Regulation and function of the Shox homeobox 2 gene in the developing mouse embryo

Abstract

The Shox homeobox 2 gene codes for a homeodomain transcription factor which is a critical mediator of proximal limb and hindbrain development, with a loss of expression in the limb and central nervous system resulting in a severe truncation of the developing stylopod and postnatal lethality, respectively. Shox2 expression is regulated by a combination of enhancers in the Shox2 regulatory landscape including the limb hindbrain control region (LHB) and over 19 other enhancers within the downstream Shox2 gene desert (S2GD). Here, we characterize the function and regulation of Shox2 in embryonic mouse development, expanding the mammalian limb development model and contributing to a better understanding of combinatorial gene regulation. First, the function of Shox2 in the patterning and outgrowth of the proximal limb is explored through single-cell RNA sequencing analysis of datasets from wild-type and Shox2-null embryonic limb buds. A combined approach of clustering and RNA velocity comparisons of these datasets reveal broad-scale changes in mesenchymal, progenitor, and chondrocyte populations in the early limb bud and altered trajectories of progenitor cells in the early embryonic limb bud. Second, the regulatory contribution of the S2GD and individual LHB enhancer were explored through the characterization of knockouts with a loss of S2GD activity in the limb and a genomic loss of the LHB enhancer, respectively. Inter-landmark calculations of stylopod lengths on adult reconstructions from µCT scans reveal that a loss of S2GD activity in the early limb mesenchyme results in a significant truncation of the humerus and femur, which is less severe, but phenotypically similar to the truncation in corresponding Shox2 mutants. Despite exhaustive phenotyping including geometric morphometrics, no significant change in length or morphology of the humerus or femur was detected in LHB knockout adult limbs. However, LHB knockout mice have an acoustic startle defect, likely associated with a detected loss of Shox2 expression in the embryonic hindbrain. These findings present the LHB enhancer as one of many enhancers in the S2GD which dynamically regulates expression of Shox2 uniquely in different tissues, with redundant activity in the developing limb and an essential activity contributing to Shox2 expression in the developing hindbrain.