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The role of two extracellular matrix glycoproteins in the development of the starfish Pisaster ochraceus Maghsoodi, Bita 2005

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The R o l e o f Two E x t r a c e l l u l a r M a t r i x G l y c o p r o t e i n s i n the Development of S t a r f i s h Pisaster ochraceus by B i t a Maghsoodi B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Anatomy and C e l l B i o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Jan u a r y 2005 © B i t a Maghsoodi, 2005 The role of two extracellular matrix glycoproteins in the development of the starfish Pisaster ochraceus Abstract: Cell-extracellular matrix (ECM) interactions are crucial for the development of the embryos. E C M provides an environment through which cells can migrate, and a substratum for their adhesion and guidance. In this study, cell-ECM interactions were investigated in early morphogenesis of the starfish Pisaster ochraceus. Two monoclonal antibodies (HL-1 and PM-2) generated against E C M components of the hyaline layer and the blastocoel, respectively, were used to identify, localize and characterize these components. Immunocytochemical studies using HL-1 antibody shows that HL-1 epitope is synthesized by the ectodermal cells from the early blastula stage to the bipinnaria stage, and is secreted into the hyaline layer. The epitope was first observed around the Golgi apparatus and deglycosylation of the epitope resulted in the loss of its antigenicity, implicating HL-1 as a glycoprotein. When embryos were incubated in seawater containing an antibody to block the function of the HL-1 antigen, show that the hyaline layer was exfoliated. The results indicated that this E C M component may play a role in development and maintenance of the structural integrity of the hyaline layer. Furthermore, embryos exposed to seawater containing the HL-1 antibody were unable to swim and failed to develop a proper GI tract. This indicated that the HL-1 epitope is necessary for ciliary activity and GI tract development. ii PM-2 epitope also showed the characteristics of a glycoprotein and it too appeared to be important in the development of starfish embryos. Immunofluorescence and immunogold studies showed that the PM-2 epitope was present in the cortical granules and it was synthesized by the early blastomeres through to the bipinnaria stage. These studies also showed that the PM-2 epitope was concentrated in the blastocoel, in the hyaline layer and in the lumen of the Gl tract. Functional blocking experiments using anti-PM-2 antibody revealed that the PM-2-perturbed embryos contained very few migratory mesenchyme cells and failed to develop a proper G l tract. This indicated that the PM-2 epitope may play a role in mesenchyme cell migration, development of the Gl tract and overall growth ofthe embryo. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Figures vi Abbreviations vii Acknowledgements x Chapter 1- Introduction 1 • Extracellular matrix 1 • Extracellular matrix components 2 • Fibrous proteins 3 • Glycosaminoglycans 7 • Proteoglycans 9 • Role of proteoglycans 11 • Cell-ECM interactions 14 • E C M receptors 15 • E C M in development 16 • Echinoderm embryos as a model system 17 • Starfish development 19 • Echinoderm E C M during development 23 • Hyaline layer 23 • E C M lining the GI tract 32 • Basement membrane 32 • Blastocoel 34 • Rationale and thesis objectives 37 Chapter 2- Materials and Methods 39 • Rearing of Pisaster ochraceus embryos 39 • Fixation and embedding of embryos 40 • Monoclonal antibody production 41 • Purification of PM-2 and HL-1 antibodies 43 • Immunohistochemistry 45 • Preparation of colloidal gold antibody 45 • Polyacrylamide gel electrophoresis (PAGE) and Western blotting 48 • H L extraction 48 • Gels and blotting membranes 49 • Silver stain 50 • Western blots 50 • Purification of antigens 51 • In vivo perturbation studies 52 • Immunostaining of perturbed embryos 55 • Tunicamycin and (3 xyloside treatments 56 • Statistical analysis 56 • Deglycosylation of antigens 56 iv • Videomicroscopy of HL-1 treated embryos 57 Chapter 3- Results 58 HL-1 • Distribution and localization of HL-1 epitope during development 58 • Effects of perturbation by the HL-1 antibody 61 • Electron microscopy studies of exfoliation of the hyaline layer 64 • Electron microscopy studies of the hyaline layer regeneration 70 • Biochemical studies of HL-1 78 • Inhibition of ciliary movement 78 P M - 2 • Distribution and localization of PM-2 epitope during development 81 • Immunogold 84 • Western blot analysis of the PM-2 antigen in early development 91 • Perturbation experiments 93 • Localization of PM-2, following PM-2 antibody treatment 102 • Perturbation of development with tunicamycin and f3-xyloside 109 Chapter 4- Discussion 111 H L - 1 • HL-1 111 • Other developmental roles of the HL-1 antigen 112 • Regeneration of the hyaline layer 115 • Biochemistry of HL-1 containing molecule 116 • Conclusions 118 P M - 2 • Localization of PM-2 epitope during development 119 • PM-2 epitope classification 120 • Western blot analysis 121 • The effects of anti PM-2 antibody on development 122 • Does the antibody enter the blastocoel? 126 • Conclusions 130 General Summary 132 References 136 v List of Figures: Fig. 1. Starfish embryonic development 21 Fig. 2. Transmission electron micrograph of a mature hyaline layer 26 Fig. 3. Diagram showing the development of the hyaline layer 30 Fig. 4. Immunolocalization of the HL-1 antigen during embryonic development 59 Fig. 5. Effects of the HL-1 antibody on the embryonic development 62 Fig. 6. Electron micrographs of the exfoliating hyaline layer 65-69 Fig.7. Electron micrographs of embryos regenerating new hyaline layer 72-77 Fig.8. Western blot analysis of purified HL-1 and deglycosylated purified HL-1 79 Fig. 9. Immunofluorescent localization of PM-2 in early development 82 Fig. 10. Immunogold localization of the PM-2 antigen at the T E M level 85-90 throughout development Fig. 11. SDS-PAGE and western blot analysis of the PM-2 antigen in early 92 development Fig. 12. Effects of PM-2 antibody on fertilization 94 Fig. 13. Effects of one dose of PM-2 antibody on developing embryos in vivo. 95 Fig. 14. Effects of multiple doses of PM-2 antibody on developing embryos. 98 Fig. 15. Effects of different concentrations of PM-2 antibody on developing 100 embryos in vivo. Fig. 16. Localization of PM-2 antibody following PM-2 antibody treatment. 104 Fig. 17. Electron micrographs of embryos incubated in PM-2. 106-108 Fig. 18. Immunofluorescent studies of embryos treated with either tunicamycin 110 or (3-xyloside. List of abbreviations: (3-xyloside (3-D-xylopyranoside B L basal lamina B M basement membrane Brij-56 polyoxyethylene 10 cetyl ether CAPS 3-(Cyclohexylamino)-l-propanesulfonic acid C C D Charge Coupled Device Con A Concavalin A D A B C O 1,4-diazabicyclo[2.2.2]octane DIC diffraction interference phase contrast dH 2 0 distilled water D M E M Dulbeccos modified Eagle medium DMSO dimethyl sulfoxide E C M extracellular matrix E C L enhanced chemiluminescence (ECL is a trademark of Amersham Pharmacia Biotech) E D T A ethylenediamine tetraacetic acid E G T A [ethyleneglycol-bis-(p-amino ethyl ether) N,N,N,N tetraacetic acid Endo F endoglycosidase F FACIT fibril-associated collagens with interrupted triple helices FCS fetal calf serum FITC fluorescene isothiocyanate g gravity GAGs glycosaminoglycans gal galactose vii galNAc N-acetyl-D-galactosamine GI gastrointestinal tract glcNAc N-acetyl-D-glucosamine HAT hypoxanthine aminopterin thymidine HI first sublayer of the supporting layer of the hyaline layer H2 second sublayer of the supporting layer of the hyaline layer H3 third sublayer of the supporting layer of the hyaline layer H L hyaline layer H L L hyaline layer-like HRP horseradish peroxidase IgG immunoglobulin G IgM immunoglobulin M JVC Victor Company of Japan, Ltd. kDa kiloDaltons M molar (moles per liter) M M molecular mass MW molecular weight N-linked asparagine linked O-linked serine/threonine linked PAGE polyacrylamide gel electrophoresis PAS periodic acid Schiff PBS phosphate buffered saline PEG polyethylene glycol PM-1 Pisaster matrix-1 PM-2 Pisaster matrix-2 PMSF phenylmethyldisulfonyl fluoride PVDF polyvinylidene difluoride SDS sodium dodecyl sulfate TBS Tris buffered saline TBST Tris buffered saline with Tween T E M transmission electron microscopy T E M E D N,N,N',N'-tetramethylethylenediamine Tris tris(hydroxymethyl)aminomethane VBS veronal buffered saline WGA wheat germ agglutinin VCR video cassette recorder Acknowledgements: I would like to thank my supervisor, Dr. Bruce Crawford for giving me a chance to work with him and for his continued support and guidance throughout my graduate program. His encouragement and demand for excellence has made my graduate education second to none. I would also like to thank the members of my supervisory committee, Dr. Timothy O'Connor, Dr Linda Matsuuchi and Dr. Ravi Shah for their commitment to help me through my Ph.D., their guidance and their critical evaluation of my work. Special thanks to my friends and colleagues, particularly Dr. Maria Glavas, Dr. Louise Liang and Mr. Morten Labo for their continued support through my Ph.D. Finally I am indebted to my family mom, dad, Mona and Bobby without whom I would not have a chance to pursue what I like most - my education. x Chapter 1- Introduction Extracellular matrix: Mature tissues are integrated units that consist of both cellular and extracellular components (Gossler et al., 1998). To perform many of their functions, cells bind to other cells or to material in their surrounding environment. These molecules collectively called the "extracellular matrix" (ECM) form the scaffold upon which tissues and organs are built. In the past, the extracellular matrix was thought to act only as a scaffold to support tissues (Manasek, 1975). It is clear now that the E C M plays a complex and active role in regulating the behavior of the cells and important developmental processes (Hynes, 1999; Humphries, 2000; Assoian et al., 2001; Danen et al., 2001; Schwartz, et al., 2001; Bottaro et al., 2002). Cells secrete E C M , which allows them to regulate certain properties of their environment and can also be influenced by the environment (Chekenya et al., 2002; Wilberg et al., 2002; Lin et al., 2003). Aberrant interactions between cells and E C M could lead to a wide range of anomalous cellular phenomena, including invasion and metastasis of tumor cells (D'souza-Schorey et al., 1998; Voura et al., 2001; Liu et al., 2002; Theocharis et al., 2003). E C M is a main constituent of embryonic tissues and it occupies vast spaces and cavities that are formed from the blastula stage forward. E C M also plays an important role in embryonic development (Juliano and Haskill, 1993; Lin et al., 2003; Zoltan-Jones et al., 2003). In a developing mammalian embryo, E C M molecules are synthesized and secreted by the cells that reside in them. Embryonic cells are in turn, in close contact with both cellular and extracellular components. E C M interacts with developing tissues influencing cellular differentiation, migration, proliferation, shape and metabolic functions (Bernstein and Liotta, 1994; Broverman et al., 1998; Perssinotto et al., 2000; Wight, 2002; Pisano et al., 2003). These events involve interactions between different components of E C M such as fibronectin, vitronectin, collagen and 1 the integrin superfamily of transmembrane receptors (Danen et al., 2001; van der Flier et al., 2001). Integrin-ligand interactions trigger a cascade of events including phosphorylation of proteins at adhesion sites and the enrollment of various cytoskeletal proteins to form focal adhesions (Coussen et al., 2002). These interactions are essential for proper embryonic development as well as proper maintenance of the architecture of tissues (Hay, 1981; Thiery et al, 1983; Ekbolm et al, 1986; Adams and Watt, 1993; Boudreau and Bissell, 1998; Freire et al., 2002; Burke et al., 2004; Fujiwara et al., 2004). Although the composition and function of E C M has been well described in vertebrate embryos and reasonably well in some invertebrate embryos specially in sea urchin, very little is known about the composition of the E C M in many invertebrates such as starfish embryos and larvae. The following thesis will examine the composition, structure and function of two components of the starfish E C M in order to better understand the role of these E C M components during starfish development and also during development in general. Extracellular matrix components: The extracellular components of tissues and organs consist of water, ions and macromolecules such as polysaccharides and proteins (Hay, 1991). Together these molecules form the extracellular matrix. The two key classes of the extracellular matrix macromolecules are fibrous proteins and polysaccharide glycosaminoglycans (GAGs), which are attached to proteins to form proteoglycans (Hay, 1991). Glycosaminoglycans and proteoglycans, form hydrated gels which interact with fibrous proteins. Fibrous proteins (elastin or collagen) mainly contribute to organizing the matrix, however, both laminin and fibronectin in addition to having structural roles, have adhesive functions as well. Collagen fibers reinforce and organize the matrix. Elastin fibers make the matrix flexible, and adhesive proteins help cells attach to the E C M . 2 Fibrous proteins: Fibrous proteins are a major component of the extracellular matrix. They reside in a hydrated polysaccharide gel. Four main types of fibrous proteins are collagen, elastin, fibronectin and laminin. Collagen: One of the major constituents of the E C M is collagen, which is secreted primarily by connective tissue cells. So far, 26 genetically distinct collagens have been identified (Myllyharju and Kivirikko, 2001; Koch et al., 2001; Fitzgerald and Batesman, 2001; Bosman et al., 2003). All collagen complexes are composed of a triple helix of monomers, approximately 300 r|m in length and 1.5 r\m in diameter. Collagens can be divided into several subfamilies according to their primary structure and/or their molecular organization. Fibrillar collagens (types I-III, V and X) have the same general triple helical arrangement and form cross-striated fibrils (Exposito et al., 2002; Geise et al., 2003). Another group in the collagen family is the nonfibrillar collagens. This group does not form large fibril bundles and consists of type IV collagen found in the basement membranes, type VI which has beaded filaments, type VII which forms attached fibrils involved in anchoring of the basement membrane to the underlying E C M . Collagen types VIII and X are non-fibrillar collagens and form hexagonal networks. Collagen types IX, XII, XIV, X X , and XXI are associated collagens and are also referred to as "fibril-associated collagens with interrupted triple helices" (FACIT), which presumably play a role in regulating the diameter of collagen fibrils (van der Rest et al., 1991). Collagens that associate with FACIT are types XVI and XIX. Types XIII and XVII are found in the plasma membrane and lastly collagens X V and XVIII which contain multiple triple domains and interruptions (Exposito et al., 2002, Geise et al., 2003). 3 The role of collagen as a scaffold has been known for sometime, however, we now know that collagen also controls cell shape, differentiation, and migration (Toole, 1982; Depreter et al., 1998; Iruela-Arispe, 1996; Sanders et al., 2003). A prime example of this is the regeneration of broken bones and wound healing (Junge et al., 2002; Robins, 2003; Hansen et al., 2003). The collagen lattice provides the road map for blood vessels to grow and feed healing areas. In the embryo, collagen appears first in the basement membrane of the ectoderm and endoderm and then in the mesoderm and the neural tube (Hay, 1991). This collagen, (type IV), provides an adhering structure for the epithelial and the mesenchymal cells to attach. Fibrillar collagen (type II) first appears around the notochord and provides structural strength as well as mediates certain inductive effects of the notochord on growth and differentiation (Hay, 1991; McAlinden et al., 2002). Collagen I, appears at around the time of neural crest cell formation and appears to be involved in promoting migration of these cells (McCarthy and Hay, 1991; Soto-Suazo et al., 2004). In addition to providing tensile strength, adhesion and a migratory medium, collagen is also responsible for promoting differentiation of embryonic cells (Hay, 1991; Gerecht-Nir et al., 2003). Elastin: Tissues have the ability to recoil after stretching mainly because of a component of the extracellular matrix, the elastic fibers. These fibers have the ability to extend. Along with tensile strength, certain tissues such as lungs and blood vessels require elasticity for normal function (Kielty et al., 2002). Elastic fibers are composed of two distinct components: centrally located elastin is surrounded peripherally by microfibrils (Robb et al., 1999). Elastin and microfibrils are different in their morphology and chemical composition (Cleary et al., 1996). Elastin is very hydrophobic and highly insoluble. It is formed from its soluble precursor, tropoelastin. Both tropoelastin and insoluble elastin are rich in hydrophobic amino acids such as 4 glycine, alanine, proline and valine. Because elastin molecules are hydrophobic they are able to "slide" over one another or "stretch" to maintain structural integrity and provide recoil (Debelle et al., 1999). Microfibrils mainly consist of fibrillins and other structural glycoproteins such as fibulin-5 and emilin (Cleary et al., 1996). These proteins have a high content of aspartate, glutamate and cysteine. Elastin molecules are secreted into the extracellular matrix, where they cross-link together to form an extensive network (Haas et al, 1991; Parks et al., 1993; Vrhavski et al., 1998; Hinek et al., 2000). There is little synthesis of elastic fibers in adult tissues. Unlike most components of the extracellular matrix, elastic fibers are synthesized only during development (Davis 1993a). This makes the synthesis of this protein vital as the amount and orientation of the molecule should function for a lifetime (Robb et al, 1999). Elastin molecules are absent in invertebrates and some basal group of vertebrates. However, studies have shown that the microfibrilar component of elastic fibers appear to be present and confer to non-linear stress/strain properties of the connective tissues in certain invertebrate species (Faury, 2001). Fibronectin: The extracellular matrix contains many adhesive glycoproteins that bind cells to other matrix macromolecules. One of the best known adhesive glycoproteins is fibronectin, which helps to mediate cell-matrix adhesions. Fibronectin is deposited by a variety of cells to make up a meshwork of adhesive proteins and it affects many cellular functions such as growth, differentiation, cytoskeletal organization and migration (von der Mark et al., 1989; Tabata et al., 2003; Cousin et al., 2004). Fibronectin is a multifunctional molecule composed of two analogous subunits of 220-250 kDa. It consists of a series of folded modular domains known as fibronectin repeats I, II and III joined by disulfide bonds (Hynes, 1999). Different domains play different roles. For 5 instance, while one domain binds to heparin another binds to collagen, another binds to certain receptors on the cell surface (Garcia et al., 2002; East et al., 2002). This way, fibronectin contributes to the organization of the matrix and provides an attachment for cells to the matrix. Fibronectin is categorized as either plasma fibronectin or cellular fibronectin. Plasma fibronectin is produced primarily by hepatocytes, whereas, cellular fibronectin is produced by many other cell types. Both types of fibronectin are produced by alternative splicing of a common mRNA precursor. Fibronectin mRNA has three alternative splicing sites [extra domain A (EDA), extra domain B (EDB), and type III connecting segment (IIICS)]. Alternative splicing of these regions allow for synthesis of 20 different forms of fibronectin mRNA (Kumazaki et al., 1999). Fibronectin is important for cell migration. In vertebrate and invertebrate embryos, large amounts of fibronectin are found on pathways where cells migrate. Experiments in which synthetic peptides and recombinant proteins were used to identify the functional domains needed for human fibroblast migration over fibronectin, show that fibronectin domains containing the Arginine-Glycine-Aspartate (RGD) sequence, support maximal fibroblast attachment (Clark et al., 2003). Cell migration, apparently is promoted by the matrix binding activity of fibronectin. Specific sequences within the IIICS region of fibronectin are required for migration (Clark et al., 2003). In vitro studies where cells have been grown on fibronectin matrices, cellular growth and differentiation were accelerated compared to those grown on collagen I, collagen IV and laminin-1, (Tabata et al., 2003). Fibronectin has also been shown to be important in cellular differentiation. 6 Laminin: Laminin is a major component of the basement membrane and is involved in migration, spreading, adhesion, growth and differentiation of various cell types cells (McCarthy et al., 1983; Dillner et al., 1988; Panayotou et al., 1989; Albini et al., 1991; Chun et al., 2003; Fujiwara et al., 2004; Silva et al., 2004). Regulated expression of laminin, both temporally and spatially, during development, is important for the morphogenesis of the nervous system (Nurcombe, 1992; Libby et al., 1999). During development, Schwann cells that lack laminin yl are unable to differentiate and synthesize myelin proteins (Chen et al., 2003). Laminin is also known for its ability to promote neurite outgrowth in cultured neuronal cells grown on a laminin substrate (Baron-van Evercooren et al., 1982; Manthorpe et al., 1983; Rogers et al., 1983; Freire et al., 2002). Laminin is a family of five a, four p\ and three y chains that assemble to form at least 15 different a|3y heterotrimers (Colognato et al., 2000). Several laminin isoforms have been described, each distributed in a distinctive tissue. Knockout experiments in mice show that cc5 plays a role in the development of the brain, limb, placenta, kidney and the lungs. Similar experiments reveal that (32 is important in neuromuscular and glomerular function and y in endoderm differentiation (Thyboll et al., 2002). Like fibronectin, laminin also contains a number of functional domains; one binds to collagen, another to heparan sulfate, and one or more to laminin receptor proteins on cell surfaces (Timpl and Brown, 1996). Glycosaminoglycans (GAGs): Glycosaminoglycans (GAGs) are unbranched polymers of repeating disaccharide units, each of which consists of an amino sugar (N-acetyl-glucosamine or N-acetyl-galactosamine) and a uronic acid (glucuronic or iduronic acid) (Wight et al., 1991; Davies et al., 2001). The type of 7 disaccharide unit varies among different GAGs and with the exception of hyaluronates, the saccharide of all GAGs are sulfated in various positions (Varki et al., 1999). To date four main classes of GAGs have been described: hyaluronic acid, chrondoitin / dermatan sulfate, keratan sulfate and heparan sulfate. Among these, only hyaluronic acid does not maintain a covalent bond to a protein core (Couchman and Woods, 1993). Hyaluronic acid is also synthesized and secreted as free chains (Toole, 1990). Other GAGs bind to core proteins to form proteoglycans, which can either be located at the cell membrane (eg. syndecans, versicans) or in the extracellular matrix (eg. aggrecan and versican) (Perrimon et al., 2000; Selleck, 2000). GAGs also tend to bind to GAG-binding proteins and hence, participate in many cellular events such as cell adhesion, migration, proliferation and apoptosis (Perrimon et al., 2000). These cellular events are mediated mainly by the interaction of GAGs and their receptors (often integrins) and their corresponding intracellular signaling events. In vitro studies have shown that an increase in the hyaluronan production results in epithelial-mesenchymal transformation (Zoltan-Jones et al., 2003). Many of the genes that affect growth and morphogenesis often encode individual proteoglycans or G A G biosynthetic enzymes (Prydz and Dalen, 2000). This is probably because these molecules serve as growth factor co-receptors as well as a reservoir for growth factors and cytokines. GAGs tend to form highly extended random coil conformations, occupying large volumes compared to their mass. They have the ability to form gels even at very low concentrations. Being negatively charged, GAGs attract positively charged ions, hence creating an osmotic environment, causing large amounts of water to be carried into the matrix (Hardingham and Bayliss, 1990). This generates a pressure from the matrix, which allows it to withstand compressive forces. Even at low concentrations, GAGs, are able to fill most of the extracellular space, hence, providing mechanical support to tissues. 