A temporally‐restricted pattern of endothelial cell collagen 4 alpha 1 expression during embryonic development determined with a novel knockin Col4a1‐P2A‐eGFP mouse line

Abstract

SummaryClassical collagen type IV comprising of a heterotrimer of two collagen IV alpha 1 chains and one collagen IV alpha 2 chain is the principal type of collagen synthesized by endothelial cells (EC) and is a major constituent of vascular basement membranes. In mouse and man, mutations in genes that encode collagen IV alpha 1 and alpha 2 result in vascular dysfunction. In addition, mutations in genes that encode the Ephrin receptor B4 (EPHB4) and the p120 Ras GTPase‐activating protein (RASA1) that cause increased activation of the Ras mitogen‐activated protein kinase (MAPK) signaling pathway in EC result in vascular dysfunction as a consequence of impaired export of collagen IV. To understand the pathogenesis of collagen IV‐related vascular diseases and phenotypes it is necessary to identify at which times collagen IV is actively synthesized by EC. For this purpose, we used CRISPR/Cas9 targeting in mice to include immediately after the terminal Col4a1 codon a sequence that specifies a P2A peptide followed by enhanced green fluorescent protein (eGFP). Analysis of eGFP expression in Col4a1‐P2A‐eGFP mice revealed active embryonic EC synthesis of collagen IV alpha 1 through mid to late gestation followed by a sharp decline before birth. These results provide a contextual framework for understanding the basis for the varied vascular abnormalities resulting from perturbation of EC expression and export of functional collagen IV.