Biomedical subjects
Publications and source records attributed to J Beckers.
Fistula formation between a coronary artery bypass graft and a cardiac cavity is an uncommon finding. We describe a case of transcatheter occlusion of a coronary artery bypass graft to right ventricle fistula in a patient presenting with a subacute inferior myocardial infarction.
The Notch signaling pathway has pleiotropic functions during mammalian embryogenesis. It is required for the patterning and differentiation of the presomitic and somitic paraxial mesoderm and of the neural tube. We used DNA-chip expression profiling and 2D-gel electrophoresis combined with peptide mass fingerprinting to identify genes and proteins differentially regulated in E10.5 Dll1 (delta-like 1, Delta1) mutant embryos. The differential expression profiling approach identified 47 regulated transcripts and 40 differentially expressed proteins. The majority of these genes has until now not been associated with Notch signaling. Subsequent whole-mount in situ hybridization confirmed that a subset of the identified transcripts has restricted and distinct patterns of expression in E10.5 mouse embryos. For most genes these expression patterns were affected in the presomitic mesoderm, in differentiating somites of Dll1 mutant embryos and in the neural tube and cells differentiating from it. Similar effects were observed in embryos homozygous for the Headturner (Htu) and pudgy (pu) mutations, which are alleles of the Notch ligands Jag1 and Dll3. The regulated expression of a subset of the proteins was validated by immunoblots. Remarkably six of the proteins down-regulated in Dll1 mutant embryos are proteasome subunits. The large set of regulated genes identified in this differential expression profiling approach is an important resource for further functional studies.
A major advantage of the mouse model lies in the increasing information on its genome, transcriptome, and proteome, as well as in the availability of a fast growing number of targeted and induced mutant alleles. However, data from comparative transcriptome and proteome analyses in this model organism are very limited. We use DNA chip-based RNA expression profiling and 2D gel electrophoresis, combined with peptide mass fingerprinting of liver and kidney, to explore the feasibility of such comprehensive gene expression analyses. Although protein analyses mostly identify known metabolic enzymes and structural proteins, transcriptome analyses reveal the differential expression of functionally diverse and not yet described genes. The comparative analysis suggests correlation between transcriptional and translational expression for the majority of genes. Significant exceptions from this correlation confirm the complementarities of both approaches. Based on RNA expression data from the 200 most differentially expressed genes, we identify chromosomal colocalization of known, as well as not yet described, gene clusters. The determination of 29 such clusters may suggest that coexpression of colocalizing genes is probably rather common.
The Delta1 gene encodes one of the Notch ligands in mice. Delta1 is expressed during early embryogenesis in a complex and dynamic pattern in the paraxial mesoderm and neuroectoderm, and is essential for normal somitogenesis and neuronal differentiation. Molecular components thought to act in response to ligand binding and Notch activation have been identified in different species. In contrast, little is known about the transcriptional regulation of Notch receptors and their ligands. As a first step to identify upstream factors regulating Delta1 expression in different tissues, we searched for cis-regulatory regions in the Delta1 promoter able to direct heterologous gene expression in a tissue specific manner in transgenic mice. Our results show that a 4.3 kb genomic DNA fragment of the Delta1 gene is sufficient in a lacZ reporter transgene to reproduce most aspects of Delta1 expression from the primitive streak stage to early organogenesis. Using a minimal Delta1 promoter we also show that this upstream region contains distinct regulatory modules that individually direct tissue-specific transgene expression in subdomains of the endogenous expression pattern. It appears that expression in the paraxial mesoderm depends on the interaction of multiple positive and negative regulatory elements. We also find that at least some regulatory sequences required for transgene expression in subdomains of the neural tube have been maintained during the evolution of mammals and teleost fish, suggesting that part of the regulatory network that controls expression of Delta genes may be conserved.
