A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2

ABSTRACT During development cell fates are specified by tightly controlled gene expression programs. PBX TALE transcription factors control gene regulatory networks (GRN) that direct vertebrate tissue patterning and organ morphogenesis. How PBX1/2 proteins acquire context-specific functions, despite widespread embryonic expression of Pbx1/2 , remains elusive. In mouse limb buds, mesenchymal-specific loss of PBX1/2 or of the transcriptional regulator HAND2 results in similar phenotypes, suggesting that PBX1/2- and HAND2-dependent programs converge to control limb development. To investigate this scenario, we combined tissue-specific and temporally-controlled mutagenesis with multi-omics approaches using the murine hindlimb bud as a model. We reconstructed a GRN collaboratively directed by PBX1/2 and HAND2, demonstrating that Pbx1 - Hand2 genetically interact in vivo during hindlimb patterning, with PBX1 concomitantly acting as an upstream regulator of Hand2 . At organismal-level resolution the GRN is active within restricted subsets of posterior-proximal hindlimb mesenchymal cells, wherein Pbx1/2 and Hand2 are co-expressed with their target genes. Genome-wide profiling of PBX1 binding across multiple tissues further revealed that HAND2 selects a subset of PBX-bound regions to impart limb patterning functionality. This research elucidates mechanisms underlying limb bud-specific functions by PBX1/2, while informing general principles by which promiscuous transcription factors cooperate with select cofactors to instruct distinct developmental programs.