Laminin-511 is an epithelial message promoting dermal papilla development and function during early hair morphogenesis

Abstract

Hair morphogenesis takes place through reciprocal epithelial and mesenchymal signaling; however, the mechanisms controlling signal exchange are poorly understood. Laminins are extracellular proteins that play critical roles in adhesion and signaling. Here we demonstrate the mechanism of how laminin-511 controls hair morphogenesis. Dermal papilla (DP) from laminin-511 mutants showed developmental defects by E16.5, including a failure to maintain expression of the key morphogen noggin. This maintenance was critical as exogenous introduction of noggin or sonic hedgehog (Shh) produced downstream from noggin was sufficient to restore hair follicle development in lama5(-/-) (laminin-511-null) skin. Hair development required the beta1 integrin binding but not the heparin binding domain of laminin-511. Previous studies demonstrated that Shh signaling requires primary cilia, microtubule-based signaling organelles. Laminin-511 mutant DP showed decreased length and structure of primary cilia in vitro and in vivo. Laminin-511, but not laminin-111, restored primary cilia formation in lama5(-/-) mesenchyme and triggered noggin expression in an Shh- and PDGF-dependent manner. Inhibition of laminin-511 receptor beta1 integrin disrupted DP primary cilia formation as well as hair development. These studies show that epithelial-derived laminin-511 is a critical early signal that directs ciliary function and DP maintenance as a requirement for hair follicle downgrowth.

Figure 1.

Arrested hair morphogenesis in lama5^−/− mice. (A) Typical morphology of hair placodes and associated developing dermal condensates/papillas (arrows) in E14.5 and E16.5 wild-type (WT) and lama5^−/− (null) skin. (B) NBI/BCIP detects alkaline phosphatase (AP) activity in dermal condensates of both null and wild type. Quantification of hair follicles in E14.5, E16.5 wild-type and lama5^−/− (null) skin. (HP) Hair placodes; (HG) hair germs; (PEG) hair pegs.

Figure 2.

lama5^−/− skin shows defective hair mesenchymal–epithelial signaling. (A) Semiquantitative RT–PCR analysis of critical signaling molecules involved in early hair morphogenesis, from mRNA isolated from E14.5 and E16.5 wild-type (WT) or lama5^−/− dorsal skin. (B) The same mRNA was reviewed for the expression level of Shh, Gli1, and Gli2 by quantitative RT–PCR. (C–E) Dual-label IF microscopy of E16.5 wild-type or lama5^−/− (null) dorsal skin, using indicated antibodies to CD133, laminin α5 pAb (Lm10), noggin, phospho-SMAD, and perlecan. The color of the text indicates the secondary antibody conjugates used: (green) fluorescein; (red) Texas Red. All sections were costained with nuclear marker (DAPI, blue). Arrow in C shows colocalization of CD133 staining cells with laminin-511. (F) Western blot-evaluated protein levels of Lef1 in E16.5 dorsal skin of both genotypes. GAPDH was a control for equal protein loadings. (G, left) Topgal activity detected by X-gal staining in E16.5 Topgal/lama5^−/− and Topgal/wild-type skin. (Right) Hematoxylin illustrated the hair germ morphology of same skin sections. Dotted lines indicate skin basement membrane (BMZ). (H,I) IF microscopy of E16.5 dorsal wild-type skin on the left, or lama5^−/− skin on the right, using antibodies to E-cadherin and P-cadherin, using DAPI nuclear stain.

Figure 3.

Exogenous noggin or SHH, but not by Wnt3a, can rescue hair development in the absence of epithelial laminin-511. (A) Hematoxylin-eosin staining of skin harvested 2 wk following hair reconstitution assay using either wild-type (WT) or lama5^−/− (null) E16.5 keratinocytes combined with wild-type dermal cells. (Right) Quantification of hair follicles in both conditions. (B) Full thickness E16.5 lama5^−/− dorsal skin was incubated overnight with purified Wnt3a protein, Shh adenovirus (SHH), or noggin and analyzed by hematoxylin-eosin staining 9 d after grafting to nude mice. (Top right) Quantification of hair follicles under six different conditions for hair restoration experiments, including PBS control. (C) Level of β-catenin in E16.5 lama5^−/− skin after overnight treatment with indicated quantity of purified Wnt3a (in ng/mL) demonstrating the activity of recombinant Wnt3a. (D) Immunofluorescence microscopy of adjacent frozen sections confirming hair follicle formation in absence of laminin-511 in grafted skin treated with noggin. Laminin-511 negative hair follicles (arrows) were identified as clusters of cells (nuclear antibody, DAPI) showing positive keratin 17 and perlecan expression, and negative Pecam expression, distinguishing them from blood vessels. The expression of laminin-511 was visualized by laminin α5 pAb (L511); white dots indicate the border between graft and host skin.

Figure 4.

Role of laminin-511 and β1 integrin in hair follicle downgrowth and DP maturation. (A) Wild-type (WT) or lama5^−/− (null) E16.5 skin was examined using antibodies to laminin-511 (L511) and activated β1 integrin at the time points indicated. Note colocalization of laminin-511 and β1 integrin at interface between follicular epithelium and DP (arrows). (DP) Dermal papilla; (DC) dermal condensate; (IRS) inner root sheath; (ORS) outer root sheath. (B) Hematoxylin-eosin sections of lama5^−/− skin grafts 9 d after treatment with purified laminin proteins. The ΔG45 deletion mutant of laminin-511 contains β1 integrin binding sites but lacks the major heparin binding domain. The ΔG45 deletion mutant of laminin-511 lacks the β1 integrin-binding site and the major heparin binding domain. Lam-111 represents laminin-111. (Bottom right panel) Quantification of hair follicles in each of the conditions in B.

