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Ontogeny of the basal lamina in the sea urchin embryo
DEVELOPMENTAL
BIOLOGY
103,235-245 (19&Q)
Ontogeny of the Basal Lamina in the Sea Urchin Embryo GARY M. WESSEL,* RICHARD B. MARCHASE,* AND DAVID R. MCCLAYF’ *Lkpartment qf Anatomy, Duke University l&dim1 Center, and flkprtmat
of Zoology,Duke University, Durham, North Caroling 27706
Received July 26, 1983;accepted in revised form Jarwarp 9, 1984 The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen typesI, III, and IV, and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 pm) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de luluo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.
the cell surfaces of the sea urchin embryo. Evidence for the presence of glycosaminoglycans, including heparan Nearly 20 years ago Gustafson and Wolpert (1967) sulfate and chondroitin sulfate, was suggested by biopostulated that interactions between the basal lamina chemical data (Solursh and Katow, 1982). The existence and blastomeres were crucial to gastrulation in the sea of collagen in the sea urchin embryo was suggested by urchin embryo. At the mesenchyme blastula stage of ultrastructural data (Crise-Benson and Benson, 1979; development, primary mesenchyme cells pass through Spiegel and Spiegel, 1979), by detection of the collagen the basal lamina to invade the blastocoelic compartment. processing enzyme prolyl hydroxylase (Benson and SesThese cells then use the basal lamina as a substrate for sions, 1980; Mizoguchi and Yasumasu, 1983), and by the a series of characteristic migrations that culminate with presence of hydroxyproline in macromolecules (Puccithe cells coalescing at specific sites to produce the larval Minafra et CL, 1972; Golob et aL, 1974; Gould and Benson, skeleton. Meanwhile, midway through archenteron for1978). There is not, however, any direct biochemical evmation secondary mesenchyme cells extend long filoidence to demonstrate the presence of collagen in the podia that contact and attach to the basal lamina. Gussea urchin embryo. tafson and Wolpert (1967) postulated that these contacts, The purpose of the present study is to trace the followed by mesenchyme cell contraction, were meexpression of extracellular matrix (ECM) components chanically important for completion of the archenteron. through embryonic development and to ascertain the Several recent studies have suggested that the basal general pattern of basal lamina formation. An immulamina-extracellular matrix complex that lines the nofluorescent technique was employed on paraffin-secblastocoel in the sea urchin embryo is similar to that tioned material using polyclonal antibodies (PcAb) and found in vertebrates. The presence of a basal lamina in monoclonal antibodies (McAb). The PcAb used were the sea urchin embryo was first suggested by electron specific for the known vertebrate extracellular matrix microscopic studies (Endo and Uno, 1960; Wolpert and molecules: fibronectin, heparan sulfate proteoglycan, Mercer, 1963; Okazaki and Niijima, 1964; Gibbons et uL, laminin, and collagen types I, III, and IV. The McAb 1969) and was identified as a lo-nm-thick extracellular used were generated against sea urchin embryonic comstructure found along the blastocoel wall. Immunofluponents. The components studied were selected on the orescent data have suggested that presence of both a basis of antibody binding to the basal lamina in the fibronectin-like molecule (Spiegel et aL, 1980; Katow et pluteus larval stage. Most components examined were oL, 1982; Spiegel et CCL, 1983) and a laminin-like molecule found within the unfertilized egg. Our results suggest (Spiegel et aL, 1983) within the basal lamina and along a coordinated pattern of assembly, redistribution, and perhaps synthesis of these antigens during early de1To whom correspondence should be addressed. velopment leading to the basal laminar structure. We INTRODUCTION
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0012-X06/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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also report two basal laminar antigens expressed de luyuo by the mesenchyme cells at the mesenchyme blastula stage. METHODS
Gametes of Lqtechinus variegates were obtained by injection with 0.5 iM KCl. Eggs were washed five times in artificial seawater (Dawson, 1969) and fertilized with a dilute sperm suspension. Source of Antibodies Pol&mal antibodies. Antibodies to human plasma fibronectin were purchased from Cappel Laboratories. Antibodies to collagen types I, III, and IV and to laminin and heparan sulfate proteoglycan were gifts from H. Kleinman and G. R. Martin of the National Institute of Dental Health. These antibodies were raised in rabbits or sheep, and the specificity of each antibody had been previously verified by RIA, ELISA, Ouchterlony immunodiffusion, immunoelectrophoresis, and/or immunofluorescent blocking experiments as described (Timpl et aC, 1977,1979a,b, Wick et d, 1979; Foidart et a& 1980; Hassell et a+!, 1980). Monoclmal antibodies. BALB c/j3 mice were injected intraperitoneally with 100 pg of the insoluble fraction of Lytechinus variegates (52 hr pluteus) embryos following detergent extraction (0.1% Triton X-100,10 mM NaHCOs, 0.01% PMSF, and 15 pg/ml DNase). The detergent extraction solubilized cells but much of the extracellular matrix of the embryo remained intact, thus allowing for an enrichment of components of the basal lamina, and/or macromolecules attached to the basal lamina. After 10 days the mice were boosted with a second 100 pg injection of the immunogen. Some mice were injected on a monthly basis for 3 months, and in each case mice were sacrificed 3 days after a boost injection. Spleens were obtained from mice by sterile dissection. Lymphocyte-myeloma hybridomas were generated according to the protocol of Galfre et al. (1977). The myeloma parent was a HAT-sensitive P3X63AgS line obtained from Dr. George Eisenbarth of the Harvard School of Medicine. Hybridomas were raised in HAT medium according to Littlefield (1964). Supernatants of hybrid colonies were screened for anti-sea urchin extracellular matrix antibody production using an ELISA assay (Engvall and Perlmann, 1972) with the original immunogen and/or by immunofluorescence, and the resulting positive hybridomas were cloned.
Embryos were fixed in Bouins solution for 2 hr, dehydrated through an ethanol series, embedded in par-
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affin at 6O”C, and serially sectioned at 4 pm. Rehydrated slides were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBS-TWBO) and incubated (30 min, 23’C) with a polyclonal or monoclonal antibody in a humid chamber. After washing in PBSTW20, a fluorescein-conjugated secondary antibody appropriate for the primary antibody (rabbit anti-sheep Ig, goat anti-rabbit Ig, or rabbit anti-mouse Ig, Cappel Laboratories; each diluted l/30 in PBS-TW20) was layered onto the slide. The slides were incubated in a humid chamber (30 min, 23’C), washed (PBS-TW20), and then mounted with a glycerol/PBS (9:l) medium. This immunohistological technique identified more than 75% of the antigens which were detectable by the monoclonal antibodies in ELISA assays utilizing the original immunogen. Immunofluorescence was assessed and photographed with a Leitz fluorescent microscope equipped for epifluorescence. McAb cell culture supernatants were used without dilution. PcAb were diluted from stock as follows: rabbit anti-fibronectin l/40; sheep anti-laminin l/80; rabbit anti-heparan sulfate proteoglycan i/400; sheep anti-type I collagen l/80; sheep anti-type III collagen l/80; and sheep anti-type IV collagen 11320. Th,e titers used for each PcAb were chosen so as to achieve the greatest photographic resolution in the ph.&&stage and were then used throughout the developmental sequence of study. The resolution of the immunofluorescence technique is insufficient to distinguish the basal lamina from closely apposed cell membrane antigens or blastocoel material, so that the precise location of an antigen described as “basal laminar” remains undetermined. Nevertheless, the antigens chosen for this study were all primarily localized to the area which included the basal lamina. All experiments described included negative controls using fluorescein-conjugated secondary antibody with primary antibody from preimmune sera, parent myeloma supernatant, nonrelevant McAb, and, in the cases of laminin and fibronectin, PcAb absorbed with the original immunogen. Representative negative controls are shown only in Fig. 1, but all experiments described included these same controls. Other PcAb and McAb specific for nonbasal laminar components were used as a control to ensure that antibodies do not bind nonspecifically to the ECM. These include a cortical granule PcAb and McAbs as described (McClay et &, 1983). Molecules identified by the PcAb in the sea urchin embryo will be referred to as the antigen used in generating the antisera: i.e., laminin, fibronectin, etc. This terminology will simplify the discussion but should not imply any more than a molecular cross-reactivity to known antigen composition. It does suggest the presence of a similar antigen though which may warrant biochemical confirmation.
