The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays. Consistent with these findings, Dll3 does not bind to cells expressing any of the four N receptors, and N1 does not bind Dll3-expressing cells. However, in a cell-autonomous manner, Dll3 suppressed N signaling, as was found for other DSL ligands. Therefore, Dll3 functions not as an activator as previously reported but rather as a dedicated inhibitor of N signaling. As an N antagonist, Dll3 promoted Xenopus laevis neurogenesis and inhibited glial differentiation of mouse neural progenitors. Finally, together with the modulator lunatic fringe, Dll3 altered N signaling levels that were induced by other DSL ligands.     

Possible roles of DLK1 in the Notch pathway during development and disease

The Delta-Notch pathway is an evolutionarily conserved signaling pathway which controls a broad range of developmental processes including cell fate determination, terminal differentiation and proliferation. In mammals, four Notch receptors (NOTCH1-4) and five activating canonical ligands (JAGGED1, JAGGED2, DLL1, DLL3 and DLL4) have been described. The precise function of noncanonical Notch ligands remains unclear. Delta-like 1 homolog (DLK1), the best studied noncanonical Notch ligand, has been shown to act as an inhibitor of Notch signaling in vitro, but its function in vivo is poorly understood. In this review we summarize Notch signaling during development and highlight recent studies in DLK1expression that reveal new insights into its function.     

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.     

Disruption of the somitic molecular clock causes abnormal vertebral segmentation

Somites are the precursors of the vertebral column. They segment from the presomitic mesoderm (PSM) that is caudally located and newly generated from the tailbud. Somites form in synchrony on either side of the embryonic midline in a reiterative manner. A molecular clock that operates in the PSM drives this reiterative process. Genetic manipulation in mouse, chick and zebrafish has revealed that the molecular clock controls the activity of the Notch and WNT signaling pathways in the PSM. Disruption of the molecular clock impacts on somite formation causing abnormal vertebral segmentation (AVS). A number of dysmorphic syndromes manifest AVS defects. Interaction between developmental biologists and clinicians has lead to groundbreaking research in this area with the identification that spondylocostal dysostosis (SCD) is caused by mutation in Delta-like 3 (DLL3), Mesoderm posterior 2 (MESP2), and Lunatic fringe (LFNG); three genes that are components of the Notch signaling pathway. This review describes our current understanding of the somitic molecular clock and highlights how key findings in developmental biology can impact on clinical practice.     

Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced NOTCH signaling

NOTCH signaling induced by Delta1 (DLL1) and Jagged1 (JAG1) NOTCH ligands is modulated by the β3N-acetylglucosaminyl transferase Fringe. LFNG (Lunatic Fringe) and MFNG (Manic Fringe) transfer N-acetylglucosamine (GlcNAc) to O-fucose attached to EGF-like repeats of NOTCH receptors. In co-culture NOTCH signaling assays, LFNG generally enhances DLL1-induced, but inhibits JAG1-induced, NOTCH signaling. In mutant Chinese hamster ovary (CHO) cells that do not add galactose (Gal) to the GlcNAc transferred by Fringe, JAG1-induced NOTCH signaling is not inhibited by LFNG or MFNG. In mouse embryos lacking B4galt1, NOTCH signaling is subtly reduced during somitogenesis. Here we show that DLL1-induced NOTCH signaling in CHO cells was enhanced by LFNG, but this did not occur in either Lec8 or Lec20 CHO mutants lacking Gal on O-fucose glycans. Lec20 mutants corrected with a B4galt1 cDNA became responsive to LFNG. By contrast, MFNG promoted DLL1-induced NOTCH signaling better in the absence of Gal than in its presence. This effect was reversed in Lec8 cells corrected by expression of a UDP-Gal transporter cDNA. The MFNG effect was abolished by a DDD to DDA mutation that inactivates MFNG GlcNAc transferase activity. The binding of soluble NOTCH ligands and NOTCH1/EGF1-36 generally reflected changes in NOTCH signaling caused by LFNG and MFNG. Therefore, the presence of Gal on O-fucose glycans differentially affects DLL1-induced NOTCH signaling modulated by LFNG versus MFNG. Gal enhances the effect of LFNG but inhibits the effect of MFNG on DLL1-induced NOTCH signaling, with functional consequences for regulating the strength of NOTCH signaling.     

A cell autonomous role for the Notch ligand Delta-like 3 in αβ T-cell development

Notch signalling is critical to help direct T-cell lineage commitment in early T-cell progenitors and in the development of αβ T-cells. Epithelial and stromal cell populations in the thymus express the Notch DSL (Delta, Serrate and Lag2)ligands Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged 1 and Jagged 2, and induce Notch signalling in thymocytes that express the Notch receptor. At present there is nothing known about the role of the Delta-like 3 (Dll3) ligand in the immune system. Here we describe a novel cell autonomous role for Dll3 in αβ T-cell development. We show that Dll3 cannot activate Notch when expressed in trans but like other Notch ligands it can inhibit Notch signalling when expressed in cis with the receptor. The loss of Dll3 leads to an increase in Hes5 expression in double positive thymocytes and their increased production of mature CD4(+) and CD8(+) T cells. Studies using competitive irradiation chimeras proved that Dll3 acts in a cell autonomous manner to regulate positive selection but not negative selection of autoreactive T cells. Our results indicate that Dll3 has a unique function during T-cell development that is distinct from the role played by the other DSL ligands of Notch and is in keeping with other recent studies indicating that Dll1 and Dll3 ligands have non-overlapping roles during embryonic development.     

