a SPECIAL ISSUE REVIEWS-A PEER REVIEWED FORUM

A Glimpse Into Dorso-Ventral Patterningof the Drosophila EyeAmit Singh,1,2,3* Meghana Tare,1 Oorvashi Roy Puli,1 and Madhuri Kango-Singh1,2,3*

During organogenesis in all multi-cellular organisms, axial patterning is required to transform a singlelayer organ primordium into a three-dimensional organ. The Drosophila eye model serves as an excellentmodel to study axial patterning. Dorso-ventral (DV) axis determination is the first lineage restrictionevent during axial patterning of the Drosophila eye. The early Drosophila eye primordium has adefault ventral fate, and the dorsal eye fate is established by onset of dorsal selector gene pannier (pnr)expression in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral com-partments called the equator is the site for Notch (N) activation, which triggers cell proliferation and dif-ferentiation. This review will focus on (1) chronology of events during DV axis determination; (2)how early division of eye into dorsal and ventral compartments contributes towards the growth and pat-terning of the fly retina, and (3) functions of DV patterning genes. Developmental Dynamics 241:69–84,2012. VC 2011 Wiley Periodicals, Inc.

Key words: Drosophila eye; eye development; axial patterning; dorso-ventral patterning; dorsal selector; ventral eyegenes; Lobe; Serrate; Pannier; Homothorax; Teashirt; Wingless

Accepted 6 September 2011

INTRODUCTION

During development, the patterningsignals progressively restrict cellfates by subdividing a large develop-ing field into smaller fields with lim-ited developmental potential. Thesesmaller fields that correspond to thedomains of expression of selectorgenes are referred to as compart-ments. The selective spatio-temporalexpression pattern of the cell fate se-lector genes is responsible for the for-mation of compartments (Blair, 2001;Curtiss et al., 2002; Held, 2002; Dah-mann et al., 2011). The boundarybetween the compartments, wheretwo different cell types are juxta-posed, is responsible for generating

new signaling centers to regulate pat-terning, growth and differentiation ofa developing field (Meinhardt, 1983;Blair, 2001). Thus, formation of thedevelopmental boundaries is crucialfor maintaining the downstream pat-terning events (Blair, 2001; Curtisset al., 2002; Dahmann et al., 2011).Therefore, an important question indevelopmental biology is how theseboundaries are generated and main-tained during development. The aimof this review is to provide an over-view of recent advances in our under-standing of generation of the bound-ary between the dorsal and ventralcompartment of the eye and its impli-cations on development of the eye as

an organ. This process is referred toas Dorso-Ventral (DV) patterning ofthe Drosophila eye. The Drosophilaeye, an ideal model system for study-ing organogenesis, has been exten-sively used to investigate tissue pat-terning and cell–cell communicationduring axial patterning. Further-more, Drosophila eye serves as anexcellent model to understand thegenetic mechanism responsible for di-vision of a developing field into sev-eral smaller fields with positional faterestrictions (Singh et al., 2005b). Thegenetic machinery that controlsDrosophila eye development closelyresembles that of higher vertebrates,suggesting conservation of certain

1Department of Biology, University of Dayton, Dayton, Ohio2Premedical Program, University of Dayton, Dayton, Ohio3Center for Tissue Regeneration and Engineering at Dayton (TREND), University of Dayton, Dayton, OhioGrant sponsor: NIH; Grant number: 1R15 HD064557-01; Grant sponsor: University of Dayton.*Correspondence to: Amit Singh or Madhuri Kango-Singh, Department of Biology, University of Dayton, Dayton, OH45469. E-mail: amit.singh@notes.udayton.edu or kangosmz@notes.udayton.edu

DOI 10.1002/dvdy.22764Published online 27 October 2011 in Wiley Online Library (wileyonlinelibrary.com).

DEVELOPMENTAL DYNAMICS 241:69–84, 2012

VC 2011 Wiley Periodicals, Inc.

Dev

elop

men

tal D

ynam

ics

genetic pathways throughout evolu-tion (Wawersik and Maas, 2000;Gehring, 2005; Erclik et al., 2009;Kumar, 2009). The axial pattern ofthe eye disc has not been well studieduntil recently due to the complexity ofeye development.

Axial patterning, which is essentialfor organogenesis in all the multi-cel-lular organisms, involves formation ofAntero-Posterior (AP), Dorso-Ventral(DV), and Proximo-Distal (PD) com-partments (Cohen et al., 1993; Cohen,1993). During axial patterning of thewing- and the leg-imaginal discs, thesequence of events involves the divi-sion of a field into anterior and poste-rior compartments of independent celllineages, followed by subdivision ofthese imaginal discs into dorsal andventral compartments. Interestingly,this sequence of division is not fol-lowed in the developing eye imaginaldisc because it does not have analo-gous anterior-posterior axis. Instead,DV patterning is the first lineagerestriction in the developing eyeimaginal disc (Singh and Choi, 2003;Singh et al., 2005b). Despite the dif-ferences in the sequence of events,evidence suggests that some aspectsof the DV patterning mechanism arehighly conserved in the developingeye and the wing. An important com-mon conclusion is that the borderbetween DV compartments is a centerfor organizing the growth and pat-terning of the disc. In subsequent sec-tions of the review, we will focus onthe mechanism involved in generationof DV domains in the developing eye,and the genetic basis for the estab-lishment of the DV pattern.

EYE IMAGINAL DISC

In Drosophila, a holometabolousinsect, the primordia of all the adultstructures are sequestered in thelarva as epidermal invaginations thatare called imaginal discs (Bodent-stein, 1950; Ferris, 1950; Atkins andMardon, 2009). The adult eye,antenna, head cuticle, and head struc-tures develop from a common develop-ing field called an eye-antennal imagi-nal disc (Cohen, 1993; Held, 2002).The regions of the eye-antennal imag-inal disc, which give rise to headstructures including ptilinum, frons,and maxillary palpus, originate from

five embryonic segments and theacron (Jurgens and Hartenstein,1993; Younossi-Hartenstein and Har-tenstein, 1993). The monolayer epi-thelium does not accurately reflect thesac-like anatomy of the imaginal discs(Gibson and Schubiger, 2001). Dro-sophila imaginal discs are a contigu-ous cell sheet of flattened epithelialcells with two opposing surfaces, a co-lumnar epithelium called disc proper(DP) and a squamous peripodial epi-thelium called the peripodial mem-brane (PM) (McClure and Schubiger,2005; Atkins and Mardon, 2009). TheDrosophila retina develops from theDP while the PM of the eye-antennaldisc gives rise to the adult head struc-tures (Fig. 1; Milner et al., 1983; Hay-nie and Bryant, 1986; Atkins andMardon, 2009). The eye-antennalimaginal disc emerges from an embry-onic imaginal primordium, which isan anterior-dorsal sac comprisingapproximately 20 cells that are setaside during mid-embryogenesis(Poulson, 1950; Garcia-Bellido andMerriam, 1969; Yamamoto, 1996).The embryonic precursors for imagi-nal discs grow asynchronously fromthe rest of the developing embryo(Anderson, 1972a,b; Crick and Law-rence, 1975; Cohen, 1993; Held, 2002;Kumar, 2011). In the first two larvalstages, the eye imaginal disc cellsdivide and grow. The distinctionbetween the developing antenna andthe eye field begins to appear duringthe second instar larvae (Kumar andMoses, 2001; Kenyon et al., 2003;Dominguez and Casares, 2005; Atkinsand Mardon, 2009). The developingeye field is further divided into pre-cursors for the eye proper, head cuti-cle, and the ocelli while the antennalfield divides into precursors for theantenna and head cuticle. Retinal dif-ferentiation begins from the posteriormargin of the eye imaginal disc dur-ing late second instar or early thirdinstar stage of larval development(Ready et al., 1976). Since the retinaldifferentiation is synchronous in na-ture, it appears like a wave of differ-entiation initiating at the posteriormargin of eye imaginal disc, whichthen proceeds anteriorly. The wave ofdifferentiation is referred to as themorphogenetic furrow (MF, Fig. 1 A,A’, arrowhead). It results in transitionof an undifferentiated epithelium to

differentiated cell types comprised ofregularly spaced photoreceptor clus-ters (Ready et al., 1976; Wolff andReady, 1993). Posterior to the furrow,photoreceptor clusters are generatedby a sequence of events including theselection of the R8 founder neuronand recruitments of additional photo-receptor precursors in the order of R2/5, R3/4, and R1/6/7 (Wolff and Ready,1993; Kumar, 2011). Thus, if the eyeimaginal disc is largely undifferenti-ated until second instar of develop-ment, an interesting issue is how com-partments are identified in theDrosophila eye imaginal disc.

DORSAL AND VENTRAL

COMPARTMENTS AND

THE EQUATOR

The adult compound eye is comprisedof approximately 800 unit eyes orommatidia (Fig. 1D). Each ommati-dium consists of eight photoreceptorneurons assembled in an asymmetrictrapezoidal pattern, and when viewedfrom the top it resembles a honey-comb-like, hexagonal facet. The sur-rounding cell types are non-neuronaland include pigment, cone cells, andmechanosensory bristles (Fig. 1E, F;Wolff and Ready, 1993). This patternof organization is repeated in allommatidia of the eye. However, de-spite the similarity in the cellularcomposition of each ommatidium, thespatial arrangement of ommatidia inthe eye is organized in two orienta-tions (Fig. 1E, F). These two orienta-tions also serve as a marker to distin-guish the dorsal and the ventralhalves of adult eye. The photorecep-tors are arranged in a trapezoidalfashion within an ommatidium. Theommatidial clusters within an eye areorganized in a mirror asymmetry asthey are polarized in the oppositedirections (Fig. 1E, F). The boundarybetween the ommatidia of the dorsalhalf and their mirror image ventralommatidia is referred to as an equa-tor (Fig. 1E, F). This mirror symme-try, which corresponds to DV axis orcompartments of the adult eye, hasbeen described in many insect eyes(Dietrich, 1909). Since the develop-mental mechanisms underlying theDV pattern have not been studied indetail, it raises an interesting

Dev

elop

men

tal D

ynam

ics

70 SINGH ET AL.

question of how the dorsal and ven-tral pattern is established.

