SPECIAL ISSUE RESEARCH ARTICLE

Restriction of Ectopic Eye Formation byDrosophila Teashirt and Tiptop to theDeveloping AntennaRhea R. Datta, Jessica M. Lurye, and Justin P. Kumar*

In Drosophila, the retinal determination network comprises a set of nuclear factors whose loss-of-functionphenotypes often include the complete or near total elimination of the developing eye. These genes alsoshare the ability of being able to induce ectopic eye formation when forcibly expressed in nonretinal tissuessuch as the antennae, legs, halteres, wings, and genitals. However, it appears that the ability to redirect andtransform tissue fates is limited; not all tissues and cell populations can be forced into adopting an eye fate.In this report, we demonstrate that ectopic eye formation by teashirt and its paralog tiptop, a potential neweye specification gene, is restricted to the developing antennae. Of interest, tiptop appears to be a moreeffective inducer of retinal formation than teashirt. A genetic screen for interacting proteins failed toidentify paralog-specific relationships suggesting that the differences between these two genes may beattributed instead to structural differences between the duplicates. We also demonstrate that in addition tobeing expressed in coincident patterns within the developing eye, both paralogs are transcribed at verysimilar levels. Developmental Dynamics 238:2202–2210, 2009. © 2009 Wiley-Liss, Inc.

Key words: teashirt; tiptop; Drosophila; retina; eye specification

Accepted 18 February 2009

INTRODUCTION

In Drosophila, early eye developmentis governed by a set of DNA bindingproteins and transcriptional coactiva-tors that collectively are termed theretinal determination (RD) network.The core factors are encoded by thePax6 genes eyeless (ey, Quiring et al.,1994; and twin of eyeless [toy], Czernyet al., 1999), the SIX class transcrip-tion factor sine oculis (so, Cheyette etal., 1994; Serikaku and O’Tousa,1994), the transcriptional coactivatorand protein tyrosine phosphatase eyesabsent (eya, Bonini et al., 1993) and adistant relative of the Ski/Sno pro-tooncogene dachshund (dac, Mardon

et al., 1994). Removal of any of thesegenes leads to the complete elimina-tion of the adult compound eye (re-viewed in Kumar, 2008). Remarkably,forced expression of any memberwithin nonretinal tissues such as theantennae, legs, halters, and wings canlead to the formation of ectopic eyes(Halder et al., 1995; Bonini et al.,1997; Shen and Mardon, 1997; Czernyet al., 1999; Weasner et al., 2007). As acassette, these factors function duringeye development in a broad range oforganisms including vertebrates (re-viewed in Treisman, 1999; Wawersikand Maas, 2000; Hanson, 2001). Inaddition to this core network of genes,

there are several other factors thatinteract with and modulate the activ-ities of these genes, which are alsorequired for early eye development orcan induce ectopic eye formation.These genes include the Pax6(5a)genes eyegone (eyg, Jun et al., 1998)and twin of eyegone (toe, Jang et al.,2003), the SIX transcription factor op-tix (Seimiya and Gehring, 2000), theMeis1 homolog homothorax (hth, Paiet al., 1997), the serine-threonine ki-nase nemo (nmo, Braid and Verheyen,2008), the pipsqueak motif containingDNA binding proteins distal antenna(dan) and distal antenna related(danr, Curtiss et al., 2007) and the

Department of Biology, Indiana University, Bloomington, IndianaGrant sponsor: National Institutes of Health; Grant number: 1R01 EY014863.*Correspondence to: Justin P. Kumar, Department of Biology, Indiana University, Bloomington, IN 47405.E-mail: jkumar@indiana.edu

DOI 10.1002/dvdy.21927Published online 3 April 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 238:2202–2210, 2009

© 2009 Wiley-Liss, Inc.

zinc finger transcription factor teash-irt (tsh, Pan and Rubin, 1998).

