INTRODUCTION Drosophila eye although structurally distinct from theve - PDF document

INTRODUCTION Drosophila eye, although structurally distinct from thevertebrate eye, shows striking parallels at the molecular level.Many genes that function in eye formation in the ßy havehomologs that are expressed during vertebrate eye develop-ment (Quiring et al., 1994; Zuker, 1994; Oliver et al., 1995).Drosophilaeyes absent eyaencodes a nuclear protein that, in the ßy, functions prior to theÞrst notable differentiation event Ð morphogenetic furrowformation Ð in eye progenitor cell development (Bonini et al.,eya SUMMARY Drosophila eyes absent gene directs ectopic eye formation in a pathwayconserved between ßies and vertebrates Nancy M. Bonini*, Quang T. Bui, Gladys L. Gray-Board and John M. Warrick Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA*Author for correspondence (e-mail: nbonini@sas.upenn.edu) GAL4 was made by subcloning a fragment which reports eyelessstaining in eye progenitor cells (Quiring et al., 1994), into the GAL4vector (Brand and Perrimon, 1993), and transforming the constructDrosophila eyamutant alleles are as previously described (Bonini et al.,eyelessmutant strain was provided by courtesy of Dr Walterwhich express in the eye portion of the eye-antennal disc. is expressed in the imaginal discs in an expression pattern similar toeyeless-lacZlines are as described by Quiring et al.UAS-eyelesslines are as described by Halder et al. (1995a). ImmunohistologyTissue preparations were Þxed with 2% paraformaldehyde in TBS,permeabilized with 0.5% Triton X-100, and stained in primaryantibody overnight. Primary antibodies were anti-Eya (Bonini et al.,was stained with secondary antibodies conjugated to ßuorescein orcyanine-3 (Jackson ImmunoResearch Laboratory), rinsed in TBS,then mounted in PDA-glycerol, as previously described (Bonini et al.,eyeless-lacZ expressionpattern was performed as described by Quiring et al. (1994). Forviewing ßies by scanning electron microscopy, ßies were critical pointdried and scanned at 5 kV. For sections of eye tissue, ßies were ÞxedRESULTS A vertebrate Eya homolog is able to functionallyreplace the ßy gene To test potential functional homology between the vertebrateeya genes, we asked whether a vertebrate was capable of replacing the ßy eya gene in the eye develop-mental pathway. To do this, we required functional replace-eya activity in the eyaeyamutant is a viable, eye-speciÞc null for eyagene activity in eyeprogenitor cells anterior to the furrow (Bonini et al., 1993).These mutant ßies are completely eyeless due to complete lossof eye progenitor cells by cell death (Fig. 1A). Thus, anysurvival and development of ommatidia to the adult eye in thismutant background would indicate functional replacement ofeya gene activity. To express the vertebrate gene, the GAL4-UAS system oftissue-speciÞc targetting was used (Brand and Perrimon, 1993).UAS-cDNA that is predicted to encode a full-length proteinhomolog shows an overall identity of 49% with the predictedßy protein sequence. To express the gene sufficiently early ineye progenitor cell development, we constructed an eyelessGAL4 transgenic line that drives GAL4 expression in eye pro-genitor cells prior to furrow formation. Remarkably, the mouse gene restored eye formation toeyamutant ßies (Fig. 1A,B). Sections through the eyesreplace the ßy gene in eye formation. These data demonstratethat molecular features of the eye developmental pathwayeya gene activity are conserved betweenßies and vertebrates. eya function is essential for eyeless Given this striking level of conservation of eya eyelessgene activity and eya gene activity suggested by expressionstudies in vertebrates. To determine whethereya expression occurred upon eye formation direc

