FIGURE 2.

Altered proliferation pattern and dynamics of expression of Notch signaling operands HES5 and DLL1 during differentiation of RTT female patient-derived dorsal organoids. (A) Schematic representation of SOX2⁺ ventricular zone (VZ) and TUJ1⁺ neuronal layer measurement in cortical structures. For each cortical structure, three measurements were taken for the post-mitotic neuronal zone. (B) Representative images of the VZ delineated by white dashes and the neuronal layer containing TUJ1⁺ cells at day 41. Scale bars, 50 μm. (C) Mean value of the thicknesses of the neuronal zone layer (μm). Three measurements were performed for each zone in each structure. (n = independent experiments; healthy control: n = 10 and 19 cortical structures from at least 10 individual organoids; IC-MeCP2:R255X: n = 5 and 14 cortical structures from at least 10 individual organoids; MeCP2:R255X: n = 4 and 25 cortical structures from at least 10 individual organoids). (D) Representative immunofluorescence images of KI67 proliferative marker and TUJ1⁺ neuronal marker at day 41. Scale bars, 50 μm. (E) Quantification of the% of KI67⁺ cells as determined by flow cytometry analysis. (F) qRT-PCR analysis of the expression of (i) the Notch target and the transcription repressor of proneuronal genes HES5 (days 41) and (ii) the Notch ligand DLL1 (day 41). mRNA levels relative to GAPDH and normalized to the control condition. (n = independent experiments; WT: n = 6; IC-MeCP2:R255X: n = 4; MeCP2:R255X: n = 3). (G) Representative images of in situ hybridization of (i) HES5 and (ii) DLL1 as RNA probes and their co-localization with TUJ1 performed by Immunofluorescence. Scale bars, 50 μm. (H) Representative immunofluorescence images of HES5 staining and the co-localization with the progenitor marker SOX2. Scale bars, 10 μm. For all graphics depicted, Student’s t-test (two-tailed) statistics, was applied *p < 0.05, **p < 0.01, ***p < 0.001; error bars represent SEM.