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Parent stem cells can serve as niches for their daughter cells

Subjects

Abstract

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance1,2, while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation3,4,5,6. Recently, stem cells have been shown to regulate themselves using an autocrine mechanism7. The existence of a ‘stem cell niche’ was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells1. Since then, an increasing body of work has focused on defining stem cell niches1,2,3,4,5,6. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium, basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells8,9. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells9,10 . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly, this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation, lineage tracing and signalling pathway modulation, we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals, the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus, a parent stem/progenitor cell can serve as a functional daughter cell niche.

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Figure 1: Secretory progenitor cells differentiate into ciliated cells following basal stem/progenitor cell ablation.
Figure 2: Secretory progenitor cells show tonic Notch2 activity at steady state.
Figure 3: Tonic Notch2 activity is required to maintain secretory cells by preventing their differentiation into ciliated cells.
Figure 4: Basal cell Jagged 2 expression is required to maintain secretory progenitors and prevent their differentiation into ciliated cells.

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Acknowledgements

We thank A. Glick for providing the CK5rtTA mice, B. Hogan for providing CK5creER and SCGB1A1creER mice and Y.-Y. Kong for sharing the Mib1 floxed mice. B. Z. Stanger provided the RBPjk floxed mice and shared protocols for the immunohistochemical detection of Notch components. We also thank B. Stripp for providing the goat anti-SCGB1A1 antibody. We wish to extend our thanks to all of the members of the Rajagopal laboratory and the HSCI flow cytometry core facility. This research was supported by the New York Stem Cell Foundation (J.R. is a New York Stem Cell Foundation-Robertson Investigator), by a National Institutes of Health-National Heart, Lung, and Blood Institute Early Career Research New Faculty (P30) award (5P30HL101287-02), an RO1 (RO1HL118185) from NIH-NHLBI (to J.R.) and a Harvard Stem Cell Institute (HSCI) Junior Investigator Grant (to J.R.). J.R. is also the Maroni Research Scholar at Massachusetts General Hospital.

Author information

Authors and Affiliations

Authors

Contributions

A.P.-S. designed and performed the experiments and co-wrote the manuscript; P.R.T. performed the ablation experiments and edited the manuscript; B.M.L. optimized the immunodetection of N2ICD, analysed the phenotype of RBPjk and Mib1 deletion in vivo and co-wrote the manuscript; B.S. performed flow cytometry experiments and analysis, contributed to the in vitro experiments and edited the manuscript; R.D.-W.C. and M.P. helped with the analysis of the in vivo experiments; T.G. contributed to Jag2 deletion in vivo experiments; J.R. suggested and co-designed the study and co-wrote the manuscript.

Corresponding author

Correspondence to Jayaraj Rajagopal.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Parent stem/progenitor cells can serve as niches for their own daughter cells.

a, Schematic representation of the airway epithelial cell lineage. Basal stem/progenitor cells give rise to secretory progenitor cells that, in turn, give rise to terminally differentiated ciliated cells. b, Basal cells expressing Notch ligands provide a tonic forward Notch signal to neighbouring secretory daughter cells. Blocking this forward signal prevents Notch activation in secretory cells and results in their differentiation into ciliated cells. c, A schematic of the traditional arrangement of a stem cell that is maintained in a stem cell niche (left) and a schematic that further illustrates that stem cells can themselves serve as daughter cell niches, in which the parent stem cell itself is required for the maintenance of its own progeny (right). d, Schematic of the types of signals that occur between cells within a lineage and the theoretical modes of cell regulation that they imply. Blue arrows indicate a lineage relationship. Red arrows represent signals.