Rib-vertebrae (rv) is an autosomal recessive mutation in mouse that affects the morphogenesis of the vertebral column. Axial skeleton defects vary along the anterior-posterior body axis, and include split vertebrae and neural arches, and fusions of adjacent segments. Here, we show that defective somite patterning underlies the vertebral malformations and altered Notch signaling may contribute to the phenotype. Somites in affected regions are irregular in size and shape, epithelial morphology is disrupted, and anterior-posterior somite patterning is abnormal, reminiscent of somite defects obtained in loss-of-function alleles of Notch signaling pathway components. Expression of Dll1, Dll3, Lfng and Notch1 is altered in rv mutant embryos, and rv and Dll1(lacZ), a null allele of the Notch ligand Delta1, genetically interact. Mice double heterozygous for rv and Dll1(lacZ), show vertebral defects, and one copy of Dll1(lacZ) on the homozygous rv background enhances the mutant phenotype and is lethal in the majority of cases. However, fine genetic mapping places rv into an interval on chromosome seven that does not contain a gene encoding a known component of the Notch signaling pathway.
When the developing embryo implants into the uterine wall, resident maternal immune cells may encounter antigens present on the fetal tissues. The nature and constituents of the ensuing maternal immune response, and its regulation, are of considerable interest in understanding normal and abnormal pregnancy. Here, we report the presence of natural killer (NK)1.1(+) alpha beta T cells in the murine periimplantation uterus. These cells account for a large portion of both the T-cell and natural killer cell populations in early pregnancy, while their numbers in the non-pregnant uterus and later in pregnancy are greatly reduced. Phenotypically, these NK1.1+ alpha beta T cells belong to a previously described subset of cells that bear a V alpha 14-J alpha 281-encoded T-cell receptor. Unlike other organs, where both CD4(+) and CD4(-)/CD8(-) NK1.1(+) alpha beta T cells are found, the placental/decidual population appears to be entirely CD4(-)/CD8(-). The V beta repertoire of the placental/decidual population is also altered from that of other organs, with a majority of cells expressing V beta 3. Together, these features suggest the possibility of local development. NK1.1(+) alpha beta T cells are known to recognize the class I-like CD1 molecule. Consistent with this association, we demonstrate CD1 expression by tissues within the pregnant uterus. Our findings define an additional organ-specific immune environment where NK1.1(+) alpha beta T cells may play a role, and continue to demonstrate the specialized nature of the maternal intrauterine immune system during pregnancy.
Somitic segmentation provides the framework on which the segmental pattern of the vertebrae, some muscles and the peripheral nervous system is established. Recent evidence indicates that a molecular oscillator, the 'segmentation clock', operates in the presomitic mesoderm (PSM) to direct periodic expression of c-hairy1 and lunatic fringe (l-fng). Here, we report the identification and characterisation of a second avian hairy-related gene, c-hairy2, which also cycles in the PSM and whose sequence is closely related to the mammalian HES1 gene, a downstream target of Notch signalling in vertebrates. We show that HES1 mRNA is also expressed in a cyclic fashion in the mouse PSM, similar to that observed for c-hairy1 and c-hairy2 in the chick. In HES1 mutant mouse embryos, the periodic expression of l-fng is maintained, suggesting that HES1 is not a critical component of the oscillator mechanism. In contrast, dynamic HES1 expression is lost in mice mutant for Delta1, which are defective for Notch signalling. These results suggest that Notch signalling is required for hairy-like genes cyclic expression in the PSM.
Genes of the HoxD complex have a crucial role in the morphogenesis of vertebrate limbs. During development, their functional domains are colinear with their genomic positions within the HoxD cluster such that Hoxd13 and Hoxd12 are necessary for digit development, whereas Hoxd11 and Hoxd10 are involved in making forearms. Mutational analyses of these genes have demonstrated their importance and illustrated the requirement for a precise control of their expression during early limb morphogenesis. To study the nature of this control, we have scanned the posterior part of the HoxD complex with a targeted reporter transgene and analyzed the response of this foreign promoter to limb regulatory influences. The results suggest that this regulation is achieved through the opposite effects of two enhancer elements which would compete with each other for interacting with nearby-located promoters. The physical position of a given gene within this genomic interval of opposite regulations might thus determine its final expression pattern. This model provides a conceptual link between the morphology of the future limb and the genetic organization of the Hox gene cluster, a translation of a genomic context into a morphogenetic topology.