Figure 5.

Cooperation of laminin-511, Shh, and PDGFrα in mesenchymal development. (A, right) Real-time RT–PCR-evaluated mRNA levels of DP markers Cspg2 (versican), nestin, nexin, snail2, and Wnt5a in E16.5 wild-type (WT) and lama5^−/− (null) skin. (Left) Real-time RT–PCR evaluated the mRNA level of major developing DP cell surface receptors, Fgfr2, Pdgfrα, and Tgfrb1. (B) Functional blocking anti-PDGFrα antibody-treated lama5^+/− skin. E16.5 lama5^+/− skin was incubated overnight in anti-PDGFrα antibody or control goat IgG and analyzed 9 d following grafting to nude mice. Quantification of hair follicle formation is shown in right panel. (C) IF microscopy of E16.5 wild-type skin before (left panel) or after dispase digestion (middle and right panels), using CD133, type VII collagen (ColVII), and laminin-511 (Lm10) antibodies. Note loss of laminin-511 but preservation of CD133-expressing DP cells following dispase digestion. (DP) Developing DP; (BMZ) basement membrane zone marked by white dots; (Epi) epidermis; (Der) dermis. (D) Quantitative-PCR-evaluated induction of noggin mRNA expression in dispase-treated E16.5 mesenchyme under five conditions: laminin-111 + PDGF-AA, laminin-511 + PDGF-AA, laminin-111 + Shh + PDGF-AA, laminin-511 + Shh + PDGF-AA, and laminin-511 + PDGF-AA.

Figure 6.

Laminin-511 promotes primary cilia formation in the DP of E16.5 skin and in dermal mesenchymal cells in vitro. (A) Expression of primary cilium structural proteins (acetylated α-tubulin and polaris), basement membrane proteins, perlecan and laminin-511 (Lm511), DP marker CD133, and proliferation marker Ki67 as analyzed by immunofluorescent microscopy at E16.5 and E18.5 time points. The names and corresponding colors of antibodies in combination with blue nuclear stain (DAPI) are indicated on the panels. At the right, the total number of primary cilium in both wild-type (WT) and lama5^−/− (null), and the total number of CD133-positive cells per wild type and lama5^−/− DP are quantified. (B) Fresh isolated E16.5 lama5^−/− (null) and wild-type (WT) primary mouse mesenchymal cells after 12 h of culture were stained with antibodies (names and corresponding colors indicated on the panels) including acytylated tubulin (tb), Ki67, polaris and PDGFRα, and phospho-PDGFRαY⁷⁵⁴ and nuclear stain (DAPI, blue) and analyzed by IF microscopy. (C) Fresh isolated E16.5 null and wild-type primary mesenchymal cells were cultured with either laminin-511 or laminin-111 for 12 h, then stained with acetylated tubulin (tb) and β1 integrin antibodies. Insets show higher magnification of primary cilia structures. The table on the right shows quantification of primary cilia formation under the different indicated conditions.

Figure 7.

Blockade of a major laminin-511 receptor, β1 integrin, inhibits primary cilia formation in the DP leading to an arrest of hair follicle development. (A) Freshly isolated E16.5 wild-type skin, following overnight incubation with either β1 or β3 integrin blocking antibodies or control antibody as indicated (2C9.G2; BD Biosciences) at 4°C, was analyzed by immunofluorescence microscopy using antibodies to the DP marker CD133, the primary cilia marker α-acetylated tubulin (tb), and nuclear stain (DAPI). (B, left) Quantification of primary cilia formation as seen by α-acetylated tubulin staining in CD133-positive DP; each graph is the average of multiple high-power fields. (Right) Quantification of hair follicles in the skin of 9-d grafts following overnight treatment with control of β1 integrin antibodies. Each graph represents the average of multiple low-power fields. Error bars represent the standard deviation. (C, top panel) Hematoxylin and eosin staining shows hair formation in 9-d skin grafts of E16.5 wild-type skin, after overnight treatment with either β1 integrin blocking or control hamster IgM. (Bottom panel) NBT/BCIP staining shows AP activity in 9-d skin grafts. Conditions are as indicated in images.

Figure 8.

Schematic of laminin-511’s role in hair development. Laminin-511 is secreted by the follicular epithelium into basement membrane separating follicular epithelium and DP, where it interacts with β1 integrin receptors on the DP cell surface. This leads to primary cilia development, which in turn amplifies Shh signaling, through Shh effector proteins including patched (PTCH), smoothened (SMO), and Gli. Epithelilal derived PDGF interacts with PDGFRα on DP primary cilia, which, together with Shh, promotes the expression and secretion of noggin in mesenchymal cells. Noggin promotes epithelial BMP inhibition leading to Lef-1 expression, which, coupled with Wnt/β-catenin-induced activation, drives further Shh expression. Red arrows indicate a Shh-noggin-positive amplification loop that laminin-511 facilitates.