Notch signaling regulates venous arterialization during zebrafish fin regeneration

To protect against blood pressure, a mature artery is supported by mural cells which include vascular smooth muscle cells and pericytes. To regenerate a functional vascular system, arteries should be properly reconstructed with mural cells although the mechanisms underlying artery reconstruction remain unclear. In this study, we examined the process of artery reconstruction during regeneration of the zebrafish caudal fin as a model to study arterial formation in an adult setting. During fin regeneration, the arteries and veins form a net‐like vasculature called the vascular plexus, and this plexus undergoes remodeling to form a new artery and two flanking veins. We found that the new vascular plexus originates mainly from venous cells in the stump but very rarely from the arterial cells. Interestingly, these vein‐derived cells contributed to the reconstructed arteries. This arterialization was dependent on Notch signaling, and further analysis showed that Notch signaling was required for the initiation of arterial gene expression. In contrast, venous remodeling did not require Notch signaling. These results provide new insights toward understanding mechanisms of vascular regeneration and illustrate the utility of the adult zebrafish fin to study this process.     

Notch signaling in lymphocyte development

Cytokine and antigen receptor signals play well-characterized roles in promoting the survival and maturation of T and B lymphocyte progenitors through sequential developmental stages. Emerging studies suggest equally important roles for more ancient signaling pathways that evolved prior to the adaptive immune system in jawed vertebrates. In particular, there are at least two essential functions for the highly conserved Notch signaling pathway in lymphocyte development. First, Notch signals are essential for the development of T cell progenitors in the thymus and intestinal epithelium. Second, Notch signals are required to suppress B cell development in the thymus. This review will focus on focus on recent advances in our understanding of how Notch signaling regulates this developmental switch, as well as how Notch might regulate subsequent survival and cell fate decisions in developing T cells.     

Notch 1 signaling regulates peripheral T cell activation

Notch signaling has been identified as an important regulator of leukocyte differentiation and thymic maturation. Less is known about the role of Notch signaling in regulating mature T cells. We examined the role of Notch 1 in regulating peripheral T cell activity in vitro and in vivo. Coligation of Notch 1 together with TCR and CD28 resulted in a dramatic inhibition of T cell activation, proliferation, and cytokine production. This effect was dependent on presenilin activity and induced the expression of HES-1, suggestive of Notch 1 signaling. Biochemical analysis demonstrated an inhibition of AKT and GSK3beta phosphorylation following Notch 1 engagement while other biochemical signals such as TCR and ERK phosphorylation remained intact. Similar effects were observed in vivo in an adoptive transfer model. Therefore, Notch 1 signaling may play an important role in regulating naive T cell activation and homeostasis.     

Overexpression of miR-34c inhibits high glucose-induced apoptosis in podocytes by targeting Notch signaling pathways

Recent findings have shown that microRNAs play critical roles in the pathogenesis of diabetic nephropathy. miR-34c has been found to inhibit fibrosis and the epithelial-mesenchymal transition of kidney cells. However, the role of miR-34c in diabetic nephropathy has not been well studied. The current study was designed to investigate the role and potential underlying mechanism of miR-34c in regulating diabetic nephropathy. After treating podocytes with high glucose (HG) in vitro, we found that miR-34c was downregulated and that overexpression of miR-34c inhibited HG-induced podocyte apoptosis. The direct interaction between miR-34c and the 3’-untranslated region (UTR) of Notch1 and Jagged1 was validated by dual-luciferase reporter assay. Moreover, Notch1 and Jagged1 as putative targets of miR-34c were downregulated by miR-34c overexpression in HG-treated podocytes. Overexpression of miR-34c inhibited HG-induced Notch signaling pathway activation, as indicated by decreased expression of the Notch intracellular domain (NICD) and downstream genes including Hes1 and Hey1. Furthermore, miR-34c overexpression increased the expression of the anti-apoptotic gene Bcl-2, and decreased the expression of the pro-apoptotic protein Bax and cleaved Caspase-3. Additionally, the phosphorylation of p53 was also downregulated by miR-34c overexpression. Taken together, our findings suggest that miR-34c overexpression inhibits the Notch signaling pathway by targeting Notch1 and Jaggged1 in HG-treated podocytes, representing a novel and potential therapeutic target for the treatment of diabetic nephropathy.     

Notch信号系统调控骨髓间充质干细胞向心肌样细胞的分化

背景：Notch信号系统在调控骨髓间充质干细胞的定向分化中起关键作用,但尚无涉及干细胞分化为心肌细胞及其分化机制的报道。 目的：分析Notch信号系统在骨髓间充质干细胞分化为心肌细胞过程中的调控作用。 方法：将分离培养的骨髓间充质干细胞与心肌细胞共培养,Jagged1组加入Notch激活剂Jagged1,DAPT组加入Notch激活剂Jagged1和抑制剂DAPT,对照组加入PBS缓冲液。用反转录-聚合酶链反应、免疫组化等方法检测干细胞分化为心肌细胞的情况及Notch信号系统的表达。 结果与结论：骨髓间充质干细胞在体外可分化为心肌细胞,与DAPT组及对照组相比,Jagged1组的干细胞分化为心肌细胞的比率提高,心肌标志物表达增多,并且Notch1和Jagged1表达增强。证实Notch信号系统对骨髓间充质干细胞分化为心肌细胞起正向调控作用。     

Notch signaling in T cell development

Notch signaling regulates cell fate decisions during development. Recent experiments suggest that Notch signaling is essential for initial commitment to the T cell lineage and may function together with signals from the pre-TCR and the TCR to regulate subsequent steps of T cell development.     