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inar antigens are not present in the egg and are expressed ok nova in development by mesenchyme cells; Basal Lam&u Compcments are Localized to Granules these will be described later. Within 30 min following in the Un&rtilized Egg insemination, many of the egg granules appeared to Most of the basal laminar antigens identified in this disintegrate and each of the antigens became diffuse study by PcAbs (i.e., fibronectin, heparan sulfate pro- throughout the egg cytoplasm (Fig. lB), except in the teoglycan, laminin, and collagen types I, III, and IV) cortical region of the egg where reduced staining formed a peripheral halo. This pattern was observed for all the and the antigens recognized by McAbs (i.e., ‘7Cll,lBlO, basal laminar antigens found within the egg. The egg lG9, and 8D8) are found within the unfertilized egg. granules containing basal laminar components are disThese molecules were localized to granules ranging in tinct from the cortical granules (Fig. 1D) and from some size from 0.5 to 2 pm and were uniformly distributed throughout the egg cytoplasm (Fig. 1A). Two basal lam- molecules destined for embryonic regions other than RESULTS
FIG. 1. Embryonic extracellular matrix (ECM) components are found within granules of the unfertilized egg. (A, anti-heparan sulfate proteoglycan immunofluorescence). The granules range in size from 0.5 to 2.0 pm and are uniformly distributed throughout the egg cytoplasm. Granules recognized by each of the PcAb and McAb to ECM components are similar in the egg. After fertilization, the granules disperse (B, 30 min postinsemination, anti-heparan sulfate proteoglycan immunofluorescence) and a peripheral halo of reduced stain occurs in the cortical region. To contrast the granule disintegration seen with the antibodies to basal laminar molecules, a molecule identified by McAb 3G5, destined for the embryonic cell surface does not diffuse from its granules until later in development, (C, 1 hr postinsemination, dispersion of granule contents does not occur until the morula stage). Egg granules are distinct from cortical granules (D, anti-hyalin immunofluorescence on unfertilized eggs). Representative negative controls for the antibodies used in the immunofluorescent technique are shown (E and F). Lytechinus variegates unfertilized egg with brightfield insert (E) and gastrula stage with brightfield insert (F) using rabbit anti-fibronectin absorbed with fibronectin. All experiments described included negative controls using fluorescein-conjugated secondary antibody with primary antibody from preimmune sera, parent myeloma supernatant, nonrelevant McAb and in the case of laminin and fibronectin, PcAb absorbed with the original immunogen. X300.
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the basal lamina (e.g., the endoderm). The latter retain their intracellular granular appearance until later in development (Fig. 1C; see also McClay et aL, 1983 for other examples). Figures 1E and F show representative negative controls for the immunofluorescent techniques used. Egg granules containing any one basal laminar component appeared similar to granules containing any other basal laminar components. This suggests that the antigens could be colocalized to the same type of egg granules. As a test of this hypothesis eggs were centrifuged in an isopycnic sucrose gradient to stratify cytoplasmic components using the methods of Harvey (1956). When centrifuged eggs were examined by immunofluorescence, the granules recognized by each of the polyclonal antibodies to basal laminar molecules had relocalized to the centrifugal end of the egg (Fig. 2A). The redistribution of granules was complete within 3 min of centrifugation (10,000~) and was uniform for the six PcAb tested. Granules recognized by basal lamina-directed McAb were redistributed to the same region of the egg (Fig. 2B) in a manner indistinguishable from the patterns of the polyclonal antibodies. However, polyclonal antibodies or McAb specific for molecules destined for embryonic regions other than the basal lamina, such as the cell surface or the hyaline layer, recognized granules that either did not move or were relocalized differently in the centrifuged eggs (Figs. 2C,
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D). These data support the notion that molecules destined for similar embryonic regions are colocalized to similar granules within the unfertilized egg. The Sequence of Basal Lamina Development: Mwula Stage The apparent colocalization of basal laminar molecules in egg granules suggested that these components might arrive at the developing basal lamina at the same time. This possibility is shown not to be the case at the morula stage, when some but not all of the basal laminar components appeared in the lining of the blastocoel. For example, the molecules identified by McAb lBl0 (Fig. 3A) were clearly concentrated to the blastocoel wall. However, molecules recognized by McAb Xl1 (Fig. 3B) were not found in the developing basal lamina at this stage; instead molecules were located preferentially within the apical half of the blastomeres. Other components recognized by the McAb and PcAb redistributed in early development similar to one of these two patterns. Among those in the first group were fibronectin, laminin, types I, III, and IV collagen, and the molecules identified by McAbs lB10, lG9, and SDS. Molecules not found along the blastocoel wall in the morula stage were heparan sulfate proteoglycan, and the molecule identified by McAb7Cll. Thus there appears to be an asynchrony in the addition of components to the developing basal lamina.
FIG. 2. Ieopynic centrifugation of the unfertilized egg pellets the granules containing basal laminar components to the centrifugal end of the dumbbell-shaped egg (A, anti-heparan sulfate proteoglycan immunofluorescence with brightileld insert; B, McAb IBlO immunofluorescence with brightfield insert). The behaviors of the granules recognized by the polyclonal antibodies to heparan sulfate proteoglycan, ilbronectin, laminin, and collagen types I, III, and IV, and the granules recognized by the monoclonal antibodies directed against sea urchin embryonic extracellular matrix components are indistinguishable from each other in the centrifuged egg. In contrast to the behavior of basal laminar antigens are the antigens within granules which are destined for embryonic regions other than the basal lamina (C, McAb 3G5), these granules are not affected by centrifugation. Polyclonal antibodies to hyalin stain the cortical granules of the egg (D), which are not affected by centrifugation. Note: the diffuse fluorescence seen in the centrifugal end of the eggs in C and D is due to autofluorescence of pigment granules. This autofluorescence is not reproduced photographically in A and B due to the intensity of the pelleted egg granules. X300.
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In contrast to the components just described, the molecules identified by McAb 5H5 and IG8 are not present in the blastula (Fig. 45). These antigens do not appear in development until the mesenchyme blastula stage (12 hr) and will be described later. Mesenchgww Blastula Stage
FIG. 3. Morula embryos (4 hr) (A) McAb lB10, antigens are concentrated along the blastocoel wall. (B) McAb 7Cl1, antigens are predominantly within the apical cell regions, and not along the blastocoel wall. Note that nuclei are not stained. X300.
Blustula Stage By the time a mature blastula has formed ECM materials have accumulated at the basal laminar region and/or in the apical region of the embryo (Fig. 4). These staining patterns cannot provide quantitative comparisons of antigen abundance between the antibodies employed because of differences in antibody titers, antigen/ antibody affinity, and retention of antigenicity after fixation. However several distributional similarities and differences can be noted. Some molecules appeared to be distributed both in the basal laminar region and in the apical region of the embryos (Fig. 4). The basal laminar staining in these examples appeared to line the blastocoel wall uniformly. The apical staining of McAb lBl0 (Fig. 4H) and of type IV collagen (Fig. 4F) is confined to intracellular granules. Type III collagen is present along the apical surface of blastomeres (Fig. 4E) as well as the molecule identified by the McAb 7Cll (Fig. 4G). The components identified by McAb lG9 and 8D8 were specific for the basal laminar region (Fig. 41). No apical staining was seen in these embryos and amplification of McAb binding with an additional fluoresceinated step (fluorescein-conjugated goat anti-rabbit to the existing fluorescein-conjugated rabbit anti-mouse McAb) did not detect components outside of the basal lamina but did intensify the basal laminar staining. Heparan sulfate proteoglycan distribution in the blastula stage was unique in that no basal laminar staining was detected (Fig. 4B). Even when higher concentrations of antibodies were used, heparan sulfate proteoglycan was detected only along the outer surface of the embryo.
The ingression of 1” mesenchyme cells into the blastocoel was accompanied by an intensified staining of the basal lamina for most of the antigens (Fig. 5; note that the order of antibody presentation is the same for Figs. 4, 5, and 6). In most cases 1” mesenchyme cells were surrounded by the antigen. In addition there was significant intracellular staining of 1” mesenchyme cells by antifibronectin (Fig. 5A), McAb IG9 (Fig. 51), and McAb’7Cll (Fig. 5G). The immunofluorescent technique is not capable of demonstrating which cells synthesize the basal laminar components, but the significant intracellular staining of mesenchyme cells for these antigens at least suggests that these cells may be in the process,,of antigen synthesis. Heparan sulfate proteoglycan not detected in the basal lamina of the blastula stage (Fig. 4B) becomes prominent on the$nesenchyme cell surfaces and along the basal lamina (Fig. 5B). Pluteus Larval Stage The basal lamina of the pluteus larva (32 hr) appeared as a homogeneous extracellular layer beneath the epithelial layers of endoderm and ectoderm (Fig. 6). It is most clearly delineated by antibodies to fibronectin (Fig. 6A), laminin (Fig. 6C), and type IV collagen (Fig. 6F) and by McAbs lBl0 (Fig. 6H) and lG9 (Fig. 61). The molecules identified by McAb lG9 were exclusive to the basal lamina (Fig. 61). Laminin, type IV collagen, and the molecules identified by McAb lBl0 were concentrated in the basal lamina but had intracellular deposits of antigens as well. These intracellular apical deposits are found in both ectoderm and endoderm. McAb Xl1 (Fig. 6G) and anti-heparan sulfate proteoglycan (Fig. 6B) stain the basal lamina and much of the mesenchymal matrix. The apical location of both of these molecules, most pronounced in the blastula, was not detected in the pluteus stage. A less defined staining pattern was found for type I and type III collagen. Type I collagen retained its apical presence and had a diffuse appearance in the basal laminar region (Fig. 6D). Type III collagen appeared to have decreased in intensity from the mesenchyme blastula stage (Fig. 6E). A novel pattern of expression was observed for the antigen stained by McAb 8D8 (Fig. 6J). This molecule, present throughout the basal lamina in the mesenchyme
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FIG. 4. Blastula embryos (8 br). Immunofluorescence using PcAb to (A) fibronectin, (B) beparan sulfate proteoglycan, (C) laminin, (D) type I collagen, (E) type III collagen, (F) type IV collagen, and (G) McAb Wll, (H) McAb lB10, (I) McAb lG9 (McAb 8D8 not shown, but indistinguishable at this stage from McAb lG9), and (J) McAb 5H5 with brightfield insert (same for McAb lG8). X300. Some molecules have both a basal and apical localization: (A) anti-fibronectin, (C) anti-laminin, (D) anti-type I collagen, (E) anti-type III collagen, (F) anti-type IV collagen, (G) McAb Wll, and (H) McAb 1BlO. Heparan sulfate proteoglycan is not detected within the basal lamina of the blastula (B), whereas the molecules identified by McAba lG9 and 8D8 (I) are already specific to the basal lamina. Some basal laminar molecules are not expressed until later in development, McAb 5H5 and lG8 (J).