The pioneering studies to discernthe relation between the equator andthe DV compartmental boundary inthe Drosophila eye suggested that theequator is not determined as theboundary between the dorsal and ven-tral cell lineages (Ready et al., 1976).Even though the result from thisstudy does not exclude the possibilitythat the dorsal and the ventraldomains of the eye derive from two in-dependent cell lineages, the lineageboundary may not precisely corre-spond to the equator. A series of ele-gant genetic analysis experimentsinvolving a large number of mosaicclones in the adult eye and the headsupported this idea (Baker, 1978).These experiments demonstrated thatclones strictly follow the DV bound-ary, and they do not intermingle nearthe DV border (Held, 2002). Theseresults validated the hypothesis thatDrosophila eye derives from DV com-partments. The wing imaginal disc isdivided into anterior and posteriorgroups of cells in its early stage of de-velopment, which is followed by fur-ther partitioning into dorsal and ven-tral compartments (Lawrence andMorata, 1976). To analyze whetherthe eye and the head are also subdi-vided into different domains by se-quential compartmentalization as inthe wing, another mosaic analysiswas carried out. Nearly all clones(96%) were restricted to either dorsalor ventral domains of the eye, therebyconfirming the presence of a boundarybetween the dorsal and ventral cells.Thus, clones generated in the dorsalor ventral compartment did not crossthe DV boundary. A few clones (4%)did cross the DV border, which wasprobably due to the fact that suchclones might have been induced priorto compartmentalization (formation ofdorsal and ventral compartmentboundary) or two independent dorsaland ventral clones might have juxta-posed at the equator region therebygiving a false notion of a single clonenot respecting the DV boundary(Baker, 1978). The DV lineage restric-tion observed in the adult eye wasalso confirmed in the developing eyeimaginal disc where large clones donot cross the DV midline and showeda sharp outline along the DV midline,

and the clones located within the dor-sal or ventral domain had wiggly bor-ders (Dominguez and de Celis, 1998).Unlike the DV lineage restriction thatis established in the first instar larvaleye imaginal discs, there is evidencethat anterior-posterior restrictionoccurs a little later in the secondinstar eye imaginal disc. Thus, thesignificance of this anterior-posteriorrestriction remains to be seen as themorphological distinction between theanterior and posterior regions. In sub-sequent sections, we discuss the roleof DV patterning genes and the gener-ation of pattern along the DV axis ofthe eye.

GENESIS OF THE EYE

The DV boundary has been suggestedas the site for the activation of Notch(N) signaling in the eye imaginal discto promote growth (de Celis et al.,1996; Go et al., 1998; Baonza andGarcia-Bellido, 2000). However, if DVpatterning occurs as late as seen inthe adult eye based on the orientationof the photoreceptors, then it may notbe crucial for the growth of the eye.Thus, efforts were channeled towardsinvestigating whether DV patterningtakes place earlier during eye devel-opment. Seminal reports from threedifferent groups established that DVlineage restriction takes place earlierduring larval eye development due todomain-specific expression of the DVpatterning genes (Cho and Choi,1998; Dominguez and de Celis, 1998;Papayannopoulos et al., 1998). TheDV patterning genes are a class ofgenes involved in generating andmaintaining the DV lineage in theeye. These reports identified a newtime line for the initiation of DV pat-terning to early larval development.They also identified the genes whoseexpression and/or function was re-stricted either to the dorsal or ventralcompartment of the eye. Thus, it wasproposed that DV patterning in theeye is generated by the domain-re-stricted expression of the dorsal andthe ventral eye genes (Cho and Choi,1998; Dominguez and de Celis, 1998;Papayannopoulos et al., 1998; Choet al., 2000).

The basic question was what is thedefault state of the early eye primor-

dium? To answer this question, sev-eral groups directed their efforts toidentify the genes that are expressedin the early eye primordium. Duringembryonic development, the eye pri-mordium begins as a homogenousgroup of cells that continue to growduring first larval instar to form theeye imaginal disc. All the cells of thefirst-instar eye imaginal disc uni-formly express Lobe (L), a geneknown to be involved in ventral eyedevelopment (Singh and Choi, 2003).These studies revealed that until thelate first instar of larval eye develop-ment, the entire eye primordium isventral in fate and depends on thefunction of ventral genes like L andits downstream target Serrate (Ser)(Singh and Choi, 2003; Singh et al.,2005b; Kumar, 2011). Loss-of-functionof the L gene, which is expressedubiquitously in the eye imaginal disc(see Fig. 3A), results in selectivegrowth defects in the ventral half ofthe eye (Figs. 2, 3C,D). The loss-of-function studies suggested that therequirement of L function evolvesalong the temporal axis (Singh andChoi, 2003; Singh et al., 2005b). Dur-ing early eye development, loss-of-function of L results in the completeloss of eye field (Fig. 3B). However,loss of L gene function later duringeye development causes selective lossof the ventral half of the eye (Singhet al., 2005b). Loss-of-function of Seralso results in the similar loss of theventral eye phenotype (Table 1; Singhand Choi, 2003; Singh et al., 2005b).Interestingly, the timing of restrictionof L/Ser functional domain from theentire developing eye field (Fig. 3D,E) to only the ventral half of the eye(Fig. 3B, C) corresponds to the onsetof pannier (pnr) gene expressionalong the dorsal margin of the eye(Table 1; Fig. 2). During late firstinstar of eye development, the entirehomogenous population of the ventralcells of the eye primordium transitioninto two distinct dorsal and ventrallineages with the onset of pnr expres-sion on the dorsal eye margin (Singhand Choi, 2003). This suggests thatthe ventral fate is the ground state ofthe larval eye imaginal disc, and Land Ser are essential for survival and/or maintenance of this ventral state(Singh and Choi, 2003; Singh et al.,2005b, 2006).

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 71

Fig. 1.

Fig. 2.

Dev

elop

men

tal D

ynam

ics

72 SINGH ET AL.

DV PATTERNING IN THE

EYE

Genes Regulating Ventral

Eye Growth

L, a gene involved in ventral eye de-velopment (Table 1), was firstreported in 1925, as a gene requiredfor eye growth (Morgan et al., 1925)and was cloned in 2002 (Chern andChoi, 2002). L encodes an ortholog ofPRAS40 (Oshiro et al., 2007; VanderHaar et al., 2007; Wang and Huang,2009), and it is required during allstages of larval eye development(Chern and Choi, 2002; Singh et al.,2005b). L protein is expressed in bothdorsal and ventral domains through-out eye imaginal disc development(Fig. 3A). Even though L is expresseduniformly in the entire eye field, Lfunction is not required for growthand differentiation in the dorsalregion of the eye (Chern and Choi,2002; Singh et al., 2005b). Evidencefor this ‘‘domain-specific’’ function ofL (ventral specificity) came fromgenetic mosaic analysis using a nullmutation (Chern and Choi, 2002;Singh and Choi, 2003). Loss-of-func-tion clones of L show an eye suppres-sion phenotype specifically in the ven-tral eye; however, the dorsal clones donot suppress eye fate and exhibit

well-organized photoreceptor clusters(Chern and Choi, 2002; Singh andChoi, 2003). Further genetic analysisrevealed that this domain-specific Lfunction in growth was downstreamto N-signaling, mediating N functioneither in the same or parallel path-way (Chern and Choi, 2002). Thus,the growth of early eye disc is con-trolled asymmetrically in the dorsaland ventral domains. Expression of Lin both dorsal and ventral domains ispuzzling since its function is onlyrequired for growth of the ventral do-main. It is possible that an unidenti-fied partner that is expressed in thedorsal domain of the eye imaginaldisc antagonizes the L function, or Lmay be selectively activated by someyet to be identified partner in the ven-tral domain.

Another candidate gene that maybe contributing to ventral eye devel-opment is decapentaplegic (dpp), amember of the TGF-b family of pro-teins, which acts as a long-rangesecreted morphogen (Table 1; Nellenet al., 1996; Chanut and Heberlein,1997). Dpp forms a gradient in theearly eye anlage (anterior brain andeye field) that transverses from dorsalto ventral (Chang et al., 2001). In theearly eye imaginal disc, Dpp is prefer-entially expressed in the ventral eye

domain (Cho et al., 2000). In dppmutants, the ventral part of early eyedisc exhibits similar pattern defectsas seen in L mutants. This dpp pheno-type may be an outcome of ectopicinduction of pnr or wg expression inthe ventral domain as observed in Lmutants (Singh et al., 2005a). In theDP of early eye imaginal disc Dpp,Hedgehog (Hh) and Wg signalingfrom the peripodial membrane isrequired to trigger N activation. Simi-lar to limb patterning and develop-ment (Brook and Cohen, 1996; Pentonand Hoffmann, 1996; Theisen et al.,1996), during eye imaginal disc devel-opment, Dpp antagonizes Wg. Thisantagonistic interaction occurs in theperipodial membrane across the DVborder (Cho et al., 2000). Thus, Dppsignaling plays a role in inducing DVpolarity from peripodial membrane.Ser is the N ligand in the ventral

domain of the eye imaginal disc (Table1; Speicher et al., 1994; Cho and Choi,1998; Dominguez and de Celis, 1998;Papayannopoulos et al., 1998; Domi-nguez and Casares, 2005). Ser actsdownstream of L in the ventral eye asSer transcription is repressed in earlyeye discs from Lsi homozygous larvae.Evidence for this conclusion comesfrom the Ser-lacZ reporter expres-sion. Interestingly, Ser-lacZ expres-sion in the posterior medial regionremains at a significant level. Loss-of-function clones of L cause strongreduction of Ser in the ventral eyewhereas increased levels of L usingthe ‘‘flp out’’ approach induce Serexpression even in the dorsal domainof eye imaginal disc. Thus, L that actsdownstream to N may be involved ininducing the Ser expression in thedeveloping eye imaginal disc (Chernand Choi, 2002). Hypomorphic allelesof Ser exhibit reduced eye size sug-gesting a role for Ser in eye develop-ment. However, Ser loss-of-functionclones do not exhibit any phenotypesin the eye (Sun and Artavanis-Tsako-nas, 1996; Papayannopoulos et al.,1998; Chern and Choi, 2002), but mis-expression of the dominant-negativeform of Ser (SerDN) causes severegrowth defects in the eye imaginaldisc (Kumar and Moses, 2001; Singhand Choi, 2003). It is possible that Serfunction may be compensated byanother factor, or Ser may somehowbe secreted or transendocytosed into

Fig. 1. Drosophila eye is a highly organized structure. A–C: Eye antennal imaginal disc of athird-instar larva. B: Eye imaginal disc stained for membrane marker Disc large (Dlg: green) andElav (red), a pan-neuronal marker that marks the photoreceptor neurons are shown. Arrowheadmarks the position of the morphogenetic furrow (MF). C, C’: Eye imaginal disc stained with Bar (B:green) to mark DV polarity. D: Scanning electron micrograph (SEM) of the wild-type Drosophilaeye. Adult eye is a compound eye which is made up of 750–800 unit eyes, each termed an omma-tidium (Wolff and Ready, 1993). All ommatidia are organized as a regularly spaced hexagonarranged in mirror image symmetry along the dorso-ventral (DV) axis. E: Cross section of the adultcompound eye showing arrangement of ommatidial clusters along DV axis (Wolff and Ready,1993). Note that in each ommatidium, eight rhabdomeres are organized in an asymmetric trape-zoidal fashion. All these ommatidia within a compound eye are organized in two basic clustersbased on the orientation of the trapezoid. If the R3 rhabdomere points up, it is dorsal, whereas thereverse is ventral. F: Cartoon showing the mirror image symmetry of ommatidial cluster orientationin the dorsal and ventral half of the adult eye. All the blue arrows mark the ommatidia in the dorsalhalf of the eye, while the red ones mark the ventral half of the eye. All images are oriented as Dor-sal (up), Ventral (down), Anterior (right), and Posterior (left). AN, antenna.

