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The role of cellular and extracellular factors during mouth formation in embryos of the starfish Pisaster… Abed, Mona 1984

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THE ROLE OF CELLULAR AND EXTRACELLULAR FACTORS DURING MOUTH FORMATION IN EMBRYOS OF THE STARFISH PISASTER OCHRACEUS By MONA ABED B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Anatomy, U n i v e r s i t y of B r i t i s h Columbia We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH ""COLUMBIA December 1984 © Mona Abed, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 3E-6 (3/81) ABSTRACT The morphological changes in arrangement of both c e l l u l a r and e x t r a c e l l u l a r material (ECM) during mouth formation in embryos of the asteroid Pisaster ochraceus, have been studied using LM, TEM, and SEM. In early gastrula, the ECM consists of small v e s i c l e s and amorphous material of an intermediate s t a i n i n g density, and "beaded strands" c o n s i s t i n g of scattered intermediately stained fialments associated with densely stained granules. I n i t i a l l y , the ECM i s found in the blastocoel immediately adjacent to the ectoderm and endoderm c e l l s . In l a t e r stages, i t extends further into the b l a s t o c o e l , u n t i l i t bridges the gap between the ectoderm and endoderm. As t h i s occurs, less amorphous material i s seen and increasing numbers of "beaded strands" are present throughout the region occupied by the ECM. These are p a r t i c u l a r l y evident and appear better organized in the quadrant of the embryo in which the mouth w i l l form. Between 59 and 90 hrs a f t e r f e r t i l i z a t i o n , several events occur in rapid succession. The f i r s t involves the migration of c e l l s from the endodermal epithelium at the t i p of the archenteron into the blastocoel to form mesenchyme c e l l s . Their departure leaves a hole in the endodermal epithelium of the archenteron t i p which i s covered by the endodermal basal lamina. Shortly a f t e r t h i s , the presumptive stomodeal c e l l s send filamentous and co n i c a l c e l l u l a r processes into the b l a s t o c o e l . The endodermal basal lamina covering the hole extends as a b l i s t e r into the b l a s t o c o e l . At the same time the archenteron has become bent toward the presumptive stomodeal region. In the b l a s t o c o e l , mesenchyme c e l l s are enmeshed in "beaded strands" located in the presumptive mouth region. At this stage, the strands are highly branched, and tend to be r a d i a l l y arranged, and have become almost completely encrusted with densely stained material.- F i l o p o d i a of the mesenchyme c e l l s connect with the filamentous ectodermal processes described above. The c e l l bodies of the mesenchyme c e l l s appear to be connected with either the basal lamina b l i s t e r i t s e l f , or with scattered endoderm c e l l s located within the b l i s t e r , through small gaps in i t . At a s l i g h t l y l a t e r stage, the basal lamina b l i s t e r i s seen to be in contact with the c o n i c a l ectodermal processes. S t i l l l a t e r , a complete tube of basal lamina has formed between the ectoderm and endoderm. I n i t i a l l y , only scattered c e l l s are associated with the surface of the tube. Later, the tube i s occupied by endodermal c e l l s and invaginated ectodermal c e l l s forming the stomodeum . For approximately 24 hrs a f t e r i t i s formed, the mouth i s plugged with an o r a l plate c o n s i s t i n g of both ectodermal and endodermal c e l l s . These c e l l s eventually round up, loose t h e i r connection with t h e i r neighbors, and are l o s t to complete the formation of the mouth. The above observations suggest that the ECM components are secreted into the blastocoel by both the ectoderm and endoderm c e l l s . The components then appear to undergo a self-assembly into a filamentous meshwork. This meshwork appears to form a s c a f f o l d i n which the mesenchyme c e l l s migrate. The change in o r i e n t a t i o n of the f i b e r s i n the meshwork and the increase in densely stained material associated with i t a f t e r mesenchyme c e l l s migrate through i t , suggests that they may be responsible, at least i n part, for these changes. Mesenchyme c e l l s tend to be l o c a l i z e d to the quadrant of the embryo in which the mouth w i l l form, before and during mouth formation. This i s also the region in which the ECM i s highly organized. The increased organization of the ECM may guide and concentrate the mesenchyme c e l l s to the presumptive mouth region where they form associations with both the filamentous processes of the stomodeal ectoderm and the b l i s t e r of basal lamina, either i v d i r e c t l y or v i a connections through the basal lamina to c e l l s within i t . Once this contact has been made, c o n t r a c t i l e filaments, known to be located i n the f i l o p o d i a , could serve to p u l l the basal lamina b l i s t e r across the blastocoel to make contact with the coni c a l ectodermal spikes. This appears to be followed by fusion of the b l i s t e r of the basal lamina with that of the stomodeum forming the endodermal tube. Once formed, the endodermal tube appears to provide a framework for the organization of both the ectodermal and endodermal c e l l s which w i l l form the mouth. V TABLE OF CONTENTS Page Abstract i i L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements x i i i Introduction 1 Materials and Methods 11 I. C o l l e c t i o n and Maintenance of Adult S t a r f i s h 11 II . C o l l e c t i o n and Preservation of Sea Water 11 II I . Glassware 12 IV. Rearing of Embryos 12 (1) I s o l a t i o n and Preparation of the Gametes (2) F e r t i l i z a t i o n of Eggs and Culture of Normal Embryos V. L i C l Treatment 13 VI. Preparation of Embryos for Microscopy 13 (1) Staging the Embryos (2) F i x a t i o n and Embedding VII. Microscopy 15 (1) Light Microscopy (LM) (2) Transmission Electron Microscopy (TEM) (3) Scanning Electron Microscopy (SEM) Results 16 I. Factors A f f e c t i n g Normal Development 16 I I . Normal Development 17 I I I . Mouth Formation 28 (1) The Basal Lamina v i Page (2) The E x t r a c e l l u l a r M a t r i x (ECM) (3) The Hyaline Membrane IV. E x o g a s t r u l a t i o n Studies 108 (1) Development of Exogastrulae (2) The E x t r a c e l l u l a r M a t r i x (ECM) (3) The Hyaline Membrane Di s c u s s i o n and Conclusions 146 I. The E f f e c t of Sea Water on Normal Development 146 I I . Stages of Normal Development 147 I I I . Mouth Formation 148 (1) The Role of the Basal Lamina (2) The Role of the E x t r a c e l l u l a r M a t r i x Summary 176 Bib l i o g r a p h y 178 v i i LIST OF TABLES Table Page 1 The rate of development of P. ochraceus embryos at 11.5-12°C 27 2 Changes in the ECM and the hyaline membrane of normal and 143 exogastrulated P. ochraceus embryos v i i i LIST OF FIGURES Figure Page 1 U n f e r t i l i z e d egg of P. ochraceus before maturation 19 2 U n f e r t i l i z e d egg of P. ochraceus a f t e r maturation 19 3 F e r t i l i z e d egg of P.ochraceus 19 4 Two early cleavage stages 19 5 Blastula stage - 36 hrs , 19 6 Early gastrula stage - 40-47 hrs 19 7 Early-mid gastrula stage - 55 hrs 22 8 Mid gastrula stage - 59 hrs 22 9 Mid-late gastrula stage - 67 hrs 22 10 Early b i p i n n a r i a stage - 90 hrs 22 11 A TEM l o n g i t u d i n a l section through the archenteron - 50-55 hrs.... 24 12 A TEM l o n g i t u d i n a l section through the archenteron - 55-59 hrs.... 24 13 Rate of Development of P. ochraceus embryos at 11.5-12°C 26 14 A wholemount of a mid-gastrula stage archenteron with a hole in i t s t i p 30 15 A LM cross-section through the t i p of a mid-gastrula stage archenteron 30 16 A TEM of the hole in the archenteron showing the endodermal basal lamina.... 30 17 The beak-like p r o f i l e of a bent archenteron; 1 um section 32 18 Scattered c e l l s i n the presumptive mouth region 32 19 A SEM of the bent archenteron and the adjacent endoderm 34 20 A SEM of the ectodermal c e l l processes 34 21 A TEM of the basal lamina b l i s t e r opposite the presumptive stomodeal ectoderm 37 22 A high magnification TEM of a co n i c a l ectoderm process opposite the basal'lamina b l i s t e r 37 i x Figure Page 23 A TEM of the filamentous ectodermal processes 39 24 A LM of the basal lamina b l i s t e r and associated c e l l s 41 25 A TEM of the basal lamina b l i s t e r and associated c e l l s 41 26 A LM demonstating possible contact between a mesenchyme and endoderm c e l l through the basal lamina b l i s t e r 43 27 A SEM depicting possible contact between endoderm and mesenchyme and between mesenchyme and ectoderm 43 28 Contact between the basal lamina b l i s t e r and the ectoderm - 1 um grazing section 46 29 Contact between the b l i s t e r and an ectodermal process 1 um section 46 30 Contact between the basal lamina b l i s t e r and co n i c a l ectodermal process - TEM thin section 48 31 A high magnification TEM of the ectodermal process at the point of contact with the basal lamina b l i s t e r 48 32 A LM cross-section through the beginning stages of basal lamina tube formation 50 33 A TEM of the redundancy of the basal lamina between the two points of contact with the ectoderm 53 34 The basal lamina tube l i n e d with endodermal c e l l s 1 um section.... 55 35 A SEM showing the continuity of the endodermal basal lamina with the ectodermal basal lamina 57 36 A TEM of the inner surface of the stomoderm showing the absence of basal lamina 59 37 A LM of the o r a l plate : 61 38 A TEM of the o r a l plate 61 39 A CM showing the opening of the mouth 64 40 A TEM showing the mucous plug in the opening of the mouth 64 41 ECM concentrated along the ectoderm and endoderm of a 42 hr gastrula - 1 um section 66 42 A LM cross-section through the blastocoel showing the ECM of a 46 hr gastrula 66 X Figure Page 43 The l i g h t and dark s t a i n i n g material of a 42 hr early gastrula -TEM l o n g i t u d i n a l section 68 44 A TEM of the close association of the ectoderm and the ECM in a 42 hr embryo 68 45 A high magnification TEM of the l i g h t s t a i n i n g component of the ECM in a 42 hr embryo 70 46 A high magnification TEM of the dense s t a i n i n g component of the ECM in a 42 hr embryo 70 47 A 51 hr early-mid gastrula showing the ECM throughout the blastocoel -LM l o n g i t u d i n a l section ..... 73 48 A TEM of the u l t r a s t r u c t u r e of an ECM strand of a 51 hr embryo.... 75 49 A TEM showing the branched ECM strands of a 51 hr embryo 75 50 Beaded strands occupy one quadrant of a 55 hr embryo 1 urn cross-section 77 51 The complex u l t r a s t r u c t u r e of a t y p i c a l ECM strand of a 55 hr embryo 77 52 The r a d i a l pattern of ECM strands in the presumptive mouth region of a 59 hr embryo as seen with the LM .... 80 53 A TEM of the branched appearance and u l t r a s t r u c t u r e of a t y p i c a l ECM strand of a 59 hr embryo 80 54 A LM of a 71 hr embryo showing the densely stained granules of the ECM 82 55 Two d i f f e r e n t arrangements of ECM strands wihtin the blastocoel of an 83 hr embryo as seen with the LM 84 56 A high magnification LM cross-section through the archenteron of an 83 hr embryo showing the r a d i a l pattern of the ECM 84 57 A TEM through the presumptive mouth region of an 83 hr embryo showing the r a d i a l pattern of ECM strands 86 58 The web-like structure of the ECM of an 83 hr embryo as seen with the TEM 86 59 A space beneath a crescent shaped region 89 60 A crescent shaped region without a space beneath i t 89 X I Figure Page 61 Not a l l holes i n the ectoderm are associated with a crescent shaped region 89 62 Patches of ECM along the endoderm as seen with the TEM 91 63 Patches of ECM associated with spaces i n the endoderm as seen with the TEM 91 64 A high magnification TEM showing the components of the ECM patches of the endoderm 93 65 A TEM of the ECM patch along the ectoderm 93 66 A LM of a mid-late gastrula fixed in the presence of Alcian blue for 1 hr showing the l o c a l i z a t i o n of the ECM 96 67 A TEM of a mid-late gastrula fixed in the presence of Al c i a n blue for 1 hr showing the l i g h t l y stained ECM 96 68 The hyaline membrane surrounds the surface of the ectoderm - 1 u lo n g i t u d i n a l section .. 98 69 A low magnification TEM of the layers of the hyaline membrane 100 70 A high magnification TEM of the u l t r a s t r u c t u r e of the hyaline membrane 100 71 The i n t a c t hyaline membrane of the invaginating archenteron as seen with the LM 103 72 The d i s i n t e g r a t i n g hyaline membrane in the expanding t i p of the archenteron as seen with the LM 103 73 A low magnification of the u l t r a s t r u c t u r e of the expanding archenteron t i p 105 74 A close up of the c e l l s i n the expanding archenteron t i p 105 75 A TEM of the stomodeal invagination 107 76 A Nomarski interference phase-contrast of the L i C l induced early exogastrula 110 77 A LM of a n a t u r a l l y occuring early exogastrula 110 78 The c e l l u l a r debris of L i C l induced exogastrula as seen with the TEM 112 79 Mesenchyme c e l l s in the blastocoel of an 81 hr "natural" exogastrula as seen with the LM 115 x i i Figure Page 80 A LM lo n g i t u d i n a l section through the segmented archenteron of a L i C l induced exogastrula 117 81 A close up of the embryonic stomodeum of an exogastrulated embryo. 117 82 A close up of the opening in the t i p of an evaginated archenteron. 117 83 The d i s t r i b t i o n of the ECM of an early natural exogastrula 1 urn section 119 84 A LiCL induced early exogastrula showing the ECM and c e l l debris 1 um section 122 85 A TEM of the ECM associated with the ectoderm of a L i C l induced early exogastrula... 124 86 A TEM of the ECM associated with the endoderm of a L i C l induced early exogastrula 124 87 The d i s t r i b u t i o n of the ECM in a 55 hr "natural" exogastrula 1 um section 126 88 A TEM of the t y p i c a l ECM of a 67 hr "natural" exogastrula i n the region of the ectoderm 128 89 A TEM of the t y p i c a l ECM of a 67 hr "natural" exogastrula i n the region of the endoderm 128 90 A LM of the ECM of a 71 hr L i C l induced exogastrula 131 91 A TEM of the ECM of a 71 hr L i C l induced exogastrula 133 92 The ECM of a 83 hr L i C l induced exogastrula i n the region of the ectoderm - TEM thin section 135 93 The ECM of an 83 hr L i C l induced exogastrula i n the region of the endoderm - TEM thin section 135 94 A LM lon g i t u d i n a l section showing the complete hyaline membrane around a "natural" early exogastrula 138 95 A LM of the evaginated t i p of a 55 hr "natural" exogastrula 138 96 A TEM showing the discontinuous hyaline membrane of the evaginated t i p of a L i C l induced early exogastrula 140 97 A TEM showing the remnants of the hyaline membrane of a 67 hr "natural" mid-exogastrula 140 98 A TEM showing the remnants of the hyaline membrane of an 83 hr L i C l induced exogastrula 142 x i i i ACKNOWLEDGEMENT S It has been a great p r i v i l e d g e for me to have had the opportunity to do my work i n a department of such high standards. F i r s t , I would l i k e to give honor to my supervisor, Dr. Bruce Crawford. It was h i s tremendous knowledge of embryology and microscopy, his patience, persistance and good advice that helped make th i s research a success. I f e e l I have acquired an excellent t r a i n i n g i n electron microscopy and in wr i t i n g and presenting s c i e n t i f i c papers. Second, I would l i k e to give thanks and appreciation to Ursula V i e l k i n d . Her understanding of "the f r u s t r a t i o n s of a grad student" was most valuable i n helping me cope with "what appeared to be" setbacks and delays. Third, I would l i k e to express my sincere gratefulness to my parents whose great investment i n me and th e i r desire to see me e x c e l l , provided much of the inner motivation to succeed at achieving this degree. To them I dedicate my contribution to s c i e n t i f i c research. F i n a l l y , a spec i a l thank you to my many friends, e s p e c i a l l y Keith and Anne Coleman, Sheila McLachlan, Ted S u l l i v a n and Sharon McCann, for th e i r many prayers and t h e i r unending support and encouragement. Above a l l however, I give the praise and the glory to my Lord, Jesus Christ for guiding me and ordering my steps according to His perfect w i l l : "He who began a good work i n me has completed i t to the end". My hope i s that t h i s work w i l l continue to the glory of God. Maranathal INTRODUCTION E a r l y c e l l m i g r a t i o n and the c e l l groups so formed, determine the general form of the embryo and l a y the foundations of i t s organ systems. This process of the generation of form or the assumption of a new shape i s c a l l e d morphogenesis. Changing form e n t a i l s the organized s h i f t i n g of c e l l s i n t o a new environment i n which they can be used i n the ensuing steps of development. C e l l movements of t h i s type involved w i t h the development of embryonic form are often r e f e r r e d to as morphogenetic movements. Morphogenetic movements range from mere indentations of c e l l sheets as i n the formation of the o p t i c v e s i c l e , to t r a n s l o c a t i o n of c e l l s as i n the m i g r a t i o n of neural c r e s t c e l l s . The broad o u t l i n e s of such movements are w e l l known f o r g a s t r u l a t i o n and organogenesis i n a number of organisms both v e r t e b r a t e s and i n v e r t e b r a t e s , i n c l u d i n g c h i c k , amphibian, t e l e o s t s and echinoderms (Trinkaus, 1965). D i s t i n c t patterns of c e l l movement during morphogenesis (such as the migration of neural c r e s t c e l l s ) have a l s o been known f o r many years, but i t i s only r e c e n t l y that we have begun to understand how such movements may be d i r e c t e d and c o n t r o l l e d . One of the e a r l y t h e o r i e s put forward to e x p l a i n the c o n t r o l of morphogenetic movements was that of contact guidance pos t u l a t e d by Weiss (1947, 1961). This mechanism was based on t r a n s i t o r y c e l l c o n t a c t s . According to the author, the d i r e c t i o n i n which a c e l l w i l l move i s a r e s u l t of (1) a tug of war, determined by the stre n g t h of attachment of and the tension i n the pseudopod; and (2) one side having a greater adhesiveness than another f o r the p a r t i c u l a r pseudopod. Other f a c t o r s thought to play a r o l e i n the c o n t r o l of morphogenetic events are s p e c i f i c adhesive r e c o g n i t i o n between l i k e c e l l s promoted by c e l l surface r e c e p t o r s ; c y t o s k e l e t a l elements such as microtubules and m i c r o f i l a m e n t s ; 2 l o c a l i z e d c e l l d i v i s i o n ; and programmed c e l l death (Oppenheimer, 1980). The idea that the embryonic c e l l s themselves produce and are mutually dependent on an e x t r a c e l l u l a r m a t r i x has repeatedly been put forward and has i t s o r i g i n e a r l y i n t h i s century ( S z i l y , 1904; Studnicka, 1911; B a i t s e l l , 1925; Weiss, 1933). More r e c e n t l y , however, the e x t r a c e l l u l a r m a t r i x (ECM) has been recognized by many i n v e s t i g a t o r s , such as Hay, P o r t e r , Weston and Toole ( f o r review see Hay, 1981), to be an important f a c t o r i n the c o n t r o l of c e l l movement. A great deal of our knowledge about the ECM has come from extensive studies on c e l l s during v e r t e b r a t e embryogenesis. Neural c r e s t c e l l s f o r example, are known to be i n t i m a t e l y a s s o c i a t e d w i t h the e x t r a c e l l u l a r matrix which creates a s c a f f o l d through which the c e l l s can migrate (Ebendal, 1977; Tosney, 1978; Weston, 1983; E r i c k s o n and Turley, 1983; Spieth and K e l l e r , 1984). E p i t h e l i a l t i s s u e s , such as the e a r l y ectoderm and endoderm secrete matrices of c o l l a g e n , g l y c o p r o t e i n s , and glycosaminoglycans (GAG) (Cohen and Hay, 1971; Manasek, 1973), which are then assembled to form a s c a f f o l d to support m i g r a t i o n of mesenchyme c e l l s ( B a i t s e l l , 1925; Gross, 1961; Hay, 1968; Hay and Revel, 1969). In order to understand how these components may i n t e r a c t , a b r i e f i n t r o d u c t i o n of the GAG i s necessary. Glycosaminoglycans, formerly c a l l e d mucopolysaccharides, are unbranched polysaccharide chains composed of r e peating d i s s a c h a r i d e u n i t s . They are h i g h l y n e g a t i v e l y charged due to the presence of s u l f a t e and/or carboxyl groups on many of the sugar groups. Seven groups have been d i s t i n g u i s h e d i n v e r t e b r a t e s according to the type of residue l i n k a g e s , and the number and l o c a t i o n of s u l f a t e groups ( A l b e r t , et a l . , 1983). They are h y a l u r o n i c a c i d , c h o n d r o i t i n 4 - s u l f a t e , c h o n d r o i t i n 6 - s u l f a t e , dermatan s u l f a t e , heparan s u l f a t e , h e p a r i n , and keratan s u l f a t e . 