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In the first part of our review of cardiac development,1 we discussed the initial changes involved in transformation of the heart forming regions of the embryo into the great veins, the atrial and ventricular chambers, and the arterial trunks. We showed that this first part of cardiac development could be divided into phases of formation of the primary myocardial tube, looping of the tube, during which additional parts are added that give the future compartments their definitive topography, and the assembly of these components into the cardiac chambers and arterial trunks. In this second review, we discuss the processes that complete the separation of the two sides of the definitive heart, for the most part involving septation of the parts of the primary tube not themselves directly involved in formation of the chamber-specific compartments of the atriums and ventricles. In this respect, when concluding our first review, we explained how the arterial trunks were also formed by septation within the solitary outflow tract of the primary heart tube. We also showed, however, that subsequent to formation of the two arterial trunks, there was disappearance of the cushions that initially divided them. Thus, in the definitive heart, the proximal parts of the aorta and pulmonary trunk, along with the sinuses of the arterial roots and the subpulmonary infundibulum, possess their own discrete walls, separated by extra-cardiac space. Although septation by fusion of endocardial cushions will be a topic included in this second review, septation and separation of the outflow tracts is sufficiently complicated to require special treatment. Because of this, we will devote a third review specifically to the mechanisms underscoring the remodelling of the outflow tracts. In this review, therefore, we will confine our considerations to the formation of the atrial, atrioventricular, and ventricular septal structures. As will become …

Article de synthese sur le metabolisme de l'azote et la synthese d'uree chez les mammiferes. Degradation des proteines. Transport des acides-amines entre les organes et a travers la membrane plasmique des hepatocytes. Cycle de l'ornithine et distribution des enzymes de ce cycle dans le foie. Ontogenese de la synthese d'uree. Pathophysiologie de l'hyperammonemie

In recent years, the rat genes encoding glutamate dehydrogenase (GLUD; Das et al. 1993), glutamine synthetase (glutamateammonia ligase, GLUL; van de Zande et al. 1990) and carbamoylphosphate synthetase 1 (CPS; Van den Hoff et al. 1995) have been isolated. These enzymes have important functions in ammonia metabolism, and each of them is encoded by a single gene. GLUD (E.C. 1.4.1.3) catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia, using NAD+ or NADP+ as cofactor. GLUL (E.C. 6.3.1.2) catalyzes the synthesis of glutamine from glutamate, thereby hydrolyzing ATP to ADP. CPS (E.C. 6.3.4.16) is the first and rate-determining enzyme of the ornithine cycle and catalyzes the production of carbamoylphosphate from ammonia, bicarbonate and ATP. In the present communication, we mapped the position of these three genes in the rat by FISH and by somatic cell hybrids. In addition, we determined the chromosomal location of the human GLUL gene and of the mouse, Cps1 gene by FISH. For the FISH mapping of the human glutamine synthetase gene (also called glutamate-ammonia ligase, approved human gene symbol GLUL), a human cDNA (total 2738 bp in pBluescript; Van den Hoff et al. 1991) was used. As shown in Fig.1a, the gene could be unequivocally mapped to Chromosome (Chr) (HSA) 1, band q25. The three genes were mapped in the rat both with a rat-mouse somatic cell hybrid panel (Szpirer et al. 1984; Klinga Levan et al. 1993) and with FISH. For the mappings with the cell hybrid panel, cDNA probes of approximately 1000 bp from the 38 ends of the genes were used (Glul 1111 bp, van de Zande et al. 1990; Glud1 957 bp, Das et al. 1989; Cps1 883 bp, De Groot et al. 1986). The probes were labeled with radioactivity by use of a-P-CTP and the random priming method. They were subsequently hybridized to filters containing 15 mg of genomic DNA from each hybrid restricted with EcoRI or BamHI. In each case the rat hybridizing fragments could be distinguished from the mouse bands, and the genes were assigned as follows: Glud1 to rat Chr (RNO) 16, Cps1 to RNO9, and Glul to RNO13. In the rat, the FISH results corroborated and refined the findings from the somatic cell hybrid panel. Longer probes are preferred in FISH analysis, and for the regional mapping of the Glul gene with FISH, two genomic clones were used (pgGS2, 5000 bp including exons 2–6, and pgGS4, 4500 bp including exon 1; van de Zande et al. 1990). Both probes gave very clear signals at the same chromosomal location, and rat Glul could be sublocalized to RNO13q22 (Fig. 1b). The rat G-band nomenclature is according to Levan (1974); for an updated recent version of the idiogram, see RATMAP database (URL http://ratmap.gen.gu.se/ratmap/WWW Nomen/RNOIdiogrRev96new.GIF). For the mapping of rat Glud1, two clones of genomic DNA (pgGDH6d, 6700 bp containing exon 1, and pgGDH2u, 5800 bp containing exons 8–12; Das et al. 1993) were used. The results from each of the probes were the same, and Glud1 could be sublocalized to RNO16p16 (Fig. 1c). Since RNO16 is a metacentric chromosome in which both chromosome arms have very similar stainability and banding pattern except in optimal metaphases, we wanted to check our conclusion with respect to which chromosome arm carried the Glud1 gene. Sasaki and associates (1994) have published excellent pictures of FISH mapping of the Atp7b gene (Wilson Disease gene homolog), and convincingly assigned this gene to RNO16q12.3. We used the same probe (designated pWD4) in simultaneous hybridizations with the Glud1 probe and could show that the two genes were located on opposite chromosome arms (Fig. 1d), thus verifying our conclusion that Glud1 is at RNO16p16. For the FISH mapping of the rat carbamoyl-phosphate synthetase gene (Cps1), a full-length cDNA probe (cCPSf.1, 5500 bp; De Groot et al. 1986) was used. The findings corroborated the previous hybrid panel mapping, and the Cps1 gene could be sublocalized to 9q34 (Fig. 1e). Since the Cps1 gene had not been mapped in the mouse, we attempted to map it by FISH with the rat cDNA probe. This worked out well, and the mouse Cps1 gene could be assigned to mouse Chr (MMU) 1, band C3 (Fig. 1f). Comparative mapping shows that the human GLUL gene is comprised in a region spanning bands HSA 1q22–1q42 and containing 11 human genes for which there are homologous rat genes on RNO13 (Table 1). Only seven of these genes have been mapped also in the mouse, but they are all situated distally in MMU1 (spanning about 26 cM from C4bp at map position 68 to Atp1a2 at map position 94; mouse data from Mouse Genome Database, MGD). The human homolog of the rat Glud1 gene is located at HSA 10q23.3, and the mouse homolog is on MMU14. Glud1 is included in a group of three genes (also comprising Rbp3 and Sftp1) that is conserved on these chromosomes. In contrast, the human and mouse genes homologous to the Atp7b gene on the long arm of RNO16 are on HSA13 and MMU8, respectively, as is the Atp4b gene, which is also located on RNO16. Thus, it looks as if RNO16 resulted from the fusion of two chromosome segments that are on separate chromosomes in both humans and mice, and, therefore, occurred after the separation of the rat lineage from human and mouse lineages. The human homolog of Cps1 is on HSA2q33–36. In total, there are nine genes on HSA2q (spanning the segment 2q32–2q37) that have their homologs on RNO9 (Table 1). A corresponding segment in the mouse is located on MMU1 (spanning 31 cM from Slc9a2 at map position 21 to Ugt1a1 and Akp3 at position 52). We have pointed out earlier that Correspondence to: G. Levan Mammalian Genome 8, 362–364 (1997).

