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A Serrate–Notch–Canoe complex mediates essentialinteractions between glia and neuroepithelial cellsduring Drosophila optic lobe development

Raquel Perez-Gomez1,*, Jana Slovakova1,`,§, Noemı Rives-Quinto1,`, Alena Krejci2 and Ana Carmena1,"

1Instituto de Neurociencias-CSIC/UMH, 03550-Sant Joan d’Alacant, Alicante, Spain2University of South Bohemia, Faculty of Science and Institute of Entomology, Biology Centre ASCR, Branisovska 31, Ceske Budejovice,Czech Republic

*Present address: University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic`These authors contributed equally to this work§Present address: IST Austria, Am Campus 1, A-3400 Klosterneuburg, Austria"Author for correspondence (acarmena@umh.es)

Accepted 12 August 2013Journal of Cell Science 126, 4873–4884� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.125617

SummaryIt is firmly established that interactions between neurons and glia are fundamental across species for the correct establishment of a functional

brain. Here, we found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of theoptic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions that generate neuroblasts. The proneural gene lethal of scute (l9sc) is transiently activated by the epidermal growth

factor receptor (EGFR)–Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateralneuroepithelium, promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium ofmultiple signaling pathways, including EGFR–Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia

and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex iscrucial for interactions between glia and neuroepithelial cells during optic lobe development. Ser is tissue-autonomously required in the gliawhere it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling toavoid the premature activation of the EGFR–Ras pathway and hence of L9sc. Interestingly, different Notch activity reporters showed very

different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote theexpression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling.

Key words: Glia, Serrate-Notch signaling, Optic lobe, Canoe, Drosophila

IntroductionGlial cells are not a mere structural filler within the brain but they

perform multiple and vital tasks for the proper development and

functioning of the nervous system. The roles that glial cells display

during the development of the nervous system are as diverse as the

multiple glial types specified, including axon ensheathment, axon

guidance, phagocytosis and the establishment of the blood–brain

barrier (Banerjee and Bhat, 2007; Bundgaard and Abbott, 2008;

Edenfeld et al., 2005; Lemke, 2001; Nave and Trapp, 2008; Parker

and Auld, 2006). In Drosophila, three main different types of glial

cells have been very well characterized in the larval brain, namely

surface glia (subdivided into the outermost perineurial glia and the

underlying subperineurial glia), cortex glia and neuropile glia

(Hartenstein, 2011; Pereanu et al., 2005; Stork et al., 2012).

Embryonic neuroblasts (NBs), specifically neuro-glioblasts, give

rise to the precursors of the larval glia that will increase in number

throughout the larval life, mainly at late larval stages and

fundamentally from neuroglioblast division, although the mitosis

of differentiated glia also contributes (Pereanu et al., 2005). These

three types of glia perform crucial functions during the

development of the Drosophila brain. For example, surface glial

cells provide signals at early stages of the larval period to induce

embryonic quiescent NBs to resume proliferation (Ebens et al.,

1993). Cortex glial cells have important trophic functions for

neurons, and neuropile and surface glia act as key intermediate

targets during axon pathfinding in the brain (Hidalgo, 2003; Hoyle

et al., 1986; Pielage and Klambt, 2001; Poeck et al., 2001; Sepp

et al., 2001; Tayler and Garrity, 2003). Glial processes engulf NBs

and neurons in the Drosophila larval brain, which is formed by the

central brain and the optic lobes (Hartenstein et al., 2008).

The optic lobes, which are located at the lateral side of both

brain hemispheres, form part of the Drosophila visual system.

They derive from neuroectodermal placodes in the embryonic

head that invaginate, lose contact with the epidermis and attach to

the brain (Green et al., 1993). At the beginning of the larval life,

just after larval hatching, cells of the optic lobe start to proliferate

and they separate into an outer proliferation center (OPC), which

will give rise to the outer medulla and lamina neurons, and an

inner proliferation center (IPC), which generates the inner

medulla, the lobula and the lobula plate neurons (Hofbauer and

Campos-Ortega, 1990; Meinertzhagen and Hanson, 1993). The

OPC anlage is formed by neuroepithelial (NE) cells, which

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proliferate by symmetric cell division until the OPC reaches aproper size, at third larval instar. At this point, NE cells switch to

asymmetric differentiative divisions generating medulla NBs atthe medial edge of the OPC anlage (Egger et al., 2007).Differentiation of NE cells to medulla NBs progresses from themedial to the lateral edge of the OPC in a ‘proneural wave’,

which was recently identified by the transient and localexpression at the wave front of the proneural gene lethal of

scute (l9sc) (Yasugi et al., 2008). Over the past few years multiple

signaling pathways have proved to be essential for regulating theproneural wave progression within the neuroepithelium in atissue-autonomous way, including Notch, JAK–STAT, EGFR–

Ras–PointedP1 (PntP1) and Fat–Hippo signaling pathways(Egger et al., 2010; Ngo et al., 2010; Reddy et al., 2010;Yasugi et al., 2010; Yasugi et al., 2008). However, not much isknown about non-autonomous regulatory mechanisms. Given

that glial cells proliferate and differentiate in close contact withthe OPC and IPC neuroepithelia, the glia might influence thedevelopment of the optic lobe, including the progression of the

proneural wave.

In this work, we have analyzed the relationships between NEcells of the optic lobe and the surrounding glia. We found that the

PDZ (PSD-95, Discs Large, ZO-1)-domain-containing proteinCanoe (Cno) (Miyamoto et al., 1995) is expressed in the NE cellsof the OPC and IPC. Cno and its vertebrate homologues AF-6/

Afadin are scaffolding proteins that are predominant at adherensjunctions (AJs) where they regulate the linkage of AJs to the actincytoskeleton (Lorger and Moelling, 2006; Mandai et al., 1997;Matsuo et al., 1999; Miyamoto et al., 1995; Sawyer et al., 2009).

Cno also performs AJ-independent functions regulating neuron–glia interactions and asymmetric cell division (Slovakova andCarmena, 2011; Speicher et al., 2008). In addition, Cno acts as an

integration hub of different signaling pathways, including Rasand Notch during muscle and heart progenitor specification(Carmena et al., 2006).