ted by the eyelessgene, we generated ectopic eyes with eyelesstissue for ectopic expression of Eya protein. Flies bearing aUAS-eyelessinsert (Halder et al., 1995a) were crossed to a-GAL4 insert line, which expresses GAL4 in the imaginaldisc expression pattern of animals generated ectopic eyes on the legs, wings and antennalregion of the head (Fig. 2A,C; Table 1). Immunostaining of third-instar larval imaginal discsconÞrmed that Eya was indeed ectopically expressed in regionseyelessdirected ectopic eye formation: the antennalportion of the eye-antennal disc, leg and wing discs (Fig. 2F,I, gene functionally complements the ßy eyamutant. (A) Scanning electron micrograph of the head of an eyamutant ßy. This allele of eyaeyain eyeprogenitor cells prior to furrow formation, and results in completelack of eyes (Bonini et al., 1993). (B) Scanning electron micrographof an eye generated by the mouse eyaeyaUAS-Eya2eyeless-GAL4. The eyeshows a pattern of ommatidial units with bristle cells similar to thenormal ßy compound eye. (C) Tangential section of a ßy eyeeyaUAS-eyeless-GAL4 ßy. Photoreceptor cells have developed in a patterntypical of the compound eye (compare to Fig. 3C). In this section, itis possible to see all photoreceptor cells: to the right, ommatidiawith R1-R6, plus R7 are present; to the left, ommatidia with R1-R6,plus R8 are seen. The ommatidia are surrounded by a pigmentlattice, and the equator can be seen running through the eye. Bar, 50Table 1. function is essential for ectopic eye formation Structure with ectopicUAS-eyelesseya; UAS-eyeless eye formation Antennae100%0%Legs100% 0% Wing100%0%Haltere24% 0%50 animals scored for each data point; ectopic eye development is scored*Animals are completely eyeless in addition to lacking ectopic eye  Legs truncated and abnormally shaped (see Fig. 2C-E). directs eye developmentdata not shown). Of these imaginal tissues, Eya is normallyexpressed only in eye and ocellar progenitor cells of the eye-Eya is not normally expressed in cells of the antennal, leg orwing disc proper (Fig. 2H,K, data not shown; Bonini et al.,1993). Activation of eyagene expression by eyelesseya may indeed be required for formation of eyes byeyelesseyanormal compound eye development. To test whether eyagene activity was essential for eyelessdriven ectopic eye formation, we attempted to induce ectopiceyes with UAS-eyeless -GAL4, but now in theeyaeyapletely eyeless and null for the early eye function of the eyagene. If eye formation by eyeless eyagene activity, then ectopic eye formation should fail in the eyamutant background. Eye formation indeed failed in all tissuesof the ßy where ectopic eyes had previously developed: thelegs, wings and antennal segments of the head (Table 1, Fig.protein expression was detectable in the antennal, leg or wingdiscs of animals bearing UAS-eyelesstranseyamutant background (Fig. 2G,J). These experimentsalso demonstrated that UAS-eyeless was not able to restorenormal eye formation to the eyaeyeless gene activity cannot replace or substitute for theeyain eye development. Taken together, these dateeyaeyelessto form eyes; the eyalogical target of eyelessgene activity in eye formation. eya gene directs ectopic eye formation Given the high conservation demonstrated above of the eyapathway at the functional level between ßies and vertebrates,and given the essential role of eyaeyelesswell extend between ßies and vertebrates, we were interested eyagene function is essential for ectopic eye formation by the eyeless gene. (A) Ectopic eye formation in the antennal lobe (arrow) of aUAS-eyeless -GAL4 adult ßy. (B) Lack of normal and ectopic eye formation in an eyamutant animal bearing UAS-eyelessGAL4 insertions. (C) First leg of a UAS-e

yeless-GAL4 ßy. The leg is stunted in length, and eye tissue is present in the distal portion(arrow). (D) First leg of a UAS-eyelesseyamutant background. No eye tissue is present, however the leg is gnarled andstunted in length. (E) First leg of a normal ßy, for comparison. (F,I) Expression of Eya in animals of genotype UAS-eyeless Strong ectopic expression of Eya occurs in the antennal lobe (F, white arrows), and leg discs (I, arrow). Compare to normal patexpression in H and K. (G, J) Expression of Eya in animals of genotype UAS-eyelesseyaeyamutant, no Eya expression is detectable in the eye portion of the disc except for the ocellar progenitors (arrow; the ocelli foeyamutant). No ectopic Eya protein is detectable in the antennal region of the disc (G) or the leg discs (J); the leg discs appearmorphologically abnormal. (H,K) Expression of Eya in the (H) eye-antennal and (K) leg discs of a normal animal. Eya expression the eye portion of the eye-antennal disc, and is present both anterior and posterior to the furrow (arrow). Expression also occregion of the eye disc far anterior to the furrow. Eya is not expressed in the leg discs. Anterior is to the right for F-H. Bar to determine what were potential effects of the eyafor eye formation. Could eya, like eyeless, mediate eyeformation? Previously, we had expressed the eyasuch expression could restore the eyes to eyalacked eye formation, no other consistent effects were observedin the rescued animals. Nevertheless, we attempted to expresseyaat higher levels using the GAL4-UAS system (Brand andPerrimon, 1993), to determine whether it was possible toinduce dominant phenotypes that would yield clues to theUAS-eyaconstruct was made with the eya cDNA. Todetermine whether the construct was functional, we attemptedeyamutant phenotype using various GAL4 linesthat express in the eye progenitor Þeld prior to furroweyelesstions, when crossed to a UAS-eya eyamutant eyes up to three quarters of normal size. Given thatrescue was partial, we attempted to increase eye size by increas-UAS-eya UAS-eyatrans lines were lethal in two doses of the transgenes; for those linesthat were lethal at the late pupal stage (homozygous animals could be observed by dissection of thepupae. This analysis showed that these lines displayed not onlyrescue of the eye, but also ectopic eye formation in other regionsof the animals where GAL4 was expressed with these con-UAS-eyaeyainto a normal background for additional analysis. We focusedon expression of UAS-eyadriven by bination led to late pupal lethals that could be readily observed;in these animals, GAL4 expression occurs in the imaginal discexpression pattern of , in the eye and antennal portions ofthe eye-antennal disc, the leg and wing discs, among otherIn a wild-type background, UAS-eyadppcopy generated rare examples of ectopic eyes, which resemblednormal eyes, on the antennal segment (10% of the animals, Fig.3A,B). Tangential sections of the ectopic eyes formed indicatedthat photoreceptor cells developed in a pattern similar to thatof the normal compound eye (Fig. 3C). With two copies ofUAS-eya dpp-GAL4, ectopic eye formation was induced in theantennal region of the head in almost all animals (96% ectopiceye formation on antennae); 80% also showed ommatidialformation on the legs, and occasionally on the wings. Glass, aphotoreceptor-speciÞc gene, was used as a marker to detectdevelopment of retinal tissue in the larval imaginal discs. Glassexpression is normally restricted to the eye portion of the eye-other imaginal discs (Ellis et al., 1993). Ectopic expression ofGlasswas seen in the antennal and leg imaginal discs; in thesetissues, rosettes of developing photoreceptor clusters similar toeyahas the capacity to fu

nction as a master regulatory genefor eye formation. Requirement for eyelessin ectopic eyes producedby eya These observations raised questions regarding the relationshipeya eyeless gene functions during eyeeyawas essential for ectopic eye formationeyeless (see Table 1; Fig. 2), was eyeless essential for ectopic eye formation by eya? To address this, weÞrst asked whether eyeless gene expression was induced duringectopic eye formation directed by the eyagene. Normally,eyeless expression is restricted to the eye portion of the eye-antennal imaginal disc (Fig. 4A; Quiring et al., 1994). In UAS-eya dpp-GAL4 animals, expression of eyeless cally in the antennal region of the eye-antennal disc, in theregion where eya directed ectopic eye formation (Fig. 4B).eyaalso directed ectopic eye formation in the legeyelessexpression was not detectable in thateya expression (Fig. 4C); eyelesswas capable ofautoregulation in leg discs when eyeless itself was ectopicallyexpressed (Fig. 4D). This suggested that eyeless eya activity to form eyes in some tissues, but dis-pensible in others. We also determined that eyeless expressed in the eye progenitor cells of the eya eyagene directs ectopic eye formation. (A,B) Scanningelectron micrographs of ectopic eyes (arrows) formed on the antennalsegment of ßies heterozygous for UAS-eyadpp-GAL4. The regularnormal eye. (C) Tangential eye section through the normal eye (right)and the ectopic eye (left) formed on the antennal segment of a ßyheterozygous for UAS-eya dpp-GAL4. (D,E) Ectopic expression ofthe photoreceptor-speciÞc