Title: Interconversion of early VSX2WT and VSX2R200Q hiPSC-OV phenotypes by pharmacological manipulation of WNT signaling.
Legend: Pharmacological manipulation of the WNT signaling pathway during in vitro retinogenesis in hiPSCs affects the cell fate decision between retinal pigment epithelium (RPE) and retinal neurons (neuroretina). In wild type human iPS cells (hiPSCs), augmenting WNT signaling increases RPE at the expense of neuroretina while in hiPSCs derived from a microphthalmic patient (VSX2R200Q), which causes a neuroretina to RPE cell fate switch, inhibiting WNT signaling rescues the phenotype. Immunocytochemistry analysis on d18 VSX2WT hiPSC-OVs treated with the WNT agonist CHIR99201 (A-C) or vehicle (D-F) showing VSX2 (red) and MITF (green) coexpression (A-C) or lack thereof (D-F). RT-qPCR analysis of d30 VSX2WT hiPSC-OVs treated from d14-d20 with vehicle or CHIR99201 (G). Immunocytochemistry analysis on d18 VSX2R200Q hiPSC-OVs treated with the WNT inhibitor IWP2 (H-J) or vehicle (K-M) showing lack of coexpression (H-J) or coexpression (K-M) of VSX2 (red) and MITF (green). (N) RT-qPCR analysis of d30 VSX2R200Q hiPSC-OVs treated from d12-d20 with vehicle or inhibitor. Nuclei are shown in blue. *p< 0.01; **p<0.001; ***p<0.0001. Scale bars = 50 µm.
Citation: Capowski EE, Wright LS, Liang K, Phillips MJ, Wallace K, Petelinsek A, Hagstrom A, Pinilla I, Borys K, Lien J, Min JH, Keles S, Thomson JA, Gamm DM (2016). Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells. Stem Cells. In press. doi: 10.1002/stem.2414. [Epub ahead of print] PMID: 27301076
Abstract: Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP-seq and ChIP-PCR assays on early stage optic vesicle-like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC-OVs from a patient with a functional null mutation in VSX2 revealed mis-expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSC-OVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC-OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents.
About the Lab: Gamm’s laboratory at the Waisman Center utilizes stem cell technology to investigate the cellular and molecular events that occur during human retinal differentiation and generate cells for use in human retinal disease modeling and cell-based rescue or replacement strategies. To meet these goals, Gamm utilizes a variety of human cell types, including ES and iPS cells, which have the capacity to mimic retinal development and disease, as well as to delineate the genetic “checkpoints” necessary to produce particular retinal cell types. By understanding the behavior of these cell types in vitro and in vivo, Gamm hopes to optimize strategies to delay or reverse the effects of blinding disorders such as retinitis pigmentosa and age–related macular degeneration.