Wounding increases nuclear ploidy in wound-proximal epidermal cells of the Drosophila pupal notum

Abstract

After injury, tissues must replace cell mass and genome copy number. The mitotic cycle is one mechanism for replacement, but non-mitotic strategies have been observed in quiescent tissues to restore tissue ploidy after wounding. Here we report that nuclei of the mitotically capable Drosophila pupal notum enlarged following nearby laser ablation. Measuring DNA content, we determined that nuclei within 100 µm of a laser-wound increased their ploidy to ~8C, consistent with one extra S-phase. These data indicate non-mitotic repair strategies are not exclusively utilized by quiescent tissues and may be an underexplored wound repair strategy in mitotic tissues.

Figure 1. Wounding affects the cell-cycle and ploidy in the Drosophila pupal notum

A: FUCCI fly indicating cell cycle state pre-wound, 3 hr, and 6 hr post wound in the Drosophila pupal notum (green nuclei = G1, red = S-phase, yellow = G2/M) Most nuclei are in G2 before wounding. Bar is 50 µm; inset bar in A ⁱⁱⁱ is 5 µm. B: Nuclear volume of G2 (yellow) FUCCI nuclei segmented in 3D using NIS elements from 3 independent samples. Mean and standard deviation shown (black bars), Mann-Whitney test indicates p < 0.0001 (****) between 0 min and 6 hr post wounding. C: DNA content of nuclei at 3 h after wounding. DAPI intensity was measured in 3D-segmented nuclei then normalized to DAPI intensity in haploid spermatids. Each dot represents a nucleus, and data from 4 biological replicates (wounded nota) are combined. Results are binned into 50 µm distances from wound center with n total nuclei within each bin shown. For reference, ploidy was measured in an unwounded control sample. Solid lines indicate the mean of each category, dotted line indicated 4C cutoff. A one-way ANOVA with multiple comparisons comparing the unwounded control to distance bins resulted in p < 0.0001 to each bin. D : Nuclei increase in DNA content and size closer to the wound. D ⁱ shows DAPI-stained nuclei 3 hr post-wounding. W=wound center. D ⁱⁱ shows DAPI-stained spermatids used to normalize DNA content. Both images are max intensity projections, preprocessed with rolling ball correction before projection, 19 Z-slices in D ⁱ and 24 Z-slices in D ⁱⁱ . Bar is 50 µm and applies to D ⁱ and D ⁱⁱ .