Studies on the Role of Non-canonical Wnt/JNK Signaling During Early Anterior-Posterior Axis Formation in Sea Urchin Embryos

Abstract

The establishment of embryonic body axes is a crucial event during early metazoan embryogenesis, determining the correct shape of the adult body plan. Wnt signaling pathways are conserved cascades that regulate important aspects of embryonic development in animals. Our lab has previously reported that the Wnt/β-catenin, Wnt/JNK, and Wnt/Ca2+ pathways integrate to specify and pattern the primary germ layers gene regulatory network (GRN) along the anterior-posterior (AP) axis in sea urchin embryos. Previously, we had shown that Wnt1/Wnt8-Fzl5/8-JNK signaling governs the downregulation of the anterior neuroectoderm (ANE) GRN at lower ectoderm cells (termed equatorial ectoderm) to establish the ANE territory around the anterior pole. However, the exact downstream mechanism of this pathway and the regulated GRN remains unclear. We used a combination of advanced molecular approaches to investigate this, revealing the role of transcription factors activated by Wnt/JNK signaling during early AP axis specification and patterning. We discovered two crucial transcription factors downstream of Wnt/JNK signaling that play a role in early germ layers positioning. First, we found that Sp5 transcription factor plays a crucial role in two essential developmental processes in sea urchin embryos: endoderm specification, which is a crucial part of the endoderm kernel, and AP neuroectoderm patterning, which is a downstream factor of Wnt/JNK signaling. This is the first study to demonstrate the significance of Sp5 signaling in non-chordate deuterostome embryos. Furthermore, our findings suggest that the roles of Sp5 during early vertebrate embryogenesis are similar to their functions in sea urchin embryos. Next, our findings demonstrate the critical role of ATF2 as a downstream factor of Wnt/JNK signaling in the proper positioning of germ layers during early embryonic development in sea urchins. Specifically, ATF2 is involved in the activation of important factors such as Sp5 and Wnt8, which restrict the ANE GRN to the anterior pole of the embryo. In addition, our analysis suggests that ATF2 is necessary for the maintenance of endoderm and mesoderm GRNs territories in sea urchin embryos. Additionally, while the negative regulation of Wnt signaling at extracellular and intracellular levels has been documented, the membrane-based mechanism of control was previously unknown. In this study, we present a phylogenetic analysis and expression pattern of the Znrf3/Rspo/Lgr complex, which may regulate the receptors and co-receptors of Wnt signaling pathways. Together, our findings shed light on the extended mechanism of the Wnt/JNK signaling pathway during the AP axis patterning of early sea urchin embryos, supporting the notion that the early primary axis patterning mechanism is evolutionarily conserved among bilaterians.