Extended Data Figure 2 Ablation of ciliated cells has no effect on airway cell proliferation, mesenchymal cell types, mesenchymal morphology and airway stem and progenitor cell replication over time.

a, Immunostaining for basal (CK5 (green)), ciliated (FOXJ1 (red)), and secretory cells (SCGB1A1 (white)) on either control (left panels) or tamoxifen (Tam)-treated FOXJ1-DTA mice (right panels) 5, 15, 30, 45, 60 and 150 days after ciliated cell ablation (n = 3 mice). b, Quantification of absolute cell numbers of basal CK5+ cells (top graph) and secretory SCGB1A1+ cells (bottom graph) per trachea on control (black bars) or Tam-treated (white bars) mice over time (n = 3 mice). c, Immunostaining for ciliated cells (FOXJ1 (green)) and proliferating cells (Ki67 (red)) on either control (upper panel) or tamoxifen (Tam)-treated FOXJ1-DTA mice (lower panel) (n = 6 mice). On the right, quantification of the percentage of Ki67+ cells per total DAPI+ cells in tracheal sections from control (C) or Tam-treated mice 3 days after cell ablation (n = 3 mice). d, Quantification of the percentage of proliferating Ki67+ cells relative to total DAPI+ cells in control (black bars) or Tam-treated (white bars) mice over time (n = 3 mice). e, Immunostaining for ciliated cells (AcTub (green)) and cells that have undergone proliferation (BrdU (red)) on either control (upper panel) or Tam-treated mice (lower panel) at day 3 (n = 6 mice). On the right, quantification of the percentage of BrdU+ cells per total DAPI+ cells in tracheal sections from control (C) or Tam treated mice (n = 3 mice). f, Haematoxylin & eosin (H&E) staining of tracheal sections 3 days after ciliated cell ablation. g, Immunostaining for CD45+ haematopoietic cells (left panels), CD31+ endothelial cells (middle panels) and SMA+ smooth muscle cells (right panels) (green) three days after cell ablation (n = 6 mice). Nuclei stained with DAPI (blue). The ns indicates that the cell number comparisons are not statistically significant. n = biological replicates/condition (two independent experiments). Data shown in the graphs are means ± s.e.m. Scale bar, 20 μm.

Extended Data Figure 3 Basal stem/progenitor cell ablation promotes the differentiation of secretory cells into ciliated cells without affecting the mesenchyme.

a, Immunostaining for YFP lineage label (green) and the ciliated cell marker c-MYB (red) in SCGB1A1-YFP; CK5-DTA mice (n = 3 mice). White arrowheads point to double positive cells. b, Immunostaining for YFP lineage label (green) and the ciliated cell marker AcTub (red) using SCGB1A1-YFP; CK5-DTA mice (n = 3 mice). c, Flow cytometry analysis for lineage-labelled YFP+ cells (x axis) and CD24+ ciliated cells (y axis) cells from control iPBS-treated or doxycycline-treated SCG1A1-YFP; CK5-DTA mice. d, Quantification of the percentage of FOXJ1+ cells per total DAPI+ cells in tracheal sections from control iPBS-treated or iDOX-treated SCG1A1-YFP; CK5-DTA mice (n = 3 mice). On the right, absolute numbers of FOXJ1+ cells per tracheal section (n = 3 mice). e, H&E staining of tracheal sections following basal cell ablation. f, Immunostaining for CD45+ haematopoietic cells (left panels), CD31+ endothelial cells (middle panels) and SMA+ smooth muscle cells (right panels) (green) in control or basal cell-ablated trachea (n = 3 mice). All analyses were performed 3 days after cell ablation. Nuclei stained with DAPI (blue). n = biological replicates/condition (two independent experiments). **P < 0.01. Data shown in the graphs are means ± s.e.m. Scale bar, 20 μm.

Extended Data Figure 4 Characterization of Notch pathway components in the steady-state murine tracheal epithelium.

a, Relative mRNA expression of Notch1, Notch3 and Notch4 assessed by qRT–PCR in pure sorted populations of airway epithelial cells (n = 3 mice). Relative expression is normalized to baseline transcript levels in secretory progenitor cells. b, Immunostaining for N1ICD (red) in combination with the basal cell marker p63 (top panel), the secretory cell marker SSEA-1 (middle panel) and the ciliated cell marker FOXJ1 (bottom panel) (green). c, Immunostaining for N3ICD (red) in combination with the basal cell marker podoplanin (PDPN) (top panel), the secretory cell marker SSEA-1 (middle panel) and the ciliated cell marker FOXJ1 (bottom panel) (green). d, Relative mRNA expression of Hes1, Hey1 and HeyL assessed by qRT–PCR in pure sorted populations of airway epithelial cells (n = 3 mice). Relative expression is normalized to baseline transcript levels in secretory progenitor cells. n = biological replicates/condition. **P < 0.01; ***P < 0.001. nd indicates lack of detection. Data shown in the graphs are means ± s.e.m. Nuclei stained with DAPI (blue). White arrowheads point to double positive cells. Scale bar, 20 μm.

