Identiﬁcation of Novel Regulators of atonal Expression in ...

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.093302

Identification of Novel Regulators of atonal Expressionin the Developing Drosophila Retina

David Melicharek,*,1 Arpit Shah,†,1 Ginnene DiStefano,* Andrew J. Gangemi,†

Andrew Orapallo,† Alysia D. Vrailas-Mortimer‡ and Daniel R. Marenda*,2

*Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104,†Department of Biological Sciences, University of the Sciences in Philadelphia, Philadelphia,

Pennsylvania 19104 and ‡Department of Cell Biology, EmoryUniversity School of Medicine, Atlanta, Georgia 30322

Manuscript received July 1, 2008Accepted for publication September 17, 2008

ABSTRACT

Atonal is a Drosophila proneural protein required for the proper formation of the R8 photoreceptorcell, the founding photoreceptor cell in the developing retina. Proper expression and refinement of theAtonal protein is essential for the proper formation of the Drosophila adult eye. In vertebrates, expressionof transcription factors orthologous to Drosophila Atonal (MATH5/Atoh7, XATH5, and ATH5) and theirprogressive restriction are also involved in specifying the retinal ganglion cell, the founding neural celltype in the mammalian retina. Thus, identifying factors that are involved in regulating the expression ofAtonal during development are important to fully understand how retinal neurogenesis is accomplished.We have performed a chemical mutagenesis screen for autosomal dominant enhancers of a loss-of-function atonal eye phenotype. We report here the identification of five genes required for proper Atonalexpression, three of which are novel regulators of Atonal expression in the Drosophila retina. Wecharacterize the role of the daughterless, kismet, and roughened eye genes on atonal transcriptional regulationin the developing retina and show that each gene regulates atonal transcription differently within thecontext of retinal development. Our results provide additional insights into the regulation of Atonalexpression in the developing Drosophila retina.

A fundamental question in developmental biologyis the control of neurogenesis. Proper neural

development underlies the basic cellular processesrequired within all cells of the mature nervous system.Neurophysiology, and even broader processes such asconsciousness or intelligence intimately depend uponproper developmental control of cells within the nervoussystem. The developing eye of the fruit fly Drosophilamelanogaster serves as an excellent system to model howneurogenesis is controlled within a developing nervoustissue.TheadultDrosophilaeye consistsof�800regularlyspaced unit eyes, called ommatidia. Each ommatidiumcontains 20 cells: 8 photoreceptor neurons (R1–R8) and12 accessory cells (cone, pigment, and bristle cells)arranged in a stereotypical array (Ready et al. 1976;Hsiung and Moses 2002; Mollereau and Domingos

2005), and each of which requires exquisite precision intheir morphology for proper function. This morpho-logical exactness requires precision in development.Development of the eye begins as a monolayer fieldof undifferentiated columnar epithelial cells (the eye/

antennal imaginal disc). These cells grow by randomproliferation until, during the early third instar larvalstage, a wave of differentiation, marked by a band ofcells with constricted apical actin cytoskeletal rings (themorphogenetic furrow) begins at the posterior marginof the presumptive eye disc and sweeps anteriorly acrossthe eye field. As the furrow moves across the disc, newcolumns of precisely spaced retinal founder cells (the R8photoreceptor cell) are specified roughly every 2 hours(Ready et al. 1976; Basler and Hafen 1989; Wolff

and Ready 1993). The central event in R8 foundercell specification is the initial expression and eventualrefinement of the proneural transcription factor Atonal(Ato) within the developing R8 neuron ( Jarman et al.1994; White and Jarman 2000).

Ato protein is initially expressed in a broad stripe ofcells just anterior to the morphogenetic furrow (Figure1A) ( Jarman et al. 1994). At the leading edge of thefurrow, Ato expression is refined to small clusters of�20 cells, the ‘‘intermediate groups’’ (arrows in Figure1B) ( Jarman et al. 1995). Later, within the furrow,Ato expression is refined to single cells, the future R8photoreceptor cells (arrowheads in Figure 1B) ( Jarman

et al. 1995; Baker et al. 1996). Ato expression is even-tually lost in these founder cells as the furrow advancesmore anteriorly, although the R8 cell fate is maintained

1These authors contributed equally to this work.2Corresponding author: Department of Bioscience and Biotechnology,

Drexel University, 3141 Chestnut St., Philadelphia, PA 19104.E-mail: [email protected]

Genetics 180: 2095–2110 (December 2008)

through the expression of Senseless within the R8(Frankfort et al. 2001). atonal transcriptional regula-tion is very dynamic. atonal transcription is regulated bytwo separate control regions near the atonal gene (Sun

et al. 1998). Genomic DNA flanking the 59 end of the

gene regulates the late phase of atonal expression in theintermediate groups and single R8s (Figure 1C) (Sun

et al. 1998), while genomic DNA flanking the 39 end ofthe gene regulates the early phase of atonal expressionin the initial broad stripe anterior to the furrow (Figure1D) (Sun et al. 1998).

After R8 founder cell specification, the remainingneurons (R1–R7) are recruited into the developingommatidia by inductive signals through the epidermalgrowth factor receptor (Egfr)/Ras/MAP kinase pathway(Figure 1E) reviewed in Freeman (1997, 1998). Eachphotoreceptor neuron is distinguishable from theothers within the ommatidium by the order in whichthe neuron is specified and by the position it takeswithin the developing ommatidial cluster. Thus, R2/R5are recruited into the ommatidium first, followed byR3/R4, R1/R6, and lastly R7 (Ready et al. 1976;Tomlinson and Ready 1987). The correct expressionand spacing of Ato protein is crucial to this properdevelopmental progression of photoreceptor develop-ment, and in ato mutants, no photoreceptors areformed, and surviving adults are nearly eyeless ( Jarman

et al. 1994, 1995).In review, retinal neurogenesis in the developing fly

eye may be considered in three phases: 0, 1, and 2(Figure 1). In phase 0, ahead of the morphogeneticfurrow, cells are undifferentiated and randomly pro-liferating. Phase 1 is marked by the establishment of theommatidial founder cells (the R8 neuron), throughprogressively restricted expression of Atonal. In phase 2,posterior to the morphogenetic furrow, the remainingneurons of the ommatidial cluster are recruited in a setpattern and sequence. For the eye to function normally,these developmental events must be precisely andfaithfully carried out. Any abnormality or deviation inthis process disrupts this delicate dance of developmentand further, may be detected through disruption of theexternal morphology of the adult eye (so-called ‘‘rougheye’’ phenotypes).

There are striking similarities between fly and verte-brate eye development, such as the process of specify-ing and spacing the first retinal neural cell type. Invertebrates, expression of bHLH transcription factorsorthologous to Drosophila Atonal (MATH5/Atoh7,XATH5, and ATH5) and their progressive restrictionare also involved in specifying the retinal ganglion cells,the founding neural cell type in mammals, in a processsimilar to Drosophila R8 photoreceptor development(Brown et al. 1998, 2001; Perron et al. 1998; Hassan

and Bellen 2000; Perron and Harris 2000; Hutcheson

and Vetter 2001; Vetter and Brown 2001; Wang et al.2001; Yan 2005). Thus, a more complete understandingof what transcription factors are required for R8 specifi-cation in Drosophila eye development, and how andwhen these transcription factors regulate Atonal expres-sion, will likely be of very broad relevance to ourunderstanding of the process of mammalian retinal

Figure 1.—The three phases of Drosophila larval retinaldevelopment. (A–D) Late third instar eye imaginal discs, an-terior right. (A) Atonal protein expression. Phase 0 indicatesthe domain of gene expression anterior to Atonal expression.Phase 1 indicates the domain of gene expression concurrentwith Atonal expression. Phase 2 indicates the domain of geneexpression posterior to Atonal expression. (B) Atonal expres-sion is controlled by two regulatory elements. The 39 regula-tory element controls Atonal expression ahead of themorphogenetic furrow (region in red). The 59 regulatory el-ement controls Atonal expression within the intermediategroups (arrows) and R8 nuclei (arrowheads) within and pos-terior to the morphogenetic furrow (region in green). (C) b-galactosidase protein expression as directed by the 59 atonalenhancer element. (D) b-galactosidase protein expressionas directed by the 39 atonal enhancer element. (E) Schematicof the three phases of retinal development in Drosophila.Events anterior to Atonal expression (phase 0), during Atonalexpression (phase 1), and posterior to Atonal expression(phase 2) are indicated.

