Figure 3. DNA damage delays migration and migrating stem cells with MCDD are more sensitive to ionising radiation.

(A) Experimental scheme showing worms pre-exposed to irradiation (5 and 10 Gy) followed by a shielded irradiation and amputation after 24 hr. Worms are fixed at 4 dpa and 6 dpa (dpa = days post-amputation). Box represents the migratory region, represented in the figure below. (B, C) FISH showing worms pre-exposed to IR (5 and 10 Gy) show delayed stem cell migration after 4 and 6 dpa. Dotted line represents anterior boundary of the shield. Scale bar: 350 μm. (D) Distance migrated by 10 most distant cells are counted from individual worms (n = 5 per condition). Results are expressed as mean ± SD. Statistical significance determined by multiple t-test using the Holm–Sidak method, *p<0.05. (E) Schematic of experimental set up to study sensitivity of migrating cells to IR. In addition to the initial shielded irradiation, the worms were irradiated with a low dose of IR (5 Gy, whole body) when MCDD is high (7 dpa) and are fixed after 24 hr to check the survival of the migratory stem cells to IR. (F–K) Representative smedwi-1 FISH showing migrating cells are more sensitive to IR than the cells in the shielded region. The region counted for analysis is marked with a box (bold: migratory region, dotted: shielded region). (n = 5 per condition, scale bar: 200 μm F, I; ;100 μm G, H, J, K). (L) Quantification of smedwi-1⁺ cells/mm² cells in the shielded region and in the migratory region. The decrease in cells/ mm² in the migratory field is significant compared to the decrease in the shielded region indicating that MCDD sensitises cells to IR. Cartoon showing the region counted for analysis. Each dot represents number of surviving cells from individual worms, n = 5. Statistical significance determined by two-way ANOVA using Tukey’s multiple comparison test (*p<0.05). (M) Distance migrated by stem cells showing that cells are more sensitive to low-dose IR the further they have migrated. Each dot represents the distance migrated by individual cells. Distance migrated by 11 most distant cells are counted from individual worms (n = 5 per condition). Results are expressed as mean ± SD (student’s t-test; *p<0.0001, ns = not significant).

Figure 3—figure supplement 1. Stem cells pre-loaded with damage before wounding show delays in migration.

(A) Representative FISH showing the distribution of stem cells in the shield after 5 and 10 Gy of IR followed by the shielded irradiation assay (3 day post-IR). [] brackets indicate the shielded area. Scale bar: 400 μm. (B) The stem cells can eventually migrate and rescue the whole animal. Representative FISH showing the stem cells migrate and reach the wound and rescue the worm (25 day post-IR). Scale bar: 400 μm. (C–P) Worms are irradiated with a low dose of IR (5 Gy) when cells start to migrate (4 dpa, C–H) and cells reach the wound (20 dpa, K–P). Worms are fixed after 24 hr to check the survival of the migratory stem cells. Representative smedwi-1 FISH showing the sensitivity of migrating cells and cells in the shielded region. The region counted for analysis is marked with a box (bold: migratory region, dotted: shielded region). (n = 5 per condition). (I) Distance migrated by stem cells suggests no significant sensitivity of stem cells to IR (at least 11 distant cells are counted from individual worms and plotted in the graph. Results are expressed as mean ± SD (p=0.48, ns = not significant, student’s t-test) n = 5 worms/condition. (J) Quantification of smedwi-1⁺ cells/mm² cells (yellow) in the shielded region and in the migratory region. Rate of decrease in the migratory field and in the shielded region is plotted in the graph. The region counted for analysis is marked with a box (bold: migratory region G, J, M, P; dotted: shielded region H, K, N, Q). Cells are counted by making a 300 × 300 box anterior to the shield (counted as migratory field) and 300 × 300 box posterior to the shield (counted as shielded region). Results are expressed as mean ± SD and Tukey’s multiple comparison test is used to check for significance. Each dot corresponds to smedwi-1⁺ cells/mm² in individual worm, n = 5, *p<0.05, ns = not significant).