FoxC1 is essential for vascular basement membrane integrity and hyaloid vessel morphogenesis

Purpose: Alterations in FOXC1 dosage lead to a spectrum of highly penetrant, ocular anterior segment dysgenesis phenotypes. The most serious outcome is development of glaucoma, but this only occurs in 50-75% of patients. Therefore, the need to identify specific pathways and genes that interact with FOXC1 to promote glaucoma is great. In this study, we investigated loss of foxC1 in the zebrafish to characterize phenotypes and gene interactions that may impact glaucoma pathogenesis. Methods: Morpholino knockdown in zebrafish, RNA and protein marker analyses, transgenic reporter lines, and angiography, along with histology and transmission electron microscopy were used to study foxC1 function and gene interactions. Results: Zebrafish foxC1 genes were expressed dynamically in the developing vasculature and periocular mesenchyme during development. Multiple ocular and vascular defects were found after knockdown of foxC1. Defects in the hyaloid vasculature, arterial-venous malformations, and coarctation of the aorta were observed with maximal depletion of foxC1. Partial loss of foxC1 resulted in CNS and ocular hemorrhages, defects in intersegmental vessel patterning, and increased vascular permeability. To investigate the basis for these disruptions, ultrastructure of foxC1-depleted hyaloid vascular cells was studied. These experiments, along with Laminin-111 immunoreactivity, revealed disruptions in basement membrane integrity. Finally, co-depletion of laminin alpha-1 and foxC1 uncovered a genetic interaction between these genes during development. Conclusions: Genetic interactions between FOXC1 and basement membrane components influence vascular stability and may impact glaucoma development and increase stroke risk in FOXC1 patients.