An enhancer-based gene-therapy strategy for spatiotemporal control of cargoes during tissue repair.

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.

Vitamin D Stimulates Cardiomyocyte Proliferation and Controls Organ Size and Regeneration in Zebrafish.

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 anti-proliferative, 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.

Characterization of the three zebrafish orthologs of the mitochondrial GTPase Miro/Rhot.

Mitochondria exhibit dynamic locomotion and spatial rearrangement. This movement is necessary for a cell to maintain basic metabolic functions, and disruption of motility often results in cell death. Miro is a mitochondrial outer membrane Rho GTPase essential for mitochondrial movement and distribution in diverse systems, including yeast, animals, and plants. We sought to study the previously uncharacterized Miro protein family in zebrafish. We confirmed that, like human Miro, the zebrafish Miro proteins (Rhot1a, Rhot1b, and Rhot2) localize to mitochondria in mammalian tissue culture cells by both biochemical fractionation and immunofluorescent colocalization. In addition, using whole mount in situ hybridization, we observed ubiquitous expression of all three mRNAs throughout development. By microinjecting three antisense morpholino oligonucleotides targeted to each of the rhot genes, we knocked down all three proteins simultaneously in developing zebrafish embryos. The triple morphants demonstrated a dose-dependent defect in posterior body-axis elongation, while a single knockdown of each protein at the same dose produced no effect. This phenotype could be rescued with human Miro1 mRNA and is most likely due to increased cell death. Taken altogether, this research demonstrates the importance of the Rhot proteins during vertebrate development.