Non-steady-state electrophoretic processes can be estimated by a repeated application of a steady-state model based on the electroneutrality equation, the modified version of Ohm's law, and the mass balances of the co- and counterions. With such a mathematical model, all parameters in sample zones in capillary zone electrophoresis (CZE) can be calculated. The relationships between the calculated parameters for sample zones in CZE, such as the pH, concentrations of co- and counterions, and the ratio E1m1/E2m2 versus the mobilities of both anionic and cationic analytes can be visualized in a SystChart, a set of eight panels, for a given background electrolyte (BGE). All properties of a zone, such as the fronting/tailing character and the question of peaks/dips can be read from such a SystChart. Applying n coions, n-1 discontinuities are present in such a SystChart, indicating the presence of system peaks applying that BGE. For BGEs with one coion, no system peaks (discontinuities) exist at moderate pH values. SystCharts calculated for BGEs with a low pH do show discontinuities, however, which indicates that system peaks are present in electropherograms applying BGEs at low pH. Experimentally, it is shown that system peaks are indeed present in electropherograms applying BGEs with one coion at low pH and the mobilities of the system peaks generally increase with decreasing pH. Hydrogen ions seem to act as a second coionic species. Of course, these system peaks are only visible in the UV signal if the BGE has UV-absorbing properties.
Cell-to-cell communication mediated by the evolutionary conserved Notch signalling pathway regulates cell fate decisions and patterning in various tissues in diverse organisms (Artavanis-Tsakonas et al., 1995, Science 268, 225-232). Signalling between neighboring cells is transduced by binding of DSL and Notch proteins which interact as ligand (DSL) and receptor (Notch). Mouse Delta1 (delta-like 1; Dll1) encodes one of the four known mammalian DSL proteins and is essential for normal somitogenesis and neuronal differentiation. Here, we describe Delta1 expression during organogenesis and fetal development using the highly sensitive histochemical detection of the lacZ gene product expressed from a targeted Delta1:lacZ knock-in allele (Dll1(lacZ)). We find that Delta1 is expressed in epithelial ducts of several organs, skeletal and smooth muscles, the central nervous system, as well as some sensory epithelia.
Extensive sequencing in the HoxD complex of several vertebrate species has revealed a set of conserved DNA sequences interspersed between neighboring Hox genes. Their high degree of conservation strongly suggested that they are used for regulatory purposes, a hypothesis that was largely confirmed by using "classical transgenesis" or in vivo mutagenesis through the embryonic stem (ES) cell technology. Here, we show that this is not always the case. We report that the deletion of a conserved regulatory sequence located in the HoxD complex gives different results, depending on the transgenic approach that was used. In "conventional" transgenesis, this sequence was necessary for proper expression in a subdomain of the developing limb. However, a deletion of this sequence in complexo did not confirm this effect, thereby creating an important discrepancy between the classical transgenic and the ES cell-based, targeted mutagenesis. This unexpected observation may show the limitations of the former technology. Alternatively, it could illustrate a redundancy in regulatory circuits and, thus, justify the combination of parallel strategies.
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Vertebrate Hoxd genes are essential determinants of limb morphogenesis. In order to understand the genetic control of their complex expression patterns, we have used a combined approach involving interspecies sequence alignments in parallel with transgenic analyses, followed by in vivo mutagenesis. Here, we report on the identification of a regulatory element that is located in the vicinity of the Hoxd-12 gene. While this element is well conserved in tetrapods, little sequence similarity was scored when compared to the cognate fish DNA. The regulatory potential of this region XI (RXI) was first assayed in the context of a Hoxd-12/lacZ reporter transgene and shown to direct reporter gene expression in posterior limb buds. A deletion of this region was generated by targeted mutagenesis in ES cells and introduced into mice. Analyses of animals homozygous for the HoxDRXI mutant allele revealed the function of this region in controlling Hoxd-12 expression in the presumptive posterior zeugopod where it genetically interacts with Hoxa-11. Downregulation of Hoxd-12 expression was also detected in the trunk suggesting that RXI may mediate a rather general function in the activation of Hoxd-12. These results support a model whereby global as well as local regulatory influences are necessary to build up the complex expression patterns of Hoxd genes during limb development.