Extended Data Figure 5 Downregulation of Notch signalling transduction following RBPjk deletion in secretory progenitor cells induces their conversion into ciliated cells.

a, Immunostaining for lineage-labelled YFP+ cells (green) in combination with RBPjk (red) in Tam-treated SCGB1A1-RBPjkfl/+ control mice (upper panels) and Tam-treated SCGB1A1-RBPjkfl/fl mice (lower panels). White arrowheads point to lineage-labelled RBPjk cells. The yellow arrows point to lineage-labelled cells that have not undergone recombination. b, Quantification of the percentage of RBPjk+ cells per total YFP+ cells at experimental day 15 following tamoxifen administration to SCGB1A1-RBPjkfl/+ control (black bar) and SCGB1A1-RBPjkfl/fl mice (white bar) (n = 6 mice). c, Relative mRNA expression of Notch signalling component genes (RBPjk, Hes1, HeyL) analysed by qRT–PCR in sorted YFP+ cells from Tam-treated SCGB1A1-RBPjk+/+ control mice (black bars) (n = 3 mice) and Tam-treated SCGB1A1-RBPjkfl/fl mice (white bars) (n = 4 mice). Relative expression is normalized to baseline transcript levels in YFP+ control cells. d, Immunostaining for YFP lineage label (green) and the secretory progenitor cell markers SCGB3A2 (left panels) and SSEA-1 (right panels) (red) in Tam-treated SCGB1A1-RBPjkfl/+ mice (control) (top panels) and SCGB1A1-RBPjkfl/fl mice (bottom panels). e, Immunostaining for YFP lineage label (green) and the ciliated cell markers AcTub (left panels) and c-MYB (right panels) (red) in Tam-treated SCGB1A1-RBPjkfl/+ mice (control) (top panels) and SCGB1A1-RBPjkfl/fl mice (bottom panels). White arrowheads point to lineage-labelled secretory cells that differentiated into ciliated cells following RBPjk deletion. f, Immunostaining for lineage-labelled YFP+ cells (green) and the basal cell marker CK5 (red) on either Tam-treated SCGB1A1-RBPjkfl/+ control mice (upper panel) or Tam-treated SCGB1A1-RBPjkfl/fl mice (lower panel). g, Quantification of the percentage of CK5+ cells per total YFP+ cells in Tam-treated SCGB1A1-RBPjkfl/fl mice compared to control mice. h, Flow cytometry analysis of EpCAM+ YFP+ CD24+ lineage-labelled ciliated cells and EpCAM+YFP+CD24 SSEA-1+ lineage-labelled secretory cells or EpCAM+YFP+CD24GSIβ4+ lineage-labelled basal cells in airways from either control or Tam-treated SCGB1A1-RBPjkfl/fl mice. i, Quantification of the percentage of epithelial (EpCAM+) lineage-labelled (YFP+) basal, secretory and ciliated cells in either Tam-treated SCGB1A1-RBPjk+/+ control or SCGB1A1-RBPjkfl/fl mice by flow cytometry (n = 3 mice). The analysis was performed 10 days after the last tamoxifen injection. Images are representative of n = 6 mice per condition (biological replicates) repeated three times. Nuclei stained with DAPI (blue). **P < 0.01; ***P < 0.001. Data shown in the graphs are means ± s.e.m. Scale bar, 20 μm.

Extended Data Figure 6 Lineage-labelled ciliated cells demonstrate long term persistence after RBPjk deletion without a change in epithelial cell proliferation and apoptosis.