blastula, had become localized to the anterior aspect of the embryo associated with the stomadeum. This pattern was unique among the basal laminar antigens studied, and provides further evidence for molecular heterogeneity in the basal laminar environment. The de nmo Appearance of Two Basal Lmninar Components Two components of the basal lamina appeared de nauo in development (Fig. 7). These components were not present in the unfertilized egg, nor during early development (Fig. 7A). They were first expressed by primary mesenchyme cells completing the process of ingression (Fig. ?B, McAb lG8; Fig. 7C McAb 5H5). In the mesenchyme blastula stage, these antigens were detected intracellularly, along the cell surface of the mesenchyme cells, and along the blastocoel wall. In gastrulae, the de
nova molecules detected by McAbs 5H5 and lG8 lined the basal lamina of the endoderm and ectoderm and covered the mesenchymal matrix (Fig. 7D). Western blot analysis indicates that these antigens are distinct from each other (in preparation). Thus two germ layer specific antigens appear within the basal lamina simultaneously at a critical time in development. DISCUSSION
The ontogeny of several basal laminar associated components in the sea urchin embryo has been described by immunofluorescence. Most of the components were found localized to discrete granules within the unfertilized egg. Fertilization initiated the specific trafficking of these antigens through early development. Extracellular deposition of certain basal laminar components began during cleavage, and a complete lining of the
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FIG. 5. Mesenchyme blastula stage embryos (12 hr). Immunofluorescence using PcAb to (A) fibronectin, (B) heparan sulfate proteoglycan, (C) laminin, (D) type I collagen, (E) type III collagen, (F) type IV collagen, and (G) McAb 7Cl1, (H) McAb lB10, (I) McAb lG9, and (J) McAb 8D8, (mag 300X). There is an intensified staining of the basal lamina here for most antigens compared to the blastula stage (Fig. 4). Significant intracellular staining of primary mesenchyme cells is seen for anti-fibronectin (A), McAb 7Cll (G), and McAb lG9 (I). Note the presence of anti-heparan sulfate proteoglycan staining in the basal lamina of the mesenchyme blastula (B), but not in the blastula (Fig. 4B).
blastocoel wall had formed by the blastula stage. Sequential addition of new components to the ECM occurred in the blastula and mesenchyme blastula so that by the pluteus stage the basal lamina was different quantitatively and qualitatively from the earlier stages. Antibodies to laminin and collagen type IV, both known to be specific for the basal lamina in vertebrates, were also localized to the basal lamina of the sea urchin. Other antigens associated with the ECM in vertebrates, i.e., collagen types I and III, fibronectin, and heparan sulfate proteoglycan were similarly localized to the sea urchin ECM. However, PcAb directed against non-basal laminar components (e.g. anticortical granule PcAb) did not stain the basal lamina, though they did stain other embryonic regions. Thus, although the basal laminar components of the sea urchin could be quite different from their vertebrate counterparts, there is a highly conserved histological specificity for each of the crossreacting antibodies.
The ECM and basal laminar components were found to be localized to 2 pm deposits within the unfertilized egg. It is probable that these depositions are surrounded by membranes, but since no limiting membrane could be detected with the technique used the term “granule” has been used here to describe these localizations. Definitive data on the granule structure and heterogeneity will require immunoelectron microscopy. The centrifugation study revealed that the granules of basal laminar components are not anchored within the cytoplasm of the egg since they are easily moved in a centrifugal field. The granules may be similar to the membranebound vesicles termed “heavy bodies” by Afzelius (1957). He described these organelles as dense aggregates of ribonucleoprotein found in the centrifugal end of centrifuged eggs (Afzelius, 1957). The origin and function of heavy bodies is not known, but the RNA of heavy bodies has been proposed to be a source of masked mRNA which functions during early development (Harris, 1967,
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FIG. 6. Pluteus Larvae (32 hr). Immunofluorescence using PcAb to (A) fibronectin, (B) heparan sulfate proteoglycan, (C) laminin, (D) type I collagen, (E) type III collagen, (F) type IV collagen, and (G) McAb 7Cl1, (H) McAb lB10, (I) McAb lG9, and (J) McAb 8D8. X300. Basal laminar staining is most pronounced by antibodies to fibronectin (A), laminin (C), type IV collagen (F), McAb lBl0 (H), and McAb lG9 (I). Apical intracellular deposits of antigens are present in pun&ate localizations [type IV collagen (F), and McAb lBl0 (H)] or have a diffuse appearance [laminin (C) and type I collagen (D)]. The molecules identified by McAb 8D8 (J) are localized to the anterior aspect of the embryo associated with stomadeum.
FIG. 7. The de nbuo expression of two basal laminar antigens. These antigens are not present in the egg nor during early development. (A, hlastula embryo, McAb 5H5 with brightfield insert). The expression of molecules identified by McAb lG8 (B) and McAb 5H5 (C) (mesenchyme blastula, 12 hr) is limited to mesenchyme cells, first apparent during 1” mesenchyme cell ingression. The antigens line the blastocoel wall during the early mesenchyme migration phase, and remain mesenchyme specific through later development (D, McAb 5H5, gastrula). X300.
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Sanchez, 1968). Heavy bodies and the egg granules described here are of the same estimated size (2 pm), both disperse after fertilization (Harris, 196’7),both are mobile within a centrifugal field (Anderson, 1970), and both migrate to the centrifugal end of the egg upon centrifugation (Afzelius, 1957). The fact that the granules containing basal laminar components have a higher density than most other cytoplasmic components is a finding that might be utilized for the isolation of the granules and the characterization of their pre-basal laminar antigens. The redistribution of the basal laminar molecules in early development is an unexplained problem of intracellular trafficking and secretion since initiation of transport of the antigens is delayed until after fertilization and secretion into the extracellular space is deferred until blastulation. The first step in the secretory process is associated with the conversion of the granules to a diffuse intracellular staining pattern. How this change is related to intracellular trafficking is not yet known. The antigens could be partitioned into smaller vesicles or into some diffuse membranous system. The diffuse staining after fertilization does not appear to be an artifact because other antigens in different egg granules (e.g., presumptive endoderm antigens) (Fig. 1D) retained their granular appearance until much later in development. Even though the centrifugation data suggest that the basal laminar components may be colocalized to similar granules in the egg, they appear to be secreted asynchronously into the extracellular matrix. The best evidence for this was seen in the morula stage (Fig. 3). Some antigens have accumulated in the developing basal lamina of the morula (i.e., McAb lBlO), whereas other antigens have not (i.e., McAb i’Cl1, heparan sulfate proteoglycan). Evidence for asynchronous deposition is also seen in the mesenchyme blastula stage. Heparan sulfate proteoglycan, not found within the basal lamina of the blastula, becomes prominent in the basal lamina of the mesenchyme blastula. In addition two molecules, identified by McAbs 5H5 and lG8, are expressed de nouo at the mesenchyme blastula stage. Thus, appearance of molecules in the basal lamina occurs asynchronously from the initial formation of a blastocoel in early cleavage to the ingression of primary mesenchyme cells at the mesenchyme blastula stage. In addition to the basal laminar staining expected of the ECM antigens in the pluteus, some components were found also in either intracellular granules (McAb lB10, type IV collagen, laminin) or on the apical surface of the epithelium (McAb 7Cl1, type I and type III collagens). The intracellular compartments may represent a storage pool of the antigens available for future basal lamina deposition. The antigens appearing on the surface
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of the embryo may be relevant to the “apical” lamina described by Hall and Vacquier (1982). The apical lamina is an extracellular layer on the extraembryonic surface distinct from the hyaline layer. By its association with both the hyaline layer and the apical cell surface, the apical lamina is proposed to be an extracellular substrate for use in morphogenic movements (Hall and Vacquier, 1982). Furthermore, Spiegel and Spiegel have reported the presence of collagenous fibers and glycosaminoglycans (Spiegel and Spiegel, 1979) associated with the hyaline layer. Thus, some ECM components may have a dual interaction with epithelial cells as both apical and basal substrates. Some of the antigens described here have been reported in sea urchin embryos by other investigators. Fibronectin has been identified by immunofluorescence on whole Sphaerechinus granularis embryos (Spiegel et aL, 1980; 1983). After treatment of the embryos with calcium-magnesium-free sea water to remove the hyaline layer, cross-reactivity to anti-human plasma fibronectin was detected at the embryonic surface and between the cells of the blastula and early grastrula. Katow et al (1982), in their immunofluorescent experiments on fixed, halved Lytechinus p&us embryos, found fibronectin associated exclusively with the primary mesenthyme cells early in the migration phase. The conflicting results between Spiegel et al (1980,1983) and Katow et al. (1982) may be due to the different species used, to the different antibodies used, or to differences in antigen accessibilities as a result of specimen preparation. The technique used in the present study (paraffin sections) circumvents an antigen exposure problem since the entire embryo is equally exposed to the antibody. Our results add to the findings of Spiegel et cd (1980, 1983) and Katow et al. (1982) by extending observations from the unfertilized egg to the pluteus larva. The present data support the previous observations in that a basal laminar staining of fibronectin (Spiegel et a& 1983) was present, and a concentration of fibronectin was seen associated with the lo mesenchyme cells (Katow et a.l, 1982). Evidence for the presence of laminin in the sea urchin embryo has also been reported (Spiegel et aL, 1983). Immunofluorescent staining detected laminin in isolated basal laminae and on whole embryos of the prism stage of Sphaerechinus granularis and Arbacia punctuluta, The present data support the finding of laminin in the basal lamina and extend the findings by establishing the developmental distribution from egg to pluteus. Intracellular laminin could not have been detected in the Spiegel et al. (1983) experiments since the epithelial cells were removed in order to expose the basal lamina of the prism-staged embryo. It has been shown in classical sea urchin developmental studies that sulfate is a necessary constituent
