Genetic knockout suggests Isl1 enhancer redundancy in mouse hindlimb development

Isl1 encodes a LIM homeodomain transcription factor, which is expressed in hindlimb progenitor cells in the lateral plate mesoderm and is required for initiating hindlimb development. Previous studies by lacZ transgenesis in mouse embryos identified a cis-element located 3’ to the Isl1 gene, which could drive lacZ reporter expression in hindlimb progenitor cells and the branchial arch ectoderm. We refer to this cis-element as the Isl1 hindlimb progenitor enhancer (HLPE). Our previous study also showed that SALL4, a transcription factor, is enriched at the Isl1 HLPE, suggesting that SALL4 may regulate Isl1 expression through Isl1 HLPE. We sought to determine whether Isl1 HLPE regulates Isl1 expression and created mutant mice that lack the Isl1 HLPE sequence by CRISPR/Cas9. The Isl1 HLPE^−/− mouse lines, established after breeding with wild-type mice, did not exhibit gross morphological defects, except that their long bones are shorter than those of wild type. The shorter long bone phenotype was observed in the mid-gestation stage and was associated with misregulation of the expression of several chondrogenic genes, suggesting that the deletion of HLPE affects chondrogenesis. Although Sall4 regulation of Isl1 through Isl1 HLPE was suspected, skeletal analysis did not exhibit any synergy between Isl1 HLPE^−/− and conditional Sall4 mutation. In situ hybridization showed seemingly normal expression of Isl1 and its downstream gene Tbx4 in Isl1 HLPE^−/−, TCre; Sall4^fl/fl mutants, and Isl1 HLPE^−/−; TCre; Sall4^fl/fl mutants. Finally, by quantitative gene expression analysis, Isl1 expression is reduced but not abolished in Isl1 HLPE^−/− and Isl1^+/−; Isl1 HLPE ^± embryos, compared to wild-type embryos. Similarly, quantitative imaging analysis after immunostaining showed reduced ISL1 signals in the branchial arch ectoderm in Isl1 HLPE^−/− embryos. These results support our notion that the Isl1 HLPE sequence functions as an enhancer for Isl1 expression in hindlimb progenitor cells and branchial arch ectoderm cells and suggest that multiple redundant enhancers co-regulate Isl1 expression.