C. elegans genes lin-12 and glp-1 are members of the family. They act as receptors in developmental cell interactions and their functions overlap. The gene lag-2 encodes a transmembrane protein with similar amino-terminal regions to Delta and Serrate, and acts as a ligand for lin-12 and glp-1 receptors (Tax, 1994).

Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates. In C. elegans, two DSL genes, lag-2 and apx-1, influence different cell fate decisions during development. APX-1 can fully substitute for LAG-2 when expressed under the control of lag-2 regulatory sequences. In addition, truncated forms lacking the transmembrane and intracellular domains of both LAG-2 and APX-1 can also substitute for endogenous lag-2 activity. Moreover, these truncated forms are secreted and able to activate LIN-12 and GLP-1 ectopically. Expression of a secreted DSL domain alone may enhance endogenous LAG-2 signaling. These data suggest ways that activated forms of DSL ligands in other systems may be created (Fitzgerald, 1995).

Xenopus X-Serrate-1 encodes 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 is found ubiquitous from unfertilized egg to tadpole, but an upregulation occurs in the tailbud stage embryo. Adult expression is found in eye, brain, kidney, heart, spleen and ovary. Organ-related expression occurs in eye, brain, heart and kidney from an early stage of rudiment formation. Overexpression of X-Serrate-1 leads to a reduction of primary neurons, whereas an intracellularly deleted form of X-Serrate-1 increases the number of primary neurons. Although the function of X-Serrate-1 in primary neurogenesis is quite similar to that of X-Delta-1, expression of X-Serrate-1 and X-Delta-1 do not affect each other. Co-injection experiments have shown that wild-type X-Serrate-1 and X-Delta-1 suppress 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 (Kiyota, 2001).

The Notch ligands, Delta/Serrate/Lag-2 (DSL) proteins, mediate the Notch signaling pathway in 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. The role of the intracellular domain of Xenopus Serrate (XSICD) was examined in Xenopus embryos. X-Serrate-1 has a putative nuclear localization sequence (NLS) 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 shows 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 shows the inhibitory effect on primary neurogenesis. However, a point mutation in the NLSs of XSICD inhibits the nuclear localization of XSICD, which causes the induction of a neurogenic phenotype. Animal cap assay reveals that X-Serrate-1 suppresses primary neurogenesis in neuralized animal caps, but X-Delta-1 does not. Moreover, XSICD cannot 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 ligand-mediated activation of Notch receptor is involved in the primary neurogenesis. Moreover, a bi-directional signaling pathway is proposed, mediated by X-Serrate-1 in Notch signaling (Kiyota, 2004).

These findings suggest that X-Serrate-1 is involved in a specific signal transduction pathway on neural determination distinguishable from the canonical Notch signaling in this system. It is proposed that there are two pathways for the action of X-Serrate-1. One is the canonical Notch-mediated signaling pathway that occurs by binding of the DSL domain of X-Serrate-1 to EGF motif of Notch receptor, because X-Serrate-1 induces the activation of the Notch target gene, XESR-1. The other pathway is that mediated by XSICD itself. In the X-Serrate-1-expressing cell, XSICD is cleaved by an unknown protease (X) and translocated in the nucleus. XSICD does not activate the expression of XESR-1 in contrast to the case of full-length X-Serrate-1. The inhibitory effect of XSICD may not be related to the canonical Notch-mediated signaling pathway. Therefore, XSICD causes the inhibitory effect on primary neurogenesis by two putative pathways: XSICD may suppress directly the expression of proneuronal gene; XSICD may activate some genes that repress the neuronal differentiation. In the cytoplasmic pathway, XSICD may associate with some Notch-related factors, because the neurogenic phenotype is induced by XSICDmt. The NLS mutant of X-Serrate-1 (XSermt) also induces the neurogenic phenotype similar to XSICDmt, which suggests that the nuclear translocation of XSICD is important. Because the effect of XSermt is lower than that of XSICDmt on primary neurogenesis and XSermtGFP is detected only on the plasma membrane, XSermt might not be proteolytically cleaved to produce the XSICDmt fragment because the fragment is not translocated in the nucleus. In any case, XSICD by itself can show an inhibitory effect on primary neurogenesis, which is completely different from the case of XDICD. Although it is still incertain whether XSICD has an inhibitory effect cell-autonomously and whether another molecule (e.g. Notch protein in the neighboring cell) is necessary for the generation of XSICD, a bi-directional pathway is proposed mediated by the Serrate molecule in Notch signaling (Kiyota, 2004).

