Determining the function of Tbx1 in the pharyngeal endoderm for aortic arch development

/ABSTRACT: TBX1, encodes a critical gene for the etiology of aortic arch defects in patients with 22q11.2 deletion syndrome. These defects derive from the 4th pharyngeal arch artery (4th PAA) in mammalian embryos. Formation of the 4th PAA requires tissue interactions that include neural crest cells (NCCs). Although Tbx1 is widely expressed in the pharyngeal arches, including the endoderm that forms the pharyngeal pouches, it is not expressed in NCCs in mouse models. It was previously found that inactivation of Tbx1 in the embryonic pharyngeal endoderm, using the Sox172a-iCre allele, causes malformations of the caudal endodermal pouches and aortic arch anomalies. As preliminary data, we used the same mice to inactivate Tbx1 and confirmed these mutant embryos had absent caudal pharyngeal pouches and found that they have 4th PAA defects. Additionally, we performed a pilot single- cell RNA-sequencing (scRNA-seq) experiment from control and Tbx1 endoderm conditional null mutants using Sox172a-iCre, at E9.5. I analyzed the data and found differentially expressed genes (DEGs) including some related to cell adhesion and signaling that might explain the basis of these phenotypes. We therefore hypothesize that the function of Tbx1 in the endoderm is to regulate the expression of genes for cell adhesion needed for endodermal pouch formation as well as signaling genes needed for NCC development to form the 4th PAAs. The constitutive Sox172a-iCre line has some endothelial expression, as expected. We recently obtained another Cre line that is tamoxifen inducible, termed Sox17Cre-ERT2 which results in exclusive endoderm expression with E6.5 injection. In Aim 1, I will inactivate Tbx1 using this line and will compare the phenotypes between this and the constitutive Cre line. Then I will determine the cellular basis of pharyngeal pouch and NCC defects. I will validate expression of some of the most interesting preliminary DEGs for cell adhesion and signaling molecules by in vivo studies of control and endodermal (constitutive and tamoxifen inducible) mutant embryos. These phenotypes and cellular changes will be compared to inactivation of Tbx1 by Tbx1Cre, in which all cell types that express Tbx1 will be affected. In Aim 2, I will generate three replicates of multi-omic (scRNA-seq and scATAC- seq) data at E9.5 and E10.5 to identify DEGs and altered chromatin accessibility in the endoderm and NCCs in control and mutant embryos. This will allow me to create an endodermal Tbx1-dependent gene regulatory network. I will also use cell-cell communication software to identify altered ligand receptor pairs and downstream pathway genes perturbed by the loss of endodermal Tbx1 to add to the network. Genes within this network will be prioritized through a detailed filtering strategy and I will validate a subset by in vivo analysis. I will also test the functional relevance of the top 1-2 candidates regulated by Tbx1 through phenotyping studies. Upon completion of these aims, this project will uncover cellular and molecular mechanisms regulated by Tbx1 on a single-cell level that mediate aortic arch anomalies in mouse models and human patients with 22q11.2 deletion syndrome as well as for sporadic occurrences in the general population. Project Number: 1F31HL180025-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Kevyn Jackson | Institution: ALBERT EINSTEIN COLLEGE OF MEDICINE, BRONX, NY | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F10A-R (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31HL18002501