Tsh was first initially identified as aspecifier of trunk identity and seg-mentation in the embryo, where lossof tsh function resulted in the trans-formation of the prothoracic segmentto a labial identity (Fasano et al.,1991; Roder et al., 1992). A potentialrole for tsh during early eye develop-ment was first hinted at when it wasrecovered in an enhancer/promoter(EP) screen for genes that could forcenonretinal tissues into adopting aneye fate (Pan and Rubin, 1998). Tshprotein is distributed throughout theregions of the developing eye that lieanterior to the advancing morphoge-netic furrow (MF). It functions to pro-mote cell proliferation and dependingupon the context can either promoteor inhibit eye development (Bessa etal., 2002; Singh et al., 2002, 2004;Bessa and Casares, 2005). Retinal mo-saic clones that completely lack tshdevelop normally thereby promptingthe suggestion that a second gene isacting in a functionally redundantmanner to tsh in the eye (Pan andRubin, 1998).

The tsh paralog, tiptop (tio) wasidentified as a regulator of Drosophilaembryogenesis (Laugier et al., 2005).tio null mutants are viable, fertile anddisplay no retinal abnormalities. tshand tio have different expression pat-terns until stage 17 of embryonic de-velopment, after which point theyhave shared expression domains. In-terestingly, these genes are not com-pletely redundant to each other asknockdown experiments with RNAiconstructs can induce retinal defects.It appears that both genes are main-tained, in part, because each gene iscapable of repressing its own expres-sion and that of its paralog (Laugier etal., 2005; Bessa et al., 2009). Thus, itseems that correct development of theretina is not dependent upon the ab-solute expression levels of each indi-vidual gene. Rather, it is suggestedthat the eye simply requires certaincombined levels of this zinc finger sub-family (Bessa et al., 2009).

In this study, we have attempted tofurther elucidate the roles of Tsh andTio in eye development. We find thatboth genes are expressed in identicalpatterns in the eye–antennal disc andthat their expression levels are not

significantly different from eachother. We have also analyzed the abil-ity of the duplicates to direct eye spec-ification and demonstrate that whiletsh and tio can induce ectopic eye for-mation (Pan and Rubin, 1998; Bessaet al., 2009), this activity is limited inthat both genes are capable of onlyredirecting the fate of the developingantennal imaginal disc. Additionally,we observe that tio is more effectivethan tsh in inducing ectopic eye for-mation.

RESULTS

tsh and tio Are Expressed atSimilar Levels in the Eye

Tsh/Tio proteins belong to a subclassof Zn finger DNA binding proteins.Tsh contains three such motifs whileTio, like its orthologs in basal insectsand vertebrate species, contains fourdomains (Fig. 1A; Fasano et al.,1991; Laugier et al., 2005). Both tioand tsh are expressed in coincidentpatterns ahead of the advancingmorphogenetic furrow (Fig. 1B,C;Pan and Rubin, 1998; Bessa et al.,2009). tio null mutants and tsh nullretinal clones do not appear to ad-versely affect eye development (Panand Rubin, 1998; Laugier et al.,2005). This has prompted the sug-gestion that each gene can substi-tute for the other during retinal de-velopment. In support of thishypothesis, is the demonstrationthat each gene negatively regulatesthe transcription of the other. Theloss of either gene would result inthe de-repression of the other para-log, thus compensating for the initialdeficit in expression of either tsh ortio (Laugier et al., 2005; Bessa et al.,2009). One expectation from thismodel is that the transcriptional lev-els of both genes should be approxi-mately equal. To test this hypothe-sis, we used quantitative reversetranscriptase-polymerase chain re-action (RT-PCR) to determine andcompare the transcriptional levels ofboth tsh and tio in the developing eyedisc. We recovered RNA from eye–antennal imaginal discs and observethat a typical wild-type eye–anten-nal disc contains approximately8,356 tsh and 7,787 tio transcripts,respectively. These differences

(across five samples and with multi-ple primer pairs) are not statisticallysignificant (P � 0.627). We concludethat both genes are not expressed atsignificantly different levels fromeach other (Fig. 1D). These resultsfurther support the premise that tshand tio are likely to share many com-mon functions during normal eye de-velopment.

