Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity
1998; Springer Nature; Volume: 17; Issue: 24 Linguagem: Inglês
10.1093/emboj/17.24.7351
ISSN1460-2075
Autores Tópico(s)Cerebrovascular and genetic disorders
ResumoArticle15 December 1998free access Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity Julien Royet Julien Royet European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Tewis Bouwmeester Tewis Bouwmeester European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Stephen M. Cohen Corresponding Author Stephen M. Cohen European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Julien Royet Julien Royet European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Tewis Bouwmeester Tewis Bouwmeester European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Stephen M. Cohen Corresponding Author Stephen M. Cohen European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Author Information Julien Royet1, Tewis Bouwmeester1 and Stephen M. Cohen 1 1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7351-7360https://doi.org/10.1093/emboj/17.24.7351 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity. Introduction Signaling mediated by Notch-family receptors is involved in controlling the choice between alternative cell fates (reviewed in Artavanis-Tsakonas et al., 1995; Gridley, 1997; Kimble and Simpson, 1997; Robey, 1997). In primary neurogenesis, Notch signaling directs cells to adopt an epidermal fate as opposed to the default state of neural differentiation (Heitzler and Simpson, 1991; Chitnis et al., 1995; Henrique et al., 1995). Later, in the peripheral nervous system, Notch signaling distinguishes between neural and accessory cell fate (Guo et al., 1996). In some cases Notch is thought to have a permissive function, rendering cells insensitive to other signals which trigger differentiation (Fortini et al., 1993). Notch signaling can also serve an instructive role, for example controlling mitotic division in the Caenorhabditis elegans germ line (reviewed in Kimble and Simpson, 1997), or establishing the dorsal-ventral boundary of the Drosophila wing imaginal disc by directing localized expression of wingless, vestigial and cut (Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Kim et al., 1995; Rulifson and Blair, 1995; Neumann and Cohen, 1996; de Celis and Bray, 1997; Micchelli et al., 1997). Notch encodes a large transmembrane protein which serves as a signal-transducing receptor for the EGFrepeat containing ligands of the Delta-Serrate-LAG2 family. Truncation of the extracellular domain of Drosophila, Xenopus or mouse Notch proteins generates ligand-independent, activated receptors that have constitutive signaling activity (Coffman et al., 1993; Lieber et al., 1993; Rebay et al., 1993; Struhl et al., 1993; Kopan et al., 1994). When expressed without a transmembrane domain the intracellular portion of Notch concentrates in the nucleus (Lieber et al., 1993; Struhl et al., 1993). Expression of an extracellularly-truncated form of mouse Notch in cultured cells leads to spontaneous intracellular cleavage which allows the intracellular domain to localize to the nucleus, where it can activate transcription of Notch target genes together with CBF1 (Jarriault et al., 1995; Kopan et al., 1996; Schroeter et al., 1998). CBF1 is the vertebrate homologue of Suppressor of Hairless, Su(H), a DNA binding protein required for Notch signal transduction (Fortini and Artavanis-Tsakonas, 1994; Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995). Together, this family of Notch binding proteins is called CSL for CBF1, Su(H) and LAG1. Although Drosophila Notch cannot be detected in the nucleus under normal conditions in vivo, recent studies using Notch-GAL4 fusion proteins present strong evidence that cleavage of Notch liberates a fragment of the protein that can translocate to the nucleus and act there to regulate transcription of GAL4-dependent target genes (Struhl and Adachi, 1998). Another recent study has shown that mouse Notch cleavage can be stimulated by ligand binding in cell culture, leading to release of an intracellular fragment that binds to the CSL protein (Schroeter et al., 1998). Using mutants that reduce ligand dependent proteolytic processing of Notch, Schroeter et al. (1998) have shown that the efficiency of processing correlates with the ability to stimulate Notch target gene expression. CSL binding may serve to target Notch to specific DNA sequences in the control regions of Notch-regulated target genes, such as the vertebrate HES1 gene or the vestigial boundary enhancer (Jarriault et al., 1995; Kim et al., 1996; Schroeter et al., 1998). Several proteins have been identified as modifiers of the activity of Notch-family