lvato l by c e n t r i f u g a t i o n (5000 x g fo r 20 min) or decantat ion . 5. A l i q u o t and store at -20°C. - 4 1 -Appendix #5: Preparat ion o f C o l l o i d a l Gold (S lo t & Geuze, 1985) To make 100 mis: 1. Stock S o l u t i o n s : So lut ion I : 1 ml of 1% HAuCl 4 in 79 mis ddH 2 0. So lut ion I I : Reducing mixture 4 ml IX t r i - sod ium c i t r a t e 2H2O 0-5 ml IX tannic ac id 25mM K2CO3 ddH20 to br ing volume to 20 ml 2. Bring both I & II to 60°C separate ly . 3. Add reducing mixture to so lut ion I qu ick ly while s t i r r i n g ; keep at 60° to avoid a heterodisperse s o l . 4. A f t e r sol has turned red , heat to b o i l i n g and store at 277K. NB: react ion time w i l l increase a to p a r t i c l e s i ze of g o l d . 5. The p a r t i c l e s i z e can be c o n t r o l l e d by adding d i f f e r e n t amounts of tannic ac id as fo l lows : Volume IX Tannic Ac id Volume 25mM K2CO3 P a r t i c l e S ize 2.0 ml 2 ml 4nm 0.5 ml / 6nm 0.125 ml / 10-15nm 0.03 ml / 20-25nm 0.0 ml / 20-25nm -42-Appendix #6: MICROTITRATION ASSAY FOR DETERMINATION OF OPTIMAL [PROTEIN] REQUIRED TO STABILIZE COLLOIDAL GOLD. (Hor isberger , 1981) 1. Add 100 ul dH20 to m i c r o t i t e r w e l l . 2. Add 100 ul of 1 mg/ml prote in in dH20 to f i r s t w e l l . 3. S e r i a l l y d i l u t e to 9 t h w e l l ; leave 1 0 t h well p rote in f r e e . 4. Adjust gold sol to appropr iate pH f o r prote in using 0.2 M K 2 C 0 3 . * 5. Add 500ul gold sol to each well and mix by p i p e t t i n g up and down several t imes. Al low to stand for 15 min. 6. To assess the res i s tance of the mixture to sa l t - induced f l o c c u l a t i o n , add lOOuls of 10% NaCl and l e t stand for 5 min. The NaCl so lu t ion w i l l coagulate the u n s t a b i l i z e d gold p a r t i c l e s , i . e . those p a r t i c l e s not s u f f i c i e n t l y coated with p r o t e i n . 7. Coagulat ion i s judged v i s u a l l y by the colour changing from red to v i o l e t and f i n a l l y to l i g h t b lue . The l a s t well which remains red represents the d i l u t e d endpoint, i . e . that concentrat ion of p rote in which i s j u s t able to s t a b i l i z e the c o l l o i d a l go ld .** 8. The f i n a l concentrat ion of prote in used for conjugation should be double the amount determined in step 7 to ensure s u f f i c i e n t p ro te in to bind up a l l a v a i l a b l e s i t e s on the gold c o l l o i d . * The pH value o f the c o l l o i d a l gold i s a c r u c i a l parameter when assess ing the success of conjuat ion . Strong adsorpt ion of macromolecules r e s u l t i n g in stable probes occurs at values c lose to or s l i g h t l y basic to the i s o e l e c t r i c point ( p i ) of a given p r o t e i n , because at these pH va lues , the z w i t t e r i o n form of the prote in i s dominant and the i n t e r f a c i a l tension i s maxiamal. ** I f tannic ac id i s used in c o l l o i d a l gold prep, co lor change may be slow, because of the masking e f f e c t of polymers formed by the excess TA. The excess TA can be broken down by adding 0.1 - 0.2% H 202 to the so ls a f t e r which s a l t induced f l o c c u l a t i o n i s f a s t and c l e a r . -43-Appendix #7: PREPARATION OF LECTIN-GOLD CONJUGATES. (Morris & Sae l inger , 1986; De May, 1983) 1. At room temperature, adjust pH of 10 mis gold sol at or j u s t to the basic s ide of the p i fo r the l e c t i n to be conjugated (See tab le 2 ) . 2. O p t i o n a l : Centr i fuge gold sol 5 min. x 7000g to remove large aggregates. 3. Prepare the l e c t i n so lut ions by d i s s o l v i n g s a l t - f r e e l e c t i n s in dH20 immediately p r i o r to use. I f the l e c t i n s are a l ready in s o l u t i o n , they should be d ia lyzed f i r s t against water or against very low molar i ty NaCl, in order to prevent s a l t s from i n t e r f e r i n g with adsorpt ion to the gold c o l l o i d . For concentrat ions used in t h i s study, see tab le 2. 4. Add 0.5 ml of l e c t i n so lu t ion a l l at once to 10 ml gold sol and s t i r 30 min. at room temperature. Add i t ion o f a s t a b i l i z e r (M PEG 20,000 MW to give f i n a l concentrat ion of 0.5mg/ml) at t h i s point i s opt ional and i f poss ib le should be put o f f u n t i l a f t e r c e n t r i f u g a t i o n , as the PEG causes aggregate format ion. 5. Centr i fuge 30 min. x 15,000g at 277K ( fo r A u£o ) . or u n t i l supernatent i s no longer red . NB: smal ler gold p a r t i c l e s ize w i l l requi re longer c e n t r i f u g a t i o n time. 6. C a r e f u l l y a s p i r a t e the supernatant conta in ing f ree p r o t e i n . Then, resuspend the loose dark red coloured p e l l e t in 5mM phosphate buf fer at appropr iate pH. T i g h t l y p e l l e t e d material should not be resuspended as t h i s introduces large aggregates in to the conjugate s o l u t i o n . 7. Repeat steps 4 & 5 twice, to ensure removal of f ree prote in and gold p a r t i c l e s which have not been f u l l y s t a b i l i z e d . 8. Add 1% PEG (MW 20,000) to give f i n a l concentrat ion o f 0.5 mg/ml (approx. 7 5 u l / l . 5 m l s ) . This s t a b i l i z e r minimizes aggregation and blocks remaining adsorpt ion s i t e s . 9. D ia lyze conjugate against buf fers* of inc reas ing strength over a 24 hour per iod (4°C) to br ing i t up to p h y s i o l o g i c a l s t rength: a) 50 mM T r i s b) 50 mM T r i s with 50 mM NaCl c) 50 mM T r i s with 100 mM NaCl d) 50 mM T r i s with 150 mM NaCl Found to be successful fo r a l l l e c t i n s accept PNA, which was d i a l y s e d only to 50 mM T r i s with 100 mM NaCl. * a l l buf fers adjusted to appropr iate pH. 10. Store at 4°C and use with in 1 - 4 weeks. 11. Just p r i o r to use, centr i fuge conjugates fo r 5 min. x 7000g (277K) to remove large aggregates. -44-3. RESULTS 3.1 FORMATION OF THE ESOPHAGEAL MUSCULATURE The c e l l s which form the esophageal musculature are der ived from a populat ion of u n d i f f e r e n t i a t e d mesenchymal c e l l s , which bud o f f from the t i p of the archenteron and form coeloms at about 3 1/2 days of development. These mesenchymal c e l l s migrate through the ECM-rich b l a s t o c o e l , while the development o f the a l imentary canal cont inues. A f t e r 5 1/2 days of development, the endoderm has become segmented to form the stomodeum, esophagus, stomach and i n t e s t i n e ( F i g . l ) . At t h i s t ime, some of the mesenchymal c e l l s come to rest e x c l u s i v e l y on the presumptive esophageal endoderm ( F i g . 2 ) . With continued d i f f e r e n t i a t i o n , the mesenchymal c e l l s begin to e x h i b i t muscle f i b e r s and a t t a i n a more int imate contact with the esophageal BL v i a evaginat ions ( F i g . 3 ) . These evaginations appear to break through the BL in some areas ( F i g . 4 ) . By 15 days of growth, the esophageal muscle wall i s complete, and i s character ized by an i n t e r l o c k i n g o f muscle c e l l s forming a smooth muscle sheath around the esophageal endoderm ( F i g . 5 ) . -45-F i g . 1: A l .Ou saggi ta l sect ion of a g lutara ldehyde/a lc ian b l u e - f i x e d 5 1/2 day embryo, showing the segmented al imentary canal c o n s i s t i n g of the stomodeum ( S t ) , esophagus (Es) , stomach ( S ) , and i n t e s t i n e ( I n ) . Note the mesenchymal c e l l s (arrows) s e t t l i n g on the esophagus. x300 -46-- 4 7 -Fig. 2: A TEM through the esophagus of a 5 day embryo f ixed with g lutara ldehyde/a lc ian b lue, showing two mesenchymal c e l l s (Me) s e t t l i n g on the esophageal endoderm (En) . x l2,000 Fig. 3: A TEM through the esophagus of a 6 day embryo f i xed with g lutara ldehyde/a lc ian b lue , showing the d i f f e r e n t i a t i n g mesenchymal c e l l s (Me) at taching to the esophagus ( E s ) . Of i n t e r e s t are the evaginations of the mesenchymal c e l l s , some of which contact the esophageal BL (arrowheads). Muscle f i b e r s are beginning to appear in the mesenchymal c e l l s , and are seen here in X-sect ion (square) . x23,100 -49-Fig. 4: A TEM through the esophagus of a 6 day embryo f ixed with g lutara ldehyde/a lc ian b lue, showing the i n t e r a c t i o n s of two mesenchymal c e l l s (Me) with the esophagus ( E s ) . Of i n t e r e s t are the mesenchymal evaginations which in some cases appear to t raverse the BL (arrows) and make contact with the esophageal endoderm. x57,600 50 - 5 1 -Fig. 5a: A TEM of a c r o s s - s e c t i o n through the esophagus of a 15 day embryo f i xed with g lutara ldehyde/a lc ian b lue , showing the esophageal endodermal c e l l s on the i n s i d e of the tube, and the muscle c e l l s (Mu) wrapping around the outs ide of the tube. x2300 Fig. 5b: A higher magni f icat ion of the above, showing a muscle c e l l in X - s e c t i o n . The muscle f i b e r s are r e a d i l y v i s i b l e (arrows). X19 .800 52 - 5 3 -3.2 FIXATION L ight Microscopy Several f i x a t i o n s were attempted in order to achieve a balance between optimal c e l l u l a r preservat ion and maximal carbohydrate p r e c i p i t a t i o n . F ixa t i ves conta in ing glutaraldehyde were not used in the study because glutaraldehyde creates an induced f luorescence in t i ssues as a r e s u l t of i t s i n t e r a c t i o n s with a r y l e t h y l amines, which i n t e r f e r e d with observat ion of F I T C - l e c t i n l a b e l l i n g . The 3 f i x a t i v e s , 95% ethanol , paraformaldehyde in sea water, and paraformaldehyde/CPC in sea water were a l l s a t i s f a c t o r y , but each one had i t s strengths and weaknesses. Although 95% ethanol gave very poor c e l l p r e s e r v a t i o n , inc lud ing poor BM and ECM f i x a t i o n , i t preserved the hya l ine l a y e r , an outer ECM surrounding the embryo, very w e l l . The hya l ine layer of t i s s u e f ixed with 95% ethanol showed intense f luorescence a f t e r s t a i n i n g with SBA, RCA and Con A, but v i r t u a l l y no s t a i n i n g with WGA was noted. Fresh ly prepared paraformaldehyde in e i t h e r sea water or M i l l o n i g ' s phosphate buf fer gave better c e l l u l a r preservat ion than did the ethano l . There was l i t t l e or no d i f f e r e n c e in t i s s u e preservat ion between the f i x a t i v e buffered in sea water and that in M i l l o n i g ' s , but l a b e l l i n g was more intense in t i s sues f i xed with the former, and i t therefore became the buf fer of choice . Add i t ion of CPC to the t h i s f i x a t i v e gave better preservat ion of the BM and the ECM (F igs .6a ,6b) than an e i t h e r paraformaldehyde or glutaraldehyde a lone, although the c e l l u l a r preservat ion was poor. Con A and WGA l a b e l l i n g was observed fo l lowing t h i s f i x a t i o n , however SBA l a b e l l e d very poor ly and RCA did not label at a l 1 . The use of paraformaldehyde with 1% a l c i a n blue in 80% sea water gave e x c e l l e n t preservat ion of c e l l s and ECM, but t i s s u e preserved in -54-Fig. 6a: A l.Oia sect ion of a g lutara ldehyde/a lc ian b l u e - f i x e d embryo showing the ectoderm ( E c ) , the stomach endoderm (En), and some hint of ECM in the b l a s t o c o e l . Note that the BM i s not r e a d i l y v i s u a l i z e d in t h i s preparat ion . x2000 Fig. 6b: A l .Ou sect ion of a paraformaldehyde/CPC-fixed embryo, showing the ectoderm ( E c ) , and a mesenchymal c e l l (Me). Note that in t h i s preparat ion , we l l -de f ined strands of ECM can be v i s u a l i z e d in the b lastocoel (arrowheads). x2200 55 -56-t h i s manner did not label with any of the FITC-1 e c t i n s . T issue f i xed in Bouin's was genera l ly not very recept ive to l e c t i n b ind ing; however, some granules in the c e l l s l a b e l l e d i n t e n s e l y with both Con A and WGA. The general t i s s u