Sall genes regulate hindlimb initiation in mouse embryos.

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which trigger molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5) and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing one allele Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.