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

Background

Descending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.

Methods

We studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.

Results

Western blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.

Conclusions

Notch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.     

Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1^Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1^Dll4ki/+ mice, the Dll1^Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.     

OSM-11 facilitates LIN-12 Notch signaling during Caenorhabditis elegans vulval development

Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.     

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.     

Selenium binding protein 1 inhibits tumor angiogenesis in colorectal cancers by blocking the Delta-like ligand 4/Notch1 signaling pathway

BackgroundSelenium binding protein 1 (SELENBP1) is frequently downregulated in malignancies such as colorectal cancer (CRC), however, whether it is involved in tumor angiogenesis is still unknown.MethodsWe analyzed the expression and localization of SELENBP1 in vessels from CRC and neighboring tissues. We investigated the in vitro and in vivo activity of SELENBP1 in angiogenesis and explored the underlying mechanism.ResultsSELENBP1 was localized to endothelial cells in addition to glandular cells, while its vascular expression was decreased in tumor vessels compared to that in vessels from neighboring non-tumor tissues. Gain-of-function and loss-of-function experiments demonstrated that SELENBP1 inhibited angiogenesis in vitro, and blocked communications between HUVECs and CRC cells. Overexpression of SELENBP1 in CRC cells inhibited tumor growth and angiogenesis, and enhanced bevacizumab-sensitivity in a mouse subcutaneous xenograft model. Mechanic analyses revealed that SELENBP1 may suppress tumor angiogenesis by binding with Delta-like ligand 4 (DLL4) and antagonizing the DLL4/Notch1 signaling pathway. The inhibitory effects of SELENBP1 on in vitro angiogenesis could largely be rescued by DLL4.ConclusionThese results revealed a novel role of SELENBP1 as a potential tumor suppressor that antagonizes tumor angiogenesis in CRC by intervening the DLL4/Notch1 signaling pathway.     

Notch expression patterns in the retina: An eye on receptor-ligand distribution during angiogenesis

The critical contribution of the Notch signaling pathway to vascular morphogenesis has been underscored by loss-of-function studies in mouse and zebrafish. Nonetheless, a comprehensive understanding as to how this signaling system influences the formation of blood vessels at the cellular and molecular level is far from reached. Here, we provide a detailed analysis of the distribution of active Notch1 in relation to its DSL (Delta, Serrate, Lag2) ligands, Jagged1, Delta-like1, and Delta-like4, during progressive stages of vascular morphogenesis and maturation. Important differences in the cellular distribution of Notch ligands were found. Jagged1 (Jag1) was detected in "stalk cells" of the leading vasculature and at arterial branch points, a site where Delta-like4 (Dll4) was clearly absent. Dll4 was the only ligand expressed in "tip cells" at the end of the growing vascular sprouts. It was also present in stalk cells, capillaries, arterial endothelium, and in mural cells of mature arteries in a homogenous manner. Delta-like1 (Dll1) was observed in both arteries and veins of the developing network, but was also excluded from mature arterial branch points. These findings support alternative and distinct roles for Notch ligands during the angiogenic process.     

Characterization of Two Distinct Lymphoproliferative Diseases Caused by Ectopic Expression of the Notch Ligand DLL4 on T Cells

Notch signaling is essential for the development of T cell progenitors through the interaction of NOTCH1 receptor on their surface with the ligand, Delta-like 4 (DLL4), which is expressed by the thymic epithelial cells. Notch signaling is quickly shut down once the cells pass β-selection, and CD4/CD8 double positive (DP) cells are unresponsive to Notch. Over the past two decades a number of papers reported that over-activation of Notch signaling causes T cell acute lymphoblastic leukemia (T-ALL), a cancer that prominently features circulating monoclonal CD4/CD8 double positive T cells in different mouse models. However, the possible outcomes of Notch over-activation at different stages of T cell development are unknown, and the fine timing of Notch signaling that results in T-ALL is poorly understood. Here we report, by using a murine model that ectopically expresses DLL4 on developing T cells, that the T-ALL onset is highly dependent on a sustained Notch activity throughout the DP stage, which induces additional mutations to further boost the signaling. In contrast, a shorter period of Notch activation that terminates at the DP stage causes a polyclonal, non-transmissible lymphoproliferative disorder that is also lethal. These observations resolved the discrepancy of previous papers on DLL4 driven hematological diseases in mice, and show the critical importance of the timing and duration of Notch activity.