A chick Serrate homologue, C-Serrate-1, is expressed in the central nervous system, as well as in the cranial placodes, nephric epithelium, vascular system, and distal limb-bud mesenchyme. In most of these sites, its expression is associated with expression of C-Notch-1 and C- Delta-1. All three genes are expressed in the ventricular zone of the hindbrain and spinal cord, throughout the period when neurons are being born. Within this zone, C-Delta-1 and C-Serrate-1 are expressed in complementary subsets of nondividing cells that appear to be nascent neurons: C- Serrate-1 expression is restricted to specific locations along the dorsoventral axis, forming narrow bands extending from the anterior hindbrain to the tail. These observations strongly suggest that Delta-Notch signalling delivers lateral inhibition not only early but throughout vertebrate neurogenesis to regulate neuronal commitment, and that Serrate-Notch signalling may act similarly in this process. By analogy with its role in Drosophila wing patterning, C-Serrate-1 may also have a role in organising the dorso-ventral pattern of the neural tube. Signalling via Notch maintains neurogenesis, both in vertebrates and in flies, by keeping a proportion of the neuroepithelial cells in an uncommitted stem-cell-like state (Myat, 1996).

The roles of Notch, Delta, and Serrate were studied in vertebrate epithelial appendage morphogenesis using the feather as a model. The following observations were made:

  1. C-Notch-1, C-Delta-1, and C-Serrate-1 are not expressed at the early placode stage and are therefore not involved in the determination of bud versus interbud compartments.
  2. From symmetric short buds to asymmetric long buds, C-Delta-1 and C-Serrate-1 are expressed in the posterior bud mesenchyme in a nested fashion, while C-Notch-1 is expressed as a stripe perpendicular to the anterior-posterior (A-P) axis and positioned posterior to the midpoint.
  3. Epithelial-mesenchymal recombination with rotation leads to the disappearance of protein products of these genes followed by their reappearance with new positions appearing to predict their new morphological orientation.
  4. Conditions leading to branched buds (e.g., recombination of later buds) show polarized staining patterns before branching occurs.
  5. The condition leading to symmetrical round buds (e.g., treatment with the protein kinase A agonist forskolin) suppresses expression of all three genes.
These results have led to the hypothesis that Notch, Delta, and Serrate are involved in establishing the A-P asymmetry of feather buds (Chen, 1997).

Starting from probes resembling that of Drosophila Serrate, a full-length human cDNA was assembled (termed human Jagged2) from overlapping cDNA clones. The cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. Expression of the murine Jagged2 homolog is found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript is most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Coexpression of Jagged2 and Notch1 occurs within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibits myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 leads to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induce increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by the binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates (Luo, 1997).

The human and murine Jagged2 (Jag2), a Serrate-like gene, have been cloned, and murine Jag2 pattern during embryogenesis has been studied . Jag2 is expressed as early as E9 in the surface ectoderm of the branchial arches and in the apical ectodermal ridge (AER) of the developing limb. At E12.5, Jag2 expression is upregulated in differentiated neurons of the central and peripheral nervous system and in the inner neuroblastic layer of the developing retina. Outside the nervous system, Jag2 is expressed in the developing vibrissae follicles, tooth buds, thymus, submandibular gland and stomach. These findings suggest the involvement of Jagged2 in the development of the mammalian limb, branchial arches, central and peripheral nervous systems and several tissues whose development depends upon epithelial-mesenchymal interactions (Valsecchi, 1997).

