Cell-specific alterations in the activation of the Pitx1 regulatory landscape caused by the loss of a single enhancer (scRNA-seq)

Developmental genes often reply on multiple transcriptional enhancers to achieve correct expression during embryogenesis. These enhancers can have partially redundant activities, thus it is difficult to ascertain the direct impact of the loss of a single one of them as often only mild, if any, phenotypes are seen. In this work we use the testbed locus Pitx1 to characterise the regulatory and cell specific alterations following the loss of one of its enhancers Pen in vivo. We combine single cell transcriptomics and in embryo cell tracing and observe the global decrease perviously observed in deletion of Pen hindlimbs results from an accumulation of low to non-expressing cells that affects more proximal rather than distal limb and leads to a clubfoot phenotype product of a localised heterochrony and loss of irregular connective tissue. We also analyse the contribution of the enhancer to the overall locus structure and activity and we suggest that in some cases redundant enhancers may locally enforce a robust activation of their gene’s regulatory landscape. Whole forelimb and hindlimb buds were prepared for single cell RNA-seq analysis to identify which cell types make up the developing limbs of Mus musculus.