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. 1998 Aug 18;95(17):9956-61.
doi: 10.1073/pnas.95.17.9956.

Evidence for regulation of cartilage differentiation by the homeobox gene Hoxc-8

Y G Yueh  1 , D P Gardner, C Kappen
Affiliations

Evidence for regulation of cartilage differentiation by the homeobox gene Hoxc-8

Y G Yueh et al. Proc Natl Acad Sci U S A. .

Abstract

Homeobox genes of the Hox class are required for proper patterning of skeletal elements, but how they regulate the differentiation of specific tissues is unclear. We show here that overexpression of a Hoxc-8 transgene causes cartilage defects whose severity depends on transgene dosage. The abnormal cartilage is characterized by an accumulation of proliferating chondrocytes and reduced maturation. Since Hoxc-8 is normally expressed in chondrocytes, these results suggest that Hoxc-8 continues to regulate skeletal development well beyond pattern formation in a tissue-specific manner, presumably by controlling the progression of cells along the chondrocyte differentiation pathway. The comparison to Hoxd-4 and Isl-1 indicates that this role in chondrogenesis is specific to proteins of the Hox class. Their capacity for regulation of cartilage differentiation suggests that Hox genes could also be involved in human chondrodysplasias or other cartilage disorders.

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Figures

Figure 1
Figure 1
Expression of homeobox transgenes and Hoxc-8 in the developing skeleton. Our binary transgenic mouse system (A) is based on the potent viral transactivator VP16. The combination of two transgenes, namely the transactivator (TA) and a transresponder (TR) transgene, in the same individual leads to activation of the TR transgene. (B) Sites of IE-LacZ transgene activation indicated by β-galactosidase activity. VP16 expression under control of the Hoxc-8 promoter leads to TR gene expression in growth zones and cartilage of the skeleton at 17.5 days of development. (C) Section through a rib of the embryo in B showing staining for β-galactosidase in proliferating (p), prehypertrophic (ph), and some hypertrophic (h) chondrocytes. (D and E) Sections through two ribs at different planes of the respective growth zones with prehypertrophic cells (D) and hypertrophic cells (E) exhibiting β-galactosidase activity. (F) β-Galactosidase expression in proliferating cells in the prospective neural arch of a vertebra. Notably, there is no detectable transgene activation in perichondrial cells (arrows). (G–J) In situ hybridizations with a Hoxc-8 antisense probe to parasagittal sections of a mouse embryo at 15.5 days (G and H) and 18.5 days (I and J). Cells of the developing rib cartilage (bright-field exposure in G and I) express Hoxc-8, whereas the surrounding tissue is negative (H and J). Sense probe for Hoxc-8 gave no appreciable signal (not shown).
Figure 2
Figure 2
Cartilage abnormalities in Hox gene transgenic mice. Skeletons from newborn mice were stained with Alizarin red (bone) and Alcian blue (cartilage). (A–D) Cartilage defects upon overexpression of Hoxc-8 (B and C) or Hoxd-4 (D) transgenes. (A) Skeleton of a wild-type newborn FVB mouse. (B and C) Reduced Alcian blue staining in ribs and knee cartilage of a Hoxc-8 transgenic mouse that died shortly after birth. (D) Alcian blue staining was reduced in ribs and vertebral column of a Hoxd-4 transgenic animal. Note that the cartilaginous portions of the ribs were present (arrows in D, compare with B) and that tracheal cartilage was normal. (E–P) Severity of cartilage abnormalities increased with transgene dosage. The skeleton from an animal hemizygous for both TA and TR loci resembled staining of the wild-type situation (compare E to A). The thoracic (E–H), lumbar (I–L), and sacral regions (M–P) of the skeletons from animals with the genotypes TA/+ TR/+ (E, I, M), TA/+ TR/TR (F, J, N), TA/TA TR/+ (G, K, O), and TA/TA TR/TR (H, L, P) are shown. All animals with transgene loci in excess of hemizygosity died shortly after birth or were dead at birth. The distortion and flexibility of the skeleton were highest in the animal homozygous for both transgenes.
Figure 3
Figure 3
Alterations in cartilage differentiation in Hoxc-8 transgenic mice. Embryos (genotypes TA/+ +/+ and TA/TA TR/+) were isolated at 16.5 days of development. Corresponding sections at the lumbar level were stained with hematoxylin/eosin (A–D), Alcian blue (E and F), or an antibody that detects proliferating cell nuclear antigen (PCNA; G–J). For enumeration of cells, photographs I and J were enlarged and every cell with a nucleus within the vertebral center was counted (K and L). (M and N) In situ hybridization for collagen II mRNA to sections from 17.5 day embryos (genotypes +/+ TR/+ and TA/TA TR/TR) that cut through more than one developing vertebra at the lower thoracic level. Both photographs were taken under identical exposure conditions. Sections hybridized with the sense probe were negative (not shown).
Figure 4
Figure 4
Model for the regulation of cartilage differentiation by Hoxc-8. Hoxc-8 leads to accumulation of proliferating precursor cells, thus interfering negatively with the progression of differentiation. The signaling molecule Indian hedgehog (ihh) has been shown to negatively modulate the rate of differentiation at the level of prehypertrophic cells. Because patched, the receptor for ihh, is expressed in the adjoining perichondrium, this signalling pathway acts indirectly (broken line) on chondrocyte differentiation (26). Hoxc-8 is expressed in and directly affects chondrocytes prior to the prehypertrophic stage upstream of ihh.
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