JAGGED Research Articles

Cancer-wide in silico analyses using differentially expressed genes demonstrate the functions and clinical relevance of JAG, DLL, and NOTCH.

Notch ligands [jagged (JAG) and, delta-like (DLL) families] and receptors [NOTCH family] are key regulators of Notch signaling. NOTCH signaling contributes to vascular development, tissue homeostasis, angiogenesis, and cancer progression. To elucidate the universal functions of the JAG, DLL, and NOTCH families and their connections with various biological functions, we examined 15 types of cancer using The Cancer Genome Atlas clinical database. We selected the differentially expressed genes (DEGs), which were positively correlated to the JAG, DLL, and NOTCH families in each cancer. We selected positive and negative hallmark signatures across cancer types. These indicated biological features associated with angiogenesis, hypoxia, KRAS signaling, cell cycle, and MYC targets by gene ontology and gene set enrichment analyses using DEGs. Furthermore, we analyzed single-cell RNA sequencing data to examine the expression of JAG, DLL, and NOTCH families and enrichment of hallmark signatures. Positive signatures identified using DEGs, such as KRAS signaling and hypoxia, were enriched in clusters with high expression of JAG, DLL, and NOTCH families. We subsequently validated the correlation between the JAG, DLL, and NOTCH families and clinical stages, including treatment response, metastasis, and recurrence. In addition, we performed survival analysis to identify hallmark signatures that critically affect patient survival when combining the expression of JAG, DLL, and NOTCH families. By combining the DEG enrichment and hallmark signature enrichment in survival analysis, we suggested unexplored regulatory functions and synergistic effects causing synthetic lethality. Taken together, our observations demonstrate the functions of JAG, DLL, and NOTCH families in cancer malignancy and provide insights into their molecular regulatory mechanisms.

Overexpression of the Liriodendron tulipifera BOP2 Gene (LtuBOP2) Affects Leaf Margin Development in Transgenic Arabidopsis thaliana

BLADE-ON-PETIOLE 2 (BOP2) plays a pivotal role in leaf morphogenesis. Liriodendron tulipifera is a suitable model for exploring the molecular mechanisms underlying leaf serration formation, which are largely unknown. Here, we isolated the full-length LtuBOP2 gene and its promoter from L. tulipifera and characterized its function in leaf morphogenesis through multidimensional approaches. The spatiotemporal expression pattern of LtuBOP2 indicated the high expression of LtuBOP2 in stems and leaf buds. We constructed LtuBOP2 promoter, fused the promoter sequences to the β-glucuronidase (GUS) gene, and then transformed them into Arabidopsis thaliana. Histochemical GUS staining results indicated that GUS activity was higher in petioles and the main vein. LtuBOP2 overexpression in A. thaliana caused moderate serration in the leaf tip, owing to the increased number of abnormal lamina epidermal cells and defective vascular tissue, thus indicating a novel role of BOP2. The ectopic expression of LtuBOP2 in A. thaliana promoted the expression of the lateral organ boundary gene ASYMMETRIC LEAVES2 (AS2) and inhibited JAGGED (JAG) and CUP-SHAPED COTYLEDON2 (CUC2) expression to establish leaf proximal-distal polarity. Moreover, LtuBOP2 participated in leaf serration formation by promoting the antagonistic relationship between KNOX I and hormones during leaf margin development. Our findings revealed the role of LtuBOP2 in the proximal-distal polarity formation and development of leaf margin morphology, providing new insights into the regulatory mechanisms of the leaf formation development of L. tulipifera.

A “notch” in the cellular communication network in response to anoxia by wood frog (Rana sylvatica)

