The histogenesis of the separation between atrial and ventricular myocardium at the atrioventricular junction in the developing human heart has been investigated immunohistochemically by using monoclonal antibodies specific for atrioventricular cushion tissue, mesenchymal cells, atrial and ventricular myocardium, and myocardium of the primary ring. It was found that the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This process takes place at the ventricular margin of the myocardium of the atrioventricular canal. The separation of atrial and ventricular myocardium starts at approximately 7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. The only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction. Our findings show that the nonmuscular part of the developing leaflets of the atrioventricular valves derives from the atrioventricular cushions and that the tissues of the atrioventricular groove do not contribute to the development of these leaflets.

(1) The expression of neurofilaments can be used to delineate the nodal area in the intact SAN but is not sufficiently sensitive for characterizing all individual isolated nodal cells. (2) A fundamentally different organization of the SAN is presented: The gradual increase in density of atrial cells from the dominant area toward the crista terminalis in the SAN causes a gradual increase of atrial electrotonic influence that may be an important cause of the gradual transition of the nodal to the atrial type of action potential.

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of ␤-catenin from mice exhibiting constitutive activated ␤-catenin in the liver. Adenovirus-mediated expression of oncogenic ␤-catenin was used to isolate early targets of ␤-catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell-derived chemotaxin 2 (LECT2) gene as a direct target of ␤-catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated ␤-catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a ␤-catenin-independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by ␤-catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up-regulation of LECT2 in human liver tumors that expressed aberrant activation of ␤-catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutro- H epatocellular carcinoma (HCC), the major primary liver cancer, is becoming increasingly common worldwide. 1 The prognosis for patients with HCC is rather poor. The molecular changes underlying HCC remain largely unknown despite the fact that major risk factors, such as chronic hepatitis B or C infection and exposure to hepatocarcinogens like aflatoxin B1, are well recognized. Several genetic changes have been implicated in at least 3 pathways of carcinogenesis, specifically, the p53, RB and Wnt/␤-catenin signaling pathways. 2 Deregulation of the Wnt pathway appears to be most frequent of these changes in human HCC; it occurs in about 30% to 40% of patients. 2,3 It also occurs in more than 90% of hepatoblastomas, which are rare embryonal liver tumors. 4 Mutations affecting 2 partners of the Wnt pathway have been found in liver cancers. One is a mutation that activates the ␤-catenin gene. Such mutations occur mainly in hepatitis B-negative HCC 5 and in more than 50% of hepatoblastomas. 6,7 The other is a mutation that inactivates the axin 1, and, less commonly, the axin 2 gene. 5,8,9 Mutations that activate the Wnt pathway result in ␤-catenin accumulation in the nucleus. This process, in association with LEF/TCF transcription factors, modulates the transcription of target genes. 10,11 It is now clear that the genetic program triggered by activation of ␤-catenin signaling depends on the cellular context. The ␤-catenin target genes c-myc and cyclin D1 are well

A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated G1N2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i.e., 4 1/2 weeks of development) these G1N2-expressing cells are localized in the myocardium that surrounds the foramen between the embryonic left and right ventricle. In the lesser curvature of the cardiac loop this "primary" ring occupies the lower part of the wall of the atrioventricular canal. During subsequent development, G1N2-expressing cells continue to identify the entrance to the right ventricle, but the shape of the ring changes as a result of the tissue remodelling that underlies cardiac septation. During the initial phases of this process the staining remains recognizable as a continuous band of cells in the myocardium that surrounds the developing right portion of the atrioventricular canal, subendocardially in the developing interventricular septum and around the junction of the embryonic left ventricle with the subaortic portion of the outflow tract. During the later stages of cardiac septation, the latter part of the ring discontinues to express G1N2, while upon the completion of septation, no G1N2-expressing cardiomyocytes can be detected anymore. The topographic distribution pattern of G1N suggests that the definitive ventricular conduction system derives from a ring of cells that initially surrounds the "primary" interventricular foramen. The results indicate that the atrioventricular bundle and bundle branches develop from G1N2-expressing myocytes in the interventricular septum, while the "compact" atrioventricular node develops at the junction of the band of G1N2-positive cells in the right atrioventricular junction (the right atrioventricular ring bundle) and the ("penetrating") atrioventricular bundle. A "dead-end tract" represents remnants of conductive tissue in the anterior part of the top of the interventricular septum. The location of the various components of the avian conduction system is topographically homologous with that of the G1N2-ring in the human embryonic heart, indicating a phylogenetically conserved origin of the conduction system in vertebrates.