8 Proteoglycans: By definition, a proteoglycan is a molecule, which is composed of at least one glycosaminoglycan side chain covalently attached to the protein core of the molecule. Proteoglycans are present in various species such as sponges (Misevic and Burger, 1990), sea cucumbers (Vieira and Mourao, 1988; Kariya et al, 1990), fruit flies (Brower et al., 1987) and vertebrates (Nareyeck et al., 2004). PGs reside in the extracellular matrices, on cell surfaces and intracellularly in granules (Couchman, 2003). The sizes and structures of PGs vary enormously, which give rise to numerous biological functions. These include: cell adhesion, cell migration, matrix assembly and growth factor sequestration (Couchman and Woods, 1993). Different classes of GAGs and different lengths of G A G chains add to the complexity of the proteoglycans. For this reason, they are very difficult to sequence. However, recent progress in molecular biology has advanced this field and so far the core proteins of many proteoglycans (PG) have been sequenced. Based on their topographical distribution and their relationship to the core protein, PGs, can be grouped into five classes. These classes are: 1. Secretory PGs 2. Small leucine-rich PGs, 3. Cell surface PGs, 4. Large aggregating PGs, 5. Basement membrane PGs, (Couchman and Woods, 1993). The smallest core protein (~20 kDa) sequenced is that of PG serglycin, a member of the secretory PG family (Bourdon et al., 1985; Humphries et al., 1992). This protein contains a sequence of 24 uninterrupted repeats of serine-glycine. This repeat sequence establishes the location of the G A G side chains. Serglycin may be involved with hemopoietic cell differentiation (Maillet et al, 1992). Recently, serglycin was also shown to act as a ligand for lymphocyte adhesion molecule CD44 and, therefore, it may be involved with adhesion and activation of lymphoid cells (Toyama-Sorimachi et al, 1995). Decorin, a member of small leucine-rich family of PGs, has a protein core of 36 kDa. It binds to collagen fibrils (Krusius and Ruoslahti, 1986) and aids in organizing many connective 9 tissue structures. It has been proposed that decorin is a bidentate ligand that attaches to two parallel neighboring collagen molecules in the fibril, helping to stabilize fibrils and orient fibrillogenesis (Scott, 1996; Reed et al., 2002). Decorin may also be important in the organogenesis (Scholzen et al. 1994). Decorin's protein core has been shown to bind transforming growth factor beta (TGF-p1), a multifunctional superfamily of proteins that regulate growth, differentiation, migration, adhesion, and apoptosis of a wide variety of cells. Hence interactions between decorin with TGF-(3 may result in regulation of cell division and differentiation in embryonic tissues (Yamaguchi et al. 1990). The family of syndecans, members of cell surface PG family, now consists of at least four different types: syndecan 1, syndecan 2 (fibroglycan), syndecan 3 (N-syndecan) and syndecan 4 (ryudocan, amphiglycan) (Wilcox-Adelman et al., 2002; Couchman, 2003). They have a core protein of ~32 kDa and contain a COOH-terminal cytoplasmic domain, a hydrophobic domain and an extracellular domain, which carry the G A G chains. Many cell types can express more than one type of syndecan, and the expression levels may change during development and differentiation (Elenius et al, 1991). It has been shown that syndecan 2 can promote the formation of dendritic spines in neurons (Ethell et al., 2001). It has also been suggested that syndecan 4 may regulate certain cytoskeletal events that are required for the formation of the focal adhesions (Couchman, 2003). Syndecans bind collagens with a high affinity and specificity, they can also bind the C-terminal, heparin-binding domain of fibronectin (Bernfield and Sanderson, 1990), thrombospondin (Bernfield and Sanderson, 1990), tenascin (Salmivirta et al, 1991) and bFGF (Chernousov and Carey, 1993). Aggrecan, with the largest core protein, 210 kDa, are part of the large aggregating PG family. They are abundant in cartilage and form aggregates. The core protein is composed of terminal globular domains (G). Globular domains G l (331 amino acids) and G2 (200 amino acids) are located in the amino terminal end of the protein core separated by a short interglobular 10 domain. G3 domain is located in the carboxy-terminal domain. GI domain forms a triple-loop structure that is able to form a non-covalent bond to hyaluronan (Perkins et al, 1989). G2 domain has high homology with GI domain, but obviously cannot participate the interactions between the aggrecan and hyaluronan. The G3 domain (222 amino acids) contains several cysteine residues and it has been suggested to be involved in the interactions between aggrecan and extracellular matrix (Halberg et al, 1988; Yamaguchi, 2000). Perlecan, a member of the basement membrane PGs, contains 5-7 variable-length globular domains (Olsen, 1999) and has been found in all basement membranes. Perlecans core protein contains domains homologous to L D L receptor, laminin, neuronal cell adhesion molecule (N-CAM) and epidermal growth factor (EGF). Based on its structure, perlecan may have several interactions with the components of basement membranes. Interactions between heparan sulphate chains and laminin may be important for its binding in the basement membrane. Perlecan may also be involved in cell adhesion through fibronectin (Singer et al, 1987). In vitro studies indicate that perlecan can stimulate early stages of cartilage differentiation (Gomes et al., 2004) and are involved in suppressing smooth muscle cell proliferation through activation of a cascade of intracellular signaling (Walker et al., 2003). Role of proteoglycans: Sequestering growth factors: The presence of diverse core proteins and dissimilar lengths of individual G A G chains on proteoglycan core proteins indicates that these macromolecules are very complex structures. These intricacies enable proteoglycans to become involved in a variety of biological functions (Perin et al., 1988; Yamaguchi et al., 1990). Accurate regulation of the amount of active growth factors is essential for proper tissue maintenance. PGs can bind to growth factors, alter their 11 activity and hence provide local tissue bound pool for growth factors. It is believed that sulfated PGs have a higher affinity for binding to growth factors (Perin et al., 1988). In vitro studies using mice hippocampal neurons show that chondroitin sulfate chains from various marine organisms exhibit growth factor-binding and neurite outgrowth-promoting activities (Bao et al., 2004). Other studies have shown that perlecan, a heparin sulfate PG, binds to fibroblast growth factor at the mouse blood-brain barrier (Deguchi et al., 2002). In addition, syndecans have also been extensively studied in this respect and have been shown to possess the ability to bind to fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) (Bosman et al., 2003; McQuade et al., 2003). Assembly ofthe extracellular matrix: Proteoglycans have also been shown to play a part in the assembly of other E C M components. There are indications that proteoglycan-collagen interactions are important in the regulation of collagen fibril formation and matrix assembly (Hascall and Hascall, 1981; Hardingham and Fosang, 1992; Wilberg et al., 2002; Reed et al., 2002; Day et al., 2004). Studies have shown that when synthesis of corneal dermatan sulfate proteoglycans was disturbed, the organization of the corneal stroma was disrupted, including alterations in collagen fibril packing (Hahn and Birk, 1992). Biglycan, a leucine rich PG, interacts with N-terminal part of collagen IV and organizes it into hexagonal-like networks (Wilberg et al., 2002). Proteoglycans have also been shown to link other matrix proteins together. The conserved N-terminal globular G l domain of aggrecan binds to hyaluronan (Hardingham and Bayliss, 1990; Day et al., 2004) and modulates assembly of the extracellular matrix (Day et al., 2004). 12 Cellular migration: Many studies have indicated that extracellular and cell surface proteoglycans are important for cell migration. For instance migratory endothelial cells exhibit increased chondroitin sulfate and dermatan sulfate proteoglycan synthesis when compared to sessile cells in vitro (Kinsella and Wight, 1986). Another study has shown that the proteoglycan perlecan, associated with laminin in the basement membrane can promote neurite outgrowth (Hantaz-Ambroise et al., 1987). Biglycan induces morphological and cytoskeletal changes in fibroblasts. They form long protruding filamentous processes, resulting in an increase in their migration (Tufvesson et al., 2003). In vitro studies using skeletal muscle satellite cells has shown that dermatan sulfate enhances growth factor dependent migration in these cells (Villena et al., 2004). In sea urchin, primary mesenchymal cell migration is inhibited when proteoglycans synthesis is disrupted (Akasaka et al., 1980; Solursh et al., 1986). This could be due to disruption of chondroitin sulfate proteoglycans, which have been implicated in migration of these cells (Lane and Solursh, 1991). Cellular proliferation: Proteoglycans may also be involved in the control of cellular proliferation. Certain motifs on proteoglycans have been implicated in proliferation of various cells. Epidermal growth factor-like motifs in some proteoglycans such as aggrecans may alter the proliferative and metabolic activity of chondrocytes and fibroblasts (Hardingham and Fosang, 1992). Similar functions have been ascribed to glycoproteins such as laminin and tenascin containing these domains (Engel, 1991). In cell culture, versican is expressed when cells are actively proliferating, and once cells reach confluence, versican expression decreases (Zhang et al., 2001). Recent experiments have shown that versican G3 (C-terminal globular domain) 13 expression constructs, placed in cultured bovine intervertebral disc cells, result in a greater cellular proliferation (Yang et al., 2003). Similarly, a mutant G3 construct, lacking the E G F motif, inhibits cell proliferation (Wu et al., 2001). Cell-ECM Interactions: As reviewed above, many of the essential cellular processes are initiated and regulated as a result of presiding interactions between cells and the E C M . The E C M can dynamically regulate and control certain cellular processes such as growth, adhesion, migration, invasion, gene expression, differentiation and apoptosis (Ingber and Folkman, 1989; Juliano and Haskill, 1993; Assoian et al., 1996; Joly et al., 2003; Alfandari et al., 2003; Wadsworth et al., 2004). In turn, these cellular events can regulate certain physiological events such as embryonic development, tissue morphogenesis, and angiogenesis and possibly even pathological processes such as metastasis (Ingber and Folkman, 1989; Yurchenco et al., 1990; Damsky et al., 1992; Juliano and Haskill, 1993; Assoian et al., 1996; Tonnesen et a., 2000; Joly et al., 2003; Dettman et al., 2003). E C M provides a substratum for migration and guidance for cells and a medium on which cells can migrate (Hay, 1991; Adams and Watt, 1993; Lin and Bissell, 1993; Roskelley et al., 1995; Tufvesson et al., 2003). Many E C M components are vital mediators in developmental processes (Grobstein, 1967; Wessels, 1977; Hay, 1981, 1984). Induction and morphogenesis of the corneal epithelium (Toole, 1981; Svoboda et al., 1999; Zhang et al., 1999), epithelial branching of lungs, kidneys and salivary glands (Hardman et al., 1993; Morris et al., 2003; Raitz, 2003; Sakai et al., 2003), induction of chick vertebral cartilage synthesis by notochord (Hay, 1981), are all examples in which E C M elements play a role in development. 14 ECM Receptors: Cells interact with E C M molecules through their E C M receptors. One major family of E C M receptors is the integrins. These receptors were appropriately named integrins by Buck and co-workers (1987), as they integrate intracellular and extracellular scaffolds, allowing them to work together. Integrin proteins have been found to span the cell membrane of most cell types. They are heterodimeric transmembrane molecules that consist of one a and one (3 subunit. The family of human integrins consists of 24 members. Each integrin is a heterodimer composed of 1 of 18 alpha and 1 of 8 beta subunits (reviewed by Berman et al., 2003). The two subunits interact and bind to their ligands in the extracellular matrix. Ligand specificity is based on both heterodimer arrangement and cellular context (Horwitz, 1997). The strength of ligand-integrin interaction is dependant upon receptor clustering and integrin association with accessory molecules (Bazzoni and Hemler, 1998; Liu et al., 2000; van der Flier and Sonnenberg, 2001). Attachment of integrins to E C M molecules activates a variety of signal transduction pathways such as the activation of Ras, M A P kinase, focal adhesion kinase, Src, Rac and Rho, (Miyamoto et al., 1996; Aplin et al., 1998; Schwartz et al., 2000; Ridley, 2000; Eliceiri, 2001; Hood et al., 2003; del Pozo et al., 2004). In turn these pathways induce cellular responses such as migration (Giancotti and Ruoslahti, 1990; Bauer et al., 1992), proliferation (Giancotti and Ruoslahti, 1990; Schreiner et al., 1991; Liu et al., 2004), differentiation (Whittaker et al., 1993; Zhang et al., 2003), tissue specific gene expression and apoptosis (Roskelley et al., 1994; Boudreau et al., 1996; Kheradmand et al., 1998; Wang et al., 1998; reviewed by Stupack et al., 2003). Integrins also respond to signals from within the cells (Inoue et al., 2003). These messages render integrins to become more or less adhesive towards their binding partners in the E C M (Horwitz, 1997). 15 ECM in Development: During development, regulated interactions between E C M molecules and embryonic cells are necessary. The role of E C M is frequently studied by examining where and when its components are produced and secreted, and what morphogenetic changes are occurring nearby both temporally and spatially. Cells synthesize and deposit E C M molecules at very early stages of embryonic development (Eyal-Giladi, 1995; personal observations). This E C M is not static but changes constantly throughout development. Cells secrete new molecules into it and modify the existing E C M molecules (Vafa et al., 1996) thus the extracellular matrix can become differentiated at the level of its molecular composition (Novak et al., 1998; Adelsman et al., 1999). In addition, physical characteristics of E C M can also be changed by the migration of cells through it (Hay, 1995). The distribution of new molecules in the E C M is frequently correlated with regions of morphogenetic change (Busch-Nentwich, 2004). For instance, monoclonal antibodies have identified specific E C M components that are localized to the stomodeum of the gastrula stage sea urchin embryos (Wessel et al., 1984). A polyclonal antibody against an E C M molecule identified as E C M 3, is deposited through the basement membrane except at the site of stomodeum formation (Wessel et al., 1995). Yet another E C M molecule, E C M 1, is selectively accumulated around the invaginating archenteron and later in development concentrates at the site of stomodeum formation (Ingersoll et al., 1994). These new E C M molecules may have a direct role in the changes that appear in the cellular organizations. In sea urchins, experiments in which, tunicamycin and (3-D-xylosides have been used to block the synthesis of GPs (Heifetz and Lennarz, 1979) and GAGs (Benson et al., 1990) respectively, blocked embryonic differentiation and morphogenesis. Similarly, inhibiting the biosynthesis and processing of collagens also blocks differentiation of embryonic cells (Butler et al., 1987). In addition, cells cultured on a Matrigel E C M , express significantly more of the 16 endoderm-specific marker (Endo-1), than cells cultured on a non-ECM substrata (Chen and Wessel, 1996), suggesting that E C M is involved in the differentiation of endoderm. Echinoderm embryos as a model system: It is difficult to find a simple model system in higher organisms, with which to approach the complex problem of morphogenesis. This has been overcome to a certain extent by the use of cell and tissue culture techniques. These techniques provide a simple and reproducible in vitro system to study cellular interactions (Lee et al., 2001). Cells can be grown on gels that mimic part or all of the E C M . As powerful as these techniques are, often, cells in culture do not respond and behave identically to cells in vivo. This is mainly because, not all the components of the E C M , or signaling molecules are present in the artificial environment. Morphological changes caused by cell-cell and cell-ECM interactions can also be studied using living systems (in vivo). Several tunicates, annelids, and nematodes have been used to investigate cell-cell communications during morphogenesis (Miya et al., 1997). These provide a simplified system for in vivo studies of development. In addition, these and other invertebrate embryos are also easily maintained at a minimal cost and can be grown in large numbers in synchronous cultures. Starfish embryos and the early larvae offer several added advantages, which when combined, make them a desirable system to study embryology. Their simple morphology during development consists of a layer of ectoderm cells, which are separated from the endoderm cells by the blastocoel. The blastocoel consists of an extensive clear gel-like E C M and in older embryos also contains mesenchyme cells, which are thought to organize and secrete the matrix (Abed and Crawford, 1986; Strathmann, 1989; Crawford, 1990). The blastocoel contains many alcianophilic fibers (Crawford, 1989, 1990), which are similar to those found in vertebrates and sea urchin (Endo and Noda, 1977; Katow and Solursh, 1979; Kawabe et al., 1981), however, unlike sea urchin embryos, starfish embryos do not form calcium carbonate 17 spicules, which makes them more appropriate for electron microscopy studies. Starfish embryos are also transparent, which makes visualization of cell movement and migration during growth, possible. In addition due to their transparent nature, the embryos/larvae can be raised in large synchronous cultures, which makes biochemical and molecular studies using this system much easier. These qualities make it an excellent organism for studying the role of E C M during early embryonic development. Several techniques have been used to study the role of E C M in echinoderm development. One such method is the injection of macromolecules such as enzymes and antibodies into the blastocoel of embryos (Burke and Tamboline, 1990; Ingresoll and Ettensohn, 1994). However, this is very tedious and only a limited number of embryos can be tested at any one point (Kaneko et al., 1995). It was later observed that if echinoderm embryos were raised in a hypertonic seawater environment, macromolecules could passively enter the blastocoel through intercellular junctions (Dan-Sohkawa et al., 1995). The negative aspect of this method is that the molecules that change the osmolarity of the seawater and make it hypertonic could potentially cause side effects to the embryonic development. In search of a better technique to study the starfish embryonic E C M , Kaneko et al. (1995) observed that incubating the embryos for 15 minutes in Ca + 2 free seawater opens the ectodermal and endodermal septate junctions and allows for macromolecules and enzymes to enter the blastocoel. However, this technique jeopardizes the integrity of both ectoderm and endoderm cells, and embryos cannot be incubated in this solution for more than 15 minutes. A technique developed in this laboratory (Reimer, 1994) in which starfish embryos are incubated in normal seawater containing the experimental macromolecules has shown that at least some of these macromolecules are taken up by the intact embryo. This technique is advantageous since it allows for a large number of embryos to be treated homogenously and allows studying the role of different E C M components in development of starfish embryos. 18 Starfish development: Pisaster ochraceus oocytes are 150-160 \xm in diameter and are pale orange in color (Strathmann, 1987) (Fig. 1). They are surrounded by a jelly coat (Schroeder et al., 1979) and a vitelline membrane (Schroeder, 1981). The latter, lies immediately adjacent to the plasmalemma in an unfertilized egg, and forms part of the fertilization membrane. Once fertilized, starfish eggs undergo many cleavage stages to form a blastula, a hollow ball of cells, which hatches out of the fertilization membrane at about 40-45 h at 12°C. The hyaline layer, an E C M layer that surrounds the later embryonic and larval stages, also starts to form at fertilization, but its formation is not completed until embryos are hatched (Pang et al., 2002). In sea urchin, a related echinoderm species, some of the vegetal cells forming the blastula begin to thicken and flatten. They form contracting, long processes called pseudopods (Gustafson and Wolpert, 1999) and begin to dissociate from the remaining vegetal cells to form the larval skeleton. These cells are called the primary mesenchyme cells. Starfish only have one type of mesenchyme cells. In starfish, the vegetal cells continue to invaginate into the blastocoel as a sheet and form a blind-ended tube, which is known as the archenteron (Fig. 1). The hyaline layer, which is the external E C M surrounding the embryo, is also pulled into the archenteron along with these cells. At this point, the external hyaline layer and an equivalent layer on the surface of the archenteron cells are both morphologically (Crawford and Abed, 1986) and immunologically (Crawford et al., 1997) indistinguishable. However, by the time the embryo has reached the late gastrula stage, antibodies that react with the hyaline layer covering the ectoderm cells no longer react with the E C M layer covering the Gl tract (Pang et al., 2003). The archenteron elongates in the blastocoel and its tip becomes expanded. At around 3 days post fertilization, some of the endodermal cells at the tip of the archenteron undergo an epithelial-mesenchymal 19 transformation. These cells lose their adhesion with adjacent cells and to the underlying hyaline layer and migrate into the blastocoel as mesenchymal cells. A similar epithelial-mesenchymal transformation takes place in sea urchin where the cells are referred to as secondary mesenchyme cells. It has been suggested that in starfish the mesenchyme cells are involved in reorganization of the E C M in the blastocoel and along with ectodermal and endodermal cells they secrete many components of this E C M (Crawford and Abed, 1986; Reimer, 1994). They may also be involved in coelom and mouth formation (Crawford and Chia, 1978; Crawford and Abed, 1983, 1986). Mesenchymal cells ultimately form a variety of structures such as the neurons and muscle cells in the larval stages. Shortly after epithelial-mesenchymal transformation, a blister of basement membrane is formed at the tip of the archenteron (Abed and Crawford, 1986). This appears to be guided across the blastocoel by mesenchyme cells and fuses with the basement membrane of the ectoderm of the presumptive stomodeum (Crawford and Abed, 1983; Abed and Crawford, 1986). This newly formed tube of basement membrane then acts as a guide for the migratory endodermal and ectodermal cells of the stomodeum. Around this time (5-6 days at 12°C), the lateral tip of the archenteron also forms two out pockets which give rise to the coeloms. The mouth forms between 5 and 6 days post-fertilization, at this time the larva has reached the bipinnaria stage. Once the mouth is formed, the archenteron becomes regionalized into a short esophagus, a round stomach and an intestine. At this stage the larvae appears capable of feeding. By 7 days post-fertilization, the anal opening moves from the posterior end to a more ventral position. By now, the coeloms have detached from the gut, assumed a more dorsal position and remain suspended in the blastocoel. In addition the left coelomic vesicle sends out a canal near the dorsal side, at the border of esophagus and stomach and communicates with the outside through a pore. The larva eventually grows arms becoming a brachiolaria larva. It can now swim and feed on plankton for up to 3-4 months (Strathmann, 1987). At the end of 20 this period, the larva becomes fixed to a suitable substratum and undergoes metamorphosis to form a juvenile starfish. Fig.l. Starfish embryonic development (A) A photograph of an unfertilized egg. It can be differentiated from the zygote by the presence of a large, germinal vesicle (nucleus) and by the lack of a fertilization membrane. n= nucleus; Bar=50 um. (B) A photograph of a zygote (fertilized egg). It is recognized by the presence of the fertilization membrane (fm) surrounding it. Bar=50 um. (C) A photograph of cleavage stage embryos. They are characterized by a solid mass of blastomeres that forms when the zygote cleaves. Bar=50 um. (D) A photograph of a late blastula. It is characterized by a single layer of cells surrounding the central hollow area - the blastocoel (bl). The blastomeres are seen to be smaller and are individually not as obvious. Bar=50 um. (E) A photograph of an early gastrula. Some of the cells from the surface of the embryo move to the interior and form and archenteron (ar). They replicate, and form new layers of cells. Bar=50 um. (F) A photograph of a mid to late gastrula. The archenteron elongates further into the blastocoel and mesenchymal cells (arrows) migrate away from the tip of archenteron (ar) into the blastocoel (bl). Bar=50 um. (G) A photograph of a seven-day-old bipinnaria. The gastrointestinal tract is formed and the esophagus (es), the stomach (s) as well as the intestine (in) are well defined. Bar=100 urn. (H) A photograph of an early brachiolaria. The arms are starting to form (arrows). Bar=120 urn. (I) A photograph of adult starfish. Bar= 0.5 cm (Photographs courtesy of Dr. B.J. Crawford laboratory collection). 