Wood frogs (Rana sylvatica) experience months of whole-body freezing during winter. Anoxia is one of the side stresses along with cell dehydration and hyperglycemia. Among multicellular organisms, communication and coordination is essential between neighbouring cells, particularly under stress conditions. Notch signaling is an effective communication channel between cells and regulates multiple pro-survival pathways. Signaling initiates when membrane-bound ligands Delta-like (DLL) or Jagged (JAG) interact with notch receptors. Activated receptor undergoes cleavage to release intracellular domain (NICD) in the cytoplasm. NICD translocates to the nucleus and forms a transcriptional complex with MAML and RBPJ that interacts with promoter regions and activates stress-specific genes. The role of notch signaling in enduring anoxia was assessed by studying the pathway components using immunoblots, TF-ELISA, and qPCR on treated samples of liver and heart. Bioinformatics tool Pymol was used to prepare structures based on available protein sequences for ligands, NOTCH receptor and the transcriptional complex. The results showed an increase in the levels of both ligands and receptors but decreased levels of RBPJ, suggesting an effective transmission of stress signal but suppressed gene transcription that goes in accordance with lowering energy expense required in energy crisis during anoxia. The study suggests Notch-independent activation of HES1 and HES5. Tissue-specific response of HES1, HES5, and MAML implies energy conservation and myocardial protection. The current study is the first analysis of the regulation of notch signaling in amphibians on encountering anoxic conditions that present multiple future directions.

Overexpression of Liriodendron tulipifera JAG Gene (LtuJAG) Changes Leaf Shapes in Transgenic Arabidopsis thaliana.

In Arabidopsis thaliana, JAGGED (JAG) is a transcription inhibitor that controls the development of leaf polarity and regulates the expression of genes controlling lateral organ formation. Liriodendron tulipifera is an ornamental tree with extraordinary tulip-shaped flowers and goose web-like leaves, this is one of the suitable plants for morphological development research. To investigate the potential functions of the LtuJAG gene, we isolated the full-length LtuJAG from L. tulipifera, transferred it into A. thaliana via agrobacterium-mediated transformation, and monitored its expression pattern. Subcellular localization showed that LtuJAG was located in the nucleus. RT-qPCR assays indicated that LtuJAG was expressed mainly in leaf buds and flowers, but not in mature leaves and stems. GUS staining results showed that LtuJAG was expressed in the shoot apical meristem (SAM). Overexpressing LtuJAG changed A. thaliana leaf shapes, causing a moderate serration and a slight asymmetric distribution in the medio-lateral and proximal-distal axes. Ectopic expression of LtuJAG induced the expression of lateral organ boundary suppressors JAGGED LATERAL ORGANS (JLO) and ARABIDOPSIS THALIANA HOMEOBOX1 (ATH1). It also repressed the expression of the apical meristem suppressor class-1 KNOX gene (KNOX I) and altered endogenous hormone levels. Our results suggest that LtuJAG plays a role in negatively regulating leaf polarity formation in L. tulipifera.

Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2

Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4-NOTCH1 activation was more than twice that of DLL4-NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1-NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes.

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum–valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.

Comparative transcriptome analysis reveals the regulatory networks of cytokinin in promoting the floral feminization in the oil plant Sapium sebiferum

BackgroundSapium sebiferum, whose seeds contain high level of fatty acids, has been considered as one of the most important oil plants. However, the high male to female flower ratio limited the seed yield improvement and its industrial potentials. Thus, the study of the sex determination in S. sebiferum is of significant importance in increasing the seed yield.ResultsIn this study, we demonstrated that in S. sebiferum, cytokinin (CK) had strong feminization effects on the floral development. Exogenous application with 6-benzylaminopurine (6-BA) or thidiazuron (TDZ) significantly induced the development of female flowers and increased the fruit number. Interestingly, the feminization effects of cytokinin were also detected on the androecious genotype of S. sebiferum which only produce male flowers. To further investigate the mechanism underlying the role of cytokinin in the flower development and sex differentiation, we performed the comparative transcriptome analysis of the floral buds of the androecious plants subjected to 6-BA. The results showed that there were separately 129, 352 and 642 genes differentially expressed at 6 h, 12 h and 24 h after 6-BA treatment. Functional analysis of the differentially expressed genes (DEGs) showed that many genes are related to the hormonal biosynthesis and signaling, nutrients translocation and cell cycle. Moreover, there were twenty one flowering-related genes identified to be differentially regulated by 6-BA treatment. Specifically, the gynoecium development-related genes SPATULA (SPT), KANADI 2 (KAN2), JAGGED (JAG) and Cytochrome P450 78A9 (CYP79A9) were significantly up-regulated, whereas the expression of PISTILLATA (PI), TATA Box Associated Factor II 59 (TAFII59) and MYB Domain Protein 108 (MYB108) that were important for male organ development was down-regulated in response to 6-BA treatment, demonstrating that cytokinin could directly target the floral organ identity genes to regulate the flower sex.ConclusionsOur work demonstrated that cytokinin is a potential regulator in female flower development in S. sebiferum. The transcriptome analysis of the floral sex transition from androecious to monoecious in response to cytokinin treatment on the androecious S. sebiferum provided valuable information related to the mechanism of sex determination in the perennial woody plants.

