As a result of transgenic insertional mutagenesis, heterozygous Fused toes (Ft) mice display a syndactyly of forelimbs and a thymic hyperplasia. Homozygous Ft/Ft embryos die in midgestation, exhibiting a deformation of craniofacial structures, a syndactyly and a polydactyly of fore- and hindlimbs, a disorganization of the ventral spinal cord, and defects in left-right axis formation. Here we report on our structural analyses of the Ft mutation. We established a physical as well as a gene map of the Ft locus, showing that the transgene integration resulted in a deletion of 1.6 Mb of genomic sequences on mouse Chromosome 8. Owing to this deletion, six genes, including the entire IroquoisB (IrxB) gene cluster, are directly affected by the Ft mutation.

Iroquois proteins comprise a conserved family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissues in both vertebrates and invertebrates. Earlier studies identified four murine Iroquois (Irx) genes. Here we report the isolation of two additional members of the murine gene family, Irx5 and Irx6. Phylogenetic analysis of the Irx gene family revealed distinct clades for fly and vertebrate genes, and vertebrate members themselves were classified into three pairs of cognate genes. Mapping of the murine Irx genes identified two gene clusters located on mouse chromosomes 8 and 13, respectively. Each gene cluster is represented by three Irx genes whose relative positions within both clusters are strictly conserved. Combined results from phylogenetic, linkage, and physical mapping studies provide evidence for the evolution of two Irx gene clusters by duplication of a larger chromosomal region and dispersion to two chromosomal locations. The maintenance of two cognate Irx gene clusters during vertebrate evolution suggests that their genomic organization is important for the regulation, expression, and function of Irx genes during embryonic development.

The Fused toes (Ft) mouse mutation was created by insertional mutagenesis, resulting in the deletion of several hundred kb of genomic sequences of mouse Chromosome (Chr) 8. Mice heterozygous for the Ft mutation are characterized by partial syndactyly of forelimbs and massive thymic hyperplasia indicating that programmed cell death is affected. Homozygous Ft/Ft embryos die at midgestation and show severe malformations of craniofacial structures. Furthermore, establishment of left-right asymmetry is random. Here we report on the positional cloning of a novel gene by exon trap analysis of a genomic clone encoding wild-type sequences corresponding to parts of the deletion in Ft mutants. RT-PCR experiments demonstrated that the newly identified gene, Fatso (Fto), is expressed throughout embryonic development. Wide expression was also found in tissues of adult mice. We show that expression of Fto is completely absent in mouse embryonic fibroblasts homozygous for the Ft mutation. In addition, we isolated the full-length cDNA which encodes a putative 58-kDa protein showing no similarities to known proteins or protein motifs. The expression data of Fto define it as a candidate gene involved in processes such as programmed cell death, craniofacial development, and establishment of left-right asymmetry.

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