e morphology was not as good as that of t i s sues f i xed in paraformaldehyde. The e f f e c t of each f i x a t i v e on the c e l l preservat ion and l e c t i n binding are summarized in table 5. TABLE 5: THE EFFECT OF VARIOUS FIXATIVES ON FITC-LECTIN BINDING SITES FIXATIVES (1) Paraformaldehyde/CPC in sea water (2) Paraformaldehyde/alcian blue in sea water (3) Paraformaldehyde/CPC in Mi l lon igs (4) 1°: Paraformaldehyde in sea water 2° : Paraformaldehyde/CPC in sea water (5) Bouins (6) 95% Ethanol COMMENTS •poor preservat ion of c e l l s (due to CPC) •good s ta in ing of BL and ECM with WGA & Con A •poor s ta in ing of HL accept with WGA •moderate s ta in ing of granules with WGA only •good preservat ion of c e l l s , BL, ECM & HL •no s ta in ing with any l e c t i n s •similar to ( 1 ) , but decreased s ta in ing of BL & ECM with WGA & Con A •staining s i m i l a r to (1) , with no improvement of c e l l u l a r preservat ion . poor c e l l preservat ion •excellent s ta in ing of granules for WGA & Con A poor s ta in ing of HL, ECM & BM for NGA, Con A, SBA & RCA very poor c e l l preservat ion excel lent s ta in ing of HL with RCA, SBA, & Con A moderate s ta in ing of HL with WGA poor s ta in ing of BM with Con A & WGA moderate s ta in ing of ECM with Con A & WGA -58-Fixation-TEM Since g lutara ldehyde-re la ted induced f luorescence was not a concern with the TEM s t u d i e s , glutaraldehyde formed the basis f o r the two f i x a t i o n s used. The standard f i x a t i v e quoted in the l i t e r a t u r e fo r use when process ing t i s s u e in Lowicryl K4M r e s i n i s 0.1% glutara ldehyde, 2% paraformaldehyde in PBS. This f i x a t i v e was t r i e d i n i t i a l l y , but was not used for the major i ty of the l e c t i n - b i n d i n g studies because there was no d i f f e r e n c e in l e c t i n - b i n d i n g between t h i s f i x a t i o n and those described below, and the l a t t e r f i x a t i v e s gave better u l t r a s t r u c t u r a l preservat ion of t h i s m a t e r i a l . The 2 f i x a t i o n s which were used f o r the study were Dunlap's phosphate buffered g lutara ldehyde, and glutaraldehyde with a l c i a n blue in sea water. In genera l , Dunlap's f i x a t i v e preserved the t i s s u e very w e l l , e s p e c i a l l y the BL and ECM. F ixat ion of the hyal ine layer was very poor, however, and consisted of a th in band of m a t e r i a l , i n d i c a t i n g that qu i te a b i t was l o s t during the process ing . A l l l e c t i n - b i n d i n g s i t e s were preserved well with t h i s f i x a t i v e , and in f a c t , l a b e l l i n g of the basal lamina and ECM appeared to be s l i g h t y more intense with Con A, DBA and LFA a f t e r t h i s f i x a t i o n than a f t e r f i x a t i o n in g lutara ldehyde/a lc ian b lue . The combined glutaraldehyde and a l c i a n blue f i x a t i v e was selected f o r use as i t gave better u l t r a s t r u c t u r a l preservat ion of the t i s s u e in general and of the hyal ine l ayer , basal lamina and ECM in p a r t i c u l a r . Good l a b e l l i n g was achieved using t i s s u e preserved with t h i s f i x a t i v e suggesting that the a l c i a n blue did not i n t e r f e r e s i g n i f i c a n t l y with the l e c t i n - b i n d i n g s i t e s . In a d d i t i o n , i t was found that t h i s f i x a t i v e gave bet ter u l t r a s t r u c t u r a l preservat ion of the hyal ine l a y e r , than did -59-Dunlap's f i x a t i v e . 3.3 FITC-LECTIN LABELLING Lect in L a b e l l i n g Patterns of the Basement Membrane and ECM Although the s t ructure of the basement membrane (BM) in t h i s t i s s u e i s too th in to be s a t i s f a c t o r a l l y resolved with the LM a f t e r s ta in ing with R ichardson 's , a s t ructure located in the region of the BM i s r e a d i l y v i s u a l i z e d a f t e r s t a i n i n g with FITC l a b e l l e d reagents. Some d i f f i c u l t y a r i s e s , however, when d i f f e r e n t i a t i n g l a b e l l i n g of the BM from that of the dense ECM associated with i t , and therefore no attempt w i l l be made to separate the two in the d e s c r i p t i o n . L a b e l l i n g with both Con A and WGA was observed in PF/CPC-fixed t i s s u e . Con A densely l a b e l l e d the ectodermal and endodermal BM in a l l regions of the embryo, except fo r the esophagus (both dorsal and v e n t r a l ) and the p o s t e r i o r aspect of the stomach ( F i g . 7 a ) . Very intense l a b e l l i n g of the BM and assoc iated ECM was present along the dorsal ectoderm ( F i g . 8 a ) , which was quite d i f f e r e n t than the s t a i n i n g pattern along the stomodeum and esophagus. Along the stomodeum, l a b e l l i n g of the BM and ECM exhib i ted a patchy appearance ( F i g . 8 c ) , while l i t t l e or no l a b e l l i n g was present along the BM of the esophagus ( F i g . 8 b ) . At higher m a g n i f i c a t i o n , l a b e l l i n g of the ECM resembled granules or beads arranged along an " i n v i s i b l e s t r i n g " ( F i g . 8 d ) . The major i ty o f the BM and assoc iated ECM a lso l a b e l l e d densely with WGA, again with the exception of the dorsal and ventral esophageal regions and the region p o s t e r i o r to the stomach ( F i g . 9 a ) . As with Con A, dense l a b e l l i n g of the BM/ECM underly ing the dorsal ectoderm was observed ( F i g . 1 0 a ) . As w e l l , l a b e l l i n g continued around to the area of the stomodeum, where i t exhib i ted a l i n e a r appearance rather than the -60-patchy one seen with Con A. In the region of the esophagus, the BM exh ib i ted l i t t l e or no s t a i n i n g , as with Con A. L i t t l e s ta in ing was a lso noted beneath the endoderm of the p o s t e r i o r stomach ( F i g . 10c). The s t a i n i n g of the ECM in a l l regions of the embryo appeared f i lamentous and not granular as was the s t a i n i n g with Con A. In the region between the esophagus and ectoderm, there was intense l a b e l l i n g of a "web" of matrix both in the dorsal and ventral reg ion . No l a b e l l i n g of the BM or assoc iated ECM was observed a f t e r s ta in ing with e i t h e r SBA or RCA. Contro ls In a l l cases the l e c t i n s d id not bind s p e c i f i c a l l y to any t i s s u e in the embryos when sect ions were incubated with the control s ta ins (0.1M i n h i b i t o r y sugar so lu t ion incubated with F I T C - l e c t i n conjugate) . - 6 1 -Fig. 7a: A sagg i ta l sect ion of a 6 day embryo, f i xed with PF/CPC, and sta ined with FITC-Con A. The regions immediately beneath the dorsal ectoderm and dorsal stomach ( large arrowheads) label densely, while moderate l a b e l l i n g i s present beneath the e p i t h e l i a in the other areas. Of i n t e r e s t i s the esophageal reg ion , which in t h i s case shows l i t t l e or no l a b e l l i n g , both d o r s a l l y and v e n t r a l l y (small arrowheads), where the BM i s l o c a t e d . x480 Fig. 7b: An adjacent saggi ta l sect ion of the above, stained with the control sugar/conjugate s o l u t i o n , mannose/FITC-Con A. x480 62 - 6 3 -F i g . 8a-d are a l l sect ions f i xed with PF/CPC as above, and stained with FITC-Con A 8a: The dorsal ectoderm, showing the densely l a b e l l e d BM and i t s assoc iated ECM (arrowheads). x l l 2 0 8b: A region of the al imentary c a n a l , showing the esophagus (Es) , with mesenchymal c e l l s (Me) attached to i t , and the stomach ( S ) , which i s devoid of mesenchymal c e l l s . Of i n t e r e s t i s the f a i n t l y l a b e l l e d esophageal BM (arrows), which d i f f e r s markedly from the heav i ly l a b e l l e d ECM found between the stomach epithel ium and the dorsal ectoderm (arrowheads), xl 120 8c: A region o f the stomodeum ( S t ) , showing the patchy s ta in ing of the BM and assoc iated ECM (arrowheads). This i s in contrast to the l i n e a r - t y p e s t a i n i n g of the ECM assoc iated with the ectoderm (Ec) in other regions of the embyro ( F i g . 8 a ) . xl120 8d: The region between the ventral esophagus (Es) and ectoderm (Ec) showing the web-l ike arrangement of ECM c o n s i s t i n g of amorphous regions a l igned in to organized l i n e a r pat terns . X2000 8e: A region of the dorsal ectoderm, stained with the control sugar conjugate, mannose/FITC-Con A. xl120 64 -65-Fig. 9a: A saggi ta l sect ion of a 6 day embryo, f i xed with PF/CPC, and stained with FITC-WGA. The BM region i s very densely l a b e l l e d in most areas of the embryo (arrows), with the exception of the esophagus (arrowheads) and d i s t a l area of the stomach, which exh ib i t very weak s t a i n i n g . x480 Fig. 9b: An adjacent saggita l sect ion o f the above, stained with the control sugar conjugate, galNAc/FITC-WGA. x480 66 - 6 7 -F i g . 10a-10d are higher magni f icat ions of d i f f e r e n t embryonic reg ions , f i xed as above, and stained with FITC-WGA. 10a: The a n t e r i o r ectoderm, showing the densely s t a i n i n g BM region and assoc iated ECM (arrows) under ly ing the ep i the l ium, xl120 10b: A region of the al imentary cana l , showing the esophageal endoderm ( E s ) , the f a i n t l y l a b e l l i n g esophageal BM (arrow-heads), and the c h a r a c t e r i s t i c mesenchymal c e l l s s e t t l i n g on i t . Shown a lso i s a part of the dorsal stomach ( S ) , and the dense BM/ECM assoc iated with i t (arrows), xl120 10c: A region of the p o s t e r i o r end of the embryo, showing the stomach epithel ium (S) with i t s f a i n t l y s t a i n i n g BM (arrow-heads), xl 120 lOd: A region of the ventral esophagus ( E s ) , showing the web-l ike appearance of ECM located between the esophagus and ectoderm ( E c ) . xl120 lOe: A region of the ventral esophagus, stained with the sugar control fo r the above, (glcNAc/FITC-WGA). xl120 68 -69-3.4 TEM-LECTIN LABELLING STUDIES E f f e c t of Embedding Media on L a b e l l i n g Although conventional embedding in Epon resu l ted in good preservat ion of c e l l u l t r a s t r u c t u r e , sect ions of Epon-embedded material d id not label with l e c t i n - g o l d preparat ions probably because Epon i s h igh ly c r o s s l i n k e d and hydrophobic in nature. Mater ia l embedded in Epon could be used i f i t was exposed to the l e c t i n - g o l d conjugate p r i o r to embedding; however the s ta in did not penetrate well i n to the t i s s u e . While the s t a i n could be admitted by c u t t i n g the embryo open, i t did not appear to d i f f u s e well through the b l a s t o c o e l , meaning that the exposure to the s t a i n could e a s i l y be d i f f e r e n t in d i f f e r e n t parts of the embryo. Although embedding in Lowicryl resu l ted in poor c e l l p r e s e r v a t i o n , i t was adequate for the u l t r a s t r u c t u r a l preservat ion of the c e l l u a r and e x t r a c e l l u l a r components ( F i g . 1 1 a , l i b ) . In contrast to Epon, Lowicryl undergoes only secondary c r o s s l i n k i n g during po lymer i zat ion , and i s po lar in nature. Because of t h i s , sect ions of Lowicryl-embedded material could be stained d i r e c t l y , which meant that a l l regions of the t i s s u e were exposed to the l e c t i n . In a d d i t i o n , with t h i s technique, d i f f e r e n t l e c t i n s could be tested on adjacent s e r i a l sect ions o f t i s s u e . Since the object was to compare the l a b e l l i n g pattern of l e c t i n s in d i f f e r e n t regions of the embryo, mater ial embedded in Lowicryl was used for the major i ty of the work. The basal lamina o f Dunlap's f ixed-Lowicry l processed t i s s u e cons isted of a lamina densa, approximately 80nm in width, and an e lec t ron lucent area of about the same width, the lamina luc ida ( F i g . 