Abstract
The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.
Highlights
NK3 homeobox 2 (Nkx3.2, Bapx1) is an evolutionarily conserved gene encoding a homeodomain-containing transcription factor that is involved in cartilage growth and differentiation in gnathostomes
Zebrafish nkx3.2uu2803/uu2803 line generated with Clustered Regulatory Interspaced Short Palindromic (CRISPR)/CRISPR-associated protein 9 (Cas9) survives to adulthood and has open mouth phenotype
The last eight amino acids encoded by the first exon and all amino acids encoded by the second exon including the entire DNA-binding homeodomain of Nkx3.2 are predicted to be absent in this shortened protein and these changes generate a severe loss-of-function allele
Summary
NK3 homeobox 2 (Nkx3.2, Bapx1) is an evolutionarily conserved gene encoding a homeodomain-containing transcription factor that is involved in cartilage growth and differentiation in gnathostomes. It was first described in Drosophila (bagpipe, bap), where it plays a major role in the visceral mesoderm during the formation of the midgut musculature [1]. During vertebrate evolution Nkx3.2 expression was incorporated into the intermediate domain of the first pharyngeal arch. This event has been proposed to be crucial for jaw joint formation during the transition from jawless to jawed vertebrates [2]. Endothelin-1 signaling through its receptor directs the dorsoventral patterning of the migrating neural crest cells that form the skeleton of the first
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