protein Glass in antennal (D, arrowhead)and leg (E, arrow) imaginal discs of larval animals of genotype UAS-eya dpponly in the eye portion of the eye-antennal disc (Ellis et al., 1993),posterior to the furrow (arrow in D). Reverse images of ßuorescent- directs eye developmenteya normal expression pattern of eyelessTo address a functional requirement for eyeless, we investi-eyeless eyagenes. Such experiments are limited by theeyelesscurrently available Ð there are no null mutantsfor the eye function of eyeless which would allow us to removeeyeless gene activity completely (Quiring et al., 1994). Never-theless, we attempted to induce ectopic eye formation witheyaeyeless gene activity, by using theeyelesseyelessmutant ßies show a range ofreduced eye phenotypes, with about 30% of the ßies missing atleast one eye completely. Directed expression of the eyaeyelessbackground, did not result in ectopic eye formationin the antennal segments of the head, or the legs (data not shown).eyelessgene activity in theeyato direct eye development both in the head and inthe legs. Thus, eya appears to function both downstream andeyelessgene activity in eye formation. Potentiation between eyelesseyain eyeformation This regulatory relationship between the two genes promptedbetween the genes. To do this, we examined the ability ofeyelessto direct eye formation when combined with additionaleya gene activity. Animals bearing UAS-eya trans-GAL4 show limited dominant effects (see above andTable 2); in animals bearing UAS-eyelessGAL4, Eya protein is already highly expressed (see Fig. 2).Nevertheless, ectopic eye formation by eyelesswas dramati-eyagene activity was provided(Table 2, Fig. 5). The ectopic eyes were larger and formed witheyelesseyaalone, and eyeformation now occurred on the genitalia, a condition never pre-viously observed in individuals with either gene alone (Table2 and Fig 5B,E). This effect did not appear additive (Table 2).Rather, these data suggest functional synergy between eyelesseyagene activities in eye formation. Our data reveal an active role of the eyagene in eye formation,and suggest a model of gene regulatory interactions betweeneyeless eya

in eye formation in the ßy that may extend toConservation of eya function between vertebratesWe found that a vertebrate homolog of eyagene, can functionally replace the ßy gene in eye formation.eya gene in eyeformation has been conserved through evolution, between ßiesand vertebrates, despite dramatic differences in eye structurebetween the two (see Zuker, 1994). Such functional homologyhas been shown for various Pax-6eyeless (Halder et al., 1995a; Glardon et al., 1997): we have extendedeyaand its homologs, a second gene of the eyedevelopmental pathway. The vertebrateÞed to date are all expressed in the developing or adult eye,suggesting all homologs may function in aspects of vertebrateeye formation and maintenance (Duncan et al., 1997; Xu et al.,1997; Zimmerman et al., 1997). For the demonstrate here a homologous role in the eye developmentalpathway. eya gene activity to eyelessWe have addressed and clariÞed the relationship of eya Ectopic eye formation by eyaeyelessgene expression.eyeless in eye progenitor cells of a normal eye-eyelessexpression is detected with a galactosidase reporter construct (Quiring et al., 1994). Arrowindicates position of the morphogenetic furrow. (B,C) Ectopicexpression of eyelesseyagenotype UAS-eyadpp-eyelessexpression occurredin the antennal region of the eye-antennal imaginal disc (B,arrowhead; small arrow indicates the position of the morphogeneticfurrow). In the leg discs (C), however, ectopic eyelesseya expression. (D) When eyeless expressed, eyelessexpression is detectable in leg discs (D) as well asin the antennal portion of the eye-antennal disc (not shown),consistent with autoregulation of the eyelesseyeless; UAS-eyelesstranseyeless expression is present in the eye progenitoreyamutant discs. The eye portion of the disc is reduced insize due to loss of the eye progenitor cells by programmed cell death,no morphogenetic furrow is present (Bonini et al., 1993). Anterior toTable 2. Functional synergy between ectopic eye formation Structures with ectopic UAS-eyeless eye