22 Echinoderm ECM during development: The E C M in planktotrophic echinoderm larvae consists of four major structures. Two that are located outside the embryos; the hyaline layer (HL) which covers the ectodermal cells and surrounds the embryos (Vacquier et al., 1968) and an extension of this layer which lines the GI tract and it is similar to the hyaline layer, but in Pisaster ochraceus appears to be different immunologically (Pang et al., 2002). The latter is called hyaline like layer (HLL) E C M . The basement membrane lines the basal surfaces of the ectoderm and endoderm and a gel-like E C M that fills the blastocoel and the larval body cavity are found within the embryo/larvae (Crawford et al., 1997). Hyaline layer: The outer surfaces of the ectodermal cells of various marine embryos are covered with a layer of E C M (Spiegel et al., 1989; Campbell et al., 1991; Cerra, 1999). In echinoderm embryos/larvae this is a clear jelly-like layer, which is referred to as the hyaline layer. The hyaline layer of the sea urchin is thought to contain various components that are similar or identical to vertebrate E C M components including collagen, fibronectin and laminin (Spiegel et al., 1979; Spiegel et al., 1983). Upon fertilization, many of the components stored in the cortical granules of the unfertilized egg are secreted into the perivitelline space elevating the fertilization membrane and are assembled to form the hyaline layer (and the basement membrane) (Kane and Hersh, 1959; Endo, 1961; Runnstrom, 1966; Anderson, 1968; Holland, 1979, 1980; Hylander and Summers, 1982; Mayne and Robinson, 2002). More recently, immunofluorescent studies using antibodies against sea urchin hyaline layer and basement membrane components identified five storage compartments in the egg: cortical granules, basement membrane vesicles, apical 23 vesicles, echinonectin vesicles and a fifth compartment for the maternal cadherin (Matese et al., 1997). The release of these compartments onto their appropriate destination is highly regulated. Cortical granules and basement membrane vesicles exocytose within 30 seconds post-fertilization whereas the apical vesicles release their contents within 5 minutes after fertilization. Echinonectin and cadherin are seen on the cell surface 15 minutes and 30 minutes post-fertilization, respectively (Matese et al., 1997). Components: Morphological and immunocytochemical studies, indicate that the sea urchin hyaline layer is composed of a variety of macromolecules (Spiegel et al., 1989; Reimer and Crawford, 1990). In echinoids the hyaline layer has been shown to contain the glycoprotein hyalin (Alliegro and McClay, 1988; Stephens and Kane, 1970; Adelson et al., 1992), as well as collagen, fibronectin, laminin and proteoglycans (Alliegro et al, 1988; Spiegel and Spiegel, 1979; Spiegel et al., 1980; 1989). Results from immunocytochemical studies of echinoids have revealed that hyalin, a 330 kDa glycoprotein, is the major constituent of the echinoid hyaline layer and is potentially responsible for normal morphogenesis and gastrulation (Adelson and Humphreys, 1988). Echinonectin, a putative substrate adhesion molecule, is another macromolecule isolated from the hyaline layer, which may also be responsible for early morphogenetic events (Alliegro et al., 1988; Veno et al., 1990; Alliegro and Alliegro, 1991). Functions: Examination of sea urchin (Morill, J. personal communications) and starfish embryos (Campbell and Crawford, 1991), preserved by freeze substitution, shows that the hyaline layer of 24 both groups is composed of a multilayered E C M . Numerous functions have been proposed for this multilayered structure. The hyaline layer appears to function as a substrate for cell adhesion (Fink and McClay, 1982; Fink and McClay, 1985; Wessel et al., 1998) and is necessary for initiation of primary invagination and morphogenesis (Adelson and Humphreys, 1988; Kimberly and Hardin, 1998). Studies in which sea urchin embryos were incubated in an antibody against a collagenase component present in the hyaline layer resulted in delayed gut formation and spicule elongation (Mayne and Robinson, 2002), suggesting that collagen is present in the hyaline layer and that this layer may play a role in morphogenesis. Furthermore, the hyaline layer may act as a protection against mechanical stress and it may also lubricate the embryo (Lundgren, 1973; Crawford and Abed, 1986). In addition, it could act as a filter for organic molecules and may prevent the entery of macromolecules into the blastocoel (Spiegel et al., 1989) and it has been shown to play a role in the feeding behavior of the larva (Cerra and Byrne, 1995; Cerra et al., 1997). It seems possible that the numerous functions of the hyaline layer may correspond to different regions and sublayers of this complex E C M structure. Structure: Studies by Campbell et al. (1991) and Pang et al. (2002; 2003) demonstrated that when preserved by freeze substitution, the mature hyaline layer of starfish is composed of four major layers. From the outer surface of the ectoderm cells outward these are, the intervillous layer, the supporting layer, a boundary layer, and course outer meshwork (Fig. 2) (Campbell et al., 1991). The intervillous layer is the sub-layer bordering the epithelium (Fig. 2) and as the name suggests, is located between the microvilli and under the T E M it consists of a loose network of fibers. It consists of heterogeneously arranged E C M components and it has been suggested that the material in this layer may take up water and may act like a hydrostatic cushion to maintain the outer sublayers in an expanded state (Campbell et al., 1991). The fibrous meshwork 25 component of the intervillous layer may represent the apical lamina described in the echinoids (Hall and Vacquier, 1982; Burke et al., 1998). Fig. 2. Transmission electron micrograph of a mature hyaline layer The hyaline layer surrounds the embryo. co= coarse outer meshwork; b= boundary layer; IV=intervillous layer; HI, H2, H3 make up the supporting layer. Bar= 0.5 um. Courtesy of Pang et al. (2002) 26 The supporting layer is located near the tops of the microvilli and appears to be very dense (Fig. 2). It is composed of HI, H2 and H3 sub-layers. Morphologically, HI is very dense and compact. This sub-layer has been implicated in providing structural support for the coarse outer meshwork and the boundary layer. Some of the fibrous elements of the boundary layer appear to be embedded in the HI layer suggesting that HI may be the anchoring site for these fibers. Observations by Campbell et al. (1991) and Crawford et al. (1993) have shown that HI sublayer forms a cap of dense material over the tips of the microvilli, which also implies that microvilli may be anchored there. The H2 sub-layer underlies the HI and probably represents a less hydrated form of E C M than the one present in the blastocoel matrix or the one in the coarse outer meshwork (Campbell, 1990). It appears to be the most stable layer and when fixing sea urchin and starfish embryos for T E M using conventional techniques, it is often the only layer preserved (Crawford and Campbell, 1993). It has been suggested that this sub-layer could function as a filter (Spiegel et al., 1989). Sub-layer H3, underlies H2 and is potentially a transition between the supporting layer and the intervillous layer. The H3 sublayer is represented by a series of thin electron dense plaques located between the upper border of the intervillous layer and the H2 layer. Examination of this layer in detail shows that it is very tenuous and is not always present. The boundary layer lies outside of the supporting layer (Fig. 2). T E M observations of this region show that it appears to consist of numerous fibers whose bases are buried in the HI layer and that extend more or less vertically with their terminal regions ending in the fibers that make up the course outer meshwork, suggesting that it may anchor the outer meshwork to the rest of the hyaline layer. It appears to be a repeating series of E C M loops, and provides an increased surface area for attachment (Campbell, 1990). When preserved by freeze substitution method, the coarse outer meshwork is homogenous and consists of a thick meshwork of fibrous material that appears to be interleaved 27 with the apex of the fibers of the boundary layer (Campbell et al., 1991). The thickness and appearance of these fibers is very similar to those found in the boundary layer and indeed may represent fibers that have detached from this layer. It is suggested that the coarse outer meshwork may act as a lubricant to help the animal move through the water (Crawford and Abed, 1986) and may protect the entire embryo, predominantly the inner layers of the hyaline layer, from mechanical perturbation (Lundgren, 1973). Often the multilayered hyaline layer also contains cilia and microvilli. The latter extend from the epithelial cells through to the boundary layer of the hyaline layer. The cilia extend all the way through the hyaline layer and into the surrounding seawater where they are involved in locomotion (Crawford and Campbell, 1993). T E M studies of starfish hyaline layer by Crawford and Campbell (1993) have shown that the each cilium is surrounded by several microvilli. Microvilli are in turn, joined together by the hyaline layer to form a collar at the base of the cilium (Crawford and Campbell, 1993). During hyaline layer organization, apical surfaces of the blastomeres bind firmly to the hyaline layer through microvilli (Katow and Solursh, 1980), which terminate in the boundary layer (Campbell and Crawford, 1991). It has been suggested that starfish embryonic microvilli play a role in phagocytosis (Norrenvang and Wingstrand, 1970) and since they surround the cilia, microvilli may also play a role in guiding the direction and extent of the ciliary stroke (Crawford and Campbell, 1993). Development: Pang et al. (2002) extensively studied the development of the hyaline layer in Pisaster ochraceus embryos (Fig. 3). Development of this multilayered structure begins at fertilization when some materials from the cortical granules are released into the perivitelline space and fuse with the vitelline membrane to help in forming the fertilization membrane while other components appear to become associated with the outer surface of the egg plasma membrane 28 forming plaques of dense material (Fig. 3A) (Pang et al., 2002). Over the course of the next 48 hours the asteroid hyaline layer gradually becomes organized into a series of sublayers. This organization is not random and occurs in a definite sequence. By the time the embryo reaches 18 hours post-fertilization stage, the fertilization membrane appears as a trilaminar structure composed of a thicker inner layer in between two thin electron dense layers. By 20 hours post fertilization, many microvilli are observed on the surfaces of the blastomeres and by 22 hours post fertilization the dense material is associated with the tips of short microvilli located on the outer surface of the blastomeres (Fig. 3B) (Pang et al., 2002). At around 28 hours post fertilization the tips of some of these microvilli start to join together by a single strand of E C M which appears to be the HI sublayer of the supporting layer (Fig. 3C) (Pang et al., 2002). HI sublayer spreads to eventually completely surround the entire embryo (Fig. 3D) (Pang et al., 2002). At this stage, often, alternating thick regions of E C M at the tip of the microvilli, and single stranded E C M extending between the microvilli are observed. The thicker regions of E C M are composed of four regions of the hyaline layer (HI, H2, H3 of the supporting layer and the boundary layer). As this is occurring, components of the boundary layer extend out from the tips of the microvilli and the microvilli appear to elongate. This is accompanied by thickening of the HI sublayer. At this time all sublayers of the supporting layer completely surround the embryo (Fig. 3E) (Pang et al., 2002). By the time the embryo reaches the bipinnaria stage, all the layers of the hyaline layer are thickened and have become more organized (Fig. 3F) (Pang et al., 2002). It appears that the dense material may induce both the organization of the elements of the hyaline layer as well as the formation of the microvilli that support them (Pang et al., 2002). 29 30 Fig. 3. A diagram showing the development of the hyaline layer. Hyaline layer development begins at fertilization with secretion of cortical granules (eg) into the perivitelline space (pv) located between the egg plasmalemma and the fertilization membrane (f). Dense plaques (d) can be seen on the surface of the plasmalemma (A). At the early cleavage stages (B), these dense plaques are associated with the tip of microvilli (mv). At the blastula stage, (C) microvilli have elongated and HI sublayer joins the tips of microvilli together. A developing boundary layer (b) is also associated with some of regions of HI sublayer. By the time embryo hatches (D), the fertilization membrane (f) and the jelly coat (jc) have been lost. The HI sublayer surrounds the embryo, the boundary layer (b) is complete and the intervillous layer (iv) as well as the coarse outer meshwork (co) are organized. At the gastrula stage (E), the three sublayers of the supporting layer (HI, H2, H3) can be seen (E). The layers become more organized and thicken by the time the embryo reaches the bipinnaria stages (F). (Courtesy of Pang et al., 2002). 31 ECM lining the Gl tract: The E C M that lines the Gl tract, the hyaline-like layer (HLL) is an extension of the hyaline layer and although immunologically different, it is morphologically very similar to that surrounding the ectodermal cells. The number and types of sublayers varies from region to region of the Gl tract. H L L in the esophagus is composed of all sublayers of the hyaline layer with the exception of H3 sublayer and the boundary layer. The latter appears to be very rudimentary or absent altogether (Pang et al., 2002). Similarly, the E C M lining the stomach and the intestines is composed of the intervillous layer, and shorts segments of H2 sublayer attached to the tips of microvilli as well as the outer meshwork. Neither the HI nor the H3 sublayers of the hyaline layer, nor the boundary layer are observed in the lining of the stomach and the intestines (Pang et al., 2002). Basement membrane: The echinoid basement membrane is a specialized form of the E C M that acts as a semi-permeable membrane to most macromolecules (Farquhar, 1981). In the gastrulating echinoid, tbasement membrane separates the basal epithelial surfaces and the basal surfaces of the archenteron, and the coelomic pouches from the connective tissue (Endo and Uno, 1960; Wolpert and Mercer, 1963; Okazaki and Niijima, 1964; Gibbons et al., 1969; Leblond and Inoue, 1989). The basement membrane has been suggested to be a loosely-knit network of fibrils and hydrated gel, which better facilitates the diffusion of molecules between the epithelia and the blastocoel than a tightly packed mat of E C M . Biochemically, most basement membranes contain a common set of proteins that include laminin, entactin/nidogen, collagen IV and the heparan sulfate and chondroitin sulfate proteoglycans (Zagris, 2001). The structural 32 arrangement of the basement membrane is not uniform (Zagris, 2001). Early formation of the basement membrane consists of an orderly assembly of its components (Dziadek and Timpl, 1985). For instance, in the mouse embryos, laminin and heparan sulfate proteoglycans are present on the basement membrane at the two-cell stage (Wu et al., 1983; Zagris et al., 2001). Entactin/nidogen are first detected at the morula stage, and collagen IV and fibronectin are first detected at the blastocyst stage (Adamson and Ayers, 1979; Leivo et al., 1980). It has been shown that the sea urchin basement membrane also contains laminin (Spiegel et al., 1983), fibronectin (DeSimone et al., 1985) and collagen (Suzuki et al., 1997). In addition Pamlin, a sea urchin-specific E C M molecule in the ectodermal basement membrane has been described to promote cell migration in Hemicentrotus pulcherrimus (Katow, 1995) and P1-200K, an adhesive protein in the basement membrane of Paracetrotus lividus appears to play a role in skeletogenesis (Tesoro et al., 1998). Ultrastructural observations of the basement membrane of starfish embryos that have been prepared by conventional techniques show that it consists of lamina lucida and lamina densa (Crawford, 1989). Ultrastructural studies of isolated basement membranes (Crawford, 1989) and immunocytochemical studies with lectins demonstrated that the thickness and the amount of certain carbohydrate groups varied in different regions of the larval basement membranes (Reimer et al., 1992). It was suggested that these differences could guide morphogenetic events. Reimer et al. (1992) have suggested that thinning in the basement membrane of the esophageal endoderm and the mesenchymal cells of the starfish embryos may be determining where muscle differentiation is to occur. Basement membrane may also act to provide a path for presumptive esophageal muscle cells to migrate on and it also may determine the shape and position of the esophagus (Crawford and Abed, 1983). In addition, as described earlier, the basement membrane has been implicated in the formation of the mouth in starfish P. ochraceus (Crawford and Abed, 1983). 33 Blastocoel: In the starfish Pisaster ochraceus, the largest amount of E C M is found in the blastocoel. Starfish E C M occupies the entire cavity of the blastula and later of the gastrula and the bipinnaria larva (Crawford and Abed, 1986). E C M of the blastocoel appears to be initially secreted by ectoderm and endoderm cells (Crawford et al., 1997). Later when the mesenchyme cells migrate into the blastocoel, they also contribute to the secretion of the E C M in the blastocoel (Reimer and Crawford, 1997). The starfish and echinoid blastocoel cavity contains amorphous E C M gel and a web of fibers (Crawford and Chia, 1978; Abed and Crawford, 1986; Crawford, 1989; Cherr et al., 1992; Crawford et al., 1997). The fibers form dense webs beneath the ectoderm, especially on the dorsal surface, they also radiate from the esophagus to the inner portion of the surrounding ectoderm (Crawford, 1990). These fibers may be involved in the migration of the mesenchyme cells in addition to maintaining the constriction at the middle of the embryo. It has been hypothesized that the gel permits the embryo to take on various shapes (Strathmann, 1987; Crawford et al., 1990). These shapes ultimately facilitate feeding by positioning the ciliary bands in optimal locations for food retrieval (Strathmann, 1987). In addition to this, the viscosity of the gel makes development of the opposing muscles unnecessary (Strathmann, 1987; Crawford, 1990). This latter point is important, because in asteroids migrating mesenchymal cells wrap themselves around the esophagus, to form a continuous muscular tube in order to constrict the lumen (Crawford, 1990), however, there is no corresponding musculature to open the lumen. Once the esophagus constricts, esophageal fibers attached to it and to the ectoderm, draw in the ectoderm and distort the gel. During relaxation, the gel allows for rebounding of the ectoderm, and the esophageal fibers, pulls at the esophagus 34 and the esophageal musculature causing the lumen to dilate. (Strathmann, 1987; Crawford, 1990). It has been suggested that mesenchymal cells, have the ability to dissolve and reform the gel. This allows for a greater freedom of cellular distribution and therefore a wider spectrum for creating shapes (Strathmann, 1989). In the asteroid embryos, mesenchymal cells appear'to use fibrous E C M elements as guide-wires to travel along the blastocoel (Crawford and Chia, 1982). The mesenchymal cells, that do not form muscle cells, migrate along these fibers and dissolve and re-secrete both the surrounding gel and the fibers (Crawford and Abed, 1986). Antibodies against different components of the starfish P. ochraceus blastocoel have shown that it contains a variety of large macromolecules. Some of these molecules begin to be synthesized as early as in the unfertilized egg stage but are not released into the blastocoel until the blastula and the later stages of development. PM-1, a proteoglycan-like G A G is first observed in the endodermal cells of the early gastrula. It is secreted into the blastocoel and at the late gastrula stage is present in the lumen of the GI tract (Reimer and Crawford, 1997). PM-1 has been suggested to play a role in the development and differentiation of the GI tract (Reimer and Crawford, 1997). Another E C M component of the starfish blastocoel PM-2 has also been previously shown to stain the blastocoel, part of the E C M lining the gut as well as a portion of the hyaline layer (Crawford et al., 1997). HL-1 is a major component of the hyaline layer, however, Crawford et al. (1997) have shown that it also reacts with some material that lines the early GI tract as well as (to a lesser extent) with the blastocoel matrix. Many of the components of the blastocoel show similar staining pattern. In the unfertilized eggs, a third monoclonal antibody PC3H2 is present in the cortical granules as well as the cytoplasmic granules (Reimer and Crawford, 1995). Upon fertilization, PC3H2 is secreted into the perivitelline space and at the blastula stage it can be seen in the blastocoel (Reimer and Crawford, 1995). The early staining pattern of PC3H2 is very similar to PM-2. The latter 35 however shows stronger staining pattern in the blastocoel of the gastrula and the bipinnaria embryos suggesting that the two antigens are different. In the developing echinoids, the largest amount of E C M is also found in the blastocoel. The fibrous E C M components found in the blastocoel are synthesized during gastrulation and are confined by the walls of the embryonic cavity (Endo and Noda, 1977; Katow and Solursh, 1979; Galileo and Morrill, 1985; Morrill and Santos, 1985; Berg et al., 1996; Ingersoll and Ettensohn, 1994). Spaces between the fibers, in the echinoid E C M , are composed of several macromolecules including GAGs (Karp and Solursh, 1974; Solursh and Katow, 1982; Kariya et al., 1997; Ameye et al., 2001), proteoglycans (Oguri and Yamagata, 1978; Reimer and Crawford, 1997; Tomita et al., 2000; Tomita et al., 2002), collagen (Pucci-Minafra et al., 1972; Golob et al., 1974; Crise-Benson and Benson, 1979; Wessel and McClay, 1987; Beson et al. 1990; Wessel et al., 1991; Seid et al., 1997), fibronectin and laminin (Spiegel et al., 1980, 1983; Katow et al., 1982; Wessel et al, 1984; McCarthy and Burger, 1987; Katow et al., 1990). These components together form a gel-like material. 36 Rationale and thesis objectives: Although first thought to be relatively unimportant in development, components of the E C M have recently proven to be intimately involved in many morphogenic events. Many of the studies that demonstrated the roles of E C M in development were carried out by the use of inhibitors of post-translational modifications of proteins, such as inhibitors of glycoprotein and proteoglycan synthesis, G A G chain sulfation and collagen cross linking (Solursh et al., 1986; Wessel and McClay, 1987; Thurmond et al., 1997; Clayton et al., 2001; Khatri, 2003). These studies although informative on the role of E C M in development, can be difficult to interpret mainly because the inhibitors are not completely specific and they can affect different developmental events. Antibodies are more specific with respect to studying the function of molecular determinants. The use of antibodies to block the function of specific E C M components has been carried out in both vertebrate and invertebrate embryos. However so far, very few experiments have been carried out in starfish using antibodies against E C M components. In this thesis monoclonal antibodies developed in mice against different E C M components in starfish embryos by Campbell and Crawford (1991) and Reimer and Crawford (1995) have been used to study two E C M components. One such antibody is anti-HL-1, which reacts against an epitope present in all sublayers of the hyaline layer except the H2 sublayer of the supporting layer. A second antibody, anti-PM-2, reacts against an epitope in the HI sublayer of the supporting layer as well as an epitope in the blastocoel and the lumen of the Gl tract. The overall objective of this study was to understand the role of E C M in the development of P. ochraceus embryos and to gain a greater understanding of the role of E C M in development in general. The main objective was to determine the role of two E C M components, HL-1 and PM-2, in the development of starfish. To achieve this, the second objective was to determine the location and pattern of the synthesis of the antigens during development. Fluorescent microscopy 37 and colloidal gold-labeled immunoelectron microscopy was used on material prepared by freeze substitution. The results were then compared with significant events during morphogenesis to determine how the synthesis and secretion of these molecules might correlate with these events. The third objective was to learn about the biochemical nature of the epitope. To accomplish this, immunoblotting, electrophoresis and enzymatic digestion on the HL-1 and the PM-2 epitopes were carried out. The fourth objective was to gain a more detailed knowledge of the roles that HL-1 and PM-2 epitopes may play in development. In order to do so, the HL-1 and the PM-2 antibodies were used as blocking agents and embryos/larvae of various stages were incubated in seawater containing the purified antibodies for different periods of time. The results were analyzed at both the light and the T E M levels in an attempt to further define the roles of the E C M molecules in the hyaline layer and the blastocoel regions of the embryos/larvae. The final objective was to learn about the mechanism by which embryos exposed to the PM-2 antibody transferred this antibody into the blastocoel. To achieve this immunofluorescence and immunogold electron microscopy was used to follow the path that the antibody has taken to enter the blastocoel. Insights gained from studying HL-1 and PM-2 components of the extracellular matrix and their interactions with cells during early development of starfish embryos should be helpful in understanding starfish development and may also help to define the role of different E C M components in other developmental systems. 