2096 D. Melicharek et al.

development and are important to fully understandhow retinal neurogenesis is accomplished.

We have taken a genetic approach to understandingthe mechanisms that control Drosophila retinal neuro-genesis in the morphogenetic furrow (phase 1), byundertaking a genetic modifier screen based on anato loss-of-function genotype that displays a rough eyephenotype. We screened for dominant enhancers of thisphenotype and report here the identification of fivegenes, three of which are novel ato regulators. We showthat daughterless, a gene previously identified to affectAtonal protein expression, regulates atonal transcrip-tion both negatively and positively from the two differ-ent atonal enhancer elements. We further show thatkismet gene function is positively required for atonaltranscription at both atonal enhancer elements. Weshow that kismet gene function is precisely required forevents within the morphogenetic furrow, but is dispens-able for the expression of genes required for retinaldevelopment anterior or posterior to the furrow. Im-portantly, we show that mutations in other chromatinremodeling factors (brahma, osa, snr1) do not affectAtonal expression, while mutations in Trithorax do. Asmany of these chromatin remodeling factors affecteye development both anterior and posterior to thefurrow, our findings provide an intriguing specificity toKismet-mediated chromatin remodeling in retinal de-velopment. Finally, we characterize the expression andcontribution of the Roughened Eye protein in atonaltranscriptional regulation. We show that roughened eyegene function positively regulates atonal transcriptionfrom only one of the two known atonal genomic en-hancer elements. Our results provide additional in-sights into the regulation of atonal expression within themorphogenetic furrow, further illustrating the complexdevelopmental regulation this proneural gene under-goes during eye development. Further, as each of thegenes we identified in our screen in Drosophila havemammalian homologs, our results may also implicatethese genes in mammalian retinal development as well.

MATERIALS AND METHODS

Drosophila stocks: Unless otherwise noted, all crosses werecarried out at 25� on standard cornmeal–molasses–agarmedium. BL number refers to Bloomington Stock Centerstock number. Stocks used: ato1090 (also called atots, describedhere), Df(3R)p13 [BL 1943 ato1 and ato3 ( Jarman et al. 1994,1995)], atonal-lacZ enhancer trap lines (both 59F:9.3 and39F:5.8) are described in Sun et al. (1998), hhAC (Lee et al.1992), Egfr E1 (Baker and Rubin 1989, 1992), lilliXS407 (gift fromAmy Tang) (Tang et al. 2001), lilliXS575 (gift from Amy Tang)(Tang et al. 2001), lilliA17-2 (gift from Arno Muller) (Muller

et al. 2005), lilliS35 (Neufeld et al. 1998a), kis1 (Kennison andTamkun 1988), kisk13416 (Roch et al. 1998; Srinivasan et al.2005), roe1 (Ma et al. 1996), roe3 (St Pierre et al. 2002), rn19 (St

Pierre et al. 2002), rn16 (St Pierre et al. 2002), rn5 (Agnel et al.1989), smo3 P{neo FRT} 40A (Vrailas and Moses 2006), tkv8

P{neo FRT} 40A (Vrailas and Moses 2006), smo3, tkv8 P{neoFRT} 40A (Vrailas and Moses 2006), daUX P{neo FRT} 40A

(synonym da10, gift from Claire Cronmiller) (Brown et al.1996), da1 (Bell 1954), eya2 (Bonini et al. 1993), Star1 (Lewis

1945; Higson et al. 1993), DlRevF10 SerRX82 P{neoFRT}82B (Zeng

et al. 1998), osa308 P{neoFRT}82B (Treisman et al. 1997;Janody et al. 2004), trx E2 P{neoFRT}82B ( Janody et al.2004), brmT485 P{neoFRT}80B ( Janody et al. 2004), and snr1R3

P{neoFRT}82B (Zraly et al. 2003).Mosaic clones: Mosaic clones were generated using ey:FLP

(Newsome et al. 2000), as described (Xu and Rubin 1993).Flip stocks (1) y�, w�, ey:FLP; Ubi-GFP, P{neoFRT}80B; (2) y�, w�,ey:FLP ; Ubi-GFP, P{neoFRT}82B; and (3) y�, w�, ey:FLP; Ubi-GFP,P{neoFRT}40A were crossed to the following stocks as appro-priate to generate mutant clones:

1. w�; kisLM27, P{neoFRT}40A/Cyo2. w�; kisLM27, P{neoFRT}40A, 59 ato-lacZ/Cyo3. w�; kisLM27, P{neoFRT}40A, 39 ato-lacZ/Cyo4. w�; kisEC1, P{neoFRT}40A/Cyo5. w�; smo3 P{neo FRT} 40A/Cyo6. w�; tkv8 P{neo FRT} 40A/Cyo7. w�; smo3, tkv8, P{neo FRT} 40A/Cyo8. DlRevF10 Ser RX82 P{neoFRT}82B/TM6B9. w�; daUX P{neo FRT} 40A/Cyo

10. w�; daUX P{neo FRT} 40A, 59 ato-lacZ/Cyo11. w�; daUX P{neo FRT} 40A, 39 ato-lacZ/Cyo12. w�; osa308 P{neoFRT}82B/TM6B13. w�; brmT485 P{neoFRT}80B/TM6B14. w�; trxE2 P{neoFRT}82B/TM6B15. w�; snr1R3 P{neoFRT}82B/TM6B

Mutagenesis screen: A schematic of the genetic screen usedis provided in supplemental Figure 2. For chemical muta-genesis, isogenic males of the genotype w�; p[w1]/p[w1];Df(3R)p13/TM3 [where the Df(3R)p13 chromosome removesthe atonal locus] were treated with 25 mm EMS (Sigma) asdescribed (Ashburner 1989). These males were then crossedto w�; ato1090/ato1090 virgin females at 25�, and F1 males or virginfemales were scored under Leica dissecting microscopes forenhancement of the base Df(3R)p13/ato1090 eye phenotype at25� (see Figure 2E). Df(3R)p13 corresponds to BloomingtonStock no. 1943. p[w1] refers to the transposable elementinsertion P{EP}EP2178 from the Szeged Drosophila StockCentre, which itself does not modify the base Df(3R)p13/ato1090

eye phenotype at 25�. Mutations that affected this P-insertion(resulting in white-eyed flies) were used as a measure of theefficiency of mutagenesis. F1 males or virgin females of thegenotype w�; p[w1], */1; Df(3R)p13, e, */ato1090, where *indicates the presence of the modifying mutation, were thencrossed to w�; ato1090/TM6B, and those mutants that retainedthe observed enhancement in these F1 progeny were used tocreate independent stocks. Those mutants that failed toreproduce the observed enhancement were discarded. A fulldescription of the genetic mapping is described in thesupplemental Results.

Atonal antibody preparation: The ato reading frame fusedto the His6 tag and inserted into the pRSET vector (a gift fromAndrew Jarman, see Jarman et al. 1995) was used to expressand isolate Atonal protein. The protein was purified andused to immunize two guinea pigs (Covance) and two rabbits(Zymed).

For Roe antibody production, genomic antibodies wereproduced from Strategic Diagnostics (http://www.sdix.com/)according to the manufacturer’s protocol. The followingamino acid sequence was used from the Roe N-terminalspecific sequence: VSSSSGSSSTAGPAPTGSTRGRKSRIYPNPNQHIIVTSNSVDNGGIRMQNATLNERNSQNSSAGAAGVGNVTTAAGSGNGISSSTSSPHHMTQLDVKDTK.

