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Identification of rare, transient post-mitotic cell states that are induced by injury and required for whole-body regeneration in Schmidtea mediterranea

Abstract

Regeneration requires the coordination of stem cells, their progeny and distant differentiated tissues. Here, we present a comprehensive atlas of whole-body regeneration in Schmidtea mediterranea and identify wound-induced cell states. An analysis of 299,998 single-cell transcriptomes captured from regeneration-competent and regeneration-incompetent fragments identified transient regeneration-activated cell states (TRACS) in the muscle, epidermis and intestine. TRACS were independent of stem cell division with distinct spatiotemporal distributions, and RNAi depletion of TRACS-enriched genes produced regeneration defects. Muscle expression of notum, follistatin, evi/wls, glypican-1 and junctophilin-1 was required for tissue polarity. Epidermal expression of agat-1/2/3, cyp3142a1, zfhx3 and atp1a1 was important for stem cell proliferation. Finally, expression of spectrinβ and atp12a in intestinal basal cells, and lrrk2, cathepsinB, myosin1e, polybromo-1 and talin-1 in intestinal enterocytes regulated stem cell proliferation and tissue remodelling, respectively. Our results identify cell types and molecules that are important for regeneration, indicating that regenerative ability can emerge from coordinated transcriptional plasticity across all three germ layers.

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Fig. 1: A single-cell reconstruction of successful and unsuccessful planarian regeneration.
Fig. 2: Regulators of tissue homeostasis were identified in irradiation-sensitive stem cell clusters.
Fig. 3: Amputation-specific clusters are non-uniformly distributed across tissue lineages.
Fig. 4: Five amputation-specific states were identified, visualized and quantified in the muscle, epidermis and intestine.
Fig. 5: Amputation-specific cell states arise from pre-existing post-mitotic cells.
Fig. 6: Muscle TRACS express genes that are required for tissue polarity.
Fig. 7: Epidermal TRACS express genes that are required for the regulation of stem cell proliferation.
Fig. 8: Intestinal TRACS express genes that are required for stem cell maintenance and tissue remodelling.

Data availability

All scRNA-seq data supporting the findings of this study have been deposited at the GEO under accession code GSE146685. scRNA-seq data can also be explored in our Shiny app at: https://simrcompbio.shinyapps.io/bbp_app/. Previously published sequencing data that were reanalysed here are available under accession codes GSE111764 (ref. 24) and GSE107874 (ref. 49). A list of SMEDIDs for all of the cloned genes is provided in Supplementary Table 15 and sequence information are available online (https://planosphere.stowers.org/find/genes). All other data supporting the findings of this study are available from the corresponding author on reasonable request or can be accessed from the Stowers Original Data Repository (http://www.stowers.org/research/publications/libpb-1513). Source data are provided with this paper.

Code availability

Original scripts used for the analysis and visualization of single-cell sequencing data are available at GitHub (https://github.com/0x644BE25/smedSPLiT-seq).

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Acknowledgements

We thank S. McKinney for assistance with automated confocal imaging; the members of the ASA laboratory for discussions and advice; and the members of the Stowers cytometry and molecular biology core facilities for technical contributions and methods development. A.S.A. is an investigator of the Howard Hughes Medical Institute (HHMI) and the Stowers Institute for Medical Research. B.W.B.-P. is a Jane Coffin Childs Memorial Fund Postdoctoral Fellow. F.G.M. is a HHMI Postdoctoral Fellow. This work was supported in part by NIH R37GM057260 to A.S.A.

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Authors

Contributions

Conceptualization and data interpretation: B.W.B.-P. and A.S.A. Data analysis: B.W.B.-P., C.E.B. and S.C. Acquisition of data: B.W.B.-P., A.M.K., A.R.S. and A.C.B. Cloning of planarian gene transcripts: F.G.M. Writing the original manuscript: B.W.B.-P. Supervision and funding acquisition: A.S.A. All of the authors revised and edited the manuscript.

Corresponding authors

Correspondence to Blair W. Benham-Pyle or Alejandro Sánchez Alvarado.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Identification of optimal biopsy sizes and treatments for single cell reconstruction.

