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.

Chiehfu Jeff Peng,Athula H Wikramanayake

doi:10.1371/journal.pone.0080693

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.

Highlights

How animal body plans are established during embryogenesis is a central question in developmental biology
Dsh is broadly expressed during early embryogenesis, but the protein is highly enriched at the vegetal cortex
Experimental studies have provided compelling evidence that the vegetal cortex of the sea urchin egg is required for canonical Wnt (cWnt)-mediated activation of the endomesodermal gene regulatory network (EGRN) in vegetal blastomeres, but the underlying mechanisms have been unclear

Summary

Introduction

How animal body plans are established during embryogenesis is a central question in developmental biology. But in bilaterians, which comprise the vast majority of metazoan taxa, the body plans are built around three distinct embryonic coordinates that define the anterior-posterior (AP), the dorsal-ventral (DV) and the left-right (LR) axes. The relationship between the AV egg polarity and the AP axis was first reported in the early 19th century by Karl Ernst von Baer [3]. Many studies have since confirmed that the eggs of most metazoans have an AV axis, and that the relationship between this egg polarity and patterning of the AP axis first observed in amphibians by Baer is conserved in many other bilaterians [1,2,4,5,6,7,8,9,10]. How the asymmetric activation of this pathway is influenced by the AV axis of the egg is still not well understood in most metazoans and as such it is an important area of investigation [5,6,11,12]

Methods

Results

Conclusion