Differences in Ectopic EyeFormation Are Not Due toExpression Patterns orLevels

Despite multiple similarities, thesegenes are unlikely to be completelyfunctionally redundant as tio can onlypartially rescue the trunk denticlephenotype of tsh null mutants (Lau-gier et al., 2005). As retinal develop-ment in tio null mutants and tsh nullmutant clones is relatively normal weset out to determine whether there areany differences between the abilitiesof either Tsh and Tio to induce ectopiceye development. Previous reportshave demonstrated that both genescan induce ectopic eyes when drivenby dpp-GAL4 (Pan and Rubin, 1998;Bessa et al., 2009). We extended theseresults by forcibly expressing tsh andtio (individually) throughout develop-ment with 219 different GAL4 driversand observe that both genes can coaxonly cells within the antennal discinto adopting a retinal fate (see be-low). Coexpression of both genes didnot lead to an expansion of tissuesthat can be converted the retinal tis-sue—ectopic eyes are still restrictedto the antennal disc. We found thatexpression of tsh could induce ectopiceyes with four GAL4 drivers (Fig.2A–D) while tio could induce ectopiceyes when expressed with eight driv-ers (Fig. 2A–H). To attribute this re-sult to an actual property of the gene,we tested (1) temporal and spatial dif-ferences in the driver expression pat-tern; (2) the transcriptional level ofeach UAS line; and/or (3) the tran-scriptional strength of each GAL4line. We first compared the expressionpatterns of the drivers and do not seeany correlation between the pattern ofexpression and the induction of ec-topic eyes. For example, tsh can in-duce ectopic eyes when expressed withcb41-GAL4 but not with MJ33a-GAL4

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even though they are expressed invery similar patterns within the an-tennal disc (Fig. 2C,G). All eight driv-ers are expressed in both the secondand third-instar discs; thus, it doesnot appear that temporal cues arecritical.

We then measured the levels ofGAL4 transcription using quantita-tive RT-PCR. Although we observesignificant differences between indi-vidual driver lines, there is no corre-lation between these differences and

Fig. 1.

Fig. 2.

Fig. 1. Expression patterns and RNA levels oftsh and tio in the developing eye of Drosophila.A: Schematic diagram of Tsh and Tio proteins.B: Confocal images of third-instar imaginaldiscs. Both discs are of wild-type animals. An-terior is to the right and the visualized mole-cules are listed at the bottom right of eachpanel. C: Determination of tsh and tio transcriptlevels in the developing eye imaginal disc usingquantitative reverse transcriptase-polymerasechain reaction.

Fig. 2. Expression patterns and transcriptionlevels of GAL4 drivers. A–H: Confocal images ofthird-instar imaginal discs. The identity of theGAL4 driver is listed at the bottom right of eachpanel. All lines were crossed to a UAS-GFPresponder. F-actin is visualized in red, whilegreen fluorescent protein (GFP) is visualized inblue. Anterior is to the right. I: A table describingthe average copy number of GAL4 transcriptsfor each driver line. J: Determination of GAL4transcript levels in the developing eye–antennaldisc of different GAL4 lines using quantitativereverse transcriptase-polymerase chain reac-tion.