Immunohistochemistry and microscopy: Eye disc prepara-tions were as described (Tio and Moses 1997) mounted in

Drosophila atonal Genetic Screen 2097

Vectashield (Vector Laboratories, H-1000), and imaged byconfocal microscopy using a Nikon Eclipse TE2000-U laser-scanning confocal microscope. Primary antibodies: guinea piganti-Atonal (1:1000, described in this study), rabbit anti-Roughened Eye (1:50, described in this study), rat anti-ELAV(1:500, 7E8A10 from Developmental Studies Hybridoma Bank),rabbit anti-Kismet-L (1:100, gift of J. Tamkun; Srinivasan et al.2005), mouse anti-Daughterless (1:50, gift of C. Cronmiller;Brown et al. 1996), and rabbit anti-b-galactosidase (1:1000,Cortex Biochem CA2190). Secondary antibodies were fromJackson ImmunoResearch: goat anti-mouse Cy5 (1:500, 115-175-003) and goat anti-rabbit TRITC (1:250, 111-025-003).

Adult eyes were immersed in 100% ethanol, and digitalphotographs were taken under a Leica mZ 12.5 stereomicro-scope, using an attached Leica digital camera.

RESULTS

Characterization of a temperature-sensitive loss-of-function atonal genetic background: The ato1090 muta-tion (henceforth referred to as atots, a generous gift from

V. Rodrigues) was initially isolated in an EMS screendesigned to identify second-site modifiers of a lozengephenotype and was subsequently tested for complemen-tation with the Df(3R)p13 deficiency [henceforth re-ferred to as Df(ato)], which removes the atonal locus.Heterozygous atots/1 flies show no dominant effect onadult eye morphology at either 18� or 29� (Figure 2B).Similarly, eyes from heterozygous flies containing Df(ato)also show no dominant effect on adult eye morphologyat 29� (Figure 2C). However, when atots is crossed in transto Df(ato), adult eye morphology is severely disruptedat 29� (Figure 2D), showing loss of nearly all photo-receptors and a strongly reduced eye size. When theseflies are raised at either 25� or 18�, eye size andmorphology are strongly rescued (Figures 2E and datanot shown), although these eyes still display a rough eyephenotype. Df(ato) has been previously used to examineatonal-specific loss-of-function effects in transheterozy-gous mutant combinations ( Jarman et al. 1994, 1995;

Figure 2.—The ato1090 allele (referred to asatots) is a temperature-sensitive mutation that af-fects Atonal protein expression. (A–F) Stereo-microscope pictures of adult compound eyes,anterior right, dorsal up, same magnification.Genotypes are listed in lower right of each panel.Temperature cultured is listed in upper right ofeach section. (A) Wild-type adult eye from fly cul-tured at 29� shows normal appearance. (B) Adulteye from heterozygous atots/1 mutant fly cul-tured at 29� shows wild-type appearance. (C)Adult eye from heterozygous Df(3R)p13/1 [re-ferred to as Df(ato)] mutant fly cultured at 29�shows wild-type appearance. (D) Adult eye fromtransheterozygous atots/Df(ato) mutant fly cul-tured at 29� shows virtually no eye. (E) Adulteye from transheterozygous atots/Df(ato) mutantfly cultured at 25� shows a small, rough eye.(F) Adult eye from transheterozygous atots/ato1

mutant fly cultured at 25� shows a small, rougheye similar to that of atots/Df(ato) flies culturedat 25�. (G–I) Third instar eye imaginal discs show-ing Atonal protein expression, anterior right.(G) Wild-type eye disc cultured at 29� shows nor-mal Atonal expression pattern. Arrows indicateAtonal protein expression in single R8 nuclei.(H) Transheterozygous atots/Df(ato) mutant eyedisc from fly cultured at 25� shows decreasedAtonal protein expression, particularly posteriorto the furrow in R8 nuclei (arrowhead). (I)Transheterozygous atots/Df(ato) mutant eye discfrom fly cultured at 29� shows strongly decreasedAtonal protein expression, both posterior to thefurrow in R8 nuclei (arrowhead), and anterior tothe furrow in the broad Atonal stripe (arrow).( J–L) Stereomicroscope pictures of adult com-pound eyes, anterior right, dorsal up, same mag-nification. All flies were cultured at 25�. ( J) hhAC/1;atots/Df(ato) mutant eye. The eye appears smallerthan atots/Df(ato) eyes alone at this temperature,indicating dominant enhancement by hhAC. (K)

Egfr E1/1 adult eyes show a slightly rough and small eye alone. (L) Egfr E1/1; atots/Df(ato) mutant eye. The eye appears smallerthan atots/Df(ato) eyes alone at this temperature, indicating dominant enhancement by Egfr E1.


Baker et al. 1996). However, Df(ato) deletes genes otherthan atonal. To verify that the phenotypic effects ob-served from our atots allele were due to a decreasespecifically in atonal gene function, we analyzed trans-heterozygous combinations of atots with two other atonalmutants (ato1 and ato3). Both of these alleles are strongloss-of-function mutations in the atonal gene ( Jarman

et al. 1994, 1995). In each case, transheterozygousmutant combinations of atots/ato1 and atots/ato3 pro-duced small rough eyes at 25� very similar to thephenotype observed in the atots/Df(ato) genotype at25� (Figure 2F and data not shown). Thus, these datasuggest that the small rough eye phenotype of the atots/Df(ato) genotype is due to loss of atonal gene functionwithin the Df(ato) stock and not due to effects fromremoval of other loci within this deficiency.

We next analyzed Atonal protein expression in theatots/Df(ato) mutant background at two different tem-peratures (25� and 29�) and found that the preciseexpression and refinement of Atonal is disrupted. At 25�there is a defect in the refinement of Atonal proteininto R8 cells (compare arrows in Figure 2G to arrow-head in Figure 2H). This is consistent with what hasbeen previously reported for Atonal protein and atonalmRNA in the ato1/Df(ato) loss-of-function atonal geneticbackground ( Jarman et al. 1995). At 29� this effect ismore dramatic, with a reduction in Atonal expressionboth ahead of the furrow (arrow in Figure 2I) and noexpression within R8 cells posterior to the furrow(arrowhead in Figure 2I).

Although they are reduced and morphologicallyabnormal, adult eyes still form in the atots/Df(ato) mutantbackgrounds, even though the only functional copy ofthe atonal gene comes from the atots mutant. Takentogether, these data suggested that (1) the atots allele is atemperature-sensitive, loss-of-function allele that stillproduces functional Atonal protein at both 25� and 29�;(2) the atots/Df(ato) atonal eye phenotype is due todecreased (although not absent) Atonal protein expres-sion, where R8 refinement within the developing retinais strongly impaired; and (3) the rough eye phenotypeof transheterozygous atots/Df(ato) flies is not saturated at25� and could potentially be enhanced at this temper-ature by second-site dominant modifier mutations (atleast to the severity of the phenotype at 29�).

To examine whether the atots/Df(ato) rough eyephenotype at 25� (hereafter referred to as the atophenotype) could be dominantly modified as describedabove, we first examined whether loss-of-function muta-tions in genes known to be involved in ato generegulation could dominantly enhance this phenotype.The Hedgehog (hh) signal transduction pathway isinvolved in numerous cellular processes (Ingham andMcMahon 2001; Stark 2002; Lum and Beachy 2004),including resolution of Atonal protein expression inthe developing fly eye (Borod and Heberlein 1998;Dominguez 1999; White and Jarman 2000). We ana-

lyzed the effect of loss of a single copy of the hh gene onthe ato phenotype at 25�. We found that a null mutationin the hh gene (hhAC, which alone has no heterozygousdominant adult eye phenotype) shows a dominant gen-etic enhancement of the ato phenotype (Figure 2J),indicating that this phenotype is indeed sensitive toloss-of-function mutations in cellular components thatregulate Atonal protein expression.

