Growth Factors

Growth factors, like the ECM, have been shown to fulfil vital developmental roles in many embryonic systems. Among them, transforming growth factor-beta (TGF-p) superfamily members appear to have diversified greatly with the evolution of vertebrates, but, although they are believed to be widely distributed among the animal kingdom, only a few invertebrate deuterostome TGF-p molecules have been identified so far. Sequence comparisons suggest an early origin and an evolutionary conservation of the molecular conformation of the members of TGF-p superfamily (see reviews by Hogan et al. 1994; Kingsley 1994; Chin et al. 2004). Among echinoderms, the first gene encoding a member of the TGF-p superfamily to be reported was identified in the sea urchin embryo, and it was named univin (Stenzel et al. 1994). Sequence comparisons placed univin in the bone morphogenetic protein (BMP) group of the TGF-p superfamily along with the vertebrate BMPs, decapentaplegic protein from Drosophila and Vg-1 from Xenopus. Recently, we have shown the involvement of this growth factor in the ectoderm inductive signals needed for the skeleton morphogenesis in the sea urchin embryo (Zito et al. 2003). In this system, skeletogenesis is a fully documented example of ecto-mesoderm induction, since PMCs, although already determined to only synthesise the skeleton (Okazaki 1975), need signals from the surrounding ectoderm to direct skeleton growth and patterning (Armstrong et al. 1993; Ettensohn and Malinda 1993; Guss and Ettensohn 1997). As previously mentioned, we found that the inhibition of Pl-nectin interaction with ectoderm cells produced perturbed embryos, in which only skeleton elongation and patterning were specifically affected. Interestingly, we observed that univin expression was strongly inhibited in skeleton-defective embryos, while its misexpression, obtained by univin mRNA injection, rescued skeletogenesis. These findings are in agreement with the hypothesis of the involvement of diffusible molecules in the ecto-mesoderm induction suggested by other researchers (Kiy-omoto and Tsukahara 1991; Page and Benson 1992; Ettensohn and Malinda 1993; Guss and Ettensohn 1997). Our data, in particular, support a model in which some ectodermal cells secrete processed univin or a related growth factor into the blastocoel, where it signals PMCs to synthesise the spicule matrix proteins required for spicule growth (Fig. 9). The ability of ectodermal cells to produce the signal depends on their association with Pl-nectin on the apical ECM. Since by our preliminary results the interaction between ectodermal cells and Pl-nectin seems mediated by an integrin receptor, it is reasonable to hypothesise the involvement of one of the signalling pathways already known for integrins in other systems. Studies aimed at the identification of such a pathway are in progress in our laboratory. Furthermore, this model predicts

Fig. 9. Model to explain ecto-mesoderm induction in the sea urchin embryo. Ectoderm cells properly interacting with the outer Pl-nectin secrete into the blastocoel the growth factor univin, which signals PMCs to synthesise the spicule. The interaction of ectoderm cells with Pl-nectin, possibly mediated by an integrin receptor, activates a yet unknown signalling pathway. The model predicts also the expression of a putative TGF-p receptor on PMCs and thus a signalling pathway

that PMCs should express a TGF-p receptor. Recently, one similar to vertebrate type I receptor,Alk2,has been found to be expressed in ingressed PMCs of S.purpuratus embryos,which can mediate both activin and BMP signals (L. Angerer, unpubl. observ.). However, whether this receptor mediates the skeleton-promoting signal is not yet known.

Two very recent papers have shown the involvement of a MAP kinase signalling pathway in the development of the micromere lineage and mes-enchyme differentiation (Fernandez-Serra et al. 2004; Rottinger et al. 2004). Whether a MAP kinase signalling is required for the ecto-mesoderm induction guiding skeletogenesis remains to be demonstrated.

Other BMPs homologues have been recently cloned in the sea urchin embryo, specifically SpBMP5-7 (Ponce et al. 1999), TgBMP2/4 (Hwang et al. 1999) and LvBMP2/4 (Angerer et al. 2000), and AtBMP2/4 in the starfish (Shih et al. 2002). Their developmental expression patterns have been described by Northern blotting and in situ hybridisation experiments. Functional studies altering LvBMP2/4 mRNA levels showed the involvement of this growth factor in regulating cell fate allocation along the sea urchin animal-vegetal embryonic axis (Angerer et al. 2000). Evidence of the presence of growth-factor-like molecules belonging to other families than TGF-ps has also been reported. In 1987, a cDNA clone whose protein product displayed striking homology to the EGF family of proteins was identified and characterised in the sea urchin embryo (Hursh et al. 1987). The presence of other growth factors in echinoderms has been shown by indirect methods. Platelet-derived growth factor-BB (PDGF-BB) and TGF-a have been reported to be involved in development by using human recombinant proteins, which rescued sea urchin embryo abnormalities induced by ECM disrupters (Ramachandran et al. 1993), or using anti-human PDGF-B and TGF-a antibodies which affected embryo development (Govindarajan et al. 1995). Antibodies against mammalian receptors for PDGF and EGF, or a dominant/negative RNA for PDGF receptor, have been reported to be involved in early differentiation and morphogenesis of the embryo (Ramachandran et al. 1995,1997). A new member of the fibroblast growth factor receptor (FGFR) family has been cloned in S. purpuratus and it has been shown to contain a series of domains characteristic of the family, as three immunoglobulin-like motifs, an acid box, a transmembrane domain, a relatively long juxtamembrane sequence, a split tyro-sine kinase domain and two conserved intracellular tyrosine residues (McCoon et al. 1996). These authors detected SpFGFR protein only in muscle cells of the embryo, suggesting that its function may be required to support the proliferation, migration and/or differentiation of myoblasts, rather than being involved in commitment to a muscle fate (McCoon et al. 1998).

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