To ascertain whether the small hio liver was functional, we took advantage of a reporter system based on PED6, a fluorescent phospholipid.21 When WT medaka ingest PED6, endogenous lipase find more activity and the rapid transport of cleavage products results in intense gallbladder fluorescence.4 We observed equivalent levels of green fluorescence in the gallbladders of WT and hio embryos treated with PED6 at stage 36 (Fig. 4B), indicating that hio livers have a normal capacity to metabolize lipids. Taken together, our results show that loss of raldh2 function initially impairs liver specification and retards liver development but does not impair hepatic cell differentiation

or liver functions at later stages of embryogenesis. Stafford and Prince22 have reported that hepatic and pancreatic cell markers are undetectable in zebrafish nls embryos. To investigate whether the hio mutation affected pancreas development in medaka, we carried out in situ hybridization using probes for the pdx1 and insulin genes. We observed pdx1-expressing cells in the pancreatic primordium region in both WT and hio embryos at stage 28 (Fig. 4C). Bortezomib nmr Furthermore, insulin-expressing cells were present in both WT and hio embryos at stage

30 (Fig. 4D). Thus, unlike its effects on liver development, the medaka hio mutation does not appear to affect pancreas development. This result stands in contrast to the zebrafish nls mutation, which severely impairs the development of both the liver and the pancreas. It has been shown in zebrafish that mesodermal wnt2bb expression promotes liver specification.17 It is also known that the wnt2ba gene acts downstream of RA signaling and regulates pectoral fin development in zebrafish.7, 20 The wnt2ba and wnt2bb

genes are both members of wnt2b gene family that exists in both zebrafish and medaka. These observations suggested CHIR99021 to us that Wnt2bb might be a good candidate for the key molecule regulating piscine liver specification downstream of RA signaling. To explore this hypothesis, we examined wnt2bb expression in hio embryos. At stage 22, no wnt2bb expression in the LPM was observed in either WT or hio embryos (Supporting Fig. 4). However, by stage 24, wnt2bb expression in the LPM directly adjacent to the liver-forming endoderm was induced in WT embryos but not in hio embryos (Fig. 5A, left panel). These results suggest that the hio mutation causes a loss of wnt2bb gene expression. Interestingly, wnt2bb still had not been expressed in the hio LPM at stage 29, when the liver bud forms (Fig. 5A, right panel). Thus, the small livers that eventually appear in medaka hio mutants seem to form independently of Wnt2bb signaling, just as occurs in zebrafish prt mutants.