Geng, X.;Mahamud, M.;Rosikiewicz, W.;Ent, M.;Zawieja, S.;Chen, H.;Cleuren, A.;Li, C.;Davis, M.;Srinivasan, R.

The two-hit hypothesis provides a novel framework for incompletely penetrant vascular disorders. Emberger syndrome, caused by heterozygous loss-of-function mutations in mechanosensitive transcription factor (TF) GATA2, is associated with lymphedema in a subset of patients, suggesting that modifiers influence disease development. Heterozygous loss-of-function mutations in mechanosensitive TF FOXC2 also cause lymphedema. Complete deletion of either factor in lymphatic endothelial cells (LECs) causes severe, overlapping defects, including complete loss of lymphatic valves, whereas single heterozygous mutants exhibit milder phenotypes, suggesting genetic buffering. However, whether GATA2 and FOXC2 interact with each other and with shear stress-dependent transcriptional programs remains unclear. Here, we show that Gata2 +/- ;Foxc2 +/- mice develop profound lymphatic vascular defects, including absent lymphatic valves, perinatal lethality, and valve dysfunction, revealing dosage-dependent cooperation between GATA2 and FOXC2. Using a Cre-dependent model of LEC-specific GATA2 overexpression, we found that increased GATA2 dosage downregulated PROX1 and disrupted lymphatic vascular development. ATAC-seq and bulk RNA-seq of shear-exposed human LECs showed that PROX1 governs shear-responsive genes, including KLF2 and KLF4, whereas FOXC2 modulates PROX1-dependent and independent gene subsets. Together, these findings identify PROX1 as a central integrator of shear stress-responsive transcriptional programs and reveal that balanced GATA2 and FOXC2 dosage preserves this network during lymphatic vascular development.