We compared the Selleckchem MLN0128 sequence of a genomic fragment encoding the WT medaka raldh2 gene with the sequences of the corresponding fragments from four independent homozygous hio embryos. We found an A to G transversion in hio alleles that would cause the threonine 468 residue in the WT RALDH2 enzyme to be replaced

by alanine (Fig. 1B). A comparison of the predicted WT RALDH2 amino acid sequences among medaka, human, xenopus, and zebrafish revealed an overall amino acid sequence identity of 81% (between medaka and human or xenopus) and 84% (between medaka and zebrafish) (Fig. 1C). The threonine 468 residue was conserved among all species examined. Moreover, threonine 468 lies within the catalytic domain of WT RALDH2 (Fig. 1C). These results suggest that the mutant RALDH2 protein produced in hio mutants is

inactive. It has been well established that the defects of RA signaling lead to the impairment of fin development in zebrafish.7, 8, 10, 16 We showed that the injection of RALDH2-MO into WT embryos results in the BGB324 solubility dmso impairment of fin development, and the injection of raldh2 mRNA or exogenous RA rescued the defects of fin development of hio mutant (Supporting Fig. 1). These results indicate that RALDH2 and RA regulate fin development in medaka. In addition, hio embryos lacked tbx5 and wnt2ba expression, which acted downstream of RA during fin development (Supporting Fig. 2). Taken together, we concluded that RA signaling plays important roles in fin development in medaka. We have previously reported that the medaka hio mutation results in a small and malformed liver.3 To examine the role of raldh2-dependent signaling in liver formation in medaka, we employed three approaches. First, to investigate whether loss-of-function of raldh2 could account for this liver defect, we injected raldh2-MO into WT embryos and inspected the developing liver. We found that the raldh2 morphants had the same undersized livers as the hio

mutants (Fig. 2A). Estimation of liver size via in situ hybridization using a gata6 probe confirmed the reduced liver size in the raldh2 morphants (Fig. 2B). Second, to determine whether the hio/raldh2 mutation was responsible for the small livers of these mutants, we 上海皓元 injected in vitro transcribed raldh2 mRNA into the cytoplasm of one-cell stage embryos that were the progeny of intercrossed hio heterozygotes and used gata6 in situ hybridization to assay these embryos for rescue of liver size. As expected, 25% of the progeny of intercrossed hio heterozygotes (uninjected controls) had small livers. In contrast, the percentage of progeny with decreased liver size was reduced to 14% after injection of raldh2 mRNA (Fig. 2C). Finally, we investigated whether treatment with exogenous RA, the bulk of which is synthesized by RALDH2, could rescue the liver defects caused by the hio mutation.