Fig. 2. Ventral is the default state of the early eye imaginal disc. Larval eye primordium com-prising a few cells require the function of ventral genes L/Ser for growth and proliferation (Singhand Choi, 2003). Loss-of-function phenotype of L/Ser in the developing eye imaginal discevolves along the temporal scale. During early first instar of larval development, loss-of-functionof L/Ser results in complete loss of the eye field. However, after the onset of pnr expression dur-ing early second larval instar, which results in generation of DV lineage in the developing eyeimaginal disc, loss of Drosophila results in loss of only the ventral half of the eye. However, inthe late third-instar stage of development when retinal differentiation is almost complete, loss ofL/Ser does not have a significant affect on overall adult eye morphology. Based on these results,it was proposed that the entire early eye primordium, prior to onset of pnr expression, is ventralin fate (Singh and Choi, 2003).

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 73

TABLE 1. Genes Involved in Dorso-Ventral Patterning and Domain-Specific Expression and Growth

Drosophila

Vertebrate

homolog Nature Function in eye References

Ventral genesfringe (fng) Lunatic

fringeGlycosyl

transferaseSecreted signaling protein,

DV boundary formationIrvine and Wieschaus, 1994;

Cho and Choi, 1998; Dominguezand de Celis, 1998;Papayannopoulos et al., 1998

Serrate (Ser) Jagged-1 Ventral N ligand Ventral eye growthand development

Speicher et al., 1994; Cho and Choi,1998; Dominguez and de Celis,1998; Papayannopoulos et al.,1998; Cho et al., 2000

Chip Nli/Ldb1/Clim-2

Transcriptionco-factor

Define ventral eyeboundary

Roignant et al., 2010

Sloppy paired(Slp)

BF-1 (notcompletehomology)

Forkheadtranscriptionfactor

Ventral eye growth Sato and Tomlinson, 2007

decapentaplegic(dpp)

BMP TGF-b Ventral growth Chanut and Heberlein, 1997;Singh et al., 2005b

Dorsal genespannier (pnr) GATA-1 Zinc finger,

GATA familyDorsal eye fate selector Ramain et al., 1993;

Maurel-Zaffran and Treisman,2000; Gomez-Skarmeta andModolell, 2002; Singh et al.,2005b; Oros et al., 2010

araucan (ara) Irx 1, 3 Homeodomain Dorsal eye fate selector Gomez-Skarmeta and Modolell,1996; Cavodeassi et al., 1999;Pichaud and Casares, 2000;Gomez-Skarmeta andModolell, 2002

Caupolican (caup) Irx2, 5 Homeodomain Dorsal eye fate selector Gomez-Skarmeta and Modolell,1996; Cavodeassi et al., 1999;Pichaud and Casares, 2000;Gomez-Skarmeta andModolell, 2002

mirror (mirr) Irx 4, 6 Homeodomain Dorsal eye fate selector McNeill et al., 1997;Heberlein et al., 1998;Kehl et al., 1998; Yang et al.,1999; Singh et al., 2005b

Delta (Dl) Delta like3 (DLL3)

TransmembraneNotch Ligand

Dorsal Notch(N) Ligand

Cho and Choi, 1998; Dominguez andde Celis, 1998; Papayannopouloset al., 1998; Cho et al., 2000;Dominguez and Casares, 2005;Singh et al., 2005b

Genes with DV asymmetric responsehomothorax

(hth)Meis Homeodomain Ventral eye suppression Rieckhof et al., 1997; Pai et al., 1998;

Ryoo et al., 1999; Pichaud andCasares, 2000; Dominguez andCasares, 2005; Singh et al., 2005b;Bessa et al., 2008

teashirt (tsh) TSH1, TSH2,TSH3

C2H2 zinc fingertranscription factor

Dorsal eye growth,ventral eye suppression

Fasano et al., 1991; Pan andRubin, 1998; Bessa et al., 2002;Singh et al., 2002, 2004;Datta et al., 2009

Lobe (L) PRAS40(Ortholog)

Proline rich Aktsubstrate

Ventral eye growth,no affect on dorsal eye

Chern and Choi, 2002; Singh andChoi, 2003; Singh et al., 2005a,b,2006; Wang and Huang, 2009

Expression on both marginsoptomotor

blind (omb)Tbx5 Transcription factor Expressed on dorsal

and ventral eyemargin. Not known.

Calleja et al., 1996; Singh et al., 2004

Dev

elop

men

tal D

ynam

ics

74 SINGH ET AL.

neighboring cells as shown in experi-ments performed using a cell culturesystem (Klueg and Muskavitch, 1999;Kumar and Moses, 2001; Singh et al.,2005b). This may explain the appa-rent lack of phenotype in Ser mutantclones. Misexpression of SerDN inearly eye imaginal disc using ey-Gal4(Hazelett et al., 1998) results in eitherpreferential loss of ventral eye or lossof the entire eye (Singh and Choi,2003). Misexpression of SerDN in ran-dom gain-of-function clones generatedby the ‘‘flp-out’’ method (Pignoni andZipursky, 1997) results in suppressionof eye fate in the ventral half of theeye. The similar phenotypes of SerDN

misexpression and L mutants in theeye disc further validate that L andSer work in the same pathway to reg-ulate the growth of the ventral eyedomain.

It is known that Ser can activate Nonly at the DV border since Ser-Ninteraction is prevented by Fringe(Fng) in the ventral domain cellsaway from the DV border of the eyeimaginal disc. Fng is a glucosaminyl-transferase that elongates O-linkedfucose residues to the EGF domains ofN (Okajima and Irvine, 2002). Fng isknown to bind N to promote N-Delta(Dl) interaction and is required torestrict N activation at the DV border(Irvine and Wieschaus, 1994; Kimet al., 1995; Fleming et al., 1997).Contrary to the positive function ofFng in N-Dl interaction, Fng inhibitsSer-N interaction when it is bound toN protein (Ju et al., 2000; Singh et al.,2005b). As a result, the N activationby Dl is enhanced only at the DV bor-der. The expression pattern of theseDV-patterning genes changes dynam-ically in the developing eye imaginaldisc, thereby showing striking differ-ences before and after the initiation ofretinal differentiation. Initially, fng isexpressed in the ventral domain ofthe eye imaginal disc, but as the eyeimaginal disc undergoes retinal dif-ferentiation and the morphogeneticfurrow proceeds anteriorly, fngexpression further evolves. At thisstage, fng exhibits preferential local-ization anterior to the furrow in boththe dorsal and ventral eye domain.These results validate the conclusionof genetic mosaic studies, which sug-gested that DV pattern is establishedduring early eye development prior to

retinal differentiation. The essentialrole of Fng in DV patterning wasdemonstrated by analysis of fng mu-tant clones. In loss-of-function clonesof fng in the ventral eye, DV polarityis reorganized near the ectopic fngþ/fng� border resulting in non-autono-mous polarity reversals. This leads tothe generation of de novo equatorsand ectopic localized activation of Nat the fngþ/fng� boundary (de Celiset al., 1996; Cho and Choi, 1998; Goet al., 1998; Baonza and Garcia-Bel-lido, 2000).

The DV axis in the wing imaginaldisc is inverted in comparison to theeye imaginal disc. In the eye imaginaldisc, Dl and Ser are preferentiallyexpressed in the dorsal and ventraldomains, respectively. However, thelocalization of Dl and Ser preferentialexpression is reversed in the develop-ing wing imaginal disc. The inversionof the DV axis in the eye and the wingdisc may be due to the fact that theeye disc rotates 180� during embryo-genesis (Struhl, 1981). Therefore, Serfunctions as an N ligand in the dorsalcells, whereas Dl is the N ligand inthe ventral cells. Not surprisingly, fngis ventral-specific in the eye but dor-sal-specific in the wing imaginal disc.

Other candidate genes involved inventral eye development are Chip andsloppy paired (slp) (Table 1). Chip, atranscriptional co-factor, is requiredfor ventral eye development (Roignantet al., 2010). Chip, a ubiquitous tran-scriptional co-factor, interacts withclasses of transcription factors duringneural development. Chip has beenreported to establish the ventralboundary of the eye and the head tis-sue (Roignant et al., 2010). Slp belongsto the forkhead family of transcriptionfactors, which are required for embry-onic patterning (Grossniklaus et al.,1992). Slp locus has two transcriptionunits. Both of them are expressed inthe ventral eye and are functionallyredundant (Sato and Tomlinson, 2007).Slp and Iro-C proteins have beenshown to repress each other at the DVmidline. N signaling at the DV midlinesuppresses Slp at the midline (Satoand Tomlinson, 2007).

Dorsal Selector Genes

It is known that compartment boun-daries are defined by the spatio-tem-

poral expression of genes (Blair, 2001;Curtiss et al., 2002; Dahmann et al.,2011). For example, engrailed (en)and apterous (ap) are expressed in theposterior and the dorsal compart-ments of the wing imaginal disc,respectively (Brower, 1986; Cohenet al., 1992; Hidalgo, 1998; Held,2002). Thus, ‘‘Selector’’ genes wereidentified that assign a unique prop-erty to the cells within their expres-sion domains, which results in the for-mation of unique territories (Blair,2001; Curtiss et al., 2002). Moreover,loss-of-function of these selector genesresults in the loss of that particularfate. In the Drosophila eye, the pres-ence of these selector genes (which ex-hibit dorsal or ventral domain specificexpression) became apparent in theearlier enhancer trap screens (Bieret al., 1989; Bhojwani et al., 1995;Sun et al., 1995). Enhancer trap linescontaining mini-white (w) and lacZreporter gene (P-lacW) (Bellen et al.,1989; Bier et al., 1989; Wilson et al.,1989; Bhojwani et al., 1995; Sunet al., 1995) that show domain-specificexpression in the eye were isolated inthe screens. Interestingly, some ofthese lines have wþ expression re-stricted only to the dorsal half of theadult eye. These enhancer trap lineshave made significant contributionstowards understanding the DV pat-terning in the eye (Choi et al., 1996;Sun and Artavanis-Tsakonas, 1996;McNeill et al., 1997; Kehl et al., 1998;Morrison and Halder, 2010). Most ofthese dorsal-specific P insertion lineswere mapped to the chromosomalregion 69CD, identifying this regionas a hot spot for P-lacW insertionsthat show dorsal eye-specific expres-sion. The molecular characterizationof this 69CD chromosomal regionrevealed the existence of a cluster ofhomeobox genes, araucan (ara), cau-polican (caup), and mirror (mirr)(Gomez-Skarmeta and Modolell,1996; McNeill et al., 1997; Grillenzoniet al., 1998; Heberlein et al., 1998;Kehl et al., 1998; Singh et al., 2005b).This cluster of homeobox genes thatare located within an approximately140-Kb region (Netter et al., 1998) areexpressed in the dorsal half of theeye. They are referred to as Iroquois-complex (Iro-C) because the mutationin these genes lacks lateral thoracicbristles and resembles the hair style

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 75

Fig. 3. Lobe (L) and Serrate (Ser) are required for cell survival in the developing eye imaginal disc. A: In the wild-type eye imaginal disc, L (green)expression is ubiquitous. Elav (red) marks the photoreceptor neurons. B: Wild-type adult eye. C, D: Loss of L results in the preferential loss of ven-tral half of the (C) developing eye imaginal disc, and (D) the adult eye. C: Eye imaginal discs stained for Wg (green) to identify dorsal versus ventraleye imaginal disc compartment. The boundary of the eye field is as outlined in C (white) and D (black) showing preferential loss of the ventral eye.E, F: Early loss-of-function of Ser by misexpressing dominant-negative form of Ser in the entire eye imaginal disc (Kumar and Moses, 2001; Singhand Choi, 2003) using an ey-Gal4 driver (Hazelett et al., 1998; Singh and Choi, 2003) results in complete loss of the eye field both in (E) the eyeimaginal disc and (E) the adult.

Fig. 4.

Dev

elop

men

tal D

ynam

ics

of the Indian tribe, the Iroquois, alsoreferred to as Mohawks (a nativetribe that shaved all but a medialstripe of hairs on the head) (Gomez-Skarmeta and Modolell, 1996; Leynset al., 1996). They named the genesAraucan and Caupolican in honor ofAmerindian tribes: Aracaunians andone of their heroes Caupolican.

The members of Iro-C are highlyconserved, essential genes and exhibitsignificant differences in their expres-sion pattern (Gomez-Skarmeta andModolell, 2002). Mirr is strongly anddynamically expressed in the CNS(Netter et al., 1998; Urbach and Tech-nau, 2003) and it is essential for fol-licle cell patterning (Jordan et al.,2000) while Ara and Caup are prefer-entially expressed in mesodermal tis-sues in the embryos (Netter et al.,1998). However, in the eye imaginaldisc, all three Iro-C members areexpressed in the dorsal half (Fig. 4E),raising a possibility that they mightbe functionally redundant. Loss-of-function of the mirre48 allele showsweak but significant defects of non-autonomous DV polarity reversals incomparison to mirrþ ommatidia in thedorsal half of the eye (McNeill et al.,1997). Compartments of different celllineages do not intermingle due to dif-ferences in cell identities and affin-ities (Garcia-Bellido et al., 1973;Irvine, 1999; Dahmann et al., 2011).Somatic clones of cells lacking mirrfunction in the dorsal half of the eyeexhibit smooth clone borders, indicat-ing that cells lacking mirr avoid mix-ing with the neighboring mirr-expressing cells. However, the clonesin the ventral half where mirr is notexpressed show wiggly clone borders(Yang et al., 1999). This analysis sug-gests that mirr functions as a dorsal

fate selector. Since the phenotype ofmirr clones was not strong enough, itraised the possibility that ara andcaup, the other two members of Iro-C,can partly compensate for the loss ofmirr function in the eye. The issue offunctional redundancy got resolvedwhen a deficiency iroDMF3, whichuncovers all three Iro-C genes by thedeletion of ara and caup as well as a50-region of mirr (Gomez-Skarmataet al., 1996; Diez del Corral et al.,1999), was employed for clonal analy-sis. Loss-of-function clones of iroDMF3

in the eye showed repolarization ofthe ommatidial polarity in the dorsalclones along with dorsal eye enlarge-ment or formation of an ectopic eyefield on the dorsal margin. There wasno phenotype in the ventral half ofthe eye (Fig. 4F, G). These results fur-ther highlighted the importance of aboundary between the dorsal and ven-tral cell types. These results stronglysupport that the three Iro-C genes arepartially redundant, and the Iro-C asa whole is required for organizing theDV polarity pattern and growth of theeye.

Loss-of-function of iroDMF3 also sug-gested that Iro-C genes function asdorsal selectors for head structures aswell since mutant clones in the dorsalregion induce the formation of ventralhead structures (Cavodeassi et al.,2000). Ectopic ventral head tissuesthat resulted from loss of Iro-C genesare cell-autonomous and, therefore,accompanied by loss of correspondingdorsal structures. In contrast, ectopicventral eyes are generated non-cell-autonomously since reversals of DVommatidial polarity are detected inIro-Cþ wild-type region adjacent tothe mutant clones. This also supportsthe idea that the DV boundary is an

organizing center for DV pattern andgrowth in the eye imaginal disc.Furthermore, DV patterning of theeye occurs in earlier larval stagesthan the head patterning. In theDrosophila eye, pnr, another dorsalgene, which is expressed in the dorsaleye margin (Fig. 4A), exhibits similarloss-of-function (Table 1; Fig. 4B, C)and gain-of-function (Fig. 4D) pheno-types as observed with Iro-C in theeye and the head (Maurel-Zaffran andTreisman, 2000; Pichaud andCasares, 2000; Singh et al., 2005b;Oros et al., 2010). Pnr, a GATA-1 tran-scription factor, plays an importantrole in dorsal eye development, andacts as a selector for dorsal eye fate(Ramain et al., 1993; Maurel-Zaffranand Treisman, 2000; Pichaud andCasares, 2000; Dominguez andCasares, 2005; Singh et al., 2005b;Oros et al., 2010). In the hierarchy ofdorsal genes, pnr is the top-mostgene, and it induces Wingless (Wg),which, in turn, induces the expressionof downstream target genes mirr inthe dorsal half of the eye (Maurel-Zaf-fran and Treisman, 2000; Dominguezand Casares, 2005; Singh et al.,2005b). During later stages of devel-opment, which correspond to the reti-nal differentiation stage in late sec-ond instar and third instar of larvaleye development, pnr is involved indefining the dorsal eye margin by reg-ulating the retinal determination(RD) genes (Oros et al., 2010).Wg, a secretory protein and a

morphogen, is expressed along theantero-lateral margins of the third-instar eye imaginal disc (Table 1;Baker, 1988). Wg plays multiple rolesduring eye development. One of theseroles of Wg is to promote growth ofthe early eye imaginal disc. Duringearly eye development, Wg expressionis restricted to the dorsal eye domain(Cho et al., 2000; Chang et al., 2001).During retinal differentiation stage,Wg is known to prevent ectopic induc-tion of retinal differentiation from thelateral eye imaginal disc margin (Maand Moses, 1995; Treisman andRubin, 1995). Thus, Wg that acts as anegative regulator of the eye duringretinal differentiation functions as adorsal eye fate gene. Dl, an N ligandin the dorsal eye imaginal disc, hasbeen assigned to the dorsal gene cate-gory in the early eye imaginal disc

Fig. 4. Pnr and Iro-C members function as dorsal eye fate selectors. A: Pnr expression (green) isrestricted to the dorsal eye margin of the developing eye imaginal disc (Maurel-Zaffran and Treis-man, 2000; Pichaud and Casares, 2000; Singh and Choi, 2003). Elav (red) marks the photorecep-tor neurons. B, C: Loss-of-function clones of pnr result in the enlargement of the existing dorsaleye field (e.g., in the clone outlined in B) in the eye imaginal disc (B) and adult eye (C). B: Note thatthere is a non-autonomous eye enlargement in the eye imaginal disc, which is attributed to gener-ation of a de novo equator in the dorsal compartment of the eye imaginal disc (Maurel-Zaffran andTreisman, 2000; Oros et al., 2010). D: Misexpression of pnr (ey>pnrD4) in the eye imaginal discsuppresses eye fate, validating a late function of pnr in defining the eye field boundary (Oros et al.,2010). E: The expression domain of the members of Iroquois complex (Iro-C>GFP, green) spansthe dorsal region of the eye imaginal disc. F, G: Loss-of-function of Iro-C causes dorsal eyeenlargements in the (F) eye imaginal disc and in (G) adult eye. These phenotypes are similar to the(B, C) pnr loss-of-function phenotypes. H: Misexpression of ara, a member of Iro-C, in the entireeye imaginal disc (ey>ara) results in small eye. D, Dorsal; V, ventral.

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 77

(Table 1). Dl is preferentiallyexpressed in the dorsal domain of eyeimaginal discs during first- and sec-ond-instar stages.

GENES WITH DOMAIN

SPECIFIC GROWTH

RESPONSE: ROLE OF

TEASHIRT (TSH) AND

HOMOTHORAX (HTH)

In addition to the genes that exhibitDV domain-specific expression duringpatterning, there is a group of genes,which show differential functions inthe dorsal-ventral compartments, butare not expressed in a DV-specific pat-tern. These genes can be broadly clas-sified into two groups: (1) Genesexpressed uniformly in the eye imagi-nal disc but their functional domain isrestricted only to the ventral half ofthe eye, for example L and hth (Table1); (2) Genes that are expressed uni-formly in the early eye imaginal discfunction differently in the dorsal andventral half of the eye, for example,tsh (Table 1).

