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ABSTRACT Heterozygous variants in SOX10 cause congenital 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, previously undescribed 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. RNA sequencing 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. 

Abstract Heterozygous variants in the gene encoding the SOX10 transcription factor cause congenital syndromes affecting pigmentation, digestion, hearing, and neural function. Most of these symptoms are attributable to failed differentiation and loss of neural crest cells. Extensive research on mouse and zebrafish models has confirmed that Sox10 is essential for most non-skeletal crest derivatives, but seemingly dispensable for skeletal development. We challenge that concept here by revealing a novel requirement for Sox10 in skeletal mineralization. Neither neural crest- nor mesoderm-derived bones initiate mineralization on time in zebrafishsox10mutants, despite normal osteoblast differentiation and matrix production. We show that mutants are deficient in the ionocyte subpopulation tasked with taking up calcium from the environment through the Trpv6 epithelial calcium channel, leading to a severe calcium deficit that explains the lack of mineralization. As these ionocytes do not derive from asox10+ lineage, we hypothesized that the primary defect instead resides in a separate organ that regulates ionocyte numbers or calcium uptake at a systemic level. Screening of the endocrine hormones known to regulate calcium homeostasis in adult vertebrates revealed significantly elevated levels of stanniocalcin (Stc1a), an anti-hypercalcemic hormone, in larvalsox10mutants. Previous studies demonstrated that Stc1a inhibits calcium uptake in fish by repressingtrpv6expression and blocking proliferation of Trpv6+ ionocytes. Our epistasis assays indicate that excess Stc1a is the proximate cause of the calcium deficit insox10mutants. Lineage tracing shows that the pronephros-derived glands that synthesize Stc1a interact withsox10+ neural crest-derived cells, and that the latter are missing in mutants. We conclude that a subpopulation of Sox10+ neural crest non-cell-autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary for the initiation of bone mineralization. 

Significance The evolution of jaws in early vertebrates provided such a predatory advantage that 99% of vertebrate species living today are jawed. What is often overlooked, however, is another structural innovation that happened concurrently and may have been equally critical to the lineage’s success: the evolution of musculoskeletal gill covers to actively drive oxygenated water over the gills. Here, we identify the first essential gene for gill cover formation in modern vertebrates, Pou3f3, and uncover the genomic element that brought Pou3f3 expression into the pharynx more than 430 Mya. Remarkably, small changes in this deeply conserved sequence account for the single large gill cover in living bony fish versus the five separate covers of sharks and their brethren.