(a) Schematic of experimental design. (b) Piwi-1 expression of parent animals at time biopsy was taken after irradiation treatment (right), as well as representative images 14 days post amputation (dpa), survival curves, and scoring of regeneration of photoreceptor pigmentation of biopsies 0.75mm–1.50mm taken from comparable parent animals following irradiation treatment. Notation on representative images indicates number of fragments that regenerated photoreceptors by 14dpa out of total surviving at 14dpa (exact n is provided in source data file). (c) Representative images of biopsies from un-irradiated, sub-lethally irradiated, and lethally irradiated animals imaged 1, 2, 4, 7, 10, and 14 days post amputation. Scale = 500µm.

Source data

Extended Data Fig. 2 Optimization and acquisition of a single cell reconstruction of planarian regeneration.

(a) Schematic of experimental design using Atto-conjugated linker molecules to visualize SPLiT-seq reagents after second [2] and third [3] round barcoding, and to detect biotin tagged molecules [4]. (b) Representative images of cells/objects detected in the Hoechst+ compartment at all steps of the barcoding process and area vs. Hoechst intensity plots with Hoechst+ cell compartment highlighted from each stage of barcoding. Note the accumulation of non-nucleated debris that occurs during the barcoding process that needed to be removed prior to sequencing. As a result, Hoechst+ intact cells were sorted following barcoding using the plot depicted in B [4] as a guide. Gating strategy utilized pre-barcoding (c) and post-barcoding (d). Abundance of sorted population pre-barcoding (e) and post-barcoding (f). (g) Number of cells captured, mean nUMI/cell, and mean nGene/cell for each of the 21 conditions. (h) UMAP embeddings cells sampled from each of the 21 conditions (see materials and methods for sub-sampling methodology) illustrating the change in tissue composition and captured transcriptional states across the dataset.

Extended Data Fig. 3 Annotation of muscle, epidermal, and intestinal tissue subclusters.

UMAP embedding of global dataset with tissue highlighted and UMAP embedding of tissue cells colored by tissue subcluster ID for muscle (a), epidermis (b), or intestine (c). Scaled mean expression of cluster enriched genes by tissue and tissue subcluster for muscle (d), epidermis (e), and intestine (f) enriched genes. Whole mount in situ hybridization of tissue markers analyzed in D (g), E (h), and F (i). Scale = 500µm.

Source data

Extended Data Fig. 4 Annotation of neural and parenchymal tissue subclusters.

UMAP embedding of global dataset with nervous system (a) or parenchyma (e) highlighted. UMAP embedding of neural (b) or parenchymal (f) cells colored by tissue subcluster ID. Scaled mean expression of cluster neural-enriched (c) or parenchymal-enriched (g) genes by tissue and tissue subcluster. Whole mount in situ hybridization of tissue markers analyzed in C (d) or G (h). Scale = 500µm.

Source data

Extended Data Fig. 5 Annotation of phagocytic, protonephridial, and pharyngeal tissue subclusters.

UMAP embedding of global dataset with tissue highlighted and UMAP embedding of tissue cells colored by tissue subcluster ID for phagocytic (a) and protonephridial (d) cells. Scaled mean expression of cluster enriched genes by tissue and tissue subcluster for phagocytic (b) and protonephridia (e) enriched genes. Whole mount in situ hybridization of tissue markers analyzed in B (c) and E (f). (g) UMAP embedding of global dataset with pharyngeal clusters highlighted. (h) UMAP embedding of pharyngeal cells. (i) Whole mount in situ hybridization of pharynx-enriched genes. Scale = 500µm.

Source data

Extended Data Fig. 6 Additional Data supporting tissue annotations and identification of TRACS.

(a) Tissue annotation prediction made using Fincher et al. tissue annotations transferred to SPLiT-seq dataset using Seurat’s TransferData function. (b) Proportion of cells from each global tissue cluster assigned to tissue lineages by TransferData. UMAP embedding of all neural (a), parenchymal (c), phagocytic (e), protonephridial (g), and pharyngeal (i) cells, colored by time. Scaled proportion of cells from each neural (b), parenchymal (d), phagocytic (f), protonephridial (h), or pharyngeal (j) subcluster across sampled conditions, normalized to the sample in which the subcluster had maximum representation.

