Mutations in the T (brachyury) gene cause a novel syndrome consisting of sacral agenesis, abnormal ossification of the vertebral bodies and a persistent notochordal canal.

BACKGROUND: The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates. RESULTS: We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development. CONCLUSIONS: We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

Follistatin-like 1 in vertebrate development.

Follistatin-like 1 (Fstl1) is a member of the secreted protein acidic rich in cysteins (SPARC) family and has been implicated in many different signaling pathways, including bone morphogenetic protein (BMP) signaling. In many different developmental processes like, dorso-ventral axis establishment, skeletal, lung and ureter development, loss of function experiments have unveiled an important role for Fstl1. Fstl1 largely functions through inhibiting interactions with the BMP signaling pathway, although, in various disease models, different signaling pathways, like activation of pAKT, pAMPK, Na/K-ATPase, or innate immune responses, are linked to Fstl1. How Fstl1 inhibits BMP signaling remains unclear, although it is known that Fstl1 does not function through a scavenging mechanism, like the other known extracellular BMP inhibitors such as noggin. It has been proposed that Fstl1 interferes with BMP receptor complex formation and as such inhibits propagation of the BMP signal into the cell. Future challenges will encompass the identification of the factors that determine the mechanisms that underlie the fact that Fstl1 acts by interfering with BMP signaling during development, but through other signaling pathways during disease.

Ebstein's anomaly may be caused by mutations in the sarcomere protein gene MYH7.

Ebstein's anomaly is a rare congenital heart malformation characterised by adherence of the septal and posterior leaflets of the tricuspid valve to the underlying myocardium. Associated abnormalities of left ventricular morphology and function including left ventricular noncompaction (LVNC) have been observed. An association between Ebstein's anomaly with LVNC and mutations in the sarcomeric protein gene MYH7, encoding ß-myosin heavy chain, has been shown by recent studies. This might represent a specific subtype of Ebstein's anomaly with a Mendelian inheritance pattern. In this review we discuss the association of MYH7 mutations with Ebstein's anomaly and LVNC and its implications for the clinical care for patients and their family members.

Sox4 mediates Tbx3 transcriptional regulation of the gap junction protein Cx43.

Tbx3, a T-box transcription factor, regulates key steps in development of the heart and other organ systems. Here, we identify Sox4 as an interacting partner of Tbx3. Pull-down and nuclear retention assays verify this interaction and in situ hybridization reveals Tbx3 and Sox4 to co-localize extensively in the embryo including the atrioventricular and outflow tract cushion mesenchyme and a small area of interventricular myocardium. Tbx3, SOX4, and SOX2 ChIP data, identify a region in intron 1 of Gja1 bound by all tree proteins and subsequent ChIP experiments verify that this sequence is bound, in vivo, in the developing heart. In a luciferase reporter assay, this element displays a synergistic antagonistic response to co-transfection of Tbx3 and Sox4 and in vivo, in zebrafish, drives expression of a reporter in the heart, confirming its function as a cardiac enhancer. Mechanistically, we postulate that Sox4 is a mediator of Tbx3 transcriptional activity.

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components.

OBJECTIVE: In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. DESIGN: Rats were treated with AM or Dron and the expression of TRalpha 1, TRalpha 2, TRbeta 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. METHODS: Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRalpha 1, TRalpha 2, TRbeta 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. RESULTS: AM and Dron affected TR expression in the RA similarly by decreasing TRalpha 1 and beta 1 expression by about 50%. In the LVW, AM and Dron decreased TRbeta 1 and, interestingly, AM increased TRalpha 1. In the apex, AM also increased TRalpha 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. CONCLUSION: Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.

Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart.

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRalpha1, TRalpha2 and TRbeta1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRalpha1, TRalpha2 and TRbeta1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRbeta1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRalpha1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRalpha1 and TRbeta1 isoforms are co-expressed. We also found the heterogeneous expression of the TRalpha1, TRalpha2 and TRbeta1 isoforms in the developing mouse heart, which, in the case of the TRbeta1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRbeta1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRalpha1 and TRbeta1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.

Multiple transcriptional domains, with distinct left and right components, in the atrial chambers of the developing heart.

