Epigenetics of Development

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

As described in the goals and objectives section of this report, this project consists of three specific aims: Isolation and characterization of tissue-specific epigenetic regulators Genetic screens carried out in Drosophila and C. elegans have been highly successful in identifying genes regulating cell-type specific epigenetic gene regulation in invertebrates, but the molecular mechanisms involved in organ- and tissue-specific epigenetic regulation in vertebrates are still relatively unknown. We have developed a novel EpiTag zebrafish transgenic reporter line that allows us to monitor dynamic changes in DNA methylation-based epigenetic regulation in intact animals during development. Using this transgenic line, we have performed the first large-scale F3 genetic screen in a vertebrate to identify recessive mutants in regulators of epigenetic gene silencing or activation, yielding a number of mutants defective in ubiquitous or tissue-specific epigenetic gene silencing or activation. The isolated mutants show a wide array of phenotypes ranging from complete lack of GFP expression to gain or loss of GFP expression in specific organs such as liver, brain, blood cells and eyes. Using RNAseq-based mapping, we have already successfully mapped most of these mutants and are actively performing additional work to conclusively identify and further characterize the mutated genes. These genes include a liver-specific gene in a nucleotide biosynthetic pathway whose disruption causes epigenetic dysregulation and that results in fatty liver disease. Further functional characterization of the mutated genes identified in our ongoing genetic screen is likely to yield many important and valuable new insights into epigenetic regulation in vertebrates, just as comparable powerful genetic screens carried out in invertebrates have done. Epigenetic regulation of regeneration and reproduction In addition to serving as a highly effective reporter for our successful genetic screen for epigenetic regulators, we also discovered that the EpiTag line shows a remarkable strong activation in, and serves as a superb marker for, cells at early stages of gametogenesis and regenerating cells in the fin, heart, and trunk muscle. EpiTag expression marks very early stages where epigenetic reprograming is taking place in both contexts. We are pursuing follow-up studies on these observations as well. We are using the well-studied fin regeneration model to study epigenetic regulators of regeneration. A variety of evidence points to the importance of DNA methylation and other epigenetic changes during zebrafish fin regeneration. However, other recent work has suggested that global DNA methylation does not change dramatically during regeneration, although these studies rely on samples containing mixed populations of regenerating cells at different stages as well as non-regenerating cells. We are using the EpiTag transgenic line to isolate and characterize cells at very early stages of regeneration to determine what epigenomic changes are specifically associated with early EpiTag+ cells by transcriptomic and epigenetic profiling of EpiTag+ cells isolated from regenerating fins. We are also examining the identity of cells cycling through the early EpiTag+ stages of regeneration by performing scRNAseq at multiple regenerating stages to determine what cells contribute to EpiTag+ cell clusters, what they become, and how they relate to cell types in non-regenerating fins. Although the blastema was historically thought to be derived from a stem-like multipotent cell population, recent studies have shown that lineage-restricted cells retaining memory of their original identity contribute to regeneration in zebrafish, salamanders, and even mice. Our new studies are helping to better understand which cells contribute to regeneration, and how. Epigenetic changes in cavefish adaptation and evolution In addition to eye and pigment loss and other adaptations, Astyanax cavefish have extreme and unusual tissue and metabolic adaptations that allow them to survive darkness, cold, hypoxia, and food deprivation. These include vascular changes and altered wound healing. We hypothesize that in a similar manner to loss of eyes, changes in epigenetic gene regulation may also underlie other cavefish adaptations. We are currently generating vascular-specific transgenic reporter lines in Astyanax and studying the function of gas-exchange endothelium in this useful complementary fish species.