Characterization of a putative late acting enhancer of Shox2

Various enhancers often collectively regulate their target gene during development. Nevertheless, it is still unclear how exactly enhancers cooperate to control gene expression and how they drive defined spatiotemporal transcription in the different tissues. Here we investigate the role of the PLE4 enhancer in regulating the Shox2 gene transcription in the late development phases of mouse limbs. First, by analyzing the enhancer patterns of activity within the limbs with a transgenic LacZ reporter system, we show that PLE4 bears a late limb activity that corresponds to Shox2 expression. Secondly, we established in a Shox2 fluorescent dsmCherry reporter background, via CRISPR/Cas9, Shox2 PLE4-/PLE4-;dsmCherry/+ transgenic mouse embryos to study the impact of PLE4 deletion on Shox2 expression. FACS and WISH assay on limbs from Shox2 PLE4-/PLE4-;dsmCherry/+ embryos revealed minor changes in the cell population expressing Shox2 but resulted in the remodeling of the Shox2 expression pattern. These results suggest PLE4 involvement in Shox2 regulation in the late stages of limb development