a notch-independent function of suppressor of hairless...

INTRODUCTION

Notch signalling plays a fundamental role during a greatnumber of developmental processes in multicellular animals(Artavanis-Tsakonas et al., 1999; Mumm and Kopan, 2000). Itmediates communication between adjacent cells and is oftenemployed for binary fate decisions. Interactions between Notchand its ligands trigger the proteolytic cleavage of the Notchreceptor, releasing the intracellular domain that travels to thenucleus. In the nucleus, the intracellular domain binds to theSu(H)/CBF transcription factor to activate the expression oftarget genes (Barolo et al., 2002; Furriols and Bray, 2001;Morel and Schweisguth, 2000). During its activation, Notch iscleaved twice. The first cleavage is ligand dependent andis performed by an ADAM metalloprotease encoded inDrosophilaby the kuzbaniangene (kuz) (Klein, 2002; Lieberet al., 2002; Pan and Rubin, 1997; Sotillos et al., 1997; Wen etal., 1997). Kuz cleaves Notch in the extracellular part adjacentto the transmembrane domain. The resulting fragment withinthe membrane is then cleaved a second time by a proteaseencoded by the Presenilingene (Psn). The cleavage occurs inthe transmembrane region and releases the intracellular domaininto the cell (Brou et al., 2000; Lecourtois and Schweisguth,1998; Qi et al., 1999; Schroeter et al., 1998; Struhl and Adachi,1998; Struhl and Greenwald, 1999).

In many developmental processes Su(H) seems to act as arepressor of the expression of target genes in the absence ofthe Notch signal (Barolo et al., 2002; Furriols and Bray, 2000;Morel and Schweisguth, 2000). For this ‘default repression’ itrequires the Hairless protein (H), which acts as a bridgebetween Su(H) and its co-repressors CtBP and Groucho(Barolo et al., 2002; Morel et al., 2001).

One intensely studied process in which the Notchpathwayplays an important role is the development of the bristle senseorgan of the adult peripheral nervous system (PNS) ofDrosophila (Modolell and Campuzano, 1998). These bristlesare simple mechanosensory organs that consist of only fourcells. All four cells are generated by a single precursor, referredto as the sensory organ precursor cell (SOP) (Fig. 1A). TheSOP is selected from a cluster of cells that are defined by theexpression patterns of the proneural genes, such as the genesof the achaete-scutecomplex (AS-C) (Fig. 1B,C). The activityof the proneural genes enables all cells of a cluster (proneuralcluster) to develop as SOPs. The selection of the SOP in theproneural cluster occurs through a process called lateral ormutual inhibition and is mediated by the Notch signallingpathway. Lateral inhibition ensures that only a defined numberof cells of a proneural cluster develop as SOP, whereas the restswitch fate and develop to epidermoblasts. During lateralinhibition, the SOP sends an inhibitory signal via the Notch

1973Development 130, 1973-1988 © 2003 The Company of Biologists Ltddoi:10.1242/dev.00426

Su(H)/CBF1 is a key component of the evolutionaryconserved Notch signalling pathway. It is a transcriptionfactor that acts as a repressor in the absence of the Notchsignal. If Notch signalling is activated, it associates withthe released intracellular domain of the Notchreceptorand acts as an activator of transcription. During thedevelopment of the mechanosensory bristles of Drosophila,a selection process called lateral inhibition assures that onlya few cells are selected out of a group to become sensoryorgan precursors (SOP). During this process, the SOP cellis thought to suppress the same fate in its surroundingneighbours via the activation of the Notch/Su(H) pathwayin these cells. We show that, although Su(H) is required toprevent the SOP fate during lateral inhibition, it is alsorequired to promote the further development of the SOPonce it is selected. Importantly, in this situation Su(H)

appears to act independently of the Notchsignallingpathway. We find that loss of Su(H) function leads to anarrest of SOP development because of the loss of sensexpression in the SOP. Our results suggest that Su(H) actsas a repressor that suppresses the activity of one or morenegative regulator(s) of sensexpression. We show that thisrepressor activity is encoded by one or several genes ofthe E(spl)-complex. Our results further suggest that theposition of the SOP in a proneural cluster is determinedby very precise positional cues, which render the SOPinsensitive to Dl.

Key words: Suppressor of Hairless, Enhancer of split complex,Notch-signalling, Bristle development, Presenilin, Kuzbanian,Senseless

SUMMARY

A Notch-independent function of Suppressor of Hairless during the

development of the bristle sensory organ precursor cell of Drosophila

Stefan Koelzer and Thomas Klein*

Institut für Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany*Author for correspondence (e-mail: [email protected])

Accepted 30 January 2003

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ligand encoded by Delta (Dl) to its neighbours to activate theexpression of the genes of the Enhancer of split-complex[E(spl)-C] in these cells (Bailey and Posakony, 1995; Hinz etal., 1994; Jennings et al., 1994; Lecourtois and Schweisguth,1995; deCelis et al., 1996; Lai et al., 2000a; Lai et al., 2000b).

The activity of the genes of the E(spl)-C antagonizes that ofthe proneural genes (Knust et al., 1992; Nakao and Campos-Ortega, 1996). As a result the neighbouring cells switch fateand develop as epidermal precursors. The expression of thegenes of the E(spl)-Cis directly activated by a transcription

S. Koelzer and T. Klein

Fig. 1.The development of themechanosensory bristle organ ofthe adult peripheral nervous systemof Drosophila. (A) An adult bristle(machrochaete) stained forneurA101-lacZ to reveal the nucleusof the socket cell (blue) and anti22C10 antibody staining to markthe neurone (brown). The bristlesense organ consists of two morecells: the prominent bristle cell anda sheath or thecogen cell, which isnot visible in the picture. (B) Awing imaginal disc of the late thirdlarval instar stage, stained with antiHnt antibody (red) to reveal theSOPs of the machrochaete. Thedisc contains a scaGal4 insertionthat activates UAS GFP in the cellsof the proneural clusters (green).The double staining reveals that theclusters are arranged in astereotypic pattern that allows theidentification of each clusterindividually. ANP and PNP,anterior and posterior notopleural;APA and PPA, anterior andposterior postalar; DC,dorsocentral; SC, scutellar clusters.(C) Development of the bristlesense organ. The SOP is selectedfrom a proneural cluster during theprocess of lateral inhibition, whichis mediated by the Notchsignallingpathway (not shown). The SOP,recognizable by the high level ofexpression of the proneural proteinAc, signals through the Notchligand Delta to its neighbours (pinklines). Activation of the pathwayresults in the Su(H)-dependentswitch to the epidermal fate in theneighbours of the SOP. The highlevels of Ac and Sc proteins in theSOP are achieved through theactivation of the SOP-E of the scgene (Culi and Modolell, 1998).Once the SOP is selected, itswitches off the expression of theproneural genes and initiates expression of neurA101-lacZ, sensand hnt. It then divides to generate the second order precursor cells pIIa andpIIb. pIIa divides to give rise to the socket and bristle cells. pIIb divides to generate a third-order precursor pIIIb and a glial cell. The glial cellmigrates away and does not contribute to the formation of the sense organ. pIIIb further divides to give rise to the neurone and the sheath cellthat protects the neurone. In this lineage, the Notch-signalling pathway is employed several times to help the cells to choose the correct fate. Inthe first step, pIIb sends a Notch-mediated inhibitory signal (pink line) that prevents pIIa from joining the pIIb fate and developing the pIIa fate.Later Notch is required to send an inhibitory signal from the bristle to the socket and from the neurone to the sheath cell to prevent the receivingcells from choosing the same fate as the sending cell. The differentiated neurone can be detected through the expression of the neurone specific22C10 and Elav marker. (D) The consequence of loss of Notchfunction during bristle development. Owing to the lack of Notch signalling, allcells of a Notchmutant proneural cluster choose the SOP fate. As a result of the missing communication between the progenies of the SOP, anexcess of neurones develops at the expense of the other fates of the sensillum. These supernumerary neurones can be visualized by anti 22C10or anti Elav antibody staining.

1975Function of Su(H) during SOP development

complex that includes Su(H) as the DNA-binding part (Baileyand Posakony, 1995; Hinz et al., 1994; Jennings et al., 1994;Lecourtois and Schweisguth, 1995; deCelis et al., 1996; Lai etal., 2000a; Lai et al., 2000b).

