Cell fate choices by Tbx1 in forming the mammalian heart

ABSTRACT Tbx1 encodes a T-box transcription factor that is a critical gene responsible for the etiology of congenital heart disease in individuals affected with 22q11.2 deletion syndrome (22q11.2DS). This syndrome occurs in 1/4000 live births and 1/1000 fetuses. Approximately 60-70% of 22q11.2DS patients have congenital heart disease, mostly affecting formation of the cardiac outflow tract (OFT). In addition, individuals with 22q11.2DS also have hypernasal speech and feeding difficulties due to hypotonia of the branchiomeric muscles. Mutations in TBX1 have been identified in patients that have congenital heart disease but do not have a 22q11.2 deletion, confirming its status as a human disease gene. Tbx1 is expressed and required in mouse models for cardiac OFT formation and branchiomeric muscle specification in the mesoderm of the pharyngeal apparatus, termed the cardiopharyngeal mesoderm. In the original program, we performed single cell RNA-sequencing to understand the function of Tbx1 in the cardiopharyngeal mesoderm using our mouse models. We identified cell populations such as the anterior and posterior second heart fields that contribute cells to different components of the heart and branchiomeric muscles. We also discovered a previously uncharacterized progenitor population, which strongly expresses Tbx1, that we termed multilineage primed progenitors (MLPs). These MLPs simultaneously express genes of the anterior and lateral part of the posterior second heart field cell types. Expression of genes that promote cell fate progression were reduced when Tbx1 was inactivated and other, non-mesodermal genes were ectopically expressed in MLPs in mutant embryos. This suggests that the role of Tbx1 in MLPs is to promote normal heart cell lineage maturation while restricting activation of atypical cell lineages in MLPs. The anterior and posterior second heart field cells are, in part, specified at gastrulation. Our hypothesis is that MLPs are an intermediate dynamic population that provides progenitor cells whose normal differentiation progress depends on Tbx1 function. In this application, we will thoroughly determine the function of Tbx1 in MLPs by mouse genetic and single cell multiomic studies in Aim 1. Tbx1 is also strongly expressed in the anterior and lateral part of the posterior second heart fields and we will investigate its specific functions in these cells in Aim 2. Single cell technology, including single cell RNA-seq, ATAC-seq and spatial transcriptomics combined with utilization of CreERT2 mouse lines will allow for timed and cell type specific studies of Tbx1 function. In order to have a mechanistic understanding of how Tbx1 can regulate cell fate progression in these cell types, we will examine distant-acting enhancers, uncovered from our omics data, by mouse transgenesis that includes knockin and knockout strategies in Aim 3. The overall impact of the project will be a deep understanding of the molecular genetic mechanisms for cardiac cell fate acquisition by Tbx1 on individual progenitor cell types in early to mid- gestation mouse embryos.