In liver, most genes are expressed with a porto-central gradient. The transcription factor hepatic nuclear-factor4␣ (HNF4␣) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4␣-response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase-I and interacted with a protein that is recognized by HNF4␣-specific antiserum. Chromatin-immunoprecipitation assays of HNF4␣-deficient (H4LivKO) and control (H4Flox) livers with HNF4␣ antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone-deacetylase1 (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone-receptor ␤1 ( T he development and maintenance of liver architecture and function is regulated by liver-enriched transcription factors. 1 One of these, hepatic nuclear factor 4␣ (HNF4␣; NR2A1) is expressed at high levels in liver, kidney, intestine, and pancreas 2,3 and binds to the promoter of 12% of genes that are expressed in adult liver. 4 HNF4␣ is an orphan member of the nuclear-receptor superfamily. 2 Depending on chain length and degree of saturation, 5 fatty acyl-coenzyme A thioesters may act as agonistic or antagonistic factors, but whether or not these thioesters function as ligands remains unsettled. 2,[6][7][8] Transcriptional regulation by HNF4␣ is accomplished by interactions with coactivator or corepressor mediators (e.g., GRIP1, SRC-1, CBP/p300, SMRT). 6,7,9,10 The resulting coactivator or corepressor complexes have intrinsic histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity, respectively. Histone modifications play an important role in the regulation of the

Highlights d Colonic Gli1+ cells are fibroblasts with epithelial stem cell supporting properties d CD90 is upregulated in Gli1+ cells and marks fibroblasts in the colon stem cell niche d CD90+ fibroblasts produce class 3 semaphorins (Sema3) d The effect of CD90+ fibroblasts on epithelial proliferation is Nrp2 dependent

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.

Glutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. Glutamine serves, with alanine, as a major nontoxic interorgan ammonia carrier. Elimination of hepatic GS expression in mice causes only mild hyperammonemia and hypoglutaminemia but a pronounced decrease in the whole-body muscle-to-fat ratio with increased myostatin expression in muscle. Using GS-knockout/liver and control mice and stepwise increments of enterally infused ammonia, we show that 35% of this ammonia is detoxified by hepatic GS and 35% by urea-cycle enzymes, while 30% is not cleared by the liver, independent of portal ammonia concentrations £ 2 mmol/L. Using both genetic (GSknockout/liver and GS-knockout/muscle) and pharmacological (methionine sulfoximine and dexamethasone) approaches to modulate GS activity, we further show that detoxification of stepwise increments of intravenously (jugular vein) infused ammonia is almost totally dependent on GS activity. Maximal ammonia-detoxifying capacity through either the enteral or the intravenous route is 160 lmol/hour in control mice. Using stable isotopes, we show that disposal of glutaminebound ammonia to urea (through mitochondrial glutaminase and carbamoylphosphate synthetase) depends on the rate of glutamine synthesis and increases from 7% in methionine sulfoximine-treated mice to 500% in dexamethasone-treated mice (control mice, 100%), without difference in total urea synthesis. Conclusions: Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on glutamine synthesis, we hypothesize that glutamate dehydrogenase complements mitochondrial ammonia production. (HEPATOLOGY 2017;65:281-293). G lutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. (Note: NH 3 is protonated for 98% to NH 4 1 at physiological pH. We use the term "ammonia" to refer to the sum of NH 3 and NH 4 1 unless specified as either "NH 3 " or "NH 41 .") Glutamine serves, with alanine, as a major nontoxic interorgan ammonia shuttle in the body (1) and as an aminomoiety donor for the synthesis of nucleotides, amino acids, amino-sugars, and oxidized nicotinamide adenine dinucleotide. Its intracellular turnover rate exceeds that of all other amino acids. GS deficiency causes only moderate hyperammonemia, but affected humans and mice suffer from encephalopathy and die neonatally. (2,3) GS is predominantly expressed in the nervous system, kidney, and liver, that is, in established glutamine-consuming organs, (4)(5)(6) whereas skeletal muscle, with a much lower expression but large mass, is considered the main net ...