Fig 2: Loss of PLK4 blocks centriole assembly in ependymal cells.(A) Quantification of the number of centrioles per cell in disengagement (D) phase. Plk4F/F or Plk4F/+ ependymal cells transduced with AAV-Cre-GFP were analyzed at differentiation days 7 and 8. N=3, n=20. Two-tailed Welch’s t-test. (B) Images of Plk4F/F or Plk4F/+ ependymal cells transduced with AAV-Cre-GFP. Cells were stained with DEUP1 (magenta) and CEP135 to mark centrioles (white). Scale bar=5 µm. (C) Quantification of the percent of FOXJ1+Plk4F/F or Plk4F/+ ependymal cells transduced with AAV-Cre-GFP that have multiple cilia at differentiation day 12. Acetylated tubulin staining was used to identify multiciliated cells. N=3, n>300. Two-tailed Welch’s t-test. (D) Confocal images of Plk4F/F or Plk4F/+ ependymal cells transduced with AAV-Cre-GFP. Cells were stained with antibodies against FOXJ1 (magenta) and acetylated-a-tubulin (white). Scale bar=5 µm. (E, F) Expansion microscopy of Plk4F/F or Plk4F/+ ependymal cells at differentiation day 5 transduced with AAV-Cre-GFP. Cells were stained with acetylated-a-tubulin (cyan) and DEUP1 (magenta). Scale bar=0.5 µM. Data information: All data represent the means ± SEM. **p<0.01; ****<0.0001. Figure 2—source data 1.Values for biological and technical replicates for graphs in Figure 2.

Fig 3: hfNPCs display proliferative potential and express canonical NPC markers in vitro. (A) Phase contrast image from hfNPCs as neurospheres-like cultures forming primary (black arrows) and secondary (white arrows) neurospheres as a hallmark of self-renewal in the presence of mitogens at passage 1. Scale bar = 200 µm. (B) Representative phase contrast image of hfNPC culture in adherent conditions in laminin-coated wells when cultured at passages 2–5 (Scale bar = 20 µm). (C) Doubling time (t) analysis of hfNPCs (black line; data presented as the mean ± SEM (n = 5 samples)) and one rat fetal NPC sample (red line) growing in adherent conditions over five passages. (D) The pinwheel cytoarchitecture (indicated with a white dotted line) of hfNPC at passage 1 from neurosphere-like cultures is highlighted after ß-catenin (red; delimiting the cell perimeter) and ?-tubulin (green; for cilia detection) staining (scale bar = 20 µm (left panel) and 10 µm (right panel)). (E) Representative images from nuclear staining of DAPI (blue), Ki67 (red), and BrdU (green) of hfNPCs in adherent conditions (scale bar = 10 µm). Right panel: Quantification of the percentage of positive cells for BrdU and Ki67. Data presented as the mean ± SEM (n = 3 samples). (F) Left panel: Representative images of immunofluorescent staining for Notch, Pax6, Nestin, Sox2, FoxJ1, DCX, and neurogenin (scale bar = 20 µm). Right panel: Quantification of the percentage of positive cells for each canonical neural progenitor cell marker. Data presented as the mean ± SEM (n = 5 samples).

Fig 4: Pre-existing SOX2+ Cells Are the Major Cellular Source of KRT5+ Cells after PR8 InfectionSox2-CreER, Rosa-mTmG mice treated with tamoxifen to lineage label airway epithelial cells were infected with PR8 influenza virus and killed 14 and 21 post infection. Photomicrographs (A–D) showing representative lung tissue sections immunostained for KRT5 (red), lineage markers (green), and DAPI (blue) (A–D). Scale bars, 100 µm. Scatterplots and pie charts show the quantitation and relative contribution, respectively, of HOPX, SFTPC, SCGB1A1, KRT5, and SOX2 cell lineages toward airway (E) and alveolar (F) KRT5+ cells after PR8 infection. Individual dots represent cells per animal (E) or cells per pod (F). Data include means ± SEM. Photomicrographs (G and H) show representative lung tissue sections from uninjured animals immunostained for KRT5, SCGB1A1, and FOXJ1 (red), lineage marker (green), and DAPI (blue). Arrows identify SOX2+ Lin- cells. At least three animals were analyzed per time point. See also Figures S1–S3 and Table S1.

Fig 5: PLK4 is required for multicliogenesis in vivo.Confocal images of the lateral ventricle from control or Plk4F/F;Usp28F/F;NestinCre mouse brains at P21. Sections were stained with antibodies against FOXJ1 (white), CEP164 (yellow), and acetylated-a-tubulin (magenta). Scale bar=5 µm.