Genetic interactions between Shox2 and Hox genes during the regional growth and development of the mouse limb

Abstract

Homeobox genes of the Hox and Shox families have prominent roles in development and disease, with their independent mutation leading to regional shortening of the skeletal elements of the limb. In this work, we investigate whether Shox2 and Hox genes together coordinate limb development. By adapting double mRNA FISH to single embryos, we show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11, juxtaposing the distal expression of Hoxa13 and Hoxd13. Their concerted proximal limb expression is critical for limb growth as Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. These genetic interactions are likely a result of Shox2 and Hox genes together promoting chondrocyte maturation, as their individual mutation similarly leads to reduced Runx2 expression in the humerus. In addition to cooperative functions in the proximal limb, Hox genes also repress Shox2 expression in a subset of cells of the distal limb. This repression may be important for normal development, since ectopic Shox2 expression disrupts Hox-dependent autopod morphogenesis. Finally, to provide insight into the molecular role of SHOX2 during development, proteins that co-purify with SHOX2 in the limb bud were identified using immunoprecipitation followed by mass spectrometry. This approach identifies numerous potential SHOX2 interacting partners, including the chromatin regulators SMARCE1 and HDAC2.