Notch signaling represses the glial fate in fly PNS

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1 Development 128, (2001) Printed in Great Britain The Company of Biologists Limited 2001 DEV Notch signaling represses the glial fate in fly PNS Véronique Van De Bor and Angela Giangrande* Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/IGBMC/ULP/INSERM BP ILLKIRCH, c.u. de Strasbourg, France *Author for correspondence ( Accepted 19 January; published on WWW 22 March 2001 SUMMARY By using gain-of-function mutations it has been proposed that vertebrate Notch promotes the glial fate. We show in vivo that glial cells are produced at the expense of neurons in the peripheral nervous system of flies lacking Notch and that constitutively activated Notch produces the opposite phenotype. Notch acts as a genetic switch between neuronal and glial fates by negatively regulating glial cell deficient/ glial cells missing, the gene required in the glial precursor to induce gliogenesis. Moreover, Notch represses neurogenesis or gliogenesis, depending on the sensory organ type. Numb, which is asymmetrically localized in the multipotent cell that produces the glial precursor, induces glial cells at the expense of neurons. Thus, a cellautonomous mechanism inhibits Notch signaling. Key words: Notch, Glia, Sensory organ, glide/gcm, PNS, Fate choice, Drosophila INTRODUCTION The Notch (N) protein is involved in many developmental pathways that require cell communication (for a review, see Artavanis-Tsakonas et al., 1999 and references therein). In flies, N controls cell communication within clusters of cells that are competent to acquire the neural fate (proneural clusters), so that one cell becomes neural precursor while all the others adopt the epidermal fate (see Vervoort et al., 1997a; Vervoort et al., 1997b; Modolell and Campuzano, 1998; Baker, 2000 for reviews). This process allows the delamination of neural stem cells (neuroblasts or NBs) in the central nervous system (CNS) and the development of the sensory organ precursors (SOPs) in the peripheral nervous system (PNS; Doe and Goodman, 1985; Cubas et al., 1991; Heitzler and Simpson, 1991; Brand et al., 1993; Skeath and Carroll, 1993; Jan and Jan, 1998). Thus, lack of N results in CNS and PNS hyperplasia, whereas N gain-of-function mutations prevent neurogenesis (Hartenstein and Posakony, 1990; Lieber et al., 1993; Rebay et al., 1993). At later developmental stages, when neural precursors have differentiated, N is also required to promote fate choices within the lineage (Hartenstein and Posakony, 1990; Parks and Muskavitch, 1993; Spana and Doe, 1996). Finally, injection of constitutively active N into Xenopus prevents the formation of neurons, as expected if the N pathway inhibits commitment to a neural fate (Coffman et al., 1993; Dorsky et al., 1995). Thus, the molecular cues that underlie the establishment of different cell fates in the nervous system seem to be conserved throughout evolution. An important issue is to determine the role of cell communication in the differentiation of the second type of cells of the nervous system, glial cells, which, like neurons, arise from multipotent CNS and PNS precursors (Leber et al., 1990; Williams et al., 1991; Davis and Temple, 1994; Bossing et al., 1996; Schimd et al., 1999; Schmidt et al., 1997; Akiyama-Oda et al., 1999; Bernardoni et al., 1999; Gho et al., 1999; Reddy and Rodrigues, 1999b; Van De Bor et al., 2000). The effects of N mutations on glial differentiation have recently been explored in vivo and in vitro by using gain-offunction mutations that activate the N pathway constitutively (see Wang and Barres, 2000 for a review). These studies indicate that N regulates most types of glial cells in vertebrate peripheral and central nervous systems: it represses oligodendrocytes generation in vitro (Wang et al., 1998), whereas it promotes the differentiation of peripheral, radial and retinal Müller glia (Furukawa et al., 2000; Gaiano et al., 2000; Morrison et al., 2000). Two important issues however, limit our understanding of the role of N in gliogenesis: (1) Mice deficient in N signaling die before gliogenesis begins; (2) gain-of-function studies do not provide absolute evidence as to the role of a gene in vivo. We have used Drosophila melanogaster to analyze the role of N in gliogenesis. We have previously shown that two types of sensory organs exist, gliogenic and non-gliogenic, the first type producing peripheral glial cells (Van De Bor et al., 2000). The division of a gliogenic SOP generates four cells (neuron, sheath, socket and shaft cells) and a glial precursor (GP) that proliferates and produces six glial cells in average (Fig. 1). The GP, which arises through the asymmetric division of the IIb secondary order precursor, expresses glial cell deficient/glial cell missing (glide/gcm) (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996; Bernardoni et al., 1997; Bernardoni et al., 1998; Gho et al., 1999; Reddy and Rodrigues, 1999b; Van De Bor et al., 2000) and requires it in order to produce glial cells (Van De Bor et al., 2000). For the sake of simplicity, this gene will be referred to as glide, for the rest of the text.

