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V M Christoffels

Publications and source records attributed to V M Christoffels.

17 recordsLinked to original sources
PubMed2001

Pitx2 expression defines a left cardiac lineage of cells: evidence for atrial and ventricular molecular isomerism in the iv/iv mice.

The homeobox gene Pitx2 has been characterized as a mediator of left-right signaling in heart, gut, and lung morphogenesis. However, the relationship between the developmental role of Pitx2 and its expression pattern at the organ level has not been explored. In this study we focus on the role of Pitx2 in heart morphogenesis. Chicken Pitx2 transcripts are present in the left portion of the cardiac crescent and in the left side of the heart tube. Through looping Pitx2 is present in the left atrium, in the ventral portion of the ventricles and in the left-ventral part of the outflow tract. Mouse Pitx2 shows a similar developmental profile of expression. To test whether Pitx2 represents a lineage marker we have tagged the left portion of the chicken cardiac tube with fluorescent DiD. Labeled cells were found at HH16 in the left atrium and in the ventral region of the ventricles and the outflow tract. In the iv/iv mouse model of cardiac heterotaxia Pitx2 was abnormally expressed in the atrial and in the ventricular chambers. Furthermore, altered Pitx2 expression correlated with the occurrence of DORV. Our data reveal the existence of molecular isomerism not only in the atrial, but also in the ventricular compartment of the heart.

Animals↗
PubMed2001

Expression of Irx6 during mouse morphogenesis.

Iroquois (Irx) proteins comprise a family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissues in both vertebrates and invertebrates. The six murine Irx genes are organized in two clusters, each consisting of three genes. Irx1, Irx2 and Irx4 belong to the IrxA cluster on chromosome 13, whereas Irx3, Irx5 and Irx6, comprising the IrxB cluster, are located on chromosome 8 (Peters et al., Genome Res. 10 (2000) 1453). Developmental expression patterns have so far only been reported for five Irx genes. Here, we investigated the expression pattern of Irx6 during mouse morphogenesis and early organogenesis. The pattern was found to be much more restricted compared to the other five Irx genes, and its level of expression was much lower.

Animals↗
PubMed2001

Gene and cluster-specific expression of the Iroquois family members during mouse development.

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.

Animals↗
PubMed2001

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.

Animals↗
PubMed2000

A single regulatory module of the carbamoylphosphate synthetase I gene executes its hepatic program of expression.

A 469-base pair (bp) upstream regulatory fragment (URF) and the proximal promoter of the carbamoylphosphate synthetase I (CPS) gene were analyzed for their role in the regulation of spatial, developmental, and hormone-induced expression in vivo. The URF is essential and sufficient for hepatocyte-specific expression, periportal localization, perinatal activation and induction by glucocorticoids, and cAMP in transgenic mice. Before birth, the transgene is silent but can be induced by cAMP and glucocorticoids, indicating that these compounds are responsible for the activation of expression at birth. A 102-bp glucocorticoid response unit within the URF, containing binding sites for HNF3, C/EBP, and the glucocorticoid receptor, is the main determinant of the hepatocyte-specific and hormone-controlled activity. Additional sequences are required for a productive interaction between this minimal response unit and the core CPS promoter. These results show that the 469-bp URF, and probably only the 102-bp glucocorticoid response unit, functions as a regulatory module, in that it autonomously executes a correct spatial, developmental and hormonal program of CPS expression in the liver.

3T3 Cells↗
PubMed2000

Patterning the embryonic heart: identification of five mouse Iroquois homeobox genes in the developing heart.

We isolated cDNAs of mouse Iroquois-related homeobox genes Irx1, -2, -3, -4, and -5 and characterized their patterns of expression in the developing heart. Irx1 and Irx2 were found to be expressed specifically in the ventricular septum from the onset of its formation onward. In fetal stages, the expression of both genes appeared to gradually become confined to the myocardium of the atrioventricular bundle and bundle branches of the forming ventricular conduction system. Irx3 was found to be expressed specifically in the trabeculated myocardium of the ventricles. Irx4 expression was observed in a segment of the linear heart tube and the atrioventricular canal and ventricular myocardium including the inner curvature after looping, resembling the pattern of MLC2V. Transcripts for Irx5 were detected specifically in the endocardium lining the ventricular and atrial working myocardium that also expressed von Willebrand factor, but were absent from the endocardium of the endocardial cushions, i.e., the atrioventricular canal, inner curvature, and outflow tract. The spatiodevelopmental pattern of Irx5 matched that of ANF, a marker for the forming working myocardium of the chambers. Taken together, all members of the Irx gene family were found to be expressed in highly specific patterns in the developing mouse heart, suggesting a critical role in the specification of the distinct components of the four-chambered heart.

