OBJECTIVETo perform the genetic identification of cloche(172) mutant zebrafish.

METHODSThe chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to treat the AB stain male fish. Large-scale forward genetic screening was carried out to search for lyC-deficient zebrafish mutant by WISH. The morphology changes of the embryos at 3 days postfertilization (3dpf) stage were observed and the cloche(172) gene was identified by mapping and complementation test.

RESULTSWe selected 4 lyC-deficient zebrafish by WISH. cloche(172) mutant showed morphological changes similar to cloche mutant in 3dpf stage. One fourth of the embryos showed cloche phenotype as found in complementation test, and the cloche(172) gene was mapped on the telomere of zebrafish 13 chromosome where cloche gene was located. Numerous red blood cells were observed in the cloche(172) mutant, while only a few cells were found in the cloche mutant in the tail region by o-dianisdine staining.

CONCLUSIONcloche(172) gene which is responsible for the phenotype of cloche mutant may be a novel point mutation allele of the cloche mutant.

Alleles ; Animals ; Chromosome Mapping ; Cloning, Molecular ; Embryo, Nonmammalian ; embryology ; metabolism ; Ethylnitrosourea ; toxicity ; Genetic Complementation Test ; Male ; Muramidase ; genetics ; Mutation ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

PURPOSE: Genes of the HoxD cluster play a major role in vertebrate limb development, and changes that modify the Hoxd12 locus affect other genes also, suggesting that HoxD function is coordinated by a control mechanism involving multiple genes during limb morphogenesis. In this study, mutant phenotypes were produced by treatment of mice with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed mutant mice exhibiting the specific microdactyly phenotype and examined the genes affected. MATERIALS AND METHODS: We focused on phenotype characteristics including size, bone formation, and digit morphology of ENU-induced microdactyly mice. The expressions of several molecules were analyzed by genome-wide screening and quantitative real-time PCR to define the affected genes. RESULTS: We report on limb phenotypes of an ENU-induced A-to-C mutation in the Hoxd12 gene, resulting in alanine-to-serine conversion. Microdactyly mice exhibited growth defects in the zeugopod and autopod, shortening of digits, a missing tip of digit I, limb growth affected, and dramatic increases in the expressions of Fgf4 and Lmx1b. However, the expression level of Shh was not changed in Hoxd12 point mutated mice. CONCLUSION: These results suggest that point mutation rather than the entire deletion of Hoxd12, such as in knockout and transgenic mice, causes the abnormal limb phenotype in microdactyly mice. The precise nature of the spectrum of differences requires further investigation.
Animals ; Base Sequence ; DNA/genetics ; DNA Primers/genetics ; Ethylnitrosourea/toxicity ; Genes, Homeobox ; Homeodomain Proteins/*genetics ; Limb Deformities, Congenital/genetics ; Male ; Mice ; Mice, Inbred BALB C ; Mutagens/toxicity ; *Point Mutation ; Transcription Factors/*genetics