Figure 6. Expression of ZIKV-NS2A, but not DENV-NS2A, reduces proliferation and disrupts adherens junction formation of ventricular radial glial cells in human forebrain organoids.

(A) Day 45 forebrain organoids were electroporated to express GFP, GFP and ZIKV-NS2A, or GFP and DENV-NS2A, and analyzed 3 days later (45+3) after pulsing with EdU (10 μM) for 1 hr. Sample confocal images for immunostaining for GFP and PAX6, and staining for EdU and DAPI are shown (top panels). Scale bar: 100 μm. Quantifications of percentages of EdU⁺GFP⁺ cells among all GFP⁺ cells, or GFP⁺PAX6⁺EdU⁺ cells among GFP⁺PAX6⁺ cells are also shown (bottom panels). Values represent mean + SEM (n = 10 organoids; ***: P < 0.001; Student’s t-test).

(B) Sample confocal images of immunostaining for GFP, PKCλ, and staining for DAPI are shown (left panels). Scale bar: 100 μm. Arrows point to regions with discontinuous AJ formation. Quantifications of AJ continuity based on PKCλ expression are also shown (right panel). Values represent mean + SEM (n = 9 organoids; **: P < 0.01; Student’s t-test).

(C) Expression of ZIKV-NS2A, but not DENV-NS2A, or GFP alone, led to a loss of typical radial glia morphology of PAX6⁺GFP⁺ cells at 7 days after electroporation (45+7). Sample confocal images of immunostaining for GFP and PAX6, and staining for DAPI (left panels; scale bar: 100 μm) and quantification of percentages of GFP⁺PAX6⁺ cells with multipolar morphologies are shown. Values represent mean + SEM (n = 7 organoids; **: P < 0.01; Student’s t-test).

Also see Figure S6.