Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD) [1][1]. This observation predicts gene regulatory network (GRN) imbalances [2][2], but the nature of dosage-vulnerable GRNs and their contribution to human cardiogenesis and CHDs are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor TBX5 during human cardiac differentiation from induced pluripotent stem (iPS) cells. Single cell RNAseq revealed that transcriptional responses to reduced TBX5 levels are not homogeneous, and instead, discrete sub-populations of cardiomyocytes exhibit dysregulation of distinct TBX5 dose-sensitive genes related to cellular phenotypes and CHD-associated genetics. Cellular trajectory inference revealed TBX5 dosage-dependent differentiation paths, with implications for cardiac developmental identity. GRN analysis of the single cell RNAseq data identified vulnerable nodes enriched for CHD genes, implicating a critical sensitivity to TBX5 dosage in cardiac network stability. A novel GRN-predicted genetic interaction between TBX5 and MEF2C was validated in mouse, revealing a highly dosage-sensitive pathway for CHD. Our results reveal unforeseen complexity and exquisite sensitivity to TBX5 dosage in discrete sub-populations of iPSC-derived cardiomyocytes, providing mechanistic insights into human CHDs and quantitative transcriptional regulation in disease. [1]: #ref-1 [2]: #ref-2

A TBX5 dosage-sensitive gene regulatory network for human congenital heart disease