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the ability of each gene to induce ec-topic eyes (Fig. 2I,J). Among the fourdrivers that produce ectopic eyeswhen placed in combination withUAS-tsh (dpp-GAL4, cb26-GAL4,cb41-GAL4, and Ser-GAL4) the aver-age transcript level of GAL4 rangesfrom 212 to 14,825, suggesting thatthe transcription level of the theseparticular driver lines is not the lim-iting factor in the induction of ectopiceyes by tsh. In addition, expression oftsh with dpp-GAL4, cb26-GAL4, orcb41-GAL4 induces ectopic eyes butnot with T98-GAL4 even though theaverage transcript level of the latter(6,089) is substantially greater thanthose of the former lines (Fig. 2I,J).We then turned our attention to theexpression levels of the two UAS linesthemselves. Individual expression oftsh and tio in all cells posterior to thefurrow by means of the GMR-GAL4driver induces a mild roughening of theposterior portions of the adult retina(Fig. 3A,C). The retinal phenotypes arenearly identical to each other. Usingquantitative RT-PCR we measured thetranscript levels in GMR-GAL4/UAS-tsh and GMR-GAL4/UAS-tio third-instar imaginal discs (Fig. 3B,D). Thesemeasurements include not only thetranscripts that are derived from theUAS line but also the endogenous tshand tio transcript levels respectively.We then subtracted the average wild-type endogenous tsh and tio transcriptsand determined an average transcriptlevel that should only reflect transcrip-tion from the UAS lines themselves. Weobserve that GMR-GAL4/UAS-tsh andGMR-GAL4-UAS-tio eye imaginal discsproduce (directly from the UAS inser-tion) approximately 1,782 tsh and 3,330tio transcripts, respectively (Fig. 3E).These differences (across four biologicalsamples and with multiple primerpairs) are not statistically significant(P � 0.126), and we conclude that, inthis case, the transcriptional levels ofthe UAS lines themselves do not ac-count for the differences in ectopic eyeformation.

Tsh Induces Ectopic Eyes inTwo Cell Populations

We then focused on the location of theectopic eyes within the developing an-tennal segment. Within the develop-ing antennal segment two zones can

be coaxed into adopting a retinal fate.The first and most commonly trans-formed territory is the ventral-mostregion of the antennal disc (Fig. 4A–D,6; green arrow). This area normallygives rise to the head cuticle that liesadjacent to the eye disc (Haynie andBryant, 1986). It should be noted thatmost other retinal determinationgenes are capable of inducing ectopiceyes only within this zone (Halder etal., 1995; Bonini et al., 1997; Shen andMardon, 1997; Seimiya and Gehring,2000; Weasner et al., 2007; Braid andVerheyen, 2008). The second, less fre-quently converted region is within theportion of the antennal disc that willgive rise to the arista and possibly thethird antennal segment of the adultantenna (Fig. 4A, green arrowhead;Haynie and Bryant, 1986). Other thantsh and tio, the only other genes thatare known to convert these cells intophotoreceptor cells are distal antenna(dan) and distal antenna related(danr; Curtiss et al., 2007).

The ectopic eyes, particularly thosederived from along the ventral ridge ofthe antennal disc, can range in size,with some eyes containing just a hand-ful of ommatidia (Fig. 4D,H) to othersbeing almost half the size of the normalcompound eye (Fig. 4C,G). The largereyes appear to be the result of signifi-cant tissue proliferation as the antennaldiscs are greater in size than wild-typeand have an abnormal profile. This isconsistent with the role that has beenassigned to tsh in promoting cell prolif-eration in the normal eye (Bessa et al.,2002). It also appears that there is adistinct directionality to movement ofthe ectopic furrows. Instead of seeingfurrows all along the internal edge ofthe ectopic eye, as is seen in patched(ptc) and Pka loss-of-function clones aswell as hedgehog (hh) overexpressingclones (Chanut and Heberlein, 1995;Heberlein et al., 1995; Ma and Moses,1995; Pan and Rubin, 1995; Strutt etal., 1995) we observe with antibodiesdirected against DAC and EYA proteinsthat an ectopic furrow is present onlyalong a partial stretch of the internaledge (Fig. 4E,F, white arrows).

