Fig 2: Inhibition of autophagy facilitates exit from pluripotency and drives hiPSC cells toward neuroectoderm. (A) AG27 iPSCs were treated or not with the autophagy inhibitors bafilomycin A1 (BafA1) or chloroquine (CQ) for 4 h before replacing with E8 medium. Representative images of the cell morphology at the 24 h time point are shown (n = 3). Scale bar: 50 µm. (B) AG27 iPSCs were treated as in (A) and processed for RTqPCR analysis at 24 h using TaqMan probes for major pluripotency genes (KLF4, SOX2, NANOG). Untreated iPSCs served as control. The graph shows the average of three independent experiments (n = 3). Error bars represent SD. **P < 0.01, ***P < 0.001. (C) AG27 iPSCs were treated as in (A) and processed for RTqPCR analysis at 24 h using an array card (Fig. S3A) to analyze the transcript levels of genes of various germ layers. The most significant changes in transcript levels are shown and graphs represent an average of three independent experiments (n = 3). Error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001. (D) AG27 iPSCs were treated or not with BafA1 or CQ for 24 h and then processed for RTqPCR analysis using an array card (Fig. S3A) to analyze the transcript levels of genes of various germ layers. The graph shows PAX6 expression levels and represents an average of three independent experiments (n = 3). Error bars represent SD. ***P < 0.001. (E) AG27 iPSCs cells were electroporated with guide RNA for ATG7 (Table 1). The tracr-RNA was fluorescently labeled with ATTO dye. Clones were screened using restriction fragment length polymorphism (RFLP). Cell lysates from wild type (WT) and ATG7± hiPSC clones were analyzed by immunoblotting against LC3B to confirm the effect of ATG7 depletion on LC3B lipidation (LC3B-I to LC3B-II conversion) (n = 3). (F) Cells from wild type (WT) and ATG7± hiPSC were differentiated into neuroectoderm using SMAD inhibitors. Lysate was collected daily until day 6 and processed for Western blotting (n = 3). ACTB was used as a loading control. (G) WT and ATG7+/ -AG27 cells were grown as single cells in a 90 mm dish in E8 medium and observed for morphological changes over 30 days post-seeding. Medium was changed every day. Representative images of three independent experiments, taken at different time-points post-seeding are shown. Scale bar: 50 µm. (H) AG27 WT (upper panels) and ATG7± cells (lower panels) were seeded and stained with PAX6 antibodies 3 days after seeding. Nuclei were stained with Hoechst. Images are representative of three independent experiments. Scale bar: 100 µm. (I) RNA was isolated from WT and ATG7±hiPSCs cells grown in cultures for 2 weeks and then analyzed by RTqPCR (TaqMan assay) against PAX6. The graph is a representative of three independent experiments (n = 3). Error bars represent SD. ***P < 0.001. (J) AG27 WT (upper four panels) or ATG7± cells (lower four panels) were grown in cultures for 2 weeks, then fixed and stained with antibodies against TUBB3 and LC3B and analyzed by confocal microscopy. Nuclei were stained with DAPI. Images are representative of three independent experiments. Scale bar: 50 µm.

Fig 3: K263E mutation and TDP-43 knock-down exert similar effects on gene expression. A, The domain map of TDP-43. The position of the K263E mutation is shown. NTD, N-terminal domain; RPM, RNA recognition motifs; IDR, intrinsically disordered regions; CR, conserved region. B, Genomic DNA sequencing of TARDBP isogenic iPSC lines. C, Bright-field images of wild-type and TARDBP isogenic iPSCs. Scale bar: 200 µm. D, Schematic of the differentiation of iPSCs into cortical neurons. E, Fold changes in the expression of OCT4 in iPSC, PAX6 in NPC, and TUBB3/FOXG1 in neuron cultures (normalized to ACTB; n = 3). F, Quantification of the number of TUBB3+ neurons at day 36 (n = 4). G, Volcano plot showing genes with significantly altered expression in cortical neurons derived from TDP-43K263E iPSCs relative to those from wild-type iPSCs. DEGs were identified using the Wald test with a cutoff of 0.05 for Benjamini–Hochberg adjusted p values and a cutoff of 0.25 for the log2 fold change ratio. Quality control of RNA-seq data are shown in Extended Data Figure 1-1. H, Volcano plots showing genes with significantly altered expression following TDP-43 knock-down in iPSC-derived neurons and SH-SY5Y cells. The published RNA-seq datasets (Klim et al., 2019; Melamed et al., 2019) were reanalyzed. I, PCA of RNA-seq datasets based on DEGs induced by TDP-43 knock-down. J, K, Scatter plot (J) and heatmap (K) comparing the fold changes in gene expression induced by TDP-43K263E and TDP-43 knock-down. Analyzed genes were selected as intersections between the DEG lists of two independent TDP-43 knock-down experiments from studies by Klim and colleagues and Melamed and colleagues without additional filters. Shaded areas indicate 95% confidence intervals. Pearson’s correlation coefficients were calculated after excluding the TARDBP values.

Fig 4: Expression levels of neurotrophins, neurotrophin-receptors and functional neuronal markers. Overall brain gene expression levels of A neurotrophins and B neurotrophin-receptors; C gene expression levels of glutamate transporters (EAAT2, VGLUT1) and GABAa1 subunit; D expression of glutamate decarboxylases GAD65 and GAD67 and F western blot analysis; E expression levels of GABA transporters (GAT-1, GAT-2, GAT-3, VGAT); G protein levels of beta-III-tubulin (TUBB3). Bar charts represent mean values + SEM obtained in two independent experiments and analyzed together, n = 4–5 per experiment, *p < 0.05, **p < 0.01 (Mann–Whitney U-test); Control (black bars) and PACAP-treated (white bars). Western blot membranes show two representative samples of each group, and bar charts represent mean values + SEM, n = 4

Fig 5: Neurogenesis in the lateral line placodes of Q-VD-OPh-treated mice. (A,C,F,G) Reconstructions of serially sectioned mouse embryos (section interval evaluated=10 µm) show ectoderm (light grey), otic vesicle with detachment site (dark grey), epibranchial placodes (orange), lateral line placodes (blue), immunopositive cells (brown dots), developing cranial nerves (partly numbered brown contours in G). (B,D,E) Micrographs taken from serially sectioned mouse embryos. (A) Sox2+ cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #021). (B) Sox2+ mantle (m) and support (s) cells in a neuromast primordium of a middle lateral line placode. Note the apical cavity (arrowhead). (C) Ngn1+ cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #039, mirror-imaged right side). (D) Ngn1+ neuroblasts in an anterodorsal lateral line placode. (E,F) Ngn2+ neuroblasts are present in epibranchial placodes, but absent from lateral line placodes (embryo #001). (G) Tubb3+ neurons constitute the anlagen of the cranial nerves 7 and 8 (VII/VIII), 9 (IX), 10 (X), 11 (XI) and 12 (XII). Tubb3+ candidates for vestigial lateralis ganglia (asterisks) reside in close proximity to the middle lateral line placode (embryo #014). All micrographs were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars: 5 µm. ad, m, p, anterodorsal, middle, and posterior lateral line placode, respectively; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; ot, otic anlage; ov, optic vesicle; Q-VD-OPh, pan-caspase inhibitor; wec, whole embryo culture.