17, 18, 24 In this study, we showed that RALDH2 drives wnt2bb expression during liver specification in medaka (Fig. 5). Based on the proposal of Shin et al.18 that Fgf and Bmp act downstream of Wnt2bb during liver specification,

the sum total of all these results suggests that liver specification also requires a sequential RA Wnt Fgf + Bmp signaling cascade. Intriguingly, we found that RA signaling induced tbx3 expression in medaka (Supporting Fig. 5). However, our morpholino studies showed that RA signaling associated with liver formation can regulate tbx3 expression without involving Wnt2bb (Supporting Fig. 5). These data indicate that Tbx3 can act downstream of RA signaling, but it is likely that other T-box family members are involved in the putative RA Wnt Tbx Fgf + Bmp signaling cascade that drives liver MK-2206 concentration development. We are continuing our search for the identity of this transcription factor. A sequential RA Wnt Tbx Fgf + Bmp signaling cascade is indispensable for the limb induction process that underlies Crizotinib datasheet pectoral fin development. Alterations in raldh2 such as the medaka hio and zebrafish nls and nof mutations lead to an absence of pectoral fins, as does knockdown of wnt2ba

using MO in WT zebrafish.8, 10, 16 Notably, these mutants and morphants never form pectoral fins during the entire course of embryogenesis. Conversely, a sequential RA Wnt Fgf + Bmp signaling cascade is not indispensable for liver specification, because medaka hio mutants and zebrafish prt mutants are able to form a functional liver at an abnormally late stage of development. A molecule that may be able to partially compensate for a loss of RALDH2 is Fgf10, which is also induced downstream of RA signaling and involved in limb and liver formation. Loss of fgf10

prevents fin development in zebrafish,7 and Fgf10-deficient mouse embryos lack limbs and have an G protein-coupled receptor kinase abnormally small liver.25, 26 Thus, fgf10 and raldh2 functions may cooperate during embryogenesis such that their mutation results in similar phenotypes. Moreover, in zebrafish fgf10 mutants, the hepatopancreatic ductal epithelium is severely dysmorphic, and cells of the hepatopancreatic ductal system and adjacent intestine misdifferentiate and adopt a hepatic or pancreatic fate.27 These results indicate that Fgf10 functions to repress the differentiation of hepatopancreatic ductal epithelium into hepatic or pancreatic cells and thus demarcates developing organs and tissues. In our hio mutants, it may be that the observed lack of liver specification leads not only to impaired liver development but also to misdifferentiation in the hepatopancreatic ductal system that results in the formation of a small liver. Such misdifferentiation could obscure an absolute requirement of raldh2 for liver specification, and might create an obstacle to finding mutations that specifically interfere with the initial specification of the liver anlage.