We then tested whether the ato phenotype is sensitiveto gain-of-function mutations that regulate Atonal pro-tein expression as well. Increased nuclear translocationof MAP kinase, a downstream component of the Egfrpathway, has been shown to disrupt Atonal proteinexpression and spacing (Kumar et al. 2003; Vrailas et al.2006), and increased Egfr expression directed withinthe morphogenetic furrow also shows decreased Atonalexpression (Chen and Chien 1999). Further, a gain-of-function mutation in Egfr (Egfr E1) results in decreasedAtonal expression within the developing retina ( Jarman

et al. 1995). Egfr E1, which alone has slight rough eyephenotype (Figure 2K), also shows a dominant geneticenhancement of the ato phenotype (Figure 2L). Theseresults suggest that the ato phenotype is also sensitive todominant enhancement by gain-of-function mutantsas well.

A genetic screen for enhancers of the ato phenotype:Using the ato phenotype at 25�, we performed a chemical(EMS) mutagenesis screen, examining a total of 22,151flies for dominant enhancement of the phenotype. Werecovered a total of 48 dominant enhancers, 15 of whichseparated into five total complementation groups (Table1). Further, of the 48 enhancers we identified, only 1 ofthese enhancers was a mutation in the Star gene, whichitself exhibits a dominant rough eye phenotype. Thissuggests that the multiallele complementation groups weidentified in our screen most likely reflect genes directlyinvolved in regulating atonal gene function and/orexpression and are not additive or indirect effects frommutations in general modifiers of eye morphology.

The enhancers we identified in our genetic screengenerally fell within one of three groups with regard toatonal expression: (1) those that differentially regulatedatonal expression anterior and posterior to the morpho-genetic furrow, (2) those that similarly regulated atonalexpression anterior and posterior to the morphogeneticfurrow, and (3) those that only regulated atonal expres-sion posterior to the furrow.

Differential regulation of atonal expression—hedge-hog (hh) and daughterless (da): We mapped three of thethird chromosome dominant enhancers of the atophenotype to the hh locus (Table 1). hh is a segmentpolarity gene (Nusslein-Volhardand Wieschaus 1980)that encodes a secreted ligand required for Hedgehogpathway signaling and is involved in multiple develop-mental processes (Ingham and McMahon 2001; Lum

and Beachy 2004), including the progression of themorphogenetic furrow (Heberlein et al. 1993; Ma et al.


1993) and the regulation of atonal gene expression in thedeveloping fly retina (Dominguez 1999). Previous stud-ies have shown that loss of hh signaling has oppositeeffects on atonal expression in the developing retina(Dominguez 1999).

We mapped two of the second chromosome domi-nant enhancers of the ato phenotype to the da locus(Figure 3, J–L; Table 1). Both alleles of da moderatelyenhance the ato phenotype (Figure 3, J and K), and bothalleles failed to complement two independent loss-of-function da mutations (daUX, da1, Table 1). We thereforetested for dominant enhancement of the ato phenotypewith these independent loss-of-function da mutantsand found that they also dominantly enhanced the atophenotype (e.g., Figure 3L). We conclude that the twoalleles we identified in our screen are also loss-of-function da alleles.

daughterless (da) encodes the only type I basic helix-loop-helix (bHLH) transcription factor in flies (Smith

and Cronmiller 2001). Type II bHLH proteins gener-ally exhibit a more restricted expression pattern, whilethe type I bHLH family of proteins (such as Daughter-less) are more widely expressed throughout develop-ment (Massari and Murre 2000). Type II bHLHproteins bind to type I bHLH transcription factors toform functional heterodimers to regulate target geneexpression ( Jarman et al. 1993; Kophengnavong et al.2000; Massari and Murre 2000).

Mutations in da were previously shown to negativelyregulate Atonal protein expression in the developingDrosophila retina (Brown et al. 1996), but were alsosuggested to abolish R8 expression as well (Brown et al.1996). To determine the effects of da mutation on atotranscriptional regulation from the two different atoenhancer elements, we created loss-of-function homo-zygous somatic da mutant clones (Xu and Rubin 1993)and analyzed ato-lacZ reporter expression within these

clones. We used a da null allele, daUX, and the eyeless:Flipspecific recombinase to generate clones in the devel-oping retina. In these clones, we observed expandedAtonal protein expression posterior to the morphoge-netic furrow (arrows in Figure 4, A and B). We nextanalyzed atonal transcriptional regulation from the 39

genomic enhancer element within daUX mutant clonesand found that the expression of this reporter is stronglyincreased and expanded in these clones posterior to themorphogenetic furrow (Figure 4, C and D). Analysis ofatonal transcriptional regulation from the 59 genomicenhancer element showed the opposite effect, withnearly a complete loss of expression of atonal transcrip-tion within the intermediate groups and single R8nuclei (Figure 4, E and F). Taken together, these resultssuggest that da gene function is differentially requiredfor atonal transcriptional regulation, negatively regu-lating atonal transcription at the 39 atonal genomicenhancer, while simultaneously positively regulatingatonal transcription at the 59 atonal genomic enhancer.

Similar regulation of atonal expression—lilliputian(lilli): We mapped two of the second chromosomedominant enhancers of the ato phenotype to the lillilocus (Figure 3, B and C; Table 1). Both alleles showmoderate-to-strong enhancement of the ato phenotype(e.g., Figure 3B), and both alleles failed to complementBloomington Stock Collection Deficiency Df(2L)JS17,which deletes the lilliputian gene. Both alleles also failedto complement four independent loss-of-function lillimutations (lilliXS407, lilliA17-2, lilliXS575, and lilliS35) (Neufeld

et al. 1998b; Tang et al. 2001). We therefore tested fordominant enhancement of the ato phenotype with theseindependent loss-of-function lilli mutants and found thatall four alleles also dominantly enhanced the ato pheno-type, although not as severely as the two we recovered inour screen (e.g., Figure 3C). We therefore concluded thatthese two mutants were alleles of lilliputian (lilliGD17 and

TABLE 1

Complementation groups and alleles

Gene Alleles Cytogenetic map Fails to complement

lilliputian GD17, AG5 23C1 Df(2L)JS17lilliXS407

lilliXS575 (Tang et al. 2001)lilliA17-2 (Muller et al. 2005)lilliS35 (Neufeld et al. 1998b)

kismet EC1, LM27 AS21, AO9 21B4-21B5 Df(2L)ED19kis1 (Kennison and Tamkun 1988)kisk13416 (Roch et al. 1998)

daughterless AB12, IB21 daUX (Brown et al. 1996)da1 (Bell 1954)

hedgehog FS1, MM2 RM2 94E1 hhAC (Lee et al. 1992)Roughened eyes SM8, BM10 KK16, KM29 84D3 roe3 (St Pierre et al. 2002)

rn16 (Agnel et al. 1989)Star FF28 21E4 Star1 (Lewis 1945; Higson et al. 1993)Single hits 32


lilliAG5), and that both behave genetically as loss-of-function mutants. Further, we conclude that it is loss-of-function at the lilli locus, and not at a potential secondsite, that is associated with the enhancement of the atophenotype in these experiments.