The mouse syndactylism (sm) mutation impairs some of the earliest aspects of limb development and leads to subsequent abnormalities in digit formation. In sm homozygotes, the apical ectodermal ridge (AER) is hyperplastic by embryonic day 10.5, leading to abnormal dorsoventral thickening of the limb bud, subsequent merging of the skeletal condensations that give rise to cartilage and bone in the digits, and eventual fusion of digits. The AER hyperplasia and its effect on early digital patterning distinguish sm from many other syndactylies that result from later failure of cell death in the interdigital areas. Positional cloning was used to show that the gene mutated in sm mice encodes the putative Notch ligand Serrate. The results provide direct evidence that a Notch signaling pathway is involved in the earliest stages of limb-bud patterning and support the idea that an ancient genetic mechanism underlies both AER formation in vertebrates and wing-margin formation in flies. In addition to cloning the sm gene, three modifiers of sm have been mapped, for which possible candidate genes are suggested (Sidow, 1997).

The Notch signaling pathway is a conserved intercellular signaling mechanism that is essential for proper embryonic development in numerous metazoan organisms. The in vivo role of the Jagged2 (Jag2) gene (also known as Serrate 2), which encodes a ligand for the Notch family of transmembrane receptors, has been examined by making a targeted mutation that removes a domain of the Jagged2 protein required for receptor interaction. Mice homozygous for this deletion die perinatally because of defects in craniofacial morphogenesis. The mutant homozygotes exhibit cleft palate and fusion of the tongue with the palatal shelves. The mutant mice also exhibit syndactyly (digit fusions) of the fore- and hindlimbs. The apical ectodermal ridge (AER) of the limb buds of the mutant homozygotes is hyperplastic, and an expanded domain of Fgf8 expression is observed in the AER. In the foot plates of the mutant homozygotes, both Bmp2 and Bmp7 expression and apoptotic interdigital cell death are reduced. Mutant homozygotes also display defects in thymic development, exhibiting altered thymic morphology and impaired differentiation of gammadelta lineage T cells. These results demonstrate that Notch signaling mediated by Jag2 plays an essential role during limb, craniofacial, and thymic development in mice. It is suggested that the hyperplastic AER of the Jag2 knockouts produces excess FGF activity that inhibits expression of Bmp2 and Bmp7 in the foot plate, with a subsequent reduction of interdigital cell death (Jiang, 1998).

The gene Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. R-fng is homologous to Drosophila fringe. R-fng is expressed in the dorsal ectoderm and apical ectodermal ridge (AER) prior to the expression of Fgf-8, a gene thought to play a role in the formation of the AER. Abnormal limb phenotypes consisting of AER-like structures are observed in 16% of embryos infected with a replication-competent retroviral vector containing R-fng, suggesting that R-fng misexpression perturbs the normal formation of the AER. Engrailed-1 (see Drosophila Engrailed) normally expressed in the ventral ectoderm, strongly represses R-fng and Wnt-7a, suggesting that En-1 regulates dorsal-ventral polarity of the limb and the positioning of the AER. Serrate-2 is expressed in the AER from the earliest stages of its formation through at least stage 26. Chicken Notch-1 is also expressed in the AER. Thus R-fng, like its Drosophila counterpart, may act upstream of Notch signaling (Laufer, 1997 and Rodriguez-Esteban, 1997).

Activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof. The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicates that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreases myocardial gene expression and increases expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling has the opposite effect. Activation or inhibition of Notch also regulates contribution of individual cells to the myocardium. Importantly, expression of Nkx2.5 and Gata4 remains largely unaffected, indicating that Notch signaling functions downstream of heart field specification. It is concluded that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates (Rones, 2000).

Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induce jagged1 gene expression, whereas a mutant defective for transactivation does not. Importantly, jagged1 transcripts are also upregulated by endogenous NF-kappaB activation and this effect is inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers leads to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlates with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 is highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system (Bash, 1999).

