evolution of insect eye development: first insights from fruit fly

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INTEGR. COMP. BIOL., 43:508–521 (2003)

Evolution of Insect Eye Development:First Insights from Fruit Fly, Grasshopper and Flour Beetle1

MARKUS FRIEDRICH2

Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, Michigan 48202

SYNOPSIS. The molecular genetic dissection of Drosophila eye development led to the exciting discovery ofa surprisingly large panel of genes and gene activities, which are functionally conserved across phyla. Littleeffort has yet been made towards pinpointing non-conserved gene functions in the developing Drosophilaeye. This neglects the fact that Drosophila visual system development is a highly derived process. The com-parative analysis of Drosophila eye development within insects can be expected to enhance resolution andaccuracy of between phyla comparisons of eye development, and to reveal molecular developmental changesthat facilitated the evolutionary transition from hemimetabolous to holometabolous insect development. Herewe review aspects of early Drosophila eye development, which are likely to have diverged from the situationin more primitive insects, as indicated by results from work in the flour beetle Tribolium castaneum and thegrasshopper Schistocerca americana.

INTRODUCTION

The power of biological model systems equals theease of genetic analysis multiplied by applicability tohuman biology. The fruit fly Drosophila melanogasterhas performed beyond expectation under this equation.Originally chosen for reasons of affordability and shortgeneration time rather than similarity to vertebratephysiology, the analysis of the complete Drosophilagenome delivered ultimate proof that this model spe-cies also positions usefully close to humans on the treeof life if shared genes and gene functions are taken asmeasure of relatedness (Rubin et al., 2000). The sur-prising evolutionary insights that emanated from themolecular dissection of Drosophila compound eye de-velopment constitute a paradigm case of radicalchange of comparative perspective. Considering thefundamental differences between the Drosophila com-pound eye and the vertebrate lens eye, an independentorigin of insect and vertebrate visual organs seemedcertain (Salvini-Plawen and Mayr, 1977). This pre-molecular view was revolutionized by the discoveryin Walter Gehring’s laboratory that the orthologs ofthe paired class transcription factor Pax6 are essentialfor eye development in both fly and vertebrates (Quir-ing et al., 1994). Subsequent studies revealed that ho-mologs of three equally conserved gene families eyesabsent (eya), sine oculis (so) and dachshund (dac) in-teract with Pax6 in an evolutionarily conserved eyespecification regulatory network (for review see Des-plan, 1997; Pappu and Mardon, 2002). The finding ofsimilarities in the function of the proneural transcrip-tion factor atonal (ato) and the signaling moleculehedgehog (hh) in the developing retina of flies andvertebrates raised the possibility that the conservationof animal eye development extends to processes down-

1 From the Symposium Comparative and Integrative Vision Re-search presented at the Annual Meeting of the Society for Integra-tive and Comparative Biology, 4–8 January 2003, at Toronto, Can-ada.

2 E-mail: [email protected]

stream of eye specification (Kumar, 2001; Brown etal., 2001; Neumann and Nuesslein-Volhard, 2000).

The nature of Drosophila eye development conser-vation has been a topic of lively discussion (Halder etal., 1995; Hanson and Van Heyningen, 1995; Gehring,1996; Tomarev, 1997; Kumar, 2001; Pichaud et al.,2001; Pichaud and Desplan, 2002). Less attention hasbeen paid to the fact that Drosophila eye developmentalso offers the opportunity to unravel molecular de-velopmental changes underlying the emergence ofevolutionary novelty. Drosophila represents one of themost derived modes of insect eye development to theeyes of an entomologist (Fig. 1). This is in part dueto the derived life cycle and in part to unique mor-phological modifications of the juvenil instars. As istypical for holometabolous insects, the fruitfly devel-ops through a series of specialized postembryonicgrowth stages. These larval instars lack most elementsof the adult body plan including the compound eyes.The complex transformation to adult morphology be-gins in the last larval instar and completes during theresting stage of the pupa. The development of the adultDrosophila eye is thus a postembryonic process. Inprimitive insects, however, much of the adult retinadevelops already during embryogenesis (Fig. 1). Theimmature instars or nymphs of hemimetabolous insectssuch as grasshopper or cockroach for instance are bornwith adult like morphology. Elaboration of the adultmorphology during the final nymphal molt requiresonly the extension of wings in addition to differenti-ation of functional genitalia. The first instar nymphpossesses a pair of fully functional compound eyes,which are enlarged between the subsequent growthmolts and maintained into the adult. Morphologicaland molecular data firmly establish that holometabo-lous development evolved from within hemimetabo-lous insects (Kristensen, 1995). The postembryonicmode of Drosophila eye development is thus clearlyan evolutionarily derived process.

Evolutionary change of morphology requireschange of developmental programs (Carroll, 1994).

509EVOLUTION OF INSECT EYE DEVELOPMENT

FIG. 1. Life cycle and time course of compound eye development in Drosophila, Tribolium and Schistocerca. Time windows of compoundeye retina specification, determination and differentiation indicated by open, grey and black arrows respectively. Specification and determinationarrows in Tribolium box are hypothetical (see text). Cladogram to the right of the boxes indicates phylogenetic relationships between the threespecies.

Evidently, the evolution of holometabolous insectsmust have involved modifications of embryonic de-velopment to generate the derived morphology of thelarva. It is also reasonable to assume that the postem-bryogenic differentiation of adult structures startingfrom the larval body plan involved modifications ofancestrally embryonic differentiation processes. Thus,the evolutionary transition from embryonic to post-embryonic eye development most likely involved notonly a temporal shift of the onset of adult retina dif-ferentiation, but also considerable modifications ofembryonic and postembryonic visual system devel-opment. The extent to which the deeply conserved mo-lecular genetic control mechanisms of Drosophila eyedevelopment are embedded in derived patterningmechanisms is unclear. This is because our knowledgeof insect eye development outside the genus Drosoph-ila is fairly limited. Accounts on eye development ininsect species other than Drosophila are scattered(Meinertzhagen, 1973; Bate, 1978; Trujillo-Cenoz,1985; Egelhaaf, 1988; Melzer and Paulus, 1994; Fried-rich et al., 1996; Champlin and Truman, 1998). A ma-jor focus has been the comparison of the sequence ofcell differentiation events in the developing retina,which is highly conserved in insects and crustaceans(Melzer et al., 2000; Hafner and Tokarski, 2001). Thisreview discusses first evidence for evolutionary diver-gence of molecular genetic patterning events, whichlead up to the onset of differentiation of the compound

eye retina in Drosophila. Examples will be drawnfrom ongoing comparative analyses in the Americandesert locust Schistocerca americana, a hemimetabo-lous insect, and the flour beetle Tribolium castaneum,a primitive holometabolous insect.