During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.

Development of the myocardium of the atrioventricular canal and the vestibular spine in the human heart.

To establish the morphogenetic mechanisms underlying formation and separation of the atrioventricular connections, we studied the remodeling of the myocardium of the atrioventricular canal and the extracardiac mesenchymal tissue of the vestibular spine in human embryonic hearts from 4.5 to 10 weeks of development. Septation of the atrioventricular junction is brought about by downgrowth of the primary atrial septum, fusion of the endocardial cushions, and forward expansion of the vestibular spine between atrial septum and cushions. The vestibular spine subsequently myocardializes to form the ventral rim of the oval fossa. The connection of the atrioventricular canal with the atria expands evenly. In contrast, the expression patterns of creatine kinase M and GlN2, markers for the atrioventricular and interventricular junctions, respectively, show that the junction of the canal with the right ventricle forms by local growth in the inner curvature of the heart. Growth of the caudal portion of the muscular ventricular septum to make contact with the inferior endocardial cushion occurs only after the canal has expanded rightward. The atrioventricular node develops from that part of the canal myocardium that retains its continuity with the ventricular myocardium.

Regulation of the spatiotemporal pattern of expression of the glutamine synthetase gene.

Glutamine synthetase, the enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine, is expressed in a tissue-specific and developmentally controlled manner. The first part of this review focuses on its spatiotemporal pattern of expression, the factors that regulate its levels under (patho)physiological conditions, and its role in glutamine, glutamate, and ammonia metabolism in mammals. Glutamine synthetase protein stability is more than 10-fold reduced by its product glutamine and by covalent modifications. During late fetal development, translational efficiency increases more than 10-fold. Glutamine synthetase mRNA stability is negatively affected by cAMP, whereas glucocorticoids, growth hormone, insulin (all positive), and cAMP (negative) regulate its rate of transcription. The signal transduction pathways by which these factors may regulate the expression of glutamine synthetase are briefly discussed. The second part of the review focuses on the evolution, structure, and transcriptional regulation of the glutamine synthetase gene in rat and chicken. Two enhancers (at -6.5 and -2.5 kb) were identified in the upstream region and two enhancers (between +156 and +857 bp) in the first intron of the rat glutamine synthetase gene. In addition, sequence analysis suggests a regulatory role for regions in the 3' untranslated region of the gene. The immediate-upstream region of the chicken glutamine synthetase gene is responsible for its cell-specific expression, whereas the glucocorticoid-induced developmental appearance in the neural retina is governed by its far-upstream region.

Radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) content of adult rat liver.

1. Rabbit antiserum was raised against purified carbamoyl-phosphate synthase (ammonia) from rat liver. 2. The antiserum proved to be specific in double-diffusion test and reacted in an in situ immunohistochemical test on rat liver proteins fractionated on a sodium dodecyl sulphate polyacrylamide gel only in the region where carbamoyl-phosphate synthase (ammonia) migrated. 3. This antiserum was used for setting up a radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) in cetyltrimethylammonium bromide extracts of rat liver. To obtain reproducible results in this assay it was necessary to treat the unlabelled ligand with sodium dodecyl sulphate and dithiothreitol. This treatment led to a large increase in the percentage of labelled ligand displaceable by added unlabelled ligand. 4. Radioimmunochemical determination showed that adult rat liver (3-month old) contains 5.5 mg carbamoyl-phosphate synthase (ammonia) protein per gram wet weight.

Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro.

1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. The products of synthesis are short (Mr less than 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate/urea, guanidinium . HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase.

Histones of Xenopus laevis erythrocytes. Purification and characterization of the lysine-rich fractions.

Lysine-rich histones have been isolated from the terminally differentiated erythrocytes of Xenopus laevis. Three major proteins have been separated by ion-exchange chromatography. These proteins have been characterized by electrophoresis, amino acid analysis and immunochemical techniques. It is concluded that two 'typical' lysine-rich subfractions are present in Xenopus erythrocytes and, in addition, a serine-rich histone, that shares no common antigenic determinants with the other lysine-rich histones.

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl)phthalate.