The development of the atrial chambers in the human heart was investigated immunohistochemically using a set of previously described antibodies. This set included the monoclonal antibody 249-9G9, which enabled us to discriminate the endocardial cushion-derived mesenchymal tissues from those derived from extracardiac splanchnic mesoderm, and a monoclonal antibody recognizing the B isoform of creatine kinase, which allowed us to distinguish the right atrial myocardium from the left. The expression patterns obtained with these antibodies, combined with additional histological information derived from the serial sections, permitted us to describe in detail the morphogenetic events involved in the development of the primary atrial septum (septum primum) and the pulmonary vein in human embryos from Carnegie stage 14 onward. The level of expression of creatine kinase B (CK-B) was found to be consistently higher in the left atrial myocardium than in the right, with a sharp boundary between high and low expression located between the primary septum and the left venous valve indicating that the primary septum is part of the left atrial gene-expression domain. This expression pattern of CK-B is reminiscent of that of the homeobox gene Pitx2, which has recently been shown to be important for atrial septation in the mouse. This study also demonstrates a poorly appreciated role of the dorsal mesocardium in cardiac development. From the earliest stage investigated onward, the mesenchyme of the dorsal mesocardium protrudes into the dorsal wall of the primary atrial segment. This dorsal mesenchymal protrusion is continuous with a mesenchymal cap on the leading edge of the primary atrial septum. Neither the mesenchymal tissues of the dorsal protrusion nor the mesenchymal cap on the edge of the primary septum expressed the endocardial tissue antigen recognized by 249-9G9 at any of the stages investigated. The developing pulmonary vein uses the dorsal mesocardium as a conduit to reach the primary atrial segment. Initially, the pulmonary pit, which will becomes the portal of entry for the pulmonary vein, is located along the midline, flanked by two myocardial ridges. As development progresses, tissue remodeling results in the incorporation of the portal of entry of the pulmonary vein in left atrial myocardium, which is recognized because of its high level of creatine. Closure of the primary atrial foramen by the primary atrial septum occurs as a consequence of the fusion of these mesenchymal structures.