Regulation of Notch activation in CD4+ T cells

Abstract Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Mature T cells express Notch receptors 1, 2 and 3. Data from our lab and others indicate that T cell receptor (TCR) signaling activates Notch. Conventional ligand dependent Notch signaling involves, interaction of the Notch receptor with ligands such as Delta like ligand (DLL) 1,3, 4 or Jagged (JAG) 1, 2. However, in T cells it is unclear whether TCR induced activation of Notch is dependent upon engagement of Notch receptor with ligand. In-vitro stimulation of purified CD4+T cells, using antibodies against CD3 and costimulatory molecule CD28, results in activation of Notch. This occurs in absence of APC or any apparent ligand presenting cell. Thus, the mechanism that drives Notch activation during TCR stimulation is unclear. When naïve CD4+ T cells were cultured with bone marrow derived dendritic cells pulsed with antigen, we observed activation of Notch in the apparent absence of ligand on APC or activated T cells. However, when we examined ligand expression on naïve CD4+ T cell, activated by anti-CD3/CD28, we found resting T cells express constitutively low levels of DLL1 which increases with activation and is maintained through activated state. Additionally, DLL4 and JAG1 are expressed only after T cell activation and maintained only for 24hrs after activation. This upregulation is completely dependent upon signaling through CD28. However, whether the expression of ligand is sufficient to activate Notch remains unclear. Our preliminary data thus suggests that TCR mediated signaling in the absence of ligand may be is sufficient to activate Notch.

Apelin13/APJ promotes proliferation of colon carcinoma by activating Notch3 signaling pathway.

BackgroundThe link between Apelin (APL)/APL receptor (APJ) and Jagged (JAG)/Notch signaling pathways in colorectal cancer (CRC) has been poorly investigated. APL/APJ system, a potent angiogenic factor, is up-regulated in a variety of cancers. It contributes to tumor angiogenesis, and correlates with progression of malignancy. JAG/Notch signaling also contributes to progression, proliferation and metastasis of multiple cancers, including CRC. Here we tested the hypothesis that APL/APJ system promotes CRC proliferation by up-regulating Notch3, thus allowing further binding of JAG1 to Notch3.Materials and MethodsWe used a variety of methods including Western blot, RT-qPCR, gene silencing, ELISA, immunofluorescence staining, to investigate the interaction between APL/APJ system and Notch3 signaling pathway in both surgically-resected specimens and CRC cell line LS180.ResultsWe show that the expression of APL13, APJ, and Notch3 is elevated in CRC. We further demonstrate that APL13 can be secreted into culture media of LS180 cells, suggesting the existence of autocrine loop in CRC. Moreover, we found that APL13 stimulated expression of Notch3. Finally, we found that inhibition of either APJ or Notch3 prevents proliferation of LS180 cells.ConclusionsOur results suggest that APL13/APJ and JAG1/Notch3 signaling pathways are linked in CRC. These findings provide a new direction to the efforts targeting effective therapeutic and management approaches in the treatment of CRC.

Mechanism of Astragaloside prevents cholestatic liver fibrosis through inhibition of Notch signaling activation