Drosophila, Tribolium and Schistocerca: Three waysto make an insect eye

Drosophila represents one of the most complexmodes of insect compound eye development (for re-view see Wolff and Ready, 1993). First complicationsstem from the fact that the Drosophila larva is ace-phalic. It lacks head appendages and is furnished withan internalized head skeleton instead (Fig. 1). As aconsequence, the larval head components are replacedwith newly differentiated adult head capsule tissuesduring postembryonesis. The cuticle skeleton of theadult head develops from specialized ectodermal sacs,the imaginal discs. These may be considered second-ary embryonic fields that are set aside during earlyembryogenesis and remain buried deep inside the bodyduring larval development. The compound eye retinadevelops from the eye-antennal imaginal disc, whichis a derivative of the embryonic visual primordium(Fig. 2A). Only little developed at the beginning ofpostembryogenesis the eye-antennal imaginal disc pro-ceeds through continuous growth and differentiationduring larval development. By the second larval instar,the anterior part, which will give rise to the antenna,

510 MARKUS FRIEDRICH

FIG. 2. Different stages of compound eye morphogenesis in Drosophila, Tribolium and Schistocerca. A. Lateral view of Drosophila stage13 embryonic head according to Chang et al. (2001). B. Lateral view of Tribolium embryonic head at germband retraction stage according toUllmann (1966) and Heming (1982). C. Lateral view of Schistocerca embryonic head at 40% of development. D. Third instar Drosophilaantennal-eye imaginal disc. E. Lateral view of one day old Tribolium pupal head. F. Lateral view of Schistocerca first nymphal instar head.Arrows indicate position of the morphogenetic furrow. Ommatidial preclusters represented by grey circles. ant 5 antenna, cly 5 clypeolabrum,eyd 5 eye disc, eyl 5 eye lobe, eyp 5 eye placode, ilo 5 inner optic lobe (lobula), ley 5 larval eye, man 5 mandible, max 5 maxilla, lab5 labium, olo 5 outer optic lobe (medulla and lamina), pro 5 protocerebrum; anterior is left and dorsal is up.

can be morphologically discriminated from the poste-rior part, which will form the retina and additionalhead cuticle areas. Close to middle of the third andlast larval instar, retina differentiation starts at the pos-terior margin of the eye disc. This process is markedby formation of the morphogenetic furrow, which re-fers to the front of differentiation where cells are uni-formly shorter and distally constricted forming a con-spicuous indentation along the dorsoventral axis of theeye disc. The furrow moves from its posterior startpoint towards anterior (Fig. 2D). Posterior to the fur-row, ommatidial preclusters emerge in a regular arrayanticipating the regularity of the adult retina. Photo-receptor cells join the ommatidial preclusters first, fol-lowed by cone cells and pigment cells. This earlyphase of retina cell determination and differentiationcontinues until the furrow has reached its final desti-nation at about 10 hours after pupation when the eyedisc everts. Once the entire eye field has been estab-lished, the retina cells undergo terminal differentiationin a concerted manner. Hallmarks of this process arethe elimination of surplus cells by apoptosis, synthesisof screening pigments and elaboration of the photo-receptor cell rhabdomeres. Approximately two thirdsof the posterior eye disc proper differentiates as retina

while the anterior third and parts of the peripodialmembrane, which is the second tissue layer of the sac-like disc, develop into adjacent head cuticle elements(Haynie and Bryant, 1986).

Eye imaginal disc formation as seen in Drosophilais not an obligatory feature of postembryonic eye de-velopment in holometabolous insects. More basal dip-teran and holometabolous species develop through aeucephalic larva, which carries a fully developed headcapsule equipped with antennal and mouthpart ap-pendages. The larva of the flour beetle Tribolium cas-taneum exemplifies this level of evolutionary organi-zation (Fig. 1). In this case, metamorphosis is signifi-cantly less dramatic. The differentiation of the adultbody plan proceeds through reinitiated growth and ter-minal differentiation of larval structures, which fromthis perspective function as adult organ primordia. Theadult antenna for instance develops via dramaticgrowth and further differentiation of the larval anten-na. The adult Tribolium retina, however, is not formedby further differentiation of the larval eyes. It seemsto develop de novo in a region of the late larval head,which corresponds to the future field of the retina inthe adult head capsule. This ectodermal tissue com-partment has been termed ‘‘eye placode’’ (Fig. 2E)


(Marshall, 1928; Friedrich et al., 1996; Friedrich andBenzer, 2000). The beginning of Tribolium retina dif-ferentiation is marked by the initiation of the morpho-genetic furrow at the posterior margin of the eye plac-ode. The morphogenetic furrow passes through the eyeplacode in anterior direction, coming to halt shortlybefore the antenna. The timing of Tribolium retina dif-ferentiation corresponds very closely to that in Dro-sophila (Fig. 1). The progression of the morphogeneticfurrow starts during the second half of the last larvalinstar and continuous through approximately the firstthird of pupation after which terminal differentiationinitiates. It is important to note that the Tribolium eyeplacode area becomes fully elaborated in the embryotogether with all other essential compartments of theadult head (Fig. 2B). In the more primitive Tribolium,adult head compartment and for the most part organprimordia formation have remained embryonic. It isseparated in time from the postembryonic differentia-tion of the adult retina. This marks a fundamental dif-ference to the continuous postembryonic morphogen-esis of the Drosophila head from internalized imaginaldiscs, and the integration of Drosophila retina differ-entiation into this derived process.

Eye development in hemimetabolous insects such asthe grasshopper Schistocerca americana is a repetitivemulti-step process which starts in the embryo and ex-tends through postembryogenesis (Fig. 1) (Anderson,1978; Friedrich and Benzer, 2000). Almost one thirdof the adult Schistocerca compound eye retina is ofembryonic origin. This embryonic fraction is formedfrom the ectoderm of the lateral-most tissue compart-ments of the embryonic head, the eye lobes (Fig. 2C).The morphogenetic furrow initiates in the posteriormargin of the eye lobe ectoderm shortly before 35%of embryogenesis. With the morphogenetic furrowprogressing anteriorly, the embryonic retina continuesto extend in anterior direction throughout much of thesecond half of embryogenesis, which however is alsocharacterized by the terminal differentiation of the ret-ina. Most of the eye field of the first instar nymph isthus fully differentiated and functional. Except for theanterior margin where cell proliferation and differen-tiation continuously reinitiate between the postembry-onic growth molts thereby enlarging the embryonicretina field to the size of the adult retina in severalpostembryonic increments (Figs. 1 and 2F) (Anderson,1978).