We analyzed atonal transcriptional regulation fromboth the 39 and 59 atonal genomic enhancer elementsand found that transcription from both of these elementsis reduced in homozygous lilli mutant clones (data notshown), suggesting that lilli gene function is positivelyrequired for ato transcriptional regulation within andposterior to the morphogenetic furrow at both the 39 and59 atonal genomic enhancers. Characterization of thesignificance of this interaction will be described elsewhere(G. DiStefano and D. Marenda, unpublished results).

kismet (kis): We mapped four of the second chromo-some dominant enhancers of the ato phenotype to thekis locus (Figure 3, D–F; Table 1). All four alleles show amild enhancement of the ato phenotype (e.g., Figure3D), and all four alleles failed to complement ExelixisDeficiency Df(2L)ED19, which deletes the kismet gene.All four alleles also failed to complement two indepen-

dent loss-of-function kis mutations (kis1 and kisk13416)(Kennison and Tamkun 1988; Roch et al. 1998). Wetested for dominant enhancement of the ato phenotypewith these independent loss-of-function kis mutants,and found that these two alleles also dominantly en-hanced the ato phenotype (Figure 3, E and F). Wetherefore concluded that these four mutants werealleles of kismet (kisLM27, kisEC1, kisAS21, and kisAO9), and allbehave genetically as loss-of-function mutants. Further,we conclude that it is loss-of-function at the kis locus,and not at a potential second site, that is associatedwith the enhancement of the ato phenotype in theseexperiments.

kismet was initially identified in a screen for suppres-sors of a dominant homeotic phenotype associated withPolycomb (Kennison and Tamkun 1988) and has criticalfunctions in embryonic development and segmentation(Daubresse et al. 1999; Srinivasan et al. 2005). kisencodes multiple nuclear proteins that are related tochromatin remodeling factors and are largely associa-ted with transcriptionally active chromatin (Srinivasan

et al. 2005). In the Drosophila eye, mutations in kismet

Figure 3.—Genetic enhancement of the atonalloss-of-function eye phenotype. (A–L) Stereomi-croscope pictures of adult compound eyes, ante-rior right, dorsal up, same magnification. Allpanels show atots/Df(ato) mutant eyes raised at25�. Enhancer mutations listed in lower right-hand panel. (A) atots/Df(ato) mutant eye showsa small, rough appearance. (B and C) Loss-of-function mutations in lilliputian (lilli). (B) lil-liGD17/1; atots/Df(ato) mutant eye. (C) lilliS35/1;atots/Df(ato) mutant eye. Both lilli mutants domi-nantly enhance the small, rough eye phenotype.(D–F) Loss-of-function mutations in kismet (kis).(D) kisLM27/1; atots/Df(ato) mutant eye. (E) kis1/1;atots/Df(ato) mutant eye. (F) kisk13416/1; atots/Df(a-to) mutant eye. All three kis mutations enhancethe small, rough eye to varying degrees. (G–I)Loss-of-function mutations in roughened eye (roe).(G) atots/Df(ato), roeKM29 mutant eye. (H) atots/Df(ato),roeSM8 mutant eye. (I) atots/Df(ato), roeBM10 mutant eye.All three roe mutants strongly enhance the small,rough eye phenotype. ( J–L) Loss-of-function muta-tions in daughterless (da). (J) daIB21/1; atots/Df(ato)mutant eye. (K) daAB12/1; atots/Df(ato) mutant eye.(L) daUX/1; atots/Df(ato) mutant eye. All three damutations moderately enhance the small, rougheye phenotype.


were recovered as enhancers of a gain-of-function kinasesuppressor of Ras (ksr) rough eye phenotype (Therrien

et al. 2000).To better understand how kismet function is required

for atonal expression in the developing retina, we firstanalyzed Kismet protein expression in developing thirdlarval instar retinas. Using an antibody that is specificto the long form of the Kismet protein, Kismet-L, wedetected ubiquitous Kismet expression in developingthird instar retinas (Figure 5A). Kismet is expressedanterior to the morphogenetic furrow (Figure 5A),within the furrow (arrowhead in Figure 5B) and withindeveloping photoreceptor cells posterior to the furrow

(arrow in Figure 5B). Kismet is also expressed in laterdeveloping structures, including cone cells (arrow inFigure 5C). Thus, Kismet expression matches that of aprotein that may be generally required for multipleevents in Drosophila eye development and not specificto atonal expression within or posterior to the morpho-genetic furrow.

To further characterize which phase in eye develop-ment kis gene function is required, we created loss-of-function homozygous somatic kis mutant clones. Wefirst analyzed Kismet protein expression within theseclones. In kisLM27 homozygous mutant clones, Kismetprotein is absent from the mutant tissue (Figure 5, D–F),indicating that this particular allele is a protein null.This was also true for kisEC1 homozygous mutant clones(data not shown). We analyzed Atonal protein expres-sion within kisLM27 homozygous mutant clones andfound that Atonal expression is markedly reduced(Figure 5, G–I). Atonal expression was most severelyreduced in the broad stripe of Atonal expressionanterior to the morphogenetic furrow as well as withinthe intermediate groups, (arrows in Figure 5G). SomeAto expression within single R8s still remained (arrowin Figure 5I). Similar results were obtained with kisEC1

homozygous mutant clones (data not shown). Thesedata suggest that kis gene function is required fornormal Atonal protein expression.

The Kismet protein is a transcription factor, thoughtto play a role in altering gene expression through changesin chromatin structure (Srinivasan et al. 2005). There-fore, we next looked at atonal transcriptional regulation atboth the 39 and 59 regulatory elements within kis mutanttissue. In kisLM27 homozygous mutant clones, we foundthat the amount of b-galactosidase protein expressedfrom the 39 ato-lacZ element was strongly reduced (Figure5, J–L). Similarly, we found that the amount of b-galactosidase protein expressed from the 59 ato-lacZelement was also strongly reduced (Figure 5, M–O).Taken together, these data strongly suggest that kisgene function is normally required for atonal tran-scriptional activation and/or maintenance at both the39 and 59 atonal gene enhancer elements.

On the basis of Kismet protein expression within thedeveloping eye, we sought to further analyze the role ofkis gene function in other phases of eye development,both anterior and posterior to the morphogeneticfurrow. Unlike homozygous somatic mutant clones ingenes required for cell division and cell growth, homo-zygous mutant kis clones are not small and scarce withinthe developing retinas (Figure 5D), suggesting that kisgene function is not required anterior to the morpho-genetic furrow for cell division and/or growth func-tions. To further analyze gene expression anterior to thefurrow, we examined the expression of the Hairy pro-tein. Anterior to the furrow, the expression of hairy (h)marks the ‘‘preproneural domain’’ (Greenwood andStruhl 1999), which is expressed just prior to atonal

Figure 4.—daughterless functions differentially at bothatonal enhancer elements. (A–F) Third instar larval retinas,anterior right. All panels show daUX homozygous mutantclones marked by the absence of GFP (green). (A) Atonal pro-tein expression (red) in da mutant clone. Arrow denotes theexpansion of Atonal protein toward posterior within clones.(B) Atonal protein expression (white) from A. (C) b-galactosi-dase protein expression (red) as driven by the 39 atonal geno-mic enhancer element. Note the expansion of b-galactosidaseexpression in the posterior. (D) b-galactosidase protein (white)from C. (E) b-galactosidase protein expression (red) as drivenby the 59 atonal genomic enhancer element. Note the loss ofb-galactosidase expression. (F) b-galactosidase protein(white) from E.


expression in the developing retina. Within homozy-gous mutant kis clones, h expression was normal (Figure6, A–C), suggesting that kis function is also not requiredfor patterning events immediately ahead of the mor-phogenetic furrow. kis loss-of-function clones were pre-viously reported to have no effect on photoreceptordifferentiation posterior to the morphogenetic furrow( Janody et al. 2004). Consistent with these data, we alsoobserve no significant effect on ELAV expression withinkis mutant clones located posterior to the morphoge-netic furrow (data not shown), suggesting that kis genefunction is also not required for photoreceptor differ-entiation posterior to the morphogenetic furrow.

To further determine if kis function is restricted toevents occurring within and near the morphogeneticfurrow, we analyzed the expression of other proteinsexpressed within and near the furrow, including theexpression of Daughterless and Scabrous. Daughterlessprotein was previously shown to be expressed through-out the eye disc, with stronger expression within themorphogenetic furrow (Brown et al. 1996). In homo-zygous mutant kis clones, we observed that Daughterlessexpression was reduced, but not absent (Figures 6,D–F). Scabrous expression is a target of Atonal and isalso expressed within and near the intermediate groupsin the furrow (Baker et al. 1990; Mlodzik et al. 1990;Frankfort and Mardon 2002). In homozygous mutantkis clones, we observed that like Daughterless expres-sion, Scabrous expression was also reduced, but notabsent (Figure 6, G–I). Taken together, these datasuggest that kismet gene function is restricted to regu-lating the expression of factors within and near themorphogenetic furrow of the developing larval retina.

The Kismet protein belongs to the trithorax group oftranscriptional regulators (Daubresse et al. 1999),which include trithorax, kohtalo, skuld, and members ofthe Brahma complex (brm, osa, snr1, and moira) (Tamkun

et al. 1992; Dingwall et al. 1995; Elfring et al. 1998;Collins et al. 1999). Mutations in brm, snr1, and osa allaffect eye development (Treisman et al. 1997; Brumby

et al. 2002; Marenda et al. 2003), and mutations in thetrithorax genes trithorax, brahma, kohtalo, and skuld wererecently identified in a mosaic genetic screen used toidentify genes required for retinal patterning ( Janody

et al. 2004). Our identification of kis in our genetic screenled us to hypothesize that other trithorax group genesmight also be involved in regulating Atonal expression.To test this possibility, we created clones in the de-

Figure 5.—Kismet expression and function in the develop-ing retina. (A–C) Wild-type retinas. (D–O) kisLM27 homozygousmutant clones marked by the absence of GFP (green). Allpanels show third instar larval eye discs, anterior right. (A–C) Kismet protein expression in the developing eye. (A) Kis-met protein appears nuclear and is expressed throughout theretina. Arrowhead marks the morphogenetic furrow. (B) Kis-met protein expression in nuclei ahead of the furrow, withinthe morphogenetic furrow (arrowhead), and within nucleiposterior to the furrow (arrow). (C) Kismet expression withincone cells (arrow). (D–F) Kismet protein expression withinloss-of-function kisLM27 mutant clones. (D) Kismet protein ex-pression is in red. Loss of GFP expression (green) marks thecells mutant for kis. Note loss of Kismet protein within kisLM27

mutant clones. (E) GFP expression (white) from D. (F) Kis-met protein expression (white) from D. (G–I) Atonal proteinexpression within loss-of-function kisLM27 mutant clones. (G)Atonal protein (red) is lost in kis mutant clones (arrow inclone) predominantly in the broad stripe of expression ante-rior to the furrow (compare arrow in clone to arrow above innormal tissue). (H) GFP expression (white) from G. (I)Atonal expression (white) from G. Note the presence of R8nuclei within kis mutant clones (arrow). (J–L) b-galactosidaseprotein expression as driven by the 39 atonal genomic en-hancer element within loss-of-function kisLM27 mutant clones.( J) b-galactosidase protein expression (red) is reduced butnot absent in kis mutant clones. (K) GFP expression (white)

from J. (L) b-galactosidase protein expression (white) from J.(M–O) b-galactosidase protein expression as driven by the 59atonal genomic enhancer element within loss-of-functionkisLM27 mutant clones. (M) b-galactosidase protein expression(red) is reduced but not absent in kis mutant clones. (N) GFPexpression (white) from M. (O) b-galactosidase protein ex-pression (white) from M.


veloping retina for loss-of-function mutations in brahma(brmT485), osa (osa308), snr1 (snr1R3), and trithorax (trxE2).

brm mutant clones were very small, consistent withwhat has been previously reported ( Janody et al. 2004).Analysis of multiple small brm mutant clones showed acomplex regulation on Atonal protein expression (Fig-ure 7, A–F). Within brm mutant clones in and near themorphogenetic furrow, Atonal protein expression wasunaffected in both the broad stripe of expression and insingle R8 nuclei as well (arrows in Figure 7, A–C).However, within �10% of brm mutant clones posteriorto the morphogenetic furrow, Atonal protein expres-sion in single R8 nuclei was inappropriately expressed(arrows in Figure 7, D–F). For both osa and snr1 mutantclones, Atonal protein expression was unaffected (ar-rowheads in Figure 7, G–L), suggesting that althoughthese genes are required for retinal patterning, they arenot significantly affecting Atonal expression within themorphogenetic furrow. We did observe loss of Atonalprotein expression in clones of cells mutant for trx(Figure 7, M–O). Within trx mutant clones, Atonalexpression is absent at both the broad stripe as well aswithin single R8 nuclei. Taken together, our resultssuggest that of the trithorax group proteins we tested,only Kismet and Trithorax are required for Atonalexpression in the morphogenetic furrow.

Regulation of Atonal expression at a single enhan-cer—roughened eye (roe): We mapped four of the thirdchromosome dominant enhancers of the ato phenotypeto the roe locus (Figure 3, G–I; Table 1). Mutations in roe

are strong enhancers of the ato phenotype (Figure 3,G–I). Each of these alleles was on a chromosome thatalso contained the Df(ato) deletion, and these flies werecrossed to a small deletion (rn16, St Pierre et al. 2002)that removes the C terminus of the rotund gene and theentire roughened eye gene. These adults produced a smalleye phenotype that is stronger than the ato eye pheno-type used in our screen (compare Figure 3A and G–I toFigure 8, C and D). Heteroallelic mutant combinationsof the roughened eye (roe) gene produce viable adults withsmall rough eyes (Figure 8, A and B) (St Pierre et al.2002). We therefore concluded that the four alleles thatmade up this complementation group were alleles of theroughened eye gene (roeSM8, roeBM10, roeKM29, roeKK16; Table 1).

To better understand how Roe protein function isrequired for atonal expression in the developing retina,we raised an antibody to the N-terminal portion of theRoe protein and analyzed Roe protein expression indeveloping retinas. roe mRNA was previously reported asbeing expressed within a band of 4–6 cells within themorphogenetic furrow (St Pierre et al. 2002). Tomore precisely define the area within the eye disc inwhich the Roe protein is expressed, we costained wild-type developing retinas for both Roe and Atonal proteinexpression (Figure 8, E and F). Consistent with previousreports, we also detected Roe expression within a bandof 4–6 cells within the morphogenetic furrow (Figure8E). Roe protein expression is predominantly nuclear.Interestingly, Roe expression first appears just behind(posterior to) the broad stripe of Atonal expression that

Figure 6.—Kismet function in the morphoge-netic furrow. (A–F) and (H and I) show kisLM27 ho-mozygous mutant clones marked by the absenceof GFP (green). (G) Wild-type disc. All panelsshow third instar larval eye discs, anterior right.(A–C) Hairy protein expression within loss-of-function kisLM27 mutant clones. (A) Hairy expres-sion (red) is not altered in kis mutant clones. (B)GFP expression (white) from A. (C) Hairy ex-pression (white) from A. (D–F) Daughterless pro-tein expression within loss-of-function kisLM27

mutant clones. (D) Daughterless expression(red) is reduced but not absent in kis mutantclones. (E) GFP expression (white) from D. (F)Daughterless expression (white) from D. (G)Wild-type Scabrous expression (white) in normaldisc. (H) Scabrous expression (Red) in kis mu-tant clone. Scabrous expression is decreasedbut not absent. (I) Scabrous expression (white)from H.


is anterior to and within the morphogenetic furrow(arrowhead in Figure 8F). Roe protein first colocalizesboth within the first column of nascent R8 nuclei imme-diately posterior to the broad stripe of Atonal expression(black arrow in Figure 8F) as well as within those nucleisurrounding this R8 cell. Roe protein then appears to besolely expressed within those nuclei immediately sur-rounding the second column of R8 cells (blue arrow inFigure 8F) and is no longer expressed near the thirdcolumn of R8 cells (red arrow in Figure 8F).