3 These s u l f a t e d GAG are a s s o c i a t e d w i t h a p r o t e i n core forming what i s known today as the proteoglycans ( A l b e r t , et a l . , 1983). Hyaluronic a c i d , however, i s an e x c e p t i o n a l GAG because i t s sugar residues are not 3 6 s u l f a t e d and i t has a much higher molecular weight, 4x10 to 8x10 , than the other GAG. GAG chains occupy vast amounts of space because of t h e i r i n f l e x i b i l i t y and form hydrated gels due to t h e i r h y d r o p h i l i c nature and high d e n s i t y of negative charges. Even at low c o n c e n t r a t i o n s , GAG are able to create a turgor w i t h i n the ECM which r e s i s t s compressive f o r c e s , a l l o w i n g the r a p i d d i f f u s i o n of water s o l u b l e molecules and the m i g r a t i o n of c e l l s and c e l l processes ( A l b e r t , 1983). How the components of the m a t r i x are assembled, however, i s s t i l l not w e l l understood (Hay, 1981; 1983). Several f u n c t i o n s have been a t t r i b u t e d to the glycosaminoglycans. GAG are important (1) i n the c o n t r o l of c e l l p r o l i f e r a t i o n (Cohn, et a l . , 1976); (2) i n c e l l m igration (Toole, et a l . , 1972; Katow and Solursh, 1974,1979; P r a t t , et a l . , 1975); (3) i n m a i n t a i n i n g morphogenetic s t r u c t u r e s ( B e r f i e l d , et a l . , 1973; Banerjee, et a l . , 1977); and (4) i n c e l l d i f f e r e n t i a t i o n (Grobstein, 1955,1967; Konigsberg and Hauschka, 1965; Markwal, et a l . , 1975; M o r r i s , et a l . , 1977). For example, i n v i t r o s t u d i e s by Kosher and coworkers (Kosher, 1977) have shown that proteoglycans, GAG, and c o l l a g e n can e f f e c t i v e l y s u b s t i t u t e f o r l i v i n g notochord or neural tube i n s t i m u l a t i n g i s o l a t e d somites to undergo chondrogenesis. Another more organized e x t r a c e l l u l a r s t r u c t u r e that contains GAG and appears to be i n v o l v e d i n c e l l movement i s the basement membrane (as seen w i t h the l i g h t microscope), or the basal lamina (as seen w i t h the e l e c t r o n microscope). Basement membranes were f i r s t described by Todd and Bowman i n 1857 ( K e f a l i d e s et a l . , 1979), but i t was not u n t i l 1951 that Krakower and Greenspon i s o l a t e d t h i s e x t r a c e l l u l a r m a t e r i a l from r e n a l g l o m e r u l i and showed that i t contains collagenous and non-collagenous p r o t e i n components. Since then, w i t h the development of the e l e c t r o n microscope, i t has been e s t a b l i s h e d that basement membranes are f u n c t i o n a l t i s s u e u n i t s having a complex o r g a n i z a t i o n of type IV c o l l a g e n , g l y c o p r o t e i n s and proteoglycans c o n t a i n i n g heparan s u l f a t e and J other GAG. The basement membrane contains a b a s a l lamina which i s composed of f i n e f i l a m e n t s embedded i n an e l e c t r o n dense amorphous matrix ( C o t t a - P e r e i r a , and Rodrigo, 1978). Recent immunofluorescence studies have shown that both la m i n i n (Timpl, 1979) and f i b r o n e c t i n (Stenman, 1978; Hay, 1981) are c o n s t i t u e n t s of the basement membrane. Laminin i s thought to be involved i n the attachment of e p i t h e l i a l c e l l s to basement membranes (Sa k a s h i t a , 1980; Vlodavsky, 1981). F i b r o n e c t i n might f u n c t i o n as an adhesive l i g a n d s e r v i n g as a c r o s s - l i n k i n g agent between d i f f e r e n t molecules of the basement membrane and the ECM and between c e l l s and the ECM (Hay, 1981). The b a s a l lamina l i e s between the lamina l u c i d a , on the e p i t h e l i u m s i d e , and the r e t i c u l a r lamina which includes collagenous anchoring f i b r i l s and the m i c r o f i b r i l s ( K e f a l i d e s , 1979). The r e t i c u l a r lamina i s a product of connective t i s s u e c e l l s , i n most cases, and g e n e r a l l y contains f i b e r s which are t h i c k e r than those of the b a s a l lamina i t s e l f . This r e t i c u l a r l a y e r i s not n e c e s s a r i l y always present. For i n s t a n c e , where c e l l s are found on e i t h e r side of the b a s a l lamina as i n the glomerulus and embryonic t i s s u e s , there i s no r e t i c u l a r lamina ( K e f a l i d e s , 1979). With our knowledge today, s e v e r a l r o l e s have been a s c r i b e d to the b a s a l lamina. Besides a c t i n g as a semipermeable f i l t e r and a boundary between two c e l l l a y e r s , i t can provide a s c a f f o l d i n g during t i s s u e 5 remodelling or i n j u r e d t i s s u e regeneration, and may play a r o l e i n d i f f e r e n t i a t i o n . For example, some have demonstrated that fragments of b a s a l lamina i n f l u e n c e the a g g l u t i n a t i o n and spreading of e p i t h e l i a l c e l l s and f i b r o b l a s t s (Gerfaux, 1979; Wich, 1979). Today, our knowledge of morphogenetic events combined with a growing understanding of movements of c e l l s i n c u l t u r e and of mixed aggregates on the one hand, and w i t h more s o p h i s t i c a t e d techniques i n molecular b i o l o g y , biochemistry, h i s t o c h e m i s t r y , and e l e c t r o n microscopy on the other hand, has created a s i t u a t i o n i n which a n a l y s i s of the mechanisms of morphogenetic movements at the c e l l u l a r l e v e l i s p o s s i b l e . Morphogenesis i n v e r t e b r a t e s i s complex and d i f f i c u l t to study. Such studies must u s u a l l y be performed with i s o l a t e d embryonic c e l l s i n v i t r o , under c o n d i t i o n s that o f t e n bear l i t t l e resemblance to those i n the embryo. There are, however, a few simpler systems that have been studied and have helped to increase our understanding of the types of forces that shape the organism. One of these systems i s an i n v e r t e b r a t e group, the sea u r c h i n s ( e c h i n o i d s ) . U n l i k e v e r t e b r a t e embryos, which are complex, opaque, and u s u a l l y i n a c c e s s i b l e , embryos of echinoderms are simple, transparent and r e l a t i v e l y easy and inexpensive to r a i s e . The sea u r c h i n embryo has r a p i d development, the larvae being formed w i t h i n four days a f t e r f e r t i l i z a t i o n . I t i s p o s s i b l e to obtain a large number of normal embryos or embryos w i t h s p e c i f i c abnormalities at s p e c i f i c stages, since e a r l y development tends to be r e l a t i v e l y synchronous. Having o.nly two c e l l sheets, each only one c e l l l a y e r t h i c k , and a f l u i d - f i l l e d b l a s t o c o e l c o n t a i n i n g the m i g r a t i n g c e l l s , they f a c i l i t a t e the observation and i n t e r p r e t a t i o n of the events of morphogenesis at the c e l l u l a r l e v e l . In a d d i t i o n , the biochemistry of t h i s system has been e x t e n s i v e l y studied ( f o r a review see Davidson et a l . , 1982), making i t 6 p o s s i b l e to c o r r e l a t e observations on c e l l u l a r behaviour w i t h biochemical data. E a r l y s t a r f i s h ( a s t e r o i d ) embryos have s i m i l a r advantages to the echin o i d embryos. Moreover, s t a r f i s h embryos have only one type of mesenchyme c e l l e q u ivalent to the secondary mesenchyme c e l l s of the ec h i n o i d . Because they lack the primary mesenchyme c e l l s , which form the s p i c u l e s i n the sea u r c h i n , the p o s s i b i l i t y of confusion between the acti o n s of the two c e l l types, and the p o s s i b i l i t y of i n t e r f e r e n c e w i t h l i g h t m i c r o s c o p i c a l observations are minimized. In both echinoids and a s t e r o i d s , the mesenchyme c e l l s , p a r t i c u l a r l y the secondary mesenchyme c e l l s i n the e c h i n o i d , play a major r o l e i n e a r l y morphogenesis. These c e l l s leave the t i p of the i n v a g i n a t i n g endoderm s h o r t l y a f t e r g a s t r u l a t i o n and migrate f r e e l y through the b l a s t o c o e l e by extending and r e t r a c t i n g c o n t r a c t i l e f i l o p o d i a (Crawford and Chia, 1982). In the e c h i n o i d , i t has been suggested that secondary mesenchyme c e l l s are inv o l v e d i n the formation of the mouth, coelomic pouches, and that they form the esophageal musculature, pigment c e l l s and c e l l s of the p e d i c e l l a r i a i n o l d e r embryos (MacBride, 1896). M i c r o f i l a m e n t s appear to form the b a s i s f o r the c o n t r a c t i l e a c t i v i t y of mesenchyme c e l l processes i n both echinoids ( T i l n e y and Gibbins, 1969) and a s t e r o i d s (Crawford and Chia, 1978). Gustafson and Wolpert (1967) have suggested (1) that a c t i v e shortening of extended pseudopods can b r i n g about c e l l movement or s t r e t c h i n g ; and (2) that pseudopodal a c t i v i t y and the adhesiveness of the c e l l surface may be d i f f e r e n t i n d i f f e r e n t parts of the embryo. This i s i n agreement w i t h the contact guidance mechanism discussed above. Based on these concepts of adhesion and t e n s i o n , morphogenetic models have been proposed i n an attempt to describe mechanisms f o r d i r e c t e d c e l l movement, pattern formation, and 7 changes i n the shape of c e l l sheets (review by Gustafson and Wolpert, 1967). In s t a r f i s h embryos, c e l l s migrate and d i f f e r e n t i a t e i n a s i m i l a r manner to that seen i n v e r t e b r a t e embryos. A s t e r o i d mesenchyme c e l l s e x h i b i t many of the c h a r a c t e r i s t i c s described f o r v e r t e b r a t e neural c r e s t c e l l s . They migrate i n t o a space which contains ECM which appears s i m i l a r to that found i n neural c r e s t pathways i n v e r t e b r a t e embryos (Katow and Solursh, 1979). Although mesenchyme c e l l m i g r a t i o n i n i t i a l l y appears to be random, u n l i k e the m i g r a t i o n of neural c r e s t c e l l s , they e v e n t u a l l y migrate to defined regions of the embryo where they form s e v e r a l d i f f e r e n t c e l l types i n c l u d i n g muscle and pigment c e l l s (Chia, 1977). L i g h t and e l e c t r o n microscope studies at or near the time of mouth formation i n the e c h i n o i d (Immers, 1961; Immers and Runnstrom, 1965; Sugiyama, 1972; Endo & Noda, 1977; Katow and Solursh, 1979; Akasaka et a l . , 1980; Kawabe et a l . , 1981) have noted the presence of a f i b r i l l a r m a t e r i a l and a s s o c i a t e d GAG w i t h i n the b l a s t o c o e l . These GAG appear to be necessary f o r mesenchyme c e l l movement (Karp & Solursh, 1974), and i t has been suggested that t h i s m a t e r i a l may guide the movement of the archenteron (Kawabe et a l . , 1981) and the mesenchyme c e l l s (Crawford and Chia, 1981). The b a s a l lamina i s a l s o thought to be involved i n d i r e c t i n g morphogenesis i n v e r t e b r a t e s . Accumulated evidence over the years, has shown that mesenchyme c e l l s promote branching of an e p i t h e l i a l tube by removing GAG fromm the b a s a l surface of the e p i t h e l i u m ( f o r review, see B e r n f i e l d and Banerjee, 1978). Since then, the involvement of the b a s a l lamina during development has been described i n terms of basal lamina d i s c o n t i n u i t i e s or i n t e r r u p t i o n s . These would a l l o w ( l ) the 8 t r a n s l o c a t i o n of c e l l s (Katow and Solursh, 1980); and/or (2) l o c a l ectodermal p r o t r u s i o n s by which mesenchyme c e l l movement may be guided (Solursh and Revel, 1978). The b a s a l lamina i n echinoids was not described u n t i l the e l e c t r o n microscopic work of the 1960s. Endo and Uno (1960) noted that a t h i n membranous s t r u c t u r e appeared along the inner surface of the b l a s t u l a r w a l l around the eighth d i v i s i o n c y c l e i n one species of sea u r c h i n . Wolpert and Mercer (1963) working w i t h a d i f f e r e n t s p e c i e s , found a "0.1 um t h i c k fuzzy l a y e r " one hour before hatching. Okazaki and N i i j i m a (1964) were s u c c e s s f u l i n i s o l a t i n g t h i s "inner l a y e r " which they i d e n t i f i e d to be the basement membrane, since i t appeared s i m i l a r to the basement membrane seen i n amphibia at that time. They a l s o reported that i t was composed of mucopolysaccharides, but the f i b r o u s components were not discerned. In a d d i t i o n , because of i t s p o s i t i o n and e l a s t i c nature, they proposed that the f u n c t i o n of t h i s basement membrane was to act as a b a r r i e r to unwanted molecules. More r e c e n t l y , Katow and Solursh (1980) observed d i s c o n t i n u i t i e s i n the basal lamina i n the v e g e t a l p l a t e region during primary mesenchyme c e l l ingress ion i n the sea u r c h i n . They suggested that perhaps the mesenchyme c e l l s form by a d i s t i n c t mechanism i n which the f i r s t step i s the l o s s of the b a s a l lamina. A t h i r d type of ECM which i s a l s o thought to be i n v o l v e d i n morphogenesis of e c h i n o i d embryos, i s the h y a l i n e l a y e r . This membrane envelopes sea u r c h i n embryos (Spi e g e l et a l . , 1980) as w e l l as other echinoderm embryos. The h y a l i n e l a y e r i s formed at f e r t i l i z a t i o n from the f u s i o n of c o r t i c a l granules w i t h the plasma membrane of the egg (Endo, 1961; Runnstrom, 1966). The surface of the f e r t i l i z e d egg becomes f i r m l y attached to the h y a l i n e l a y e r by means of c e l l surface m i c r o v i l l i (Dan and Ono, 1952; Dan, 1960; Wolpert and Mercer, 1963). Composed of 9 polysaccharides and p r o t e i n (Vasseur, 1948; Nakano and Ohachi, 1954; Vacquier, 1969), the h y a l i n e l a y e r , when sta i n e d w i t h Ruthenium red and viewed w i t h the transmission e l e c t r o n microscope, appears as a complex f i b r i l l a r m a t r i x resembling the ECM of v e r t e b r a t e s ( S p i e g e l and S p i e g e l , 1979). Several i n v e s t i g a t o r s have studied the h y a l i n e l a y e r w i t h respect to i t s composition and the l o c a l i z a t i o n of i t s components i n u n f e r t i l i z e d and f e r t i l i z e d eggs, as w e l l as i n reaggregated c e l l s of the sea u r c h i n (Endo, 1961; Kondo and Sakai, 1971; Kane, 1973; Spiegel and S p i e g e l , 1975, 1979;Banzhof, et a l . , 1980; McCarthy and S p i e g e l , 1983a,b). Because of i t s composition, l o c a l i z a t i o n and u l t r a s t r u c t u r e , the h y a l i n e l a y e r i s a l s o thought to play a major r o l e i n c e l l o r g a n i z a t i o n , adhesion and therefore morphogenesis (Herbst, 1900; Dan and Ono, 1952; Dan, 1960; Gustafson and Wolpert, 1962; Wolpert and Mercer, 1963). Mouth formation i s an e x c e l l e n t example of a morphogenetic movement found i n e a r l y a s t e r o i d embryos which can be e a s i l y s t u d i e d . C l a s s i c a l d e s c r i p t i o n s of mouth formation i n a s t e r i o d s at the l i g h t microscope l e v e l (Agassiz, 1877; Metschnikoff, 1884; Roux, 1895; MacBride, 1896; Gemmill, 1912&1914; Horstadius, 1939) show that mesenchyme c e l l s migrate from the t i p of the archenteron i n t o the b l a s t o c o e l e l e a v i n g a hole i n the archenteron t i p . This i s followed by the bending of the endodermal tube, the archenteron, toward a b l i n d inpocketing of the ectoderm, the stomodeum. These two s t r u c t u r e s e v e n t u a l l y contact one another and fuse to form the mouth, but there are few clues as to how these events are c o n t r o l l e d . Studies' of e a r l y morphogenetic events i n echinoids suggest that one c o n t r o l mechanism may i n v o l v e the c o n t r a c t i l e processes of mesenchyme c e l l s which are thought to a i d i n the e l o n g a t i o n and bending of the archenteron (Gustafson and Kinnander, 1960; Gustafson. and Wolpert, 1963a,b,1967), but these studies give no clues as to how the arrangement 10 of the c o n t r a c t i l e mesenchyme c e l l processes are d i r e c t e d so as to exert force i n the c o r r e c t d i r e c t i o n at the c o r r e c t time. Using the s t a r f i s h embryo as a model system, the present work e n t a i l s the study of a b a s i c morphogenetic event: formation of the mouth i n the s t a r f i s h P. ochraceus. L i g h t microscopy and transmission e l e c t r o n microscopy of m a t e r i a l f i x e d i n A l c i a n blue, a dye which i s known to preserve the e x t r a c e l l u l a r m a t e r i a l (Behnke and Zehlander, 1970), and scanning e l e c t r o n microscopy have been used to study the changes i n arrangement of both the c e l l s and e x t r a c e l l u l a r m a t r i x during mouth formation. P a r t i c u l a r emphasis was placed on any observed i n t e r a c t i o n between the c e l l s and the e x t r a c e l l u l a r m a t r i x i n c l u d i n g the b a s a l lamina and h y a l i n e l a y e r . A d e t a i l e d morphological study of these i n t e r a c t i o n s might give clues as to how c e l l movements are c o n t r o l l e d during mouth formation i n p a r t i c u l a r , and morphogenesis i n general. 11 MATERIALS AND METHODS I. C o l l e c t i o n and Maintenance of the Adult S t a r f i s h Ripe ad u l t P i s a s t e r ochraceus were c o l l e c t e d from the rocky i n t e r t i d a l zone at P t . Roberts, WA. , Stanley Park i n Vancouver, Copper Cove i n North Vancouver, and 10 M i l e Point i n V i c t o r i a , B r i t i s h Columbia, between March and June, 1981-83. The animals were kept i n the Department of Zoology i n aquaria s u p p l i e d w i t h running n a t u r a l sea water from a closed system at a temperature of 10-12°C. They were maintained under constant l i g h t and away from other spawning s t a r f i s h , since i t has been suggested that the photoperiod c o n t r o l s sexual a c t i v i t y (Pearce and Eanisse, 1982), and that spawning may be epidemic ( F r i d a y Harbour Laboratory Manual). I I . C o l l e c t i o n and P r e s e r v a t i o n of the Sea Water U n l i k e the adu l t animals, the embryos are very s e n s i t i v e to t h e i r environment e s p e c i a l l y during the f i r s t few days of development, and great care was taken to f u r n i s h a s u i t a b l e environment f o r t h e i r growth. The q u a l i t y of the sea water was found to be a primary f a c t o r c o n t r i b u t i n g to the success of the c u l t u r e s (see R e s u l t s ) . The sea water used i n r a i s i n g the embryos was obtained from e i t h e r the Gulf I s l a n d s , the San Juan I s l a n d s , Point Roberts, or V i c t o r i a . I t was f i l t e r e d through Whatman #1 f i l t e r paper and stored i n p l a s t i c containers at 10-12°C. The f i l t e r e d water was a l s o aerated f o r a few minutes j u s t before use. This was done with a bubbling stone connected to an a i r v a l v e . The s a l i n i t y of the sea water from a l l areas was measured using an osmette p r e c i s i o n osmometer. 12 I I I . The Glassware Because the c o n d i t i o n of the glassware used was another f a c t o r c o n t r i b u t i n g to the success of the c u l t u r e s , only new glassware was used. This glassware was designated "E" (embryonic); i t was r i n s e d only w i t h sea water or or d i n a r y tap water and used s o l e l y f o r c u l t u r e s of the embryos according to the i n s t r u c t i o n s i n the F r i d a y Harbor Laboratory Manual. IV. Rearing the Embryos 1). I s o l a t i o n and Pr e p a r a t i o n of the Gametes The a d u l t s t a r f i s h were sexed by making a s l i t i n one of the arms and examining the gonad. The ovaries appear salmon pink and the t e s t e s appear i v o r y i n c o l o r . Once one animal of each sex had been found, an e n t i r e arm, i n c l u d i n g the gonads, was removed and the gonads were d i s s e c t e d out ( F u s e l e r , 1973). The r e l a t i v e h e a l t h and s u r v i v a l of the animals was f a r b e t t e r f o r those whose gonads were obtained by removal of an e n t i r e arm than those obtained through a s l i t i n an arm as reported p r e v i o u s l y (Crawford and Chia, 1980). Once removed, the t e s t e s were stored "dry" ( i n absence of seawater) i n a covered p e t r i d i s h at 10-12°C f o r 1-2 h r s . The ovaries were incubated i n f i l t e r e d n a t u r a l sea water c o n t a i n i n g 1 mg/ml 1-methyladenine i n an uncovered p e t r i d i s h at 10-12°C f o r 1-2 hrs (Stevens, 1970). This procedure causes maturation of the eggs which i s i n d i c a t e d by the breakdown of the nucleus or germinal v e s i c l e of the egg (see R e s u l t s ) . 2). F e r t i l i z a t i o n of Eggs and Culture of Normal Embryos When more than 90% of the germinal v e s i c l e s had broken down (1-2 h r s ) , enough eggs were t r a n s f e r r e d to a c u l t u r e j a r to cover 50% of the bottom w i t h a monolayer of the gametes, to prevent overcrowding. 13 The egg suspension was then washed several times by resuspension with f i l t e r e d natural sea water and allowed to r e s e t t l e . At the same time, a sperm suspension was made up by d i l u t i n g a few of drops of dry sperm in about 50 ml of the natural sea water giving a s l i g h t l y turbid suspension. A few drops of this d i l u t e sperm suspension were added to the water containing the eggs. This amount was more than s u f f i c i e n t to f e r t i l i z e an egg cu l t u r e . After 1/2-1 hr, the f e r t i l i z e d eggs were again washed to remove the excess sperm and the embryos were allowed to develop to the desired stages at 10-12°C. The sea water was changed twice during the day to maintain optimum growth conditions. During the i n i t i a l stages of development, when the embryos were s e t t l e d at the bottom of the culture j a r s , the water was decanted and the embryos resuspended in fresh natural sea water. During the l a t t e r stages, when the embryos began to swim, a portion of the culture was poured out into another j a r , and fresh natural sea water was added to each j a r . V. L i C l Treatment Exogastrulation was induced by tr e a t i n g the embryos with L i C l according to a method by Child (1940). The L i C l was prepared by adding to sea water a stock solution of 0.5 M L i C l in d i s t i l l e d water. Embryos at 2-4 c e l l stages were placed in f i l t e r e d sea water (controls) and f i l t e r e d sea water containing ei t h e r 0.05 M L i C l or 0.10 M L i C l for a period of 5,10,12, and 15 hrs. The treated embryos were then washed 3-5 times by gentle c e n t r i f u g a t i o n and resuspension and allowed to develop in f i l t e r e d sea water at 10-12°C. VI. Preparation of Embryos for Microscopy 1). Staging the Embryos Normal and exogastrulated embryos were fixed during the course of 14 study. Embryos were grown at a c a r e f u l l y c o n t r o l l e d temperature (11.5-12°C) over a period of four days. The cultures were observed under a stereomicroscope every 6-8 hrs u n t i l the embryos began to gastrulate. Thereafter, aliquots of the cultures were fixed at 4 hr i n t e r v a l s . An average of 10 embryos was randomly selected for each time i n t e r v a l and the archenteron length to body length r a t i o (AL/BL) was calculated, using an ocular micrometer and a stage micrometer. Archenteron lengths (AL) and AL/BL r a t i o s were plotted against hours of development, in order to obtain a quantitative d e s c r i p t i o n of the rate of development, of P. ochraceous embryos. 2). F i x a t i o n and Embedding When the desired stages were reached, aliquots of the cultures were removed from the jars with a pipette and the embryos were concentrated by gentle c e n t r i f u g a t i o n i n 12 ml coni c a l centrifuge tubes in a c l i n i c a l centrifuge (1/3 speed for 1-2 min). Several drops of the concentrated embryos were then added to 2-5 ml of f i x i n g s o l u t i o n . The material was fixed at room temperature i n 1% gluteraldehyde with 1% A l c i a n Blue 8GX (Eastman Chemicals) (Behnke and Zelander, 1971) in 80% sea water, pH 7.0 for 1 hr (Crawford and Abed, 1983). (The experiments which required aliquots of the cultures to be fixed at 4 hr i n t e r v a l s however, remained i n this primary f i x a t i v e for 3-12 hrs.) This was followed by 3 rinses i n 2.5% NaHCO^ buffer, pH 7.4. P o s t - f i x a t i o n was done i n 2% OsO^ buffered at pH 7.4 with 1.25% NaHCC>3 (Wood and Luft, 1965) for 1 hr at room temperature. The animals were rinsed again in 2.5% NaHCO^ buffer, stained for 1 hr in 2% uranyl acetate, and dehydrated i n a graded series of ethanol and 3 changes of propylene oxide. After dehydration, the propylene oxide was replaced with two changes of Epon 812 (Luft, 1961) over a period of 24 hrs. The embryos were then 15 embedded in Epon and the Epon was allowed to polymerize at 60°C for 48 hrs. VII. Microscopy 1) . Light Microscopy (LM) Sections for l i g h t microscopy, ranging i n thickness from 0.25 um to 2 um, were obtained using glass knives on a Porter Blum MT-1 microtome. The sections were mounted on s l i d e s and stained with Richardson's st a i n (Richardson et a l . , 1960) and photographed on a Zeiss Universal Microscope using Adox KB-17 f i l m . The f i l m was developed for 7 min at room temperature in Kodak D-76 d i l u t e d 1:1 with d i s t i l l e d water, and fixed in Edwal Quick-Fix for 5 min. Live embryos or embryos fixed and stored in 2.5% glutaraldehyde in sea water and/or post-fixed in 2% osmium were photographed with either phase-contrast or Nomarski interference phase-contrast o p t i c s . A l c i a n blue was not used in these f i x a t i o n s . 2) . Transmission Electron Microscopy (TEM) Material for TEM was sectioned on a diamond k n i f e . S i l v e r or thin sections (50-60 nm) were picked up on carbon-coated or formvar-coated grids and stained with lead c i t r a t e (Reynolds, 1963) for 5 min at room temperature. Electronmicrographs were taken on either a P h i l l i p s 200, 300, or 301 microscope using Eastman fine grain p o s i t i v e f i l m #5302. The f i l m was developed for 5 min at room temperature in f u l l strength Kodak D-19 and fixed i n Edwal Quick-Fix for 4 min. 3) . Scanning Electron Microscopy (SEM) Material for SEM studies was dried by the c r i t i c a l point method using amylacetate and CO . Micrographs are courtesy of Dr. B.J. Crawford. 