The evolution of postembryonic eye developmentmust have required the elaboration of mechanismswhich prevent the onset of adult retina differentiationin the embryo, maintain the prospective retina fieldduring early postembryogenesis, and coordinate thepostembryogenetic differentiation of the adult retinawith the complex process of metamorphosis. Thesechanges of development represent ground state chang-es as they are prerequisite for the postembryonic de-velopment of the compound eye retina in holometab-olous species in general. Additional modificationsmust have evolved in the lineage leading to Drosoph-

ila, which, as indicated, concerned the postembryonicdevelopment of the prospective retinal field and thecoordination of this process with the de novo devel-opment of the other adult head primordia in the eye-antennal imaginal disc. Yet further modifications ofembryonic visual system development are likely tohave been enforced in Drosophila by the evolution ofits extreme mode of short germ development in com-bination with the evolution of the acephalic larval headmorphology (Melzer and Paulus, 1989). It is thus notsurprising that the molecular control of Drosophila eyedevelopment differs from that in more primitive spe-cies at numerous steps preceding the final differentia-tion of the eye field.

PATTERNING OF THE EMBRYONIC VISUAL ANLAGE

Specification of the precursor embryonic tissuewhich gives rise to the visual system is the first stepin the sequence of events leading to the formation ofthe insect eye. In Drosophila, all visual system com-ponents map to a single unpaired primordium strad-dling the dorsal midline in the anterior head region ofthe blastoderm embryo, which is traditionally consid-ered the nonsegmental tip of the embryo (acron) andin addition includes the anlagen for the first brain neu-romer, the protocerebrum (Green et al., 1993; Dum-strei et al., 1998; Namba and Minden, 1999). The Dro-sophila visual primordium is molecularly defined byexpression of the homeobox gene so (Cheyette et al.,1994; Serikaku and O’Tousa, 1994; Chang et al.,2001). Homologs of so (Six3/6) are also involved inspecifying the visual primordium in other animal phyla(Oliver et al., 1995; Jean et al., 1999). While the tran-scriptional code for Drosophila visual primordiumspecification seems highly conserved, the mechanismsresponsible for its initial spatial regulation may be ofmore recent evolutionary origin. The expression of soin the visual primordium depends on activation by theTGF-b signaling factor decapentaplegic (dpp), whichis expressed in a dorsoventral gradient in the earlyDrosophila blastoderm embryo (Chang et al., 2001).Maximal Dpp levels in the dorsal midline of the em-bryo initiate expression of the HOX-3 orthologoustranscription factor zerknuellt (zen) which represses soexpression along the dorsal midline (Fig. 3). At thesame time, lower Dpp levels continue to activate soexpression along the lateral sides of the embryo. It hasbeen noted that the resulting split of the visual anlageresembles the morphogenesis of the vertebrate anteriorplate (Chang et al., 2001). In this case, however, ho-mologs of the signaling factor gene hh, induce the me-dian split of the anterior brain anlage (Ekker et al.,1995).

A possible explanation for this discrepancy is thatthe long germ insect Drosophila exhibits considerabledifferences in the formation of the embryonic anlagencompared to the ancestral mode of short germ devel-opment. This raises the possibility that evolutionarilyderived anterior head patterning mechanisms in Dro-sophila replaced ancestral mechanisms. Indeed, two


FIG. 3. Derived role of Drosophila dpp and zen in visual primor-dium patterning. A. Schematic dorsal view of visual system com-partments in the anterior head of the Drosophila embryo early ex-tended germband stage. Zen expression (grey) is induced by Dppalong the dorsal midline. Visual primordium specifying genes (blackarea) are repressed by Zen and activated by Dpp at a distance (afterChang et al., 2001). B. Schematic dorsal view of early heart-stagegermband of Schistocerca gregaria. Dpp and Zen (grey) are coex-pressed in the necklace cells, which mark the boundary betweenamnion and serosa cells (after Dearden and Akam, 2001). Stomodealopening indicated by circle positioned on midline. Visual primor-dium indicated by black areas; Anterior is up.

lines of evidence from anterior patterning gene ex-pression in primitive short germ insects such as Tri-bolium and Schistocerca suggest that midline pattern-ing of the Drosophila head is controlled by derivedmechanisms (Fig. 3). In both of these species zen isexpressed in the extra-embryonic serosa and amnionicmembrane tissue but not in the germband proper (Fal-ciani et al., 1996; Dearden et al., 2000). The peripherallocation of this expression domain excludes an in-volvement of zen in patterning the embryonic midlineof these species. Furthermore, the expression of dppoverlaps with that of zen in the serosa and amnionicmembrane of Tribolium and Schistocerca. This sug-gests that the activation of zen by dpp is conservedduring extraembryonic tissue specification (Sanchez-Salazar et al., 1996; Dearden and Akam, 2001). Thespatial regulation of its expression pattern howevermakes it seem unlikely that dpp is involved in midlinepatterning using alternative transcriptional mediators.

Second, genes involved in patterning the anteriorDrosophila head such as tailless (tll), orthodenticle(otd), and the segmentation gene wingless (wg) aretypically expressed in circumferential domains strad-dling the dorsal ectoderm midline like so (Nagy andCarroll, 1994; Chang et al., 2001). With the onset ofgastrulation, these circumferential stripes break up intoisolated expression elements. In most cases, expressionceases in the dorsal midline reminiscent of dorsal mid-line so repression by dpp/zen. In Tribolium howeverthe earliest detectable blastoderm expression patternsof wg, tll, and otd are already split in the dorsal mid-line (Nagy and Carroll, 1994; Li et al., 1996; Schroderet al., 2000). The same is true for the expression ofwg in the orthopteran species Acheta domesticus andSchistocerca gregaria (Niwa et al., 2000; Dearden andAkam, 2001). Taken together, these data support theexistence of ancestral midline patterning mechanismsthat might have been strongly modified or lost duringDrosophila evolution.