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of sea water for gastrulation to occur (Herbst, 1897). Subsequent studies demonstrated a dramatic increase in sulfate incorporation and in synthesis of sulfated glycosaminoglycans in the basal laminar region of the late blastula (Immers, 1956; Runnstrom et aL, 1964). These results led to the hypothesis that sulfated glycosaminoglycans are important for directed mesenthyme cell migration and for the initiation of gastrulation (Karp and Solursh, 1974). Recent biochemical evidence has suggested the presence of sulfated glycosaminoglycans in the mesenchyme blastula, including a significant amount of heparan sulfate (Solursh and Katow, 1982). The immunofluorescent data in this study suggest that heparan sulfate proteoglycan first appeared within the basal lamina at the mesenchyme blastula stage intimately associated with the ingression of lo mesenchyme cells. This suggests that heparan sulfate proteoglycan may be a component implicated by earlier basal laminar sulfation experiments. Collagen has been detected in many invertebrates and is strikingly similar to vertebrate collagens by morphological and biochemical criteria (Adams, 1978). In the sea urchin embryo, evidence based on the detection of hydroxyproline in macromolecules suggests that collagen is present (Pucci-Minafre et aL, 1972; Golob et aL, 1974; Gould and Benson, 1978). In addition, prolylhydroxylase, an enzyme activity required for collagen processing, has been detected in the sea urchin embryo (Benson and Sessions, 1980; Mizoguchi and Yasumasu, 1983). Ultrastructural evidence for collagen in the sea urchin embryo has been described as well. Crise-Benson and Benson (1979) reported that collagen-like striated fibers were found within the blastocoel of prism staged embryos and Spiegel and Spiegel (1979) found striated fibers in the hyaline layer of Arbacia punctulota which were sensitive to collagenase (1 mg/ml), but this has not been supported biochemically (Hall and Vacquier, 1982). The three collagen types studied here (types I, III, and IV) were found within the blastocoel early in development. The pattern of type I collagen staining may represent the fibers reported by Crise-Benson and Benson (1979) since the characteristic banding of vertebrate fibrillar type I collagen is more apparent at the electron microscopic level than it is with other collagen types (Gross, 1974, Gay and Miller, 1978). The decreased intensity in development seen with type III collagen is unique among the patterns observed for basal laminar components and suggests that type III collagen may be important only during early development. This result supports an existing hypothesis that different collagens reflect individual tissue requirements (Adams, 1978). It may be significant that the two basal laminar antigens expressed de nova in development appear at a
VOLUME 103, 1984
time of morphogenetic significance. These new ECM molecules are correlated with the high motility and directionality of the 1” mesenchyme cells, with skeleton formation, and with the initiation of gastrulation. In in vitro cultures of primary mesenchyme cells isolated from the embryo at the 16 cell stage, these antigens were expressed synchronously with those in viva (Fink and McClay, 1984). Thus the expression of these components is programmed in mesenchyme cells by the 16 cell stage, and is controlled independent of the embryo in a pattern which may suggest a role for mesenchymedirected morphogenesis. It is generally accepted that proteins required for early development are either stored in the egg or synthesized on stored mRNAs (Davidson, 1976). The bulk of new transcription becomes activated at around the mesenchyme blastula stage in many species. If one assumes that the egg is parsimonious in what it stores and that stored elements are required early in development, then it must be of some importance to partition a significant portion of the egg volume to the storage of ECM components. If this reasoning is correct, the basal lamina must be required by the embryo well in advance of the mesenchyme blastula stage. Perhaps cavitation of the blastocoel requires the presence of a basal lamina as a substrate for the cells. Alternatively, basal laminar molecules could promote cell polarity from the presumptive basal surface of the cells. This hypothesis is dependent upon the availability of basal lamina molecules in the egg, and the rapid polarized movement of the molecules in development, but does not require the molecules to serve immediately as a substrate. Whatever the reason, it is important enough to demand significant space in the egg. We wish to thank Ms. Kathryn Cates, Ms. Barbara McPartland, and Ms. Gail Cannon for their secretarial and technical assistance and Dr. James Norman Dent for his experience. This work was supported by Grant HD 14483 from the National Institute of Health. REFERENCES ADAMS, E. (1978). Invertebrate collagens. Science X%591-598. AFZELIUS, B. A. (1957). Electron microscopy on the basophilic structure of the sea urchin eggs. Z. Zeyfih. Mikrosk. Anat. 45,660. ANDERSON, E. (1970). A cytological study of the centrifuged whole, half and quarter eggs of the sea urchin Arimku pundtita. Jl Cell Bid 47.711-733. BENSON, S. C., and SESSIONS, A. (1980). Prolyl hydroxylase activity during sea urchin development. Exp. Cell Res. 130,467-470. CRISE-BENSON, N., and BENSON, S. C. (1979). Ultrastructure of collagen in sea urchin embryos. Wilhelm Roux’s Arch 186.65-70. DAVIDSON, E. (1976). “Gene Activity in Early Development.” Academic
Press, New York. DAWSON, R. M. C. (1969). “Data for Biochemical Research.” Oxford
University Press, London. ENDO,Y., and UNO, N. (1960). Intercellular bridges in sea urchin blastula. Zod i&g. 69, 8.
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ET AL.
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ENGVALL,B. E., and PERLMAN,P. (1972). Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzymelabeled anti-immunoglobulin in antigen coated tubes. J. ImmurwL 109,129-135. FINK, R., and MCCLAY, D. R. (1984). Three cell recognition changes accompany the impression of sea urchin primary mesenchyme cells. (submitted) FOIDART,J. M., BERE, E. W., YAAR, M., RENNARD,S., GUILLINO, M., and MARTIN, G. R. (1980). Distribution and immunoelectron microscopic localization of laminin, a non-collagenous basement membrane glycoprotein. Lab Invest. 42,336~342. GALFRE,G., HOWE,S. C., MILSTEIN,C., BUTCHER,G. W., and HOWARD, J. C. (1977). Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature (London) 266, 550-552. GAY, S., and MILLER, E. J. (1978). “Collagen in the Physiology and Pathology of Connective Tissue.” Gustav-Fischer Verlag Publishers, Stuttgart. GIBBONS,J. R., TILNEY, L. G., and PORTER,IS. R. (1969). Microtubules in the formation and development of the primary mesenchyme in Arbacia punctuluta I. The distribution of microtubules. J. CeUBiol 41,201-227. GOLOB,R., CHETSANGA,C. J., and DOTY,P. (1974). The onset of collagen synthesis in sea urchin embryos. Biochim Biophys. Acta 349,135141. GOULD,D., and BENSON,S. C. (1978). Selective inhibition of collagen synthesis in sea urchin embryos by a low concentration of actinomycin D. Exp. CeU Res 112, 73-78. GROSS,J. (1974). Collagen biology, structure, degradation and disease. In “The Harvey Lecture Series,” Series 86, pp. 351-432. Academic Press, New York. GUSTAFSON, T., and WOLPERT,L. (1967).Cellular movement and contact in sea urchin morphogenesis. Biol Rev. 42,442-498. HALL, G., and VACQUIER,V. (1982). The apical lamina of the sea urchin embryo: major glycoproteins associated with the hyaline layer. Den Bid 89,160-178. HARRIS, P. (1967). Structural changes following fertilization in the sea urchin egg. Formation and dissolution of heavy bodies. Exp. Cell Res. 48, 569-581. HARVEY,E. B. (1956). “The American Arhzcziz and Other Sea Urchins.” Princeton University Press, Princeton, New Jersey. HASSEL,J. R., ROBEY,P. G., BARRACH,J. J., WILCZEK,J., RENNARD, S. I., and MARTIN, G. R. (1980). Isolation of a heparan-sulfate containing proteoglycan from basement membrane. Proc Nati Ad Sci USA 77,4494-4498.
HE-ST, C. (1897). Uber die zur entwickelung der seeigellarven nothwendigen anorganischen stoffe, ihre rolle und ihre vertret barkeit. I. Die zur entwickelung nothwendigen anorganischen &off. Wilhelm Roux’s Arch, 5,649-793.
IMMERS,J. (1956). Cytological features of the development of the eggs of Paracentrotus lividus reared in artificial sea water devoid of sulfate ions. Exp Cell Res. 10.546-548. KARP, G. C., and SOLURSH,M. (1974). Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo. Dev. BioL 41.110-123.
