Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

Abstract

Background: Heparan sulfate proteoglycan 2 (HSPG2) encodes for perlecan, a large proteoglycan that plays an important role in cartilage formation, cell adhesion, and basement membrane stability. Mutations in HSPG2 have been associated with Schwartz-Jampel syndrome and Dyssegmental Dysplasia Silverman-Handmaker Type, two disorders characterized by skeletal abnormalities. These data indicate a function for HSPG2 in cartilage development/maintenance. However, the mechanisms in which HSPG2 regulates cartilage development are not completely understood. Here, we explored the relationship between this gene and craniofacial development through morpholino-mediated knockdown of hspg2 in zebrafish. Results: Knockdown of hspg2 resulted in a fusion of the mandibular jaw joint at 5 days post fertilization (dpf). We surmised that defects in mandible development were a consequence of neural crest cell (NCC) dysfunction, as these multipotent progenitors produce the cartilage of the head. Early NCC development was normal in morphant animals as measured by distal-less homeobox 2a (dlx2a) and SRY-box transcription factor 10 (sox10) expression at 1 dpf. However, subsequent analysis at later stages of development (4 dpf) revealed a decrease in the number of Sox10 + and Collagen, type II, alpha 1a (Col2a1a)+ cells within the mandibular jaw joint region of morphants relative to random control injected embryos. Concurrently, morphants showed a decreased expression of NK3 homeobox 2 (nkx3.2), a jaw joint molecular marker at 4 dpf. Conclusions: Collectively, these data suggest a complex role for hspg2 in jaw joint formation and late stage NCC differentiation.

A novel cis-regulatory element drives early expression of Nkx3.2 in the gnathostome primary jaw joint

“…Indeed, by quantifying the jaw joint expression of nkx3.2 in zebrafish embryos by measuring in situ hybridization staining intensity, we were able to measure a ~45% decrease at 48–56 hpf in homozygous JRS1 deletion mutants relative to wild types and heterozygous mutants. Castellanos and Quintana, 2021 reported mild jaw joint phenotypes reminiscent of those seen in our homozygous JRS1 mutants in 5 dpf hspg2 morphants that were associated with a ~30% reduction in nkx3.2 gene expression measured at 4 dpf. Their results and ours are consistent with earlier work indicating that a reduction in early nkx3.2 expression levels can result in more subtle jaw joint phenotypes, increasing in severity with increasing morpholino dosage ( Miller et al, 2003 ).…”

An evolutionarily conserved cis-regulatory element of Nkx3.2 contributes to early jaw joint morphology in zebrafish

“…It also indicates that JRS1 is most important to the early jaw joint expression of nkx3.2 , prior to 6 dpf, and may contribute relatively less to later expression. Castellanos et al (2021) recently reported subtle jaw joint phenotypes reminiscent of those seen in our homozygous JRS1 mutants in 5 dpf hspg2 morphants that were associated with a ∼30% reduction in nkx3.2 gene expression at 4 dpf. Their results and ours are consistent with earlier work indicating that a reduction in nkx3.2 expression levels can result in more subtle jaw joint phenotypes, increasing in severity with increasing morpholino dosage (Miller et al, 2003).…”