Iroquois homeobox (Irx) family members, a group of highly conserved homeodomain containing transcription factors, are involved in the patterning and the proper functions of vertebrate organs. They can act as transcriptional activators or repressors in a context-dependent manner. Preliminary data indicated that Irx3 and Irx5 are functionally redundant during cardiac morphogenesis, and they physically interact with other cardiac transcription factors. At E14.5, outflow tract septation failure and ventricular septation failure were observed in Irx3/5DKO mouse hearts. Loss of Irx3/5 in neural crest and endothelial cell lineages led to outflow tract septation failure and ventricular septal defect. In adult mice, Irx3 is expressed in the atrioventricular conduction system, and loss of Irx3 leads to slower ventricular conduction velocity. qRT-PCR analysis and immunofluorescence staining revealed that the expression of gap junction proteins, Cx40 and Cx43, are affected by the loss of Irx3. Over-expression of Irx3 and a dominant repressor form of Irx3, Irx3-EnR, resulted in Cx40 upregulation, indicating that Irx3 acts as an indirect positive regulator of Cx40. Irx3-EnR over-expression in vivo resulted in postnatal onset of atrial enlargement, ventricular hypertrophy, and conduction failure. Taken together, this study demonstrates the significance of Irx3/5 in both cardiovascular development and cardiac electrophysiology.

Low to moderate level of chronic arsenic exposure contributes to cardiovascular ailments including heart disease and aneurysms. Current research on the etiology and progression of cardiovascular disease focuses mainly on adult which fails to capture the developmental origins of cardiovascular disease. Thus, disruption in morphogenetic events during early development may initiate and pattern the molecular programming of cardiovascular ailments in adulthood. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. Coronary vessel development is initiated by precursor cells that are derived from the epicardium. Epicardial derived cells undergo proliferate, migrate, and differentiate into several cardiac cell types which are the cellular components of the coronary vessels. The key cellular event occurs in this process is the epithelial to mesenchymal transition (EMT), which can also be utilized by endocardial cushion cells to form aortic and pulmonary valves. The TGFβ family of ligands and receptors are essential for developmental cardiac EMT and coronary smooth muscle cell differentiation. Whether arsenic has any impact on TGFβ mediated cardiovasculogenesis is not known. Monomethylarsonous acid [MMA(III)] is the most potent metabolite of inorganic arsenic and has been shown to partly account for arsenic induced toxicity. The fetus is exposed to relatively higher levels of MMA (III) as compared to adults probably due to deficiency in methylation of transferred inorganic arsenic from the placenta. However, the developmental toxicity of MMA (III) has not yet been studied. In this study, we exploit a novel cardiac progenitor cell line to recapitulate epicardial EMT in vitro and to study developmental toxicity caused by arsenicals. We show that chronic exposure to low level of arsenite and MMA (III) disrupts developmental EMT programming in epicardial cells causing deficits in cardiac mesenchyme production. The expression of EMT program genes is also decreased in a dose-dependent manner following exposure to arsenite and MMA (III). Smad-dependent TGFβ2 canonical signaling and the non-canonical Erk signaling pathways are abrogated as detected by decreases in phosphorylated Smad2/3, Erk1/2 and Erk5 proteins. There is also loss of nuclear accumulation of p-Smad and p-Erk5 due to arsenical exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenicals do not block TGFβ2 stimulated p38 activation. Additionally, smooth muscle cell differentiation, which is proven to be governed by p38 signaling in epicardial cells, also remains intact with arsenical exposure. Overall these results show that arsenite and MMA (III) are strong and selective cardiac silencers. The molecular mechanisms of arsenical toxicity on TGFβ-Smad signaling in epicardial cells is further explored. A relatively high level of acute arsenical exposure rapidly depletes phosphorylated nuclear Smad2/3. Restoration of the nuclear accumulation of Smads can be achieved by inhibiting the expression or activation of Smad specific exportins suggesting that arsenicals augment Smad nuclear exportation. Abrogated Smad signaling caused by arsenicals is associated with severe deficits in EMT during mouse epicardium and chick endocardial cushion development. Thus progenitor cell outgrowth, migration, invasion and vimentin filament reorganization are significantly inhibited in response to arsenical exposure. Disrupted Smad nuclear shuffling is probably caused by zinc displacement on the MH-1 DNA binding domain of Smad2/3. Thus zinc supplementation restores both nuclear content and transcriptional activities of Smad2/3. Rescued TGFβ-Smad signaling by zinc also contributes to cellular transformation and mesenchyme production in embryonic heart explants. LINE1 (L1) retrotransposons are a group of mobile DNA elements that shape the genome via novel epigenetic controls. Although expression of L1 is required for early embryo implantation and development, abnormally elevated L1 is shown to inhibit embryonic cells from transforming and differentiating during organogenesis. Cellular redox signaling, which is regulated by antioxidant responsive elements (AREs), has been shown to play a key role in L1 activation and retrotransposition. However, whether L1 can be induced by the cellular oxidative stress caused by arsenic is not known. We provide evidence showing that L1 ORF-1 and ORF-2 mRNA levels are both up-regulated by arsenic. Nuclear accumulation of L1 ORF-2 is observed in response to 30 min arsenic exposure, which may lead to active retrotransposition events in the genome. Transcriptional activity of L1 is regulated by Nrf2 as mutations in ARE regions within the L1 promoter and Nrf2 silencing using siRNA both significantly inhibit L1 transcriptional activity. Nrf2 overexpression together with arsenic exposure creates synergic induction in L1 promoter activity suggesting that arsenic mediated L1 activation is partially Nrf2 dependent. Taken together, these findings reveal a molecular mechanism responsible for arsenic cardiac toxicity and define a novel genetic toxic effect of arsenic during embryonic heart development.

