Wnt Signaling in the Cnidarian Nematostella vectensis: Insighits into the Evolution of Gastrulation

Abstract

The genesis of gastrulation was arguably a key evolutionary innovation that enabled metazoan diversification. The developmental mechanisms that induced archenteron formation and endodermal cell fate specification during gastrulation are unknown, but one crucial step was likely the co-option of a localized molecular asymmetry that was present in ancient embryos. One ancient polarity present in most metazoan eggs is the animal vegetal (AV) or primary egg axis, which is established by the asymmetric localization of maternal factors such as RNA, proteins and organelles during oogenesis. In bilaterians, which account for a majority of animal taxa, the AV axis predicts the axial properties of the embryo and the adult with the animal pole giving rise to the anterior of the organism, and the vegetal pole giving rise to the endomesoderm and the site of gastrulation. In contrast, in early diverging non-bilaterian taxa such as ctenophores and cnidarians, the animal pole gives rise to the endoderm and the site of gastrulation. These and other observations have led to the hypothesis that endoderm specification and gastrulation evolved at the animal pole, and moreover, that the mechanisms that regulate these processes were moved down to the vegetal pole in the bilaterian lineage. This idea is supported at the molecular level by the observation that endoderm specification in both bilaterians and non-bilaterians is regulated by nuclear beta-catenin signaling indicating a role for the Wnt signaling pathway in the evolution of gastrulation. In the cnidarian Nematostella vectensis, the Dishevelled (NvDsh) protein, a critical component of Wnt signaling, is enriched at the animal pole of eggs and embryos and is required for Wnt/beta-catenin signaling mediated endodermal cell fate specification but not for primary archenteron invagination. Primary archenteron invagination in Nematostella is mediated by the localized activation of Wnt/PCP signaling at the animal pole. The mechanisms coordinating Wnt signaling dependent endoderm cell fate specification and primary archenteron invagination at the animal pole in Nematostella embryos are not known, but elucidation of these mechanisms may provide critical insight into the evolution of gastrulation. There is increasing evidence to suggest that a highly conserved group of core proteins functioning in the Wnt/PCP signaling pathway play a critical role in regulating gastrulation through the asymmetric localization of key components of signaling cascades in both vertebrates and invertebrates. For my dissertation research, I focused on the role of core Wnt/PCP proteins in establishing the embryonic polarity that leads to the asymmetric activation of Wnt signaling in the blastomeres at the animal pole in Nematostella. In Nematostella, the core PCP genes NvStrabismus (NvStbm) and NvFlamingo (NvFmi) are expressed maternally and are localized to the animal pole from the egg stage throughout early development. The Frizzled homologs NvFrizzled1 (NvFz1), NvFrizzled4 (NvFz4) and NvFrizzed5 (NvFz5) are expressed maternally in dynamic expression patterns during early development. NvFz10 is expressed in the early gastrula stage at the animal pole and is restricted to the invaginating cells of the presumptive endoderm. NvPrickle (NvPk) is asymmetrically expressed at the blastula stage and at the late gastrula stage it is expressed in the pharynx and on one side of the embryo in the presumptive ectoderm. Inhibition of NvFz1 function during early development of Nematostella blocked endodermal cell fate specification but not primary archenteron invagination. In contrast, loss of NvFz10 function blocked primary archenteron invagination without affecting endodermal cell fate specification. This experimental uncoupling of Wnt/PCP signaling mediated initial archenteron invagination from Wnt/beta-catenin mediated endoderm specification in Nematostella provides further evidence for the independent evolution of these two processes during early metazoan evolution. The localized expression of NvFmi to the animal pole of eggs and embryos indicated a possible role for this core Wnt/PCP protein in coordinating the activity of the two Wnt pathways at the animal pole. This idea was tested by disrupting NvFmi function using morpholinos and dominant-negative approaches. Downregulation of NvFmi function blocked both Wnt/PCP signaling mediated archenteron invagination and Wnt/beta-catenin signaling mediated endoderm cell fate specification, while overexpressing the cytoplasmic carboxy terminus of the NvFmi protein selectively disrupted endodermal gene expression. These observations indicates that NvFmi regulates both Wnt pathways and that it may function as a scaffold to coordinate both Wnt/?-catenin and Wnt/PCP signaling to drive primary archenteron invagination and endoderm cell fate specification in Nematostella through the asymmetric localization of Wnt pathway components to the animal pole. Overall, this study has provided experimental evidence as to how different branches of Wnt signaling mediate primary archenteron invagination and cell fate specification through NvFz10 and NvFz1 respectively during gastrulation in Nematostella and possible mechanisms of coordination of these two processes in time and space through the function of NvFmi.</p