The role of connexin40 in developing atrial conduction

AbstractConnexin40 (Cx40) is the main connexin expressed in the murine atria and ventricular conduction system. We assess here the developmental role of Cx40 in atrial conduction of the mouse. Cx40 deficiency significantly prolonged activation times in embryonic day 10.5, 12.5 and 14.5 atria during spontaneous activation; the severity decreased with increasing age. In a majority of Cx40 deficient mice the impulse originated from an ectopic focus in the right atrial appendage; in such a case the activation time was even longer due to prolonged activation. Cx40 has thus an important physiological role in the developing atria

Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle.

Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues

Transcriptional dynamics elicited by a short pulse of notch activation involves feed-forward regulation by E(spl)/Hes genes.

Dynamic activity of signaling pathways, such as Notch, is vital to achieve correct development and homeostasis. However, most studies assess output many hours or days after initiation of signaling, once the outcome has been consolidated. Here we analyze genome-wide changes in transcript levels, binding of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and RNA Polymerase II (Pol II) immediately following a short pulse of Notch stimulation. A total of 154 genes showed significant differential expression (DE) over time, and their expression profiles stratified into 14 clusters based on the timing, magnitude, and direction of DE. E(spl) genes were the most rapidly upregulated, with Su(H), Pol II, and transcript levels increasing within 5-10 minutes. Other genes had a more delayed response, the timing of which was largely unaffected by more prolonged Notch activation. Neither Su(H) binding nor poised Pol II could fully explain the differences between profiles. Instead, our data indicate that regulatory interactions, driven by the early-responding E(spl)bHLH genes, are required. Proposed cross-regulatory relationships were validated in vivo and in cell culture, supporting the view that feed-forward repression by E(spl)bHLH/Hes shapes the response of late-responding genes. Based on these data, we propose a model in which Hes genes are responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining the critical functions these key regulators play in many developmental and disease contexts

Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage

Metabolism in time and space - exploring the frontier of developmental biology

Despite the fact that metabolic studies played a prominent role in the early history of developmental biology research, the field of developmental metabolism was largely ignored following the advent of modern molecular biology. Metabolism, however, has recently re-emerged as a focal point of biomedical studies and, as a result, developmental biologists are once again exploring the chemical and energetic forces that shape growth, development and maturation. In May 2017, a diverse group of scientists assembled at the EMBO/EMBL Symposium ‘Metabolism in Time and Space’ to discuss how metabolism influences cellular and developmental processes. The speakers not only described how metabolic flux adapts to the energetic needs of a developing organism, but also emphasized that metabolism can directly regulate developmental progression. Overall, and as we review here, this interdisciplinary meeting provided a valuable forum to explore the interface between developmental biology and metabolism

Dissecting the mechanisms of Notch induced hyperplasia

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Hairless-Mediated Repression of Notch Target Genes Requires the Combined Activity of Groucho and CtBP Corepressors

Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. Here we have investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, we find that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, we demonstrate that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding

The transcriptional repressor REST is a critical regulator of the neurosecretory phenotype

Release of distinct cellular cargoes in response to specific stimuli is a process fundamental to all higher eukaryotes and controlled by the regulated secretory pathway (RSP). However, the mechanism by which genes involved in the RSP are selectively expressed, leading to the establishment and appropriate functioning of regulated secretion remaining largely unknown. Using the rat pheochromocytoma cell line PC12, we provide evidence that, by controlling expression of many genes involved in the RSP, the transcriptional repressor REST can regulate this pathway and hence the neurosecretory phenotype. Introduction of REST transgenes into PC12 cells leads to the repression of many genes, the products of which are involved in regulated secretion. Moreover, chromatin immunoprecipitation assays show that many of the repressed genes recruit the recombinant REST protein to RE1 sites within their promoters and abrogation of REST function leads to reactivation of these transcripts. In addition to the observed transcriptional effects, PC12 cells expressing REST have fewer secretory granules and a reduction in the ability to store and release noradrenaline. Furthermore, an important trigger for synaptic release, influx of calcium through voltage-operated calcium channels, is compromised. This is the first demonstration of a transcription factor that directly controls expression of many major components of the RSP and provides further insight into the function of REST

Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage