a secreted delta1-fc fusion protein functions both as an activator and inhibitor of notch1 signaling

A Secreted Delta1-Fc Fusion ProteinFunctions Both As An Activator andInhibitor of Notch1 Signaling

Carol Hicks,1 Ena Ladi,2 Claire Lindsell,2 James J.-D. Hsieh,3 S. Diane Hayward,3,4

Andres Collazo,5 and Gerry Weinmaster2,6*1Department of Neurobiology, UCLA School of Medicine, Los Angeles, California2Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California3House Ear Institute, Los Angeles, California4Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California5Department of Pharmacology and Molecular Science, Johns Hopkins University School of Medicine,Baltimore, Maryland6Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland

Signaling induced through interactions between DSL(Delta, serrate, LAG-2) ligand-signaling cells and Notch-responding cells influences the developmental fate of awide variety of invertebrate and vertebrate cell types.Consistently with a requirement for direct cell–cell inter-actions, secreted DSL ligands expressed in flies do notappear to activate Notch signaling but rather producephenotypes reminiscent of losses in Notch signaling. Incontrast, secreted DSL ligands expressed in Caenorhab-ditis elegans or supplied to mammalian cells in cultureproduce effects indicative of Notch activation. In fact,engineered secreted DSL ligands have been used tostudy Notch signaling in neurogenesis, gliogenesis, he-matopoeisis, neurite morphogenesis and ligand-inducednuclear translocation of the Notch intracellular domain.Using a recombinant, secreted form of the DSL ligandDelta1, we found that antibody-induced oligomerization(termed “clustering”) was required for this soluble ligandto bind specifically to Notch1-expressing cells, undergointernalization, and activate downstream signaling. Inter-estingly, clustering with either limiting or excess antibodyled to ligand binding in the absence of Notch signaling,indicating that ligand binding is necessary but not suffi-cient for activation of Notch signaling. Moreover, suchantibody clustering conditions blocked Notch1 signalinginduced by membrane-bound DSL ligands. We proposethat multimerization influences whether ligand binding toNotch results in activation or inhibition of downstream sig-naling and suggest that differences in ligand presentationmight account for why secreted forms of DSL ligands havebeen reported to function as agonists and antagonists ofNotch signal transduction. © 2002 Wiley-Liss, Inc.

Key words: Delta; Jagged; activation; inhibition; Notchsignaling

Notch signal transduction regulates the developmentand maintenance of the nervous system in both vertebrates

and invertebrates (Gridley, 1997; Weinmaster, 1997;Artavanis-Tsakonas et al., 1999). Notch activation in-volves interactions between a DSL ligand (Delta1-4, orJagged1-2) on a signaling cell and a Notch receptor(Notch1-4) present on the surface of a responding cell.Both DSL ligands and Notch receptors are single-pass,transmembrane, cell surface proteins, and genetic studiesin Caenorhabditis elegans and Drosophila support the ideathat cell fates regulated by Notch signaling require directcell–cell interactions (Greenwald and Rubin, 1992;Greenwald, 1994); however, the molecular mechanism bywhich ligand binding leads to receptor activation anddownstream signaling is not well understood.

A model for Notch signaling has been proposed inwhich ligand binding to Notch effects proteolytic cleav-ages within Notch extracellular and intramembrane se-quences to release the intracellular domain (NICD) fromthe membrane, facilitating its translocation to the nucleus,where it regulates gene expression directly through inter-acting with DNA binding proteins collectively known as“CSL” [CBF1, Su(H), LAG-1] (Mumm and Kopan, 2000;Weinmaster, 2000). Although the exact mechanism bywhich ligand binding facilitates ectodomain and in-tramembrane proteolysis is unknown, it has been sug-gested that endocytosis may play a role (Parks et al., 2000).Evidence obtained in Drosophila for transfer of the Notchextracellular domain into Delta-signaling cells hasprompted the notion that in addition to providing ligand

Carol Hicks’s current address is: Department of Cell and Molecular Biol-ogy, Pharmacia, Kalamazoo, MI 49007.

*Correspondence to: Gerry Weinmaster, Department of Biological Chem-istry, UCLA School of Medicine, Los Angeles, CA 90095-1737.E-mail: [email protected]

Received 22 January 2002; Revised 6 March 2002; Accepted 7 March 2002

Published online 6 May 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.10263

Journal of Neuroscience Research 68:655–667 (2002)

© 2002 Wiley-Liss, Inc.

for Notch activation that the ligand-signaling cell may alsoplay an active role in removal of the repressive Notchectodomain through a process of transendocytosis (Parks etal., 2000).

These models underscore the well-established rolefor cell–cell interactions in Notch signaling, but they alsoimply that soluble forms of DSL ligands would be unableto activate Notch signaling. However, there are a numberof reports that soluble DSL ligands can activate Notchsignaling. For example, putative soluble forms of the C.elegans ligands LAG-2 or APX-1 can substitute for losses inLAG-2 activity (Fitzgerald and Greenwald, 1995), andvarious secreted forms of either Delta1 or Jagged1 havebeen used to inhibit and induce differentiation of mam-malian cell types (Li et al., 1998; Varnum-Finney et al.,1998, 2000; Wang et al., 1998; Qi et al., 1999; Sestan etal., 1999; Morrison et al., 2000; Ohishi et al., 2000). Ingeneral, the soluble DSL ligands used in these studies weregenerated through recombinant methodologies that in-volved deletion of transmembrane and intracellular se-quences to allow the secretion of soluble extracellulardomains. In addition, membrane-bound Delta has beenshown to be proteolytically cleaved by the metalloprotein-ase Kuzbanian to release a soluble extracellular form ofDelta that has biological activity (Qi et al., 1999). At oddswith soluble DSL ligands activating signaling are findingsthat membrane association of LAG-2 is required for mu-tant rescue in C. elegans (Henderson et al., 1997) and thatsecreted forms of Delta and Serrate function as antagonistsof Notch signaling in Drosophila (Fleming et al., 1997;Hukriede and Fleming, 1997; Hukriede et al., 1997; Sunand Artavanis-Tsakonas, 1997; Qi et al., 1999). Suchantagonistic signaling activities might result from nonpro-ductive interactions of secreted DSL ligands with Notchthat preclude productive interactions with endogenousmembrane-bound DSL ligands. Taken together, thesefindings suggest that soluble extracellular forms of DSLligands can function to regulate Notch signal transductioneither positively or negatively.

In this study we used an engineered, secreted form ofthe DSL ligand rat Delta1 to address the activity of solubleDSL ligands and evaluate their role as regulator of Notchsignaling. We hypothesized that the conflicting data re-ported for secreted DSL ligand activities might be due tosoluble ligands forming different multivalent complexesthat differ in their abilities to activate or inhibit Notchsignaling. To test this idea, we designed a chimeric pro-tein, designated Dl1Fc, in which Delta1 extracellular se-quences are fused in frame at the C-terminus with humanIgG Fc sequences such that treatment with �Fc antibodieswould promote oligomerization or “clustering” of Dl1Fc.

We found that forced oligomerization of Dl1Fc with�Fc antibodies was necessary to detect binding to Notch1;however, ligand binding did not always correlate withactivation of Notch1 signal transduction. Using differentconcentrations of �Fc antibodies to cluster Dl1Fc artifi-cially, we identified conditions that promoted both bind-ing to and activation of Notch1 as well as clustering

conditions that, though allowing binding to Notch1, didnot activate downstream signaling. Importantly, antibody-clustered Dl1Fc that bound to Notch1 but failed to acti-vate signaling antagonized Notch1 signaling induced bymembrane-bound DSL ligands in a cell coculture assay.These data suggest that the multimeric state acheivedthrough clustering secreted DSL ligands with �Fc anti-bodies determines whether ligand binding to Notch leadsto activation or inhibition of downstream signaling. More-over, our findings suggest that ligand binding is necessarybut not sufficient for Notch activation and serve to un-derscore the complexities of ligand-induced Notch signaltransduction.

MATERIALS AND METHODS

Constructs, Cell Lines, and Antibodies

pCDNA3-Dl1Fc has been previously described (Wang etal., 1998; Hicks et al., 2000; Morrison et al., 2000) and encodesthe first 467 amino acids of rat Delta1 fused in frame with theCH2 and CH3 domains of Fc (minus the hinge region) clonedin pCDNA3 (Invitrogen). pBosNotch1 (1–2,531) and pBos-OCDN1 (1–1,761) are as described elsewhere (Shawber et al.,1996a), and pBosOEDN1 encodes a deletion in Notch1 thatremoves most of the extracellular sequences (amino acids 81–1,712). pBosN1GFP encodes Notch1 with an in-frameC-terminal green fluorescent protein (GFP) tag. The mamma-lian expression vector encoding human Fc with a deletion of thehinge region was obtained from Dr. David Anderson for trans-fection into 293T cells to generate the stable cell line Fc/293Tusing hygromycin selection. Parental cell lines were obtainedfrom ATCC and propagated as indicated. The following pub-lished stable cell lines were used in this study: HEK 293T/Dl1Fc, HEK 293T/Fc, Notch1/C2C12 (N113), Jagged1/L(SN3T9), and Delta1/L (Dl18; Shawber et al., 1996a; Wang etal., 1998; Nofziger et al., 1999; Hicks et al., 2000; Morrison etal., 2000).

Generation of Conditioned Media and LigandBinding Assay

Conditioned media (CM) were prepared from confluentDl1Fc and Fc 293T cells grown in 10 cm dishes with 5 mlDMEM for 5 days, centrifuged at low speed to remove cellulardebris, and stored at 4°C (Hicks et al., 2000). Protein expressionwas analyzed by Western blot analysis with 1:2,500 anti-Fc/HRP (Jackson Immunoresearch, West Grove, PA), followed bychemiluminescence detection (ECL; Amersham, ArlingtonHeights, IL).

For the binding assay, HEK-293 cells were transfectedusing either CaPO4 or Lipofectamine2000 (Gibco, Grand Is-land, NY). A total of 10 �g DNA containing either 500 ng ofpBosNotch1, pBosOCDN1, pBosOEDN1 and 9.5 �g of pBosvector was used for CaPO4 transfections. Twenty-four hoursposttransfection, the cells were trypsinized and plated onto poly-L-lysine (PLL)-coated coverslips and incubated for an additional24 hr. Except where otherwise indicated, CM isolated fromeither Fc- or Dl1Fc-expressing cells were incubated with a1:200 final dilution of either Cy5 (�Fc-Cy5) or Texas red(�Fc-TR) conjugated anti-human Fc antibody (Jackson Immu-

656 Hicks et al.

noresearch) for 1 hr at 4°C. Unclustered and clustered CM werediluted with DMEM containing goat serum (GS) and bovineserum albumin (BSA) to final concentrations of 10% and 1%,respectively, and added directly to transfected cells for 15–45min at room temperature (RT). For the clustering antibodytitration experiments, incubations were carried out on ice toprevent nonspecific binding of the clustering antibody. Treatedcells were washed with Hank’s balanced salt solution (HBSS;Gibco) and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Cells were stained for Notch1 expressionusing either affinity-purified 93-4 (intracellular domain; Shaw-ber et al., 1996a) or 5261 (extracellular domain; Shawber et al.,1996a) antibodies and processed for immunofluorescence detec-tion using FITC-conjugated goat anti-rabbit secondary antibody(Jackson Immunoresearch) with Hoechst DNA counterstain(Molecular Probes, Eugene, OR). To detect binding of unclus-tered CM, 1:200 �Fc-Cy5 was included in the secondary anti-body incubation. At this dilution, Fc protein was readily de-tected in Fc-expressing cells (data not shown). Cells werewashed and mounted with paraphenylene-diamine andmowiol/PBS mounting medium for analysis by confocal mi-croscopy (Zeiss LSM 410). Approximately 20 optical sectionswere collected along the z-axis for each cell imaged usinghigh-numerical-aperture oil and water objectives (40�–100�).Projections of the optical sections to one plane were made usingthe Zeiss LSM software (version 3.92). The confocal pinholeaperture was closed down to create thin optical sections thatallowed resolution of different labeled structures two opticalsections apart.

CBF1 Transactivation Assay

For cocultures, N1-C2C12 (N113) or C2C12 cells werecotransfected in 2 � 3 well tissue culture dishes using Lipo-fectamine (Gibco-BRL) with 250 ng of CBF1-luciferase re-porter construct (Hsieh et al., 1996), 50 ng pRLTK (Promega,Madison, WI) expressing Renilla luciferase for normalization oftransfections, and 2.7 �g of vector DNA as previously described(Nofziger et al., 1999; Hicks et al., 2000; Bush et al., 2001).Twenty-four hours posttransfection, cells were treated withclustered Dl1Fc or Fc CM, L cells, or Jagged1- or Delta1-expressing L cells for 24–30 hr, after which lysates were col-lected and analyzed as described (Nofziger et al., 1999; Hicks etal., 2000; Bush et al., 2001). Notch activation of CBF1 isexpressed either as a ratio of normalized luciferase values in-duced by ligand-expressing cells compared with that obtainedwith parental L cells or as Dl1Fc over Fc CM, unless otherwiseindicated.

RESULTS

Delta1Fc Binding to Notch1 Requires ClusteringBecause DSL ligands are membrane bound, we de-

signed a secreted DSL ligand in which the extracellulardomain of rat Delta1 was fused in frame with human IgGFc sequences lacking the hinge region (Dl1Fc; Fig. 1A).The activity intrinsic to cell-associated ligands can bemimicked by artificially clustering the secreted Dl1Fc by�Fc antibodies as previously demonstrated for ephrins(Davis et al., 1994). HEK 293T cells were transfected with

Dl1Fc or Fc encoding constructs, and CM were collectedand analyzed by immunoblotting with �Fc antibody. Dl1Fcand Fc proteins of the expected size were secreted from cellsand released into the culture medium (Fig. 1B), and thusstable cell lines expressing these soluble proteins were gener-ated and used to produce CM for further studies.

To determine the ability of Dl1Fc to bind toNotch1, a binding assay was developed using HEK 293cells transiently transfected with plasmid DNAs encodingfull-length or mutant forms of Notch1. UntransfectedHEK 293 cells do not express detectable levels of endog-enous Notch1 and, therefore, served as internal controlsfor binding specificity. We have found that an inverserelationship exists between the amount of pBosN1 plasmidDNA transiently transfected into 293 cells and the level ofNotch1 protein expressed at the cell surface that would be

Fig. 1. Dl1Fc and Fc proteins are secreted into the medium fromexpressing HEK 293T cells. A: Schematic representation of Delta1 andrecombinant Delta1-Fc (Dl1Fc) chimeric and Fc proteins are shownwith the indicated structural motifs: DSL (Delta, Serrate, LAG-1),EGFR (epidermal growth factor-related repeats), and TM (transmem-brane domain). B: HEK-293T cells were transiently transfected withDNA encoding vector (lane 1), Dl1Fc (lane 2), or Fc (lane 3). CM werecollected and analyzed by immunoblotting using an anti-Fc antibody.Nonreduced Fc dimer is indicated by an asterisk.

Soluble Notch Ligands 657

available for ligand binding (data not shown). Transfec-tion of pBosN1 DNA over a range of 0.01–10 �gindicated that 0.5 �g of DNA was sufficient for immu-nodetection of Notch1-expressing cells and that, underthese conditions, 60% of the transfected cells boundligand. CM containing Dl1Fc or Fc were assayed forbinding to Notch1 either directly (unclustered) or fol-lowing incubation with a Cy5 conjugated anti-humanFc antibody (�Fc-Cy5-clustered). In these experiments,HEK 293 cells were transfected with 0.5 �g of eithervector (pBos) or pBosN1 DNA, and 48 hr later the cellswere incubated with clustered or unclustered CM,washed extensively, fixed, stained for Notch1 expres-sion, and imaged by confocal microscopy (see Materialsand Methods for details). Under these conditions, bind-ing of Dl1Fc preclustered with �Fc-Cy5 was detectedas a red fluorescence on Notch1-expressing cells (greenfluorescence; Fig. 2). No red signal was detected whencells were incubated first with Dl1Fc CM, followed by

�Fc-Cy5 (Fig. 2A); however, a signal for Notch1-expressing cells was detected when Dl1Fc CM was firstpreincubated with �Fc-Cy5 (Fig. 2B), indicating thatDl1Fc binding to Notch1 required preclustering with�Fc antibody. Importantly, Notch1-expressing cells in-cubated with �Fc-Cy5-clustered Fc CM did not exhibitany red fluorescence (Fig. 2C), and no Dl1Fc or Fcbinding was observed under any conditions for vector-transfected cells (Fig. 2D–F), indicating that the Dl1Fcbinding detected is specific for Notch1-expressing cells.

To demonstrate further that the signal detected withclustered Dl1Fc was due to a specific interaction withNotch1 extracellular ligand-binding sequences, cells trans-fected with mutant forms of Notch1 missing either extra-cellular (OEDN1) or intracellular (OCDN1) sequenceswere assayed. In contrast to the binding observed forNotch1-transfected cells (Fig. 2B, 3A–A��), a signal forclustered Dl1Fc was not detected for OEDN1-expressingcells (Fig. 3B). However, clustered Dl1Fc showed efficient

Fig. 2. Dl1Fc binding to Notch1 requires clustering with an anti-Fcantibody. CM from either Dl1Fc (A,B,D,E)- or Fc (C,F)-expressingcells were assayed for binding to Notch1 (A–C)- or vector (D–F)-transfected cells. Dl1Fc CM were presented either as unclustered (A,D)or clustered with �Fc-Cy5 (B,E; red fluorescence). To control fornonspecific binding of the Fc tag, Fc CM was clustered with �Fc-Cy5

and assayed for binding (C,F). After incubation with the indicated CM,cells were washed extensively, fixed, and processed for immunofluo-rescence to identify Notch1-expressing cells (green fluorescence; seeMaterials and Methods for details). Confocal images of overlays fromred and green channels are shown.

658 Hicks et al.

binding to OCDN1 (Fig. 3C), a truncated form ofNotch1 that contains only the extracellular and transmem-brane domains. Insofar as Dl1Fc binding was detected onlywith Notch1 proteins that contain extracellular sequences,

the signal detected with clustered Dl1Fc represents a spe-cific interaction with Notch1 sequences exposed at the cellsurface. Moreover, Notch1 intracellular sequences are notrequired for ligand binding and detection.

Fig. 3. Dl1Fc binding is specific for Notch1 extracellular sequences anddoes not require Notch1 intracellular sequences. HEK 293T cells weretransiently transfected with DNA encoding either full-length Notch1(A–A��), a truncated form of Notch1 missing the extracellular domainOEDN1 (B–B��), or a truncated form of Notch1 missing the intracel-lular domain OCDN1 (C–C��) and assayed for Dl1Fc binding (A–C;

red channel). After incubation with �Fc-Cy5 preclustered Dl1Fc CM,the cell monolayers were washed extensively, fixed, and processed forimmunofluorescence with either 93-4 (N1, OEDN1) or 5261(OCDN1) Notch1 affinity-purified antisera and FITC-conjugatedanti-rabbit antibodies (green channel; A�–C�). Overlays are shown inA��–C��.


Studies in C. elegans and Drosophila have indicatedthat DSL ligands can be transferred from the ligand-signaling cell into the receptor-responding cell (Hender-son et al., 1994; Klueg et al., 1998; Klueg and Muskavitch,1999; Parks et al., 2000) and that, in some cases, intracel-lular multivesicular complexes containing Delta andNotch epitopes have been identified (Fehon et al., 1991;Kooh et al., 1993; Klueg et al., 1998; Klueg andMuskavitch, 1999; Parks et al., 2000). Furthermore, ex-amination of aggregates of Delta- and Notch-expressingS2 cells has revealed that the entire Delta protein is inter-nalized by the Notch-interacting cell and that Deltatransendocytosis does not require the Delta intracellulardomain (Klueg et al., 1998). Because these studies usedmembrane-bound Delta, we asked whether secretedDl1Fc bound to Notch1-expressing cells was internalized.For these experiments, HEK 293 cells were transfectedwith a plasmid encoding a C-terminal GFP-taggedNotch1 (N1GFP) and analyzed for Dl1Fc binding follow-ing clustering with an �Fc-TR antibody. As demonstratedfor �Fc-Cy5-clustered Dl1Fc (Figs. 2B, 3A), �Fc-TR-clustered Dl1Fc bound to N1GFP-expressing cells (Fig.4). To determine whether the bound ligand was internal-ized, Dl1Fc-positive cells were serial sectioned opticallyon the confocal microscope and examined for the presenceof a Dl1Fc-derived signal within the cell. Cells showinglow levels of Dl1Fc binding were examined to avoid anynonspecific effects on internalization that might occurwhen high amounts of Dl1Fc are bound by cells. Theconfocal pinhole aperture was closed down enough toallow us to distinguish the presence of Dl1Fc:�FcTRclusters at the cell surface from those 1–2 �m within thecells. For example, the red fluorescence pattern of Dl1Fc:�FcTR is quite different in optical sections near the cellsurface (Fig. 4E) from that of optical sections as little as 2.1(Fig. 4H) or 4.2 (Fig. 4K) �m away from the sectionshown in Figure 4E (see Fig. 4C for comparison), dem-onstrating that different clusters can be resolved by depth.Dl1Fc clusters often colocalized with N1GFP, which isconsistent with this overlap representing receptor–ligandinteractions (Fig. 4B,E–P). However, in some cases, thered fluorescence intrinsic to Dl1Fc:�FcTR did not appearto overlap with the green fluorescence of N1GFP in thesame optical section (Fig. 4K, arrowhead), but, when anadjacent optical section was examined, overlap could bedetected, which becomes obvious when a composite isformed from multiple sections (Fig. 4B). Although muchof the Dl1Fc staining appears to be restricted to the cellmembrane, optical sections inside the cells at the level ofthe nucleus also reveal clusters of internalized Dl1Fc (seearrows and arrowheads in Fig. 4D,H,K). These findingssuggest endocytosis of Dl1Fc by N1GFP-expressing cellsand that this internalization does not require membrane-bound ligand.

Delta1Fc Activation of Notch1 SignalingRequires Clustering

Although we could detect a signal for Dl1Fc insidethe cell, indicative of ligand–receptor internalization, it is

not known whether such intracellular complexes representdown-regulation or activation of ligand-induced Notchsignaling. Dl1Fc has been previously shown to activateNotch signaling (Berezovska et al., 2000a,b) and to regu-late cellular differentiation (Wang et al., 1998; Morrison etal., 2000), and in some cases clustering is required, asdemonstrated here for Dl1Fc CM binding (Fig. 2). To testfurther the requirement for Dl1Fc clustering in activationof Notch1 signal transduction, clustered and unclusteredCM were assayed using the well-established CBF1 re-porter system as a measure of Notch signaling (Shawber etal., 1996b; Nofziger et al., 1999; Bush et al., 2001).Briefly, either parental C2C12 cells or stable Notch1-expressing C2C12 cells (N113) were transiently trans-fected with a CBF1-luciferase reporter construct and ei-ther cocultured with Delta1-expressing L cells (D1) orincubated with Dl1Fc preclustered with unconjugated�Fc antibody. After culturing for 24 hr, the cells wereassayed for luciferase activity. In this assay, bothmembrane-bound Delta1 (D1) and preclustered Dl1FcCM stimulated activation of the CBF1-luciferase reportergene in a Notch1- and Delta1-dependent manner (Fig. 5).In contrast, unclustered Dl1Fc CM did not activate thisreporter in N113 cells above background levels, indicatingthat secreted Dl1Fc requires preclustering with an �Fcantibody to induce Notch1 signaling, a finding that con-curs with the preclustering requirement for Dl1Fc bindingto Notch1 (Fig. 2).

Both binding and activation of Notch1 by Dl1Fcrequired pretreatment with �Fc antibodies (Figs. 2, 5),suggesting that soluble Delta1 is functional only whenoligomerized through antibody-induced clustering.

‹

Fig. 4. Dl1Fc is detected both on the surface of and inside N1GFP-expressing cells. HEK 293T cells were transiently transfected withDNA encoding full-length Notch1 tagged with GFP (N1GFP) andincubated with Dl1Fc preclustered with �Fc-TR and processed forconfocal microscopy. Two cells displaying Dl1Fc binding were opti-cally sectioned from the surface closest to the objective to the oppositesurface. Four optical sections are shown. A: Projection of all Dl1Fcfluorescence seen in the 20 optical sections collected. Note that thetotal fluorescence and its restriction to the two cells indicates a relativelylow level of Dl1Fc binding. Cross section shown in D is indicated bya line. B: Overlay of N1GFP projections (green) with the Dl1Fcprojection (red). C: Projection of all 512 cross sections of Dl1Fcfluorsecence down to a plane parallel to the line in A. Though thez-resolution is an order of magnitude less than the x-y resolution,Dl1Fc fluorsecence can still be easily separated in cross section. Linesrepresent the planes of optical sections shown in E–P. D: InternalizedDl1Fc seen in cross section. Arrows and arrowheads indicate fluorse-cence seen in H and K. E,H,K,N: Dl1Fc fluorsecence seen in singleoptical sections. Internalized Dl1Fc is indicated by arrows and arrow-heads. F,I,L,O: N1GFP fluorsecence seen in single optical sections.G,J,M,P: Dl1Fc in red and N1GFP in green shown in overlay. E–G:Surface of cells closest to objective. Optical sections 1.4 �m frombeginning of z-series. H–J: Optical sections 3.5 �m deep. K–M:Optical sections 5.6 �m deep. N–P: Surface of cells farther fromobjective at optical sections 9.8 �m deep. n, Nucleus. Scale bar equals5 �m.

660 Hicks et al.

Figure 4


This dependence on antibody-mediated oligomerizationprompted us to consider the possibility that binding andactivation might depend on the complexity of the formedDl1Fc:�Fc oligomers. In fact, according to the latticetheory, the molecular composition of antigen–antibodycomplexes varies with the concentrations of the antigenand antibody (Klein, 1982). Therefore, we reasoned thatthe complexity of the Dl1Fc:�Fc network or latticeformed through �Fc-mediated clustering might also de-pend on the amount of Dl1Fc present in the CM as wellas the concentration of the Fc antibody. To investigate thisidea further, we determined whether the concentration of�Fc antibody used to precluster Dl1Fc would affect bind-ing and activation of downstream signaling. In these ex-periments, Dl1Fc was clustered at ratios of �Fc:CM of1:10, 1:50, 1:100, and 1:500 and tested both in the bindingand in the CBF1-reporter assays (Fig. 6). Even thoughthere is a requirement for clustering Dl1Fc in our bindingassay (Fig. 2), all �Fc-clustering ratios tested facilitateddetectable binding to HEK 293 cells transiently transfectedwith Notch1 (Fig. 6A). However, when these same ratioswere tested in the CBF1-reporter assay, only �Fc-clustering ratios of 1:50 and 1:100 activated the CBF1-reporter construct, whereas clustering ratios of 1:10 and1:500 produced activities similar to those measured forclustered Fc CM at the same antibody concentrations (Fig.6B). These results are consistent with the lattice theory,which suggests that, when antibody is present in excess,antigen binding sites are saturated, breaking up multivalentcomplexes, whereas, with limiting of antibody, there isnot enough crosslinking to make multivalent complexes(Klein, 1982). Our data indicate that only specific ratios of�Fc:Dl1Fc can induce Notch signaling, even though

binding of Dl1Fc to Notch1 occurs at all antibody con-centrations tested, suggesting that ligand binding is neces-sary but not sufficient for Notch activation and down-stream signaling. Consistently with a requirement formultimerization in soluble ligand activity, monomeric anddimeric forms of human Delta1 can bind C2C12 cells, yetthis binding does not induce Notch signaling or blockmyogenesis; however, when immobilized on plasticdishes, these soluble forms are functional (Varnum-Finneyet al., 2000).

Inactive �Fc-Clustered Forms of Dl1Fc Act asAntagonists of Ligand-Induced Notch1 Signaling

The finding that �Fc-clustering ratios of 1:10 and1:500 produced Dl1Fc that bound but failed to activateCBF1 provided support for the notion that different clus-tering antibody concentrations generate different multiva-lent �Fc:Dl1Fc complexes that differ in their potential toactivate signaling despite their uniform potential to bindNotch1. Because �Fc:Dl1Fc oligomers can be generatedthat bind Notch1 but do not activate downstream signaltransduction (Fig. 6), we determined if such �Fc:Dl1Fcoligomers could function as antagonists of Notch signalinginduced by membrane-bound DSL ligands. We reasonedthat �Fc:Dl1Fc oligomers defective in CBF1-reporter ac-tivation, despite their ability to bind to Notch1, mightfunction to block ligand-induced Notch1 signalingthrough competing with cell-associated DSL ligands forNotch1 expressed on the surface of interacting cells. Totest this idea, we designed experiments to measure thelevel of CBF1 activation induced in Notch1 cells cocul-tured with DSL ligand-presenting cells, in the presence ofeither Fc or Dl1Fc CM clustered with �Fc antibodyconcentrations that support ligand binding (Fig. 6A) butdo not lead to CBF1-reporter activation (Fig. 6B).

N113 cells transfected with the CBF1 reporter con-struct were cocultured with D1 or L cells in the presenceand absence of Fc or Dl1Fc CM clustered at final �Fcantibody concentrations of 1:10 and 1:500. In addition,unclustered CM for both Dl1Fc and Fc were tested andcompared with activities measured for the respective clus-tered CM (Fig. 7A). CBF1 activation induced throughcoculturing N113 cells with D1 cells, relative to L cells,was reduced 65% when cocultures were incubated alongwith 1:10 �Fc:Dl1Fc oligomers and reduced 62% in thepresence of 1:500 �Fc:Dl1Fc clustered oligomers (Fig.7A). Importantly, when unclustered Dl1Fc was added tococultures, the levels of luciferase activity induced inN113 cells in response to D1 cells were comparable withthose in cocultures that contained either clustered or un-clustered Fc CM, indicating that the inhibition in CBF1activation measured for �Fc:Dl1Fc oligomers was depen-dent on and specific for clustered Dl1Fc CM.

Although we have shown here that Dl1Fc binds toNotch1, homotypic interactions have also been reportedfor Delta (Rebay et al., 1991), raising the possibility thatthe decreases detected in Delta1-induced Notch1 signalingcould result from �Fc:Dl1Fc oligomers binding to Delta1-presenting cells in the cocultures rather than Notch1-

Fig. 5. Dl1Fc requires antibody-mediated clustering to activateNotch1-induced CBF1 activity. C2C12 and stable Notch1-expressingC2C12 cells (N113) were transiently transfected with a CBF1-luciferase reporter construct and assayed for Notch-mediated CBF1activation following coculturing with either L cells or Delta1-expressing cells (D1) or incubation with Dl1Fc clustered at 100:1 with�Fc (Dl1Fc:�Fc) or unclustered Dl1Fc CM. Activity is represented as-fold activation by ligand (D1 cells or Dl1Fc) over that induced by Lcells or Fc CM, respectively. Values reported are the average of twoexperiments. Neither D1 cells (1.62 � 0.01) nor Dl1Fc:�Fc (1.11 �0.05) strongly activated CBF1 in C2C12 cells. CBF1 was activated inN113 cells by D1 (7.2 � 1.7) and Dl1Fc:�Fc (4.0 � 0.3) but not byunclustered Dl1Fc (1.1 � 0.005).

662 Hicks et al.

responding cells. However, we have been unable to dem-onstrate binding of �Fc:Dl1Fc oligomers to Delta1-expressing cells (data not shown) and thus favor the ideathat �Fc:Dl1Fc oligomers antagonize Notch signalingthrough nonproductive binding to Notch1-expressingcells, which prevents access and activation of Notch1 byDelta1-presenting cells. To test this idea further, we mea-sured the ability of �Fc:Dl1Fc oligomers to antagonizeNotch1 signaling induced in cocultures by the related yetstructurally distinct DSL ligand Jagged1. N113 cells trans-fected with the CBF1-reporter were cocultured withJagged1-presenting cells in the presence and absence of�Fc:Dl1Fc oligomers along with the appropriate controlsused in Figure 7A, and CBF1 activation was determined asa measure of Jagged1-induced Notch signaling. Whenassayed under these conditions, Jagged1-induced activa-tion of CBF1, relative to that produced by L cells, wasreduced by 67.3% in the presence of 1:10 �Fc:Dl1Fcoligomers and by 76.5% with 1:500 �Fc:Dl1Fc oligomers(Fig. 7B). Importantly, clustered Fc CM did not diminishJagged1-Notch1-mediated activation of CBF1, indicatingspecificity for Dl1Fc in this assay. These data support thenotion that �Fc:Dl1Fc oligomers that bind but fail toactivate Notch1 can antagonize signaling induced bymembrane-bound DSL ligands. Taken together, thesefindings suggest that the ability of Dl1Fc to function eitheras an agonist or as an antagonist of Notch signaling is likelyto depend on the multimeric state achieved through clus-tering at different �Fc antibody concentrations as well asthe presence of DSL-ligand signaling cells.

DISCUSSIONGenetic, cellular, and molecular studies have pro-

vided support for the idea that DSL ligands induce Notchsignaling via direct cell–cell interactions (Greenwald andRubin, 1992). The membrane nature of DSL ligandsrequired for cell–cell interactions would also function torestrict Notch signaling to local sites, in contrast to se-creted ligands that activate signaling at sites distant fromtheir source. However, Delta can be proteolyticallycleaved from the cell surface to generate a soluble extra-cellular form that binds Notch and displays biologicalactivity (Qi et al., 1999), suggesting that Notch signalingmight also be induced by a soluble form of Delta. It is alsopossible that, rather than activating Notch signaling, sol-uble forms of Delta function as blockers of ligand–receptorinteractions and downstream signaling. In support of thisidea, secreted forms of Delta perturb aggregation of Delta-and Notch-expressing cells (Qi et al., 1999) and blockNotch-induced repression of myogenesis (Varnum-Finney et al., 2000). Furthermore, secreted forms of Deltaand Serrate expressed in flies produce strong antimorphicphenotypes, suggesting that they block endogenousligand-induced Notch signaling (Fleming et al., 1997;Hukriede and Fleming, 1997; Hukriede et al., 1997; Sunand Artavanis-Tsakonas, 1997).

In C. elegans, characterization of putative secretedforms of the DSL ligands LAG-2 and APX-1 has alsoproduced conflicting findings and interpretations. In one

study, soluble extracellular domains of these ligands rescuemutant ligand phenotypes (Fitzgerald and Greenwald,1995), whereas another report indicates that such formsare inactive in mutant rescue unless fused to GFP se-quences (Henderson et al., 1997). Interestingly, althoughsecreted DSL ligands are strong dominant negatives whenexpressed in wild-type Drosophila (Fleming et al., 1997;Hukriede and Fleming, 1997; Hukriede et al., 1997; Sunand Artavanis-Tsakonas, 1997), in C. elegans they pro-duce phenotypes indicative of ectopic receptor activation(Fitzgerald and Greenwald, 1995).

The generation of secreted extracellular domains ofmammalian DSL ligands has proved to be a very useful toolwith which to study Notch signaling in a number of differentsystems. Secreted forms have allowed measurement of bind-ing affinities for the different DSL ligands to Notch receptors(Shimizu et al., 1999, 2000b), demonstration of enhancedligand binding by fringe-modified Notch (Bruckner et al.,2000; Hicks et al., 2000), and characterization of eventsdownstream of receptor activation, such as proteolytic cleav-age, phosphorylation, and nuclear localization of Notch (Be-rezovska et al., 2000a,b; Shimizu et al., 2000a). In addition,hematopoietic (Li et al., 1998; Varnum-Finney et al., 1998;Han et al., 2000; Karanu et al., 2000, 2001; Ohishi et al.,2000), neurogenic (Qi et al., 1999; Sestan et al., 1999; Mor-rison et al., 2000), gliogenic (Wang et al., 1998; Morrison etal., 2000), and myogenic (Varnum-Finney et al., 2000) cellfates have all been manipulated in vitro through exposure tosoluble DSL ligands, and a role for Notch signaling in regu-lating cell numbers through cell cycle regulation, prolifera-tion, and apoptosis has been identified (Han et al., 2000;Ohishi et al., 2000). Not only have studies with soluble DSLligands been enormously helpful in identifying biologicalfunctions for Notch, but the findings that engineered solubleforms of DSL ligands promote the expansion of primitivehematopoietic precursors (Han et al., 2000; Ohishi et al.,2000) holds the exciting prospect for application of thesesoluble DSL ligands in ex vivo expansion of hematopoieticstem/progenitor cells necessary for gene therapy.

As useful as secreted DSL polypeptides have provedto be, the molecular basis for the antagonistic and agonisticactivities reported for soluble extracellular forms remainsunknown. What factors might account for the observeddifferences? Perhaps ligand multimerization is an impor-tant attribute of DSL ligand activity. Consistent with thishypothesis, we found that unclustered Dl1Fc did not bindto Notch1-expressing cells and that Dl1Fc binding wasdetected only when artificially clustered with �Fc anti-body. Insofar as Dl1Fc binding required clustering, it wasnot surprising that Dl1Fc-induced activation of Notchsignaling also required clustering. However, it was surpris-ing to find that the clustering �Fc antibody concentrationdetermined whether Dl1Fc could activate Notch signal-ing. Specifically, we identified conditions under whichclustered Dl1Fc bound to Notch1, but this binding failedto induce Notch signaling, as measured by activation of aCBF1-reporter construct. These data suggest that ligandbinding is necessary but not sufficient for activation of


Fig. 6. Dl1Fc binding to Notch1 is necessary but not sufficient forCBF1 activation. A (a–d�): Dl1Fc CM clustered with �Fc dilutions of1:10, 1:150, 1:100, or 1:500 were analyzed for binding to Notch1-transfected cells as described for Figure 3. B: N113 cells were tran-siently transfected with a CBF1-luciferase reporter construct and incu-bated for 48 hr with either Dl1Fc- or Fc-CM clustered with �Fc

dilutions of 1:10, 1:150, 1:100, or 1:500 and assayed for CBF1-drivenluciferase activity and normalized for transfection efficiency. Dl1Fcclustered at 1:50 or 1:100 stimulated CBF1 activation by 3.3- and4.7-fold, respectively, over Fc CM clustered under the same conditions.In contrast, Dl1Fc clustered at 1:10 or 1:500 showed weak CBF1activation. Values reported are for single representative experiments.

Notch signal transduction; however, the mechanistic roleligand plays in addition to receptor binding is unknown.

Current models for Notch signaling have proposedroles for DSL ligands beyond simple binding to Notch(Parks et al., 2000). Whether ligand binding serves tostabilize ligand–Notch interactions, induce conforma-tional changes in Notch that expose cleavage sites forspecific proteases, or dissociate the Notch extracellulardomain to effect Notch activation and downstream sig-naling is unknown. According to the transendocytosismodel (Parks et al., 2000), ligand-mediated endocytosis ofNotch is an important aspect of Notch activation, and thisis likely to depend on ligand presentation within theplasma membrane. However, we find that soluble ligands,albeit artificially clustered forms, bind specifically toNotch1, are internalized in Notch1-expressing cells, andinduce transactivation of CBF1, a read-out of Notch sig-naling. Based on the fact that we and others (Varnum-Finney et al., 1998; Varnum-Finney et al., 2000) haveidentified a requirement for oligomerization of solubleligands for biological activity, multimerization of ligandwithin the plasma membrane could be an important eventin ligand-induced Notch signaling and may well accountfor the membrane nature of DSL ligands. Why clusteringof Dl1Fc is important for receptor binding and activationis unknown; it may be that ligand oligomerization isnecessary to promote Notch dimerization and/or cluster-ing, which could facilitate proteolytic events required foractivation of downstream signaling.

Consistent with our findings, a secreted ephrin-B1/Fc fusion protein requires clustering for full activitywhen presented to primary endothelial cells expressingendogenous EphB1 (Stein et al., 1998). Ephrins aremembrane-bound proteins whose soluble Fc-tagged formsare active only when artificially clustered with an �Fcantibody (Davis et al., 1994), as found here for the DSL

ligand Delta1. Fractionation studies of �Fc antibody-clustered ephrin-B1/Fc have identified different oli-gomerized forms of ephrin-B1/Fc that have distinct prop-erties for EphB1-binding and receptor activation (Stein etal., 1998). Specifically, higher order ephrin B1 oligomers(tetramers) are more biologically active than dimeric formsof this ligand. Importantly, as reported here for Dl1Fc–Notch1 interactions, ephrin-B1/Fc binding does not cor-relate with full activation of the EphB1 receptor. Therequirement for clustering identified for ephrins and DSLligands likely reflects the transmembrane structure of theseligands such that ligand multimerization within the plasmamembrane would facilitate binding to cognate receptors aswell as activation of downstream signal transduction.

Studies with a soluble human Delta1 have reportedthat monomeric and dimeric forms can bind endogenousreceptors expressed in C2 cells, but myogenic differenti-ation is repressed only when these secreted forms areimmobilized on plastic plates, suggesting that aggregationor multimerization is also important for induction of

Fig. 7. Dl1Fc antagonizes Notch1 activation of CBF1 by DSL ligands.A: N113 cells were transiently transfected with a CBF1-luciferasereporter construct and cultured with either L or D1 cells in the presenceof either Fc or Dl1Fc CM unclustered or clustered at �Fc dilutions of1:10 and 1:500 and assayed for luciferase activity. Activation is ex-pressed as percentage -fold activation induced by membrane-boundligands. B: Transfected N113 cells cultured with either L or Jagged1-expressing cells (J1) in the presence of either Fc or Dl1Fc CM clusteredat of 1:10 and 1:500 and assayed for CBF1 activation. Values reportedare for single representative experiments.

Figure 6. (Continued.)


Notch signaling in this system (Varnum-Finney et al.,2000). However, other studies have not found a require-ment for clustering, but the use of highly concentrated andpurified secreted DSL proteins (Shimizu et al., 1999,2000a,b; Han et al., 2000) or synthetic DSL peptides (Li etal., 1998) likely provides an opportunity for protein ag-gregation that could induce ligand multimerization re-quired for Notch binding and receptor activation. In fact,we have previously reported that Dl1Fc activates Notch1to inhibit oligodendrocyte precursor cell differentiation inthe absence of antibody-induced clustering (Wang et al.,1998); however, in these experiments, the Dl1Fc CM wasconcentrated approximately 20-fold, conditions that alsoproduce the formation of large-molecular-weight aggre-gates (data not shown). In another study, we found thatDl1Fc could not induce the differentiation of Schwanncells from neural crest cells unless it was preclustered(Morrison et al., 2000).

With regard to conditions that antagonize ligand-induced Notch signaling, we found that clustering condi-tions that promoted Dl1Fc binding in the absence ofNotch1 activation were effective at inhibiting Notch sig-naling induced by membrane-bound DSL ligands. Al-though we have not characterized the structure of thevarious Dl1Fc:�Fc complexes used in this study, we spec-ulate that the antagonistic and agonistic activities demon-strated here for Dl1Fc are a consequence of differentclustering antibody concentrations generating differentmultimeric forms, all of which bind Notch1 yet havedifferent activation capabilities. For example, in situationsin which ligand binding to Notch1 did not activate down-stream signaling, such ligand binding by Notch couldconceivably preclude interactions with membrane-boundDSL ligands and activation of downstream signaling. Inthis regard, our findings are reminiscent of reports ofsoluble DSL ligands inducing antimorphic effects in the fly(Fleming et al., 1997; Hukriede and Fleming, 1997;Hukriede et al., 1997; Sun and Artavanis-Tsakonas, 1997),perturbing Delta-Notch-mediated aggregation of S2 cells(Qi et al., 1999) and inhibiting Notch-induced myogenicrepression (Varnum-Finney et al., 2000). Perhaps all thesedominant-negative effects are the consequence of solubleligands competitively inhibiting Notch signaling.

Given the conflicting data reported in the literatureregarding the activities of various soluble DSL ligands,both in vertebrate and in invertebrate systems, it will beimportant to determine the precise molecular nature andmechanism by which secreted DSL ligands exert theireffects on Notch signaling, whether positive or negative.Importantly, the biological relevance of soluble DSL li-gands to Notch signal transduction in whole animals needsto be addressed. In fact, even though proteolytic fragmentsof the Delta extracellular domain have been identified inDrosophila embryos, larvae, and cultured cells, their func-tion and physiological role remain to be determined(Klueg et al., 1998). Arguing against soluble ligands acti-vating Notch signaling in vivo are the strong Notch an-timorphic phenotypes reported for secreted forms of Deltaand Serrate expressed in Drosophila (Fleming et al., 1997;

Hukriede and Fleming, 1997; Hukriede et al., 1997; Sun andArtavanis-Tsakonas, 1997). Nonetheless, Drosophila Kuzba-nian (Kuz), an ADAM family member, has been shown torelease functional ligand from the surface of cultured Delta-expressing cells (Qi et al., 1999), and Kuz mutants displayphenotypes indicative of defects in Notch signaling (Pan andRubin, 1997; Sotillos et al., 1997). Based on these findings, ithas been proposed that DSL ligands proteolytically releasedfrom cells might activate Notch signaling in vivo (Qi et al.,1999).

Because we find that Notch signaling induced by asecreted form of Delta1 is sensitive to clustering condi-tions, we speculate that ligand oligomerization plays animportant role in influencing the conformational changesrequired for Notch proteolysis and downstream signaling.Given that many aspects of the Notch signaling modelremain to be tested, development of soluble DSL ligandsprovides an additional tool with which to study not onlythe cell types regulated by Notch but also the molecularmechanism of ligand-induced Notch signaling. The po-tential use of secreted DSL proteins both as physiologicalregulators of Notch signaling and as therapeutic agentsunderscores the importance of soluble DSL ligand studies.

ACKNOWLEDGMENTSWe thank Larry Zipursky and Alison Miyamoto for

helpful comments and critical reading of the manuscript,Luisa Iruela-Arispe and Wendy Walwyn for help withconfocal analysis, Guy diSibio for construction of Dl1Fc,Gay Bush for generation of Dl1Fc- and Fc-expressing 293cell lines, and David Anderson for the Fc construct. Thiswork was supported by National Institutes of Health grantR01-NS31885-09 and the Stop Cancer Foundation (JonssonComprehensive Cancer Center) to G.W., NIH grant R01-DC04061 to A.C., and NIH training grants to C.H. and E.L.

REFERENCESArtavanis-Tsakonas S, Rand MD, Lake RJ. 1999. Notch signaling: cell fate

control and signal integration in development. Science 284:770–776.Berezovska O, Jack C, McLean P, Aster JC, Hicks C, Xia W, Wolfe MS,

Kimberly WT, Weinmaster G, Selkoe DJ, Hyman BT. 2000a. Aspartatemutations in presenilin and gamma-secretase inhibitors both impairnotch1 proteolysis and nuclear translocation with relative preservation ofnotch1 signaling. J Neurochem 75:583–593.

Berezovska O, Jack C, McLean P, Aster JC, Hicks C, Xia W, Wolfe MS,Weinmaster G, Selkoe DJ, Hyman BT. 2000b. Rapid Notch1 nucleartranslocation after ligand binding depends on presenilin-associatedgamma-secretase activity. Ann NY Acad Sci 920:223–226.

Bruckner K, Perez L, Clausen H, Cohen S. 2000. Glycosyltransferaseactivity of Fringe modulates Notch–Delta interactions. Nature 406:411–415.

Bush G, diSibio G, Miyamoto A, Denault JB, Leduc R, Weinmaster G.2001. Ligand-induced signaling in the absence of furin processing ofNotch1. Dev Biol 229:494–502.

Davis S, Gale NW, Aldrich TH, Maisonpierre PC, Lhotak V, Pawson T,Goldfarb M, Yancopoulos GD. 1994. Ligands for EPH-related receptortyrosine kinases that require membrane attachment or clustering foractivity. Science 266:816–819.

Fehon RG, Johansen K, Rebay I, Artavanis-Tsakonas S. 1991. Complexcellular and subcellular regulation of notch expression during embryonicand imaginal development of Drosophila: implications for notch function.J Cell Biol 113:657–669.

666 Hicks et al.

Fitzgerald K, Greenwald I. 1995. Interchangeability of Caenorhabditis elegansDSL proteins and intrinsic signalling activity of their extracellular domainsin vivo. Development 121:4275–4282.

Fleming RJ, Gu Y, Hukriede NA. 1997. Serrate-mediated activation ofNotch is specifically blocked by the product of the gene fringe in thedorsal compartment of the Drosophila wing imaginal disc. Development124:2973–2981.

Greenwald I. 1994. Structure/function studies of lin-12/Notch proteins.Curr Opin Genet Dev 4:556–562.

Greenwald I, Rubin GM. 1992. Making a difference: the role of cell-cellinteractions in establishing separate identities for equivalent cells. Cell68:271–281.

Gridley T. 1997. Notch signaling in vertebrate development and disease.Mol Cell Neurosci 9:103–108.

Han W, Ye Q, Moore MA. 2000. A soluble form of human delta-like-1inhibits differentiation of hematopoietic progenitor cells. Blood 95:1616–1625.

Henderson ST, Gao D, Lambie EJ, Kimble J. 1994. lag-2 May encode asignaling ligand for the GLP-1 and LIN-12 receptors of C. elegans.Development 120:2913–2924.

Henderson ST, Gao D, Christensen S, Kimble J. 1997. Functional domainsof LAG-2, a putative signaling ligand for LIN-12 and GLP-1 receptors inCaenorhabditis elegans. Mol Biol Cell 8:1751–1762.

Hicks C, Johnston SH, diSibio G, Collazo A, Vogt TF, Weinmaster G.2000. Fringe differentially modulates Jagged1 and Delta1 signallingthrough Notch1 and Notch2. Nat Cell Biol 2:515–520.

