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. 2021 Nov 15;10:e68584. doi: 10.7554/eLife.68584

Figure 2. Tiny limb staged regenerates have increased proliferation, decreased cell death, and smaller cell sizes than uninjured or completely regenerated limbs.

Transverse sections through the zeugopod of limbs at different stages of regeneration were analyzed for cell proliferation (A), cell death (B), cell size (C), and extracellular matrix (ECM) size (D). (A and B) Cell proliferation and death were analyzed in the epidermis, soft tissue, and skeletal elements. (A) Cell proliferation was analyzed by EdU labeling (n = 5). (B) Cell death was analyzed using TUNEL labeling (n = 4). (C and D) Cell and ECM size measurements were quantified in the epidermis, muscle, and skeletal elements. (C) Cell size was quantified using fluorescently tagged wheat germ agglutinin (plasma membrane) for epidermal and muscular analysis and Alcian blue staining (collagen) for skeletal analysis (n = 4). (D) ECM area was calculated by [(tissue area – cellular area)/tissue area] (n = 4). Error bars = SEM. p-Values calculated by ANOVA and the Tukey post hoc test. * = p < 0.05 ** = p < 0.005.

Figure 2.

Figure 2—figure supplement 1. Measurement of cell and extracellular matrix (ECM) size.

Figure 2—figure supplement 1.

Cell size and ECM area were quantified using 7 μm cross sections through the zeugopod. Skeletal tissue was analyzed using the histology stain of hematoxylin, eosin, and Alcian blue. The epidermis and muscle (represented) were analyzed using fluorescent stains of wheat germ agglutinin (WGA – magenta) and DAPI (blue). (A) Average cell size was quantified by measuring the area of nucleated cells. (B) Normalized ECM area was quantified by finding the sum of all the cellular area in a tissue. The sum was then subtracted from the total tissue area (red arrow indicating tissue boarder) and divided by the total tissue area to provide the normalized ECM area.