38 Chapter 2- Materials and Methods: Rearing of Pisaster ochraceus embryos: Adult starfish (Pisaster ochraceus) were collected from Stanley Park, Vancouver, British Columbia, Canada and kept in seawater tanks at 12°C in the Department of Anatomy and Cell Biology, University of British Columbia for several months. They were fed either fish or mussels. When gametes were required, the gonads were dissected out (Fuseler, 1973) and the ovaries were rinsed once in filtered seawater. The ovaries were then incubated in filtered seawater containing 1 u,g/ml of 1-methyl-adenine (Stevens, 1970) until most of the eggs had lost their germinal vesicles (nuclei), indicating that the oocytes had reached full maturity. The eggs were then washed in freshly aerated seawater and allowed to settle through 300 ml of fresh seawater in a one liter plastic beaker. Testes were removed as above and stored in a dry, covered petri dish at 4°C. One to two drops of "dry undiluted sperm" were diluted in approximately 5 ml seawater to give a slightly cloudy suspension. After a few minutes the sperm were checked for motility by microscopic observation. Sufficient eggs to cover 50% of the surface area of the bottom of a one liter plastic beaker were transferred from the washing beaker into a second one liter beaker containing 300-400 mis of aerated sea water and were fertilized with 0.5-1.0 ml dilute sperm solution. Once the fertilization was completed the embryos were allowed to develop at 12°C. Cultures were maintained at sufficient density to cover 50% of the bottom of the culture dishes with a monolayer of eggs. Embryos at various developmental stages were harvested by gentle centifugation (125 x g for 5 minutes on a clinical centrifuge), the majority of seawater was removed and the embryos were either stored at -70°C for use in biochemical studies, or fixed by freeze-substitution (see below) for immunohistochemical or immunocytochemical studies. 39 Fixation and embedding of embryos: Cryoprotection: Embryos that were to be preserved by the freeze substitution method (see below) were often cryoprotected prior to freezing in order to prevent damage from ice crystal formation. To accomplish this they were cryoprotected with 15% 2-3 butanediol in seawater. Embryos of different stages were centrifuged (125 x g for 3 minutes), excess water was removed and they were incubated in the cryoprotectant for 20-30 minutes prior to freezing. Freeze substitution: In order to retain good tissue morphology and immunogenicity, embryos/larvae were fixed by freeze substitution according to the method of Campbell et al. (1991). Uncryoprotected or cryoprotected embryos were allowed to settle in conical glass centrifuge tubes at 0-4°C for 5-10 minutes. The majority of the supernatant was removed. One microliter of packed embryos was placed on freeze fracture copper grids using a 20 ul Pipettman. The embryos were spread out to form a monolayer and the residual liquid was removed with a triangle of filter paper. Following this, the grid was quickly plunged into liquid propane that had been cooled in liquid nitrogen to just above the freezing point (-192°C). The frozen embryos were then transferred to liquid nitrogen for about a minute. Following this, the grids were either placed in a 1 ml cryogenic freezer vial and stored in liquid nitrogen for further use or, they were placed in absolute ethanol at - 8 5 ° C to begin the substitution process. The embryos were maintained in 100% ethanol at - 8 5 ° C for 5 days. During this time the water ice was gradually replaced by absolute ethanol. Subsequently, they were placed at -20°C for approximately 4 hours, then at 4°C for another 4 hours and finally they were placed at room temperature for further 4 hours 40 before being embedded in a plastic resin. Two resins were used: either JB4 (Polysciences) or LR White (JBS). Embedding was as per manufacturers instructions. Material embedded in JB4 was sectioned at 1-1.5 |xm and stained for immunofluorescence as described below. Material in LR White was sectioned at 50-60 rjm for T E M on a Porter Blum MK1 microtome fitted with a diamond knife. The sections were stained using the colloidal gold technique followed by uranyl acetate and lead citrate and examined and photographed on a Philips 300 T E M . Chemical fixation: Packed embryos were immersed in 1% gluteraldehyde in 80% seawater (pH 7.0), saturated with alcian blue, at room temperature for 4 hours (Behnke and Zelander, 1970). Following a rinse in 1.25% NaHC0 3 buffer (pH 7.2) the embryos were post fixed in 2% Os0 4 (pH 7.4) in the same buffer for 1 hour. Embryos were then washed with buffer and stained en bloc with 2% uranyl acetate for 45 minutes, washed in distilled water, dehydrated with increasing concentrations of ethanol followed by two changes of 100% propylene oxide and embedded in Epon 812 (Luft, 1961). Monoclonal antibody production: Culture of hybridoma cells: Hybridoma cells developed by Dr. B. Crawford, Dr. S. Campbell and Dr. C. Reimer, were stored in 1 ml cryovials under liquid nitrogen until required (Reimer and Crawford, 1995). One vial of hybridoma cells that secreted the required antibody was removed from the liquid nitrogen, thawed in a 37°C water bath, transferred to a 50 ml conical plastic centrifuge tube and centrifuged for 10 minutes at 800 g. The supernatant was removed and the cells were resuspended in 10 mis of D M E M (Dulbecco's modified Eagle media) without fetal calf serum 41 (FCS) and centrifuged as above. The cells were then resuspended in 1 ml of D M E M with 20% FCS and transferred to 10 ml plastic petri plates containing 10 ml D M E M supplemented with 20% FCS and the plates were transferred to a humidified 37°C incubator gassed with 5% C 0 2 in air. Cells were allowed to grow undisturbed until they covered approximately 50-70% of the surface of the petri plate. Once this had occurred, they were split into more plates. To achieve this the plates were swirled to suspend the cells and half of the cells and medium were transferred to a new petri plate. Each plate was then supplied with 10% FCS and 90% D M E M . They were allowed once again to grow undisturbed in the 37°C incubator until they were ready to be split again. To harvest the medium, 90% of the liquid including many of the cells, was removed. The medium was centrifuged at 800 x g for 10 minutes. The supernatant was removed and stored at -20°C. The remaining cells in the plates were fed D M E M with 5 or 10 % FCS, and were allowed to re-grow. The supernatant harvest was repeated until approximately 1 L of conditioned medium had been collected for each of the two monoclonal antibodies involved. Ascites production: The PM-1 and PM-2 hybridomas were grown in culture to densities of roughly 1 x 106 cells per ml. For ascites production, the cells were collected by centrifugation, washed in sterile D M E M without FCS and resuspended in 1ml of sterile D M E M . Ascites tumors were induced in pristane-primed BALB/c mice by intraperitoneal injection of 0.5-1.0 ml of D M E M containing 5-10 x 106 hybridoma cells. The mice were examined twice each day to assess them for abdominal distention. Once the abdomens were distended but before the mice showed signs of respiratory distress (approximately 7-10 days post injection), mice were put under mild sedation with Halothane and ascites fluid was drained from their peritoneal cavities using 20 gauge sterile 42 needles. The mice were then euthanized by over anesthetizing them with halothane. The ascites fluid was clarified by centrifugation (10 min. x 10000 g), and defatted as follows: To 5 ml ascites fluid, 5 ml VBS (veronal buffered saline; 4 mM barbitone, 0.15 M NaCl, 0.8 mM Mg + 2 , 0.3 mM Ca + 2), pH 7.2, were added, along with 150 mg silicon dioxide powder. The mixture was incubated at room temperature for 30 minutes with occasional shaking, and then centrifuged 2000 x g for 20 minutes (Neoh et al., 1986). The supernatant was removed and stored at - 2 0 ° C in 1.5 ml conical polypropylene centrifuge tubes. Purification of PM-2 and HL-1 antibodies: Purification of HL-1 (IgM) and PM-2 (IgG) antibodies was accomplished using HiTrap IgM and IgG purification kits. The chromatography columns were mounted on a Pharmacia UV-1 detector that was attached to a pen chart recorder. HiTrap IgM purification kits were used to purify The HL-1 antibody. Hi Trap IgM kit is a thiophilic medium composed of 2-mercaptopyridine coupled to Sepharose High Performance (Porath et al. 1985). Thiophilic interactions are based on electron donating and accepting action of the ligand (Hutchens and Porath, 1987) or hydrophilic and hydrophobic interactions between the ligand and the protein (Finger et al., 1996). To achieve this, HL-1 antibody was adjusted in (NH4) 2S0 4 to a final concentration of 0.8 M and filtered through a 0.45 u.m filter before being applied to the column. The column was then washed with 5 ml volumes of each buffer A, B, and C supplied by Amersham Pharmacia Biotech. Buffer A consisted of 20 mM NaP0 4, 0.8 M (NH4) 2S0 4, pH 7.5. Buffer B consisted of 20 mM Na 3P0 4 , pH 7.5 and buffer C consisted of 20 mM NaP0 4, pH 7.5 with 30% isopropanol. The column was then equilibrated with 5 ml buffer A. The sample was applied to the column, and then washed with 15 ml of buffer A until no material appeared in the effluent. HL-1 antibody was then eluted with 15 ml buffer B. The column was regenerated with 43 10 ml buffer C and finally re-equilibrated with 5 ml buffer A. The column was stored in 20% ethanol at 4°C. Purification of PM-2 was performed using columns, which were filled with Protein G Sepharose High Performance (Hi Trap Protein G Columns). These columns are designed for purification of monoclonal and polyclonal IgG from a variety of sources. Protein G which is a cell surface protein of group G streptococci acts by binding to the Fc region of IgG. This interaction occurs over a wide range of pH, however the affinity between Protein G and IgG is strongest at pH 7.0. Elution of the IgG is achieved by lowering the pH of the sample to about 2.5-3.0 (Amersham Pharmacia Biotech). Water and chemicals used in the buffer preparation were passed through a 0.45 \im filter before use. Collection tubes were prepared by adding 100 u.1 of neutralizing buffer (1 M Tris-HCl pH 9.0) per ml of fraction to be collected. A 10 ml syringe was filled with binding buffer (20 mM sodium phosphate, pH 7.0) and connected to the column. This solution was passed through the column drop by drop (approximately at a rate of 1 ml/min) to avoid introducing air into the column. Following this, 1 ml of PM-2 antibody sample was applied to the column using a syringe. The column was then washed with 10 ml of binding buffer until no material appeared in the effluent; 5 ml of elution buffer (0.1 M Glycine-HCl, pH 2.5) were then applied to the column and the eluted material was stored in neutralizing buffer. This process was repeated several times, and all the eluted material was pooled and concentrated using Centricon 50 centrifuge tubes. The concentrated antibodies were stored at - 8 0 ° C . The column was then washed with 10 ml of binding buffer and rinsed and stored with 20% ethanol at 4°C to prevent microbial growth. 44 Immunohistochemistry: Immunofluorescence staining: To localize the antigen during normal development, thin sections (1-1.5 u.m) of JB4 or LRWhite (LRW) embedded, freeze substituted embryos were cut on a Porter-Blum MT-1 ultra-microtome, using a dry glass knife. Each section was placed on a glass slide, covered by a drop of distilled water and warmed on a hot plate to evaporate the water and ensure good adherence of the section to the slide. To ensure blocking of non-specific binding sites, each section was incubated in PBS containing 0.5% BSA (bovine serum albumin) or with 2% PBS/blotto (phosphate buffered saline [PBS], pH 7.4, containing 2% Lucerne skim milk powder and 0.1% NaN 3 [Sigma] for 30 min. Then, a drop of undiluted hybridoma supernatant was applied to the sections for one hour. Following this, the sections were washed twice with PBS, incubated for one hour in fluorescein isothiocyanate (FITC)-conjugated secondary antibody that was prepared in rabbit or goat against mouse IgG or IgM. The section was then washed in PBS and mounted with 16% gelvatol, 0.4% D A B C O (1,4 diazabicyclo [2,2,2] octane; Aldrich) 30% glycerol in PBS, pH 7.2 (Taylor and Heimer, 1974), and photographed with Kodak T M A X 3200 or T M A X 400 on a Zeiss Axiophot Photomicroscope equipped with epifluorescence optics. Control sections were stained as above with normal mouse IgG and IgM replacing the primary antibody. All incubations were carried out in a moist chamber. Preparation of colloidal gold antibody (Slot and Geuze, 1985): Colloidal gold solutions: Solution 1 (chloroauric acid) consisting of 1% HAuCl 4 in distilled water, and solution 2 (citric acid reducing solution) consisting of 1% tri-sodium citrate, 1% tannic acid in distilled water were prepared as follows: Both solutions 1 and 2 were heated to 60°C, in a water bath. 45 Solution 2 was quickly added to solution 1. The resulting mixture was maintained at 60°C with continuous stirring until the solution turned red. Once this had occurred the solution was removed from the heat and allowed to cool to room temperature. The final colloidal gold solution was stored at 4°C. Microtitration assay for determination of the correct protein concentration for gold solution stabilization: One mg/ml of protein to be assayed was dialyzed in 2 mM sodium borate pH 9.0. Following this, 100 u.1 of distilled water was added to ten, 1.5 ml Eppendorf tubes, 100 ul of the protein were then added to the first tube and serial dilutions were carried out for the rest of the tubes. The 10th tube was left protein free. The pH of the colloidal gold was adjusted to the pi of the protein being assayed using 0.2 M K 2 C 0 3 , 500 u.1 of colloidal gold were added to each tube and the tubes were stirred and allowed to stand for 15 minutes. One hundred u.1 of 10% NaCl were then added to each tube to assess the resistance of the mixture to salt-induced flocculations. The tubes were examined for a color change from red to blue. The last tube to maintain a red color represented the end point for protein-gold stabilization. To ensure proper stabilization of the protein-gold conjugates the protein stabilization concentration of the end-point tube was calculated and doubled for experimental stabilizing concentration. Conjugation of gold solution to IgG I IgM (Hodges et al, 1984): One hundred ul of stock goat anti-rabbit IgG or IgM were diluted to 500 ul in 2 mM sodium borate-HCl, pH 9.0 and dialyzed against borate buffer overnight. About 0.5 ml of dialyzed protein was added to 10 ml stirring gold solution (pH adjusted with 0.2 M K 2 C 0 3 to 9.0) and stirring was continued for 30 minutes. To the stirring gold solution 0.5 ml 10% BSA 46 was added and stirring was continued for another 5 minutes. Once the conjugation was complete the colloidal gold conjugate was washed. To achieve this, the conjugated gold solution was centrifuged at 15000 x g for 45 minutes and resuspended in 10 mM Tris with 50 mM NaCl, 0.1% BSA (pH 8.2). Following this, the colloidal gold solution was dialyzed against an increasing concentration of NaCl (up to 150 mM) for 24 hrs. To inhibit bacterial growth 0.02% sodium azide was added and the solution was stored at 4°C. Immunogold Staining: Ultrathin sections (50-60 r\m) as determined by silver-gold color of LRW embedded material was cut using a Diatome diamond knife on a Porter-Blum MT-1 ultra microtome. They were picked up on 400 mesh nickel grids (Marivac) and stained as follows: Each grid was floated on a drop of the pre-incubation buffer consisting of 10% normal serum of the secondary host (either goat or rabbit serum) in PBS/blotto for one hour. This was followed by 90 minutes incubation in undiluted hybridoma supernatant. The grids were then washed 2 times over 30 minutes with filtered PBS/blotto, and floated on anti mouse IgG or IgM colloidal gold conjugate prepared as above. The grids were washed in distilled water and stained with saturated aqueous uranyl acetate for 10 min, followed by lead citrate for another 10 minutes (Reynolds, 1963). Electron microscopy was performed on a Philips 300 or 301 T E M . Control grids were stained as above with normal mouse IgG replacing the primary antibody. All PBS/blotto used in this protocol was filtered prior to use with a Millipore Millex 0.45 um filter attached to a Becton-Dickinson 5 ml syringe (Fisher). To localize the primary antibody in embryos that had been exposed to seawater containing these antibodies (see below), the embryos were sectioned as above and stained with 47 colloidal gold conjugated to the secondary only. This was followed by staining with uranyl acetate and lead citrate. The sections were then observed and photographed as above. Poly aery lamide gel electrophoresis (PAGE) and Western blotting: Sample preparation: Two to three ml of the following embryonic stages (oocyte, morula, blastula, gastrula and bipinnaria larva) were collected and placed in a 10 ml dounce tissue homogenizer on ice along with an equal volume of homogenizing buffer (20 mM Tris, 0.5 M NaCl, pH 7.4) containing a mixture of protease inhibitors (1 mg/ml pepstatin A [Sigma], and 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% Brij-56 (polyoxythylene 10 cetyl ether; Sigma). Brij 56 is a non-ionic detergent that aids to break up the cellular membranes, yet does not contain phenol rings and therefore does not interfere with protein monitoring using UV absorbance at 280 r\m. The tissue/buffer mixture was then homogenized with 10 plunges of the dounce homogenizer and sonicated with a Fisher probe sonic dismembrator set to 45% for 30 seconds and allowed to incubate on ice for 30 minutes. The homogenate was then centrifuged in a Beckman Optima T L X Ultracentrifuge with a Beckman TLA-100 rotor at 50000 x g for 60 minutes at 4°C. The supernatant was stored at -70°C. Protein concentrations were determined using the BioRad D C Microplate Protein Assay using 2%, 5% and 10% dilutions of the supernatant. This assay is based on the Lowry colorimetric assay (Lowry et al, 1951). HL extraction: H L was extracted either by using HL-1 antibody or by using EDTA/Glycine solution. 48 Extraction with HL-1 antibody: Seven-day-old embryos were incubated in the presence of 0.7 u.g/ml of HL-1 for 2 days. This antibody causes the embryonic H L to slough off. This process took 2 days and was monitored by phase contrast microscopy. Once the H L had sloughed off, the embryos were pelleted by centrifugation at 1000 x g for 10 minutes. The pellet was removed and stored at -70°C. The supernatant was further centrifuged with a Beckman Optima T L X Ultracentrifuge using a Beckman TLA-100 rotor at 50000 x g for 30 minutes at 4°C. The supernatant was discarded and the second pellet was stored at -70°C. Both the first and the second pellets were analysed using SDS-PAGE and Western blots and protein concentrations were determined using the BioRad DC Microplate Protein Assay using 2%, 5% and 10% dilutions of the supernatant. Extraction with EDTA/Glycine (Crawford, 1989): Half ml of packed seven-day-old embryos were incubated in 20 ml of 0.1% E D T A in 1 M glycine (pH 8.0-8.1). This solution dissolves H L in less than 20 minutes and dissociates the cells. Embryos and dissociated cells were collected by centrifugation as above and the supernatant was collected and stored at -70°C for further analysis by SDS-PAGE and Western blots. Gels and blotting membranes: Analysis of purified antigens and embryo homogenates, were performed on 4-10% gradient gels in a Biorad vertical mini-slab system utilizing the buffer system described by Laemmli (1970). Gradient acrylamide gels (4-10%) were used because they facilitated the separation of high molecular weight macromolecules better than the low percentage conventional acrylamide gels while retaining the low molecular weight material. For total 49 protein visualization and Western blot analysis, lanes were loaded with approximately 10 fxg/ml protein/lane as determined by BioRad DC Protein Assay. For purified PM2 or HL-1 antigens, 1 Ug/ml protein/lane was used. The separated proteins / glycoproteins were visualized with silver stain. Gels were equilibrated for 15 minutes in transfer buffer and proteins were electroeluted onto BioRad polyvinylidene difluoride (PVDF) membranes for 1 hour at 100 v. Membranes were rinsed for 15 minutes in distilled water and then either stained for total protein with 0.025% Coomassie Brilliant Blue R-250 (Sigma) in 40% methanol, or processed for immunoblotting (see below). Silver stain: For visualization of lower concentrations of proteins, gels were stained with a silver solution (Morrissey, 1981). To fix the proteins/glycoproteins, gels were initially incubated in 30% ethanol, 10% acetic acid for 3 hr. Following this, they were removed and placed in 30% ethanol for 30 minutes at room temperature. To remove SDS, gels were then washed three times for 10 minutes each in distilled water and placed in 0.1% AgN0 3 for 30 minutes. They were once again washed with distilled water for 20 seconds (to remove excess AgN0 3 ) and incubated in 2.5% NaC0 3and 0.02% formaldehyde until bands became apparent. Western blots: PVDF membranes containing proteins for immunoblotting were treated to visualize immunoreactive bands as follows: Membranes were incubated for 1 hour at room temperature in 10 mM Tris, 0.15 M NaCl, 1 M HC1, 0.2% Tween 20 (TBST) with 5% milk powder to block non specific binding sites. Following this the membranes were rinsed in washing buffer (TBS with 50 0.1% milk powder), and incubated for 2 hours in a 1:1 dilution of primary hybridoma supernatant in TBST. The membranes were then washed 3 times for 15 minutes in TBS washing buffer, and incubated for another hour in goat anti mouse IgG-HRP (Pierce) diluted in TBST. After a final wash, immunoreactive bands were visualized by E C L detection kit (Amersham Pharmacia Biotech). The E C L protein detection kit is an enhanced chemiluminescence detection system. A combination of horseradish peroxidase, hydrogen peroxide and alkaline conditions, catalytically oxidizes luminol, a cyclic diacylhydrazide. Following this oxidation, luminol, which is in an excited state, will now return to ground state it will release photons by doing so. Phenols are added to this system to enhance the chemiluminescent emission of luminol. The maximum light emission is at 428 nm (blue) and is detected by blue-sensitive film. Apparent molecular masses for SDS-PAGE and Western blots were estimated by comparison with Sigma high molecular weight standards. Purification of antigens: Antigen purification was done using HiTrap (N- hydroxysuccinamide) NHS-activated columns on a Pharmacia Biotech UV-1 detector attached to a Kipp & Zonnen BD-40 chart recorder. HiTrap columns are packed with 1 ml NHS-activated Sepharose High Performance which covalently bind to ligands containing primary amino groups. The columns were prepared by washing them with 5 ml of 1 mM ice-cold HC1, with a flow rate not exceeding 1 ml/min. One mg/ml of the purified PM-2 or HL-1 (1ml volume) antibodies in 0.2 M NaHC0 3 and 0.5 M NaCl (pH 8.3) were then added to the columns with a 5 ml syringe. Following this the columns were sealed and kept at room temperature for 30 minutes to allow the antibodies to bind to the beads. To deactivate protein binding, the column was washed three times with alternating solutions of 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 and 0.1 M acetate, 0.5 M NaCl pH 4.0. The column was then washed with 3 ml of PBS pH 7.4 followed by 3 ml of 0.1 M glycine pH 51 3.0 and finally by another 3 ml PBS pH 7.4. Embryo homogenates were then passed through the column using a syringe. The syringe was fitted with a 0.45 \xm filter to remove particulate matter. The column was then washed with 5 ml PBS and the bound antigen was eluted with 0.1 M Glycine pH 3.0. The peak was monitored with a 280 rim ultraviolet detector (Pharmacia). The fractions under the peak were pooled and concentrated using Centricon centrifugal filters (Amicon). Approximate protein concentrations were determined using BioRad DC Microplate Protein Assay and the samples were analyzed by SDS-PAGE as described above. In vivo perturbation studies: To obtain the optimum antibody concentration for treatment experiments, unfertilized oocyte and embryos were raised in seawater containing different concentrations of PM-2 and HL-1 antibodies. The highest concentration of the antibody that caused the most severe developmental/morphological defects without killing the embryos was chosen. To determine the effects of PM-2 and HL-1 antibodies on the development of live Pisaster ochraceus embryos, 50 ul of packed embryos were placed into each well of a 24-well tissue culture plates, containing 3 ml of freshly aerated millipore filtered sea water with 0.06 mg/ml gentamycin sulfate (Sigma), and 10 u.g/ml of purified PM-2 or 7 \iglm\ HL-1 antibodies. In each experiment three plates were run simultaneously. Control embryos were raised in seawater containing gentamycin sulfate with no additional antibody or in seawater containing gentamycin sulfate plus 10 u.g/ml of pooled mouse IgG or IgM. The embryos were raised at 12°C, and the development of both experimental and control embryos were monitored with the light microscope. After the addition of the antibodies, samples of the embryos were removed at different stages (hatched blastula, gastrula, bipinnaria and brachiolaria stages). They were washed once with seawater and fixed by rapid freezing followed 52 by freeze substitution in 100% ethanol (see above) and embedded in JB4 or LRW (Campbell et al, 1991). The embedded embryos were then sectioned for either light microscopy or T E M and stained with only the secondary antibodies to reveal the location of the primary antibody in the embryo. Other groups of the embryos at the same stages described above were fixed in 1% gluteraldehyde in seawater, mounted and photographed on T M A X 100 film with a 16x objective, using a Zeiss photomicroscope with DIC optics. PM-2 perturbation experiments using unfertilized oocyte: Fifty ul of packed unfertilized oocytes were incubated in each well of a 24-well tissue culture plates. Ten u.g/ml of purified PM2 antibody and 10 u.1 of dilute sperm solution were added to each well and the eggs were allowed to fertilize. Fertilized eggs were viewed under the light microscope for the presence of the fertilization membrane. Once the fertilization membrane was observed, 10 y\ of packed fertilized eggs were fixed using both gluteraldehyde as well as the rapid freezing techniques as described above. This experiment was repeated 8 times. PM-2 perturbation experiments using fertilized eggs: Fifty ul of packed fertilized eggs were incubated in each well of a 24-well tissue culture plates. Ten ug/ml of purified PM2 antibody were added one hour post fertilization to each well and the embryos were allowed to grow at 12°C until they reached the hatched blastula, gastrula, bipinnaria and brachiolaria stages. Embryos were monitored with a light microscope and 10 ul of packed embryos were fixed at each stage using either 1% gluteraldehyde in seawater or rapid freezing techniques. This experiment was repeated 8 times. 