The legless transgene insertional mutation was found in a screen of hundreds of independent transgenic lines of mice. Hemizygous animals appear normal, but homozygous legless mutants display a remarkable phenotype. Hindlimbs are truncated at the knees, with no distal structures present. Forelimbs show variable expressivity, with differing numbers of anterior elements missing from one individual to the next. Sometimes just the thumb is missing, sometimes the thumb and forefinger, and sometimes the entire paw and the radius of the forearm are missing, with the ulna present and normal. Furthermore, the brains of mutants are malformed, with the most anterior components, the olfactory lobes, entirely absent, and the cerebral hemispheres show necrotic regions of variable severity. We also observed that 50% of the legless mutants had situs inversus, or reversed left-right asymmetry. A gene required for correct lateralization has apparently been disrupted, allowing the process to randomize. Interestingly, we have shown that during embryogenesis the legless mutants are hypersensitive to very low doses of retinoic acid, with the limb and brain defects dramatically enhanced in severity. These doses of retinoic acid have little or no effect on hemizygous and wild type littermates, suggesting that the mutation has somehow impacted the retinoid pathway. Furthermore, complementation tests with the classical situs inversus viscerum gene indicated that the transgene insertion likely mutated at least two genes, with one involving lateralization. We have now characterized several genes located in the transgene disrupted region. One of these, designated Sp-4, is an outstanding candidate for involvement in the legless phenotype. The expression pattern of this gene correlates extremely well with the phenotype with transcripts most abundant in the developing limbs and anterior regions of the brain. Moreover, Sp-4 encodes a zinc finger transcription factor, and transcription factor genes often play important regulatory roles in development. And finally, the DNA binding target sequence of Sp-4 is found in the promoter regions of several genes of the retinoid pathway, providing a molecular link between the mutation and the observed retinoid hypersensitivity. We hypothesize that the Sp-4 gene, and another gene of some promise designated 10B, play an important role in limb and/or brain development. We propose to directly test this by conducting structural and functional characterizations that will include individual gene targeting. We also propose to continue the search for additional genes of interest, such as the situs inversus gene, which we suspect remain to be found. The long range goal of this work is to understand the molecular roles that the disrupted genes play in limb and brain development, and lateralization.
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