Molecular Separation of Two Signaling Pathways for the Receptor, Notch

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Developmental Biology 313 (2008) 556–567 www.elsevier.com/developmentalbiology

Molecular separation of two signaling pathways for the receptor, Notch

Maude Le Gall 1, Cordell De Mattei 2, Edward Giniger ∗

Axon Guidance and Neural Connectivity Unit, Basic Neuroscience Program, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health, Bldg. 37, Rm. 1016, 37 Convent Drive, Bethesda, MD 20892, USA National Human Genome Research Institute (NHGRI), National Institutes of Health, Bldg. 37, Rm. 1016, 37 Convent Drive, Bethesda, MD 20892, USA Received for publication 30 April 2007; revised 18 October 2007; accepted 23 October 2007 Available online 11 December 2007

Abstract

Notch is required for many aspects of cell fate specification and morphogenesis during development, including neurogenesis and axon guidance. We here provide genetic and biochemical evidence that Notch directs axon growth and guidance in Drosophila via a “non-canonical”, i.e. non-Su(H)-mediated, signaling pathway, characterized by association with the adaptor protein, Disabled, and Trio, an accessory factor of the Abl tyrosine kinase. We find that forms of Notch lacking the binding sites for its canonical effector, Su(H), are nearly inactive for the cell fate function of the receptor, but largely or fully active in axon patterning. Conversely, deletion from Notch of the binding site for Disabled impairs its action in axon patterning without disturbing cell fate control. Finally, we show by co-immunoprecipitation that Notch protein is physically associated in vivo with both Disabled and Trio. Together, these data provide evidence for an alternate Notch signaling pathway that mediates a postmitotic, morphogenetic function of the receptor. © 2007 Elsevier Inc. All rights reserved.

Keywords: Notch; Signal transduction; Non-canonical signalling; Abl; Disabled; Trio; Axon guidance; CSL

Introduction the “canonical” β-catenin pathway and at least two “non- canonical” signaling mechanisms (Boutros et al., 1998; Kuhl et We have come to appreciate that a small handful of al., 2000; Pandur et al., 2002; Yoshikawa et al., 2003). ubiquitous, highly conserved signal transduction pathways Presumably, this diversity of signaling pathways is used, in together account for a remarkable fraction of the patterning of a various combinations, to elicit different cellular outcomes in developing animal. In different tissues, Hedgehogs, Wnts, response to an input Wnt signal. Multiple signaling pathways TGFβs, receptor tyrosine kinases and a few other key signaling have also been described downstream of other receptor families, modules specify cell identities, set developmental boundaries, such as the receptor tyrosine kinases (Fantl et al., 1992). direct cell migration and shape tissue morphogenesis (Gerhart The receptor Notch, together with its ligands, Delta and and Kirschner, 1997; Ptashne and Gann, 2002). In each case, Serrate, define another of these common, multipotent develop- however, it remains mysterious how a single signal can produce mental signaling pathways (Artavanis-Tsakonis et al., 1999; such varied effects. In the case of Wnt signaling, some of the Frisen and Lendahl, 2001). First studied in Drosophila, Notch diversity of biological readouts seems to arise from the orthologs have been found in nearly all metazoan phyla, where existence of at least three different Wnt signaling pathways, they define the boundaries of developmental compartments, distinguish the developmental potentials of sibling cells and ∗ Corresponding author. Axon Guidance and Neural Connectivity Unit, Basic limit the segregation of differentiated cells from among fields of Neuroscience Program, National Institute of Neurological Disorders and Stroke equipotent progenitors. A conserved signaling mechanism has (NINDS), National Institutes of Health, Bldg. 37, Rm. 1016, 37 Convent Drive, been described for Notch proteins, whereby ligand activation Bethesda, MD 20892, USA. Fax: +1 301 451 5368. causes Notch to be cleaved proteolytically at the inner edge of E-mail address: [email protected] (E. Giniger). 1 UMRS872, Inserm, UPMC, UPD, Centre de Recherche des Cordeliers, the plasma membrane, releasing an intracellular fragment that Paris, France. transits to the nucleus to form a transcription control complex in 2 Dendritic Nanotechnologies, Inc., Mount Pleasant, MI 48858. association with at least two cofactors, the DNA-binding protein