53 PM-2 perturbation experiments using hatched blastula stage embryos: Perturbation in one dose ofthe PM-2 antibody: Fifty u.1 of packed fertilized eggs were incubated in each well of a 24-well tissue culture plate. At around 40 hours post fertilization (hatched blastula stage), Ten u.g/ml of purified PM-2 antibody were added to each well and the embryos were allowed to grow at 12°C for 16 days post perturbation. Embryos were closely monitored with a light microscope. Ten ul of packed embryos were fixed at 2 hours post perturbation. In addition, 10 ul of packed embryos were fixed each day for the next 14 days. The embryos/larvae were fixed using 1% gluteraldehyde in seawater or rapid freezing techniques. This experiment was repeated 4 times. Perturbation in multiple doses ofthe PM-2 antibody: The above experiment was repeated to observe the effects of the multiple doses of the PM-2 antibody on the embryos. When the embryos reached the hatched blastula stage, 10 u.g/ml of purified PM-2 antibody were added to each well. On the subsequent days an additional 5 u.g/ml of the PM-2 antibody were added everyday to each well and the embryos were raised at 12°C for as long as they lived (usually 7 days post perturbation). Ten u.1 of packed embryos was fixed everyday using either 1% gluteraldehyde in seawater or rapid freezing techniques. This experiment was repeated 4 times. HL-1 perturbation experiments using hatched blastula stage embryos: These experiments were carried out in the same manner as those described for PM-2 perturbation studies, except the HL-1 antibody was substituted for the PM-2 antibody. As with the PM-2 experiments, control embryos were raised in seawater containing gentamycin sulfate with no additional antibody or in seawater containing gentamycin sulfate plus 10 u.g/ml of 54 pooled mouse IgG or IgM. After several trials with different concentrations of the antibody, the concentration that resulted in the most extreme abnormal phenotype without killing the embryos was chosen. Perturbation in one dose of the HL-1 antibody: Fifty ul of packed fertilized eggs were incubated in each well of a 24-well tissue culture plates. At around 40 hours post fertilization (hatched blastula stage), 7 u,g/ml of purified HL-1 antibody were added to each well and the embryos were allowed to grow at 12°C for 16 days. Embryos were closely monitored with a light microscope. Ten ul of packed embryos were fixed at 2 hours post perturbation and another 10 ul were fixed each day for the next 14 days. Fixation consisted of either 1% gluteraldehyde in seawater or rapid freezing techniques followed by freeze substitution. This experiment was repeated 4 times. Immunostaining of perturbed embryos: To determine the location of the antibody following the treatments described above, embryos which had been raised in seawater containing PM-2 or HL-1 antibodies or pooled normal mouse IgG or IgM, were washed once in seawater, then freeze substituted, embedded and sectioned as above. The sections were pre-incubated with 0.5% BSA in PBS for 30 minutes and stained with only the secondary antibody for 1 hour. They were mounted and viewed for fluorescence as described above. Control embryos were incubated in seawater containing IgG or IgM. They were also stained with only the secondary antibody and were viewed using a Zeiss Axiophot Photomicroscope equipped with epifluorescence optics. 55 Tunicamycin and B xyloside treatments: Early gastrula embryos were incubated in 2 fig/m\ concentration of tunicamycin or 5 mM concentration of (3-xyloside for one day. They were then freeze-substituted in ethanol, sectioned and stained with the PM-2 antibody and a FITC-labeled secondary antibody (as described above). Control embryos were incubated in fresh seawater until they reached late gastrula/early bipinnaria stage and were fixed and stained as above. Five embryos from each category were examined. This experiment was repeated twice. Statistical analysis: The size of the blastocoel, archenteron and the GI tract of the perturbed experimental groups and the controls were analyzed and variations in the size of the structures described above were determined by comparing the photographs of 10-20 embryos from each group. Statistical differences were compared by paired r-test using Statistica program. Statistical significance was defined as P<0.5. Deglycosylation of Antigens: Deglycosylation of PM-2 and HL-1 antigens was carried out using GlycoPro Deglycosylation Kit (ProZyme, San Leandro, CA). This enzymatic method removes all N and O-linked oligosaccharides without degrading the proteins. PNGase F is the most effective method of removing all N-linked oligosaccharides from glycoproteins and a combination of sialidase, P (1-4) galactosidase and N-acetylglucosaminidase is used to remove O-linked sugars from glycoproteins. To remove the carbohydrate moieties, 10 ul of 5x reaction buffer 7 were added to 20 \ig or less of each purified PM-2 and HL-1 antigens. The solution was heated up to 100°C and then cooled to room temperature to overcome steric hinderance, which slows or 56 inhibits the action of PNGase F on certain residues of glycoproteins. About 2.5 u.1 of Triton X-100, along with 1 ul of each endo-o-glycosidase, sialidase, PNGase, 13 (1-4) galactosidase and glucosaminase were added to the solution. This mixture was incubated at 37°C for 3 hours. The samples were kept at -70°C and analyzed by SDS-PAGE and Western blots. The above was repeated for BSA samples (2 u,g/ml), which served as a control for the deglycosylation experiments. The BSA samples were incubated from 3 to 5 days at 37°C and stored as above. Videomicroscopy of HL-1 treated embryos: Half ml of seawater containing early gastrula embryos was placed on a slide. Ten pig/ml of HL-1 antibody in seawater were added adjacent to the seawater containing the embryos (on the slide) and the two were allowed to merge. The effect of the antibody on embryos, specifically their ciliary movement, was monitored using a Zeiss photoscope II light microscope with differential interference phase contrast (DIC) optics and a Javelin ultrachip high resolution Charge Coupled Device (CCD) and the results were recorded on a Victor Company of Japan, Ltd. (JVC) video cassette recorder (VCR) black and white camera. 57 Chapter 3- Results: Part A- HL-1 Distribution and localization of HL-1 epitope during development: To study the role of starfish E C M components in development, a number of monoclonal antibodies against starfish E C M were used. The HL-1 antibody recognizes a major component of the hyaline layer and it was used to localize the HL-1 epitope. HL-1 immunoreactivity was not apparent until approximately 20 hours post fertilization at which time, the fertilization membrane began to show staining against the HL-1 antibody (Fig. 4A). Staining was observed in the form of small dense granules in the cytoplasm of the cells. Some staining was also present at the apical surfaces of the cells (associated with the hyaline layer) as well as in the intercellular spaces (Fig. 4A). At 27 hours, the hyaline layer was stained more intensely (Fig. 4B). Granules in the cytoplasm of the blastomeres were stained, as were the intercellular spaces. Some staining was present between the cells especially at the bases, and light staining was present in the blastocoel. At the mid blastula stage, at around 35 hours post fertilization, similar staining pattern was observed to that seen in the 27 hour blastula stage embryos (Fig. 4C). Immunoreactivity in the hyaline layer was more intense in the hatched blastula stage (Fig. 4D). At the gastrula stage, fluorescence was mainly observed in the hyaline layer and on the apical surfaces of the ectodermal cells (Fig. 4E). Diffuse patches in the apical cytoplasm of the cells, also showed some light staining. This staining pattern continued to the bipinnaria stage (Fig. 4F). In addition, hyaline layer-like covering the lumen of the gastrointestinal tract at the bipinnaria stage was now lightly stained (results not shown). By the time the embryo reached the later bipinnaria stages, the HL-1 immunoreactivity was no longer apparent in the E C M of the GI tract. 58 59 Fig. 4. Immunolocalization of HL-1 antigen during embryonic development. Embryos of various developmental stages were freeze-substituted in ethanol, embedded in JB4 resin and thinly sectioned for fluorescent microscopy. These sections were stained with HL-1 antibody followed by FITC-labeled rabbit anti mouse IgG antibody. e= ectoderm cells. (A) 20 hours post-fertilization, the blastomeres contain fluorescent granules (arrow heads). The fertilization membrane is lightly stained (arrow). In addition stained material is seen at the lateral cell borders and between the blastomeres. Bar = 10 um. (B) 27 hours post-fertilization, the early blastomeres contain some fluorescent granules, and the hyaline layer (arrow) is more intensely stained. The basal surface of the blastomeres (arrow head) is also lightly stained, bl = blastocoel; Bar = 25 um. (C) 35 hours post-fertilization, the mid-blastula contains a few stained granules in the blastomeres (small arrow heads). The fertilization membrane (large arrow head) is faintly stained, and the hyaline layer is stained very strongly (arrow). bl=blastocoel; Bar = 25 um. (D) 40 hours post-fertilization, the hatched blastulae, contains positively stained granules in the blastomeres and there is some fluorescence in the spaces between the cells (arrow heads). At this stage, the hyaline layer stains very strongly and seemed thicker than that seen in the previous stages (arrow). The blastocoel (bl) is also stained. Bar = 25 um. (E) 70 hours post-fertilization. Diffusely stained areas (arrow heads) are seen in the cell apices, regions normally occupied by the Golgi apparatus. The hyaline layer is also stained (arrow). Bar = 10 um. (F) Five days post-fertilization, the hyaline layer (arrow) and the intercellular spaces (arrow head) of the bipinnaria larvae are stained, suggesting the release of the HL-1 epitope from the cells into these two separate areas. Bar = 25 um. 60 Effects of perturbation by the HL-1 antibody: In an attempt to block the function of the HL-1 antigen, the embryos were raised in the presence of the HL-1 antibody. Addition of the antibody to the seawater prior to hatching (2 days post fertilization) had no obvious effect on the development of the embryos. When 2 day old post hatched embryos were incubated in seawater containing the antibody and were examined at the late gastrula stage (4 days post-fertilization) and at the bipinnaria stage (7 days post-fertilization) (Fig. 5), the overall embryonic/larval stature and the Gl tract development appeared to be inhibited. At the late gastrula stage the archenterons of the perturbed embryos were 14% shorter (180±3 um vs. 207±4 urn; n=20, p< 0.05) and the overall embryonic stature was 20% shorter than the controls (295±3 um vs. 367±3 urn; n=20, p< 0.05). Once these embryos reached 7 days of development, they showed abnormal development of the gastrointestinal tracts (Gl). The Gl tracts of the perturbed embryos were 20% shorter than their control counterparts (322±6 um vs. 400±3 urn; n=20, p< 0.05) and the overall size of the embryos were 35% shorter than the controls (347±5 urn vs. 524±6 um; n=20, p< 0.05). Twenty embryos from each category were examined and the average of the results within each category was recorded. When bipinnaria/larvae were exposed to seawater containing 7 fig/m\ of the HL-1 antibody, they lost their ability to swim within 15 minutes and sank to the bottom of the dish. In less than one hour in the presence of the HL-1 antibody, the hyaline layer appeared to be exfoliating (data not shown) and one day post treatment, most of the hyaline layer had exfoliated. This was observed under the light microscope as an extended process from the embryo (Fig. 5 B and D). 61 62 Fig. 5 Effects of the HL-1 antibody on the embryonic development. These embryos (20 in each set) were incubated in the HL-1 antibody for 2 days (B) or 5 days (D) and were fixed in 1% gluteraldehyde. They were then photographed using DIC phase contrast optics. bl=blastocoel; ar=archenteron; c=coeloms; es=esophagus; s=stomach; in=intestine; mo=mouth. (A) Late gastrula control embryo showing migrating mesenchymal cells (arrows). Bar = 40 um. (B) Late gastrula embryo incubated in the HL-1 antibody for 2 days. Note that material is being sloughed from the exterior of the embryo (arrow). The overall stature of the embryo was stunted compared to the controls (295±3 um vs. 367±3 um) and the perturbed embryos had shorter archenterons (180±3 umvs. 207±4 um), n = 20; Bar = 40 um. (C) Control bipinnaria larva incubated in fresh seawater for 7 days. Esophagus (es), stomach (s) and intestines (in) have been developed. Bar = 40 urn. (D) Bipinnaria larva raised in seawater containing 7 ug/ml HL-1 for 5 days. E C M that appears to be part of the hyaline layer is being exfoliated (arrow). On average the overall stature of perturbed embryos were shorter (347±5 um vs. 524±6 um). These embryos also had shorter GI tracts compared to the controls (322±6 um vs. 400±3 um). n = 20; Bar = 40 um. 63 Electron microscopy studies of exfoliation of the hyaline layer: To determine if the material being sloughed from the surface of the cells was the hyaline layer and if so which layers of the hyaline layer were being exfoliated, the pattern of hyaline layer exfoliation was studied at the T E M level. To achieve this, detailed electron microscopy was carried out on embryos that had been incubated in seawater containing 7 u,g/ml HL-1 antibody for 5 hours, 10 hours, 24 hours, 36 hours and 48 hours. Ten embryos in each category were studied and the results were identical within each category. After 5 hours in seawater containing the HL-1 antibody, the outer meshwork formed aggregation-like structures and appeared to be exfoliating (Fig. 6A). Certain areas of HI supporting layer and the boundary layer also appeared to be starting to break up. After 10 hours of exposure to the antibody most of the aggregations of the outer meshwork had been lost (Fig. 6B). The boundary layer was no longer visible and the supporting layer was incomplete. However, a few microvilli were still present at this stage. The microvilli extended from the ectodermal cells to the supporting layer. In addition, the intervillous layer appears disorganized and its density was reduced. Following 24 hours of treatment (Fig. 6C), most of the hyaline layer had been exfoliated. The outer meshwork and the boundary layers were lost and only the intervillous layer, and part of the supporting layer were present at this time. Some microvilli were still present and were associated with the supporting layer (Fig. 6C). By 36 hours, the remnants of the hyaline layer had thinned further, leaving only a few strands of the supporting layer and the intervillous layers (Fig. 6D). Finally after 2 days of exposure to seawater containing the HL-1 antibody, all of the hyaline layer sub-layers have been exfoliated except a few fibers of what appeared to be part of the intervillous layer. In addition, the microvilli had been almost completely lost (Fig. 6E). It was interesting to note that although the hyaline layer was removed, the cell junctions were still present and the embryos remained intact. 64 Fig. 6. Electron micrographs of the exfoliating hyaline layer. T E M of the embryos that were exposed to 7 p.g/ml of HL-1 for 5 hours (A), 10 hours. (B), 24 hours. ( C ) , 36 hours. (D), and 48 hours (E). OM= outer meshwork; B= boundary layer; IV= intervillous layer; HI, H2 and H3 together form the supporting layer of the hyaline layer. (A)5 hours post treatment. The outer meshwork has aggregated and it appears to be sloughing (white arrows). Some areas of H3 of the supporting layer along with their overlying boundary layer also appear to be becoming disorganized (white arrow head). n=10; Bar = 0.5 um. 65 (B) Ten hours post treatment. Most of the outer meshwork, the boundary layer (long arrow) and parts of the supporting layer (short arrow) have been lost. Microvilli (mv) are still associated with the supporting layer. n=10; Bar = 0.5 um. 66 (C ) Twenty hours post treatment. Most of the hyaline layer has been sloughed off. The intervillous layer (IV) as well as a remnant of dense fibers that may represent part of the supporting layer (arrow) still exist in some areas. A few microvilli (mv) can be seen which extend from the ectodermal cells to the supporting layer. mv=microvilli; n=10; Bar = 0.6 um. 67 (D)Thirty six hours post treatment. After 36 hours of exposure to the HL-1 antibody, only parts of the outer membrane (OM), the intervillous layer (IV) and few fragments of the supporting layer remained (arrow). Some microvilli (mv) can still be seen. c=cilia; n=10; Bar = 0.5 urn. 68 (E) Forth eight hours post treatment. Embryos incubated in the HL-1 antibody for 48 hours exhibit only a few components of the intervillous layer (arrows). All other sublayers have been lost and only a few short microvilli (mv) can be seen. Although structural details are not visible at this magnification, junctions (j) still appear to be present between adjacent cells. n=10; Bar = 0.5 um. 69 Electron microscopy studies of the hyaline layer regeneration: It was observed that when HL-1 perturbed embryos were transferred to fresh seawater, they had the ability to regenerate a new hyaline layer. To observe the extent of this regeneration, embryos that had been stripped of their hyaline layer for two days, were transferred to fresh seawater and allowed to reform / regenerate a new hyaline layer. Embryos were transferred to fresh seawater for 10, 20, 30, 40, 45 and 50 hours post treatment. Stages prior to 10 hours and beyond 50 hours post treatment were not examined. Detailed electron microscopy was carried out on 10 embryos from each category and the results were identical within each category. After 10 hours in fresh seawater, the intervillous layer could be visualized (Fig. 7A). The HI sub-layer of the supporting layer appeared to be present and surrounded most of the embryo. The H2 and H3 sublayers of the supporting layer were also present in some regions. Sections of the boundary layer were also present above the thickened regions of the supporting layer. A few microvilli were observed in the intervillous layer, reaching up to the supporting layer of the hyaline layer. Although an outer meshwork was present, it was very thin and at this time it did not show any sign of organization. Embryos that were transferred to fresh seawater for 20 hours showed a much more organized hyaline layer (Fig. 7B). Most areas had all of the sub-layers of the supporting layer and when present, they were associated with a well-formed boundary layer. The intervillous layer and the outer meshwork were more complex and contained many more thread-like fibers within them than were present at 10 hours. After 30 hours in fresh seawater, all sub-layers of a "mature hyaline layer" were present (Fig. IC). Although the intervillous layer appeared to be similar to that seen in the control group, it was slightly more dense. It consisted of fibrous material within which the microvilli were embedded. HI, H2 and H3 sub-layers of the hyaline layer were almost completely formed 70 and the boundary layer also covered the entire surface of the embryo. The material of the boundary layer seemed aggregated at this stage and the outer meshwork was very thick and fibrous. After 40 hours in fresh seawater, the entire hyaline layer looked more organized (Fig. 7D). The microvilli extended through the intervillous layer and reached the supporting layer. The supporting layer was not much thicker than the previous stages but the HI sublayer was very clear. The boundary layer was also well organized and shows a more distinct fibrous structure. This layer was denser than the controls. Light microscopic studies did not show any of the apical aggregates seen in the previous stage. The outer meshwork was composed of coarse fibers and covered the surface of the embryo. A t 45 hours, the hyaline layer had not changed very much from that seen at 40 hours. Microv i l l i were visible in the intervillous layer. The supporting, boundary, and the outer meshwork also appeared completely formed and well organized (Fig. 7E). After 50 hours in fresh seawater, the intervillous layer, supporting layer and the outer meshwork looked very similar to their counterparts (Fig. 7F). The boundary layer appeared thread-like or fibrous and had a finer structure than before. It is now indistinguishable from the boundary layer of the control embryos. 