Epicardial Loss of Crk and CrkL Alters Cardiac Cytokeratin Expression
Location
Philadelphia, PA
Start Date
17-4-2026 1:30 PM
End Date
17-4-2026 2:30 PM
Description
Introduction
Crk and CrkL are adaptor proteins that regulate signaling pathways to control cell migration, proliferation, apoptosis and differentiation. They have been implicated in congenital heart disease. Our lab observed Crk and CrkL expression in the epicardium and epicardium-derived cells (EPDCs), including coronary vascular cells, in adult and embryonic mouse hearts. While biological roles for Crk and CrkL have been established in cardiac neural crest cells during outflow tract septation and atrioventricular valve development, their function within the epicardium and EPDCs has not been defined. Crk and CrkL are expressed in the embryonic mouse epicardium and EPDCs, including coronary vascular progenitors. This suggests their potential role in epicardial signaling and lineage specification. KRT7 and KRT19 encode epithelial intermediate filament proteins that serve as markers of epicardial identity, epithelial integrity, cytoskeletal organization, and maintenance of cell-cell adhesion. Their expression is dynamically regulated during epithelial-to-mesenchymal transition (EMT) in development. Mature cardiomyocytes lack significant expression of these markers. We aim to investigate the role of Crk and CrkL in the epicardium and EPDCs and their impact on cardiac cytokeratin expression.
Methods
We generated mutant mice with epicardial-specific deletion of Crk-CrkL (Crk-CrkL-epidel/+). We isolated total RNA from control and mutant Crk-CrkL-epidel/+ mouse hearts and conducted transcriptome analysis via RNA sequencing (RNAseq) to examine differences in mRNA expression between them. Our bioinformatic analysis of the RNAseq data was performed to classify the differentially expressed transcripts by gene ontology. The RNAscope technique was used to examine expression of 2 transcripts by hybridization of specific probes to these transcripts on sectioned control and mutant hearts. RNAscope-hybridized tissues were imaged on a Motic scanner to localize regions of expression within sectioned hearts. Transcripts were quantified by region in each sectioned tissue using Image J software. We began validating mRNA expression of these transcripts by quantitative PCR (qPCR) with transcript-specific probes.
Results
Our RNAseq analysis identified 1544 transcripts that were differentially expressed between control and mutant mice. Several of these altered transcripts were involved in developmental biology, epithelial cell differentiation and cytoskeleton organization. Two transcripts, KRT7 and KRT19, are type I and II cytokeratins that compose intermediate filament proteins responsible for structural integrity of epithelial cells and are expressed in simple epithelia. We observed a 4.5-fold and a 5.8-fold increase in KRT7 and KRT19 expression in mutant vs. control hearts, respectively, via RNAseq analysis. Specifically, we detected punctate and clustered mRNA expression of these transcripts in the epicardium and myocardium of our Crk-CrkL mouse hearts by RNAscope analysis. Variations in expression were observed between control and mutant mice. qPCR analysis of RNA isolated from Crk-CrkL mouse hearts is being conducted to validate mRNA expression of these two transcripts.
Discussion
Given the established role of Crk and CrkL in cytoskeletal remodeling and cell state transitions, we believe these adaptor proteins function upstream of epicardial gene programs that include KRT7 and KRT19. Crk and CrkL have the potential to link intracellular signaling pathways to maintenance of epithelial identity and EMT regulation in the developi
Embargo Period
5-19-2026
Epicardial Loss of Crk and CrkL Alters Cardiac Cytokeratin Expression
Philadelphia, PA
Introduction
Crk and CrkL are adaptor proteins that regulate signaling pathways to control cell migration, proliferation, apoptosis and differentiation. They have been implicated in congenital heart disease. Our lab observed Crk and CrkL expression in the epicardium and epicardium-derived cells (EPDCs), including coronary vascular cells, in adult and embryonic mouse hearts. While biological roles for Crk and CrkL have been established in cardiac neural crest cells during outflow tract septation and atrioventricular valve development, their function within the epicardium and EPDCs has not been defined. Crk and CrkL are expressed in the embryonic mouse epicardium and EPDCs, including coronary vascular progenitors. This suggests their potential role in epicardial signaling and lineage specification. KRT7 and KRT19 encode epithelial intermediate filament proteins that serve as markers of epicardial identity, epithelial integrity, cytoskeletal organization, and maintenance of cell-cell adhesion. Their expression is dynamically regulated during epithelial-to-mesenchymal transition (EMT) in development. Mature cardiomyocytes lack significant expression of these markers. We aim to investigate the role of Crk and CrkL in the epicardium and EPDCs and their impact on cardiac cytokeratin expression.
Methods
We generated mutant mice with epicardial-specific deletion of Crk-CrkL (Crk-CrkL-epidel/+). We isolated total RNA from control and mutant Crk-CrkL-epidel/+ mouse hearts and conducted transcriptome analysis via RNA sequencing (RNAseq) to examine differences in mRNA expression between them. Our bioinformatic analysis of the RNAseq data was performed to classify the differentially expressed transcripts by gene ontology. The RNAscope technique was used to examine expression of 2 transcripts by hybridization of specific probes to these transcripts on sectioned control and mutant hearts. RNAscope-hybridized tissues were imaged on a Motic scanner to localize regions of expression within sectioned hearts. Transcripts were quantified by region in each sectioned tissue using Image J software. We began validating mRNA expression of these transcripts by quantitative PCR (qPCR) with transcript-specific probes.
Results
Our RNAseq analysis identified 1544 transcripts that were differentially expressed between control and mutant mice. Several of these altered transcripts were involved in developmental biology, epithelial cell differentiation and cytoskeleton organization. Two transcripts, KRT7 and KRT19, are type I and II cytokeratins that compose intermediate filament proteins responsible for structural integrity of epithelial cells and are expressed in simple epithelia. We observed a 4.5-fold and a 5.8-fold increase in KRT7 and KRT19 expression in mutant vs. control hearts, respectively, via RNAseq analysis. Specifically, we detected punctate and clustered mRNA expression of these transcripts in the epicardium and myocardium of our Crk-CrkL mouse hearts by RNAscope analysis. Variations in expression were observed between control and mutant mice. qPCR analysis of RNA isolated from Crk-CrkL mouse hearts is being conducted to validate mRNA expression of these two transcripts.
Discussion
Given the established role of Crk and CrkL in cytoskeletal remodeling and cell state transitions, we believe these adaptor proteins function upstream of epicardial gene programs that include KRT7 and KRT19. Crk and CrkL have the potential to link intracellular signaling pathways to maintenance of epithelial identity and EMT regulation in the developi