The Ca(++)-dependent cell adhesion molecule E-cadherin is expressed throughout mouse development and in adult tissues. Classical gene targeting has demonstrated that E-cadherin-deficient embryos die at the blastocyst stage. To study the involvement of E-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage P1 recombinase Cre/loxP system. Mice with homozygous loxP sites in both alleles of the E-cadherin (Cdh1) gene were generated and these mice were crossed with transgenic mice with the Cre recombinase under the control of the hormone-inducible MMTV promoter. This resulted in deletion of the E-cadherin gene in the differentiating alveolar epithelial cells of the mammary gland. The mutant mammary gland developed normally up to 16-18 days of pregnancy but exhibited a dramatic phenotype around parturition. The production of milk proteins was so drastically reduced that adult mutant mothers could not suckle their offspring. Thus, the lack of E-cadherin affected the terminal differentiation program of the lactating mammary gland. In concordance with this finding, the prolactin-dependent activation of the transcription factor Stat5a was initiated but not maintained in the mutant gland. Instead, without E-cadherin massive cell death was observed at parturition and the mutant mammary gland at this stage resembled that of the involuted gland normally seen after weaning. These results demonstrate an essential role for E-cadherin in the function of differentiated alveolar epithelial cells. No tumors were detected in mutant glands lacking E-cadherin.

Using Cre/loxP, we conditionally inactivated the beta-catenin gene in cells of structures that exhibit important embryonic organizer functions: the visceral endoderm, the node, the notochord, and the definitive endoderm. Mesoderm formation was not affected in the mutant embryos, but the node was missing, patterning of the head and trunk was affected, and no notochord or somites were formed. Surprisingly, deletion of beta-catenin in the definitive endoderm led to the formation of multiple hearts all along the anterior-posterior (A/P) axis of the embryo. Ectopic hearts developed in parallel with the normal heart in regions of ectopic Bmp2 expression. We provide evidence that ablation of beta-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos.

('bgr;)-Catenin is a central component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. We have investigated the role of (β)-catenin during brain morphogenesis, by specifically inactivating the (β)-catenin gene in the region of Wnt1 expression. To achieve this, mice with a conditional ('floxed') allele of (β)-catenin with required exons flanked by loxP recombination sequences were intercrossed with transgenic mice that expressed Cre recombinase under control of Wnt1 regulatory sequences. (β)-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 (β)-catenin in controlling midbrain-hindbrain development. The craniofacial phenotype, not observed in embryos that lack Wnt1, indicates a role for (β)-catenin in the fate of neural crest cells. Analysis of neural tube explants shows that (β)-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 (β)-catenin affects neural crest cell survival and/or differentiation. Our results demonstrate the pivotal role of (β)-catenin in morphogenetic processes during brain and craniofacial 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.

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-γ, 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-γ 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.

Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and human ␣1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the ␣1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.Tissues use specific sets of collagens, often synthesized simultaneously, to achieve and maintain particular functional properties. Most collagens are secreted and assembled within the extracellular environment; however, a growing subclass of collagens are transmembranous and inserted into the plasma membrane in a type II orientation to extend their extracellular collagenous domains from the cell surface.The subclass of transmembranous collagens currently includes types XIII, XVII, XXIII, and XXV, summarized in a recent review (1). The group has also been referred to as the MACITs for membrane-associated collagens with interrupted triple helices (2). Type XIII collagen, the first member of the group identified (3), has an important function in muscle tissue. Engineered genetic mutations in the Col13a1 gene in one case causes cardiovascular defects (4) and in another causes abnormal skeletal muscle myofibers with progressive myopathy that is worsened by exercise (5). Type XIII collagen mediates cell attachment through integrin ␣1␤1 but does not interact through another common collagen receptor, ␣2␤1 (6). The second transmembranous collagen to be identified was type XVII (7), which was known for many years as bullous pemphigoid antigen 2 and BP180 prior to the elucidation of its collagenous nature. Being a component of the hemidesmosome (8), type XVII collagen provides structural integrity to the cornea (9 -11) and skin (12), clearly demonstrated by mutations in the COL17A1 gene that cause epidermolysis bullosa (13, 14). Our laboratories added collagen XXIII to the family of transmembranous collagens by identifying a human EST 3 and using its sequence to clone a fragment of chicken collagen XXIII cDNA to examine its expression in cornea (15). We also cloned the full-length mouse collagen XXIII cDNA sequence and deposited it into GenBan...

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 murineMycobacterium 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.

Collagen XXIII belongs to the class of type II orientated transmembrane collagens. A common feature of these proteins is the presence of two forms of the molecule: a membrane-bound form and a shed form. Here we demonstrate that, in mouse lung, collagen XXIII is found predominantly as the full-length form, whereas in brain, it is present mostly as the shed form, suggesting that shedding is tissue-specific and tissue-regulated. To analyze the shedding process of collagen XXIII, a cell culture model was established. Mutations introduced into two putative proprotein convertase cleavage sites showed that altering the second cleavage site inactivated much of the shedding. This supports the idea that furin, a major physiological protease, is predominantly responsible for shedding. Furthermore, our studies indicate that collagen XXIII is localized in lipid rafts in the plasma membrane and that ectodomain shedding is altered by a cholesterol-dependent mechanism. Moreover, newly synthesized collagen XXIII either is cleaved inside the Golgi/trans-Golgi network or reaches the cell surface, where it becomes protected from processing by being localized in lipid rafts. These mechanisms allow the cell to regulate the amounts of cell surface-bound and secreted collagen XXIII.The group of collagenous transmembrane proteins consists of type XIII, XVII, XXIII, and XXV collagens and several related proteins such as ectodysplasin A, the class A macrophage scavenger receptors, the MARCO1 receptor, and the group of colmedins. These are type II transmembrane proteins that contain at least one collagenous triple helical domain (summarized in Ref. 1). Collagens XIII, XXIII, and XXV are of unknown function and consist of three collagenous domains that are flanked and separated by noncollagenous domains. Whereas collagen XVII is more distantly related, types XIII, XVII, XXIII, and XXV all exist in two forms: a transmembrane form and a shed ectodomain form. Whereas collagen XVII is shed from the surface by TACE (tumor necrosis factor-␣-converting enzyme), a member of the ADAM (a disintegrin and metalloproteinase) family (2), mutation analysis of collagens XIII and XXV demonstrated that the protease furin produces the shed forms (3, 4). Initial cell culture studies suggested an involvement of furin either directly or indirectly in the cleavage of collagen XXIII as well (5). Furthermore, the co-existence in tissues of both the transmembrane and shed forms of collagen XXIII was suggested from immunoblot analyses (6).The shedding of an ectodomain amplifies the possible functional role of a protein because different forms, i.e. full-length cell surface-bound or soluble, likely have different biological activity. The "sheddase" furin is a member of a proprotein convertase family. Among other functions, furin participates in the maturation and activation of proteins at the cell surface, endowing the cell with the ability to change its functional behavior (7-9). Moreover, conversion by proteolytic cleavage can be tightly regulated (10, 11). Furin-de...