Source data

Extended Data Fig. 7 Data supported the visualization and quantitation of TRACS.

(a) Selected images of whole mount double-fluorescent in situ hybridizations of wound-induced genes (yellow) and tissue-specific markers (magenta). (b) Schematic representation of TRACS quantitation strategy. (c) Selected images of whole mount in situ hybridization of piwi-1+ stem cells and immunohistochemistry of H3P+ mitotic cells. (d) Quantitation of density of H3P+ cells in control and colchicine-treated animals. n = 9 (0%), 7 (0.10%), 10 (0.15%), 9 (0.20%), and 9 (0.25%) biologically independent animals. Data are presented in box plots of min, max, and median, with the bounds of the box at the first and third quartile and whiskers extending from quartile to the minimum or maximum. P values are two-sided unpaired t-tests compared to 0%, with no corrections for multiple comparison. Scale = 500µm.

Source data

Extended Data Fig. 8 Additional data supporting wound-induced muscle cluster and M16-enriched genes requires for tissue maintenance and regeneration.

UMAP embedding of all muscle cells, colored by time after amputation and split by cells from biopsies taken from un-irradiated (a), sub-lethally irradiated (b), or lethally irradiated (c) animals. (d) Scaled proportion of cells from each muscle subcluster across sampled conditions, normalized to sample in which subcluster had maximum representation. (e) Scaled mean expression of muscle cluster 16 enriched genes (black arrow) by tissue, muscle subcluster, and sample. (f) Gene expression of screened muscle genes in bulk RNA-seq dataset of planarian regeneration63. (g) UMAP feature plots and gene expression patterns of grp78, CaATPase, and tubulin-β visualized by fluorescent in situ hybridization in intact animals and regenerating fragments 2 days post amputation. (h) Representative images RNAi-treated animals 3 days post feeding. (i) Survival of RNAi-treated animals shown in H (n = 20 for each condition). (j) Representative images of homeostatic (21 days post feeding, 10 animals) and regeneration phenotypes (14dpa, 20 animals) in unc-22 and tubulin-β RNAi-treated animals. (k) Gene expression patterns of M16-enriched genes visualized by fluorescent in situ hybridization in intact animals and regenerating fragments 2 days post amputation. P values are log-rank test (I) compared to unc-22 control. Scale = 500µm.

Source data

Extended Data Fig. 9 Additional data supporting amputation-specific epidermal clusters and epidermal genes requires for stem cell proliferation.

UMAP embedding of all epidermal cells, colored by time after amputation and split by cells from biopsies taken from un-irradiated (a), sub-lethally irradiated (b), or lethally irradiated (c) animals. (d) Scaled proportion of cells from each epidermal subcluster across sampled conditions, normalized to sample in which subcluster had maximum representation. (e) Scaled mean expression of E2/E20 enriched genes (black arrows) by tissue, muscle subcluster, and sample. (f) Gene expression of screened epidermal genes in bulk RNA-seq dataset of planarian regeneration63. (g) Gene expression patterns of epidermal genes visualized by fluorescent in situ hybridization in intact animals and regenerating fragments 2 days post amputation. Selected images of whole mount in situ hybridization of piwi-1+ stem cells and immunohistochemistry of H3P+ mitotic cells in RNAi-treated animals 7 days post feeding (h), or 7 days post amputation (j). H3P+ cell density in RNAi-treated animals 7 days post feeding (I) or 7 days post amputation (K). n (7dpf) = 9 (unc-22), 8 (agat-1), 9 (agat-3), 10 (agat-2), 6 (cyp3142a1), and 10 (atp1a1) biologically independent animals. n (7dpa) = 7 (unc-22), 11 (agat-1), 7 (zfhx3), 9 (agat-3), 10 (agat-2), 8 (cyp3142a1), and 7 (atp1a1) biologically independent animals. Data are presented in box plots of min, max, and median, with the bounds of the box at the first and third quartile and whiskers extending from quartile to the minimum or maximum. P values are two-sided unpaired t-tests with no corrections for multiple comparison compared to unc-22 control. Scale = 500µm.

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Extended Data Fig. 10 Additional data supporting amputation-induced intestinal clusters and intestinal genes required for tissue homeostasis and regeneration.

