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B. A. Link, F. B. Berry, M. A. Walter, J. M. Skarie; FOXC1.1 and FOXC1.2 Influence Ocular Vascular Development and Cellular Stress Levels in Developing Zebrafish. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5910.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in FOXC1, a forkhead domain transcription factor, cause dominantly inherited Axenfeld-Rieger Syndrome (ARS) in humans. ARS is a congenital disorder affecting development of many tissues including eye, teeth and heart. The ocular phenotype is an anterior segment dysgenesis, with 50% of patients subsequently developing glaucoma. Recently, examination of FoxC1 knockout mice has revealed defects in cardiovascular development, including defects in arterial specification and cardiac outflow, along with disorganized yolk vessels. Another study revealed that direct targets of FOXC1, such as FOXO1a, are involved in cellular stress responses. We have previously shown that loss of the zebrafish foxC1 genes, foxC1.1 and foxC1.2 show similar gross phenotypes to other models. In this study, we examine the role of FoxC1 in ocular vascular development and cellular stress responses.
Translation-blocking morpholino oligonucleotides were used to knock-down activity of foxC1 genes independently and together. Phenotype analysis was conducted on morphants using light microscopy, histology, in situ hybridization, angiography, and immuno-based markers. Transgenic fish lines and confocal microscopy were utilized to observe vascular tissue and cellular stress levels.
Knockdown of foxC1.1 or foxC1.1/foxC1.2, but not foxC1.2 resulted in CNS and ocular hemorrhaging, accompanied by a reduction in heart rate and blood flow. A dilation of tail veins and arterial-venous malformations along the cardinal vein and dorsal aorta were also noted. Within the eyes, an accumulation of blood cells and apparent lack of hyaloid vessels were noted. Subsequent morphant analysis in the fli1:GFP transgenic fish, which marks vascular cells, showed a dysmorphic hyaloid vascular system and disorganization of major axial vessels. Markers of cellular stress were found to be upregulated in foxC1.1/foxC1.2 morphants. Acridine orange staining showed increased cell death throughout the embryo, and knockdown of foxC1 in the hsp70:GFP transgenic fish revealed upregulation of this cellular stress reporter.
Vascular development is impaired in foxC1 morphant zebrafish, including the ocular hyaloid system. Cellular stress levels and cell death was also increased in foxC1 morphants. The combined or individual effects of these developmental and cell homeostatic defects may contribute to the susceptibility of developing glaucoma with loss of FoxC1.
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