Apical Cell Surface

It has been reported that the first ECM structure developing soon after fertilisation of the egg is the external one. As viewed by electron microscopy after ruthenium staining (Lundgren 1973), the presence of at least two distinct layers is evident: an inner apical lamina more tightly associated with the apical plasma membrane, which is the thickest, and an outer layer, the hyaline layer, associated with the tips of microvilli (see Fig. 6).

Fig. 6. Schematic diagram of the ultrastructure of the epithelium of the blastula embryo. (Adapted from Hardin 1996)

The Apical Lamina

The apical lamina is a fibrous layer surrounding the embryo, which remains on the apical surface after removal of hyalin from the hyaline layer (Hall and Vacquier 1982; Burke et al. 1998), and is composed principally of fibropellins (Bisgrove et al. 1991; Burke et al. 1991). The gene coding for one of the three known fibropellins has been sequenced and, like other extracellular molecules, it contains a series of up to 20 epidermal growth-factor-like repeats (Delgadillo-Reynoso et al. 1989; Grimwade et al. 1991; Bisgrove and Raff 1993). In vitro experiments showed that dissociated cells adhere to affinity-purified fibropellins in a temperature-, time- and dose-dependent manner, suggesting the role as a substrate for cell adhesion (Burke et al. 1998). Their in vivo functional role has been investigated using monoclonal antibodies, which were shown to interfere with the initial phase of gastrulation (Naka-jima and Burke 1996). At this time no further proteins have been described as belonging to this layer.

The Hyaline Layer

Historically, the first function assigned to the hyaline layer has been to hold blastomeres together (Osanai 1960; Vacquier and Mazia 1968). In agreement with this hypothesis, Citkowicz (1971) showed that the hyaline layer forms a shell that remains structurally intact after removal of epithelial cells using hyperosmotic solutions, demonstrating that it is a structure independent from cells that lie underneath. A number of proteins constituting the hyaline layer have been identified and characterised. The major component is hyalin, a high molecular weight glycoprotein secreted by the cortical granules, which can be isolated by successive steps of solubilisation and precipitation through removal and re-addition of Ca2+ to seawater or other osmotically balanced media (Kane 1970). Hyalin has been extensively studied both structurally and functionally in sea urchin and sea star embryos (Vacquier 1969; Kane and Stephens 1969; Spiegel and Spiegel 1979; Adelson et al. 1992) and partial cDNAs have been isolated by screening expression libraries with monoclonal antibodies to hyalin (Wessel et al. 1998). Its sequence contains consensus calcium-binding motifs, in agreement with its capacity to interact with calcium biochemically (Robinson et al. 1992), and modular repeats similar to that known in other large ECM molecules (McClay 1991). Hyalin has been shown to support cell adhesion in vitro (McClay and Fink 1982) and to be involved in sea urchin morphogenesis in vivo (Adelson and Humphreys 1988). Interestingly, a recent study has demonstrated that two mammalian cortical granule envelope proteins share common antigenic epitope(s) with echinoderm hyalin and, like hyalin, play a role in early embryogenesis (Hoodbhoy et al. 2000).

Pl-Nectin: An Example ofSignal(s) from Outside to Inside the Embryo

About 10 years ago, with the aim of purifying fibronectin from the sea urchin embryo, we isolated and characterised another ECM protein localised in the hyaline layer, namely Pl-nectin (Fig. 6). Although the protocol used for its purification was that originally developed for fibronectin, the molecular weight and other features indicated that this protein was a new molecular species. Pl-nectin has been isolated from the species Paracentrotus lividus (Matranga et al. 1992) and Temnopleurus hardwickii (Th-nectin; Yokota et al. 1994) as a collagen-binding molecule and, like other ECM components, it has been found stored in specific cytoplasmic vesicles, recently named necto-somes, in the unfertilised egg (Kato et al. 2004). The genesis and distribution of the nectosome during oogenesis, its translocation to the cortex and gradual secretion into the hyaline layer after fertilisation have recently been fully documented by using immunoelectron microscopy in the Japanese species T. hardwickii (Kato et al. 2004). At later stages, Pl-nectin has been found to be localised on the apical surface of ectoderm and endoderm cells. By in vitro assays, the protein has been shown to mediate cell-substrate adhesion in a dose-dependent fashion, suggesting a functional role during development (Matranga et al. 1992). Thus it was crucial to investigate the in vivo Pl-nectin function by morphogenetic assays in which embryos were cultured in the