RETINAL FATE COMMITMENT (I): EYE VERSUS ANTENNA

The Drosophila visual system consists of severalcomponents. The information received by the regularsensory array of the facetted retina is processed in theoptic neuropil layers lamina, medulla, and lobula,which together constitute the optic lobe. In addition,the fruit fly possesses three ocelli, comparatively sim-ple photosensory organs that are centered between thecompound eyes at the dorsal midline of adult head,and an extra set of simple larval eyes, the Bolwig or-gans. Except for the ocelli, the tissues of all Drosoph-ila visual system components derive from compart-mentalization of the early embryonic visual anlage atstage 12 of Drosophila embryonic development (Fig.2A). At the stage of visual primordium partitioning,the population of cells, which will give rise to the pri-mordium of the adult retina, the eye imaginal disc, ischaracterized by specific expression of the master reg-ulatory gene ey (Chang et al., 2001; Kumar and Mo-ses, 2001c). This would suggest early commitment ofthe Drosophila eye disc, in line with the fate mappingsupported view that the adult Drosophila retina is de-termined during embryogenesis (Postlethwait andSchneiderman, 1971; Younossi-Hartenstein et al.,1993). However, partitioning of precursor tissue maynot be correlated with terminal commitment for organfate. The differentiation of the Drosophila retina is theendpoint of a series of discrete stages of tissue com-mitment. This was discovered studying of the role ofNotch (N) and Epidermal growth factor receptor (Egfr)signaling during early eye-antennal disc development,which revealed that the posterior part of the eye-an-tennal imaginal disc remains competent to adopt an-tennal fate until the second half of the second larvalinstar (Fig. 1) (Kumar and Moses, 2001a). At thisstage, the commitment decision of eye versus antennafate is under antagonistic control of the two signalingpathways with N signaling promoting retina fate ver-sus Egfr signaling promoting antenna fate. Consistentwith the experimental evidence for postembryonic de-termination of the retina field primordium, the regu-latory genes essential for Drosophila retina develop-ment are not coexpressed in the eye-antennal disc be-fore the second larval instar. Induction of Drosophilaretinal fate results from the concerted action of seventranscriptional regulators: the recently duplicated Dro-sophila Pax6 orthologs ey and twin of eyeless (toy),the related paired domain protein eye gone (eyg), andthe transcription factors dac, eya, so and optix (re-viewed in Kumar, 2001; Pappu and Mardon, 2002).Only toy and eyg are coexpressed with ey in the eye-antennal disc primordium following compartmentali-zation of the embryonic visual anlage (Kumar and Mo-ses, 2001c). This step controls the specification of theretinal precursor tissue, which is followed by post-embryonic determination. In a consistent manner, alsothe delay of antenna primordium determination cor-relates with absence of the antenna fate specifying


FIG. 4. Expression of wg and fng during dorsoventral compartmentformation in the early Drosophila eye disc. A. First instar eye-an-tennal imaginal disc. B. Third instar eye disc. Area filled with di-agonal grey bars rising from left to right indicates fng expressiondomain. Area filled with diagonal grey bars rising from right to leftindicates wg expression domain. Orientation of ommatidial clustersafter rotation indicated by arrows. Position of morphogenetic furrowindicated by lateral indentations. Dorsal is up and anterior is left.

transcription factor distal-less (dll) in the embryonicantennal anlage (Kumar and Moses, 2001a).

From a comparative perspective it is important tonote that the absence of adult retina and antenna dif-ferentiation in the Drosophila embryo correlates withthe lack of expression of transcription factors, whichare essential for the determination of the retina andantenna primordia. In non-holometabolous insectssuch as Schistocerca, the primordia of both the adultantenna and eye are formed during embryonic devel-opment (Fig. 2C). This implies that all of the requireddetermination genes need to be expressed already inthe respective embryonic anlagen. Transcription fac-tors, which are known to function in Drosophila an-tenna determination such as dll, spalt-major (salm) andextradenticle (exd) (Dong et al., 2002), are indeed ex-pressed in the embryonic grasshopper antenna (Fried-rich, unpublished observation). One prediction fromthe Drosophila model is therefore that also the eyedetermination genes are coexpressed in the posterioreye lobe margin of the grasshopper prior to the initi-ation of retina differentiation. Preliminary analyses ofeya and so expression during grasshopper embryonicdevelopment have yielded results that are consistentwith such scenario (Dong and Friedrich, unpublished).

Hypothesis building regarding the onset of eye de-termination transcription factor coexpression duringvisual system development in Tribolium is lessstraightforward. Two scenarios are conceivable. Evi-dently, the primordia of the adult antennae are formedduring embryogenesis in the form of the larval anten-nae, which also holds for most other head structuresin the eucephalic Tribolium (Fig. 2B). By analogy, thecompound eye retina primordium, i.e., the eye placode,may also be determined in the embryo but preventedfrom initiating differentiation. The determination stepmay involve coexpression of the retinal determinationgenes in the embryonic retinal primordium. However,morphological or molecular evidence for embryonicdetermination of the Tribolium eye placode is yetmissing. Alternatively, the shift of retina determinationnetwork gene coexpression into postembryogenesisseen in Drosophila may represent a mechanism, whichevolved early in holometabolous insects to precludeembryonic differentiation of adult retina. For this hy-pothesis to hold true, the members of the Triboliumeye specification transcription factor network shouldnot be coexpressed during embryonic development butin the eye placode during postembryonic development(Fig. 1). The comprehensive analysis of eye determi-nation gene expression in Tribolium and Schistocercawill provide the data necessary for the correct evolu-tionary interpretation of postembryonic retina deter-mination in Drosophila.

AXIS AND COMPARTMENT SPECIFICATION

The Drosophila eye-antennal imaginal disc pro-ceeds through continuous patterning and growth dur-ing larval development. Fundamental patterning stepsconcern the establishment of the anteroposterior and

dorsoventral axes (Lee and Treisman, 2001). In addi-tion, the retina field is divided into dorsal and ventralcompartments, which meet exactly along the midlineof the disc forming a border that has been termed theequator. In developmental terms, the equator is an Nsignaling based organizing center, which stimulatesgrowth of the eye disc prior to the onset of retina dif-ferentiation, and provides signals necessary for planarcell polarity patterning in the differentiating retina(Cho and Choi, 1998; Dominguez and de Celis, 1998;Papayannopoulos et al., 1998; Reifegerste and Moses,1999; Cho et al., 2000). Morphologically, the Dro-sophila equator becomes manifest by a 90 degree ro-tation of the developing ommatidia, which occurs inmirror image orientation in the dorsal and ventral halfof the retina field (Fig. 4B) (Dietrich, 1909). The dor-soventral compartmentalization of the Drosophila ret-ina field also affects early patterning and growth of theeye disc. Genetic ablation of the Lobe gene for in-stance leads to ventral specific loss of retina growth(Chern and Choi, 2002). Loss of homothorax (hth)gene activity leads to ectopic retina differentiation inthe ventral but not dorsal compartment of the eye disc(Pichaud and Casares, 2000). The zinc finger transcrip-tion factor teashirt (tsh) represses retina differentiationin the ventral compartment but promotes retina differ-entiation in the dorsal compartment (Singh et al.,2002).

The occurrence of similar dorsoventral compartmentpattern elements in the retina of other arthropod spe-cies as well as the general need for tissue growth inthe developing retina would lead one to expect thatformation of the equatorial organizing center by dor-soventral compartment formation is a conserved aspectof Drosophila eye disc development (Friedrich et al.,1996). This hypothesis can be tested as the gene net-work involved in dorsoventral compartment formationis well understood (Cho and Choi, 1998; Dominguezand de Celis, 1998; Papayannopoulos et al., 1998; Choet al., 2000). One of the first steps involves the dorsal


compartment specific activation of wg expression bythe transcription factor pannier (pnr) (Maurel-Zaffranand Treisman, 2000). Wg in turn activates expressionof the homeobox transcription factor mirror (mirr) thefunction of which is to suppress the expression offringe (fng) in the dorsal part of the disc (Yang et al.,1999). As a consequence, fng expression is restrictedto the ventral compartment. This leads to specific ac-tivation of N signaling along the midline where fngenhances the activation of N by its ligand Delta (Dl)while suppressing N activation by the second N ligandSerrate (Ser) in the ventral part of the disc. In sum-mary, dorsal expression of pnr, wg and mirr, versusventral expression of fng are essential elements of dor-soventral compartment formation in the Drosophilaeye disc (Fig. 4).

Interestingly, wg is not expressed in a dorsalizedmanner throughout development of the early grasshop-per embryonic retina although the polar expression ofwg at the anterior retina margin is conserved (Fig. 6)(Friedrich and Benzer, 2000). Complementary to this,the grasshopper eye lobe also lacks ventrally restrictedexpression of fng while its expression in and anteriorto the furrow is conserved (Dong and Friedrich, un-published; Dearden and Akam, 2000). These resultssuggest the absence of an N signaling based equatorialorganizing center in the grasshopper retina. Althoughsurprising at first glance, this is consistent with thelack of evidence for planar cell polarity patterning inthe grasshopper retina (Wilson et al., 1978). Further-more, while the N pathway is an essential upstreamgrowth activator in the Drosophila eye disc, many ad-ditional signaling factors stimulate proliferation in theDrosophila eye disc such as Dpp, Egfr and Wg (Burkeand Basler, 1996; Halfar et al., 2001; Lee and Treis-man, 2001). It is therefore conceivable that differentsignaling pathways support retina growth in grasshop-per without participation of N. To determine if the Nsignaling based dorsoventral compartment formation isa derived aspect of Drosophila eye imaginal disc de-velopment it will be necessary to analyze the relevantgenes in other insect species with dorsoventral specificpattern elements. Shared genetic mechanisms wouldindicate the function of an evolutionarily conservedpatterning mechanism that was lost during the evolu-tion leading to grasshopper. Lack of dorsoventral com-partment specific expression of wg and fng on the oth-er hand would suggest that dorsoventral patterningmechanisms evolved multiple times independently.

While the mechanisms related to dorsoventral pat-terning are suspect of evolutionary change, establish-ment of the anterior posterior axis in the Drosophilaeye disc is likely to follow ancient paths. The primarydeterminant of the anteroposterior axis is the expres-sion of wg, which changes from dorsal specific ex-pression in the second larval instar to a pair of dorsaland ventral domains at the anterior margin of the eyefield with beginning of the third instar (Fig. 4). As Wginhibits furrow initiation, movement and neuronal dif-ferentiation, the initiation of retina differentiation is

forced to occur at maximal distance to these expres-sion domains at the midline of the posterior eye lobemargin (Ma and Moses, 1995; Treisman and Rubin,1995). Furthermore, ectopic activation of Wg signalingcan enforce anterior compartment identity to the pos-terior of the retinal eye field (Lee and Treisman, 2001).Polar expression of wg in front of the retina field con-sistent with this patterning function was found con-served in several arthropod species ranging fromSchistocerca and Tribolium to crustacean and myria-pod species (Friedrich and Benzer, 2000; Niwa et al.,2000; Duman-Scheel et al., 2002; Hughes and Kauf-man, 2002).

INITIATION OF DIFFERENTIATION:WITH OR WITHOUT DPP?

The initiation of retina differentiation in the Dro-sophila eye disc has been genetically dissected intotwo discrete phases (Kumar and Moses, 2001b). Phaseone or ‘‘furrow birth,’’ which involves the primordialactivation of morphogenetic furrow formation andphotoreceptor differentiation at the posterior eye discmargin, requires activity of the N, EgfR and Hh sig-naling pathways (Dominguez and Hafen, 1997; Borodand Heberlein, 1998; Kumar and Moses, 2001b). Thesecond phase begins with the continuous initiation ofdifferentiation along the disc margins necessary forlateral increase of the eye field size. This process,dubbed ‘‘reincarnation,’’ depends on input from theEgfr and Dpp signaling pathways (Chanut and Heber-lein, 1997; Kumar et al., 1998; Curtiss and Mlodzik,2000). In the grasshopper eye lobe, furrow initiationoccurs also at the posterior margin centered at the mid-line followed by anterior progression of the furrow andlateral extension of the eye field (Fig. 2C). Consideringthe conserved spatial dynamics of retina differentiationit seems likely that the same regulatory mechanismsare at work in the grasshopper eye. However, compar-ative expression pattern analyses of the key player dppindicate partial evolutionary divergence at the molec-ular regulatory level. In Drosophila, dpp is stronglyexpressed along the posterior and lateral disc marginsbefore furrow birth and continues to be expressedalong the lateral eye disc margins during furrow re-incarnation (Blackman et al., 1991; Cho et al., 2000).This is consistent with functional evidence for Dppinvolvement in furrow initiation (Blackman et al.,1991; Chanut and Heberlein, 1997; Pignoni and Zi-pursky, 1997). In situ hybridization experiments inSchistocerca failed to detect expression of dpp in theposterior and lateral grasshopper eye lobe ectodermmargins prior to and following furrow initiation, whichargues against a role of dpp in both phases of grass-hopper furrow initiation (Friedrich and Benzer, 2000).

It has been proposed that the underlying cause forthis discrepancy between grasshopper and Drosophilaeye development may be the divergence of mecha-nisms that control the expression of wg in the eye field(Friedrich and Benzer, 2000). In Drosophila, dpp isessential for transforming the dorsal compartment spe-


FIG. 5. Signaling and transcription factor domains along the an-teroposterior axis of the differentiating retinal field in Drosophila,Tribolium and Schistocerca. Empty bars: Signaling factors. Brightbars: Furrow progression activating transcription factors. Black bars:Furrow progression inhibiting transcription factors. I: Anterior mosteye disc region developing into head cuticle. II: Anterior eye discregion developing into retina. PPN: Preproneural zone. MF: mor-phogenetic furrow. IV: Differentiating retina.

cific expression of wg into the anterior polar domains.The dorsal expression domain of the morphogeneticfurrow inhibitor wg extends initially to the posteriormargin of the disc during the dorsoventral compart-ment patterning phase (Fig. 4A) (Cho et al., 2000).During the late second larval instar, wg becomes re-pressed at the posterior margin by dpp. In the thirdinstar eye disc, dpp continues to be required for re-pression of wg transcription in both the dorsal and ven-tral margin (Royet and Finkelstein, 1997; Cho et al.,2000). A similar wg expression pattern change is notobserved during grasshopper eye lobe developmentwhere wg is expressed in anterior polar domains fromvery early on (Friedrich and Benzer, 2000). The wgand dpp expression patterns described in the devel-oping Tribolium eye lobes match that in the Schisto-cerca eye lobes (Nagy and Carroll, 1994; Sanchez-Salazar et al., 1996). The lack of dpp expression inthe grasshopper eye lobes prior to the initiation of themorphogenetic furrow thus correlates with a funda-mental difference regarding the emergence of the con-served expression of wg in the anterior eye field ofprimitive insects. It is therefore possible that the re-quirement of dpp for furrow initiation along the lateralmargins of the Drosophila eye disc evolved in con-junction with its apparently derived role of suppressingwg from these regions.