On the basis of expression of the Roe protein, wepredicted that Roe protein function with regard to atonalexpression may be limited to the 59 atonal regulatoryelement. As an initial test of this hypothesis, we analyzedAtonal protein expression in a strong roe loss-of-functionbackground that had been previously used to analyze lossof roe function in the developing retina (roe3/rn16, Figure8B) (St Pierre et al. 2002). This genetic backgroundincludes both a deletion of the rotund locus that removesfunction for both the rotund and roe genes (rn16) and also apoint mutation that specifically removes roe gene functionalone (roe3) (St Pierre et al. 2002). In this background,Atonal protein expression is reduced, but not absent.Atonal protein is still present in a broad stripe anterior tothe furrow (Figure 8H), however, refinement of Atonalinto single R8 nuclei is nearly absent (compare Figure 8,G and H), suggesting that mutation of the roe genespecifically affects atonal expression from the 59 regula-tory element. To verify this, we analyzed atonal transcrip-tional regulation at both the 39 and 59 regulatoryelements within the roe loss-of-function background(Figure 8, I–L). ato transcription as assayed from the 39

atonal regulatory element appears normal in roe loss-of-function eye discs (Figure 8, I and J). However,transcription from the 59 atonal regulatory element isseverely reduced (Figure 8, K and L), and ato transcrip-tion in single R8 cells is absent, consistent with what weobserve with Atonal protein expression in this back-ground. When taken together, these results suggest thatthe Roe protein is necessary for atonal transcriptionalregulation specifically at the 59 atonal regulatory elementwithin the morphogenetic furrow, and that in roe mutants,single R8 cells are not specified.

DISCUSSION

We performed an autosomal mutagenesis screen inDrosophila looking for dominant second-site mutationsthat could enhance the phenotype of a loss-of-functiontransheterozygous atonal genotype. We recovered a totalof 48 dominant enhancers, which separated into fivetotal complementation groups (Table 1). Each of theseenhancers fell within one of three groups: (1) thosethat regulated atonal expression differently at the twodifferent enhancer elements, (2) those that regulatedatonal expression similarly at the two different enhancer

Figure 7.—Atonal expression in trithorax group loss-of-function mutant clones. (A–O) Third instar eye discs showingAtonal protein expression in different loss-of-function back-grounds. Mutant clones are marked by loss of GFP expression(green) in all panels. (A–F) Atonal protein expression (red)in brmT485 mutant clones. (A) Atonal expression is still presentin brm mutant clones within the broad stripe of Atonal expres-sion. Arrows denote Atonal expression in clone. (B) Atonalexpression is still present in single R8 nuclei in brm mutantclones. Arrows denote Atonal expression in brm clones. (C)Atonal expression (white) from B. (D) Atonal expression isnot reduced in R8 nuclei (arrow) posterior to the furrow.(E) GFP (white) expression from D. (F) Atonal expression(white) from D. Arrows denote two ectopic Atonal positive nu-clei posterior to the morphogenetic furrow. (G–I) Atonal pro-tein expression (red) in osa308 mutant clones. Atonalexpression is still present within the clones both ahead ofthe furrow (arrow) and within R8 nuclei (arrowheads). (H)GFP (white) expression from G. (I) Atonal expression (white)from G. ( J–L) Atonal protein expression (red) in snr1R3 mu-tant clones. Atonal expression is still present within the clonesboth ahead of the furrow and within R8 nuclei (arrowheads).(K) GFP (white) expression from J. (L) Atonal expression(white) from J. (M–O) Atonal protein expression (red) intrxE2 mutant clones. Atonal expression is lost within theseclones (arrows in M and O). (N) GFP (white) expression fromM. (O) Atonal expression (white) from M.


elements, and (3) those that only affected one enhancerelement.

Differential regulation of atonal transcription: In ourscreen, we identified mutations in both the hedgehog anddaughterless genes as enhancers of our loss-of-functionatonal phenotype. Both of these mutants alter Atonalprotein expression differentially anterior to the mor-phogenetic furrow as compared to posterior to themorphogenetic furrow. Regulation of atonal expressionby the hedgehog pathway is well described (Dominguez

1999; Suzuki and Saigo 2000; White and Jarman

2000; Lim et al. 2008). Lim et al. (2008) describe recentevidence for Daughterless-mediated atonal expressionin the developing retina that may further explain theresults we have observed in our study.

We have shown that daughterless function is requiredto repress atonal transcription at the 39 atonal enhancer.Importantly, this repression is only required for 39 atonaltranscription posterior to the morphogenetic furrow,as 39 atonal enhancer expression is not upregulatedanterior to the furrow in da mutant clones (Figure 4). Atthe 59 atonal enhancer, daughterless function is requiredto promote atonal transcription. Atonal forms hetero-dimers with Daughterless ( Jarman et al. 1993), and it isthis heterodimerization that is required for bHLHprotein function (Kophengnavong et al. 2000; Massari

and Murre 2000). Since the 59 atonal enhancer ele-ments undergo autoregulation (Sun et al. 1998), thissuggests that Daughterless protein function at the 59

element may play an important role in this autoregula-tion. However, our data suggest that posterior to themorphogenetic furrow, while the Daughterless pro-

tein is required to form heterodimers with Atonal topositively regulate atonal transcription from the 59 atonalenhancer, it is simultaneously required to transcription-ally repress atonal expression at the 39 atonal enhancerelement. Lim et al. (2008) have recently suggested thatDaughterless forms homodimers and/or heterodimerswith unknown bHLH proteins to repress atonal expres-sion. Our data fit well with this model of atonal regu-lation and suggest that Daughterless homodimers maybe required in cells surrounding developing R8 photo-receptors to repress atonal transcription from the 39

atonal enhancer element.Similar regulation of atonal transcription: In our

screen, we identified mutations in both the lilliputianand kismet genes as enhancers or our loss-of-functionatonal phenotype. Mutations in both of these genes havea similar effect on atonal transcriptional regulation atthe two atonal enhancer elements, as each is required topromote atonal transcription at both enhancers. In-terestingly, mutations in both of the human homologsof these genes are involved in forms of mental re-tardation. Mutation in Fmr2/AF4 gene family members(the human homolog of lilliputian) are involved inboth acute lymphoblastic leukemia and FRAXE non-syndromic X-linked mental retardation syndrome (Gu

et al. 1996; Gu and Nelson 2003). Mutations in Chd7(the human homolog of kismet) are estimated to becausative for nearly two-thirds of all CHARGE syndromediagnoses (Sanlaville and Verloes 2007), a rare,autosomal dominant disorder. Of further interest,patients with CHARGE syndrome also display eye andear abnormalities, including coloboma, a symptom that

Figure 8.—roughened eye function in regulatingAtonal expression in the eye. (A–D) Stereomicro-scope pictures of adult eyes, anterior right, dorsalup. (A) roe1/roe3 adult eyes show a mild small andrough eye phenotype. (B) roe3/rn16 adult eyesshow a moderate small and rough eye phenotype.(C) roeSM8, Df(ato)/rn16 and (D) roeBM10, Df(ato)/rn16 both show an enhanced small and rougheye compared to roe3/rn16 adult eyes. (E–L) Thirdinstar larval eye discs from wild-type (E–G) androe3/rn16 (H–L) genotypes. (E and F) Colocaliza-tion of wild-type Atonal (green) and Roe (red)protein expression. (F) Arrowhead denotesAtonal expression anterior to Roe expression.Black arrows denote first column of R8 nucleiwhere Atonal and Roe protein are colocalized.Blue arrow denotes second column of R8 nucleithat are surrounded by Roe protein expression.Red arrow denotes third column of R8 nuclei.(G) Wild-type Atonal expression (white). (H)Atonal expression (white) in roe3/rn16 back-ground. Note loss of Atonal expression in R8nuclei. (I and J) Atonal expression (green) andb-galactosidase protein expression (red) as

driven by the 39 atonal genomic enhancer element in roe3/rn16 mutant retinas. ( J) Magnified view of I. (K and L) Atonal expression(green) and b-galactosidase protein expression (red) as driven by the 59 atonal genomic enhancer element in roe3/rn16 mutantretinas. (L) Magnified view of K.


is also attributed to mutations in the mammalian homo-log of the hedgehog gene (Schimmenti et al. 2003; Fuerst

et al. 2007). The human homolog of atonal (math5) isexpressed and critically required for both proper retinaland auditory development (Brown et al. 1998, 2001;Wang et al. 2001; Yang et al. 2003; Le et al. 2006;Hufnagel et al. 2007; Saul et al. 2008) and thus maybe a good candidate for a critical target gene that isdisrupted in CHARGE syndrome.