Fig. 7A, B. Skeleton defects obtained after culturing embryos in the presence of high concentrations of monoclonal antibody to Pl-nectin. A Control embryo cultured in the presence of unrelated IgGs; B treated embryo showing strong skeleton defects presence of monoclonal antibodies to Pl-nectin. To our surprise, after this treatment, we observed the presence of a high number of embryos with dramatic skeleton defects, but with normally developed ectoderm and endoderm structures (Fig. 7; Zito et al. 1998,2000). In addition, depending on the amount of antibody used, it was possible to obtain embryos with different degrees of skeleton defects, which were classified on an arbitrary scale (Zito et al. 2003). These data suggested that outer ectoderm-Pl-nectin interaction is indirectly involved in inner skeleton formation. For more detailed description on this issue, see Section 10. The partial sequence of the coding region of the Pl-nectin gene (accession no. AJ578435) has revealed its similarity with another component of the hyaline layer, namely echinonectin, purified from the species Lytechinus variegatus. This protein has a lectin-binding activity that allowed its chromatographic purification and, like Pl-nectin, it is an adhesive substratum for cells (Alliegro et al. 1988; 1990). Although in a previous report no cross-reaction between the two molecules was found, it is very likely that Pl-nectin and echinonectin belong to the same family of ECM components.

Other Apical ECM Components

Concerning the presence of fibronectin-like molecules in the sea urchin embryo, first evidence was obtained by immunofluorescence experiments using antibodies against human fibronectin, even if the cross-reactive material was described as being differently distributed in the embryo, i.e. on the outer cell surface of the epithelial layer (Spiegel et al. 1983), on the basement membrane (Spiegel et al. 1980; DeSimone et al. 1985), and also on the surface of migrating PMCs (Katow et al. 1982). A sea urchin fibronectin-like molecule has been purified for the first time from ovaries of Pseudocentrotus depressus by Iwata and Nakano (1981,1983). The same authors have also demonstrated that this protein is involved in migration, adhesion and spicule synthesis of in vitro-cultured micromeres (Miyachi et al. 1984). Later, fibronectin-like proteins were purified from five different species living in Mediterranean and

Pacific seawaters and their biochemical and immunological relationships have been analysed for the purpose of comparative phylogenetic studies (Matranga et al. 1995). These proteins differed in their binding affinity to gelatin but shared different epitopes, suggesting that they are members of a sea urchin fibronectin superfamily. Furthermore, analysis of the different features of fibronectin-like proteins and sea urchin nectins, i.e. Pl-nectin and Th-nectin, has been carried out, since essentially the same method was utilised to purify both groups of molecules (Yokota et al. 1994). It has been shown that these two groups of proteins belong to different ECM protein families since they differ in their affinity to collagen as well as in their localisation inside the embryo.

A gene coding for a new ECM protein has been sequenced in the direct developer Heliocidaris erythrogramma. By the use of polyclonal antibodies raised against the protein, it has been demonstrated that it is localised on the apical surface of ectoderm, in tight association with the plasma membrane. The protein has been named apextrin and it has been proposed to be involved in apical cell adhesion (Haag et al. 1999).

Although a reasonable amount of information is currently available on the hyaline layer and apical lamina components, the spatial relationship between them is still unclear. An approach to such studies could come from experiments of co-immunoprecipitation after transient cross-linking of surface molecules in live embryos. Alternatively, electron microscopic observations of rotary shadowed purified molecules in diluted solutions, or in combination with other potential partners, would serve to solve the intricate meshwork that surrounds the embryo.

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