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Basement Membranes in Skin Are Differently Affected by Lack of Nidogen 1 and 2  Sharada Mokkapati, Anke Baranowsky, Nicolae Mirancea, Neil Smyth, Dirk.

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Presentation on theme: "Basement Membranes in Skin Are Differently Affected by Lack of Nidogen 1 and 2  Sharada Mokkapati, Anke Baranowsky, Nicolae Mirancea, Neil Smyth, Dirk."— Presentation transcript:

1 Basement Membranes in Skin Are Differently Affected by Lack of Nidogen 1 and 2 
Sharada Mokkapati, Anke Baranowsky, Nicolae Mirancea, Neil Smyth, Dirk Breitkreutz, Roswitha Nischt  Journal of Investigative Dermatology  Volume 128, Issue 9, Pages (September 2008) DOI: /jid Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Epithelial organization of skin from nidogen double-null embryos is not altered in the absence of nidogens. In histological sections, embryonic skin (E18.5) of (a) wild-type and (b) nidogen double-null mice show a comparable epidermal phenotype. Immunofluorescence on (c, e, and g) wild-type and (d, f, and h) nidogen double-null samples confirms regular epidermal differentiation by distinct staining for (c and d) the basal K14, (e and f) the suprabasal K10, and (g and h) the late marker loricrin. Arrows mark the dermo-epidermal interface. Bars (a and b)=90μm, (c–h)=100μm, insets (c–h)=45μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Composition of the dermo-epidermal BM remains unaltered in the absence of both nidogen isoforms. Immunofluorescence reveals continuous deposition of major components in the BM of (a, c, e, and g) wild-type control and (b, d, f, and h) double-null embryos, demonstrated for (a and b) the laminin γ1 chain (staining both laminin-511 (α5β1γ1) and laminin-411 (α4β1γ1)), (c and d) the laminin α5 chain (staining laminin-511), (e and f) collagen IV, and (g and h) perlecan. Note that the laminin α5 chain is not detectable in capillaries but is clearly visible in (c) larger blood vessels. Vascular staining of both α5 and γ1 is reduced in (b and d) nidogen-deficient mice, whereas there is no striking change for (e-h) collagen IV and perlecan. Bar=100μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Immunofluorescence and western blot analysis of laminin-332 and integrin chains. Skin of (a, c, and e) wild-type and (b, d, and f) nidogen double-null embryos reveal comparable staining patterns for the (a and b) laminin γ2 chain (present in laminin-332 (α3β3γ2)), and the (c and d) integrin chains β1 (insets, bar=45μm) and (e and f) β4. Bar (a–f)=100μm. (g) Western blot analysis demonstrates similar levels of the laminin γ1 and integrin chains, analyzing extracts of three individual skin samples of wild-type (lanes 1–3) and double-null embryos (lanes 4–6), except of a marked reduction of integrin chains in lane 6. Thirty micrograms of total protein per lane was resolved on 4–12% SDS-polyacrylamide gradient gels under reducing (laminin γ1) and nonreducing (integrin β1 and β4 chains) conditions. Loading control, actin. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Electron microscopy of epidermal surface and the dermo-epidermal junction. (a and b) Ultrastructurally epidermal differentiation is indistinguishable between (a and c) normal and (b and d) double-null mice, both showing a prominent stratum granulosum and stratum corneum, typical for this stage. Also the (c and d) lamina densa of the BM is largely continuous in nidogen-deficient mice, although it appears locally slightly fuzzy, giving rise to (d) microblisters (arrow) and a more ragged dermo-epidermal interface. Furthermore, (c and d) hemidesmosomes (Hd) on the average are smaller in size and slightly less abundant (insets, Hd; Ds, regular desmosomes). BC, basal cell; Nu, nucleus; SC, stratum corneum; SG, stratum granulosum. Bars (a and b)=5μm, (c and d)=1μm, (c and d, insets)=0.5μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Capillary BMs are leaky and deficient in laminin-411 in the absence of nidogens. (a and b) Giemsa staining shows that (a, arrows) in wild-type skin, red blood cells remain generally within the vessels, whereas (b, arrows) in nidogen-deficient skin, many erythrocytes leak from vessels into the dermal matrix. (c and d) Double-fluorescence, using antibodies against PECAM-1 (green, endothelial marker) and the laminin α4 chain (red, representative of laminin-411 (α4β1γ1), the only laminin in embryonic capillaries), shows (c, merged, yellow areas) complete co-distribution in all blood vessels in wild-type skin, but a (d) very dramatic loss of α4 in capillary walls in nidogen double-null skin; confocal images. Bars=100μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Dramatic reduction of BM components around capillaries in the absence of nidogens. To visualize deposition of the major BM components (red) in the capillary walls of (a–c, g–i, m–o) wild-type and (d–f, j–l, p–r) nidogen double-null skin, vessels were co-stained with antibodies against the endothelial marker PECAM-1 (green). Whereas in wild-type skin, staining for the (a–f) laminin α4 and (g–l) γ1 chains and (m–r) collagen IV demonstrates (c, i, and o, merged, yellow) extensive co-distribution, in double-null skin, this is merely marginal for (f) laminin α4, moderate for (l) γ1, and comparably intense but irregular and patchy for (r) collagen IV. Confocal images; bar=10μm. (s) Detection of the laminin α4 chain by western blot analysis shows in comparison with two different wild-type samples (lane 1, 2) a strong, though variable, reduction of α4 in skin extracts from nidogen double-null mice (lanes 3–7; five individual embryos). Protein extracts were separated on a 4–12% SDS-polyacrylamide gradient gel under reducing conditions. Loading control, actin. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Ultrastructure of blood vessels in the upper dermis. (a–c) Regular small vessels from control mice show a continuous BM at their outer surface and also around (c) associated pericytes. In nidogen-deficient mice, the microvasculature reveals several anomalies including a drastic reduction of regular pericytes, which are in part replaced by (d–g) abnormal perivascular cells, (d) extravasated or (f) penetrating red blood cells, (d and e) lack of distinct BM structures (showing only some diffuse electron dense material), and (g) abundant inflated microvessels with enlarged lumina and thin, fragile vessel walls. Arrows, pointing at (a–c, inset) BM or (e) diffuse BM-like material. EJ, endothelial cell–cell junction; L, vascular lumen; Pc, pericytes; Pv, perivascular cells; RB, red blood cells. Bars (a and e)=2μm, (b and c, inset)=0.5μm, (c)=1μm, (b, g, and f)=5μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

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