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Eric Clark, Ruchira Singh, Molly Wilson, Jackie Meyer, David Kuai, Joe Phillips, David M Gamm; Elucidating the role of FGF signaling using an iPS cell model of retinal development.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1381.
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© ARVO (1962-2015); The Authors (2016-present)
Studies in animal models have implicated a regulatory loop between FGF signaling and the transcription factor Visual System Homeobox 2 (VSX2) in the maintenance of neural retinal progenitor cell (NRPC) identity and regulation of retinogenesis. However, the specific roles of FGF family members in these processes remain to be elucidated. We used a human induced pluripotent stem cell (hiPSC) model of retinogenesis to examine the expression and function of specific FGFs during neural retina (NR) development.
hiPSCs derived from a patient with a functional null mutation in VSX2 (R200Q) and an unaffected sibling underwent targeted differentiation to obtain optic vesicle structures (hiPSC-OVs). qRT-PCR was used to evaluate transcript levels of specific FGFs and FGF receptors over time. qRT-PCR, Western blots, and ICC were employed to evaluate the effect of exogenous FGF and neutralizing antibody treatment on the expression of selected genes and proteins involved in retinal development. Lastly, phosphorylation assays were performed to determine the effect of FGFs on ERK1/2 activation.
Time course studies revealed biphasic expression profiles of FGF3, 8, and 9 in differentiating hiPSC-OVs, with peaks corresponding to the formation of the eye field and OV. Furthermore, expression levels of FGF3, 9, 19 were higher in WT vs. (R200Q)VSX2 hiPSC-OVs. Continuous application of exogenous FGF9 to (R200Q)VSX2 hiPSC-OVs from d20-d90 led to increased expression of NR genes and decreased expression of RPE markers. Treatment of (R200Q)VSX2 hiPSC-OVs with exogenous FGF during specific developmental time windows revealed a role for FGF9 in proliferation and differentiation of NRPCs. In contrast, treatment of WT hiPSC-OVs with neutralizing antibody did not have a significant effect on the expression of NRPC markers, suggesting that FGF9 deficiency is tolerated in the presence of functional VSX2. Phosphorylation assays indicated that FGF9-mediated effects on (R200Q)VSX2 hiPSC-OVs are likely mediated by ERK 1/2 activation.
Our results demonstrate that both VSX2 and FGF9 play roles in the proliferation and differentiation of NRPCs, and that functional loss of VSX2 can be overcome by FGF9. These insights increase our understanding of human retinogenesis and may prove useful in the production of retinal cell types for therapeutic purposes.
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