ad, Immunostaining for the lineage label YFP (green) in combination with the secretory cell markers SCGB1A1 (a), SCGB3A2 (b) or the ciliated cell markers FOXJ1 (c) and AcTub (d) (red) on either Tam-treated SCGB1A1-RBPjkfl/+ control mice (upper panels) or Tam-treated SCGB1A1-RBPjkfl/fl mice (lower panels) 30 days after the last tamoxifen injection (n = 3 mice). White arrowheads point to lineage-labelled ciliated cells. e, Quantification of the percentage of each cell type per YFP+ cells on either control mice (black bars) or Tam-treated SCGB1A1-RBPjkfl/fl mice (white bars) at day 30. f, Quantification of the percentage of ciliated FOXJ1+ cells that incorporate BrdU after continuous BrdU administration to Tam-treated SCGB1A1-RBPjkfl/fl mice (n = 3 mice). g, Immunostaining for Ki67 (red) to assess overall proliferation in either Tam-treated SCGB1A1-RBPjkfl/+ control mice (upper panel) or Tam-treated SCGB1A1-RBPjkfl/fl mice (lower panel) (n = 3 mice). h, i, Immunostaining for FOXJ1 (green) and BrdU (red) in combination with YFP (cyan) (h) or alone (i) on Tam-treated SCGB1A1-RBPjkfl/fl mice that received continuous BrdU (n = 3 mice). j, Immunostaining to detect apoptotic cells by TUNEL assay (red) in combination with YFP lineage-labelled cells (green) in either Tam-treated SCGB1A1-RBPjkfl/+ control mice (upper panel) or Tam-treated SCGB1A1-RBPjkfl/fl mice (lower panel) (n = 3 mice). k, Immunostaining for activated caspase3 (green) in control and Tam-treated SCGB1A1-RBPjkfl/fl mice (n = 3 mice). fk, Analysis conducted 10 days after induction. Nuclei stained with DAPI (blue). n = biological replicates per condition. ***P < 0.001. Data shown in the graph are means ± s.e.m. Scale bar, 20 μm.

Extended Data Figure 7 Efficient deletion of Notch2 in secretory progenitor cells and its effect on cell type distribution.

a, Relative mRNA expression of Notch2 in YFP+ cells from Tam-treated SCGB1A1-Notch2+/+ control mice and Tam-treated SCGB1A1-Notch2fl/fl experimental mice assessed by qRT–PCR (n = 3 mice). b, Relative mRNA expression of the Notch target genes (Hes1, HeyL) in YFP+ cells from control mice and Tam-treated SCGB1A1-Notch2fl/fl experimental mice (n = 3 mice). Relative expression is normalized to baseline transcript levels in lineage-labelled YFP+ control cells. c, Immunostaining for lineage label YFP (green) in combination with N2ICD (red) on control mice (Tam-treated SCGB1A1-Notch2+/+) and experimental airways (Tam-treated SCGB1A1-Notch2 fl/fl). White arrowheads point to lineage-labelled cells that had lost Notch2 and therefore do not show N2ICD expression. d, Quantification of the percentage of N2ICD+ cells per total YFP+ cells in Tam-treated SCGB1A1-Notch2fl/fl mice compared to control (n = 7 mice). e, Immunostaining for YFP lineage label (green) and the secretory progenitor cell markers SCGB3A2 (left panels) and SSEA-1 (right panels) (red) in control (top panels) and experimental (bottom panels) mice. f, Immunostaining for YFP lineage label (green) and the ciliated cell markers AcTub (left panels) and c-MYB (right panels) (red) in control (top panels) and experimental (bottom panels) mice. White arrowheads point to lineage-labelled secretory cells that differentiated into ciliated cells following Notch2 deletion. Yellow arrows point to actual cilia (green) in lineage-labelled cells. g, Flow cytometry analysis of EpCAM+YFP+CD24+ lineage-labelled ciliated cells and EpCAM+ YFP+CD24SSEA-1+ lineage-labelled secretory cells or EpCAM+YFP+CD24GSIβ4+ lineage-labelled basal cells in airways from either Tam-treated SCGB1A1-Notch2+/+ control mice or Tam-treated SCGB1A1-Notch2fl/fl mice. h, Quantification of the percentage of epithelial (EpCAM+) lineage-labelled (YFP+) basal, secretory and ciliated cells in either Tam-treated SCGB1A1-Notch2+/+ control (n = 4 mice) or SCGB1A1-Notch2fl/fl mice (n = 6 mice) by flow cytometry. i, Immunostaining for the basal cell transcription factor p63 (red) on control or SCGB1A1-Notch2fl/fl airways. j, Quantification of the percentage of p63+ cells per total DAPI+ cells on tracheal sections from control or experimental mice (n = 7 mice). Analysis performed 10 days after induction. Images are representative of n = 7 mice per condition (biological replicates) repeated three times (three independent experiments). Nuclei stained with DAPI (blue). *P < 0.05; ***P < 0.001. Data shown in the graphs are means ± s.e.m. Scale bar, 20 μm.