Hsieh JJ, Henkel T, Salmon P, Robey E, Peterson MG, Hayward SD.1996. Truncated mammalian Notch1 activates CBF1/RBPJk-repressedgenes by a mechanism resembling that of Epstein-Barr virus EBNA2. MolCell Biol 16:952–959.

Hukriede NA, Fleming RJ. 1997. Beaded of Goldschmidt, an antimorphicallele of Serrate, encodes a protein lacking transmembrane and intracel-lular domains. Genetics 145:359–374.

Hukriede NA, Gu Y, Fleming RJ. 1997. A dominant-negative form ofSerrate acts as a general antagonist of Notch activation. Development124:3427–3437.

Karanu FN, Murdoch B, Gallacher L, Wu DM, Koremoto M, Sakano S,Bhatia M. 2000. The notch ligand jagged-1 represents a novel growthfactor of human hematopoietic stem cells. J Exp Med 192:1365–1372.

Karanu FN, Murdoch B, Miyabayashi T, Ohno M, Koremoto M, Galla-cher L, Wu D, Itoh A, Sakano S, Bhatia M. 2001. Human homologuesof Delta-1 and Delta-4 function as mitogenic regulators of primitivehuman hematopoietic cells. Blood 97:1960–1967.

Klein J. 1982. Immunology: the science of self-nonself discrimination: JohnWiley & Sons.

Klueg KM, Muskavitch MA. 1999. Ligand-receptor interactions and trans-endocytosis of Delta, Serrate and Notch: members of the Notch signallingpathway in Drosophila. J Cell Sci 112:3289–3297.

Klueg KM, Parody TR, Muskavitch MA. 1998. Complex proteolyticprocessing acts on Delta, a transmembrane ligand for Notch, duringDrosophila development. Mol Biol Cell 9:1709–1723.

Kooh PJ, Fehon RG, Muskavitch MA. 1993. Implications of dynamicpatterns of Delta and Notch expression for cellular interactions duringDrosophila development. Development 117:493–507.

Li L, Milner LA, Deng Y, Iwata M, Banta A, Graf L, Marcovina S,Friedman C, Trask BJ, Hood L, Torok-Storb B. 1998. The humanhomolog of rat Jagged1 expressed by marrow stroma inhibits differenti-ation of 32D cells through interaction with Notch1. Immunity 8:43–55.

Morrison SJ, Perez SE, Qiao Z, Verdi JM, Hicks C, Weinmaster G,Anderson DJ. 2000. Transient Notch activation initiates an irreversibleswitch from neurogenesis to gliogenesis by neural crest stem cells. Cell101:499–510.

Mumm JS, Kopan R. 2000. Notch signaling: from the outside in. Dev Biol228:151–165.

Nofziger D, Miyamoto A, Lyons KM, Weinmaster G. 1999. Notch sig-naling imposes two distinct blocks in the differentiation of C2C12 myo-blasts. Development 126:1689–1702.

Ohishi K, Varnum-Finney B, Flowers D, Anasetti C, Myerson D, Bern-stein ID. 2000. Monocytes express high amounts of Notch and undergocytokine specific apoptosis following interaction with the Notch ligand,Delta-1. Blood 95:2847–2854.

Pan D, Rubin GM. 1997. Kuzbanian controls proteolytic processing ofNotch and mediates lateral inhibition during Drosophila and vertebrateneurogenesis. Cell 90:271–280.

Parks AL, Klueg KM, Stout JR, Muskavitch MA. 2000. Ligand endocytosisdrives receptor dissociation and activation in the Notch pathway. Devel-opment 127:1373–1385.

Qi H, Rand MD, Wu X, Sestan N, Wang W, Rakic P, Xu T, Artavanis-Tsakonas S. 1999. Processing of the notch ligand delta by the metallo-protease Kuzbanian. Science 283:91–94.

Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsakonas S. 1991. Specific EGF repeats of Notch mediate interactionswith Delta and Serrate: implications for Notch as a multifunctionalreceptor. Cell 67:687–699.

Sestan N, Artavanis-Tsakonas S, Rakic P. 1999. Contact-dependent inhi-bition of cortical neurite growth mediated by notch signaling. Science286:741–746.

Shawber C, Nofziger D, Hsieh JJ-D, Lindsell C, Bogler O, Hayward D,Weinmaster G. 1996a. Notch signaling inhibits muscle cell differentiationthrough a CBF1-independent pathway. Development 122:3765–3773.

Shawber C, Boulter J, Lindsell CE, Weinmaster G. 1996b. Jagged2: aserrate-like gene expressed during rat embryogenesis. Dev Biol 180:370–376.

Shimizu K, Chiba S, Kumano K, Hosoya N, Takahashi T, Kanda Y,Hamada Y, Yazaki Y, Hirai H. 1999. Mouse Jagged1 physically interactswith Notch2 and other notch receptors. Assessment by quantitativemethods. J Biol Chem 274:32961–32969.

Shimizu K, Chiba S, Hosoya N, Kumano K, Saito T, Kurokawa M, KandaY, Hamada Y, Hirai H. 2000a. Binding of Delta1, Jagged1, and Jagged2to Notch2 rapidly induces cleavage, nuclear translocation, and hyperphos-phorylation of Notch2. Mol Cell Biol 20:6913–6922.

Shimizu K, Chiba S, Saito T, Kumano K, Hirai H. 2000b. Physicalinteraction of Delta1, Jagged1, and Jagged2 with Notch1 and Notch3receptors. Biochem Biophys Res Commun 276:385–389.

Sotillos S, Roch F, Campuzano S. 1997. The metalloprotease-disintegrinKuzbanian participates in Notch activation during growth and patterningof Drosophila imaginal discs. Development 124:4769–4779.

Stein E, Lane AA, Cerretti DP, Schoecklmann HO, Schroff AD, Van EttenRL, Daniel TO. 1998. Eph receptors discriminate specific ligand oli-gomers to determine alternative signaling complexes, attachment, andassembly responses. Genes Dev 12:667–678.

Sun X, Artavanis-Tsakonas S. 1997. Secreted forms of Delta and Serratedefine antagonists of Notch signaling in Drosophila. Development 124:3439–3448.

Varnum-Finney B, Purton LE, Yu M, Brashem-Stein C, Flowers D, StaatsS, Moore KA, Le Roux I, Mann R, Gray G, Artavanis-Tsakonas S,Bernstein ID. 1998. The Notch ligand, Jagged-1, influences the devel-opment of primitive hematopoietic precursor cells. Blood 91:4084–4091.

Varnum-Finney B, Wu L, Yu M, Brashem-Stein C, Staats S, Flowers D,Griffin JD, Bernstein ID. 2000. Immobilization of Notch ligand, Delta-1,is required for induction of notch signaling. J Cell Sci 113:4313–4318.

Wang S, Sdrulla AD, diSibio G, Bush G, Nofziger D, Hicks C, WeinmasterG, Barres B. 1998. Notch receptor activation inhibits oligodendrocytedifferentiation. Neuron 21:63–75.

Weinmaster G. 1997. The ins and outs of notch signaling. Mol CellNeurosci 9:91–102.

Weinmaster G. 2000. Notch signal transduction: a real rip and more. CurrOpin Genet Dev 10:363–369.


a secreted delta1-fc fusion protein functions both as an activator and inhibitor of notch1 signaling

Documents