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.

Spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD) is an autosomal recessive skeletal dysplasia, characterized by disproportionate short stature with a short and stiff neck and trunk. SMMD is caused by inactivating mutations in NKX3-2, which encodes a homeobox-containing protein. Because of the rarity of the disorder, the diagnostic feature has not been fully established yet. We describe an affected newborn with dysmorphic facial features and severe short trunk. The patient required immediate intubation at the delivery room and duodenal atresia was detected during his course in neonatal intensive care unit. Skeletal survey revealed total absence of the ossification of the vertebral bodies, pubis, and ischia. Mainly the femora was short and broad with mild flaring of the metaphyses. The downward sloping or tented appearance of the ribs was distinctive. A diagnosis of SMMD was made on clinical and radiological grounds. Molecular analysis revealed homozygosity for a novel mutation, c.507-508delCA (p.Gly171Cysfs*55) in exon 2 of NKX3-2. The patient was operated on postnatal day 7 for duodenal atresia. In the post-operative period he developed sepsis and respiratory failure and he died on postnatal day 14. Although no neuroradiologic imaging could be performed, the findings of clubfoot, neuromuscular respiratory insufficiency requiring invasive mechanical ventilation and downward sloping or tented appearance of the ribs were suggestive of very early cervical cord compression leading to perinatal mortality. To our knowledge this patient yet represents one of the most severe postnatal phenotypes of SMMD.