only very low levels of fluorescence were seen in the cytoplasm. Since the mRNA coding for the Dsh::GFP fusion protein is injected into 6807310 zygotes and it is likely incorporated into all blastomeres, the low levels of fluorescence outside the VCD in Dsh::GFP mRNA-injected zygotes and Pyrroloquinolinequinone disodium salt site embryos suggests that the fusion protein is unstable when not localized to the VCD. The degradation of the Dsh protein after disruption of microfilaments suggests that the actin cytoskeleton may play a role in regulating the stability of this protein in the egg. The Dsh protein has a defined actin-binding site and it is known to interact with actin, and while it is logical to invoke a transport or tethering function for the cytoskeleton in regulating this protein, the degradation of all Dsh pools following cytochalasin treatment is puzzling. This effect does not reflect a general effect on all egg proteins since the disruption of microfilaments does not lead to the degradation of tubulin or actin. Moreover, pulse-treating eggs with cytochalasin D, which has a reversible effect on the actin cytoskeleton, for just five minutes also led to Dsh degradation in eggs following the two hour incubation period. Further studies are needed to determine how disruption of the cytoskeleton affects the stability of Dsh in the sea urchin egg, and to determine if the stability of other regulatory proteins in the cWnt pathway is affected by microfilament disruption. The potential role of the VCD in selectively activating Dsh-mediated cWnt signaling in vegetal blastomeres The recent sequencing of the S. purpuratus genome showed that there are eleven Wnt ligands, four Frizzled receptors and one LRP 5/6 receptor in this species. Subsequent studies have shown that none of the mRNAs coding for the maternally expressed ligands and receptors is enriched at the vegetal pole of the egg or early embryo. While it is possible that there are unidentified transmembrane cell surface components in the cWnt pathway that are localized to the vegetal pole, there is now compelling evidence that the determinants that selectively activate cWnt signaling intracellularly in vegetal blastomeres during endomesoderm specification are firmly attached to the VCD. Early insights into the cortical localization of regulatory factors in echinoderm ova came from the work of experimentalists who showed that the removal of vegetal deep cytoplasm had little effect on gastrulation in sea urchins. In sharp contrast, sea urchin and sea star embryos developing from eggs where the vegetal cortex is selectively extirpated become severely animalized and do not gastrulate. Analysis of multiple molecular marker expression in P. lividus embryos developing from VC eggs showed that they do not form endoderm or mesoderm. Croce et al. went on to show that VC embryos did not activate the cWnt pathway in vegetal blastomeres and moreover, showed that the animalized phenotype could be rescued by overexpression of activated catenin. Overexpression of other Wnt pathway components such as Wnt6, a dominant-negative form of GSK and overexpression of Dsh could also rescue endomesoderm in these embryos, but to a lesser extent. Unlike VC embryos rescued with activated -catenin, however, which had a relatively normal larval morphology, 11414653 the VC embryos where endomesoderm was rescued by overexpression of Wnt6, dnGSK and wild-type Dsh had severely disrupted larval morphology. Additionally, these experiments did not determine if the rescue in endo

Statistical differences were determined by paired Student’s t test

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