Hox genes encode transcription factors that regulate patterning of the body plan of animals. The murine Hoxa13 gene is critical for digital and reproductive tract morphogenesis. The N-terminal, non-homeodomain segment (NTD) of vertebrate HOXA13 protein orthologues is highly conserved. A subset of the orthologue-specific conserved NTD residues are shared among paralogue group 13 HOX proteins and implies the existence of ancient and distinct functional activities for the NTDs of HOX proteins. We hypothesize that conserved residues within the NTD of HOXA13 are critical for transcriptional activity and for interactions with normal protein partners of HOXA13. In Aim I we will use an in vitro transient transfection assay system with a cellular promoter, that we have shown to be activated 20-fold by HOXA13, to test the function of NTD-mutant HOXA13 proteins. The in vitro system will provide a direct interpretation of the effect of a conserved sequence alteration on normal transcription factor function. These experiments will allow us to define the role of conserved amino acids and peptide domains efficiently, and to determine the specific functional effect of a modification on normal HOXA13 activity. In Aim II we will use the yeast two-hybrid system to isolate and study proteins specifically interacting with three different domains of HOXA13. Specificity of binding will be verified with in vivo and in vitro methods and amino acids essential for binding will be determined. The effect of the protein alterations described in Aim I on binding by the candidate cofactors will be determined. Functional assessment of isolated proteins will be tested in the in vitro transfection assay. In Aim III we will use homologous recombination in ES cells to modify evolutionarily-conserved HOXA13 amino acid motifs known from work in Aim I to be critical for specific transcriptional activities in vitro. We will inject these modified ES cells into blastocysts to create chimeric mice, obtain germline transmission of the mutant alleles, and characterize the limb phenotypes associated with these mutations.