Fig. 4. TFEB does not regulate OPC differentiation into distinct MOL subpopulations during development and the OL lineage heterogeneity observed in vivo is not modeled in 3D culture conditions.

a, b Confocal representative images show that MOL2 (Klk6⁺-Aspa⁺ cells) and MOL5/6 (Ptgds⁺-Aspa⁺ cells) in the juvenile (P20) gray matter of the spinal cord of the Olig2::Cre⁻-TFEB^fl/fl (a) and Olig2::Cre⁺-TFEB^fl/fl (b) mice. Scale bar = 20 μm. c, e Quantification of the MOL2 (Klk6⁺-Aspa⁺ cells, c), and MOL5/6 (Ptgds⁺-Aspa⁺ cells, d) populations in the gray and white matter of the juvenile (P20) spinal cord, and summary table (e). Percentages of the populations are calculated on the total number of mature OLs (Aspa⁺ cells) in the analyzed region. Data are presented as mean ± SEM. n = 3−6 animals per genotype, and values of the individual data points are reported in the Source Data file. Asterisks indicate a significant difference between conditions (**p ≤ 0.01, ***p ≤ 0.0001, two-way ANOVA with Sidak’s correction). Exact p values are reported in the Source Data file. f, g Confocal representative images show the MBP⁺ and Ptgds⁺-MBP⁺ cells derived from brain (f) and spinal cord (g) OPCs cultured on microfibers for 3, 7, or 14 days of differentiation in vitro. Scale bar = 50 μm. h, i Quantification of the MBP⁺ (h) and Ptgds⁺/MBP⁺ (i) cells cultured on microfibers for 3, 7 or 14 days of differentiation in vitro. Percentages of the populations are calculated on the total number of nuclei (h) or MBP⁺ cells (i). Data are presented as mean ± SEM. n = 3−11 independent experiments. Each experiment was a biological replicate. Tissue from n = 4−10 P7 pups was used for each biological replicate. OPC oligodendrocyte progenitor cell, Ctrl Olig2::Cre⁻-TFEB^fl/fl, cKO Olig2::Cre⁺-TFEB^fl/fl, GM gray matter, WM white matter. j, k Representative images of MBP⁺ cell from brain OPCs 3 days in vitro with process extension on top of microfibers (j) and 14 days in vitro with some processes enwrapping microfibers (k). OPC oligodendrocyte progenitor cell. Source data are provided as a Source Data file.