receptors. Deltex binds to the CDC10 repeats and positively regulates Notch (Diederich et al., 1994; Matsuno et al., 1995). Numb, Dishevelled and SEL-10 binding reduce Notch activity (Axelrod et al., 1996; Frise et al., 1996; Guo et al., 1996; Hubbard et al., 1997). Numb binds to the juxtamembrane and C-terminal regions of the Notch intracellular domain and inhibits Notch during specification of cell fates in the PNS (Guo et al., 1996). Dishevelled binds to the C-terminal portion of the cytoplasmic domain of Notch and reduces Notch activity in mediating the choice between neural and epidermal cell fates (Axelrod et al., 1996). sel-10 was identified as a negative regulator of lin-12 activity in C.elegans. sel-10 encodes a protein with F-box and WD40 repeats that binds to the intracellular domain of Notch. Based on similarity to yeast CDC4, SEL-10 may be a component of a ubiquitin E3-ligase that targets Notch for degradation. In this report we present genetic and molecular characterization of a new regulator of Notch signaling activity. The gene was identified in a screen for dominant modifiers of a Notch mutant phenotype in the Drosophila wing. The mutant dominantly suppresses the wing notching phenotype of notchoid mutations and so we call it Notchless. Notchless encodes a novel protein containing WD40-repeats that binds to the cytoplasmic domain of Notch. Notchless modifies Notch signaling activity in a variety of Notch-dependent signaling processes in Drosophila and Xenopus embryos. Results Genetic characterization of a novel modifier of Notch activity notchoid1 (nd1) is a viable mutant allele of Notch that causes scalloping of the wing (Figure 1C). The severity of the nd1 phenotype is sensitive to the level of activity at other loci encoding components of both the Notch and Wingless signaling pathways (Couso and Martinez Arias, 1994; Fortini and Artavanis-Tsakonas, 1994; Hing et al., 1994). Thus nd1 provides a sensitized genetic background in which to screen for modifiers of Notch signaling activity. The BDGP collection of P-element induced lethal mutations (Spradling et al., 1995) was screened for dominant modifiers of the nd1 phenotype. Several P-element induced mutants were found to enhance the severity of nd1 (not shown). One P-element induced mutant, l(2)k13714, was found that suppresses the scalloping of nd1 wings (Figure 1C and D). On the basis of its ability to dominantly suppress scalloping of the wing, we call the gene identified by the l(2)k13714 P-element Notchless (Nle). Figure 1.Genetic interactions between Notchless and notchoid. (A) Cuticle preparation of a wild-type wing. (B) Wingless protein expression in a wild-type wing imaginal disc visualized by antibody staining. Only the wing pouch is shown. The arrow indicates the stripe of Wingless at the dorsal-ventral boundary. (C) Cuticle preparation of a nd1 wing (genotype nd1/Y; note that Notch is on the X-chromosome so males carry only one copy of the gene). Note the notches of the wing (loss of tissue) and mild thickening of the wing veins (e.g. arrow). (D) nd1/Y; Nlek13714/+ wing. The notching of the wing is completely suppressed. Thickening of the veins is suppressed. Note that veins are interrupted or shortened in this genotype (e.g. arrow), suggesting overactivation of the Notch pathway. The same result was obtained using nd1 and the NleΔ8 allele and also using nd2. (E) nd1/Y; Su(H)AR9/+. Removing one copy of the Su(H) gene enhances the severity of the notching of the wing. Wingless expression in a disc of the same genotype is shown at right. Wg is reduced and irregular at the dorsal-ventral boundary. (F) nd1/Y; Su(H)AR9/Nlek13714 wing. Removing one copy of Nle suppresses the notching of the nd1/Y; Su(H)/+ wing and enhances the loss of veins. Wingless expression is restored to normal. (G) ndfa/Y wing. (H) ndfa /Y; Nlek13714/+ wing. The notching of the wing margin is completely suppressed. Veins are normal in this genotype. The same result was obtained using the NleΔ8 allele. Download figure Download PowerPoint To verify that the gene mutated by the P-element is responsible for the mutant phenotype we generated strains from which the original P-element was removed by transposase-mediated excision. These chromosomes differ from the original l(2)k13714 chromosome only by the lack of the P-element and fail to suppress the nd1 phenotype (data not shown). Although l(2)k13714 comes from a collection of P-elements that are supposed to be lethal mutations, we noted that homozygous mutant individuals are recovered in this stock. They are morphologically normal, though males are sterile. The scalloping of nd1 mutant wings is thought to be caused by reduced Wingless activity because overexpression of Wingless can