A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage

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Abstract

The classical model of tissue renewal posits that small numbers of quiescent stem cells (SCs) give rise to proliferating transit-amplifying cells before terminal differentiation. However, many organs house pools of SCs with proliferative and differentiation potentials that diverge from this template. Resolving SC identity and organization is therefore central to understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNA-seq), mouse genetics and tissue injury approaches, we uncover cellular hierarchies and mechanisms that underlie the maintenance and repair of the continuously growing mouse incisor. Our results reveal that, during homeostasis, a group of actively cycling epithelial progenitors generates enamel-producing ameloblasts and adjacent layers of non-ameloblast cells. After injury, tissue repair was achieved through transient increases in progenitor-cell proliferation and through direct conversion of Notch1-expressing cells to ameloblasts. We elucidate epithelial SC identity, position and function, providing a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage.

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Fig. 1: Identification of distinct cell types by single-cell transcriptomic analysis and delineation of their spatial organization in the incisor epithelium.
Fig. 2: An unbiased catalogue of cell types during amelogenesis.
Fig. 3: Mapping of class 3 cell types to spatial regions of the incisor-epithelium growth region.
Fig. 4: Class 1 cells house progenitor cells that give rise to class 2 and class 3 populations
Fig. 5: Proliferation dynamics of the incisor epithelium growth region.
Fig. 6: Single-cell transcriptomic analysis reveals changes in cell cycle and differentiation program after injury.
Fig. 7: SI cells undergo direct conversion to ameloblasts to contribute to the recovery of injured incisor epithelium.

Data availability

The scRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE131204. Source data for figures are provided in Supplementary Table 11. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

The custom code developed for the whole-mount proliferation assay (Fig. 5d–f) is provided at https://github.com/kephale/incisor-cell-segmentation. The code for selected single cell RNAseq data analyses including SPRING visualizations is available as Jupyter notebooks at https:///www.github.com/rapolaszilionis/Sharir_et_al_2019.

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Acknowledgements

We thank A. Rathnayake, R. D’Urso, N. Wang, B. Hoehn and S. Alto for technical assistance, L. Jimenez-Rojo for mouse surgeries, K. Lindquist for consulting on GSEA analyses and J. Bush as well as members of the O.D.K. and A.M.K. laboratories for helpful discussion. This work was supported by the NIH (R35-DE026602 and R01-DE027620 to O.D.K). A.S. was funded by the NIH (K08-DE026219). J.H. was funded by the NIH (K99-DE025874). A.M.K. was funded by the NIH (5R33CA212697). UCSF Parnassus Flow Cytometry Core is supported by the NIH (P30DK063720 and 1S10OD021822-01). UCSF Core Center for Musculoskeletal Biology and Medicine is supported by the NIH (P30AR066262).

Author information

A.S. conceived the study, designed and performed experiments, and analysed data. P.M., D.C.-A., M.W., T.W., J.H., L.E., K.H, and P.P. performed experiments and analysed data. R.Z., K.K. and T.M. analysed data. C.W.S. contributed essential reagents. O.D.K. and A.M.K. directed the study and wrote the paper with A.S., P.M., R.Z., and J.H.

Correspondence to Allon M. Klein or Ophir D. Klein.

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Competing interests

A.M.K. is a founder of 1CellBio. C.W.S. is employed by Genentech.

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Integrated supplementary information

Supplementary Figure 1 The incisor epithelium is in steady state between 8 and 16 weeks of age.