Amino Acid Sequence↗
PubMed2000

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium.

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.

Animals↗
PubMed2000

Chamber formation and morphogenesis in the developing mammalian heart.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and beta-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of "primary" myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.

Animals↗
PubMed1999

A mechanistic model for the development and maintenance of portocentral gradients in gene expression in the liver.

In the liver, genes are expressed along a portocentral gradient. Based on their adaptive behavior, a gradient versus compartment type, and a dynamic versus stable type of gradient have been recognized. To understand at least in principle the development and maintenance of these gradients in gene expression in relation to the limited number of signal gradients, we propose a simple and testable model. The model uses portocentral gradients of signal molecules as input, while the output depends on two gene-specific variables, viz., the affinity of the gene for its regulatory factors and the degree of cooperativity that determines the response in the signal-transduction pathways. As a preliminary validity test for its performance, the model was tested on control and hormonally induced expression patterns of phosphoenolpyruvate carboxykinase (PCK), carbamoylphosphate synthetase I (CPS), and glutamine synthetase (GS). Affinity was found to determine the overall steepness of the gradient, whereas cooperativity causes these gradients to steepen locally, as is necessary for a compartment-like expression pattern. Interaction between two or more different signal gradients is necessary to ensure a stable expression pattern under different conditions. The diversity in sequence and arrangement of related DNA-response elements of genes appears to account for the gene-specific shape of the portocentral gradients in expression. The feasibility of testing the function of hepatocyte-specific DNA-response units in vivo is demonstrated by integrating such units into a ubiquitously active promoter/enhancer and analyzing the pattern of expression of these constructs in transgenic mice.

Animals↗
PubMed1999

Involvement of a cis-acting element in the suppression of carbamoyl phosphate synthetase I gene expression in the liver of carnitine-deficient mice.

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (-12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from -161 to +138) and enhancer (469 bp around -6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5'-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein-protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.

Animals↗
PubMed1998

Hypoglycemia-associated hyperammonemia caused by impaired expression of ornithine cycle enzyme genes in C/EBPalpha knockout mice.

Ammonia produced by amino acid metabolism is detoxified through conversion into urea by the ornithine cycle in the liver, whereas carbon skeletons of amino acids are converted to glucose by gluconeogenic enzymes. Promoter and enhancer sequences of several genes for ornithine cycle enzymes interact with members of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family. Disruption of the C/EBPalpha gene in mice causes hypoglycemia associated with the impaired expression of gluconeogenic enzymes. Here we examined the expression of ornithine cycle enzyme genes in the livers of C/EBPalpha-deficient mice. mRNA levels for the first, third, fourth, and fifth enzymes of five enzymes in the cycle were decreased in C/EBPalpha-deficient mice. Protein levels for the first, second, fourth, and fifth enzymes were also decreased. In situ hybridization analysis revealed that the enzyme mRNAs were distributed normally in the periportal region but were disordered in C/EBPalpha-deficient mice with relatively higher mRNA levels in the midlobular region. Blood ammonia concentrations in the mutant mice were severalfold higher than in wild-type mice. Thus, C/EBPalpha is crucial for ammonia detoxification by ornithine cycle enzymes and for coordination of gluconeogenesis and urea synthesis.

Ammonia↗
PubMed1998

Glucocorticoid receptor, C/EBP, HNF3, and protein kinase A coordinately activate the glucocorticoid response unit of the carbamoylphosphate synthetase I gene.

A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPalpha-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPbeta knockout mice and in HNF3alpha and -gamma double-knockout mice. (The role of HNFbeta could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.

Animals↗
PubMed1996

The upstream regulatory region of the carbamoyl-phosphate synthetase I gene controls its tissue-specific, developmental, and hormonal regulation in vivo.