Recent studies however demonstrated that dpp pro-motes morphogenetic furrow initiation by activatingthe expression of retina determination genes such aseya (Curtiss and Mlodzik, 2000). Provided the respec-tive Dpp signal is not secreted by extraretinal tissuesin the grasshopper (see below), or replaced by relatedTGF-ß related signal transduction pathways, the dif-ferences between the regulatory networks controllingfurrow initiation in Drosophila and Schistocerca mighteven be deeper.

PROGRESSION OF DIFFERENTIATION

Once initiated, the furrow moves from posterior toanterior laying out the regular array of developing om-matidial precursor clusters in the posterior eye field.In Drosophila, the progression of this dynamic differ-entiation border is maintained by the combined actionof at least three signal transduction pathways, whichcoordinate the transcriptional control of retina differ-entiation (Bessa et al., 2002) (Fig. 5). The transcriptionfactors Hth, Tsh and Ey, which are coexpressed in theanterior disc region, have been proposed to interactphysically and keep the anterior eye disc tissue in anundifferentiated growth state (Bessa et al., 2002). Thelong-range signaling factor Dpp, expressed in the fur-row, instructs cells far anterior to the furrow to ad-vance from this state into a preproneural state (PPN),which is characterized by repression of retina differ-entiation antagonist hth and expression of the pro-neural transcription factor daughterless (da). Preco-cious neural differentiation in the PPN zone is pre-vented by the helix-loop-helix transcription factorhairy (h), which is also activated by dpp. Exit from

the PPN state is induced via activation of the N sig-naling pathway by the short-range factor Dl, which isalso expressed in the furrow. This leads to repressionof the neuronal specification antagonists extramach-rochaete (emc) and h, allowing for expression of atoin the furrow. The initial uniform expression of ato inthe furrow refines to R8 photoreceptor cells posteriorto the furrow. This is followed by rapid recruitment ofadditional photoreceptors cells to the newly emergingommatidia. The positive feedback loop between dif-ferentiation and induction of furrow progression isclosed by the expression of Hh in the maturing pho-toreceptor cells, which diffuses anteriorly and activatesN and dpp transcription in the furrow.

As the basic cellular morphology of the morpho-genetic furrow is conserved in diverse arthropods thereis little reason to suspect evolutionary divergence ofthe regulatory network driving furrow progression.


This is furthermore suggested by the strikingly similarinvolvement of hh and ato in vertebrate eye develop-ment indicating evolutionary conservation at a deepphylogenetic level (Neumann and Nuesslein-Volhard,2000; Brown et al., 2001). Nonetheless, comparativeexpression analysis in the developing retina of Schis-tocerca and Tribolium suggests partial evolutionary di-versification (Fig. 5). In Drosophila dpp begins toclear from the posterior margin and to increase ex-pression in the morphogenetic furrow after furrow ini-tiation (Blackman et al., 1991). In Schistocerca dpp isexpressed at low levels throughout the anterior eyefield ectoderm in addition to an expression domainposterior to the furrow, which is associated with thearea of the eye lobe retina where the photoreceptoraxons project from the retina into the lamina (Friedrichand Benzer, 2000). This finding has implications re-garding possible mechanisms involved in grasshopperfurrow progression by comparison to Drosophila.Grasshopper dpp may either instruct the entire anteriorretina field to enter the PPN state or carry out an en-tirely different function. Ongoing studies in our labshow that the homeodomain transcription factor exd,the essential dimerization partner of hth, is expressedneither in the grasshopper eye nor in regions imme-diately adjacent to it. This implies the absence of hthmediated transcriptional control in the anterior of thegrasshopper eye lobe (Dong and Friedrich, unpub-lished). The lack of a Drosophila PPN related tran-scriptional switch of hth expression in the grasshoppereye field points to the possibility that the furrow pro-gression promoting function of dpp is not conserved.Interestingly, Drosophila dpp is not essential for fur-row progression as hh on its own can induce the PPNstate although at a slower pace (Burke and Basler,1996; Greenwood and Struhl, 1999). It has been pro-posed that the expression of dpp in the Drosophilamorphogenetic furrow evolved to accelerate furrowprogression in this fast developing species (Friedrichand Benzer, 2000; Bessa et al., 2002). This idea wasbased on the assumption of an otherwise largely con-served network of transcriptional control of retina dif-ferentiation. The correlated lack of hth/exd in thegrasshopper eye lobe however raises the possibilitythat the divergence of dpp expression is connected tomore fundamental differences in the early control ofretina differentiation. This may be related to the evo-lutionarily derived dynamic determination of head ver-sus retina fate in the Drosophila eye disc.

RETINAL FATE COMMITMENT (II):EYE VERSUS HEAD CUTICLE

Coexpression of the seven essential eye specifica-tion master genes during the second larval instar pois-es cells in the Drosophila eye disc to adopt retinal fate(Kumar and Moses, 2001a). However, only approxi-mately two thirds of the posterior disc will differenti-ate into retina while cells in the anterior margin giverise to adjacent head cuticle elements (Haynie andBryant, 1986). Consistent with this, the domain of eye

specification transcription factor coexpression is cen-tered in the posterior of the eye disc and does notextend through the anterior disc (Bessa et al., 2002).At the time of furrow progression, for instance, theexpression of dac and eya does not extend more an-teriorly than to the anterior border of the PPN zonedue to hth mediated repression. The expression domainof hth on the other hand which initially covers theentire early eye-antennal imaginal disc becomes pro-gressively restricted to the posterior eye disc due torepression by Dpp emanating from the moving furrow(Pichaud and Casares, 2000; Bessa et al., 2002). Thereare thus two commitment stages through which cellsof the eye disc pass. During the second larval instarcells become committed to participating in retina overantenna associated head compartment formation. Dur-ing the third larval instar, cells adopt retina fate if theyfall within the reach of the morphogenetic furrowwhile developing into head cuticle components other-wise. At this stage, retina fate is promoted in the pos-terior disc by the combined action of the dpp, hh, Nand Egfr signaling pathways. The specification of headcuticle fate is in the realm of the anterior polar ex-pression domains of wg, which besides activating hthexpression in the anterior eye disc field, is a negativeregulator of photoreceptor differentiation itself (Maand Moses, 1995; Treisman and Rubin, 1995; Pichaudand Casares, 2000). In addition, there is evidence thatWg signaling can induce head cuticle morphogenesisas opposed to retina differentiation (Royet and Fin-kelstein, 1997; Baonza and Freeman, 2002). In sum-mary, wg is not only essential for determining the an-terior border to the progression furrow but also for themorphogenesis of major parts of the dorsal (vertex)and ventral (gena) Drosophila head. Consistent withthis, the respective eye disc anlagen reside within theexpression domains of hth and wg (Pichaud and Cas-ares, 2000).

Although the expression of wg in two polar domainsanterior to the developing retina is highly conserved,the fate map of wg expressing cells in the developinggrasshopper head shows significant differences withthat in the Drosophila eye disc (Fig. 6). Already theearly lateral wg expression domains reside within theanterior eye field of the eye lobe but do not extendinto areas outside the eye lobes. The discrepancy ismost obvious with regards to the dorsal head regions,which give rise to the lateral and median ocelli. Theselie within the wg expression domains in Drosophilabut are remote from the wg expression domains in thegrasshopper eye lobe. Further support for evolutionarydivergence of wg related patterning of the adult Dro-sophila head is indicated by the lack of Wg signalingdownstream target gene expression in the grasshopperhead. The transcription factor engrailed, which is ac-tivated by wg via otd, is required for ocelli formationin Drosophila but not expressed in the differentiatingocelli of the grasshopper embryo (unpublished obser-vation) (Royet and Finkelstein, 1995). The expressionof wg in the grasshopper eye lobe is thus compatible


FIG. 6. Comparison of Drosophila and Schistocerca dorsal head fate map. Left: Left half of grasshopper embryonic head at 40% of devel-opment. Middle: Morphology of generalized dorsal insect head. Right: Drosophila third instar antennal-eye imaginal disc. Grey shaded areas:wg expression domains. Homologous ocelli indicated by arrows. ant 5 antennal disc, eyd 5 eye disc, eyl 5 eye lobe, fro 5 frons, ver 5vertex.

with a role in negatively regulating furrow progressionand thereby organizing the anterior border of the ret-ina, but not with patterning adjacent head cuticle re-gions to the extent it is occurring in Drosophila. Thisdifference in wg patterning functions correlates withthe fact that partitioning of head versus retina field byformation of the eye lobes has been completed beforefurrow initiation during grasshopper embryonic headdevelopment. The integration of retina differentiationand adult head cuticle partitioning under antagonisticcontrol by wg and dpp represents a derived aspect ofDrosophila eye disc development.

CONTROL AT A DISTANCE (I):EXTRARETINAL SIGNALING SOURCES

The antagonism of anterior head cuticle versus ret-ina differentiation is only one example for the coor-dination of retina differentiation with adult head de-velopment in the Drosophila eye-antennal imaginaldisc. To embrace the entire scope of this patterningaspect, it is important to recall the double layer natureof the sac-like eye-antennal imaginal disc. The apicalsurface of the disc proper, the posterior compartmentof which forms the retina, is overlaid by the peripodialmembrane, which contributes a considerable part ofthe posterior dorsal head cuticle (Haynie and Bryant,1986). There is thus not only a need to coordinategrowth and differentiation between the anterior andposterior pole of the disc proper but also between theperipodial membrane and the disc proper. Recent stud-ies have provided evidence of regulatory communi-cation between these tissue layers by exchange of sig-naling factors across the lumen of the disc. Hh, Dppand Wg for instance are expressed in the peripodialmembrane of the early eye-antennal disc from wherethey instruct dorsoventral compartment formation inthe disc proper (Cho et al., 2000). Similarly, Fng andSer have been reported to be expressed in the peri-podial membrane instead of the disc proper in the con-text of dorsoventral compartment formation (Gibsonand Schubiger, 2000). Information exchange seems toproceed in both directions as Dpp expressed in the discproper furrow has been reported to maintain cell sur-vival in the peripodial membrane (Gibson et al., 2002).

Although results from these studies differ in some de-tail, Ser for instance has also been described as discproper located target of thh signaling (Cho et al.,2000), the evidence for signal exchange between discproper and peripodial membrane is overall consistentand compelling. Active and passive transport mecha-nisms have been proposed to be involved in the ex-change of signaling molecules between the two tissues.Dpp, expressed in the furrow of the disc proper hasbeen found to accumulate in the disc lumen (Gibsonet al., 2002). Cellular processes reaching from the per-ipodial membrane to the disc proper have been pro-posed to transport Hh, Wg and Dpp protein (Cho etal., 2000; Gibson and Schubiger, 2000).

From an evolutionary perspective, the regulatory in-teractions between peripodial membrane and discproper represent a derived patterning aspect of theDrosophila eye-antennal disc. In the more primitiveTribolium eye placode no peripodial membrane isformed and the differentiating retina cells contact thecuticle of the larval head (Friedrich et al., 1996). Thereis thus no candidate non-retinal signaling source facingthe Tribolium eye placode. Likewise, no peripodialmembrane equivalent tissue exists in the developinggrasshopper embryonic head. Nonetheless, extraem-bryonic tissues of the grasshopper embryo could serveas analogous extraretinal patterning sources. From gas-trulation to about 35% of development, the amnionicmembrane covers most of the grasshopper embryoreaching from the dorsal margins ventrally. The eyelobe ectoderm in particular develops in contact withthe amnionic membrane (Friedrich and Benzer, 2000).Interestingly, dpp is expressed in the dorsal edges ofthe grasshopper amnionic membrane from where itcould diffuse to the eye lobes (personal observation).The amnionic membrane will therefore have to be con-sidered as potential signaling source in future analysesof eye development in the grasshopper and non-holo-metabolous insects in general.

CONTROL AT A DISTANCE (II):HORMONAL REGULATION OF RETINA DIFFERENTIATION

In addition to local signals hormonal instructionsaffect the progression of the Drosophila morphoge-