a,b, In vivo micro-computed tomography (µCT) images of mouse hemi-mandibles immediately after a small notch was made in the left incisor (a) and 4 days after (b). Boxed areas (orange) are enlarged on the right. The change in distance between the notch and the tooth’s labial surface (red and green lines) corresponds to the amount of enamel growth in 4 days. Scale bars, 3 mm. c, µCT images showing the left hemi-mandible at different ages and the associated changes in size of the incisor growth region. Scale bars, 5 mm in left panels; 100 μm in right panels. d, Immunostaining for BrdU in the incisor growth region. Mice were dosed three times with BrdU over 3 days before harvesting to assess cell division. Nuclei were counterstained with DAPI. Scale bars, 100 μm. e, Schematic models showing broadly distributed cycling cells in the incisor growth region of young mice (<5 weeks, top) versus the restriction of cycling cells in the IEE and the adjacent SR in older mice (bottom). f, TUNEL staining revealed few (1-2 cells/section) apoptotic cells in cervical loops from 8 and 16 weeks old mice. Yellow arrowheads mark TUNEL signals. Scale bars, 100 μm. g, Quantification of the rate of incisor growth per week and the incisor growth region volume, both assessed by µCT. N=3 animals. h, Percentage of BrdU+ cells/section. Dashed lines outline epithelium. N=3 animals. Scatter plots indicate the mean of samples and error bars represent ± S.D. of the mean. Normally distributed data were analyzed using parametric tests including one-way ANOVA with Tukey–Kramer post hoc test. Significance was taken as P<0.05 with a confidence interval of 95%. * P< 0.05, ** P < 0.01, ** P < 0.01, *** P < 0.001.

Supplementary Figure 2 Previously reported markers are broadly expressed in the incisor growth region.

RNAscope in situ hybridization in mouse incisor growth region for Sox2 (a), Gli1 (d), Bmi1 (g) and Lrig1 (j); transcripts are visualized as red dots. Enlargements of the boxed insets from the dorsal (green) and ventral (orange) OEE, and the middle portion of the IEE (yellow) regions for each marker are shown on the right. Schematic view of the incisor growth region highlights the expression domains of the above genes. Sox2 (a’), Gli1 (d’) and Bmi1 (g’) are expressed throughout the proximal dental epithelium, including the OEE, IEE and the SR, whereas Lrig1 (j’) is present almost exclusively in the OEE and the OSR adjacent to the OEE. Representative images of RFP (Sox2, Lrig1 and Bmi1) or βgal (Gli1) immunostaining in the incisor growth region of Sox2CreER;R26tdTomato (b), Bmi1CreER;R26tdTomato (h), Lrig1CreER;R26tdTomato (k) and Gli1CreER;R26lacZ (e) mice 24 hours (top) or 21 days (bottom) after a single, low-dose tamoxifen injection. The initial labelling of all these markers was consistent with our in situ hybridization results, indicating that the progeny of Sox2, Gli1 and Bmi1 cells may originate from cells in any of the above regions. In contrast, cells labelled with Lrig1 were found exclusively in the OEE and condensed SR. Green, yellow and orange arrowheads in 24 hours chase mark cells in the dorsal, ventral OEE and IEE, respectively. Dashed lines outline incisor epithelium. SPRING plots of the entire control (top right) and of Class 1 after regressing out the cell cycle effect (right bottom), overlaid with gene expression of the above markers (Sox2 (c), Gli1 (f), Bmi1 (i) and Lrig1 (l); Color scale as in Fig. 1g. Scale bars, 100 μm.

Supplementary Figure 3 Extended analysis of the transcriptomic signatures of the incisor epithelium during homeostasis.

a, b, Flow cytometry gating used for sorting the control (a) and recovery (b) incisor epithelial cells. Experiment performed once. c, SPRING plot of the entire control dataset before filtering. d, Examples of genes specific to populations identified as mesenchymal (dark red) and immune (light blue) cells. e, SPRING plot of the control incisor epithelium dataset after removal of mesenchymal and immune cells. f, SPRING plots overlaid with gene expression patterns for the markers described in Fig. 1h. g, SPRING plots overlaid with gene expression patterns for the markers described in Fig. 2c–f. h, SPRING plots overlaid with gene expression patterns for the markers described in Fig. 3.

