Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered antiproliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.

The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.

Background: Vitamin D is an essential nutrient that has long been known to regulate skeletal growth and integrity. In models of major appendage regeneration, treatment with vitamin D analogs has been reported to improve aspects of zebrafish fin regeneration in specific disease or gene misexpression contexts, but also to disrupt pattern in regenerating salamander limbs. Recently, we reported strong mitogenic roles for vitamin D signaling in several zebrafish tissues throughout life stages, including epidermal cells and osteoblasts of adult fins. To our knowledge, molecular genetic approaches to dissect vitamin D function in appendage regeneration have not been described. Results: Using a knock-in GFP reporter for the expression of the vitamin D target gene and negative regulator cyp24a1, we identified active vitamin D signaling in adult zebrafish fins during tissue homeostasis and regeneration.Transgenic expression of cyp24a1 or a dominant-negative vitamin D receptor (VDR) inhibited regeneration of amputated fins, whereas global vitamin D treatment accelerated regeneration. Using tissue regeneration enhancer elements, we found that local enhancement of VDR expression could improve regeneration with low doses of a vitamin D analog.Conclusions: Vitamin D signaling enhances the efficacy of fin regeneration in zebrafish.