Distinct Roles of Hand2 in Initiating Polarity and Posterior Shh Expression during the Onset of Mouse Limb Bud Development

Abstract

The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. In wild-type limb buds, Hand2 is part of the protein complexes containing Hoxd13, another essential regulator of Shh activation in limb buds. Chromatin immunoprecipitation shows that Hand2-containing chromatin complexes are bound to the far upstream cis-regulatory region (ZRS), which is specifically required for Shh expression in the limb bud. Cell-biochemical studies indicate that Hand2 and Hoxd13 can efficiently transactivate gene expression via the ZRS, while the Gli3 repressor isoform interferes with this positive transcriptional regulation. Indeed, analysis of mouse forelimb buds lacking both Hand2 and Gli3 reveals the complete absence of antero-posterior (AP) polarity along the entire proximo-distal axis and extreme digit polydactyly without AP identities. Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer.

Author Summary

During early limb bud development, posterior mesenchymal cells are selected to express Sonic Hedgehog ( Shh), which controls antero-posterior (AP) limb axis formation (axis from thumb to little finger). We generated a conditional loss-of-function Hand2 allele to inactivate Hand2 specifically in mouse limb buds. This genetic analysis reveals the pivotal role of Hand2 in setting up limb bud asymmetry as initiation of posterior identity and establishment of the Shh expression domain are completely disrupted in Hand2 deficient limb buds. The resulting loss of the ulna and digits mirror the skeletal malformations observed in Shh-deficient limbs. We show that Hand2 is part of the chromatin complexes that are bound to the cis-regulatory region that controls Shh expression specifically in limb buds. In addition, we show that Hand2 is part of a protein complex containing Hoxd13, which also participates in limb bud mesenchymal activation of Shh expression. Indeed, Hand2 and Hoxd13 stimulate ZRS–mediated transactivation in cells, while the Gli3 repressor form (Gli3R) interferes with this up-regulation. Interestingly, limb buds lacking both Hand2 and Gli3 lack AP asymmetry and are severely polydactylous. Molecular analysis reveals some of the key interactions and hierarchies that govern establishment of AP limb asymmetries upstream of SHH.