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 the Cre/loxP system we conditionally inactivated β-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured β-catenin −/− endothelial cells showed a different organization of intercellular junctions with a decrease in α-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell–cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of β-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.
Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs).After infection of a susceptible cell, translation of the picornavirus plus-strand RNA genome is controlled by the internal ribosome entry site (IRES). This cis-regulatory RNA element of about 450 nucleotides folds into complex and highly conserved secondary structures and facilitates translation by direct binding of ribosomes to an internal site of the viral RNA. IRES elements have been found in various viral RNAs and also in cellular mRNAs (for reviews, see references 1 and 16).Internal initiation of picornavirus translation seems to require all canonical eukaryotic initiation factors (eIFs) also involved in cellular cap-dependent translation (28, 33), except for the actual cap-binding protein eIF4E. For the majority of eukaryotic mRNAs that are translated by a ribosome-scanning mechanism (13,18,38), the initiation of translation is the most important point of regulation in the overall process of protein synthesis (29). Modulation of the activity of these eIFs alters the general rate of protein synthesis, and the signal transduction routes leading to the eIFs are becoming clear now (36), supporting the idea that translational control substantially contributes to the regulation of gene expression in eukaryotes.For picornaviruses, the initiation of translation also appears to be a major point of control. In vitro studies revealed that additional noncanonical translation initiation factors are involved in picornavirus IRES-mediated translation (1, 41). Accordingly, there is significant genetic evidence that picornavirus IRES elements contain determinants of cell specificity.Analysis of poliovirus IRES mutants showed that translation defects could be cell type specific, since a decreased translation capacity of mutant templates was evident in cell extracts of neuronal origin, but not in HeLa cells (26). Cell-specific determinants were recently demonstrated to exist in the poliovirus 5Ј-untranslated region (UTR) by experiments using viruses with chimeric genomes. When the poliovirus IRES was replaced with that of human rhinovirus (HRV), neuropathogenicity in a mouse model was abrogated (15). The differential translation of wild-type and attenuated Sabin vaccine strains of poliovirus in different cell types indicates that cellular factors influencing translational activity may be differentially expressed, consistent with the idea that factors distinct from the standard initiation factors determine picornavirus translation efficiency (for review, see reference 1). The general idea that IRES elements may allow fine-tuning of gene expression was also supported for cellular IRES elements. The ornithine decarboxylase and p58 PISTLRE IRES elements specifically function during the G 2 /M period (8, 37), the Ultrabithorax and Antennapedia IRES activities exhibit a high degree of developmental regulatio...
('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.
The tumor necrosis factors (TNF-alpha and lymphotoxin, or LT-alpha) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-alpha/TNF-alpha knockout mice and compared mice having one or two functional LT-alpha/TNF-alpha alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-alpha levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D-galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-alpha levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.
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