Supplementary Figure 4 Quantitative analysis of incisor epithelial kinetics using the TetOn-H2B-GFP system.

a, Frontal images and µCT rendering of the hemi-mandible from control (top), tetOff (middle) and tetON (bottom)-H2B-GFP mice. tetOff (middle) mice possessed chalky white incisors with blunted tips and missing enamel (yellow arrowhead), as compared to the yellow, sharp and intact incisors in controls (top) and TetOn-H2B-GFP (bottom) mice. µCT analysis shows that enamel is compromised (pink arrowheads) in the tetOff-H2B-GFP mice. Coronal sections at a region distal to the first molar [red dotted lines in (b)] show reduced density (blue arrowheads) and defective enamel (red arrowhead) in TetOff-H2B-GFP mice. Scale bars, 5 mm (in a- 3D),1 mm (in coronal section). b, Quantification of the incisor volume (top) and enamel density (bottom) assessed by µCT. N=3 animals. Bar charts indicate the mean of samples and error bars represent ± S.D. of the mean. Data were analyzed using unpaired t-test with Welch's correction. Significance was taken as P<0.05 with a confidence interval of 95%. * P< 0.05. c, Immunostaining for GFP in the incisor growth region. Prior to Dox induction, the incisor epithelium in TetOn-H2B-GFP is GFP-negative. Scale bar, 100 µm. d, Immunostaining for GFP in the incisor growth region of TetOn-H2B-GFP mice pulsed (Dox) between postnatal day (P) 2 and 4 and chased for 8 weeks. The OEE and the adjacent SR maintained high levels of GFP, which persisted for at least 8 weeks. Scale bars, 100 µm. e, Immunostaining for PAX7 (red) and GFP (green) in the anterior tibialis muscle of TetOn-H2B-GFP mice. Scale bars, 10 µm. f, Immunostaining for GFP in the incisor growth region of TetOn-H2B-GFP mice pulsed (Dox) for 21 days starting at 8 weeks of age and chased for 6 (top) and 12 (bottom) months. The IEE and the rest of the proliferating regions of the incisor epithelium are negative for GFP (yellow insets), while the lower regions of the OEE and the adjacent SR (orange insets) maintain high levels of GFP. The layers of cells ventral to the SI and the distal ameloblasts layer (pink insets) also contain GFP-positive cells. Scale bars, 100 µm.

Supplementary Figure 5 Extended analysis of the transcriptomic signatures of the incisor epithelium during recovery.

a, TUNEL (red) and EdU (green) staining revealed high levels of apoptotic cell death in the cycling regions of the incisor epithelium during 5FU treatment (day 2), while the non-cycling regions remained unaffected. Yellow (IEE) and orange (OEE) boxed area are enlarged on the bottom. Scale bars, 50 µm. b, c, SPRING plots combining control and recovering incisor epithelial cell dataset. Colors are the same as depicted in Fig. 4g, h. d, Top 20 enriched genes for each of the cell populations defined in the injury condition (top map) show similar expression patterns (annotated in doted green vertical lines) as their counterparts in control (bottom map). Cells are colored as in Fig. 1d for control (bottom right), and by classification results (see “Defining cell populations observed in single-cell RNAseq data” in methods) for injury (top right). e-g, SPRING plot shown in panel c with the two conditions displayed side-by-side and colored by gene expression of markers shown Fig. 4i-k: cycling cell markers Ccnb1 and Birc5, pre-ameloblasts markers Igfbpl1 and Ank2, and injury-enriched markers Sfrp5 and Cldn10. h, I, Equivalents of Supplementary Fig. 3e, f for Class 1 cells in injury.

Supplementary Figure 6 Examination of the effect of 5FU treatment on Notch signaling.

a, Notch1 activity as assessed by NICD immunostaining. Scale bars, 100 μm. b, RNAscope in situ hybridization shows changes in expression patterns of Ank2 (Class 2) and Ccnb1 (Class1) during recovery from 5FU. Scale bars, 100 μm. c, RNAscope in situ hybridization shows changes in expression patterns of Notch1 and target genes during recovery from 5FU. Scale bars, 100 μm. d, Heatmap showing the average expression of Notch signaling pathway components per Class 3 populations in control and recovery day 3 states.

