Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain

Abstract

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

FIGURE 1.

Proliferation of basal keratinocytes is increased in the skin of γ1III4-deficient mice. In histological sections, embryonic skin (E18.5) of control (a) and γ1III4-deficient (b) mice show a comparable epidermal morphology, including skin appendages. Immunohistochemistry of skin sections with antibodies against Ki67 (c and d) shows statistically significant higher numbers of proliferating cells in the basal compartment of the interfollicular epidermis of mutant mice (d). The number of proliferating cells was quantified by counting Ki67-positive cells in 10 high power fields (40×) per section in 10 individual control and mutant mice (g, n = 10; p = 0,0001). White bar, controls (γ1III4^+/+); black bar, mutants (γ1III4^−/−). Immunohistochemistry of skin sections with antibodies against keratin K14 shows a uniform staining of the basal cell layer in control (e) and mutant (f) mice; E, epidermis; D, dermis. The hair follicle is indicated by an arrow in b. Scale bars, 100 μm (a–d) and 25 μm (e and f).

FIGURE 2.

Epidermal differentiation is not altered in γ1III4-deficient mice. Immunofluorescence of control (a–d) and γ1III4-deficient (e–h) embryonic skin sections (E18.5) shows comparable epidermal differentiation patterns by staining for basal K14 (a and e, and insets), suprabasal K10 (b and f), and the late markers loricrin (c and g) and filaggrin (d and h). Scale bar, 100 μm; insets, 3-fold magnification.

FIGURE 3.

Nidogen 1 deposition is impaired in skin BMs of γ1III4-deficient mice. A, immunofluorescence staining for nidogen 1 (a and e), nidogen 2 (d and h), laminin γ3 chain (b and f) of skin sections of E18.5 control (a–d) and γ1III4-deficient (e–h) mice. Continuous nidogen 1 staining is seen in control sections (a) at the dermoepidermal junction (arrowheads), around capillaries (open arrows), and hair follicles (filled arrows), whereas in the mutant mice (e), it is largely or completely lost in the dermoepidermal BM and around capillaries, respectively (asterisks, unspecific staining of the stratum corneum). Co-localization of nidogen 1 (a and e) and the laminin γ3 chain (b and f) is restricted to the BM of hair follicles in γ1III4-deficient mice (merge, c and g). Nidogen 2 staining appears unchanged (d and h). Scale bar, 100 μm (d and h) and 20 μm (a–c and e–g). B, Western blots of protein extracts from γ1III4-deficient skin show strongly reduced nidogen 1 (nd1) levels when compared with control littermates (γ1III4^+/+), whereas nidogen 2 (nd2) is not altered. Three individual samples of control and γ1III4-deficient mouse skin were resolved on SDS-polyacrylamide gradient gels under reducing (nd1, actin as loading control) and nonreducing (nd2, ponceau staining) conditions. C, cell lysates of γ1III4^+/+ (lane 1, input) or γ1III4^−/− (lane 2, input) fibroblasts were incubated with His-tagged nidogen 2 bound to Ni-NTA-agarose beads (lanes 5 and 6, respectively). Cell lysates of γ1III4^+/+ (lane 3) and γ1III4^−/− (lane 4) fibroblasts incubated with uncoupled Ni-NTA-agarose beads. After washing, the beads were boiled, and the released proteins were analyzed by Western blots with antibodies against the His tag to detect nidogen 2 (middle panel) and against the laminin γ1 chain (Lγ1, bottom panel). Coommassie blue staining (top panel), molecular masses (kDa) are indicated to the right.

FIGURE 4.

Additional deletion of the laminin γ3 chain in γ1III4-deficient mice further reduces nidogen 1 deposition. Immunofluorescence staining for nidogen 1 (a and b) and nidogen 2 (c and d) on skin sections of mice lacking the laminin γ3 chain (LAMC3^−/−; a and c) or both the γ3 chain and the nidogen binding module γ1III4 (γ1III4^−/−/LAMC3^−/−; b and d). In contrast to nidogen 1, nidogen 2 staining is not changed in the double knock-out mice. Scale bar, 50 μm.

FIGURE 5.

Deposition of other major BM components and receptors are unaffected in γ1III4-deficient skin. Immunofluorescence reveals normal distribution of major BM components and receptors in control (a–d) and γ1III4-deficient (e–h) embryos, as demonstrated for the laminin γ1 (laminin 511 and 411; a and e) and γ2 (laminin 332) chains (b and f), and the integrin chains β1 (c and g) and β4 (d and h). Scale bar, 100 μm. Western blot analysis (i) shows comparable protein levels for the integrin chains (intg) β1 and β4 and the laminin γ1 chain (Lγ1) between control and γ1III4-deficient skin samples. Loading control, actin; molecular masses (kDa) are indicated to the right.

FIGURE 6.

Comparable ultrastructure of the upper part of epidermis and BMs in skin of control and γ1III4-deficient mice. Electron microscopy of the epidermis (a–d) reveals neither striking alterations in upper layers (a and b: SG, stratum granulosum; SC, stratum corneum) nor at the dermoepidermal junction (c and d) of γ1III4-deficient mice including hemidesmosomal fine structure (insets: HD, hemidesmosome; DA, dermal anchorage of BM; DS, desmosomal cell-cell junction). An arrow indicates occasional slight BM discontinuity. Scale bars, 4 μm (a), 3 μm (b), and 1 μm (c and d). Ultrastructure of small blood vessels in the upper dermis of both control (e and g) and γ1III4-deficient mice (f and h) shows distinct BMs at the vessel walls (arrows) and associations with pericyte extensions or perivascular cells (asterisks). CF, collagen fibrils; EJ, endothelial junction; PC, pericyte; PV, perivascular cell; RB, red blood cell; VL, vascular lumen. Scale bars, 2 μm (e) and 1 μm (f–h and insets).

FIGURE 7.

Nidogen 1 but not nidogen 2 deposition is impaired in heart and lung of γ1III4-deficient mice. Immunofluorescence of heart sections from control (a and c) and γ1III4-deficient embryos (b and d) at E18.5 and of lung sections from control (e and g) and γ1III4-deficient (f and h) mice at birth with antibodies against nidogen 1 (a, b, e, and f) and nidogen 2 (c, d, g, and h). At birth, neonatal lungs from control pups show typically dilated peripheral airway saccules (e and g), whereas lungs from mutant pups appear immature (f and h). Scale bars, 100 μm (a, b, and f), 50 μm (c, d, e, g, and h). Open arrows, autofluorescence by elastic and collagenic fibers (a, b, and f); asterisks and arrowheads, terminal airways (e–h); filled arrows, small blood vessels in the myocardium (c and d).