formationUAS-eyaUAS-eyelessUAS-eyaAntennae10%95%100%Proboscis0%13%91%Legs0%100%100%Wing0%100%100%Haltere0%33%93%Genitalia0%0%55% data points. Ectopic eye development is scored by the presence of ommatidia with pigment. Expression of the UAS constructs driven with activity to that of the eyelessgene. Previous data indicate that,normally, eyeless expression precedes that of eya in eye prog-eyelessis expressed in the eye pri-eyaexpression is initiated during the mid-larval stages (Bonini etal., 1993). We found that eyagene activity was essential foreye formation by eyeless; these data, along with the observa-eyelessremains expressed in ßy eyaeyais downstream of eyeless expression was induced upon ectopiceyeless expression; mammalian expression is also affectedPax-6eya/Eyathus appears to be essential for eye formation byeyeless/Pax-EYAgene activity may be a criticalimproper eye formation. eya mutant background, we note that eyelessactivitywas not completely ineffective Ð although ectopic eyeformation did not occur, leg development remained severelyaffected (see Fig. 2 D,J). This suggests that eyeless is activat-eyaing with normal leg development. These additional genes mayeya in the eye developmental pathway, or in adifferent branch of the eye developmental pathway (see belowand Fig. 6). Furthermore, target genes of eyelessmay havefunctions in addition to those associated with eye development.For example, the dachshund) gene is a target of eyelessgene activity that functions both in eye and leg developmenteyaas a master control gene for eye formation We found that eyaeyelessas a Ômaster control geneÕ for eye formation. By this term, werefer to the fact that eya priate genetic pro

gram of the many genes required for eyedevelopment (Halder et al., 1995a). Using loss-of-functioneyelessmutants, we found evidence that eyeless activity is eyaactivity functionally enhances ectopic eye formationeyelessgene. (A-C) Ectopic eye formation on ananimal of genotype UAS-eyelesstrans -GAL4. Ectopic eyesform on the antennal segment of the head (A, arrow), the wings andlegs (C, arrows). No ectopic eye formation occurs on the genitalia(B). (D-F) Ectopic eye formation on an animal of genotype UAS-eyelesstransto UAS-eya dpp-GAL4. Ectopic eyes which form onthe antennal segment (D, arrows), wings and legs (F, arrows) arelarger than without UAS-eya. Ectopic eye tissue is now also observedon the genitalia (E, arrow). Bar, 100 C, E-F. eya/EYAeye development eyelessPax-6eya/EYA in eye development. eya is placed downstream ofeyelessdata indicate that expression of eyeless prior to expression of eya(Bonini et al., 1993) in normal eyedevelopment. However, a loop between eya eyelesseyaactivity essential foreyeless function, but also eyelesseyaeyaeyeless together are more effective in eyeeyaeyelessleast partially distinct pathways (curved arrow to the left and thepathway through eya), both of which are critical for eye formation.We propose these same gene interactions may exist for themammalian counterparts, given the conservation of function ofeyeless Seyshown previously (Halder et al., 1995a), andfunctional conservation between mammalian eyashown directs eye developmenteyato form eyes Ð similar to the requirement foreya gene activity in the proper eyelessfunction in eyeformation. Thus, the activities of the eyaeyeless appear connected by a regulatory loop, with each functionallyrequired by the other in eye formation (Fig. 6). One qualiÞca-tion of these conclusions is that in leg discs, we were unableeyeless expression upon ectopic eyaactivity. Never-eyeless indicated that eye formation by eyain legs appeared dependenteyeless gene activity. Thus, we suggest that eyelessis required for ectopic eye formation by the eyaeya downstream of, but connected back to, eyelessgene function. Genetically, there is little to argue which geneis Þrst; however, the normal expression patterns of the geneseyelessexpression temporally precedes that of eyaduring normal eye formation (Bonini et al., 1993; Quiring etal., 1994). In ectopic eye formation, the genes are eachMoreover, we found that eyaeyeless tional synergy in eye formation Ð the same dosage of eyelesswas potentiated when combined with additional eya function. This synergy was observed with a construct, and whether other regulatory elements will mediatea