Extended Data Figure 8 Proliferation and apoptosis following deletion of Notch2 in secretory progenitor cells.

a, Immunostaining for lineage label YFP (green), FOXJ1 (cyan) and N2ICD (red) in Tam-treated SCGB1A1-Notch2fl/fl mice. White arrowhead points to a lineage-labelled cell co-expressing markers for secretory and ciliated cell fates. The inset shows the single stain for FOXJ1 of the indicated region. b, Immunostaining for lineage label YFP (green), FOXJ1 (cyan) and SSEA-1 (red) in Tam-treated SCGB1A1-Notch2fl/fl mice. White arrowhead points to a lineage-labelled transitional cell. c, Immunostaining for BrdU (green), p63 (red) and Ki67 (cyan) to assess overall proliferation on either Tam-treated SCGB1A1-Notch2+/+ control mice (upper panels) or Tam-treated SCGB1A1-Notch2fl/fl mice (lower panels). d, Quantification of the percentage of ciliated FOXJ1+ cells that incorporate BrdU after continuous BrdU administration to Tam-treated SCGB1A1-Notch2fl/fl mice (n = 4 mice). e, Immunostaining for FOXJ1 (green) and BrdU (red) on Tam-treated SCGB1A1-Notch2fl/fl mice that received continuous BrdU (n = 4 mice). f, Immunostaining to detect apoptotic cells by TUNEL assay (green) on either Tam-treated SCGB1A1-Notch2+/+ control mice (upper panel) or Tam-treated SCGB1A1-Notch2fl/fl mice (lower panel). g, Immunostaining for YFP (green) in combination with activated caspase3 (red) on control mice (upper panel) or Tam-treated SCGB1A1-Notch2fl/fl mice (lower panel). Analysis performed 10 days after induction. Images are representative of n = 7 mice per condition (biological replicates) repeated three times (three independent experiments). Nuclei stained with DAPI (blue). Scale bar, 20 μm.

Extended Data Figure 9 Loss of Notch ligands in basal stem cells promotes secretory cell differentiation into ciliated cells without affecting proliferation or apoptosis.

a, Quantification of the percentage of basal PDPN+ cells that express Mib1 (left graph) on either Dox-treated CK5-Mib1+/+ control mice or Dox-treated CK5-Mib1fl/fl mice (n = 4 mice). Right graph, percentage of basal cells in which Mib1 was deleted in Dox-treated CK5-Mib1fl/fl mice (n = 4 mice). b, Immunostaining for Mib1 (red) and the basal cell marker CK5 (green). White arrowheads point to Mib1+ basal cells. c, Immunostaining for the secretory cell markers SCGB3A2 (left panels) and SSEA-1 (right panels) (red) in control (top panels) and experimental (bottom panels) mice. d, Immunostaining for the ciliated cell markers AcTub (left panels) and c-MYB (right panels) (green) in control (top panels) and experimental (bottom panels) mice. e, Flow cytometry analysis of EpCAM+ CD24+ ciliated cells and EpCAM+ SSEA-1+ secretory cells from control and experimental mice. f, Percentage of epithelial (EpCAM+) basal, secretory and ciliated cells on both groups by flow cytometry (n = 3 mice). g, Immunostaining for Ki67 (green) and the secretory cell marker SCGB1A1 (red) on control (top panel) or Dox-treated CK5-Mib1fl/fl mice (bottom panel). h, Immunostaining for BrdU (green) in combination with the basal cell transcription factor p63 (red) on both groups. i, Immunostaining for FOXJ1 (green) and BrdU (red) on Dox-treated CK5-Mib1fl/fl mice that received continuous BrdU. j, Percentage of ciliated FOXJ1+ cells that incorporate BrdU after continuous BrdU administration to Dox-treated CK5-Mib1fl/fl mice (n = 4 mice). k, Immunostaining to detect apoptotic cells by TUNEL assay (green) on either control (upper panel) or experimental mice (lower panel). l, Immunostaining for activated caspase3 (green) on both groups). m, Immunostaining for N2ICD (red), SCGB1A1 and SCGB3A2 (red), or FOXJ1 and AcTub (green) in control (top panels) or experimental mice (bottom panels) after five weeks of continuous doxycycline treatment (n = 4 mice). al, Analysis performed 2 weeks after the beginning of Dox induction. Images are representative of n = 4 mice per condition (biological replicates) repeated twice. *P < 0.05; ***P < 0.001. Data shown in the graphs are means ± s.e.m. Nuclei, DAPI (blue). Scale bar, 20 μm.