Tio Is a More EffectiveInducer of Ectopic Eyes

Expression of tio appears to transformthe same two antennal cell popula-

tions into ectopic eyes (Fig. 5A–G,green arrows and arrowheads). Simi-lar to those induced by tsh, the ectopiceyes that result from tio expressioncan also range in size from just a fewommatidia (Fig. 5D,E,G,K) to nearlyhalf the size of the normal eye (Fig.5H,J,K). Expression of tio also ap-pears to promote tissue proliferationas the antennal discs appear distortedand significantly larger in size thanwild-type (Fig. 5C,F–K). However, de-spite these similarities there are alsoseveral differences between the twogenes. First, in addition to the fourGAL4 drivers that induced ectopiceyes with tsh (Figs. 2A–D, 5A–D), tioinduced ectopic eyes when expressedwith four additional GAL4 drivers(Figs. 2E–H, 5E–G). Of interest, whentio is expressed with one of these fourdrivers (T98-GAL4), we fail to observeectopic eyes in third-instar imaginaldiscs but do see them in adult animals(data not shown). It is likely that thefinal steps of eye development (i.e.,cell fate specification) are executedduring the early stages of pupal devel-opment. Second, unlike tsh, expres-sion of tio often induces multiple ec-topic eyes within a single antennaldisc (Fig. 5E,I,K). And third, we ob-serve that, in many instances that anectopic furrow forms along the entireinternal edge of the ectopic retina(Fig. 5H, arrow). Simultaneous ex-pression of both tsh and tio did notincrease the number of GAL4 driversthat could induce ectopic eyes. In fact,ectopic retinal development remainedlimited to the eight GAL4 drivers de-scribed in Figure 2.

In an attempt to identify paralog-specific genetic interactions thatmight explain the distinctions be-tween tio and tsh, we conducted mod-ifying genetic screens on flies thatwere expressing either tsh or tio in alldeveloping photoreceptors by meansof the GMR-GAL4 driver. Expressionof either gene leads to a near identicalmild to moderate roughening of theposterior portion of the retina (Fig.3A,C). Underlying this roughening isa nonspecific loss of photoreceptorcells within each ommatidium (datanot shown). We conducted two geneticscreens, identified several interactinggenes including wg and homothorax(hth) but failed to identify any para-log-specific interactions. The identifi-

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Fig. 3.

Fig. 4.

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cation of genetic interactions with hthare consistent with recent demonstra-tions that expression of tio, like that oftsh, activates and maintains hth ex-pression (Bessa et al., 2009). Our fail-ure to identify such interactions does

Fig. 5.

Fig. 6.

Fig. 3. Transcript levels and phenotypic effectsof UAS-tsh and UAS-tio expression. A,C: Scan-ning electron microscopy of adult compoundeyes. Genotype is listed at the bottom right ofeach panel. Anterior is to the right. B,D: Deter-mination of the tsh and tio transcript levels inthe developing eye–antennal discs. Note thatthese quantifications include both endogenousand exogenous transcript levels. E: Determina-tion of tsh and tio transcript levels that are justderived from GMR-GAL4 overexpression. Theendogenous wild-type values were subtractedfrom the total values in B and D.

Fig. 4. Induction of ectopic eyes by Tsh is re-stricted to the antennal disc. A–F: Confocal im-ages of third-instar imaginal discs. The identityof the GAL4 driver is listed at the bottom right ofeach panel. All lines were crossed to a UAS-tshresponder. Visualized molecules are listed inthe top right of each panel. Green arrows andarrowheads denote the two cell populationsthat can be converted into developing photore-ceptors. White arrow denotes area where a newmorphogenetic furrow is observed. Pink arrowindicates areas where there is no new furrow.Anterior is to the right. G,H: Light microscopeimages of adult animals. The identity of theGAL4 driver is listed at the bottom right of eachpanel. Both lines were crossed to a UAS-tshresponder. Anterior is to the left.

Fig. 5. Induction of ectopic eyes by Tio is re-stricted to the antennal disc. A–G: Confocalimages of third-instar imaginal discs. The iden-tity of the GAL4 driver is listed at the bottomright of each panel. All lines were crossed to aUAS-tio responder. Please note that we wereunable to detect ELAV-positive cells within theantennal disc of animals in which UAS-tio wasexpressed by the T98-GAL4 line (despite thefact that we see ectopic eyes in the adult). Thefinal stages of photoreceptor developmentlikely take place during the pupal stage. Visual-ized molecules are listed in the top right of eachpanel. Green arrows and arrowheads denotethe two cell populations that can be convertedinto developing photoreceptors. White arrowmarks a morphogenetic furrow that extendsalong the entire length of the internal edge of anectopic eye. Anterior is to the right.

