Expression patterns of Wnt genes in mouse gut development.

Wnt signaling regulates cell fate decisions and cell proliferation during development and in adult tissues in both invertebrates and vertebrates. Here we describe the identification of Wnt genes, Wnt2a, 4, 5a, 5b, 6 and 11, expressed in mouse embryonic gut development. Each of these genes exhibits a characteristic and regional-specific expression pattern along the anterior-posterior axis of the digestive tube between embryonic day (E) 12.5 and 16.5 of embryonic development. The expression of Wnt5a is confined to the mesenchymal compartment, while expression of Wnt4 is found both in the intestinal epithelium and the mesenteric anlage. Wnt11 is expressed in the epithelium of esophagus and colon, but also in mesenchymal cells of the stomach. Wnt5b and Wnt6 exhibit restricted expression in the epithelium of the esophagus. A characteristic regionalized expression pattern is observed in the developing stomach. Wnt5a is expressed in the mesenchymal layer of the prospective gland region but becomes restricted to the tip of the gland region by E14.5. Wnt11 is highly expressed at the gastro-esophageal junctions, while Wnt4 is found in the epithelium lining the pyloric region of the stomach but not in the epithelium of the prospective gland region.

Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development.

beta-Catenin is a central component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. We have investigated the role of beta-catenin during brain morphogenesis, by specifically inactivating the beta-catenin gene in the region of Wnt1 expression. To achieve this, mice with a conditional ('floxed') allele of beta-catenin with required exons flanked by loxP recombination sequences were intercrossed with transgenic mice that expressed Cre recombinase under control of Wnt1 regulatory sequences. beta-Catenin gene deletion resulted in dramatic brain malformation and failure of craniofacial development. Absence of part of the midbrain and all of the cerebellum is reminiscent of the conventional Wnt1 knockout (Wnt1(-/-)), suggesting that Wnt1 acts through beta-catenin in controlling midbrain-hindbrain development. The craniofacial phenotype, not observed in embryos that lack Wnt1, indicates a role for beta-catenin in the fate of neural crest cells. Analysis of neural tube explants shows that (beta-catenin is efficiently deleted in migrating neural crest cell precursors. This, together with an increased apoptosis in cells migrating to the cranial ganglia and in areas of prechondrogenic condensations, suggests that removal of beta-catenin affects neural crest cell survival and/or differentiation. Our results demonstrate the pivotal role of beta-catenin in morphogenetic processes during brain and craniofacial development.

Lethal granuloma disintegration in mycobacteria-infected TNFRp55-/- mice is dependent on T cells and IL-12.

Genetically susceptible, TNFRp55 gene-deficient (TNFRp55-/-) mice succumb to infection with Mycobacterium avium. Before their death, M. avium-infected TNFRp55-/- mice develop granulomatous lesions that, in contrast to granulomas in wild-type syngeneic mice, undergo acute disintegration. To determine the factors involved in these events, we depleted T cell subsets or neutralized the inflammatory cytokines IFN-gamma, IL-12, or TNF in TNFRp55-/- mice infected i.v. with M. avium. Infected TNFRp55-/- mice treated with a control mAb became moribund between days 26 and 34 postinfection, showing widespread inflammatory cell apoptosis within disintegrating granulomas. In contrast, TNFRp55-/- mice depleted of either CD4+ or CD8+ cells after granuloma initiation stayed healthy until at least day 38 postinfection and showed no signs of granuloma destruction. Neutralization of IL-12, but not of IFN-gamma or TNF, also protected M. avium-infected TNFRp55-/- mice from granuloma decomposition and from premature death. Treatment with dexamethasone or with a specific inhibitor of inducible NO synthase did not prevent granuloma dissolution or death of TNFRp55-/- mice. In conclusion, granuloma disintegration in TNFRp55-/- mice is a lethal event that is dependent on IL-12 and that is mediated by an excess of T cells.

NOS2-derived nitric oxide regulates the size, quantity and quality of granuloma formation in Mycobacterium avium-infected mice without affecting bacterial loads.

