X-Serrate-1 is involved in primary neurogenesis in Xenopus laevis in a complementary manner with X-Delta-1

Notch, Delta and Serrate encode transmembrane proteins that function in cell fate specification in the Drosophila melanogaster embryo. Here we report gene expression patterns and functional characterization of a Xenopus Serrate homolog, X-Serrate-1. The isolated cDNA encoded a transmembrane protein with a Delta/Serrate/LAG-2 domain, 16 epidermal growth factor-like repeats and a cysteine-rich region. Expression of X-Serrate-1 was observed ubiquitously from unfertilized egg to tadpole, but an upregulation occurred in the tailbud stage embryo. Adult expression was found in eye, brain, kidney, heart, spleen and ovary. Whole-mount in situ hybridization revealed that the organ-related expression in eye, brain, heart and kidney occurred from an early stage of rudiment formation. Overexpression of X-Serrate-1 led to a reduction of primary neurons, whereas an intracellularly deleted form of X-Serrate-1 increased the number of primary neurons. Although the function of X-Serrate-1 in primary neurogenesis was quite similar to that of X-Delta-1, expression of X-Serrate-1 and X-Delta-1 did not affect each other. Co-injection experiments showed that wild-type X-Serrate-1 and X-Delta-1 suppressed overproduction of primary neurons induced by dominant-negative forms of X-Delta-1 and X-Serrate-1, respectively. These results suggest that X-Serrate-1 regulates the patterning of primary neurons in a complementary manner with X-Delta-1-mediated Notch signaling.     

The intracellular domain of X-Serrate-1 is cleaved and suppresses primary neurogenesis in Xenopus laevis

The Notch ligands, Delta/Serrate/Lag-2 (DSL) proteins, mediate the Notch signaling pathway in a numerous developmental processes in multicellular organisms. Although the ligands induce the activation of the Notch receptor, the intracellular domain-deleted forms of the ligands cause dominant-negative phenotypes, implying that the intracellular domain is necessary for the Notch signal transduction. Here we examined the role of the intracellular domain of Xenopus Serrate (XSICD) in Xenopus embryos. X-Serrate-1 has the putative nuclear localization sequence (NLS) in downstream of the transmembrane domain. Biochemical analysis revealed that XSICD fragments are cleaved from the C-terminus side of X-Serrate-1. Fluorescence microscopic analysis showed that the nuclear localization of XSICD occurs in the neuroectoderm of the embryo injected with the full-length X-Serrate-1/GFP. Overexpression of XSICD showed the inhibitory effect on primary neurogenesis. However, a point mutation in the NLSs of XSICD inhibited the nuclear localization of XSICD, which caused the induction of a neurogenic phenotype. The animal cap assay revealed that X-Serrate-1 suppresses primary neurogenesis in neuralized animal cap, but X-Delta-1 does not. Moreover, XSICD could not activate the expression of the canonical Notch target gene, XESR-1 in contrast to the case of full-length X-Serrate-1. These results suggest that the combination of XSICD-mediated intracellular signaling and the extracellular domain of Notch ligands-mediated activation of Notch receptor is involved in the primary neurogenesis. Moreover, we propose a bi-directional signaling pathway mediated by X-Serrate-1 in Notch signaling.     

XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos

Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.     

XMam1, Xenopus Mastermind1, induces neural gene expression in a Notch-independent manner

Mastermind, which is a Notch signal component, is a nuclear protein and is thought to contribute to the transactivation of target genes. Previously we showed that XMam1, Xenopus Mastermind1, was essential in the transactivation of a Notch target gene, XESR-1, and was involved in primary neurogenesis. To examine the function of XMam1 during Xenopus early development in detail, XMam1-overexpressed embryos were analyzed. Overexpression of XMam1 ectopically caused the formation of a cell mass with pigmentation on the surface of embryos and expressed nrp-1. The nrp-1-positive cell mass was produced by XMam1 without expression of the Notch target gene, XESR-1, and not by the activation form of Notch, NICD. The ectopic expression of nrp-1 was not inhibited by co-injection of XMam1 with a molecule known to inhibit Notch signaling. The nrp-1 expression was also recognized in the animal cap injected with XMam1DeltaN, which lacks the basic domain necessary for interacting with NICD and Su(H). These results show that XMam1 has the ability to induce the cell fate into the neurogenic lineage in a Notch-independent manner.     

