Impact of mutations of cartilage matrix genes on matrix structure, gene activity and chondrogenesis.

OBJECTIVE: Chondrocytes in the growth plate at different stages of differentiation synthesize characteristic extracellular matrix (ECM) components. Mutations in some ECM genes result in chondrodysplasia in humans and mice. We aimed to evaluate the impact of loss- and gain-of-function mutations of ECM genes on matrix structure, gene expression and formation of the growth plate. DESIGN: We review information on the impact of deficiencies in proteoglycans, and types X and II collagens on skeletal development. Additionally, we compare the impact of a glycine904 to cysteine (G904C) mutation in the triple helical coding domain of mouse Col2a1 with two previously reported Col2a1 mutations (exon7 deletion (Del1) and G85C). The G904C Col2a1 gene was introduced as a transgene into mice. Transgenic newborn mice were examined for skeletal development. The histology of the epiphyseal cartilage and the growth plate, and the ultrastructure of chondrocytes and collagen fibrillar morphology in the ECM were studied in 18.5-day transgenic and wild-type fetuses. The distribution of the mRNAs for Col2a1, Col11a1, Col9a1, Matn1, Agc and Ihh in the growth plate of 18.5-day G904C/G904C and wild type fetuses were compared by in situ hybridization. RESULTS: Heterozygous transgenic mice harbouring five copies of the G904C Col2a1 transgene developed skeletal abnormalities and dwarfism. Homozygous G904C/G904C mice died at birth, showing cleft palate, disrupted zonation of chondrocytes and reduction of the zone of hypertrophic chondrocytes. Fewer collagen fibrils were found in ECM of the cartilage. Rough endoplasmic reticulum of the chondrocytes of G904C/+ and G904C/G904C mice was distended. In G904C/G904C mutant mice, Agc gene activity was extended to the hypertrophic zone. Expression of the other genes studied was unchanged. Calcified materials that were not found normally in the maturing and only at low abundance in the hypertrophic zones of the wild type growth plate, were present in these zones in G904C/G904C mice. Despite phenotypic similarities for the G904C and Del1 mice, reduced expression of types I, II, IX, X collagens and aggrecan were reported for the latter mutation. Changes in gene activity and matrix organization in the growth plate also accompanied deficiencies in aggrecan, perlecan and collagen II. CONCLUSIONS: The data suggest that a single amino acid alteration in collagen II could lead to skeletal abnormalities through multiple secondary effects on the synthesis and assembly of ECM components. The functional impact of mutations of ECM genes reveals that chondrodysplasia is caused not just by the formation of abnormal matrix molecules, but that the alteration of one ECM component may lead to a cascade of disruption of other gene activities in chondrocytes which collectively contribute to the pathological changes in the architecture of the growth plate.

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development.

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis-regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.

Methylation of hen erythrocyte DNA.

We have analysed the 5-methylcytosine content of hen erythrocyte DNA and found it to be lower than that of DNA from other chick tissues analysed. Erythrocyte DNA is also a better substrate for DNA methylase having a five-fold lower Km than DNA from white blood cells. This is probably because it contains a large number of hemimethylated sites. Thus the inverse correlation between methylation and gene expression does not apply to the chick red blood cell.