Mechanical and Transcriptional Roles for Primary Cilia during Heart Development

点击翻译按钮获取中文摘要

Abstract: Congenital heart defects (CHD) are the most common birth defects, affecting 1% of all live births. Of the known genetic contributors to CHD, the pathogenic mechanism is undetermined for most. Our lab has identified de novo mutations in kruppel-like factors 2 and 4 (KLF2, 4) in CHD patients presenting with hypoplastic left heart syndrome (HLHS). Klf2 and 4 are mechanosensitive transcription factors that regulate endothelial to mesenchymal transition (EndoMT), a process necessary for cushion (heart valve progenitor) development. Understanding how EndoMT is spatially and temporally controlled by mechanical modulation of Klf2/4 will provide insights into the heart valve defects present in HLHS patients. Our lab has implicated primary cilia (non-motile, rod-like mechanosensors) on the endocardium (EC) in cardiogenesis beyond their known role in left-right asymmetry. However, whether EC primary cilia transduce mechanical signals into regulation of Klf2/4 during cushion development has yet to be shown. The goal of this proposal is to discover the relationship between mechanical forces, EC primary cilia and Klf2/4 during heart cushion formation in vivo. Based on my preliminary results, the overall hypothesis of this proposal is that EC primary cilia link mechanical forces and Klf2/4 transcription in a Ca2+ and cytoskeletal-dependent manner during endocardial cushion formation in vivo. To address this hypothesis, we will utilize both mouse and zebrafish models. Mouse hearts are more comparable to humans (zebrafish have two chambers), but the genetic programs governing cardiogenesis are conserved. Events that may be lost in static mouse samples can instead be observed using live zebrafish in time-lapse microscopy due to their optical clarity and external development. Aim 1 will investigate how EC primary cilia and Klf2/4 expression change spatially over heart cushion development, utilizing in situ hybridization and immunofluorescence in mice, and various transgenic lines marking endocardium, cilia, and the promoter regions of Klf2/4 in live zebrafish. Aim 2 will test whether Klf2/4 depend on cilia, ciliary Ca2+ and/or mechanical forces through use of various mutant backgrounds, such as Ift20 and Kif3a (cilia KOs), Ncx/silent heart morpholino (heartbeat KO, mouse and zebrafish, respectively), and Pkd2 (ciliary Ca2+ KO). Our data provides insight into the mechanism controlling Klf2/4 mechanosensitivity during heart cushion formation, and highlight a ciliary role as mechanosensors during cardiogenesis. This proposal addresses the NIH-NHLBI's objectives 1 and 2 in regards to investigating the genetic and mechanical mechanism behind the valve defects present in many CHD patients. Our findings could also lead to treatments to ameliorate the progressive valve dysfunction experienced by many CHD patients. My use of live, whole- mount imaging will extend beyond the observations possible in static, fixed embryos and bring a rare perspective of ciliary-dependent transcription in vivo. The role of mechanical forces during development is a rapidly expanding field, and our results will contribute to both a basic and translational biological perspective.