Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Abstract

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

FIG. 1

Targeted disruption of mouse Crtm encoding matrilin 1. (A) Structure of wild-type allele, targeting construct, and recombinant locus. Dark boxes represent exons of Crtm. The expected fragment sizes after EcoRI digestion are 17 kb for the wild-type allele and 6 kb for the recombinant allele. E, EcoRI; Nh, NheI; Ns, NsiI; X, XhoI. (B) Southern blot analysis of mouse tail DNA isolated from the progeny of a mating between heterozygous parents. DNAs were digested with EcoRI and hybridized with the probe indicated in panel A. +/+, wild-type mouse; +/−, heterozygous mouse; −/−, homozygous mutant mouse. (C) Northern blot analysis of total RNA from limb cartilage, derived from 3-day-old wild-type (+/+) and homozygous mutant mice. The same filter was hybridized with cDNA probes specific for matrilin 1 (mat1), matrilin 2 (mat2), matrilin 3 (mat3), and glyceraldehyde phosphodehydrogenase (GAPDH).

FIG. 2

Analysis of the skeleton in wild-type (+/+) and matrilin 1-deficient (−/−) mice. (A) X-ray of an 8-week-old mutant mouse shows no gross skeletal abnormalities compared to a normal littermate. (B to E) Hematoxylin-eosin staining of knee region (B and C) and tibia (D and E) from 3-day-old wild-type (B and D) and homozygous mutant (C and E) mice. h, hypertophic zone; p, proliferative zone; so, center of secondary ossification. Bars, 200 μm for panels B and C and 50 μm for panels D and E.

FIG. 3

Immunostaining of cartilage. Consecutive sections of the tibia from wild-type (+/+ [A, C, E, G, and I]) and matrilin 1-deficient (−/− [B, D, F, H, and J]) newborn littermates were stained with specific antibodies against matrilin 1 (mat1), matrilin 2 (mat2), matrilin 3 (mat3), aggrecan (agn), and type II collagen (col2). Bar, 100 μm.

FIG. 4

Biochemical analysis of wild type (+/+) and mutant (−/−) epiphyseal and tracheal cartilages. (A) Western blot analysis of matrilin 1 (mat1) and matrilin 3 (mat3) expression in pooled 4 M GuHCl extracts from 3-day-old mouse epiphyseal cartilage. The identities of the matrilin 1 trimers (m1)₃ and matrilin 3 trimers (m3)₃ and tetramers (m3)₄ were determined by comparison with the migration position of protein molecular mass markers (kilodaltons). (B and C) Coomassie-stained SDS–5% polyacrylamide gel of collagens sequentially extracted with neutral salt (0.15 M NaCl), 4 M GuHCl, and pepsin from pooled 7-day-old mouse epiphyseal (B) and tracheal (C) cartilages. The identities of the cartilage collagen α1(II) band and the covalently cross-linked collagen β-components are indicated.