16 RESULTS I. Factors A f f e c t i n g Normal Development The ova of s t a r f i s h c o l l e c t e d from the various areas and i n d i f f e r e n t years demonstrated marked d i f f e r e n c e s i n t h e i r a b i l i t y to undergo development a f t e r f e r t i l i z a t i o n . Ova obtained from s t a r f i s h c o l l e c t e d at Copper Cove i n 1981 and 1982 developed normally i n sea water from V i c t o r i a and San Juan I s l a n d s . In c o n t r a s t , ova of animals obtained from Copper Cove and other lower mainland s i t e s i n 1983 f a i l e d to develop normally i n sea water from V i c t o r i a . Ova of s t a r f i s h obtained from the V i c t o r i a area developed normally i n sea water from Galiano (Gulf Islands) and V i c t o r i a , but not i n sea water from the lower mainland i n 1983. The s a l i n i t y of the sea water around the lower mainland and Point Roberts area was i n the range of 500-600 m i l l i o s m o l e s . The sea water from the V i c t o r i a area and the i s l a n d s had a s a l i n i t y i n the range of 850-1000 m i l l i o s m o l e s . The ova of animals c o l l e c t e d from the lower mainland were smaller and more i r r e g u l a r l y shaped than those c o l l e c t e d at V i c t o r i a . These eggs re q u i r e d 4-5 hrs of incubation i n 0.1 mg/ml 1-methyladenine to cause breakdown of t h e i r germinal v e s i c l e s , whereas those from the V i c t o r i a area r e q u i r e d only 1-2 h r s . The sperm of s t a r f i s h obtained from a l l l o c a t i o n s were normal i n appearance and m o t i l i t y and i n t h e i r a b i l i t y to f e r t i l i z e mature ova. In the poor c u l t u r e s , i n which embryos appeared abnormal, cleavages were i r r e g u l a r and asymmetrical and most of the embryos d i d not reach the b l a s t u l a stage. Of those that d i d , the b l a s t u l a appeared w r i n k l e d and the b l a s t o c o e l was f i l l e d w i t h d e b r i s , which appeared to c o n s i s t of c e l l fragments. A few of them proceeded beyond the b l a s t u l a stage. Some reached the mid-gastrula stage, but again the embryos were abnormal. 17 These were stunted i n growth, and had b l a s t o c o e l i n f o l d i n g s and d e b r i s . Some had exogastrulated. In no case d i d development proceed beyond the p o i n t where mesenchyme c e l l s appeared i n the b l a s t o c o e l . I I . Normal Development The b a s i c developmental p a t t e r n was s i m i l a r to that described i n other p l a n k t o t r o p h i c species and to that described f o r P i s a s t e r  ochraceous by Strathman ( F r i d a y Harbour Laboratory Manual). The eggs are approximately 160 um i n diameter and are covered with a j e l l y coat. Immediately upon r e l e a s e from the ovary, the eggs contained a large germinal v e s i c l e (FIG 1). A f t e r maturation i n 1-methyladenine f o r v a r y i n g periods (see above), the germinal v e s i c l e was no longer v i s i b l e (FIG 2). The sperm were much smaller i n s i z e and became a c t i v e swimmers upon contact w i t h the sea water. Under our c u l t u r e c o n d i t i o n s , a c l e a r f e r t i l i z a t i o n membrane formed w i t h i n 10-15 min a f t e r f e r t i l i z a t i o n (FIG 3). Within 2-4 h r s , the c e l l completed i t s m e i o t i c d i v i s i o n s r e l e a s i n g 2-3 p o l a r bodies (FIG 3), and proceeded to cleavage. Cleavage was equal (FIG 4) and w i t h i n 12 hrs gave r i s e to a hollow b l a s t u l a (FIG 5). By 20 h r s , the surface c e l l s were c i l i a t e d and the animal began to r o t a t e w i t h i n the f e r t i l i z a t i o n membrane. The embryo broke out of i t s f e r t i l i z a t i o n membrane (hatched) 24-28 hrs a f t e r f e r t i l i z a t i o n , and began to swim a c t i v e l y . By 36 h r s , the l a t e b l a s t u l a stage had been reached. This c o n s i s t e d of a b l a s t u l a w i t h thickened c e l l s at the v e g e t a l p o l e . Over the next 5-10 h r s , the v e g e t a l pole invaginated and the endoderm began to advance across the b l a s t o c o e l (FIG 6), g i v i n g r i s e to the archenteron and the b l a s t o p o r e . 18 Figure 1 An u n f e r t i l i z e d egg of the s t a r f i s h P i s a s t e r ochraceus before maturation, showing the germinal v e s i c l e (GV). x400 Figure 2 An u n f e r t i l i z e d egg a f t e r i ncubation i n 1-methyladenine f o r 1-2 hours to promote a c t i v a t i o n . The germinal v e s i c l e (GV) has broken down and the egg i s now ready to be f e r t i l i z e d . x400 Figure 3 An egg, 2-4 hours a f t e r f e r t i l i z a t i o n . The f e r t i l i z a t i o n membrane (FM) enc l o s i n g the egg and 3 p o l a r bodies (arrowheads) can be seen. x400 Figure 4 Two e a r l y stages of cleavage: (a) 8 c e l l stage (b) 16 c e l l stage showing the beginning of the formation of a hollow c a v i t y , the b l a s t o c o e l e (B). x250 Figure 5 B l a s t u l a stage, 36 hours a f t e r f e r t i l i z a t i o n . This c o n s i s t s of a s i n g l e layered blastoderm (arrow) surrounding a f l u i d f i l l e d b l a s t o c o e l e ( B ). x500 Figure 6 An e a r l y g a s t r u l a 40-47 hours p o s t - f e r t i l i z a t i o n . The ve g e t a l pole i s i n v a g i n a t i n g i n t o the b l a s t o c o e l e (B) to form the archenteron (A) and the blastopore (BP). x300 20 By 55 h r s , the r a t i o of archenteron length to body length (AL/BL) was 0.579 and the b l i n d end of the endoderm was beginning to expand, although as yet there was no change i n i t s thickness (FIG 7). The c e l l s of the archenteron t i p at t h i s stage appeared more elongated w i t h a broad apex and a narrower base as revealed by TEM t h i n s e c t i o n s (FIG 11). By 59 h r s , the AL/BL was 0.600, the archenteron t i p had expanded f u r t h e r and had become t h i n - w a l l e d (FIG 8 ) . TEM t h i n s e c t i o n s through the archenteron at t h i s stage showed short cuboidal c e l l s of uniform base and apex widths (FIG 12). At 63 hrs (AL/BL = 0.608), c e l l processes were extending from c e l l s at the t i p of the archenteron and by 67 hrs ( A l / B l = 0.633), mesenchyme c e l l s were v i s i b l e i n the b l a s t o c o e l (FIG 9). At l a t e r stages, the archenteron elongated i n an a n t e r i o r to p o s t e r i o r d i r e c t i o n more s l o w l y , while body growth continued more r a p i d l y . This change was evidenced by lower AL/BL r a t i o s i n the subsequent stages. At 71 h r s , the archenteron began to bend toward the ectoderm i n the presumptive mouth r e g i o n , and two c e l l u l a r outpocketings of the d i s t a l end of the archenteron formed the coelomic pouches. Within 87.5 h r s , a f t e r f e r t i l i z a t i o n , the ectoderm had invaginated to form the stomodeum, and had j o i n e d w i t h the endoderm to form the o r a l p l a t e of the mouth. By 90 h r s , the e a r l y b i p i n n a r i a stage was c h a r a c t e r i z e d by a completely segmented gut: mouth, esophagus, and stomach; two w e l l - d e f i n e d coelomes and the beginning of an o r a l hood (FIG 10). An early-mid g a s t r u l a stage, 55 hours p o s t - f e r t i l i z a t i o n . The archenteron (A) has grown i n t o the b l a s t o c o e l e (B) and the whole embryo has elongated. The archenteron t i p ( a r r o w s ) i s beginning to expand, but i s not yet t h i n - w a l l e d . x575 Figure 8 A mid-gastrula stage, 59 hours p o s t - f e r t i l i z a t i o n , showing the expanding t i p of the archenteron and i t s surrounding t h i n w a l l of c e l l s (arrows). B = b l a s t o c o e l ; L = Archenteron Lumen x575 Figure 9 An embryo approximately 67 hours a f t e r f e r t i l i z a t i o n showing mesenchyme c e l l s and c e l l processes (arrows). The ectoderm (EC), endoderm (EN) and mesenchyme (M) c e l l s can now be d e f i n e d . B = b l a s t o c o e l e x550 Figure 10 An e a r l y b i p i n n a r i a stage ( l a t e r a l v i e w ), 90 hours i n t o development. The gut i s now segmented i n t o the mouth ( o r a l p l a t e , OP), esophagus ( E ) , stomach ( S ) , and coelomic pouches (CP) and the stomodeal i n v a g i n a t i o n (S) can be i d e n t i f i e d . The blastopore has become the anus (arrow). x250 23 Figure 11 A TEM of a l o n g i t u d i n a l s e c t i o n through the archenteron between 50-55 hours, showing the t y p i c a l elongated c e l l s of the endoderm before the expansion of the archenteron t i p . B = b l a s t o c o e l ; L = archenteron lumen x5500 Figure 12 A TEM of a l o n g i t u d i n a l - s e c t i o n through the archenteron between 55-59 hours showing the t y p i c a l cuboidal endodermal c e l l s of the expanding archenteron t i p . B = b l a s t o c o e l ; L = archenteron lumen x9200 25 A p l o t of the AL/BL r a t i o s and the ALs against hours of development (FIG 13) produced two curves which showed that growth of the archenteron was r a p i d between 40 and 65 hrs p o s t - f e r t i l i z a t i o n , or u n t i l mesenchyme c e l l s appeared i n the b l a s t o c o e l . A slower r a t e of development between 65 and 90 hrs was evidenced by a tapering o f f of the curves. The r a t e of development of the e a r l y s t a r f i s h embryo at a temperature of 11.5-12°C w i t h respect to the archenteron length to body length r a t i o s (AL/BL) and the archenteron lengths (AL) from e a r l y to l a t e g a s t r u l a , are summarized i n Table 1. 26 FIG 13 •• Rots of Development of P. ocfiroceous Embryos at 11.5-12'C: Development of Hie Mouth 0 CO s If CO .0.700 -0.600 --0.500 --0.400 --0.300 0.200 0.100 4-0.000 / © © / /' C O 0 ' / e EHRLY MID LATE GfiSTRULflTION o_<n — z z o — rr a U J z >-ui z CD U J X >- 2 in <r I x z l-O D — SI a 1 7T 0 30 40 50 60 70 Development (hrs.) 80 90 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 © 27 TABLE 1: THE RATE OF DEVELOPMENT OF PISASTER OCHRACEOUS EMBRYOS AT 11.5-12 C: ARCHENTERON LENGTH TO BODY LENGTH RATIOS (AL/BL) . AND ARCHENTERON LENGTHS (AL) DURING EARLY DEVELOPMENT. . STAGE TIME (hrs) AL/BL AL (u) F e r t i l i z e d Egg - f e r t i l i z a t i o n membrane .25 -p o l a r Bodies 2-4 E a r l y B l a s t u l a 12 Mid B l a s t u l a 20 Hatching 24-28 Late B l a s t u l a 36 -vege t a l pole thickened E a r l y G a s t r u l a 42 0.375 60 -vegetal pole i n v a g i n a t i n g Early-Mid G a s t r u l a 47 0.500 90 Early-Mid G a s t r u l a 51 0.474 90 Mid G a s t r u l a 55 0.579 110 -archenteron t i p expanding Mid G a s t r u l a 59 0.600 120 -archenteron t i p expanded , Mid G a s t r u l a 63 0.608 124 - c e l l processes at t i p Mid G a s t r u l a 67 0.633 138 -mesenchyme c e l l s Mid-Late G a s t r u l a 71 0.581 136 -archenteron bending Mid-Late G a s t r u l a 74 0.598 140 Mid-Late G a s t r u l a 81 0.574 140 Mid-Late G a s t r u l a 83 0.577 142 Late G a s t r u l a 87.5 0.586 150 - o r a l p l a t e formed E a r l y B i p i n n a r i a 90 -mouth formed 28 I I I . Mouth Formation l ) . The Basal Lamina Examination of embryos i n the mid-gastrula stage at the LM l e v e l , showed that c e l l s were coming o f f the t i p of the archenteron and mi g r a t i n g i n t o the b l a s t o c o e l to become mesenchyme c e l l s . A hole was oft e n present i n the expanded t i p of the archenteron. This was not loc a t e d p r e c i s e l y at the t i p but u s u a l l y s l i g h t l y to one side (FIG 14). One um t h i c k c r o s s - s e c t i o n s through t h i s region revealed that i t was bounded by a membrane (FIG 15), and upon c l o s e r examination w i t h the TEM, the membrane appeared to be a c o n t i n u a t i o n o f , and i d e n t i c a l i n s t r u c t u r e t o , the lamina densa of the endodermal basal lamina (FIG 16). No r e t i c u l a r lamina was present. Examination of LM s e c t i o n s of a s l i g h t l y l a t e r stage demonstrated that the archenteron was bent so that the opening i n the t i p came to r e s t d i r e c t l y opposite the ectoderm c e l l s which formed the presumptive stomodeum (FIG 17). S e r i a l LM cross and l o n g i t u d i n a l s e c t i o n s of t h i s stage showed that the b a s a l lamina over the opening had bulged from the t i p of the archenteron i n t o the b l a s t o c o e l forming a b l i s t e r . This b l i s t e r extended toward the ectoderm i n the presumptive mouth r e g i o n ( F i g 17). Whole mounts of a s i m i l a r stage revealed numerous c e l l s s c a t t e r e d w i t h i n the b l a s t o c o e l i n the area between the bending archenteron t i p and the presumptive stomodeal ectoderm (FIG 18). SEM's of a s i m i l a r stage showed the ectoderm i n the presumptive mouth region to be a s s o c i a t e d w i t h numerous c e l l processes on i t s inner surface (FIG 19). This i s i n c o n t r a s t to the r e s t of the ectoderm which was r e l a t i v e l y smooth. A higher m a g n i f i c a t i o n SEM of the presumptive stomodeal ectoderm e x h i b i t e d a v a r i e t y of processes (FIG 20): some were large and c o n i c a l ; o t h e r s , were smaller and more filamentous and were 29 Figure 14 A wholemount of a mid g a s t r u l a stage embryo, showing the ectoderm (EC), the archenteron w i t h a hole i n i t s t i p ( l a r g e arrowheads) and mesenchyme c e l l s ( s m a l l arrowheads). B = b l a s t o c o e l ; EC = ectoderm x250 Figure 15 A lum c r o s s - s e c t i o n through the t i p of the archenteron of an embryo at the same stage as the one seen i n F i g . 14. The hole seen i n F i g 14 i s bound by a membrane (arrows). EC = ectoderm; B = b l a s t o c o e l ; En = encoderm c e l l s x550 Figure 16 A TEM t h i n s e c t i o n through the t i p of the archenteron at a s l i g h t l y l a t e r stage. The membrane i s part of the endodermal ba s a l lamina (arrows) which at t h i s stage i s b u l g i n g i n t o the b l a s t o c o e l e ( B ). L = archenteron lumen x4000 30 B 1 6 31 Figure 17 A 1 um l o n g i t u d i n a l s e c t i o n of an embryo at a s i m i l a r stage to that i n F i g . 16. The archenteron i s bent toward one side of the ectoderm (EC) and has developed a b e a k - l i k e p r o f i l e d i r e c t l y above which i s the b a s a l lamina b l i s t e r (arrow). Endodermal c e l l s (EN) can be seen on the i n s i d e of the b a s a l lamina and mesenchyme c e l l s (M) and e x t r a c e l l u l a r m a t e r i a l (ECM) are present i n the b l a s t o c o e l e . The ECM i s l o c a l i z e d i n one h a l f of t h i s embryo which has been f i x e d f o r one hour i n the presence of A l c i a n blue. x280 Figure 18 A Nomarski i n t e r f e r e n c e , phase-contrast photomicrograph of an osmium shadowed wholemount at a s i m i l a r stage as F i g . 17 showing the presump-t i v e mouth r e g i o n . Numerous mesenchyme-like c e l l s (arrows) are s c a t t e r e d between the archenteron (A) and the ectoderm (EC). x560 32 33 Figure 19 An SEM of the archenteron t i p and adjacent ectoderm (EC) at the same stage as F i g . 17. The b a s a l lamina ( l a r g e arrows) p a r t i a l l y covers the opening i n the t i p although large holes are present i n i t . The archenteron (EN) i s bent toward the ectoderm (EC) and spikes (small arrows) are present on the inner surface of the ectoderm d i r e c t l y opposite the beak of the archenteron. The r e s t of the ectoderm appears smooth. xlOOO Figure 20 An SEM of the ectoderm (EC) d i r e c t l y opposite the bent archenteron (A) on the inner surface of the ectoderm showing both c o n i c a l ( l a r g e arrows) and filamentous processes ( f i l o p o d i a ) on the inner surface of the ectoderm. x2000 35 s i m i l a r i n appearance to the f i l o p o d i a of the mesenchyme c e l l s . Examination of t h i s r e g ion of the ectoderm w i t h the TEM revealed that at l e a s t some of these processes were ectodermal i n o r i g i n . Both the large c o n i c a l and the smaller filamentous types were seen i n t h i n s e c t i o n s . The large c o n i c a l spikes were continuous w i t h the cytoplasm and t h e i r bases spanned almost the e n t i r e b a s a l surface of the ectodermal c e l l (FIGS 21,22). The smaller filamentous processes were a l s o cytoplasmic p r o j e c t i o n s of the ectodermal c e l l s , but they occupied only a small p o r t i o n of the ba s a l surface of a p a r t i c u l a r ectoderm c e l l (FIG 23). Both types of processes were devoid of a ba s a l lamina, except around t h e i r bases, and were located opposite the ba s a l lamina b l i s t e r of the archenteron. L i g h t microscope s e c t i o n s through the t i p of the archenteron c o n s i s t e n t l y showed the basal lamina b l i s t e r a s s o c i a t e d w i t h s c a t t e r e d c e l l s on both s i d e s , i e . on the surface f a c i n g the endoderm and on that f a c i n g the b l a s t o c o e l (FIG 24). Often, one of the endodermal c e l l s was observed at the d i s t a l end of the b l i s t e r ( l e a d c e l l ) . TEM t h i n s e c t i o n s at the same l e v e l a l s o showed the c e l l s of the endoderm and mesenchyme to be c l o s e l y r e l a t e d to the ba s a l lamina b l i s t e r (FIG 25). In many cases, they were opposite each other w i t h the basal lamina between them. In a d d i t i o n , c a r e f u l examination of the basal lamina i t s e l f revealed that i t was not continuous, but that t i n y holes were present i n i t through which endodermal c e l l processes were seen to extend (FIG 25). L i g h t micrographs a l s o showed the extended b a s a l lamina b l i s t e r a s s o c i a t e d w i t h what appeared to be an i s o l a t e d endodermal c e l l on i t s endodermal surface immediately opposite a p u t a t i v e mesenchyme c e l l on the b l a s t o c o e l surface of the ba s a l lamina (FIG 26). Small c e l l processes extended from the mesenchyme c e l l and appeared to make contact w i t h the 3 6 Figure 21 A TEM t h i n s e c t i o n showing the presumptive mouth region i n an embryo at the same stage as F i g . 20. The b a s a l lamina b l i s t e r (small arrows) i s opposite a c o n i c a l ectodermal spike ( l a r g e arrow). The base of what appears to be a second spike i s a l s o v i s i b l e (double arrow). EC = ectoderm B = b l a s t o c o e l EN = endoderm L = archenteron lumen Figure 22 A high m a g n i f i c a t i o n TEM of the base of a c o n i c a l ectodermal c e l l process ( l a r g e arrow) d i r e c t l y opposite the b a s a l lamina b l i s t e r ( s m a l l arrowhead). The b a s a l lamina appears discontinuous around the t i p of the spike (small arrows). A cross s e c t i o n of a c e l l process ( l a r g e arrowheads) i s seen at the t i p of the b l i s t e r . B = b l a s t o c o e l L = archenteron lumen EC = ectoderm x8000 38 Figure 23 A TEM of the filamentous c e l l processes along the inner aspect of the ectoderm i n the presumptive mouth r e g i o n . At l e a s t 3 of these ( l a r g e arrows) are true cytoplasmic p r o j e c t i o n s through the ectodermal b a s a l lamina ( s m a l l arrows) i n t o the b l a s t o c o e l e (B). xl6,750 40 Figure 24 A LM cross s e c t i o n through the archenteron b l i s t e r of an embryo at the same stage as F i g . 18. C e l l s are associated w i t h the ba s a l lamina b l i s t e r (arrows) on i t s endodermal aspect. E x t r a c e l l u l a r m a t e r i a l (ECM) i s present i n the b l a s t o c o e l e and the endodermal lumen ( L ) . x875 Figure 25 A t h i n s e c t i o n through the basal lamina b l i s t e r at the same l e v e l as F i g . 24 showing s c a t t e r e d endodermal c e l l s (EN) along the ba s a l lamina. One endodermal c e l l i s at the lead ( l a r g e arrowhead) i n f r o n t of a cross s e c t i o n of a c e l l process (small arrowhead) i n the b l a s t o c o e l e (B). Another.endodermal c e l l (or c e l l process) ( l a r g e arrow) i s l y i n g next to a mesenchyme c e l l (M) separated by the bas a l lamina ( b l ) . The ba s a l lamina i s folded i n some places and o c c a s s i o n a l l y a small hole can be seen (small arrow) through which an endodermal c e l l process may poke i n t o the b l a s t o c o e l . E x t r a c e l l u l a r m a t e r i a l i s present both i n the b l a s t o c o e l and the lumen of the archenteron ( L ) . x3000 42 Figure 26 A LM at a s l i g h t l y l a t e r stage than F i g . 24 showing the base of an ectodermal spike ( l a r g e arrowhead), the endodermal b l i s t e r ( s m a l l arrowheads) and what appears to be contact between a mesenchyme c e l l and endoderm c e l l p o s s i b l y through holes i n the b a s a l lamina ( l a r g e arrow). Mesenchyme f i l o p o d i a ( s m a l l arrows) are extending toward the ectoderm (EC). EN = endoderm c e l l s x800 Figure 27 A SEM of an embryo at a s l i g h t l y l a t e r stage than F i g . 27 showing what may be endoderm (EN) and mesenchyme c e l l s (M) making contact w i t h each other (arrowheads). Mesenchyme f i l o p o d i a appear to be making contact w i t h the t h i n filamentour spikes of the ectoderm (arrows). A b a s a l lamina ( b l ) sheet i s s t r e t c h e d across the hole i n the archenteron. EC = ectoderm x3000 44 endodermal c e l l , p o s s i b l y through holes i n the b a s a l lamina, s i m i l a r to those seen i n FIG 25. At the same time, the mesenchyme c e l l e x h i b i t e d f i l o p o d i a which extended towards the ectoderm i n the presumptive mouth region (FIG 26). A SEM of a s i m i l a r stage depicted what appeared to be various c e l l - c e l l contacts (FIG 27). The ba s a l lamina sheet was st r e t c h e d over the archenteron t i p and p o s s i b l y over two prominent p u t a t i v e endodermal c e l l s . What may be mesenchyme c e l l s appeared to make contact w i t h the endodermal c e l l s , and mesenchyme f i l o p o d i a extended and contacted the ectoderm. Closer examination of t h i s ectoderm revealed what appeared to be t h i n filamentous processes making contact w i t h the f i l o p o d i a . One um s e c t i o n s of a s l i g h t l y l a t e r stage, showed that the ba s a l lamina was i n contact w i t h the ectoderm (FIG 28), u s u a l l y v i a an ectodermal c e l l process (FIG 29). Examination of t h i s stage w i t h the TEM revealed that the ectodermal process that was i n contact w i t h the b a s a l lamina was of the l a r g e c o n i c a l type (FIG 30). A higher m a g n i f i c a t i o n of t h i s point of contact (FIG 31) showed that the ba s a l lamina of the b l i s t e r was folded where i t was i n contact w i t h the d i s t a l p o r t i o n of the process. The ectodermal b a s a l lamina was continuous up to the base of the process on e i t h e r s i d e , and was a l s o h i g h l y folded p a r t i c u l a r l y along the surface of the ectodermal c e l l w i t h the cytoplasmic process (FIG 31). The f l a t t e n e d t i p of the spike and i t s l a t e r a l borders, however, were devoid of a continuous basal lamina but were as s o c i a t e d w i t h what were e i t h e r pieces of ba s a l lamina or e x t r a c e l l u l a r m a t e r i a l . One um LM s e c t i o n s of what appeared to be a s l i g h t l y l a t e r stage showed that the ba s a l lamina b l i s t e r was i n contact w i t h at l e a s t two large c e l l processes of the presumptive stomodeal ectoderm (FIG 32). The ectoderm i n the region of the spikes appeared thickened and was somewhat 45 Figure 28 A t a n g e n t i a l LM s e c t i o n through the ba s a l p o r t i o n of the ba s a l lamina b l i s t e r at a s l i g h t l y l a t e r stage showing the basal lamina i n contact w i t h the ectoderm i n the presumptive mouth region ( l a r g e arrowhead). ECM i s present i n d i s t i n c t strands (small arrowheads). M = mesenchyme c e l l x300 Figure 29 A 1 um s e c t i o n through the middle of the b a s a l presumptive mouth r e g i o n , at the same stage as i n contact (arrow) w i t h an ectodermal sp i k e . (EN) Endodermal c e l l s (M) Mesenchyme c e l l s x750 lamina b l i s t e r i n the F i g . 28. The b l i s t e r 47 Figure 30 A TEM through the t i p of the archenteron at the same stage as F i g . 29. The b a s a l lamina b l i s t e r i s i n contact (arrows) with a c o n i c a l ectodermal spike (arrowhead). x3600 Figure 31 A high m a g n i f i c a t i o n TEM of the c o n i c a l spike seen i n F i g . 