During normal development, the SOP arises in a distinctposition in each proneural cluster, indicating that the cells atthese positions have a bias to develop the SOP fate (Cubas etal., 1991; Cubas and Modollel, 1992). It is thought that thisslight bias is amplified during lateral inhibition through afeedback loop between the activity of Notch and theexpression of Dl(Heitzler et al., 1996; Schweisguth, 1995):the more Notch is active in a given cell, the less it expressesDl. Hence, the ability of that cell to inhibit their neighboursdecreases over time. Conversely, a cell where Notch is lessactive expresses higher levels of Dl and can inhibit itsneighbours more efficiently, and its inhibiting ability increasesover time.

Once a SOP is selected through lateral inhibition, it startsto express genes such as asense (ase),neuralized (neur),senseless(sens; Ly – FlyBase) and hindsight (hnt; peb –FlyBase). The expression of these markers is important for thecorrect development of the SOP. In particular, sensappears tobe essential for the normal development of the sensillum (Noloet al., 2000). It encodes a zinc-finger transcription factor andis activated in the SOP by the proneural proteins Achaete (Ac)and Scute (Sc) (Nolo et al., 2000). Upon ectopic expression,Sens is able to induce supernumerary SOPs, indicating that itis sufficient to initiate the development of sensory organs (Noloet al., 2000). At the time when sensand neurexpression isinitiated, the expression of the proneural genes is switched offin the SOP (Cubas et al., 1991).

The SOP subsequently divides to generate two second-orderprecursors, pIIa and pIIb (Hartenstein and Posakony, 1990)(Fig. 1C). pIIa divides once more and generates the bristle andsocket cell. pIIb divides to give rise to a pIIIb precursor celland a glia cell (Gho et al., 1999) (Fig. 1C). The glia cellmigrates away from the developing sense organ. The pIIIbdivides another time to give rise to the neurone and a sheathcell (Fig. 1C).

The Notch pathway is required repeatedly during thefurther development of the bristle sensillum (Hartenstein andPosakony, 1990) (Fig. 1C,D): It sends an inhibitory signal fromthe second order precursor cell pIIb to pIIa that prevents pIIafrom choosing the pIIb fate. Later, the Notchpathway is againrequired to send a signal from the bristle to the socket cell andfrom the neuron to the sheath cell to prevent the receiving cellsfrom choosing the same fates as the sending cell (Fig. 1C).Thus, loss of Notch function results in the development of allcells of a proneural cluster into SOP cells. These SOP cellsthen generate an excess of neurones at the expense of the otherfates (Fig. 1D). This scenario implies that loss of functionmutants of all genes that are involved in the Notch pathwayshould display an excess of SOPs that subsequently generatedan excess of neurones (Fig. 1D), a phenotype namedneurogenic.

Schweisguth and Posakony (Schweisguth and Posakony,1992) have reported that in Su(H)mutant wing imaginal discsnot all proneural clusters can be detected with the SOP-specificneurA101-lacZ marker (neurA101). By contrast, cells of allclusters express this marker in kuzmutant wing discs (Sotilloset al., 1997). The differences in the mutant phenotype of kuz

and Su(H)raise the possibility that Su(H) might have a functionduring SOP development that is independent from its functionduring Notchsignalling. To test this possibility, we comparedthe consequences of loss-of-function mutations of genes thatare involved in the Notchpathway on the development of theSOP, and the differentiation of neurones. We found that inSu(H)mutants, the development of the SOPs arrests during anearly phase. This is not observed in Psn, Notchand kuzmutants, and suggests that Su(H) is required for SOPdevelopment in a Notchindependent fashion. We provideevidence that this arrest is caused by the loss of the activityof the gene senseless(sens), which is crucial for SOPdevelopment. Our results suggest that Su(H) acts as a repressorof one or more members of the E(spl)-complex that in turnrepress the expression ofsens.

We further find that Su(H)mutant cells are unable to preventthe SOP fate in normal neighbours that are located at theposition of the proneural cluster, where the SOP normallyforms. It appears that cells at this position are insensitive to theDl signal. This observation suggests that the positionalinformation within a proneural cluster is more precise thananticipated and that the position of the SOP is stronglydetermined.

MATERIALS AND METHODS

Fly strainsThe following alleles were used in this work: Su(H)∆47 P(B)FRT40A(a null mutant) (Morel and Schweisguth, 2000), Su(H)AR9, Su(H)SF8

(Schweisguth and Posakony, 1992), PsnC1 [null mutant described byStruhl and Greenwald (Struhl and Greenwald, 1999)], PsnI2 [strongmutation described by Ye et al. (Ye et al., 1999)], kuz1405, kuz1403

[strong mutants, see Sotillos et al. (Sotillos et al., 1997)], Df(1)N81K

FRT101 (null mutation) (Brennan et al., 1997) and neurA101 (Huanget al., 1991). The Df(3R)E(spl)b32.2 is described by Schrons et al.(Schrons et al., 1992).

Reporter strains were E(spl)m8-lacZ(Lecourtois and Schweisguth,1995; Nakao and Campos-Ortega, 1996), E(spl)mβCD2 (de Celis etal., 1998), SOP-E (Culi and Modolell, 1998) and Gbe+Su(H) (Furriolsand Bray, 2001).

UAS stocks were UASsens(Nolo et al., 2000), UASSu(H) (Kleinet al., 2000), UAS Su(H)∆H(Furriols and Bray, 2000) and UAS GFP[a gift from S. Bahri and Yeh et al. (Yeh et al., 1995)].

Gal4 drivers wre scaGal4 (Hinz et al., 1994) and dppGal4 (a giftfrom S. Carroll).

HistochemistryAntibody staining was performed according to standard protocols.The anti Dl, anti Wg and anti Hnt antibodies were obtained fromthe Developmental Studies Hybridoma Bank developed under theauspices of the NICHD and maintained by the University of Iowa,Department of Biological Sciences, Iowa City, IA 52242. The antiSens antibody was a gift of H. Bellen (Nolo et al., 2000). The anti22C10 and anti Elav antibody were a gift of C. Klämbt.

Fluorochrome-conjugated antibodies were purchased fromMolecular Probes.

RESULTS

The wing imaginal disc of Drosophilagives rise to the wingproper and one half of the mesothorax (notum) of the fly. In a

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wing imaginal disc of the late third larval instar stage, theproneural clusters in the notum have fully developed and thefirst SOPs are recognizable (Fig. 1B). These SOP and theircorresponding proneural clusters are arranged in a stereotypedpattern and form the large bristles, which are calledmachrochaete. The development of the machrochaete providesa classical model for sensillum development in insects.

To find out whether there is a difference in the phenotypesof Su(H) and mutants of other genes involved in the Notchpathway, we have compared the development of the SOPs ofthe machrochaete in wing imaginal discs that were mutant forof Su(H), Notch, Psn and kuz. We used two other markers,besides neurA101-lacZ, that specifically label SOPs of the wing

imaginal disc (sensand hnt). Expression of both genes isrestricted to the SOPs in the notum (Fig. 2A,E,J) (Nolo et al.,2000; Pickup et al., 2002).

We found that in Notchand kuzmutant wing imaginal discsall proneural clusters of the notum could be detected withneurA101-lacZ (Fig. 2A,B,D). As previously reported, thesituation is different in Su(H)mutants (Schweisguth andPosakony, 1992). For example, the cells of the dorsocentralcluster (arrow in Fig. 2A-D; see Fig. 1B for the naming of theclusters) strongly express neurA101 in kuz discs and Notchmutant clones (Fig. 2B,D; data not shown), whereas expressionof this marker is strongly reduced or absent in the cells of thecorresponding cluster in Su(H)mutants (Fig. 2C). Likewise, the


Fig. 2.Expression of the SOP markers neurA101-lacZ, Sens and Hnt in Notchmutant cell clones as well as in Psn, Su(H)and kuzmutant wingimaginal discs. Expression of the SOP marker is detected by antibody staining. Alleles used in this analysis are: Df(1)N81K, PsnC1, Su(H)∆47

and the combination kuz1405/kuz1403. They are amorphic or strong alleles of the corresponding genes. Anterior is towards the left, ventraltowards the bottom. (A-D) neurA101-lacZexpression. The wing disc shown in A is also stained with an anti Wg antibody to visualize theexpression of Wg (red). neurA101-lacZexpression in A is shown in green. The arrows indicate the SOPs that have formed. (E-I) Expression ofSens. (J-N) Expression of Hnt. (A,E,J) Expression of neurA101-lacZ, Sens and Hnt in wild-type wing imaginal discs of the late third larval instarstage. (B,F,K) Wing imaginal bearing Notchmutant clones that are labelled by the absence of the green GFP marker. Expression of thecorresponding SOP marker is shown in red. (G,L) PsnC1 mutant wing imaginal discs. (C,H,M) Su(H)∆47 mutant wing imaginal discs.(D,I,N) kuz1405/kuz1403mutant wing imaginal discs. In the wild type, neurA101-lacZ, Sens and Hnt are expressed in all SOPs of the wingimaginal disc. Sens is also expressed in cells along the wing margin (wm in E). Arrows in A indicate the position of each individual SOP. Thefigure reveals that cells of all proneural clusters in the notum that are mutant for Notch, Psnor kuzexpress the three SOP markers (arrows in F-I,K-N). The situation is different in Su(H) mutants: only a few cells of the DC, the ANP and ANP cluster weakly express neurA101-lacZ(arrows). Moreover, expression of Sens (H) and Hnt (M) is lost in almost all cells of the notal clusters. We occasionally found weak expressionof Sens in some cells of the ANP and PNP cluster (arrow).


proneural clusters for the ANP and PNP (arrowheads in C,D)are neurA101positive in Notchmutant clones and in kuz, but notin Su(H) mutant discs (Fig. 2C,D). The difference betweenSu(H)mutants and mutants of other genes of the Notchpathwaywere even clearer if we looked at the expression of sensandhnt. All proneural clusters are detectable with these two markersin notae of Notch, Psnand kuzmutant wing imaginal discs (Fig.2E-G,I,J-L,N highlighted by arrows). However, expression ofsensand hnt was strongly reduced or absent in the cells of theclusters of Su(H)mutant wing discs (Fig. 2H,M).

During the course of our experiments, we noticed that theloss of Psnfunction causes the strongest phenotype. We foundthat the proneural clusters are larger than in other mutants andoften fused (see Fig. 2G,L). This observation is in agreementwith the results of the analysis of Psn mutants during wingdevelopment, where loss of its function also causes thestrongest phenotype (Klein et al., 2000).

Absence of neurones in Su(H) mutant wing imaginaldiscsTo look at the consequences of loss of the SOP markers forneural differentiation of the progenies of Su(H)mutant SOPs,we monitored the expression of 22C10(futsch) and elav, whichare specific for mature neurones (Fig. 3). Although all cells ofPsn and Notch mutant proneural clusters express 22C10andsome in addition express elav (Fig. 3A,B,D), the expression ofthese markers was not detectable in cells of Su(H)mutantclusters (Fig. 3C,E). This indicates that the loss of expressionof SOP marker such as sens, hntand neurA101 wasaccompanied by a lack of neural differentiation in Su(H)mutant wing imaginal discs.

The phenotype of Su(H) is epistatic over that of PsnThe results presented so far reveal a qualitative differencebetween the phenotypes of Su(H)mutants and mutants of othergenes of the Notchpathway, including Notch. This suggeststhat Su(H) has a function during the development of the SOP

of the machrochaete that is independent from the Notchpathway. To confirm this conclusion, we analysed thephenotype of Su(H); Psndouble mutant proneural clusters. Wefound that Su(H); Psndouble mutant discs display a phenotypesimilar to the Su(H) mutant one: they express early SOPmarkers such as the SOP-E, but fail to express sensor 22C10in cells of most proneural clusters (Fig. 4A-D). Furthermore,expression of UAS Su(H)in these double mutant clusters withscaGal4, re-establishes the expression of sensand 22C10(Fig.4E). These results indicate that the mutant phenotype of Su(H)is epistatic over that of Psn. They further confirm theconclusion that Su(H) is required for the development of theSOP in a Notch-independent manner.

The cells of the proneural clusters are present inSu(H) mutantsThe lack of expression of SOP markers in proneural clustersin Su(H)mutants could be caused by the loss of the cells of acluster or by an arrest in their early development as a SOP. Todiscriminate between these possibilities, we monitored theexpression of genes that are expressed in proneural clusters,such as E(spl)m8, and the activity of the earliest marker for theSOP, the achaete-scuteSOP enhancer (SOP-E) (Culi andModolell, 1998), in Su(H) mutant wing imaginal discs. Wefurther used the expression of Dl, which is, as we have found,strongly expressed in cells of proneural clusters of Psnmutants(data not shown). All these markers were expressed in cells ofproneural clusters in the notum of Su(H) mutant wing discs(Fig. 5A-C). Likewise, many cells of the proneural clusters ofSu(H); Psndouble mutant wing discs also express the SOP-E(Fig. 4B). In summary, the cells of proneural clusters of Su(H)mutant notae are present and many of them express early SOPmarkers, such as the SOP-E and neurA101, but they fail toexpress later markers such as sensand hnt. Therefore, weconclude that in Su(H)mutants, the cells of the proneuralclusters arrest their development during an early phase of SOPdevelopment (see Fig. 5F).

Fig. 3.Expression of a neurone-specific marker incells of proneural clusters in Notch, Su(H)and Psnwing imaginal discs of the late third larval instarstage. Anterior is towards the left, ventral towardsthe bottom. (A-C) Wing imaginal discs stained withanti 22C10 antibody. (D,E) Wing imaginal discsstained with anti Elav antibody. (A) Df(1)N81K

mutant clones in the notum of a wing imaginal discrevealed by the lack of the GFP marker. The arrowsindicate proneural clusters where the cells express22C10. (B) Likewise, cells of the proneural clustersof PsnC1 mutant wing imaginal discs express22C10 (arrows). By contrast, no expression of22C10 is detectable in the Su(H)∆47mutant wingdisc (C). (D) Expression of Elav in PsnC1 mutants.The cells of some clusters, such as the APA+tr1,PPA and PSA clusters, which are fused to one bigcluster (arrow), do express Elav. (E) By contrast,Su(H)∆47mutant wing imaginal discs are devoid ofany Elav expression in cells of the notum. Theresults suggest that the loss of SOP markers in thecells of Su(H)mutant proneural clusters isaccompanied by a loss of neural differentiationmarkers.

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The strong expression of E(spl)m8in SOP in Su(H)mutantwing imaginal discs is surprising because expression of thisgene is switched of in the SOP during normal development(Nolo et al., 2000) (Fig. 5D,E). Thus, it appears that Su(H) isalso required to switch off expression of this gene in the SOPduring normal development.

Forced expression of sens can re-establishexpression of SOP markers normally absent in cellsof Su(H) mutant proneural clustersensencodes a zinc-finger containing transcription factor thatis essential for SOP development (Nolo et al., 2000). Thus, wewondered whether it is the loss of sensactivity that causes thearrest in SOP development in Su(H)mutant wing imaginaldiscs. To test this hypothesis, we looked to see if forcedexpression of senscould restore expression of neural markerssuch as 22c10and elavand SOP markers such as hnt and theSOP-E in Su(H)mutant discs (Fig. 6). In the first series ofexperiments, we expressed UAS senswith dppGal4 (Fig. 6A-G). We found that most cells that express Sens were able toactivate the expression of the SOP-E, as well as hnt(Fig.6A,C,F,G). Most sens-expressing cells of the notum alsoinitiated 22C10 expression (Fig. 6B,C). The ability of Sens toactivate these genes ectopically was restricted to the notum.

Sens was also able to activate the expression of elav.However, activation of elavexpression was more restricted andappeared in clusters of cells that mapped to regions of theproneural clusters (Fig. 6D,E). This indicates that the activityof Sens is required for the expression of elav but, in contrastto 22C10, hnt and the SOP-E, is not sufficient. Similar resultswere found when UAS senswas expressed with scaGal4 (datanot shown).

In summary, the results show that Sens is able to activate theexpression of those genes that are normally absent in Su(H)mutant proneural clusters and suggests that the loss of sens

activity causes the arrest in SOP development in Su(H)mutants. Hence, Su(H) appears to be required in the SOP toactivate the expression of sensin a Notch-independent manner.

SOP development in Su(H) mutant cell clones We also analysed the function of Su(H) during SOPdevelopment by inducing mutant cell clones in the notumduring first larval instar [24-48 hours after egg laying (ael)](Fig. 7). Using this type of analysis, we found a spectrum ofphenotypes. In several cases, the mutant cells express earlymarkers such as neurA101or the SOP-E, but fail to express hnt.The arrowhead in Fig. 7A-E points to such an example. In thiscase one Su(H)positive cell lies in the cluster. It is this cell thatexpresses SOP-E and hnt. By contrast, the mutant cells of thecluster express only the SOP-E. In other cases, we found avarying fraction of Su(H)mutant cells that express the earlymarker and also hnt, suggesting that some of the mutant cellsdo not arrest their development (arrows in Fig. 7B-E; Fig. 7F-I). However, many of the cells of this class expressed lowerlevels of hntthan normal (see Fig. 7H,I). Altogether, theseobservations confirm our conclusion that Su(H) is required forthe development of the SOP.

We observed that the fraction of cells that expresses hntvaries among the clusters of the notum. We concentrated onthe SC and DC clusters: in six out of seven cases, we foundhnt-positive cells in a fraction of Su(H)mutant cells of the SCcluster. By contrast, we found only one case out of 15, whereone cell weakly expresses hnt in clones that include parts ofthe DC cluster (Fig. 7F,G). In this case, the hnt-expressing cellis at a position where the pDC would normally arise. At theposition of the aDC, two cells express hnt (Fig. 7F), but do notexpress the SOP-E (inset in Fig. 7F). This suggests that theaDC has already divided to give rise to pIIa and pIIb. Bycontrast, the pDC still express the SOP-E and weakly hnt andhas not divided, indicating that its development is delayed incomparison with that of the aDC. During normal development,the aDC develops later than the pDC and switches off the SOP-E before dividing (Huang et al., 1991). Thus, it appears thatthe pDC is strongly delayed in its development. This


Fig. 4.Analysis of SOP development inSu(H)∆47; PsnI2 double mutant wing imaginaldiscs. Anterior is towards the left, ventraltowards the bottom. Expression of Sens and22C10is detected by antibody staining.(A) Sens expression is lost in all clusters withthe exception of some cells of the PNP clusterthat have residual expression (arrowhead).(B) Expression of the SOP-E in the same discas shown in A. The cells of the notalproneural clusters of the double mutant discsstill express the SOP-E (arrows), indicatingthat the absence of Sens expression is notcaused by the loss of the cells. (C) Merge ofthe pictures A and B, showing Sens

expression in red and expression of the SOP-E in green. (D) Similar toSu(H)mutants, Su(H)∆47; PsnI2 double mutants have lost the expressionof 22C10, indicating that the cells of the proneural clusters fail todifferentiate neurone-specific traits. (E) Expression of a weak UASSu(H)line in the proneural clusters of Su(H) Psndouble mutant wing imaginaldiscs with scaGal4. The expression of UASSu(H)re-establishes theexpression of Sens and 22C10in the cells of the proneural clusters.


observation further supports our conclusion from theanalysis of the homozygous mutants, that Su(H) mutantproneural cells arrest SOP development at an early stage.

For the other clusters, where we have less casesexamined, we found that in four out of six cases of clones,including parts of the pPA/tr2/aPA+tr1 cluster we found afraction cells weakly expressing hnt. In one out of the fourobserved cases of the PSA cluster, we found weak hntexpression in one cell. In the two clones we found for theANP/PNP cluster no expression was observed.

As expected, kuzmutant cell clones that include regions ofproneural clusters contain big clusters of hnt-expressing cells,indicating that cells of kuzmutant proneural clusters canprogress in their development as SOP (Fig. 7J).

The SOP is insensitive to the Dl signal from its Su(H)mutant neighboursDuring the course of the clonal analysis of Su(H), we very oftenfound that a single enlarged and Su(H)-positive cell adjacentor nearly surrounded by mutant cells that express the SOP-E(Fig. 7B-E; Fig. 8I-M). Invariantly, this Su(H)-positive cellexpressed hnt (Fig. 7B-E) and was located at the position ofthe cluster, where a SOP would normally arise (Fig. 7B-G).This observation contradicts the lateral inhibition model, whichpredicts that cells in which Notch is least active have thehighest potential to inhibit their neighbours from adopting theSOP fate. Hence, cells in a cluster that are defective in Notchsignal reception should be very potent to inhibit their wild-typeneighbours and a Su(H)-positive cell should therefore neveradopt the SOP fate, if adjacent to Su(H)mutant cells.

To investigate this paradox further, we examined if the Su(H)mutant cells express high levels of Dl as we have observed inhomozygous mutant discs (see Fig. 5A). Indeed, we found thatSu(H)mutant cells in proneural clusters do express high levels

of Dl (Fig. 8A-D). This raises the possibility that Dl protein inSu(H)mutant cells might not be active. We therefore looked tosee whether the Su(H)mutant cells of a proneural cluster areable to activate Notch in adjacent Su(H)-positive cells. As ameasure of Notchactivity, we used the activity of theGbe+Su(H)-lacZconstruct [Gbe+Su(H)] (Furriols and Bray,2001). The activity of this construct is dependent on Su(H) andthe presence of a functional Notchreceptor (Furriols and Bray,2001). The expression pattern of Gbe+Su(H) faithfully revealsthe activity domains of Notch in the wing imaginal disc(Furriols and Bray, 2001). In the notal region of the disc,Gbe+Su(H) is active in a pattern that is similar to that of Dl(Fig. 8E-G). We observed that expression of Gbe+Su(H) isswitched off in SOPs. As an example the posterior SOP of theDC cluster is shown in Fig. 8H-J. At the stage when the SOPinitiates expression of hnt, an upregulation of the expression ofGbe+Su(H) can be observed in the adjacent cells (Fig. 8H-J).The SOP itself does not express the construct (Fig. 8H-J). Theexpression of Gbe+Su(H), including the ring of higherexpression of around the SOP, is strictly dependent on theactivity of Notchand Su(H)(data not shown, Fig. 8M-O). Thisobservation is in agreement with the model of lateral inhibitionand suggests that the SOP sends a signal that activates theNotchpathway in its immediate neighbours.

Fig. 5.Expression of Dl, the SOP-E andE(spl)m8-lacZin Su(H)∆47mutant wingimaginal disc of the late third larval instar stage.Anterior is towards the left, ventral towards thebottom. (A) Expression of Dl, revealed by antiDl antibody staining, is elevated in the cells ofthe proneural clusters (highlighted by thearrows). (B) Expression of the SOP-E inSu(H)∆47mutant discs indicates that most of thecells of the proneural clusters express thisenhancer (arrows). (C) The same Su(H)∆47

mutant wing disc as shown in A and B showingexpression of Dl (red) and anti β-Gal antibody(green) to visualize the expression of E(spl)m8.Expression of E(spl)m8-lacZis detectable in the

cells of the proneural clusters.(D,E) Expression of E(spl)m8-lacZ in the notal region of awild-type wing imaginal discs.(D) Expression of E(spl)m8-lacZ, (E) Expression of Sens(red) and E(spl)m8-lacZ(green). The comparisonbetween D and E reveals thatexpression of E(spl)m8-lacZisswitched off in SOPs at thetime when they initiateexpression of Sens (Nolo et al.,2000). The arrows in D and Eindicate the developing SOPs.(F) Results summary. Cells ofthe proneural cluster in Su(H)mutant notae are present butarrest their development beforeexpression of sensand hntisinitiated.

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We found that Su(H)mutant cells of a proneural cluster canactivate the Gbe+Su(H) construct in their Su(H)-positiveneighbours. This is indicated by the upregulation of theconstruct in these cells (Fig. 8K-O, arrowhead in N, O). Hence,the cells of Su(H) mutant proneural cluster seem to express anactive form of Dl. However, one exception was observed:Gbe+Su(H) is not activated in the hnt expressing, Su(H)positive SOPs that are located next to mutant cells (arrows inFig. 8K,M,O). Expression of Notch itself in Su(H)mutant cellswas normal (data not shown). These observations indicate that

Su(H) mutant cells, although expressing active Dl, cannotactivate the Notchpathway in cells that are located at positionswhere the SOP develops. Thus, it appears that the position ofthe SOP within a proneural cluster is strongly determined.Cells at this position appear to be insensitive to lateralinhibition.

The conclusion that the position of the SOP within aproneural cluster is pre-determined is further supported byanother observation: We found three cases where a Su(H)mutant clone includes almost all cells of a proneural cluster. Inthese cluster, one or two cells of the cluster weakly express Hnt(arrows in Fig. 7B,E). These cells are located at positions,where the SOP would be expected to arise. Thus, it appearsthat cells at certain positions in a cluster are strongly biasedtowards the SOP fate.

The repressor function of Su(H) is required forexpression of sens in the SOPThe results above suggest that Su(H) is required for the proper


Fig. 6.Forced expression of UAS sensin Su(H)∆47

mutant wing imaginal discs re-establishes theexpression of the SOP-E, hnt, 22C10 and elavincells of the proneural cluster. Anterior is towards theleft, ventral is towards the bottom. (A-G) Expressionof UAS senswith dppGal4 in Su(H)mutant wingimaginal discs. (A) Expression of the SOP-E isactivated in a stripe of cells (arrow) in the notum thatcorresponds to the dppGal4 expression domain.(B) Expression of 22C10 (arrow). (C) The same discas in B showing expression of UASGFP in green and22C10 in red. The double staining reveals that mostof the cells in the notum that express Sens initiateexpression of the neurone-specific marker 22C10(arrow). The arrowhead in A-C indicates theboundary between the notum and the wing andhighlights the fact that the ability of Sens to activatethe SOP-E and 22C10 is restricted to cells of thenotum. (D) Expression of elavrevealed by anti Elavstaining. (E) The same disc as in D showing theexpression of UAS GFP in green and that of Elav inred. The double staining reveals that although Sens isexpressed in a broad stripe in the notum, elavexpression is activated only in clusters of cells thatare located at positions of the proneural clusters(arrows). This observation suggests that other, locallyrestricted factors are in addition required to initiatethe expression of elav. As in the case of 22C10, Senscan activate expression of Hnt in all cells of the

notum where it is expressed (see F,G). (F) Expression of Hnt.(G) Expression of UAS GFP (green) and Hnt (red) in the samedisc as shown in F. Arrowheads in F and G indicate thewing/notum boundary and that the ability of Sens to activatehnt is again restricted to cells of the notum. (H) Resultssummary. Sens seems to be required to activate the expressionof hnt, and the neurone-specific genes 22C10and elavin thedeveloping SOP. This suggests that Sens coordinates thedevelopment and differentiation of the SOP. Furthermore, Sensseems to be required for the maintenance of the expression ofthe SOP-E and thus, the maintenance of high proneural geneactivity in the SOP. Hence, the results suggest that loss of Sensactivity is the cause for the observed arrest in development inSu(H)mutants.


expression of sensin the SOP. Su(H) could activate theexpression of sensby binding directly to its promoter. Such aNotch-independent activation of target genes by Su(H) hasrecently been discovered (Barolo et al., 2000; Klein et al.,2000). Alternatively, Su(H) could act as a repressor thatswitches off the expression of a factor that in turn represses theexpression of sens. Default repression by Su(H) in absence ofNotch-signalling seems to be a common mechanism to silenceexpression of Notch target genes in the absence of Notchactivity (Barolo et al., 2002). We have performed the followingexperiments to discriminate between the two possibilities.First, we made use of a construct in which a VP16 trans-activation domain is fused to Su(H) (Su(H)VP16) (Kidd et al.,1998). UAS Su(H)VP16acts exclusively as an transcriptional

activator that activates all Notchtarget genes in the embryo andwing, similar to activated forms of Notch (UAS Nintra) (Kiddet al., 1998; Klein et al., 2000). If Su(H) is a direct activatorof senstranscription, expression of UAS Su(H)VP16mightactivate sensin cells where it is expressed. However, if Su(H)represses the expression of a repressor, UAS Su(H)VP16should activate expression of this repressor and thus sensexpression should be lost. We observed that expression of UASSu(H)VP16with dppGal4 did not induce expression of sens inthe notum. By contrast, Su(H)VP16appears to suppress itsexpression in most parts of the notum (Fig. 9D,E). This resultfavours the possibility that the repressor function of Su(H) isrequired for sensexpression. However, we cannot rule out that,in this experiment, the loss of sens expression is caused

Fig. 7. Clonal analysis of Su(H)during SOPdevelopment of the machrochaete. (A-E) Anexample of a large clone that encompasses part ofthe PPA, tr2/APA+tr1 and DC clusters.(A) Overview. Expression of the SOP-E is shown inblue and that of Hnt in red. The clones are labelledby the absence of the GFP marker. The arrowheadindicates the region that is shown at highermagnification in B-E. (B) Expression of Hnt isdetected in single cells highlighted by the arrowheadand arrows. The cell highlighted by the arrowheadexpresses high levels of Hnt. (C) Channel revealingthe clone area by the absence of GFP. Arrowheadindicates a large GFP-positive cell that is located inthe mutant territory. (D) Expression of the SOP-E isdetected in groups of cells. (E) The pseudo-colourcomposite of the single channels shown in B-D.Green, GFP expression; red, Hnt expression; blue,SOP-E expression. The picture reveals that the Su(H)mutant cells of the DC cluster do express the SOP-Ebut not Hnt. Hnt is restricted to the green wild-typecell at the boundary of the clone. In the two otherclusters, only one mutant cell (arrows in B,E) weaklyexpress Hnt. This shows that the activity of Su(H) isrequired for the SOP to express Hnt. However, therequirement for Su(H)among cells of a proneuralcluster seems to vary, as indicated by the weakexpression of Hnt in a single mutant cell in the twocluster labelled by the arrows. Another importantobservation revealed by this figure is that wild-typecells can develop as SOP, even if located adjacent toSu(H)mutant cells. The arrowheads in B-E label awild-type cell that, as the SOP-E expression reveals,is part of the otherwise Su(H)mutant DC cluster.This cell is the only cell that expresses Hnt, indicating that it has chosen the SOP fate, although surrounded by mutant cells. Hence, the Su(H)mutant cells are not able to inhibit the cells from adopting the SOP fate. The expression of Hnt in one cells of each of the two mutant clusters(arrows) also suggest that cells at specific positions in the clusters have a higher inclination to adopt the SOP fate. (F,G) Cells at specificpositions within a proneural cluster are determined to adopt the SOP fate. (F) Expression of Hnt in cells of the DC cluster of the late third larvalinstar stage, where the posterior part consists of Su(H)mutant cells. The expression of SOP-E in this disc is shown in the insert. (G) Expressionof the SOP-E (blue), hnt (red) in the same disc shown in F. The Su(H)mutant area is labelled by the absence of GFP (green). Two hnt-positivecells are detectable at the aDC position. These cells have switched off the SOP-E, indicating that these cells are the second order precursors ofthe aDC. In the mutant territory a single hnt-expressing cell is detectable. This cell is located at the position of the pDC and still expresses theSOP-E. The pDC develops earlier than aDC during normal development. Hence, it appears that the Su(H)mutant SOP has arrested itsdevelopment at an early stage. For further information, see text. The phenotype of Su(H)mutant clones varies also among proneural clusters.An example is the SC cluster shown in H,I. (H) Expression of neurA101 in a Su(H)mutant clone that includes the SC cluster. Many, if not allcells of the cluster express this early marker. (I) By contrast, only a fraction of these cells also express Hnt, often at low levels. (J) Expression ofHnt in kuzES24mutant proneural clusters. Expression of Hnt is shown in red, the clones are revealed by the absence of the green GFP marker.Hnt is expressed in probably all mutant cells of the proneural clusters. This suggests that, as in the case of homozygous kuzmutants, the cells ofkuzmutant clusters do not arrest their development as SOP.

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indirectly through the suppression of the determination of theSOP by UAS Su(H)VP16.

To explore the possibility that Su(H) might act as a repressorfurther, we have analysed SOP development in a Hairless(H)mutant background. H is a crucial part of the repressor complexthat assembles around Su(H) to repress target gene expression(Klein et al., 2000; Furriols and Bray, 2000; Morel at al., 2001;Barolo et al., 2002). If Su(H) acts as a repressor, H should alsobe required. Hence, removal of Hfunction in Psnmutant wingimaginal discs should lead to the loss of sensexpression in asimilar way to that observed for Su(H)or Su(H); Psn double

mutants (see above). We found that this is indeed the case (Fig.9A-C). In Psn Hdouble mutant wing discs, expression of senswas dramatically reduced or lost in proneural clusters (Fig.9A,C). However, the cells of the clusters still express the SOP-E (Fig. 9B,C). This observation shows that the double mutantcells have not died, but fail to develop. It appears that the lossof H function in Psnmutants leads to an arrest of SOPdevelopment in a similar manner as in Su(H)and in Su(H); Psndouble mutants.

We further expressed a form of Su(H) that cannot bind Hbecause it lacks the H-binding domain (Furriols and Bray,


Fig. 8. (A-D) Cells of Su(H)mutant proneuralcluster express high levels of Dl. (A) Expression ofDl (red) and the SOP-E (blue) in a disc, bearingSu(H)mutant clones labelled by the absence of thegreen GFP marker. The arrow indicates the regionshown in higher magnification in B-D.(B) Expression of Dl. (C) Expression of the SOP-E.(D) Pseudo-colour composite of the disc shown inA-C, showing expression of Dl in red, of the SOP-Ein blue. The Su(H)mutant territory is revealed bythe absence of GFP. The picture reveals that the cellsthat express the SOP-E and thus belong to aproneural cluster, also express high levels of Dl.(E-J) Expression of the Gbe+Su(H) construct[Gbe+Su(H)], Dl and Hnt in the notum of a wild-type wing imaginal disc of the late third larval instarstage. (E) Expression of Dl (red) and Gbe+Su(H)(green) occurs in similar domains. Hnt expression(also in red) reveals some of the SOPs present at thistime. Arrow indicates the region of the pDC SOP.This region is shown at higher magnification in H-J.(F) Expression of Gbe+Su(H) in the notum of awild-type disc. (G) Expression of Dl in the samedisc as shown in F. The comparison of F and Gfurther reveals the similarity of the expressionpattern of Gbe+Su(H) and Dl. (H-J) Highermagnification of the region of the DC cluster,highlighted by the arrow in E. (H) Expression ofGbe+Su(H). (I) Expression of Dl and Hnt. At thisfocal plane expression of Hnt, but not Dl is visible.Dl is located on the apical side of the cell, which isout of focus. (J) Pseudo-colour image showing theexpression of nuclear GFP in green to reveal allcells, of Hnt/Dl in red and the Gbe+Su(H) in blue.Comparison with I,J reveals that the expression ofthe Gbe+Su(H) is elevated in cells that areimmediate neighbours of the SOP (arrowheads inH,J). The SOP itself does not express Gbe+Su(H).This suggests that the SOP sends a signal thatactivates the Notchpathway in its neighbours.(K-O) Su(H) mutant cells can activate the expression of Gbe+Su(H) in their wild-type neighbours. (K) Expression of Dl/hnt (red) and theGbe+Su(H) (blue) in a wing disc, bearing Su(H)mutant clones, labelled by the absence of the green GFP marker. The arrow indicates theregion shown at higher magnification in L-O. (L) Expression of Dl and Hnt. A group of cells express Dl in its membrane. One cell of this groupexpresses in addition Hnt, which is located in the nucleus (arrow). (M) Su(H)mutant area, revealed by the absence of GFP. Arrow indicates asingle wild-type cell that is located in the Su(H)mutant territory. (N) Expression of Gbe+Su(H). The expression is lost in the mutant territory,indicating that it is dependent on the activity of Su(H).Arrowhead indicates the stripe of elevated expression of the Gbe+Su(H) in the wild-typecells at the clone boundary. (O) Pseudo-colour composite picture of the region shown in L-N. Expression of Dl and Hnt is shown in red,expression of Gbe+Su(H) is shown in blue. The clone is labelled by the absence of GFP. The arrow highlights the Hnt-expressing cell, whichexpresses GFP and is therefore Su(H)positive. Comparison with N reveals that this cell does not express the Gbe+Su(H), although it issurrounded by Su(H)mutant cells that strongly express Dl. At the left clone boundary, where the GFP positive wild-type cells are adjacent tothe mutant cells (arrowhead) the expression of Gbe+Su(H) is elevated (revealed by the arrowheads in N,O). This suggests that the Su(H)mutantcells can activate the Notchpathway in their wild-type neighbours. Thus, Dl expressed in Su(H)mutant cells appears to be active. Nevertheless,the Notchpathway is not active in the wild-type SOP, suggesting that it is insensitive to Dl.


2000). Overexpression of this UAS Su(H)∆H construct byscaGal4 abolished hntexpression in Psnmutant wing imaginaldiscs (Fig. 9F). As in the H Psndouble mutants, the cells ofthe proneural clusters were present, as visualized by theexpression of UAS GFP driven by scaGal4. Thus, expressionof UAS Su(H)∆Hin cells of the proneural clusters appears tocause an arrest of SOP development as observed in Su(H)mutants. In summary, these results support the conclusion thatSu(H) requires H for its function in SOP development (see Fig.9H).

The arrest of SOP development in Su(H) mutants iscaused by one or more members of the E(spl)-complexSu(H) is directly required for the activation of the genes of theE(spl)-C (Bailey and Posakony, 1995; Hinz et al., 1994;Jennings et al., 1994; Lecourtois and Schweisguth, 1995;deCelis et al., 1996; Lai et al., 2000a; Lai et al., 2000b). Sevenof the genes of this complex encode bHLH repressor proteinsthat are required for the suppression of the SOP fate in the cellsof the proneural clusters during the process of lateral inhibition

(Knust et al., 1992; Nakao and Campos-Ortega, 1996) (Fig.10A). Four other genes of the complex encode members of thebeardedprotein family (Lai et al., 2000a; Lai et al., 2000b)(Fig. 10A). The data raise the possibility that the repressor ofSOP development is encoded by one or more genes of theE(spl)-C.

Initially, we tested this possibility by monitoring theexpression of sensand hntin Su(H)mutant wing imaginal discsthat carried a deletion for the whole E(spl)-C, Df(3R)E(spl)b32.2(gro+) (Fig. 10B,C). As a result these discs had onlyhalf the number of the genes of the complex. To our surprise,we found that in these Su(H)∆47; Df(3R)b32.2(gro+)/+ discs, thecells of all proneural clusters expressed high levels of sensandhnt (Fig. 10B,C), indicating that reducing the activity of thegenes of the E(spl)-Cby half is sufficient to remove the blockin SOP development in Su(H)mutant wing discs. Hence, oneor more genes of the complex seem to encode the repressorsthat prevent the expression of sensand the development of theSOPs in absence ofSu(H).

We then tried to determine, which of the genes of thecomplex encode the repressor. From previous work, it is known

Fig. 9. (A-C) Analysis of SOP development in thenotum of PsnC1 HE31 double mutant wing imaginaldiscs. Anterior is towards the left, ventral is to thebottom. Expression of Sens is revealed by antibodystaining. (A) Expression of Sens is strongly reducedor absent in the PsnC1 HE31 double mutant proneuralclusters. (B) The cells of the double mutant clustersstill express the SOP-E (arrows), indicating that theyare present. (C) The same disc as in A and B,showing expression of the SOP-E in green and ofSens in red. The analysis indicates that, in the doublemutant discs, SOP development arrests in a similarmanner as in Su(H)mutants. (D,E) Expression ofUAS Su(H)VP16with dppGal4 in wing imaginaldiscs of the late third larval instar stage.(D) Expression of sens. Expression of Sens is lost inmost SOPs of the notum. Compare with Fig. 2E fornormal expression of sens. The arrow indicatesectopic expression of Sens in the wing area, which isa result of the ectopic induction of the wing marginand bristles by UAS Su(H)VP16 (Klein et al., 2000;Furriols et al., 2000). (E) Expression of UAS GFP ina disc of the same genotype as in D, showing that theexpression of dppGal4 has expanded over most of thenotum. (F) A PsnC1 mutant wing imaginal disc whereUAS Su(H)∆His expressed with scaGal4. The discsis stained by anti Hnt antibody staining. Noexpression of Hnt is observed in the region of thenotum. (G) The same disc as in F showing expressionof Hnt in red and of GFP in green. The expression ofthe GFP marks the cells of the proneural clusters.This result shows that although the cells of theclusters are present, they fail to develop to the stageat which Hnt expression is initiated. Thus, theexpression of UAS Su(H)∆Hresult in an arrest ofSOP development in a similar manner as in Psn Hdouble mutants. (H) Summary of the results shown inthis picture. The results suggest that Su(H)suppresses the expression of a negative regulator ofsens. For this function, Su(H) requires the presenceof H. Recent evidence by Barolo et al. (Barolo et al., 2002) indicates that H acts as a bridge between Su(H) and its co-repressors Groucho (Gro)and CtBP. It is likely that Gro and CtBP are also part of the repressor complex required for the proper expression of sens.

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that only one of the bHlH proteins, E(spl)m8, and two of thebearded-like proteins, Μαand M4, are expressed in cells ofSu(H)mutant proneural clusters (Bailey and Posakony, 1995;deCelis et al., 1996; Lai et al., 2000a; Lai et al., 2000b) (Fig.10A). The other members of the complex are either notexpressed in the notal region of the wing imaginal disc, or theexpression is lost in Su(H)mutant cells (Bailey and Posakony,1995; deCelis et al., 1996; Lai et al., 2000a; Lai et al., 2000b).Hence, it is likely that persistent expression of one of thethree proteins expressed in cells of Su(H)mutant proneuralclusters causes the arrest of SOP development. However,forced expression of UAS m4 or UAS mαin cells of theproneural clusters with scaGal4 results in the formation ofsupernumerary bristles (Lai et al., 2000b). This suggeststhat these proteins stimulate rather than preventing SOPdevelopment.

By contrast, the expression of E(spl)m8is switched off inthe SOP during normal development (Nolo et al., 2000) (Fig.5D,E). Thus, Su(H) appears to be required to switch off theexpression of E(spl)m8. Furthermore, expression of UASE(spl)m8with scaGal4 prevents SOP development in Su(H)or

Psn mutant proneural clusters (Klein et al., 2000) (data notshown). These facts suggest that the abnormal persistentexpression of E(spl)m8in Su(H) mutant proneural clustersmight cause the arrest in SOP development. One prediction forthis hypothesis is that E(spl)m8should not be abnormallyexpressed in SOPs of mutants that are involved in Notchsignalling, but do not affect the formation of the Su(H)/Hrepressor complex. One such an example is the mutants of Psn.Thus, we monitored the expression of E(spl)m8in Psnmutantwing discs. In addition we looked at the expression of theE(spl)mβgene, which is expressed in a broader domain andseems to include all regions of the wing imaginal discs whereNotch is active (de Celis et al., 1996) (Fig. 10E). We found thatE(spl)m8is strongly expressed in cells of Psnmutant proneuralclusters in a similar way to expression in Su(H)mutants(compare Fig. 10D with Fig. 5C). Hence, it is unlikely that theabnormal expression of E(spl)m8alone causes the arrest inSOP development observed in Su(H) mutants. As expected,expression of E(spl)mβwas reduced in both mutants in asimilar manner and is not elevated in cells of the proneuralclusters (Fig. 10E,F; data not shown).

Altogether, the results indicate that therepressor of SOP development in Su(H)mutantsis encoded by one or more genes of the E(spl)-C. At the moment, it is difficult to determinewhether the repressor activity is encoded by onemember of the complex or by a combination ofE(spl)m8with one or both of the beaded-likeproteins.


Fig. 10.One or more members of the E(spl)-Cencode for the repressor activity that arrests SOPdevelopment in cells of Su(H)mutant proneuralclusters. (A) Organization of the E(spl)-C. Thediagram is based on previous work (Schrons et al.,1992; Bailey and Posakony, 1995; deCelis et al.,1996; Lai et al., 2000a; Lai et al., 2000b). Blue boxeslabel the genes that encode bearded-like genes; redlabels the genes that encode bHLH repressors. TheDf(3R)E(spl)b32.2uncovers all genes of the complex.The diagram reveals that six genes of the complex areexpressed in the notal region of the wing imaginaldisc. The expression of three of these genes [mα, m4and E(spl)m8] is upregulated in cells of Su(H)mutantproneural clusters. Overexpression of mα and m4causes neurogenic and E(spl)m8anti-neurogenicphenotypes. (B,C) Expression of Sens (B) and Hnt(C) in Su(H)mutant wing imaginal discs, which carryone copy of the deficiency Df(3R)E(spl)b32.2in theirgenome. The expression of the markers is revealed byantibody staining. (B,C) A reduction of the number ofthe genes of the E(spl)-Cby half is sufficient toregain expression of hntand sensin cells of Su(H)mutant proneural clusters. This suggests that therepressor function that is repressed by Su(H) isencoded by one or more members of the complex.(D) Expression of E(spl)m8-lacZin PsnC1 mutantwing imaginal discs. Arrows indicate the proneuralclusters. The cells of the clusters express high levelsof E(spl)m8-lacZ. (D,E) Expression of E(spl)mβ-CD2in wild-type (D), Su(H)∆47 mutant wing imaginaldiscs (E). Expression of E(spl)mβ-CD2 is reduced inthe Su(H)mutant wing imaginal discs. A similarreduction is observed in Psnmutant discs (not shown)


DISCUSSION

The Notch pathway is one of the fundamental signallingsystems that are conserved throughout the animal kingdom.Therefore, it is important to gain information about thefunction of its core members. We here have identified a newrole of Su(H) during a fundamental differentiation process ofone Drosophilacell type, the development of the SOP of themechanosensory bristles. Our results suggest that Su(H) isrequired to promote SOP development. This is based on thefact that most cells of proneural clusters in the notum that lackSu(H) function do not express SOP markers such as Sens, Hntand partially neurA101-lacZ. Loss of neurA101-lacZ expressionin some proneural clusters of Su(H) mutant discs has beenobserved before (Schweisguth and Posakony, 1992). This losshas been attributed to a ‘general sickness’ of the mutant discs,as the lack of neurA101-lacZ expression was only observed inthe late developing proneural clusters (Schweisguth andPosakony, 1992). Our data argue against such an explanation:Psnmutant wing imaginal discs exhibit a stronger neurogenicphenotype than do Su(H)mutants. Similar to Su(H)mutants,homozygous Psn mutant animals also die during the earlypupal phase. Nevertheless, the cells of the proneural clustersof these mutants express all tested markers, indicating that SOPdevelopment is not affected. The same is true for kuzmutants,whose mutant phenotype is comparable with that of Su(H)mutants. Hence, general sickness of the wing imaginal disccells is not likely to explain the arrest of SOP development inSu(H)mutants.

A role of Su(H) in development of the SOP is surprising,because it is a core element of the Notchsignalling pathwayand the activity of this pathway is required to prevent SOPdevelopment in cells of the proneural clusters (Schweisguthand Posakony, 1992; de Celis et al., 1996; Heitzler et al., 1996;Nakao and Campos-Ortega, 1996; Klein et al., 2000).Importantly, in this new role, Su(H) seems to functionindependently of the Notchsignalling pathway. This isindicated by the finding that the Su(H) mutant phenotype isepistatic over that of Psnmutants.

The data presented here indicate that Su(H) appears to berequired to suppress the activity of one or more members ofthe E(spl)-C, that in turn suppress the expression of genessuch as hntand sens. This conclusion is based on: (1) thefailure of Su(H)VP16to activate sens, (2) the fact that Psn Hdouble mutants display a similar loss or reduction of sensexpression as Su(H)and Su(H); Psndouble mutants and (3)the fact that expression of a Su(H)construct that is unableto bind H (UAS Su(H)∆H) leads to an arrest of SOPdevelopment in Psnmutant wing imaginal discs. Severalreports show that H is involved in Su(H)-related suppressionof gene expression in the absence of Notch signalling(Furriols and Bray, 2001; Klein et al., 2000; Morel andSchweisguth, 2001; Barolo et al., 2002). Recently, it has beenshown that H acts as a bridge between Su(H) and the generalco-repressors CtBP and Gro (Barolo et al., 2002; Morel andSchweisguth, 2001). It is therefore likely, that thisSu(H)/H/Gro/dCtBP complex mediates the repressor functionrequired during SOP development.

Repression by Su(H) is not strictly required in all proneuralclusters to allow expression of sens and other late SOPmarkers. Examples are the clusters in the wing region, such as

the clusters of the dorsal radius. However, even in theseclusters, sensand hnt are not expressed in all cells that expressearly markers, such as neurA101 (e.g. compare wing imaginaldiscs in Fig. 2C with 2H,M; clonal data not shown). Therefore,it appears that the activity of Su(H) promotes SOPdevelopment also in these clusters. The clusters of the dorsalradius give rise to other types of sense organs, such ascampaniforme sensilla, and it is possible that there are differentrequirements for the activity of Su(H)for the development ofthe different types of sense organs

The repressor activity requires the activity of one ormore genes of the E(spl) complex We show that the removal of one copy of the E(spl)-C isalready sufficient to relieve the block in SOP development inSu(H)mutants, indicating that the arrest is probably caused bythe abnormal expression of one or more members of thecomplex. Although the complex encodes for several well-characterized repressors of neural development, we were notable to pinpoint the repressor function to any particular gene.Many studies by various groups have studied the regulation ofthe genes of the E(spl)-C(Bailey and Posakony, 1995; deCeliset al., 1996; Lai et al., 2000a; Lai et al., 2000b). From thesestudies, it is clear that only three genes of the complex areexpressed in the cells of Su(H)mutant proneural clusters. Allother members are either not expressed in the notal region ofthe wing imaginal disc or their expression is lost in the mutantcells. Previous studies have shown that both bearded-likeproteins that are expressed in Su(H)mutant proneural clusterspromote SOP development (Lai et al., 2000a; Lai et al., 2000b).Hence, it is unlikely that the abnormal expression of thesegenes causes the observed arrest in SOP development. To oursurprise, we found that the strongest candidate, the bHLHrepressor encoded by E(spl)m8, is also abnormally expressedin Psn mutants, where SOP development proceeds and theSu(H)/H-containing complex is intact. The observation isinteresting, because it suggests that the activity of the wholeNotch pathway is required to switch off the expression ofE(spl)m8 in the SOP, but it also indicates that abnormalexpression of the gene cannot be the reason for the arrest inSOP development in Su(H)mutant cells. Thus, the repressoractivity might not be encoded by a specific member of theE(spl)-C.

One possibility is that the combination of the threeabnormally expressed genes of the complex generates therepressing activity. An alternative is that Su(H) controls theexpression of other genes that act in combination with theupregulated members of the complex to suppress SOPdevelopment. Another possibility is that more genes of thecomplex are de-repressed in Su(H)mutants at a level notdetectable by the currently available methods. In this scenario,the weak expression of several bHLH-encoding genes will sumup to a level of repressor activity sufficient to stop SOPdevelopment. Using currently available techniques, it is verydifficult to discriminate between these possibilities.

The stability of the Su(H) proteinWe found that in Su(H)mutant cell clones induced during thefirst larval instar stage, hntis expressed in a fraction of cellsof specific proneural clusters, such as the scutellar cluster, butabsent or strongly reduced in other clusters. We further found

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that in Su(H)mutant wing imaginal discs, expression of sensand hnt is lost or stronger reduced than in mutant cell clonesinduced during the first larval instar.

A high stability of the Su(H) protein is a possibleexplanation for this discrepancy. In favour of this explanationis the observation that the maternal component of Su(H)issufficient to allow the development of homozygous animalsuntil early pupal stages (Lecourtois and Schweisguth, 1995).Furthermore, we found that vestigial (vg), a target gene of theNotch/Su(H)pathway during wing development (Kim et al.,1996) is expressed in Su(H)mutant wing imaginal disc ofthe early third larval instar stage (S.K. and T.K., unpublished).This indicates the presence of Su(H)activity at this stage. Thisresidual activity of Su(H) must be provided by the maternalcomponent. Both observations suggest that the Su(H) proteinis degraded slowly and thus persists in mutant cells for a longtime. It is therefore likely that Su(H) mutant cells, induced atthe first larval instar, contain residual amount of Su(H). Thisresidual amount of Su(H) might be sufficient to activateexpression of late SOP marker in cells of certain proneuralclusters.

An alternative explanation for the milder phenotypeobserved in the Su(H)mutant clones is that it requires time toaccumulate a sufficient level of activity of the repressor(s) ofthe E(spl)-C to stop SOP development. Hence, in the case ofthe clonal analysis, the loss of Su(H)activity occurs later thanin homozygous mutant wing imaginal discs and lower levelsof repressor activity would be present in cells of the proneuralclusters of the machrochaete.

Determination of the sensory organ precursor cellThe development of the machrochaete is a paradigm for theassignment of different fates to initially equivalent cells.Proneural genes are expressed in clusters of cells and conferon these cells the potential to become SOPs. Carefulexamination has revealed that the SOPs of the machrochaetearise at the same positions within the proneural cluster (Cubasand Modollel, 1992), indicating that the selection of the SOPis not random. It is thought that other factors, such asExtramachrochaete, Pannier and Wingless introduce smalldifferences in proneural activity that favour cells at specificpositions within the cluster to become the SOP (Cubas andModollel, 1991) (reviewed by Simpson, 1997). These smalldifferences in proneural activity are enhanced through theactivity of the Notchpathway: a cell with high proneuralactivity expresses high levels of Dl and is therefore potent toinhibit its neighbours (Heitzler and Simpson, 1991; Hinz etal., 1994). Cells with a high activity of Notch have lessproneural activity and Dl. Thus, they are less potent to inhibitits neighbours (reviewed by Simpson, 1997). In this scenario,the Notch pathway is required to amplify initially smalldifferences in proneural activity in cells among a cluster. Thisamplification eventually results in the accumulation of highlevels of activity in the SOP and loss of activity in theneighbours. In this way, the pathway acts to resolve a crudepre-pattern to the level of a single cell. According to thismodel, cells defective in Notch signal reception should bevery potent in lateral inhibition. However, we here found theopposite: A cell that is located at the position where the SOParises is able to adopt the SOP fate, even if surrounded bySu(H) mutant cells. It can do so despite the fact that the

mutant neighbours accumulate high levels of proneuralactivity (indicated by the expression of the SOP-E), aswell as Dl. We here show that Dl, expressed in the Su(H)mutant cells at high level, is active and can activate Notchsignalling in wild-type cells with the exception of the SOP.Thus, although the SOP was adjacent to cells with anextremely high potency for lateral inhibition during the wholelive of a proneural cluster, it has succeeded in adopting theSOP fate. It appears that the SOP is determined by positionalcues that are much more precise than anticipated. Thesecues render the cell at the correct position in the clusterinsensitive to lateral inhibition. This suggests that smalldifferences in proneural activity are not the crucial biasimposed on cells within a proneural cluster and that lateralinhibition might not be required to resolve a crude pre-pattern.

Nevertheless, the big clusters of SOPs observed in otherneurogenic mutants, indicate that the Notch pathway has afunction in preventing the SOP fate in all cells of a proneuralcluster and also in cells that are located further away from theSOP.

Furthermore, we observed a ring of high expression ofthe Gbe+Su(H) construct around the SOP during normaldevelopment, suggesting that the SOP sends a signal thatactivates the Notchpathway in its immediate neighbours. Thislateral inhibition is relatively late, as we observe it only aroundSOPs that already express hnt. It also occurs only in the cellsadjacent to the SOP.

Altogether, these observations suggest that the Notchpathway might have two separable functions during SOPdevelopment. During early phases of a proneural cluster, theactivity of the pathway keeps the cells of the cluster undecided,perhaps by mutual repression. Owing to positional cues, onecell becomes insensitive to the inhibitory signal and adoptsthe SOP fate. Subsequently the SOP inhibits its immediateneighbours by sending an inhibitory signal through Dl. Asimilar function of the Notchpathway has recently beenproposed for the segregation of the embryonic neuroblasts ofDrosophila(Seugnet et al., 1997).

Schweisguth (Schweisguth, 1995) reported that, duringmichrochaete development, cells can adopt the SOP fate, evenif they are located adjacent to Su(H)mutant cells, suggestingthat, also during development of this bristle type, the mutantcells cannot inhibit its normal neighbours. It was suggested thatthe Su(H)mutant cells might loose contact with neighbouringwild-type cells and, because Dl/Notch signalling depends oncell contact, this could prevent the activation of the Notchpathway in the wild-type cells. Our data suggest that this is notthe case: the mutant cells can activate the pathway in adjacentwild-type cells. Hence, the results of Schweisguth(Schweisguth, 1995) suggest that also during michrochaetedevelopment positional cues might be important for thedetermination of the SOP.

We thank S. Bray, S. Campuzano, J. Campos-Ortega, J. Modolell,A. Martinez-Arias, H. Bellen, N. Baker, G. Struhl, M. Fortini. C.Klaembt and F. Schweisguth for sending stocks and reagents. We alsothank R. Wilson, M. Kaspar and K. Reiners for critical comments onthe manuscript. This work was supported by the DeutscheForschungsgemeinschaft through SFB 572 and the ARC program ofthe DAAD.



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