Fig. 6: Optogenetic spatial modulation of polarized WNT3A organizing centers in 2D and 3D cultures.

A Engineering of HEK-293 cells for light-controlled expression of Wnt3a combined with chemically-inducible Cdh3 expression (HEK^Cdh3-OptoWnt cells). The engineered cell lines harbor four genomic constructs mediating the tetracycline-controlled expression of the mouse endothelial cadherin Cdh3 for clustering and mCherry (top), and optogenetically-regulated expression and secretion of mouse Wnt3a using either a blue-light or red-light optogenetic gene-switch. B Quantitative RT-PCR analysis of optogenetic Wnt3a expression in HEK^Cdh3-OptoWnt cells. HEK^Cdh3 cells with tetracycline-inducible Cdh3 expression were further engineered for blue or red light-inducible expression of Wnt3a using the BLUE_SINGLE or RED_E system, respectively. Cells were illuminated with the corresponding wavelengths for 24 h prior to RNA extraction and qPCR analysis. Data represent mean values with one standard deviation of three technical replicates compared with two-sided independent Student’s t-tests. ns, 5e-2 <p ≤ 1; **, 1e-3 <p ≤ 1e-2; ***, 1e-4 <p ≤ 1e-3. p-values: BLUE-Tet, 2.407e-4; BLUE+Tet, 1.287e-3; RED-Tet, 2.163e-3; RED+Tet, 6.885e-2. Source data are provided in this paper. C Optogenetic WNT3A-signaling across cell populations. HEK^Cdh3-OptoWnt cells were induced with tetracycline for endothelial cadherin-mediated self-clustering and mCherry expression and mixed with TOP-GFP sensor cells for WNT3A perception and detection via EGFP expression. Cell cultures were illuminated for 24 h with 10 µmol m⁻² s⁻¹ blue light (overall illumination) using LEDs, or kept in darkness. Representative images of four experiments are shown. Scale bar, 100 µm. D Three-dimensional optogenetic WNT3A organizing centers formed from HEK^Cdh3-OptoWnt cells inducing WNT3A-signaling in proximal TOP-GFP spheroids. Assembloids were formed from 3000 or 6000 tetracycline-induced HEK^Cdh3-OptoWnt cells and the same number of TOP-GFP cells were illuminated with 20 µmol m⁻² s⁻¹ blue light (overall illumination) using LEDs for 30 h, or kept in darkness. Representative images of one out of three similar experiments with 18 illuminated and four dark-incubated samples are shown. Scale bar, 100 µm. E Spatial induction of WNT3A signaling in a two-dimensional HEK^Cdh3-OptoWnt and TOP-GFP co-culture using a photomask. Experiment as in (C) but the cells were illuminated from below through a 3D-printed photomask covering half of the sample area. A representative area of the acquired stitched wide-field region of a single experiment is shown. Scale bar, 100 µm. F Selective patterned activation of a distinct 3D optogenetic WNT3A organizing center and signaling perception by a TOP-GFP spheroid assembled with multiple organizers. Experiment as in (D) but using 2× 4000 HEK^Cdh3-OptoWnt and 8000 TOP-GFP cells and digital mirror-controlled 450 nm blue light illumination at the microscope. A 4X objective was used for the initial image and pattern projection of a single selected sample which successfully formed a tripartite assembloid. The final image was acquired using a 10X objective. Scale bar, 100 µm. A, B Tet, tetracycline.