Investigation Into the Epicardial Requirement for Crk and CrkL Adaptor Proteins in the Mammalian Heart
Location
Philadelphia, PA
Start Date
30-4-2025 1:00 PM
End Date
30-4-2025 4:00 PM
Description
INTRODUCTION: Congenital heart defects occur in ~1% of the population and increase the risk of cardiovascular disease. During cardiac development, epicardial cells delaminate into the subepicardium where they undergo epithelial-mesenchymal transformation (EMT) to differentiate into mesenchymal cells that become epicardium-derived cells (EPDCs). These EPDCs form coronary vascular cells. These EPDCs secrete paracrine factors that are essential for fetal myocardial growth and coronary vessel patterning. Epicardial EMT is involved in cardiac repair and regeneration. The epicardium retains the ability to differentiate into various cardiac cell phenotypes following myocardial injury or ischemia, thus contributing to cardiac tissue regeneration. Crk and CrkL are adaptor proteins that mediate key signaling pathways in mammalian development, including heart valve formation, angiogenesis for vascular remodeling, and neural crest cell maturation necessary for pharyngeal arch artery development and outflow tract septation. Crk and CrkL are expressed during mouse heart development in the epicardium, myocardium, endocardium and coronary vessels. Global deletion of Crk-CrkL in mice causes embryonic lethality due to cardiac defects. However, the specific roles of Crk and CrkL in the epicardium and coronary vessels have not been identified.
OBJECTIVE: Our goal was to identify the expression requirement of Crk and CrkL adaptor proteins in the heart during epicardium and coronary vessel formation.
METHODS: We used a conditional knockout strategy to generate mouse embryos with Wt1-Cre-driven deletion of Crk and CrkL from the epicardium (Crk-CrkL epi+/-). This approach enabled us to bypass the embryonic lethality caused by global Crk-CrkL deletion. Embryos were isolated at embryonic day (E) 13.5, 15.5, and 17.5 to assess cardiovascular development. We used hematoxylin and eosin staining for histological evaluation of cardiac structure. Also, we used immunofluorescence staining with antibodies to evaluate markers for epicardial (Wt1), myocardial (cardiac actin), vascular smooth muscle (SM22α), and endothelial (VE-cadherin) cell lineages. RNA sequencing was conducted on control and Crk-CrkL deletion hearts to identify differentially expressed genes and potential pathways targeted by Crk-CrkL.
RESULTS: Our analysis of Wt1 expression in E13.5 Crk-CrkL epi+/- hearts versus controls indicated a decrease in EPDC migration from the epicardium and reduced invasion into the compact myocardium. This deficit was resolved by E15.5. Immunofluorescence for VE-cadherin in E13.5 Crk-CrkL epi+/- hearts revealed superficially displaced primitive coronary vessels that failed to embed properly into the compact myocardium. However, this was resolved by E15.5 when the VE-cadherin expression pattern was similar in control embryos. This suggests coronary vessel patterning was restored after a brief developmental delay. Expression of the vascular smooth muscle marker, SM22α, in coronary vessels was consistent with VE-cadherin expression by E15.5.
CONCLUSION: Our data suggest Crk and CrkL play a critical role in early epicardial formation and EPDC migration, particularly in guiding coronary vessel patterning. Coronary vascular developmental defects were transient. This implies compensation occurs to restore epicardium and coronary vessel formation by later stages of development. These findings enable us to understand mechanisms of Crk and CrkL function in cardiac biology and shed light on the role of the epicardium in cardiac repair and regeneration.
Embargo Period
5-20-2025
Investigation Into the Epicardial Requirement for Crk and CrkL Adaptor Proteins in the Mammalian Heart
Philadelphia, PA
INTRODUCTION: Congenital heart defects occur in ~1% of the population and increase the risk of cardiovascular disease. During cardiac development, epicardial cells delaminate into the subepicardium where they undergo epithelial-mesenchymal transformation (EMT) to differentiate into mesenchymal cells that become epicardium-derived cells (EPDCs). These EPDCs form coronary vascular cells. These EPDCs secrete paracrine factors that are essential for fetal myocardial growth and coronary vessel patterning. Epicardial EMT is involved in cardiac repair and regeneration. The epicardium retains the ability to differentiate into various cardiac cell phenotypes following myocardial injury or ischemia, thus contributing to cardiac tissue regeneration. Crk and CrkL are adaptor proteins that mediate key signaling pathways in mammalian development, including heart valve formation, angiogenesis for vascular remodeling, and neural crest cell maturation necessary for pharyngeal arch artery development and outflow tract septation. Crk and CrkL are expressed during mouse heart development in the epicardium, myocardium, endocardium and coronary vessels. Global deletion of Crk-CrkL in mice causes embryonic lethality due to cardiac defects. However, the specific roles of Crk and CrkL in the epicardium and coronary vessels have not been identified.
OBJECTIVE: Our goal was to identify the expression requirement of Crk and CrkL adaptor proteins in the heart during epicardium and coronary vessel formation.
METHODS: We used a conditional knockout strategy to generate mouse embryos with Wt1-Cre-driven deletion of Crk and CrkL from the epicardium (Crk-CrkL epi+/-). This approach enabled us to bypass the embryonic lethality caused by global Crk-CrkL deletion. Embryos were isolated at embryonic day (E) 13.5, 15.5, and 17.5 to assess cardiovascular development. We used hematoxylin and eosin staining for histological evaluation of cardiac structure. Also, we used immunofluorescence staining with antibodies to evaluate markers for epicardial (Wt1), myocardial (cardiac actin), vascular smooth muscle (SM22α), and endothelial (VE-cadherin) cell lineages. RNA sequencing was conducted on control and Crk-CrkL deletion hearts to identify differentially expressed genes and potential pathways targeted by Crk-CrkL.
RESULTS: Our analysis of Wt1 expression in E13.5 Crk-CrkL epi+/- hearts versus controls indicated a decrease in EPDC migration from the epicardium and reduced invasion into the compact myocardium. This deficit was resolved by E15.5. Immunofluorescence for VE-cadherin in E13.5 Crk-CrkL epi+/- hearts revealed superficially displaced primitive coronary vessels that failed to embed properly into the compact myocardium. However, this was resolved by E15.5 when the VE-cadherin expression pattern was similar in control embryos. This suggests coronary vessel patterning was restored after a brief developmental delay. Expression of the vascular smooth muscle marker, SM22α, in coronary vessels was consistent with VE-cadherin expression by E15.5.
CONCLUSION: Our data suggest Crk and CrkL play a critical role in early epicardial formation and EPDC migration, particularly in guiding coronary vessel patterning. Coronary vascular developmental defects were transient. This implies compensation occurs to restore epicardium and coronary vessel formation by later stages of development. These findings enable us to understand mechanisms of Crk and CrkL function in cardiac biology and shed light on the role of the epicardium in cardiac repair and regeneration.