2 1382 V. Van De Bor and A. Giangrande The division of the other IIb daughter, also called IIIb, produces the neuron and the sheath cell. By analyzing developing individual lineages, we have found that sensory organs lacking N are devoid of neurons and contain additional glial cells. The induction of constitutively active N produces sensory organs with the reverse phenotype. Thus, N regulates the choice between neuronal and glial fates and thereby represses gliogenesis. We also show that N acts by repressing the expression of glide. Finally, the cell fate determinant Numb (Uemura et al., 1989; Rhyu et al., 1994; Guo et al., 1996; Frise et al., 1996; Wang et al., 1997), which inhibits N activity, activates the glial fate. Our data also put the bases for the identification of other players in the N cascade that represses gliogenesis. Finally, the analysis of gliogenic and non-gliogenic sensory organs shows that the readout of the N signaling depends on the developmental context, namely the type of sensory organ. MATERIALS AND METHODS Fly strains The wild-type strain was Sevelen. N loss- and gain-of-function phenotypes were obtained using y, N ts1 (Shellenbarger and Mohler, 1975), and puas-n intra [w 1118 ; P(UAS-N intra w + )/TM3,Sb,Ser] (Seugnet et al., 1997) strains, respectively. y, N ts1 strain was kept at 18 C. Development at 18 C was estimated to be twice as long as at 25 C. Numb was overexpressed by using the yw, hs-numb 2.4 C strain provided by L. Y. Jan and Y. N. Jan (Rhyu et al., 1994). Suppressor of hairless (Su(H)) was overexpressed using (independently) the w 1118 ; P(hs-Su(H)-4 w + ), w 1118 ; P(hs-Su(H)-8 w + ) and w 1118 ; P(UAS- Su(H)-J2 w + ) strains provided by J. Posakony (Schweisguth and Posakony, 1994). Enhancer of split m8 (E(spl)m8) gene was overexpressed using the w 1118 ;+; P(UAS-m8 w + ) strain provided by C. Delidakis (Ligoxygakis et al., 1999). The hs-gal4 (w; P(hs-Gal4 w + )) strain was used to drive ubiquitous expression. Immunohistochemistry Fixation, dissection and antibody incubation were as described (Giangrande et al., 1993). Wings were mounted in Vectashield medium (Vector). Staging of third instar larvae was performed as described (Maroni and Stamey, 1983). The following primary antibodies were used: rat anti-elav (1:2000) (provided by G. Rubin), rabbit anti-repo (1:8000) (provided by A. Travers), rat anti-glide (1:1000), mouse anti-cut (1:100) (DSHB), rabbit anti-pros (1:500) (provided by H. Vaessin) rabbit anti-ph3 (1:50000) (Upstate Biotechnology). Secondary antibodies coupled with Oregon Green (Molecular Probes), Cy3, Cy5 and FITC (Jackson) were used at 1:400. Heat shock induction of loss- and gain-of-function phenotypes N intra, Su(H) and E(spl)m8 ubiquitous expression were obtained by crossing the w; P(hs-Gal4 w + ) flies with w 1118 ; P(UAS-N intra w + )/TM3,Sb,Ser, w 1118 ; P(UAS-Su(H)-J2 w + ) and w 1118 ;+; P(UASm8 w + ) lines, respectively. Prepupae or larvae coming out of these crosses were collected and heat shocked for 1 hour at 38 C at different stages around sensory organ birth. Wings were dissected at 24 hours after puparium formation (APF) and labeled as above. Ectopic expression of Su(H) was also induced using the w 1118 ; P(hs-Su(H)-4 w + ) or the w 1118 ; P(hs-Su(H)-8 w + ) strains in the same conditions as above. To induce the loss-of-function phenotype, y, N ts1 larvae and pupae were collected and incubated at 30 C for a period of 6 to 12 hours, which covers sensory organ development. To ubiquitously express Numb, hs-numb white pupae were collected and heat shocked at 6 hours APF for 2 hours at 37 C. RESULTS Lack of N induces glial differentiation in the adult fly peripheral nervous system N is involved in all the fate decisions taken during the development of mechanosensory organs composed of four cells (Fig. 1) (Hartenstein and Posakony, 1990; Parks and Muskavitch, 1993; Guo et al., 1996). Early and transient lack of N in the division of the SOP cell, which normally produces the IIa and the IIb secondary order precursors, leads to IIb duplication and thereby to sensory organs that contain two neurons. Lack of N at later stages leads to fate transformations in the progenies of the IIa and IIb cells (effects on IIa: tormogen to trichogen transformation; effects on IIb: thecogen to neuron transformation). The continuous absence of N results in sensory organs composed of four neurons. Opposite results are obtained upon constitutive activation of the N pathway (Fig. 1). More recent studies have shown that peripheral glia in the notum and in the wing also arise from sensory organs (Gho et al., 1999; Reddy and Rodrigues, 1999b; Van De Bor et al., 2000). Indeed, two types of mechanosensory organs, gliogenic and non-gliogenic, exist in the wing, in addition to the classic sensory organ composed of four cells (Van De Bor et al., 2000). In gliogenic mechanosensory organs, the SOP divides and produces IIa and IIb. IIa produces the tormogen and the trichogen whereas IIb generates a IIIb precursor and a cell that transiently expresses glide. This cell, which differentiates into a glial precursor cell or GP, starts to proliferate and migrate along the axons as it ceases to express glide (Van De Bor et al., 2000). The GP division, which takes place well after the other divisions of the sensory organ lineage, is symmetric and produces two cells that divide again. At the end of wing development, six glial cells originate on average from the GP. The IIIb precursor divides and produces the neuron and the thecogen cell. In the non-gliogenic sensory organ, IIb produces a sheath cell and a IIIb precursor that produces two neurons. In the case of taste receptors present on the wing margin, additional divisions produce a multiply innervated gliogenic sensory organ (Ray et al., 1993; Nottebohm et al., 1994; Van de Bor et al., 2000 and data not shown). These data call for a new analysis of the role of N in the sensory organ development and glial differentiation. In order to determine the role of N in gliogenesis, we used a thermosensitive allele, N ts1, and challenged animals with different heat shock regimens. The model tissue was the wing, which contains glial cells arising from mechano- and chemosensory organs located on L1 (anterior margin) and L3 veins (Giangrande et al., 1993; Giangrande, 1994; Giangrande, 1995; Van De Bor et al., 2000). The reverse polarity (repo) gene, a target of glide that codes for an homeobox containing protein (Campbell et al., 1994; Xiong et al., 1994; Halter et al., 1995), is expressed in the GP as well as in its progeny and thus was used as a glial-specific marker (Fig. 2). The initial regimen consisted of a heat pulse between 1 and 13 hours after puparium formation (APF), a period during which L1 and L3 SOPs are still forming and dividing (Huang et al., 1991; Fig. 3B). This shock results in a dramatic increase of glial cells compared with wild-type wings (Fig. 2A,C). We quantified the effects of the mutation by comparing the number of Repopositive cells on L1 and L3 nerves with those observed in a wild-type wing of a similar stage that had undergone the same

3 Notch represses the glial fate in fly PNS 1383 heat-shock treatment. At 24 hours APF, N ts1 heat-shocked wings contain some 30 glial cells on L3, compared with the 11 glial cells found in the wild type. On L1, which normally carries in average seventy-four glial cells, the estimation of the glial cell number in N ts1 wings was more difficult, owing to the accumulation of labeled nuclei; however, at least 300 cells were counted. We can exclude that the increase in glial cell number is due to the presence of ectopic, N loss-of-function induced, sensory organs for the following reason. Sensory organ lineages can be recognized by using the Cut antibody (Blochlinger et al., 1990). Counting of the clusters of cutpositive cells on L1 and L3 veins revealed that most treated wings contained the normal number of sensory organs (Fig. 2 B,D; data not shown). Thus, the N ts1 mutation results in average in a three- to fourfold excess of glial cells, indicating that the N pathway controls glial differentiation. N triggers the fate choice between neurons and glial cells in the sensory organ lineage The supernumerary glial cells observed in treated N ts1 wings might originate through two processes: overproliferation of the glial precursor or fate transformation within the sensory organ lineage. Indeed, by 24 hours APF, the stage at which wings were analyzed, glial migration and proliferation have already started. To discriminate between the two possibilities, we analyzed wings at early stages, when the GP is still associated with the sensory organ cluster and has not started to proliferate, and asked how many Repo-positive cells are associated with a sensory organ. N ts1 and wild-type animals were submitted to a 1-10 hour APF shock and double labeled with anti-repo and anti-cut to recognize glial and sensory organ cells, respectively. One Repo-positive cell is normally present in each chemosensory organ, whereas clusters of two to five cells express repo in mutant sensory organs. The cluster contains the same number of cells in wild-type and in mutants wings, confirming that supernumerary glial cells do not arise through overproliferation of the glial precursor (Fig. 2 B,D). Thus, the phenotype observed at late stages is due to a fate transformation within the sensory organ lineage. We then asked whether supernumerary Repo-positive cells behave like GP cells and proliferate. Heat-shocked wings were double labeled with anti-repo and anti-ph3, an antibody that recognizes the phosphorylated form of histone H3 present in the chromatin of dividing cells (Su et al., 1998). Mutant wings contain an excess of dividing Repo-positive cells, compared with wild-type wings, indicating that proliferation increases in N ts1 wings (data not shown). Thus, lack of N triggers a complete cell fate transformation. N affects several decisions in the gliogenic lineage To determine the precise role of N, for the rest of our analyses we focused on the development of the three gliogenic mechanosensory organs on L3, the developmental profile of which has already been established (Murray et al., 1984; Hartenstein and Posakony, 1989; Huang et al., 1991; Blair et al., 1992; Van De Bor et al., 2000). The precursors of these sensory organs, also called late sensory organs, single out at around 6 hours APF and reach the five cell stage by 10 hours APF (Fig. 3B). Thus, a 12 hour shock at 1-13 hours APF eliminates N throughout the early steps of sensory organ development. More than 80% of late sensory organs displayed an increase of glial cell number (Figs 3A, 4). The most common phenotype, observed in 40% of the cases, was the presence of sensory organs composed of six cells, all expressing Repo. At a minor frequency, we also observed less extreme phenotypes such as sensory organs composed of five to six cells, and containing two to five Repo-positive cells (Figs 3, 4). These phenotypes suggest that fate transformations have taken place during the different divisions of the sensory organ lineage. Two Repo-positive cells are most likely generated by the duplication of the IIb cell, as also suggested by the presence of two neurons in those sensory organs (Fig. 4C). The production of three glial cells is most likely due to the fact that the IIIb cell, which normally divides and produces the neuron and the sheath cell, requires N. In its absence, IIIb undergoes a fate transformation into GP but retains its proliferative potentials and thereby produces two Repo-positive cells. No neurons are present in these sensory organs (Fig. 4D). Six glial cells are most probably generated by sensory organs in which several divisions have been affected so that the two SOP daughters have adopted the IIb fate and the two IIIb daughters have adopted the GP fate (Figs 3, 4B). In no case did we detect Fig. 1. Role of Notch in PNS cell fate specification. (A) A classical sensory organ lineage. SOP indicates the sensory organ precursor cell; IIb, IIa and IIIb represent the PIIb, PIIa and PIIIb secondary precursor cells, respectively; To, Tr, n and Th indicate the tormogen, trichogen, neuron and thecogen cell, respectively. From left to right: wild-type classic lineage (WT), N ts1 sensory organ composed of four neurons, and N intra sensory organ composed of four tormogen cells. Double arrows indicate a role of Notch in the cell fate choices. (B) Shows the gliogenic sensory organ lineage; GP indicates the glial precursor cell.

4 1384 V. Van De Bor and A. Giangrande composed of six cells, five of which express glide. These cells are indistinguishable, in that they all express glide at similar levels. By analyzing mutant sensory organs at different stages during development, we observed that one cell expresses glide at the same time as in the wild type and most likely corresponds to the normal GP. Additional glide-expressing cells are only detectable at later stages (data not shown), owing to cell fate reprogramming. These data support the hypothesis that the N pathway represses glial differentiation by regulating glide expression in the sensory organ lineage. Thus, the decision of expressing glide is regulated by cell-cell communication. Fig. 2. Lack of Notch induces glial differentiation in the fly PNS. Heat shocked, wild-type (WT; A,B) and N ts1 (C,D) wings labeled with anti-cut (green) and the glial specific marker anti-repo (red). In this and all the subsequent figures, anterior is towards the top and distal towards the right. (A,C) 24 hours APF wings. Compared with wild-type, the N ts1 wing displays an increased number of glial nuclei along L1 and L3 veins (L1 and L3). (B,D) High magnification of wing margins at 10 hours APF. In the wild-type, differentiating dorsal (d) and ventral (v) chemosensory organs contain one Repopositive cell (arrowheads) per cluster, whereas in N ts1, chemosensory organs contain two to four Repo-positive cells per cluster. Broken lines indicate a ventral chemosensory cluster. Scale bars: 51 µm in A,C; 8 µm in B,D. sensory organs composed of four cells and containing only Repo-positive cells (data not shown). To determine the time window during which N is required, short heat pulses were provided at different developmental stages. Shock at 0-6 hours APF mostly led to the transformation of IIa into IIb (Fig. 3). The second most frequent phenotype was the presence of three Repo-positive cells. In the case of shock at 6-12 hours APF, a high percentage of sensory organs were not affected; however, a higher variety of phenotypes could be observed compared with the early shock. For example, some sensory organs contained six Repopositive cells. Shock at 3-9 hours APF led to phenotypes that were intermediate to those observed with early (0-6 hours APF) and late (6-12 hours APF) shocks. However, a 9-15 hour APF shock produced sensory organs composed of five cells and containing one glial cell and two neurons (Fig. 4E). Thus, lack of N at this stage triggers a sheath-to-neuron transformation in the daughters of IIIb. Finally, no effects were observed when a hour APF shock was provided (data not shown). These results indicate that N is required throughout the development of gliogenic sensory organs. Lack of N induces the expression of gcm The above results suggest that N acts in same cascade as glide, the glial-promoting factor expressed in the GP. To understand the mode of action of N, we asked whether glide expression was affected in sensory organs lacking N. Gliogenic sensory organs were submitted to 1-13 hour APF heat shocks and double labeled with anti-glide and anti-cut. Two to five glideexpressing cells could be detected by the end of sensory organ development. Fig. 5 shows an example of sensory organ Constitutively active N represses glial differentiation Activation of the N signaling by Delta triggers N proteolysis and its translocation into the nucleus (Lieber et al., 1993; Lecourtois and Schweisguth, 1998; Struhl and Adachi, 1998; Struhl and Greenwald, 1999). Here N interacts with the Su(H) protein and regulates the expression of target genes (Schweisguth and Posakony, 1992; Schweisguth and Posakony, 1994; Fortini and Artavanis-Tsakonas, 1994; Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995). A truncated N molecule that does not contain the extracellular ligand binding domain (N intra ) produces a constitutively active receptor (Lieber et al., 1993; Struhl et al., 1993; Seugnet et al., 1997). Expression of this mutated N molecule produces a gainof-function phenotype of neural hypoplasia, similar to that observed by overexpressing N, owing to a cell fate conversion reciprocal to that observed in N null mutants (Hartenstein and Posakony, 1990; Lieber et al., 1993; Rebay et al., 1993). We therefore assessed the effects of an excess of N on glial differentiation by using a strain carrying the hs-gal4 and the UAS-N intra transgenes. Gliogenic and non gliogenic sensory organs alternate along the L3 nerve. From distal to proximal the sequence is gliogenic (L3-3), non gliogenic (L3-2), gliogenic (L3-1), non gliogenic (ACV), gliogenic (L3-v) (Fig. 6; data not shown). Induction of a constitutively active form of N produced wings lacking glial cells (Fig. 6; data not shown). The strongest effects were seen upon N induction at the time of SOP birth. Upon this regimen all L3 glial cells are missing, some L1 glial cells are still present (Fig. 6A,B). The most likely explanation for this result is the presence of two populations of L1 glial cells arising at different stages. Indeed, some L1 glial cells arise from chemosensory organs, which differentiate at the same time as the L3 gliogenic sensory organs, some arise from mechanosensory organs, which differentiate at later stages, and are most probably unaffected by the heat shock shown in Fig. 6. When analyzed simultaneously with markers specific for the sheath cell (Prospero (Pros); Spana and Doe, 1995), for the neuron (Elav; Robinow and White, 1988) and for the whole sensory organ (Cut), several defects were observed. The most frequent was a IIb to IIa transformation that resulted in a four cell-containing sensory organ devoid of neuron and sheath cell (Fig. 6B). In other cases, the GP was transformed into a IIIb, producing a six cell-containing sensory organ composed of two neurons and two sheath cells (Fig. 6C). Finally, a neuron-tosheath transformation was also observed in six cell-containing sensory organs composed of four sheath cells (Fig. 6D). Thus, transient expression of a constitutively active N molecule completely represses the glial fate in the fly PNS.

5 % of sensory organ A Notch represses the glial fate in fly PNS B 1385 SOP 6h IIa IIb 7h 40 IIIb GP 8h To Tr 9h 10 Th n 10h 0 1 WT HS 0-6h HS 3-9h HS 6-12h HS 1-13h number of repo + cells / sensory organ GP GP 20h Fig. 3. Effects of different heat shock regimens on gliogenesis in N ts1 wings. (A) Histogram representing the phenotypes observed in N ts1 gliogenic sensory organs after different heat-shock treatments: 0-6, 3-9, 6-12 and 1-13 hours APF, which are indicated along the horizontal axis. For each regimen of heat shock (HS), we analyzed at least 40 gliogenic sensory organs. Wings were analyzed at 15 hours APF. The first column shows that in wild-type flies (WT), regardless of whether they were heat shocked or not, we observed one Repo-positive cell per gliogenic sensory organ in all cases. In N ts1 wings, heat shocks produce mutant sensory organs composed of five to six cells and containing one to six Repo-positive cells. Each heat shock regimen produces a panoply of phenotypes. The strength and the frequency of such phenotypes depend on the stage at which the shock was given. Color coding from white to black indicates the number of Repo-positive cells (one, white; six, black) observed in the mutant sensory organs. Vertical columns indicate the percentage at which the different phenotypes were observed using a given shock. (B) A wild-type gliogenic lineage during pupal development. h indicates the number of hours APF; SOP indicates the sensory organ precursor cell; IIb, IIa and IIIb represent the PIIb, PIIa and PIIIb secondary precursor cells, respectively; To, Tr, n and Th indicate the tormogen, trichogen, neuron and the thecogen cell, respectively; GP indicates glial precursors. Gain-of-function phenotypes also confirm the role of N in all the divisions of the sensory organ lineage. The N cascade required for the establishment of the glial fate Cell fate decisions in the sensory organ lineage depend on the integration of autonomous and regulatory processes. It has been shown that Numb acts by antagonizing N activity in the sensory organ lineage, most likely through direct proteinprotein interaction (Uemura et al., 1989; Rhyu et al., 1994; Guo et al., 1996; Frise et al., 1996; Wang et al., 1997). This triggers a bias in the cell-cell communication process and thereby in the establishment of different cell fates. The Numb protein asymmetrically segregates to one daughter cell, the presumptive IIb, during SOP division (Rhyu et al., 1994). As later on Numb is again asymmetrically distributed in IIb and preferentially accumulates in the GP, i.e. the glide-expressing cell (Gho et al., 1999; Van De Bor et al., 2000), we asked whether Numb affects glial differentiation. To perturb Numb activity we used the hs-numb strain and induced heat shocks at different stages of sensory organ development. numb gain-of-function phenotypes resembled those observed with N ts1. A 1 hour shock at the time of SOP formation induced an increased number of glial cells in most of lineages analyzed (more than 80%). Variable effects were observed which recapitulate all N loss-of-function phenotypes. In the most extreme case we found sensory organs composed of six cells that were all Repo-positive. Thus, the effects of Numb ectopic expression can most likely be accounted for by the repression of the N cascade in the GP cell. Within the proneural cluster, N represses the proneural genes of the Achaete-scute Complex (ASC), as a result of the activation of the neurogenic E(spl)m8 gene (de Celis et al., 1996; Nakao and Campos-Ortega, 1996; Alifragis et al., 1997; Giebel and Campos-Ortega, 1997; Jiménez and Ish-Horowicz, 1997) via the transcription factor Su(H) (Oellers et al., 1994; Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995). Gain-of-function experiments performed with hs-su(h), UAS-Su(H) or UAS-E(spl)m8 transgenes (Schweisguth and Posakony, 1994; Ligoxygakis et al., 1999) did not produce the fate transformation observed with N intra (52 sensory organs analyzed for Su(H), 25 for E(spl)m8). Thus, neither Su(H) nor E(spl)m8 seem to be involved in the N pathway that directs the fate choice between neuron and glia. Effects of N mutations in non gliogenic lineages The sensory organ lineage in which the role of N had been previously analyzed contains four cells: a neuron, a sheath cell, a tormogen and a trichogen (Hartenstein and Posakony, 1990; Lieber et al., 1993; Rebay et al., 1993). However, wing nongliogenic sensory organs contain five cells, two of which are neurons (Van De Bor et al., 2000). These data prompted us to analyze the effects of N mutations on the development of such sensory organs. N ts1 flies were submitted to two heat shock

6 1386 V. Van De Bor and A. Giangrande Fig. 4. Inactivation of the Notch pathway induces a fate transformation. Sensory organs from heat shocked 15 hours APF wings, triple labeled with anti-cut (green), anti-repo (red) and the neuronal specific marker anti-elav (blue) (Robinow and White, 1998). First and second columns show the cut and the triple labeling, respectively. (A) Wild-type sensory organ composed of five cells, one of which is Elav-positive (black arrowhead) and the most proximal one is Repo-positive (white arrowhead). (B-E) N ts1 sensory organs. (B) Lineage composed of six Repo-positive cells (white arrowheads). (C) Lineage composed of six cells, two of which are Elav-positive (black arrowheads) and two others are Repo-positive (white arrowheads). (D) Lineage formed by five cells, three of which are Repo-positive (white arrowheads). Note the absence of Elav labeling in this sensory organ. (E) Lineage formed by five cells, two of which are Elav-positive (black arrowheads) and one is Repo-positive (white arrowhead). (F) hs-numb sensory organ composed of six Repopositive cells (white arrowheads). Right-most panels show schematic representations of sensory organs. SOP indicates the sensory organ precursor cell; IIb, IIa and IIIb represent the PIIb, PIIa and PIIIb secondary precursor cells, respectively; To, Tr, n, g and Th indicate the tormogen, trichogen, neuron, glia and the thecogen cell, respectively; To/Tr indicates a Tormogen or Trichogen fate. Scale bar: 2.25 µm in A,C; 3 µm in B; 1.9 µm in B-F. regimens: either between 12 hours before puparium formation (BPF) and white pupa (WP), or between 18 hours BPF and 6 hours BPF. Both shocks cover the stage at which SOP differentiates, in addition the early shock also affects the first division, which takes place by 4 hours BPF. In both cases, 40-50% of the sensory organs showed supernumerary neurons. The strongest phenotype is represented by a sensory organ composed of six neurons (Fig. 7). Intermediate effects involve a thecogen to neuron or a thecogen to IIIb transformation (data not shown). Reciprocal defects were observed upon constitutive activation of the N pathway, namely partial or total transformation of neurons into sheath cells, the strongest defect being a sensory organ composed of six cells, four of which are thecogen cells (data not shown). Intermediate effects involve the transformation of a IIIb daughter into a sheath cells. Thus, as in the case of gliogenic and classic sensory organs, all fate decisions are affected by N in non-gliogenic sensory organs. DISCUSSION N represses the glial fate in the fly PNS N has been involved in many aspects of cell differentiation that require cell communication and cell fate choices (for a review, see Artavanis-Tsakonas et al., 1999). In many cases, the N pathway consolidates the bias conferred by intrinsic signals that are asymmetrically distributed in the multipotent precursor cells (Guo et al., 1996; for a review see Jan and Jan, 1998). In the nervous system, it represses the formation of mature and functional neurons. Previous papers had shown that proneural and neurogenic genes, including N, control glial differentiation in flies (Giangrande, 1994; Giangrande, 1995; Nelson and Laughon, 1994), however, the precise role of N in this process had not been established, due to the lack of appropriate tools. We show that N signaling represses glial differentiation and induces the neuronal pathway. We also show that N acts as a genetic switch between the two fates by repressing glide Fig. 5. Lack of Notch induces glide expression. Sensory organs from 10 hours APF heat-shocked wings labeled with anti-cut (green) and anti-glide (red). Right-most panels show double exposures. (A) Wild-type wing gliogenic sensory organ composed of five cells, one of which is Glidepositive (arrowhead). (B) N ts1 sensory organ composed of six cells, five of which are Glidepositive (arrowheads). Asterisk indicates the Glidenegative cell. Scale bar: 2.5 µm.

