Deciphering transcriptional and epigenomic regulation of early cardiogenesis

Transcriptional networks governing early cardiac precursor cell (CPC) specification are incompletely understood due in part to the difficulty of distinguishing CPCs from their mesoderm germ layer of origin in early gastrulation. Cardiogenesis in the gastrulating embryo begins when mesoderm progenitor cells emerge from the primitive streak and migrate anterior-laterally to coalesce at the anterior midline. Errors during CPC specification and patterning can cause devastating Congenital Heart Defects (CHDs). Occurring in 1-2% of live births, CHDs often require surgical interventions and can result in secondary heart disease. The genetic etiology of CHDs indicates that genes encoding transcription factors (TFs) are overrepresented as causative and are predominantly haploinsufficient, indicating that fine dysregulation of gene expression is a driving mechanism for disease. Thus, understanding the transcriptional regulatory networks governing early cardiac specification is paramount for understanding CHDs and necessary to develop novel therapeutic strategies. Our comprehension of transcriptional regulation at the initiation of cardiogenesis is hindered in part by the paucity of molecular tools capable of distinguishing the emerging cardiac lineage from the surrounding mesoderm. Prior studies leveraged lineage tracing from the basic-helix-loop-helix (bHLH) TF Mesp1, however as this lineage contributes to other mesodermal derivatives beyond the heart the method is insufficient for isolation of early CPCs. To overcome this challenge and investigate the cardiac lineage distinctly from the surrounding mesoderm, we leveraged a pan-cardiac enhancer transgene reporter, Smarcd3-F6, that restrictively marks emerging, early CPC populations within the mesoderm. We utilized bioinformatic detection of fluorescent reporter transgenes tracking both the Mesp1 lineage and Smarcd3-F6 expression in whole embryo single cell transcriptomic data to interrogate the heterogeneity of CPC transcriptional profiles in an in vivo mouse gastrulation time course. The dataset we generated towards this goal represents a valuable resource for investigations of the early cardiac mesoderm and for broader questions of cell fate allocation from germ layers during gastrulation. We further leveraged the Smarcd3-F6 enhancer sequence as an experimental discovery platform for the identification of regulatory network logic during early cardiogenesis. We identified specific GATA and T-box motif sites necessary for a minimal Smarcd3-F6 sub-region’s enhancer activity. This in vivo enhancer study provides a framework for functional characterization of transcriptional regulatory networks during development. Lastly, we utilized single cell transcriptomic and chromatin accessibility sequencing to define the resilience and vulnerability of cardiac specification in embryos deficient for Mesp1, the early-expressed and often-posited ‘cardiac master regulator’. Our results distinguish Mesp1-independent and dependent processes in early cardiogenesis, showing that Mesp1 deficient CPCs progress through cardiogenesis until lateral plate mesoderm stages, at which point their disrupted regulatory landscape prohibits maturation further into patterned cardiac progenitor and cardiomyocyte fates. Collectively, these results illustrate the complex transcriptional and epigenomic interdependence of regulatory networks during lineage specification and further advance our fundamental understanding of the processes governing cardiac specification in vivo at single cell resolution. The investigative frameworks and the interpretations of findings described in this dissertation illuminate generalizable principles for the regulatory logic guiding the allocation and subsequent differentiation of precursor cells towards distinct, functional cell types during gastrulation