Investigating COUP-TFII in human vein development

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY The cardiovascular system is one of the earliest systems to develop and is critical for the survival of nearly all tissues and is comprised of four vessel types: arteries, veins, lymphatic vessels, and capillaries. Although they are all lined internally with a single-cell layer of endothelial cells (ECs), these ECs have cell identities specific to their vessel. Little is known about how the endothelial cell subtypes specialize, including veins. Disruptions in vein EC specialization and function can lead and/or contribute to conditions including arteriovenous malformations, cancers, or metabolic diseases. Previous research in mouse and zebrafish models revealed that a transcription factor, COUP-TFII, is required for vein EC identity specification. COUP-TFII is considered an orphan receptor since a physiological ligand has not been identified and is considered a key marker for venous identity. However, it remains unclear whether it is required for human vein development and critical aspects of its mechanisms of action are unknown. This is due to the lack of human culture systems for vein EC development and the difficulty of obtaining enough vascular cells for biochemical experiments from model organism blood vessels. Recently, our collaborators in the labs of Drs. Lay Teng Ang and Kyle Loh have established a stem cell differentiation protocol for vein and artery ECs from human pluripotent cells. The protocol results in the efficient (>85%) generation of highly pure vein ECs and recapitulates the steps of early embryonic cell differentiation. Leveraging this protocol, I aim to study COUP-TFII during human vein development. In Aim 1, I will establish if COUP-TFII is required for human vein EC specification, and if so, what protein domains of COUP-TFII are required. I will use and generate COUP-TFII loss-of-function CRISPR-modified human embryonic stem cell (hESC) lines to determine if markers of venous identity are disrupted in their gene and protein expression. I have already found that vein ECs generated from a COUP-TFII knockout hESC line has differential gene expression when compared to wildtype and that this is recapitulated in the COUP-TFII ligand binding domain truncation, suggesting that ligand binding plays an important role in vein EC development. In Aim 2, I will investigate if COUP-TFII regulated genes are required for venous EC specification, and what, if any, protein binding partners COUP-TFII is interacting with during human vein EC development. To do this, I will use COUP-TFII hESC lines that are tagged with FLAG, BioID and TurboID to perform immunoprecipitation and mass spectrometry experiments. Lastly, I will identify COUP-TFII ligands in Aim 3 through proximity labeling and mass spectrometry. Together, these aims will provide insight into the molecular dynamics of COUP-TFII in human vein EC development and allow me to gain technical and intellectual skills in experimental design and prepare for competitive postdoctoral positions.