To obtain homozygous hio mutant embryos, heterozygous carriers of the hio mutation were mated. Typically, the eggs were spawned synchronously every morning. Embryos were raised at 30°C, and embryonic stages were determined based on morphological features, as previously described.6 The hio mutation was induced in the Cab-Kyoto line of medaka.3 The Kaga-Kyoto line of medaka was used for polymorphic marker-based genetic mapping.3 Genetic mapping and chromosome walking were performed essentially as described.19 Partial or click here full-length complementary DNAs of the raldh2 (Accession number AB439727), tbx5 (AB439834), wnt2bb (AB439835),

wnt2ba, cp, prox1, insulin, and tbx3 genes were generated by reverse-transcription polymerase chain reaction of messenger RNAs (mRNAs) from various stages of medaka embryos (Supporting Table 1). Alignment was performed using MultAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html). WT raldh2 mRNA (400 pg), obtained by in vitro transcription of a pBS-KS(−)-raldh2 clone, was injected into the cytoplasm

of one-cell stage embryos that were the progeny of intercrossed hio heterozygotes. Morpholino oligonucleotides (MOs) were synthesized by Gene-Tools, LLC (Corvallis, OR). MOs (0.8 pmol) were injected into the cytoplasm NVP-AUY922 price of one-cell stage WT medaka embryos. The sequences of MOs used were as follows: raldh2 MO, 5′-ATGACTGCCGTGGCTGCGCTGCTGT-3′; wnt2bb MO, 5′-ATATACCTGAGAGTGTCCAGAACAG-3′. Embryos resulting from hio heterozygote intercrosses were incubated in the dark from stage 21 onward in various dilutions of a 10−2 M all-trans RA (Sigma) stock solution in dimethylsulfoxide. The diluent Methane monooxygenase was 1× balanced salt solution composed of 110 mM NaCl, 5 mM KCl, 1 mM CaCl2, and 2.2 mM MgSO4, pH7.5. Teratogenic effects (such as disrupted heart and AP axis) were observed at 10−8 M all-trans RA and above. Whole- mount in situ hybridization was

performed as previously described,3 using antisense DIG-labeled riboprobes generated from medaka tbx5, wnt2ba, prox1, cp, insulin, wnt2bb, tbx3, or raldh2 partial or full-length complementary DNAs. Probes used to detect gata6, foxA3, ck19, and pdx1 expression were as previously described.4 Medaka embryos at stage 36 were placed in 0.5 mL 1× balanced salt solution containing 0.3 mg/mL N-([6-(2,4-dinitro-phenyl)amino]hexanoyl)-1-palmitoyl-2-BODIPY-FL-pentanoyl-sn-glycero-3-phosphoethanolamine (PED6) and incubated in the dark for 4 hours at 28°C. The treated embryos were rinsed with 1× balanced salt solution and placed in a glass depression slide. PED6 fluorescence was detected using a Zeiss Axioplan 2 microscope. Using bulked segregation analysis, we performed positional cloning and mapped hio between restriction fragment length polymorphisms OLc2806f and Scaf21_1.0M on LG3 (Fig. 1A). This region includes a sequence with homology to the mammalian and zebrafish raldh2 genes.