Fig. 6. Transformation of the antenna into eyeby RD genes. A schematic diagram of a third-instar eye–antennal imaginal disc. Nearly all ret-inal determination genes, including Tsh and Tio,can convert a population of cells (labeled in red,arrowhead) lying within the ventral most sectionof the antenna. Both genes, in addition to danand danr, are also able to transform a secondpopulation of cells (labeled in pink, arrow).

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not rule out their existence, but it doessuggest that such interactions are notexceedingly common and might makeonly a minor contribution to their sub-tle functional differences.

DISCUSSION

In this report, we describe several find-ings that shed light on the function oftsh and tio, two zinc finger containingtranscription factors that appear tofunction during eye development inDrosophila. Our first result focuses onthe transcript levels of both genes dur-ing normal retinal formation and wefind that both paralogs are expressed atequivalent levels. Previous reports havedemonstrated that loss-of-function tionull mutants and loss-of-function tshnull retinal clones have negligible reti-nal defects (Pan and Rubin, 1998: Lau-gier et al., 2005). Both genes are ex-pressed in identical patterns anterior tothe furrow (Bessa et al., 2009) and havethe ability to repress their own and eachother’s expression (Laugier et al., 2005;Bessa et al., 2009). From these data, amodel has emerged in which tsh and tioshare responsibilities and are partiallyredundant in the eye, where the loss ofone gene leads to the transcriptional de-repression of the paralog. The result iscompensation by one paralog for theloss of the other. In the simplest incar-nation of this model, both genes shouldbe expressed at relatively equivalentlevels. If either gene is expressed at sig-nificantly higher levels than its paralog,then mutations in the lesser-expressedgene should result in a visible pheno-type. For example, the paralogs eyegone(eyg) and twin of eyegone (toe) are ex-pressed in identical patterns during eyedevelopment. However, their transcriptlevels vary, with eyg and toe transcriptsmake up 87% and 13% of the totalPax6(5a) transcript pool. Mutations ineyg have drastically reduced eyes,whereas knockdown of toe expressionby RNAi has minimal if any phenotypiceffects (Yao et al., 2008). Having bothgenes (tsh and tio) expressed at similarlevels supports the observations thatloss of either gene has minimal pheno-typic effects and that both genes represseach other’s transcription without ex-tinguishing each other’s expression.

The second result is focused on theability of tsh and tio to induce ectopiceye development in comparison with

other RD genes. Prior reports havedemonstrated that, when expressed inthe dpp expression pattern, bothgenes are capable of inducing ectopiceye formation (Pan and Rubin, 1998;Bessa et al., 2009). We used 219unique GAL4 drivers to express UAS-tsh and UAS-tio responder lines andfind that the ability of both tsh and tioto induce ectopic eye formation differssignificantly from ey, which can pro-mote eye development in a plethora ofnonretinal tissues including the legs,antennae, wings, and halteres (Halderet al., 1995). In contrast, ectopic eyeformation, induced by the expressionof tsh and tio, is limited to just thedeveloping antennal segment. An-other difference between tsh, tio, andthe other eye specification genes is inthe nonretinal cell populations thatthey can convert into eye tissue. Un-like other eye specification genes,which can only transform a single cellpopulation within the antenna(Halder et al., 1995; Bonini et al.,1997; Shen and Mardon, 1997; Se-imiya and Gehring, 2000; Weasner etal., 2007; Braid and Verheyen, 2008),tsh and tio can coax two separate cellpopulations into adopting a retinalfate (Fig. 6). These results suggest adevelopmental paradox. On one hand,tsh and tio appear more effective thanother eye specification genes includingey in converting cells within the an-tenna toward a retinal fate. On theother hand, however, both genes aremuch more restricted than genes likeey and eya in inducing ectopic eyes inother nonretinal tissues.