30. The b a s a l lamina b l i s t e r at the point of contact w i t h the spike i s folded and extends p a r t i a l l y along the sides of the process ( s m a l l arrowheads). There, the b a s a l lamina i s broken up, but i s continuous w i t h the ectodermal b a s a l lamina from the base of the spike along the c e l l s of the ectoderm (arrows). Note that the f l a t t e n e d t i p of the spike i s devoid of b a s a l lamina ( l a r g e arrowhead). ECM i s present i n c l o s e a s s o c i a t i o n w i t h the b a s a l lamina. xll.OOO 49 Figure 32 A LM c r o s s - s e c t i o n of a s l i g h t l y l a t e r stage through the top p o r t i o n of the archenteron. The basal lamina i s i n contact w i t h the ectoderm i n at l e a s t 2 places (arrows) g i v i n g r i s e to a b a s a l lamina tube. Four c o n i c a l ectodermal processes (arrowheads) extend from the ectoderm and at l e a s t 2 of these are i n contact w i t h the b l i s t e r on e i t h e r s i d e . The presumptive stormodeal (S) ectoderm i n the region of the processes i s thickened and s l i g h t l y bent inward. L = archenteron lumen b l = b a s a l lamina x950 50 51 bent inward forming what appeared to be the beginning of stomodeal i n v a g i n a t i o n . O c c a s s i o n a l l y , a t h i r d and a f o u r t h spike were seen on the inner surface of t h i s presumptive stomodeal ectoderm (FIG 32). Examination of a s i m i l a r stage with the TEM showed the b a s a l lamina b l i s t e r to be i n contact w i t h a c o n i c a l c e l l process at one end (FIG 33) i n a manner analogous to that described i n FIG 31. On the other s i d e , the b a s a l lamina b l i s t e r was a s s o c i a t e d w i t h what may be rounded c e l l s or c r o s s - s e c t i o n s of c e l l processes (FIG 33). The presumptive stomodeal b a s a l lamina was seen as a t h i n l i g h t s t a i n i n g l i n e adhering c l o s e l y to the b a s a l surface of the c e l l s but appeared to be h i g h l y redundant i n one region (FIG 33). The b a s a l lamina of the b l i s t e r was s t r e t c h e d between the two points of contact d i r e c t l y above the presumptive stomodeal ectoderm (FIG 33). A high m a g n i f i c a t i o n SEM of a s l i g h t l y l a t e r stage showed that there was c o n t i n u i t y between the endodermal and ectodermal basal laminae. In other words, the b a s a l laminae had formed a tube. Further observations revealed that presumptive endoderm c e l l s were l o o s e l y s c a t t e r e d along the endodermal aspect of the tube, while mesenchyme c e l l s were a s s o c i a t e d w i t h the b l a s t o c o e l or inner surface (FIGS 34,35). One um s e c t i o n s showed that i n some animals at t h i s stage, the ectodermal c e l l s w i t h i n the boundaries of the tube had a l s o begun to elongate to form the stomodeum (FIG 34). There was no basal lamina at the inner surface of these stomodeal c e l l s (FIG 36). W i t h i n 5 hrs of tube formation, the tube of basal lamina was completely l i n e d w i t h endodermal c e l l s , forming a low columnar e p i t h e l i u m , and a plug of c e l l s , the o r a l p l a t e was formed (FIG 37). Thin s e c t i o n s revealed that the o r a l p l a t e c o n s i s t e d of both ectodermal and endodermal c e l l s . There was no b a s a l lamina between the two l a y e r s Figure 33 A TEM t h i n s e c t i o n through the presumptive mouth region at a s i m i l a r stage as i n F i g . 32. The endodermal ba s a l lamina ( s m a l l arrowheads) i s seen as a t h i n l i n e extending on both sides towards a c o n i c a l spike on one side ( l ) and what appears to be c r o s s - s e c t i o n s of c e l l processes on the other side ( 2 ) . On side 1 the b a s a l lamina spike contact ( s m a l l arrows) appears s i m i l a r to that described i n F i g . 32. On side 2 the b a s a l lamina i s a l s o i n contact w i t h the c e l l processes ( s m a l l arrows). The ectodermal b a s a l lamina ( l a r g e arrowheads) between areas 1 and 2 appears i n t a c t but i s h i g h l y folded i n region ( 2 ) . A p o r t i o n of the b l i s t e r i s a l s o s t r e t c h e d across the b l a s t o c o e l e between areas 1 and 2 ( s m a l l arrowheads) w i t h what appears to be a c r o s s - s e c t i o n of a c e l l process (CP) r e s t i n g i n the center. ECM i s c l o s e l y a s s o c i a t e d w i t h the b a s a l lamina. L = archenteron lumen x3200 53 54 gure 34 A LM of a l a t e r stage shows the b a s a l lamina (arrows) tube a s s o c i a t e d w i t h c e l l s on i t s endodermal aspect ( L ) . A mesenchyme c e l l (M) i s a l s o seen r e s t i n g on i t s inner (or b l a s t o c o e l e ) surface. The c e l l s of the stomodeum (S) are elongated. ECM i s present i n the b l a s t o c o e l e (B). CP = coelomic pouch EN =endodermal c e l l s x850 55 56 gure 35 A high m a g n i f i c a t i o n SEM of the same stage as F i g . 34 showing the c o n t i n u i t y of the endodermal basal lamina ( b l ) w i t h the ectodermal b a s a l lamina ( l a r g e arrows). P u t a t i v e endoderm (EN) c e l l s are embedded i n the laminar sheet and a mesenchyme c e l l (M) s i t s on i t s inner surface next to the ectodermal w a l l (EC). Note a l s o the filamentous processes ( s m a l l arrows) of the ectoderm. L = archenteron lumen x3000 58 gure 36 A t h i n s e c t i o n through the upper p o r t i o n of the developing mouth at the same stage as F i g 34 and 35 showing the contact between the b a s a l laminae of the ectoderm and the i n v a g i n a t i n g stomodeal c e l l s . While the ectodermal and endodermal basal laminae have fused f o r the most p a r t , some d i s c o n t i n u i t y of the bas a l lamina i s s t i l l present on both sides of the stomoderm. The basal lamina a l s o appears folded ( s m a l l arrowheads) i n some areas. No basal lamina i s present along the inner surface of the stomodeal c e l l s ( l a r g e arrowheads). x4000 59 60 Figure 37 A LM of a 90 hour embryo. The stomodeum (S) i s completely l i n e d w i t h c e l l s and the o r a l p l a t e (OP) i s formed by a plug of c e l l s . Coelomes (CP) are a l s o v i s i b l e . L = archenteron lumen x650 Figure 38 A TEM of the same stage as F i g . 37 which shows that the o r a l p l a t e i s made up of an endodermal l a y e r (EN) and a ectodermal l a y e r (EC) w i t h no b a s a l lamina between them. Two elongated ectodermal c e l l s (arrows) have t h i n c e l l processes which extend along the endodermal aspect of the b a s a l lamina. ECM = e x t r a c e l l u l a r m a t e r i a l M - mesenchyme L = archenteron lumen arrowheads = continuous b a s a l lamina x2600 62 (FIG 38). Two elongated ectodermal c e l l s w i t h long processes were a l s o c o n s i s t a n t l y observed on e i t h e r side of the stomodeal i n v a g i n a t i o n . Their processes extended along the sides of the o r a l p l a t e and contacted the b a s a l lamina on t h e i r l a t e r a l aspect (FIG 38). L i g h t micrographs, of embryos f i x e d 5-10 hrs l a t e r , e x h i b i t e d an opening where the s o l i d o r a l p l a t e had been (FIG 39). Thin s e c t i o n s through t h i s r e g ion demonstrated a few rounded loose c e l l s trapped i n a sheet of densely s t a i n e d f i b r o u s ECM (mucus) (FIG 40), in s t e a d of the organized array of the c e l l s of the o r a l p l a t e . In many cases, the opening was complete and no r e s i d u a l c e l l s were present. 2). The E x t r a c e l l u l a r M a t r i x In order to examine the changes i n the e x t r a c e l l u l a r matrix during e a r l y development, s e r i a l s e c t i o n s of staged embryos were obtained and examined under both the TEM and the LM. LM sect i o n s of an e a r l y g a s t r u l a , 42 hrs p o s t - f e r t i l i z a t i o n , showed that the e x t r a c e l l u l a r m a t r i x (ECM) c o n s i s t e d of what appeared to be a few l i g h t l y s t a i n e d strands of v a r y i n g thickness which were concentrated i n regions along the ectoderm and around the t i p of the archenteron. L i t t l e m a t e r i a l was i n the b l a s t o c o e l space i t s e l f (FIG 41). Four hours l a t e r (46 hrs p o s t - f e r t i l i z a t i o n ) , more ECM appeared to be present i n the center of the b l a s t o c o e l , but the m a j o r i t y of i t was found c l o s e to the ectoderm (FIG 42) and the endoderm. At t h i s p o i n t , the m a t e r i a l had the appearance of f i n e grains and a few short strands. Thin s e c t i o n s of the same stage, showed the ECM to be made up of both a l i g h t s t a i n i n g and a dark s t a i n i n g m a t e r i a l some of which was as s o c i a t e d w i t h the ba s a l lamina of the endoderm and ectoderm (FIGS 43,44). I t appeared that more was present around the endoderm than the ectoderm, p a r t i c u l a r l y i n the space between the l a t e r a l borders of the endoderm and the ectoderm; however, 63 Figure 39 A LM c r o s s - s e c t i o n through the mouth of an e a r l y b i p i n n a r i a showing the mouth (M), eosophagus (0) and coelomic pouches (CP). Rounded c e l l s are present i n the region formerly occupied by the o r a l p l a t e (arrows). OH = o r a l hood x200 Figure 40 A TEM c r o s s - s e c t i o n through the mouth of an e a r l y b i p i n n a r i a showing rounded c e l l s embedded i n a sheet of e x t r a c e l l u l a r m a t e r i a l (m) i n the region formerly occupied by the o r a l p l a t e (arrows). x4000 64 65 Figure 41 A l o n g i t u d i n a l s e c t i o n of an e a r l y g a s t r u l a , 42 hours a f t e r f e r t i l i z a t i o n showing the ectoderm (EC) and the i n v a g i n a t i n g archenteron (A) i n t o the b l a s t o c o e l e ( B ) . E x t r a c e l l u l a r m a t e r i a l (ECM) i s concentrated along regions of the ectoderm and endoderm (arrows). x775 Figure 42 A c r o s s - s e c t i o n of a 46 hour e a r l y g a s t r u l a showing the s t a i n e d strands and granules of the e x t r a c e l l u l a r m a t e r i a l (arrows) occupying more of the b l a s t o c o e l e space (B). Note that ECM i s not present i n the centre of the b l a s t o c o e l at t h i s stage. x800 Figure 43 A TEM of a l o n g i t u d i n a l - s e c t i o n of a 42 hour e a r l y g a s t r u l a showing the l i g h t and dark s t a i n i n g m a t e r i a l (arrowheads) of the ECM. The ECM i s a s s o c i a t e d c l o s e l y w i t h the basal laminae (arrows) of both the archenteron (A) and the ectoderm (EC). x9500 Figure 44 A low m a g n i f i c a t i o n TEM of the ectoderm of a 42 hour embryo showing the cl o s e a s s o c i a t i o n of the ECM to the ectodermal c e l l s (EC) with respect to the r e s t of the b l a s t o c o e l e space during t h i s e a r l y stage. Compare w i t h F i g . 41. x2500 69 Figure 45 A high m a g n i f i c a t i o n TEM of the l i g h t s t a i n i n g component of the ECM i n the 42 hour embryo. I t i s composed of v e s i c l e s and an amorphous m a t e r i a l of intermediate s t a i n i n g d e n s i t y . x23,000 Figure 46 A high m a g n i f i c a t i o n TEM of the dense s t a i n i n g component of the ECM i n the 42 hour embryo. Note the l i n e a r arrangement where the l i g h t and dark components are a s s o c i a t e d together. A l s o note the presence of the filamentous m a t e r i a l (arrows) that appear to be c l o s e l y a s s o c i a t e d w i t h the dense component. x30,000 71 t h i s may have been due to the i n v a g i n a t i o n of the v e g e t a l pole pushing the ECM i n t o a smaller space. A higher m a g n i f i c a t i o n of t h i s r e g i o n , revealed that the l i g h t s t a i n i n g m a t e r i a l was composed p r i m a r i l y of v e s i c l e - l i k e s t r u c t u r e s i n no p a r t i c u l a r arrangement (FIG 45). However, where t h i s m a t e r i a l was associated w i t h the dark s t a i n i n g , dense substance, the dark granules were often arrayed i n a l i n e a r f ashion (FIG 46). A filamentous m a t e r i a l could a l s o be seen a s s o c i a t e d w i t h the dense granules. L o n g i t u d i n a l t h i c k s e c t i o n s of an embryo 51 hrs p o s t - f e r t i l i z a t i o n , showed that the ECM was now located throughout the b l a s t o c o e l w i t h some m a t e r i a l s t i l l a s s o c i a t e d w i t h regions along the ectoderm and endoderm (FIG 47). The m a t e r i a l was d a r k l y s t a i n e d and had a s i m i l a r appearance at the LM l e v e l of beads or short strands as seen p r e v i o u s l y . The same stage examined under the TEM showed d i s t i n c t strands of v a r i o u s lengths and complexity (FIG 48,49). These were of at l e a s t three components: v e s i c u l a r s t r u c t u r e s , a very dense s t a i n i n g m a t e r i a l and i n t e r m e d i a t e l y s t a i n e d filamentous strands ( i n s e t ) . The b a s a l lamina c o n s i s t e d of s i m i l a r components at t h i s stage (FIG 49). At 55 hrs a f t e r f e r t i l i z a t i o n , examination of 1 um c r o s s - s e c t i o n s at the LM l e v e l d i s c l o s e d numerous d i s t i n c t strands of ECM having a beaded appearance (FIG 50). The strands were more c l o s e l y a s s o c i a t e d w i t h the archenteron and were p r i m a r i l y located i n one quadrant of the embryo. A high m a g n i f i c a t i o n TEM of a t y p i c a l strand seen at t h i s stage (FIG 51), revealed a l i g h t s t a i n i n g filamentous core a s s o c i a t e d w i t h a dense substance and small concentrations of an amorphous m a t e r i a l which s t a i n e d w i t h an intermediate d e n s i t y along i t s length. Few v e s i c u l a r s t r u c t u r e s were seen at t h i s stage (FIG 51). Figure 47 A LM of a l o n g i t u d i n a l - s e c t i o n of a 51 hour early-mid g a s t r u l a , showing the ectoderm ( E ) , the archenteron (A) and the s t a i n e d ECM w i t h i n the b l a s t o c o e l e ( B). The ECM has a s i m i l a r appearance of beads and short strands as i n F i g s . 41 and 42, but i t i s throughout the b l a s t o c o e l e spac at t h i s stage. x750 73 74 Figure 48 A TEM showing the ectoderm c e l l s , the bas a l lamina ( b l ) and the short ECM strands of an early-mid g a s t r u l a (51 hours). The strand i s composed of at l e a s t 3 components ( I n s e t ) : (a) an intermediate s t a i n i n g filamentous core ( s m a l l arrows) (b) l i g h t s t a i n i n g v e s i c u l a r s t r u c t u r e s ( s m a l l arrowheads) (c) a dense s t a i n i n g substance ( l a r g e arrowhead). The ectodermal b a s a l lamina ( l a r g e arrow) a l s o possesses v e s i c u l a r s t r u c t u r e s and densely s t a i n e d regions (arrows), (pm) = plasma membrane x23,000 Inset x33,000 Figure 49 A TEM of a s e c t i o n through a 51 hour embryo showing the greater complexity of the ECM strands, and the c l o s e a s s o c i a t i o n of some of the strands to the b a s a l lamina of the endoderm c e l l s (arrows). xl3,000 76 Figure 50 A 1 um t h i c k c r o s s - s e c t i o n through the expanding t i p of the archenteron (A) of a 55 hour embryo. "Beaded" strands of ECM are more d i s t i n c t i n one quadrant of the embryo (arrows). EC = ectoderm x800 Figure 51 A high m a g n i f i c a t i o n TEM of a t y p i c a l ECM strand at the same stage as i n F i g . 50. The strand c o n s i s t s of a t h i n filamentous ( s m a l l arrow) core. A l i g h t s t a i n i n g amorphous m a t e r i a l i s associated w i t h the strand ( l a r g e arrowheads) but few v e s i c l e s are seen. Thick i r r e g u l a r l y shaped, densely s t a i n e d regions are a s s o c i a t e d w i t h the strand (small arrowheads). They are probably r e s p o s i b l e f o r g i v i n g the strands a "beaded" appearance when viewed with the l i g h t microscope. x27,600 78 At a s l i g h t l y l a t e r stage (59 h r s ) , the ECM was more evenly dispersed w i t h i n the b l a s t o c o e l . L i g h t s t a i n i n g strands beaded with dense s t a i n i n g granules extended from the archenteron toward the ectoderm and appeared more d i s t i n c t i n the presumptive mouth region of the b l a s t o c o e l (FIG 52). A high m a g n i f i c a t i o n TEM of the t y p i c a l ECM at the same stage demonstrated a s i m i l a r morphology as the previous stage, but the strands were more branched (FIG 53). Few v e s i c u l a r s t r u c t u r e s were seen at t h i s p o i n t . By 71 hrs p o s t - f e r t i l i z a t i o n , mesenchyme c e l l s were present i n the b l a s t o c o e l (FIG 54) and the ECM was s i m i l a r i n appearance at the LM l e v e l to that seen i n 59 hr embryos. At 83 h r s , a c r o s s - s e c t i o n through the b l a s t o c o e l d i s p l a y e d an extensive network of beaded i n t e r l a c i n g strands (FIG 55). These strands, however, were l e s s densely i n t e r l a c e d i n the c e n t r a l p o r t i o n of the b l a s t o c o e l but appeared to be a s s o c i a t e d w i t h numerous l a r g e , dark s t a i n i n g granules i n t h i s r e g i o n . This was i n c o n t r a s t to the m a t e r i a l i n the periphery of the b l a s t o c o e l which was composed of a denser meshwork of l i g h t s t a i n i n g strands and fewer, and somewhat s m a l l e r , dark s t a i n i n g granules. A c r o s s - s e c t i o n of the same stage through the archenteron revealed strands that extended from the endoderm towards the ectoderm and formed a r a d i a l p a t t e r n which was more d i s t i n c t i n the presumptive mouth region (FIG 56). O c c a s s i o n a l l y , strands were ass o c i a t e d w i t h the endodermal ba s a l lamina. The t y p i c a l beaded appearance of the strands seen i n the LM at e a r l i e r stages was not as d i s t i n c t i n most cases, although some granules were observed. Instead, e n t i r e strands appeared somewhat more densely s t a i n e d and t h i c k e r than those i n previous stages. Cross-sections through the embryo at the l e v e l of the b a s a l lamina b l i s t e r seen w i t h the TEM a l s o e x h i b i t e d a r a d i a l 79 Figure 52 A LM of a c r o s s - s e c t i o n of a 59 hour embryo showing the ectoderm (EC), the archenteron t i p (A) and the ECM i n the b l a s t o c o e l e . The ECM i s an extensive network of beaded strands extending from the archenteron toward the ectoderm e s p e c i a l l y w i t h i n the presumptive mouth (PM) r e g i o n . x640 Figure 53 A TEM of the ECM at m i d - g a s t r u l a t i o n (59 hrs) showing a s i m i l a r morphology as that seen i n the previous stage ( F i g . 51). The strands are more branched at t h i s stage and few v e s i c l e s are present. x58,000 81 gure 54 A LM of 71 hr embryo showing mesenchyme c e l l s ( l a r g e arrows) i n the b l a s t o c o e l e . More densley s t a i n e d granules (small arrows) appear i n the e x t r a c e l l u l a r m a t r i x at t h i s stage compared w i t h previous stages. The bend i n the ectoderm (EC) i s due to damage during f i x a t i o n . x750 82 83 Figure 55 A c r o s s - s e c t i o n through the b l a s t o c o e l e of an 83 hr mid-late g a s t r u l a showing the ectoderm (EC) and mesenchyme c e l l s (M) and the extensive network of the ECM. Loosely i n t e r l a c i n g beaded strands occupy the c e n t r a l (C) p o r t i o n of the b l a s t o c o e l e , while a denser meshwork covers i t s periphery ( P ) . A l s o , note the l a r g e r and denser granules that are a s s o c i a t e d w i t h the strands i n the c e n t r a l p o r t i o n (arrows). x750 Figure 56 A LM of a c r o s s - s e c t i o n through the presumptive mouth region of an embryo at the same stage as i n F i g . 55. The ECM strands extend from the archenteron (A) and a t h i c k basal lamina b l i s t e r ( l a r g e arrow) towards the ectoderm (E) i n a d i s t i n c t r a d i a l p a t t e r n ( s m a l l arrows). Note the increased thickness of the strands compared to previous stages. x2000 85 Figure 57 A t h i n s e c t i o n through the presumptive mouth region of an 83 hour embryo showing the b a s a l lamina b l i s t e r ( l a r g e arrow) and i t s r e l a t i o n s h i p to the ECM strands. The strands are extending r a d i a l l y ( s m a l l arrows) from the b l i s t e r toward the ectoderm. EN = endoderm c e l l B =blastocoel x6200 Figure 58 A high m a g n i f i c a t i o n TEM of the t y p i c a l ECM seen i n mid-late g a s t r u l a e (83 h r s ) . Note the web-like s t r u c t u r e and i t s t h i c k n e s s . The filamentous core and the v e s i c l e s are not obvious. Small dense blebs protude along the length of the strand (arrows). The l i g h t s t a i n i n g amorphous m a t e r i a l a s s o c i a t e d w i t h the strands at e a r l i e r stages i s not present. x30,000 87 p a t t e r n of the ECM strands (FIG 57). Branched and s i n g l e dense strands of v a r y i n g lengths extended from the b l i s t e r toward the presumptive stomodeal ectoderm. Some strands were associated w i t h the b l i s t e r of basa l lamina and appeared to extend from i t . A high m a g n i f i c a t i o n TEM of t y p i c a l ECM strands of the same stage i n the v i c i n i t y of the b l i s t e r revealed a very densely s t a i n e d web-like s t r u c t u r e (FIG 58). Instead of the l i g h t s t a i n i n g f i l a m e n t s , t h i c k e r strands were observed a s s o c i a t e d wit h s t i l l darker regions and what appeared to be small blebs that a l t e r n a t e d along the length of the strand. A few v e s i c l e s were a l s o present. Other LM observations made w i t h respect to the ECM revealed crescent shaped regions of densely s t a i n e d m a t e r i a l present i n patches along the inner surface of the ectoderm (FIGS 59,60,61). These u s u a l l y occurred i n the e a r l y stages of g a s t r u l a t i o n , and sometimes they were observed when mesenchyme c e l l s were present i n the b l a s t o c o e l . In some cases, a hole or space i n the ectoderm was oft e n seen d i r e c t l y beneath these patches (FIG 59); i n other i n s t a n c e s , i t was not seen (FIG 60). Other spaces w i t h i n the ectoderm, however, were not a s s o c i a t e d w i t h any d a r k l y s t a i n e d m a t e r i a l (FIG 61). Examination w i t h the TEM showed a patchy d i s t r i b u t i o n of a l i g h t l y s t a i n e d m a t e r i a l along the b l a s t o c o e l surface of the endoderm (FIGS 62,63). A space was u s u a l l y present beneath these areas. In some cases, the bas a l lamina appeared discontinuous over the space, and a l i g h t l y s t a i n e d m a t e r i a l was located w i t h i n the space and i n a region of the b l a s t o c o e l immediately adjacent to i t (FIGS 62,63)-. A c l o s e r examination of t h i s m a t e r i a l showed that i t was composed of v e s i c u l a r s t r u c t u r e s , t h i n filamentous strands and an amorphous m a t e r i a l , s i m i l a r to some of the elements of the ECM seen during the e a r l i e r stages described above (FIG 64). Thin s e c t i o n s of the patchy regions along the 88 Figures 59-61 These LMs of densely s t a i n e d crescent shaped regions (arrows) observed along the inner surface of the ectoderm (EC); showing the d i f f e r e n t c o n f i g u r a t i o n of the ectodermal c e l l s immediately adjacent to them. Figure 59 shows a space beneath the dense patch. x2000 In Figure 60 there i s no space a s s o c i a t e d w i t h the densely s t a i n e d r e g i o n . xlOOO Figure 61 demonstrates that not a l l holes i n the ectoderm are ass o c i a t e d w i t h t h i s densely s t a i n e d m a t e r i a l . xl800 Note that i n a l l three cases, the outside h y a l i n e membrane i s i n t a c t and there i s no apparent damage to the ectoderm (small arrows). B = b l a s t o c o e l 89 90 Figures 62 & 63 TEMs of l o n g i t u d i n a l t h i n - s e c t i o n s through the archenteron showing crescent shaped l i g h t l y s t a i n e d patches along the b l a s t o c o e l (B) surface of the endoderm (EN). Note the densely s t a i n e d b a s a l lamina (sma l l arrow) and the spaces (S) beneath with the patches. Breaks i n the b a s a l lamina ( l a r g e arrows), u s u a l l y over a space (s) are a s s o c i a t e d w i t h a l i g h t s t a i n i n g m a t e r i a l w i t h i n the space and i n a region of the b l a s t o c o e l adjacent to i t . xl9,000 92 Figure 64 A high m a g n i f i c a t i o n TEM of a l i g h t s t a i n i n g patch along the endoderm (EN). I t i s composed of l i g h t l y s t a i n e d t h i n filamentous strands, v e s i c u l a r s t r u c t u r e s (small arrows) and an amorphous m a t e r i a l ( l a r g e arrows). Densely s t a i n e d bodies ( l a r g e arrowheads) are i n the immediate v i c i n i t y . The r e g u l a r spacing of the dense granules of the bas a l lamina i s l a c k i n g i n the region d i r e c t l y below the patch (white arrow), pm = plasma membrane b l = basal lamina x44,000 Figure 65 A TEM of the dense s t a i n i n g region along the inner surface' of the ectoderm. There i s a large i n t e r c e l l u l a r space (s) below the patch and the densely s t a i n e d m a t e r i a l i s more concentrated i n the area immediately next to the bas a l lamina (arrows). Note the continuous h y a l i n e membrane on the outer surface of the ectoderm (arrowheads). x5000 94 ectoderm revealed a very densely s t a i n e d m a t e r i a l s i m i l a r i n appearance to the dense component of the ECM (FIG 65). The ECM was best observed i n embryos that were f i x e d i n the presence of A l c i a n Blue f o r more than 1 h r. L i g h t micrographs of embryos at m i d - g a s t r u l a t i o n f i x e d f o r only one hour, showed a poorer p r e s e r v a t i o n of the m a t e r i a l , i n that the ECM appeared to be l o c a l i z e d only i n one h a l f of the embryo, presumably i n the presumptive mouth region (FIGS 17, 66). TEM t h i n s e c t i o n s of the mesenchyme c e l l stage revealed a uniform meshwork of a l i g h t l y s t a i n e d m a t e r i a l (FIG 67). LMs of embryos, at an equivalent stage, f i x e d i n the presence of A l c i a n Blue f o r three or more hours, showed the ECM to be d i s t r i b u t e d throughout the b l a s t o c o e l (FIGS 47, 52, 54-56) and TEM t h i n s e c t i o n s (FIGS 12, 43, 44, 48, 57) revealed a combination of l i g h t l y and d a r k l y s t a i n e d m a t e r i a l i n a more complex u l t r a s t r u c t u r e , as has been described above. In a d d i t i o n , i t was discovered that the ECM was not v i s i b l e i n embryos kept overnight i n 70% a l c o h o l . L i g h t micrographs of these embryos showed an empty b l a s t o c o e l and a b a r e l y v i s i b l e endodermal ba s a l lamina b l i s t e r . 