similar level of synergy remains to be determined. However,eyelesshas functions in addition to activating eyaactivityis also suggested by the severely affected leg morphologyobserved upon eyelessexpression in the eya ground (see above). These data suggest that eyeless eyamay function in at least partially distinct pathways for eyeformation (Fig. 6). By such a model, expression of either genealone, if expressed strongly enough, will eventually drive bothpathways because they form part of a regulatory loop.However, when both genes are expressed strongly, bothpathways will be strongly driven, leading to an enhancementof eye formation compared to that with either gene alone. Taken together, these data suggest that early events of eyeformation proceed not by a simple linear pathway, but ratherno single Ômaster control geneÕ for eye formation, but acomplex regulatory network of gene activities required totrigger the biological event of eye development. These initialevents of eye formation include additional genes, such as in this regulatory pathwayhas also been shown able to directeye formation (Shen

and Mardon, 1997). Given the increase ineyelessexpression upon -directed eye formation (Shen andlikely also requires eyeless gene activityto form eyes. This suggests that, minimally, eyaconnected through common regulation of, and regulation by,eyelessgene activity. eyaeyeless central to eye formation conserved in both ßies and vertebrates,(Cheyette et al., 1994; Serikaku andOÕTousa, 1994; Oliver et al., 1995), are also of great interest.Moreover, how these early events of eye determination subse-quently merge with pattern formation events of furrowmovement (Heberlein and Moses, 1994) and cell cycle regula-tion (Thomas and Zipursky, 1994), are key aspects of gener-ating a patterned neural structure like an eye. Somehow, thesedifferent aspects of the eye developmental process must betriggered by the eye differentiation pathway and coordinatelyregulated, to achieve this exquisitely organized neural center. The role of the eya geneeya gene has additional roles in development. In ßieseya can be embryonic lethal (NŸsslein-Volhard etin defects in gonad formation (Boyle et al., 1997), whereasEYA1 show developmental defects of thebranchial arches, ear and kidney (Abdelhak et al., 1997). Thus,eya gene has roles in development of the animal in additionto a function in eye formation. It is thus of interest to determinewhether expression of eyahas consequences over and aboveectopic eye formation. Expression in other tissues or at othertimes in development may lead to elucidation of additionalexpression for eye formation. Toward this end, it is rather sur-prising that genes like eyeless, eya animal in addition to eye formation, should induce eye devel-opment when ectopically expressed. What leads to this speci-Þcity is of particular interest. With respect to the role of eya eye formation, loss-of-function eyamutants show death of eyeTaken together, these data indicate that, although theeya in eye differentiation is coupled to both differ-entiation and survival, the most dramatic effects of the geneupon strong expression are in the differentiation pathway.eyaactivity withcell death will be indirect, through an effect on the pathway ofdifferentiation. What gene activites might be altered in eyamutants, such that the cells become directed down a deathpathway, remain to be deÞned. With respect to the eye developmental pathway, the biolog-ical activities of eyeless eya in eye formation extend to theirPax-6Sey has been shown to function in the ßy (Halder et al., 1995a) andwe have shown that a mouse homolog of eya functionally replace the endogenous ßy gene in eye develop-ment. These data indicate a remarkable level of conservationof gene function in eye formation between ßies and mammals.et al., 1995a,b; see Zuker, 1994) that common geneticpathways may be used for the formation of eyes of widelydivergent structure in organisms as evolutionarily distant asWe thank our many colleagues in the ßy and vertebrate communi-ties, especially in the laboratories of Dr Walter Gehring, NorbertDrosophilaCenters, who have generously provided reagents. We thank Drs LauraLillien, Anthony Cashmore and Mark Fortini for critical comments.Eye Institute (EY11259), the John Merck Fund, the University ofPennsylvania Research Foundation (to N. M. B.), and a Vision CenterTraining Grant (to J. W.). Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C.,Weil, D., Cruaud, C., Shaly, I., Leibovici, M., et al. Drosophila eyes absent Renal (BOR) syndrome and identiÞes a novel gene family. Nature Genet.Blackman, R., Sanicola, M., Raftery, L., Gillevet, T. and Gelbart, W.(1991). An extensive 3-regulatory region directs the imaginal diskexpression of decapentaplegicfamily inDrosophilaDevelopmentBonini,