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KATOW, H., YAMADA, K. M., and SOLURSH,M. (1982). Occurrence of fibronectin on the primary mesenchyme cell surface during migration 22,120-124. in the sea urchin embryo. ~ermtiutim LI~EFIELD, J. W. (1964). Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710. M&LAY, D. R., CANNON,G. W., WESSEL,G. M., FINK, R. D., and MARCHASE,R. B. (1983). Patterns of Antigenic Expression in Early Sea Urchin Development. In “Time, Space, and Pattern in Embryonic Development” (W. Jeffries and R. Raff, eds.), pp. 157-169. Liss, New York. MIZOGUCHI,H., and YAZUMASU,I. (1983). Inhibition of archenteron formation of the inhibitors of prolyl hydroxylase in sea urchin embryos. Cell m 12, 225-231. OKAZAKI, K., and NIIJIMA, L. (1964). “Basement membrane” in sea urchin larvae. EmLnyologia 8,89-100. PUCCI-MINAFRA,I., CASANO,C., and LA ROSA,C. (1972). Collagen synthesis and spicule formation in sea urchin embryos. Cell D$ 1, 157-165. RLJNNSTROM, J., HORSTADIUS,S., IMMERS,J., and FUDGE-MESTF~ANGELO, M. (1964). An analysis of the role of sulfate in the embryonic differentiation of the sea urchin (Paracentrotua lividzcs). Rev. Suisse ZOOL71,21. SANCHEZ,S. (1968). Effects de l’actinomycine D sur lea constituents cellulaires et la metabolisme de I’ARN de l’ovocyte d’oursin (Paracentrotos lividus). Exp. Cell Res. 50, 19-31. SOLURSH,M., and KATOW,H. (1982). Initial characterization of sulfated macromolecules in blastocoels of mesenchyme blastulae of S&ong&nxntrotus purpuratus and Lytechinus pi&us. Dev. Biol 94,326336. SPIEGEL,E., and SPIEGEL,M. (1979). The hyaline layer is a collagencontaining extracellular matrix in sea urchin embryos and reaggregating cells. Exp. Cell Res. 123, 434-441. SPIEGEL,E., BURGER,M., and SPIEGEL,M. (1980). Fibronectin in the developing sea urchin. J. CeU Biol 87, 309-313. SPIEGEL,E., BURGER,M., and SPIEGEL, M. (1983). Fibronectin and Laminin in the Extracellular Matrix and Basement Membrane of Sea Urchin Embryos. Exp. CeU Res. 144,47-55. TIMPL, R., WICK, G., and GAY, S. (1977). Antibodies to distinct types of collagens and their application in immunohistology. J. ImmunoL Methods 18, 165-182. TIMPL, R., GLANVILLE, R. W., WICK, G., and MARTIN, G. R. (1979a). Immunochemical study on basement membrane (type IV) collagens. Immunology 38,109-116. TIMPL, T., ROHDE,H., ROBEY,P. G., RENNARD,S. I., FOIDART,J. M., and MARTIN, G. R. (1979b). Laminin-a glycoprotein from basement membranes. J. BioL Chem 254,9933-9937. WICK, G., GLANVILLE, R. W., and TIMPL, T. (1979). Characterization of antibodies to basement membrane (types IV) collagen in immunohistological studies. Immwwbi&gg 156, 372-381. WOLPERT,L., and MERCER,E. H. (1963). An electronmicroscope study of the development of the blastula of the sea urchin embryo and its radial polarity. Ezp. Cell. Res. 30, 230-300.
BIOLOGY
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Ontogeny of the Basal Lamina in the Sea Urchin Embryo GARY M. WESSEL,* RICHARD B. MARCHASE,* AND DAVID R. MCCLAYF’ *Lkpartment qf Anatomy, Duke University l&dim1 Center, and flkprtmat
of Zoology,Duke University, Durham, North Caroling 27706
Received July 26, 1983;accepted in revised form Jarwarp 9, 1984 The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen typesI, III, and IV, and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 pm) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de luluo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.
the cell surfaces of the sea urchin embryo. Evidence for the presence of glycosaminoglycans, including heparan Nearly 20 years ago Gustafson and Wolpert (1967) sulfate and chondroitin sulfate, was suggested by biopostulated that interactions between the basal lamina chemical data (Solursh and Katow, 1982). The existence and blastomeres were crucial to gastrulation in the sea of collagen in the sea urchin embryo was suggested by urchin embryo. At the mesenchyme blastula stage of ultrastructural data (Crise-Benson and Benson, 1979; development, primary mesenchyme cells pass through Spiegel and Spiegel, 1979), by detection of the collagen the basal lamina to invade the blastocoelic compartment. processing enzyme prolyl hydroxylase (Benson and SesThese cells then use the basal lamina as a substrate for sions, 1980; Mizoguchi and Yasumasu, 1983), and by the a series of characteristic migrations that culminate with presence of hydroxyproline in macromolecules (Puccithe cells coalescing at specific sites to produce the larval Minafra et CL, 1972; Golob et aL, 1974; Gould and Benson, skeleton. Meanwhile, midway through archenteron for1978). There is not, however, any direct biochemical evmation secondary mesenchyme cells extend long filoidence to demonstrate the presence of collagen in the podia that contact and attach to the basal lamina. Gussea urchin embryo. tafson and Wolpert (1967) postulated that these contacts, The purpose of the present study is to trace the followed by mesenchyme cell contraction, were meexpression of extracellular matrix (ECM) components chanically important for completion of the archenteron. through embryonic development and to ascertain the Several recent studies have suggested that the basal general pattern of basal lamina formation. An immulamina-extracellular matrix complex that lines the nofluorescent technique was employed on paraffin-secblastocoel in the sea urchin embryo is similar to that tioned material using polyclonal antibodies (PcAb) and found in vertebrates. The presence of a basal lamina in monoclonal antibodies (McAb). The PcAb used were the sea urchin embryo was first suggested by electron specific for the known vertebrate extracellular matrix microscopic studies (Endo and Uno, 1960; Wolpert and molecules: fibronectin, heparan sulfate proteoglycan, Mercer, 1963; Okazaki and Niijima, 1964; Gibbons et uL, laminin, and collagen types I, III, and IV. The McAb 1969) and was identified as a lo-nm-thick extracellular used were generated against sea urchin embryonic comstructure found along the blastocoel wall. Immunofluponents. The components studied were selected on the orescent data have suggested that presence of both a basis of antibody binding to the basal lamina in the fibronectin-like molecule (Spiegel et aL, 1980; Katow et pluteus larval stage. Most components examined were oL, 1982; Spiegel et CCL, 1983) and a laminin-like molecule found within the unfertilized egg. Our results suggest (Spiegel et aL, 1983) within the basal lamina and along a coordinated pattern of assembly, redistribution, and perhaps synthesis of these antigens during early de1To whom correspondence should be addressed. velopment leading to the basal laminar structure. We INTRODUCTION
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also report two basal laminar antigens expressed de luyuo by the mesenchyme cells at the mesenchyme blastula stage. METHODS
Gametes of Lqtechinus variegates were obtained by injection with 0.5 iM KCl. Eggs were washed five times in artificial seawater (Dawson, 1969) and fertilized with a dilute sperm suspension. Source of Antibodies Pol&mal antibodies. Antibodies to human plasma fibronectin were purchased from Cappel Laboratories. Antibodies to collagen types I, III, and IV and to laminin and heparan sulfate proteoglycan were gifts from H. Kleinman and G. R. Martin of the National Institute of Dental Health. These antibodies were raised in rabbits or sheep, and the specificity of each antibody had been previously verified by RIA, ELISA, Ouchterlony immunodiffusion, immunoelectrophoresis, and/or immunofluorescent blocking experiments as described (Timpl et aC, 1977,1979a,b, Wick et d, 1979; Foidart et a& 1980; Hassell et a+!, 1980). Monoclmal antibodies. BALB c/j3 mice were injected intraperitoneally with 100 pg of the insoluble fraction of Lytechinus variegates (52 hr pluteus) embryos following detergent extraction (0.1% Triton X-100,10 mM NaHCOs, 0.01% PMSF, and 15 pg/ml DNase). The detergent extraction solubilized cells but much of the extracellular matrix of the embryo remained intact, thus allowing for an enrichment of components of the basal lamina, and/or macromolecules attached to the basal lamina. After 10 days the mice were boosted with a second 100 pg injection of the immunogen. Some mice were injected on a monthly basis for 3 months, and in each case mice were sacrificed 3 days after a boost injection. Spleens were obtained from mice by sterile dissection. Lymphocyte-myeloma hybridomas were generated according to the protocol of Galfre et al. (1977). The myeloma parent was a HAT-sensitive P3X63AgS line obtained from Dr. George Eisenbarth of the Harvard School of Medicine. Hybridomas were raised in HAT medium according to Littlefield (1964). Supernatants of hybrid colonies were screened for anti-sea urchin extracellular matrix antibody production using an ELISA assay (Engvall and Perlmann, 1972) with the original immunogen and/or by immunofluorescence, and the resulting positive hybridomas were cloned.