3 ). The Hyaline Membrane A h y a l i n e l a y e r which s t a i n e d w i t h A l c i a n Blue, encapsulated the outside surface of a l l embryos observed, up to and i n c l u d i n g the e a r l y b i p i n n a r i a stage. In l i g h t micrographs, the h y a l i n e membrane appeared as an i n t e r m e d i a t e l y s t a i n e d fuzzy l a y e r (FIG 68). Clear v e s i c l e s are present i n the region of the c e l l s immediately adjacent to the h y a l i n e membrane around the periphery of the ectoderm (FIG 68). Examination of the h y a l i n e membrane w i t h the TEM revealed an outermost meshwork of very short dark and l i g h t strands (FIG 69). These short strands were l i n e a r l y arranged at the periphery forming a boundary 95 Figure 66 A c r o s s - s e c t i o n below the b l i s t e r through a mid-late g a s t r u l a f i x e d i n 1% glutaraldehyde and 1% A l c i a n blue f o r 1 hour, showing the ectoderm (EC), the endoderm (EN) and mesenchyme c e l l s (M). The ECM i s l o c a l i z e d i n the presumptive mouth (PM) region of the embryo. Note that the mesenchyme c e l l s w i t h i n the b l a s t o c o e l are a s s o c i a t e d w i t h the ECM. The break i n the ectoderm i s due to damage during embedding. Compare w i t h FIG 55. x750 Figure 67 A TEM of a c r o s s - s e c t i o n through the presumptive mouth region of a mid-l a t e g a s t r u l a f i x e d i n 1% glutaraldehyde and 1% A l c i a n blue f o r 1 hour. The ECM i s a l i g h t l y s t a i n e d amorphous m a t e r i a l and the ba s a l laminae (arrows) are t h i n and l i g h t l y s t a i n e d . Dense granules and strands are not seen. Compare w i t h FIG 57. x2100 EN = endodermal c e l l s EC = ectoderm 97 Figure 68 A 1 um l o n g i t u d i n a l - s e c t i o n of an e a r l y g a s t r u l a shows the h y a l i n e l a y e r surrounding the surface of the ectoderm (arrowheads). At t h i s LM l e v e l the h y a l i n e membrane appears as a fuzzy i n t e r m e d i a t e l y s t a i n e d l a y e r . Clear v e s i c l e s can be seen w i t h i n the c e l l s (arrows) adjacent to the h y a l i n e membrane. x320 EC = ectoderm A = archenteron B = b l a s o c o e l > 98 99 Figure 69 A low m a g n i f i c a t i o n TEM shows the la y e r s of the h y a l i n e membrane (regions 1,2 and 3). Small c e l l u l a r v e s i c l e s (arrows) c o n t a i n i n g a l i g h t s t a i n i n g m a t e r i a l l i e c l o s e to the outer plasma membrane.of the ectodermal c e l l s . The bas a l lamina ( b l ) can be seen at the inner surface of the ectoderm. x9,000 B = b l a s t o c o e l c = c i l i a EC = ectoderm Figure 70 A high m a g n i f i c a t i o n TEM showing the u l t r a s t r u c t u r e of the h y a l i n e l a y e r . Region 1 i s composed of as outermost boundary of l i n e a r l y arranged s t a i n e d f i b r i l s ( s m a l l arrowheads), below which i s a loose meshwork of s t a i n e d f i b r i l s . Region 2 i s composed of a dense l i n e . On one side i t i s as s o c i a t e d w i t h the s t a i n e d f i b r i l s of region 1, and on the other side i t i s i n contact w i t h the t i p s of m i c r o v i l l i (arrows). The t h i r d r e g ion appears to be a space c o n t a i n i n g the m i c r o v i l l i and some sta i n e d f i b r i l s ( l a r g e arrowheads). xl4,000 c = c i l i a 100 101 f o r the meshwork. This outermost region was followed by a t h i c k densly s t a i n e d l i n e . One side of the l i n e was a s s o c i a t e d w i t h the f i b e r s of the meshwork; the other side was associated w i t h the m i c r o v i l l i of the ectoderm c e l l s (FIG 70). The space below the l i n e contained m i c r o v i l l i and strands s i m i l i a r to those of the meshwork (FIG 70). Small v e s i c l e s c o n t a i n i n g v a r y i n g amounts of a l i g h t l y s t a i n e d m a t e r i a l were a l i g n e d along the a p i c a l surface of the ectodermal c e l l s (FIGS 69,70). Cross-sections of c i l i a were seen embedded w i t h i n the h y a l i n e meshwork (FIG 70), at the periphery of t h i s l a y e r (FIG 69), and j u s t outside of i t (FIG 69). Upon g a s t r u l a t i o n , the h y a l i n e l a y e r surrounding the embryo invaginated along w i t h the i n v a g i n a t i n g vegetal. pole c e l l s . The developing archenteron lumen was l i n e d w i t h the h y a l i n e m a t e r i a l i n the e a r l y g a s t r u l a (42-51 hrs) (FIG 71). One um l o n g i t u d i n a l s e c t i o n s through the expanding archenteron t i p (55 hrs) revealed a l i g h t l y s t a i n e d m a t e r i a l i n the t i p and a mass of dense m a t e r i a l below i t (FIG 72). TEM t h i n s e c t i o n s revealed a t h i n n e r and a l i g h t e r meshwork around the inner surface of the t i p which became p r o g r e s s i v e l y darker and t h i c k e r along the main body of the archenteron (FIG 73). The h y a l i n e membrane i n t h i s region however, lacked i t s usual compact and organized form. In a d d i t i o n , the a p i c a l surface of the c e l l s where the h y a l i n e membrane had almost disappeared, appeared more rounded compared to those c e l l s i n which the h y a l i n e membrane was p a r t i a l l y i n t a c t and fewer c e l l u l a r v e s i c l e s were present where the h y a l i n e l a y e r was unstructured (FIG 74). The lumen of the t i p contained what appeared to be empty membranous v e s i c l e s of d i f f e r e n t shapes and s i z e s and c r o s s - s e c t i o n of c i l i a 102 Figure 71 A LM of an e a r l y g a s t r u l a showing the ectoderm (EC) and the i n v a g i n a t i n g archenteron (A). Note the dense s t a i n i n g of the h y a l i n e membrane w i t h i n the lumen of the archenteron (L) and the i n t a c t boundary of i t s outermost l a y e r e s p e c i a l l y at the t i p of the archenteron (arrows). , x640 B = b l a s t o c o e l Figure 72 A LM of the expanding t i p of the archenteron (A) of a m i d - g a s t r u l a . The archenteron lumen (L) contains both a dark s t a i n i n g m a t e r i a l and a l i g h t s t a i n i n g m a t e r i a l (arrows). There i s no d i s t i n c t h y a l i n e l a y e r i n the expanding t i p of the archenteron, as seen i n the previous stage F i g 71). x800 104 Figure 73 A low m a g n i f i c a t i o n TEM showing the u l t r a s t r u c t u r e of the expanding t i p of a mid-gastrula archenteron and i t s lumen ( L ) . The t r i l a m i n a r s t r u c t u r e of the h y a l i n e membrane i s not as w e l l d e f i n e d . The outermost meshwork (1) i s d i s o r g a n i z e d and not as compact as seen p r e v i o u s l y ( F i g . 69). The dense l i n e f o l l o w i n g i s very l i g h t l y s t ained and much thinner ( 2 ) . The innermost l a y e r (3) contains some dense m a t e r i a l , small c l e a r and dense v e s i c l e s and m i c r o v i l l i . Numerous c e l l u l a r v e s i c l e s appear i n the lower region of the archenteron, but these decrease i n number w i t h i n the c e l l s of the very t i p (#1-5). The c e n t r a l p o r t i o n of the lumen contains many cr o s s - s e c t i o n s of c i l i a (c) and numerous membranous v e s i c l e s ( v ) . B = b l a s t o c o e l x2200 b l = b a s a l lamina Figure 74 A close-up of a p o r t i o n of F i g . 73 ( i n c l u d i n g c e l l s 1-5). Note that these c e l l s contain.fewer c e l l u l a r v e s i c l e s , are rounded up, and appear to have l o s t t h e i r j u n c t i o n a l complexes. x4200 106 gure 75 A low m a g n i f i c a t i o n TEM of the stomodeal i n v a g i n a t i o n ( S ) , showing the h y a l i n e membrane i n v a g i n a t i n g along w i t h the ectoderm (EC). The three regions of the h y a l i n e l a y e r are i n t a c t although the f i r s t appears t h i c k e r . This however, i s probably due to the i n v a g i n a t i n g c e l l s , or the plane of s e c t i o n . x3000 B = b l a s t o c o e l 107 108 (FIG 73). TEM t h i n s e c t i o n s through the stomodeum of an o l d e r embryo showed the h y a l i n e l a y e r of the ectoderm i n v a g i n a t i n g along w i t h the ectodermal c e l l s (FIG 75). At the p o i n t of i n v a g i n a t i o n , the h y a l i n e membrane appeared t h i c k e r , but i t s o v e r a l l s t r u c t u r e was i n t a c t . IV. E x o g a s t r u l a t i o n Studies 1). Development of Exogastrulae Embryos incubated i n 0.05 M L i C l g a s t r u l a t e d normally, that i s the endoderm invaginated i n the normal manner. The m a j o r i t y of those incubated i n 0.10 M L i C l f o r 5 and 10 hrs a l s o g a s t r u l a t e d normally, although t h e i r b l a s t o c o e l contained what appeared to be c e l l u l a r d e b r i s which was not present i n the c o n t r o l embryos. Development of embryos incubated f o r 12 and 15 hrs i n 0.1 M L i C l , appeared normal u n t i l the l a t e b l a s t u l a stage. A f t e r a 2-3 hr delay i n hatching, the c e l l s at the v e g e t a l pole became more elongated than those i n the c o n t r o l s and began to bulge outward, that i s , the embryos began to exogastrulate (FIG 76). The c e l l s at the animal pole were a l s o more elongated than t h e i r normal counterparts (FIG 77). The b l a s t o c o e l and the surrounding ectoderm appeared to be g r e a t l y reduced i n s i z e . The evaginated endoderm appeared enlarged and was accompanied by a c a v i t y that was continuous w i t h the b l a s t o c o e l of the ectoderm. A b a s a l lamina l i n e d the b a s a l surfaces of the ectoderm and the endoderm, and a meshwork / of e x t r a c e l l u l a r m a t e r i a l f i l l e d both the b l a s t o c o e l and the endodermal lumen. The b l a s t o c o e l a l s o contained m a t e r i a l that was not apparent i n the normal embryos or i n n a t u r a l l y o c c u r r i n g exogastrulae (see below). TEM t h i n s e c t i o n s revealed t h i s m a t e r i a l to be c e l l u l a r d e b r i s made up of small c l e a r v e s i c l e s and what appeared to be membrane bound cytoplasmic fragments and granules (FIG 78). 109 Figure 76 A L i C l induced e a r l y exogastrula showing the evaginated archenteron (arrows) and the b l a s t o c o e l e (B). x400 Figure 77 A LM of a " n a t u r a l " e a r l y exogastrula showing the elongated c e l l s of the ectoderm (arrows). The b l a s t o c o e l extends i n t o the evaginated archenteron (arrowhead). x400 A = evaginated archenteron I l l F igure 78 A TEM showing the c e l l u l a r d ebris of L i C l induced exogastrulae. Note the membrane enclosed cyloplasmic fragments and granules, and small v e s i c l e s among the l i g h t s t a i n i n g m a t e r i a l of the e x t r a c e l l u l a r m a t r i x . x5700 112 113 O c c a s s i o n a l l y , a few exogastrulated embryos were found w i t h i n c o n t r o l c u l t u r e s . Examination of these " n a t u r a l " exogastrulae w i t h the TEM revealed that there was no e x t r a c e l l u l a r m a t e r i a l i n the i n t e r c e l l u l a r spaces. A l s o , the b l a s t o c o e l contained l i t t l e or no c e l l u l a r d e b r i s as compared to the L i C l induced exogastrulae, although an e x t r a c e l l u l a r matrix was present. C e l l s were seen i n the lumen of the exogastrulated endoderm immediately adjacent to the t i p , roughly at the same time (67 hrs) the mesenchyme c e l l s appeared i n the b l a s t o c o e l of the c o n t r o l embryos (FIG 79). At the time the endodermal basal lamina b l i s t e r was formed i n the normal embryos, the exogastrulae e x h i b i t e d a h i g h l y folded b a s a l lamina i n the endodermal t i p . This was ass o c i a t e d c l o s e l y w i t h the surrounding ECM. I n d i v i d u a l loose c e l l s i n the same area showed signs of b l i s t e r i n g of t h e i r plasma membranes, but there was no evidence of an opening i n the t i p of the endoderm or a b a s a l lamina b l i s t e r anywhere w i t h i n the embryos. By 83.5 h r s , the exogastrulated gut was elongated and was bent to one s i d e . By the time the normal embryos reached the e a r l y b i p i n n a r i a stage, the exogastrulae had segmented i n t o three parts (FIG 80): a) an expanded d i s t a l t i p , b) a narrow mid-section; and c) an expanded proximal end. In some exogastrulae, a hole appeared i n the ectoderm on the same side to which the endoderm was bent (FIG 81), and a hole was seen i n the endoderm i n some cases (FIG 82). C e l l s which resembled mesenchyme c e l l s i n the normal embryo were present throughout the b l a s t o c o e l and lumen of the exogastrulated embryos. 2). The E x t r a c e l l u l a r M a t r i x (ECM) In 1 um t h i c k s e c t i o n s of a " n a t u r a l " exogastrulae at 42 hrs p o s t - f e r t i l i z a t i o n , the ECM was c l o s e l y a s s o c i a t e d w i t h the ectoderm and l i t t l e was present i n the b l a s t o c o e l (FIG 83). The ECM at the endodermal 114 Figure 79 An 81 hour " n a t u r a l " exogastrula roughly at the same time that mouth formation i s t a k i n g place i n the c o n t r o l embryos (mid-late g a s t r u l a ) . The evaginated archenteron (A) i s t h i n w alled and bent and mesenchyme - l i k e c e l l s (arrows) are i n the lumen immediately adjacent to the t i p . Also note the i r r e g u l a r surface of the evaginated endoderm (arrowheads). B = b l a s t o c o e l x800 EC = ectoderm 115 116 Figure 80 A l o n g i t u d i n a l - s e c t i o n through a L i C l induced exogastrula showing the ectoderm (EC) the segmented archenteron (1,2,3), and a mass which may represent a coelomic pouch (CP). Mesenchyme-like c e l l s appear i n the b l a s t o c o e l (B). In t h i s embryo the t i p of the archenteron has not opened to the o u t s i d e ; however, an invaginated region i n the ectoderm opposite the bend i n the archenteron has done so (arrowhead). This . region may represent the embryonic stomodeum. x350 Figure 81 A close-up of the ectoderm i n the exogastrulated embryo showing what may represent the embryonic stomodeum (arrow). Mesenchyme-like c e l l s and c e l l d e b r i s are i n the b l a s t o c o e l e (B). x700 Figure 82 A l o n g i t u d i n a l s e c t i o n of the exogastrulated embryo showing B = b l a s t o c o e l t i p of the archenteron of an an opening (arrow) i n the t i p . x700 117 118 gure 83 A l o n g i t u d i n a l - s e c t i o n through a " n a t u r a l " exogastrula showing the d i s t r i b u t i o n of the ECM at t h i s e a r l y stage (42 hours). The ECM i s c l o s e l y a s s o c i a t e d w i t h the ectoderm (arrows) and f i l l s th space of the evaginated endoderm (arrowhead). L i t t l e ECM i s present i n the b l a s t o c o e l e (B). x950 . 119 120 end, however, f i l l e d the evaginated space, the archenteron lumen. In the l a t t e r , the ECM appeared to be more concentrated probably due to the smaller s i z e of the space i t was occupying (FIG 83). At t h i s stage, the ECM had the appearance of a l i g h t s t a i n i n g granular m a t e r i a l w i t h some short f i l a m e n t s . In L i C l induced exogastrulae at 50.5 hrs p o s t - f e r t i l i z a t i o n the b l a s t o c o e l was f i l l e d w i t h ECM (FIG 84). C e l l u l a r d ebris i n the form of granules and cytoplasmic fragments was a l s o present i n a meshwork of e x t r a c e l l u l a r m a t e r i a l composed of l i g h t l y s t a i n e d strands and d a r k l y s t a i n e d f i n e granules (FIG 84). In some p l a c e s , ECM strands appeared to be i n contact w i t h the inner surface of the ectoderm and extended out i n t o the b l a s t o c o e l space. TEM t h i n s e c t i o n s through these same exogastrulae showed a very dense meshwork of a l i g h t l y s t a i n e d m a t e r i a l that appeared not only i n the b l a s t o c o e l but a l s o i n the spaces between the c e l l s both w i t h i n the ectoderm and the endoderm (FIG 85,86). This m a t e r i a l was i n t e r s p e r s e d w i t h a few d a r k l y s t a i n e d granules mostly i n the b l a s t o c o e l and endodermal lumen. The b a s a l lamina i n both regions appeared to be composed of the same l i g h t l y s t a i n e d m a t e r i a l and the dense granules as the ECM. The v e s i c u l a r s t r u c t u r e s seen i n the m a t r i x of a normal e a r l y g a s t r u l a were not observed here. At 55 hrs p o s t - f e r t i l i z a t i o n , e x t r a c e l l u l a r m a t e r i a l of a " n a t u r a l " exogastrula was s t i l l p r i m a r i l y located along the inner aspect of the ectoderm, but more was present i n the b l a s t o c o e l than at the e a r l i e r stage. A large amount of m a t e r i a l appeared to be concentrated at the endodermal end i n a s i m i l a r manner to that described at 42 hrs (FIG 87). The m a t r i x at t h i s stage, e x h i b i t e d short beaded strands as revealed by l i g h t microscopy. 121 Figure 84 A l o n g i t u d i n a l - s e c t i o n through a L i C l induced exogastrula at 50.5 hrs of developement. The b l a s t o c o e l e i s f i l l e d w i t h c e l l d e bris (d) and ECM. The ECM at t h i s LM l e v e l appears as s t a i n e d strands and granules (arrows). A = evaginated archenteron x775 EC = ectoderm 123 Figures 85 & 86 A TEM of the exogastrula i n F i g . 84 showing ectoderm ( F i g . 85) and evaginated endoderm ( F i g . 86) and i t s associated ECM. The ECM i s a l i g h t s t a i n i n g f i n e l y granular m a t e r i a l , although i t appears more f l o c c u l e n t i n the region of the endoderm, and i s i n t e r s p e r s e d w i t h small darker s t a i n i n g granules. Thin filamentous strands and v e s i c u l a r s t r u c t u r e s seen i n the normal embryo at the same stage are not present here. The ba s a l lamina (arrows) appears as a dotted l i n e a s s o c i a t e d w i t h the amorphous m a t e r i a l of the ECM. Note that the ECM i s present not only i n the b l a s t o c o e l e ( B ) , but a l s o w i t h i n the i n t e r c e l l u l a r spaces. F i g . 85 x9500 F i g . 86 x4600 124 125 gure 87 A l o n g i t u d i n a l - s e c t i o n of a " n a t u r a l " exogastrulated embryo (55 hrs) showing the d i s t r i b u t i o n of the ECM. Some m a t e r i a l i s s t i l l c l o s e l y a s s o c i a t e d with, the ectoderm (arrows), but more i s present i n the b l a s t o c o e l (B) space i t s e l f . A large amount of ECM i s concentrated around the evaginated endoderm and the archenteron lumen (arrowheads). Note the granular appearance of the strands. x800 126 127 Figures 88 & 89 Thin s e c t i o n s through a 67 hour " n a t u r a l " exogastrula showing the u l t r a s t r u c t u r e of the ECM i n the region of the ectoderm ( F i g . 88) and the endoderm ( F i g 89). The ECM i n the b l a s t o c o e l e of the ectoderm i s s i m i l a r to that i n the normal embryo at 59 hours. I t c o n s i s t s of very short densely s t a i n e d strands a s s o c i a t e d w i t h v e s i c u l a r s t r u c t u r e s . . The i n t e r m e d i a t e l y s t a i n e d filamentous strands are short and not as d i s t i n c t . The ECM a s s o c i a t e d w i t h the evaginated endoderm c o n s i s t s of very short densely s t a i n e d strands. Few v e s i c l e s are present. Note the densely s t a i n e d b a s a l lamina ( b l ) and the few a s s o c i a t e d v e s i c l e s ( F i g . 89). EN = evaginated endoderm F i g . 88 xl3,800 F i g . 89 xl2,400 129 Examination of the matrix of a 67 hr " n a t u r a l " exogastrula w i t h the TEM revealed a s i m i l a r composition as that observed i n the normal embryos at 59 hrs p o s t - f e r t i l i z a t i o n . The ECM i n the region of the ectoderm showed very dense strands of v a r y i n g lengths a s s o c i a t e d w i t h l i g h t l y s t a i n e d , v e s i c u l a r s t r u c t u r e s and i n t e r m e d i a t e l y s t a i n e d , short filamentous strands (FIG 88). The ECM i n the endodermal region was a l s o composed of very dense, short strands, however, fewer v e s i c l e s and filamentous strands were v i s i b l e (FIG 89). The bas a l lamina l i n i n g both the endoderm and the ectoderm was densely s t a i n e d and i n some areas was ass o c i a t e d w i t h the surrounding ECM (FIG 89). Branched strands seen i n the normal embryos were r a r e l y observed i n the exo g a s t r u l a . At 71 hrs p o s t - f e r t i l i z a t i o n (FIG 90), the p o r t i o n of the b l a s t o c o e l a s s o c i a t e d w i t h the ectoderm and that a s s o c i a t e d w i t h the archenteron of a L i C l induced e x o g a s t r u l a , were completely f i l l e d w i t h a s t a i n e d s t r a n d - l i k e m a t e r i a l which formed a very dense meshwork. TEM t h i n s e c t i o n s of the ectodermal b l a s t o c o e l at t h i s stage (FIG 91) revealed s h o r t , d a r k l y s t a i n e d strands which were as s o c i a t e d w i t h a l i g h t l y s t a i n e d , f l o c c u l e n t m a t e r i a l along t h e i r e n t i r e length. There was no apparent o r g a n i z a t i o n of the strands. The bas a l lamina appeared t h i c k e r and was more densely s t a i n e d . Strands of ECM were attached to the bas a l lamina i n some areas. At a l a t e r stage, 83 h r s , TEM t h i n s e c t i o n s revealed a mass of f l o c c u l e n t m a t e r i a l i n t e r s p e r s e d w i t h d a r k l y s t a i n e d granules i n the b l a s t o c o e l of the ectoderm (FIG 92). The bas a l lamina appeared p r i m a r i l y as a l i g h t l y s t a i n e d s t r u c t u r e and therefore was not very d i s t i n c t i n r e l a t i o n to the surrounding ECM. The ECM at the endodermal end showed l e s s l i g h t l y s t a i n e d m a t e r i a l and more densely s t a i n e d m a t e r i a l i n the form.of short beaded strands. The basal lamina appeared to be associated 130 gure 90 A l o n g i t u d i n a l - s e c t i o n through a 71 hr L i C l induced e x o g a s t r u l a . The st a i n e d strands of the ECM form a dense meshwork i n the b l a s t o c o e l e and the anchenteron lumen. x350 A = evaginated archenteron EC = ectoderm 131 132 Figure 91 A TEM of the t y p i c a l e x t r a c e l l u l a r m a t e r i a l of the embryo i n F i g . 