Figure 5.
Tissue Stretching or Compaction Can Increase or Decrease ERK Activity Ectopically
(A, D, and E) Pupal notum expressing endo-Ecad::GFP and miniCic::mCherry during a spontaneous phase of tissue stretching orthogonal to the antero-posterior axis (A; blue arrows) or during stretching induced by laser wounding (E) schematic of the experiment in (D); orange arrows show tissue displacement; A, anterior right; P, Posterior left. From left to right and top to bottom: (1) miniCic::mCherry fluorescent signal; (2) inverted endo-Ecad::GFP; (3) overlay of compaction rate and PIV images averaged over 50 min (PIV biggest arrow: 5 μm/hr in A and 10 μm/hr in E; compaction rate scale bar −0.35 to 0.7 hr−1; blue, stretching; red, compaction); and (4) pseudo-image of the local cross-correlation value between compaction rate and miniCic signal derivative for a lag time of 0 min. The peak of correlation appears in the posterior central region (white region) in (A) and anterior to the wound in (E). Scale bars represent 20 μm.
(B and F) Representative plots of the compaction rate (blue) and miniCic intensity derivative (black) in a single PIV square (small red rectangles, A and E) during the spontaneous stretch (B) and during wounding (F). Insets, normalized cross-correlation of compaction rate versus the derivative of miniCic for those curves is shown.
(C and G) Mean normalized cross-correlation of compaction rate versus miniCic derivative during spontaneous stretching (C) or after wounding (G; red curves) from red square regions in (A) and (E) and control areas (in black) for several nota (n = 3) and subregions. Error bars indicate SEM.
(H) Snapshots of a cell in the stretched region anterior to the wound. Cell contour in blue shows corresponding nucleus with miniCic signal with white dotted circles. Scale bar represents 10 μm.
(I) Averaged normalized cell apical area and miniCic nuclear signal in the stretched region (n = 20 cells). Light colors: SEM. Inset: single curve for the cell shown in (H) is shown.
(J) Local projection of a live pupal notum expressing endo-Ecad::GFP (green) and miniCic::mCherry (magenta, bottom) at different times after laser sectioning of a square (blue) of 110 × 110 μm. Insets: miniCic signal from representative nuclei of the isolated piece of tissue is shown. Scale bar represents 10 μm.
(K) Segmentation of the isolated tissue prior and post-sectioning (apical area in pseudo-colors). Bottom right graph: average variation of cell apical area is shown (one dot per notum; [A final − A initial]/A initial).
(L) Averaged normalized miniCic intensity in the isolated tissue square (t0 = sectioning; 4 nota). Error bars indicate SEM.