71 Fig. 7. Electron micrographs of embryos regenerating a new hyaline layer. T E M of embryos that were incubated in seawater containing 7 u.g/ml of HL-1 for 2 days and subsequently transferred to fresh seawater for 10, 20, 30, 40, 45 and 50 hours. Each group of embryos was freeze-substituted in ethanol and embedded in LRWhite to preserve details of the hyaline layer for T E M . OM= Outer meshwork; b=boundary layer; IV= intervillous layer; mv=microvilli. (A) Ten hours in fresh seawater. Embryos that were in fresh seawater for 10 hours formed portions of the supporting layer. Segments of the boundary layer (b) were associated with thicker regions of the supporting layer (thick arrow). A few microvilli (mv) could be seen. Intervillous layer (IV) was also present. n=10; Bar = 0.3 um. 72 (B)Twenty hours in fresh seawater. After 20 hours in fresh seawater, a diffuse part of the HI and H2 sub-layers of the supporting layer had been formed (arrow head) in many regions. The boundary layer (b) was much thicker and almost completely surrounded the embryo (large arrows). The microvilli (mv) extend to the boundary layer. The outer meshwork (OM) had increased in complexity over that seen at 10 hours. n=10; Bar = 0.3 fim. 73 (C) Thirty hours in fresh seawater. After 30 hours of being transferred to fresh seawater, most of the hyaline layer had been almost completely regenerated. The intervillous layer (IV), the supporting layer (HI, H2 and H3), the boundary (b) and the outer meshwork (OM) have all been formed. However, the apical surface of the boundary layer was still segmental in appearance (arrows). n=10; Bar = 0.3 urn. 74 (D) Forty hours in fresh seawater. After 40 hours, the boundary layer (b) also appeared to have been fully formed, although it failed to show the characteristic fibrous appearance of the normal HL. mv=microvilli; n=10; Bar = 0.4 um. 7 5 (E) Forty five hours in fresh seawater. After 45 hours the hyaline layer was very similar to the hyaline layer at 40 hour. The hyaline layer at this stage was composed of all the usual layers and sub layers; from the basal to apical surface, the intervillous layer (IV), the supporting layer composed of HI , H2 and H3, the boundary layer (b), and the outer meshwork (OM) , but the boundary layer still had an amorphous rather than fibrous appearance. c=cilia; mv=microvilli; n=10; Bar = 0.4 um. 7 6 (F) Fifty hours in fresh seawater. After 50 hours in fresh seawater, the boundary layer had a fibrous appearance (arrow). The hyaline layer at this stage was completely formed and contained all the layers and sub layers: the intervillous layer (IV), the supporting layer composed of HI, H2, and H3, the boundary layer (b), and the outer meshwork (OM). Boundary layer was denser but less organized in the 40 and 45 hour post treated embryos but had the normal fibrous appearance by 50 hours post treatment. n=10; Bar = 0.4 um. 77 Biochemical studies of HL-1: Deglycosylation: To determine whether or not the HL-1 antibody recognizes a carbohydrate epitope, the immunoaffinity purified HL-1 antigen was deglycosylated using a series of enzymes (discussed in the materials and methods) that cleave both N-linked and O-linked carbohydrates. Once the antigen was deglycosylated, it was resolved on a SDS-PAGE and was then either silver stained for electrophoretic mobility of the antigen or it was transferred to a PVDF membrane for Western blotting (Fig. 8). This experiment was repeated 3 times. Silver staining of the purified antigen on the SDS-PAGE showed 4 bands. Two bands at higher molecular masses than 400 kDa, a band at 220 kDa and another band at 150 kDa (Fig 8A). These bands were also present when affinity-purified HL-1 antigen was transferred to a blot and was immunostained (Fig. 8B). Deglycosylated HL-1 antigen was also separated on a SDS-PAGE and transferred to a blot. No positive staining was observed for the deglycosylated material (Fig. 8C). Inhibition of ciliary movement: It was noticed that one of the effects of the HL-1 antibody on the embryos was the loss of mobility and swimming activity. To study this effect further, embryos were placed on a slide under a light microscope, the HL-1 antibody was added to them and the ciliary mobility was recorded and observed. Detailed observation of the effects of the HL-1 antibody on the ciliary movement, showed that, within 2-3 minutes, the cilia stopped beating. In fact, when the antibody reached the embryos, the cilia stopped at the contact region on the surface of the embryo and from there, this effect was spread to cover the entire surface of the embryo so that 78 all ciliary activity was stopped. When the antibody was washed, the embryos recovered and started swimming again within 15 minutes. Controls in which approximately the same number of embryos were placed on a slide and were incubated with the same concentration of normal mouse IgM, continued swimming and did not show any inhibition to the ciliary movement. Twenty embryos from each group were examined and the experiment was repeated ten times. Fig. 8. Western blot analysis of purified HL-1 and deglycosylated purified HL-1. Immunoaffinity purified HL-1 antigen containing 7 pig/ml of protein/lane was separated by electrophoresis on 4-10% gradient gels. The material was then either silver stained (A) or transferred to a PVDF membrane and stained for HL-1 immunoreactivity (B). Four bands were present two at molecular masses greater than 400 kDa, one at ~220 kDa and another at ~150 kDa. Purified HL-1 was deglycosylated and was also separated on 4-10% gradient gels. It was transferred to a PVDF membrane and stained against HL-1 (C). The two heavy bands of molecular masses greater than 400 kDa were faintly present, possibly indicating that the HL-1 79 epitope may be a carbohydrate which is cleaved by deglycosylation of the antigen. However. A lower molecular mass species was not evident. This could indicate that the HL-1 epitope included a carbohydrate moiety. 80 Part B- PM-2: Distribution and localization of the PM-2 epitope during development: To study the localization of the PM-2 epitope during development, JB4 sections of unfertilized oocytes, fertilized eggs, blastulae, gastrulae and bipinnaria larvae were stained with anti-PM-2 and FITC labeled secondary antibody (Fig. 9). PM-2 immunoreactivity was observed throughout all the developmental stages of the Pisaster ochraceous embryos. PM-2 stained material was found in numerous brightly labeled granules that were distributed throughout the cytoplasm of the unfertilized eggs (Fig. 9A). Following fertilization, stained material was primarily observed in the developing fertilization membrane (Fig. 9B). However, many granules remained in the cytoplasm and continued to be present at the morula stage (Fig. 9C). At this stage the cell membranes and the fertilization membrane, were also lightly stained. In the blastula stage, a few fluorescent granules were still present in the blastomeres (Fig. 9D). A small amount of fluorescence was observed in the blastocoel and the fertilization membrane showed only light staining. At the gastrula stage (Fig. 9E), fluorescence was mainly present in the blastocoel, in the archenteron cells, and in the lumen of the archenteron. Light staining was seen in the apex of the ectodermal cells. The hyaline layer was also stained lightly. The bipinnaria larvae were intensly immunostained (Fig. 9F). The blastocoel, lumen of the Gl tract and the hyaline layer were strongly stained and the ectodermal and endodermal cell membranes also showed fluorescence. Some fluorescence was also present in some of the mesenchyme cells and the cells of the coeloms at this stage. Controls stained with either only the primary or the secondary antibody showed no fluorescence. 81 82 Fig. 9. Immunofluorescent localization of PM-2 in early development. One um cross sections through different stages of embryonic development. These embryos were fixed by freeze-substitution in ethanol and embedded in plastic (JB4). Embedded tissues were sectioned and stained with anti-PM-2 antibody followed by FITC labeled goat anti mouse secondary antibody. (A) In the unfertilized oocyte, PM-2 stained granules were observed in both central (long arrow) and cortical (short arrow) cytoplasm. n=nucleus; Bar = 50 um. (B) In the fertilized egg, PM-2 immunoreactivity was mainly observed in the cortical granules (short arrow) and the fertilization membrane (arrow head). It was also present in granules located in the central cytoplasm (long arrow). The jelly coat (jc) was also lightly stained. Bar = 20 um. (C) In the morula, the blastomeres contained positively stained granules. In addition the plasma membranes (arrow) and in the developing hyaline layer (arrow head) were also stained. Bar = 30 um. (D) In the blastula, a few fluorescent granules were present in the blastomeres (arrow). The blastocoel (bl), the hyaline layer and fertilization membrane (arrow head) were starting to show some fluorescence. Bar = 30 um. (E) In the gastrula stage embryos, the hyaline layer was stained (arrow head). The E C M in the blastocoel (bl) was very intensely stained. Granules located in the lumen of the archenteron (ar) and the ectoderm cells (e) were also stained positively. Bar = 25 um. (F) In the bipinnaria, the E C M located in the blastocoel (bl), the hyaline layer (arrow head) and the E C M lining the coeloms and gastrointestinal tract (large arrows) were stained intensely with the PM-2 antibody. PM-2 positive granules were also located in the GI tract (en), ectoderm (e) coelom (c) and mesoderm cells (small arrow). Bar = 20 um. 83 Immunogold: To better localize the PM-2 antigen, immunogold electron microscopy was used to examine eggs and embryos. Gold particles were first seen in the cortical granules of the unfertilized eggs (Fig. 10A). Once the eggs were fertilized, gold particles were seen in the perivitelline space (Fig. 10B). At the early blastula stage, gold particles were mainly observed within the blastomeres at the trans-Golgi and in Golgi-associated vesicles (Fig. IOC). Some gold was also observed in the blastocoel. At the gastrula stage (Fig. 10D), the concentration of gold in the blastocoel had increased. Golgi and Golgi-associated vesicles were also positively stained with gold particles. At the bipinnaria stage, gold was present in the blastoceol (Fig. IOE) as well as in the hyaline layer (Fig. 10F). In the blastocoel gold particles were associated with amorphous material in the matrix and not with the major strands present here. In the hyaline layer, gold particles were present only in the intervillous layer (Fig. 10F). Within the cells, gold was associated with trans-Golgi regions as well as Golgi-associated vesicles. The Golgi apparatus of all endoderm, mesenchyme and ectoderm cells was stained with gold particles, indicating that all of these cell types synthesize PM-2 and exocytose it into the extracellular matrix of the blastocoel and the hyaline layer. Five embryos from each of the different developmental stages mentioned above were examined in detail. Sections of embryos stained with either only the primary, the secondary or pooled mouse IgG antibodies did not show positive staining. 84 4& , y Fig. 1 0 . Immunogold localization of the PM -2 antigen at the T E M level throughout development. The embryos were fixed by freeze-substitution in ethanol and embedded in LR-White resin and stained with PM-2 antibody conjugated to colloidal gold. To maintain maximum antigenicity, osmium was not included in the fixation protocol. (A) A section through an unfertilized oocyte showing colloidal gold particles in a cortical granule (arrows). n=5; Bar = 0.3 um. 85 (B) A section through a fertilized egg showing positive staining in the perivitelline layer (arrows). n=5; Bar = 0.3 um. 86 (C) A section through a blastula stage showing positive staining for PM-2 in trans-Golgi and Golgi associated vesicles (arrows). n=5; Bar = 4.0 um. 87 (D) A section through a gastrula stage embryo, showing positive staining in the trans-Golgi and Golgi associated vesicles (arrows-inset). Note that the blastocoel (BL) is also positively stained. n=5; Bar = 0.5 um ; inset bar = 0.4 um. 88 (E) A section through a mesenchyme cell of a bipinnaria larva. The Golgi and Golgi-associated vesicles were labeled with colloidal gold (arrow). The amorphous material in the blastocoel (BL) was also highly labeled indicating that mesenchyme cells may be synthesizing and secreting the PM-2 antigen into the blastocoel. n=5; Bar = 0.5 um. 8 9 (F) A section through an ectoderm cell with the apical E C M (HL). The intervillous layer of the hyaline layer (arrows) shows strong labeling. Golgi (arrow head-inset) and Golgi-associated vesicles were also labeled indicating that ectodermal cells are synthesizing the PM-2 epitope and that they may be secreting it to the hyaline layer. n=5; Bar (original and inset) = 0.4 um; G = Golgi. 90 Western blot analysis of the PM-2 epitope in early development: To examine some of the biochemical aspects of the PM-2 epitope, total embryo homogenates of oocyte, blastulae, gastrulae and bipinnaria were subjected to SDS-PAGE and probed with the PM-2 antibody for the Western blot (Fig. 11A and B). Although the PM-2 antigen was present in all the developmental stages, it exhibited a dramatic change in the apparent molecular masses of extractable material during development. In the oocyte, 5 different cross-reactive protein bands were observed at 130, 122, 100, 70 and 50 kDa. The 50 kDa band was present at roughly the same intensity in all the developmental stages except for the gastrula stage where it was somewhat weaker. The 130 kDa band was present only in the oocyte. The bands at 122 and 100 kDa decreased in staining as the embryo grew older. The 70 kDa band was much fainter in the blastula but it showed a stronger signal by the bipinnaria stage. Overall, this band was still much fainter than the band present at the oocyte stage, however, other additional bands also became more evident as the embryo grew older. Two bands of molecular masses higher than 201 kDa, and another band at 150 kDa, first became apparent at the blastula stage and showed a stronger signal in the gastrula and the bipinnaria stages. SDS-PAGE and Western blot analysis for the PM-2 epitope were conducted 5 times. 91 Coomassie Blue Western blot Fig. 11. SDS-PAGE and Western blot analysis of the PM-2 antigen in early development. Crude embryonic extracts containing 10 fAg/ml of protein/lane from either oocyte, blastula, gastrula and bipinnaria were separated by electrophoresis on 4-10% gradient gels. (A) The SDS-PAGE was stained with Coomassie blue showing a number of very high molecular weight bands and numerous other faint bands. (B) The SDS-PAGE gel was transferred to PVDF membrane and stained for PM-2 immunoreactivity. Five separate bands of molecular masses 130, 122, 100, 70 and 50 kDa were observed in the oocyte. These bands faded at the blastula, gastrula and the bipinnaria stages and were replaced with heavier bands of molecular masses 150, 201 and one greater than 400 kDa. MW = Molecular mass. 92 Perturbation experiments: In an attempt to block the function of the PM-2 antigen, the embryos were raised in the presence of the PM-2 antibody. This antibody was added either prior to fertilization, right after fertilization or at the hatched blastula stage. If fertilization was allowed to occur in the presence of 10 u,g/ml antibody, 90% of the eggs died after going through several cleavage stages. The surface of these eggs treated with PM-2 looked very uneven and the fertilization membranes were wrinkled (Fig. 12). Fifty perturbed eggs were studied in detail and were compared to the control group. When fertilized eggs were incubated in 10 u.g/ml of the PM-2 antibody, no morphological differences were observed up to hatching and the experimental embryos looked similar to the control group. This group was not studied in detail. Embryos raised in seawater containing one dose of 10 u.g/ml of the PM-2 antibody, which was added after they had hatched, were able to develop coeloms (Fig. 13 D), but the archenterons of these embryos at the late gastrula stage were 15% shorter (204 ±4 urn vs. 242±6 urn; n=50, p< 0.05) and the overall stature of these embryos was 25% more stunted compared to those in the control group (319±4 umvs. 430±2 um; n=50, p< 0.05) (Fig. 13 E). As well, these embryos appeared to have fewer migratory mesenchymal cells relative to control embryos (it is difficult to determine the number of mesenchymal cells since they can not all be focused on the same plane, hence their number was not counted in these experiments). Mesenchyme cells of the PM-2 treated embryos appeared to form clusters around the anterior pole of the archenteron. By the bipinnaria stage, PM-2 treated embryos had 22% shorter Gl tracts (340±7 um vs. 439+3 um; n=50, p< 0.05) and were 16% shorter than their control counterparts (471±6 urn vs. 559±4 urn; n=50, p< 0.05) (Fig. 13 F). While these embryos did not develop normally, they could 93 survive for up to 20 days. Fifty perturbed embryos from each group were studied in detail and were compared to the control group. Fig. 12. Effects of the PM -2 antibody on fertilization. This figure demonstrates the effects of incubating unfertilized oocytes in the PM-2 antibody and allowing fertilization to occur. In this experiment, PM2 was added directly to seawater. Fertilized eggs were photographed using DIC phase contrast optics. Fifty fertilized eggs in each category were observed and the following results were obtained in 90% of the eggs that survived. n=50; Bar = 30 urn. (A) A control egg fertilized in fresh seawater. Normal fertilization membrane is shown with arrows. (B) An egg which was fertilized while being incubated in seawater containing the PM-2 antibody. Note the uneven fertilization membrane (arrows). 94 Fig. 13. Effects of one dose of the P M - 2 antibody on developing embryos in vivo. This figure demonstrates the effects of incubating early gastrula stage embryos in seawater containing 10 |ag/ml of PM-2 antibody. In this experiment, one dose of PM-2 antibody was added directly to the seawater of hatched blastula/early gastrula stage embryos (circa 2.5 days). The experimental and control embryos were closely monitored until they reached the early 95 bipinnaria stage (5 days). The following results were observed in 100% of the embryos tested. 50 embryos in each category were examined. These embryos were photographed using DIC phase contrast optics. n=50; Bar = 50 urn. (A) Mid gastrula control embryo. The archenteron (ar) has expanded midway through the embryo. Mesenchymal cells have started migrating away from the tip of the archenteron (arrow). (B) Late gastrula control embryo. The archenteron (ar) shows segmentations (arrows) and coeloms (c) are present in the blastocoel. (C) Bipinnaria control embryo. The mouth (mo), esophagus (es), stomach (s) and the intestine (i) have been formed. (D) Early gastrula embryos that were exposed to the PM-2 antibody for one day. The tip of the archenteron was not expanded like those of the control (arrow) but coeloms have started to develop (arrow head). (E) Late gastrula embryos that had been exposed to PM-2 antibody for 2 days. Very little elongation of the embryo is seen. These embryos are in fact, 25% shorter than their control counterparts (359±4 um vs. 478±2 um; n=50, p< 0.05). The archenteron was not segmented (arrows) and there were few migratory mesenchymal cells (small arrows). The tip of the archenteron exhibits a cluster of disorganized cells (arrow head). (F) The PM-2 treated embryos that grew to the equivalent stage of the bipinnaria exhibit stunted gastrointestinal tracts (Gl) (340±7 um vs 439±3 um; n=50, p< 0.05). This suggests that PM-2 is necessary for proper formation of the Gl tract. The overall size of the embryo was also stunted by 16% (471±6 umvs 559±5 um). 96 When hatched blastula embryos were exposed to daily doses of the PM-2 antibody, after 3 days the entire blastocoelic cavity remained small (52±2 um 2 vs 224±3 urn 2; n=25, p< 0.05) (Fig. 14C). In addition both endodermal and ectodermal cells were larger (Fig. 14C) than those of control embryos (40±1 umvs 16±1 um; n=25, p< 0.05) (Fig. 14A), and very few mesenchyme cells were present. PM-2 treated embryos did not form mouths, esophagi, stomachs and intestines. In cases where embryos where exposed to the antibody for 5 days, the entire blastocoelic cavity was essentially obliterated (Fig. 14D). The ectoderm and endoderm cells were larger (57±2 um vs 12±3 um; n=25, p< 0.05) and columnar (usually these cells are squamous) in shape and were very granular in appearance, which indicated the presence of numerous vesicles. There appeared to be a few mesenchyme cells (located between endoderm and ectoderm) that are trapped between these enlarged endoderm and ectoderm cells. The ectoderm and endoderm cells of the control group were cuboidal and much smaller. The control embryos have also formed a complete GI tract (Fig. 14B). Twenty five embryos from each category were observed. The size of two ectoderm and two endoderm cells from each embryo was measured. The results are averages of these measurements within each category. Embryos raised in one dose of 5 ug/ml of the PM-2 antibody for 5 days (Fig. 15B), showed slight abnormalities to the GI tract development and they were 20% shorter than the controls (338±4 um vs. 422±4 urn; n=20, p< 0.05). In addition, the overall stature of embryo was truncated by 25% (393±5 umvs. 524±3 um; n=20, p< 0.05) and mesenchyme cells seemed to be scattered around the esophagus. At 10 ug/ml, the GI tract was less segmented (Fig. 15C). It appeared as if these embryos were blocked at the early stages of GI tract development. If the PM-2 antibody concentration was increased to 15 ug/ml, the embryos were less differentiated (Fig. 15D). They appeared to have been blocked at the late gastrula stage with mesenchyme cells clustering at the tip of the archenteron. Under conditions, which the PM-2 concentration 97 was increased to 20 ug/ml, the embryonic differentiation was arrested at late gastrula stage, and the Gl tracts had not developed any further (Fig. 15E). Fig. 14. Effects of multiple doses of PM-2 antibody on developing embryos in vivo. Sections through embryos that have been incubated in fresh seawater (A and B) or were exposed to daily doses of 10 [xg/ml of the PM-2 antibody for either 3 days (C) or 5 days (D). These results were observed in 100% of the embryos tested. n=25; Bar = 50 urn. (A) A thin section through a 5 day old control embryo showing a segmented GI tract (arrows). Esophagus (es), stomach (s), and intestines (in) have been formed. Mesenchyme cells are seen in the blastocoel. e = ectoderm; co = coelom; bl = blastocoel (B) A section through a 7 day old control embryo showing the esophagus (es), coeloms (co) stomach (s), and intestine and numerous mesenchyme cells (m) in the blastocoel (bl). (C) A section of an embryo, which has been incubated for 3 days in the PM-2 antibody. There were a few mesenchyme cells in the blastocoel (arrows). Both the ectoderm (e) and archenteron cells (ar) were thickened and the hyaline layer in many places had been separated from the ectoderm cells (arrow heads). The archenteron is also unsegmented. (D) A section of an embryo that has been incubated in the PM-2 antibody for 5 days. There appears to be a few putative mesenchyme cells (short arrow) that are trapped between the enlarged endoderm (ar) and ectoderm cells (e). The ectoderm and the GI tract are undifferentiated and the blastocoel is essentally non-existent (long arrows). 99 Control Perturbed Perturbed Perturbed Perturbed 0 ug/ml 5 ug/ml 10 ug/ml 15 ug/ml 20 ug/ml Fig. 15. Effects of different concentrations of PM-2 antibody on developing embryos in vivo. The effects of incubating hatched blastula/early gastrula embryos in seawater containing different concentrations of PM-2 antibody. One dose of the PM-2 antibody was added to seawater containing hatched blastula/early gastrula embryos. Embryos were allowed to reach the 7 day bipinnaria stage and were then fixed in gluteraldehyde and photographed using DIC phase contrast optics. These results were observed in 100% of the embryos tested, es = esophagus; s = stomach; in = intestine; ar = archenteron. n=20 100 (A) Control embryos incubated in seawater lacking antibody or incubated in pooled mouse IgG. Arrows point to mesenchyme cells. Bar = 60 urn. (B) Embryos exposed to one dose of the PM-2 antibody at 5 ug/ml concentration. The overall embryonic stature was truncated (393±5 vs 524±3; n=20; p<0.05) and the Gl tract was much smaller (338±4 vs 422±4; n=20; p<0.05). Mesenchyme cells seemed to be scattered around the esophagus (arrows). Bar = 60 um. (C) Embryos exposed to one dose of the PM-2 antibody at 10 u.g/ml concentration. In addition to a short embryonic stature, the Gl tract was much less developed and less differentiated than in B. The embryos seemed to have been arrested in the early stages of Gl tract development. Bar = 60 um. (D) Embryos exposed to one dose of the PM-2 antibody at 15 pig/ml concentration. These embryos lacked any sign of Gl tract segmentation and appeared to have been arrested in the late gastrula stage. A cluster of mesenchyme cells (arrows) was present at the tip of the archenteron. Bar = 50 um. (E) Embryos exposed to one dose of the PM-2 antibody at 20 u,g/ml concentration. Embryo development appeared to have been arrested at the late gastrula stage. A few mesenchyme cells have been migrated in the blastocoel and have reached the overlying ectoderm cells, es = esophagus, s = stomach, in = intestine, ar = archenteron, ec = ectoderm. Bar = 50 \im. 101 Localization of PM-2, following PM-2 antibody treatment: Since the PM-2 antibody was added to the seawater in which embryos were growing, rather than administered directly into the blastocoel by injection, it was necessary to determine whether it had successfully entered the blastocoel. To examine this, some of the perturbed embryos were fixed for light and electron microscopy. These embryos were stained with a secondary antibody labeled with either FITC or colloidal gold and showed positive staining in the blastocoel, the result of which will be shown and discussed below. Control embryos were incubated in fresh seawater or seawater containing normal mouse IgG. These embryos did not show staining in the blastocoel. Immunofluorescent studies of hatched blastula stage embryos incubated in the PM-2 antibody for 2 hours, 2 days, 6 days and 16 days shows that after 2 hours of exposure to the antibody (Fig. 16A), fluorescence could be observed in small granules in the cells of the archenteron. The blastocoel was also lightly stained. Two days after adding the PM-2 antibody (Fig. 16B), fluorescence was observed in the ectodermal and endodermal cells, as well as in the blastocoel and the hyaline layer. The fluorescence in the embryos was brighter than that seen after 2 hours of exposure to the antibody. These fluorescent granules were scattered randomly in the endoderm cells but were more apically localized in the ectoderm cells. Fluorescent granules were not present in the mesenchyme cells. By 6 days (Fig. 16C), large fluorescent granules were present in the endoderm and ectoderm cells. At this stage, stained granules were also present in the mesenchyme cells. The fluorescent granules were much larger and intensely stained at this stage compared to the previous stages. The hyaline layer was not stained at this stage and although some fluorescent staining was present in the blastocoel, its concentration was drastically reduced compared with that seen at 2 days. Sixteen days after the exposure to the PM-2 antibody, fluorescent staining was diminished to a few granules (Fig. 16D). These were primarily located in the mesenchyme cells and to a lesser extend in the ectoderm and endoderm 102 cells. Neither the blastocoel nor the hyaline layer showed any fluorescent staining at this stage. Control embryos did not show any positive staining (Fig. 16E and F). T E M observations of embryos treated with PM-2 antibody show that even after 2 hours of exposure to this antibody, colloidal gold particles were present in small to intermediate vesicles inside the cells (Fig. 17A). Mesenchyme cells of the embryos incubated in PM-2 antibody for 6 days were filled with large heterogeneous vesicles containing gold particles (Fig. 17B). After incubating the embryos for 16 days in the PM-2 antibody (Fig. 17C), the mesenchyme cells were packed with heterogeneous vesicles, very few of these contained gold particles. These vesicles were much more numerous and much more enlarged over those from the control embryos and they appeared to occupy most of the mesenchyme cell volume. 