In addition to its role in neurogenesis, myogenesis, angiogenesis and retinal cell development, the Notch signaling pathway has also been implicated in hematopoiesis and in immune cell malignancies. Consistent with this notion, the expression of Jagged1 promotes the development of primitive hematopoietic precursor cells, whereas activated forms of Notch1 and Notch2 influence the differentiation of myeloid progenitors in response to different cytokines. Overexpression of an activated form of Notch1 influences T-cell differentiation during thymic development. This process recently has been proposed to involve the silencing of CD4 gene expression by HES-1. The mapping of three human Notch genes to chromosomal locations associated with leukemia, lymphoma and myeloproliferative disorders has also suggested a role in immune cell proliferation and malignancy. The demonstration that constitutively active forms of Notch1 induce T-cell leukemia/lymphoma in mice confirms this prediction. The jagged1 locus has been mapped to human chromosome 20p12. Mutations at this locus are associated with Alagille syndrome. Although alterations in immune or hematopoietic function have not been reported, it remains to be determined whether the disease results from haploinsufficiency or from a dominant negative effect exerted by the mutant protein. The ability of the Notch signaling pathway to influence the differentiation and proliferation of different cell lineages may also depend on different inducing signals and on the cellular microenvironment (Bash, 1999 and references).

Jagged1 is highly expressed in the B-cell areas of the spleen, particularly in the splenic marginal zone that is rich in plasma and memory B cells. Consistent with these results, a correlation is found between the expression of Jagged1 and c-Rel in purified mouse splenic B cells. Although the function of Jagged1 in secondary lymphoid organs remains to be determined, both Notch1 and Notch2 are also expressed in the spleen. This raises the possibility of a role for Jagged1-mediated signaling through Notch in the pathways that control the later stages of B-lymphocyte activation, differentiation and/or immune function. The ability of the EBNA-2 protein of Epstein-Barr virus to induce expression of the B-cell activation marker CD23 through its association with the Notch effector CBF1 in transformed B lymphocytes agrees with a role for Notch signaling in B cells. Co-cultivation assays demonstrating that Jagged1-expressing cells induce CD23 gene expression in neighboring B cells is consistent with this hypothesis. Future studies will help to define the role of Jagged1 in the splenic microenvironment, and to clarify whether it signals through Notch in the context of a heterotypic cell-cell interaction or in a cell-autonomous fashion. Recent work suggesting that soluble forms of Notch ligands can trigger signaling through Notch in vivo would be compatible with either possibility (Bash, 1999 and references).

c-rel knock-out mice are impaired for B-cell activation and antibody production. The observation that all known B-cell growth factors fail to rescue the proliferative defect of these cells suggests that c-Rel may regulate the expression of genes, other than those for cytokines and growth factors, which are crucial for the activation and proliferation of B lymphocytes. The finding that c-Rel can trigger the expression of Jagged1 raises the possibility of a connection between the Rel/NF-kappaB and Notch signaling pathways in secondary lymphoid organs. Thus, in addition to controlling the expression of cytokines, immunoregulatory and adhesion molecules, Rel/NF-kappaB factors may also trigger a Notch signaling cascade important for lymphocyte activation and immune function (Bash, 1999 and references).

Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a beta 3N-acetylglucosaminyltransferase (beta 3GlcNAcT) that transfers GlcNAc to O-fucose in epidermal growth factor-like repeats of Notch. Five different Chinese hamster ovary cell glycosylation mutants were used to identify a key aspect of the mechanism of fringe action. Although the beta 3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAc beta 3Fuc, is not elongated. The trisaccharide, Gal beta 4GlcNAc beta 3Fuc, is the minimal O-fucose glycan to support fringe modulation of Notch signaling. Of six beta 4galactosyltransferases (beta 4GalT) in Chinese hamster ovary cells, only beta 4GalT-1 is required to add Gal to GlcNAc beta 3Fuc, identifying beta 4GalT-1 as a new modulator of Notch signaling (Chen, 2001).