Extended Data Figure 10 Disruption of Jag2 in basal stem/progenitor cells causes the differentiation of secretory progenitor cells into ciliated cells without affecting proliferation or apoptosis.

a, Schematic representation of Jag2 inhibition using lentiviruses (LV) carrying shRNAs. Infected GFP+ cells were cultured in ALI culture system for 23 days, when they were collected, sorted and analysed. b, Relative mRNA expression of Jag2 in tracheal epithelial cells infected with mock vector (control) or with vectors carrying 4 different shRNAs targeting Jag2 72 h after infection. c, Relative mRNA expression of Jag2 in tracheal epithelial basal cells infected with mock vector (control) or with lentivirus targeting Jag2 (shJag2 877) after 23 days in ALI. d, Relative mRNA expression of the secretory genes (Scgb1a1 and Scgb3a2) and the ciliated cell genes (FoxJ1 and c-myb) in mock (black bars) and shJag2 877 (grey bars) infected cells 23 days after ALI initiation. Relative expression is normalized to baseline transcript levels in mock-infected cells. e, Relative mRNA expression of Jag2 on sorted recombined (YFP+) basal cells and unrecombined YFP basal cells from Tam-treated CK5-Jag2fl/fl mice (n = 3 mice). Relative expression is normalized to baseline transcript levels in YFP cells. f, Percentage of YFP+ cells per total DAPI+ cells (efficiency of recombination) on either Tam-treated CK5-Jag2+/+ control (black bars) or Tam-treated CK5-Jag2fl/fl (white bars) mice assessed by manual counting (left graph) (n = 5 mice) or by flow cytometry (right graph) (n = 3 mice). g, Immunostaining for SCGB3A2 (left panels) and SSEA-1 (right panels) (red) in combination with YFP (green) in control (top panels) and experimental (bottom panels) mice. h, Immunostaining for AcTub (left panels) and c-MYB (right panels) (red) in combination with YFP (green) in control (top panels) and experimental (bottom panels) mice. i, Flow cytometry analysis of EpCAM+CD24+ ciliated cells and EpCAM+SSEA-1+ secretory cells in control and experimental mice. j, Percentage of epithelial (EpCAM+) basal, secretory and ciliated cells from both groups assessed by flow cytometry (n = 3 mice). k, Immunostaining for p63 (red) on control (top panel) and experimental mice (bottom panel). l, Percentage of p63+ cells per total DAPI+ cells on both groups. m, Immunostaining for FOXJ1 (green), N2ICD (red) and SCGB1A1 (cyan). n, Immunostaining for BrdU (green), p63 (red) and Ki67 (cyan) in either control (upper panels) or experimental mice (lower panels). o, Percentage of ciliated FOXJ1+ cells that incorporate BrdU after continuous administration of BrdU to Tam-treated CK5-Jag2fl/fl mice (n = 3 mice). p, Immunostaining for FOXJ1 (green) and BrdU (red) on Tam-treated CK5-Jag2fl/fl mice that received continuous BrdU (n = 3 mice). q, Immunostaining to detect apoptotic cells by TUNEL assay (green) on both groups. r, Immunostaining for YFP (green) in combination with activated caspase3 (red) on control (upper panel) or experimental mice (lower panel). fr, Analysis performed 10 days after induction. Images are representative of n = 5 mice per condition (biological replicates) repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. Data shown in the graphs are means ± s.e.m. Nuclei, DAPI (blue). Scale bar, 20 μm.

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Pardo-Saganta, A., Tata, P., Law, B. et al. Parent stem cells can serve as niches for their daughter cells. Nature 523, 597–601 (2015). https://doi.org/10.1038/nature14553

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