Supplementary Figure 7 Assessment of the effect of 5FU treatment on Notch1-expressing cells.

a, Schematic of 5FU and tamoxifen (Tam) treatment schedule. Notch1CreER;R26tdTomato mice were dosed three times with Tam to lineage-trace Notch1-expressing cells and their descendants in steady state and during recovery from 5FU injury. b, Immunostaining for tdTomato (red) in control (left) and 5FU-treated (right two panels) mice analyzed 5 days and 21 days after the Tam induction. Scale bar,100 µm. c, Schematic of mechanical injury schedule. d, The distal portion of the incisor of Notch1CreER;R26mTmG mice was mechanically severed and the proximal region grown under the kidney capsule of a host mouse to lineage-trace Notch1-expressing cells and their descendants in steady state and during recovery from mechanical injury. Intact incisors were used as control. Bottom panel: Confocal images of control (left) and injured (right) mice analyzed 10 days after the treatment. Cells that underwent Cre recombination were permanently labeled with membrane GFP (mG). Very few cells reached the ameloblast layer in control (yellow arrowheads), while numerous traced cells reached the ameloblast layer during recovery from mechanical injury (yellow arrowheads). Scale bars,100 µm. e, TUNEL (red) and GFP (mG;green) staining revealed high levels of apoptotic cell death in the SI region of the incisor epithelium of Notch1CreER; RosaDTA/+ 2 days after TAM induction. Scale bar,100 µm. f, Schematic of the hierarchy of the three cell classes during homeostasis and injury repair. Actively cycling IEE cells (blue) generate both the enamel-producing ameloblasts (grey) and the adjacent non-ameloblast epithelial cells (orange). During injury repair, additional progenitors enter cell cycle, and SI cells are converted to become ameloblasts.

Supplementary information

Supplementary Information

Supplementary Figures 1–7, titles and legends for Supplementary tables.

Reporting Summary

Supplementary Table 1

Top 100 enriched genes per cluster in the control dataset. Fold changes are indicated for each gene, and all values are ordered per cluster.

Supplementary Table 2

Top 20 enriched genes in the control cells after the cell cycle effect was regressed out. Fold changes are indicated for each gene and all values are ordered per cluster.

Supplementary Table 3

Expression matrix of genes in the ameloblast trajectory.

Supplementary Table 4

Expression matrix of key markers used to identify cell populations in the non-ameloblast epithelium.

Supplementary Table 5

Summary of the GSEA for each cluster of the control dataset. Results are presented as individual tabs for each cluster.

Supplementary Table 6

Top 20 enriched genes in the clusters of the recovery dataset. Fold changes are indicated for each gene, and all values are ordered per cluster.

Supplementary Table 7

GSEA of control and recovery datasets.

Supplementary Table 8

Top 20 enriched genes in the recovering dataset after the cell cycle effect was regressed out. Fold changes are indicated for each gene and all values are ordered per cluster.

Supplementary Table 9

Expression matrix for genes of the Notch signaling pathway in control and recovery datasets.

Supplementary Table 10

Hyperlinks to Interactive explorers of resulting SPRING plots.

Supplementary Table 11

Source data.

Supplementary Video 1

Two-photon time-lapse live imaging shows Notch1-expressing cells in the ameloblast region of control explant.

Supplementary Video 2

Two-photon time-lapse live imaging shows Notch1-expressing cells in the ameloblast region of 5FU treated explant.

Supplementary Video 3

Two-photon time-lapse live imaging shows Notch1-expressing cells in the IEE of control explant.

Supplementary Video 4

Two-photon time-lapse live imaging shows Notch1-expressing cells in the IEE of 5FU treated explant.

Supplementary Note

Additional methods and theory.

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Sharir, A., Marangoni, P., Zilionis, R. et al. A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage. Nat Cell Biol 21, 1102–1112 (2019) doi:10.1038/s41556-019-0378-2

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