The Ras pathway triggered by the EGFR ultimately activatesthe ETS transcription factor Pnt that exists in two isoforms, PntP1and PntP2, the former acting in the optic lobe (Klambt, 1993;Yasugi et al., 2010). PntP1 is very locally activated at the

transition zone, where, in turn, it induces the local expression ofL9sc (Yasugi et al., 2010). The Notch receptor is activated by twodifferent ligands, Delta (Dl) and Serrate (Ser), which trigger the

proteolytic cleavage of the intracellular domain of Notch. Thisdomain translocates into the nucleus where it associates withSuppressor of Hairless [Su(H)] and activates target genes. The

best characterized Notch targets are the genes of the Enhancer ofSplit-Complex [E(spl)-C], which comprises seven genes thatencode basic-helix-loop-helix (b-HLH) transcription factors,

namely md, mc, mb, m3, m5, m7 and m8 (Bailey andPosakony, 1995; Delidakis and Artavanis-Tsakonas, 1992;Jennings et al., 1994; Knust et al., 1992; Lecourtois andSchweisguth, 1995; Rebay et al., 1991).

The data presented in this work strongly suggest that acomplex between Cno, Notch (present in NE cells) and Ser(present in the glia) is key for interactions of NE cells and glia

during the development of the optic lobe. We show that theNotch ligand Ser displays tissue-autonomous and non-autonomous effects in the glia and in the neuroepithelium,

activating different Notch E(spl) target genes in each tissue. Byactivating Notch in the glia, Ser regulates its proliferation and, bytriggering Notch signaling in NE cells, Ser restricts the activation

of Ras–PntP1 signaling, and hence the activation of L9sc, to the

transition zone.

ResultsCno localizes at the AJs of NE cells in the optic lobeproliferation centersCno is expressed in Drosophila embryonic neuroectoderm and inthe delaminated NBs where Cno displays an essential role inasymmetric NB division (Speicher et al., 2008). In an attempt to

characterize a potential function of Cno in the differentiation of NEcells to medulla NBs during the development of the larval opticlobe, we first analyzed Cno expression in this tissue. Cno was highlyenriched at the apical most region of NE cells in the OPC and IPC

(Fig. 1B–E9). Cno and its vertebrate orthologs are present at the AJsof different epithelial tissues (Mandai et al., 1997; Matsuo et al.,1999; Sawyer et al., 2009). Indeed, Cno colocalized at the apical

region of NE cells with Bazooka (Baz), another well-knowncomponent of the zonula adherens, where Baz colocalizes with theDE-cadherin Shotgun (Shg) (Krahn et al., 2010) (Fig. 1F–G0).

Cno is required for a correct progression of theproneural waveNext, we wanted to analyze the effect in the optic lobe of knockingdown cno. With that purpose, we overexpressed a cnoRNAi plus the

gene encoding the Dicer2 enzyme under the neuroepithelia-specificGal4 driver c855a (Manseau et al., 1997). The loss of Cno, as testedby immunofluorescence, was complete and specific (Fig. 2E–F0).

We analyzed anterior views (see Fig. 1A), in which changes in theproneural wave progression respect to well-defined landmarks, suchas the lamina furrow, are easily detected (Fig. 2A–A0). In UAS-

cnoRNAi optic lobes, the proneural wave was advanced comparedwith control optic lobes (68%; n541 brain hemispheres), evenreaching the lamina region at some points (Fig. 2A–C0). The

overexpression of cno caused the opposite phenotype (100%;n517), with a marked delay in the progression of the proneuralwave (Fig. 2D–D0) and the concomitant decrease in thedifferentiation of medulla NBs (compare Fig. 2A and D). To

further prove a specific role of cno in this process, we carried outmosaic analysis with repressible cell marker (MARCM) clones inthe neuroepithelium using armadillo (arm)-Gal4 as a driver. Clones

for the null allele cnoR2, labeled with CD8-GFP, revealed L9sc-expressing cells within the clone in an advanced position relative tothe cno+ cells outside the clone (40%; n520) (Fig. 2G,G9).

Cno colocalizes with Notch at the AJs of NE cellsNotch signaling is required to regulate the progression of theproneural wave (Egger et al., 2010; Ngo et al., 2010; Reddy et al.,2010; Wang et al., 2011; Weng et al., 2012; Yasugi et al., 2010).

Given that Cno and Notch functionally interact in other systems(Carmena et al., 2006; Miyamoto et al., 1995) and that cno

mutant phenotypes were very reminiscent of those previously

described for Notch mutants in the neuroepithelium (Yasugi et al.,2010), we decided to analyze the relative localization of Cno andNotch in the optic lobe. Confocal analysis of double

immunofluorescence for Cno and Notch showed colocalizationof these proteins at the AJs of NE cells (Fig. 3A–B0).

E(spl) Notch target genes are differentially expressed inthe optic lobe and in the surrounding gliaNext, we wondered whether Cno had some effect on the Notchactivity, which is required in NE cells to maintain their fate

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(Egger et al., 2010; Wang et al., 2011; Weng et al., 2012). First,

we wanted to analyze the Notch activity in wild-type brains. For

this analysis we used different Notch activity reporters, because it

has been previously shown that different E(spl) Notch target

genes display distinct expression patterns in other tissues (Cooper

et al., 2000; de Celis et al., 1996; Wech et al., 1999). Specifically,

we looked at Gbe+Su(H)lacZ (see the Materials and Methods)

(Furriols and Bray, 2001), E(spl)mb-CD2 (mb-CD2 hereafter) (de

Celis et al., 1998), E(spl)md-lacZ (md-lacZ) (Cooper et al., 2000)

and E(spl)m7-nuclacZ (m7-nuclacZ) (Pines et al., 2010). At third

larval instar, Gbe+Su(H)lacZ was barely detected in NE cells

(Fig. 3C–F9); it started to be highly expressed at the transition

zone where the Notch ligand Dl is enriched in a characteristic

punctuated pattern (Fig. 3C–D0) (Weng et al., 2012; Yasugi et al.,

2010). L9sc is also very locally activated at the transition zone, in

progenitor I (PI) and progenitor II (PII) cells; the latter

juxtaposed to the emerging medulla NBs (Yasugi et al., 2010)

(Fig. 3F,K). Gbe+Su(H)lacZ was highly detected in PI but its

expression dropped markedly in PII (Fig. 3F,F9,K), and it was

again highly activated in NBs along with the Notch-responding

gene deadpan (dpn) (Krejcı et al., 2009) (Fig. 3E,E9,K). A

similar expression pattern was shown for the Notch activity

reporter mc–GFP (Weng et al., 2012) (Fig. 3K). A weak

expression of Gbe+Su(H)lacZ was detected in the surface glia

in close contact with NE cells (Fig. 3D,D0,K). Interestingly, the

mb-CD2 reporter was highly expressed in these surface glia, the

same location in which the md-lacZ reporter was detected,

although at much lower levels (Fig. 3G,G9,I,I9,K); none of them

was clearly present in NE cells (Fig. 3G,G9,I,I9,K), but the mb-

CD2 reporter was also detected in medulla NBs (Fig. 3H,H9,K).

The only reporter that was expressed in the neuroepithelium was

m7-nuclacZ, which also showed a uniform expression in the

transition zone and in the emerging NBs (Fig. 3J–K). Specific

effects of Notch loss and gain of function on the expression of

these reporters were observed in the optic lobe and in the

surrounding glia, confirming them as bona fide Notch target

genes in the brain (supplementary material Fig. S1).

The Notch ligand Ser is expressed in the glia

Given this pattern of Notch activity and the fact that Dl is

enriched at the transition zone with almost undetectable levels in

NE cells or in the surrounding glia (Fig. 3C,C9,D,D9) (Weng

et al., 2012; Yasugi et al., 2010), we wondered whether Ser,

another ligand of Notch that activates it in different tissues and

cell contexts (Fleming et al., 1990; Rebay et al., 1991; Thomas

et al., 1991), would be expressed in the optic lobe. A UAS-

CD8::GFP construct under a Ser-Gal4 driver revealed that,

whereas Dl was highly restricted to the transition zone, Ser was

active in what seemed to be the brain glia, with no detectable

expression in NE cells (Fig. 4A–B9). Double labeling with the

glial marker Nervana (Nrv) confirmed the expression of Ser

(Ser-Gal4..UAS-CD8::GFP) in the glia (Fig. 4C–C0). Given

that CD8-GFP is a membrane protein that reflects where Ser-

Gal4 is expressed but not the real localization of the Ser protein

within the glia, we used a specific Ser antibody to analyze Ser

distribution. Unlike CD8-GFP, Ser was not uniformly present

around all glial cell membranes but it was particularly

concentrated at the glial membranes in contact with the apical

side of NE cells, where Ser colocalized with Notch (Fig. 4D–

E0).

Fig. 1. Cno localizes at the AJs of NE cells in the optic lobe.

(A) Schematic of a larval CNS that includes two brain hemispheres

and the ventral ganglia. The optic lobe is shown in color in one hemisphere.

Medulla NBs are represented in red, NE cells in green and the lamina (L)

in blue. Two frontal views are shown, one more anterior (a; highlighted in

orange) and another one more posterior (p; highlighted in purple). At this

posterior view, medulla NBs are detected both at medial (mNBs) and

lateral position (lNBs); OPC, outer proliferation center; IPC, inner

proliferation center; D, Dorsal; V, ventral; m, medial; l, lateral; LF, lamina

furrow. (B–E9) Brain hemisphere showing the optic lobe in green (c855a-

Gal4..UAS-CD8::GFP) and Cno in red in an anterior (B–C9) and a

posterior view (D–E9). The higher magnifications (C,C9,E,E9) show in

detail the location of Cno at the apical most part of NE cells. (F–F0) Baz

(red) colocalizes with Shg (green) at the AJs of NE cells. (G–G0) Cno (red)

colocalizes with Baz (blue) at the AJs of NE cells (arrows).

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Cno forms a complex with Notch and Ser in vivo

Cno and Notch, as well as Notch and glial Ser, stronglycolocalized at the most apical side of NE cells, at the interface

with the surrounding glia. Hence, we speculated that these

proteins might be in a complex. To test this hypothesis, we

performed co-immunoprecipitations (co-IPs) from larval head

extracts and found that Cno co-immunoprecipitated with both

Notch and Ser (Fig. 4F). Hence, Ser, Notch and Cno exist in a

complex in vivo.

Knockdown of cno in NE cells affects Notch and Serlocation and Notch activity

Colocalization and co-IP experiments suggested that Cno could

contribute to stabilize Notch at the AJs of NE cells to facilitate

Notch binding to its ligand Ser in the adjacent glia. Indeed, we

observed that not only Notch but also Ser were highly reduced

after knockdown of cno (UAS-cnoRNAi) in NE cells (63%;n519), compared with controls (Fig. 5A–B0). Then, we reasoned

that the Notch activity should be also affected in this cno mutant

condition. In fact, the analysis of the Notch reporter m7-nuclacZ

showed a slight but consistent reduction of its expression in the

neuroepithelium (67%; n59) (Fig. 5C–D9).

Ser loss of function in the glia non-autonomously affectsthe proneural wave progression

To analyze in more detail the effect of glial Ser in the

development of the neuroepithelium, we expressed in the glia a

dominant-negative form of Ser (DNSer) called Bd E24 (recently

renamed Sersec), which is a strong dominant-negative form

(Fleming et al., 2013). The expression of DNSer in the glia

caused a marked advance of the proneural wave (100%; n515)

(Fig. 5E,E9,H,H9), a phenotype reminiscent of that displayed

when cno is knocked down in the neuroepithelium. The

expression of DNSer in the glia also caused ectopic expression

of L9sc within the neuroepithelium (100%; n521)

(Fig. 5F,F9,I,I9). Moreover, PntP1 was also ectopically activated

in DNSer (100%; n511), colocalizing with L9sc in several cells,

indicating that the Ras pathway was misregulated

(Fig. 5G,G9,J,J9). Hence, Ser in the glia is crucial for

determining the correct spatial activation of the Ras–PntP1

pathway in the neuroepithelium. The expression of DNSer in the

glia caused a significant depletion in the glial levels of the Notch

reporter mb–CD2 (100%; n512) (Fig. 5K,L), expression that

was rescued after the simultaneous expression of DNSer and an

activated form of Notch, Nintra (Ni), in the glia (Fig. 5M,M9).

Fig. 2. Cno is required for the normal proneural wave

progression. (A–D9) Brain hemispheres in an anterior view

stained for L9sc (red) and Dpn (green); GFP is in blue. c855a-

Gal4 drives the expression of UAS-CD8::GFP in A–C9 and of

UAS-cno::GFP in D,D9. Higher magnifications of A–D (average

projections of several confocal planes of Z-stacks) are shown in

A9–D9 (single confocal projections from Z-stacks). The

knockdown of cno (UAS-cnoRNAi) causes an advance in the

proneural wave compared with control optic lobes (A–C9); two

examples are shown, one more extreme (C) than the other (B).

Arrows indicate the lamina furrow (LF, dotted line); arrowheads

point to NBs that are reaching the lamina (L). The overexpression

of cno (UAS-cno::GFP) causes a delay in the proneural wave

progression (D,D9); compare the width of the bands

corresponding to NBs and NE cells (arrow with double

arrowheads) with the control (A,A9, D,D9). (A0–D0) Schematics

representing the progression of the proneural wave (black arrow)

in the different genetic backgrounds specified. L9sc (red) appears

at the leading edge of the proneural wave. (E–F0) In control optic

lobes, Cno is detected at the AJs of NE cells (E,E9) along with

Baz (E,E0). GFP, in green, reveals the expression of UAS-CD8

under the c855a-Gal4 driver. In UAS-cnoRNAi optic lobes, Cno

completely disappears from NE cells (F,F9), whereas Baz is still

detected (F,F0). (G–G9) cnoR2 clone is labeled with CD8-GFP.

L9sc+ (red) cells normally at the proneural wave front (labeled

with a red dotted line) appear in advanced positions (arrowheads)

within the clone (LF, lamina furrow, white dotted line); Dlg labels

NE cells (blue).

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However, the expression of DNSer plus Ni in the glia did not

rescue the phenotype of ectopic L9sc+ and PntP1+ cells in the

neuroepithelium (Fig. 5N,N9). Intriguingly, the overexpression of

DNSer in the glia led to a striking reduction in the expression of the

Notch activity reporter m7-nuclacZ in NE cells (100%; n57)

(Fig. 5O-P9). To further support the role of glial Ser in the

underlying neuroepithelium, we performed mosaic analysis with

MARCM clones in the glia using the nrv2-Gal4 driver. Clear

ectopic L9sc was observed underlying glial SerRX82 mutant clones

in 69% of the brain hemispheres analyzed (n526) (Fig. 5Q). Thus,

altogether, these results strongly suggest that, in normal

conditions, Ser non-autonomously triggers Notch signaling in the

neuroepithelium and this activity avoids the premature activation

of Ras–PntP1 signaling in NE cells.

Ser–Notch signaling in the glia regulates glia proliferation

By using Repo, as a specific glial marker, and phospho-histone3

(PH3), as an indicator of mitotic cells, we found that the

expression of DNSer in the glia led to a statistically significant

decrease in the number of Repo+ and PH3+ cells, compared with

control optic lobes (Fig. 6D–D0,G,H). This phenotype was

opposite to the overexpression of the constitutive active form

Ni in the glia (Fig. 6E–E0,G,H). Interestingly, the expression of

UAS-cnoRNAi in NE cells showed a similar phenotype to the

expression of DNSer in the glia (Fig. 6B–B0,H): a significant

decrease in the number of glial PH3+ cells. In addition, the

overexpression of cno in NE cells showed, similar to the

overexpression of Ni in the glia, a significant increase in Repo+ and

PH3+ cells, compared with control optic lobes (Fig. 6C–C0,G,H).

Fig. 3. Cno colocalizes with Notch, which is

active in NE cells and in the surrounding

glia. (A–B0) Cno (in red) and Notch (in green)

colocalize at the apical AJs of NE cells in the

optic lobe. An anterior view (A–A0) and a

posterior view (B–B0) are shown. NE,

neuroepithelium; LF, lamina furrow; L,

lamina. (C–F9) The Notch activity reporter

Gbe+Su(H)lacZ (in green) is shown along with

Dl (red) in an anterior view (C–C0) or a

posterior view (D–D0), along with Dpn (red;

E,E9) or L9sc (red; F,F9). NE (Shg or

CD8::GFP expression under the control of the

c855a-Gal4 driver) is in blue; TZ, transition

zone. Green arrows indicate Gbe+Su(H)lacZ

expression in NE cells at the TZ and green

arrowheads show its expression in medulla

NBs (mNBs and lNBs in the posterior view

shown in D,D0). Red arrows indicate the

enrichment of Dl at the TZ (C,C9,D,D9), Dpn

expression in the TZ and in NBs (E) or L9sc at

the TZ (F). Asterisks (D,D0,E–F9) indicate the

PII cell at the TZ that does not express

Gbe+Su(H)lacZ and expresses L9sc (red

arrowhead in F). No expression of

Gbe+Su(H)lacZ is detected in NE cells outside

the TZ (D,D0,E–F9). (G-H9) The Notch activity

reporter mb-CD2 (in red) is expressed in the

glia along with the glial marker Nrv (in green),

but is not detected in NE cells (in blue)

(G,G9). mb-CD2 is also present in the NBs

along with Dpn (in green; H,H9). (I,I9) The

Notch activity reporter md-lacZ (in red)

localizes at low levels in the glia labeled with

Nrv (in green). (J,J9) The Notch activity

reporter m7-nuclacZ (in green) is expressed

throughout the neuroepithelium (NE, in blue),

including the TZ labeled by L9sc (in red) and

in the emerging medulla NBs, adjacent to

L9sc-expressing cells. (K) Schematic

summarizing the expression of the different

Notch activity reporters analyzed.

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Indeed, in UAS-cnoRNAi, the Notch reporter mb-CD2 was

downregulated in the surface glia, specifically in the subperineurial

glia, in close contact with the neuroepithelium (53%; n519)

(Fig. 6K–L9) and the overexpression of cno in the neuroepithelium

led to an upregulation of the Notch activity reporter Gbe+

Su(H)lacZ in the glia (100%; n512), whereas, in normal

conditions, it is barely detected (Fig. 6I–J9; see also Discussion).