7 Notch represses the glial fate in fly PNS 1387 Fig. 6. Constitutive activation of the Notch pathway represses glial differentiation. 24 hours APF wings that were heat shocked at 6 hours APF for one hour at 38 C, and simultaneously labeled with anti-cut (green), anti-repo (red) and anti-elav (blue). (A) In the wild type, glial nuclei are present along L1 and L3 veins (L1,3). Five neurons are present on the L3 vein: L3-1, L3-2 and L3-3 indicate the neurons of the three campaniform sensilla on L3; E2 indicates the extra neuron coming from the L3-2 lineage; ACV indicates the anterior cross vein neuron; arrowheads indicate Cut-positive cells of the L3 sensory organs. (B) In a N intra wing, glial cells are completely absent in L3, their number being severely reduced in L1. Asterisks indicate sensory organs composed by four cells that express Cut at high level. Notice the absence of Elav labeling in these sensory organs. (C,D) 24 hours APF N intra sensory organs, triple labeled with anti-cut (green), anti-prospero (red) and anti-elav (blue). (C) Lineage composed of six cells, two of which express Prospero (black arrowheads) and two Elav (white arrows). (D) Lineage composed of six cells, four of which express Prospero (black arrowheads), none Elav. Right-most panels show schematic representation of wild-type and N intra sensory organs. SOP indicates the sensory organ precursor cell; IIb, IIa and IIIb represent the PIIb, PIIa and PIIIb secondary precursor cells, respectively; To, Tr, n, g and Th indicate the tormogen, trichogen, neuron, glia and the thecogen cell, respectively; To/Tr indicates a Tormogen or Trichogen fate. Scale bars: A, 37 µm in A,B; 2.5 µm in C,D. expression. Strikingly, N seems to act in opposite directions in fly and some vertebrate peripheral glial cells (Furukawa et al., 2000; Gaiano et al., 2000; Morrison et al., 2000). Indeed, gain-of-function N mutations promote differentiation of Müller, radial and Schwann glial cells. Two possible explanations can account for these results: (1) the genetic switch between neuronal and glial fates has different requirements in flies and vertebrates; (2) the role of N depends on the subtype of glial cell. The analysis of other classes of fly glial cells will help us elucidate this point. Preliminary analyses on the embryonic CNS, however, suggest that the response of central glial cells to N depends on the subtype (data not shown). The observation that oligodendrocyte differentiation, like fly peripheral glial cells, is repressed by N, also argues in favor of the second hypothesis (Wang et al., 1998). Our data indicate that the initial commitment to the glial fate does not require the presence of neurons (Van De Bor et al., 2000; the present study). Indeed, glide is expressed well before neurons are born, at a stage where only three cells are present in sensory organ lineage, GP, IIIb and IIa. Moreover, numb is even required earlier, in the IIb (present data and Uemura et al., 1989; Rhyu et al., 1994). Thus, although neurons may play a role in the maintenance of the glial fate by expressing signals that regulate glial proliferation and/or survival, they do not seem to be involved in the first step of gliogenesis. It will be interesting to identify the cells that express the N ligand during vertebrate nervous system development. In particular, it will be important to define whether, in vertebrates, the N cascade that controls glial versus neuronal differentiation involves cells of different lineages. Indeed, as neurogenesis starts before gliogenesis, it had been proposed that the early born cell type signals to induce the late glial fate in multipotent precursors (Morrison et al., 2000). Alternatively, N may act as in flies, in daughter cells of a multipotent precursor, possibly by reinforcing autonomous decisions imposed by Numb-like molecules (Zhong et al., 1996; Zhong et al., 2000; Wakamatsu et al., 2000). Fig. 7. (A,B) Role of N in non-gliogenic sensory organs. L3-2 sensory organ from wild-type (A) or N ts1 (B) wings, triple labeled with anti-cut (green), anti-pros (red) and anti-elav (blue). As shown in Van De Bor et al. (2000), Prospero is expressed in the sheath cell (black arrowhead) as well as in the neurons (white arrowheads). Note the presence of six neurons expressing Elav and Prospero in B compared with the two observed in the wild-type (A). Scale bar: 2.5 µm.

