Role for the Axin-RGS domain during embryonic development: maternal vs. zygotic functions

Upon sperm entry, the vertebrate egg undergoes a series of cell divisions that create a number of smaller cells without increasing the embryonic mass. This induces an elevation of intracellular calcium transient that is conserved across species. In zebrafish, fertilization occurs through an opening in the chorion, the micropyle and in Xenopus it can occur anywhere in the animal hemisphere. Wnt signaling activation is required during dorsal-ventral axis specification and it needs to be suppressed during the regionalization of the brain. Axin is a negative regulator of Wnt signaling and contains an RGS (Regulator of G Protein Signaling) domain. RGS domains are typical of RGS proteins, which are involved in a distinct signaling pathway, G-protein signaling. RGS proteins exert a negative effect of G-protein signaling by accelerating the GTPase activity (GAP) of the Gα subunit, thus turning off the signaling. Axin contains an RGS domain, however, it is not clear whether Axin is directly involved in G-protein signaling. We will also present a work performed using another negative regulator of the Wnt signaling network called naked cuticle (Nkd). Nkd has been shown to modulate β-catenin dependent and independent Wnt signaling. In chapter 2, we will show that the Axin-RGS like function is dispensable during the formation of the dorsal-ventral axis. We manipulated this protein by creating a point mutation in a critical aminoacid within the Axin-RGS domain, known to be detrimental for the GAP function of RGS proteins, Axin1Q162A. Maternal depletion of Axin1 in Xenopus oocytes causes hyperactivation of Wnt signaling and results in dorsalization. Axin1Q162A is able to suppress the dorsalization of maternally depleted embryo and restore normal dorsa-ventral axis formation.

In chapter 3, we will describe the role of Axin during the patterning of the vertebrate brain. We show that the point mutant is not able to restore normal brain development in zebrafish embryos after Axin knockdown. We hypothesize that Axin-RGS like function is necessary during the patterning of the vertebrate brain that occurs after zygotic transcription has been initiated. Moreover, we show that Axin-RGS like activity may be dispensable during this stage of development. Finally, we demonstrate that Axin1Q162A localization differs from the wildtype Axin1 and Axin1 but not Axin1Q162A is localized to the plasma membrane upon Gα overexpression in zebrafish embryos.

Embryonic organ laterality is preceded by molecular and physiological asymmetries. In chapter 4 we describe the role of another Wnt antagonis, Nkd cuticle, during left-right patterning. Prior to organogenesis, a group of cells called Dorsal Forerunner Cells, (DFCs), migrate ahead of the dorsal blastoderm during gastrulation to form the Kupffer's vesicle (KV). This vesicle will trigger a signaling cascade that will culminate with left-right determination. We show data that support the requirement of Nkd in organ laterality and convergence and extension movements using zebrafish and Xenopus laevis.