Embryos were fixed in Bouins solution for 2 hr, dehydrated through an ethanol series, embedded in par-
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affin at 6O”C, and serially sectioned at 4 pm. Rehydrated slides were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBS-TWBO) and incubated (30 min, 23’C) with a polyclonal or monoclonal antibody in a humid chamber. After washing in PBSTW20, a fluorescein-conjugated secondary antibody appropriate for the primary antibody (rabbit anti-sheep Ig, goat anti-rabbit Ig, or rabbit anti-mouse Ig, Cappel Laboratories; each diluted l/30 in PBS-TW20) was layered onto the slide. The slides were incubated in a humid chamber (30 min, 23’C), washed (PBS-TW20), and then mounted with a glycerol/PBS (9:l) medium. This immunohistological technique identified more than 75% of the antigens which were detectable by the monoclonal antibodies in ELISA assays utilizing the original immunogen. Immunofluorescence was assessed and photographed with a Leitz fluorescent microscope equipped for epifluorescence. McAb cell culture supernatants were used without dilution. PcAb were diluted from stock as follows: rabbit anti-fibronectin l/40; sheep anti-laminin l/80; rabbit anti-heparan sulfate proteoglycan i/400; sheep anti-type I collagen l/80; sheep anti-type III collagen l/80; and sheep anti-type IV collagen 11320. Th,e titers used for each PcAb were chosen so as to achieve the greatest photographic resolution in the ph.&&stage and were then used throughout the developmental sequence of study. The resolution of the immunofluorescence technique is insufficient to distinguish the basal lamina from closely apposed cell membrane antigens or blastocoel material, so that the precise location of an antigen described as “basal laminar” remains undetermined. Nevertheless, the antigens chosen for this study were all primarily localized to the area which included the basal lamina. All experiments described included negative controls using fluorescein-conjugated secondary antibody with primary antibody from preimmune sera, parent myeloma supernatant, nonrelevant McAb, and, in the cases of laminin and fibronectin, PcAb absorbed with the original immunogen. Representative negative controls are shown only in Fig. 1, but all experiments described included these same controls. Other PcAb and McAb specific for nonbasal laminar components were used as a control to ensure that antibodies do not bind nonspecifically to the ECM. These include a cortical granule PcAb and McAbs as described (McClay et &, 1983). Molecules identified by the PcAb in the sea urchin embryo will be referred to as the antigen used in generating the antisera: i.e., laminin, fibronectin, etc. This terminology will simplify the discussion but should not imply any more than a molecular cross-reactivity to known antigen composition. It does suggest the presence of a similar antigen though which may warrant biochemical confirmation.
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inar antigens are not present in the egg and are expressed ok nova in development by mesenchyme cells; Basal Lam&u Compcments are Localized to Granules these will be described later. Within 30 min following in the Un&rtilized Egg insemination, many of the egg granules appeared to Most of the basal laminar antigens identified in this disintegrate and each of the antigens became diffuse study by PcAbs (i.e., fibronectin, heparan sulfate pro- throughout the egg cytoplasm (Fig. lB), except in the teoglycan, laminin, and collagen types I, III, and IV) cortical region of the egg where reduced staining formed a peripheral halo. This pattern was observed for all the and the antigens recognized by McAbs (i.e., ‘7Cll,lBlO, basal laminar antigens found within the egg. The egg lG9, and 8D8) are found within the unfertilized egg. granules containing basal laminar components are disThese molecules were localized to granules ranging in tinct from the cortical granules (Fig. 1D) and from some size from 0.5 to 2 pm and were uniformly distributed throughout the egg cytoplasm (Fig. 1A). Two basal lam- molecules destined for embryonic regions other than RESULTS
FIG. 1. Embryonic extracellular matrix (ECM) components are found within granules of the unfertilized egg. (A, anti-heparan sulfate proteoglycan immunofluorescence). The granules range in size from 0.5 to 2.0 pm and are uniformly distributed throughout the egg cytoplasm. Granules recognized by each of the PcAb and McAb to ECM components are similar in the egg. After fertilization, the granules disperse (B, 30 min postinsemination, anti-heparan sulfate proteoglycan immunofluorescence) and a peripheral halo of reduced stain occurs in the cortical region. To contrast the granule disintegration seen with the antibodies to basal laminar molecules, a molecule identified by McAb 3G5, destined for the embryonic cell surface does not diffuse from its granules until later in development, (C, 1 hr postinsemination, dispersion of granule contents does not occur until the morula stage). Egg granules are distinct from cortical granules (D, anti-hyalin immunofluorescence on unfertilized eggs). Representative negative controls for the antibodies used in the immunofluorescent technique are shown (E and F). Lytechinus variegates unfertilized egg with brightfield insert (E) and gastrula stage with brightfield insert (F) using rabbit anti-fibronectin absorbed with fibronectin. All experiments described included negative controls using fluorescein-conjugated secondary antibody with primary antibody from preimmune sera, parent myeloma supernatant, nonrelevant McAb and in the case of laminin and fibronectin, PcAb absorbed with the original immunogen. X300.
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the basal lamina (e.g., the endoderm). The latter retain their intracellular granular appearance until later in development (Fig. 1C; see also McClay et aL, 1983 for other examples). Figures 1E and F show representative negative controls for the immunofluorescent techniques used. Egg granules containing any one basal laminar component appeared similar to granules containing any other basal laminar components. This suggests that the antigens could be colocalized to the same type of egg granules. As a test of this hypothesis eggs were centrifuged in an isopycnic sucrose gradient to stratify cytoplasmic components using the methods of Harvey (1956). When centrifuged eggs were examined by immunofluorescence, the granules recognized by each of the polyclonal antibodies to basal laminar molecules had relocalized to the centrifugal end of the egg (Fig. 2A). The redistribution of granules was complete within 3 min of centrifugation (10,000~) and was uniform for the six PcAb tested. Granules recognized by basal lamina-directed McAb were redistributed to the same region of the egg (Fig. 2B) in a manner indistinguishable from the patterns of the polyclonal antibodies. However, polyclonal antibodies or McAb specific for molecules destined for embryonic regions other than the basal lamina, such as the cell surface or the hyaline layer, recognized granules that either did not move or were relocalized differently in the centrifuged eggs (Figs. 2C,
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D). These data support the notion that molecules destined for similar embryonic regions are colocalized to similar granules within the unfertilized egg. The Sequence of Basal Lamina Development: Mwula Stage The apparent colocalization of basal laminar molecules in egg granules suggested that these components might arrive at the developing basal lamina at the same time. This possibility is shown not to be the case at the morula stage, when some but not all of the basal laminar components appeared in the lining of the blastocoel. For example, the molecules identified by McAb lBl0 (Fig. 3A) were clearly concentrated to the blastocoel wall. However, molecules recognized by McAb Xl1 (Fig. 3B) were not found in the developing basal lamina at this stage; instead molecules were located preferentially within the apical half of the blastomeres. Other components recognized by the McAb and PcAb redistributed in early development similar to one of these two patterns. Among those in the first group were fibronectin, laminin, types I, III, and IV collagen, and the molecules identified by McAbs lB10, lG9, and SDS. Molecules not found along the blastocoel wall in the morula stage were heparan sulfate proteoglycan, and the molecule identified by McAb7Cll. Thus there appears to be an asynchrony in the addition of components to the developing basal lamina.
FIG. 2. Ieopynic centrifugation of the unfertilized egg pellets the granules containing basal laminar components to the centrifugal end of the dumbbell-shaped egg (A, anti-heparan sulfate proteoglycan immunofluorescence with brightileld insert; B, McAb IBlO immunofluorescence with brightfield insert). The behaviors of the granules recognized by the polyclonal antibodies to heparan sulfate proteoglycan, ilbronectin, laminin, and collagen types I, III, and IV, and the granules recognized by the monoclonal antibodies directed against sea urchin embryonic extracellular matrix components are indistinguishable from each other in the centrifuged egg. In contrast to the behavior of basal laminar antigens are the antigens within granules which are destined for embryonic regions other than the basal lamina (C, McAb 3G5), these granules are not affected by centrifugation. Polyclonal antibodies to hyalin stain the cortical granules of the egg (D), which are not affected by centrifugation. Note: the diffuse fluorescence seen in the centrifugal end of the eggs in C and D is due to autofluorescence of pigment granules. This autofluorescence is not reproduced photographically in A and B due to the intensity of the pelleted egg granules. X300.
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In contrast to the components just described, the molecules identified by McAb 5H5 and IG8 are not present in the blastula (Fig. 45). These antigens do not appear in development until the mesenchyme blastula stage (12 hr) and will be described later. Mesenchgww Blastula Stage
FIG. 3. Morula embryos (4 hr) (A) McAb lB10, antigens are concentrated along the blastocoel wall. (B) McAb 7Cl1, antigens are predominantly within the apical cell regions, and not along the blastocoel wall. Note that nuclei are not stained. X300.
