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.

Keywords: SHOX; chondrogenesis; epistasis; fluorescence in situ hybridization; perichondrium.

Figure 1

Characterization of Shox2 and Hox gene expression. (A–C) Fluorescence in situ hybridization of Shox2 and Hox genes in single embryos, with yellow signal in the merged image showing coexpression. (A) At E10.5, Hox genes are expressed in a nested pattern within the Shox2 expression domain, which occupies the majority of the limb bud. The inset for the Shox2/Hoxd13 series shows a single optical section through the limb bud, using confocal microscopy. (B) At E11.5, Shox2 expression is confined to the proximal limb and shares a similar distal expression border to that of the Hoxd9 and Hoxa11 expression domains. Hoxd13 is exclusively expressed in the distal limb bud. (C) At E12.5, Shox2 expression is maintained in the proximal limb. Hoxd9 and Hoxa11 are expressed in the zeugopod, with Hoxd9 additionally being expressed in the developing digits. Hoxd13 is expressed in the developing digits, and Hoxa13 is expressed in the developing digits and carpals. (D) Chromogenic in situ hybridization at E12.5 and E13.5 shows Shox2 and the Hox genes are expressed both outside and within the elements of the stylopod and zeugopod. Shox2 and Hoxa11 are each expressed in the proliferating chondroctyes within their respective expression domains. Expression outlining each element corresponds to the perichondrium. H, humerus; R, radius; U, ulna. Bar, 0.25 mm.

Figure 2

Epistasis between Shox2 loss-of-function mutations and Hox genes during growth of the stylopodal and zeugopodal elements. (A) Deletion of a single copy of Shox2 has no effect on the newborn limb in an otherwise wild-type background, but leads to a shortened humerus in a HoxD^−/− background (n = 4–6 for each genotype). Arrowhead points to humerus. (B) Deletion of both copies of Shox2 has a stronger effect on the growth of the ulna in a HoxA^c/c background than in an otherwise wild-type background (n = 4–6 for each genotype). Arrowhead points to ulna. (A′ and B′) Interaction plots showing disproportionate effects, as shown by nonparallel lines, of the respective Shox2 mutation in each Hox background. Data are plotted as means ± SEM. Effect of Shox2 mutation is significantly different in each Hox background compared to an otherwise wild-type background. P < 0.001. (A′′ and B′′) Genotype–phenotype maps showing the effects of Shox2 mutations on skeletal length in multiple Hox backgrounds, arranged in decreasing dose, in both the fore- and hindlimbs.

Figure 3

Epistasis between Shox2 gain-of-function and Hox genes during growth of the stylopod. (A) Shox2 ISH on wild-type and RosaShox2 limbs at E12.5. (B) Real-time PCR determining the relative Shox2 levels in wild-type and RosaShox2 animals in whole E10.5 forelimb buds and proximal E12.5 forelimb buds. (C) Newborn forelimb skeletons showing the effects of Shox2 overexpression in wild-type and Shox2^c/- backgrounds. Arrowheads point to humerus. (C′) Interaction plot displaying the effects of overexpressing Shox2 in wild-type, Shox2^c/−, HoxA^c/+; HoxD^−/−, and HoxA^c/c; HoxD^−/− backgrounds (n = 3–6 for each genotype). Data are plotted as means ± SEM. Effect of RosaShox2 is significantly different in each mutant background compared to an otherwise wild-type background. P < 0.001. (D) Newborn forelimb skeletons and quantification of the effect of modulating Shox2 dose in a HoxA^c/c; HoxD^−/− background (n = 3–6 for each genotype). Arrowheads point to humerus. P < 0.05.

Figure 4

Independent mutation of Shox2 and Hox genes results in a similar delay in humeral chondrocyte maturation. Shown are in situ hybridization and histological staining of E14.5 forelimb sections. (A) Wild-type, Shox2^c/−, and HoxA^c/c; HoxD^−/− humeri all show Col2a1 expression, with wild-type limbs additionally showing downregulation of expression in the middle of the element. Wild-type humeri have prominent Runx2 and Col10a1 expression, while Shox2^c/− and HoxA^c/c; HoxD^−/− animals show reduced Runx2 levels and a lack of Col10a1 expression. Arrowheads point to the middle of the humerus. sc, scapula. (B) Hematoxylin and eosin staining showing that wild-type limbs contain hypertrophic chondrocytes, while Shox2^c/− and HoxA^c/c; HoxD^−/− animals lack these cells.

Figure 5

Disruption of Hox genes gives a transient decrease in Shox2 expression in the early limb bud and a lack of Shox2 expression in the perichondrium, as seen through in situ hybridization. (A) At E10.5, HoxA^c/c; HoxD^−/− animals have a decrease in Shox2 expression, compared to wild-type, HoxA^c/c, and HoxD^−/− animals (n = 3/3). (B) At E11, HoxA^c/c; HoxD^−/− animals have similar Shox2 levels to those of wild-type controls (n = 2/2). (C) At E14.5, Shox2 is expressed in the proliferating chondrocytes (arrowhead) and the perichondrium (arrow) of wild-type animals, but expression is selectively absent in the perichondrium of HoxA^c/c; HoxD^−/− animals (n = 3/3). ch, chondrocytes; pc, perichondrium.