mediate cell–substrate adhesion in a dose-dependent fashion (Fig. 8.5c), suggesting
a functional role during development (Matranga et al. 1992). A homologous protein
has been purified from Temnopleurus hardwickii, and named Th-nectin (Yokota
et al. 1994). In the outer ECM, two other proteins called fibropellins are present
after fertilization and throughout early development. At the blastula stage,
fibropellins become organized into distinct fibers which form a mesh-like network
over the surface of the embryo (Bisgrove et al. 1991).
The function of Pl-nectin in vivo has been studied by morphogenetic assays in
which embryos were cultured in the presence of a McAb to Pl-nectin (Fig. 8.5c). A
high number of embryos with serious skeleton defects, but with normally developed
ectoderm and endoderm structures, were found (Zito et al. 1998, 2003). It was
demonstrated that skeleton deficiency was not due to a reduction in the number of
PMCs ingressing the blastocoel, which were found as normal in number (usually 32
cells in P. lividus) and regularly arranged in a subequatorial ring (Zito et al. 2003).
On the other hand, skeleton-deficient embryos showed a decrease in the expression
levels of SM30 (Zito et al. 2003), while SM50 and MSP130 expression were not
affected by the treatment with a McAb to Pl-nectin (Zito, personal communication).
Concurrently, a strong reduction in the expression levels of univin, a TGF-b family
growth factor, was found, while its mis-expression obtained by the injection of its
mRNA was sufficient to rescue defects in skeleton elongation and SM30 expression
(Zito et al. 2003). Based on these results, we proposed for the first time, a working
model in which the secretion of univin, or other growth factor(s), into the blastocoel
by ectodermal cells drives PMCs to synthesize SM30 and other spicule matrix
proteins required for spicule growth. The competence of these ectodermal cells to
produce the signal depends on their binding to Pl-nectin (see Fig. 8.6).
Some parts of the proposed model have been later confirmed by our laboratory
and a few others. Recently, it has been shown by in vitro immunoprecipitation and
affinity chromatography experiments that Pl-nectin binds to a bC integrin subunit,
suggesting that the interaction of Pl-nectin with ectoderm cells is mediated by a
bC-containing integrin receptor (Zito et al. 2010). Interestingly, an integrin recognition motif (LDT) has been found in the Pl-nectin protein sequence (Costa et al.
2010). An LDT motif has been originally identified in the human mucosal addressin
cell adhesion molecule (MAdCAM-1) as the binding site of the a4/b7 integrin
receptor. Thus, the binding of Pl-nectin with ectoderm cells surface could result
from the interaction of the LDT motif with the identified integrin receptor. Adhesion assays using competing synthetic peptides (LDT, and others) are in progress in
our laboratory to confirm this hypothesis. In addition, the structure of Pl-nectin,
consisting of six tandemly repeated discoidin domains, provides it with the ability
to: (1) bind to ECM molecules bearing galactose and N-acetylglucosamine carbohydrate moieties (including collagen, or cell membrane surface glycoproteins), (2)
bind to cell surface proteins, such as tyrosine kinase receptors and G proteincoupled receptors, and (3) form multimeric structures by the discoidin domains
self-binding (Costa et al. 2010). The last property would explain the nectosome
structures observed by TEM described in T. hardwikii (Kato et al. 2004).
8 Echinoderms as Blueprints for Biocalcification
237
a functional role during development (Matranga et al. 1992). A homologous protein
has been purified from Temnopleurus hardwickii, and named Th-nectin (Yokota
et al. 1994). In the outer ECM, two other proteins called fibropellins are present
after fertilization and throughout early development. At the blastula stage,
fibropellins become organized into distinct fibers which form a mesh-like network
over the surface of the embryo (Bisgrove et al. 1991).
The function of Pl-nectin in vivo has been studied by morphogenetic assays in
which embryos were cultured in the presence of a McAb to Pl-nectin (Fig. 8.5c). A
high number of embryos with serious skeleton defects, but with normally developed
ectoderm and endoderm structures, were found (Zito et al. 1998, 2003). It was
demonstrated that skeleton deficiency was not due to a reduction in the number of
PMCs ingressing the blastocoel, which were found as normal in number (usually 32
cells in P. lividus) and regularly arranged in a subequatorial ring (Zito et al. 2003).
On the other hand, skeleton-deficient embryos showed a decrease in the expression
levels of SM30 (Zito et al. 2003), while SM50 and MSP130 expression were not
affected by the treatment with a McAb to Pl-nectin (Zito, personal communication).
Concurrently, a strong reduction in the expression levels of univin, a TGF-b family
growth factor, was found, while its mis-expression obtained by the injection of its
mRNA was sufficient to rescue defects in skeleton elongation and SM30 expression
(Zito et al. 2003). Based on these results, we proposed for the first time, a working
model in which the secretion of univin, or other growth factor(s), into the blastocoel
by ectodermal cells drives PMCs to synthesize SM30 and other spicule matrix
proteins required for spicule growth. The competence of these ectodermal cells to
produce the signal depends on their binding to Pl-nectin (see Fig. 8.6).
Some parts of the proposed model have been later confirmed by our laboratory
and a few others. Recently, it has been shown by in vitro immunoprecipitation and
affinity chromatography experiments that Pl-nectin binds to a bC integrin subunit,
suggesting that the interaction of Pl-nectin with ectoderm cells is mediated by a
bC-containing integrin receptor (Zito et al. 2010). Interestingly, an integrin recognition motif (LDT) has been found in the Pl-nectin protein sequence (Costa et al.
2010). An LDT motif has been originally identified in the human mucosal addressin
cell adhesion molecule (MAdCAM-1) as the binding site of the a4/b7 integrin
receptor. Thus, the binding of Pl-nectin with ectoderm cells surface could result
from the interaction of the LDT motif with the identified integrin receptor. Adhesion assays using competing synthetic peptides (LDT, and others) are in progress in
our laboratory to confirm this hypothesis. In addition, the structure of Pl-nectin,
consisting of six tandemly repeated discoidin domains, provides it with the ability
to: (1) bind to ECM molecules bearing galactose and N-acetylglucosamine carbohydrate moieties (including collagen, or cell membrane surface glycoproteins), (2)
bind to cell surface proteins, such as tyrosine kinase receptors and G proteincoupled receptors, and (3) form multimeric structures by the discoidin domains
self-binding (Costa et al. 2010). The last property would explain the nectosome
structures observed by TEM described in T. hardwikii (Kato et al. 2004).
8 Echinoderms as Blueprints for Biocalcification
237