Axial Hox9 activity establishes the posterior field in the developing forelimb

Current models hold that the early limb field becomes polarized into anterior and posterior domains by the opposing activities of Hand2 and Gli3. This polarization is essential for the initiation of Shh expression in the posterior margin of the limb bud, but how this polarity is established is not clear. Here we show that initial anteroposterior polarization of the early forelimb field requires the function of all four Hox9 paralogs (Hoxa9, Hoxb9, Hoxc9, and Hoxd9). This is unexpected, given that only HoxA and HoxD AbdB group genes have been shown to play a role in forelimb patterning, regulating the activation and maintenance of Shh expression and subsequent proximal-distal patterning of the forelimb. Our analysis of Hox9 quadruple mutants demonstrates that Hox9 function is required for the expression of Hand2 in the posterior limb field. Subsequently, Gli3 expression is not repressed posteriorly, Shh expression is not initiated, and collinear expression of HoxA/D10-13 is not established, resulting in severely malformed forelimbs lacking all posterior, Shh-regulated elements. This Hox9 mutant phenotype is restricted to the forelimbs; mutant hindlimbs are normal, revealing fundamental differences in the patterning mechanisms governing the establishment of forelimb and hindlimb fields.