Sox10 is required for systemic initiation of bone mineralization

Heterozygous variants in SOX10 cause syndromes affecting pigmentation, digestion, hearing, and neural development, primarily attributable to failed differentiation or loss of non-skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest- nor mesoderm-derived bones initiate mineralization on time in zebrafish sox10 mutants, despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment, resulting in severe calcium deficiency. As these ionocytes derive from ectoderm, not crest, we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin (Stc1a), an anti-hypercalcemic hormone, in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros-derived glands that synthesize Stc1a interact with sox10+ cells, but these cells are missing in mutants. We conclude that sox10+ crest-derived cells non-autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA-seq was performed on pooled whole-body zebrafish wild-type and sox10 mutant larvae at 45 hpf, 4 dpf and 7 dpf. Total of 18 samples. 45 hpf (3 replicates: controls n = 10, 20 & 25; mutants n = 10,15 & 15 embryos), 4 dpf (3 replicates: controls n = 10, 15 & 12; mutants n = 10,11 & 12 embryos) and 7 dpf (3 replicates: controls n = 12, 30 & 18; mutants = 18, 12 & 7 embryos)