Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice.

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.

Growth selection in mice reveals conserved and redundant expression patterns of the insulin-like growth factor system.

Transgenic and knockout models have been used successfully in order to attribute specific functions to distinct growth factors. However, it is not clear which from the different IGF-components are actually altered when growth is affected. Furthermore it is not clear if unique or redundant patterns of IGF-component expression are present under conditions of elevated or reduced growth. To address these questions we have used a unique set of mouse models generated by divergent selection for high and low body growth. The set of mouse models consisted of eight mouse lines established in different laboratories. We have studied systemic and local expression of growth relevant genes in these mouse lines highly diverging for body and carcass weights but also for nose-rump lengths. As a strictly conserved pattern, serum IGF-I levels were dramatically increased in all H-lines if compared with the respective L-lines. By contrast serum IGFBP concentrations did not reveal clear patterns of expression in response to growth selection: IGFBP-3 was elevated in some H-lines, IGFBP-2 was increased in H- or L-lines and IGFBP-4 was similar in H- and L-lines. The fact that IGFBP-2 was the only IGFBP elevated in part of the L-lines, identifies IGFBP-2 as an exclusive although facultative negative effector for growth in the circulation among all other IGFBPs. In muscle tissue from selected breeding groups characterized by specific increases of the carcass weights we found redundant patterns of gene expression indicating the absence of tissue-specific or uniquely fixed expression patterns during growth selection within muscle tissue. The finding that serum but not tissue IGF-I levels were strictly positively correlated with growth during growth selection argues for an important role of endocrine IGF-I for postnatal growth in mice.

Insulin-like growth factor-binding protein-2 (IGFBP-2) overexpression negatively regulates bone size and mass, but not density, in the absence and presence of growth hormone/IGF-I excess in transgenic mice.

Insulin-like growth factor-binding protein-2 (IGFBP-2) has been suggested to be a negative regulator of bone growth and maintenance. The objective of this study was to characterize the effect of elevated IGFBP-2 on the skeletal phenotype of adult transgenic mice, in the absence and presence of growth hormone (GH) excess. 43 male mice were examined at an age of 4 months (7 IGFBP-2 transgenic mice, 12 GH transgenic mice, 10 mice carrying both transgenes, and 14 controls). The bone mineral content of the total skeleton and of isolated bones was quantified by dual energy X-ray absorptiometry (DXA), after validation versus ash analysis. Cortical and trabecular bone was quantified by peripheral quantitative computed tomography (pQCT), after validation versus microCT. A strong linear relationship was found between DXA and ash weight, and between pQCT and micro CT ( r>0.95). Bone size and bone mineral content were significantly reduced in IGFBP-2 transgenic mice, the magnitude of the effect varying between skeletal sites and between bone compartments. Elevated IGFBP-2 negatively modulated the GH-stimulated increase in bone size and mineral content, and completely blocked GH-effects at cortical sites. Notably, bone density was not decreased in IGFBP-2 transgenic animals compared with controls. In conclusion, IGFBP-2 is identified as a potent negative regulator of normal and GH-stimulated bone growth in vivo. Interestingly, elevated IGFBP-2 levels did not lead to a decrease in bone density, suggesting that IGFBP-2 negatively affects bone size and mineral content, but not bone maintenance in adult mice.

Insulin-like growth factor binding protein 2 (IGFBP-2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF-I excess on adrenocortical cells in vivo.

GH and IGF-I are capable of inducing cellular hypertrophy and/or hyperplasia. Chronic overexpression of GH in transgenic mice results in systemically and locally increased IGF-I levels and in disproportionate overgrowth, including adrenocortical enlargement and corticosterone hypersecretion. Using PEPCK-bovine GH transgenic (G) mice, we demonstrate that adrenal enlargement involves both hypertrophy (44%) and hyperplasia (50%) of zona fasciculata cells. To clarify whether IGFBP-2 affected cell volume and number, we crossed hemizygous G mice with hemizygous CMV-IGFBP-2 transgenic (B) mice, generating G mice, B mice, GB double transgenic mice, and nontransgenic controls (C). The absolute weight of the adrenal glands was significantly increased in 5-wk- and 4-month-old G mice vs. C and B mice. IGFBP-2 overexpression in GB mice reduced this effect of GH excess by 26% and 37% in 5-wk- and 4-month-old animals, respectively. GH-induced hypertrophy of zona fasciculata cells was completely abolished by IGFBP-2 overexpression in GB mice whereas hyperplasia was not affected. Basal and ACTH-induced plasma corticosterone levels of 4-month-old G mice, but not of GB mice, were two- to threefold increased compared with C mice. Plasma ACTH levels were similar in all groups. Our data show that IGFBP-2 potently separates hypertrophic and hyperplastic effects of GH/IGF-I excess on adrenocortical cells.

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice.

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.