Granuloma formation in response to mycobacterial infections is associated with increased expression of inducible nitric oxide synthase (NOS2) within granuloma macrophages and increased levels of nitrate/nitrite in the sera of infected mice. Continuous treatment with 5 mm or 10 mm l-N6-(1-imino-ethyl)-lysine (L-NIL), a selective NOS2-inhibitor, in acidified drinking water for up to 7 weeks consistently reduced infection-induced nitrate/nitrite to background levels in mycobacteria-infected BALB/c mice. Oral treatment with 5 mm L-NIL initiated at the time of infection significantly exacerbated growth of Mycobacterium tuberculosis, but had no effect on Mycobacterium avium colony-forming unit development in the liver, spleen and lungs of intravenously infected mice. In order to examine the role of nitric oxide in mycobacteria-induced granulomatous inflammation in the absence of any effect on the bacterial load, M. avium-infected mice were treated with 5 mm L-NIL from day 1 through 38 and the development of granulomatous lesions in the liver was assessed by histology, immunohistology and reverse-transcription-polymerase chain reaction (RT-PCR). Computer- and video-assisted morphometry performed at 4 and 7 weeks post-infection showed that treatment with L-NIL led to markedly increased number, cellularity and size of granulomatous lesions in infected mice regardless of the virulence of the M. avium isolate used for infection. Immunohistology of the liver revealed that in mice treated with L-NIL, the numbers of CD3+ T cells, CD21/35+ B cells, CD11b+ macrophages and RB6-8C5+ granulocytes associated with granulomatous lesions was increased. RT-PCR of the liver showed that in L-NIL-treated mice infected with M. avium, mRNA levels of tumour necrosis factor, interleukin-12p40, interferon-gamma, interleukin-10 and interferon-gamma-inducible protein-10 (IP-10) were up-regulated, while mRNA levels of interleukin-4, monocyte chemotactic protein-1 (MCP-1) and MCP-5 were similar to those in untreated control infected mice. When M. avium-infected mice were treated with 5 mm L-NIL between the 5th and 12th weeks of infection, similar changes in granuloma number and size were found in the absence of any effect on the bacterial load. These findings demonstrate that nitric oxide regulates the number, size and cellular composition of M. avium-induced granulomas independently of antibacterial effects by modulating the cytokine profile within infected tissues.

Fatal granuloma necrosis without exacerbated mycobacterial growth in tumor necrosis factor receptor p55 gene-deficient mice intravenously infected with Mycobacterium avium.

The pathogenesis of mycobacterial infections is associated with the formation of granulomas in which both antibacterial protection and tissue damage take place concomitantly. We used murine Mycobacterium avium infection to compare the development of granulomatous lesions in intravenously infected tumor necrosis factor receptor p55 (TNFRp55) gene-deficient (p55(-/-)) mice to the development of granulomatous lesions in M. avium-infected syngeneic C57BL/6 (p55(+/+)) mice. Up to 5 weeks after infection with either the highly virulent M. avium strain TMC724 or the intermediately virulent M. avium strain SE01, bacterial counts in the liver, spleen, and lung of p55(-/-) mice were identical to or lower than those in infected p55(+/+) mice. However, the formation of mononuclear cell foci in the liver was delayed by approximately 2 to 3 weeks in p55(-/-) mice compared to the results obtained for infected p55(+/+) mice. Despite comparable bacterial loads, granulomas in p55(-/-) mice underwent progressive necrosis, causing damage to the surrounding liver tissue. The appearance of necrotizing granulomas was associated with the death of all infected p55(-/-) mice, regardless of the virulence of the M. avium strain used for infection. Granulomatous lesions in the liver contained three times as many CD3(+) cells in p55(-/-) mice yet appeared more diffuse than in p55(+/+) mice. Semiquantitative reverse transcription-PCR studies revealed that prior to mouse death, interleukin-12 (IL-12) and gamma interferon mRNA levels were up regulated in the livers of infected p55(-/-) mice, while mRNA levels for tumor necrosis factor, the inducible isoform of nitric-oxide synthase (iNOS), and IL-10 were similar to those found in infected p55(+/+) mice. In response to persistent mycobacterial infection, the absence of TNFRp55 causes the disregulation of T-cell-macrophage interactions and results in fatal granuloma necrosis even when adequate antibacterial functions are maintained.