Fig 2: Imaging modality to whole animal in vivo fluorescence. (a) Immunofluorescent staining showing the expression of basal epithelial marker (K5 and K14) in mammary organoids generated from Rosa26 tdTomato mammary gland. The Red fluorescence is tdTomato endogenous fluorescence (Scale bar = 100 µm). (b) Schematic showing imaging modalities to whole gland imaging and whole animal imaging. Animals were anaesthetized after 4 weeks post transplantation and imaged for tdTomato fluorescence at the left side (site for transplantation) and also checked for mammary fatpad imaging through ultrasound imaging. The mammary glands were dissected out and checked for the endogenous tdTomato fluorescence to visualize ductal trees, and also performed whole-gland immunostaining to check tdTomato-derived mammary epithelial cells. (c) In vivo whole animal fluorescence (Xenogen IVIS fluorescence imager) reveals tdTomato expression at the site of transplantation. The right side was used as control (n = 5 pregnant mice). The Radiant efficiency scale in the whole animal fluorescence is expressed as (p/sec/cm2/sr)/(µW/cm2). Color scale: Min = 1.00e8 and Max = 1.00e9. (d) Ultrasound imaging reveals the formation of mammary fat pad and complete absence of the lymph node (LN) confirming efficient mammary fat pad clearing and the ability of mammary organoids to reconstitute mammary gland (n = 5 pregnant mice). (e) Tissue clearing enables 3D confocal imaging to visualize mammary ductal tree formation. The mammary glands were surgically dissected after 4 weeks post transplantation (virgin) and incubated in Fructose-Glycerol clearing Solution (FGCS) for 24 h, making the whole tissue translucent for efficient imaging. Tissue clearing can also be performed for 3–7 days (Pregnant mice) depending on the stage of mammary gland development. Confocal microscopy of the whole gland was subsequently imaged to check for the presence of ductal trees, and the endogenous tdTomato fluorescence confirms organoids are able to regenerate mammary gland ductal tree in vivo (Scale bar = 1 mm).

Fig 3: Representative images showing how FGF7p-treated mice have limited KRT14 staining and no ectopic lumenal foci compared to Vehicle 1 (Veh1)-treated mice 28 days after cyclophosphamide. (a–d) IF for KRT14 (white) shows that the Vehicle 1-treated and injured mouse (a) has widespread numbers of basal KRT14+ cells (arrowheads) and an ectopic lumenal KRT14+ cell (arrow). A higher power magnification of the boxed region in panel a re-demonstrates the ectopic KRT14+ cell (arrow) arising from a column of KRT14+ cells (between concave arrowheads) that are attached to basal KRT14+ cells (arrowhead) in the Vehicle 1-treated mouse. A lower power magnification (c) and higher power magnification from the boxed region in panel c (d) show that the FGF7p-treated and injured mouse has many fewer basal KRT14+ cells (arrowheads) and no ectopic lumenal foci compared to the Vehicle 1-treated mouse. Blue (a–d) = DAPI. L (a–d) = lumen. Scale bars (a–d) = 50 µm. (e) Graph showing that the mean number of ectopic lumenal KRT14+ foci is higher in the Vehicle 1-treated mice than FGF7p-treated mice 28 days after injury. N = 6 (3 bladders with 2 planes assessed per mouse). *p < 0.05.