suppress the phenotype (Couso and Martinez Arias, 1994) and because further reducing wingless activity enhances the nd1 phenotype (Hing et al., 1994). Removing one copy of the Su(H) gene enhances the severity of the nd1 phenotype and causes an obvious reduction of Wingless expression at the DV boundary [relative to the level in wild-type; compare Figure 1B with E; nd1 Su(H)/+]. Wingless is restored to wild-type levels and the loss of wing tissue is completely suppressed when the Notchless mutant is introduced in this background [Figure 1F; nd1 Su(H)/Nle]. Notchless also suppresses the phenotypes of ndfa (Figure 1G and H) and nd2 (data not shown), indicating that the genetic interaction is not specific to one particular allele of Notch. The scalloping phenotype of nd alleles is thought to be due to reduced Notch function. Notch signaling through Su(H) is required to induce Wingless at the wing margin (Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Rulifson and Blair, 1995; Neumann and Cohen, 1996). Reducing Su(H) gene dosage enhances the nd1 phenotype. Introducing one copy of the Notchless mutant restores Wingless expression in the nd1 Su(H)/+ background. This suggests that reducing Notchless activity increases Notch activity at the DV boundary of the wing disc. Cloning the Notchless gene The P-element insertion in l(2)k13714 was mapped to cytological position 21C7-8 by the BDGP (Flybase), between the breakpoints of two large deletions Df(2L)al and Df(2L)ast1 (Figure 2A). Neither of these deletions acts as a dominant suppressor of nd1 (data not shown), suggesting that the Notchless gene lies in the interval between them. DNA flanking the l(2)k13714 P-element was cloned by plasmid rescue and hybridized to a chromosomal walk spanning the 100 kb between the deletion breakpoints. The rescued DNA hybridized to a 3.3 kb EcoRI fragment of λ phage Y2-6. Sequencing of the genomic flank identified transcription units on both sides of the P-element insertion (Figure 2A). The 1.5 kb transcript was identified as the Notchless gene by two criteria: (i) the 15 kb SalI fragment of phage Y2-6 was able to restore Notchless activity when introduced into a nd1 Nle/+ mutant background (data not shown). The transgene contains all of the 1.5 kb transcription unit but only part of the other transcription unit; and (ii) expression of the 1.5 kb cDNA under GAL4 control restores full Nle activity. nd1; Nle/+ mutant flies carrying a GAL4 driver show the suppressed nd1 phenotype (Figure 2B). The wing notching phenotype is restored when the 1.5 kb transcript is expressed in the wing disc under GAL4 control in the nd1; Nle/+ mutant (compare Figure 2B with C). Thus, increasing the amount of Notchless product using GAL4 counteracts the effects of the Nle mutant and alleviates the suppression of the nd1 mutant phenotype. This indicates that the Nle mutant phenotype is due to reduced gene activity. Figure 2.Cloning the Notchless gene. (A) schematic representation of the Nle locus. The l(2)k13714 P-element was mapped to 21C8 in the interval between Df(2)al and Df(2)ast1. A chromosomal walk of ∼100 kb (kindly provided by M.Noll) spans this interval. The P-element is inserted in a 3.3 kb EcoRI fragment of phage Y2-6 (map positions of EcoRI, BamHI and SalI sites are indicated). Transcription units were identified on both sides of the P-element by sequence analysis (indicated by arrows below). The 5′ ends of both transcripts are located close to the P-element. Genomic rescue fragment indicates the ∼15 kb SalI fragment. A transgene containing this fragment restores Nle activity (i.e. reverts suppression of the nd1 phenotype by the Nle mutant; data not shown). This result excludes the transcript depicted at left as a candidate to encode Nle because it is only partially contained within the rescue fragment. In situ hybridization showed uniform low level expression of the Nle transcript in imaginal discs (not shown). Δ8 indicates the deletion generated by imprecise excision of the K13714 P-element. Quantitation of Southern blots indicates that the 3.3 kb EcoRI fragment is entirely deleted in Δ8 (not shown). The Nle gene and the adjacent transcription unit are disrupted. The end points of the deletion have not been mapped. It is likely that other genes are affected. (B) Notchless phenotype (suppressed nd1 phenotype) produced when one copy of Nle is mutated in a nd1 fly. In this example the fly also carried the GAL4 driver-line C765 on the third chromosome. (C) Wing from a fly of the genotype as in (B), which also carried a UAS-Nle transgene on the second chromosome. Placing the 1.5 kb transcript under C765-GAL4 regulation restores the nd1 phenotype (arrow). Download figure Download PowerPoint The P-element