The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium.

To resolve an apparent discrepancy in the developmental appearance of glutamine synthetase (GS) protein in rat [Gaasbeek Janzen et al. (1987) J. Histochem, Cytochem., 35:49-54] and mouse [Bennett et al. (1987) J. Cell Biol., 105:1073-1085] liver, we have investigated its expression during liver development in the mouse and compared it with that of carbamoylphosphate synthetase I (CPS). The expression of glutamate dehydrogenase was used as a marker to identify all hepatocytes in these strongly hematopoietic livers. GS protein accumulation starts in mouse hepatocytes at embryonic day (ED) 15. The first hepatocytes in which the enzyme accumulates were found around the major hepatic veins. CPS protein was found to accumulate in mouse hepatocytes from ED 13 onward: first, at the center of the median and lateral lobes, but temporarily not at the periphery of these lobes and not at the caudate lobe. The initial phase of accumulation of GS and CPS protein was characterized by a heterogeneity in enzyme content between hepatocytes. By ED 17, both enzymes were detectable in all hepatocytes at the center of the median and lateral lobes. This event marked the onset of the development of the complementary distribution of the enzymes typical of zonal heterogeneity in the adult mammalian liver. However, during the perinatal period, the pericentral hepatocytes temporarily accumulated CPS protein. We also observed heterochrony between species in the appearance of CPS protein in the small intestine.

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetic in difficult imaging situations, such as low fluorescence signals and high absorbance signals.

Previous studies showed that acetylcholinesterase (AChE) activity is present in the downstream (arterial) part of the embryonic chick and rat heart, but its functional significance was unclear. To establish whether other components of a cholinergic signal-transduction pathway are present in the embryonic heart, we localised the mRNAs encoding choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the muscarinic receptor isoforms (mAChRs; m1-m5).Messenger RNA detection and localisation by in situ hybridisation and reverse transcriptase-polymerase chain reaction were employed.Expression of ChAT and AChE mRNAs was observed from 15 embryonic days onward in the neural tissue covering the dorsocranial wall of the atria. Muscarinic receptors (m1, m2, m4) were observed at the same localisation as AChE and ChAT mRNAs, both during embryogenesis and after birth. In addition, m1 and m4 mAChRs showed a low level of expression in the atrial myocardium during the fetal period. No expression of the m3 or the m5 mAChRs was observed in or near the embryonic hearts. ChAT, AChE, and mAChRs (m1, m2, m4) mRNAs always colocalised in the cardiac ganglia. However, none of these mRNAs was found at a detectable level in the outflow tract and/or the ventricular trabeculations.The AChE activity in the arterial part of the embryonic heart is probably synthesised elsewhere and subserves a function different from the hydrolysis of locally produced acetylcholine.

The valve function of the ureterovesical-junction (UVJ) is responsible for protection of the low pressure upper urinary tract from the refluxing of urine from the bladder. Controversy about the microanatomy of the human ureterovesical-junction persists.Ten (3 male and 7 female) fresh cadaveric bladders (mean age 70 years old) were studied. The bladders were fixed within 24 hours postmortem, frozen, and serially sectioned. Acetyl- and butyryl- (nonspecific) cholinesterase activity were visualised as described by Karnovsky and Roots. The three-dimensional distribution of the different muscle groups participating in the formation of the UVJ was reconstructed.Three different muscle groups were identified: (1) the detrusor muscle and the deep trigone were mainly acetylcholinesterase-positive, (2) the inner and outer layer of the ureteric muscle were butyrylcholinesterase-positive and merged into a single longitudinal layer at the level of the UVJ and form the superficial trigone distally to the ureteric orifices, and (3) the muscularis mucosae is a discontinuous butyrylcholinesterase-positive layer in the bladder that is absent from the trigone. No evidence of any muscular connection was found between the ureter and bladder musculature.The anatomy of the UVJ as observed by us suggests the following model of the ureteric peristalsis. The urine bolus arrives in the ureteric lumen at the UVJ level. The ureter can only shorten its length, slides freely in its tunnel, and discharges the urine bolus in the bladder cavity. Ureteric constriction due to the peristalsis and thickening of the contracted portion of the ureter prevents the upstream leakage. Distal spreading of the ureteric "peristalsis" in the superficial trigone increases the submucosal ureteric length and prevents reflux.

Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene was isolated and characterized. The gene is 110 kb in length and contains 38 exons. The basal promoter comprises the first 161 nucleotides upstream of the transcription-initiation site. Determination of the state of methylation of the 5' portion of the gene identified a CCGG sequence at -6.3 kb that is selectively demethylated in adult tissues which express CbmPS. This site remains methylated before birth, however, despite recruitment of all hepatocytes for CbmPS synthesis, indicating that its demethylation is a consequence of rather than a condition for expression of CbmPS. Transient expression assays revealed that the region surrounding the CCGG site at 6.3 kb functions as an enhancer. In FTO-2B hepatoma cells and Rat-1 fibroblasts, this enhancer is constitutively active when tested in front of the basal viral thymidine kinase promoter. When tested in front of the basal CbmPS promoter in hepatoma cells, however, the activity of this enhancer is dependent on the presence of glucocorticoids. In Rat-1 fibroblasts, the presence of both glucocorticoids and cyclic AMP is required for full activity, suggesting that the hepatocyte-specific expression of CbmPS is related to tissue-specific differences in the sensitivity to cyclic AMP. Matrix-attachment regions (MAR) are present upstream and downstream of the CbmPS gene. The downstream MAR defines the 3' boundary of the gene. The upstream MAR is located midway between the basal promoter and the enhancer, and may function as a hinge point to facilitate the positioning of the enhancer in the vicinity of the basal promoter.

Many enzymes are distributed heterogeneously within the liver lobule. The factors that play a determining role in the establishment and maintenance of these heterogeneous expression patterns have not yet been identified. To investigate whether the composition of the afferent hepatic blood plays a crucial role in the maintenance of the heterogeneity of gene expression of the parenchymal cells within the liver lobule, we changed the source of the afferent hepatic blood by microsurgical techniques. Three different groups of experimental animals were studied: rats with livers that are perfused with portal blood only (ligation of the hepatic artery), with caval blood only (portocaval transposition and ligation of the hepatic artery) and arterial blood only (portocaval shunt, arterialization of the distal end of the portal vein and ligation of the hepatic artery). To study differences in gene expression patterns, we chose enzymes that have a heterogeneous expression pattern within the liver lobule: the periportally located enzymes carbamoylphosphate synthase, succinate dehydrogenase, phosphoenolpyruvate carboxykinase and the pericentrally located enzymes glutamine synthase, glutamate dehydrogenase and NADPH-cytochrome P-450 reductase. To eliminate the potential interference of the long half-lives of some of these proteins on the interpretation of the results, we also studied the distribution of the mRNAs of carbamoylphosphate synthase, glutamine synthase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase. The animals were studied 2 wk after the operations. On the basis of their changes in body weight the animals were in steady state for at least a week. The patterns of gene expression of the enzymes studied did not change, regardless of the source of the altered afferent hepatic blood. The changes in gene expression that were observed in animals that did not regain their preoperative weight were shown to be caused by a limited intake of food. This study demonstrates that the physiological position of the liver within the circulation (i.e., between the gastrointestinal tract and the systemic circulation) is not as critical as is often stated and is certainly not essential for the maintenance of liver cell heterogeneity. The data suggest that the direction of the bloodstream (i.e., the existence of an upstream and a downstream compartment) is a major determinant of zonation of gene expression.

The spatial distribution of alpha- and beta-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1) In all stages examined, beta-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while alpha-MHC is the main isoform in the atria. In addition, alpha-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses alpha-MHC whereas only the lower part expresses beta-MHC. The separation of atria and ventricles by the fibrous annulus takes place at the ventricular margin of the AVC wall. Hence, the beta-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of alpha-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined alpha- and beta-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized at the periphery of the SA node.

Using monoclonal antibodies against the M and B subunit isoforms of creatine kinase (CK) we have investigated their distribution in developing human skeletal and cardiac muscle immunohistochemically. It is demonstrated that in skeletal muscle, a switch from CK-B to CK-M takes place around the week 8 of development, whereas in the developing heart, CK-M is the predominant isoform from the earliest stage examined onward (i.e., 4 1/2 weeks of development). In all hearts examined, local differences in concentration of the CK isoforms are observed. The CK-M expression in the developing outflow tract (OFT) and conduction system is described in detail. Between the weeks 5 and 7 of development, the distal portion of the OFT is characterized by low CK-M expression, whereas around the week 8-10 of development the myocardium around the developing semilunar valves in the OFT expresses a very high level of CK-M. At all stages examined, a relatively low CK-M level is observed in those regions in which the "slow" components of the conduction system do develop (e.g., the sinoatrial junction and atrioventricular junction), whereas a relatively high concentration of CK-M is observed in those areas that are destined to become the "fast" components, i.e., the subendocardial myocardium of the ventricles. The high expression of CK-M in the developing "fast components" of the conduction system contrasts with the relatively low expression of CK-M in the force-producing myocardium of the interventricular septum and free ventricular wall.