Objective: The Notch signaling pathway is closely related to biliary fibrosis. Previous studies have shown that Astragaloside (AS) can prevent the progression of cholestatic liver fibrosis. The purpose of this study is to observe the effect of AS on the regulation of Notch signaling pathway in biliary fibrosis. Methods: Cholestatic liver fibrosis was established by common bile duct ligation (BDL) in rats. Two weeks after BDL, the rats were randomly divided into a model group (i.e., BDL), an Astragalosides group (AS), and a sorafenib (SORA) positive control group and treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Protein and gene expression were determined by immunostaining, immunoblotting and RT-PCR, respectively. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, -4, Jagged (JAG)1, Delta like (DLL)-1, -3, -4, Hes1, Numb and RBP-Jκ. Statistical analysis of variance analysis, q test, P < 0.05 showed that the difference was statistically significant. Results: (1) AS significantly reduced the deposition of collagen and the Hyp content of liver tissue (500.15 ± 86.10 vs. 625.72 ± 105.62, P = 0.031), and inhibited the activation of hepatic stellate cells. (2) AS significantly decreased the protein and mRNA expressions of transforming growth factor (TGF)-β1 (1.02±0.15 vs. 1.89±0.36, P = 0.007; 1.17±0.18 vs. 1.68±0.29, P = 0.013, respectively) and α-smooth muscle actin (α-SMA, 0.41±0.11 vs. 0.72±0.16, P = 0.003; 1.71±0.57 vs. 2.68±0.46, P = 0.008, respectively) compared with BDL group. In contrast, AS significantly enhanced expression of the Smad 7 protein compared with the BDL group (0.72±0.008 vs. 0.33±0.001, P = 0.005). AS also reduced biliary epithelial cell proliferation. AS reduced the mRNA levels of CK7, CK8 and CK18 (1.31±0.39 vs. 2.63±0.82, P = 0.009; 0.71±0.09 vs. 0.87±0.08, P = 0.031; 2.56±0.32 vs. 3.41±0.39, P = 0.010, respectively) and reduced the positive areas of CK19 and OV6 (62 337.17±21 873.38 vs. 22 5472.67±26 933.63, P = 0.000; 92 237.43±15 894.11 vs. 171 298.13±61 761.37, P = 0.000, respectively). (3) The mRNA expression of Notch-2, -3, -4 and JAG1 were significantly reduced in the AS group compared to the BDL group (1.07±0.19 vs. 1.51±0.28, P = 0.044; 0.99±0.24 vs. 1.18±0.10, P = 0.043; 1.36±0.42 vs. 3.40±0.44, P = 0.048; 2.62±0.43 vs. 3.73±0.83, P = 0.046, respectively). In contrast, the mRNA level of Numb was clearly enhanced after AS treatment (0.90±0.05 vs. 0.75±0.11, P = 0.019). In addition, consistent with the mRNA levels, the protein expressions of Notch-2, -3, -4 and JAG1 were reduced significantly (1.27±0.18 vs. 1.71±0.26, P = 0.004; 0.99±0.11 vs. 4.38±0.60, P = 0.001; 1.76±0.32 vs. 4.01±0.74, P = 0.002; 1.62±0.33 vs. 2.74±0.63, P = 0.002) and the Numb protein level was increased significantly (1.50±0.15 vs. 0.85±0.11, P = 0.001) in AS group compared with BDL group. Conclusion: AS may prevent cholestatic liver fibrosis via inhibition of the Notch signaling pathway, thereby inhibiting the abnormal proliferation of biliary epithelial cells. Results indicate that AS may be a potential treatment for cholestatic liver disease.

Suppression of sucrose synthase affects auxin signaling and leaf morphology in tomato.

Metabolic enzymes have been found to play roles in plant development. Sucrose synthase (SUS) is one of the two enzyme families involved in sucrose cleavage in plants. In tomato, six SUS genes have been found. We generated transgenic tomato plants with RNAi suppression of SlSUS1, SlSUS3 and SlSUS4 genes. Independent transgenic lines with RNAi suppression of more than one SUS gene exhibited morphological effects on their cotyledons and leaf structure, but there were no significant effects on their carbohydrate levels, demonstrating that SUS has a developmental function, in addition to its metabolic function. Shoot apices of the transgenic lines showed elevated expression of JAGGED (JAG) and the auxin transporter PIN1. In a PIN1-GFP fusion reporter/SUS-RNAi hybrid, PIN1-GFP patterns were altered in developing leaves (as compared to control plants), indicating that SlSUS suppression alters auxin signaling. These results suggest possible roles for SUS in the regulation of plant growth and leaf morphology, in association with the auxin-signaling pathway.