insertion that causes the mutation is located 310 bp 5′ to the start of the Nle open reading frame. It is therefore likely that the P-element mutant reduces the level of Nle expression. To obtain a deletion that removes the Nle locus, we identified mutants generated by mobilization of the P-element. An excision mutant named NleΔ8 deletes sequences on both sides of the insertion (Figure 2A). To determine whether the NleΔ8 deletion allele would produce a stronger increase in Notch activity than the l(2)k13714 P-element insertion mutant, we first examined suppression of the nd1 phenotype. We observed no difference in the extent of suppression of nd1 (data not shown). The NleΔ8 deletion is embryonic lethal when homozygous, but deletes at least one additional transcription unit. Bearing in mind that any phenotypes produced by the deletion could be attributed to its being mutated in more than one gene, we examined homozygous NleΔ8 embryos and clones of NleΔ8 mutant cells for neurogenic phenotypes. No difference was detected between mutant and wild-type embryos in the developing PNS and CNS, visualized by 22C10 antibody (data not shown). Likewise, we did not observe any bristle pattern abnormality in the notum or wing of homozygous NleΔ8 mutant clones (data not shown). Flies heterozygous for the P-element insertion and the NleΔ8 deletion are viable, morphologically normal and male sterile, like the homozygous P-element mutant. Together these observations suggest that the original P-element mutant may be a null allele of Nle. The lethality caused by the NleΔ8 deletion is likely to be due to another gene. Notchless enhances the effects of mutants that increase Notch activity Certain Abruptex alleles of Notch have been classified as mutations that increase Notch activity. Their phenotypes are enhanced by increasing the level of wild-type Notch gene product and are suppressed by reducing it (de Celis and Garcia-Bellido, 1994; Brennan et al., 1997). Like other gain-of-function Abruptex alleles, Ax28 flies show reduced numbers of some bristles on the head and thorax, as well as shortening of wing veins (Figure 3A and B). These phenotypes are made more severe by introducing an extra copy of the wild-type Notch gene (data not shown). They are also enhanced by removing one copy of the Notchless gene (Figure 3C). The shortening of the wing veins is more pronounced in Ax28 Nle/+ flies (arrows). Ax28 Nle/+ flies show increased loss of both small bristles in the thorax (note the large bare patch outlined in red in Figure 3C) and of large bristles in the head compared with Ax28 flies. Blue shading on the head indicates the cluster of orbital bristles. There are three in wild-type flies, one or two in Ax28 flies and none in Ax28 Nle/+ flies. Thus removing one copy of Nle enhances the severity of the phenotypes caused by increased Notch activity in Ax28 flies. Figure 3.Notchless enhances Abruptex mutant phenotypes. (A) Wild-type wing, thorax and head cuticles. Veins 1-5 are numbered. The red arrows in the central panel indicate two of the large bristles on the thorax. The blue shading in the right panel indicates the cluster of three orbital bristles above the eye. (B) Abruptex28 mutant wing, thorax and head cuticles. Note that veins 4 and 5 are incomplete and do not extend to the wing margin. The number of large bristles is reduced in the thorax (red arrow). Only one or two orbital bristles are found in the head. (C) Abruptex28 NleΔ8/+ mutant wing thorax and head cuticles. The loss of veins is more severe in the wing (arrows). Note also the extensive loss of small bristles in the thorax (red outline). Orbital bristles are absent in the head (blue shading). The same results were obtained using the Nlek13714 allele. Download figure Download PowerPoint We observed that wing veins are reduced in mutant combinations involving nd1 and Nle/+ (Figure 1D and F). Similar results were obtained with nd2 (data not shown). This phenotype is likely to reflect increased Notch activity. Matsuno et al. (1995) have observed loss of wing veins in nd1 heterozygous flies (which are themselves morphologically normal) when a low level of the activated form of Notch is expressed under heat-shock control. Together, these observations suggest that the nd1 mutation shows an abnormal increase in Notch activity in wing vein formation. By analogy to the effects of expressing the activated form of Notch (Matsuno et al., 1995), it is probable that the effect of the Nle mutation is to further increase the aberrant Notch activity in the nd1 mutation. We note that these results appear to be at odds with the observation that the nd1 mutation reduces Notch function at the wing margin (Figure 1). This suggests that the nd1 mutation