The simultaneous expression of DNSer and Ni in the glia rescued the

DNSer phenotype in the glia, but not in the neuroepithelium

(Fig. 6F–H). Indeed, the expression of mb-CD2, which was

downregulated in the glia after expressing DNSer in this tissue, was

also rescued when DNSer and Ni were simultaneously expressed in

the glia (Fig. 5M,M9). Hence, Ser is required autonomously in the

glia to regulate their proliferation.

Working model

Taking all our data together, we propose a working model in

which the brain glia would display an important role during the

progression of the proneural wave in the optic lobe (Fig. 7H).

Ser, present in the glia in close contact with the neuroepithelium,

can activate Notch in the glia, as detected by the Notch

activity reporters mb-CD2, md-lacZ and, to a lesser degree,

Gbe+Su(H)lacZ contributing to regulate glia proliferation. Ser

can also activate Notch in the neuroepithelium, as revealed by the

Fig. 4. Ser is expressed in the glia and forms a complex

with Notch and Cno in vivo. (A–B9) In Ser-

Gal4..UAS-CD8::GFP optic lobes, CD8::GFP is

detected in glial membranes (green) with no presence in

NE cells (labeled with Shg, in blue), whereas Dl (in red) is

highly restricted to the transition zone (red arrows) in the

neuroepithelium (NE). An anterior view (A,A9) and a

posterior view (B,B9) are shown. White arrows indicate

the lamina furrow (LF, dotted line in A9). (C–C0) Detail of

a Ser-Gal4..UAS-CD8::GFP optic lobe, in a posterior

view. CD8::GFP colocalizes with the glial marker Nrv (in

red); Shg, labeling the NE, is in blue. (D–E0) The Ser

protein (in red) accumulates strongly in the glia at the

interface between glial membranes and NE cells (arrows)

(D–D0), where it colocalizes with Notch (green in E–E0).

(F) c855a-Gal4..UAS-Notch::GFP larval lysates were

subject to immunoprecipitation (IP) with an anti-GFP

antibody and probed on immunoblots (IB) with anti-Cno,

with anti-GFP (as an IP control) and with anti-Ser (as a

positive control). c855a-Gal4..UAS-Cno::GFP larval

lysates were subject to IP with anti-GFP and probed on IB

with anti-Ser and with anti-GFP (as an IP control). c855a-

Gal4..UAS-GFP larval lysates were used as a negative

control in all cases.

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Fig. 5. Ser loss of function in the glia non-autonomously affects the proneural wave progression. (A–D9) cno loss of function in NE cells affects Notch and Ser

location and Notch activity. A detail of an optic lobe neuroepithelium in a posterior view is shown in all panels. In control optic lobes, Ser (in red) and Notch (in green)

highly colocalize at the interface between glia and NE cells (in blue) (A–A0). In UAS-cnoRNAi optic lobes, the amount of Ser and Notch drops significantly at the

glia–NE cell interface (arrows in B–B0; compare with A–A0). Compared with control optic lobes, in UAS-cnoRNAi optic lobes the Notch activity decreases in NE

cells, as revealed with the reporter m7-nuclacZ (C–D9; L9sc is in red). (E–J9) Brain hemispheres in an anterior view (E,E9,H,H9) and a posterior view

(F–G9, I–J9) where E9,H9,F9 and I9 are details at higher magnification of the images shown in E,H,F and I, respectively; NE cells (c855a-Gal4..UAS-CD8::GFP or

Dlg) and the glia (repo-Gal4..UAS-CD8::GFP) appear in blue in E–G9 and H–J9. With Ser loss of function in the glia (repo-Gal4..UAS-DNSer), there is a

strong advance of the proneural wave (arrowheads in H,H9; compare with control E,E9). In a posterior view, ectopic L9sc-expressing cells are detected in the middle of

the neuroepithelium (NE; arrowhead in I,I9; compare with F,F9). The white arrows indicate the normal position of L9sc at the medial (m) and lateral (l) position (note

that in I the proneural wave is so advanced in the most ventral part that no NE cells are detected). Most of these L9sc+ cells also ectopically express PntP1

(J,J9; compare with G;G9). (K,L) With Ser loss of function in the glia, the Notch reporter mb-CD2 significantly drops in the glia compared with the control (arrows).

The expression of mb-CD2 in the NBs is less affected because the expression of nrv2-Gal4 is weaker in NBs (arrowheads; see blue channel). (M,N9) The simultaneous

expression of DNSer and Ni in the glia (repo-Gal4..UAS-SerDN+UAS-Ni) rescues the expression of mb-CD2 in the glia (M,M9) but does not rescue the

ectopic L9sc+/PntP1+ cells present in the neuroepithelium (N,N9). (O–P9) With Ser loss of function in the glia (repo-Gal4..UAS-DNSer) there is, along with the

ectopic expression of L9sc (P), a high decrease in the expression of the Notch activity reporter m7-nuclacZ in NE cells (P,P9; compare with control O,O9). (Q) In

SerRX82 glial mutant clones, labeled with GFP, ectopic L9sc is detected in the underlying NE cells.

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Notch activity reporter m7-nuclacZ, activity that restricts the

EGFR–Ras–PntP1 signaling and hence L9sc expression to the

transition zone. Cno in a complex with Notch at the AJs of NE cells

somehow stabilizes Notch at the membrane, favoring the binding of

its ligand Ser present in the adjacent glia. Dl, which is enriched at

the NE cells of the transition zone (Fig. 7A,A9), could preferentially

activate the Notch target gene Gbe+Su(H)lacZ at this location (and

in the emerging NBs). Indeed, the ectopic activation of Dl in all NE

cells (Fig. 7B,B9) led to a concomitant ectopic activation of the

Gbe+Su(H)lacZ reporter along with Dpn (100%; n512)

(Fig. 7D,E), as well as to a repression of the m7-nuclaz reporter

in NE cells (100%; n57) (Fig. 7F,G). Intriguingly, when Dl was

ectopically activated in the whole surface glia, it was exclusively

detected in the perineurial glia (Fig. 7C–C90), being completely

absent in the subperineurial glia where Ser accumulates strongly in

contact with neuroepithelial Notch (Fig. 7A; see the Discussion).

DiscussionGlial cells are key players during the development of the nervous

system, and their number is indicative of nervous system

complexity (Nave and Trapp, 2008). In Drosophila, larval glia

are essential during the development of the brain, where they

display multiple functions (Hartenstein, 2011). In this work, we

provide evidence that glia play a key role during optic lobe

development and proneural wave progression.

A Ser–Notch–Cno complex at the interface between theglia and NE cells

Cno and its vertebrate homologues AF-6/Afadin localize at

epithelial AJs where they regulate the linkage of AJs to the actin

cytoskeleton by binding both actin and nectin family proteins

(Lorger and Moelling, 2006; Mandai et al., 1997; Matsuo et al.,

1999; Sawyer et al., 2009; Takahashi et al., 1998). Here, we have

Fig. 6. Notch activity in the glia promoted by Ser

regulates glia proliferation. (A–F0) Detail of an optic

lobe and the surrounding glia with Repo in red and

PH3 in green. Ser loss of function in the glia (repo-

Gal4 . .UAS-DNSer) causes a statistically

significant decrease of Repo and PH3-positive cells in

the glia (D–D0,G,H). The overexpression of Ni in the

glia (repo-Gal4..UAS-Ni) causes the opposite

phenotype: a significant increase in the number of

Repo+ cells, as well as in the number of PH3+ cells in

the glia compared with the control

(A–A0,E–E0,G,H). The simultaneous overexpression in

the glia of SerDN and Ni rescues the phenotype of

SerDN in the glia (F–H). cno knockdown (c855a-

Gal4..UAS-cnoRNAi) and overexpression (c855a-

Gal4..UAS-cno::GFP) in NE cells display similar

phenotypes to those seen with the loss and gain of

Notch signaling (B–C0,G,H). (G,H) Quantification of

the phenotypes analyzed; average Repo+ cell number

(G) and percentage (mean 6 s.e.m.) of glial PH3+ cells

(H) per optical section analyzed in the genotypes

specified. n5number of total optical sections analyzed

from different optic lobes. *P,0.05; **P,0.01;

***P,0.001. Repo+ cells with knockdown of cno

(UAS-cnoRNAi) were not analyzed because of the

disorganization of glial cells intermingled with the

neuroepithelium. (I–J9) Overexpression of cno in the

neuroepithelium leads to an increase of Notch activity

in the glia, as revealed with the Notch reporter

Gbe+Su(H)lacZ (arrows in J,J9), which is barely

detected in the glia in control embryos (arrows in I,I9).

There is also an expansion of the Gbe+Su(H)lacZ

expression domain in the transition zone (TZ) where

more Dpn-positive cells (‘PI cells’) are detected.

(K–L9) The knockdown of cno in NE cells (c855a-

Gal4..UAS-cnoRNAi) leads to a decrease in the

levels of the Notch activity reporter mb–CD2 in the

subperineurial glia, in contact with the

neuroepithelium (arrows in L9, compare with K9).

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found that Cno colocalizes with Notch at the AJs of NE cells in

the optic lobe proliferation centers. Notch also colocalized with

its ligand Ser; they accumulated strongly at the interface between

NE cells and the surrounding glia. Our co-immunoprecipitation

experiments indicate the formation of a Ser–Notch–Cno complex

in vivo, and the mutant analysis shows the functional relevance of

such a complex at the glia–neuroepithelium interface. The data

presented in this study support the hypothesis that Cno stabilizes

Notch at the AJs of NE cells, favoring the binding of Ser present

in the adjacent glial cells. Indeed, in cno loss-of-function

mutants, both Notch and Ser distribution is affected; this

alteration is accompanied by an abnormally advanced proneural

wave, a phenotype reminiscent of that shown by optic lobes with

Notch loss of function (Yasugi et al., 2010), and also with Ser

loss of function, as we show here. Activation of the Notch

pathway is essential to maintain the integrity of the

neuroepithelium and to allow the correct progression of the

proneural wave (Egger et al., 2010; Wang et al., 2011; Weng

et al., 2012). Our results show that glial Ser is responsible for

such activation, promoting the expression of the m7-nuclacZ

reporter in NE cells. In fact, the reduction of glial Ser either by

knocking down epithelial cno or by expressing DNSer in the glia

led to a decrease in the expression of the m7-nuclacZ reporter in

NE cells and to an ectopic activation of the Ras–PntP1 pathway

and of L9sc. We propose that this is ultimately the cause of the

proneural wave advance observed in those genotypes. Thus, the

activation of Notch in the neuroepithelium by glial Ser, in normal

conditions, would be essential to avoid a premature activation of

the EGFR–Ras–PntP1 pathway and hence of L9sc. Indeed, Notch

has been shown to downregulate different EGFR–Ras signaling

pathway components such as Rhomboid (Rho), required for the

processing of the EGFR ligand Spitz, in other developmental

contexts where both pathways are actively cross-talking (Carmena

et al., 2002). Therefore, Notch activity in NE cells could be

contributing to inhibit Rho, restricting its presence to the transition

zone where Rho is very locally expressed (Yasugi et al., 2010).

We observed that, in wild-type conditions, Ser is present in all

surface glia (perineurial and subperineurial), as shown by the

expression of CD8::GFP (SerGal4..UAS-CD8::GFP). Notch,

as tested by different reporters, is active in this tissue and highly

reduced with Ser loss of function in the glia. This suggests the

existence of Ser–Notch-mediated intercellular communication

between the glial cells that comprise both the perineurial

and subperineurial glia. Intriguingly, the knockdown and

overexpression of cno in NE cells also had a clear effect on

Notch activity in the glia: a reduction and an increase, respectively.

This is more challenging to explain. Because the cno loss of

function in the NE led to a high reduction of both neuroepithelial

Fig. 7. Dl and Ser locate at different domains and

activate different Notch target genes during optic lobe

development. (A–B9, D–G) The overexpression of Dl

within the neuroepithelium extends the Dl domain

(B,B9), normally restricted to the transition zone

(A,A9), causing ectopic activation of the Notch activity

reporter Gbe+Su(H)lacZ along with Dpn (E, compare with

the control D) and a repression of the reporter m7-nuclacZ

(G, compare with the control F). (C–C90) Overexpression of

Dl in the glia leads to an accumulation of Dl in the

perineurial glia but it is not detected in the subperineurial

glia in tight contact with the neuroepithelium (arrowheads),

where Ser is normally accumulated (A). (H) Working

model. Glial Ser autonomously triggers Notch activity in the

glia, as detected with the Notch reporters mb-CD2, md-lacZ

(both in red) and Gbe+Su(H)lacZ (in green), and non-

autonomously in the neuroepithelium, as visualized with the

Notch reporter m7-nuclacZ (purple). Cno binds Notch at the

AJs of NE cells and Notch binds glial Ser. This complex is

essential for the activation of m7-nuclacZ in the

neuroepithelium by Ser. This allows the local activation of

the Ras–PntP1 pathway only at the TZ where Ras–PntP1

signaling, in turn, locally activates L9sc. Dl activates the

Notch activity reporter Gbe+Su(H)lacZ at the TZ. The

emerging medulla NBs express three of the four Notch

activity reporters analyzed: Gbe+Su(H)lacZ, mb-CD2

and m7-nuclacZ.

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Notch and glial Ser, the easiest explanation is that an ‘excess’ of

unbound glial Ser is degraded and this impinges on the generalthresholds of glial Ser, therefore causing a general reduction in theNotch activity in this tissue. This will be an interesting area to

explore in detail and we leave the question open for futureinvestigation.

Notch target genes are differentially expressed throughoutoptic lobe and glia development

The activity of Notch in the neuroepithelium and in medulla NBsseems controversial. For example, Notch has been shown to beactive in the neuroepithelium at low or null levels (Wang et al.,

2011; Weng et al., 2012; Yasugi et al., 2010) or in a ‘salt andpepper’ pattern (Egger et al., 2011). Weak or no activity of Notchin NBs has also been reported (Egger et al., 2010; Yasugi et al.,

2010), as well as a high activation (Ngo et al., 2010; Weng et al.,2012). One possibility to conciliate all these results and apparentlycontradictory data is that different Notch target genes used as

Notch activity reporters are, in fact, differentially activated inparticular regions or tissues. Our results support this proposal.Here, we have used four different Notch reporters,Gbe+Su(H)lacZ, E(spl)mb-CD2, E(spl)md-lacZ and E(spl)m7-

nuclacZ. Whereas m7-nuclacZ was expressed throughout theneuroepithelium, Gbe+Su(H)lacZ was restricted to the transitionzone, although both were expressed in medulla NBs along with

mb-CD2. In addition, mb-CD2 was strongly activated in the glia,whereas the Gbe+Su(H)lacZ and the md-lacZ reporters wereexpressed at much lower levels at this location. Differential

activation of Notch target genes has been previously reported andtissue-specific factors could contribute to this differentialexpression (Cooper et al., 2000; de Celis et al., 1996; Wech

et al., 1999). This is an intriguing scenario to analyze in the future.The in-depth analysis of other Notch reporter genes in thedeveloping optic lobe would contribute to clarify this issue.

Dl and Ser: two ligands for Notch during optic lobe andglia development

At third larval instar during optic lobe development, Dl ishighly restricted to two to three cells at the transition zone in the

neuroepithelium, where Dl activates Notch (Ngo et al., 2010;Weng et al., 2012; Yasugi et al., 2010). In this work, we havefound that the other ligand of Notch, Ser, is expressed in the

surrounding glia at this larval stage and it is strongly accumulatedat the interface with NE cells. Ser activates Notch in theneuroepithelium and this, in turn, would contribute to restrict the

activation of the Ras–PntP1 pathway and L9sc to the transitionzone. Intriguingly, we have observed that Ser preferentiallyactivates the Notch target gene m7-nuclacZ in theneuroepithelium, whereas Dl activates other Notch target genes,

including Gbe+Su(H)lacZ, in the transition zone. For example,the overexpression of Dl in NE cells caused an ectopic activationof Gbe+Su(H)lacZ throughout the neuroepithelium, along with

dpn, which also behaves as a Notch target in other systems(Krejcı et al., 2009), and a repression of m7-nuclacZ (Fig. 7D–G). In addition, the loss of function of Ser in the glia caused a

striking decrease in the expression of m7-nuclacZ in theneuroepithelium (Fig. 5O–P9). One possibility to explain theseobservations is that the pool of Notch associated with the AJs and

activated by glial Ser is subject to particular post-translationalmodifications or/and is associated with other AJ proteins(including Cno) that somehow make Notch more receptive to

Ser and able to activate specific target genes (i.e. m7). In this

regard, it is interesting to note that Dl ectopically expressed in theglia (i.e. repoGal4..UAS-Dl) was not detected at the interfacewith NE cells, where glial Ser is strongly localized in contact with

Notch (Fig. 4E–E0), but Dl was restricted to the outermost surfaceglia (perineurial glia) (Fig. 7C–C90). This result strongly indicatesthat Dl cannot bind or has very low affinity for this pool of Notch atthe AJs, hence it is actively degraded in the subperineurial glia.

The low affinity of Dl for Notch at this location further suggeststhat this pool of Notch at the AJs must be endowed with particularcharacteristics, as mentioned above, that ultimately could alter the

activity properties of Su(H), explaining in turn the distinctexpression pattern of Notch target genes. Another possibility,which is not necessarily exclusive, to explain the differential

activation of the Notch reporters is that they respond to differentNotch thresholds. For example, m7-nuclacZ could require very lowlevels of Notch activation, whereas Gbe+Su(H)lacZ might require

high amounts of Notch signaling in NE cells. All these questionsremain open for further investigation.

