Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase diminishes the survival and size of chondrocytes during orofacial morphogenesis in zebrafish

Abstract

Objectives: The 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) is the enzyme controlling the rate-limiting step in the synthesis of cholesterol, sterols, and isoprenoids in the mevalonate biosynthetic pathway. Impaired function of HMGCR in zebrafish produces craniofacial malformations and orofacial cleft, mainly affecting the post-migratory neural crest cells with little earlier effect. Here we investigate morphogenetic and cellular mechanisms underlying the generation of these malformations.

Methods: The morphology of chondrocytes in the lower jaw and the proliferation/apoptosis in the ethmoid plate were analysed in hmgcr1b mutants and in embryos treated with atorvastatin. In the ceratohyal of treated embryos, we measured the number and dimensions of chondrocytes. In the ethmoid plate, we performed proliferation and apoptosis assays to quantify the number of cells undergoing each process in both hmgcr1b mutants and pharmacologically treated embryos. All embryos were imaged using confocal microscopy and processed to obtain maximum intensity z-projection.

Results: The shortening of the ceratohyal is produced by a moderate reduction in the number of cells combined with isometric shrinkage of the chondrocytes. At the same time, the shortening of the ethmoid plate is due to a combination of a slightly diminished proliferation with massive abnormal apoptosis at the proliferation front.

Conclusion: HMGCR function is necessary for the normal survival and morphology of chondrocytes during condensation and chondrogenesis in the developing palate and jaw. Further studies are required to establish the pathways through which HMGCR acts on apoptosis, proliferation, and cell size during normal craniofacial development.

Keywords: HMGCR; mevalonate pathway; orofacial cleft; statins; zebrafish.