Critical role for OX40 signaling in Thymic and Peripheral Treg Expansion

Abstract Regulatory T-cells (Treg) play a pivotal role in suppressing self-reactive effector T-cells (Teff) and help maintain the critical balance between self-tolerance and autoimmunity. Restoration of homeostatic balance between Teff/Treg cells has been shown to ameliorate many autoimmune diseases including type-1 diabetes. Recently, we have reported a novel strategy for the selective expansion of Tregs using OX40L (a TNF-superfamily ligand) and Jagged (JAG)-1 (a Notch family ligand) treatment independent of canonical TCR-signaling, but mediated through specific interaction between OX40L-OX40 and JAG1-Notch3 preferentially expressed on Tregs. OX40L-JAG1 treatment of NOD mice with soluble OX40L and JAG1 increased proliferation of peripheral Tregs and delayed the onset of diabetes. In the present study, we show that OX40L treatment significantly increased Tregs in the thymus by inducing proliferation of thymic Tregs independent of TCR-stimulation upon addition of exogenous IL-2. OX40−/− mice had reduced numbers of thymic Tregs compared to wild type (WT) controls. The reduction in thymic Treg numbers likely due to impaired ability of proliferation in response to OX40L. However, we did not observe any change in IL-2 sensitivity of thymocytes in OX40−/− mice. Mechanistic studies suggested that OX40L-IL-2 induced thymic Treg proliferation was mediated through NF-kB and AKT-mTOR-S6 signaling. Further, phenotypic characterization studies revealed a significant decrease in TIGIT+Treg population, often associated with Treg activation, in both thymus and spleen of OX40−/− mice compared to WT controls. Taken together, these results suggest a critical role for OX40L/OX40 signaling in the development as well as function of Tregs.

Huang Qi Decoction Prevents BDL-Induced Liver Fibrosis Through Inhibition of Notch Signaling Activation.

Notch signaling has been demonstrated to be involved in ductular reactions and fibrosis. Previous studies have shown that Huang Qi Decoction (HQD) can prevent the progression of cholestatic liver fibrosis (CLF). However, whether HQD affects the Notch signaling pathway is unclear. In this study, CLF was established by common bile duct ligation (BDL) in rats. At the end of the first week, the rats were randomly divided into a model group (i.e., BDL), an HQD group, and a sorafenib positive control group (SORA) and were treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, and -4, Jagged (JAG) 1, and Delta like (DLL)-1, -3, and -4. The results showed that HQD significantly reduced the deposition of collagen and the Hyp content of liver tissue and inhibited the activation of HSCs compared with the BDL group. In addition, HQD significantly decreased the protein and mRNA expressions of TGF-[Formula: see text]1 and [Formula: see text]-SMA. In contrast, HQD significantly enhanced expression of the Smad 7 protein. HQD also reduced biliary epithelial cell proliferation, and reduced the mRNA levels of CK7, CK8, CK18, SRY-related high mobility group-box gene (SOX) 9, epithelial cell adhesion molecule (EpCAM) and the positive areas of CK19 and OV6. In addition, the mRNA and protein expressions of Notch-3, -4, JAG1, and DLL-1, -3 were significantly reduced in the HQD compared to the BDL group. These results demonstrated that HQD may prevent biliary liver fibrosis through inhibition of the Notch signaling pathway, and it may be a potential treatment for cholestatic liver disease.

HANABA TARANU (HAN) Bridges Meristem and Organ Primordia Boundaries through PINHEAD, JAGGED, BLADE-ON-PETIOLE2 and CYTOKININ OXIDASE 3 during Flower Development in Arabidopsis.