behaves as a loss-of-function allele in one context and as a gain-of-function allele in another (see Discussion). Note that ndfa shows only the phenotypes thought to be due to reduced Notch activity, loss of wing margin and vein thickening, and that these phenotypes are suppressed by removing one copy of Nle (Figure 1G and H). Notchless opposes deltex function Deltex is thought to function as a positive regulator of Notch activity (Diederich et al., 1994; Matsuno et al., 1995). deltex mutant flies show a phenotype resembling a reduction of Notch activity: nicking of the distal region of the wing blade and thickening of the wing veins (Figure 4A). Removing one copy of Notchless restores the deltex mutant wing to normal (Figure 4B). Thus the effects of reducing deltex activity can be compensated for by simultaneously reducing Notchless activity. Likewise, removing one copy of Notchless enhances the effects of overexpressing Deltex using a heat-shock deltex transgene (Matsuno et al., 1995). Under conditions where Deltex overexpression produces no visible abnormality in an otherwise wild-type wing (Figure 4C), it causes loss of veins in a Nle/+ background (Figure 4D, arrow). This resembles the effects of increasing Notch activity in Abruptex mutants. These results suggest that Deltex and Notchless act in opposite directions as modifiers of Notch activity in wing development. Nle also shows genetic interaction with the Notch pathway genes Su(H) and groucho, but not with Serrate, Delta, Hairless or strawberry Notch (data not shown). Figure 4.Genetic interactions between deltex and Notchless. (A) deltex1 mutant wing. Note the slight notching of the wing tip (arrowhead) and the thickened veins (e.g. arrow). (B) deltex1 NleΔ8/+ mutant wing. Wing shape and vein pattern are completely restored to normal. The same result was obtained using the Nlek13714 allele. (C) Heat-shock Deltex overexpression under mild conditions produces no phenotype in an otherwise wild-type wing (see also Matsuno et al., 1995). Two 1 h treatments at 37°C were given between 0 and 24 h after pupation. (D) Comparable heat-shock Deltex treatment causes loss of veins in a NleΔ8/+ wing (arrow). The same result was obtained using the Nlek13714 allele. Download figure Download PowerPoint Notchless encodes a novel WD40-repeat-containing protein The predicted Notchless protein has a novel highly conserved N-terminal domain followed by nine WD40 repeats (Figure 5A). The WD40 repeat is found in a wide variety of proteins of diverse function and is thought to be a protein interaction domain (reviewed in Neer et al., 1994). Typically WD40 proteins contain seven repeats. Structure analysis of β-transducin suggests that these form a propeller-like structure and that seven repeats can pack to make a flat cylinder (Neer and Smith, 1996). Notchless is unusual in that it appears to contain nine WD40 repeats. Repeats 5 and 6, though recognizable as WD motifs, appear quite divergent in that they lack particular signature residues of the WD40 repeat (not shown). Figure 5.Molecular features of Notchless protein. (A) Schematic representation of Notchless protein and its orthologues. The conserved Nle domain is indicated in dark gray. WD40 repeats are numbered 1-9 (white numbers). Percent identity to the Drosophila protein are indicated for the Nle domain and for individual WD40 repeats. DDBJ/EMBL/GenBank accession Nos for the sequences are Drosophila Nle (AJ012588); Xenopus Nle (AF069737); mouse EST (AA396500); Human EST (AA341327); S.cerevisiae (1351791); C.elegans sequence was compiled from multiple clones (C48486, D70156, C35601 and M89091) and has a gap in the sixth WD40 repeat. (B) Comparison of Nle domains. Sequence identity is highlighted in black, similarity in gray. Similarities are not highlighted if shared by fewer than four proteins. Dashes indicate gaps introduced to accommodate extra residues in the yeast protein. '+15 aa' indicates a larger insertion. As Notch homologues have not been reported in yeast, it is possible that the yeast Nle protein has a different function, reflected in the more divergent structure of this domain. Download figure Download PowerPoint BLAST searches using the N-terminal sequence (before the first WD repeat) identified closely related sequences in yeast, C.elegans, man and mouse. In all cases the N-terminal domain is followed by WD repeats. The human and mouse ESTs extend far enough to show the start of the first WD repeat. Degenerate PCR using primers directed against conserved sequences in the N-terminal domain of the mouse and human proteins was used to isolate a Xenopus Nle cDNA. The Xenopus protein also contains nine WD repeats with strong similarity to the Drosophila and C.elegans