Fig 2: Gating strategy used to study neural progenitors by flow cytometrySVZs from 12 to 15 mice at P5 were obtained by dissection (Figures 1 and 2), pooled together, dissociated into single cells and then stained for flow cytometry.(A–C) Single, viable cells were identified using the strategy shown in the panels (A–C). First, cells were separated from debris using side-scatter (SSC-A) versus forward scatter (FSC-A) gating (A). From that gated population single cells were separated from doublets (SSC-H vs SSC-A) (B). Viable cells were then identified based on negative DAPI (or Live/Dead) staining (C).(E) From these viable cells a double gating strategy was used to classify subpopulations of SVZ neural progenitors. Cells were first characterized based on their expression of CD140a and NG2 (E).(D) Three subsets P1 (green), P2 (red) and P3 (yellow) were defined based on gates set using isotype controls (D).(F–I) Next, populations P1 to P3 were individually analyzed for expression of CD133 and LeX (F, H, I), where positively stained cells were, again, gated based on isotype controls (G). This strategy produced the following stem cell and progenitor populations: Within P1: MP2 (CD133+/Lex+), GRP2 (CD133-/Lex-) and BNAP/GRP1 (CD133-/Lex+) (I). Within P2, MP4 (CD133+/Lex+), GRP3 (CD133-/Lex-) and PFMP (CD133-/Lex+) (F). Within P3, NCS (CD133+/Lex+) and MP1 (CD133-/Lex+) (H). The full antigenic profile and frequency of each of these progenitors is provided in Table 1. Proliferative status was evaluated in each population by subsequently gating for EdU incorporation as shown in panel K for BNAP/GRP1s.(J) Cells unstained for EdU but stained for the other markers (EdU-control) were used to set the gate for EdU+ cells (J).

Fig 3: Survival and recurrence analysis based on the gene expression of CD29, FAP, and periostin in our HCC cohort and bioinformatics analysis of other CAF markers in the GEPIA online database. (A–L) Overall survival and disease-free rate based on the gene expression of CD29, FAP, and periostin in tumor tissue or tumor-free liver tissue of our HCC patients (Kaplan–Meier analysis, N = 75). (M) Bioinformatics analysis of PDGFRB, α-SMA, S100A4, COL1A1, PDGFRA, CXCL12, CAV1, and vimentin in the GEPIA database. The gene expression of these markers in tumor and normal liver tissue (CCA: n = 9 for normal liver tissue; n = 36 for tumor tissue; HCC: n = 160 for normal liver tissue; n = 369 for tumor tissue) was assessed by 1-way analysis of variance. Gene expression of these markers in different stages of liver tumors (CCA: n = 36; HCC: n = 369) was assessed by 1-way analysis of variance. The differences in survival related to CAF markers PDGFRB, α-SMA, FSP1, COL1A1, PDGFRA, CXCL12, CAV1, and vimentin messenger RNA expression were compared in each group involving all patients (Log-rank test, n = 36 for CCA, n = 364 for HCC). (-) Without a statistically significant difference. CAV1, caveolin 1; COL1A1, collagen type I α 1; CXCL12, C-X-C motif chemokine ligand 12; FSP1, fibroblast-specific protein 1; N, normal liver tissue; OS, overall survival; PDGFRB, platelet-derived growth factor receptor β; T, tumor tissue; TF, tumor free.

Fig 4: Bulk RNA-seq analysis of cells sorted by surface marker.a In situ hybridization of E16.5 mouse embryo showing dermis. eYFP, Pdgfra, and Thy1 signals were observed at dermis while only DAPI (blue color) was observed at epidermis. b Section staining of E16.5 mouse embryo showing dermis. eYFP, Pdgfra, and Crabp1 signals were observed at dermis while only DAPI (blue color) was observed at epidermis. c Section staining of E16.5 mouse embryo showing muscle fiber and brown adipose tissue. While DAPI and YFP signal were observed at both locations, muscle and brown adipose tissue were respectively stained by MF20 and Pparg antibodies. d In vitro culture assay of E14.5 YFP+ cells sorted by the combination of Pdgfra and Thy1 antibodies. Scale bar, 100 µm. e Bar plot of Oil Red O intensity between cultured samples. Student’s t-test was performed between two culture conditions. Values are means ± SEM, n = 3; ns not significant, ***P < 0.001. f Culture schematic and RT-qPCR result of E14.5 Pdgfra+/Thy1− cells (LP) before and after TGFβ1 ligand treatment. Two-way ANOVA was performed followed by Dunnett’s multiple comparisons test. Values are means ± SEM, n = 3; ns, not significant; **P < 0.01,***P < 0.001, ****P < 0.0001. g Venn diagrams of highly variable gene sets between sorted and cultured samples. Highly variable genes between two populations were selected. Key genes found in the corresponding sets are listed. Genes with normalized expression data > 0.3 were kept as set in the diagram.

Fig 5: β1‐tubulin expression in the developing thyroid and deleterious effect of TUBB1 mutations A TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with four tissues per stage. Results are reported as mean ± SEM. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differences.B Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5 and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with four tissues per stage. Results are reported as mean ± SEM. Statistical comparisons versus E13.5 (black), **P < 0.01 using ANOVA test.CSorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam‐positive cells), epithelial cells (EpCAM), fibroblasts (Pdgfra) and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to EpCAM‐positive cells at adult stage and peptidylprolyl isomerase A). Mean ± SEM of three independent cell‐sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *P < 0.05, ***P < 0.001 using ANOVA test.DImmunohistofluorescence of β1‐tubulin (in red), thyroglobulin (TG, in green) and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown below.EStructural representation of wild‐type and p.P160L mutated β1‐tubulin showing the change in conformation surrounding helix H4. Top: proline at position 160 in pink; bottom: substituting leucine at the same position in blue.FLocalization of transfected Myc‐tagged wild‐type and mutant (P160L) β1‐tubulin in Nthy cells. Immunostaining with anti‐Myc antibody (in green) and microtubule cytoskeleton stained with α‐tubulin antibody (in red). Note that β1‐tubulin ‐P160L is found throughout the cytoplasm showing puncta appearance, whereas wild‐type β1‐tubulin colocalizes with the microtubules.