The carbamoyl-phosphate synthetase I gene is expressed in the periportal region of the liver, where it is activated by glucocorticosteroids and glucagon (via cyclic AMP), and in the crypts of the intestinal mucosa. The enhancer of the gene is located 6.3 kilobase pairs upstream of the transcription start site and has been shown to direct the hormone-dependent hepatocyte-specific expression in vitro. To analyze the function of the upstream region in vivo, three groups of transgenic mice were generated. In the first group the promoter drives expression of the reporter gene, whereas the promoter and upstream region including the far upstream enhancer drive expression of the reporter gene in the second group. In the third group the far upstream enhancer was directly coupled to a minimized promoter fragment. Reporter-gene expression was virtually undetectable in the first group. In the second group spatial, temporal, and hormonal regulation of expression of the reporter gene and the endogenous carbamoyl-phosphate synthetase gene were identical. The third group showed liver-specific periportal reporter gene expression, but failed to activate expression in the intestine. These results show that the upstream region of the carbamoyl-phosphate synthetase gene controls four characteristics of its expression: tissue specificity, spatial pattern of expression within the liver and intestine, hormone sensitivity, and developmental regulation. Within the upstream region, the far upstream enhancer at -6.3 kilobase pairs is the determinant of the characteristic hepatocyte-specific periportal expression pattern of carbamoyl-phosphate synthetase.

Animals↗
PubMed1995

The far-upstream enhancer of the carbamoyl-phosphate synthetase I gene is responsible for the tissue specificity and hormone inducibility of its expression.

The role of the proximal promoter and the far-upstream enhancer in the hepatocyte-specific and hormonal regulation of the carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes, hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5'-untranslated region of the mRNA is a major, non-tissue specific stimulator of expression in FTO-2B hepatoma cells, acting at the post-transcriptional level. A 469-base pair DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral thymidine kinase promoter. Sequences similar to a cyclic AMP-responsive element and a glucocorticosteroid-responsive element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity.

Animals↗
PubMed1994

A gene of Acinetobacter calcoaceticus BD413 encodes a periplasmic peptidyl-prolyl cis-trans isomerase of the cyclophilin sub-class that is not essential for growth.

Downstream of the Acinetobacter calcoaceticus estA gene, encoding a cell-bound esterase, an open reading frame (orf) was identified, which may encode a protein with a mass of 20.4 kDa. This protein shows extensive similarity to both prokaryotic and eukaryotic peptidyl-prolyl cis-trans isomerases (PPIases) of the cyclophilin sub-class, especially to the periplasmic rotamase (RotA) of Escherichia coli. A putative signal sequence suggests that the product of the Acinetobacter gene, we termed rotA, is located outside the cytoplasm. Transcription of the gene is initiated from a promoter, just upstream of the rotA orf. The observation that two A. calcoaceticus rotA deletion mutants display no apparent mutant phenotype, suggests that this PPIase is not essential for growth of the organism. These mutants, to our knowledge, are the first prokaryotic PPIase mutants reported.

Acinetobacter calcoaceticus↗
PubMed1993

Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases.

Acinetobacter calcoaceticus BD413, when grown in batch culture in nutrient broth, produces both extracellular lipase activity and cell-bound esterase activity during and after the transition between exponential growth and the stationary phase. From a library of A. calcoaceticus DNA in Escherichia coli, plasmids were isolated that enabled E. coli to grow on media with tributyrin as the sole carbon source. Assays with model substrates classified the product of the cloned gene as an esterase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to contain one open reading frame, termed estA, which encodes a protein of 40.0 kDa. The amino acid sequence of this protein shows homology to a number of lipolytic enzymes, most notably to esterases. Deletion of estA only partially abolished cell-bound esterase activity in A. calcoaceticus, indicating that BD413 forms at least two esterases. Both esterases show the same temporal regulation of expression. beta-Galactosidase activity was measured in strains in which a promoterless lacZ gene was inserted into estA. Induction of lacZ expression in these strains also occurred at the end of exponential growth in batch cultures, indicating that production of the esterase is regulated at the genetic level.

Acinetobacter calcoaceticus↗