Fig 4: H2BGFP-dilution analysis in tail epidermis.a Structure of the mouse tail epidermis. 3D reconstruction of confocal z stacks (top panel) and orthogonal xyz view (mid panel). Apical blister-shaped regions (scale) alternate with deeper regions (interscale) (boundaries delineated by dotted lines), being arranged in lines separated by hair follicles (asterisks). Green: H2BGFP expression; white: KRT14 immunostaining (as a marker of basal layer); red: CD45 immunostaining; blue: DAPI. Scale bars, 200 µm. Bottom panel: Cartoon illustrating the tail skin structure. b Representative confocal z stacks of scale and interscale regions of tail epidermis during H2BGFP chase experiments in R26M2rtTA/TetO-H2BGFP mice, showing H2BGFP (green), immunostaining for KRT14 (white) and immunostaining for pan-leukocyte marker CD45 (red). Images from the same time point correspond to different mice to illustrate inter-animal variation. Label-retaining cells (LRCs) are highlighted with arrowheads (CD45+ cells) or full arrows (CD45- cells) (details in inserts; blue: DAPI). Scale bars, 20 µm. c Time course of basal-layer keratinocyte H2BGFP intensity distributions from scale and interscale regions of tail. Experimental data shown as boxplots per individual biologically independent mouse (intensities normalized to average keratinocyte intensity at time 0, raw H2B-GFP intensity values are given in Supplementary Data 2). n = 3 animals at each time point except 18 days where n = 2 mice. Centre line of box is median value, box indicates 25th and 75th centiles and whiskers indicate minimum and maximum values. Solid black lines: average H2BGFP-dilution rates (within 95% CI limits—shaded gray areas—where the value of ? = 1.2/week reported by Mascre et al.4 falls). Insert: Detail of H2BGFP intensity distributions separated per field of view for the single tissue found to be bimodal in the overall, per-animal modality test (see Fig. 3d). Analyses per field of view all resulted non-significant (unimodality).

Fig 5: Plexin-B1/Plexin-B2 inhibit YAP activity in response to mechanical forces.a Confocal images of murine epidermis at E15.5 and E16.5 immunostained for active YAP (red) and K14 (green). Scale bar, 25 µm. b, c Quantification of the data in a (percentage of basal cells) (mean ± s.d.; E15.5: n = 5 mice per genotype, E16.5: n = 4 mice per genotype; b p = 0.0047, c p = 0.0078; two-sided unpaired t-test). d, e Primary mouse keratinocytes on d circular or e square micropatterns immunostained for Yap (green) after 3 h of 1.8 mM Ca2+. Representative images are shown on the left, quantifications of Yap localization are shown on the right (mean ± s.d.; circles: control: n = 73 micropatterns from 5 mice, PlexDKO: n = 73 micropatterns from four mice; N > C: p = 0.000008, N = C: p = 0.012038, N < C: p = 0.000008; squares: control: n = 69 micropatterns from five mice, PlexDKO: n = 69 micropatterns from four mice; N > C: p < 0.000001, N = C: p < 0.000001, N < C: p = 0.000050; two-sided unpaired t-test). Blue: DAPI. Scale bars, 25 µm. f Schematic illustration of abrupt crowding experiments. Confluent monolayers were exposed to 120% static uniaxial stretch, which was abruptly released. Cells were analyzed at the indicated time points (R5’: 5 min after release; R15’: 15 min after release). g, h Representative YAP immunofluorescence images g and quantification of nuclear YAP h upon crowding (mean ± s.d.; n = 5 independent experiments with >200 cells/condition/experiment; *p = 0.0358, ***p = 0.0004, one-way ANOVA, Tukey test; values are normalized to control 120% stretch; statistics have been done from the non-normalized values; scale bars 20 µm). i–l Pregnant females were injected at E15.5 with vehicle control (DMSO) or verteporfin (i.p., 100 µg/g body weight). Embryos were harvested at E16.5. Shown are in i representative H&E-stained histological sections, in j the quantification of epidermal thickness (mean ± s.e.m.; control DMSO-treated: n = 4 embryos, PlexDKO DMSO-treated: n = 4 embryos, control verteporfin-treated: n = 7 embryos, PlexDKO verteporfin-treated: n = 8 embryos; p = 0.011 for DMSO, p = 0.165 for verteporfin; two-sided unpaired t-test), in k representative images of immunohistochemical stainings of the embryonic epidermis using an anti-BrdU antibody (brown), and in l the quantification of BrdU-positive cells (mean ± s.e.m.; control DMSO-treated: n = 4 embryos, PlexDKO DMSO-treated: n = 4 embryos, control verteporfin-treated: n = 7 embryos, PlexDKO verteporfin-treated: n = 7 embryos; p = 0.016 for DMSO, p = 0.345 for verteporfin; two-sided unpaired t-test).