1. Hth: Hth a vertebrate homolog ofmurine proto-oncogene MEIS1(Moskow et al., 1995), encodes ahomeodomain transcription factorof the three-amino-acid extensionloop (TALE) sub-family (Rieckhofet al., 1997). Like L, hth isexpressed in the entire early eyeprimordium. However, with theonset of differentiation in the eye,Hth expression gets restricted tothe cells anterior to the furrow(Fig. 5A, A’; Pai et al., 1998;Pichaud and Casares, 2000; Bessaet al., 2002; Singh et al., 2002a).Even though hth is expressed an-terior to the furrow both in thedorsal and ventral half of the eyeimaginal disc (Fig. 5A, A’), theloss-of-function of hth causes eyeenlargement only in the ventraleye margin (Fig. 5B, B’). However,loss-of-function clones of hth inthe dorsal compartment do notshow any phenotype in the eyeimaginal disc. Furthermore, hthmutant cells do not survive in theanterior eye (Pichaud andCasares, 2000; Bessa et al., 2002,2008). As expected, misexpressionof hth suppresses the eye fate (Paiet al., 1998). Moreover, eye sup-

pression function of Hth is inde-pendent of any domain constraint.Hth is involved in multiple func-tions during development, and itis required for nuclear localization

of a homeoprotein Extradenticle(Exd). hth encodes a protein withnuclear localization signal (NLS)and two conserved domains: the Nterminal evolutionarily conserved

Fig. 5. Domain-specific function of hth is restricted to the ventral eye margin. A, A’: Hth (green)is expressed in the entire peripodial membrane (PM) whereas disc proper specific expression ofHth is restricted anterior to the furrow both in the dorsal as well as the ventral domain of theeye imaginal disc (Rieckhof et al., 1997; Pai et al., 1998; Pichaud and Casares, 2000; Bessaet al., 2002; Singh et al., 2002a). ELAV (red), a pan neural marker, marks the photoreceptorsneurons in the eye imaginal disc. B, B’: Loss-of-function clones of hth marked by the absenceof the GFP reporter (clonal boundary marked by white dotted line) in the ventral eye results ineye enlargement, whereas in the dorsal eye these clones do not have any effect. C, D: Misex-pression of hth in the entire eye using ey-Gal4 driver (ey>>hth) results in a reduced eye field asseen in (C) the eye imaginal disc and (D) adult eye (Pai et al., 1998).

Dev

elop

men

tal D

ynam

ics

78 SINGH ET AL.

MH domain (for Meis and Hth),and a C-terminal region includingthe homeodomain (HD) (Rieckhofet al., 1997; Kurant et al., 1998;

Pai et al., 1998; Noro et al., 2006).Alternative splicing is known toprovide additional complexity tothe genes encoding the Hth tran-

scription factors (Glazov et al.,2005; Noro et al., 2006). Hth formsa heterodimer with Exd throughits MH domain and translocatesinto the nucleus to regulate tran-scription (Ryoo et al., 1999; Jawet al., 2000; Stevens and Mann,2007). Since Exd is present in theentire eye, the ventral specificfunction of Hth has been proposedthrough its interaction with Wgand Tsh. Together they areinvolved in suppression of eye fateon the ventral margin.

2. Tsh: The homeotic gene tsh enco-des a C2H2 zinc-finger transcrip-tion factor with three widelyspaced Zinc finger domains

Fig. 6. DV asymmetric function of homeotic gene teashirt (tsh) in the developing eye imaginaldisc depends on the spatial cues provided by other genes that are expressed in DV asymmetricfashion (Singh et al., 2004). Gain-of-function of Tsh suppresses the eye fate in the ventral eye,whereas it promotes dorsal eye enlargement. It has been shown that the DV asymmetric func-tion of Tsh in the developing eye imaginal disc depends on the domain-specific cues providedby genes that are either expressed or function in a DV-asymmetric fashion. Tsh in collaborationwith Wg and Ser can induce Hth in the ventral eye to suppress the eye fate. The ventral eyesuppression function of tsh is independent of other ventral-specific genes like fng and L. In thedorsal eye, tsh is known to promote growth and eye in collaboration with Iro-C members and anN ligand in the dorsal eye, Dl (Singh et al., 2002, 2004, 2005b). D, Dorsal; V, ventral.

Fig. 7.

Fig. 7. Genes with DV-asymmetric expres-sion or function are involved in regulating DVpatterning in the Drosophila eye. (I) The earlyeye imaginal disc primordium comprises a ho-mogeneous population of ventral eye fatecells whose growth and survival depend on L/Ser function (Singh and Choi, 2003; Singhet al., 2005b, 2006). (II) Later during develop-ment, DV lineage is generated by onset ofexpression of dorsal gene pnr (Singh andChoi, 2003). Pnr acts upstream of Wg, whichin turn triggers the expression of downstreamIro-C members (Maurel-Zaffran and Treisman,2000). Dl, an N ligand, is involved in dorsaleye development. The default ventral eye fateis maintained by function of L and Ser (Singhand Choi, 2003; Singh et al., 2005b). Thereare three other players involved in ventral eyedevelopment, namely, fringe (fng) (Cho andChoi, 1998; Dominguez and de Celis, 1998;Papayannopoulos et al., 1998; Irvine, 1999;Cho et al., 2000; Dominguez and Casares,2005), Chip (Roignant et al., 2010), andSloppy paired (Slp) (Sato and Tomlinson,2007). L is known to act antagonistically tothe dorsal genes to define the boundarybetween the dorsal and ventral compartmentof the eye (Singh et al., 2005a). L and Ser alsointeract antagonistically with hth on the ventraleye margin to define the ventral eye marginboundary (Singh et al., 2005b). There is a pos-itive feedback loop between Wg and Hth onthe ventral eye margin (Pichaud and Casares,2000; Singh et al., 2004; Dominguez andCasares, 2005; Singh et al., 2005b). Chip actsindependently of hth (Roignant et al., 2010).Sloppy paired (Slp) is involved in ventral eyedevelopment by repressing Iro-C at the DVmidline (Sato and Tomlinson, 2007). TheseDV-patterning genes work together and con-tribute towards sculpting the final shape andsize of the adult eye. (III) The ommatidial clus-ters within an adult eye are organized into twomirror symmetric orientations that are polar-ized in the opposite directions in the dorsaland the ventral half. The dorsal selectors ex-hibit a dorsal-specific expression of mini-whitereporter genes in the adult eye.

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 79

(Fasano et al., 1991). Tsh plays animportant role during Drosophilaeye development (Pan and Rubin,1998; Bessa et al., 2002; Singhet al., 2002a, 2005b; Datta et al.,2009; Kumar, 2009, 2010, 2011).tsh is expressed both in dorsal andventral eye anterior to the furrow,and it exhibits a DV constraint inits function. In the ventral eye,tsh acts as a repressor of eye fate,whereas in the dorsal eye, it pro-motes eye development (Fig. 6;Singh et al., 2002b, 2004). Inter-estingly, the DV constraint in tshfunction in the eye stems from thepartners with which it collabo-rates in the dorsal or the ventraleye disc (Fig. 6; Singh et al.,2004). It was shown that Tsh coop-erates with Iro-Complex membersand Dl in the dorsal eye for itsgrowth promotion function (Fig. 6;Singh et al., 2004).The function oftsh in the ventral eye is dependenton Hth and Ser. The expression oftsh overlaps with hth in the eyeimaginal disc, and like hth, tshexpression also evolves duringlarval eye development. Initially,in the first-instar eye imaginaldisc, tsh is expressed in the entireeye imaginal disc but its expres-sion retracts anteriorly to nearlythree-quarters of the eye imaginaldisc when the retinal differentia-tion begins (Bessa et al., 2002;Singh et al., 2002a). Furthermore,Tsh and Hth physically interactwith each other [along with Pax-6homolog, Eyeless (Ey)] to repressthe expression of downstream tar-get genes (Bessa et al., 2002; Dom-inguez and Casares, 2005).Further insights into the potentialmechanism of tsh and hth in regu-lating growth and differentiationin the eye came initially fromanalysis of expression patterns ofthe retinal determination (RD)gene network members (Bessaet al., 2002). It has been proposedthat Tsh, Hth, and Ey coexpressin the proliferating cells anteriorto furrow to block precocious reti-nal differentiation and promotecell proliferation (Bessa et al.,2002; Singh et al., 2002a; Domi-nguez and Casares, 2005). Allthese studies suggest thatDV patterning genes contribute

towards the growth of the eyefield.

BOUNDARY FORMATION

DURING ORGANOGENESIS

One of the important questions is howorgan size and growth are regulatedby DV patterning genes in the eye.The dorsal selector genes assign adorsal fate, and, thereby, generate agroup of cells with unique propertiesthat make them different from thedefault ventral state cells of the devel-oping eye disc. Interestingly, theboundary between the dorsal and ven-tral cells is maintained by the antago-nistic interactions between the genesrequired for the growth and develop-ment of the dorsal and ventraldomains of the eye (Fig. 7; Singhet al., 2005a). It has been shown thatL is essential for growth of the ventraleye tissue, but it is dispensable in thedorsal region specified by pnr function(Singh and Choi 2003). In addition toa boundary between the dorsal andventral compartment within the eye,a boundary is defined between thedeveloping eye field and the sur-rounding head cuticle on the dorsaland ventral margins (Fig. 7). Sincethe adult eye, head cuticle, and othermouthparts are generated from theeye-antennal imaginal disc, there is asequential fate restriction betweenthe developing eye and head cuticle.Interestingly, these DV-patterninggenes play an important role of defin-ing the boundary of the eye field onthe dorsal and the ventral margins(Oros et al., 2010).

The boundary between the eye fieldand the head cuticle on the dorsalmargin is regulated by pnr (Fig. 7). Ithas been shown that pnr functionevolves during eye development. Dur-ing early second instar of develop-ment, pnris required for defining thedorsal lineage, before the onset of reti-nal differentiation, by inducing Wgand members of the Iro-C complex(Maurel-Zaffran and Treisman, 2000;Singh and Choi, 2003; Singh et al.,2005b; Oros et al., 2010). However,later during the late second- instarstage of eye development, when themorphogenetic furrow (MF) is initi-ated, pnrsuppresses the photorecep-tor differentiation at the dorsal eye

margin (Oros et al., 2010). The endog-enous expression of pnris only in theperipodial membrane of the dorsaleye margin, which gives rise to theadult head cuticle. Loss-of-functionclones of pnrexhibit ectopic dorsaleyes, which are restricted within theclones, and suggests that absence ofpnrfunction promotes ectopic eye for-mation in the dorsal eye margin.Thus, Pnr defines the boundarybetween the head cuticle and the dor-sal margin of the developing eye field(Fig. 7; Oros et al., 2010). Since pnrisnot expressed in the ventral eye,there is a different mechanism todefine the boundary of the eye field onthe ventral margin. The boundary ofthe eye field on the ventral eye mar-gin is defined by the antagonisticinteraction of L with hth (Singh et al.,2011). In the ventral eye, transcrip-tional co-factor Chip interacts withthe LIM-homeodomain proteins todefine the boundary of the eye field(Roignant and Treisman, 2009). Inter-estingly, Chip-mediated regulation ofthe ventral eye boundary is independ-ent of hth (Roignant and Treisman,2009). Thus, the genetic cascade thatregulates the boundary of the eyefield on the dorsal and the ventralmargin of the eye is different.