90. (Fixed i n the presence of A l c i a n blue f o r 3 hrs) The ECM c o n s i s t s of a dense meshwork of short densely s t a i n e d strands and a l i g h t s t a i n i n g f l o c c u l e n t m a t e r i a l . The bas a l lamina ( b l ) i s a l s o densely s t a i n e d and as s o c i a t e d w i t h the f l o c c u l e n t m a t e r i a l . x l l , 0 0 0 EC = ectoderm 133 134 Figure 92 A t h i n s e c t i o n through the ectoderm (EC) of an 83 hr L i C l induced exogastrulated embryo ( f i x e d i n the presence of A l c i a n blue f o r 1 hr.) showing the ECM. I t c o n s i s t s of densely s t a i n e d granules and a l i g h t l y s t a i n e d f l o c c u l e n t m a t e r i a l s i m i l a r to that seen i n the 50.5 hour L i C l induced exogastrula ( F i g . 85). Strands, as seen i n Fig.91 are not present and the basal lamina ( b l ) i s l e s s densely s t a i n e d than that i n F i g . 91. x8600 Figure 93 A t h i n s e c t i o n through the evaginated endoderm of an 83 hr L i C l induced exogastrula showing the beaded appearance of the ECM strands (arrowheads). The l i g h t l y s t a i n e d f l o c c u l e n t m a t e r i a l appears washed out. Only the dense granules of the basal lamina (arrows) can be seen. x6000 M = mesenchyme c e l l 135 136 w i t h more dense granules and therefore was more obvious i n t h i s region (FIG 93). 3 ) . The Hyaline Membrane The h y a l i n e l a y e r surrounding the L i C l induced and " n a t u r a l " exogastrulae appeared to have the normal s t r u c t u r e observed w i t h the LM i n the c o n t r o l embryos during e a r l y g a s t r u l a t i o n (FIG 94). L i g h t micropraphs of the evaginated t i p of a 55 hr exogastrula revealed an i r r e g u l a r e x t e r n a l surface l a c k i n g the d i s t i n c t h y a l i n e l a y e r seen i n the r e s t of the embryo (FIG 95). TEM t h i n s e c t i o n s of a 50 hr exogastrula showed that the h y a l i n e membrane i n t h i s region was discontinuous (FIG 96). Thin s e c t i o n s of l a t e r exogastrula (67 hrs) revealed an almost naked endoderm t i p (FIG 97). Some of the c e l l s were as s o c i a t e d w i t h pieces of dense s t a i n i n g h y a l i n e m a t e r i a l , small cytoplasmic processes and i n t r a c e l l u l a r v e s i c l e s , while others demonstrated naked surf a c e s , smooth plasma membranes, and fewer i n t r a c e l l u l a r v e s i c l e s . Examination of t h i s region i n a l a t e r exogastrula (83 hrs) showed that the h y a l i n e l a y e r was e n t i r e l y m i s s i n g , and c e l l processes extended i n t o the outside space (FIG 98). Table 2 summarizes the changes that occurred i n the ECM and h y a l i n e membrane during the e a r l y development of the s t a r f i s h embryo. 137 Figure 94 A l o n g i t u d i n a l - s e c t i o n through a " n a t u r a l " e a r l y exogastrula (42 hrs) showing a complete h y a l i n e membrane around the e n t i r e outer surface of the embryo (arrowhead). Clear c e l l u l a r v e s i c l e s can be seen at the a p i c a l (outer) surface of most c e l l s . x700 Figure 95 A LM of a " n a t u r a l " exogastrulated embryo (55 hrs) showing the t i p of the evaginated endoderm. Note the i r r e g u l a r surface of the d i s t a l p o r t i o n of the endodermal t i p (arrows). The h y a l i n e membrane i s not as w e l l defined as that i n F i g . 94, but the c l e a r c e l l u l a r v e s i c l e s can s t i l l be seen. x750 139 Figure 96 A TEM of the evaginated endodermal t i p of a L i C l induced exogastrula (50.5 hrs) showing pieces of h y a l i n e membrane attached to the c e l l s urfaces (arrows). Few small c e l l u l a r v e s i c l e s are present. x6800 Figure 97 A TEM of the evaginated endodermal t i p of a 67 hr " n a t u r a l " exogastrula showing remnants of the h y a l i n e membrane. The arrow points to an area which has completely l o s t i t s h y a l i n e l a y e r . x9000 C = c i l i a 141 Figure 98 A t h i n s e c t i o n through the evaginated endoderm of an 83 hr L i C l induced exogastrula showing remnants of the dense l i n e a s s o c i a t e d w i t h the m i c r o v i l l u s t i p s (arrows). x22,000 142 98 143 TABLE 2: CHANGES IN THE EXTRACELLULAR MATRIX AND THE HYALINE MEMBRANE OF ALCIAN BLUE STAINED NORMAL AND EXOGASTRULATED P. OCHRACEUS EMBRYOS DURING EARLY DEVELOPMENT. TIME (hrs) NORMAL TIME (hrs) EXOGASTRULA 42 -concentrated along 42 regions of ectoderm and endoderm; - l i t t l e ECM in b l a s t o c o e l ; -appears as a stained material in the blastocoel as seen with the LM; -composed of l i g h t l y stained v e s i c l e s and a densely stained substance, associated i n a l i n e a r fashion as seen with the TEM; -a complete hyaline membrane surrounded the surface of the embryo; 51 -ECM f i l l e d more of the bl a s t o c o e l ; -strands of various length and complexity composed of v e s i c u l a r structures, intermediately stained filamentous strands and a densely stained material (TEM); 55 -"beaded" strands located 55 pri m a r i l y in the presumptive mouth region of embryo (LM); -strands consisted of a l i g h t l y stained filamentous core associated with a densely stained material and small concentrations of a stained amorphous material along i t s length; few v e s i c l e s were present (TEM); -concentrated along regions of ectoderm and -occupies space within evaginated endoderm; - l i t t l e ECM in b l a s t o c o e l ; -composed of short strands and granular material as seen with the LM; -not examined with the TEM at t h i s stage; -a hyaline membrane surrounded the exogastrulated embryo as in the normal; 50.5 - c e l l u l a r debris and ECM completely f i l l e d the bl a s t o c o e l ; -composed of a l i g h t l y stained f l o c c u l e n t material and some small densely stained granules i n no p a r t i c u l a r arrangement (TEM); d i s t i n c t strands were absent; -more of the ECM appears composed of stained short strands (LM); 144 TIME (hrs) NORMAL TIME (hrs) EXOGASTRULA -hyaline membrane structure began to break down within lumen of the t i p of the archenteron; 59 -beaded strands extended from archenteron to ectoderm e s p e c i a l l y in the presumptive mouth region (LM) ; -morphology s i m i l a r to 55 hr embryos; but was more highly branched and fewer v e s i c l e s were present; -hyaline membrane began to break up around the evaginated t i p of the archenteron; 67 -short ECM stands randomly f i l l e d the blastocoel and lumen; "Natural": -consists primarily of very short strands composed of a densely stained material which was associated with fewer v e s i c u l a r structures and filamentous strands; 71 -hyaline membrane was l o s t beneath the c e l l s i n the archenteron t i p ; - s i m i l a r to 59 hr embryos; 71 -branched strands were not seen; -pieces of hyaline membrane were attached to the evaginated endodermal c e l l s ; L i C l induced: -a dense meshwork of short densely stained strands associated with a l i g h t l y stained f l o c c u l e n t m a t e r i a l ; -no v e s i c l e s or filamentous strands present; 83 -blastocoel contained both a network of beaded loosely i n t e r l a c i n g strands in the center and a denser mesh-work of short strands at the periphery;' -no apparent organization seen; 83 L i C l induced: -the ectodermal region contained a mass of flocculent l i g h t l y stained material interspersed with small dense granules; 145 TIME (hrs) NORMAL TIME (hrs) EXOGASTRULA -strands extended from the archenteron to the ectoderm in a d i s t i n c t r a d i a l pattern within the presumptive mouth region; -the endodermal lumen contained beaded strands of varying length associated with a l i g h t l y stained material; -the strands were more densely stained and thicker and were associated with small darkly stained blebs; few v e s i c l e s were seen; -the hyaline membrane was continuous around the embryo; was also found i n the lumen of the gut and the opening of the mouth; -the hyaline membrane was completely missing around the evaginated endoderm; 146 DISCUSSION AND CONCLUSIONS I. The E f f e c t of Sea Water on Normal Development During the e a r l y course of t h i s study, considerable d i f f i c u l t y i n ob t a i n i n g c o n s i s t e n t c u l t u r e s of good healthy embryos was experienced. Since embryos are known to be extremely s e n s i t i v e to the sea water i n which they are c u l t u r e d , eggs were c u l t u r e d i n sea water from s e v e r a l d i f f e r e n t geographical l o c a t i o n s . In a d d i t i o n , gametes of animals c o l l e c t e d from v a r i o u s areas were a l s o tested w i t h sea water from each l o c a t i o n . Eggs from s t a r f i s h obtained from mainland areas such as Pt. Roberts, Stanley Park, and Copper Cove near Horseshoe Bay, i n v a r i a b l y gave c u l t u r e s i n which the embryos contained c e l l d e bris and most d i d not develop beyond the mid-gastrula stage (mesenchyme c e l l r e l e a s e ) , no matter what sea water they were incubated i n . Eggs from females, c o l l e c t e d at V i c t o r i a gave healthy c u l t u r e s when grown i n sea water c o l l e c t e d from V i c t o r i a , Galiano I s l a n d or the West Coast of Vancouver I s l a n d , but showed poor development when c u l t u r e d i n sea water from the mainland s i t e s mentioned above. Measurement of the osmo l a r i t y of the sea water from these regions demonstrated that sea water from the mainland had an osmolarity below 700 moms. and that f o r the i s l a n d s and V i c t o r i a , over 850 moms. Decreased os m o l a r i t y i s probably due to the fr e s h water outflow of the Fraser R i v e r . The r e s u l t s suggest that development w i l l be poor i n sea water w i t h an osmo l a r i t y below 850-900 moms, and that one should ob t a i n sea water from areas f a r from sources of fr e s h water f o r s u c c e s s f u l c u l t u r e s . Not only was development of embryos a f f e c t e d , but there appeared to have been an e f f e c t on the female a d u l t s . The ova from animals taken 147 from Copper Cove and Pt. Roberts were more i r r e g u l a r i n shape and required much longer for GV breakdown. This suggests that the development and/or maturation of the eggs may also be affected by e i t h e r low s a l i n i t y or pollutants present within the Fraser outflow. Further research i s needed to c l a r i f y t his point. I I . Stages of Normal Development Although a basic set of stages for the development of Pisaster  ochraceus embryos has been outlined previously by Strathman (Friday Harbour Laboratory Manual), an even more accurate method of staging was attempted for the present study p a r t i c u l a r l y during mouth formation. The pl o t of AL/BL r a t i o s and ALs versus time provides a useful tool for describing the stages leading up to mouth formation, as evidenced by an i n i t i a l sharp r i s e i n the curve. This i s i n d i c a t i v e of rapid change which i s manifested as an increase in the rate of development of the archenteron. On the other hand, in i t s l a t t e r portion, the graph tapers o f f , i n d i c a t i n g l i t t l e or no change in the rate of development as measured by AL and AL/BL r a t i o s . This implies that during mouth formation there i s l i t t l e archenteron elongation. Whereas in the f i r s t 25 hrs of g a s t r u l a t i o n (42-67 hrs) the archenteron more than doubles i t s length, reaching a height of 138 um, i t only grows 12 um i n the following 23 hrs (67-90 hrs) of development. The late gastrula stages encompass the 19 hrs between bending of the archenteron at 71 hrs and the complete formation of the mouth at 90 hrs. During t h i s time, growth i s more toward the development of form and shape that give r i s e to the rudimentary structures of the gut. The body length of the embryo however, continues to elongate, thereby producing a s l i g h t decrease i n the AL/BL r a t i o s . 148 For p r a c t i c a l purposes, therefore, Table 1 and the graph (FIG 13) are useful tools for determining at what stage in development the embryos are with respect to body and archenteron lengths, but they are not so accurate in pinpointing the events of mouth formation i t s e l f i f body and archenteron lengths are used as the determining f a c t o r s . I I I . Mouth Formation 1). The Role of the Basal Lamina Preliminary observations of mouth formation i n l i v i n g embryos and whole mounts of the s t a r f i s h P. ochraceus have been described previously (Crawford and Chia, 1978). A more de t a i l e d examination of mouth formation in these embryos was undertaken in t h i s study using l i g h t microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The dynamic process of mouth formation w i l l be described here based on a series of s t a t i c pictures obtained from over 100 s e r i a l l y sectioned embryos through the mouth region. The sequence of events i s postulated to be as follows: Some c e l l s are l o s t from the t i p of the archenteron leaving behind a hole bounded by the endodermal basal lamina. This naked basal lamina expands to form a b l i s t e r which extends into the blastocoel and the archenteron bends, bringing the hole d i r e c t l y opposite the presumptive stomodeal ectoderm. Conical and filamentous c e l l processes appear at t h i s stage on the inner surface of the ectoderm only in the presumptive mouth region. Mesenchyme c e l l s may contact some of the endodermal c e l l s associated with the b l i s t e r through holes in the basal lamina, and/or attach to the basal lamina b l i s t e r i t s e l f . In addition, they contact the filamentous spikes of the ectoderm. I n i t i a l contact of the expanded basal lamina i s with one of the c o n i c a l ectodermal spikes which i s followed by contact with at le a s t a second one. Fusion of the ectodermal and endodermal basal 149 laminae coupled with loss of both over the presumptive stomodeal region, forms a tube which i s associated at t h i s point with scattered endodermal c e l l s . This tube w i l l eventually give r i s e to the l a t e r a l walls of the esophagus. The ectoderm within the tube invaginates to form the stomodeum and there appears to be an outward migration of single endoderm c e l l s which eventually forms a continuous tube of c e l l s along the former b l i s t e r of basal lamina. The endoderm and the ectoderm j o i n to form an o r a l plate which di s s o c i a t e s a f t e r a further 24 hrs to complete the l a r v a l d i g e s t i v e t r a c t . Although mouth formation represents a continuous process, for convenience of discussion the events involved can be divided into four major topics: the bending of the archenteron, the ectodermal spikes, the basal lamina tube, and the stomodeal invagination. The f i r s t section involves the expansion of the archenteron t i p and the loss of c e l l s from the archenteron t i p , b l i s t e r i n g of the endodermal basal lamina and bending of the archenteron which have been observed to occur rather simultaneously over a short period of time (4 hrs) during mid-late g a s t r u l a t i o n . During the expansion of the archenteron t i p , the c e l l s i n the t i p , some of which are presumptive mesenchyme c e l l s , change from a columnar to a cuboidal epithelium and c e l l processes extend into the b l a s t o c o e l . Through scanning electron microscopy (SEM) studies, Chia (1977) has also demonstrated that at the time of mesenchyme c e l l formation, the t i p of the archenteron i s expanded and the c e l l s i n t h i s region are wedge-shaped and possess long f i l o p o d i a . This change in c e l l shape may simply be the r e s u l t of the mechanical p u l l i n g on the c e l l s as the t i p expands, causing them to contract and thin out. Another p o s s i b i l i t y i s that the c e l l s may be d i f f e r e n t i a t i n g and preparing to be released into the blastocoel to function as mesenchyme c e l l s , thereby 150 i n i t i a t i n g a cascade of events leading to the formation of the mouth. This idea i s supported by two other observations made i n the a s t e r o i d . L i g h t micrographs show that at t h i s stage, only the c e l l s i n the t i p extend c e l l processes i n t o the b l a s t o c o e l space; and time-lapse cinematography at t h i s time shows a p u l s a t o r y a c t i v i t y of these c e l l s (Crawford and. Chia, 1978;1982). Other i n v e s t i g a t o r s have made s i m i l a r observations i n both i n v e r t e b r a t e s and v e r t e b r a t e s . Primary mesenchyme c e l l s i n the e c h i n o i d demonstrate t h i s p u l s a t i n g a c t i o n during t h e i r r e l e a s e from the v e g e t a l pole (Gustafson and Kinnander, 1956a), and "plump ba s a l p r o t r u s i o n s " are observed during epiblast-mesoblast t r a n s i t i o n i n the c h i c k embryo ( B a l i n s k y and Walther, 1961), suggesting that s i m i l a r a c t i v i t i e s may be t a k i n g place here. In a d d i t i o n , Franke and co-workers (1983) have reported that during primary mesenchyme c e l l formation i n the mouse embryo, d r a s t i c morphological changes, many of which are s i m i l a r to those observed i n the expanding archenteron t i p , take place as these c e l l s d i f f e r e n t i a t e . The mesenchyme c e l l s lose the a p i c o - b a s o l a t e r a l p o l a r i t y t y p i c a l of e p i t h e l i a l - c e l l s , desmosomes, c y t o k e r a t i n f i l a m e n t s and a surrounding b a s a l lamina. Instead, they express vimentin f i l a m e n t s and produce numerous f i l o p o d i a - l i k e processes g i v i n g the impression of a h i g h l y m o t i l e c e l l type which f r e q u e n t l y changes shape as i t moves down the mesenchymal space. Basal lamina d i s c o n t i n u i t i e s , s i m i l a r to those seen during mesenchyme c e l l r e l e a s e i n the a s t e r o i d , are a l s o a s s o c i a t e d w i t h mesenchyme c e l l r e l e a s e and l o c a l c e l l p r o t r u s i o n s i n the p r i m i t i v e streak region during mouse embryogenesis (Sol u r s h and Revel, 1978; Franke, et a l . , 1983). P u l s a t o r y a c t i v i t y i s a l s o observed during coelomic pouch formation. Time-lapse cinematography s t u d i e s c o r r e l a t e d w i t h transmission e l e c t r o n microscopy (Crawford and Chia, 1978) have demonstrated that c e l l s w i t h 151 active pulsation and movements contain arrays of 50A microfilaments which are not seen i n quiescent c e l l s . Treatment with cytochalasin B, which i s known to disrupt arrays of 50A microfilaments associated with c o n t r a c t i l e a c t i v i t i e s in a number of c e l l s (Carter, 1967; Schroeder, 1970; Cloney, 1972; Crawford, et a l . , 1972), r e s u l t s in the cessation of coelomic pouch formation. In addition, when the mesenchyme c e l l s are exposed to cytochalasin B for one hour, they round up and lose t h e i r m o t i l i t y (Crawford and Chia, 1978). These observations suggest that pulsations which occur before and during mesenchyme c e l l release, may be mediated by c o n t r a c t i l e microfilaments, and that these pulsations may be aiding the breakup of the basal lamina to allow the c e l l s to migrate into the bl a s t o c o e l . This would suggest that a continuous basal lamina sheet over the t i p of the archenteron may serve to r e s t r a i n c e l l migration. Large holes i n the basal lamina at this stage have been seen i n SEM studies (Crawford and Chia, 1981), however this i s most l i k e l y due to a r t i f a c t , e s p e c i a l l y since the SEM material was not preserved with A l c i a n blue. Smaller holes in the basal lamina have been observed i n thin sections. Many of these have f i l o p o d i a extending through them. The loss of c e l l adhesion also appears to play a r o l e i n loosening the presumptive mesenchyme c e l l s from the archenteron t i p . A decrease i n c e l l adhesion with respect to morphogenetic movement has been correlated with at least three d i f f e r e n t parameters: (1) the appearance of new surface antigens (McClay, et a l . , 1977); (2) an increase i n surface charge (Schaeffer, et a l . , 1973); and (3) the lack of a hyaline membrane (Gustafson and Wolpert, 1962). The f i r s t two factors suggest that the c e l l surface membrane plays a r o l e i n determining both the nature of c e l l contacts and pseudopodal a c t i v i t y by expressing new surface proteins, which i n turn i s i n d i c a t i v e of c e l l d i f f e r e n t i a t i o n . The t h i r d factor, 152 the hyaline membrane, was assumed to play an important r o l e i n changes in c e l l adhesion and c e l l shapes when Gustafson and Wolpert (1962) reported that Ca-Mg free sea water, which weakens the hyaline membrane as well as c e l l - c e l l contacts, r e s u l t s in pulsatory a c t i v i t y i n sea urchin gastrulae. As the s t a r f i s h embryo prepares for mouth formation by expansion of the archenteron t i p , the hyaline layer eneath the single c e l l layer i n the t i p begins to d i s i n t e g r a t e . A s i m i l a r r e s u l t was observed at the vegetal pole of the sea urchin blastulae during primary mesenchyme c e l l release (Dan, 1952). It i s probable that the lack of an i n t a c t hyaline layer may be involved in the increased pulsatory a c t i v i t y and the loss of c e l l adhesion observed in the archenteron t i p as presumptive mesenchyme c e l l s separate themselves from other endodermal c e l l s and move into the b l a s t o c o e l . It has been suggested that the hyaline layer attaches to the f e r t i l i z e d egg i n the sea urchin by c e l l surface m i c r o v i l l i (Dan, 1960; Wolpert and Mercer, 1963). The m i c r o v i l l i seen attached to the dense l i n e of the hyaline layer described in the s t a r f i s h embryo could very well function, at least in part, to hold the ectoderm together. Therefore, the loss of the hyaline membrane due to c e l l d i f f e r e n t i a t i o n , for example, coupled with the loss of c e l l junctions, perhaps also due to c e l l d i f f e r e n t i a t i o n , would r e s u l t in the loss of c e l l - c e l l contacts and weakening of the area in question. When the a p i c a l surfaces of c e l l s in the archenteron t i p lose t h e i r hyaline layer and round up, there appears to be a decrease in the number of clear v e s i c l e s in the apex of these c e l l s . It i s possible that these c e l l u l a r v e s i c l e s may be involved either in the synthesis of the hyaline layer or in i t s degradation. The apparent decrease i n the number of v e s i c l e s i s again i n d i c a t i v e of a change in the function of the presumptive mesenchyme c e l l s . However, 153 u n t i l the behaviour and composition of these v e s i c l e s i s better understood through biochemical experimentation, i t i s d i f f i c u l t to c l e a r l y define t h e i r r o l e . It i s i n t e r e s t i n g to note