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C2XXGG(S/T)C3 consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site is useful for making predictions. The presence of O-fucose on Notch ligands raises the question of whether Fringe, an O-fucose specific ß1,3-N-acetylglucosaminyltransferase, is capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C2XXGGS/TC3 consensus site. A revised, broad consensus site, C2X3-5S/TC3 (where X3-5 are any 3-5 amino acid residues), is proposed (Panin, 2002).

The cleavage of Notch by presenilin (PS)/gamma-secretase is a salient example of regulated intramembrane proteolysis, an unusual mechanism of signal transduction. This cleavage is preceded by the binding of protein ligands to the Notch ectodomain, activating its shedding. It was hypothesized that the Notch ligands, Delta and Jagged, themselves undergo PS-mediated regulated intramembrane proteolysis. The ectodomain of mammalian Jagged is shown to be cleaved by an A disintegrin and metalloprotease (ADAM) 17-like activity in cultured cells and in vivo, similar to the known cleavage of Drosophila Delta by Kuzbanian. The ectodomain shedding of ligand can be stimulated by Notch and yields membrane-tethered C-terminal fragments (CTFs) of Jagged and Delta that accumulate in cells expressing a dominant-negative form of PS or treated with gamma-secretase inhibitors. PS forms stable complexes with Delta and Jagged and with their respective CTFs. PS/gamma-secretase then mediates the cleavage of the latter to release the Delta and Jagged intracellular domains, a portion of which can enter the nucleus. The ligand CTFs compete with an activated form of Notch for cleavage by gamma-secretase and can thus inhibit Notch signaling in vitro. The soluble Jagged intracellular domain can activate gene expression via the transcription factor AP1, and this effect is counteracted by the co-expression of the gamma-secretase-cleaved product of Notch, Notch intracellular domain. It is concluded that Delta and Jagged undergo ADAM-mediated ectodomain processing followed by PS-mediated intramembrane proteolysis to release signaling fragments. Thus, Notch and its cognate ligands are processed by the same molecular machinery and may antagonistically regulate each other's signaling (LaVoie, 2003).

The evolutionary conserved Notch signaling pathway is involved in cell fate specification and mediated by molecular interactions between the Notch receptors and the Notch ligands -- Delta, Serrate, and Jagged. Like Notch, Delta1 and Jagged2 are subject to presenilin (PS)-dependent, intramembranous 'gamma-secretase' processing, resulting in the production of soluble intracellular derivatives. Moreover, and paralleling the observation that expression of familial Alzheimer's disease-linked mutant PS1 compromises production of Notch S3/NICD, the PS-dependent production of Delta1 cytoplasmic derivatives are also reduced in cells expressing mutant PS1. These studies led to the conclusion that a similar molecular apparatus is responsible for intramembranous processing of Notch and it's ligands. To assess the potential role of the cytoplasmic derivative on nuclear transcriptional events, a Delta1-Gal4VP16 chimera was expressed and marked transcriptional stimulation of a luciferase-based reporter was demonstrated. These findings suggest that Delta1 and Jagged2 play dual roles as activators of Notch receptor signaling and as receptors that mediate nuclear signaling events via gamma-secretase-generated cytoplasmic domains (Ikeuchi, 2003).

Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is the major mesenchyme-derived regulator of ureteric budding and branching during nephrogenesis. The ligand activates on the ureteric bud epithelium a receptor complex composed of Ret and GFRα1. The upstream regulators of the GDNF receptors are poorly known. A Notch ligand, Jagged1 (Jag1), co-localises with GDNF and its receptors during early kidney morphogenesis. Both in vitro and in vivo models were used to study the possible regulatory relationship of Ret and Notch pathways. Urogenital blocks were exposed to exogenous GDNF, which promotes supernumerary ureteric budding from the Wolffian duct. GDNF-induced ectopic buds express Jag1, which suggests that GDNF can, directly or indirectly, up-regulate Jag1 through Ret/GFRα1 signalling. The role of Jag1 in nephrogenesis was studied by transgenic mice constitutively expressing human Jag1 in Wolffian duct and its derivatives under HoxB7 promoter. Jag1 transgenic mice show a spectrum of renal defects ranging from aplasia to hypoplasia. Ret and GFRα1 are normally downregulated in the Wolffian duct, but they are persistently expressed in the entire transgenic duct. Simultaneously, GDNF expression remains unexpectedly low in the metanephric mesenchyme. In vitro, exogenous GDNF restores the budding and branching defects in transgenic urogenital blocks. Renal differentiation apparently fails because of perturbed stimulation of primary ureteric budding and subsequent branching. Thus, the data provide evidence for a novel crosstalk between Notch and Ret/GFRα1 signalling during early nephrogenesis (Kuure, 2005).

Mammalian Serrate homologs: Role of Serrate in ear development

The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components show both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the Kolliker's organ immunostain for the presence of both Notch1 and Jag1, while the area of hair cell precursors express neither Notch1 nor Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominates in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreases in hair cells while the pattern of Notch1 expression now includes both supporting cells and hair cells. The normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium results in an increase in the number of hair cells. These data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation (Zine, 2000).

The sensory patches in the vertebrate ear can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell-cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. Here, the arguments are reviewed for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and the role of Notch signaling in each system is examined more closely. Using viral vectors to misexpress components of the Notch pathway in the chick ear, it has been shown that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition, is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help in the understanding of the vertebrate (Eddison, 2000).

The pattern of production of hair cells and supporting cells cannot be determined simply by the pattern of expression of Notch ligands, in the manner proposed by the simple model of lateral inhibition with feedback. The cells that become hair cells are not selected to do so by escape from exposure to Ser1 (they are constantly exposed), Dl1 (its ectopic expression does not change cell fate), or Ser2 (the knockout has only a mild effect). However hair cells contain Numb, which can block Notch activation, supporting the idea that hair cells escape the inhibitory effect of Notch activation not because of lack of ligands from their neighbors, but because they are deaf to the signal delivered by the ligands (Eddison, 2000).

Why are hair cells and supporting cells produced in the observed ratio? This cannot be accounted for simply in terms of the rules of asymmetric inheritance of Numb. If each cell in the developing sensory patch went through a final asymmetric division, yielding one daughter that inherited Numb and one daughter that did not, the result would be a 1:1 ratio of hair cells to supporting cells, whereas the measured ratio (in chick basilar papilla) ranges from 1:1.7 to 1:3.9. The level of Numb in the prospective hair cells as opposed to supporting cells may be controlled in some more complex way or through more complex sequences of cell divisions, or some molecule other than Numb and its asymmetrically located companion proteins may confer immunity to lateral inhibition and serve as the key determinant of cell fate (Eddison, 2000).

Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches. The Notch ligand Serrate1 (Jag1 in mouse and humans) is expressed in the patches from an early stage and may provide Notch activation during the prosensory phase. This study tested whether Notch signalling is actually required for prosensory patch development. When Notch activation was blocked in the chick embryo using the gamma-secretase inhibitor DAPT, a complete loss of prosensory epithelial cells was seen in the anterior otocyst, where they are diverted into a neuroblast fate via failure of Delta1-dependent lateral inhibition. The cells of the posterior prosensory patch remain epithelial, but expression of Sox2 and Bmp4 is drastically reduced. Expression of Serrate1 here is initially almost normal, but subsequently regresses. The patches of sensory hair cells that eventually develop are few and small. It is suggested that, in normal development, factors other than Notch activity initiate Serrate1 expression. Serrate1, by activating Notch, then drives the expression of Sox2 and Bmp4, as well as expression of the Serrate1 gene itself. The positive feedback maintains Notch activation and thereby preserves and perhaps extends the prosensory state, leading eventually to the development of normal sensory patches (Daudet, 2007).

Serrate: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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