Genetic Interactions Between Shox2 and Hox Genes During the Regional Growth and Development of the Mouse Limb

Abstract

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb.T HE vertebrate limb is a valuable model for studying the genetic coordination of a complex developing structure. The proximodistal axis of the limb is composed of discrete segments, the growth and development of which are selectively perturbed when individual, or combinations of, homeobox genes are disrupted. In mice, mutations of the paralogous Hox9 and Hox10 genes result in shortened stylopodal elements (containing the humerus and femur) (FromentalRamain et al. 1996a;Wellik and Capecchi 2003), deletions of Hox11 genes result in truncated zeugopodal elements (radius/ ulna and fibula/tibia) (Davis et al. 1995;Wellik and Capecchi 2003), and disruption of Hox13 genes results in agenesis of the autopod (metacarpals/metatarsals and the digits) (FromentalRamain et al. 1996b). Mutation of short stature homeobox (Shox) genes similarly gives rise to the disproportionate shortening of certain limb regions. In humans, loss of SHOX leads to the truncated zeugopod elements found in people with LeriWeill, Turner, and Langer syndromes (Rao et al. 1997;Belin et al. 1998;Shears et al. 1998;Zinn et al. 2002). While rodents have uniquely lost the Shox gene among mammals (Gianfrancesco et al. 2001), disruption of the widely conserved Shox2 gene results in severely shortened stylopodal elements in mice (Cobb et al. 2006). Thus, Hox and Shox gene perturbations each give ...

A unique stylopod patterning mechanism by Shox2 controlled osteogenesis

“…Together with the fact that the perichondrial expression of Hox13 was also proposed to pattern the autopodial skeleton (Villavicencio-Lorini et al, 2010), our study indicates that the patterning can be exerted by controlling the specification and differentiation of the perichondrial osteogenic precursor cells to simultaneously modulate the patterning of cartilaginous mode and bone formation. Interestingly, in the HoxA/D compound mutant, the expression of Shox2 in the embryonic perichondrium, but not chondrocytes, is severely affected (Neufeld et al, 2014), indicating that Shox2 itself is a key effector for Hox-TALE patterning system in the stylopod and further supports the idea that Hox-TALE patterns skeletal elements by commissioning perichondrial cells. Given the instructive role of perichondrial cells on chondrocyte differentiation and maturation (Ohbayashi et al, 2002), further dissection of Shox2-specific targets in the osteogenic lineage would further highlight key factors involved in such patterning function.…”

“…Strikingly, Shox2 mutation causes loss of the stylopod in both forelimbs and hindlimbs, which was thought to be attributed to the direct function of Shox2 in chondrogenesis (Bobick and Cobb, 2012;Yu et al, 2007). However, an epistatic additive interaction between HoxA/D genes and Shox2 was seen in limb development (Neufeld et al, 2014), suggesting an involvement of Shox2 in the Hox-TALE patterning system.…”

A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

“…S1; Rincon-Limas et al 1999). On the other hand, some proximal gene expression, including Shox2 and Hoxa11 (Neufeld et al 2014), was clearly gained within the distal domain, suggestive of an extension of proximal identity into the most distal limb aspect ( Fig. 1B,D; Supplemental Fig.…”

Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage

“…Mice with a loss of Shox2 function exhibited severe truncation of the stylopod bone of fore‐ and hindlimbs in addition to hypoplasia of the hindlimb zeugopodal bone (Bobick & Cobb, ; Cobb et al., ). Shox2 showed overlapping expression with Hoxa9‐11 in the mesenchyme of the stylopod and zeugopod then in the outer layer of the perichondrium and proliferating chondrocytes but not in the autopod (Neufeld, Wang, & Cobb, ; Swinehart, Schlientz, Quintanilla, Mortlock, & Wellik, ). Genetic analysis revealed that Shox2 is a downstream gene of Hox11 (Gross, Krause, Wuelling, & Vortkamp, ).…”

“…SHOX2 also bind to this CHBRL (Supporting Information Figure C) indicating that SHOX2 participate self‐regulation in coordination with HOX11. Interestingly, genetic analysis also suggested that Hox and Shox2 coordinate during development of the stylopodal and zeugopodal cartilage (Neufeld et al., ). Supporting this observation, we showed that SHOX2 also shares common binding regions of some Hox target genes with HOXA11 and HOXA13 (Figures and , Supporting Information Figure and Table ).…”