Blustula Stage By the time a mature blastula has formed ECM materials have accumulated at the basal laminar region and/or in the apical region of the embryo (Fig. 4). These staining patterns cannot provide quantitative comparisons of antigen abundance between the antibodies employed because of differences in antibody titers, antigen/ antibody affinity, and retention of antigenicity after fixation. However several distributional similarities and differences can be noted. Some molecules appeared to be distributed both in the basal laminar region and in the apical region of the embryos (Fig. 4). The basal laminar staining in these examples appeared to line the blastocoel wall uniformly. The apical staining of McAb lBl0 (Fig. 4H) and of type IV collagen (Fig. 4F) is confined to intracellular granules. Type III collagen is present along the apical surface of blastomeres (Fig. 4E) as well as the molecule identified by the McAb 7Cll (Fig. 4G). The components identified by McAb lG9 and 8D8 were specific for the basal laminar region (Fig. 41). No apical staining was seen in these embryos and amplification of McAb binding with an additional fluoresceinated step (fluorescein-conjugated goat anti-rabbit to the existing fluorescein-conjugated rabbit anti-mouse McAb) did not detect components outside of the basal lamina but did intensify the basal laminar staining. Heparan sulfate proteoglycan distribution in the blastula stage was unique in that no basal laminar staining was detected (Fig. 4B). Even when higher concentrations of antibodies were used, heparan sulfate proteoglycan was detected only along the outer surface of the embryo.
The ingression of 1” mesenchyme cells into the blastocoel was accompanied by an intensified staining of the basal lamina for most of the antigens (Fig. 5; note that the order of antibody presentation is the same for Figs. 4, 5, and 6). In most cases 1” mesenchyme cells were surrounded by the antigen. In addition there was significant intracellular staining of 1” mesenchyme cells by antifibronectin (Fig. 5A), McAb IG9 (Fig. 51), and McAb’7Cll (Fig. 5G). The immunofluorescent technique is not capable of demonstrating which cells synthesize the basal laminar components, but the significant intracellular staining of mesenchyme cells for these antigens at least suggests that these cells may be in the process,,of antigen synthesis. Heparan sulfate proteoglycan not detected in the basal lamina of the blastula stage (Fig. 4B) becomes prominent on the$nesenchyme cell surfaces and along the basal lamina (Fig. 5B). Pluteus Larval Stage The basal lamina of the pluteus larva (32 hr) appeared as a homogeneous extracellular layer beneath the epithelial layers of endoderm and ectoderm (Fig. 6). It is most clearly delineated by antibodies to fibronectin (Fig. 6A), laminin (Fig. 6C), and type IV collagen (Fig. 6F) and by McAbs lBl0 (Fig. 6H) and lG9 (Fig. 61). The molecules identified by McAb lG9 were exclusive to the basal lamina (Fig. 61). Laminin, type IV collagen, and the molecules identified by McAb lBl0 were concentrated in the basal lamina but had intracellular deposits of antigens as well. These intracellular apical deposits are found in both ectoderm and endoderm. McAb Xl1 (Fig. 6G) and anti-heparan sulfate proteoglycan (Fig. 6B) stain the basal lamina and much of the mesenchymal matrix. The apical location of both of these molecules, most pronounced in the blastula, was not detected in the pluteus stage. A less defined staining pattern was found for type I and type III collagen. Type I collagen retained its apical presence and had a diffuse appearance in the basal laminar region (Fig. 6D). Type III collagen appeared to have decreased in intensity from the mesenchyme blastula stage (Fig. 6E). A novel pattern of expression was observed for the antigen stained by McAb 8D8 (Fig. 6J). This molecule, present throughout the basal lamina in the mesenchyme
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FIG. 4. Blastula embryos (8 br). Immunofluorescence using PcAb to (A) fibronectin, (B) beparan sulfate proteoglycan, (C) laminin, (D) type I collagen, (E) type III collagen, (F) type IV collagen, and (G) McAb Wll, (H) McAb lB10, (I) McAb lG9 (McAb 8D8 not shown, but indistinguishable at this stage from McAb lG9), and (J) McAb 5H5 with brightfield insert (same for McAb lG8). X300. Some molecules have both a basal and apical localization: (A) anti-fibronectin, (C) anti-laminin, (D) anti-type I collagen, (E) anti-type III collagen, (F) anti-type IV collagen, (G) McAb Wll, and (H) McAb 1BlO. Heparan sulfate proteoglycan is not detected within the basal lamina of the blastula (B), whereas the molecules identified by McAba lG9 and 8D8 (I) are already specific to the basal lamina. Some basal laminar molecules are not expressed until later in development, McAb 5H5 and lG8 (J).
blastula, had become localized to the anterior aspect of the embryo associated with the stomadeum. This pattern was unique among the basal laminar antigens studied, and provides further evidence for molecular heterogeneity in the basal laminar environment. The de nmo Appearance of Two Basal Lmninar Components Two components of the basal lamina appeared de nauo in development (Fig. 7). These components were not present in the unfertilized egg, nor during early development (Fig. 7A). They were first expressed by primary mesenchyme cells completing the process of ingression (Fig. ?B, McAb lG8; Fig. 7C McAb 5H5). In the mesenchyme blastula stage, these antigens were detected intracellularly, along the cell surface of the mesenchyme cells, and along the blastocoel wall. In gastrulae, the de
nova molecules detected by McAbs 5H5 and lG8 lined the basal lamina of the endoderm and ectoderm and covered the mesenchymal matrix (Fig. 7D). Western blot analysis indicates that these antigens are distinct from each other (in preparation). Thus two germ layer specific antigens appear within the basal lamina simultaneously at a critical time in development. DISCUSSION
The ontogeny of several basal laminar associated components in the sea urchin embryo has been described by immunofluorescence. Most of the components were found localized to discrete granules within the unfertilized egg. Fertilization initiated the specific trafficking of these antigens through early development. Extracellular deposition of certain basal laminar components began during cleavage, and a complete lining of the
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FIG. 5. Mesenchyme blastula stage embryos (12 hr). Immunofluorescence using PcAb to (A) fibronectin, (B) heparan sulfate proteoglycan, (C) laminin, (D) type I collagen, (E) type III collagen, (F) type IV collagen, and (G) McAb 7Cl1, (H) McAb lB10, (I) McAb lG9, and (J) McAb 8D8, (mag 300X). There is an intensified staining of the basal lamina here for most antigens compared to the blastula stage (Fig. 4). Significant intracellular staining of primary mesenchyme cells is seen for anti-fibronectin (A), McAb 7Cll (G), and McAb lG9 (I). Note the presence of anti-heparan sulfate proteoglycan staining in the basal lamina of the mesenchyme blastula (B), but not in the blastula (Fig. 4B).
blastocoel wall had formed by the blastula stage. Sequential addition of new components to the ECM occurred in the blastula and mesenchyme blastula so that by the pluteus stage the basal lamina was different quantitatively and qualitatively from the earlier stages. Antibodies to laminin and collagen type IV, both known to be specific for the basal lamina in vertebrates, were also localized to the basal lamina of the sea urchin. Other antigens associated with the ECM in vertebrates, i.e., collagen types I and III, fibronectin, and heparan sulfate proteoglycan were similarly localized to the sea urchin ECM. However, PcAb directed against non-basal laminar components (e.g. anticortical granule PcAb) did not stain the basal lamina, though they did stain other embryonic regions. Thus, although the basal laminar components of the sea urchin could be quite different from their vertebrate counterparts, there is a highly conserved histological specificity for each of the crossreacting antibodies.
The ECM and basal laminar components were found to be localized to 2 pm deposits within the unfertilized egg. It is probable that these depositions are surrounded by membranes, but since no limiting membrane could be detected with the technique used the term “granule” has been used here to describe these localizations. Definitive data on the granule structure and heterogeneity will require immunoelectron microscopy. The centrifugation study revealed that the granules of basal laminar components are not anchored within the cytoplasm of the egg since they are easily moved in a centrifugal field. The granules may be similar to the membranebound vesicles termed “heavy bodies” by Afzelius (1957). He described these organelles as dense aggregates of ribonucleoprotein found in the centrifugal end of centrifuged eggs (Afzelius, 1957). The origin and function of heavy bodies is not known, but the RNA of heavy bodies has been proposed to be a source of masked mRNA which functions during early development (Harris, 1967,
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FIG. 6. Pluteus Larvae (32 hr). Immunofluorescence using PcAb to (A) fibronectin, (B) heparan sulfate proteoglycan, (C) laminin, (D) type I collagen, (E) type III collagen, (F) type IV collagen, and (G) McAb 7Cl1, (H) McAb lB10, (I) McAb lG9, and (J) McAb 8D8. X300. Basal laminar staining is most pronounced by antibodies to fibronectin (A), laminin (C), type IV collagen (F), McAb lBl0 (H), and McAb lG9 (I). Apical intracellular deposits of antigens are present in pun&ate localizations [type IV collagen (F), and McAb lBl0 (H)] or have a diffuse appearance [laminin (C) and type I collagen (D)]. The molecules identified by McAb 8D8 (J) are localized to the anterior aspect of the embryo associated with stomadeum.
FIG. 7. The de nbuo expression of two basal laminar antigens. These antigens are not present in the egg nor during early development. (A, hlastula embryo, McAb 5H5 with brightfield insert). The expression of molecules identified by McAb lG8 (B) and McAb 5H5 (C) (mesenchyme blastula, 12 hr) is limited to mesenchyme cells, first apparent during 1” mesenchyme cell ingression. The antigens line the blastocoel wall during the early mesenchyme migration phase, and remain mesenchyme specific through later development (D, McAb 5H5, gastrula). X300.