103 D 16 d in PM-2 bl endo F 6 d in IgG endo E 6 d control e endo Fig. 16. Localization of PM-2 antibody, following PM-2 antibody treatment. Thin sections of hatched blastula stage embryos which have been exposed to one dose of 10 ug/ml of PM-2 antibody and allowed to develop for a further 2 hours, 2 days, 6 days and 16 days. They were probed with a secondary antibody to localize the PM-2 antibody. Ten embryos 104 from each group were observed, e = ectoderm, endo=endoderm, bl=blastocoel, ar=archenteron. n=10. (A) Hatched blastula stage embryos incubated in the PM-2 antibody for 2 hours, contained fluorescent granules in the archenteron (ar) (arrow) and some fluorescent material in the blastocoel. Bar = 20 um. (B) Hatched blastula stage embryos that have been given one dose of the PM-2 antibody in seawater and were allowed to develop for another 2 days had fluorescent granules in ectoderm (e) (short arrow) and endoderm (endo). There was also some fluorescence present in the basal lamina of the ectoderm (long arrows) and the blastocoel. Bar = 25 um. (C) 6 days post incubation. There were many large fluorescent granules in the ectoderm (e), and endoderm (endo). Mesenchyme cells (arrows) also exhibit large fluorescent granules at this stage and the concentration of fluorescence in the blastocoel (bl) had decreased. Bar = 20 um. (D) 16 days after being exposed to the PM-2 antibody, the concentration of fluorescence in the blastocoel (bl) had decreased. Very few ectoderm (e) and endoderm (endo) cells were positively stained for the PM-2 antibody but the majority of fluorescence was located in the mesenchyme cells (arrows) at this stage. Bar = 25 um. (E) A section of a 6 day old control bipinnaria. The embryo was incubated in fresh seawater. When sectioned and stained with an anti-mouse FITC labeled secondary antibody, no positive staining was observed in the embryo, e = ectoderm; endo = endoderm; Bar = 20 um. (F) A section of a 6 day old bipinnaria. The embryo was raised in seawater containing normal mouse IgG. This embryo was stained with rabbit anti-mouse IgG (FITC labeled) 105 secondary antibody. There is no positive staining present in the embryo, e = ectoderm; endo = endoderm; Bar = 20 um. Fig. 17. Electron micrographs of embryos incubated in the PM -2 antibody. (A) A transmission electron micrograph showing a section through the ectoderm of an embryo that has been incubated in the PM-2 antibody for 2 hours and probed with a colloidal gold labeled secondary anti-mouse IgG antibody (15 r\m). Colloidal gold is present in intermediate-sized vesicles (arrow). Note that intercellular spaces (arrow head) lack any gold particles. n=5; Bar = 0.5 um. 106 (B) A transmission electron micrograph showing a section through mesenchyme cells of embryos that have been incubated in the PM-2 antibody for 6 days and stained with colloidal gold labeled secondary anti-mouse IgG antibody. Mesenchyme cells in these embryos are filled with heterogeneous vesicles, some of which contained gold particles (arrows), indicating that these cells have phagocytosed the antibody. n=5; Bar = 1.0 um. 107 (C) A transmission electron micrograph of a mesenchyme cell, 16 days after incubation in the PM-2 antibody. Very large heterogeneous vesicles (arrows) devoid of gold particles can be observed. These vesicles probably represent residual bodies. n=5; Bar = 0.5 um. 108 Perturbation of development with tunicamycin and |3-xyloside: To characterize whether or not the PM-2 antibody recognizes a carbohydrate epitope, and whether the carbohydrate is N-linked or O-linked to the core protein, embryos were exposed to 2 u,g/ml of tunicamycin or 5 mM (3-xyloside from the early gastrulation stage to the late gastrulation stage. Perturbed embryos with tunicamycin appeared similar to the PM-2 treated embryos. They were characterized by a shortened archenteron (Fig. 18B) and showed fewer mesenchymal cells than controls. When tunicamycin treated embryos at the late gastrula stage were sectioned and stained with the PM-2 antibody, they showed significant reduction in the amount of staining in the blastocoel. There was also little or no staining on the surface of the ectodermal cells (hyaline layer) when compared to the controls. The ectoderm, mesenchyme and endoderm cells showed little or no staining against the PM-2 antibody indicating that tunicamycin had interfered with the addition of N-linked carbohydrates to the protein core. Gastrula stage embryos, which were incubated in 5 mM (3-xyloside (Fig. 18C) appeared very similar to the controls. The archenteron was elongated and there were numerous mesenchyme cells migrating from the tip of the archenteron into the blastocoel. The E C M of the blastocoel in these embryos stained strongly with the PM-2 antibody and the pattern of staining was similar to that seen in the control embryos. In both treated with (3-xyloside and control embryos, positive staining was present in the hyaline layer and in the intercellular spaces. Five embryos from each category were observed. 109 Fig. 18. Immunofluorescent studies of embryos treated with either tunicumycin or |3-xyloside. Early gastrula embryos were incubated in either tunicamycin or |3-xyloside or in fresh seawater for one day. They were then freeze-substituted in ethanol, sectioned and stained with the PM-2 antibody and a FITC labeled secondary antibody, e = ectoderm; bl = blastocoel; ar = archenteron; n=5. (A) Control gastrula embryo stained with the PM-2 antibody. The lumen of the archenteron (ar) and the blastocoel (b) are positively stained. Ectoderm cells (e) show some fluorescent granules. Bar = 20 um (B) Embryos in tunicamycin exhibited reduced fluorescence in the blastocoel (bl) compared to the controls. The tip of the archenteron has not expanded very much and only a few mesenchymal cells appear in the blastocoel (large arrows). In addition, these embryos lack staining on the surfaces of the cells. There are a few weak fluorescent granules present in the cells of the archenteron (small arrows). Bar = 30 urn. (C) Embryos incubated in the f3-xyloside appeared relatively normal. The archenteron, ectoderm and blastocoel were stained for PM-2 and numerous mesenchyme cells were present at the tip of the archenteron (arrows). Bar = 30 um. 110 Chapter 4-Discussion: Part A- HL-1 HL-1: To date, several hyaline layer components in sea urchin have been characterized. To name a few, echinonectin, a 220 kDa dimer (Alliegro et al., 1988), apextrin, a 74 kDa protein (Haag et al., 1999) and hyalin, a fibrillar glycoprotein of 330 kDa (Citkowitz, 1971), all have been characterized and have been shown to provide substrates for cell adhesion. In this study a major component of the hyaline layer in starfish embryos (HL-1) has been partially characterized and it appears that the perturbed embryos lacking HL-1 maintain their adhesion via junctional complexes. The fact that the embryos/larvae remain intact following removal of the hyaline layer suggests that the HL-1 may not be necessary for cell-cell adhesion, although it could provide a substrate for cell adhesion necessary for morphogenesis. Light microscopy and immunogold electron microscopy show that the HL-1 epitope is found on an E C M component that is located throughout the mature and developing hyaline layer (Pang et al., 2002). This component is not present in the cortical granules or at the time of cortical granule release, but it first appears to be associated with the Golgi apparatus and the early hyaline layer at 20 hours post fertilization, a time when the structure of the hyaline layer is beginning to organize (Pang et al , 2002). Colloidal gold studies show that the HL-1 epitope remains to be associated with the Golgi apparatus and Golgi associated vesicles in the ectoderm cells until the late bipinnaria stage (Pang et al., 2002), indicating that the increase in the thickness of the hyaline layer seen over this period is due in part to the continued secretion of the HL-1 antigen by the ectoderm cells. I l l The HL-1 epitope may also play a role in maintaining the structural integrity of the hyaline layer. Exposure of embryos/larvae to the HL-1 antibody causes a gradual loss of the hyaline layer, which indicates that the HL-1 epitope may play a role in maintaining the layered structure. This is in accordance with numerous mammalian E C M components that have been implicated to be involved in maintaining the structural integrity of the E C M . A recent study by Yan and coworkers (2003) showed that a secreted glycoprotein (SPARC) is necessary for maintaining the structural integrity of the basement membranes in murine lens capsule. In another study, TSG-6, a secreted protein with binding sites for hyaluronan has been implicated in maintaining the integrity of cumulus-oocyte complex during development of ovarian follicles in mammals (Carrette et al., 2001). Since immunogold studies demonstrate that the HL-1 epitope appears in the hyaline layer coincident with the organization of that structure into layers (Pang et al., 2000) and that exposure of embryos to anti HL-1 causes loss of the layer, suggest that the HL-1 epirope may be involved in both organizing and also maintaining the hyaline layer structure. A detailed study of the H L -1 epitope will be necessary to understand the involvement of this molecule in maintaining the architectural integrity of the hyaline layer as well as its possible interactions with other E C M molecules. Other developmental roles of the HL-1 antigen: Freeze substitution studies have demonstrated that like the starfish hyaline layer, the mature hyaline layer in sea urchin is also multilayered (Morrill, personal communications). In sea urchin, numerous functions have been proposed for this multilayered structure. The hyaline layer appears to function as a substrate for cell adhesion (McClay and Fink, 1982; Fink and McClay, 1985; Wessel et al., 1998) and is necessary for initiation of primary invagination and 112 morphogenesis (Adelson and Humphreys, 1988; Kimberly and Hardin, 1998). Studies in which sea urchin embryos were incubated in an antibody against a collagenase component present in the hyaline layer resulted in delayed gut formation and spicule elongation (Mayne and Robinson, 2002), which indicate a role for hyaline layer in morphogenesis. It has also been suggested that the hyaline layer may protect and lubricate the embryo (Lundgren, 1973; Crawford and Abed, 1986) and that it may play a role in the larval feeding behavior (Cerra and Byrne, 1995; Cerra et al., 1997). It seems possible that the multiple functions of the hyaline layer correspond to different layers of this structure. GI Tract development and morphogenesis: The inhibition of starfish embryo morphogenesis by a monoclonal antibody that specifically recognizes the HL-1 epitope provides new evidence that the hyaline layer plays an important role in morphogenesis. Ideas of this nature are persistent in the literature of sea urchin development (Gustafson et al., 1967; Citkowitz, 1971). Gustafson and Wolpert (1967) speculated that differences between adhesiveness of cells to cells and cells to hyaline layer could account for the primary invagination of the archenteron. Citkowitz (1971) provided evidence based on nonspecific proteolytic digestion of embryo surfaces that the hyaline layer was important for gastrulation. Similarly, experiments in which a monoclonal antibody against hyaline - a component of the sea urchin hyaline layer, was used to block the function of this glycoprotein, both gastrulation and morphogenesis were disrupted (Adelson et al., 1988). Recent studies in sea urchin continue to support the idea that the hyaline layer is necessary for proper embryonic development. Zito et al. (1998; 2000) have suggested that loss of interactions between Pl-nectin, a component of the sea urchin hyaline layer, and ectodermal cells causes loss of signals from ectodermal cells to mesenchymal cells and therefore causes disruption of morphogenesis. Several diffusible growth factors of the TGFp family have been proposed as 113 mediators of this ecto-mesodermal induction (Stenzel et al., 1994; Ponce et al., 1999; Angerer et al., 2000). The experiments presented in this thesis indicate that the starfish hyaline layer and the HL-1 epitope in particular may also play a role in the control of morphogenesis of the larval GI tract. In starfish, when hatched blastula embryos are incubated in seawater containing HL-1 antibody, morphogenesis is disturbed. By the late gastrula stage, these embryos are 20% shorter than their control counterparts and they have shorter GI tracts. If allowed to grow to the bipinnaria stage, the abnormalities are even more pronounced. The overall stature is 35% shorter and the GI tracts are 20% shorter than the controls. It is possible that the HL-1 epitope plays an important role in the development of the GI tract. HL-1 epitope may interact with other E C M molecules and/or with the ectoderm cells that induce patterning cues necessary for proper development of the embryo and the GI tract in a similar manner to that described for Pl nectin in the sea urchin (Zito et al., 1998; 2000). Alternatively, poor development of the GI tract may be related to the overall size of the perturbed embryos. The small space available in the blastocoel of the experimental embryos may not allow for the proper formation/development of the GI tract. Loss of microvilli and ciliary activity: Observations by Campbell and Crawford (1991) and Pang et al. (2002), have shown that in the "mature" hyaline layer, the microvilli extend through the intervillous layer, and the supporting layers and terminate in the boundary layer. Studies of the hyaline layer development (Pang et al., 2002) indicate that components of the developing hyaline layer may induce formation of the microvilli. Experiments presented here show that the loss of the boundary layer and the supporting layer coincide with the loss of microvilli from the hyaline layer. Campbell and Crawford (1991) have suggested that the microvilli support the hyaline layer. Together the 114 results indicate that the microvilli and the hyaline layer are interdependent and that both are required for the proper formation of the microvilli and a functional hyaline layer. Previous studies have demonstrated that the hyaline layer, along with a circle of microvilli, form part of a collar around the base of each cilium (Crawford and Campbell, 1993). Examination of this collar suggests that it is flexible and that it could serve to help regenerate and or coordinate the pattern of beating of each cilium (Crawford and Campbell, 1993). When embryos are exposed to seawater containing anti HL-1, ciliary action stops immediately. Ciliary activity returns within 15 minutes of transferring embryos to fresh seawater. The results strongly indicate that the HL-1 epitope is necessary for ciliary activity however the mechanism of this activity is not known. It is also possible that the antibody cross-links the molecules of the hyaline layer causing it to stiffen and that the increased mechanical resistance is enough to stop the ciliary activity. Alternatively, it is possible that the interaction between anti HL-1 and the HL-1 epitope in the collar provides a physiological effect that stops ciliary movement. Rapid recovery of ciliary movement after removal of the antibody indicates that the affected component represents one element of the hyaline layer that it can be rapidly replaced. Regeneration of the hyaline layer: When the larvae have been stripped of their hyaline layer, and were placed in fresh seawater without the antibody, the microvilli regenerated, the embryos regained ciliary movement and were able to form a new hyaline layer. Abed and Crawford (1986) have suggested that the hyaline layer may undergo continuous replacement under normal conditions. The fact that the hyaline layer is shown in the present study to be capable of complete regeneration/reformation lends strength to this idea. In addition, the pattern of regeneration is remarkably similar to that seen during the original development of the hyaline layer (Pang et al., 115 2002). This further indicates that the hyaline layer has the ability to undergo continuous replacement. In many ways the structure and function of the hyaline layer can be compared to the mammalian epidermis, which in addition of being composed of many layers/strata, it also provides the first barrier of protection to the underlying tissues, as well it has the ability to regenerate continuously. Biochemistry of the HL-1 containing molecule: When affinity purified HL-1 antigen, isolated from whole embryo homogenates was examined on a Western blot with anti HL-1 as the probe, at least 3 major immunoreactive bands were present. These consisted of a double band at 400 kDa, a band at 220 kDa and a fourth band at 150 kDa. In early work, when whole embryo homogenates were subjected to reduction using P-mercaptoethanol, the 220 kDa was lost and the HL-1 positive bands were found as a double band at 400 kDa and a single band at 150 kDa (data not presented). Although all work to date suggests that the anti-HL-1 antibody is a monoclonal antibody, it reacts with several large proteins instead of one. As mentioned previously, the HL-1 epitope was first observed in the Golgi and in the Golgi associated vesicles. Since these organelles are responsible for adding and modifying carbohydrate structures on proteins (Kornfield and Kornfield, 1985), the results suggest that the HL-1 epitope is a carbohydrate structure or contains some sugars. Deglycosylation of this E C M component resulted in the loss of antigenicity suggesting that the epitope contained a carbohydrate group. In subsequent tests the enzymes failed to degrade bovine serum albumin, suggesting that it was not active against proteins. It is probable that the HL-1 epitope is a carbohydrate chain or is dependant on the presence of sugars in order to express antigenicity. If the anti HL-1 antibody were against a carbohydrate group it would explain why the antigen is present on several different molecules. It would further indicate that the molecules were glycoproteins or proteoglycans in nature. 116 The molecules that contain the HL-1 epitope all appear to be high molecular weight proteins. It is likely that like many E C M molecules that first exhibit antigenicity in the Golgi apparatus, the protein core of HL-1 is probably synthesized in the rough endoplasmic reticulum, modified in the Golgi apparatus where the carbohydrate epitope is added and finally secreted into the E C M (Hortsch et al., 1986; Meyer et al., 1982; Zimmerman et al., 1986; Griffiths et al., 1986; Matlin, 1986). 117 Conclusions: HL-1 epitope is likely a carbohydrate structure, which is first seen in the cells of the 18 hour blastula and begins to be secreted into the hyaline layer at around 20 hours post fertilization and remains present in the hyaline layer until at least the bipinnaria stage. The HL-1 epitope in the hyaline layer is associated with all the sublayers of the hyaline layer with the exception of H2 sublayer of the supporting layer. This secreted form of HL-1 corresponds to a 220 kDa band and it is almost certainly glycosylated. Although HL-1 is not present in the cortical granules it begins to appear during the cleavage stages, a time at which the hyaline layer begins the process of organizing itself into layers. Since interference with this molecule causes loss of hyaline layer organization and exfoliation of this outer extracellular layer, the HL-1 epitope probably plays a role in maintaining the structure of this E C M and may possibly be involved in organizing this structure. The HL-1 epitope may also play a role in ciliary movement and embryonic growth as well as development of the GI tract. It is likely that once the hyaline layer is sloughed, ectoderm-hyaline layer interactions are inhibited. These interactions may be necessary to induce a series of signals into the blastocoel, which will ultimately affect the growth of the embryo as well as the proper development of the GI tract. A more detailed study of the possible molecular interactions between the HL-1 epitope and other E C M molecules in the hyaline layer as well as possible interactions between the HL-1 epitope and the ectoderm cells may be necessary to further understand how HL-1 present in the outer E C M , surrounding the embryo, could be responsible for the morphological effects observed here. 118 Part B- PM-2 Localization of PM-2 epitope during development: The cytoplasm of an unfertilized sea urchin oocyte contains two populations of cortical-like granules. Cortical granules that reside in the cortex and cytoplasmic granules that are of similar size and appearance to cortical granules but are scattered throughout the cytoplasm (Vaquier, 1975; Inoue et al., 1970). Upon fertilization, the majority of the cortical granules are released into a potential space between the egg plasmalemma and the perivitelline membrane and the contents appear to help elevate the fertilization membrane. Those located deeper in the egg cytoplasm, do not appear to be released into the perivitelline space at fertilization but remain in the cytoplasm. Work with two monoclonal antibodies that stain the cortical granules, PC3H2 (Reimer and Crawford, 1995) and PM-2 antibodies, show that two populations of cortical-like granules also exist in P. ochraceus eggs and undergo similar fates as the sea urchin eggs at fertilization. Temporal and spatial expression of specific molecules during echinoid development, indicate unique roles for the E C M components (Ingresoll et al., 1994; Wessel et al., 1995). In sea urchin embryos, aSU2 integrin expression correlates with changes in the adhesive properties during cleavage and gastrulation (Hertzler et al., 1999). In this study, immunofluorescent and immunogold studies showed that the PM-2 epitope localizes first in the cortical granules and then in the fertilization membrane, indicating that this molecule is secreted and contributes to the formation of the fertilization membrane. Further secretion of this molecule does not recur until the early stages of hyaline layer organization. At this point the PM-2 epitope is secreted basally into the blastocoel and apically into the hyaline layer and contributes to the formation of a specific portion of the hyaline layer, the intervillous layer. Throughout development, PM-2 119 containing molecules appear to be continually secreted into the blastocoel. In the blastocoel, the PM-2 epitope is associated with amorphous material located throughout the E C M present there. Staining against the PM-2 epitope in the blastocoel intensified at later developmental stages, which may indicate that this molecule is needed at a higher concentration during these later stages of development. Immunofluorescent microscopy showed that during the early gastrula stage, the PM-2 epitope is mainly localized to granules in the endodermal cells. Later, numerous positively stained granules were seen in the ectodermal and mesodermal cells, suggesting that PM-2 is either synthesized and/or phagocytosed by these cells. Immunoelectron micrographs of embryos at stages following fertilization show that the colloidal gold is associated with the Golgi and Golgi-associated vesicles in both cell types. This strongly indicates that the PM-2 epitope is being synthesized (or modified) in the Golgi apparatus. Molecules exhibiting the PM-2 epitope are probably packaged into vesicles, which are seen around the Golgi apparatus and are also stained with colloidal gold. These vesicles are likely involved in transporting the molecules containing the epitope to the various extracellular spaces. PM-2 epitope classification: PM-2 first appears in the Golgi apparatus and is seen in these organelles throughout much of the early development. This inndicates that like HL-1, the PM-2 epitope may be carbohydrate in nature. Previous experiments have shown that carbohydrates play a crucial role in echinoid development and different carbohydrate moieties may have different roles in gastrulation. In sea urchin, D-mannose-like residues are suggested to function in archenteron development while N-acetyl-D-glucosamine-like groups may contribute to control of primary mesenchyme positioning (Latham et al., 1999). To determine whether PM-2 contains a carbohydrate epitope, immunoaffinity purified PM-2 antigen was deglycosylated using a 120 deglycosylation kit (Prozyme). Western blot analysis of the deglycosylated samples shows loss of the PM-2 antigenicity, indicating that the epitope could be of carbohydrate nature or at least contains carbohydrate moieties within the epitope. In addition, experiments in which sea urchin embryos were injected with an antibody to the carbohydrate moiety of an E C M glycoprotein (ECM-1), resulted in short unsegmented archenterons (Ingersoll and Ettensohn, 1994). This is similar to results obtained in this thesis, where repeated exposure to anti- PM-2 during formation of the Gl tract also leads to the formation of short unsegmented archenterons. The PM-2 results further supports the idea that the epitope is carbohydrate in nature and further supports the hypothesis that the carbohydrate moieties may play a major role in these morphogenetic events. Carbohydrates are usually linked to proteins through either an O or an N linkage. To determine if such a linkage is present, embryos were raised in the presence of either tunicamycin, which blocks N-linked carbohydrates or |3-xyloside, which blocks O-linkages. Tunicamycin incubated embryos show reduced fluorescence in the blastocoel further suggesting that PM-2 antibody may recognize N-linked carbohydrate groups. Both the Western blot analysis of deglycosylated PM-2 samples as well as the tunicamycin-treated embryos, support the suggestion that the PM-2 antibody recognizes an epitope that contains carbohydrates. Western blot analysis: Western blot analysis of the PM-2 epitope shows 5 low molecular mass bands in the oocyte, but by the hatched blastula stage, the 130 kDa band had disappeared. It is possible that this band represents materials found in the cortical granules, which are released upon fertilization and contribute to the development of the hyaline layer and the fertilization membrane. The other four bands decrease in intensity but remain present throughout development. This could simply be due to the dilution of the antigen in the samples, which is expected if the material is no longer 121 being synthesized during later stages of development. It is also possible that there is a continual regeneration of the PM-2 epitope. Immunoelectron micrographs of embryos showed that the epitope is present in the Golgi throughout development, indicating that continual synthesis of the epitope occurs throughout development. The effects of anti PM-2 antibody on development: To understand the possible functional roles of the PM-2 epitope during development, embryos were raised in the presence of the PM-2 antibody. Depending on the concentration of the antibody and the duration of exposure to the antibody, the effects on the eggs and embryos vary. Secretion from cortical granules appears to be necessary for raising and thickening the fertilization membrane in sea urchin (Eddy et al., 1979; Weidman et al., 1985). Unfertilized eggs raised in seawater containing the PM-2 antibody, are viable for only a few hours. They show poor formation of the fertilization membrane, which appears to be raised much higher than the fertilization membrane in the control embryos. PM-2 is secreted into the perivitelline space and is incorporated into the fertilization membrane. This indicates that PM-2 plays an important role in formation of the fertilization membrane in these embryos. It is possible that the PM-2 antigen is a receptor for sperm. A heavy molecular weight glycoprotein (Dhume and Lennarz, 1995) in sea urchin has also been identified and associated with cortical granules and plasma membrane and it too appears to be responsible for sperm binding activity (Ruiz-Bravo et al., 1986; Ohlendieck et al., 1993; Hirohashi and Lennarz, 1998). Antibodies against sea urchin sperm binding glycoprotein have shown to completely block fertilization. Exposure of the starfish unfertilized eggs to a higher concentration of the PM-2 antibody might do the same and prevent fertilization. 