1 2 ) . In some areas, strands of ECM were assoc iated with the lamina densa. The ECM cons isted of strands approximately 40nm in width, with i r r e g u l a r l y shaped amorphous regions arranged along the strands. These -70-F i g . 11a: A TEM through the stomach of a 6 day embryo, f ixed with g l u t / a l c i a n blue and embedded in Epon. Preservat ion of c e l l u l t r a s t r u c t u r e i s good, and c e l l organel les such as endoplasmic ret iculum ( e r ) , nucle i (nu) , and mitochondria (m) are r e a d i l y v i s u a l i z e d . x l2,000 F i g . l i b : A TEM through the stomach of a 6 day embryo, f i xed as above, but embedded in Lowicryl K4M. U l t r a s t r u c t u r e in t h i s t i s s u e i s not as r e a d i l y apparent, as in the convent iona l ly prepared t i s s u e . The BL, nuclei (nu), and i n t r a c e l l u l a r granules (G) are , however, r e a d i l y v i s u a l i z e d . x l4,000 71 -72-Fig. 12: A TEM through the stomach of a 6 day embryo, f i xed with g lutara ldehyde/a lc ian blue and embedded in Lowicryl K4M. At t h i s higher magn i f i ca t ion , two components of the BL are ev ident : an e lect ron dense lamina densa (LD), and an e lec t ron lucent lamina l u c i d a ( L L ) . The ECM i s composed of strands *40nm in width with i r r e g u l a r l y shaped amorphous regions arranged along t h e i r length (arrowheads). These amorphous regions range from 50-200 nm in diameter. In some areas, the ECM i s assoc iated and continuous with the lamina densa of the BL (arrows). x74,300 73 -74-amorphous regions ranged from 50 to 200 nm in diameter ( F i g . 1 2 ) . Au 2 I - -Lec t in L a b e l l i n g Patterns of the Basal Lamina and ECM L a b e l l i n g of both the basal lamina and ECM was observed with the l e c t i n - g o l d conjugates A u 2 5 - C o n A, Au 2 5 ~LFA and Au 2 5 -DBA ( F i g . 1 3 , 1 4 , 1 5 ) , while l a b e l l i n g of ECM alone was observed with Au 2 5-WGA and Au 2 5 -SBA ( F i g . 1 6 , 1 7 ) . There was substant ia l v a r i a t i o n in both the dens i ty of the label as well as in the u l t r a s t r u c t u r a l l o c a l i z a t i o n o f the label among the d i f f e r e n t l e c t i n s . Of the 3 l e c t i n s , l a b e l l i n g over the BL was heaviest with DBA, a ga lNAc-a l ,3-ga lNAc-b ind ing l e c t i n : The gold p a r t i c l e s were arranged in c l u s t e r s conta in ing from 3 - 1 2 p a r t i c l e s per c l u s t e r ( F i g . 1 3 ) , and were located over the e n t i r e width of the basal lamina, inc lud ing the lamina l u c i d a , the lamina densa and the dense f i b e r s o f ECM associated with the lamina densa. L a b e l l i n g of the ECM occurred p r i m a r i l y over the amorphous reg ions , and not over the th in strands ( F i g . 1 3 ) . Another l e c t i n which l a b e l l e d both the basal lamina and ECM was A u 2 5 - C o n A, a galactose/ mannose binding l e c t i n . In t h i s case, label was predominantly found over the lamina densa, with only an occasional gold p a r t i c l e located over the lamina l u c i d a . The p a r t i c l e s formed small c l u s t e r s in some areas, but were p r i m a r i l y present as s i n g l e units ( F i g . 1 4 a ) . There was some l a b e l l i n g of the amorphous regions of the ECM as well ( F i g . 1 4 ) , although i t was not as dense as that seen with DBA. A u 2 5 - L F A , a s i a l i c ac id-b ind ing l e c t i n , was the 3rd l e c t i n which l a b e l l e d the basal lamina and ECM s p e c i f i c a l l y . Unl ike the previous 2 l e c t i n - g o l d conjugates, there appeared to be more l a b e l l i n g over the lamina l u c i d a , as well as some over the lamina densa ( F i g . 1 5 a ) . The gold p a r t i c l e s were most f requent ly v i s u a l i z e d in large c l u s t e r s - 7 5 -conta in ing 10 - 20 p a r t i c l e s per c l u s t e r . L a b e l l i n g of the ECM was d i s t r i b u t e d over both the th in f i b e r s and the la rger amorphous regions ( F i g . 1 5 a ) . The l e c t i n - g o l d conjugates Au 2 5-WGA, Au 2 5~SBA and Au 2 5 -PNA showed no s p e c i f i c l a b e l l i n g of the basal lamina, however there was some scattered l a b e l l i n g of the ECM with WGA and SBA. While the SBA l a b e l l i n g appeared s p e c i f i c a l l y over the amorphous regions of the ECM rather than along the strands ( F i g . 1 6 a ) , such a d i s t i n c t i o n could not be made with WGA, which was d i s t r i b u t e d more evenly over both elements of the ECM ( F i g . 1 7 a ) . With both l e c t i n s , the gold p a r t i c l e s were present mostly as s i n g l e uni ts or small c l u s t e r s of 2 - 3. Contro ls Sect ions were incubated with control s t a i n s , which consisted o f 0.1M i n h i b i t o r y sugar conjugated with the A u 2 5 - l e c t i n . Contro ls were run for a l l l e c t i n s accept Au 2 5 -DBA; t h i s l e c t i n binds to the terminal d i s a c c h a r i d e , galNAcal ,3-galNAc, which was not a v a i l a b l e fo r the study. The monosaccharide galNAc was attempted as a control fo r DBA, but t h i s sugar did not s i g n i f i c a n t l y reduce the amount of l a b e l l i n g . The contro ls fo r the l e c t i n s Con A, LFA, WGA, and SBA showed a very minimal amount of background l a b e l l i n g . This i s documented in Figures 14b,15b,16b,& 17b. -76-F i g . 13 - 17 are a l l u l t r a t h i n sect ions o f 6 day embryos, which have been f i xed with g lutara ldehyde/a lc ian blue and embedded in Lowicryl K4M. Fig. 13: • A TEM through the ectoderm stained with AU2 5-DBA, showing heavy l a b e l l i n g of the BL and ECM. L a b e l l i n g of the BL occurs predominantly over the lamina densa (LD), while l a b e l l i n g of the ECM appears to be concentrated over the amorphous regions (arrows). The gold p a r t i c l e s are f requent ly seen as c l u s t e r s , averaging 8 p a r t i c l e s . x33,600 77 -78-F i g . 14a: A TEM through the ectoderm stained with Au2 5-Con A, showing l a b e l l i n g of the BL and ECM. L a b e l l i n g occurs predominantly over the lamina densa (arrowheads), although an occasional p a r t i c l e i s seen over the lamina luc ida (arrow). Very scattered l a b e l l i n g of the ECM i s observed. S p e c i f i c l a b e l l i n g of the i n t r a c e l l u l a r granules (G) i s a l so present with Au 2 5~Con A. x49,500 F i g . 14b: A TEM through the ectoderm, stained with the control sugar/conjugate so lu t ion of the above, 0.1M mannose/ Au 9 [--Con A. X42.000 79 -80-Fig. 15a: A TEM through the stomach region stained with AU25-LFA, showing s p e c i f i c l a b e l l i n g over the BL and ECM. Labe l l ing of the BL occurs predominantly over the lamina l u c i d a (arrows); in the ECM, gold p a r t i c l e s are over both the strands and amorphous reg ions . x37,800 Fig. 15b: A TEM through the esophagus stained with the control sugar/ conjugate so lu t ion of the above, 0.1M s i a l i c ac id/ Au 9 I --LFA. X35.700 81 -82-F i g . 16a: A TEM through the ectoderm stained with Au 2 5 -WGA, showing scattered yet s p e c i f i c l a b e l l i n g o f the ECM (arrows). L a b e l l i n g i s a l so observed over the two i n t r a c e l l u l a r granules (G) shown in t h i s s e c t i o n . Although the granules appear to d i f f e r in t h e i r e lect ron dens i ty , they are both heav i l y l a b e l l e d . x37,800 F i g . 16b: A TEM through the ectoderm stained with the control sugar/ conjugate so lu t ion of the above, 0.1M glcNAc/Au 2 5-WGA. X38 .500 83 -84-F i g . 17a: A TEM through the esophagus stained with A u 2 5 - S B A , showing s p e c i f i c l a b e l l i n g over the amorphous regions of the ECM (arrows). x29,700 F i g . 17b: A TEM through the ectoderm stained with the control sugar/ conjugate so lu t ion of the above, 0.1M galNAc/Au 2 5 -SBA. x32,000 -86-A Regional Comparison of Lect in Labe l l ing Over the Basal Lamina A morphometric ana lys i s of l a b e l l i n g over the basal lamina revealed that with 2 of the l e c t i n s , there were s i g n i f i c a n t regional d i f fe rences in the amount of l a b e l l i n g present over the basal lamina. With the l e c t i n s Con A and LFA, there was a reduct ion in the amount of label over the esophageal basal lamina when compared to the basal lamina underlying the stomach, i n t e s t i n e or ectoderm. This was determined by performing a 1-way ana lys i s of var iance tes t on the data obta ined. With Con A, the t o t a l number o f p a r t i c l e s over the basal lamina of the esophagus, which averaged 45.5 par t i cles/6.67]am l i n e a r BL, was s i g n i f i c a n t l y less (p<0.001) than the amount over the stomach, which averaged 80.8 par t i cles/6 .6vim l i n e a r BL, the i n t e s t i n e , which averaged 75 p a r t i c l e s / 6 . 6um l i n e a r BL, and the ectoderm, which averaged 77 p a r t i c l e s / 6 . 6um l i n e a r BL. These r e s u l t s are summarized in table 6. TABLE 6: NUMBER OF GOLD PARTICLES BOUND/LENGTH OF BASAL LAMINA AFTER LABELLING WITH Au25~Con A. (Data are X + MSE, with n in parenthesis) REGION GOLD PARTICLES/6.67mm BL stomach 80.8 + 1.68 (5) -esophagus -»45.5 ± 3.32 (8) ectoderm 77.0 ± 4.29 (4) i n t e s t i n e 75.2 ± 1.60 (4) A s i m i l a r r e s u l t was noted with LFA. In t h i s case a morphometric ana lys i s revealed that l a b e l l i n g over the esophageal basal lamina (169.0/3.3um) was s i g n i f i c a n t l y less (p<0.05) than l a b e l l i n g over the ectodermal basal lamina (253.5/3.3um). Although the amount of l a b e l l i n g over the basal lamina of the stomach (221.67/3.3um) and i n t e s t i n a l (229.0/3.3yim) endoderm was less than that in the region of - 8 7 -the ectoderm, i t d id not d i f f e r s i g n i f i c a n t l y (P<0.05) from i t . These r e s u l t s are l i s t e d in tab le 7. TABLE 7: NUMBER OF GOLD PARTICLES BOUND/LENGTH OF BASAL LAMINA AFTER LABELLING WITH LFA. (Data are X + SE, with n in parenthesis) REGION GOLD PARTICLES/3.33mm BL stomach 221.67 ± 7.77 (6) -esophagus -169.00 ± 11.88 (7) A f t e r DBA l a b e l l i n g of the basal lamina, the to ta l number of p a r t i c l e s averaged =051/6.67vim l i n e a r BL. There was no s i g n i f i c a n t d i f f e r e n c e (p<0.05) in average p a r t i c l e counts over the basal lamina of the esophagus, stomach, i n t e s t i n e and ectoderm with t h i s l e c t i n . These r e s u l t s are l i s t e d in tab le 8. TABLE 8: NUMBER OF GOLD PARTICLES BOUND/LENGTH OF BASAL LAMINA AFTER LABELLING WITH DBA. (Data are X + SE, with n in parenthesis) ectoderm i n t e s t i n e 253.50 + 22.23 (6) 229.00 ± 14.66 (5) REGION GOLD PARTICLES/6.67mm BL stomach esophagus ectoderm i n t e s t i n e 157.89 ± 5.13 (9) 161.78 + 4.20 (9) 145.88 ± 6.89 (8) 140 00 ± 10.20 (4) -88-3.5 INTRACELLULAR GRANULES At l eas t two d i f f e r e n t populat ions of granules were seen in c e l l s f i xed in glutaraldehyde with a l c i a n b lue . Under the TEM, one populat ion sta ined more densely ( F i g . 1 4 , 1 6 ) . Both the darker and l i g h t e r - s t a i n i n g granules appeared predominantly as spher ica l s t ructures ranging in s ize from 0 .5-3 .0 urn in diameter. The two populat ions of granules were present in approximately equal numbers, and were d i s t r i b u t e d in a l l c e l l types o f the embryo, inc lud ing the e p i t h e l i a l c e l l s of the ectoderm and endoderm, as well as the u n d i f f e r e n t i a t e d mesenchyme c e l l s . However the c e l l s of the endoderm appeared to have a greater number of granules that the ectodermal c e l l s . At both the LM and TEM l e v e l , only the l e c t i n s WGA and Con A, l a b e l l e d the granules . At the LM l e v e l , B o u i n 1 s - f i x e d material gave optimal preservat ion of the granules , which were v i s u a l i z e d as b r i g h t l y - l a b e l l e d s t ructures in the c e l l s of the ectoderm, endoderm and mesenchyme ( F i g . 