Notch signaling in development and disease

Notch receptors and ligands were first identified in flies and worms, where they were shown to regulate cell proliferation, cell differentiation, and, in particular, binary cell fate decisions in a variety of developmental contexts. The first mammalian Notch homolog was discovered to be a partner in a chromosomal translocation in a subset of human T-cell leukemias. Subsequent studies in mice and humans have shown that Notch signaling plays essential roles at multiple stages of hematopoiesis, and also regulates the development or homeostasis of cells in many tissues and organs. Thus, it is not surprising that mutations which disrupt Notch signaling cause a wide range of cancers and developmental disorders. Perhaps because it is so widely used, Notch signaling is subject to many unusual forms of regulation. In this review, we will first outline key aspects of Notch signaling and its regulation by endocytosis, glycosylation, and ubiquitination. We will then overview recent literature elucidating how Notch regulates cell-lineage decisions in a variety of developmental contexts. Finally, we will describe the roles of dysregulated Notch signaling in causing several types of cancer and other pathologies.     

Notch1通过非经典的Notch信号通路调节胰腺星形细胞的活化

目的:探讨Notch1在胰腺星形细胞(pancreatic stellate cells,PSCs)活化中的作用。方法:利用免疫组织化学法与免疫荧光双标法检测Notch1在人胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)组织的表达情况;原代分离培养小鼠PSCs,利用油红O染色、Western blot及RT-qPCR法对其进行鉴定,并利用Western blot及RT-qPCR检测Notch1及其下游关键分子HES1的表达情况;转染Notch1小干扰RNA(Notch1 siRNA)至小鼠PSCs后,利用Western blot检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤连蛋白(fibronectin)、I型胶原(collagen typeⅠ,ColⅠ)、Notch1及HES1的表达情况;利用划痕实验与CCK-8实验检测Notch1 siRNA对小鼠PSCs迁移与细胞活力的影响。结果:免疫组化与免疫荧光双标染色结果显示,Notch1表达在α-SMA阳性的PDAC间质细胞中;成功培养了小鼠PSCs细胞,且...     

Notch信号通路调控哮喘小鼠气道上皮下纤维化

 目的:探讨Notch信号通路在哮喘上皮下纤维化中的调控作用。方法:首先针对I型胶原蛋白启动子进行生物信息分析,了解有无Notch信号通路结合位点;其次通过DNA-pull down及Western blot 实验来证实Notch信号通路下游关键分子Hes1与人和小鼠I型胶原蛋白亚型基因有无结合;最后建立哮喘小鼠模型,用免疫荧光检测哮喘小鼠肺中I型胶原蛋白2个亚型在肺组织中的分布情况,并予Notch信号通路抑制剂KyoT2腺病毒载体经鼻干预,通过EVG染色观察哮喘气道上皮下纤维化情况。结果:生物信息学分析证实I型胶原蛋白2个亚型的启动子转录位点附近均存在Notch下游关键分子Hes1的结合位点。DNA-pull down及Western blot实验证实Hes1蛋白结合于I型胶原蛋白启动子转录位点附近。哮喘小鼠模型中I型胶原蛋白的2个亚型在肺组织中的表达较正常对照组增多,差异有统计学显著性(P<0.05)。哮喘小鼠模型经Notch抑制剂干预后肺组织中胶原纤维减少,气道上皮下纤维化得到缓解。结论:Notch能调控哮喘小鼠气道上皮下纤维化这一气道重构现象,抑制Notch信号通路减轻善哮喘小鼠气道上皮下纤维化。     

Regulation of Notch signaling during T- and B-cell development by O-fucose glycans

Summary:  Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O -fucose glycans of Notch cell-autonomously modulate Notch–ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O -fucosyltransferase 1 (Pofut1), and elongated by the transfer of N -acetylglucosamine (GlcNAc) to the fucose by β1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O -fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhancing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O -fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O -fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.     

Notch signaling in lymphocyte development and function

Over the past few years, the crucial role of Notch signaling in multiple stages of T-cell development has become apparent. Recent studies have helped to define more precisely the functions and components of the Notch signaling pathway in T-cell development, including during the T versus B fate decision and in early CD4(-)CD8(-)double-negative thymocytes. In addition, new evidence points to a requirement for Notch2 in the development of marginal zone B cells. Finally, recent studies have provided our first glimpse into the complex and paradoxical roles of Notch signaling in the activation and differentiation of mature T cells.     

