Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Brueckner, Martina
Abstract
The first organ to develop in vertebrates is the heart, a necessary process for all other organ systems that follow. Proper development of the embryonic heart relies on the ability for intracardiac cells to sense and translate complex mechanical forces into transcriptional signals. Failure to do so results in numerous cardiac abnormalities, such as thinned compact myocardium, stunted trabeculations and atrioventricular canal defects. These structural abnormalities are a common occurrence in congenital heart disease (CHD), which affects more than 1% of all live births. Nonetheless, the mechanism by which mechanical forces are generated, sensed, and integrated into cardiac development remains poorly understood. This body of work describes a method by which blood flow is converted into spatial regulation of endocardial cushion formation through a known mechanosensor, the primary cilium.Though many forces exist in the developing heart, blood flow-derived wall shear stress (WSS) is the most critical for heart valve formation. Here, we uncover a WSS-specific mechanosensor, the primary cilium, on endocardial cells lining the heart lumen in regions of valve development in both mouse and zebrafish embryos. We identify a role for these primary cilia in the spatial regulation of cushion formation, the first stage of valve development, by regionally restricting endothelial to mesenchymal transition (EndoMT) via modulation of Kruppel-like Factor 4 (Klf4) in mouse. KLF4 is a mechanosensitive transcription factor that we find negatively correlates with EndoMT progression. Our results indicate that endocardial cells that experience high WSS lose their cilia, correlating with KLF4 downregulation and permissive EndoMT only in high WSS regions. Mouse embryos constitutively lacking cilia (cilia KOs) paradoxically exhibit a blood-flow dependent increase in KLF4 expression, independent of upstream left-right abnormalities. These results suggest that initial cilia presence and subsequent loss on endocardium in response to cardiac function is needed for downregulation of KLF4. Overabundance of KLF4 in cilia KOs results in significantly impaired EndoMT progression and cushion cellularization. Single-Nuc RNAseq on isolated e9.5 wild-type and cilia KO hearts revealed that hearts lacking cilia fail to progress from mid- to late-EndoMT, a pseudo-stage that corresponds with Klf4 downregulation in wild-type hearts. Cilia KO hearts retain endothelial markers during EndoMT and fail to express mesenchymal and EndoMT genes that are negatively regulated by KLF4. Finally, Gene Ontology terms for mesenchymal and valve development are downregulated in cilia KO hearts, while terms for vascular identity/integrity are upregulated. Taken together, these data identify a novel mechanosensory role for EC primary cilia in regulating EndoMT localization and progression via KLF4 expression during cushion development.
Recommended Citation
Berg, Kathryn, "Mechanical and Transcriptional Roles for Primary Cilia during Intracardiac Cushion EndoMT" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1360.
https://elischolar.library.yale.edu/gsas_dissertations/1360
