Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells

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. Stem Cells 2016;34:2625-2634.

Keywords: Human induced pluripotent stem cells (hiPSCs); VSX2; WNT; optic vesicles.

Figure 1. VSX2 ChIP-seq and ChIP-PCR analyses demonstrating binding of VSX2 to WNT pathway genes in d30 VSX2^WT but not VSX2^R200Q hiPSC-OVs

Confirmation of the VSX2 consensus binding motif in VSX2^WT hiPSC-OV ChIP-seq target sequences (A). Distribution of high confidence VSX2 DNA binding targets in VSX2^WT hiPSC-OVs as categorized by genomic location (B) or RNA species (C). List of GO terms with greater than 3-fold enrichment generated via DAVID functional analysis of high confidence ChIP-seq peaks (D). Peak localization and genomic coverage maps (± 200 bp toward the 5' or 3' end) for WLS, AXIN2, and WNT1 (red and blue lines represent forward and reverse DNA strand reads, respectively). The top panel designates ChIP coverage and the lower panel shows the input control for each gene (E). VSX2 ChIP-PCR from VSX2^WT or VSX2^R200Q d30 hiPSC-OVs confirming direct binding of VSX2^WT, but not VSX2^R200Q, to targets identified proximal to the WNT signaling pathway genes WLS, AXIN2, and WNT1 (F). Regions in the MITF-H promoter previously shown by ChIP-PCR to be bound (+) or not bound (−) by VSX2 served as positive and negative controls, respectively (amplified from the same chromatin preparation presented in panel F) (G). See also Fig. S1.

Figure 2. Comparison of VSX2, MITF, and WLS localization in VSX2^WT and VSX2^R200Q hiPSC-OVs

D18 OVs from VSX2^WT (A–C) or VSX2^R200Q (D–F) hiPSCs were immunostained for VSX2 (red) and MITF (green) (A, D), MITF (red) and WLS (green) (B, E), or VSX2 (red) and WLS (green) (C, F). D35 and d50 VSX2^WT hiPSC-OVs (G–I) were examined for VSX2 and MITF (G,I) or VSX2 and WLS (H) expression and compared to d35 (J,K) and d50 (L) VSX2^R200Q hiPSC-OVs. Identical images with DAPI-labeled nuclei are shown in Fig. S2. Scale bars = 50 μm.

Figure 3. βCATENIN localization in d14 VSX2^WT and VSX2^R200Q hiPSC-OVs

D14 VSX2^WT (A–H) or VSX2^R200Q (I–P) hiPSC-OVs were immunolabeled with βCATENIN (green), VSX2 (red), and MITF (blue) primary antibodies. Examples of βCATENIN nuclear localization are designated by white arrows (E–H and M–P). Panels E–H and M–P are cropped magnifications of the outlined areas in panels A–D and I–J, respectively. Also see Fig. S3 for identical images with βCATENIN and DAPI-labeled nuclei. (Q) Graph of percent of βCATENIN nuclear localization in VSX2+ nuclei for WT and R200Q d14 hiPSC-OVs. **p=0.01. Scale bars = 50 μm.

Figure 4. Interconversion of early VSX2^WT and VSX2^R200Q hiPSC-OV phenotypes by pharmacological manipulation of WNT signaling

Immunocytochemistry analysis on d18 VSX2^WT 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 VSX2^WT hiPSC-OVs treated from d14–d20 with vehicle or CHIR99201 (G). Immunocytochemistry analysis on d18 VSX2^R200Q 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 VSX2^R200Q 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.

Figure 5. Schematic model depicting the impact of VSX2-mediated regulation of WNT signaling on early retinal patterning in wild-type hiPSC-OVs, and the consequence of the (R200Q)VSX2 mutation

VSX2^WT acts in multiple ways to maintain NR identity. On a gene expression level, VSX2 antagonizes expression of WNT pathway genes and MITF, resulting in the promotion of NR fate at the expense of RPE in hiPSC-OVs (A). In the absence of functional VSX2 (i.e. VSX2^R200Q hiPSC-OVs), expression of WNT pathway genes and MITF are unchecked, leading to RPE production over NR (B).