CONCLUDING REMARKS

Our understanding of the axial pat-terning of the Drosophila eye is farfrom complete. In this review, wehave described the overview of keydevelopmental events and genesinvolved in early DV patterning. TheDV compartment formation is a keyto initiate patterning and growth inthe early eye imaginal disc. The pres-ent information clearly illustratesthat DV patterning is required to ini-tiate the generation of heterogeneouspopulation (dorsal and ventral cellfate) of cells within a homogenous(default ventral fate) early eye pri-mordial. Although our knowledge onthe DV patterning in the eye has dra-matically increased in recent years,we still do not know the molecularinteractions important for the regula-tion of DV patterning. Moreover,many more genes (both known andnovel) are expected to be involved inDV patterning, and future studiesusing novel genetic and

Dev

elop

men

tal D

ynam

ics

80 SINGH ET AL.

bioinformatics approaches shouldhelp in defining the full complementof genes involved in this intricate pro-cess. Identification and functionalanalysis of more molecular playersinvolved in this process will help pro-vide a better picture of how a smallnumber of cells in the disc primor-dium grow to form a precise patternof mirror symmetry in the compoundeye. Furthermore, the possibility ofcrosstalk of the DV patterning path-way with other signaling pathways toregulate growth during the earlyphase of eye development cannot beruled out. All this information will laya foundation about understanding theprocess of organogenesis, as loss-of-function of the genes involved in DVpatterning results in loss of the eyefield or a part of the eye field. Thecomplexity and precision of the neuralconnectivity in the adult visual sys-tem has fascinated researchers for along time. The DV polarity of the ret-ina is responsible for controlling thetargeting of the retinal axon projec-tions to the brain in humans andother higher vertebrates. Thus, DVpatterning genes also contributetowards the wiring of the brain to theretina. How all these different facetswork together to define the final formof this complex eye structure is anopen question and is of fundamentalimportance.

SIMILARITIES WITH

VERTEBRATE EYE

The basic sensory epithelium designof the vertebrate and most inverte-brate eyes including Drosophila eye issimilar (Charlton-Perkins and Cook,2010; Sanes and Zipursky, 2010). Themorphogenetic furrow (MF) in the flyeye is analogous to the wave of neuro-geneis in the vertebrate retina (Neu-mann and Nuesslein-Volhard, 2000;Hartenstein and Reh, 2002). Recentstudies in the vertebrate visual sys-tems have identified several genesthat are expressed in a DV domain–specific manner in the retina. BMP4,a TGF-Beta closely related to Dpp,has been implicated in developmentof progenitor cells in the dorsal half ofthe eye and in establishment of theDV axis of the retina in Xenopus(Papalopulu and Kintner, 1996). Thedorsal selectors of the vertebrate eye,

BMP-4 and TbX5, act to restrict theexpression of Vax2 and Pax2 to theventral domain of the eye (Koshiba-Takeuchi et al., 2000; Mui et al., 2002;Peters, 2002; Peters and Cepko,2002). These DV expression domainscorrespond to the developmental com-partments (Peters, 2002; Peters andCepko, 2002). The DV patterningplays an important role in retinotectalprojection pattern (Koshiba-Takeuchiet al., 2000; McLaughlin et al., 2003).The R-cell projections form a precisetopographic connection with the opticlobe, and are referred to as retinotopy,which is common to both the verte-brate and the insect visual system(Gaul, 2002). Furthermore, Jagged-1(Jag1), a vertebrate homolog of theDrosophila ventral eye gene Ser,shows a DV asymmetric expressionpattern in the retina. Moreover, theloss-of-function of Jag1 results in Ala-gille’s syndrome, which also affectsthe eye (Oda et al., 1997; Xue et al.,1999; Kim and Fulton, 2007). Inter-estingly, it has been shown thatmouse retina also begins with adefault ventral-like state (Muraliet al., 2005). Therefore, the DVboundary may play conserved roles inorganizing growth and pattern of vis-ual system in higher animals, andstudies in Drosophila will further ourknowledge in the area of animal de-velopment mechanisms and help tounravel the genetic underpinnings ofdevelopmental defects caused bymutations in human homologs of Dro-sophila DV patterning genes.

ACKNOWLEDGMENTSThe authors thank members of Singhand Kango-Singh lab for their helpand comments on the manuscript. Weapologize to all authors whose workcould not be cited due to space limita-tions. A.S. is supported by an NIHgrant (1R15 HD064557-01). A.S. andM.K.S. are supported by start-upfunds from theUniversity of Dayton.

REFERENCES

Anderson DT. 1972a. The development ofhemimetabolous insects. In: Counce S,Waddington CH, editors. Developmentalsystems: insects. New York: AcademicPress. p 165–242.

Anderson DT. 1972b. The development ofhemimetabolous insects. In: Counce S,Waddington CH, editors. Developmental

systems: insects. New York: AcademicPress. p 96–163.

Atkins M, Mardon G. 2009. Signaling inthe third dimension: the peripodial epi-thelium in eye disc development. DevDyn 238:2139–2148.

Baker NE. 1988. Embryonic and imaginalrequirements for wingless, a segment-polarity gene in Drosophila. Dev Biol125:96–108.

Baker WK. 1978. A clonal analysisreveals early developmental restrictionsin the Drosophila head. Dev Biol 62:447–463.

Baonza A, Garcia-Bellido A. 2000. Notchsignaling directly controls cell prolifera-tion in the Drosophila wing disc. ProcNatl Acad Sci USA 97:2609–2614.

Bellen HJ, O’Kane CJ, Wilson C, Grossni-klaus U, Pearson RK, Gehring WJ.1989. P-element-mediated enhancerdetection: a versatile method to studydevelopment in Drosophila. Genes Dev3:1288–1300.

Bessa J, Gebelein B, Pichaud F, CasaresF, Mann RS. 2002. Combinatorial con-trol of Drosophila eye development byeyeless, homothorax, and teashirt.Genes Dev 16:2415–2427.

Bessa J, Tavares MJ, Santos J, Kikuta H,Laplante M, Becker TS, Gomez-Skar-meta JL, Casares F. 2008. meis1 regu-lates cyclin D1 and c-myc expression,and controls the proliferation of themultipotent cells in the early develop-ing zebrafish eye. Development 135:799–803.

Bhojwani J, Singh A, Misquitta L, MishraA, Sinha P. 1995. Search for the Dro-sophila genes based on patternedexpression of mini-white reporter geneof a P lacW vector in adult eyes. Roux’sArch Dev Biol 205:114–121.

Bier E, Vaessin H, Shepherd S, Lee K,McCall K, Barbel S, Ackerman L, Car-retto R, Uemura T, Grell E, Jan LY,Jan YN. 1989. Searching for patternand mutation in the Drosophila genomewith a P-lacZ vector. Genes Dev 3:1273–1287.

Blair SS. 2001. Cell lineage: compart-ments and Capricious. Curr Biol 11:R1017–1021.

Bodentstein D. 1950. The Postembryonicdevelopment of Drosophila melanogaster.In: Demerec M, editor. Biology of Dro-sophila. New York: Wiley. p 275–367.

Brook WJ, Cohen SM. 1996. Antagonisticinteractions between wingless anddecapentaplegic responsible for dorsal-ventral pattern in the Drosophila leg.Science 273:1373–1377.

Brower DL. 1986. Engrailed gene expres-sion in Drosophila imaginal discs.EMBO J 5:2649–2656.

Calleja M, Moreno E, Pelaz S, Morata G.1996. Visualization of gene expressionin living adult Drosophila. Science274:252–255.

Cavodeassi F, Modolell J, Campuzano S.2000. The Iroquois homeobox genesfunction as dorsal selectors in the Dro-sophila head. Development 127:1921–1929.

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 81

Chang T, Mazotta J, Dumstrei K, Dumi-trescu A, Hartenstein V. 2001. Dpp andHh signaling in the Drosophila embry-onic eye field. Development 128:4691–4704.

Chanut F, Heberlein U. 1997. Role ofdecapentaplegic in initiation and pro-gression of the morphogenetic furrow inthe developing Drosophila retina. De-velopment 124:559–567.

Charlton-Perkins M, Cook TA. 2010.Building a fly eye: terminal differentia-tion events of the retina, corneal lens,and pigmented epithelia. Curr Top DevBiol 93:129–173.

Chern JJ, Choi KW. 2002. Lobe mediatesNotch signaling to control domain-spe-cific growth in the Drosophila eye disc.Development 129:4005–4013.

Cho KO, Choi KW. 1998. Fringe is essen-tial for mirror symmetry and morpho-genesis in the Drosophila eye. Nature396:272–276.

Cho KO, Chern J, Izaddoost S, Choi KW.2000. Novel signaling from the peripo-dial membrane is essential for eye discpatterning in Drosophila. Cell 103:331–342.

Choi KW, Mozer B, Benzer S. 1996. Inde-pendent determination of symmetryand polarity in the Drosophila eye. ProcNatl Acad Sci USA 93:5737–5741.

Cohen B, McGuffin ME, Pfeifle C, SegalD, Cohen SM. 1992. apterous, a generequired for imaginal disc developmentin Drosophila encodes a member of theLIM family of developmental regulatoryproteins. Genes Dev 6:715–729.

Cohen B, Simcox AA, Cohen SM. 1993.Allocation of the thoracic imaginal pri-mordia in the Drosophila embryo. De-velopment 117:597–608.

Cohen SM. 1993. Imaginal disc develop-ment In: Bate M, Martinez-Arias A, edi-tors. The development of Drosophilamelanogaster. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press.

Crick FH, Lawrence PA. 1975. Compart-ments and polyclones in insect develop-ment. Science 189:340–347.

Curtiss J, Halder G, Mlodzik M. 2002. Se-lector and signalling molecules cooper-ate in organ patterning. Nat Cell Biol4:E48–51.

Dahmann C, Oates AC, Brand M. 2011.Boundary formation and maintenancein tissue development. Nat Rev Genet12:43–55.

Datta RR, Lurye JM, Kumar JP. 2009.Restriction of ectopic eye formation byDrosophila teashirt and tiptop to thedeveloping antenna. Dev Dyn 238:2202–2210.

de Celis JF, Garcia-Bellido A, Bray SJ.1996. Activation and function of Notchat the dorsal-ventral boundary of thewing imaginal disc. Development 122:359–369.

Dietrich W. 1909. Die Facettenaugen derDipteran. Z Wiss Zool 92:465–539.