Recent analysis of Kismet protein on salivary glandchromosomes suggests that Kis plays a global role intranscription by Pol II (Srinivasan et al. 2005). Consis-tent with the idea of global transcription, we observe Kisstaining in all cells of the developing Drosophila retina,suggesting that Kis would also play a role in globaltranscription in the eye. However, our analysis of kismetmutant clones suggests that kis gene function is limitedto events occurring within and near the morphogeneticfurrow. We observe effects on Atonal, Daughterless,and Scabrous protein expression within the furrow, butsee no effects on marker expression within kis mutantclones either posterior or anterior to the furrow. Wehave analyzed Kis protein expression within these clonesand found that it is absent, suggesting that these mutantsare effectively protein nulls within this tissue, and thusthe normal expression of these markers is not likely dueto residual kismet gene expression. kis mutants also showa limited number of phenotypes within Drosophilaembryos (Daubresse et al. 1999). Taken together, thesedata suggest that (1) there may be redundant factorswithin the developing retina that can compensate for kisgene function within cells posterior and anterior to thefurrow or (2) that Kismet protein function is onlyrequired in cells within and near the furrow, and doesnot have a function in areas outside these cells.

Members of the trithorax group of proteins appear tohave a variety of functions within the developingDrosophila retina ( Janody et al. 2004). Further, manyof the trithorax genes required for specific cellularprocesses in the eye have a ubiquitous expressionpattern in the eye (Kuzin et al. 1994; Treisman et al.1997; Janody et al. 2003; Zraly et al. 2003), much likethe Kismet protein. This expression data might suggesta dynamic functional and developmental regulationof these factors. In support of this, we have analyzedKismet protein expression within loss-of-function ge-netic backgrounds of developmental signals known toregulate eye development and Atonal expression, in-cluding the Notch and Hedgehog pathways, as wellas in daughterless and eyes absent mutants and find nodifference in Kismet expression in any of these mutants(supplemental Figure 1). Thus, the rapid transitions ingene expression between different phases of retinaldevelopment (anterior and posterior to the furrow)must involve functional regulation of these ubiquitouslyexpressed transcription factors and not the regulationof their gene expression within different phases.

While mutations in kis show no affect on photo-receptor differentiation posterior to the furrow, or onpatterning events anterior to the furrow, mutations inother ubiquitously expressed trithorax group genesshow differential effects in these areas. Mutations intrithorax group genes brahma, moira, skd, and kto all showloss of photoreceptor differentiation posterior to thefurrow, while mutations in the trithorax group gene osashow less severe effects on photoreceptor differentia-tion in this area of the retina ( Janody et al. 2004).Ahead of the furrow, brm and osa mutants have no effecton the expression of the anterior markers hth, ey, and tsh,while mutations in trx, skd, and kto all significantly affectthese markers ( Janody et al. 2004). Further, while we donot observe any effect on Atonal expression in thefurrow within brm, osa, or snr1 mutant clones, we dosee decreased Atonal expression in trx mutant clones.Immediately ahead of the furrow, within the prepro-neural domain, only trx, kto, and skd show effects on themarkers hairy and eya, and only trx mutants showed aneffect on dac expression ( Janody et al. 2004). Similarly,recent studies have shown atonal expression is affectedin clones mutant for skd and kto (Lim et al. 2007). Withinskd and kto clones, single R8 neurons are selected evenwithout fully resolved intermediate groups (Lim et al.2007), an occurrence that also happens in kis mutantclones (Figure 5).

Taken together with our results, these data now beginto separate specific functions within the trithorax groupof proteins into a developmental requirement forspecific events during retinal development. Thus, aheadof the furrow, only trx, skd, and kto appear to be requiredfor specific transcriptional regulation of the targetstested so far. Within the furrow, trx, skd, kto, and kis allshow an effect on atonal expression, while brm, osa, andsnr1 do not. Finally, posterior to the furrow, trithoraxgroup members brm, moira, osa, skd, kto, and trx are allrequired for photoreceptor differentiation.

Transcriptional regulation in the developing retinarequires rapid changes in gene expression as the mo-rphogenetic furrow moves across the eye disc (Moses

1991, 2003; Kumar and Moses 1997). The movement ofthe morphogenetic furrow allows for a new column ofphotoreceptor cells to be specified roughly every 2 hrfor uniform movement of the morphogenetic furrow(Campos-Ortega and Hofbauer 1977), and every 70min for nonuniform movement (Basler and Hafen

1989). Thus, the developmental regulation of differenttarget-specific transcription factors must be tightlycontrolled, as cell type specific transcription will rapidlychange as the morphogenetic furrow moves across theretina. Regulated changes in chromatin structure musttherefore also be tightly regulated, as the morphoge-netic furrow moves across the retina. The differentrequirement for trithorax group proteins ahead of,within, and posterior to the morphogenetic furrow mayreflect differential changes in chromatin structure


necessary to accommodate rapid changes in transcrip-tion during retinal development. Thus, these differenttranscriptional events may be anticipated by cells inregions of the developing retina before the morphoge-netic furrow has reached these cells.

Expression of atonal at only one enhancer element:In our screen, we identified mutations in the roughedeye gene as an enhancer or our loss-of-function atonalphenotype. We show that Roe protein expression isrestricted to the cells expressing the 59 atonal regulatoryelement marker within morphogenetic furrow, specifi-cally colocalized within and surrounding the firstcolumn of nascent R8 cells. These data suggest that roegene function is required for the proper formation ofR8 nuclei and is consistent with Roe protein expressioncolocalizing with Atonal protein expression in the R8cells. However, Roe protein is also present in nucleisurrounding the nascent R8 cells. It has been previouslyshown that Delta protein expression is disrupted in roemutant retinas (St Pierre et al. 2002), moving from apunctate expression pattern to a more diffuse pattern.Posterior to the furrow, Delta expression becomesrestricted to the R8 cell, and this activates the Notchpathway in surrounding cells to repress atonal transcrip-tion (Lee et al. 1996; Li and Baker 2001; Doroquez andRebay 2006). Thus, if the Roe protein surrounding theR8 cells functions to repress Delta expression withinthese cells, loss of Roe may lead to increased Deltaexpression within these cells and thus cause the Notchpathway to become activated in the R8 cell itself, repress-ing atonal transcription.

In the developing retina, the restricted expression ofcell-specific factors (such as roughened eye) is vital tothe proper development of a specific subset of retinalprecursor cells. However, the broader transcriptionalregulation of these cell-specific transcription factorsby ubiquitous transcription factors (such as kismet) re-mains less clear. Our results presented here suggest thatalthough these factors are expressed ubiquitously, manyof them have very specific functions in distinct phases ofretinal development. These results provide additionalinsights into the complex regulation of atonal expres-sion in the developing retina.

We thank Kevin Moses, in whose lab the atonal screen was initiallyperformed. We are indebted to Veronica Rodrigues, who graciouslyprovided us with the unpublished atonal1090 allele. We thank JohnTamkun for Kis antibodies, Claire Cronmiller for Da antibodies andstocks, Jessica Treisman, Francesca Pignoni, Amy Tang, Arno Muller,Susan Younger, Yuh Nung Jan, and Andrew Dingwall for fly stocks,Andrew Jarman for the atonal pRSET vector, the Bloomington StockCenter, and the Iowa Hybridoma Bank for reagents. We thank themembers of the Marenda lab for helpful discussions of the data, andChonnettia Jones for her contribution to Figure 1. Both D.M. and A.S.were supported by funds from the Melvin Firman Award for Un-dergraduate Research through the University of the Sciences inPhiladelphia. A.V.M. is supported by a grant from the Center ofBehavior Neurosciences, STC program of National Science Founda-tion agreement no. IBN-9876754. This work was supported by a grantfrom the National Eye Institute (EY018431) to D.R.M.

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