Identification of TOR-responsive slow-cycling neoblasts in planarians

Abstract

Epimorphic regeneration commonly relies on the activation of reserved stem cells to drive new cell production. The planarian Schmidtea mediterranea is among the best regenerators in nature, thanks to its large population of adult stem cells, called neoblasts. While neoblasts have long been known to drive regeneration, whether a subset of neoblasts is reserved for this purpose is unknown. Here, we revisit the idea of reserved neoblasts by approaching neoblast heterogeneity from a regulatory perspective. By implementing a new fluorescence-activated cell sorting strategy in planarians, we identify a population of neoblasts defined by low transcriptional activity. These RNA^low neoblasts are relatively slow-cycling at homeostasis and undergo a morphological regeneration response characterized by cell growth at 48 h post-amputation. At this time, RNA^low neoblasts proliferate in a TOR-dependent manner. Additionally, knockdown of the tumour suppressor Lrig-1, which is enriched in RNA^low neoblasts, results in RNA^low neoblast growth and hyperproliferation at homeostasis, and ultimately delays regeneration. We propose that slow-cycling RNA^low neoblasts represent a regeneration-reserved neoblast population.

Keywords: TORC1; planarians; quiescence; regeneration; stem cells.

Figure 1. Planarian neoblasts have variable rates of division

1. Whole‐mount FISH for piwi‐1 and immunostaining for BrdU at 1‐day, 1‐week and 5‐weeks post‐BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.

2. Quantification of the percentage of BrdU label‐retaining neoblasts. Data are presented as mean ± s.d. (n = 4–6 animals per time point).

3. Double‐pulse experiment showing colocalization of BrdU after a 2‐week chase period with EdU after a 1‐day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.

Data information: All images are single confocal planes taken from the prepharyngeal region as indicated in the diagrams. Source data are available online for this figure.

Figure 2. Sorting neoblasts by DNA/RNA content reveals a relatively slow‐cycling population

1. Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNA^low population. Percentages indicate the proportion of events within each gate.

2. Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm.

3. EdU pulse‐chase time course of label retention in sorted RNA^low and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue P values indicate the statistical significance of EdU dilution from 2 to 14 dpp in S/G2/M or RNA^low neoblasts, respectively. Black P values indicate the statistical significance of EdU retention in RNA^low neoblasts compared to S/G2/M neoblasts.

4. Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label‐retention time course in (C). Red boxes denote examples of EdU⁻ cells. Scale bars, 5 µm.

Data information: Data in (B and C) are presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (P values are indicated; ns, not significant [P > 0.05]). Data are representative of ≥ 3 independent experiments.

Figure EV1. Backgating and RNA‐sequencing of DNA/RNA FACS gates

1. A

   Backgating of the RNA^low, G1, S/G2/M, and PM populations from Hoechst/pyronin Y stained cells (left) onto a classic Hoechst side population plot (middle, unirradiated; right, 1 day post 60 Gy irradiation). Plots are representative of n > 3 independent experiments.

2. B

   Expression of cell cycle genes in bulk RNAseq data of RNA^low, G1 and S/G2/M cells. Heatmap scale, fold change of log₂ normalized expression compared to S/G2/M.

3. C, D

   Expression of cell cycle genes in scRNAseq of 96 RNA^low neoblasts (C) or 96 previously sequenced X1 neoblasts (D, data ref: Molinaro & Pearson, 2016). Heatmap scale, log2 normalized expression level.

Data information: In (B–D), the cell cycle phase in which each gene primarily functions is indicated by the colour bar along the left side of each heatmap.

Figure 3. RNA^low neoblasts grow during regeneration

1. Area measurements of cells from the RNA^low, G1 and S/G2/M gates in intact animals and at 6 and 48 hpa (n ≥ 28 for each sample in the RNA^low plot, n ≥ 24 in the G1 plot, n ≥ 42 in the S/G2/M plot).

2. RNA^low neoblast size in intact animals and at 48, 72 and 96 hpa (n ≥ 54).

3. RNA^low neoblast size in intact animals and at 48 h following amputation, poke or incision injuries (n ≥ 61).

4. RNA^low neoblast size in unirradiated animals and at 1, 2 and 3 dpi (12.5 Gy. of irradiation, n ≥ 52).

Data information: Data are presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (P values are indicated; ns, not significant [P > 0.05]). Data are representative of three (A) or two (B–D) independent experiments.

Figure EV2. Gate depletion kinetics following sublethal irradiation

1. Quantification of the proportion of events within the RNA^low, G1 or S/G2/M FACS gates in unirradiated controls and at 1–3 dpi (12.5 Gy of irradiation, n = 3 biological replicates). Data are normalized to unirradiated controls and presented as mean ± s.d. A one‐way ANOVA was conducted to compare the depletion of each gate at a given time point. Gate depletion was not significant at 1 dpi (F(2,6) = 1.12, P = 0.38) or 2 dpi (F(2,6) = 4.31, P = 0.069). Gate depletion was significant at 3 dpi (F(2,6) = 6.94, P = 0.027). A post‐hoc Turkey HSD test identified a significant difference between the G1 and S/G2/M gates at 3 dpi (P = 0.027).

2. Representative FACS plots of the time course in (A). Plots are representative of n = 3 independent experiments.

Figure 4. RNA^low neoblasts extend projections following amputation

1. A

   Representative images of RNA^low, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Fig 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm.