1 8 ) . FITC-WGA appeared to label the granules to a greater degree than did FITC-Con A, but as ide from the i n t e n s i t y of the l a b e l l i n g , the pattern appeared to be s i m i l a r with both l e c t i n s . The greatest number o f l a b e l l e d granules was seen in the endodermal c e l l s of the esophagus, stomach and i n t e s t i n e . There were fewer granules in the c e l l s of the ectoderm, and a g r e a t l y reduced number in the c e l l s of the stomodeum. S i m i l a r l y , under the TEM, only the two l e c t i n s Con A (F ig .14) and WGA (F ig .16) l a b e l l e d the granules in the c e l l s of the ectoderm, endoderm and mesenchyme ( F i g . 1 8 b ) . Gold p a r t i c l e s were seen over both l i g h t and d a r k - s t a i n i n g granules , although l a b e l l i n g appeared to be heavier over the d a r k - s t a i n i n g granules . -89-F i g . 18a: A th in saggi ta l sect ion through a 6 day embryo f i xed with Bou in ' s , and stained with FITC-WGA. Of p a r t i c u l a r i n t e r e s t i s the s p e c i f i c l a b e l l i n g of the i n t r a c e l l u l a r granules , which are d i s t r i b u t e d in the epi the l ium throughout the embryo. x480 F i g . 18b: A th in saggi ta l s e c t i o n , as above, f ixed with Bou in ' s , stained with FITC-Con A. The l a b e l l i n g of the i n t r a c e l l u l a r granules i s a l so noted, although i t i s not as intense as the WGA l a b e l l i n g . Note a l so the granules in the mesenchymal c e l l (Me). x480 90 - 9 1 -3.6 AUTORADIOGRAPHY 3 H-Suqars Which Were Taken Up By The Embryo 3 Of the four H-sugars placed in the sea water incubat ion medium (glucosamine, mannose, fucose, arab inose) , only glucosamine was taken up by the embryos. The grain counts over c e l l s of the embryos which were incubated in mannose, fucose, or arabinose did not exceed the background l e v e l , and i t was thus concluded that the embryos did not take up these exogenous sugars. Uptake by Glucosamine A f t e r a 3 hour chase in "co ld" sea water, most of the grains were located in the region of the stomach ( F i g . 1 9 a ) . A l l of the stomach e p i t h e l i a l c e l l s had grains over them, averaging 4-6 g r a i n s / c e l l ( F i g . 1 9 b ) . This was not the case for other e p i t h e l i a l c e l l s of the a l imentary canal however; i n t e s t i n a l and esophageal c e l l s had scattered l a b e l l i n g ( F i g . 1 9 c , 1 9 d ) , some c e l l s conta in ing up to 2 g r a i n s / c e l l , but most others not having any. The material i n s i d e the al imentary c a n a l , which i s continuous with the hyal ine l a y e r , was a lso l a b e l l e d ( F i g . 1 9 b ) , whereas few, i f any, grains were found over the hya l ine l a y e r . The ectodermal epi thel ium was s i m i l a r to the i n t e s t i n e and esophagus, showing only a few scattered g r a i n s . V i r t u a l l y no grains were observed over areas of ECM ( F i g . l 9 d ) . The control sect ion showed a very low background level ( F i g . l 9 e ) . Over the course of a 9 and 18 hour chase in cold sea water, there was l i t t l e change observed in the d i s t r i b u t i o n of glucosamine. The only change of any s i g n i f i c a n c e was in the i n t e s t i n a l ep i the l ium, where an increase in the number of grains was observed, so that at 18 hours, most c e l l s , as apposed to only some c e l l s , had 1-2 grains over them. -92-These r e s u l t s are summarized in table 9. TABLE 9: DISTRIBUTION OF 3H-GLUCOSAMINE AFTER A 6 HR LABEL IN THE LATE GASTRULA EMBRYO OF THE STARFISH PISASTER OCHRACEUS EPITHELIAL REGIONS WHERE GRAINS COUNTED* CHASE TIME (HR) ECTODERM ESOPHAGUS STOMACH INTESTINE ECM 3 + + +++ + 0 9 + + +++ +++ 0 18 + + +++ ++ 0 *LEGEND: + = 1-2 g r a i n s / c e l l in some c e l l s ++ = 2-4 g r a i n s / c e l l in most c e l l s +++ = 4-6 g r a i n s / c e l l in most c e l l s 0 = no grains -93-Fig. 19a: A saggi ta l sect ion of a 6 day embryo, processed for auto-radiography. This embryo was incubated with H-glucos-amine for 4 hours, fol lowed by a chase in cold sea water for 3 hours. At t h i s low magn i f i ca t ion , i t i s evident that the major i ty of grains are located in the stomach endothel ia l c e l l s ( S ) . Es=esophagus, In=intest ine , Ec=ectoderm. x450 Fig. 19b: A higher magni f icat ion of F ig .19a , showing the stomach endothe l ia l c e l l s which are heav i ly l a b e l l e d with grains (arrows), averaging 4-6 g r a i n s / c e l l . Grains are a l so seen over the material i n s i d e the lumen of the stomach (arrowheads). xl200 Fig. 19c: A higher magni f icat ion of F ig .19a , showing the i n t e s t i n a l (In) region of the embryo. Some scattered l a b e l l i n g of the c e l l s i s present (arrows), and as with the stomach lumen, the material i n s i d e t h i s lumen shows substant ia l l a b e l l i n g (arrowheads). xl200 Fig. 19d: A higher mani f i cat ion o f F ig .19a , showing a region of the stomodeum (St) and ventral ectoderm ( E c ) . Minimal l a b e l l i n g i s observed over the c e l l s , with only the occasional c e l l showing 1-2 g r a i n s . xl200 Fig. 19e: A control sect ion through the stomach ( S ) , showing very minimal background l a b e l l i n g . Control embryos were not 3 incubated with H-glucosamine, but were, t h e r e a f t e r , processed the same as were the experimental embyros. xl200 94 -95-4. CONCLUSIONS AND DISCUSSION 4.1 OPENING REMARKS Before d i scuss ing in d e t a i l the s p e c i f i c l a b e l i n g patterns of the l e c t i n s , a few general comments regarding l e c t i n binding studies can be made. Some researchers approach the general area of l e c t i n h is tochemistry with a great deal of skept ic i sm, and un jus t l y so, fo r i t i s not the technica l aspect of l e c t i n h is tochemistry , but rather the i n t e r p r e t a t i o n of the r e s u l t s obtained with t h i s technique, which i s often at f a u l t . As with any histochemical procedure, there i s the danger that r e s u l t s may be misrepresented and/or mis in te rpre ted . To avoid t h i s , several important points must be kept in mind when obta in ing information about t i s s u e carbohydrates using l e c t i n h is tochemist ry . F i r s t , although a p o s i t i v e r e s u l t impl ies the presence of a sugar, a negative r e s u l t does not n e c e s s a r i l y mean that the sugar i s not there , s ince the sugar could simply be i n a c c e s s i b l e to s t a i n i n g ( i . e . as sugar located i n t e r n a l l y with in a polysacchar ide chain very often w i l l not be detected by l e c t i n h i s tochemis t ry ) . Secondly, the appropr iate contro ls must be run j u s t as in any other histochemical procedure. The nonspec i f i c i n t e r a c t i o n s of l e c t i n s must be d i s t ingu i shed by sugar i n h i b i t i o n t e s t s ; fo r example, add i t ion of galactose to the FITC-RCA conjugate should render the conjugate i n e f f e c t i v e f o r s t a i n i n g , whereas the a d d i t i o n of mannose to the same conjugate should not change i t s l a b e l l i n g p r o p e r t i e s . T h i r d l y , of great importance i s the problem of f i x a t i o n - i n d u c e d changes in the molecular arrangement of t i s s u e g lycoconjugates . As discussed below, some l e c t i n - b i n d i n g s i t e s are c o n s i s t e n t l y a l t e r e d during f i x a t i o n . However the use of several d i f f e r e n t f i x a t i o n s in the experimental protocol can serve to reduce -96-experimental e r ror due to t h i s v a r i a b l e . In the present study, the above three points were taken into cons iderat ion before the data was i n t e r p r e t e d : Controls in a l l cases were negat ive , suggesting that the react ions were r e l a t i v e l y s p e c i f i c , and the use o f a range of f i x a t i o n s ensured that mis in te rpre ta t ions of the r e s u l t s due to f i xa t ion- induced changes of t i s s u e carbohydrates were minimized 4.2 FIXATION The mechanism respons ib le for the f i x a t i o n of l e c t i n receptor s i t e s i s very l i k e l y s i m i l a r to that of carbohydrates in genera l , which i s achieved through the c r o s s - l i n k i n g of prote in components of complex g lycoconjugates , rather than v i a the sugars themselves ( A l l i s o n , 1987). F i x a t i v e s d i f f e r g r e a t l y in the way in which they c r o s s - l i n k p r o t e i n s , and because of t h i s , the binding of l e c t i n s i s in f luenced s t rong ly by the f i x a t i o n method used. While some f i x a t i v e s may cause conf igura t iona l changes in the prote in part o f the glycoconjugate, making c e r t a i n sugar residues no longer a c c e s s i b l e to the l e c t i n s , others may expose new sugar residues fo r l e c t i n b ind ing . Although l e c t i n - b i n d i n g i s inf luenced by the f i x a t i o n method used, some l e c t i n s are more s e n s i t i v e to the mode of f i x a t i o n than others . For example, in the present study, RCA and SBA l a b e l l e d only those t i ssues f ixed with ethanol and g lutara ldehyde, whereas Con A and WGA were less s e n s i t i v e to the f i x a t i o n method used, and l a b e l l e d t i s s u e s f i xed in ethanol , Bou in ' s , g lutaraldehyde and paraformaldehyde. Ethanol i s considered to be among the best of f i x a t i v e s fo r LM l e c t i n binding s t u d i e s . I t i s thought to c r o s s l i n k prote ins l o o s e l y , which probably leaves more s i t e s exposed in the glycoconjugates to -97-accomodate b ind ing , p a r t i c u l a r l y by those l e c t i n s with mu l t ip le binding s i t e s (Rittman & Mackenzie, 1983; A l l i s o n , 1987). These pred ic t ions were bourne out by the present study, where f i x a t i o n with ethanol gave the best s t a i n i n g r e s u l t s fo r FITC-RCA, FITC-SBA and FITC-Con A. There i s a problem in using ethanol as a f i x a t i v e , however, in that c e l l morphology i s poor ly preserved. C h a r a c t e r i s t i c a l l y , the cytoplasm shrinks and may p i l e up against one side of the c e l l opposite the side at which the f i x a t i v e enters ; nuclei may a lso be misshapen. This became a problem in the present study in that the BM and ECM were d isp laced to the center of the embyro a f t e r f ixaton with ethanol . However, the hya l ine layer (the ECM on the outer surface of the ectoderm), appeared to be preserved opt ima l l y with t h i s f i x a t i o n . Although i t i s not true f o r a l l l e c t i n s , the decreased s ta in ing of t i s s u e s t ructures with several l e c t i n s a f t e r f i x a t i o n in paraform-aldehyde i s a common observat ion ( A l l i s o n , 1987; Malmi & Soderstrom, 1988). Formaldehyde reacts with prote ins in several d i f f e r e n t ways, the most important being the react ion with uncharged amino groups in amino a c i d s , which condense with amide or other groups to y i e l d methylene cross-br idges between prote ins (Pearse, 1960). The cross br idg ing i s thought to cause conformational changes in the sugar- contain ing molecules which could block access to the FITC-conjugated l e c t i n s and thus reduce or el imate s t a i n i n g . While embryonic a s t e r o i d t i ssues f ixed in 95% ethanol e x h i b i t strong l a b e l l i n g patterns with the l e c t i n conjugates FITC-RCA and FITC-SBA, l a b e l l i n g i s weak or non-existant on t i s s u e f i xed in paraformaldehyde. The decreased l a b e l l i n g i s probably due to formaldehyde induced conf igurat iona l changes o f the g lycoprote ins of the type descr ibed above, which mask the terminal sugar res idues . -98-CPC s e l e c t i v e l y p r e c