8 1388 V. Van De Bor and A. Giangrande Fig. 8. Role of N and Numb in gliogenesis. (A,B), (C,D) and (E) show schematic representations of classic, gliogenic and nongliogenic lineages, respectively. Color coding indicate cells in which the requirement of N (pink circles) and Numb (orange) has been shown by this and previous studies. SOP indicates the sensory organ precursor cell; IIb, IIa and IIIb represent the PIIb, PIIa and PIIIb secondary precursor cells, respectively; To, Tr, n, g and Th indicate the tormogen, trichogen, neuron, glia and the thecogen cell, respectively The N pathway controls the choice between neuronal and glial fates The observation that N signaling represses glide allows us to identify the first upstream regulator of the glial promoting factor and to integrate intrinsic and extrinsic mechanisms in the process that induce the glial fate. This is further confirmed by the result that the choice between the IIIb and the GP fates requires N as well as numb. The most likely explanation is that Numb accumulation in the GP represses the N pathway in that cell. This in turn activates the N pathway in the adjacent IIIb cell, via a feedback mechanism that reinforces the initial asymmetry between the IIb daughters. Such feedback mechanisms have already been observed in other processes involving the N cascade (for reviews, see Bray, 1998; Irvine, 1999; Wu and Rao, 1999). Surprisingly, although lack of N induces a fate transformation, it does not alter the rate of proliferation of the transformed cell, as the presumptive IIIb cell divides at the same stage as in wild-type sensory organs and produces two Repo-positive cells. We initially hypothesized that the fate transformation into glia had occurred in the daughter cells, neuron and thecogen. However, the neuronal duplication observed upon late heat shock (Fig. 4E) indicates that lack of Notch in the PIIIb progeny leads to a thecogen to neuron transformation. For this reason, we propose that the heat-shock regimens used for the N ts1 experiments were not sufficient to affect the proliferation rate. It is interesting to notice that, while transient activation of the N pathway results in the transformation of the GP into a IIIb cell, lack of Glide never results in this phenotype; rather, it leads the GP to adopt a default, neuronal fate (Van De Bor et al., 2000). These results further validate the hypothesis that glide acts in fate choice between neurons and glial cells in CNS stem cells, but does not have this role in IIb cells (Akiyama- Oda et al., 1999; Bernardoni et al., 1999; Van De Bor et al., 2000). In the PNS, the role of glide is merely to implement the glial fate in one of the IIb daughter cells in response to Numb asymmetric segregation, to the repression of the N pathway in the GP and possibly to other cell-specific cues. One of the most striking results is that repression of the N pathway throughout the development of the sensory organ (obtained by N loss-of-function mutations or by Numb overexpression) leads to sensory organs composed of six glial cells. Previous data have indicated that the competence to adopt the glial fate is restricted to some cells of the sensory organ lineage; the strongest phenotype observed upon overexpression of glide throughout the lineage is the differentiation of a sensory organ composed of five cells, three of which are Repopositive (Van De Bor et al., 2000). Thus, glide is not sufficient to induce a IIa into IIb transformation. This indicates that the pathway mediated by N and numb affects the competence of sensory organ cells to adopt the glial fate. In molecular terms, this implies the control of expression of glide regulators, positive co-factors and/or repressors. It will be interesting to determine the N pathway that acts on gliogenesis. In the case of the proneural cluster, one well defined target of the N pathway is the ASC, which is composed of four bhlh-containing genes: scute, achaete, lethal of scute and asense (Artavanis-Tsakonas et al., 1995; Modolell and Campuzano, 1998 and references therein). None of the members of the complex, however, seems to be expressed in GP, as the latest expression so far described in the sensory organ lineage is that of asense, which is specific to the SOP and to IIb (Gonzalez et al., 1989; Campuzano and Modolell, 1992; Brand et al., 1993; Dominguez and Campuzano, 1993; Giangrande, 1995). N and context dependency The role of N in gliogenic sensory organs is to trigger the fate choice between neurons and glia. Our data strongly suggest that the N pathway is normally inactive in the GP, which results in glial differentiation, and is active in IIIb, where it promotes neuronal differentiation. Thus, the N pathway is active in the precursor of neurons and sheath cells of gliogenic sensory organs, whereas in non-gliogenic lineages it is not. This role of N is somewhat intriguing when compared with the overall function of this protein in neuronal development. Indeed, N represses the neuronal fate in non-gliogenic lineages (Van De Bor et al., 2000; Fig. 8) by being inactive in the IIIb cell that produces neurons (IIb for the lineages composed of four cells; Hartenstein and Posakony, 1989). Moreover, it also represses the neural fate within the cells of the proneural cluster and represses neuronal differentiation in vertebrate nervous system (Furukawa et al., 2000; Gaiano et al., 2000; Morrison et al., 2000). A similar discrepancy between gliogenic and nongliogenic sensory organs has also been shown for Numb. These results suggest that Numb-mediated repression of N responds to cues that dictate lineage identity, so that in gliogenic sensory organs it promotes the glial fate, in the non-gliogenic ones the neuronal fate. In the absence of cell division, the precursor of embryonic

9 Notch represses the glial fate in fly PNS 1389 sensory organs, which are not gliogenic and contain four cells, adopt a default neuronal fate (Vervoort et al., 1997a; Vervoort et al., 1997b). In the future, it will be interesting to determine the default fate of gliogenic sensory organs. Finally, the identification of the intrinsic and/or extrinsic cues that control lineage identities will make it possible to clarify the molecular mechanisms that underlie cell fate choices during the development of the nervous system. We thank C. Dambly-Chaudière, C. Delidakis, M. Haenlin, P. Heitzler, L. Y. Jan, Y. N. Jan, J. Posakony, G. Rubin, A. Travers and A. Vaessin for flies and antibodies. We also thank J. L. Vonesch and D. Hentsch for their precious help with the confocal microscope and imaging. Confocal microscopy was developed with the aid of a subvention from the French MESR (95.V.0015). We thank C. Dambly-Chaudière for comments on the manuscript. 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