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.     

Exon 6 of human JAG1 encodes a conserved structural unit

Background  

Notch signaling drives developmental processes in all metazoans. The receptor binding region of the human Notch ligand Jagged-1 is made of a DSL (Delta/Serrate/Lag-2) domain and two atypical epidermal growth factor (EGF) repeats encoded by two exons, exon 5 and 6, which are out of phase with respect to the EGF domain boundaries.     

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.     

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.     

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.     

A growing family of Notch ligands

Signaling through Notch-like receptors is an evolutionarily well-conserved mechanism for cell-cell communication. Transmembrane ligands of the DSL (Delta, Serrate, LAG-2) family signal to Notch receptors on a neighboring cell, which results in an intracellular signaling cascade, influencing cellular differentiation. Recently published data shed new light on the repertoire of ligands and on processing of Notch receptors. One report provides evidence for a novel, more distantly related ligand of the Delta-type in mouse, Dll3 (Delta-like 3)¹. Ectopic expression of Dll3 perturbs primary neurogenesis in frog embryos in a manner expected for a bona fide Notch ligand. Two reports provide new information about processing of Notch receptors. A novel protease, Kuzbanian, is identified, which cleaves the Notch receptor at the extracellular side². Biochemical experiments show that the cleavage probably occurs during intracellular trafficking, and that only processed Notch receptors appear at the cell surface³. Taken together, these reports extend our knowledge about an important event in cell-cell communication—how Notch ligands and receptors meet and interact. BioEssays 20:103–107, 1998. © 1998 John Wiley & Sons, Inc.     

Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca(2+)-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.     

The extracellular matrix protein MAGP-2 interacts with Jagged1 and induces its shedding from the cell surface

Elastic fibers are composed of the protein elastin and a network of 10-12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1. MAGP-2 was found complexed with the Jagged1 extracellular domain shed from 293T cells and COS-7 cells coexpressing full-length Jagged1 and MAGP-2. MAGP-2 shedding of the Jagged1 extracellular domain was decreased by the metalloproteinase hydroxamate inhibitor BB3103 implicating proteolysis in its release. Although MAGP-2 also interacted with the other DSL ligands, Jagged2 and Delta1, they were not found associated with MAGP-2 in the conditioned media, identifying differential effects of MAGP-2 on DSL ligand shedding. The related microfibrillar protein MAGP-1 was also found to interact with DSL ligands but, unlike MAGP-2, was unable to facilitate the shedding of Jagged1. Our findings suggest that in addition to its role in microfibrils, MAGP-2 may also affect cellular differentiation through modulating the Notch signaling pathway either by binding to cell surface DSL ligands or by facilitating release and/or stabilization of a soluble extracellular form of Jagged1.     

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.     

Physical interaction of Delta1, Jagged1, and Jagged2 with Notch1 and Notch3 receptors

The Delta/Serrate/LAG-2 (DSL) domain-containing proteins, Delta1, Jagged1, and Jagged2, are considered to be ligands for Notch receptors. However, the physical interaction between the three DSL proteins and respective Notch receptors remained largely unknown. In this study, we investigated this issue through the targeting of Notch1 and Notch3 in two experimental systems using fusion proteins comprising their extracellular portions. Cell-binding assays showed that soluble forms of Notch1 and Notch3 proteins physically bound to the three DSL proteins on the cell surface. In solid-phase binding assays using immobilized soluble Notch1 and Notch3 proteins, it was revealed that each DSL protein directly bound to the soluble Notch proteins with different affinities. All interactions between the DSL proteins and soluble Notch proteins were dependent on Ca(2+). Taken together, these results suggest that Delta1, Jagged1, and Jagged2 are ligands for Notch1 and Notch3 receptors.