Two Types of Etiological Mutation in the Limb-Specific Enhancer of .

An enhancer named MFCS1 regulates Sonic hedgehog () expression in the posterior mesenchyme of limb buds. Several mutations in MFCS1 induce ectopic expression in the anterior limb bud, and these result in preaxial polydactyly (PPD). However, the molecular basis of ectopic expression remains elusive, although some mutations are known to disrupt the negative regulation of expression in the anterior limb bud. Here, we analyzed the molecular mechanism of ectopic expression in PPD including in a mouse mutation-hemimelic extra toes ()-and in other MFCS1 mutations in different species. First, we generated transgenic mouse lines with a reporter cassette flanked with tandem repeats of 40 bp MFCS1 fragments harboring a mutation. The transgenic mouse line with the -type fragment showed reporter expression exclusively in the anterior, but not in the posterior margins of limb buds. In contrast, no specific expression was observed in lines carrying the MFCS1 fragment with other mutations. Yeast one-hybrid assays revealed that the msh-like homeodomain protein, MSX1, bound specifically to the sequence of MFCS1. Thus, PPD caused by mutations in MFCS1 has two major types of molecular etiology: loss of a -motif for negative regulation of , and acquisition of a new -motif binding to a preexisting transcription factor, as represented by the mutation.