Su(H) (a member of the CSL family of proteins), and the (Gertler et al., 1993; Liebl et al., 2003). In addition to these transcriptional co-activator Mastermind (Mam) (Artavanis- cooperating factors, Abl works in concert with a specific Tsakonis et al., 1995; Hansson et al., 2004; Jeffries et al., antagonist, the signaling protein Enabled (Ena; Gertler et al., 2002; Petcherski and Kimble, 2000). 1995). Despite intensive study, however, to date it has been Over the past several years, however, experiments in a variety difficult to discern the detailed mechanism by which Abl of vertebrate and invertebrate systems have hinted at the pathway proteins guide axon growth. For example, there have existence of a “non-canonical”, Su(H)-independent, signaling been only a handful of cases where it has been possible to pathway for Notch (Brennan and Gardner, 2002; Ordentlich et demonstrate physical association of Abl pathway effector al., 1998; Ramain et al., 2001; Shawber et al., 1996; Wang et al., proteins with growth cone receptors in vivo, and to correlate 1997; Zecchini et al., 1999). In Drosophila, for example, it those associations with specific axon patterning decisions seems that a Su(H)-independent Notch activity regulates the (Bashaw et al., 2000; Forsthoefel et al., 2005). morphogenetic movements of dorsal closure (Zecchini et al., We present here genetic, molecular and biochemical evidence 1999), while in vertebrates, CSL-independent Notch signaling for a non-canonical, i.e. non-Su(H) mediated, Notch signaling appears to block muscle differentiation of myoblasts (Shawber et pathway employing Abl signaling components to promote axon al., 1996), inhibit transcriptional activation by E-proteins (E47) growth and guidance in Drosophila. We prepare Notch in B-cells (Ordentlich et al., 1998) and promote survival of derivatives that are inactive for the Su(H)-dependent cell fate neural stem cells and embryonic stem cells (Androutsellis- function of Notch but find that they nonetheless are largely or Theotokis et al., 2006). While these studies have been fully active for promoting Notch-dependent axon patterning, suggestive, however, it has been difficult to test thoroughly the while conversely, removing the binding site on Notch for notion of a non-canonical Notch signaling pathway since in most Disabled impairs axon patterning without affecting cell fate cases the molecular properties of the postulated alternate control. We also show that Notch co-immunoprecipitates from pathway have not been clearly defined. wild type fly lysates in association with Disabled, and with the Non-canonical Notch signaling has also been invoked in Abl pathway effector, Trio. Together, these data suggest a axon guidance in Drosophila (Crowner et al., 2003; Giniger, molecular basis for the genetic interaction of Notch and Abl that 1998), and we have suggested that the Abl tyrosine kinase and its promotes embryonic axon patterning in Drosophila, as well as associated signaling pathway provides a good candidate for an providing reagents and diagnostic properties that can be used to alternate Notch signaling mechanism. This hypothesis was identify the contribution of this pathway at other times and based upon two major findings, that Notch interacts genetically places in development. with gain- and loss-of-function manipulations of abl and its cofactors to direct the growth of particular axons, and that a Results protein interaction domain of one postulated Abl accessory factor, the adaptor protein Disabled (Dab), can bind directly to Notch domains required for axon patterning Notch in vitro. A link between Notch and Abl is of interest not just because of its implications for Notch, but also for the light it Previous experiments have shown that inactivating Notch with might shed on Abl signaling. Abl is a cytoplasmic tyrosine a temperature-sensitive mutation (Notchts1) shortly before CNS kinase that has been studied for more than two decades as one of longitudinal axon tracts are pioneered in the developing fly the first cellular genes clearly implicated in the etiology of a embryo yields a ‘disconnected’ phenotype in which axons fail to common human cancer (Druker et al., 2001; Fainstein et al., grow between segments of the ventral nerve cord, and those axons 1987; Goff et al., 1980). Moreover, a central role for Abl in axon can be restored by expressing a wild type Notch transgene in patterning has been solidly established by extensive genetic postmitotic neurons (Figs. 1B–H, quantified in Fig. 1N; Giniger, experiments, particularly in Drosophila (Hoffmann, 1991). 1998; Giniger et al., 1993). We therefore expressed a series of Here, Abl contributes to the growth and guidance of many, if modified Notch transgenes in the Notchts1 background to map not most, developing axons, apparently by locally controlling domains that are required for axonal function of the protein. The actin organization and dynamics (Baum and Perrimon, 2001; same derivatives were also tested for their ability to perform Bear et al., 2000; Gertler et al., 1989; Grevengoed et al., 2001; “lateral inhibition”, the classic Notch cell fate function that limits Luo, 2000; Wills et al., 1999). the number of neurons in the embryo, and whose absence Abl performs its functions in cooperation with a constella- produces the severe neural hyperplasia characteristic of strong tion of conserved accessory proteins. Three cooperating factors Notch alleles (known as the “neurogenic phenotype” (Lehmann were originally identified in Drosophila as genetic loci that et al., 1983), Figs. 1I–M; (Le Gall and Giniger, 2004)). enhance the phenotype of an abl mutant. These were trio, Deletion of the binding region for the ligands Delta and which encodes a guanine nucleotide exchange factor (GEF) for Serrate (EGF repeats 10-12; Lieber et al., 1993; Rebay et al., Rho GTPases, fax, encoding a protein found in axon bundles, 1991) from the Notch extracellular domain completely ablated and neurotactin (nrt), encoding a cell-surface adhesion protein the ability of a Notch transgene to restore axon patterning (Fig. (Gertler et al., 1993; Hill et al., 1995; Hortsch et al., 1990; Liebl 1G; quantification in panel N), consistent with prior experi- et al., 2000, 2003). An additional putative cooperating factor, ments showing that a Delta mutation produces the same axonal disabled (dab), was originally identified as a multicopy defects as does a Notch mutation (Crowner et al., 2003; Giniger suppressor of both fax abl and abl nrt mutant combinations et al., 1993). Intracellular sequences of Notch were also 558 M. Le Gall et al. / Developmental Biology 313 (2008) 556–567

