Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

Abstract

Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved cytoarchitectural domain that specifies the AV axis in metazoan ova.

Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

“…Many past experiments have supported the existence of a single domain accumulated at the vegetal pole, which contains conserved polarity effectors, such as Dishevelled (Figure 4B) (Boveri, 1901; Dan, 1979; Hörstadius, 1928). This domain may influence MT forces to drive vegetal asymmetric divisions at the 4 th cleavage (Dan, 1979; Leonard and Ettensohn, 2007; Peng and Wikramanayake, 2013; Tanaka, 1976). Yet, although this domain is assembled before fertilization, cell shape appears to override its effect in the first three rounds of cleavages, suggesting it only weakly influences MT forces in these stages (Minc et al, 2011).…”

“…The protocol for sea urchin was modified to include the addition of an antibody against Dishevelled, kindly provided by Pr Wikramanayake, used at 1/2000 (Peng and Wikramanayake, 2013). Zebrafish embryos expressing the microtubules marker EMTB-3×GFP, Tg(actb2:Hsa.MAP7-EGFP) (Wuhr et al, 2011), were fixed with (0.25 to 0.50% FA, 0.1% GA, 80 mM K Pipes, pH 6.8, 1 mM MgCl2, 5 mM EGTA, 0.2% Triton X-100) (slightly modified from (Becker and Gard, 2006)).…”

“… (A) Early developmental patterns and interphase aster organization of sea urchin embryos (adapted from (Gilbert, 2010; Minc et al, 2011; Peng and Wikramanayake, 2013)). (B) Immunostaining images of sea urchin early development, showing MT interphase asters (green), Dishevelled (red, arrows) and DNA (blue).…”

microRNAs regulate β-catenin of the Wnt signaling pathway in early sea urchin development

“…The binding interactions of β-catenin is also regulated by the transforming growth factor β (TGFβ), epidermal growth factor receptor (EGFR), hepatocyte growth factor, erythroblastic leukemia viral oncogene-2 and Janus Kinase 2 (JAK2)-mediated signaling pathways (Blume-Jensen et al, 1998; Gonzalez and Medici, 2014; Incassati et al, 2010; Liu et al, 2010; Paul et al, 2013; Zeng et al, 2006). Early in development, β-catenin may be predominantly regulated by activated Disheveled that disrupts the targeted degradation of β-catenin in the vegetal blastomeres (Peng and Wikramanayake, 2013), while miRNAs may facilitate the clearing of β-catenin later at and after the blastula stage.…”

Germ Line Versus Soma in the Transition from Egg to Embryo

“…However, the earliest activation of the pathway in the sea urchin embryo may occur independently of a Wnt ligand. Maternally-supplied Disheveled protein is enriched in the vegetal cortex of the sea urchin egg and, based on gel mobility, is modified in a form that perhaps indicates constitutive activation (Peng & Wikramanayake, 2013). In effect, the pathway may be hard-wired, implying that the early embryo activates differential axial specification autonomously, and not until later does inductive signaling influence cell fate decisions.…”