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Sanchez, 1968). Heavy bodies and the egg granules described here are of the same estimated size (2 pm), both disperse after fertilization (Harris, 196’7),both are mobile within a centrifugal field (Anderson, 1970), and both migrate to the centrifugal end of the egg upon centrifugation (Afzelius, 1957). The fact that the granules containing basal laminar components have a higher density than most other cytoplasmic components is a finding that might be utilized for the isolation of the granules and the characterization of their pre-basal laminar antigens. The redistribution of the basal laminar molecules in early development is an unexplained problem of intracellular trafficking and secretion since initiation of transport of the antigens is delayed until after fertilization and secretion into the extracellular space is deferred until blastulation. The first step in the secretory process is associated with the conversion of the granules to a diffuse intracellular staining pattern. How this change is related to intracellular trafficking is not yet known. The antigens could be partitioned into smaller vesicles or into some diffuse membranous system. The diffuse staining after fertilization does not appear to be an artifact because other antigens in different egg granules (e.g., presumptive endoderm antigens) (Fig. 1D) retained their granular appearance until much later in development. Even though the centrifugation data suggest that the basal laminar components may be colocalized to similar granules in the egg, they appear to be secreted asynchronously into the extracellular matrix. The best evidence for this was seen in the morula stage (Fig. 3). Some antigens have accumulated in the developing basal lamina of the morula (i.e., McAb lBlO), whereas other antigens have not (i.e., McAb i’Cl1, heparan sulfate proteoglycan). Evidence for asynchronous deposition is also seen in the mesenchyme blastula stage. Heparan sulfate proteoglycan, not found within the basal lamina of the blastula, becomes prominent in the basal lamina of the mesenchyme blastula. In addition two molecules, identified by McAbs 5H5 and lG8, are expressed de nouo at the mesenchyme blastula stage. Thus, appearance of molecules in the basal lamina occurs asynchronously from the initial formation of a blastocoel in early cleavage to the ingression of primary mesenchyme cells at the mesenchyme blastula stage. In addition to the basal laminar staining expected of the ECM antigens in the pluteus, some components were found also in either intracellular granules (McAb lB10, type IV collagen, laminin) or on the apical surface of the epithelium (McAb 7Cl1, type I and type III collagens). The intracellular compartments may represent a storage pool of the antigens available for future basal lamina deposition. The antigens appearing on the surface
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of the embryo may be relevant to the “apical” lamina described by Hall and Vacquier (1982). The apical lamina is an extracellular layer on the extraembryonic surface distinct from the hyaline layer. By its association with both the hyaline layer and the apical cell surface, the apical lamina is proposed to be an extracellular substrate for use in morphogenic movements (Hall and Vacquier, 1982). Furthermore, Spiegel and Spiegel have reported the presence of collagenous fibers and glycosaminoglycans (Spiegel and Spiegel, 1979) associated with the hyaline layer. Thus, some ECM components may have a dual interaction with epithelial cells as both apical and basal substrates. Some of the antigens described here have been reported in sea urchin embryos by other investigators. Fibronectin has been identified by immunofluorescence on whole Sphaerechinus granularis embryos (Spiegel et aL, 1980; 1983). After treatment of the embryos with calcium-magnesium-free sea water to remove the hyaline layer, cross-reactivity to anti-human plasma fibronectin was detected at the embryonic surface and between the cells of the blastula and early grastrula. Katow et al (1982), in their immunofluorescent experiments on fixed, halved Lytechinus p&us embryos, found fibronectin associated exclusively with the primary mesenthyme cells early in the migration phase. The conflicting results between Spiegel et al (1980,1983) and Katow et al. (1982) may be due to the different species used, to the different antibodies used, or to differences in antigen accessibilities as a result of specimen preparation. The technique used in the present study (paraffin sections) circumvents an antigen exposure problem since the entire embryo is equally exposed to the antibody. Our results add to the findings of Spiegel et cd (1980, 1983) and Katow et al. (1982) by extending observations from the unfertilized egg to the pluteus larva. The present data support the previous observations in that a basal laminar staining of fibronectin (Spiegel et a& 1983) was present, and a concentration of fibronectin was seen associated with the lo mesenchyme cells (Katow et a.l, 1982). Evidence for the presence of laminin in the sea urchin embryo has also been reported (Spiegel et aL, 1983). Immunofluorescent staining detected laminin in isolated basal laminae and on whole embryos of the prism stage of Sphaerechinus granularis and Arbacia punctuluta, The present data support the finding of laminin in the basal lamina and extend the findings by establishing the developmental distribution from egg to pluteus. Intracellular laminin could not have been detected in the Spiegel et al. (1983) experiments since the epithelial cells were removed in order to expose the basal lamina of the prism-staged embryo. It has been shown in classical sea urchin developmental studies that sulfate is a necessary constituent
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of sea water for gastrulation to occur (Herbst, 1897). Subsequent studies demonstrated a dramatic increase in sulfate incorporation and in synthesis of sulfated glycosaminoglycans in the basal laminar region of the late blastula (Immers, 1956; Runnstrom et aL, 1964). These results led to the hypothesis that sulfated glycosaminoglycans are important for directed mesenthyme cell migration and for the initiation of gastrulation (Karp and Solursh, 1974). Recent biochemical evidence has suggested the presence of sulfated glycosaminoglycans in the mesenchyme blastula, including a significant amount of heparan sulfate (Solursh and Katow, 1982). The immunofluorescent data in this study suggest that heparan sulfate proteoglycan first appeared within the basal lamina at the mesenchyme blastula stage intimately associated with the ingression of lo mesenchyme cells. This suggests that heparan sulfate proteoglycan may be a component implicated by earlier basal laminar sulfation experiments. Collagen has been detected in many invertebrates and is strikingly similar to vertebrate collagens by morphological and biochemical criteria (Adams, 1978). In the sea urchin embryo, evidence based on the detection of hydroxyproline in macromolecules suggests that collagen is present (Pucci-Minafre et aL, 1972; Golob et aL, 1974; Gould and Benson, 1978). In addition, prolylhydroxylase, an enzyme activity required for collagen processing, has been detected in the sea urchin embryo (Benson and Sessions, 1980; Mizoguchi and Yasumasu, 1983). Ultrastructural evidence for collagen in the sea urchin embryo has been described as well. Crise-Benson and Benson (1979) reported that collagen-like striated fibers were found within the blastocoel of prism staged embryos and Spiegel and Spiegel (1979) found striated fibers in the hyaline layer of Arbacia punctulota which were sensitive to collagenase (1 mg/ml), but this has not been supported biochemically (Hall and Vacquier, 1982). The three collagen types studied here (types I, III, and IV) were found within the blastocoel early in development. The pattern of type I collagen staining may represent the fibers reported by Crise-Benson and Benson (1979) since the characteristic banding of vertebrate fibrillar type I collagen is more apparent at the electron microscopic level than it is with other collagen types (Gross, 1974, Gay and Miller, 1978). The decreased intensity in development seen with type III collagen is unique among the patterns observed for basal laminar components and suggests that type III collagen may be important only during early development. This result supports an existing hypothesis that different collagens reflect individual tissue requirements (Adams, 1978). It may be significant that the two basal laminar antigens expressed de nova in development appear at a
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time of morphogenetic significance. These new ECM molecules are correlated with the high motility and directionality of the 1” mesenchyme cells, with skeleton formation, and with the initiation of gastrulation. In in vitro cultures of primary mesenchyme cells isolated from the embryo at the 16 cell stage, these antigens were expressed synchronously with those in viva (Fink and McClay, 1984). Thus the expression of these components is programmed in mesenchyme cells by the 16 cell stage, and is controlled independent of the embryo in a pattern which may suggest a role for mesenchymedirected morphogenesis. It is generally accepted that proteins required for early development are either stored in the egg or synthesized on stored mRNAs (Davidson, 1976). The bulk of new transcription becomes activated at around the mesenchyme blastula stage in many species. If one assumes that the egg is parsimonious in what it stores and that stored elements are required early in development, then it must be of some importance to partition a significant portion of the egg volume to the storage of ECM components. If this reasoning is correct, the basal lamina must be required by the embryo well in advance of the mesenchyme blastula stage. Perhaps cavitation of the blastocoel requires the presence of a basal lamina as a substrate for the cells. Alternatively, basal laminar molecules could promote cell polarity from the presumptive basal surface of the cells. This hypothesis is dependent upon the availability of basal lamina molecules in the egg, and the rapid polarized movement of the molecules in development, but does not require the molecules to serve immediately as a substrate. Whatever the reason, it is important enough to demand significant space in the egg. We wish to thank Ms. Kathryn Cates, Ms. Barbara McPartland, and Ms. Gail Cannon for their secretarial and technical assistance and Dr. James Norman Dent for his experience. This work was supported by Grant HD 14483 from the National Institute of Health. REFERENCES ADAMS, E. (1978). Invertebrate collagens. Science X%591-598. AFZELIUS, B. A. (1957). Electron microscopy on the basophilic structure of the sea urchin eggs. Z. Zeyfih. Mikrosk. Anat. 45,660. ANDERSON, E. (1970). A cytological study of the centrifuged whole, half and quarter eggs of the sea urchin Arimku pundtita. Jl Cell Bid 47.711-733. BENSON, S. C., and SESSIONS, A. (1980). Prolyl hydroxylase activity during sea urchin development. Exp. Cell Res. 130,467-470. CRISE-BENSON, N., and BENSON, S. C. (1979). Ultrastructure of collagen in sea urchin embryos. Wilhelm Roux’s Arch 186.65-70. DAVIDSON, E. (1976). “Gene Activity in Early Development.” Academic
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