2. B

   Average projection length in RNA^low, G1 and S/G2/M cells from intact animals and at 6 and 48 hpa (n ≥ 13).

3. C–E

   Quantification of the number of projections per cell (blue data) and the sum total projection length per cell (orange data) in RNA^low (C), G1 (D) or S/G2/M (E) cells isolated from intact animals and at 6 and 48 hpa (n ≥ 23 for projections/cell, n ≥ 10 for Σ projection length/cell).

Data information: Data are presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (P values are indicated; ns, not significant [P > 0.05]). Data are representative of two independent experiments.

Figure EV3. The RNA^low gate likely contains a mix of lineage‐committed and pluripotent neoblasts

1. A, B

   Expression levels of general and lineage‐committed neoblast markers in scRNAseq data of cells from the RNA^low gate (A) or previously sequenced X1 neoblasts (B, data ref: Molinaro & Pearson, 2016).Heatmap scale, log2 normalized expression level. Blue box, putative neural progenitor cluster; purple box, putative epithelial progenitor cluster.

2. C

   FACS plots showing TSPAN‐1 immunostaining of whole dissociated animals without (top) or with (bottom) anti‐TSPAN‐1 antibody.

3. D

   FACS plots showing TSPAN‐1 immunostaining of cells from the RNA^low, G1, S/G2/M, or PM gates.

Data information: In (C and D), plots are representative of n = 4 independent experiments. SSC, side scatter.

Figure 5. RNA^low neoblasts exhibit TOR‐dependent cell cycle entry during regeneration

1. Schematic of the experimental design.

2. FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment.

3. RNA^low neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55).

4. Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5–10).

5. RNA^low neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54).

6. FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.

Data information: Data in (C–E) are presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (P values are indicated; ns, not significant [P > 0.05]). In (C and D), animals were amputated at 5fd3. In (E), Animals were amputated at 7fd3. Data are representative of three (B) or two (C–F) independent experiments.

Figure EV4. fst and egfr‐3 are not required for the RNA^low neoblast growth response to injury

1. RNA^low neoblast size in intact animals and at 48‐hpa in control or fst KD animals (n ≥ 56).

2. RNA^low neoblast size in intact animals and at 48‐hpa in control or egfr‐3 KD animals (n ≥ 43).

Data information: Data presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (P values are indicated; ns, not significant [P > 0.05]). In (A), animals were amputated at 4fd3. In (B), animals were amputated at 8fd3. The data are representative of 3 (A) or 2 (B) independent experiments.

Figure 6. Lrig‐1 is required for sustained regeneration

1. Representative WISH images for Lrig‐1.

2. RNA^low neoblast size in intact animals following control or Lrig‐1 knockdown (n ≥ 53 technical replicates).

3. Quantification of H3P immunostaining in intact animals following control or Lrig‐1 knockdown (n = 9–10 technical replicates).

4. Quantification of H3P immunostaining at homeostasis (fixed at 7 days post‐RNAi, white region) and at 48 hpa (amputated at 5 days post‐RNAi and fixed at 7 days post‐RNAi, yellow region) during the first round of regeneration in control or Lrig‐1 KD animals (n = 10–17).

5. Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig‐1 KD animals from 2 to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all time points. Representative images from 7 dpa are shown. Scale bars, 500 µm.

6. Quantification of H3P immunostaining at homeostasis (fixed at 14 days post‐RNAi, white region) and at 48 hpa (amputated at 12 days post‐RNAi and fixed at 14 days post‐RNAi, yellow region) during a second round of regeneration in control or Lrig‐1 KD animals (n = 10–18).

7. Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig‐1 KD animals from 2 to 7 dpa during the second round of regeneration (n = 9–10 animals per condition per time point). The same animals were used for all time points. Representative images from 7 dpa are shown. Scale bars, 500 µm.

Data information: Data in (B–G) are presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (P values are indicated; ns, not significant [P > 0.05]). In (B and C), data were obtained at 5fd5. In (D–G), animals were initially amputated at 5fd5. Second amputations were performed at 7 dpa following the first amputation. Data are representative of three (B and C) or two (D‐G) independent experiments (with the exception of the homeostatic data in (D and F), for which a single experiment was performed). In (D–G), combined data from both independent experiments is shown.

Figure EV5. No effect of Lrig‐1 knockdown on differentiation at homeostasis

1. A–C

   Representative confocal images and quantifications of chat ⁺ neurons (A), piwi‐1 ⁺ neoblasts (B) and cell nuclei in the epidermis marked by DAPI (C) following control, ccng2, or Lrig‐1 knockdown at homeostasis (n = 8 technical replicates for all). Scale bars, 100 µm in (A); 250 µm in (B) (top panels) and 100 µm in (B) (bottom panels); 50 µm in (C).

Data information: Data presented as mean ± s.d. Statistical significance was assessed using Welch's t‐tests (ns, not significant [P > 0.05]). Animals were fixed at 5fd5. In (A and B), images are maximum projections and bottom panels are cross‐sections taken from the region indicated by the dashed white lines in the corresponding top panel. In (C), images are single confocal planes at the level of the epidermis. The data are representative of one independent experiment.

Figure 7. Proposed model of RNA^low neoblast regulation

At homeostasis, RNA^low neoblasts are relatively slow‐cycling and their size is restricted by Lrig‐1. During regeneration, RNA^low neoblasts grow, extend projections, and enter the cell cycle by 48 hpa in a TORC1‐dependent manner.