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.

cDNA sequence of the long mRNA for human glutamine synthase.

Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp.

Developmental and hormonal regulation of carbamoyl-phosphate synthase gene expression in rat liver: evidence for control mechanisms at different levels in the perinatal period.

Carbamoyl-phosphate synthase gene expression is found to be primarily regulated by conditions that enhance hepatic glucocorticosteroid levels (hormone injections) and cyclic AMP levels (induction of diabetes). After birth, changes in the level of carbamoyl-phosphate synthase protein follow changes in the level of carbamoylphosphate synthase mRNA, suggesting a pretranslational control mechanism. In fetal rats, carbamoyl-phosphate synthase gene expression is regulated by the same factors as in adults. However, both the level to which carbamoyl-phosphate synthase mRNA can accumulate and the extent to which mRNA can be translated appear to be limited, indicating control mechanisms at the pretranslational and translational level. Finally, in the immediate postnatal period, a transient but pronounced decrease in the rate of degradation of carbamoyl-phosphate synthase protein may play a role in the accumulation of the enzyme.

The immunohistochemical localization of the non-specific lipid transfer protein (sterol carrier protein-2) in rat small intestine enterocytes.

A 13 kDa protein was isolated from rabbit small intestine brush-border membrane vesicles that was postulated to be involved in intestinal phosphatidylcholine (PC) and cholesterol uptake. This protein has cholesterol and PC-transfer activity in vitro (Turnhofer, H. et al. (1991) Biochim. Biophys. Acta 1064, 275-286) and has a molecular mass and isoelectric point similar to that of the non-specific lipid transfer protein (nsL-TP, identical to sterol carrier protein-2). In addition, the first 28 N-terminal amino acid residues of the 13 kDa protein are nearly identical to nsL-TP from different species (Lipka, G. et al. (1995) J. Biol. Chem. 270, 5917-5925). In view of its possible role in intestinal lipid absorption, the localization of nsL-TP in rat small intestine was investigated using immunohistochemistry and immunoblotting. It is shown that nsLTP is predominantly localized in a subapical zone of the enterocyte but not in the brush-border membrane, thereby excluding a role in lipid uptake of this protein at the level of the plasma membrane. nsL-TP co-localized with the peroxisomal marker PMP70, underscoring earlier observations that nsL-TP is a peroxisomal protein. nsL-TP was found to be present along the entire length of the small intestine. The 58 kDa cross-reactive protein that was recently identified as a peroxisomal thiolase was shown to be present only in a small segment approximately halfway down the jejunum. The close apposition of the peroxisomes with the apical membrane and the discrete distribution of the 58 kDa protein may indicate that these organelles play a role in the intracellular processing of absorbed lipids.

Reciprocal regulation of glutamine synthetase and carbamoylphosphate synthetase levels in rat liver.

In glucocorticosteroid-treated diabetic rats, glutamine synthetase enzyme levels in the liver are decreased 3-fold, whereas carbamoylphosphate synthetase enzyme levels are increased 2.3-fold. In addition, immunohistochemistry shows that under these conditions the distribution of carbamoylphosphate synthetase is expanded over the entire liver acinus, whereas that of glutamine synthetase is reduced to very few cells bordering the central (terminal hepatic) veins. Using a newly isolated cDNA complementary to rat liver glutamine synthetase mRNA, we show that this regulation is primarily effected at a pretranslational level. (For data on carbamoylphosphate synthetase mRNA levels, see De Groot et al. (1986) Biochim. Biophys. Acta 866, 61-67). Furthermore, hybridization studies show stimulatory effects of both glucocorticosteroids and thyroid hormone on the glutamine synthetase mRNA level. Attempts to localize glutamine synthetase mRNA within the liver acinus by selective destruction of the pericentral zone failed because of generally low levels of liver mRNAs after CCl4 poisoning. In contrast to the situation after birth, significantly higher glutamine synthetase mRNA/enzyme activity ratios in fetal rat liver point to the presence of additional post-transcriptional control mechanisms before birth. These findings complement similar observations on carbamoylphosphate synthetase gene expression (De Groot et al. (1986) Biochim. Biophys. Acta 866, 61-67).

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.