Axin2 regulates chondrocyte maturation and axial skeletal development

Abstract

Axis inhibition proteins 1 and 2 (Axin1 and Axin2) are scaffolding proteins that modulate at least two signaling pathways that are crucial in skeletogenesis: the Wnt/beta-catenin and TGF-beta signaling pathways. To determine whether Axin2 is important in skeletogenesis, we examined the skeletal phenotype of Axin2-null mice in a wild-type or Axin1(+/-) background. Animals with disrupted Axin2 expression displayed a runt phenotype when compared to heterozygous littermates. Whole-mount and tissue beta-galactosidase staining of Axin2(LacZ/LacZ) mice revealed that Axin2 is expressed in cartilage tissue, and histological sections from knockout animals showed shorter hypertrophic zones in the growth plate. Primary chondrocytes were isolated from Axin2-null and wild-type mice, cultured, and assayed for type X collagen gene expression. While type II collagen levels were depressed in cells from Axin2-deficient animals, type X collagen gene expression was enhanced. There was no difference in BrdU incorporation between null and heterozygous mice, suggesting that loss of Axin2 does not alter chondrocyte proliferation. Taken together, these findings reveal that disruption of Axin2 expression results in accelerated chondrocyte maturation. In the presence of a heterozygous deficiency of Axin1, Axin2 was also shown to play a critical role in craniofacial and axial skeleton development.

Figure 1

One-week-old Axin2-null mice (n = 11) display a runt phenotype compared to heterozygous littermates (n = 12). (A) Plain x-ray; (B) body length in centimeters; (C) mass in grams. (B) and (C) are shown as the mean ± SD. *p<0.05 using unpaired t-test.

Figure 2

Axin2 is expressed in cartilage during development. (A) β-galactosidase staining of whole wild-type or Axin2-null embryos at day E13.5. Arrowhead indicates β-galactosidase activity. Representative photomicrographs (original magnification, × 100) of β-galactosidase stained frozen sections: tibia (B) and ribs (C).

Figure 3

Accelerated chondrocyte maturation in Axin2^−/− mice. Representative photomicrographs (original magnification, ×40) of distal femurs in 1-week-old Axin2^+/− [n = 13 (A)] and Axin2^−/− [n = 7 (B)]. Histomorphometric measurements of hypertrophic zone (HZ) thickness (C) and columnar zone (CZ) thickness (D). (E) Quantification of BrdU-positive cells in the epiphyseal region. Real-time RT-PCR analysis of type X collagen (F) and type II collagen (G) gene expression in primary chondrocytes isolated from Axin2^+/− and Axin2^−/− mice. Results are shown as mean ± SD. *p<0.05 using unpaired t-test.

Figure 4

Profound abnormalities in Axin 3/4 knockout embryos. (A) Whole mounts of representative E14.5 embryos lacking one or more copies of the Axin1 or Axin2 genes. Clockwise from the upper left, the genotypes are: Axin2^+/−, Axin2^−/−, Axin1^+/−; Axin2^+/−, and Axin 3/4 knockout. Ventral (B) and dorsal (C) views of E16.5 Axin 3/4 knockout embryos. Skeletal staining of E18.5 Axin1^+/− Axin2^+/− (D), Axin2^−/− (E), and Axin 3/4 knockout (F) embryos.

Figure 5

Defects of the mineralized skeleton in E18.5 Axin 3/4 knockout embryos. Ventral (A, B) or profile view (C, D) of double-heterozygous skeleton (A, C) or Axin 3/4 knockout skeleton (B, D) as rendered by micro-CT analysis.