Genes of the HoxD complex related to the Drosophila Abd-B gene are involved in the morphogenesis of vertebrate paired appendages. Hoxd-11, for instance, is necessary in combination with other Hox genes for the proper development of different parts of the tetrapod limbs. Sequence comparisons between the mouse, chicken, and zebrafish Hoxd-11 loci have revealed the conservation of several blocks of DNA sequence which may be of importance for the regulation of Hoxd-11 expression. We have used transgenic mice to show that one of these conserved elements specifically drives expression in a proximal-posterior part of developing forelimbs. Production of mice transgenic for a full fish Hoxd-11 construct as well as for mouse-fish Hoxd-11 chimeric constructs shows that the fish counterpart of this sequence is able to elicit expression in mouse forelimbs as well, though in a slightly different domain. However, this fish element requires the presence of the mouse promoter and does not work in its own context. These results are discussed in light of both the control of Hoxd gene expression during limb development and the use of a comparative interspecies approach to understand the regulation of genes involved in vertebrate development.
Herpesvirus saimiri is an oncogenic virus causing rapid T-cell lymphomas in New World primates and rabbits. Deletion analysis of one strain of H saimiri has indicated an open reading frame, StpA, necessary for oncongenicity in monkeys. We have investigated the function of StpA in tumor induction by the generation of transgenic mice. Expression of two different constructs caused the development of peripheral lymphomas. The infiltrating cells were of T-cell origin, expressing mainly the CD4 phenotype and restricted sets of V beta chains. Thus, StpA is not only necessary for the oncogenicity of Herpesvirus saimiri, but is also sufficient for the induction of peripheral pleomorphic T-cell lymphomas.
Between February 1991 and January 1992, elevated alanine aminotransferase (ALT) levels were observed in several hemodialysis patients in a dialysis center in Dendermonde, Belgium. By the end of 1992, 25 out of 68 patients had seroconverted for HCV antibodies. The HCV strains from 23 of these seroconverters were genotyped and classified as genotype 1b. Sequence analysis of the HCV Core region was carried out in 12 patients, 9 of whom were infected with a strain bearing a unique sequence motif as compared with the currently known HCV 1b strains. A new 5' UR/Core line probe assay was designed to screen for such variations. Twenty patients tested positively for this special sequence motif, while the other 3 showed the regular subtype 1b sequence. Phylogenetic analysis of the Core sequences further revealed that the latter three were neither related to the main special strain of the infection, nor to each other. These three strains could be traced to two patients already infected at the time of residence in other dialysis units and to one patient who already showed ALT elevations in 1989. Epidemiological studies revealed no traceable source for this outbreak. In conclusion, molecular analysis demonstrates nosocomial transmissions by a peculiar genotype 1b strain in a dialysis center. Three other genotype 1b strains were also present in the unit, but were not responsible for the outbreak.
Human C-reactive protein (hCRP) is a major acute-phase reactant in man. The regulation of the hCRP gene in transgenic mice is similar to that in humans. To map DNA regions required for the correct regulation of the hCRP gene, several constructs have been generated, and their expression in transgenic mice has been analyzed. Constructs lacking DNA regions surrounding the poly(A) site of the gene are not expressed either before or after induction in transgenic mice. Minimal regions 540 base pairs upstream and 1.2 kilobases downstream of the hCRP gene are sufficient for liver-specific expression. Extended 5'- and 3'-flanking regions are required to silence the expression prior to induction. Our findings demonstrate that regulatory sequences shown to confer inducible expression of the hCRP gene in hepatoma cell lines are insufficient in transgenic mice.
To investigate the role of herpesviral genes in tumourigenesis, transgenic mice were generated expressing STP-C, a transformation associated protein of the lymphoma inducing herpesvirus saimiri. Epithelial tumours developed in the salivary gland, pancreas, thymus and liver of transgenic mice within the first weeks of life. Thus, the target cells for tumour formation in the transgenic mice were surprisingly different from those of the herpesvirus from which the oncogene was derived. Our results identify STP-C as a herpesvirus oncogene sufficient for tumour induction without the cooperation of other viral gene products. Furthermore, the results demonstrate pleiotropic transforming capabilities of the STP-C oncogene and suggest that the specificity of lymphoma induction by the virus is determined by factors other than the oncogene itself.