We found that, like hio embryos, WT medaka embryos that had been injected with wnt2bb-MO lacked prox1 expression (Fig. 5B). These results suggest that Wnt2bb signaling is responsible for liver specification in medaka. In conclusion, our study has shown that the hio mutation in medaka impairs liver specification by abrogating wnt2bb expression. Our data are thus the first

genetic evidence that RA signaling positively regulates liver specification by inducing wnt2bb expression. In this study, we examined the role of RA signaling during embryogenesis by characterizing medaka hio mutants. These mutants bear Alectinib chemical structure an alteration to the raldh2 gene (Fig. 1) that encodes the enzyme principally responsible for RA synthesis, and we interpret that this is a nearly null mutation because the phenotypes of hio mutant are similar to that of RALDH2 morphants (Fig. 2 and

Supporting Fig. 1). MI-503 The hio mutants exhibit two prominent phenotypes: missing pectoral fins and a small liver (Fig. 2 and Supporting Fig. 1). Work in mouse, chick, and zebrafish has shown that RA signaling from the somitic mesoderm is essential for limb induction and is mediated by the expression of downstream factors such as wnt2ba and tbx5.7–14 We show that the hio mutation in medaka leads to defects in pectoral fin development and tbx5 and wnt2ba expression (Supporting Fig. 2). Thus, our results indicate that RA signaling is crucial for fin specification in medaka and show that limb induction MCE signaling is conserved across a broad range of species (Fig. 6, right part). Significantly, our work has also uncovered a role for RA signaling in liver development. We have demonstrated that the hio mutation retards the formation of hepatic buds from the foregut (Fig. 3A) and causes a profound defect in liver specification (Fig. 3B). In addition, we

have shown that the wnt2bb expression required for the regulation of liver specification is undetectable in the LPM of hio embryos (Fig. 5A). Our data constitute the first genetic evidence that RA signaling regulates vertebrate liver specification by inducing wnt2bb gene expression (Fig. 6, left part). Previously, Wang et al.23 reported that liver growth is severely affected in RALDH2-deficient mouse embryos. Thus, RA signaling in liver specification may be conserved among other species. There are several similarities in the signaling pathways governing pectoral fin and liver organogenesis. During zebrafish pectoral fin development, RA signaling induces wnt2ba expression, which in turn induces tbx5 expression. Tbx5 is a key molecule that regulates the expression of downstream effectors such as the fgf and bmp family members fgf24, fgf10, and bmp2b.7, 16 Thus, limb induction requires a sequential RA Wnt Tbx Fgf + Bmp signaling cascade. A parallel situation may exist for liver specification in medaka.