Title

Reelin Signaling in Cardiovascular Development

Date of Award

2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Cathy J Hatcher, PhD

Second Advisor

Ruth C Borghaei, PhD

Third Advisor

Heather L Montie, PhD

Abstract

The epicardium and coronary vessels are derived from a cell lineage that descends from the primitive proepicardium during cardiovascular development. These structures are formed through developmentally linked processes that are regulated by a network of transcription factors that control cell behavior and molecular genetic expression. Previously, we demonstrated that the Tbx5 transcription factor contributes to epicardial cell adhesion and coronary vasculogenesis in embryonic mice. We speculate that Tbx5 mediates these functions through transcriptional regulation of Reln gene expression. Reelin, which is encoded by the Reln gene, is a secreted extracellular matrix glycoprotein. Upon binding to its receptors, reelin activates multiple intracellular proteins to mediate cell functions. Reelin has an established role in neural and lymphatic system development where it regulates cell migration, polarity and adhesion. However, its role in cardiovascular development has yet to be determined. Our goal is to identify the functional contributions of reelin signaling to the structural formation of the developing heart and embryonic cardiac cell behavior. We examined reelin expression in the developing mouse heart through immunofluorescent staining. Reelin expression is initially detected in the proepicardium, and endocardium as well as the newly formed epicardium of the developing mouse heart as early as embryonic day (E) 9.5. Through immunofluorescent staining with antibodies that recognize cell-specific markers, we were able to determine that reelin expression becomes localized to epicardial cells and begins to appear in nascent coronary vessels by (E) 13.5. Expression of reelin in endothelial cells, which comprise the nascent coronary vessels, is evident at (E) 18.5 just prior to birth. In addition, the expression pattern of Crk, which is involved in the canonical signaling pathway of reelin, closely resembles that of reelin during cardiovascular development. At (E) E13.5, prominent staining of Crk in the epicardium as well as the nascent coronary vessels is observed. Crk is also expressed in the mature coronary vessels at (E) 18.5 at which time its expression in the epicardium becomes faint. These findings prompted us to explore the physiological role of reelin in endothelial cells by assessing its contributions to in vitro cell behavior in the human dermal microvascular endothelial cell (HDMEC) line that models aspects of coronary vasculogenesis. Primary cultured HDMECs were treated with Reln-specific small interfering RNAs (siRNAs) to knock down endogenous reelin expression. Our results indicate that reelin knockdown significantly alters in vitro cell behavior by significantly delaying the cell migratory ability of Reln siRNA-treated cells in comparison to negative control siRNA-treated cells. These in vitro findings are consistent with the delayed epicardial cell adhesion observed in hearts of our embryonic Tbx5-deficient mice that exhibit reduced cardiac Reln mRNA expression. Together, the results of these assays provide insight into the developmental expression of reelin and its functional contributions to cellular mechanisms required for cardiovascular development. This will comprise a platform of knowledge that may lead to the discovery of novel treatment approaches, to revascularize the diseased heart and improve the quality of life for CHD patients.

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