i p i t a t e s po lyanionic molecules, and when added to paraformaldehyde, i t improves the retent ion of carbohydrate conta in ing compounds (Wil l iams & Jackson, 1956). CPC, while e f f e c t i v e with the LM, has not found widespread use at the u l t r a s t r u c t u r a l l e v e l , s ince i t r e s u l t s in poor u l t r a s t r u c t u r a l preservat ion and tends to leave contaminating deposits (Sanders, 1986a). Work on a s t e r o i d embryos confirmed these observat ions . A f t e r add i t ion of the detergent compound CPC to paraformaldehyde, a tremendous increase in l a b e l l i n g i s observed with the l e c t i n s FITC-Con A and FITC-WGA. Unfortunately however, as with vertebrate t i ssues descr ibed above (Sanders, 1986a), u l t r a s t r u c t u r a l preservat ion i s very poor. Attempts to overcome t h i s by adding CPC to the post f i x a t i v e are unsuccessful 1, as no apprec iable d i f f e r e n c e between material f i xed in t h i s manner from that in which CPC i s used in the primary f i x a t i v e i s observed. Another f i x a t i v e used in these studies was Bouin's f i x a t i v e , which gives bet ter morphological preservat ion than ethanol . Although the mechanism of f i x a t i o n by Bouin's i s not e n t i r e l y understood, one of the main components, p i c r i c a c i d , i s known to react with prote ins to form p i c r a t e s . In t h i s study, Bouin's f ixed material demonstrated strong l a b e l l i n g of the i n t r a c e l l u l a r granules with FITC-Con A and FITC-WGA, granules which do not label c l e a r l y in paraformaldehyde/CPC-fixed t i s s u e . This d i f f e r e n c e in f i x a t i o n i s probably due to the fac t that Bou in ' s , l i k e e thano l , does not change the t e r t i a r y conf igurat ion of the molecules as extens ive ly as does paraformaldehyde. Glutaraldehyde i s a 5-carbon s t r a i g h t chain dialdehyde with a molecular weight o f 100. It i s used extens ive ly as a f i x a t i v e in e lec t ron microscopy because of i t s a b i l i t y to preserve u l t r a s t r u c t u r e . The mechanism of glutaraldehyde f i x a t i o n appears to be very s i m i l a r to -99-that of formaldehyde (Chambers et a l . , 1968). At pH 7.0 or above, the main react ion product i s an imine which forms between a f ree aldehyde group and an amino group (such as the e-amino group of a l y s i n e ) . A l c i a n blue was f i r s t used as a f i x a t i v e with glutaraldehyde by Leak (1967) to preserve carbohydrates. Although the mechanism of a l c i a n blue/glutaraldehyde f i x a t i o n i s not yet e s t a b l i s h e d , i t i s h igh ly probable that the a l c i a n b lue, a c a t i o n i c dye der ived from copper p tha locyan in , reacts with var ious negat ive ly charged a c i d i c carbohydrates (Mowry, 1963), and probably forms a s a l t l inkage (Scott et a l . , 1964). The mechanism of formation of the e lectron-dense l a b e l l i n g i s completely unknown. When osmium te t rox ide i s not used in the f i x a t i v e , i t i s thought that the copper moiety of the c a t i o n i c dye i s impl icated in the e lec t ron opac i ty (Rothman, 1969). When osmium i s used in the f i x a t i o n , i t has been suggested that an osmioph i l i c dye-mucosubstance complex i s formed, which accounts fo r the e lect ron opac i ty (Behnke and Zelander, 1970). G lutara ldehyde/alc ian blue i s the f i x a t i v e of choice in the present TEM studies because i t gives moderately good u l t r a s t r u c t u r a l preservat ion of the t i s s u e coupled with e x c e l l e n t preservat ion of the basal lamina (BL) and ECM, while s t i l l a l lowing the gold-conjugated l e c t i n s to bind to them. Although t i ssues preserved in the presence of a l c i a n blue al low l a b e l l i n g with gold-conjugated l e c t i n s at the u l t r a s t r u c t u r a l l e v e l , FITC-conjugated l e c t i n s do not label with material preserved in a l c i a n blue/paraformaldehyde. The reason for t h i s i s present ly unknown, but i t i s probably due to a combination of f a c t o r s , inc lud ing paraformaldehyde induced changes in the t e r t i a r y s t ructure of glycoconjugates discussed above, as well as the b locking of terminal sugars with a l c i a n b lue, leading to s t e r i c hindrance of the FITC-conjugated l e c t i n s . -100-4.3 THE NATURE OF LECTIN-BINDING SITES In contrast to ant ibody-antigen h is tochemistry , where binding of a c e r t a i n antibody immediately uncovers the locat ion of a s p e c i f i c molecule, the r e s u l t s of l e c t i n h istochemistry are much more d i f f i c u l t to i n t e r p r e t because of the d iverse nature of glycoconjugates in t i s s u e s . Two major kinds of glycoconjugates present in the e x t r a c e l l u l a r matrix are g lycoprote ins and proteoglycans. The carbohydrate conta in ing component of proteoglycans are the glycosaminoglycans (GAGs), of which there are four major c l a s s e s : hyaluronic ac id (HA), chondro i t in s u l f a t e (CS) , k e r a t i n s u l f a t e (KS) and heparan s u l f a t e (HS). Chondroit in s u l f a t e i s d iv ided in to c h o n d r o i t i n - 4 - s u l f a t e (CS4), c h o n d r o i t i n - 6 - s u l f a t e (CS6), and dermatan s u l f a t e (DS); the l a t t e r contains iduronate residues in place of D-glucuronic a c i d . In genera l , the s t ructure of GAGs inc ludes repeating d isacchar ides u n i t s , with a c h a r a c t e r i s t i c o l igosacchar ide l inkage region that contains g lucuronic a c i d , galactose and xy lose , where the GAGs are attached to the core prote in (Hascal l & Kimura, 1982). GAGs represent the most probable l e c t i n binding s i t e s in t i ssues s ince t h e i r carbohydrate content i s so h igh , due to the repeat ing sugar uni ts i n t r i n s i c in t h e i r s t r u c t u r e . WGA, a glcNAc-binding l e c t i n , therefore tends to bind to HA because HA has in i t s s t r u c t u r e , repeating uni ts o f glcNAc. L ikewise, SBA, a galNAc-binding l e c t i n , would bind to CS and DS, s ince they both contain a l t e r n a t i n g uni ts of galNAc. On the other hand, PNA and RCA are ga lactose-b ind ing l e c t i n s , and should therefore bind s t rong ly to keratan sulphate which contains t h i s sugar. Although, to the best of my knowledge, no one has yet tested the binding c a p a i t i e s o f PNA and RCA f o r these GAGs, Gal lager (1986a) has indeed shown that commercially a v a i l a b l e HA and CS bind WGA and SBA -101-r e s p e c t i v e l y . The proposed l e c t i n binding s p e c i f i c i t i e s to GAGs are summarized below in tabular form. TABLE 10: LECTIN-BINDING SPECIFICITIES FOR GAGs LECTIN SUGAR GAG WGA glucuronate-61,3-g lcNAcBl ,4 Hyaluronic ac id SBA g lucuronate-B l ,3-ga lNAc-B l ,4 Chondroi t in s u l f a t e SBA iduronate-B l ,3 -ga lNAc-B l ,4 Dermatan s u l f a t e PNA,RCA g lcNAc-B l ,3ga l -61 ,4 Keratan s u l f a t e While GAGs provide prospect ive binding s i t e s f o r the g lcNAc-b ind ing, galactose and galNAc-binding l e c t i n s , they do not represent potent ia l binding s i t e s fo r the mannose/glucose-binding l e c t i n s , (eg. Con A ) , nor the l e c t i n s which bind the s i a l i c ac ids (eg. LFA). There are other g lycoconjugates, however, which may be l i k e l y candidates f o r l e c t i n binding s i t e s . Studies have shown that l e c t i n s w i l l a l so bind to the sugar residues located in g l y c o p r o t e i n s , such as f i b r o n e c t i n and lamin in , as well as to co l lagen IV ( K e f a l i d e s , 1970; Yamada, 1981; Timpl & Mart in , 1982). Laminin, a large g lycoprote in of MW 1,000,000, has the conf igurat ion of an asymmetric " c r o s s " , with one long arm (77nm), and 3 i d e n t i c l e short arms (37nm) conta in ing g lobular regions on the end of each arm when viewed under the e lect ron microscope, (Rao et a l . , 1983). I t has been hypothesized that the 4-arm s t ruc ture o f laminin i s of b i o l o g i c a l importance enabl ing the molecules to "reach out" and i n t e r a c t with mul t ip le c e l l u l a r and matrix components in d i f f e r e n t d i r e c t i o n s (Hay, 1981). Lect ins which have been shown to bind laminin i s o l a t e d from mouse Engelbreth-Holm-Swarm (EHS) tumor -102-inc lude RCA, WGA and Con A. RCA binds to a galactose residue on the end g lobu lar domains located on the short arms of lamin in , while WGA and Con A bind to glcNAc and a-D-mannopyranosyl residues thought to be located on the arms themselves (Rao, et a l ; 1983). Of the l a t t e r two, Con A binds more s t rong ly to the arms. Col lagen i s another g lycoprote in which contains poss ib le l e c t i n binding s i t e s . The l e c t i n s Con A, WGA, sWGA (succ iny la ted WGA, which has a s p e c i f i c i t y fo r glcNAc on ly , whereas WGA binds to both glcNAc and s i a l i c a c i d , Monsigny, 1979), and GSA-II (a l e c t i n s p e c i f i c fo r CHg-a-D-galactose) have been shown to bind to Col lagen type I i s o l a t e d from human skin or uter ine ligaments (Soderstrom, 1987). The dens i ty of sugars in g lycoprote ins i s r e l a t i v e l y low when compared to that found in proteoglycans, and therefore l e c t i n s would be expected to bind to the g lycoprote ins ( i . e . lamin in , f i b r o n e c t i n , co l lagen) much more weakly that to GAGs. I t does appear l i k e l y , however, that Con A i s binding some v a r i e t y of g l y c o p r o t e i n , s ince no known GAG contains mannose/glucose res idues . Limax f lavus a g g l u t i n i n (LFA) i s a s i a l i c ac id -b ind ing l e c t i n which binds to N-acetyl-neuraminic ac id (Neu5Ac), as well as to the hydroxylated form, N-glycoloyl-neuraminic ac id (Neu5Gc), although binding to the former i s stronger ( M i l l e r et a l . , 1982). I t i s known that g lycoprote ins and g l y c o l i p i d s of c e l l surfaces are often s i a l a t e d , with most of the s i a l i c ac id residues occupying terminal pos i t ions of o l igosacchar ide chains ( C o r f i e l d & Schauer, 1982). However, i t i s not known what the manner of in tegrat ion of these s i a l i c ac ids in to the BL and ECM i s . I t i s worth mentioning here a note regarding the binding c h a r a c t e r i s t i c s o f Dol ichos b i f l o r u s a g g l u t i n i n . Although DBA i s -103-another galNAc-binding l e c t i n (Ho l thofer , 1983; Ribera et a l . , 1987), Baker et a l . (1983) have shown that DBA binds 20 times more s t rong ly to the o l igosacchar ide contain ing two galNAc- l inked uni ts ( g a l N A c - a l , -3galNAc), than to the monosaccharide with only one galNAc u n i t . Since in the present study, DBA did not label the same s i t e s as the galactose or galNAc-binding l e c t i n s , but instead exhib i ted a strong l a b e l l i n g pattern which was unique, i t i s suggested that DBA bound o l i g o -saccharides conta in ing two galNAc- l inked u n i t s , and that i t d id not bind o l igosacchar ides with only a s i n g l e terminal galNAc u n i t , as did the l e c t i n , SBA. The fo l lowing tab le summarizes the most probable l e c t i n binding s i t e s based on the above-mentioned evidence. TABLE 11: PROPOSED LECTIN BINDING SITES LECTIN LECTIN BINDING SITE Con A o l igosacchar ides conta in ing mannose/glucose, poss ib le inc lud ing 1° laminin and 2° col lagen LFA 1° Neu5Ac, 2° Neu5Gc DBA galNAcal ,3galNAc-contain ing o l igosacchar ide WGA 1° HA, 2° co l lagen PNA,RCA 1° KS, 2° lamin in , co l lagen SBA 1° CS, DS -104-4.4 LECTIN BINDING TO THE BASAL LAMINA AND ECM OF P. OCHRACEUS  Descr ip t ion of the BL & ECM The embryonic as te ro id BL as descr ibed by Crawford (1988) i s s i m i l a r to that descr ibed in vertebrate embyros (Hay, 1978; Sanders, 1986a). I t cons i s ts of