Shoot organ primordia are initiated from the shoot apical meristem and develop into leaves during the vegetative stage, and into flowers during the reproductive phase. Between the meristem and the newly formed organ primordia, a boundary with specialized cells is formed that separates meristematic activity from determinate organ growth. Despite interactions that have been found between boundary regulators with genes controlling meristem maintenance or primordial development, most boundary studies were performed during embryogenesis or vegetative growth, hence little is known about whether and how boundaries communicate with meristem and organ primordia during the reproductive stage. We combined genetic, molecular and biochemical tools to explore interactions between the boundary gene HANABA TARANU (HAN) and two meristem regulators BREVIPEDICELLUS (BP) and PINHEAD (PNH), and three primordia-specific genes PETAL LOSS (PTL), JAGGED (JAG) and BLADE-ON-PETIOLE (BOP) during flower development. We demonstrated the key role of HAN in determining petal number, as part of a set of complex genetic interactions. HAN and PNH transcriptionally promote each other, and biochemically interact to regulate meristem organization. HAN physically interacts with JAG, and directly stimulates the expression of JAG and BOP2 to regulate floral organ development. Further, HAN directly binds to the promoter and intron of CYTOKININ OXIDASE 3 (CKX3) to modulate cytokinin homeostasis in the boundary. Our data suggest that boundary-expressing HAN communicates with the meristem through the PNH, regulates floral organ development via JAG and BOP2, and maintains boundary morphology through CKX3 during flower development in Arabidopsis.

NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth.

A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.

LnIs a Key Regulator of Leaflet Shape and Number of Seeds per Pod in Soybean

Narrow leaflet soybean (Glycine max) varieties tend to have more seeds per pod than broad leaflet varieties. Narrow leaflet in soybean is conferred by a single recessive gene, ln. Here, we show that the transition from broad (Ln) to narrow leaflet (ln) is associated with an amino acid substitution in the EAR motif encoded by a gene (designated Gm-JAGGED1) homologous to Arabidopsis JAGGED (JAG) that regulates lateral organ development and the variant exerts a pleiotropic effect on fruit patterning. The genomic region that regulates both the traits was mapped to a 12.6-kb region containing only one gene, Gm-JAG1. Introducing the Gm-JAG1 allele into a loss-of-function Arabidopsis jagged mutant partially restored the wild-type JAG phenotypes, including leaf shape, flower opening, and fruit shape, but the Gm-jag1 (ln) and EAR-deleted Gm-JAG1 alleles in the jagged mutant did not result in an apparent phenotypic change. These observations indicate that despite some degree of functional change of Gm-JAG1 due to the divergence from Arabidopsis JAG, Gm-JAG1 complemented the functions of JAG in Arabidopsis thaliana. However, the Gm-JAG1 homoeolog, Gm-JAG2, appears to be sub- or neofunctionalized, as revealed by the differential expression of the two genes in multiple plant tissues, a complementation test, and an allelic analysis at both loci.

Antagonistic Gene Activities Determine the Formation of Pattern Elements along the Mediolateral Axis of the Arabidopsis Fruit

The Arabidopsis fruit mainly consists of a mature ovary that shows three well defined territories that are pattern elements along the mediolateral axis: the replum, located at the medial plane of the flower, and the valve and the valve margin, both of lateral nature. JAG/FIL activity, which includes the combined functions of JAGGED (JAG), FILAMENTOUS FLOWER (FIL), and YABBY3 (YAB3), contributes to the formation of the two lateral pattern elements, whereas the cooperating genes BREVIPEDICELLUS (BP) and REPLUMLESS (RPL) promote replum development. A recent model to explain pattern formation along the mediolateral axis hypothesizes that JAG/FIL activity and BP/RPL function as antagonistic lateral and medial factors, respectively, which tend to repress each other. In this work, we demonstrate the existence of mutual exclusion mechanisms between both kinds of factors, and how this determines the formation and size of the three territories. Medial factors autonomously constrain lateral factors so that they only express outside the replum, and lateral factors negatively regulate the medially expressed BP gene in a non-autonomous fashion to ensure correct replum development. We also have found that ASYMMETRIC LEAVES1 (AS1), previously shown to repress BP both in leaves and ovaries, collaborates with JAG/FIL activity, preventing its repression by BP and showing synergistic interactions with JAG/FIL activity genes. Therefore AS gene function (the function of the interacting genes AS1 and AS2) has been incorporated in the model as a new lateral factor. Our model of antagonistic factors provides explanation for mutant fruit phenotypes in Arabidopsis and also may help to understand natural variation of fruit shape in Brassicaceae and other species, since subtle changes in gene expression may cause conspicuous changes in the size of the different tissue types.

JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis

SummaryBackgroundIn all multicellular organisms, the links between patterning genes, cell growth, cell cycle, cell size homeostasis, and organ growth are poorly understood, partly due to the difficulty of dynamic, 3D analysis of cell behavior in growing organs. A crucial step in plant organogenesis is the emergence of organ primordia from the apical meristems. Here, we combined quantitative, 3D analysis of cell geometry and DNA synthesis to study the role of the transcription factor JAGGED (JAG), which functions at the interface between patterning and primordium growth in Arabidopsis flowers.ResultsThe floral meristem showed isotropic growth and tight coordination between cell volume and DNA synthesis. Sepal primordia had accelerated cell division, cell enlargement, anisotropic growth, and decoupling of DNA synthesis from cell volume, with a concomitant increase in cell size heterogeneity. All these changes in growth parameters required JAG and were genetically separable from primordium emergence. Ectopic JAG activity in the meristem promoted entry into S phase at inappropriately small cell volumes, suggesting that JAG can override a cell size checkpoint that operates in the meristem. Consistent with a role in the transition from meristem to primordium identity, JAG directly repressed the meristem regulatory genes BREVIPEDICELLUS and BELL 1 in developing flowers.ConclusionsWe define the cellular basis for the transition from meristem to organ identity and identify JAG as a key regulator of this transition. JAG promotes anisotropic growth and is required for changes in cell size homeostasis associated with accelerated growth and the onset of differentiation in organ primordia.

Prognostic significance of Notch signalling molecules and their involvement in the invasiveness of endometrial carcinoma cells

The aim of this study was to investigate the significance of the expression of Notch-related molecules in endometrial carcinoma. The expression of Notch receptors (Notch1 and 3) and Notch ligands [Jagged (JAG) 1 and Delta-like (DLL) 4] was examined immunohistochemically in 37 normal and 76 malignant endometrial tissue samples. For each section, immunohistochemical staining was scored using a positivity index (PI, full score; 200). The effects of a Notch inhibitor, DAPT, on cell proliferation, invasion and motility were investigated using endometrial carcinoma cell lines. The PIs for Notch1 (mean±SD 90.4±15.3), Notch3 (95.6 ± 20.4), JAG1 (95.5±10.0) and DLL4 (88.2±9.6), were significantly higher in endometrial carcinoma than normal endometrium. The PI for Notch1 was associated significantly with advanced International Federation of Gynecologists & Obstetricians (FIGO) stage. In addition, patients with tumours showing high expression of both Notch1 and JAG1 had a poor prognosis compared with those having double-negative carcinomas (P=0.015). DAPT suppressed invasiveness of cells derived from the endometrial carcinoma cell line KLE. The Notch1-JAG1 axis may enhance the invasive properties of endometrial carcinomas, which suggests the Notch pathway may be a promising target for the treatment of this malignancy.

Arabidopsis GenesAS1,AS2, andJAGNegatively Regulate Boundary-Specifying Genes to Promote Sepal and Petal Development

Boundary formation is crucial for organ development in multicellular eukaryotes. In higher plants, boundaries that separate the organ primordia from their surroundings have relatively low rates of cell proliferation. This cellular feature is regulated by the actions of certain boundary-specifying genes, whose ectopic expression in organs can cause inhibition of organ growth. Here, we show that the Arabidopsis thaliana ASYMMETRIC LEAVES1 and 2 (AS1 and AS2) and JAGGED (JAG) genes function in the sepal and petal primordia to repress boundary-specifying genes for normal development of the organs. Loss-of-function as1 jag and as2 jag double mutants produced extremely tiny sepals and petals. Analysis of a cell-cycle marker HISTONE4 revealed that cell division in sepal primordia of the double mutant was inhibited. Moreover, these abnormal sepals and petals exhibited ectopic overexpression of the boundary-specifying genes PETAL LOSS (PTL) and CUP-SHAPED COTYLEDON1 [corrected] and 2 (CUC1 and CUC2). Loss of PTL or CUC1 and CUC2 functions in the as1 jag background could partially rescue the tiny sepal and petal phenotypes, supporting the model that the tiny sepal/petal phenotypes are caused, at least in part, by ectopic expression of boundary-specifying genes. Together, our data reveal a previously unrecognized fundamental regulation by which AS1, AS2, and JAG act to define sepal and petal from their boundaries.