that the evaginated t i p of the exogastrulated embryo also loses i t s hyaline membrane during development. M i c r o v i l l i formed on these c e l l s i n the absence of the hyaline layer are i r r e g u l a r , i n shape, length and diameter. These may represent the m i c r o v i l l i that once served to attach to the hyaline membrane, or they may be the c e l l processes of the presumptive mesenchyme c e l l s before they are released into the b l a s t o c o e l . Although c e l l s are not seen in the space outside the embryo, i t i s assumed that c e l l adhesion i s l o s t since mesenchyme-like c e l l s do migrate into the e x t r a c e l l u l a r m a t e r i a l - f i l l e d space of the b l a s t o c o e l . The c e l l junctions may serve to hold the rest of the c e l l s together. The fact that exogastrulated endodermal c e l l s loose t h e i r hyaline layer at a stage analogous to that during which mesenchyme c e l l formation takes place in the normal, again lends i t s e l f to the idea that c e r t a i n c e l l s i n the archenteron t i p do undergo a change in t h e i r function and therefore are d i f f e r e n t i a t i n g . It also suggests that d i f f e r e n t i a t i o n of these c e l l s i s independant of i t s p o s i t i o n in the embryo and that i t has been determined at an e a r l i e r period, probably before g a s t r u l a t i o n . The loss of c e l l s from the archenteron t i p to form mesenchyme c e l l s helps to account for the hole seen i n the t i p , as suggested by Horstadius (1939). At present i t i s not c l e a r how the basal lamina over the hole expands and extends across the b l a s t o c o e l . From LMs and TEMs, the basal lamina appears to increase in i t s si z e as i t moves within the blastocoel as apposed to simply being stretched across to the ectoderm. There are at l e a s t three p o s s i b i l i t i e s as to i t s expansion. It could be simply unraveling having 154 more freedom to unfold, now that the mesenchyme c e l l s are released. It could be being synthesized de novo and added to by the endodermal and/or associated mesenchyme c e l l s . This i s suggested by the redundancy of the basal lamina as seen i n various places along i t s surface. The redundancy however, i s not observed u n t i l a f t e r the basal lamina begins to expand. Furthermore, associated ECM strands appear morphologically s i m i l a r to the basal lamina. It i s therefore possible that the basal lamina incorporates into i t strands of ECM which are associated with i t both on the blastocoel and the endodermal surface. Further studies are needed to determine whether any or a l l of these mechanisms are involved. Recently, a model proposed by Ingber (Ingber and Jamieson, 1984) to explain e p i t h e l i a l f o l d i n g during formation of the mammary gland, may give some insi g h t into a possible c o n t r o l l i n g mechanism involved i n basal lamina expansion. The model re l a t e s a l t e r a t i o n s i n basement membrane turnover with changes i n e p i t h e l i a l form during normal morphogenesis and neoplastic disorganization in mammary glands. Ingber proposes that the secretion of hyaluronic acid by mesenchyme c e l l s i n the v i c i n i t y of active e p i t h e l i a l f o l d i n g r e s u l t s in the breakdown of e p i t h e l i a l basement membrane. To compensate for t h i s , e p i t h e l i a l c e l l s begin producing basement membrane at a higher rate. This r e s u l t s i n a high rate of basement membrane turnover, which i n turn, gives r i s e to a b l i s t e r i n g or bulging of the basement membrane into the e x t r a c e l l u l a r space. Following t h i s , the e p i t h e l i a l c e l l s would organize themselves along the now enlarged basement membrane creating an outpocketing. Therefore, e p i t h e l i a l c e l l form and organization may be regulated, i n part, by physical forces a r i s i n g outside the c e l l v i a the basement membrane. An a p p l i c a t i o n of t h i s elegant model to the asteroid system may be 155 used to attempt to explain the b l i s t e r i n g observed at the t i p of the archenteron. If the e x t r a c e l l u l a r matrix which appears adjacent to the archenteron t i p contains hyaluronic acid, then i t s degradative action may allow both the release of the presumptive mesenchyme c e l l s into the b l a s t o c o e l and the b l i s t e r i n g of the basal lamina according to Ingber 1s model. Although t h i s i s by no means conclusive, i t should be considered e s p e c i a l l y since hyaluronic acid i s known to appear in large quantities during c e l l migration (Toole, 1972), and that i t i s p r i m a r i l y synthesized by the migrating c e l l s (Pintar, 1978). Growth of the archenteron appears to be a combination of c e l l d i v i s i o n and inward c e l l migration r e s u l t i n g i n invagination. The a c t i v i t y of secondary mesenchyme c e l l f i l o p o d i a appearing in the blastocoel of sea urchin embryos during t h i s stage has led to the suggestion that the archenteron may be pulled or aided by these c e l l s as they attach to and detach from the ectodermal wall (Gustafson, 1964; Dan and Okazaki, 1956; Gustafson and Wolpert, 1961a,1963b; Kinnander and Gustafson, 1960). Bending of the archenteron takes place in the exogastrulae of both asteroid (Crawford and Chia, 1980) and echinoid embryos (Burke, 1978). Howevever, aster o i d mesenchyme c e l l s do not appear in the blastocoel u n t i l a f t e r the archenteron has reached i t s maximum length, and therefore, cannot be involved in p u l l i n g the archenteron into the b l a s t o c o e l . In f a c t , exogastrulation studies in the s t a r f i s h embryo, where the mesenchyme-like c e l l s are not in a p o s i t i o n to f a c i l i t a t e bending the archenteron, have shown that the archenteron i s able to elongate and expand (Crawford and Chia, 1980). The exogastrulated archenteron bends toward the same side in which a hole i s present i n what would be the presumptive stomodeal ectoderm i n the normal embryo. This would suggest that both the bending of the archenteron and 156 the d i s s o c i a t i o n of the c e l l s of the stomodeal region are predetermined and have already been programmed by the time g a s t r u l a t i o n begins as suggested f o r s i m i l a r events i n the sea u r c h i n by Horstadius (1939) and Dan and Okazaki (1956). As the presumptive mesenchyme c e l l s are released and the b a s a l lamina b l i s t e r i s formed w i t h i n the b l a s t o c o e l , the archenteron begins to bend. At the same time, the inner aspect of the presumptive stomodeal ectodermal c e l l s are decorated with various c e l l processes ( s p i k e s ) . I f c e l l d i f f e r e n t i a t i o n and archenteron bending are predetermined events, what i s b r i n g i n g the ectodermal and the endodermal t i s s u e s i n t o r e g i s t e r ? What i s the r o l e of the b a s a l lamina b l i s t e r i n t h i s process? And what other f a c t o r s , i f any, are involved i n t h i s g u i d i n g mechanism? S e r i a l l y sectioned P. ochraceus embryos r e v e a l at l e a s t three p o s s i b l e elements that appear to be involved i n c o n t r o l l i n g or d i r e c t i n g the morphogenetic movements that give r i s e to the mouth. These are: (1) the ectodermal s p i k e s ; (2) the basal lamina; and (3) the mesenchyme c e l l s . C e l l processes seem to be involved i n many events during e a r l y echinoderm morphogenesis from egg maturation (Schroeder, 1981) through coelom and secondary mesenchyme c e l l formation (Gustafson and Wolpert, 1967; Crawford and Chia, 1978) and mouth formation. In the e a r l y development of the mouse and the r a t , e p i t h e l i a l c e l l processes protrude through the basal lamina i n t o the mesenchyme during periods of a c t i v e morphogenesis (Mathan, et a l . , 1972; C u t l e r and Chandry, 1973; Coughlin, 1975; Franke, et a l . , 1983). In the mouse, these have been described as mushroom-like p r o j e c t i o n s of the b a s a l surface membranes through the underlying b a s a l lamina (Franke, et a l . , 1983). Ectodermal b l e b s , p r o t r u s i o n s or spikes can be induced 157 experimentally in v i t r o with the removal of the basal lamina material (Surgue and Hay, 1981; Hay, 1981,1982), but how l o c a l i z e d disruptions come about in vivo to increase the surface a c t i v i t y of the c e l l s i s s t i l l not c l e a r . It may be a question of s p e c i f i c enzymes that are secreted or acti v a t e d ( L i o t t a , et a l . , 1982). "Cones of attachment", s i m i l a r in appearance to some of the ectodermal spikes described above, have been described in the echinoid (Dan and Okazaki, 1956; Kinnander and Gustafson, 1960) as regions of the ectoderm which appear to be pulled into the blastocoel l o c a l l y by the action of the mesenchymal f i l o p o d i a . The authors suggest that these may by involved in mouth formation. The present work shows that ectodermal spikes of at least two morphologies, appear on the inner surface of ectodermal c e l l s i n the presumptive mouth region of asteroids just before the basal lamina b l i s t e r contacts the presumptive stomodeal ectoderm. Scanning electron micrographs reveal large c o n i c a l spikes as well as thin f i l o p o d i a associated with a small region of the ectoderm. Light microscopy reveals that the large c o n i c a l spikes are cytoplasmic projections of the ectoderm. Although some of the filamentous processes seen in the SEM may represent ECM strands and broken mesenchyme c e l l f i l o p o d i a , TEMs show that at least some of them are a c t u a l l y ectodermal c e l l processes. It has been shown that in asteroids both types of ectodermal spikes extend through the ectodermal basal lamina into the b l a s t o c o e l , and provide attachment s i t e s of naked plasmalemma for both the basal lamina b l i s t e r , v i a the c o n i c a l spikes, and for the mesenchyme c e l l s , v i a the thin f i l o p o d i a . Occassionally, contact between mesenchymal and ectodermal c e l l s r esulted i n a s p e c i a l type of junction which appeared to occur through a l o c a l i z e d disruption of the basal lamina during mouse 158 embryogenesis (Trelsted, 1967; Franke, et a l . , 1983). Trelsted suggested that such minute and temporary junctions may control the d i r e c t i o n of tissue movements in the chick embryo. Although junctions between ectoderm and mesenchyme c e l l processes have not been observed at the TEM in asteroids, 1 um s e r i a l sections and SEM observations indicate that such an event could take place. Cross-sections of what may be c e l l processes are seen in contact with or in close proximity to the basal lamina b l i s t e r and the ectodermal spikes, often appearing between the t i p of the b l i s t e r and an ectodermal spike. The working hypothesis then, postulates that s p e c i f i c ectodermal c e l l s , predetermined to form spikes, w i l l poke t h e i r processes through the basal lamina along the ectoderm. In the meantime, mesenchymal c e l l f i l o p o d i a may attach themselves to endodermal c e l l processes and/or d i r e c t l y to the basal lamina b l i s t e r i t s e l f and proceed to " f e e l " t h e i r way along the ectodermal w a l l . Because both the spikes and the mesenchyme f i l o p o d i a are devoid of basal lamina, they are attracted to each other (Gustafson, 1975) and are able to make contact. C o n t r a c t i l e filaments known to e x i s t i n mesenchyme c e l l f i l o p o d i a (Crawford and Chia, 1978) may then provide the motor force necessary to p u l l the basal lamina into close proximity with the ectoderm. It i s t h i s combination of exploratory and c o n t r a c t i l e a c t i v i t y of mesenchymal f i l o p o d i a that i s able to l o c a l i z e the target s i t e at long distances (Gustafson and Kinnander, 1960). Since the ectodermal spikes provide regions of attachment for the basal lamina and the c e l l processes of the mesenchyme in the presumptive stomodeal region, they may serve to determine the region to which the basal lamina i s pulled thus helping to guide t h i s morphogenetic event. The a b i l i t y of the basal lamina to provide attachment s i t e s can be 159 b e t t e r understood i f one analyzes i t s composition. According to a recent review, the bas a l lamina i s a complex e x t r a c e l l u l a r s t r u c t u r e c o n t a i n i n g type IV c o l l a g e n , l a m i n i n , f i b r o n e c t i n (not always), and s p e c i f i c glycosaminoglycans (GAG), such as heparan s u l f a t e (Hay, 1983). Work c a r r i e d out during the l a s t few years has made i t c l e a r that the basal lamina i s produced by the o v e r l y i n g c e l l l a y e r w i t h which i t i s i n contact (Farquhar and Palade, 1961; Andres, 1962; Hay, 1978,1983). The most recent hypothesis, (Hay, 1983), po s t u l a t e s that c o l l a g e n and laminin have d i f f e r e n t receptor s i t e s i n the c e l l membrane which a t t a c h the bas a l lamina to the e p i t h e l i u m . In v e r t e b r a t e s , c o l l a g e n appears s t r i a t e d and co l l a g e n receptors are known to e x i s t on mesenchyme and e p i t h e l i a l c e l l s (Hay, 1983). Although the co l l a g e n i n i n v e r t e b r a t e s l a c k s the o r g a n i z a t i o n described i n vertebrates (Linsenmayer, 1981), c o l l a g e n receptor s i t e s may a l s o e x i s t on the surface of the va r i o u s c e l l types. Therefore, i t i s p o s s i b l e that mesenchyme c e l l s i n the a s t e r o i d may be at t a c h i n g to the basal lamina b l i s t e r by t h e i r s p e c i f i c or n o n - s p e c i f i c receptors f o r a bas a l lamina component, such as c o l l a g e n . The same may be true f o r the attachment of the ectodermal spike to the b a s a l lamina b l i s t e r . A l t e r n a t i v e l y , another model d e p i c t s the bas a l lamina to be arrayed w i t h Ruthenium red s t a i n e d granules along i t s l e n g t h , which supposedly a t t a c h the b a s a l lamina to the o v e r l y i n g e p i t h e l i u m (Hay, 1983). A s i m i l a r arrangement of A l c i a n blue s t a i n e d granules i s observed i n the basa l lamina of the s t a r f i s h embryo, that appear to increase i n s i z e during and a f t e r m i d - g a s t r u l a t i o n . This suggests that as the bas a l lamina detaches, b a s a l lamina receptors may become exposed to form new attachment s i t e s f o r mesenchyme c e l l s (Hay, 1983). I t may be that the subsequent b l i s t e r i n g e f f e c t of the basal lamina i s p r o v i d i n g the future 160 basal lamina attachment s i t e s for the mesenchyme c e l l s , by exposing them, and therefore making them more accessible. Previous studies i n asteroids (Gemmill, 1914; Horstadius, 1939) as well as those of mouth formation in echinoids (Gustafson and Wolpert, 1963) described numerous c e l l s located in the blastocoel of the presumptive mouth region. No d i s t i n c t i o n was made between c e l l s i n t h i s region and mesenchyme c e l l s found in other regions of the b l a s t o c o e l . However, evidence of a basal lamina b l i s t e r i n asteroids, suggests that not a l l the c e l l s are mesenchyme c e l l s , but that some are also endodermal c e l l s l y i n g within the endodermal aspect of the b l i s t e r . Where the surfaces of c e l l s are i n contact with a continuous basal lamina, they are smooth, but where they are associated with a break in the basal lamina, they send processes through. Mesenchyme c e l l s outside the basal lamina also tend to rest on i t , but do not appear to send processes through i t unless there i s an opening. The c e l l s inside the basal lamina b l i s t e r are s i m i l a r i n appearance to those outside the b l i s t e r . If the holes i n the basal lamina are of the si z e seen in the TEM, i t would suggest that the c e l l s within the basal lamina are of endodermal rather than mesenchymal o r i g i n . I f , on the other hand, the large holes in the basal lamina seen with the SEM are r e a l , there may be a free exchange between the c e l l s within the basal lamina and the mesenchyme c e l l s i n the b l a s t o c o e l . Usually, at least two co n i c a l processes are present on ectodermal c e l l s i n the presumptive stomodeal region. The b l i s t e r appears to make contact, i n i t i a l l y with one, then the other spike. Shortly a f t e r t h i s , a tube of basal lamina i s present. The ectodermal and endodermal basal laminae appear to become continuous along the outside of the spikes, and what appears to be large amounts of redundant basal lamina were seen at 161 the point of contact with the spike. The d e t a i l s of how this fusion takes place and how the fusion i s l i m i t e d to only one side of the spike i s not known. The ectodermal basal lamina along the inner surface of the stomodeal c e l l s as well as the endodermal basal lamina which formed part of the b l i s t e r appear to break down soon a f t e r . The exact d e t a i l s of t h i s process are also not known. At the time when i n i t i a l contact between the basal lamina of the endoderm and that of the ectoderm occurs, few c e l l s are seen within the cone. Later more c e l l s are present on the endodermal surface, and f i n a l l y a complete epithelium i s observed. This suggests that c e l l migration, probably of the endodermal c e l l s , may take place along the basal lamina tube. Complete invagination of the stomodeum does not usually occur i n the normal embryo u n t i l a f t e r contact with the endodermal basal lamina, and subsequent loss of both the ectodermal and endodermal basal laminae over the presumptive stomodeal region. It may be that some action of these c e l l s , e i t h e r mechanical, biochemical, or both, i s what aids the breakdown of the basal lamina. Further work involving time-lapse studies of stomodeal invagination should help to determine i f s p e c i a l i z e d c e l l movements are involved. In the echinoid, the stomodeal invagination i s supposedly brought about by the mesenchymal f i l o p o d i a (Gustafson and Kinnander, 1960). However, no such evidence was seen in the a s t e r o i d . In the asteroid, stomodeal invagination appears to be " i n t r i n s i c " . The mechanism of invagination i s not known. A rudimentary invagination of the presumptive stomodeal ectoderm in the exogastrula leads to the suggestion that perhaps the stomodeal invagination i s , at least in part, also a predetermined event. 162 The stomodeal invagination i s important in the formation of the o r a l plate which i s a temporary structure lacking a basal lamina between i t s endodermal and ectodermal c e l l layers. The o r a l plate appears to serve as a way to bring together those c e l l s which are to die, thereby completing the opening of the mouth. Programmed c e l l death i s known to be part of the morphogenetic process. C e l l death in the hand and the foot plate of tetrapod embryos, for example, i s instrumental in separating the d i g i t s from one another, and degeneration of c e l l s i n the neural tube of vertebrates contributes to the development of differences in parts of the c e n t r a l nervous system (Balinsky, 1970). It appears that c e r t a i n c e l l s are determined to die in a s p e c i f i c place and at a s p e c i f i c time during development. The loss of ectodermal c e l l s i n what would have been the stomodeal region of some exogastrulae, implies that the d i s s o c i a t i o n of the c e l l s of what would have been the o r a l plate takes place regardless of contact within the archenteron and i t s basal lamina. Again, the idea of predetermination i s evident here. Other normal events such as the formation of spikes as well as extensive changes in the configuration of the endodermal basal lamina have not been observed in the exogastrula. Whether t h i s defect i s due to an absence of a biochemical or a mechanical influence of the mesenchyme c e l l s , or simply lack of observation at the correct time, has yet to be determined. Although the broad outlines of c e l l u l a r movements during chick, mouse and sea urchin embryogenesis are well known, the d e t a i l of how these are c o n t r o l l e d at the c e l l u l a r l e v e l i s only now becoming better understood. Common observations made in recent years with respect to morphogenetic a c t i v i t i e s , suggest that perhaps we are dealing with a universal mechanism. In both vertebrates and invertebrates, various investigators 163 have reported the following to be associated with morphogenesis: ( l ) mesenchyme f i l o p o d i a ; (2) discontinuous basal laminae; and (3) ectodermal protrusions (Solursh and Revel, 1978; Katow and Solursh, 1980; Franke, et a l . , 1983). The i n t e r a c t i o n of the mesenchyme c e l l f i l o p o d i a with the endodermal and ectodermal c e l l processes proposes a possible mechanism by which the archenteron i s brought into r e g i s t e r with the presumptive stomodeal ectoderm to form the mouth. The subsequent formation of a naked basal lamina tube i s suggestive of a pathway along which c e l l s can migrate. Although, our current idea of morphogenesis involves the movement of e n t i r e c e l l sheets, by i n t e r n a l microfilament bundles and/or external forces such as mesenchyme c e l l s , the present morphological study suggests that t h i s may not be necessary in a l l cases. It i s possible that i n some cases, such as mouth formation, a developing organism f i r s t lays down a basal lamina s c a f f o l d . The c e l l s can then move and rearrange themselves within the framework to give r i s e , i n this case, to the mouth and esophagus. If t h i s i s correct, i t suggests a new mechanism by which the morphogenic movements of c e l l s may be guided. This i s not at a l l a far-fetched idea when one considers the observations of regeneration studies in kidney, muscle and nerve tissue in which the e x i s t i n g basal lamina maintains a s c a f f o l d or a framework which i s used by the regenerating c e l l s (Vracko, 1978). If this i s true, the basal lamina appears to be more than just a fixed b a r r i e r separating two c e l l types; i t may also be a dynamic structure serving as a s c a f f o l d for c e l l migration and therefore plays an i n t e g r a l r o l e i n development. 