Determining the Place of Aqp3b in Noncanonical Wnt Signaling

Abstract

During Xenopus laevis gastrulation, convergent extension is required for the mesoderm to extend into the embryo and shape the embryonic body plan. Recent results from our lab suggest that the inhibition of aquaporin3b (aqp3b) prevents convergent extension of the mesoderm and that aqp3b acts through noncanonical Wnt signaling. Wnt signaling is a key signaling pathway for embryo and tissue development. There are two types of Wnt signaling pathways, the canonical and the noncanonical pathways. There are three separate branches to noncanonical Wnt signaling. Our lab has shown that aqp3b acts through the noncanonical Wnt/Ca2+ pathway and that it acts upstream of the cytoplasmic Wnt signaling pathway member Disheveled (Dsh). The Frizzled7 (fzd7) membrane receptor is part of the noncanonical Wnt/Ca2+ pathway and also acts upstream of Disheveled (Dsh). I will test, whether in this signaling cascade, aqp3b acts upstream or downstream of fzd7. Thus, I will test whether fzd7 activates aqp3b, if aqp3b activates Fzd7, or if aqp3b is bypassed and fzd7 activates disheveled. When fzd7 is active, GFP-labeled protein kinase C (PKC-GFP) relocates from the cytoplasm to the plasma membrane. Thus, I will inject either PKC-GFP alone, PKC-GFP + fzd7, or PKC-GFP + fzd7 +aqp3bMO (morpholino oligonucleotide, which inhibits aqp3b) into two-cell Xenopus embryos and examine under a fluorescence microscope whether the PKC is bound to the membrane (active Wnt signaling: PKC + fzd7 injection or if aqp3b acts upstream of fzd7) or remains in the cytoplasm (no Wnt signaling: PKC injected alone or if aqp3b acts downstream of fzd7). With this procedure the place of aqp3b within the Wnt/Ca2+ signaling pathway will be determined.