UMAP embedding of all intestinal cells, colored by time after amputation and split by cells from biopsies taken from un-irradiated (a), sub-lethally irradiated (b), or lethally irradiated (c) animals. (d) Scaled proportion of cells from each intestinal subcluster across sampled conditions, normalized to sample in which subcluster had maximum representation. (e) Scaled mean expression of I9/I12-enriched genes (black arrows) by tissue, muscle subcluster, and sample. (f) Gene expression of screened intestinal genes in bulk RNA-seq dataset of planarian regeneration63. (g) Gene expression patterns of intestinal genes visualized by fluorescent in situ hybridization in intact animals and regenerating fragments 2 days post amputation. (H) Selected images of whole mount in situ hybridization of piwi-1+ stem cells and immunohistochemistry of H3P+ mitotic cells in RNAi-treated animals 2 days post amputation. (i) Selected images and raw x,y position of notum+ anterior pole cells visualized by whole mount in situ hybridization. (j) H3P+ cell density in RNAi-treated animals 2 days post amputation. n = 8 (unc-22), 9 (lrkk2), 8 (cathepsinB), 7 (myosin1e), 8 (polybromo-1), and 8 (talin-1) biologically independent animals. (k) Number of notum+ cells in RNAi treated animals. n = 4 (unc-22), 5 (lrrk2), 4 (CathepsinB), 3 (myosin1e); 4 (polybromo-1) and 6 (talin-1) biologically independent animals. (l) Distribution of notum+ cells in RNAi-treated animals, n = 152 (unc-22), 180 (lrrk2), 125 (cathepsinB), 92 (myosin1e); 88 (polybromo-1), and 180 (talin-1) notum+ cells. P values are a two-sided unpaired t-tests, with no adjustments for multiple comparisons. Data are presented in box plots of min, max, and median, with the bounds of the box at the first and third quartile and whiskers extending from quartile to the minimum or maximum (j) or as mean values +/- SD (k,l). Scale = 500µm (G,H) or 150µm (I).

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Supplementary Tables 1–17

Supplementary Table 1: scRNA-seq metadata. Description of single cells and associated metadata. Supplementary Table 2: global cluster markers. Global cluster enriched genes compared to the full dataset. Supplementary Table 3: metrics for tissue subclustering. Global clusters and the number of principles components used to make tissue subsets. Supplementary Table 4: tissue subcluster markers. Tissue subcluster enriched genes compared to the full dataset. Supplementary Table 5: epidermal markers. Epidermal subcluster enriched genes. Supplementary Table 6: intestinal markers. Intestinal subcluster enriched genes. Supplementary Table 7: muscle markers. Muscle subcluster enriched genes. Supplementary Table 8: neural markers. Neural subcluster enriched genes. Supplementary Table 9: parenchymal markers. Parenchymal subcluster enriched genes. Supplementary Table 10: phagocytic subcluster enriched genes. Supplementary Table 11: pharyngeal markers. Pharynx subcluster enriched genes. Supplementary Table 12: protonephridia markers. Protonephridial subcluster enriched genes. Supplementary Table 13: stem cell markers. Stem cell subcluster enriched genes. Supplementary Table 14: markers of non-differentiated cells. Non-differentiated subcluster enriched genes. Supplementary Table 15: identification of TRACS clusters. Table containing the raw cell counts for each tissue subcluster across all of the samples, as well as early time point bias and early-bias ranking for each subcluster. Supplementary Table 16: details of the primary RNAi screen. Transcript IDs, annotation (if applicable) and RNAi phenotypes of all of the transcripts that were functionally tested in the primary RNAi screen. Supplementary Table 17: details of the secondary RNAi screen. Transcript IDs, annotation (if applicable) and RNAi phenotypes of all of the transcripts that were functionally tested in the secondary RNAi screen.

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Benham-Pyle, B.W., Brewster, C.E., Kent, A.M. et al. Identification of rare, transient post-mitotic cell states that are induced by injury and required for whole-body regeneration in Schmidtea mediterranea. Nat Cell Biol 23, 939–952 (2021). https://doi.org/10.1038/s41556-021-00734-6

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