N., Leiserson, W. and Benzer, S.eyes absentGenetic control of cell survival and differentiation in the developingDrosophila eye. Boyle, M., Bonini, N. and DiNardo, S.in the development of somatic gonadal precursors within the DrosophilaDevelopmentBrand, A. H. and Perrimon, N.(1993). Targeted gene expression as a meansof altering cell fates and generating dominant phenotypes. DevelopmentCheyette, B. N. R., Green, P. J., Martin, K., Garren, H., Hartenstein, V. andZipursky, S. L.homeodomain-containing protein required for the development of the entireNeuronDuncan, M. K., Kos, L., Jenkins, N. A., Gilbert, D. J., Copeland, N. G. andTomarev, S. I.(1997). Eyes absent: a gene family found in several metazoanDrosophila glassprotein and evidence for negative regulation of its activity in non-neuronalcells by another DNA-binding protein. DevelopmentGlardon, S., Callaerts, P., Halder, G. and Gehring, W.(1997). Conservationof Pax-6 in a lower chordate, the ascidian DevelopmentGlaser, T., Walton, D. S. and Maas, R. L.evolutionary conservation and aniridia mutations in the human PAX6 gene.Nature Genet.Halder, G., Callaerts, P. and Gehring, W.(1995a). Induction of ectopic eyesby targeted expression of the eyelessDrosophilaHalder, G., Callaerts, P. and Gehring, W.(1995b). New perspectives on eyeevolution. Curr. Opin. Genet. Dev.Hanson, I. and van Heyningen, V.(1995). Pax6: more than meets the eye.Trends Genet.Heberlein, U. and Moses, K.morphogenesis: The virtues of being progressive. Hill, R., Favor, J., Hogan, B. M., Ton, C., Saunders, G., Hanson, I., Prosser,J., Jordan, T., Hastie, N. and van Heyningen, V.Small eyeresults from mutations in a paired-like homeobox-containing gene. NatureHogan, B., Horsburgh, G., Cohen, J., Hetherington, C., Fisher, G. andLyon, M.Small eyesSeychromosome 2 which affects the differentiation of both lens and nasalJ. Embryol. Exp. Morph.Jordan, T., Hanson, I., Zaletayev, D., Hodgson, S., Prosser, J., Seawright,A., Hastie, N. and van Heyningen, V.(1992). The human PAX6 gene ismutated in two patients with aniridia. Nature GeneticsLeiserson, W. M., Bonini, N. M. and Benzer, S.(1994). Transvection at theeyes absentDrosophilaNŸsslein-Volhard, C., Wiechaus, E. and Kluding, H.affecting the pattern of the larval cuticle in Drosophila melanogasterRouxÕs Arch. Dev. Biol.Oliver, G., Mailhos, A., Wehr, R., Copeland, N., Jenkins, N. and Gruss, P.most anterior border of the developing neural plate and is expressed duringeye development. DevelopmentQuiring, R., Walldorf, U., Kloter, U. and Gehring, W. J.eyelessDrosophilasmall eyeDrosophilawith transposable element vectors. Serikaku, M. A. and OÕTousa, J. E.required for Drosophila visual system development. Shen, W. and Mardon, G.(1997). Ectopic eye development in Drosophilainduced by directed dachshundexpression. DevelopmentStaehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A. H. andHoffman, F. M.factors: cell fate and gene expression changes in Drosophila decapentaplegicbut notCell Growth Differ.Thomas, B. J. and Zipursky, S. L.developing Drosophilaeye. Trends Cell Biol.Ton, C. C. T., Hirvonen, H., Miwa, H., Weil, M. M., Monaghan, P., Jordan,T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Dreschler, M.,Royer-Pokora, B., Collins, F., Swaroop, A., Strong, L. C. and Saunders,G. F.homeobox-containing gene from the aniridia region. Xu, P.-X., Woo, I., Her, H., Beier, D. and Maas, R.Drosophila eyes absentPax6for expressionDevelopmentZimmerman, J., Bui, Q., Steingrimmson, E., Nagle, D., Fu, W., Genin, A.,Spinner, N., Copeland, N., Jenkins, N., Bucan, M. and Bonini, N.Cloning and characterization of two vertebrate homologs of the Drosophilaeyes absentZuker, C. S.(1994). On the evolution of eyes: would you like it si