Fig. 1. Identification of domains of Notch protein required for axon patterning. (A) Schematic representation of Notch protein, and of derivatives used below. Functional domains are drawn approximately to scale. LNR: Lin12/Notch repeats, TM: transmembrane domain, cdc10: ankyrin/cdc10 repeat domain. Asterisks in the block diagram indicate positions of Su(H) binding sites. Thick lines represent protein coding sequences present in the indicated transgenes. (B–M) Embryos of the indicated genotypes were raised to embryonic stage 16/17 either at 25 °C (I–M) or by an appropriate temperature-shift regimen (B–H), fixed, incubated with anti-Fasciclin 2 to label particular axon tracts (B–H) or with anti-Elav to label neuronal nuclei (I–M) and visualized with peroxidase histochemistry. Experiments to assay axon patterning (C–H) employed the allele Notchts1 and used the temperature protocol described in Giniger (1998); experiments assaying lateral inhibition (J–M) employed Notch55e11. For rescue of axonal phenotypes (D–H) Notch transgene was under control of elav-GAL4; for rescue of neurogenic phenotypes (K–M) Notch transgenes were expressed under control of sca-GAL4. (B, I) wild type embryos; (C, J) Notch− (Notchts1 and Notch55e11, respectively); (D, K) Notch−; UAS-Notchwt; (E, L) Notch−; UAS-NotchΔcdc10. While this transgene gave some rescue of longitudinal axons (see quantification in panel N), rescue was incomplete and often restored intersegmental connection of only a single fascicle (arrowhead); (F, M) Notch−; UAS-NotchΔ2155; (G) Notchts; UAS-NotchΔEGF10–12. Expression of this transgene consistently produced more severe axonal defects than those observed in the parent Notchts1 stock, suggesting a dominant inhibitory activity. (H) Notchts; UAS-NotchECN. Experiments published by others have documented previously that NotchECN and NotchΔ10–12 lack all function in lateral inhibition (Jacobsen et al., 1998; Lieber et al., 1993). Arrows in panels C and H highlight breaks in longitudinal axon tracts. Anterior is to the left in all panels; dorsal is to the top in panels I–M. (N) Histogram quantifying the fraction of missing CNS longitudinal connections observed in the experiments of panels C–H. As described in the Experimental procedures, the number of broken connections between successive thoracic and abdominal hemisegments was counted, and expressed as a percentage of the total. Expression of NotchΔ10–12 consistently produced more disconnections than the Notchts1 control. Average variation in the percentage of disconnected hemisegments for a given genotype in different experiments was ±5%. NN400 hemisegments for all genotypes except Notchts1; UAS-NotchΔ10–12, for which approximately 50% of embryos had a disrupted morphology due to the antimorphic effect of the transgene, and were excluded from the quantification. essential for axon patterning as a transgene deleted for nearly significantly, however, to our surprise substantial axonal activity the entire intracellular domain (NotchECN; Jacobsen et al., 1998) remained (Figs. 1E, N; comparison to Notchts gives P≪0.001; was devoid of axonal activity (Fig. 1H). χ2). This is in contrast to the complete failure of this protein to Deletion of a portion of the intracellular domain comprising suppress the neural hyperplasia of a strong Notch mutant (Fig. the ankyrin/cdc10 repeats and the C-terminal portion of the Ram 1L, similar to the Notch null embryo in 1J), or to activate domain (NotchΔcdc10) reduced axonal activity of Notch expression of canonical Notch target genes (reviewed in Le Gall M. Le Gall et al. / Developmental Biology 313 (2008) 556–567 559 and Giniger, 2004). Remarkably, moreover, a Notch derivative 2004.) We wished to be certain that the differential rescue of truncated at the C-terminal end of the ankyrin repeat region, axonal versus cell fate phenotypes by these Notch derivatives NotchΔ2155 (Wesley and Saez, 2000), was nearly wild type for reflected the differing requirements of the two biological Notch axonal function, even though it, too, was completely processes and not peculiar properties of the Notchts1 protein. inactive for suppressing the Notch “neurogenic” phenotype In separate experiments, therefore, we also induced neurogenic (Figs. 1F, M) and has been shown previously to be incapable of defects by early temperature shifts of Notchts1 and confirmed activating expression of the Notch target gene, E(spl) (Wesley that they displayed the same pattern of rescue by various Notch and Saez, 2000). These data begin to suggest that the sequence derivatives as did the neurogenic defects of Notch55e11 (data not requirements for Notch function in axon patterning may be shown). We therefore infer (1) that the ability of Notch to different from those required for the canonical cell fate control axon patterning cannot be accounted for by its binding functions of the protein. sites for Su(H), (2) that the Disabled site, and not the Su(H) site, within the ΔA deletion is likely to be responsible for the axonal The Disabled binding site on Notch is required for full axonal deficit of NotchΔRamA protein, and (3) that the Disabled site function of Notch, while the Su(H) sites are largely dispensable does not contribute substantially to the neurogenic function of Notch. Abl tyrosine kinase has been implicated genetically in the Similar results were obtained from assay of a second Notch- mechanism by which Notch controls axon patterning, and one dependent axon guidance decision. An appropriate temperature postulated Abl accessory protein, the adaptor protein Disabled, shift of Notchts embryos blocks defasciculation of ISNb motor binds in vitro to a small region in the juxtamembrane portion of axons from the intersegmental nerve (ISN) at the first peripheral the Notch intracellular domain (Giniger, 1998; Le Gall and choice point (Crowner et al., 2003; shown schematically in Fig. Giniger, 2004). We therefore wondered whether the axon 3A). Since this is a relatively late event in embryogenesis, the patterning activity of Notch might depend, in part, on the temperature shift does not perturb CNS development, simplify- Disabled binding site, which is still present in both NotchΔcdc10 ing the phenotypic analysis and facilitating precise quantifica- and NotchΔ2155. As above, modified Notch transgenes were tion. A Notch transgene lacking just the Ram Su(H) site or all assayed for their ability to restore axon patterning in Notchts1 three Su(H) sites rescued axon patterning as well as did a wild embryos and to suppress neural hyperplasia in embryos type Notch transgene (Fig. 3B; 8% of hemisegments showing hemizygous for the strong allele, Notch55e11 (Giniger, 1998; residual axonal defects in embryos rescued with NotchRam∗ and Le Gall and Giniger, 2004). 7% with NotchRam∗Δ3, vs. 6% for wild type Notch; difference Deletion of Su(H) binding sites of Notch had only limited not significant). In contrast, the transgene lacking the Disabled effect on axon patterning despite ablating almost completely the binding site is significantly impaired in axonal activity, with ability of Notch to limit neurogenesis, while deletion of the rescue reduced by ∼40% compared to wild type (Pb0.01; Disabled site impaired axonal function with little or no effect on ANOVA). neurogenesis (Fig. 2). A derivative of full-length Notch deleted As an alternate way to assess the role of canonical Notch for the Disabled binding site, NotchΔRamA, was impaired by signaling in axon patterning we examined expression of the about 30% relative to the wild type protein in its ability to known Notch target gene, E(spl)mδ, in cells that are executing restore longitudinal growth of CNS axons when expressed in Notch-dependent guidance processes. Previous experiments Notchts1 (Fig. 2B,quantifiedinI;P≪0.001 (χ2)). The have shown that neuronal Notch activity is required for minimal deletion we have so far identified that prevents extension of the axons of the longitudinal pioneers dMP2 and Disabled binding also deletes the amino-terminal Su(H) vMP2, and for defasciculation of the ISNb pioneer RP3 binding site, thus, NotchΔRamA has a slight deficit in lateral (Giniger, 1998; Crowner et al., 2003). Examination of an E inhibition (Fig. 2F; quantified in J). However, a Notch protein (spl)mδ-lacZ reporter gene (Cooper and Bray, 1999), however, bearing a missense mutation that inactivates just that Su(H) fails to reveal any evidence for lacZ expression in these site (NotchRam∗; Le Gall and Giniger, 2004) has an identical pioneer neurons, or in surrounding neurons, at the time these deficit in neurogenesis (Fig. 2G; PN0.5, ANOVA) but is nearly Notch-dependent axon guidance events are occurring (Fig. 4). wild type for axon patterning (Figs. 2C, I), suggesting that the These data should be interpreted cautiously, of course, since axonal phenotype of NotchΔRamA is not due to the absence of even the most general Notch target genes are not used in all this Su(H) binding site. Indeed, even a Notch derivative lacking Notch-dependent processes, however, this observation is all three Su(H) binding sites (NotchRam∗Δ3) retains signifi- consistent with the idea that activation of Notch signaling cantly more axon patterning activity than does NotchΔRamA for guidance of CNS longitudinal pioneers and ISNb (Fig. 2D, Pb0.01; χ2), even though NotchRam∗Δ3 is almost motoneurons may not be associated with activation of the completely inactive in suppressing neural hyperplasia (Fig. 2H; canonical effector pathway. compare with the level of neural hyperplasia in dorsal portions Data above suggest that the Disabled binding site on Notch is of the Notch mutant embryo in Fig. 1J, and see also (Le Gall required for Notch-dependent axon patterning, a process and Giniger, 2004)), whereas NotchΔRamA is only mildly mediated by the genetic interaction of Notch with genes of impaired. (Note that the efficacy of the NotchRam∗, NotchΔRamA the Abl signaling pathway. The role of Disabled in Abl and NotchRam∗Δ3 mutations for blocking Su(H) binding to signaling, however, remains controversial due to the lack of any Notch has been documented previously; Le Gall and Giniger, disabled mutants with which to assess the loss of function 560 M. Le Gall et al. / Developmental Biology 313 (2008) 556–567

