Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton

Abstract

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

The broad role of Nkx3.2 in the development of the zebrafish axial skeleton

“…Remarkably, and consistent with other nkx3 . 2 mutant alleles [ 9 , 26 ], zebrafish homozygous for this mutation survived until adulthood, allowing us to study the mutant phenotype at a range of developmental stages up to and including adults. We employed traditional and novel techniques to characterise the effects on skeletal development caused by this mutation.…”

“…2 in the jaw joint of zebrafish [ 3 , 46 ], knockdown of nkx3 . 2 in Xenopus [ 8 ], and both knockdown and null mutants in zebrafish that result in the fusion of the jaw joint articulating elements Meckel’s cartilage and palatoquadrate accompanied by the loss of the RAP [ 3 , 9 , 26 ]. These results clearly show the importance of Nkx3.2 during primary jaw joint development.…”

“…These results further support an essential role of Nkx3.2 that is restricted to the basiventral and not the basidorsal cartilage in zebrafish. Recently described nkx3.2 mutant line displayed shortening of the rostral spine and misalignment of ribs [ 26 ], however a detailed description of the affected cervical and vertebrae associated structures was lacking making it difficult to precisely compare the phenotypes with our mutant.…”

“…Previous studies investigating other nkx3 . 2 mutant zebrafish lines also did not describe any median fin phenotypes [ 9 , 26 ]. The proximal radials of the dorsal and anal median fins develop with contributions from somite-derived cells including the sclerotome [ 77 , 78 ], while paired fins and limbs are derived from the lateral plate mesoderm [ 78 , 79 ].…”

“…These data clearly indicate a role of Nkx3.2 in the mammalian axial skeleton beyond just the middle ear that is homologous to the non-mammalian primary jaw joint. However, the function of this gene in the axial skeleton of non-mammals has been investigated very briefly [ 26 ]. In zebrafish embryos and juveniles nkx3 .…”