Finally, we focused much of our ef-forts toward understanding the extentto which the two genes differ function-ally. We have observed that, while tiocan induce ectopic eyes when ex-pressed with eight different GAL4drivers, tsh can only induce ectopiceyes when expressed with a subset ofthese drivers. Several observationssuggest that tio is a more effective in-ducer of ectopic eyes than tsh. First,tio can induced ectopic eyes when ex-pressed through a wider range ofGAL4 drivers. Second, tio appears ca-pable of inducing multiple ectopic eyeswithin a single antennal disc while tshdoes not. And third, the ectopic eyesthat are induced by tsh appear to havea fully formed morphogenetic furrowwhile only partial furrows are gener-

ated by the expression of tsh. Theredid not appear to be any correlationwith these observations and the spa-tial and temporal expression patternsof the GAL4 drivers or with the tran-scriptional levels of either the GAL4drivers or the UAS responder lines.We also attempted, but ultimatelyfailed to identify paralog-specific ge-netic interactions that could explainthis phenomenon.

The appearance of Tsh and Tio ap-pears to be a lineage-specific duplica-tion event, occurring before the Dros-ophilid diversification. It is a distinctpossibility that these paralogs havesubfunctionalized in Drosophila, al-though polymorphism data are re-quired to confirm the speculation(Shippy et al, 2008; Bessa, 2009).Studying these genes in the context ofeye development not only reveals newplayers in the RD cascade, it unearthsa new regulatory feedback loop duringimaginal disc development. We canalso show what paralogs are doing inthe nascent stages of gene evolutionby studying subtle functional changeslike those we have described here.

Despite the coincident expression oftsh and tio and the lack of retinal phe-notypes in individual null mutants,there is a singular reason to suspectthat these proteins have at least asubset of distinct functions: Tio andTsh are structurally distinct with Tshharboring three zinc fingers DNAbinding regions while Tio has four(Fasano et al., 1991; Laugier et al.,2005). A tsh/tio gene with three zincfingers appears to be specific to theDrosophilids as homologs withinbasal insects and vertebrate speciesall contain four putative DNA bindingdomains (Caubit et al., 2000, 2005;Laugier et al., 2005; Shippy et al.,2008; R.R. Datta and J.P. Kumar, un-published data). Furthermore, thereare also differences in the noncon-served segments of the proteins. Thedifferences related to the induction ofectopic eyes are likely due to dispari-ties (1) in the specificity and/or affinityof DNA binding; (2) in the number ofzinc finger domains; or (3) in the non-conserved regions of the protein. Iden-tifying, this difference is likely to shedconsiderable light onto how the Tshand Tio proteins function during nor-mal and ectopic eye development andwill also show whether coding sub-

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functionalization is occurring alongthe lengths of the paralogs.

EXPERIMENTALPROCEDURES

Fly Stocks

The following fly stocks were used inthis study: UAS-tsh (gift of AlbertCourey), UAS-tio (gift of LaurentFasano), GMR-GAL4 (gift of Larry Zi-pursky), tio-GAL4 (gift of Amit Singh),UAS-ey (gift of Walter Gehring), UAS-GFP and the DrosDel Deficiency andGAL4 collections (gifts of the Bloom-ington Drosophila Stock Center). AllGAL4 crosses were conducted at 25°C.