proteins. We note that particular WD40 repeats are more similar between species than they are to other WD40 repeats in the protein of the same species. Together, this suggests that these proteins represent true orthologues. Database searches suggest that there may only be one member of this gene family in C.elegans, mouse and human. Sequence comparison indicates that the degree of conservation in the N-terminal domain is quite high among the different family members (Figure 5B). In the 80 amino acid region corresponding to residues 27-106 of Notchless, sequence identity ranges from 33% between Drosophila and Saccharomyces cerevisiae to 61% between Drosophila and Xenopus proteins. Particular residues are identical in all species examined, suggesting that they may be important for domain structure. We propose that this be called the Nle domain. The sel-10 gene of C.elegans encodes a WD40-repeat-containing protein that modifies lin-12 function (lin-12 is a Notch homologue; Hubbard et al., 1997). Although SEL-10 and Notchless both contain WD40 repeats, they are not orthologues. Notchless has nine WD40 repeats rather than the seven repeats found in SEL-10, and does not contain the F-box that characterizes SEL-10 as a CDC4-related protein. SEL-10 does not share the conserved Nle domain in the N-terminus of Notchless. A different C.elegans predicted protein appears to be the orthologue of Notchless (Figure 5B). Notchless expression in Xenopus The Xenopus Notchless gene (XNle) is maternally transcribed and expression remains relatively constant during the early stages of embryonic development without obvious signs of localization. Elevated levels arise at the end of gastrulation and are maintained during neurulation and organogenesis (Figure 6A). Localized expression is observed in two lateral domains adjacent to the rostral neural plate, which correspond to the premigratory neural crest cells, and in a region at the anterior end of the neural plate, which corresponds to placodal precursors (Figure 6B). There is also increased expression in the involuting paraxial mesoderm and in two patches lateral to the closing slit blastopore, through which future somitic cells involute. During subsequent stages expression is evident in the somites and unsegmented paraxial mesoderm, the segmental plate. High levels are also seen in the head region; in the branchial arches, eyes and different regions of the developing brain (Figure 6B, st. 25). Later on, expression is also found in two patches on the ventral site of the embryo, the ventral blood islands which generate the hematopoietic precursors of the early embryo (Figure 6B, st. 35). The pattern of XNle expression resembles that of other components of the Notch pathway, including Delta and Kuzbanian (Chitnis et al., 1995; Pan and Rubin, 1997). These expression domains correspond to regions where Notch signaling has been implicated in cell fate specification events (Coffman et al., 1993; Chitnis et al., 1995; Jen et al., 1997). Figure 6.Expression of Xenopus Notchless during embryonic development and phenotypic effects of Notchless overexpression on formation of primary neurons. (A) Temporal expression of XNle. Total RNA isolated from the indicated stages of development was analyzed by RT-PCR analysis for expression of XNle and Histone H4 (loading control). E, egg; 4C, 4 cell stage; all other lanes are labeled with stage numbers according to Nieuwkoop and Faber (1956). (B) Spatial expression of XNle. Whole-mount in situ hybridization was used to visualize expression of XNle at neural plate stage (st. 17), tailbud stage (st. 25) and tadpole stage (st. 35). Expression patterns are described in the text. NC, neural crest; pm, paraxial mesoderm; b, brain; e, eye; ba, branchial arches; s, somites; sp, segmental plate; vbi, ventral blood islands. (C) Phenotypic consequence of overexpression of XNle, DNle and an activated form of Xenopus NotchI (XN-ICD) on primary neurogenesis. LacZ RNA was co-injected to mark the injected side. Control embryo: l, i and m denote lateral, intermediate and medial rows, respectively, of β-tubulin expressing primary neurons. Note the reduction in the number of primary neurons on the injected side in embryos injected with XNle, DNle or XN-ICD. Arrows indicate the absence of lateral and intermediate neurons in XNle and DNle injected embryos and all neurons in XN-ICD-injected embryo. In (B) and (C) anterior is to the left. Download figure Download PowerPoint Overexpressing Notchless increases Notch activity Based on the finding that reducing Nle activity increases Notch activity in Drosophila (Figures 1,2,3,4), we anticipated that overexpression of Nle would reduce N
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