Figure 2. Migration-coupled DNA damage (MCDD) in stem cells.

Representative FISH showing stem cells (smedwi-1⁺) with extended protrusions in migratory cells (A) and stationary cells from the shielded region (B). Nuclei stained with Hoechst (blue). Images are shown as single confocal Z-stack (0.32 μm). (i–iv) is the single Z-stack from top to bottom. Scale bar: 5 μm. The cartoon explains the setup of shielded irradiation assay where a lead shield is placed in the middle and a lethal dose of 30 Gy is given to these worms. Cells under the shield (Yellow) are protected from IR and starts to migrate after amputation. (C) Quantification of nuclear aspect ratio (NAR) in the migratory cells compared to stationary cells (n = 28 cells in migratory region and n = 24 cells from the shield at 7 dpa/11 dpi [shielded irradiation assay]) (*p<0.0001. Student’s t-test). (D) Immunostaining with anti-PAR (magenta) in migrating stem cells (anti-TUD-1, yellow) after 0, 4, 7, and 10 days post-amputation showing MCDD in TUD-1⁺ migrating stem cells. Box denotes the field of cells imaged for analysis. Nuclei stained with Hoechst (cyan). Scale bar: 5 μm. White arrows denote increased nuclear PAR staining in Tud1⁺ stem cells at 7 dpa, and gray arrow denotes lack of PAR fluorescence in Tud1⁻ cells. Quantification showing the PAR fluorescence normalised to the nuclear area from Tud1⁺ stem cells (E) and post-mitotic differentiated Tud1⁻ cells (F) in the migrating region compared to stem cells in the shield. The measurement of PAR fluorescence is strictly nuclear and results are expressed as mean ± SD. (*p<0.0001; one-way ANOVA using Tukey’s multiple comparison test). Brightfield images of intact (G) and wounded (I) animals at 0 dpa and 7 dpa showing the amputated migratory region and shielded region. Dotted lines denote the position of the shield. The migratory region was used for smedwi-1 FISH and the corresponding shielded region was used for COMET assay and vice versa depending on the context (refer to Figure 2—figure supplement 3). (H) smedwi-1 FISH of the migratory tissues at 7 dpa showing the presence of migrating stem cells in wounded animals compared to no migration in intact animals. Cells corresponding to the shielded region were used for COMET assay to check for the extent of DNA damage. (J) smedwi-1 FISH of the shielded tissue at 7 dpa showing the presence of stem cells under the shield in intact and wounded animals. Cells corresponding to the migratory region from the animals were used for COMET assay to check for the extent of DNA damage in migrating stem cells in wounded animals. (K) Quantification of COMET assay showing the extent of DNA breaks in migrating cells in wounded animals compared to intact animals (absence of migrating stem cells). Each dot represents the percentage of tail DNA from single cells after COMET assay (n = 624 cells from intact animals, and 597 cells in wounded animals). Results are expressed as mean ± SD (student’s t-test; *p<0.0001).

Figure 2—figure supplement 1. Shielded irradiation assay to study stem cell migration and change in nuclear aspect ratio in migrating cells.

(A) Experimental set up showing the shielded irradiation assay (i), with a top view showing the position of the shield (ii) and focusing on the shield (iii). (B) Dose distribution across the lead strip showing greater than 95% attenuation of X-ray (Abnave et al., 2017). An exposure of 30 Gy corresponds to a dose to the cells directly above the shield-protected region of less than 1.5 Gy. (C) Representative FISH of smedwi-1 showing the distribution of stem cells (yellow) after shielded irradiation assay and the migration of stem cells from the shield after 7 day post-head amputation. Brackets ‘[]’ represents the position of the shield. Scale bar = 400 μm. (D) Representative smedwi-1 FISH showing survival of stem cell after 4 day post 1.5 Gy of IR. (E) Graph represents smedwi-1⁺ cells/mm² showed no significant difference in stem cell maintenance after 1.5 Gy IR. (n = 3, p=0.3148, Student’s t-test). (F–G) Representative FISH showing stem cells (smedwi-1⁺) with extended protrusions in migratory cells (F) and stationary cells from the shielded region (E). Nuclei stained with Hoechst (blue). Images are shown as single confocal Z-stack (0.32 μm). Number represents the NAR value of individual cells, plotted in Figure 2C. Scale bar = 5 μm.

Figure 2—figure supplement 2. Migration coupled DNA damage in stem cells.

(A) Maximum projection of individual nucleus showing the localisation of nuclear PAR (magenta) surrounded by perinuclear Tudor one staining (yellow). The nucleus is stained by Hoechst (cyan). Single confocal z stacks of the representative image clearly show the Tud-1 staining to be perinuclear and evidence of strong nuclear PAR expression in the nucleus (after IR exposure) in all the stacks. The nuclear outline is measured based on the Hoechst channel, and total PAR fluorescence was then measured and normalised to the area. Nuclei is stained with Hoechst (cyan), scale bar: 5 µm. (B) Zoomed out images of Tud-1⁺ stem cells showing expression of PAR in the migratory region as compared to shielded region (related to Figure 2D). (C) Measurement of DNA damage response in Tud-1⁺ stem cells with distance travelled during migration. PAR fluorescence in stem cells at a 4 dpa, 7 dpa, and 10 dpa in the shielded irradiation assay within 40 µm slices along the anterior posterior axis starting at the most anterior cell and going back towards the shield. We observed an increase in PAR fluorescence at 7 dpa as cells migrate further away from the shield. This increasing trend of damage along the gradient is not evident in the shielded region, or at 10 days when cells have reached the wound site and stoppe migrating. Each dot represents the average of the total number of cells present in each 40 µm section within each worm (n = 5).

Figure 2—figure supplement 3. COMET assay to detect DNA breaks during stem cell migration.

(A) A schematic showing the experimental strategy to perform COMET assay coupled with smedwi-1 FISH to accurately measure DNA breaks in shielded tissue and migratory tissue. Planarian worms were amputated (Wounded) 4 days after the shielded irradiation assay and at 7 day post-amputation (7 dpa/11 dpi) the shielded tissue and the migratory tissue were amputated. Individual migratory tissue fragments from intact and wounded animals were used for COMET assay and the corresponding shielded tissue were used for smedwi-1 FISH and vice verse. Each FISH is performed in three batches (InM1, InM2, InM3) with three worms per batch. The panels are named as ‘In’ or ‘Wo’ corresponding to Intact or Wounded. ‘M’ and ‘S’ correspond to Migratory tissue or Shielded tissue. The numbers denote different batch of experiment. The corresponding shielded tissue from these worms were pooled for COMET assay. (B, D) smedwi-1 FISH from migratory tissue of intact animals showing no migration compared to migrating smedwi-1⁺ stem cells in wounded animals (D). (C) Graph represents the percentage of DNA in tail from individual comets from cells in the shielded tissue from intact and wounded animals (300 comets were analysed per condition, n = 900 comets from intact animals and n = 900 from wounded animals). (E, G) smedwi-1 FISH from shielded tissue of intact (E) and wounded animals (G) showing the accuracy of cutting the migratory region. The tip of the tail was also amputated before the COMET assay/FISH to reduce the number of dead cells from the irradiated tissue. (F) Graph represents the percentage of DNA in the tail of individual comets from cells in the migratory tissue from intact and wounded animals showing an increase in DNA breaks in cells from the migratory region compared to the shielded region. Numbers in parenthesis depicts the total number of comets analysed. The data from (F) is used in Figure 2L.