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Anna Petelinsek, Lynda S Wright, Elizabeth E Capowski, Jessica Lien, Isabel Pinilla Lozano, Jishnu Saha, Joe Phillips, David M Gamm; WNT signaling during patterning of human iPSC-derived optic vesicle-like structures. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3573.
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© ARVO (1962-2015); The Authors (2016-present)
VSX2, a homeodomain transcription factor expressed in neural retina progenitor cells (NRPCs), has a prominent role in optic vesicle (OV) patterning. Recently, we used hiPSCs to demonstrate that an R200Q mutation in the DNA binding region of VSX2 caused a partial cell fate switch from NR to RPE. RNAseq data comparing NR from wild type (WT) and (R200Q)VSX2 hiPSCs suggested that the WNT pathway was altered in VSX2 mutant OVs. To further investigate this finding, we examined the spatiotemporal expression of MITF, VSX2, and canonical WNT signaling proteins during hiPSC-OV patterning.
hiPSC-OVs were generated from an individual bearing homozygous R200Q mutations in VSX2 and an unaffected sibling using our established methods. We performed ICC to localize expression of VSX2, MITF, and the WNT markers b-catenin, WLS, and LEF1 at days 14, 18, 35 and 50 of differentiation.
Retinal differentiation of WT hiPSCs resulted in induction of a MITF+ population of OV cells at d14, a subset of which initially coexpressed VSX2. Also at d14, expression of the WNT markers WLS and LEF1, along with nuclear localization of the canonical WNT pathway effector b-catenin, were detected in MITF+ cells but seldom in VSX2+ cells. By d18 of differentiation, WT OV cells exclusively expressed either MITF or VSX2, with the latter developing as NR and the former adopting an RPE fate. However, when (R200Q)VSX2 hiPSCs were differentiated toward a retinal fate, VSX2 and MITF remained coexpressed as late as d18. Nuclear localization of b-catenin was robustly observed in VSX2+/MITF+ cells at d14 in (R200Q)VSX2 OV cells, and WLS and LEF1 displayed aberrant coexpression with VSX2 as well. However, once NR and RPE were established, differences in canonical WNT signaling were not detected between WT and mutant. WLS and LEF1 were observed in maturing RPE but not NR in both WT and mutant OVs, and nuclear localization of b-catenin was absent in both by d35.
We demonstrated that canonical WNT signaling proteins colocalized with MITF+ OV and RPE cells during early hiPSC-OV patterning, but were downregulated in NRPCs following expression of WT VSX2. Early NRPCs expressing mutant (R200Q)VSX2 displayed prolonged expression of MITF and WNT markers, along with nuclear localization of b-catenin. This data suggests that VSX2 DNA binding activity is associated with downregulation of canonical WNT signaling during NRPC specification.
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