Mutations in fam20b and xylt1 Reveal That Cartilage Matrix Controls Timing of Endochondral Ossification by Inhibiting Chondrocyte Maturation

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed “maturation,” when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development

The feelgood mutation in zebrafish dysregulates COPII-dependent secretion of select extracellular matrix proteins in skeletal morphogenesis

SUMMARY Craniofacial and skeletal dysmorphologies account for the majority of birth defects. A number of the disease phenotypes have been attributed to abnormal synthesis, maintenance and composition of extracellular matrix (ECM), yet the molecular and cellular mechanisms causing these ECM defects remain poorly understood. The zebrafish feelgood mutant manifests a severely malformed head skeleton and shortened body length due to defects in the maturation stage of chondrocyte development. In vivo analyses reveal a backlog of type II and type IV collagens in rough endoplasmic reticulum (ER) similar to those found in coat protein II complex (COPII)-deficient cells. The feelgood mutation hinders collagen deposition in the ECM, but trafficking of small cargos and other large ECM proteins such as laminin to the extracellular space is unaffected. We demonstrate that the zebrafish feelgood mutation causes a single amino acid substitution within the DNA-binding domain of transcription factor Creb3l2. We show that Creb3l2 selectively regulates the expression of genes encoding distinct COPII proteins (sec23a, sec23b and sec24d) but find no evidence for its regulation of sec24c expression. Moreover, we did not detect activation of ER stress response genes despite intracellular accumulation of collagen and prominent skeletal defects. Promoter trans-activation assays show that the Creb3l2 feelgood variant is a hypomorphic allele that retains approximately 50% of its transcriptional activity. Transgenic rescue experiments of the feelgood phenotype restore craniofacial development, illustrating that a precise level of Creb3l2 transcriptional activity is essential for skeletogenesis. Our results indicate that Creb3l2 modulates the availability of COPII machinery in a tissue- and cargo-specific manner. These findings could lead to a better understanding of the etiology of human craniofacial and skeletal birth defects as well as adult-onset diseases that are linked to dysregulated ECM deposition, such as arthritis, fibrosis or osteoporosis