Fig. 2. Roles of Su(H) binding sites and Disabled binding site in Notch-dependent lateral inhibition and CNS axon growth. Embryos of the indicated genotypes were prepared as for the experiment of Fig. 1, except that the temperature shift protocol was refined to allow more precise quantification (see Experimental procedures). A schematic of the Notch derivatives used in this experiment is included at the top of the figure; brown asterisks (∗) designate Su(H) binding sites; ‘X’ designates a missense mutation that prevents binding of Su(H) to its amino-terminal binding site; purple (#) indicates the Disabled binding site. (A–D) dorsal views of the CNS in embryos that are Notchts1 and carry elav-GAL4;(E–H) lateral views of the dorsal and lateral clusters of sensory neurons in four successive segments of embryos that are Notch55e11 and carry sca-GAL4. For comparison, panel E′ is a lower magnification view of the embryo in panel E, with a box indicating the portion included in the high-magnification panel. The UAS-Notch transgene is indicated to the left of each pair of images (A, E) Notch−; UAS-NotchWT; (B, F) Notch−; UAS-NotchRamΔA; (C, G) Notch−; UAS-NotchRam∗; (D, H) Notch−; UAS-NotchRam∗Δ3. NotchRam∗Δ3 is a full-length Notch transgene specifically deleted for all three Su(H) binding sites, NotchRam∗ bears a missense mutation inactivating just the Su(H) binding site in the Ram domain, NotchΔRamA bears a deletion of the first 35 codons of the Ram domain, thus removing both the Su(H) site and the in vitro Disabled binding site in this domain. NotchRam∗Δ3 and NotchRam∗ are described in Le Gall and Giniger (2004). Arrows in panel B highlight interruptions of the longitudinal axon tracts. (I) Histogram quantifying the percent rescue of defective longitudinal connections between hemisegments for the experiment of panels A–D. Rescue by full-length, wild type Notch was defined as 100%. Average variation in the percentage of disconnected hemisegments for a given genotype in different experiments was ±2%. P-values (χ2) for selected key comparisons are indicated: ∗∗Pb0.01; ∗∗∗P≪0.001. NN500 hemisegments for all genotypes. (J) Histogram quantifying the number of dorsal sensory neurons per hemisegment in Notch55e11 embryos rescued with the indicated Notch transgene for the experiment of panels E–H. For all genotypes, SEMb1.0 neuron (thin horizontal lines), and NN35 hemisegments. (K) Immunoblot of total lysate of wild type embryos (lane 1) or embryos expressing the indicated Notch derivative under control of elav-GAL4 (lanes 2–5). Top: anti-Notch immunostaining. Bottom: anti-β-tubulin loading control. M. Le Gall et al. / Developmental Biology 313 (2008) 556–567 561

Disabled mimics the interaction of Notch with other manipulations that enhance signaling in the Abl pathway, including overexpression of Abl or heterozygous mutation of the Abl antagonist, enabled (Crowner et al., 2003), consistent with the hypothesis that Disabled modulates Abl pathway signaling in vivo.

Co-immunoprecipitation of Notch with Disabled and Trio

In support of the genetic data described above, biochemical experiments showed that Notch co-immunoprecipitates from wild type Drosophila lysates with Disabled, and also with the Abl accessory protein, Trio. In the case of Disabled, Western blotting of anti-Disabled immunoprecipitates revealed the presence of full-length Notch (Fig. 6). Four lines of evidence suggest that the association of Notch with Disabled is authentic and specific. First, three different anti-Disabled antibodies co- precipitated Notch. These included two independent polyclonal antibodies (one is shown in Fig. 6A, lane 4, middle panel), as well as a monoclonal antibody (Fig. 6B, lane 4; characterization of anti-Disabled antibodies is documented in Supplementary Fig. S-1). Second, association was detected in two different wild type fly lysates, a total embryo lysate (Fig. 6B) and a lysate made from adult heads (Fig. 6A). Third, immunoprecipitations performed in parallel with two irrelevant mouse IgGs (anti-Islet, or normal mouse IgG) did not co-precipitate detectable Notch protein (Fig. 6A, lane 5, middle panel; Fig. 6B, lane 3; Fig. 6C, lane 3; data not shown). Fourth, immunoprecipitation of Fig. 3. Activity of Notch derivatives in turning of ISNb motor axons. (A) Disabled from extracts of Notch-overexpressing animals co- Schematic of the ISNb motor nerve. Wild type ISNb motor axons precipitated more Notch protein than did immunoprecipitation defasciculate from the main ISN trunk upon contact with Delta-expressing cells at the choice point; inactivation of Notch with a temperature-sensitive from wild type extracts done in parallel. We have not been able mutation prevents defasciculation. Numbered ovals represent muscles along to test for the presence of Disabled in anti-Notch immunopre- the ISNb path and in its target field. (B) Embryos of the indicated genotype cipitates since the current anti-Disabled antibodies, while were subjected to a temperature-shift protocol that produces a partial failure specific, are not sufficiently sensitive in Western blots (Fig. of ISNb defasciculation. Embryos were fixed, immunostained with anti- 6A, bottom panel). Fasciclin 2 antibody to label motoneurons and visualized by peroxidase histochemistry. The fraction of hemisegments in which some or all ISNb Multiple lines of evidence also demonstrate association of motor axons failed to turn at the choice point was quantified in filet Notch with Trio in wild type fly extracts. Immunoprecipitations preparations and is indicated by the horizontal bars. Thin lines indicate SEM. with anti-Notch caused co-precipitation of Trio, again, both In all cases, expression of the indicated transgene was driven with elav-GAL4. from adult head lysates (Fig. 6A, lane 3, top panel) and from The degree of rescue of the mutant phenotype was not significantly different ∗ ∗Δ wild type embryo lysates (not shown). We also reversed the among UAS-NotchWT, UAS-NotchRam and UAS-NotchRam 3; rescue by UAS-NotchRamΔA was significantly impaired (∼40% reduced; Pb0.01 experiment and found that immunoprecipitation of Trio caused (ANOVA)). co-precipitation of full-length Notch (Fig. 6C, lane 4). Finally, side-by-side co-immunoprecipitations of extracts from wild type and Notch-overexpressing animals detected more Notch phenotype of the gene (Liebl et al., 2003). To further test the co-precipitated with anti-Trio in the overexpressing lysates role of Disabled in Notch/Abl-dependent axon patterning we (Supplementary Information, Fig. S-2, compare lanes 3, 4), as therefore assayed the effect of Disabled overexpression on well as detecting more Trio in anti-Notch immunoprecipitates defasciculation of ISNb (Fig. 5). In a wild type genetic from these extracts (not shown). Together, these data show that background, overexpression of Disabled had no effect on Notch is found in complexes with both Disabled and Trio in ISNb defasciculation at the first choice point (bypass of the extract of wild type flies. Our data do not prove whether Notch choice point observed in 3±1% of hemisegments, the same as associates with Disabled and Trio simultaneously or whether in wild type embryos subjected to the temperature shift protocol these two proteins form separate complexes with Notch, (Crowner et al., 2003; Wills et al., 1999)). However, Disabled however, we note that immunoprecipitations with any of three overexpression substantially enhanced the Notch mutant anti-Disabled antibodies reliably co-precipitated Trio, both from phenotype (42±0.3% bypass in Notchts; elav-GAL4; UAS- adult (Fig. 6A, lane 4, top panel) and embryo extracts, Disabled, vs. 32±0.7% in the Notchts internal control embryos; suggesting that Trio and Disabled are indeed found in common P≪0.01, ANOVA). This synergistic interaction of Notch with complexes in vivo. 562 M. Le Gall et al. / Developmental Biology 313 (2008) 556–567

Fig. 4. E(spl)mδ is not activated during Notch-dependent guidance of CNS and ISNb pioneer axons. Wild type embryos bearing a Notch reporter transgene, E(spl)mδ-lacZ, were double-labelled with mAb22C10 and anti-β-gal to examine the CNS pioneer neurons dMP2 and vMP2 (A–C, stage 12.0), or with anti-Fasciclin 2 and anti-β-gal to examine the ISNb pioneer, RP3 (D–F, stage 15). Merged signals are shown in panels A, D; separated channels in panels B, E (anti-neuron signal, red) and C, F (anti-β-gal, green), respectively. Growth cones are indicated with orange arrows, cell body position with yellow arrows.

Discussion

Previous studies have led us to speculate that the Abl tyrosine kinase and its associated accessory factors might define an alternate, ‘non-canonical’, Su(H)-independent signaling pathway for the receptor Notch (Crowner et al., 2003; Giniger, 1998). The data reported here provide strong support for this hypothesis. In extracts of wild type Drosophila, Notch is associated with Disabled and Trio, two proteins that have been associated with the action of Abl tyrosine kinase. The functions of Notch in axon growth and guidance are likely to be executed by these complexes of Notch with Disabled and Trio, and not by its association with Su(H), since deletion of the Disabled binding site from Notch significantly impairs the axon patterning function of the receptor, whereas the Su(H) binding sites are largely dispensable for this process. Moreover, we describe two other Notch derivatives that are still capable of executing the axon patterning functions of the receptor despite being completely inactive for specifying Notch-dependent cell fates. Taken together with previous data demonstrating that the genetic interaction of Notch with multiple Abl pathway components is required specifically for Notch-dependent axon patterning, these data provide a molecular picture of a Notch signaling machinery that is distinct from the well-established mechanism by which a proteolytic fragment of Notch enters the nucleus to directly control transcription of Su(H)-dependent target genes. Fig. 5. Overexpression of Disabled interacts genetically with Notch in ISNb The key genetic data in favor of our hypothesis stem from the ts1 pathfinding. Notch embryos were generated that did, or did not, overexpress targeted construction of Notch derivatives that preferentially wildtype Disabled under control of elav-GAL4. Embryos were temperature- shifted, fixed and assayed for ISNb patterning as for the experiment of Fig. 3. impair either the canonical, cell fate function of the receptor or Thick vertical bars report the percentage of hemisegments in which ISNb axons its Abl-dependent axon patterning function, respectively. bypassed the first choice point; thin lines represent SEM. Deletion of the Su(H) binding sites from Notch progressively M. Le Gall et al. / Developmental Biology 313 (2008) 556–567 563

Fig. 6. Co-immunoprecipitation of Notch with Disabled and Trio from wild type Drosophila lysates. Total extracts were made from wild type embryos (B) or adult heads (A, C) and subjected to immunoprecipitation with the indicated antibodies. Precipitates were separated by SDS-PAGE, immunoblotted with the indicated antibodies and visualized by ECL. Small circles indicate bands attributable to the primary antibodies. Molecular weight markers (in kilodaltons) are indicated on the right in each panel. (A) Immunoprecipitations of wild type head extract with either anti-Notch (lane 3) or with polyclonal anti-Disabled (lane 4). Probing with anti-Trio (top panel) detects the ca. 250 kDa form of Trio in both Notch and Disabled immunoprecipitates (arrow; compare anti-Trio IP and input, lanes 1 and 2, respectively). A smaller Trio variant (ca. 120 kDa, arrowhead) comigrates with a prominent antibody band, so we can not determine whether it is also present in the immunoprecipitates. A very small non-specific background of Trio was often detected in control anti-Islet immunoprecipitates (lane 5), but quantification (Image J) demonstrated that the Trio signal observed in anti-Disabled and anti-Notch IPs was more than 4× above this background level. Probing the same samples with anti- Notch (middle panel) detects full length Notch in the input and in the anti-Notch IP (lanes 2, 3) and also co-precipitated with polyclonal anti-Disabled (lane 4). Probing these samples with anti-Disabled (bottom panel) detects Disabled protein only in the input and in the anti-Disabled IP (lanes 2, 4). mAB P6E11 is not sufficiently sensitive to test for Disabled protein in anti-Notch IPs. (B) Immunoprecipitation of wild type embryo extract with anti-Notch (lane 2) or monoclonal anti-Disabled (lane 4) followed by Western blotting with anti-Notch antibody detects full-length Notch (arrow), whereas immunoprecipitation with a control IgG (anti-Islet, lane 3) does not. Input lysate is shown for comparison (lane 1). (C) Immunoprecipitations from adult head extract were assayed by Western blotting with anti-Notch. Notch protein (arrow) is readily detectable in anti-Trio immunoprecipitates of wild type extract (lane 4) but not in immunoprecipitates with a control antibody (anti-Islet, lane 3). Input extracts (lane 1) and anti-Notch IP (lane 2) are shown for comparison. and dramatically reduces the ability of the receptor to limit Notch axonal phenotype would be to examine a disabled neurogenesis, but has only limited effect on growth of CNS mutant, but unfortunately no such mutants are currently longitudinal axons, and no detectable effect on Notch- available (Liebl et al., 2003). The phenotype of a trio mutant, dependent defasciculation of ISNb motor axons. In contrast, on the other hand, is consistent with the results documented here deletion of the Disabled binding site substantially reduces the (Bateman et al., 2000; Hakeda-Suzuki et al., 2002; Liebl et al., axon patterning activity of Notch (30–40%, depending on the 2000). The zygotic mutant phenotypes of trio are somewhat assay), while having no effect on cell fate function beyond what subtle, evidently because of persistence of maternally-provided can be accounted for by the known Su(H) binding site within trio RNA and protein, but they include defects in some of the the deletion. The properties of these complementary Notch CNS longitudinal axons that are affected in Notchts embryos, as mutants argue for the action of a qualitatively different Notch well as defects in ISNb motor axon guidance, while trio has not mechanism in axon patterning. Further supporting this hypoth- been reported to produce any Notch-like defects in cell fate esis is the observation that Notch co-precipitates from wildtype (Bateman et al., 2000; Liebl et al., 2000). fly extracts with two cytoplasmic signaling proteins, the Abl While deletion of the Disabled binding region of Notch cofactor, Trio and the adaptor protein, Disabled, potentially clearly reduces the axonal activity of the protein, substantial providing a molecular machinery to account for our phenotypic residual activity still remains. In the case of Su(H), residual data. In principle, a good way to further test the basis of the activity of a Notch mutant lacking all in vitro Su(H) binding 564 M. Le Gall et al. / Developmental Biology 313 (2008) 556–567 sites can be traced to an association of Su(H) with the Notch have multiple downstream targets, including nuclear gene ankyrin repeats that requires the cofactor, Mastermind (Nam et regulation in addition to cytoskeletal structure and dynamics al., 2006; Wilson and Kovall, 2006; Wu et al., 2000). By (Luo, 2000). analogy, perhaps Disabled can also associate with Notch via a The key step in canonical Notch signaling is the second site that requires a cofactor present in vivo. Consistent proteolytic cleavage of the receptor by γ-secretase to release with this idea, preliminary biochemical experiments hint that the active, intracellular moiety of the molecule, NICD. Does the RamΔA mutation does not wholly ablate recruitment of γ-secretase also play a role in Notch/Abl signaling? Since we Disabled and Trio in vivo (MLG and EG, unpublished find Disabled and Trio associated with full-length Notch prior observations), though current reagents do not allow us to assess to cleavage, and since Notch/Abl signaling in the growth cone this rigorously. If so, the ankyrin/cdc10 repeat region would be presumably targets the cortical actin cytoskeleton, one a plausible candidate for a secondary site of association. possibility is that γ-secretase cleavage terminates the Notch/ Experiments in Fig. 1 show that the ankyrin repeats, together Abl signal by separating the receptor-bound complex from with the C-terminal portion of the Ram region, contribute membrane-tethered components of the pathway such as Abl substantially to Notch-dependent axon patterning. Since our kinase and Rho GTPases. Alternatively, in contexts such as experiments clearly show that the canonical Notch signaling ISNb, perhaps displacement of Disabled and Trio away from pathway is dispensable for axonal function, the axonal the membrane is part of the mechanism by which Notch requirement for this portion of the protein cannot be traced to antagonizes Abl pathway activity. Moreover, while proteolytic its function in canonical signaling, and a contribution to activity is the most apparent function of γ-secretase there formation or activity of Notch/Abl pathway complexes would have been suggestions that the complex may also have a offer the simplest explanation. separate function in Notch trafficking, aside from cleavage. If To date, the role of Disabled in the Abl signaling pathway so, this activity could modulate Notch/Abl signaling inde- has been difficult to establish due to the lack of loss-of- pendent of any role for protease cleavage. Clearly, additional function Disabled mutant alleles. The placement of Disabled in experiments will be necessary to assess the various possible the Abl pathway was based initially largely upon the models. observation that modest overexpression of Disabled suppressed Is the interaction of Notch with Abl pathway proteins both the embryonic lethality and morphological defects limited to just a few Drosophila growth cones, or is it of produced by genetic interactions of Abl with its accessory more general biological significance? Our ability to detect genes Nrt and Fax (Gertler et al., 1993; Liebl et al., 2003). Our Notch complexes with Disabled and Trio in extract of whole data now show that deletion of the Disabled binding region of embryos argues for the latter, as does the strong phyloge- Notch specifically impairs a Notch function, axon patterning, netic conservation of all the components of the pathway. that depends on the interaction of Notch with multiple Abl Good candidates for potential Notch/Abl-dependent processes pathway components (Figs. 2 and 3; Crowner et al., 2003; are provided by those developmental contexts in which non- Giniger, 1998). Moreover, GAL4-driven overexpression of Su(H) Notch signaling has been proposed previously. In Disabled modifies the Notch ISNb phenotype in the same way Drosophila, these include organization of actin structure at as do other treatment that enhance Abl signaling, such as the D/V boundary of the developing wing (Major and Irvine, overexpression of Abl or reduction of enabled (Fig. 5; Crowner 2005); in mammals, they include myogenesis and B-cell et al., 2003). Thus, these data provide further support for development, as well as a ligand-stimulated cytoplasmic association of Disabled with the Abl signaling pathway, though signaling process of Notch that is essential for the survival a definitive demonstration awaits the generation and character- of mouse neural stem cells and human embryonic stem cells ization of a disabled mutation. (Androutsellis-Theotokis et al., 2006; Ordentlich et al., 1998; The presence of Trio in Notch complexes suggests that Shawber et al., 1996). Rho family GTPases, particularly Rho and Rac, are good Axon guidance and classic lateral inhibition seem to candidates for a downstream readout of Notch/Abl signaling represent limit cases in which the Notch signal is largely (Hakeda-Suzuki et al., 2002; Luo, 2000; Newsome et al., transduced selectively through either the Abl pathway or the 2000). Consistent with this we have observed dominant Su(H) pathway, respectively. It seems likely, however, that genetic interactions of Notch with mutations in the three in each case both pathways make some contribution to Drosophila Rac genes, but not, for example, with Cdc42 (EG, Notch function: deletion of Su(H) binding sites does have unpublished observations). Such a readout would make sense some deleterious effect on growth of CNS longitudinal in the context of the effects of Notch on growth cone axons (Fig. 2), while mutation of Abl and Nrt cause small guidance and would be consistent with previous studies of but reproducible decreases in the efficacy of the classic Drosophila Trio (Hakeda-Suzuki et al., 2002; Newsome et Notch function that discriminates the identities of sibling al., 2000) and mammalian Abl (Renshaw et al., 1996). It is cells (Giniger, 1998; EG, unpublished observations). Perhaps interesting to note that identification of Rac as an effector of two parallel Notch signals, one through the canonical Su(H) Notch/Abl signaling might suggest the possibility of Notch/ pathway and the other mediated by the Notch/Abl interaction, Abl signaling having a non-Su(H) nuclear component in some can be used in concert to provide a richer nuclear readout, or developmental contexts in addition to its cytoskeletal targets to coordinate nuclear gene regulation with cortical properties (Hall, 2005; Lim et al., 1996). Rho family GTPases typically such as cytoskeletal structure and cell adhesion. It will be of M. Le Gall et al. / Developmental Biology 313 (2008) 556–567 565 great interest to determine whether some classic functions of hybridomas; these were screened by ELISA, Western blotting, immunopreci- Notch, such as dendritic patterning (Berezovska et al., 1999; pitation and whole mount embryo staining. mAb P4D11 was found to immunoprecipitate a set of species with the published characteristics of Redmond et al., 2000; Sestan et al., 1999) or oncogenesis Disabled from extracts of wild type Drosophila; mAb P6E11 recognized on a (Raafat et al., 2007), reflect more balanced contributions both Western blot a family of high-molecular weight species of a size consistent with from canonical Notch signaling and from the Notch/Abl Disabled, and which were absent from extract of embryos deleted for the pathway. Disabled locus (Supplementary Fig. S-1). Both antibodies also labeled (albeit with some nonspecific background, especially for mAb P4D11) a pattern of tissues in whole mount embryos consistent with the Disabled expression pattern Experimental procedures and absent from dab-deficient embryos. A high concentration stock of mAb P6E11 ascites was prepared by Maine Biotechnology. Genetics, Drosophila stocks and temperature-shift protocols Quantification of phenotypes Rescue of Notch axonal and cell fate defects by UAS-Notch transgenes was performed as described previously (Crowner et al., 2003; Giniger, 1998; Le Gall Phenotypes were quantified as follows: and Giniger, 2004), with the modification that for the experiment of Fig. 2, assay For CNS longitudinal axons, Fasciclin 2-labelled embryos were examined of CNS axon extension employed the following temperature-shift protocol to either as filet preps, or in appropriately oriented whole mounts, and the number of improve the precision of quantification: embryos were collected 4 h at 18 °C; thoracic and abdominal hemisegments in which there was a complete or nearly aged 7 h, 18 °C, shifted 7 h 45′ (32 °C), then fixed. To induce neurogenic defects complete disconnection between successive segments was counted and ts1 in Notch , embryos were collected 6 h (18 °C), shifted 10 h (32 °C) and then expressed as a percentage of the total. A “nearly complete” disconnection was Δcdc10 ΔEGF10–12 fixed. UAS-Notch and UAS-Notch flies were from A. Martinez- defined as one in which the residual connection between hemisegments consisted ECN Arias (Zecchini et al., 1999), UAS-Notch was from M. Muskavitch of a single Fasciclin 2-positive axon bundle with less then ∼1/5 the staining Δ2155 (Jacobsen et al., 1998). The UAS-Notch transgene was obtained from C. intensity of a wildtype fascicle. In a single experiment, either filet preps or whole Wesley (Wesley and Saez, 2000) and transformant lines were generated by mounts were quantified; quantitative data were not compared between different 1118 injection into w . UAS-Disabled was from D. Van Vactor (Wills et al., 1999). kinds of preparations. Note that slightly different temperature-shift protocols All other mutants and UAS transgenes have been described previously were employed for the experiments of Fig. 1 vs. Fig. 2 (see above) so expressivity ΔRamA (Giniger, 1998; Le Gall and Giniger, 2004) except UAS-Notch . For this per hemisegment should not be compared between these two experiments. construct, a deletion of Notch codons T1766–R1801 was generated by PCR ISNb defasciculation was assayed in abdominal segments A2–A7 in filet and reconstructed into full-length UAS-Notch as described for other preps of Fasciclin 2-labelled embryos (Crowner et al., 2003). A hemisegment juxtamembrane derivatives (Le Gall and Giniger, 2004). was scored as an aberrant “bypass” hemisegment if at least one Fasciclin 2- positive ISNb axon failed to defasciculate from the ISN and project internally at Antibody methods the wild type choice point, immediately ventral to muscle 28, and instead projected external to muscle 28. The fraction of bypass hemisegments was Embryos for immunocytochemistry were collected, fixed and stained by scored in greater than 100 hemisegments of each of at least three separate standard methods (Bodmer and Jan, 1987). Detection was performed using experiments. Reproducibility (SEM) was ±3% for this assay. biotinylated secondary antibodies and the Vectastain Elite tertiary reagent Notch neurogenic phenotypes were quantified by counting the number of (Vector Labs), with DAB development. Embryos were examined either as Elav-immunoreactive nuclei in the dorsal cluster of sensory neurons in whole-mounts in JB4 embedding medium or as filet preps in 80% glycerol. hemisegments A1–A7. SEM was b1.0 neurons per dorsal cluster for all Fluorescence microscopy was performed on filleted samples mounted in genotypes. FluoroGuard (BioRad), using a Zeiss AxioImager and deconvolution of the resulting image stacks. Biochemical methods Antibodies, their sources and the dilutions used were as follows. For embryo immunostaining, anti-Fasciclin 2 (mAb 1D4, 1:150), anti-Sxl (mAb Extracts M114; 1:50), mAb 22C10 (1:25) and rat anti-Elav (mAb 7E8A10; 1:20) Lysis buffer for both embryo lysates and head lysates was 25 mM Tris (pH were obtained from the Developmental Studies Hybridoma Bank; rabbit 7.5), 100 mM NaCl, 0.5 mM DTT, 0.5% NP-40 (v:v), 10% glycerol (v:v), 1 mM anti-β-galactosidase (1:10,000) was from Cappel. For biochemical experi- PMSF+a 1:100 dilution of a cocktail containing 1 mg/ml leupeptin, 1 mg/ml ments, immunoprecipitations were performed using 1–10 μg of specific pepstatin, 2 mg/ml aprotinin and 10 mg/ml benzamidine (prepared in DMSO). antibody per sample, and Western blotting was performed using 10–50 μg For whole embryo lysates, embryos were collected on grape juice plates 18–24 h specific antibody per 10 ml probe solution. Anti-Notch (C17.9C6), anti-islet at 25 °C. Embryos were harvested with 0.7% NaCl/0.3% Triton X-100, (mAb40.3A4) and anti-β-tubulin (E7) were from the DSHB. Rabbit anti- dechorionated with 50% bleach, washed with NaCl/Triton and transferred to an

Trio B6 and mouse anti-Trio (mAb 9A4) were gifts from C. Hama ice-cold Dounce homogenizer. Embryos were then washed twice with cold H2O (Awasaki et al., 2000). A high-concentration stock of purified mAb9A4 was and once with cold lysis buffer. Embryos were suspended with 3 volumes of generated by the Antibody Production Facility of the Fred Hutchinson lysis buffer and lysed with 25 strokes of an ice-cold A pestle, followed by 25 Cancer Research Center, using cells provided by Dr. Hama. Non-immune strokes with a cold B pestle. Embryo lysate was transferred to microfuge tubes mouse IgG, rabbit-anti-mouse IgG, and all secondary antibodies were from and centrifuged at 15,000×g for 10′ at 4 °C. The supernatent was removed to Jackson ImmunoResearch. fresh tubes and either used immediately or frozen in aliquots on dry ice and Antibodies against Drosophila Disabled protein were prepared as follows. stored at −80 °C. The coding sequence for a fragment of Disabled corresponding to the amino- For head lysates, flies of the appropriate genotype were anaesthetized with terminal 2/3 of the protein (from the PTB domain up to an EcoRI site at nt5868 CO2, transferred to a Falcon tube and flash frozen with liquid N2. Immediately in the numbering of Gertler et al., 1993) was subcloned in frame into pGEX2T to after the nitrogen boiled off, the flies were vortexed vigorously to shake off generate a GST fusion construct, and into pRSET A to generate a His-tagged heads and appendages (taking care to keep the flies frozen), and heads were construct. After transformation of expression constructs into Escherichia coli, isolated away from bodies and legs with appropriate minisieves (Fisher): heads tagged protein was grown up at room temperature and purified on glutathione are passed by the #25 mesh but retained by the #45 mesh. Sieve assembly was beads or nickel beads, respectively, by standard methods. Several polyclonal prechilled with liquid N2 and kept cold during the separation process. Isolated anti-Disabled antisera were prepared by Dr. Liz Wayner of the FHCRC heads were transferred to a puddle of liquid N2 in a prechilled agate mortar, Antibody Development Facility by injection of either fusion protein into mice, ground to a fine powder with a chilled pestle, and transferred to an ice-cold using standard protocols. Two mice that gave ELISA-positive bleeds after Dounce. Ice-cold lysis buffer was added (using 1/5 the starting volume of whole injection with the His-tagged immunogen were further used to generate flies, corresponding to ∼3× the volume of isolated heads) and the powder was 566 M. Le Gall et al. / Developmental Biology 313 (2008) 556–567 homogenized with 25 strokes each of the A pestle and B pestle, avoiding Awasaki, T., Saito, M., Sone, M., Suzuki, E., Sakai, R., Ito, K., Hama, C., 2000. excessive frothing of the lysate. Lysate was clarified, frozen and stored as for The Drosophila trio plays an essential role in patterning of axons by embryo lysates. regulating their directional extension. Neuron 26, 119–131. Bashaw, G.J., Kidd, T., Murray, D., Pawson, T., Goodman, C.S., 2000. Immunoprecipitation Repulsive axon guidance: Abelson and Enabled play opposing roles Protein A sepharose beads (Amersham) were blocked with lysis buffer downstream of the roundabout receptor. Cell 101, 703–715. containing 0.5% BSA, prebound with Rb anti-mouse IgG (10 μg Rb anti-mouse Bateman, J., Shu, H., Van Vactor, D., 2000. The guanine nucleotide exchange per 10 μl of beads), washed 2×, bound with the appropriate primary antibody, factor trio mediates axonal development in the Drosophila embryo. Neuron and washed 2×. Extract was thawed on ice, and 10 μl of protein A/Rb anti- 26, 93–106. mouse beads was added per IP sample (150 μl of extract, diluted to 300 μl final Baum, B., Perrimon, N., 2001. Spatial control of the actin cytoskeleton in volume with lysis buffer) for pre-clearing. Fresh 0.2 mM PMSF was added to Drosophila epithelial cells. Nat. Cell Biol. 3, 883–890. thawed lysate prior to clearing. Sample was rocked at 4 °C for 1 h, then cleared Bear, J.E., Loureiro, J.J., Libova, I., Fassler, R., Wehland, J., Gertler, F.B., 2000. by centrifugation at 15,000×g,10′, 4 °C. Supernatent was removed and used Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101, immediately for co-immunoprecipitation. Beads bearing the appropriate primary 717–728. antibody (10 μl) were added per IP sample and rocked at 4 °C for 90′. Beads Berezovska, O., McLean, P., Knowles, R., Frosh, M., Lu, F.M., Lux, S.E., were spun down for 5ʺ, unbound material was removed, and the beads were Hyman, B.T., 1999. Notch1 inhibits neurite outgrowth in postmitotic washed 4× with lysis buffer. Beads were then boiled in 20 μl Laemmli sample primary neurons. Neuroscience 93, 433–439. buffer. Samples were analyzed by SDS-PAGE and Western blotting. ECL Bodmer, R., Jan, Y.N., 1987. Morphological differentiation of the embryonic detection was used, employing the Pierce SuperSignal West Pico or Femto peripheral neurons in Drosophila. Roux's Arch. Dev. Biol. 196, 69–77. reagents, or the Amersham ECL Advance system. Boutros, M., Paricio, N., Strutt, D.I., Mlodzik, M., 1998. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118. Acknowledgments Brennan, K., Gardner, P., 2002. Notching up another pathway. BioEssays 24, 405–410. We wish to thank all the members of our lab for discussions Cooper, M.T., Bray, S.J., 1999. Frizzled regulation of Notch signaling polarizes – and advice, and we particularly thank Lei Wang for help with cell fates in the Drosophila eye. Nature 397, 526 530. Crowner, D., Le Gall, M., Gates, M.A., Giniger, E., 2003. Notch steers Dro- co-IP experiments, and Dan Crowner and Lori Luke for sophila ISNb motor axons by regulating the Abl signaling pathway. Curr. outstanding technical assistance. For helpful discussions about Biol. 13, 967–972. the biochemical studies we thank Brian Howell, and for Druker, B.J., Talpaz, M., Resta, D.J., Peng, B., Buchdunger, E., Ford, J.M., comments on the manuscript we thank Bob Callahan, Ken Lydon, N.B., Kantarjian, H., Capdeville, R., Ohno-Jones, S., Sawyers, C.L., – Irvine, Chi-Hon Lee, Ron McKay and Valerie Vasioukhin. We 2001. Efficacy and safety of a specific inhibitor of the BCR ABL tyrosine kinase in chronic myeloid leukemia. N. Engl. J. Med. 344, 1031–1037. also thank the many members of the fly community who Fainstein, E., Marcelle, C., Rosner, A., Canaani, E., Gale, R.P., Dreazen, O., generously provided published and unpublished reagents and Smith, S.D., Croce, C.M., 1987. 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