a lamina densa which i s separated from the basal c e l l u l a r membrane by a lamina l u c i d a . The use of a l c i a n blue in the f i x a t i v e reveals that the lamina densa cons is ts of a f i n e fe l t -work of in termediate ly stained f i b e r s with a coarse meshwork o f t h i c k , densely sta ined and th inner , intermediate ly stained strands embedded in the inner aspect (the side adjacent to the b l a s t o c o e l ) . The l a t t e r material i s i d e n t i c a l in appearance with and connects to the ECM located in the b l a s t o c o e l . T u b u l e - l i k e s t ructures which o r i g i n a t e in the dense mater ia l assoc iated with the lamina densa, cross t h i s s t ructure and the adjacent lamina luc ida and attach to the basal plasmalemma of the e p i t h e l i a l c e l l s , thus anchoring the ECM of the b lastocoel to them. The ECM cons i s ts of strands approximately 40nm in width, with i r r e g u l a r l y shaped amorphous regions arranged along the strands. These amorphous regions range from 50 to 200 nm in diameter. Extensive biochemical and immunohistochemical studies have not been c a r r i e d out on the embryonic a s t e r o i d BL to date. However studies on embryonic BL o f the ech ino ids , another echinoderm group, have demonstrated the presence of co l l agen , f i b r o n e c t i n , laminin and HSPG ( S p i e g e l , et a l . , 1983; Wessel, et a l . , 1984; Wessel & McClay, 1987), suggesting that these proteoglycans and g lycoprote ins may a lso be present in the as te ro id basal lamina. -105-D i s t r i b u t i o n of Carbohydrates in the BL & ECM Based on the l e c t i n binding studies of the BL, we now have an understanding of the types of sugar moit ies that are present in the BL and ECM. He a l so can gain an apprec iat ion of the d i s t r i b u t i o n of these sugars throughout var ious regions of the embryo. We can, however, only speculate on the nature of the glycoconjugates in which these saccharides are incorporated . To begin wi th , both the F I T C - l e c t i n and the A u ^ - l e c t i n binding studies revealed binding of Con A, WGA, LFA, and DBA to the embryonic a s t e r o i d BL, suggesting i t i s r i c h in glycoconjugates conta in ing mannose and/or glucose mo i t i es , s i a l i c acids (Neu5Ac, Neu5Gc), and o l igosacchar ides having two galNAc- l inked uni ts (ga lNAc-a l ,3ga lNAc) . The d i s t r i b u t i o n of the l a b e l l i n g in the BL i s not e n t i r e l y uniform. The major i ty of Con A l a b e l l i n g i s found over the lamina densa; although there i s some over lamina l u c i d a , no l a b e l l i n g i s found over the assoc iated ECM f i b e r s . S ta in ing with DBA shows a s i m i l a r p a t t e r n , accept that some l a b e l l i n g i s present over the assoc iated ECM f i b e r s . The s t a i n i n g pattern with LFA i s somewhat d i f f e r e n t , in that the major i ty o f l a b e l l i n g i s over the lamina l u c i d a , and not the lamina densa. There i s a l e s s e r degree of l a b e l l i n g over the lamina densa, and no l a b e l l i n g over the assoc iated ECM f i b e r s . L e c t i n - b i n d i n g studies of the ECM in t h i s study have revealed that the ECM labe ls with Con A, DBA, SBA WGA and LFA. Con A, DBA and SBA label the i r r e g u l a r l y shaped amorphous regions e x c l u s i v e l y , while WGA and LFA label both these amorphous regions as well as the strands of ECM. While Con A and SBA give moderate l a b e l l i n g of the amorphous regions i n d i c a t i n g the presence of mannose/glucose and galNAc res idues , very strong l a b e l l i n g over the amorphous regions i s seen with DBA -106-i n d i c a t i n g a large amount of the d isacchar ide galNAc-galNAc i s present in these reg ions . The l a b e l l i n g observed with LFA i s s i m i l a r to that of WGA, in that gold p a r t i c l e s are d i s t r i b u t e d over both amorphous areas and the strands of ECM, i n d i c a t i n g the presence of a glycoconjugates conta in ing s i a l i c acids and glcNAc in these reg ions . As discussed in sect ion 4.3 of t h i s chapter, the most probable glycoconjugates represent ing the l e c t i n binding s i t e s inc lude : laminin and co l lagen (Con A ) , Neu5Ac and Neu5Gc (LFA), hyaluronic ac id and co l lagen (WGA), and galNAcal ,3galNAc-contain ing o l igosacchar ides (DBA). Immunohistochemical work on echinoid ECM has demonstrated the presence of c o l l a g e n , lamin in , f i b r o n e c t i n , and proteoglycans in the BL and ECM (Pucc i -Minafra et a l . , 1972; Oguri & Yamagat, 1978; Spiegel et a l . , 1980, 1983; Katow et a l . , 1982; Wessel et a l . , 1984). Since t h i s species i s so c l o s e l y re la ted to a s t e r o i d i a , i t would seem probable that glycoconjugates such as those present in the echinoid are present a l so in the developing a s t e r o i d . Although u l t r a s t r u c t u r a l studies of t h i s nature are r a r e , LM studies using FITC l a b e l l e d Con A and WGA have been reported with echinoid embryos (DeSimone & S p i e g e l , 1986). In t h i s case, both Con A and WGA were found to bind to the ECM in the b l a s t o c o e l . In a d d i t i o n , some of these l e c t i n - b i n d i n g prote ins have been i s o l a t e d from the ECM in the sea urchin embryos, and have been character ized to some extent. Desimone & Spiegel (1986) have found one WGA-binding prote in of MW 125,000, as well as three Con A-binding prote ins of MW 29,000, 34,000, and 37,000. In these s tud ies , the expression of the above appeared to be r e l a t e d to developmental stages, and as w e l l , are s e n s i t i v e to agents known to i n t e r f e r e with the synthesis of GAGs and g l y c o p r o t e i n s . 3 Whereas studies u t i l i z i n g H-glucosamine can provide information -107-concerning the d i s t r i b u t i o n and accumulation of newly synthesized GAGs 3 ( B e r n f i e l d & Banerjee, 1972), in t h i s study, the H-glucosamine which was taken up by the c e l l s of the GI t r a c t was not used as precursers fo r glycoconjugates in the ECM or BL, and remained predominanly in the c e l l s . There was, however, some l a b e l l i n g of the e x t r a c e l l u l a r material l i n i n g the GI t r a c t . The l a b e l l i n g of the storage granules appears to i n d i c a t e the presence of glcNAc and mannose/glucose res idues . I t i s unclear whether these moiet ies funct ion as simple metabol i tes , i . e . , as b u i l d i n g blocks f o r complex g lycoconjugates , or i f they are a lready part of complex g lycoconjugates . Both of these options are p o s s i b l e , s ince , for example, ga lac tose , mannose, glcNAc and galNAc can a l l be converted to glucose f o r metabolism, but at the same time, can a l so be incorporated d i r e c t l y / i n d i r e c t l y in to g lycoconjugates. 4.5 IMPLICATIONS OF THE RESULTS AS THEY RELATE TO DEVELOPMENT The d i s t r i b u t i o n of l e c t i n binding s i t e s in the BL throughout d i f f e r e n t regions o f the embryo, as revealed by the u l t r a s t r u c t u r a l l a b e l l i n g study (Au2g- lect in) are p a r t i c u l a r l y i n t e r e s t i n g in l i g h t of the morphogenetic event being s tud ied . While both Con A and WGA bind to the ectodermal and endodermal BM rather uniformly at the LM l e v e l , TEM studies fol lowed by a morphometric study confirm that there i s less l a b e l l i n g over the area of the esophagus with two of the l e c t i n s , Con A and LFA. The t o t a l number of Con A-bound gold p a r t i c l e s over the BL of the esophagus which averaged 45.5 part ic les/6.67pm l i n e a r BL, i s s i g n i f i c a n t l y less (p<0.001) than that over the stomach, which averaged 80.8 part ic les/6.6um l i n e a r BL, the i n t e s t i n e , which averaged 75 part ic les/6 .6nm l i n e a r BL, and the ectoderm, which averaged 77 -108-part ic les/6.6um l i n e a r BL. S i m i l a r l y , A u 2 5 - L F A was found to bind less in tense ly to the esophageal BL than to the ectodermal BL. The tota l number of p a r t i c l e s over the BL of the ectoderm averaged 253.5/3.3}jm, while that over the esophagus averaged 169.0/3.3um. Although the amount of l a b e l l i n g over the BL of the stomach (221.67/3.3um) and i n t e s t i n a l (229.0/3.3um) endoderm was less than that in the region o f the ectoderm, i t d id not d i f f e r s i g n i f i c a n t l y (P<0.05) from i t . These r e s u l t s suggest that the BL underly ing the esophageal endoderm has a decreased quant i ty of carbohydrates, as revealed by the s t a i n i n g patterns of Con A and LFA. As mentioned p r e v i o u s l y , molecules bound by the l e c t i n s are unknown. However, based on the extrapolat ions of molecules found in vertebrate ECM's, i t seems poss ib le that th i s could represent a decrease in the amount of lamin in , co l lagen and mucopolysaccharides conta in ing s i a l i c a c i d s . These l e c t i n binding studies appear to complement previous observations by Crawford (1988) that the endodermal BL underly ing the esophagus i s less a l c i a n o p h i l i c and therefore " th inner" in s t ructure than the BL of the ectoderm. Gal lagher (1986b) noted a s i m i l a r phenomena during the branching morphogenesis o f the chick lung. In her study, the l e c t i n s WGA, SBA and RCA a l l sta ined the BM of the developing chick lungs, and furthermore, th inn ing of the BM at the t i p s of newly formed bronchi was v i s u a l i z e d with a l l three l e c t i n s , and was p a r t i c u l a r l y evident with SBA. Using ruthenium red and tannic ac id to s ta in the BL f o r TEM s t u d i e s , she observed a th inn ing and sometimes discontinuous BL at the t i p s of the buds as compared with the more substant ia l BL in the interbud areas. She hypothesized that the decreased amount of l a b e l l i n g with l e c t i n s and c a t i o n i c dyes at the ac t i ve s i t e of growth represented a necessary -109-cond i t ion f o r epithelio-mesenchymal i n t e r a c t i o n s to occur . In the present study, the d i f f e r e n c e in the propert ies of the esophageal BL as compared to the BL in other regions o f the embryo could represent a developmental l y s i g n i f i c a n t event. During t h i s stage in ]\ ocraceus development, mesenchyme c e l l s are gathering at the esophagus p r i o r to d i f f e r e n t i a t i n g in to muscle. Although there are mesenchymal c e l l s in other areas of the embryo at t h i s t ime, only the ones which s e t t l e at the esophagus and only on the esophagus d i f f e r e n t i a t e in to muscle c e l l s . The end r e s u l t i s an i n t e r l o c k i n g of muscle c e l l s forming a smooth muscle sheath around the esophageal endoderm. The d i s t i n c t l o c a l i z a t i o n of one populat ion o f the mesenchyme c e l l s to t h i s region suggests that there i s some s ignal to the c e l l s t e l l i n g them "stop here" . Since the i n i t i a l i n t e r a c t i o n of the mesenchyme c e l l s appears to be with the esophageal BL, a BL which appears to d i f f e r both morphologica l ly and b iochemica l ly from those around i t , i t i s poss ib le that the stop s ignal i s l o c a l i z e d in the esophagel BL. Indeed, the r e s t r i c t i o n of movement of these c e l l s to the esophageal BL may be due to the decrease in the molecule(s) associated with the mannose and s i a l i c ac id res idues . One fu r ther i n t e r e s t i n g observat ion stems from the fac t that fo l lowing migrat ion but p r i o r to overt expression of d i f f e r e n t i a t i o n , the mesenchymal c e l l s in t h i s area develop processes which appear to break through the BL and make contact with the e p i t h e l i a l c e l l s of the esophagus. I