Drp1 embryos show delayed wound healing. (A–C) Maximum Z projections of the epidermis of control (A), Drp1 mild (B) and Drp1 strong (C) mutant embryos expressing an F-actin marker (GFP::Moesin) during wound closure. In Drp1 mild mutants (B) wounds close slower than in controls (compare B with A). In Drp1 strong mutants (C), although the wound contracts in the first 30–40 mpw, it then starts to expand (see 60–120 mpw). Later on, the wound contracts again and by 180 mpw it is almost closed. (D) Graph of average initial wound area in control and Drp1 mutant embryos (strong and mild). (E) Graph of wound closure time in control and Drp1 mutant embryos. Although the initial wound area of control and Drp1 mutants is similar (D), Drp1 mutants take longer to close their wounds (E). Unpaired t-test with Welch's correction was performed to test for significant differences between groups in D and E. ns, not significant (P>0.05), ****P≤0.0001. (F) Graph of average wound area in control, Drp1 mild and Drp1 strong mutants over time. Drp1 mild mutant wounds close slower than controls. Drp1 strong mutant wounds initially contract but start to expand after 40 mpw. At 120–130 mpw wounds start to contract again. (G) Graph of average wound area in control, Drp1-mild and Drp1-strong mutants in the first 30 mpw, corresponding to the grey region in F. Significant differences between control and Drp1 mutants start at 4 mpw in Drp1-mild mutants and at 10 mpw in Drp1-strong mutants. A two-way ANOVA with a Tukey's correction for multiple comparisons was used to test for significant differences between groups in G. Asterisks (*) refer to control and Drp1-mild mutants’ comparisons. Number signs (#) refer to control and Drp1-strong mutant comparisons. Dashed lines depict an interval of points in which the comparison between groups gives the same degree of statistical significance, given by the symbols above. #, P≤0.05, **; ##, P≤0.01; ***P≤0.001; ****P≤0.0001. Error bars represent s.e.m. Number of embryos per condition is shown in each graph. (H,I) Maximum Z projections of the epidermis of control (H) and Drp1-overexpressing (UAS-Drp1) (I) embryos expressing an F-actin marker (mCherry::Moesin) ubiquitously under the control of the da-Gal4 driver during wound closure. The wound-closure dynamics are similar between the two groups. (J) Graph of wound closure time in control and UAS-Drp1 embryos. Unpaired t-test with Welch's correction was performed to test for significant differences between groups. (K) Graph of average wound area in control and UAS-Drp1 embryos over time. No significant difference was found between control and Drp1-overexpressing embryos, neither in the time of wound closure nor the wound closure dynamics. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups. ns, not significant (P>0.05). Error bars represent s.e.m. Number of embryos per condition is shown in each graph. Scale bars: 20 µm.

Wounding does not induce major changes in mitochondrial morphology. (A–Bi) Maximum Z projections of the epidermis of control (A,Ai) and Drp1 (B,Bi) mutant embryos expressing ubiquitous mitochondrial (EYFP::mito, green) and membrane (PLCγPH::ChFP, magenta) markers. XZ and YZ sections are shown below and on the right, respectively. Insets show a zoom of the dashed region of the respective image. Scale bar: 10 µm. Inset scale bar: 5 µm. (C) Graph of average number of branches in control and Drp1 mutants, before and upon wounding. Control and Drp1 mutants show similar numbers of mitochondrial branches. Wounding leads to a reduction of branching in controls but not in Drp1 mutants. (D) Graph of average mitochondrial length in control and Drp1 mutants, before and upon wounding. Drp1 mutant mitochondrial network has increased length, compared to controls, both before and after wounding. Mitochondria from wounded epidermis show a similar length compared to unwounded, in both control and Drp1 mutants. A Mann-Whitney U test was used to test for significant differences between groups. ns, not significant (P>0.05); *P=0.0275; ***P=0.0001; ****P<0.0001. n(control)=22 cells from nine embryos, n(Drp1)=29 cells from 12 embryos. Error bars represent s.d., bw, before wounding; mpw, minutes post wounding.

