Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice

Abstract

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.

Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development

“…The ability of laminin to form gel-like aggregates in vitro in the presence of calcium ions (Yurchenco and Cheng, 1993) suggests that minimal matrices, containing laminins as major structural components, are sufficiently stable to form laminar structures and enable differentiation, proliferation and survival of cells. The hypothesis that only laminins are crucial during early development is supported by the fact that the deletion of the laminin γ1 chain caused fatal consequences during the periimplantation period (Smyth et al, 1999), but not the ablation of any other basement membrane component analyzed so far (Costell et al, 1999;Murshed et al, 2000;Schymeinsky et al, 2002). However, there is currently no ultimate model available to describe the molecular organization of basement membrane components in early embryos.…”

“…Laminins are a family of at least 15 distinct heterotrimeric molecules, which form gel-like aggregates (Colognato and Yurchenco, 2000). Nidogen-1 and -2 mediate the formation of ternary complexes between laminin and collagen IV in vitro (Fox et al, 1991;Kohfeldt et al, 1998), but their significance in vivo is not yet fully defined (Murshed et al, 2000;Schymeinsky et al, 2002). Heparan sulfate proteoglycan perlecan is integrated into the networks of laminins and collagen IV and plays important roles in the maintenance of basement membrane integrity, its filtration functions and also the local storage of growth factors .…”

“…The complexity of contributions of individual proteins to in vivo functions and their mutual interactions is further illustrated by the targeted deletion of the highly specific binding site of nidogen-1 on the laminin γ1 chain causing local disintegration of basement membranes, which leads to renal agenesis and impaired lung development . In contrast, the corresponding ablation of nidogen-1 or -2 resulted in no obvious phenotypes (Murshed et al, 2000;Schymeinsky et al, 2002). Although the differential expression of components by neighboring cells (Ekblom et al, 1994;Thomas and Dziadek, 1993) and the central role of matrix receptors in coordinating the spatial and temporal aggregation of basement membranes (Henry and Campbell, 1998;Li et al, 2002) has shed new light on the molecular mechanisms of the supramolecular assembly of basement membranes, the contributions of individual proteins is not yet fully understood.…”

Inhibition of basement membrane formation by a nidogen-binding laminin γ1-chain fragment in human skin-organotypic cocultures

“…Nidogen is constitutively located in areas of epithelial-mesenchymal interactions where it is secreted by the resident fibroblasts (Fox et al, 1991;Marinkovich et al, 1993;Ekblom et al, 1994), which has been confirmed in experimental systems in vitro (Fleischmajer et al, 1995;Smola et al, 1998). In mammals two isoforms have been discovered, nidogen-1 which is the major form, and nidogen-2 (Kohfeldt et al, 1998), and knockout mice have been generated (Murshed et al, 2000;Dong et al, 2002;Schymeinsky et al, 2002) providing a more specific functional proof. The complete lack of either nidogen-1 or -2 did not produce an obvious phenotype in basement membrane structures or related functions.…”

Laminin deposition in the extracellular matrix: a complex picture emerges

“…Mice that lack expression of either nidogen 1 or nidogen 2 have normal life spans and are relatively healthy and fertile (Murshed et al, 2000;Schymeinsky et al, 2002). Functional basement membranes are also deposited during early embryogenesis in nidogen 1 and 2 double-knockout mice, suggesting that other matrix components compensate for the lack of both nidogens, or that there is direct binding of laminin to type IV collagen that can bypass nidogens (Bader et al, 2005).…”