t i s poss ib le that the unique nature of the BL underlying the esophageal endoderm i s a necessary p r e r e q u i s i t e , f i r s t fo r th i s i n t e r a c t i o n between u n d i f f e r e n t i a t e d mesenchymal c e l l s and esophageal endodermal c e l l s to occur , and secondly, f o r subsequent muscle d i f f e r e n t i a t i o n to occur . -110-Although there appears to be a l o g i c a l sequence of events surrounding the th inn ing of the esophageal BL, several questions s t i l l remain unanswered: F i r s t l y , does the BL in t h i s region loose carbohydrates as a g e n e t i c a l l y preprogrammed event, or i s i t d e f i c i e n t in these and perhaps other molecules because i t i s the l a s t area of the BL to form and therefore may not have yet incorportated i t s f u l l complement of glyconjugates in to the BL? Secondly, i s t h i s r e l a t i v e lack of c e r t a i n sugar conta in ing molecules a requirement in order to r e s t r i c t the presumptive muscle c e l l s to the esophagus? T h i r d l y , i s i n t e r a c t i o n between the mesenchymal c e l l s and the esophageal endoderm an absolute requirement fo r muscle d i f f e r e n t i o n ? And, f o u r t h l y , i s continued contact between the mesenchymal and e p i t h e l i a l c e l l s required for t h e i r complete d i f f e r e n t i a t i o n , or i s one contact enough? Further work must be done to examine not only the molecular nature of the BL and ECM, but the d i s t r i b u t i o n o f the d i f f e r e n t l e c t i n binding s i t e s in the ECM throughout var ious regions of the embyro, as was done with the BL. In a d d i t i o n , a more d e t a i l e d ana lys i s of the components in the BL which are present in reduced q u a n t i t i e s could be attempted by i s o l a t i n g the l e c t i n binding f r a c t i o n s , fu r ther c h a r a c t e r i z i n g them, and perhaps r a i s i n g monoclonal ant ibodies to p u r i f i e d f r a c t i o n s . By i s o l a t i n g l e c t i n binding g lycoprote ins and GAGs, and p l a t i n g mesenchymal c e l l s on them, the various f r a c t i o n s can be examined f o r t h e i r a b i l i t y to promote migrat ion . Fol lowing t h i s , c h a r a c t e r i z a t i o n of the ac t i ve f r a c t i o n s could eventua l ly br ing to l i g h t some of the fac tors involved in r e g u l a t i n g c e l l migrat ion during morphogenesis. -111-5. THE HYALINE LAYER: ANOTHER EXTRACELLULAR MATRIX 5.1 INTRODUCTION The hyal ine layer (HL), i s an e x t r a c e l l u l a r layer o f material which surrounds the embryos and larvae of several c lasses of echinoderms (eg, a s t e r o i d s , ho lothuro ids , ophiuroids and echinoids Crawford & Abed, 1986) from f e r t i l i z a t i o n to metamorphosis, and i s thought to be the indispensable substratum for the overa l l s tereotyp ic o rgan izat ion of the developing embryo (Dan, 1960; Schroeder, 1988). The HL i s formed when, at f e r t i l i z a t i o n , the c o r t i c a l granules fuse with the plasma membrane of the egg and re lease t h e i r contents (Kane & Hersh, 1959; Endo, 1961; Runnstrom, 1966; Anderson, 1968). The surface of the egg i s , t h e r e a f t e r , f i r m l y attached to the HL by means of c e l l surface m i c r o v i l l i (Dan, 1960; Wolpert & Mercer, 1963). Its funct ion appears to be assoc iated with maintaining c e l l - c e l l i n t e r a c t i o n s . Herbst (1900) found that in the absence of Ca + , the sea urchin HL was s l u f f e d o f f , and subsequently, the embryo was e a s i l y d i s s o c i a b l e . More r e c e n t l y , the HL has been descr ibed as an adhesive substrate fo r c e l l s (Dan, 1960) or as an ECM (Spiegel & S p i e g e l , 1971) prov id ing an attachment s i t e fo r c e l l s during morphogenesis (Gustafson & Wolpert 1967). Dan (1960) has commented on the mutual arrangement of the blastomeres at the b l a s t u l a stage which i s maintained by t h e i r attachment to the HL, and suggests that the HL i s s i m i l a r in funct ion to an e p i t h e l i a l basement membrane. Wolpert and Mercer (1963) have confirmed Dans's observat ions and have suggested that the attachment of c e l l s to the HL v i a plasma membrane m i c r o v i l l i maintains a r a d i a l p o l a r i t y in the developing embryo u n t i l b l a s t u l a format ion. In t h i s instance , i t appears that c e l l attachment to the s h e l l - l i k e HL f i x e s the r e l a t i v e pos i t ions of blastomeres by -112-i n d i r e c t l y i n t e r l i n k i n g them, which forces the o ld surface ( i . e . the only surface with m i c r o v i l l i remaining) to face outwards, thereby d e f i n i n g the c e l l ' s ap ica l poles (Schroeder, 1988). This assoc ia t ion between m i c r o v i l l i and the HL thus integrates the e n t i r e embryo, at l e a s t during the e a r l y stages of development before the appearance of i n t e r c e l l u l a r j u c t i o n s , by automat ica l ly a l i g n i n g the blastomeres with the r a d i a l axes of the whole embryo. In sea urchin embryos, the HL has been extens ive ly character ized b iochemica l l y . The major component of the HL i s the g lycoprote in h y a l i n , a Ca + r e q u i r i n g prote in (Yazak i , 1968; Kane & Stephens, 1969) ranging in MW from 95,000 to 300,000 in d i f f e r e n t species (Hylander & Summers, 1982). Immunof1uorescent studies of sea urchin embryos have revealed that the prote in hya l in i s confined to the outer regions of the HL. Several other prote ins have been i s o l a t e d from the HL in add i t ion to h y a l i n . These inc lude two g lycoprote ins with molecular weights of 175,000 and 145,000, which Hal l & Vacquier (1982) have i s o l a t e d and c h a r a c t e r i z e d , and three other major prote ins with molecular weights of 110,000, 70,000, and 50,000, which McCarthy & Spiegel (1983) have i s o l a t e d . More r e c e n t l y , A l l i e g r o et a l . (1988) have i s o l a t e d the g lycoprote in echinonect in from the HL, which they r e f e r to as an embryonic substrate adhesion p r o t e i n , because of i t s adhesive propert ies with d i s s o c i a t e d embryonic c e l l s . In add i t ion to these prote ins i s o l a t e d from the HL, ant ibodies to several ver tebrate antigens inc lud ing c o l l a g e n , f i b r o n e c t i n , laminin and HSPG have c ross- reacted with antigens in the HL (Speigel & S p e i g e l , 1979; Spiegel et a l . , 1979; Wessel et a l . , 1984)). The HL in astero ids has not been extens ive ly invest igated b iochemica l l y ; however i t has been descr ibed morpholog ica l ly , and -113-appears to c l o s e l y resemble the echinoid HL (Crawford & Abed, 1986). A f t e r f i x a t i o n in the presence of the anionic dye a l c i a n b lue , i t i s seen to c o n s i s t of at least three l a y e r s : an i n t e r v i l l u s layer located between the m i c r o v i l l i , a supporting l a y e r , attached to the m i c r o v i l l u s t i p s , which i s equivalent to the supporting layer descr ibed in echinoids (Wolpert & Mercer, 1963; Lundgren, 1973), and an outer boundary layer composed of a coarse meshwork of ECM ( F i g . 2 0 ) . At present , i t i s unclear what r o l e these d i f f e r e n t morphological regions have in the proposed funct ion of the HL as a substrate fo r c e l l attachment, a kind of pseudo BL. -114-Fig. 20: A TEM of an u l t r a t h i n sect ion through the ectodermal ep i the l ium of a 4 day embryo f i xed with glutaraldehyde/ a l c i a n b lue , and processed for conventional TEM. The three regions of the HL are shown, inc lud ing the i n t e r v i l l u s layer ( I n ) , the supporting layer (Su), and the course outer meshwork (OM). x41,200. 115 -116-The ob jec t i ve of t h i s study was to examine the macromolecular nature o f the HL and i t s d i f f e r e n t regions using l e c t i n s as probes for carbohydrates at both the LM and the TEM l e v e l . Lect in l a b e l l i n g at the e lect ron microscopic level was essent ia l in order to show the l a b e l l i n g patterns in the d i f f e r e n t regions of the HL which were v i s u a l i z e d a f t e r f i x a t i o n in the presence of A l c i a n b lue, namely the i n t e r v i l l u s l ayer , the supporting layer and the coarse outer meshwork. In t h i s way, i t could be determined i f these morphological d i f fe rences represent biochemical (macromolecular) ones. Such information should prove useful in e l u c i d a t i n g the funct ions of the HL and may give c lues to the funct ions of each of these morphologica l ly d i f f e r e n t reg ions . The mater ia ls and methods used in t h i s study were the same as those used for the l e c t i n h istochemistry of the BL and ECM, which have a lready been descr ibed in chapter 2. 5.2 RESULTS FITC-Lect in L a b e l l i n g o f the Hyal ine Layer The HL l a b e l l e d with l e c t i n s from 4 out o f 6 of the major monosaccharide-binding groups, inc lud ing the glucose/mannose-binding group (Con A ) , the galNAc-binding group (SBA), the ga lactose-b ind ing group (RCA, PNA), and the glcNAc-binding group (WGA). UEA, a l e c t i n represent ing the fucose-binding group, did not b ind , nor d id LFA, represent ing the s i a l i c ac id -b ind ing group. Most of the l e c t i n - b i n d i n g s i t e s appeared to be preserved best with ethanol ; however WGA-binding s i t e s were most evident a f t e r f i x a t i o n with PF/CPC. Because of the l o c a t i o n of the HL on the outer-most s ide o f the embryo, i t was subjected to a degree of mechanical manipulation during the processing of the t i s s u e . This meant that in some areas of the embryo, part of the -117-HL may have been torn away and l o s t , which may have accounted for the s l i g h t v a r i a t i o n s in l a b e l l i n g i n t e n s i t i e s of the HL in d i f f e r e n t regions of the embyro. A l t e r n a t e l y , t h i s may have been due to d i f f e r e n t planes of sect ion in sect ions of embryos that were not exact ly s a g i t t a l . A r e s u l t of t h i s type i s seen in F ig .21a , where the HL showed strong l a b e l l i n g with FITC-Con A in most parts of the embryo, but weaker l a b e l l i n g in other p a r t s . The ECM material equivalent to the HL which l i n e s the al imentary canal gave patchy s t a i n i n g with Con A in the stomach and i n t e s t i n e of the ethanol f i xed m a t e r i a l , while s ta in ing was intense in the esophageal region ( F i g . 2 1 a ) . A f t e r exposure of paraformaldehyde/CPC-fixed t i s s u e , HL l a b e l l e d very i n t e n s e l y with FITC-WGA ( F i g . 2 2 a ) . As with the Con A, there was some v a r i a b i l i t y in the i n t e n s i t y of l a b e l l i n g over d i f f e r e n t regions of the embryo. There was almost no l a b e l l i n g of the ECM l i n i n g the stomach or i n t e s t i n e , although some regions of the ECM l i n i n g the esophagus l a b e l l e d heav i l y ( F i g . 2 2 a ) . -118-Th e fo l lowing f igures (21a-24b) are f luorescence micrographs showing the F I T C - l e c t i n - s t a i n i n g patterns of the HL in 6 day embryos. The embryos were embedded in JB4, and sectioned (lyO p r i o r to s t a i n i n g . F i g . 21a: A s a g i t t a l sect ion through an e thano l - f ixed embyro stained with FITC-Con A, showing the heav i ly l a b e l l e d HL (arrows) d i r e c t l y apposed to the ectodermal ep i the l ium. Of i n t e r e s t a l so i s the heavy l a b e l l i n g of the ECM material l i n i n g the esophagus ( E s ) , and to a l e s s e r degree the stomach (S) and i n t e s t i n e (In) (arrowheads). x480 F i g . 21b: A s e r i a l sect ion o f the above stained with the control sugar/conjugate so lut ion (mannose/FITC-Con A) ; f a i n t l a b e l l i n g of the HL i s observed in some areas but i t i s d r a s t i c a l l y reduced from that seen in F i g . 