Diez del Corral R, Aroca P, Gomez-Skar-meta JL, Cavodeassi F, Modolell J.1999. The Iroquois homeodomain pro-teins are required to specify body wall

identity in Drosophila. Genes Dev13:1754–1761.

Dominguez M, Casares F. 2005. Organspecification-growth control connection:new in-sights from the Drosophila eye-antennal disc. Dev Dyn 232:673–684.

Dominguez M, de Celis JF. 1998. A dor-sal/ventral boundary established byNotch controls growth and polarity inthe Drosophila eye. Nature 396:276–278.

Erclik T, Hartenstein V, McInnes RR, Lip-shitz HD. 2009. Eye evolution at highresolution: the neuron as a unit ofhomology. Dev Biol 332:70–79.

Fasano L, Roder L, Core N, Alexandre E,Vola C, Jacq B, Kerridge S. 1991. Thegene teashirt is required for the devel-opment of Drosophila embryonic trunksegments and encodes a protein withwidely spaced zinc finger motifs. Cell64:63–79.

Ferris GF. 1950. External morphology ofthe adult. In: Demerec M, editor. Biol-ogy of Drosophila. New York: Wiley. p368–419.

Fleming RJ, Gu Y, Hukriede NA. 1997.Serrate-mediated activation of Notch isspecifically blocked by the product ofthe gene fringe in the dorsal compart-ment of the Drosophila wing imaginaldisc. Development 124:2973–2981.

Garcia-Bellido A, Merriam JR. 1969. Celllineage of the imaginal discs in Dro-sophila gynandromorphs. J Exp Zool170:61–75.

Garcia-Bellido A, Ripoll P, Morata G.1973. Developmental compartmentalisa-tion of the wing disk of Drosophila. NatNew Biol 245:251–253.

Gaul U. 2002. The establishment of reti-nal connectivity. In: Moses K, editor.Drosophila eye development. Berlin:Springer-Verlag. p 205–216.

Gehring WJ. 2005. New perspectives oneye development and the evolution ofeyes and photoreceptors. J Hered 96:171–184.

Gibson MC, Schubiger G. 2001. Drosoph-ila peripodial cells, more than meetsthe eye? Bioessays 23:691–697.

Glazov EA, Pheasant M, McGraw EA,Bejerano G, Mattick JS. 2005. Ultracon-served elements in insect genomes: ahighly conserved intronic sequenceimplicated in the control of homothoraxmRNA splicing. Genome Res 15:800–808.

Go MJ, Eastman DS, Artavanis-TsakonasS. 1998. Cell proliferation control byNotch signaling in Drosophila develop-ment. Development 125:2031–2040.

Gomez-Skarmeta JL, Modolell J. 1996.araucan and caupolican provide a linkbetween compartment subdivisions andpatterning of sensory organs and veinsin the Drosophila wing. Genes Dev 10:2935–2945.

Gomez-Skarmeta JL, Modolell J. 2002. Iro-quois genes: genomic organization andfunction in vertebrate neural develop-ment. Curr Opin Genet Dev 12:403–408.

Grillenzoni N, van Helden J, Dambly-Chaudiere C, Ghysen A. 1998. The iro-

quois complex controls the somatotopy ofDrosophila notum mechanosensory pro-jections. Development 125:3563–3569.

Grossniklaus U, Pearson RK, GehringWJ. 1992. The Drosophila sloppy pairedlocus encodes two proteins involved insegmentation that show homology tomammalian transcription factors.Genes Dev 6:1030–1051.

Hartenstein V, Reh TA. 2002. Homologiesbetween vertebrate and invetebrateeyes. In: Moses K, editor. Drosophilaeye development. Berlin: Springer-Ver-lag. p 219–251.

Haynie JL, Bryant PJ. 1986. Develop-ment of the eye-antenna imaginal discand morphogenesis of the adult head inDrosophila melanogaster. J Exp Zool237:293–308.

Hazelett DJ, Bourouis M, Walldorf U,Treisman JE. 1998. decapentaplegicand wingless are regulated by eyesabsent and eyegone and interact todirect the pattern of retinal differentia-tion in the eye disc. Development 125:3741–3751.

Heberlein U, Borod ER, Chanut FA. 1998.Dorsoventral patterning in the Dro-sophila retina by wingless. Develop-ment 125:567–577.

Held LIJ. 2002. The eye disc. In: Held LI,editor. Imaginal disc. Cambridge: Cam-bridge University Press. p 197–236.

Hidalgo A. 1998. Growth and patterningfrom the engrailed interface. Int J DevBiol 42:317–324.

Irvine KD. 1999. Fringe, Notch, and mak-ing developmental boundaries. CurrOpin Genet Dev 9:434–441.

Irvine KD, Wieschaus E. 1994. fringe, aBoundary-specific signaling molecule,mediates interactions between dorsaland ventral cells during Drosophilawing development. Cell 79:595–606.

Jaw TJ, You LR, Knoepfler PS, Yao LC,Pai CY, Tang CY, Chang LP, BerthelsenJ, Blasi F, Kamps MP, Sun YH. 2000.Direct interaction of two homeopro-teins, homothorax and extradenticle, isessential for EXD nuclear localizationand function. Mech Dev 91:279–291.

Jordan KC, Clegg NJ, Blasi JA, MorimotoAM, Sen J, Stein D, McNeill H, DengWM, Tworoger M, Ruohola-Baker H.2000. The homeobox gene mirror linksEGF signalling to embryonic dorso-ven-tral axis formation through notch acti-vation. Nat Genet 24:429–433.

Ju BG, Jeong S, Bae E, Hyun S, CarrollSB, Yim J, Kim J. 2000. Fringe forms acomplex with Notch. Nature 405:191–195.

Jurgens J, Hartenstein V. 1993. Theterminal regions of body pattern. In:Martinez-Arias MBaA, editor. TheDevelopment of Drosophila mela-nogaster. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory Press. p687–746.

Kehl BT, Cho KO, Choi KW. 1998. mirror,a Drosophila homeobox gene in the Iro-quois complex, is required for sensoryorgan and alula formation. Develop-ment 125:1217–1227.

Dev

elop

men

tal D

ynam

ics

82 SINGH ET AL.

Kenyon KL, Ranade SS, Curtiss J, Mlod-zik M, Pignoni F. 2003. Coordinatingproliferation and tissue specification topromote regional identity in the Dro-sophila head. Dev Cell 5:403–414.

Kim BJ, Fulton AB. 2007. The geneticsand ocular findings of Alagille syn-drome. Semin Ophthalmol 22:205–210.

Kim J, Irvine KD, Carroll SB. 1995. Cellrecognition, signal induction, and sym-metrical gene activation at the dorsal-ventral boundary of the developing Dro-sophila wing. Cell 82:795–802.

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

Koshiba-Takeuchi K, Takeuchi JK, Ma-tsumoto K, Momose T, Uno K, HoepkerV, Ogura K, Takahashi N, NakamuraH, Yasuda K, Ogura T. 2000. Tbx5 andthe retinotectum projection. Science287:134–137.

Kumar JP. 2009. The molecular circuitrygoverning retinal determination. Bio-chim Biophys Acta 1789:306–314.

Kumar JP. 2010. Retinal determinationthe beginning of eye development. CurrTop Dev Biol 93:1–28.

Kumar JP. 2011. My what big eyes you have:How the Drosophila retina grows. DevNeurobiol [DOI: 10.1002/dneu.20921].

Kumar JP, Moses K. 2001. EGF receptorand Notch signaling act upstream ofEyeless/Pax6 to control eye specifica-tion. Cell 104:687–697.

Kurant E, Pai CY, Sharf R, Halachmi N,Sun YH, Salzberg A. 1998. Dorsotonals/homothorax, the Drosophila homologueof meis1, interacts with extradenticle inpatterning of the embryonic PNS. De-velopment 125:1037–1048.

Lawrence PA, Morata G. 1976. Compart-ments in the wing of Drosophila: astudy of the engrailed gene. Dev Biol50:321–337.

Leyns L, Gomez-Skarmeta JL, Dambly-Chaudiere C. 1996. iroquois: a prepat-tern gene that controls the formation ofbristles on the thorax of Drosophila.Mech Dev 59:63–72.

Ma C, Moses K. 1995. Wingless andpatched are negative regulators of themorphogenetic furrow and can affecttissue polarity in the developing Dro-sophila compound eye. Development121:2279–2289.

Maurel-Zaffran C, Treisman JE. 2000. pan-nier acts upstream of wingless to directdorsal eye disc development in Drosoph-ila. Development 127:1007–1016.

McClure KD, Schubiger G. 2005. Develop-mental analysis and squamous morpho-genesis of the peripodial epithelium inDrosophila imaginal discs. Development132:5033–5042.

McLaughlin T, Hindges R, O’Leary DD.2003. Regulation of axial patterning ofthe retina and its topographic mapping inthe brain. Curr Opin Neurobiol 13:57–69.

McNeill H, Yang CH, Brodsky M, UngosJ, Simon MA. 1997. mirror encodes a

novel PBX-class homeoprotein thatfunctions in the definition of the dorsal-ventral border in the Drosophila eye.Genes Dev 11:1073–1082.

Meinhardt H. 1983. Cell determinationboundaries as organizing regions forsecondary embryonic fields. Dev Biol96:375–385.

Milner M, Bleasby A, Pyott A. 1983. Therole of the peripodial membrane in themorphogenesis of the eye antennal discof Drosophila melanogaster. Roux’sArch Dev Biol 192:164–170.

Morgan TH, Bridges CB, Strutevant AH.1925. The genetics of Drosophila. Bib-liog Genet 2:1–262.

Morrison CM, Halder G. 2010. Character-ization of a dorsal-eye Gal4 line in Dro-sophila. Genesis 48:3–7.

Moskow JJ, Bullrich F, Huebner K, DaarIO, Buchberg AM. 1995. Meis1, aPBX1-related homeobox gene involvedin myeloid leukemia in BXH-2 mice.Mol Cell Biol 15:5434–5443.

Mui SH, Hindges R, O’Leary DD, LemkeG, Bertuzzi S. 2002. The homeodomainprotein Vax2 patterns the dorsoventraland nasotemporal axes of the eye. De-velopment 129:797–804.

Murali D, Yoshikawa S, Corrigan RR,Plas DJ, Crair MC, Oliver G, LyonsKM, Mishina Y, Furuta Y. 2005. Dis-tinct developmental programs requiredifferent levels of Bmp signaling duringmouse retinal development. Develop-ment 132:913–923.