122 When embryos were allowed to grow and reach the hatched blastula stage and were then exposed to the PM-2 antibody, they showed several developmental defects. These range from stunted embryonic growth to abnormal development of the gastrointestinal tract. The GI tract formation in sea urchin has been extensively studied (Wessel and McClay, 1985; Davidson et al., 2002; Brandhorst and Klein, 2002). It has been demonstrated that nuclearization of |3 catenin, a transcriptional cofactor in the wnt signaling pathway, is essential for the endoderm and mesoderm specification (Davidson et al., 2002; Brandhorst and Klein, 2002). (3 catenin accumulates in the nuclei of vegetal blastomeres from the 16 cell stage to the hatched blastula stage (Logan et al., 1999). The invaginating vegetal cells form the archenteron, which extends in the blastocoel. These cells express lineage specific genes such as Endo 1. mRNA and protein accumulation of Endo 1 begins during primary invagination and is restricted to the differentiating gut regions (Wessel and McClay, 1985). Once the archenteron is formed, the cells rearrange themselves by migrating over one another and by flattening themselves (Ettensohn, 1985). This process transforms the short, wide gut rudiment into a long thin tube. The archenteron then bends and contacts a specific "contact site" on the ectodermal wall, where eventually the mouth is formed (Hardin and McClay, 1990). The mouth fuses with the archenteron and creates the digestive tube. Embryos, which were raised in the PM-2 antibody, were stunted. As well, the esophagus and the stomach in these embryos were very much truncated compared to the control groups. Although more widely distributed than the sea urchin Endo 1 molecule, these results indicate that like sea urchin Endo 1, PM-2 may play a role in regulating the proliferation or the arrangement of the cells constituting the GI tract. As the concentration of the antibody increased, so did the defects in the GI tract development. At 10 u.g/ml, the GI tract was very poorly developed. It appeared as if the archenteron was segmented and was bent to form a mouth, but further development was blocked/stopped. Any higher concentrations of the PM-2 antibody resulted in a complete 123 inhibition of Gl tract differentiation. These embryos formed an archenteron and some migratory mesenchymal cells, but there was no sign of segmentation of the Gl tract and eventually these embryos did not survive. The development of viable embryos was affected most at 10 p-g/ml and so this concentration was used as the optimal concentration for perturbation experiments. These experiments indicated that, there is a potential concentration of the PM-2 antigen, which is necessary and has to be present at the hatched blastula stage, without which the archenteron cannot develop and form a proper Gl tract. Embryos incubated in daily doses of the PM-2 antibody for a period of 3 to 5 days, exhibited more severe developmental defects than embryos that had been incubated in one dose of the PM-2 antibody for the same time period. Five-day-old embryos exposed to multiple doses of anti PM-2 antibody contained only a few mesenchymal cells that migrate from the tip of an undifferentiated archenteron. The blastocoel cavity was also reduced. Embryos that were raised in seawater, supplied daily with the PM-2 antibody for a period of 5 days, did not form many mesenchyme cells and the blastocoel is almost non-existent. The results indicated that PM-2 may also be necessary for mesenchyme cell production and migration as well as development of the blastocoel and Gl tract differentiation. In addition to this the ectodermal and endodermal cells were enlarged, indicating that the PM-2 epitope may also function in promoting cell division. An important event in gastrulation is the migration of mesenchymal cells. Interactions between the migrating cells and the E C M in the blastocoel appear to be necessary for guiding mesenchyme cells to their target sites. In echinoderm development, appropriate contact between ectoderm cells and the hyaline layer components (Zito et al., 1998; 2000), signaling cues from the basement membrane (Marsden et al., 1998; Ffertzler et al., 1999; Katow et al., 2001), as well as many E C M components within the blastocoel (Boucaut et al., 1990; Katow, 1990; Katow et al., 2001; Gryzik et al., 2004) are essential for correct mesenchyme migration. Many of these 124 components in the blastocoel contain the (Arg-Gly-Asp) RGD sequences. Studies in which synthetic peptides of fibronectin, (RGD), were injected into the blastocoel of sea urchin embryos, mesenchymal migration and gastrulation was severely disturbed (Katow et al., 1990). Although we have no direct evidence, the fact that anti PM-2 inhibits mesenchyme cell migration indicates that PM-2 may also contain an RGD sequence. Isolation and characterization of this molecule could help to determine if this is correct. It has been suggested that mesenchyme cells may serve to direct the formation of the coeloms and the mouth (Crawford and Chia, 1978; Crawford and Abed, 1983; Abed and Crawford, 1986). In the PM-2 studies, the embryos showed an abnormal development of the gastrointestinal tract. The mouth, esophagus, stomach and the intestines were all poorly developed. The defects seen in the archenteron and the decrease and /or lack of mesenchyme cells are perhaps some of the events that lead to the subsequent defects in the gastrointestinal development at the bipinnaria stage. The PM-2 perturbed embryos also showed a reduction in the size of the blastocoelic cavity. This might be due to the fact that incubating embryos in the PM-2 antibody might interfere with the PM-2 epitope's activity/availability in the blastocoel. GAGs are highly hydrophilic and they adopt highly extended configurations, which occupy huge volumes relative to their mass. Their high density and negative charges attract cations that are osmotically active causing large amounts of water to be sucked into the matrix. Interfering with GAGs, as it is likely done in these perturbation experiments (given that PM-2 antibody is indeed taken up by cells and transferred to the blastocoel), could interfere with their extended configuration and therefore cause a reduction in the blastocoelic cavity size. 125 Does the antibody enter the blastocoel? Because of the effects of anti PM-2 antibody on the Gl tract and the mesenchyme cells, it is important to determine if these effects are obtained because the antibody has entered the blastocoel or if they are simply due to an effect caused by the antibody externally, perhaps on the hyaline layer. For this purpose, embryos that have been incubated in seawater containing the primary antibodies were fixed and stained with only the secondary antibody. At just one hour after incubation in seawater containing the anti-PM-2, this antibody appeared in the blastocoel and the staining of the E C M in the blastocoel increased over the next few hours. It appeard that the intensity of staining of the E C M in the blastocoel depended on the concentration of the antibody present in the seawater. At lower concentrations, a few FITC granules were present in the blastocoel, whereas at higher concentrations of PM-2 antibody, the blastocoel was completely filled with fluorescent material. The determining factor for the rate of transfer of the PM-2 antibody to the blastocoel may be its concentration in the seawater and / or its interaction with the surface antigen. Experiments with the starfish Asteropecten amurensis demonstrated that antibodies entered the blastocoel after the embryos/larvae had been treated with Ca+2-free seawater (Lathem et al., 1998). The authors suggested that the lack of calcium caused the septate desmosomes to leak and allowed the antibody molecules to diffuse between the ectodermal and endodermal cells of starfish embryos (Lathem et al., 1998). When P. ochraceus embryos were exposed to normal seawater containing monoclonal antibody PM-1, which was against the carbohydrate epitope of a proteoglycans-like molecule, PM-1 antigen, the antibody entered the blastocoel within 2 hours (Reimer et al., 1997). Detailed examination of such embryos/larvae following fixation and staining with FITC labeled anti-mouse IgM secondary antibody demonstrated that granules of fluorescent material were present in the cells before any staining was seen in material located in the blastocoel. In addition, little or no labeled material was seen between the lateral borders of 126 ectoderm and endoderm cells. Although it is possible that the material diffuses between the cells very quickly and is then removed from the bases of the ectoderm and endodermal cells, the presence of intact junctional complexes and the lack of fluorescent material located between the cells indicate that at the least in some cases, the antibody and/or the antigen complex is transcytosed from the apices to the bases of the ectoderm and endoderm cells and that following this, the antibody is released into the blastocoel. When embryos/larvae were exposed to normal seawater containing a second monoclonal antibody, PM-2, that recognizes a carbohydrate epitope located on molecules found in the blastocoel, it also enters the blastocoel but more slowly. To study the timing and location of the antigen containing molecules, detailed examination of PM-2 treated embryos was carried out at the light and T E M level using secondary antibodies labeled with FITC and colloidal gold, respectively. The results again indicate that like PM-1, PM-2 antibody molecules and/or the antigen-antibody complexes are being transcytosed across the ectoderm cells. In this study, the transcytosed PM-2 antibody accumulated in the blastocoel and was taken up by the mesenchymal cells. Examination of the mesenchyme cells at the T E M level demonstrated that 6 days post-treatment, mesenchyme cells contained lysosome-like granules that stained with colloidal gold. By 16 days post treatment, these lysosome-like granules were enlargened, and became more dense and heterogeneous in appearance, which suggests they could be residual bodies. These vesicles did not contain any colloidal gold particles. The results indicate that the antibody or antibody/antigen complex that had been phagocytosed by mesenchyme cells has been digested and destroyed in these vesicles and that the mesenchymal cells may be responsible for the uptake of the antibody from the blastocoel and digestion of it. The phagocytic nature of the mesenchyme cells has been also observed in sea urchin Lytechinus variegates. In these experiments yeast Saccharomyces cerevisiae was microinjected into the blastocoel of gastrula stage sea urchin embryos and the secondary mesenchymal cells 127 were observed phagocytosing them (Silva, 2000). In a similar study reported by Marsden and Burke (1998), ectoderm cells in the sea urchin embryos incubated in anti |3L integrin antibody, transcytosed the antibody to the blastocoel and mesodermal cells phagocytosed it within 2 hours. It is shown that mesenchyme cells produce E C M components (Crawford et al., 1997) and earlier observations have shown that they rearrange them. Recent work (Kaneko personal communications) indicates that mesenchyme cells can reorganize the filamentous parts of the starfish E C M . The results with the PM-2 antibody show that mesenchyme cells also phagocytose non-functional and damaged E C M components. Taken together these indicate that the E C M of the blastocoel may not be static, but turns over and supports the idea that the mesenchyme cells are involved in this turnover. The fact that both the PM-1 and the PM-2 antibodies appear to enter the blastocoel by transcytosis may indicate that all antibodies might do so. Control experiments with pooled mouse IgG and IgM, demonstrated that these molecules did not enter into the blastocoel nor did antibodies against bovine serum albumin. In addition, antibodies against the HL-1 epitope present in the hyaline layer did not get in. This indicates that antibody entry is not random. Both PM-1 and PM-2 antibodies appear to be against carbohydrate epitopes and both epitopes are located on the apices of some cells as well as in the blastocoel. Latham and coworkers (1998) have shown that the lectins, particularly concavalin A were transcytosed into the blastocoel of sea urchin. Since lectins interact with carbohydrates including those on the surface and in the blastocoel of P. ochraceus embryos and larvae (Reimer et al., 1990), there appears to be a strong possibility that interaction of the antibody with the carbohydrate epitopes that are located on molecules found primarily in the blastocoel might in some way be triggering endocytosis. Reimer et al. (1995) have shown that both FITC-labeled concavalin A (Con A), which interacts with mannose sugar, and FITC wheat germ agglutinin (WGA), which interacts with N-128 acetyl glucosamine sugar, stain the hyaline layer and the blastocoel of P. ochraceus embryos/larvae. Experiments, in which P. ochraceus embryos were incubated in both Con A and WGA show that only Con A was endocytosed (data not presented). The results indicate that interaction of a ligand with certain specific carbohydrate groups are capable of inducing endocytosis but that not all surface carbohydrate molecules are capable of doing so. In addition, the fact that the Con A did not appear in the blastocoel indicates that while certain carbohydrate structures could induce endocytosis at the surface of the cells, a second signal may be necessary for the molecule to be completely transcytosed. 129 Conclusions: This study has shown that the PM-2 epitope is attached to a large molecule(s) that exhibits carbohydrate residues. It is present in the egg and is synthesized thereafter. It is secreted into the blastocoel matrix of blastulae embryos, as well as in the hyaline layer and that it appears to be involved with the morphogenic events necessary for normal development of the embryos. Molecule(s) containing the PM-2 epitope appear to be necessary for migration of mesenchyme cells as well as in the development of the GI tract. The present observations indicate that PM-2 antibody is directed against a carbohydrate region on the molecule and by binding to this region of the PM-2 containing molecules, it inhibits GI tract development and mesenchyme cell migration. This strongly suggests that it is a carbohydrate structure on the PM-2 molecule that mediates cell migration, differentiation and digestive tract formation. This is supported by previous studies in which carbohydrate containing E C M determinants in sea urchin have been shown to play a role in gastrulation (Papakonstantinou et al., 1994), cell rearrangements during secondary invagination as well as cell movements during segmentation of the gut (Ingresoll et al., 1994). Sequential enzymatic digestion of sugars present on the PM-2 glycoprotein would provide additional knowledge about the specific sugars present in this molecule. Furthermore, isolating and sequencing PM-2 core protein would make its comparison possible with other known and sequenced glycoproteins. This comparison, could allow identification of common structural motifs, such as the argenine-glycine-asparagine cell binding domain, or regions having growth factor activity. One could also investigate PM-2 binding proteins and receptors with the use of PM-2 glycoprotein affinity columns or immunoprecipitation techniques. Knowledge about binding sites and how they are related to other known glycoproteins could aid to determine the mechanisms by which it affects mesenchyme migration and GI tract morphogenesis. In addition, observing the direct effects of PM-2 antibody on mesenchyme cells in a system where 130 these cells are isolated from the embryo would provide additional information about the PM-2 epitope and whether its acts directly on the mesenchymal cells or if the effects seen on these cells are due to the changes in the environment where mesenchyme cells normally reside. 131 General Summary: During the past several decades, researchers have used various techniques to determine the role of E C M in development. Disruption of the E C M using drugs such as (3 xyloside and collagen synthesis inhibitors have been shown to have profound effects on sea urchin development (Solursh et al., 1986; Wessel and McClay, 1987; Benson et al., 1991). Functional blocking antibodies to various E C M components have also been used to understand the role of various E C M components in eye development (Visconti et al., 2002), kidney development (Durbeej et al., 1995) and neural crest cell migration (Boucaut et al., 1984). The use of antibodies to study the role of E C M components in development has also been carried out in sea urchin embryos. Monoclonal antibodies against several E C M components in sea urchin have shown the importance of these components in gastrulation (Burke et al., 1991), spiculogenesis (Adelson and Humphreys, 1988), dorsoventral axis formation and ectoderm differentiation (Zito et al., 2000). Very few studies however have attempted to use this method to explore the role of some of the different E C M components during starfish development. The studies presented in this thesis employ antibodies against two embryonic/larval starfish E C M components, HL-1 and PM-2. Immunofluorescence, light microscopy, immunogold electron microscopy and selective blocking by incubating living embryos/larvae in seawater containing the antibodies were carried out to gain insight into the roles of these E C M components in development. Immunochemical experiments and inhibition with specific drugs were also used to examine the biochemical nature of the antigens. Immunofluorescent studies demonstrated that the HL-1 epitope was primarily found in the hyaline layer throughout most of the development. These studies further demonstrated that the HL-1 epitope first appeared in the blastomeres and in the outer surface of the embryo at around 18 hours post fertilization, a time at which the hyaline layer begins to organize itself into 132 its characteristic layers and sublayers. When embryos/larvae were raised in seawater containing the HL-1 antibody, the hyaline layer sloughed off. In addition, the transfer of the perturbed embryos to fresh seawater thus removing the antibody from their environment, resulted in complete hyaline layer regeneration. The fact that the HL-1 epitope appears at the time that the hyaline layer is organized and is present thereafter coupled with the fact that the hyaline layer is lost when the antigen is interfered with indicates that the HL-1 epitope is both necessary for maintaining the structure of the hyaline layer and it may help to organize it. Embryos incubated in the presence of the antibody were also severely shortened and their Gl tracts were less segmented than the control embryos, indicating that HL-1 could also be involved in the development of the Gl tract and the overall embryonic growth. Finally embryos, in which, the HL-1 antigen was blocked using the antibody, showed no ciliary movement and were unable to swim, indicating that HL-1 also plays a role in the ciliary movement and swimming behavior of the embryos. Analysis of the HL-1 antigen using Western blots showed that the HL-1 antibody stained multiple bands. Deglycosylation of the epitope using glycosidases resulted in complete removal of antigenicity of all the bands. The results support the hypothesis that the HL-1 epitope is either a carbohydrate common to several core proteins or the bands represent different subunits of a mature protein and that the antigen molecules are glycoproteins. Immunofluorescent studies using the PM-2 antibody show that the epitope is present in the unfertilized oocyte and remains present at least until the bipinnaria stage. It appears to be synthesized by endoderm, ectoderm and mesenchyme cells and it is secreted into the blastocoel, the lining of the Gl tract as well as into the hyaline layer. The PM-2 epitope also appears to be essential for several aspects of the embryonic development. Blocking the function of this antigen at the time of fertilization results in poor 133 development of the fertilization membrane indicating that during early stages of development the PM-2 epitope may be important for the formation of this structure. Perturbation studies post fertilization cause the disruption of mesenchymal migration and the Gl tract development. The perturbed embryos were also noticeably shorter than the controls, which means that PM-2 may be necessary for the overall growth of the embryos. In pulse chase experiments, embryos were incubated in seawater containing the PM-2 antibody and they were returned to fresh seawater for varying periods after which they were preserved by freeze substitution. They were then sectioned and probed with a fluorescent-labeled secondary antibody. This demonstrated that the PM-2 antibody (or PM-2 antibody-antigen complex) had entered into the blastocoel. Detailed examination of these embryos with both light and T E M showed that the material appeared to be transcytosed from the seawater by the ectoderm and endoderm cells into the blastocoel. Immunofluorescence and immunogold microscopy showed that following an initial rise in concentration of the antibody in the blastocoel, the concentration was reduced over time and the concentration of the antibody in the mesenchyme cells was increased. This indicates that mesenchyme cells may endocytose this material from the blastocoel. Eventually the antibody was completely removed from the blastocoel and the cells. The mesenchyme cells were filled with what appeared to be residual bodies implying that the mesenchyme cells had phagocytosed the antibody-antigen complex, indicating that they are probably involved in the turnover of the E C M in the blastocoel. The PM-2 epitope appears to be located in the Golgi apparatus, suggesting that it might be carbohydrate in nature. This is further supported by the fact that deglycosylation of this epitope causes loss of antigenicity. The results indicate that PM-2 may also be a glycoprotein. The carbohydrate moieties of glycoproteins are attached to their core protein by either N or O linkages. The drug tunicamycin blocks N linked carbohydrates while B xyloside blocks O linkages. Treatment of embryos/larvae with these drugs showed that only tunicamycin affected 134 the synthesis of the PM-2 epitope, which was indicative that PM2 epitope is most likely attached to the core protein via an N-linkage. The studies presented here show that both HL-1 and PM-2 epitopes play important and different roles in starfish development and although they are synthesized at different times, by different embryonic cells. They are concentrated in different regions in the embryo, and they both appear to be necessary for morphogenesis and development. 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