21a. x480 -120-F i g . 22a: A s a g i t t a l sect ion through a PF/CPC-fixed embryo which has been stained with FITC-WGA. L a b e l l i n g of the HL i s continuous and very intense (arrows). Some l a b e l l i n g of the ECM material l i n i n g the esophagus i s a l so noted (arrowheads). Es=esophagus, S=Stomach. x480 F i g . 22b: A s e r i a l sect ion of the above stained with the control sugar/conjugate so lut ion (glcNAc/FITC-WGA) showing negative l a b e l l i n g o f the HL. x480 121 -122-SBA and RCA both l a b e l l e d the HL in e thano l - f ixed t i s s u e . L a b e l l i n g with SBA was d iscont inuous, and appeared as p e r i o d i c d e n s i t i e s around the embryo ( F i g . 2 3 a ) . Unl ike the other l e c t i n s which appeared to label the e n t i r e HL, RCA l a b e l l e d only the innermost part o f the HL ( F i g . 2 4 b ) . With both SBA and RCA, there was l i t t l e or no l a b e l l i n g of the ECM l i n i n g the stomach or i n t e s t i n e , although the ECM l i n i n g the esophagus l a b e l l e d heav i ly with SBA ( F i g . 2 3 a ) , and l i g h t l y with RCA ( F i g . 2 4 a ) . Intense l a b e l l i n g of the mucous plug located at the junct ion between the esophagus and stomodeum was a lso noted with SBA ( F i g . 2 3 a ) . Control s ta ins c o n s i s t i n g of sugar/ lec t in conjugate so lut ions were performed on s e r i a l s e c t i o n s , and in a l l cases, l a b e l l i n g was negative (F igures 21b-24b). -123-F i g . 23a: A sag i ta l sect ion through an e thano l - f ixed embryo, stained with FITC-SBA. The l a b e l l i n g of the HL i s very intense , and has the appearance of per iod i c d e n s i t i e s along the length of the HL (arrows). The ECM material l i n i n g the i n s i d e of the esophagus (Es) labe ls very s t r o n g l y , as well as does the mucus p lug , which i s located j u s t outs ide the stomodeum (arrowheads). Some l a b e l l i n g of the ECM l i n i n g the ins ide of the stomach (S) and i n t e s t i n e (In) i s a l so ev ident . x480 F i g . 23b: A s e r i a l sect ion of the above stained with the control sugar/conjugate so lut ion (galNAc/FITC-SBA) showing no l a b e l l i n g of the HL. x480 124 -125-F i g . 24a: A s a g i t t a l sect ion through an e thano l - f ixed embryo, stained with FITC-RCA. Heavy l a b e l l i n g of the HL i s observed (arrows), and appears to label the innner region of the HL, and not the outer reg ion . Some l a b e l l i n g of the ECM l i n i n g the esophagus (Es) i s a l so f a i n t l y present (arrowheads). x480 F i g . 24b: A s e r i a l sect ion of the above stained with the control sugar/conjugate s o l u t i o n (galactose/FITC-RCA) showing no l a b e l l i n g of the HL. x480 / -127-AiUf--Lectin L a b e l l i n g of the Hyaline Layer Gold p a r t i c l e s conjugated to SBA, a galNAc-binding l e c t i n , were located over the i n t e r v i l l u s , supporting and outer meshwork regions ( F i g . 2 5 a ) . In a d d i t i o n , a l l of the ECM material l i n i n g the al imentary canal a l so l a b e l l e d with SBA-AU25-Con A, represent ing the mannose/glucose-binding l e c t i n s , did not label the HL h e a v i l y , as i t d id at the l i g h t microscopic leve l when conjugated to FITC. The somewhat scattered l a b e l l i n g was, however, above the leve l of background l a b e l l i n g , and most f requent ly was located over the supporting layer (F ig .26a ,26b) . Very few gold p a r t i c l e s were located over the ECM material l i n i n g the al imentary c a n a l , a f t e r s t a i n i n g with t h i s gold l e c t i n conjugate. The 2 l e c t i n s WGA and PNA, represent ing the glcNAc-binding and ga lactose-b ind ing group of l e c t i n s r e s p e c t i v e l y , had very s i m i l a r l a b e l l i n g patterns in the HL. Unl ike SBA, which l a b e l l e d a l l regions of the HL, l a b e l l i n g with WGA and PNA was almost e x c l u s i v e l y l im i ted to the support ing l a y e r . ( F i g . 2 7 a , 2 8 a ) . Whereas PNA l a b e l l i n g was a lso found over the ECM material l i n i n g the al imentary c a n a l , WGA l a b e l l i n g was n e g l i g i b l e over the same reg ion . The l a b e l l i n g patterns of the A u 2 5 - l e c t i n s in the HL are summarized in tab le 12. -128-TABLE 12: AU25-LECTIN LABELLING* OF THE HYALINE LAYER: A REGIONAL BREAKDOWN LECTIN REGIONS OF THE HYALINE LAYER  INTERVILLUS SUPPORTING OUTER MESHWORK WGA PNA SBA Con A + + ++ ++ ++ ++ + + / ++ / *LEGEND ++ = heavy label 1ing + = moderate l a b e l l i n g / = l i t t l e or no l a b e l l i n g Control s ta ins (O.IM blocking s u g a r / A u 2 5 - l e c t i n s conjugates) were performed on s e r i a l s e c t i o n s , and with a l l the l e c t i n s , l a b e l l i n g was negative (F ig .25b,26b,27b,28b) . -129-Fig. 25a: A TEM through the ectodermal region of the embryo, showing the hyal ine layer on the outer s ide of the ep i the l ium. This sect ion was stained with Au 2 g-SBA, and shows heavy l a b e l l i n g of the i n t e r v i l l u s ( I n ) , supporting (Su) and outer meshwork (OM) regions of the HL. x29,400 Fig. 25b: A TEM as above, stained with the control sugar/conjugate s o l u t i o n (galNAc/Au 2 5 ~SBA), showing very low background l a b e l l i n g over the HL. x31,500 Fig. 26a: A TEM through the ectodermal region of the embryo, showing the hyal ine layer which surrounds the ectodermal ep i the l ium. This sect ion was stained with Au^-Con A, and shows moderate l a b e l l i n g of the HL, predominantly over the supporting layer (arrows), but a l so over the other reg ions . X34 .000 Fig. 26b: A TEM as above, stained with the control sugar/conjugate s o l u t i o n (mannose/Au2g-Con A ) , showing very l i t t l e background l a b e l l i n g . x26,000 130 -131-F i g . 27a: A TEM through the ectodermal region of the embyro, stained with Au 2 5-WGA, showing the l a b e l l i n g pattern over the HL. The gold p a r t i c l e s are r e s t r i c t e d p r i m a r i l y to the supporting layer (Su, arrows), although an occasional p a r t i c l e i s seen over other regions of the HL. In = i n t e r v i l l u s , OM = Outer Meshwork x38,500 F i g . 27b: A TEM as above, stained with the control sugar/conjugate s o l u t i o n (glcNAc/Au^-WGA), showing a very low background leve l of l a b e l l i n g . x35,700 F i g . 28a: A TEM through the ectodermal region of the embyro, stained with Au 2 5 -PNA, showing the l a b e l l i n g pattern over the HL. L a b e l l i n g of the supporting layer i s very heavy (arrows), while l a b e l l i n g of the i n t e r v i l l u s and outer meshwork regions i s very l i g h t and s c a t t e r e d . x55,000 F i g . 28b: A TEM as above, stained with the control sugar/conjugate s o l u t i o n (ga lactose/Au 2 5 -PNA) , showing no background l a b e l l i n g . x46,200 132 -133-5.3 DISCUSSION The present study demonstrated that l e c t i n s from 4 d i f f e r e n t carbohydrate-binding groups are found to label the HL. These include galNAc-binding (SBA), ga lactose-b ind ing (RCA,PNA), glcNAC-binding (WGA), and mannose/glucose-binding (Con A) l e c t i n s . The l a b e l l i n g patterns vary from l e c t i n to l e c t i n , suggesting that the d i s t r i b u t i o n of t h e i r conjugate sugars in the HL i s not uniform. At the LM l e v e l , FITC-conjugated SBA, RCA, WGA, and Con A a l l label the HL. Although with the LM i t i s not poss ib le to reso lve the 3 regions of the HL c l e a r l y , i t appears that l a b e l l i n g with RCA i s r e s t r i c t e d to the inner region of the HL, suggesting that t h i s region i s r i c h in galatose res idues , whereas SBA, WGA and Con A l a b e l l i n g occurs over the " e n t i r e " HL, suggesting that galNac, glcNAc and mannose/glucose re i s idues are more evenly d i s t r i b u t e d . U l t r a s t r u c t u r a l studies using c o l l o i d a l gold conjugated l e c t i n s confirm the r e s u l t s descr ibed above. While a l l the layers label heav i l y with SBA, suggesting that galactosamine i s present throughout t h i s s t r u c t u r e , WGA and PNA are present almost e x c l u s i v e l y in the supporting layer suggesting that t h i s region i s much r i c h e r in glucosamine and ga lac tose . L a b e l l i n g with Con A i s moderate in i n t e n s i t y , and ind icates the presence o f some mannose/glucose residues p r i m a r i l y in the supporting region of the HL. There are some d iscrepenc ies in l a b e l l i n g patterns between LM ( F I T C - l e c t i n ) and TEM (Au2 5 ~lect in) s t a i n i n g . For example, with the LM, WGA l a b e l l i n g appears to span the e n t i r e width of the HL, whereas with the TEM, s i g n i f i c a n t l a b e l l i n g i s seen only over the supporting l a y e r . This can most l i k e l y be a t t r i b u t e d to the d i f f e r e n t f i x a t i o n s used for these two methods. It i s qui te poss ib le that the PF/CPC -134-f i x a t i o n used f o r FITC-WGA l a b e l l i n g did not f i x the e n t i r e HL, permit t ing only the supporting layer to be l a b e l l e d . More l i k e l y , l a b e l l i n g with WGA, although only present over the supporting layer , might have obscured the other regions s ince the marker, FITC, ampl i f ies the s ignal to a c e r t a i n degree. It must be remembered that the 3 regions present in the HL are r e a l l y u l t r a s t r u c t u r a l e n t i t i e s , and thus t r y i n g to d i s t i n g u i s h them with the LM i s not r e a l l y warranted. I t i s probable that the f i x a t i v e used for TEM (g lutara ldehyde/a lc ian b l u e ) , which f i x e s the e n t i r e HL, gives a more accurate representat ion of l e c t i n binding s i t e s throughout the HL than do ethanol or forml in f i x a t i v e s . As d iscussed e a r l i e r , chondro i t in s u l f a t e and dermatan s u l f a t e contain large amounts of galactosamine. Since SBA, which labe l s a l l regions o f the HL, binds s t rongly to galactosamine, i t i s poss ib le that CS and DS are present in the HL. PNA and WGA bind s t rong ly to glucosamine and galactose residues which are present in large q u a n t i t i e s in hyaluronic ac id and kerat in s u l f a t e suggesting that these may be present in the supporting l a y e r . PNA and WGA can a lso be expected to bind to laminin and c o l l a g e n , two molecules which have been found in the sea urchin HL (Spiegel et a l . , 1983). I t i s poss ib le that the support ing layer of as te ro id HL may a lso contain these substances. A l t e r n a t i v e l y , the carbohydrates may be part of new types of macromolecules. I s o l a t i o n and biochemical c h a r a c t e r i z a t i o n of some of the sugar conta in ing molecules of the HL may help to sort out th i s problem. I t has been suggested that the HL i s involved in maintaining c e l l u l a r p o l a r i t y , adhesion and organ izat ion during ear ly embryonic development (Dan, 1960; Wolpert & Mercer, 1967; Schroeder, 1988) as well -135-as p ro tec t ion (Lundgren, 1973) and l u b r i c a t i o n (Crawford & Abed, 1986). The i d e n t i f i c a t i o n of adhesive molecules in the sea urchin HL plus the c r o s s - r e a c t i v i t y of many vertebrate basal laminae ant ibodies in the HL suggests that i t serves a funct ion s i m i l a r to that of the BL. The fac t that the d i f f e r e n t morphological regions of the as te ro id HL contain d i f f e r e n t carbohydrate moieties suggests that there are d i f f e r e n t glycoconjugates in these reg ions , which may r e f l e c t d i f fe rences in adhesive propert ies within these reg ions . 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