Linking the Cytoskeleton to Transcription: A Role for Myocardin-Related Transcription Factors in Heart Development and Disease

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2017.Cellular motility and contractility is influenced by a plethora of physiological and pathological stimuli. These processes require rearrangements to the cytoskeleton and gene expression programs necessary for amplifying structural components and effectors of the contractile apparatus. Myocardin-related transcription factors (MRTFs) are mechanosensitive coactivators of serum response factor (SRF) that serve as a nexus for linking actin dynamics to transcription. This thesis highlights distinct roles for MRTFs in regulating the migration of multipotent progenitor cells in the fetal heart (Part I) and gene programs necessary for compensatory remodeling in heart failure (Part II). Part I: The epicardium is a single cell layer of mesothelium lining the heart, which provides an important pool of cardiovascular progenitor cells during heart development. Through the process of epithelial-to-mesenchymal transition (EMT), epicardial-derived cells (EPDC) invade the underlying myocardium; primarily giving rise to vascular and interstitial cardiac lineages. This thesis identifies the MRTF/SRF signaling axis as a key regulatory mechanism for controlling EPDC migration. MRTFs are expressed in a temporal and spatial manner coincident with fetal epicardial EMT. Conditional ablation of MRTFs attenuates contractile and motile gene programs in vitro, and consequently disrupts the mobilization of EPDC in vivo. Furthermore, epicardial-specific deletion of MRTFs results in a hemorrhaging phenotype characterized by defects in coronary vessel maturation. Using lineage tracing studies, we show that MRTF-mediated EPDC motility is necessary for the recruitment of epicardial-derived pericytes to coronary microvasculature. Transcriptional profiling of MRTF-depleted epicardial cells further reveals dysregulation of gene programs related to blood vessel development and axonal guidance. Together, these findings provide novel insights into the mechanisms controlling EPDC motility which could shed light on strategies to exploit this progenitor population for cardiac repair. Part II: Intercellular communication is essential for coordinating the contraction between neighboring cardiomyocytes (CM). This synchrony is established by coupling electrical activity and mechanical tension at specialized junctions called intercalated discs (IDs). This thesis uncovers a novel pool of MRTFs at IDs and highlights an indispensable role for MRTFs in regulating compensatory gene programs in heart failure. Conditional deletion of MRTFs in a mouse model of cardiac hypertrophy leads to partially penetrant lethality and rapid cardiac deterioration. This phenotype stems, in part, from a disassembly of sarcomeres, structural defects in ID integrity, and perturbations to mitochondria. Using RNA-sequencing, we find dysregulation in gene programs related to muscle contraction and the actin cytoskeleton of MRTF-depleted CM. The subcellular localization of MRTFs to regions of electromechanical stress may serve to coordinate these potent transcription factors. This thesis suggests a new paradigm for controlling MRTF activity at adhesion complexes

Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells

Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture

The yeast 14-3-3 proteins BMH1 and BMH2 differentially regulate rapamycin-mediated transcription

Synopsis 14-3-3 proteins are highly conserved and have been found in all eukaryotic organisms investigated. They are involved in many varied cellular processes, and interact with hundreds of other proteins. Among many other roles in cells, yeast 14-3-3 proteins have been implicated in rapamycin-mediated cell signalling. We determined the transcription profiles of bmh1 and bmh2 yeast after treatment with rapamycin. We found that, under these conditions, BMH1 and BMH2 are required for rapamycin-induced regulation of distinct, but overlapping sets of genes. Both Bmh1 and Bmh2 associate with the promoters of at least some of these genes. BMH2, but not BMH1, attenuates the repression of genes involved in some functions required for ribosome biogenesis. BMH2 also attenuates the activation of genes sensitive to nitrogen catabolite repression

Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.</p