Functional study of the Wnt signaling network that coordinates early anterior-posterior and dorsal-ventral axis specification and patterning in the sea urchin embryo
Type of DegreePhD Dissertation
Restriction TypeAuburn University Users
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The establishment and patterning of the different embryonic body axes are crucial events in early development of any embryo. Anterior-posterior (AP) and dorsal-ventral (DV) axis specification and patterning are fundamental developmental processes critical for establishing the correct adult body plan in metazoans. In the deuterostome sea urchin embryo AP specification and patterning depends on integrated cross-regulatory information from the Wnt/β-catenin, Wnt/JNK, and Wnt/PKC pathways, forming an interconnected Wnt signaling network. We have previously shown that a non-canonical signaling pathway involving the Fzl1/2/7 receptor antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by Wnt/β-catenin and Wnt1/Wnt8-Fzl5/8-JNK signaling. First, we studied the function of Wnt16 ligand in activating the Fzl1/2/7 signaling during early AP patterning. Our data show that Wnt16 ligand has an early function in activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism and a subsequent role in activating key regulatory endoderm GRN factors and the morphogenetic movements of gastrulation. In addition, our results show that zygotic expression of wnt16 depends on both Fzl5/8 and Wnt/β- catenin signaling during gastrulation. During early cleavage and blastula stages, the developmental mechanism involved in early AP specification and patterning by the Wnt signaling network appear to be independent of DV patterning. However, a combination of inputs from the Wnt, Nodal and BMP signaling pathways might work together in order to coordinate the proper distribution of the main germ layers along the AP and DV axis in early sea urchin development. Interestingly, our RNA-seq screen data identified several dorsal genes downregulated in Wnt signaling knockdowns at three different developmental stages. Therefore, we used morpholino perturbation approaches to study the function of the Wnt signaling network during AP and DV specification and patterning of the sea urchin embryo. Our data suggest that a non-canonical signaling is required for dorsal specification. In addition, the expression of a ciliary band maker was also affected when we perturb Wnt signaling function. Together, our results show that there is cooperative regulation from both Wnt and BMP2/4 signaling pathways during AP and DV patterning. This study establishes an important connection between the AP and DV gene regulatory networks (GRNs) and provides an important starting point for future comparisons in other deuterostome embryos. In addition, we characterize the distinct roles of the signaling molecule Siah, the potential transcriptional effector downstream of Wnt16-Fzl1/2/7 signaling NFAT and a dorsal transcriptional factor during early AP and DV axis specification and patterning of the sea urchin embryo. Our spatiotemporal expression experiments of siah as well as the Fzl1/2/7 loss-of-function results suggest that the signaling molecule Siah is activated by non-canonical Wnt16-Fzl1/2/7 signaling pathway during anterior-posterior (AP) specification and patterning in the sea urchin embryo. Next, we show that the initial input for the early expression of a specific transcription factor in the dorsal ectoderm is likely to be dependent of the non-canonical Wnt16-Fzl1/2/7 signaling pathway. Our knockdown experiments also indicate that this dorsal gene plays a role in antagonizing the ANE restriction mechanism, in addition to its well-known function in establishing the early specification of the DV GRN. Lastly, we analyze the function of the nuclear factor of activated T-cells, NFAT, as a putative transcriptional effector downstream of non-canonical Wnt16-Fzl1/2/7 signaling pathway. Our functional experiments suggest that NFAT might be required for the antagonism of the ANE positioning mechanism mediated by Wnt1/Wnt8-Fzl5/8-JNK signaling during early body axis specification and patterning of the sea urchin embryo. Taken together, this study provides a better understanding of how coordinated information from different signaling pathways is necessary for early specification and patterning along the AP and DV axes in the sea urchin embryos and provides insights for future comparisons of the shared and novel mechanisms during these crucial events in the early development of metazoan embryos.