a privileged space inside the syncytium that is topologically outside the cell,
although it is enrobed by the cell membranes of many PMC cells fused together.
A more detailed discussion of this issue can be found in Wilt and Ettensohn
(2007) and Wilt (2002).
The morphology of the spicules formed is dictated primarily by PMCs. But the
gross anatomical placement of the spicules within the blastocoel depends on the
properties of the overlying ectodermal layer of the embryo (Ettensohn and Malinda
1993; Guss and Ettensohn 1997). It has recently been shown that the initiation of
calcite formation depends on signaling by the ligand VEGF (Duloquin et al. 2007),
which emanates from a small number of ectodermal cells that immediately overlie
the congregations of PMCs in the ventrolateral aspects of the blastocoel of the early
gastrula stage. A small number of cells that express the homeobox transcription
factor, Otp, produce the VEGF signal (Dibernardo et al. 1999). PMCs express
VEGF receptors for transduction of this ectodermal signal, and initiation and
maintenance of calcification depend on this signaling.
7.2.2 Calcium
The calcium of the spicule (and presumably the Mg as well, though it has not been
studied) comes from the sea water, as shown by use of isotope studies. (Nakano
et al. 1963) Reduction of the Ca concentration of sea water (normal is ~10 mM)
below 2–4 mM causes spicules that develop to be malformed. Even lower Ca levels
result in irregular, small, mineralized masses, and a generalized deterioration of
normal embryonic development (Okazaki 1956). The PMCs must possess very
active Ca transporters, presumably with high capacity and low affinity, but they
have not been identified or characterized. Beniash et al. (1999) visualized intracellular deposits by electron microscopy that were identified as Ca precipitates,
presumably CaCO 3 . These did not display x-ray diffraction patterns and were
believed to be amorphous; after heating of the sample, these deposits diffracted
as calcite. After the initiation of triradiate spicule formation, birefringent granules
have not been observed in PMCs in living embryos or cultures (see Okazaki 1960);
so, either the imported Ca is segregated in intracellular vesicles containing supersaturated Ca solutions, or the precipitated material must be below the resolution of
the light microscope, or in a state that is not birefringent.
This role of intracellular calcium deposits was recently addressed by using the
Ca fluorophore calcein, a fluorescein derivative that is intensely fluorescent when
incorporated into calcium-containing precipitates. Wilt et al. (2008b) used calcein
pulse labeling to follow calcium delivery to spicules. They found that brief pulses of
calcein-containing sea water, followed by rinsing and culture in normal sea water,
resulted in an initial labeling of intracellular submicron sized granules, followed by
their clearance from the cell and subsequent appearance of label in developing
spicules, particularly near the tips of the extending spicules. They interpreted this as
7 Molecular Aspects of Biomineralization of the Echinoderm Endoskeleton
203
although it is enrobed by the cell membranes of many PMC cells fused together.
A more detailed discussion of this issue can be found in Wilt and Ettensohn
(2007) and Wilt (2002).
The morphology of the spicules formed is dictated primarily by PMCs. But the
gross anatomical placement of the spicules within the blastocoel depends on the
properties of the overlying ectodermal layer of the embryo (Ettensohn and Malinda
1993; Guss and Ettensohn 1997). It has recently been shown that the initiation of
calcite formation depends on signaling by the ligand VEGF (Duloquin et al. 2007),
which emanates from a small number of ectodermal cells that immediately overlie
the congregations of PMCs in the ventrolateral aspects of the blastocoel of the early
gastrula stage. A small number of cells that express the homeobox transcription
factor, Otp, produce the VEGF signal (Dibernardo et al. 1999). PMCs express
VEGF receptors for transduction of this ectodermal signal, and initiation and
maintenance of calcification depend on this signaling.
7.2.2 Calcium
The calcium of the spicule (and presumably the Mg as well, though it has not been
studied) comes from the sea water, as shown by use of isotope studies. (Nakano
et al. 1963) Reduction of the Ca concentration of sea water (normal is ~10 mM)
below 2–4 mM causes spicules that develop to be malformed. Even lower Ca levels
result in irregular, small, mineralized masses, and a generalized deterioration of
normal embryonic development (Okazaki 1956). The PMCs must possess very
active Ca transporters, presumably with high capacity and low affinity, but they
have not been identified or characterized. Beniash et al. (1999) visualized intracellular deposits by electron microscopy that were identified as Ca precipitates,
presumably CaCO 3 . These did not display x-ray diffraction patterns and were
believed to be amorphous; after heating of the sample, these deposits diffracted
as calcite. After the initiation of triradiate spicule formation, birefringent granules
have not been observed in PMCs in living embryos or cultures (see Okazaki 1960);
so, either the imported Ca is segregated in intracellular vesicles containing supersaturated Ca solutions, or the precipitated material must be below the resolution of
the light microscope, or in a state that is not birefringent.
This role of intracellular calcium deposits was recently addressed by using the
Ca fluorophore calcein, a fluorescein derivative that is intensely fluorescent when
incorporated into calcium-containing precipitates. Wilt et al. (2008b) used calcein
pulse labeling to follow calcium delivery to spicules. They found that brief pulses of
calcein-containing sea water, followed by rinsing and culture in normal sea water,
resulted in an initial labeling of intracellular submicron sized granules, followed by
their clearance from the cell and subsequent appearance of label in developing
spicules, particularly near the tips of the extending spicules. They interpreted this as
7 Molecular Aspects of Biomineralization of the Echinoderm Endoskeleton
203