netic furrow from a distance. During postembryoge-nesis, ecdysone is secreted from the larval ring glandsand metabolized into the hormone 20-hydroxyecdy-sone (20E) in peripheral tissues. Each of the three lar-val molts in Drosophila is associated with a transientpeak in 20E concentration, which instructs the epider-mal cells to secrete new cuticle. These 20 levels peaksoccur in the presence of a second hormonal mediator,juvenil hormone (JH), which preserves the growthcharacter of the molts. JH levels drop during the lastlarval instar and a moderate ecdysone peak induces thelarva to stop food uptake and to prepare for pupation.This stage is the wandering stage during which retinadifferentiation is initiated. The next ecdysone peak,which induces the pupal molt, is accompagnied by ris-ing JH levels. Juvenil hormone levels drop in the earlypupa. A final pupal 20E level peak in the absence ofJH initiates the terminal differentiation of adult struc-tures including the retina (for review see Riddiford,1993). Molecular genetic analyses have revealed thatthe progression of the morphogenetic furrow and theearly differentiation of photoreceptor clusters are sen-sitive to 20E levels as well as to the presence of com-ponents of the ecdysteroid signal transduction machin-ery. Conditional genetic ecdysteroid depletion in D.melanogaster causes stall of furrow progression, in-correct initiation of R8 founder cells, and loss of neu-ronal differentiation (Brennan et al., 1998). Somewhatconflicting results have been obtained by in vitro cul-turing studies in which morphogenetic furrow pro-gression and photoreceptor differentiation could be ob-served in the absence of ecdysteroid supplements (Liand Meinertzhagen, 1997). Nonetheless, the involve-ment of ecdysteroid signaling in the control of furrowprogression has been further substantiated by the find-ing that the furrow also stalls in tissue mutant for theecdysteroid signaling immediate early gene Broadcomplex (BR-C) isoform Z2 (Brennan et al., 2001).The canonical ecdysteroid receptor complex consistingof the ecdysteroid receptor (EcR) and the RXR ho-molog Ultraspiracle (Usp) seems not to be involved inthe transduction of the signal as the furrow is not af-fected in EcR mutant tissue (Brennan et al., 2001).Loss of Usp however causes acceleration of furrowprogression associated with ectopic expression of theBR-C isoform protein Z1 anterior to the furrow. Thediscrepancy compared to the ecdysteroid depletionphenotype is explained by the fact that Usp acts asrepressor when unbound, but as transcriptional acti-vator in the ligand bound situation (Zelhof et al., 1997;Ghbeish and McKeown, 2002). The biological signif-icance of the hormonal control of early retina differ-entiation in Drosophila remains to be determined. Onepossibility is that the hormonal clues are necessary toalign the multitude of organ differentiation events witheach other and the organism’s growth progress. Theneed for hormonal regulation of early retina differen-tiation is obvious for a second holometabolous species,in which the effect of ecdysteroid levels on retina de-velopment has been carefully investigated, the tobacco

hornmoth Manduca sexta (Champlin and Truman,1998). It is the transient halt of development duringpupal diapause in Manduca, which is controlled by thereversible block of early retina development at belowthreshold ecdysteroid levels. This responsiveness ofthe Manduca retina changes during the final pupal ec-dysteroid surge, which irreversibly terminates the pro-gression of the morphogenetic furrow and triggers ter-minal differentation throughout the retina field in aconcerted manner. The final pupal ecdysteroid peakhas the same consequences in Manduca and Drosoph-ila including rhabdomere formation, pigment synthesisand lens cuticle secretion from cone cells. In both sys-tems, these developmental responses can be triggeredin cultured retinas by application of 20E (Li and Mei-nertzhagen, 1995; Champlin and Truman, 1998). Incombination, these data suggest that the ecdysone de-pendence of terminal retina differentiation is an an-cestral aspect of postembryonic eye development inholometabolous insects. The regulatory effect of ec-dysteroid signaling on early retina differentiation mayalso trace back to the origin of holometabolous insectsbut obviously experienced lineage specific modifica-tions.

The shared ecdysteroid signaling dependence ofpostembryonic retina differentiation in Manduca andDrosophila raises the question if the hormonal controlof eye development evolved in the ancestral lineage ofholometabolous insects, or was inherited from hor-monal mechanisms already involved in the control ofretina differentiation in primitive insects. Ecdysteroidlevel peaks have long been known to induce moltingboth during embryogenesis and postembryogenesis inhemimetabolous insects including orthopterans such asSchistocerca (Lagueux et al., 1979). Less informationexisted regarding possible involvement in additionaldifferentiation processes. Cell proliferation at the post-embryonic morphogenetic furrow in Schistocerca hasbeen reported to reach highest levels between moltswhile being silent during molting, which is induced byecdysteroid level peaks (Anderson, 1978). These datasuggest that ecdysteroids may have an inhibiting effecton furrow progression during grasshopper postembry-ogenesis. In culturing experiments of embryonicSchistocerca eye lobes, however, supplementing 20Estimulated cell proliferation and the rate of morpho-genetic furrow progression (Dong et al., 2003). Alsoaspects of terminal differentiation such as screeningpigment synthesis were significantly enhanced by 20Eapplication. At the same time, base levels of furrowprogression, cell proliferation and screening pigmentsynthesis could be observed in culture even when ec-dysteroid signaling was blocked by application of ec-dysteroid antagonist Cucurbitacin B suggesting thatecdysteroid signaling is not essential for retina differ-entiation in Schistocerca (Dong et al., 2003). In com-bination, the available data from Schistocerca indicatethat retinal development is not dependent on but sen-sitive to ecdysteroid levels both during embryogenesisand postembryogenesis in primitive insects. The strin-


gent control of early retina differentiation in speciessuch as Manduca may have evolved by modificationof preexisting mechanisms of hormonal control. Thesemechanisms may already have been related to the de-velopmental control of diapause, which is also ob-served in primitive insects (Tawfik et al., 2002). Thecritical dependence of terminal differentiation on ele-vated ecdysteroid levels during pupation on the otherhand has to be added to the list of derived aspects ofDrosophila eye development when compared to prim-itive insects.

SUMMARY AND PERSPECTIVES

Given the infancy of molecular genetic studies ofeye development in non-Drosophila insects our un-derstanding of insect eye development evolution is bynecessity still incomplete and preliminary. Nonethe-less, the data accumulated so far seem to build a strongcase for the hypothesis that relatively recent evolu-tionary modifications affected many steps of the mo-lecular developmental control of Drosophila retina for-mation. Some generalization may be helpful to struc-ture the diversity of observations presented. The evo-lution of rapid embryogenesis in higher flies via anextreme form of long germband development affectedthe early patterning of the embryonic visual primor-dium. The restriction of retina differentiation to latestages of postembryonic development, which separatesholometabolous insects from primitive hemi- and ame-tabolous insects, involved changes in the timing, andperhaps logic, of retina primordium determination, andmodifications of the hormonal control of retina devel-opment. The evolution of postembryonic adult headprimordium formation from internalized imaginaldiscs, which allowed the emergence of the acephaliclarva typical for brachyceran flies, is likely to haveenforced the widest range of developmental modifi-cations. These may concern the control of initiationand progression of retina differentiation, the develop-mental communication between retina and non-retinatissues, and the dynamic partitioning of retina versusadjacent head cuticle compartments. A comprehensiveanalysis of eye development in Schistocerca and Tri-bolium holds promise of elucidating the apparentlyhighly eventful evolutionary history of Drosophila vi-sual system development. In addition, it will be nec-essary to extend the comparison to crustaceans to ver-ify the ancestral status of processes operating in prim-itive insects by outgroup comparison (Hafner and To-karski, 1998; Melzer et al., 2000; Harzsch andWalossek, 2001).

ACKNOWLEDGMENTS

I am grateful to the members of the lab for readingthe manuscript and three anonymous reviewers forhelpful comments. This research was funded by NSFgrants DBI-0070099 and DBI-0091926.

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evolution of insect eye development: first insights from fruit fly

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