Insertion of a PGK-neomycin resistance cassette into exon 3, upstream of sequences encoding the homeodomain, disrupted the gene. Western blot analysis using a monoclonal antibody specific for the most abundunt isoform of the protein did not detect any protein product in homozygous mutant E16 embryos. Immunohistochemical analysis using this same antibody did not detect nuclear or cytoplasmic expression in limb buds of E11.5 homozygous mutant embryos.