To obtain homozygous hio mutant embryos, heterozygous carriers of

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 this website 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 MCE公司 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.

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