A Window into Mammalian Basement Membrane Development: Insights from the mTurq2-Col4a1 Mouse Model

Abstract

Basement membranes (BMs) are specialized sheets of extracellular matrix that underlie epithelial and endothelial tissues. BMs regulate traffic of cells and molecules between compartments, and participate in signaling, cell migration and organogenesis. The dynamics of mammalian BMs, however, are poorly understood, largely due to a lack of models in which core BM components are endogenously labelled. Here, we describe the mTurquoise2-Col4a1 mouse, in which we fluorescently tag collagen IV, the main component of BMs. Using an innovative Planar-Sagittal live imaging technique to visualize the BM of developing skin, we directly observe BM deformation during hair follicle budding and basal progenitor cell divisions. The BM's inherent pliability enables dividing cells to remain attached to and deform the BM, rather than lose adhesion as generally thought. Using FRAP, we show BM collagen IV is extremely stable, even during periods of rapid epidermal growth. These findings demonstrate the utility of the mTurq2-Col4a1 mouse to shed new light on mammalian BM developmental dynamics.

Figure 1.. mTurq2-Col4a1 endogenously tagged reporter design and basement membrane localization in adult mouse tissues.

(A) Schematic of the COL4A1 subunit and type IV collagen trimeric protomer with an N-terminal mTurq2 fluorescent tag. (B) Schematic of the Mus musculus Col4a1 genomic locus and mTurq2 insertion site. The mTurq2 gene was inserted into Exon 2 between the coding sequences for the signal peptide and 7S domain. (C) Sagittal section of dorsal skin from mTurq2-Col4a1/+ adult mouse labelled with ColIV antibodies (magenta) and Hoechst to mark nuclei (blue; grayscale in C’-C”). (C’) mTurq2-COL4A1 (cyan) localizes to BM surrounding the hair follicle, where it overlaps with ColIV staining (C”, magenta). Scale bars 100 μm. (D-D”) High magnification view of mTurq2-COL4A1 (cyan) localization in the interfollicular epidermis from mTurq2-Col4a1/+ adult mouse labeled with ColIV (magenta) and E-Cadherin (E-Cad; grayscale). Scale bar 20 μm. (E-E”) High magnification view of mTurq2-COL4A1 (cyan) localization in the interfollicular epidermis from mTurq2-Col4a1/+ adult mouse labeled with Perlecan (magenta) and E-Cadherin (E-Cad; grayscale). Scale bar 20 μm. (F) Kidney section from mTurq2-Col4a1/+ adult mouse labelled with ColIV (magenta) and Hoechst to mark nuclei (blue). Scale bar 100 μm. (G-G”) High magnification view of mTurq2-COL4A1 (cyan) localization in the kidney of mTurq2-Col4a1/+ adult mouse labeled with ColIV (magenta in G; grayscale in G’) Hoechst (blue). mTurq2-COL4A1 expression can be seen in renal tubules (arrows), Bowman’s capsule (arrowheads) and the mesangial matrix (asterisks). Scale bar 100 μm. (H) Kidney section from mTurq2-Col4a1/+ adult mouse labeled with Perlecan (magenta) and Hoechst (blue). Scale bar 100 μm. (I-I”) High magnification view of mTurq2-COL4A1 (cyan) localization in the kidney of mTurq2-Col4a1/+ adult mouse labeled with Perlecan (magenta in G; grayscale in G’) and Hoechst (blue). mTurq2-COL4A1 expression can be seen in renal tubules (arrows), Bowman’s capsule (arrowheads), the mesangial matrix (asterisks) and the collecting duct (CD). Scale bar 100 μm.

Figure 2.. E18.5 mTurq2-Col4a1 mouse backskin shows correct and robust expression of mTurq2-COL4A1 in the dermal:epidermal junction basement membrane

(A) Average intensity projection of backskin from WT E18.5 embryo labelled with Hoechst (merge, top), anti-laminin β-1 antibody (A’) and anti-collagen IV antibody (A”). No signal is observed in the mTurq2 channel (A’”). (B) As (A) except backskin from an mTurq2-Col4a1/+ embryo. Robust mTurquoise2 signal recapitulates the labelling pattern of the collagen IV antibody. (C) Pearson’s correlation coefficient (r) measuring the correlation between laminin β-1 and collagen IV expression (antibody labelling) in WT and mTurq2-Col4a1/+ E18.5 backskins as labelled. Larger datapoints – biological replicates (n=3 embryos), smaller datapoints – individual images per replicate (n=10 per embryo). (D) Average intensity projection of backskin from WT E18.5 embryo labelled with Hoechst (merge, top), anti-laminin β-1 antibody (D’) and anti-perlecan antibody (D”). (E) As (A) except backskin from an mTurq2-Col4a1/+ embryo. (F) As (C) except measuring the correlation between laminin β-1 and perlecan. Scale bar 20 μm. ns = not significant, Mann-Whitney U-test.

Figure 3.. Normal ultrastructural organization of the epidermis and dermal-epidermal junction in mTurq2-Col4a1/+ embryonic skin.