Microscopy

The following primary antibodieswere used in this study: rat anti-ELAV (1:100, DSHB), mouse anti-DAC (1:5, DSHB), mouse anti-EYA(1:5, DSHB), rabbit anti-TSH (1:3,000,Stephen Cohen). The following sec-ondary antibodies were obtained fromJackson Laboratories: donkey anti-mouse tetrarhodamine isothiocyanate(TRITC; 1:100), donkey anti-rat fluo-rescein isothiocyanate (FITC; 1:100),goat anti-rat FITC (1:100), goat anti-rat TRITC (1:100), goat anti-rabbit (1:100). F-actin was detected with phal-loidin-TRITC (Molecular Probes).Imaginal discs were dissected in phos-phate buffer, fixed in 4% paraformal-dehyde, washed in wash buffer (0.1%Triton), and then incubated in pri-mary overnight. Secondary antibodyincubations lasted 2–3 hr after whichtissues were further dissected in washbuffer and then mounted onto slidesin Vectashield (Vector Laboratories).Tissues were examined using a ZeissAxioplan 2 compound microscope withApotome and then imaged using aZeiss Axiocam MRm camera. Adultflies with ectopic eyes were frozen at�80°C for 20 min, imaged using aZeiss Discovery light microscope andphotographed with a MRc color cam-era.

Quantitative RT-PCR

RNA from the eye–antennal discs ofthird-instar larvae from the followingstocks were isolated using the RNeasyMicro Kit (Qiagen): w1118, GMR-GAL4/UAS-tsh, GMR-GAL4, UAS-tio,

dpp-GAL4, cb26-GAL4, cb41-GAL4,Ser-GAL4, c329b-GAL4, C833-GAL4,MJ33a-GAL4, T98-GAL4. The SybrGreen Two Step RT-PCR Kit (Invitro-gen) was used for reverse transcrip-tion, cDNA synthesis and quantifica-tion. A total of 1 �g of total RNA wasused as the starting template for eachreaction. The samples were quantifiedusing the Stratagene MxPro3000PqPCR system.

RT-PCR Primer Sequences

The following primer pair was usedto detect GAL4 transcripts: 5�-TTCTTCGTCGACGATGC-3� and 5�-AATTGGTTAGAGCGGTG-3�. Thefollowing primer pairs were used todetect tsh transcripts: (primer set1) 5�-TCCGCGAGCTGAGACGAA-AAGAG-3� and 5�-CGGGGCGAAG-GCAAGGCG-3�, (primer set 2)5�-TCCGCGAGCTGAGACGAAAAG-AA-3� and 5�-CGGGGCGAAG-GCAAGGCG-3�, (primer set 3) 5�-TCTGTAGGTACCCGGAAACG-3�and 5�-TTCCAGTCAGGGAATT-GACC-3�, (primer set 4) 5�-AG-GAATCTTCAAAGCCAGCA-3� and5�-TGGCACTTCCATTTACCACA-3�.The following primer pairs were used todetect tio transcripts: (primer set 1) 5�-TGGGTCACAGATTGCAGACACG-3�and 5�-GTTAAACAGTCGGCTTCG-TAAA-3�, (primer set 2) 5�-TTGGGT-CACAGATTGCAGACACG-3� and 5�-GTTAAACAGTCGGCTTCGTAAA-3�,(primer set 3) 5�-GACAAAGCTTCCG-GTCTCTG-3� and 5�-GACGGAACTC-CAGTGTTGGT-3�, (primer set 4) 5�-AGGAATCTTCAAAGCCAGCA-3� and5�-TGGCACTTCCATTTACCACA-3�.

Gene Interaction andEctopic Eye Screens

GMR-GAL4/UAS-tsh and GMR-GAL4/UAS-tio flies were crossed tothe 356 stocks that constitute theDrosDel Deficiency Kit. F1 progenywere scored for modifications of therough eye phenotype that results fromexpressing either tsh or tip in the de-veloping eye. We then identified puta-tive interacting genes by crossing theabove screen stocks to single gene dis-ruption mutations (that are locatedbetween the breakpoints of modifyingdeficiencies) and scoring for the simi-lar modifications as seen with the

overlying larger deficiency. UAS-tshand UAS-tio were crossed to 219GAL4 drivers and adults were scoredfor the presence of ectopic eye forma-tion. All crosses were conducted at25°C.

ACKNOWLEDGMENTSWe thank Amit Singh, LaurentFasano, Albert Courey, Larry Zipur-sky, and Walter Gehring for gifts of flystocks. J.P.K. was funded by the Na-tional Institutes of Health.

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