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xiv, 78 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Myotonic Dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the protein MBNL1 (M[barbelow]uscleb[barbelow]lin[barbelow]d-l[barbelow]ike-1) is sequestered to the expanded CUG or CCUG RNAs. MBNL1 regulates the alternative splicing of a variety of pre-mRNAs and its mis-localization results in mis-splicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. I initially demonstrated that MBNL1 can bind short structured CUG and CCUG repeats with high affinity and specificity in vitro . Next, I was able to determine and articulate the first structure of a binding site of MBNL1 in an endogenous pre-mRNA that it regulates. I found that MBNL1 binds a stem-loop in the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. I determined that MBNL1 regulated exon 5 by directly competing with the essential splicing factor U2AF65 for binding upstream of exon 5. When U2AF65 is prevented from binding, factors in the spliceosome can no longer be recruited and the following exon is skipped. Furthermore, I found that MBNL1 and U2AF65 compete by binding mutually exclusive RNA structures. I also characterized a potential therapeutic approach for DM. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. Using a targeted screen of small molecules known to bind structured nucleic acids, I identified the small molecule pentamidine as a compound that disrupted MBNL1 binding to CUG repeats in vitro . I showed in cell culture that pentamidine was able to reverse the mis-splicing of two pre-mRNAs affected in DM. Pentamidine also significantly reduced the formation of RNA foci in tissue culture cells, which are characteristic of DM. MBNL1 was released from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of two pre-mRNAs in mice expressing expanded CUG repeats. This dissertation includes three previously published co-authored publications. / Committee in charge: Kenneth Prehoda, Chairperson, Chemistry; J. Andrew Berglund, Advisor, Chemistry; Victoria DeRose, Member, Chemistry; Peter von Hippel, Member, Chemistry; Alice Barkan, Outside Member, Biology

The epicardium, a cell layer enveloping the heart muscle, drives embryonic heart development and heart repair in the adult zebrafish. Previous studies found the epicardium to consist of multiple cell populations with distinct phenotypes and functions. Here, I investigated epicardial heterogeneity in the developing zebrafish heart, focusing on the developmental gene program that is also reactivated during adult heart regeneration. Transcription factor 21 (Tcf21), T-box 18 (Tbx18) and Wilms' tumor suppressor 1b (Wt1b) are often used interchangeably to identify the zebrafish epicardium. Analyzing newly generated reporter lines and endogenous gene expression, I showed that the epicardial expression of tcf21, tbx18 and wt1b during development is heterogeneous. I then collected epicardial cells from newly generated reporter lines at 5 days-post-fertilization and performed single-cell RNA sequencing. I identified three distinct epicardial subpopulations with specific gene expression profiles. The first subpopulation expressed tcf21, tbx18 and wt1b and appeared to represent the main epicardial layer. The second subpopulation expressed tbx18, but not tcf21 or wt1b. Instead, it expressed smooth muscle markers and seemed restricted to the bulbus arteriosus. The third epicardial subpopulation only expressed tcf21 and resided within the epicardial layer. I compared the single-cell subpopulations with transcriptomic bulk data and visualized the expression of marker genes to investigate their spatial distribution. Using ATAC sequencing, I additionally identified open regulatory regions located in proximity to subpopulation-specific marker genes and showed subpopulation-specific activity in vivo. My results detail distinct cell populations in the developing zebrafish epicardium, likely to fulfil distinct and specific cellular functions. Future experiments will involve targeting signature genes enriched within each epicardial subpopulation, such as those encoding Adrenomedullin a (first subpopulation), Alpha Smooth Muscle Actin (second subpopulation) and Claudin 11a (third subpopulation), employing cell type-specific genome editing to test whether and how the identified heterogeneity underlies distinct epicardial cell fates and functions. Taken together, my work adds significantly to the understanding of the cellular and molecular basis of epicardial development and can offer novel insights in the context of heart regeneration.

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Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.