Fig. 2

Wtx inhibits the canonical Wnt pathway in early zebrafish development. Whole mount in situ hybridization to detect mRNA expression changes indicative of modulated Wnt/β‐catenin signaling. The  wtx depletion phenotypes were compared with  p53‐control MO‐injected embryos and  beta‐catenin‐2 MO‐injected embryos. A, Examination of  gfp expression in zebrafish embryos heterozygous for a β‐catenin responsive transgene  Tg(TOP:dGFP) at shield stage (6 hpf). Embryos are shown from a ventral view with the animal pole at top and from an animal view with dorsal to the right. Depletion of  β‐catenin2 resulted in reduced transgene expression whereas embryos injected with the  wtx MO displayed increased number of β‐catenin responsive cells in an expanded area within the ventrolateral margin. GFP‐positive cells are recognizable as blue spots inside the areas which are marked with dashed lines in the ventral views; arrows illustrate the expansion of the transgene expressing area in  wtx morphants. The diffuse staining around the edge of the embryo is not specific. B, Quantification of the data shown in (A). Images were blinded and the numbers of  gfp positive cells were counted. Results from two independent experiments are shown. Error bars represent SD. P values according to the two‐tailed  t‐test after Bonferroni correction: ** P < .01, *** P < .001. C and D, Expression analysis of markers for lateral mesoderm and neuroectoderm by using a  pax2a/myod1/six3b probe mixture on 12 hpf zebrafish embryos. Embryos are shown in a cranial and dorsal view with anterior to the left. C, Knockdown of  β‐catenin2 caused a lateral shortening of the  pax2a expression domain in the MHB and reduced expression of  six3b in the optic primordia (white arrows). The  myod1 domain in the somites is fused (white asterisk) and absent in the somitic and presomitic mesoderm. In contrast  wtx morphants show expanded expression of  pax2a, myod1 and  six3b(white arrowheads). For “beta‐catenin‐2, “p53 control” and “wtx (ATG)” 6, 3 and 11 embryos were analyzed, respectively. (D)  Wtx (ATG) and  wtx (splice) morphants show expanded expression of  six3b (white arrows) and  myod1 (white arrowheads). For “p53 control”, “wtx (ATG)” and “wtx (splice)” 7, 13 and 9 embryos were analyzed, respectively. EF: eye field; MHB: midbrain‐hindbrain boundary; PM: presomitic mesoderm; SM: somitic mesoderm; SO: somites. Scale bar represents 150 μm