Notch signaling in T- and B-cell development

The Notch family of evolutionarily conserved proteins regulates a broad spectrum of cell-fate decisions and differentiation processes during fetal and post-natal development. The best characterized role of Notch signaling during mammalian hematopoiesis and lymphopoiesis is the essential function of the Notch1 receptor in T-cell lineage commitment. More recent studies have addressed the roles of other Notch receptors and ligands, as well as their downstream targets, revealing additional novel functions of Notch signaling in intra-thymic T-cell development, B-cell development and peripheral T-cell function.     

Notch signaling regulates the differentiation of post-mitotic intestinal epithelial cells

The intestinal epithelium comprises differentiated cells of four lineages maintained by precursor cells. As the Notch pathway controls the fate of proliferating cells in many systems, we investigated the effect of conditional expression of an activated Notch mutant in intestinal epithelium. An increase in the number of goblet cells occurs within 8 h of induction, due to an effect of Notch on post-mitotic cells, not on precursors. This observation broadens the role of Notch into controlling postmitotic differentiation and indicates that the composition of the epithelium is not solely determined by progenitor cells.     

Notch signaling: distinct ligands induce specific signals during lymphocyte development and maturation

Notch signaling is a highly conserved pathway involved in cell fate choice during development with Delta and Jagged constituting the two evolutionary conserved families of Notch ligands. These ligands are transmembrane proteins with conserved biochemical structure that share their receptors and signal through a common mechanism. Upon ligand binding Notch receptors are proteoliticaly cleaved, the intracellular domain of Notch (NICD) is released and translocated to the nucleus, where it activates target genes. In mammals, four receptors and five ligands have been described. Delta-1, Delta-3 and Delta-4 are homologues to Drosophila Delta and Jagged-1 and Jagged-2 to Drosophila Serrate. Despite strong domain homology, there is growing evidence that signals transmitted through Delta or Jagged ligands can differentially affect the target cell. At least during embryonic development, Notch receptors and Notch ligands functions cannot be compensated by other members. Knock-out mice for Notch-1, Notch-2, Delta-1 and Jagged-1 are embryonic lethal . Similarly, mice heterozygous for Delta-4 inactivation also die before birth . Invalidation of Jagged-2 results in defaults in thymus morphology and gammadelta development . Altogether, these data suggest that each Notch member can exert unique specific effects. In this review, we will thus focus on recent data about differential effects of Notch ligands on T cell development and differentiation. In light of recent biochemical and molecular advances on Notch-signaling pathway, we will examine how specific effects can be mediated by a given ligand.     

Requirements for Jag1-Rbpj mediated Notch signaling during early mouse lens development

Background: During vertebrate lens development, the lens placode in the embryonic ectoderm invaginates into a lens vesicle, which then separates from the surface epithelium, followed by two waves of fiber cell differentiation. In the mouse, multiple labs have shown that Jag1‐Notch signaling is critically required during the second wave of lens fiber cell formation. However, Notch signaling appears to play no obvious role during lens induction or morphogenesis, although multiple pathway genes are expressed at these earlier stages. Results: Here, we explored functions for Notch signaling specifically during early lens development, by using the early‐acting AP2α‐Cre driver to delete Jag1 or Rbpj. We found that Jag1 and Rbpj are not required during lens induction, but are necessary for proper lens vesicle separation from the surface ectoderm. Conclusions: We conclude that precise levels of Notch signaling are essential during lens vesicle morphogenesis. In addition, AP2α‐Cre‐mediated deletion of Rbpj resulted in embryos with cardiac outflow tract and liver deformities, and perinatal lethality. Developmental Dynamics 241:493–504, 2012. © 2012 Wiley Periodicals, Inc.     

Notch signaling is activated by TLR stimulation and regulates macrophage functions

Notch signaling is a well-conserved pathway involved in cell fate decisions, proliferation and apoptosis. We report on the involvement of Notch signaling in regulating gene expression in activated macrophages. Toll-like receptors (TLR) agonists such as bacterial lipopeptide, polyI:C, lipopolysaccharide and unmethylated CpG DNA all induced up-regulation of Notch1 in primary and macrophage-like cell lines. Notch1 up-regulation was dependent on the MyD88 pathway when stimulated through TLR2, but not TLR4. Activated Notch1 and expression of the Notch target genes, Hes1 and Deltex, were detected in activated macrophages, suggesting that Notch signaling was activated upon stimulation. Inhibiting processing of Notch receptor by gamma-secretase using a gamma-secretase inhibitor (GSI), the expression of Notch1 was down-regulated to basal levels. This treatment significantly modulated expression of TNF-alpha, IL-6, and IL-10. In addition, the amount of nitric oxide produced was significantly lower and the expression of MHC class II was up-regulated in GSI-treated cells. Treatment with GSI or silencing Notch1 resulted in decreased translocation of NF-kappaBp50 into nucleus upon stimulation. Taken together, stimulation of macrophages through the TLR signaling cascade triggered activation of Notch signaling, which in turn regulated gene expression patterns involved in pro-inflammatory responses, through activation of NF-kappaB.     

Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3–5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1‐high ES cells are prone to the mesodermal fate, while Hes1‐low ES cells are prone to the neural fate. These outcomes of Hes1‐high and Hes1‐low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.