Figure 1.
Schematic of the morphometric analysis.
l = length of the wound (blue solid line); le = length of the epidermis (yellow solid line); ae = area of the epidermis (black dotted line); aw = area of the granulation tissue (green solid line); wd = wound depth (blue dotted line); d = distance between two sections (black dotted line). Note that in case of closed wound, le = l; in case of non-epithelialized wounds (this example), le<l. In this example, every 40 sections were analyzed (see numbers on the top right corner of each picture), so d = 280 µm.
Figure 2.
Tgfß3-Cre induced recombination in the suprabasal layers of the epidermis during wound healing.
Six-mm excisional punch wounds sections were performed in Tgfb3-Cre;R26R-LacZ (a–d, f, g) or wildtype animals (e) and harvested 4 (a, d), 7 (b, e) and 11 (c) days postwounding. Tissue sections were stained for X-gal (a–c, e–g) or incubated in PBS control (d). Black arrow in (a) indicates the leading edge of the migrating keratinocytes. Note the presence of X-gal staining in the inner root sheath of the hair follicle (f) and in the subrabasal layer of the epidermis (g). Scale bar (a–e) = 100 µm; scale bar (f, g) = 50 µm.
Figure 3.
Macroscopic photomicrographs of excisional wounds.
Six-mm excisional punch wounds were performed on the back of wild type mice. One day later, wounds were treated with saline (a, e, i), TGF-ß3 and neutralizing antibody (NAB) against TGF-ß3 (b, f, j), TGF-ß3 (c, g, k), and NAB (d, h, l). Wounds were harvested 4 days (a–d), 7 days (e–h) and 11 days (i–l) post-wounding.
Figure 4.
Histological features of excisional wounds.
Hematoxylin and eosin staining of the section in the middle of the wound is shown as representative of each treatment group (saline, a, e, i; TGF-ß3+NAB, b, f, j; TGF-ß3, c, g, k; NAB, d, h, l) and time points (4 days post-wounding, a–d; 7 days post-wounding, e–h; 11 days post-wounding, i–l). Only the middle of the wound of each section is shown. Scale bar = 500 µm.
Figure 5.
TGF-ß3 is required for proper wound closure in vivo.
Morphometric analysis of serial histological sections and calculation of the total wound area (a), the epidermal area (b) and the percentage of wound closure (c). N = 6−8 per group. *P<0.05, ***P<0.001.
Table 1.
Distance between the edges of the panniculus carnosum.
Figure 6.
TGF-ß3 is required for proper keratinocyte migration and proliferation in vivo.
(a) Length of the migrating tongue in the middle of the wound. (b) Speed of migration of keratinocyte in vivo was calculated (distance of the migrating tongue divided by the time). Morphometric analysis of serial histological sections was used to calculate the epidermal volume (c). Percentage of PCNA positive basal keratinocytes (d). N = 4−8 per group. *P<0.05, ***P<0.001.
Figure 7.
TGF-ß3 is not required for epidermal homeostasis and in vitro scratch wounds.
Histological sections of skin from E17.5 wild type (a) and Tgfb3-deficient (b) embryos stained with hematoxylin and eosin. Similar sections (c, wild type; d, Tgfb3-deficient) were immunolabeled for PCNA (red) while nuclei were labeled with DAPI (blue). Dotted line indicates the basement membrane. Percentage of basal cells that were PCNA positive was calculated for both genotypes (e). (f) Interferon Regulatory Factor 6 (Irf6) expression in E17.5 cutaneous extracts of wild type and Tgfb3-deficient animals (2 samples are represented out of 6 tested). B-actin was used as loading control and densitometry used to quantify the level of Irf6 expression. (g–j) Keratinocytes were extracted from E17.5 skin and plated in culture. At confluence, in vitro scratch wounds were performed and followed over time (g, h: time point 0 h; I, j: time point 12 h). Percentage of closure was calculated for each time point (k). N = 3 per group. (f) Percentage of cells incorporating BrdU after a 2 h pulse was calculated. Scale bar = 50 µm.
Figure 8.
TGF-ß3 is required for granulation tissue repair.
Morphometric analysis of serial sections was used to calculate the wound volume (a). Wound length (b) and depth (c) were measured on sections in the middle of the wound. N = 6−8 per group. **P<0.01 (d–k) Sections of 7-day (d–g) and 11-day (h–k) wounds were immunostained for alpha-smooth muscle actin (green). Nuclear DNA was labeled with DAPI. Sections of 11-day wounds were stained with Masson’s trichrome (l–o). Panels d–o show the middle of the wound. Scale bar = 100 µm.