CONTEMPORARY GENETIC TOOLS FOR IN VIVO INVESTIGATIONS OF H3K27 DEMETHYLASES IN ZEBRAFISH CARDIOGENESIS
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Dynamic histone modification has emerged as a robust and versatile regulator of gene expression in eukaryotic cells. One such modification, the trimethylation of lysine 27 on histone H3 (H3K27me3) is facilitated by the Polycomb repressive complex 2 (PRC2) and contributes to the localized repression of transcription. Subsequently, lysine specific demethylase Kdm6b (Jmjd3) can relieve the repressive H3K27me3 mark, allowing for transcriptional activation. In vitro studies have suggested a role for Kdm6b during mesodermal and cardiovascular differentiation in mammalian embryonic stem cells; however, this relationship has yet to be characterized in vivo. I utilized the advantages of the zebrafish model to investigate the in vivo roles of Kdm6b-family demethylases during development using a reverse genetic approach. I carried out two independent loss-of-function studies to analyze the role of Kdm6b-family demethylases during embryonic development in zebrafish. By comparing genetic loss-of-function and morpholino-mediated knockdown approaches, I found that morpholino–mediated knockdown of kdm6bb transcript produces off-target effects and does not portray an accurate representation of in vivo function. I then show that, while not required for early cardiogenesis, histone demethylases kdm6ba and kdm6bb function redundantly to promote late stage proliferation during heart ventricle trabeculation. These data reveal a previously unknown functional relationship and support the hypothesis that Kdm6b-family demethylases function primarily during later stages of development. Additionally, my description of morpholino-induced off-target effects supports the need to use extreme caution when interpreting morphant phenotypes. Due to the embryonic lethality exhibited by kdm6b-deficient embryos and the limited tools available for spatiotemporal transgene control in zebrafish, I was unable to investigate demethylase function within adult animals. I attempted to circumvent these limitations by creating an inducible gene expression system that uses tissue-specific transgenes that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. I showed that these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. I then demonstrated how this technology could be used to define developmental windows of gene function by spatiotemporally controlling expression of constitutively active Notch1 in embryos. This dissertation contains previously published co-authored material.