Drp1 mutants show actin defects during wound closure. (A–D) Maximum Z projections of the epidermis of control (A,C) and Drp1 (B,D) mutant embryos expressing an F-actin (GFP::Moesin) (A,B) and a Myosin (Zip::GFP) (C,D) marker before and after wounding. Images are pseudo-colored with a gradient of fluorescence intensity, ranging from blue (low) to yellow (high). Although no differences between controls and Drp1 mutants are evident before wounding, Drp1 mutant embryos accumulate less F-actin at the wound edge than controls (compare A,B). Myosin accumulation at the wound edge seems similar between control and Drp1 mutant embryos (compare C,D). Scale bar: 20 µm. (E) Graph of average F-actin intensity at the cell cortex before wounding and at the wound edge. F-actin levels are significantly reduced in Drp1 mutants at 10 and 20 mpw. (F) Graph of average Myosin intensity at the cell cortex before wounding and at the wound edge. No significant differences were found between control and Drp1 mutants. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups in E and F. Only significant differences (P≤0.05) are represented. *P<0.05. Error bars represent s.e.m. Number of embryos per condition is shown in each graph. a.u., arbitrary units; bw, before wounding; mpw, minutes post wounding.

Drp1 mutants show altered cytosolic and mitochondrial Ca²⁺ dynamics. (A,B) Maximum Z projections of the epidermis of control (A) and Drp1 (B) mutant embryos expressing a cytosolic Ca²⁺ sensor (GCaMP6f) before and after wounding. Both control and Drp1 mutant cells around the wound dramatically increase cytosolic Ca²⁺ levels immediately upon wounding (0 mpw). Intensity returns to pre-wound levels after 15 min. Ca²⁺ levels and area of cells that respond to the wound are lower in Drp1 mutants (B, 0 mpw) compared to controls (A, 0 mpw). (C) Graph of cytosolic Ca²⁺ intensity shows that cytosolic Ca²⁺ is lower in Drp1 mutants compared to controls in the first 2.5 mpw. (D) Graph of average area of elevated cytosolic Ca²⁺ shows that the Ca²⁺ burst area is lower in Drp1 mutants compared to controls from 0 to 1 mpw. (E,F) Maximum Z projections of the epidermis of control (E) and Drp1 mutant (F) embryos expressing a mitochondrial Ca²⁺sensor (mito::GCaMP3) before and after wounding. Wounding triggers an increase in mitochondrial Ca²⁺ levels in both control and Drp1 mutant cells around the wound (E,F at 0 mpw). (G) Graph of mitochondrial Ca²⁺ intensity in control and Drp1 mutants. Drp1 mutants have a reduced mitochondrial Ca²⁺ burst at 0 mpw, compared to controls. (H) Graph of average area of elevated mitochondrial Ca²⁺ in controls and Drp1 mutant embryos. No significant differences were found between control and Drp1 mutants. Images are pseudo-colored with a gradient of fluorescence intensity, ranging from blue (low) to yellow (high). Dashed lines show the wound boundaries. Scale bar: 20 µm. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups in C, D, G and F. Only significant differences are represented: *P≤0.05, **P≤0.01, ****P≤0.0001. Error bars represent s.e.m. Number of embryos per condition is shown in each graph. bw, before wounding; mpw, minutes post wounding.

Drp1 mutant embryos show reduced mitochondrial ROS production upon wounding. (A,B) Maximum Z projections of the wound region of control (A) and Drp1-mutant (B) embryos expressing the mitochondrial ROS sensor MitoTimer ubiquitously, before and upon wounding (0 mpw). This reporter gene encodes a protein that irreversibly changes its fluorescence spectrum from green to red upon oxidation. Images show the green and red channels for each embryo, as well as the red:green ratio after image processing. Red:green ratio images are pseudo-colored with a gradient of fluorescence intensity, ranging from blue (low) to yellow (high). Scale bar: 10 µm. (C) Graph of the average red:green ratio of control and Drp1 mutant embryos, before and after wounding (0 mpw). Red:green ratio is a measure of ROS levels. Pre-wound ROS levels are similar between control and Drp1 mutants. Wounding increases ROS levels, both in controls and Drp1 mutants but this increase is lower in Drp1 mutant embryos compared to controls. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups. ns, not significant, *P=0.0170, **P=0.0049, ****P<0.0001. Error bars represent s.e.m. Number of embryos per condition is shown in the graph.