2). The Role of the E x t r a c e l l u l a r Matrix (ECM) The organization, l o c a l i z a t i o n and composition of the e x t r a c e l l u l a r matrix (ECM) has been studied extensively in embryonic vertebrates, and 164 i t seems clear that in any m u l t i c e l l u l a r organism, an ECM of one kind or another i s created to form scaffoldings (for review see Hay, 1983). Whether the matrices are simply made up of glycoproteins or more complex fibrous arrays, they and the c e l l s that produced them are mutually dependent. In order to study t h i s r e l a t i o n s h i p between the mesenchyme c e l l s and the ECM during mouth formation in the s t a r f i s h embryo, three questions were addressed: (1) When and where does the ECM f i r s t appear? (2) What changes occur in u l t r a s t r u c t u r e and d i s t r i b u t i o n of t h i s ECM during mouth formation? and (3) How might the observed changes i n structure and arrangement be r e l a t e d to the proposed function of the ECM? In an attempt to answer these questions, two sets of experiments were conducted: ( l ) a ser i e s of embryos in various stages between early and late gastrula were fixed approximately 4 hrs apart; and for reasons of convenience remained in the A l c i a n blue f i x a t i v e from 3-12 hrs; and (2) exogastrulae were induced with L i C l i n order to p h y s i c a l l y separate the ectoderm from the endoderm as much as possible, thereby decreasing any influence one might have on the other. The l a t t e r i s to f a c i l i t a t e the observations and inte r p r e t a t i o n s of the o r i g i n of the ECM and the occurrence of the strands of the matrix. LM and TEM r e s u l t s suggest that during early development in a s t e r o i d embryos, the ECM undergoes a progression i n i t s development. It appears to change i n i t s d i s t r i b u t i o n , organization and compostion. Light microscopy shows that the ECM i s present i n the early stages of g a s t r u l a t i o n . I n i t i a l l y , the ECM i s c l o s e l y associated with both the ectoderm and the endoderm , both in the normal and exogastrulated embryos, with l i t t l e ECM present i n the b l a s t o c o e l . The ECM i s composed of a l i g h t l y and densely s t a i n i n g material as revealed by the TEM. In .165 early gastrulae, these materials appear to be d i s t r i b u t e d in a random fashion. In some regions, i t consists of v e s i c l e s and a l i g h t l y stained amorphous mat e r i a l . In others, i t consists of densely stained granules. Where l i g h t and dark materials are both present in the same region of the blastocoele, a simple l i n e a r arrangement of l i g h t l y stained v e s i c l e s and densely stained granules i s often observed. In addition, in early gastrulae, the TEM observations suggest that the ectoderm secretes predominantly the densely stained material and the endoderm, the l i g h t l y stained m a t e r i a l . The above r e s u l t s point to both the endoderm and the ectoderm as being producers of the ECM, and may suggest that a f i b r i l l a r matrix may be formed only where the two materials meet within the b l a s t o c o e l . Although a general form of self-assembly may be involved, yet the idea of a concentration gradient being established by the ectoderm's production of the dense s t a i n i n g component and the endoderm's l i g h t s t a i n i n g one, i s mere speculation. This idea i s appealing, but the picture i s complicated by a number of f a c t o r s : ( l ) the l i g h t and dense substances are found very close to both the endoderm and the ectoderm before they move into the b l a s t o c o e l ; (2) the blastocoel does not show a d i s t i n c t separation between the l i g h t and dark material; and (3) although the crescent shaped patches appear r e a l , they are not observed on a consistent basis to render them s i g n i f i c a n t - for example, not a l l s e r i a l l y sectioned embryos revealed these ECM patches, but an e x t r a c e l l u l a r matrix was present in a l l . Other investigators in t h i s f i e l d have, also been addressing the question of the self-assembly of the matrix (for review, see Hay, 1981), but as of yet there have been no conclusive r e s u l t s . Therefore, u n t i l further evidence i s presented, i t s u f f i c e s to say that i n the case of the asteroid embryo, both the 166 endoderm and the ectoderm appear to be involved in the production of the ECM components beginning in early g a s t r u l a t i o n . Just p r i o r to and a f t e r mesenchyme c e l l s appear i n the b l a s t o c o e l , l i g h t microscopy shows three d i s t i n c t changes within the ECM: (1) strands begin to form; (2) the strands become more organized around the archenteron in the presumptive mouth region as compared to t h e i r i n i t i a l random arrangement within the b l a s t o c o e l ; and (3) the ECM strands appear thicker and more beaded. S i m i l a r l y , transmission electron micrographs reveal changes in the u l t r a s t r u c t u r e of the ECM at the same stages. These include (1) the appearance of a filamentous core within the ECM strands; (2) a higher degree, of branching and c r o s s - l i n k i n g between strands; and (3) an apparent increase in the densely stained substance. In addition, the r a d i a l pattern of the strands i s observed between the basal lamina b l i s t e r and the ectoderm i n the presumptive mouth region. In general, the u l t r a s t r u c t u r e of the ECM f i b e r s at mid-gastrulation i s that of a filamentous core associated with densely stained granules and v e s i c u l a r structures. Current descriptions of proteoglycan macromolecules, depict them as comprising a core protein associated with GAG side chains (Albert,1983). Because the ECM strands described above appear s i m i l a r in appearance, i t i s possible that they also represent proteoglycan molecules. How these strands are formed i s s t i l l unclear. The p o s s i b i l i t y of a s e l f - d i r e c t e d force i s discussed above. However, could outside influences also bring about the formation of the strands and therefore the matrix? In the present system, the d i s t i n c t changes within the ECM appear to take place at the time of mesenchyme c e l l a c t i v i t y ; but whether these changes are simply a matter of rearrangement of already present components, or the introduction of new components, which could come from 167 the v e s i c u l a r structures, for instance, remains to be determined. Many investigators have demonstrated that the migration of neural crest c e l l s i s influenced by the e x t r a c e l l u l a r environment (for review see Weston, 1982). The ideas presented in the l i t e r a t u r e regarding c e l l migration, could be grouped into two categories. The f i r s t , depicts the requirement of a c e l l - f r e e space and a migratory pathway, namely the e x t r a c e l l u l a r matrix for c e l l migration to occur. This would be analogous to the formation of the blastocoele in the s t a r f i s h system. The second, suggests that the migrating c e l l s , as well as the surrounding c e l l s , are able to change the migration pathway during embryogenesis. For example, oriented f i b r i l s i n the e x t r a c e l l u l a r environment may be responsible for a l i g n i n g the crest c e l l s along the migratory pathway and graded concentrations of a p a r t i c u l a r component may provide d i r e c t i o n a l i t y (Weston, 1982). In the same way, the change in strand o r i e n t a t i o n and increase in the dense component in one quadrant of the asteroid embryo, may be the manner in which the surrounding ectoderm and endoderm c e l l s as well as the migrating mesenchyme c e l l s , are causing a change in the migration pathway to provide d i r e c t i o n a l i t y . Embryos treated with L i C l showed a difference in the appearance of the ECM as compared to the n a t u r a l l y occuring exogastrulae and normal embryos. Their ECM was merely an amorphous mass lacking in form and organization. This, however, could be due d i r e c t l y to the L i C l treatment. On the other hand, the densely stained substance i s present i n short strands i n the " n a t u r a l " exogastrula at the time of mesenchyme c e l l appearance (see FIGS 88,89). The intermediately stained filaments, however, are not so obvious and the matrix lacks the web-like organization seen in the normal s i t u a t i o n . Few i f any mesenchyme c e l l s are seen i n the b l a s t o c o e l of L i C l treated embryos. This suggests that 168 i t i s the mesenchyme c e l l s that may be involved . in rearranging the web-like matrix. It i s possible that the surrounding ectoderm and endoderm c e l l s in the asteroid embryo contribute the necessary components to form the f i b r i l s , but i t i s the migrating mesenchyme c e l l s that help to orient them and form the desired pathway. Morphological differences present in the ECM during the development of the asteroid embryo, may represent differences i n the content of ECM components. A l c i a n blue, used in the present work, i s known to preserve the ECM and to s t a i n the GAG (Lev and Spicer, 1964; Behnke and Zelander, 1970; Johnson and Helmig, 1963; Derby, 1978). Therefore, i t i s possible that the observed increase in stained material leading to the apparent increase in strand thickness in the ECM of the s t a r f i s h embryos may represent an increase in the amount of GAG. It has been suggested that differences in the amount and/or type of glycosaminoglycans (GAG).present in d i f f e r e n t regions along the migration pathway, may be one c o n t r o l l i n g factor in the migration and d i f f e r e n t i a t i o n of neural crest c e l l s (Pratt, et a l . , 1975; Derby, 1978; Weston, 1983; Erickson and Turley, 1983). It i s possible that the increase in stained material represents a quantitative and/or perhaps a q u a l i t a t i v e d i f f e r e n c e in GAG in t h i s area and may help to r e t a i n the mesenchyme c e l l s i n this region of the b l a s t o c o e l . As stated above, four d i s t i n c t ECM conponents are seen in the blastocoel of embryos fixed in the presence of A l c i a n blue. These are: (1) the intermediately stained filaments; (2) the l i g h t l y stained amorphous material; (3) the v e s i c u l a r structures; and (4) the densely stained substance. Our present knowledge of the compostion of the ECM provides some clues as to what some of the observed structures could be. 169 The f i r s t of these components i s the intermediately stained filaments. Very short, f i n e filaments are f i r s t observed associated very c l o s e l y with the densely stained granules i n the early gastrula stages. Longer and thicker filaments are seen just before the mesenchyme c e l l s appear in the b l a s t o c o e l , and give r i s e to the longer, more complex strands of the matrix. Similar strands are seen i n echinoid embryos (Akasaka et a l . , 1980; Katow and Solursh, 1979), where s u l f a t e deprivation studies demonstrated that although the o v e r a l l amounts of GAG were reduced, the f i b e r s were unaffected, suggesting that they may be protein in nature. Collagen i s thought to play a major r o l e in s t r u c t u r a l support and strength (Linsenmayer, 1981). The ECM proper has been known for some time to be r i c h i n collagen as well as GAG (Hay, 1983). In vertebrates, collagen appears as a s t r i a t e d structure, but in invertebrates, i t i s usually non-striated (Hay, 1981). Therefore, the intermediately stained strands that are observed in the s t a r f i s h embryo could contain collagen. Fibronectin may also be a component of t h i s web-like structure. Since f i b r o n e c t i n i s thought to bind to s t r i a t e d collagen f i b r i l s i n vertebrate embryos (Yamada, 1981), i t i s possible that i t i s also associated with non-striated collagen in invertebrates. Fibronectin i s also a glycoprotein and has binding s i t e s for heparin and hyaluronic a c i d . It i s reasonable to assume that the "beaded strands" observed in the asteroid blastocoel may be a combination of intertwined collagen and f i b r o n e c t i n filaments with "beads" of GAG associated with them (see below). The second structure observed i n the ECM of P. ochraceus embryos i s the l i g h t l y stained amorphous material. It f i r s t appears in early g a s t r u l a t i o n but appears, to decrease and almost disappears in the l a t e r 170 stages. It i s l o c a l i z e d around the ECM strands i n small clumps, but does not appear to be attached to the strands. It i s d i f f i c u l t to determine i t s r o l e in the formation of the matrix and therefore in c e l l migration by morphological studies alone. More d e f i n i t i v e tests are needed. Because of i t s transient nature, and because morphological observations are suggestive of the self-assembly of the ECM strands, this material may represent precursor molecules of ECM components cons i s t i n g of perhaps unpolymerized f i b r o n e c t i n and/or collagen precursors which may be assembled into strands at the time of mesenchyme c e l l migration. The v e s i c u l a r structures are the t h i r d component described i n the ECM of the s t a r f i s h . These comprise almost a l l of the l i g h t s t a i n i n g material seen i n the early stages of g a s t r u l a t i o n . It appears that when the ECM strands begin to form, the v e s i c l e s disappear and only a few are observed scattered sparsely along the strands i n the l a t t e r stages of mouth formation. The v e s i c u l a r nature of these structures suggests that they contain something which i s probably released at mid-gastrulation, perhaps other precursors which contribute to the matrix. However, a desc r i p t i o n of s i m i l a r v e s i c l e s i s not apparent in the l i t e r a t u r e , nor i s biochemical data a v a i l a b l e as to what these v e s i c l e s might contain. Therefore, t h e i r i d e n t i f i c a t i o n remains unknown u n t i l further research. The l a s t of these components i s the densely stained substance. It appears in the early stages of ga s t r u l a t i o n , and remains as part of the ECM throughout g a s t r u l a t i o n . However, the material seems to become thicker and covers more of the ECM strands within the presumptive mouth region of the mid-late gastrula stage. SEM and TEM studies by Katow and Solursh (1979), have shown that early sea urchin mesenchyme blastulae contain 30 nm diameter granules aggregated i n long chains, and the gastrulae contain 30 nm diameter 171 granules attached to the surface of fine f i b e r s to form a thicker, rough surfaced complex fibrous structure. When cultured in s u l f a t e free sea water the embryos had greatly reduced numbers of 30 nm granules but the f i b e r s were normal. In other words, according to the authors, there e x i s t s a b lastocoel material whose composition changes between b l a s t u l a and gastrula stages and whose appearance i s s e l e c t i v e l y s e n s i t i v e to s u l f a t e deprivation. Histochemical and radioactive incorporation studies have shown that g a s t r u l a t i o n i s accompanied by an increase of e x t r a c e l l u l a r GAG within the blastocoel of the sea urchin (Immers and Runnstrom, 1965; Immers, 1961), and that proteoglycans are e s s e n t i a l for g a s t r u l a t i o n (Kinoshita and Saiga, 1979; Sugiyama, 1972). Although further biochemical research i s needed to i d e n t i f y the densely stained substance in the s t a r f i s h embryo, the above echinoid r e s u l t s , as well as A l c i a n blue s t a i n i n g , suggest that this material could be sulfated GAG. Although exposure to s u l f a t e - f r e e sea water removed some of the 30 nm granules present on the e x t r a c e l l u l a r f i b r i l s i n echinoid b l a s t u l a e , i t did not remove a l l of them. In the asteroid, a l l granules associated with the ECM s t a i n with A l c i a n blue at neutral pH. This suggests that the r e s i d u a l granules could contain hyaluronic acid, a non-sulfated GAG which stains at neutral pH. Toole (1971) o r i g i n a l l y postulated that an accumulation of hyaluronic acid would produce an expansion of the e x t r a c e l l u l a r space which would allow c e l l s to migrate into previously inaccessible areas. It i s possible that increased synthesis of hyaluronate during archenteron elongation could be responsible for expanding the blastocoel and for the observed changes in AL/BL. There i s increasing evidence that the appearance of hyaluronic acid during morphogenesis i s associated with c e l l migration in various tissues (Pratt, et a l . , 1975; Meier and Hay, 1973; Toole, 1971,1972; T r e l s t a d , et a l . , 1974). P r a t t et al. ( 1 9 7 5 ) , observed that the e a r l y migration of c r a n i a l neural c r e s t c e l l s occurs i n a c e l l - f r e e area r i c h i n h y a l u r o n i c a c i d . In v i t r o autoradiography s t u d i e s ( P r a t t , et a l . , 1975) on q u a i l neural c r e s t c e l l s show that although the adjacent t i s s u e s (neural tube, ectoderm and somites) synthesize h y a l u r o n i c a c i d and s u l f a t e d GAG, i t i s the mig r a t i n g c r e s t c e l l s that p r i m a r i l y synthesize h y a l u r o n i c a c i d . In the a s t e r o i d system, the hy a l u r o n i c a c i d could be secreted by the mesenchyme c e l l s , both before and a f t e r t h e i r r e l e a s e i n t o the b l a s t o c o e l . As discussed e a r l i e r i n accordance with Ingber's model (Ingber and Jamieson, 1984), the presumptive mesenchyme c e l l s may produce the h y a l u r o n i c a c i d i n order to increase the turnover r a t e of the basa l lamina. In a d d i t i o n , t h e i r presence may i n f l u e n c e the s t a b i l i t y of the matrix and therefore the i n t e r c e l l u l a r o r g a n i z a t i o n of the mesenchyme c e l l s i n the presumptive mouth r e g i o n , perhaps by making the matrix strands f i r m e r and stronger, thereby enabling c e l l m i g r a t i o n . Akasaka (1980) has suggested that some s u l f a t e d proteoglycans may be re s p o n s i b l e f o r the i n t e r c e l l u l a r o r g a n i z a t i o n of primary and secondary mesenchyme c e l l s i n e c h i n o i d s , since d i s r u p t i o n of the matrix r e s u l t s i n random d i s p e r s i o n of the mesenchyme c e l l s w i t h i n the b l a s t o c o e l (Akasaka, 1980). U l t r a s t r u c t u r a l s t u d i e s combined w i t h biochemical data suggest a c o r r e l a t i o n between the synthesis of s u l f a t e d GAG and the a b i l i t y of e c h i n o i d mesenchyme to produce f i l o p o d i a and mesenchyme c e l l movement (Immers, 1961; Karp and Solursh, 1974; Katow and Solursh, 1979,1981; Akasaka, et a l . , 1980; Kawabe, 1981). Immers (1961) suggested that i n sea u r c h i n s , the i n t e r a c t i o n of s u l f a t e d polysaccharides w i t h p r o t e i n s i s a p r e r e q u i s i t e f o r the formation of c o n t r a c t i l e f i l o p o d i a . The s u l f a t e d polysaccharides supposedly form a sheath around the f i l o p o d i a which would 173 confer upon i t the s t a b i l i t y necessary for t h e i r function. Time-lapse cinematography has demonstrated that mesenchyme c e l l s of the asteroid also produce c o n t r a c t i l e f i l o p o d i a and undergo s i m i l a r movements (Crawford and Chia, 1982) to those described i n echinoids. As in the echinoid, the f i b e r s of the asteroid are associated with A l c i a n blue stained granules which probably also contain both s u l f a t e d and nonsulfated GAG. It i s possible that some of these components are necessary for mesenchyme c e l l movement i n asteroids as w e l l . It i s i n t e r e s t i n g to note that embryos fixed in the presence of Al c i a n blue for varying lengths of time showed a difference i n the d i s t r i b u t i o n of t h e i r ECM. The unique l o c a l i z a t i o n of the ECM in the presumptive mouth region, during mid-late g a s t r u l a t i o n i n embryos fixed in A l c i a n . blue for only 1 hf, supports the idea of a d i f f e r e n t organizational pattern in the presumptive mouth region. In contrast, where the movement of c e l l s i s not extensive, such as the area of the blastocoel other than the presumptive mouth region, the ECM remains i n short strands, which may be r e a d i l y washed away during f i x a t i o n , i f the incubation period in Al c i a n blue i s less than 2-3 hrs. In addition to th e i r increased organization, ECM strands i n the presumptive mouth region were associated with more densely stained material than those located elsewhere. It has been reported in the l i t e r a t u r e , that when collagen and GAG appear i n association, both matrix components are more stable. Perhaps the reason the ECM i s more stable in the presumptive stomodeal region i s because of the l o c a l i z e d increase of GAG i n that area. Although some densely stained material i s present before mesenchyme c e l l s appear i n the bl a s t o c o e l , the f i b e r s do not become t h i c k l y encrusted with i t u n t i l a f t e r the mesenchyme c e l l s appear. Comparison of ECM i n the presumptive mouth region, a region frequented by mesenchyme 174 c e l l s , with that i n areas of the blastocoel to which mesenchyme c e l l s have not yet migrated, shows that the ECM f i b r e s from the former region have much more densely stained material associated with them and have a d i f f e r e n t arrangement than those from the l a t t e r region. This suggests that the mesenchyme c e l l s may not only rearrange them, but that they also may be responsible for producing increased amounts of GAG. During mouth formation the majority of mesenchyme a e l l s are found i n the presumptive mouth region. The observed increase i n the amount and organization of the ECM present i n th i s region may in turn be involved i n re t a i n i n g a large number of the mesenchyme c e l l s here during t h i s c r u c i a l event. The increase i n densely stained material which occurs during mid-late g a s t r u l a t i o n i n the region where mesenchyme c e l l s are occuring, further supports the idea that the mesenchyme c e l l s may be producing the ad d i t i o n a l sulfated GAG. Sea urchin embryos ra i s e d i n s u l f a t e - f r e e sea water or exposed to aryl-B-D-xyloside, which i n h i b i t s the biosynthesis of some proteoglycans, or to tunicamycin, an i n h i b i t o r of GAG, showed the following d e f i c i e n c i e s : (1) a decrease i n the 30hm diameter granules found normally i n the ECM and the basal lamina (Katow and Solursh, 1979; Katow and Solursh, 1981); (2) the lack of mesenchyme c e l l f i l o p o d i a (Karp and Solursh, 1974; Katow and Solursh, 1981; Akasaka, et a l . , 1980); and (3) a cessation i n c e l l movement (Akasaka, et a l . , 1980; Katow and Solursh, 1981). In addition, tunicamycin was found to i n h i b i t , in s t a r f i s h development, the bulging t i p of the archenteron, and the formation of coelomic pouches and the mouth (Dan-Sohkawa, et a l . , 1980). Asteroid embros cultured i n low s a l i n i t y sea water f a i l e d to develop beyond the mid-gastrula stage; i e . mouth and coelome pouch formation often f a i l to occur. This may be due to a deficiency i n s u l f a t e ions i n 175 the sea water which may be i n t e r f e r i n g with the formation of the necessary GAG involved i n mesenchyme c e l l migration and o r i e n t a t i o n . 176 Summary In summary, i t appears that events such as the bending of the archenteron and the stomodeal invagination are programmed to take place. How these are coordinated to give r i s e to the mouth seems to involve various c e l l u l a r and e x t r a c e l l u l a r i n t e r a c t i o n s . The e x t r a c e l l u l a r matrix, composed of glycosaminoglycans, f i b r o n e c t i n and .perhaps collagen, i s secreted by the c e l l s of the ectoderm and the endoderm. The r e s u l t s suggest that a self-assembly of these components takes place into a fibrous meshwork, which i s p a r t i c u l a r l y well formed i n the presumptive mouth region. The mesenchyme c e l l s , which detatch from the t i p of the endoderm into the bl a s t o c o e l , t r a v e l through t h i s meshwork, possibly being guided by i t . They may also reorganize and perhaps deposit more GAG in the presumptive mouth region, thus providing a track for other c e l l s to follow. The establishment of th i s matrix appears to define the region for the migration and the concentration of the mesenchyme c e l l s i n the correct quadrant of the embryo. The loss of mesenchyme c e l l s from the t i p of the archenteron, gives r i s e to a naked b l i s t e r of endodermal basal lamina which bulges into the bl a s t o c o e l . Ectodermal c e l l processes appear s p e c i f i c a l l y i n the presumptive stomodeal region of the ectoderm shortly thereafter. These extend through the ectodermal basal lamina and may provide p r e f e r e n t i a l points of attachment for the mesenchyme c e l l s . C o n t r a c t i l e processes of mesenchyme c e l l s located i n the presumptive mouth region, may then contact both the basal lamina b l i s t e r and/or scattered c e l l s within i t and the ectodermal c e l l processes. 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