Silvio Garofalo

Characterization of primary cultures of chondrocytes from type II collagen/β-galactosidase transgenic mice

Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.

Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human chondrodysplasias, including achondroplasia, the most common form of dwarfism in humans. From in vitro studies, the skeletal defects observed in these disorders have been attributed to constitutive activation of FGFR3. Here we show that FGF9 and FGFR3, a high‐affinity receptor for this ligand, have similar developmental expression patterns, particularly in areas of active chondrogenesis. Targeted overexpression of FGF9 to cartilage of transgenic mice disturbs postnatal skeletal development and linear bone growth. The growth plate of these mice exhibits reduced proliferation and terminal differentiation of chondrocytes similar to that observed in the human disorders. The observations provide evidence that targeted, in vivo activation of endogenous FGFR3 inhibits bone growth and demonstrate that signals derived from FGF9–FGFR3 interactions can physiologically block endochondral ossification to produce a phenotype characteristic of the achondroplasia group of human chondrodysplasias.

Targeted Expression of SHH Affects Chondrocyte Differentiation, Growth Plate Organization, and Sox9 Expression

The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation.

Y‐Position Collagen II Mutation Disrupts Cartilage Formation and Skeletal Development in a Transgenic Mouse Model of Spondyloepiphyseal Dysplasia

Mice were generated by pronuclear injection of a type II collagen transgene harboring an Arg789Cys (R789C) mutation that has been found in patients with spondyloepiphyseal dysplasia (SED). Expression was directed to cartilage by the murine Col2a1 promoter to examine the consequences of mutations involving the Y‐position of the collagen helix Gly‐X‐Y triplet on skeletogenesis. The transgenic mice had very short limbs, short trunk, short snout, and cleft palate; they died at birth. Their growth plates were disorganized and collagen fibrils were sparse in cartilage matrix. When the transgene was expressed in RCS cells, there was no evidence that R789C‐bearing collagen chains were incorporated into stable collagen molecules. Molecular modeling of the mutation raised the possibility that it destabilizes the collagen triple helix. Together our results suggest that Y‐position mutations, such as R789C, can act in a dominant negative manner to destabilize collagen molecules during assembly, reducing their availability to form fibrils, the deficiency of which profoundly disturbs the template functions of cartilage during skeletogenesis.

Skeletal development in transgenic mice expressing a mutation at Gly574Ser of type II collagen

Skeletal development of transgenic mice with a type II collagen mutation was analyzed and compared with wild‐type litter‐mates. The single base substitution in Co12a1 resulted in a glycine to serine mutation within the helical domain and corresponded to one previously identified in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:4583–4587). Skeletal staining of embryos from 14.5 through 18.5 days of gestation demonstrated a dwarf phenotype in the transgenic embryos, most notably short limb bones and vertebral column that was first detected at 15.5 days post‐coitus. In addition to the reduced length, the extent of ossification was less in the transgenic mice. The architecture of the long bone growth plate was abnormal in the transgenic tissue, in particular there was no discernible proliferative zone. There were few stacks of characteristically flattened cells and the overall length of the growth plate in the mutant embryos was reduced. At the ultrastructural level, there were fewer collagen fibrils present in the transgenic mouse cartilage compared to that of wild‐type littermates. Ultrastructural localization of collagen types II, IX and XI revealed a similar pattern between the transgenic and wild‐type pups, suggesting that the collagen fibrils present in the matrix of littermates with both phenotypes had a similar composition. Skeletal analysis and cartilage histochemistry indicated that effect of the type II collagen mutation was to reduce the density of the collagen fibrils within the cartilage matrix which was associated with delayed bone formation and resulted in a short‐limbed phenotype. Dev. Dyn. 208:170–177, 1997.