Transmission electron micrographs of ultrathin skin sections from E18.5 WT control (A, C, E and G) and mTurq2-Col4a1/+ (B, D, F, H) embryos. (A-B) Ultrastructural overview of skin architecture. Der = dermis, BL = basal layer, SL = spinous layer, GL = granular layer, SC = stratum corneum. Dotted line denotes dermal-epidermal boundary. Scale bars 10 μm. (C-D) Zoomed in view of the boundary between the basal epithelial layer and dermis. Dermal-epidermal junction (DEJ) is highlighted in pink. Dotted boxes denote regions magnified in (E) and (F). Scale bars 2 μm. (E-F) Two representative examples of DEJ region of the skin from WT (E and E’) and mTurq2-Col4a1/+ (F and F’) embryos. Nuc=nucleus. Scale bars 500 nm. (G-H) Zoomed in views of individual hemidesmosomes at the DEJ. HD=hemidesmosome, PM = plasma membrane, BM = basement membrane. LL = lamina lucida, LD = lamina densa. Scale bars 100 nm.

Figure 4.. Spatial distribution and correlation between key basement membrane components in mTurq2-Col4a1/+ embryonic backskins resembles that of WT littermates

(A) Average intensity projection (XZ optical reconstruction) of backskin from WT E15.5 embryo labelled with Hoechst (merge, top), anti-laminin β-1 antibody (A’) and anti-collagen IV antibody (A”). No signal is observed in the mTurq2 channel (A’”). (B) As (A) except backskin from an mTurq2-Col4a1/+ embryo. Robust mTurq2-COL4A1 signal recapitulates the labelling pattern of the collagen IV antibody. (C) Pearson’s correlation coefficient (r) measuring the correlation between laminin β-1 and collagen IV expression (antibody labelling) in WT and mTurq2-Col4a1/+ E15.5 backskins as labelled. Larger datapoints - biological replicate means (n=3 embryos), smaller datapoints – individual images per replicate (n=10 per embryo). (D) Fraction of laminin β-1 contribution to the laminin β-1:collagen IV overlap in E15.5 backskins; refer to main text for methodology. (E) As (D) except collagen IV contribution to laminin β-1:collagen IV overlap. (F) Average intensity projection (XZ optical reconstruction) of backskin from WT E15.5 embryo labelled with Hoechst (merge, top), anti-laminin β-1 antibody (F’) and anti-perlecan antibody (F”). (G) As (F) except backskin from an mTurq2-Col4a1/+ embryo. (H-J) As (C-E) except measuring the correlation and overlap fractions between laminin β-1 and perlecan. (K-T) As (A-J) except backskins are from E12.5 embryos. Scale bars 20 μm. ns = not significant, Mann-Whitney U-test.

Figure 5.. Planar-Sagittal (PS) Multiview imaging of mTurq2-Col4a1 mouse model

(A) Schematic of established live imaging method. The skin explant is mounted on an agarose pad and imaged using spinning disc microscopy. To visualize the tissue in Z, the image is software-reconstructed. Scale bar 50 μm. (B) Schematic of PS Multiview live imaging. The skin explant is mounted as previous, but over an agarose ridge. Both planar (XY) and sagittal (‘XZ’) focal planes are then imaged. Scale bar 50 μm. (C) Snapshots taken from a time-lapse live imaging movie of hair follicle invagination in E14.5 mTurq2-Col4a1/+;mTmG/mTmG backskin. (D) Representative image showing mean fluorescence intensity calculation methodology. Orange brackets highlight the hair follicle base region where signal intensity was measured. Magenta brackets highlight the IFE region where signal intensity was measured. Scale bar 50 μm. (E) Quantification of mTurq2-COL4A1 signal in IFE, placode, and hair germ stage. Each dot represents an individual fluorescence intensity measurement as shown in (D). Larger dots show means of independent experiments. Error bars = SEM. ***p=<0.001, Mann-Whitney U test. ns = not significant. (F) Snapshots taken from a time-lapse live imaging movie of basal cell division in E15.5 mTurq2-Col4a1/+;mTmG/mTmG backskin. Interphase, metaphase, and post-cytokinesis stages are shown (left to right). Bottom panel shows same images with membrane curvature measurements. Scale bar 10 μm. (G) Quantification of the average angle of BM deformation as shown in (F). Each dot represents an average of two angles. n=23 dividing cells. Error bars = mean+SD. *p=<0.05 (0.0366), **p=<0.01, ***p=<0.001, One-way ANOVA with Dunn’s multiple comparisons test. (H) Snapshots taken from time-lapse live imaging movies of placode, late-placode, and hair germ stages as labeled, in E14.5 and E15.5 Col4a1/+;mTmG/mTmG backskins. Bottom panel shows separate channels, note clear mTurq2-COL4A1 ‘baskets’ surrounding the developing dermal condensate. Scale bar 50 μm.

Figure 6.. mTurq2-COL4A1 is highly stable within the embryonic epidermal basement membrane.

(A) Planar overview of mTurq2-COL4A1 localization in E15.5 whole mount epidermis. White boxes indicate BM lying at the dermal-epidermal junction of the interfollicular epidermis (IFE), and along the rim of a budding hair follicle (HF). Scale bar 25 μm. See also Movie S3. (B) Still images from representative fluorescence recovery after photobleaching (FRAP) experiments of mTurq2-COL4A1 within the IFE (top panels) and rim of a hair follicle (bottom panels). Scale bars 10 μm. (C) FRAP recovery curves of mTurq2-COL4A1 within the IFE at E13.5 (grey circles; n=36), E15.5 (blue squares; n=32) and at the rim of E15.5 hair follicles (magenta triangles; n=30). Grey dotted box indicates region rescaled in (C’). Each point is the mean normalized intensity. Values were acquired across n>3 biological replicates. Error bars = SD.