Nellen D, Burke R, Struhl G, Basler K.1996. Direct and long-range action of aDPP morphogen gradient. Cell 85:357–368.

Netter S, Fauvarque MO, Diez del CorralR, Dura JM, Coen D. 1998. whiteþtransgene insertions presenting a dor-sal/ventral pattern define a single clus-ter of homeobox genes that is silencedby the polycomb-group proteins in Dro-sophila melanogaster. Genetics 149:257–275.

Neumann CJ, Nuesslein-Volhard C. 2000.Patterning of the zebrafish retina by awave of sonic hedgehog activity. Science289:2137–2139.

Noro B, Culi J, McKay DJ, Zhang W,Mann RS. 2006. Distinct functions ofhomeodomain-containing and homeodo-main-less isoforms encoded by homo-thorax. Genes Dev 20:1636–1650.

Oda T, Elkahloun AG, Pike BL, OkajimaK, Krantz ID, Genin A, Piccoli DA,Meltzer PS, Spinner NB, Collins FS,Chandrasekharappa SC. 1997. Muta-tions in the human Jagged1 gene areresponsible for Alagille syndrome. NatGenet 16:235–242.

Okajima T, Irvine KD. 2002. Regulationof notch signaling by o-linked fucose.Cell 111:893–904.

Oros SM, Tare M, Kango-Singh M, SinghA. 2010. Dorsal eye selector pannier(pnr) suppresses the eye fate to definedorsal margin of the Drosophila eye.Dev Biol 346:258–271.

Oshiro N, Takahashi R, Yoshino K, Tani-mura K, Nakashima A, Eguchi S, Miya-

moto T, Hara K, Takehana K, Avruch J,Kikkawa U, Yonezawa K. 2007. Theproline-rich Akt substrate of 40 kDa(PRAS40) is a physiological substrate ofmammalian target of rapamycin com-plex 1. J Biol Chem 282:20329–20339.

Pai CY, Kuo TS, Jaw TJ, Kurant E, ChenCT, Bessarab DA, Salzberg A, Sun YH.1998. The Homothorax homeoproteinactivates the nuclear localization ofanother homeoprotein, extradenticle,and suppresses eye development inDrosophila. Genes Dev 12:435–446.

Pan D, Rubin GM. 1998. Targeted expres-sion of teashirt induces ectopic eyes inDrosophila. Proc Natl Acad Sci USA 95:15508–15512.

Papalopulu N, Kintner C. 1996. A Xeno-pus gene, Xbr-1, defines a novel class ofhomeobox genes and is expressed in thedorsal ciliary margin of the eye. DevBiol 174:104–114.

Papayannopoulos V, Tomlinson A, PaninVM, Rauskolb C, Irvine KD. 1998. Dor-sal-ventral signaling in the Drosophilaeye. Science 281:2031–2034.

Penton A, Hoffmann FM. 1996. Decapen-taplegic restricts the domain of wing-less during Drosophila limb patterning.Nature 382:162–164.

Peters MA. 2002. Patterning the neuralretina. Curr Opin Neurobiol 12:43–48.

Peters MA, Cepko CL. 2002. The dorsal-ventral axis of the neural retina isdivided into multiple domains ofrestricted gene expression which exhibitfeatures of lineage compartments. DevBiol 251:59–73.

Pichaud F, Casares F. 2000. homothoraxand iroquois-C genes are required for theestablishment of territories within thedeveloping eye disc. Mech Dev 96:15–25.

Pignoni F, Zipursky SL. 1997. Induction ofDrosophila eye development by decapen-taplegic. Development 124:271–278.

Poulson DF. 1950. Histogenesis, oogene-sis, and differentiation in the embryo ofDrosophila melanogaster meigen. In:Demerec M, editor. Biology of Drosoph-ila. New York: Wiley. p 168–274.

Ramain P, Heitzler P, Haenlin M, SimpsonP. 1993. pannier, a negative regulator ofachaete and scute in Drosophila, enco-des a zinc finger protein with homologyto the vertebrate transcription factorGATA-1. Development 119:1277–1291.

Ready DF, Hanson TE, Benzer S. 1976. De-velopment of the Drosophila retina, a neu-rocrystalline lattice. Dev Biol 53:217–240.

Rieckhof GE, Casares F, Ryoo HD, Abu-Shaar M, Mann RS. 1997. Nucleartranslocation of extradenticle requireshomothorax, which encodes an extra-denticle-related homeodomain protein.Cell 91:171–183.

Roignant JY, Legent K, Janody F, Treis-man JE. 2010. The transcriptional co-factor Chip acts with LIM-homeodo-main proteins to set the boundary ofthe eye field in Drosophila. Develop-ment 137:273–281.

Roignant JY, Treisman JE. 2009. Patternformation in the Drosophila eye disc.Int J Dev Biol 53:795–804.

Dev

elop

men

tal D

ynam

ics

DV PATTERNING IN THE DROSOPHILA EYE 83

Ryoo HD, Marty T, Casares F, Affolter M,Mann RS. 1999. Regulation of Hox tar-get genes by a DNA bound Homo-thorax/Hox/Extradenticle complex.Development 126:5137–5148.

Sanes JR, Zipursky SL. 2010. Designprinciples of insect and vertebrate vis-ual systems. Neuron 66:15–36.

Sato A, Tomlinson A. 2007. Dorsal-ventralmidline signaling in the developingDrosophila eye. Development 134:659–667.

Singh A, Choi KW. 2003. Initial state ofthe Drosophila eye before dorsoventralspecification is equivalent to ventral.Development 130:6351–6360.

Singh A, Kango-Singh M, Sun YH. 2002a.Eye suppression, a novel function ofteashirt, requires Wingless signaling.Development 129:4271–4280.

Singh A, Kango-Singh M, Sun YH. 2002b.Eye suppression, a novel function ofteashirt, requires Wingless signaling.Development 129:4271–4280.

Singh A, Tare M, Kango-Singh M, SonWS, Cho KO, Choi KW. 2011. Opposinginteractions between homothorax andLobe define the ventral eye margin ofDrosophila eye. Dev Biol [DOI: 10.1016/j.ydbio.2011.08.017].

Singh A, Kango-Singh M, Choi KW, SunYH. 2004. Dorso-ventral asymmetricfunctions of teashirt in Drosophila eyedevelopment depend on spatial cuesprovided by early DV patterning genes.Mech Dev 121:365–370.

Singh A, Chan J, Chern JJ, Choi KW.2005a. Genetic interaction of Lobe withits modifiers in dorsoventral patterningand growth of the Drosophila eye.Genetics 171:169–183.

Singh A, Lim J, Choi K-W. 2005b. Dorso-ventral boundary is required for organiz-ing growth and planar polarity in theDrosophila eye. In: Mlodzik M, editor.Planar cell polarization during develop-ment: advances in developmental biology

and biochemistry. New York: ElsevierScience and Technology Books. p 59–91.

Singh A, Shi X, Choi KW. 2006. Lobe andSerrate are required for cell survivalduring early eye development in Dro-sophila. Development 133:4771–4781.

Speicher SA, Thomas U, Hinz U, KnustE. 1994. The Serrate locus of Drosoph-ila and its role in morphogenesis of thewing imaginal discs: control of cell pro-liferation. Development 120:535–544.

Stevens KE, Mann RS. 2007. A balancebetween two nuclear localization sequen-ces and a nuclear export sequencegoverns extradenticle subcellular local-ization. Genetics 175:1625–1636.

Struhl G. 1981. A blastoderm fate map ofcompartments and segments of the Dro-sophila head. Dev Biol 84:386–396.

Sun X, Artavanis-Tsakonas S. 1996. Theintracellular deletions of Delta and Ser-rate define dominant negative forms ofthe Drosophila Notch ligands. Develop-ment 122:2465–2474.

Sun YH, Tsai CJ, Green MM, Chao JL,Yu CT, Jaw TJ, Yeh JY, Bolshakov VN.1995. White as a reporter gene to detecttranscriptional silencers specifying posi-tion-specific gene expression duringDrosophila melanogaster eye develop-ment. Genetics 141:1075–1086.

Theisen H, Haerry TE, O’Connor MB,Marsh JL. 1996. Developmental territo-ries created by mutual antagonismbetween Wingless and Decapentaplegic.Development 122:3939–3948.

Treisman JE, Rubin GM. 1995. winglessinhibits morphogenetic furrow move-ment in the Drosophila eye disc. Devel-opment 121:3519–3527.

Urbach R, Technau GM. 2003. Segmentpolarity and DV patterning geneexpression reveals segmental organiza-tion of the Drosophila brain. Develop-ment 130:3607–3620.

VanderHaar E, Lee SI, Bandhakavi S, GriffinTJ, Kim DH. 2007. Insulin signalling to

mTOR mediated by the Akt/PKB sub-strate PRAS40. Nat Cell Biol 9:316–323.

Wang YH, Huang ML. 2009. Reduction ofLobe leads to TORC1 hypoactivationthat induces ectopic Jak/STAT signalingto impair Drosophila eye development.Mech Dev 126:781–790.

Wawersik S, Maas RL. 2000. Vertebrateeye development as modeled in Dro-sophila. Hum Mol Genet 9:917–925.

Wilson C, Pearson RK, Bellen HJ, O’KaneCJ, Grossniklaus U, Gehring WJ. 1989.P-element-mediated enhancer detection:an efficient method for isolating andcharacterizing developmentally regu-lated genes in Drosophila. Genes Dev 3:1301–1313.

Wolff T, Ready DF. 1993. Pattern formationin the Drosophila retina. In: Martinez-Arias MBaA, editor. The development ofDrosophila melanogaster. Cold SpringHarbor, NY: Cold Spring Harbor Labora-tory Press. p 1277–1325.

Xue Y, Gao X, Lindsell CE, Norton CR,Chang B, Hicks C, Gendron-MaguireM, Rand EB, Weinmaster G, Gridley T.1999. Embryonic lethality and vasculardefects in mice lacking the Notch ligandJagged1. Hum Mol Genet 8:723–730.

Yamamoto D. 1996. Architecture of theadult compound eye and the developingeye disks. In: Yamamoto D, editor. Mo-lecular dynamics in the developing dro-sophila eye. Austin, TX: Chapman andHall. p 169.

Yang CH, Simon MA, McNeill H. 1999.mirror controls planar polarity andequator formation through repression offringe expression and through controlof cell affinities. Development 126:5857–5866.

Younossi-Hartenstein A, Hartenstein V.1993. The role of the tracheae and mus-culature during pathfinding of Drosoph-ila embryonic sensory axons. Dev Biol158:430–447.

Dev

elop

men

tal D

ynam

ics

84 SINGH ET AL.