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. Author manuscript; available in PMC: 2020 Feb 11.
Published in final edited form as: Dev Cell. 2019 Jan 24;48(3):313–328.e8. doi: 10.1016/j.devcel.2018.12.018

Figure 2. MMP loss delays BM breaching time and decreases ECM removal.

Figure 2.

(A) Wild-type (left) and MMP- animals expressing laminin::GFP were scored for precise BM breaching time. Confocal imaging (ventral and lateral views of the same animal) at the early P6.p 4-cell stage show MMP- worm delayed in BM breaching. Arrows point to the BM breach (black area). The graph shows the percentage of AC invasion at each developmental time point (N ≥ 200 animals for each group, Table S1). Time after hatching at 20°C is shown. (B) 90 minute time-lapse of wild-type (top) and MMP- (bottom) animals shows BM removal after breaching is delayed in MMP- worms. Circular dotted lines show region of BM clearance at t=0. The graph shows quantification of the BM removal over time. Box plots show the average rates of BM clearing in wild-type animals (0.28 ± 0.08 μm2/min) and in MMP-mutants (0.09 ± 0.09 μm2/min, mean ± SD, p ≤ 0.01, Student’s t-test, n= 7 animals for wild-type: n=10 for MMP−). (C) Confocal sum projections of laminin::GFP (left panels) and collagen::mCh (right images) during AC invasion in wild type animals (upper) and in MMP- (lower) mutants show the progression of BM clearance by the AC. The fluorescence intensity of the BM at the perimeter of the cleared area (arrows) is increased in the absence of MMPs (~1.8 for laminin (1116 ± 352 vs 620 ± 75) and ~3.4 fold for collagen (1123 ± 426 vs 329 ± 279, mean ± SD, p≤0.01, Student’s t-test, wild-type, n=5 and 6; MMP-, n=9 and 5 for laminin::GFP and collagen::mCh, respectively)). (D) Schematic showing photoconversion of BM under the AC (green to red) before invasion. The amount of red BM physically displaced by the AC was calculated with post invasion images using regions 1–3 (see Methods). Grayscale (bottom, converted region is between red dotted lines) and spectral representation (top) of fluorescence intensity of optically converted laminin::Dendra show increased BM displacement in MMP- (right) animals (21 ± 0.09% in wild- type worms (left) versus 62 ± 35% in MMP- mutants, p≤0.05, mean ± SD, Student’s t-test, n=7 each group). Scale bars, 5μm.