Materials and MethodsDrosophila strains and geneticsAll stocks used in this study are from the Bloomington Stock Center and theVienna Drosophila RNAi Center, unless otherwise noted: Gbe+Su(H)lacZ [41 bpof the E(spl)m8 promoter that contains Grainyhead binding sites plus Su(H)binding sites] (Furriols and Bray, 2001), E(spl)mb-CD2 (de Celis et al., 1998),E(spl)md-lacZ (Cooper et al., 2000), E(spl)m7-nuclacZ (a gift from S. Bray) (Pineset al., 2010), FRT82B cnoR2 (Sawyer et al., 2009), hsFLP, FRT82B-tubGal80, arm-Gal4, c855a-Gal4 (Manseau et al., 1997), FRT82B SerRX82 (Thomas et al., 1991),repo-Gal4, nrv2-Gal4, Ser-Gal4, UAS-mCD8::GFP, UAS-cnoRNAi, UAS-cno::GFP (Slovakova and Carmena, 2011), UAS-Dicer2, UAS-BdE24 or Sersec

(DNSer, strong expressor that produces the first 1020 amino acids, up to the firstBamHI site in Serrate; a gift from Robert Fleming) (Fleming et al., 2013). UAS-Ni,UAS-NDN (Rebay et al., 1993), UAS-Notch::GFP (Kawahashi and Hayashi, 2010),UAS-Dl, UAS-GFP. The crosses Gal4xUAS were carried out at 29 C with thefollowing exception: the cross Gal4xUAS-DNSer was also carried out at less-stringent conditions, specifically at 25 C followed by an incubation overnight at29 C, for analyzing the expression of L9sc and PntP1 in the neuroepithelium.

MARCM clonesTo generate clones of cells homozygote for the null allele cnoR2, hsFLP; UAS-

CD8::GFP; FRT82B tubGal80 flies were crossed with armGal4; FRT82B cnoR2,identifying the clones by the presence of CD8::GFP. hsFLP was activated for 2hours at 37 C in first- and second-instar larvae. Clones of the null allele SerRX82

were performed in a similar way using in this case the nrv2Gal4 driver to identifyglial mutant clones.

Histology, immunofluorescence and microscopyBrains were dissected from third-instar larvae and fixed and stained withantibodies using standard protocols unless specified below. The following primaryantibodies were used: rabbit anti-Cno 1:400 (Speicher et al., 2008); rat anti-L9sc1:100 (Martın-Bermudo et al., 1991); guinea pig anti-Dpn 1:1000 (a gift from J.Skeath); rat anti-Baz 1:500 (Wodarz et al., 1999); mouse anti-Notch intracellulardomain (C17.9C6) 1:50–1:100 [Developmental Studies Hybridoma Bank(DSHB)]; mouse anti-Notch extracellular domain (C458.2H) 1:50–1:100(DSHB); mouse anti-Dl 1:40 (DSHB); rat anti-Shg 1:100 (DSHB); goat anti-Ser1:20–1:100 (Santa Cruz); mouse anti-Dlg 1:100 (DSHB); mouse anti-Repo 1:100(DSHB); mouse anti-Nrv5F7 1:600 (DSHB); rabbit anti-PH3 1:400 (Upstate);rabbit anti-PntP1 1:300 (a gift from J. Skeath); rabbit anti-bgal 1:3000 (Cappel);mouse anti-bgal 1:800 (Promega); mouse anti-CD2 1:600 (AbD Serotec).Secondary antibodies coupled to biotin (Vector Labs), Alexa Fluor 488, 546 or633 (Molecular Probes) were used. For immunostaining with the anti-Cnoantibody, brains were fixed using the heat and methanol method (Tepass, 1996).Fluorescent images were recorded using a Leica upright DM-SL microscope andassembled using Adobe Photoshop. Most of the micrographs shown in figuresrepresent single sections from confocal Z-stacks, with the exception of someaverage projections in the following figures: Fig. 2A-D,D9 and Fig. 5E,E9,H,H9.

Co-immunoprecipitationsFor in vivo Co-IPs, lysates were prepared from third-instar larval headsobtained from the following crosses: c855a-Gal4..UAS-Notch::GFP, c855a-Gal4..UAS-cno::GFP and c855a-Gal4..UAS::GFP as a negative control.

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Sectioned heads were homogenized in lysis buffer [50 mM Tris-HCl, pH 8,150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, 1 mM NaF, 100 mMNa3VO4, 2 mM PMSF and complete protease inhibitors (Roche)]. Extracts werecentrifuged for 2 minutes at 14,000 rpm (18,700 g) at 4 C. The supernatant wasincubated with Rb polyclonal antibody to GFP Sepharose beads (Abcam) for2 hours at 4 C. The beads then were washed three times with lysis buffer withoutinhibitors, resuspended in protein-set buffer (Fluka) and heated at 95 C for3 minutes. Precipitates were resolved by SDS-PAGE and immunoblotted withmouse anti-GFP (Clontech), rabbit anti-Cno (affinity purified) or goat anti-Serrate(Santa Cruz). Each experiment was performed at least twice.

Statistics

Measurement of Repo+ and PH3+ cells was made by imaging brain frontal sectionsevery 3 mm to ensure that the same cell was not counted twice. The area analyzedwas defined according to anatomical references, starting when the lobula plateneuropile appears (Ngo et al., 2010) and following in an antero-posterior directionuntil the disappearance of the outer neuroepithelium. 8–12 sections were sampledfor each brain and only surface glia were counted for statistics. Parametric andnon-parametric analyses were carried out with similar results to test differences inthe proliferation of glial cells with respect to control brains; slight differences instatistical significance were found in the genotype UAS-Ser+UAS-Ni between theparametric (**) and the non-parametric (*) tests for the percentage of PH3+ cellnumber (Fig. 6H); GLM and N-PAR1WAY procedures were performed tocompare variances between treatments and Duncan test was used for comparisonof averages using SAS v9.2.

AcknowledgementsWe thank Sarah Bray, Jim Skeath, Bob Fleming, Ben Ohlstein, IboGalindo, Mark Peifer, Andreas Wodarz, Yuh N. Jan, Utpal Banerjee,the Bloomington Drosophila Stock Center at the University ofIndiana, the Vienna Drosophila RNAi Center and the DevelopmentalStudies Hybridoma Bank at the University of Iowa for kindlyproviding fly strains and antibodies.

Author contributionsR.P.G. performed most of the experimental work and analyzed thedata; J.S. did the Co-IP experiments; N.R.Q. performed the MARCMclones; A.K. provided support and lab space to finish the work; A.C.designed and supervised the work, analyzed the data and wrote themanuscript.

FundingThis work was supported by the Spanish Government [grant numbersBFU2009-08833, BFU2012-33020 and CONSOLIDER-INGENIO2010 CSD2007-00023 to A.C.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.125617/-/DC1

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