Whereas 25%

selleck chemical of the untreated progeny of intercrossed hio heterozygotes had small livers, the percentage of progeny with a small liver was reduced to 13% after exposure to 5 × 10−9 M atRA (Fig. 2D). Thus, treatment with either WT raldh2 mRNA or exogenous RA can rescue the small liver phenotype in at least some hio mutants, although the efficiency of such rescue is much lower for the liver than for the pectoral fin. When the livers of hio mutants with treatment with either WT raldh2 mRNA or exogenous RA became as large as that of WT medaka, we judged it to be rescued. Therefore, we may have underestimated the recovery rate of liver phenotype. In any case, the loss of raldh2 function in hio mutants causes a defect not only in pectoral fin development but also in liver formation. Although the molecular mechanism by which RA signaling initiates fin development is well established,7, 20 the molecular regulation of liver development by RA signaling remains to be elucidated. To address this issue, we used in situ hybridization with a probe specific for the endodermal marker foxA3 to monitor liver development in hio embryos. Whereas hepatic buds were observed in WT medaka

at stage 25, these structures did not form in hio mutants until stage 29 (Fig. 3A). By stage 32, hepatic buds were noticeably smaller in hio embryos compared with the WT. These data indicate that the medaka hio mutation retards hepatic bud formation. Next, we determined whether the hio mutation interferes with the initial specification of liver anlage in medaka. We carried out in situ hybridization using a probe for the hepatic

specification buy 3-deazaneplanocin A marker prox1 to monitor liver specification. In WT medaka embryos, prox1 was induced in the hepatic bud starting at stage 25 (Fig. 3B, upper panel), and by stage 29, prox1-positive cells were observed only in the hepatic region. In hio embryos, the formation of the hepatic bud was delayed until stage 29 (Fig. 3A), so that prox1-positive cells were not observed in the hepatic region until this stage (Fig. 3B, bottom panel). These results indicate that Exoribonuclease the hio mutation compromises the signaling pathway required for initial hepatic fate specification. The most important cell types in the vertebrate liver are cholangiocytes (bile duct cells) and hepatocytes. To determine whether hio livers were capable of normal hepatic cell differentiation, we subjected WT and hio embryos to in situ hybridization with a probe for the cholangiocyte marker cytokeratin19 (ck19) and the hepatocyte marker ceruloplasmin (cp). At stage 28, although WT embryos showed a few ck19-positive cells in the hepatic region, hio embryos did not (Supporting Fig. 3). However, by stage 32, ck19 expression was comparable in WT and hio livers (Fig. 4A, left panel). Furthermore, cp expression was comparable in WT and hio livers at stage 34 (Fig. 4A, right panel).