Regulation of Drosophila eye development by the transcription factor Sine oculis.

Homeodomain transcription factors of the Sine oculis (SIX) family direct multiple regulatory processes throughout the metazoans. Sine oculis (So) was first characterized in the fruit fly Drosophila melanogaster, where it is both necessary and sufficient for eye development, regulating cell survival, proliferation, and differentiation. Despite its key role in development, only a few direct targets of So have been described previously. In the current study, we aim to expand our knowledge of So-mediated transcriptional regulation in the developing Drosophila eye using ChIP-seq to map So binding regions throughout the genome. We find 7,566 So enriched regions (peaks), estimated to map to 5,952 genes. Using overlap between the So ChIP-seq peak set and genes that are differentially regulated in response to loss or gain of so, we identify putative direct targets of So. We find So binding enrichment in genes not previously known to be regulated by So, including genes that encode cell junction proteins and signaling pathway components. In addition, we analyze a subset of So-bound novel genes in the eye, and find eight genes that have previously uncharacterized eye phenotypes and may be novel direct targets of So. Our study presents a greatly expanded list of candidate So targets and serves as basis for future studies of So-mediated gene regulation in the eye.

Introduction

The homeodomain transcription factor Sine oculis (So) is a member of the highly conserved Retinal Determination (RD) gene network, which consists of transcriptional regulators that are both necessary and sufficient for eye development in Drosophila (reviewed by [1]). So regulates multiple aspects of eye development and is expressed in the larval precursor structure to the adult eye – the eye imaginal disc. So expression begins in the eye imaginal disc during the second instar larval stage, when the eye disc consists of proliferating retinal progenitor cells. At the beginning of the third instar stage, an indentation called the morphogenetic furrow (MF) forms at the posterior margin of the eye disc, marking the onset of cell differentiation, and sweeps progressively across the eye disc toward the anterior margin [2]. So is expressed in a narrow domain anterior to the MF and in all the cells posterior to the MF including the differentiating cells [3], [4]. In the eye specific mutant so, So is not expressed in the eye discs. The absence of So expression blocks MF initiation as well as retinal differentiation leading to massive apoptosis of the retinal progenitor cells and adult flies without eyes [3]. Consistent with these observations, clonal analysis using so null mutant alleles indicates that so is required for MF initiation and progression, as well as the differentiation or survival of photoreceptor precursors posterior to the MF [5]. Despite the vital role of So in eye development, only a few direct So targets have been identified to date. Most of these targets encode transcriptional regulators necessary for various stages of eye development, including the RD network genes eyeless (ey) and dachshund (dac), as well as genes that direct retinal differentiation such as atonal (ato), lozenge (lz), and prospero (pros) [6], [7], [8], [9], [10]. So also regulates the expression of hedgehog (hh), which encodes a secreted ligand that drives MF progression in the eye disc [8].

In order to improve our understanding of how So regulates eye development, we have sought to identify targets of So during eye development on a genome-wide scale. To this end we have performed chromatin immunoprecipitation with an anti-So antibody followed by genome-wide sequencing (ChIP-seq) on third instar eye-antennal discs. We found 7,566 regions were enriched for So binding throughout the genome. These So-bound regions (referred to as peaks) correspond to estimated 5,952 genes. The So-enriched genes include previously characterized direct targets of So, indicating that ChIP-seq can identify biologically relevant targets of So. As expected, the genes enriched for highly significant So peaks are over-represented in Gene Ontology categories that pertain to eye development. In addition, many So peaks map to genes that have not been known to function during eye development. We have obtained mutant alleles of a subset of these So-enriched, novel genes, and have assayed their function in the eye. We have identified eight novel genes that are necessary for eye development and may act downstream of So. In addition, we have intersected our ChIP-seq data set with genome-wide changes in expression profiles due to loss or gain of so to identify candidate genes that are directly regulated by So in the eye. Together, our results greatly expand the set of putative So targets in the developing eye, and set the stage for many future studies of So function in development.

Materials and Methods

Chromatin immunoprecipitation

Chromatin immunoprecipitation protocol was performed as previously described [11]. 400 eye-antennal disc complexes (including mouth hooks, but not brains) from white wandering third instar larvae were used per replicate; two biological replicates were conducted. The chromatin sample was incubated with 1∶500 guinea pig anti-So antibody (kind gift from Ilaria Rebay; [12]), and an identical chromatin sample incubated without antibody served as negative control. ChIP-seq libraries were prepared according to the Illumina ChIP-seq library protocol. High-throughput library sequencing was performed using the Illumina Genome Analyzer IIx. The 35 bp reads from the two biological replicates were combined and then mapped to the Drosophila genome using Eland software.

Peak mapping and analysis

The reads were mapped and peaks were called using Model-based Analysis of ChIP-seq (MACS; http://liulab.dfci.harvard.edu/MACS/) [13]. Peaks with a P value of larger than 10−5 or with a fold change of less than 3 were filtered out. Gene Ontology analysis of genes with the top 10% most highly enriched peaks was performed using the publicly available Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov) [14]. De novo motif analysis of the entire ChIP-seq peak set was carried out with the publicly available Regulatory Sequence Analysis Tool (RSAT) software (http://rsat.ulb.ac.be/rsat/) [15]. So ChIP-seq data have been deposited in NCBI Gene Expression Omnibus with the accession number GSE52943 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52943).

Screen for novel putative targets of So with eye phenotypes

We ordered transposon insertions and mutant alleles of previously uncharacterized genes enriched for So binding, if available, from the Bloomington Drosophila Stock Center (BDSC) at Indiana University, Bloomington (http://flystocks.bio.indiana.edu); the Drosophila Genetic Resource Center (DGRC) at Kyoto Institute of Technology, Japan (http://www.dgrc.kit.ac.jp/en/index.html); and the Carnegie collection [16]. Homozygous lethal lines were subjected to a complementation test with a molecularly mapped deficiency ordered from BDSC that uncovers the gene of interest (goi). If a mutant failed to complement the deficiency, indicating that the lethality mapped to the region of interest, we proceeded to recombine the mutant allele onto a suitable FRT chromosome: FRT19A for X, FRT40A for 2L, FRT24D for 2R, FRT80B for 3L, and FRT82B for 3R. In case of w transposon lines, putative recombinants were identified by eye color (w animals that survived on G418), crossed individually to suitable balancer flies (FM7c/Y for the X chromosome, w; BcE/CyO for the 2nd chromosome, and w; TM3/TM6B, Tb for the 3rd), and then verified by single-fly genomic PCR using pairs of primers spanning the two transposon/genomic region junctions. For w transposon lines and point mutants, individual flies were tested by PCR (for transposon lines) or failure to complement the deficiency corresponding to the gene of interest.

The recombinant w; FRT goi/Bal males were crossed with ey-FLP; FRT cl/Bal virgins in order to test the requirement of the mutant gene in eye development, yielding progeny of the genotype ey-FLP/(w or Y); FRT goi/FRT cl. ey-FLP drives expression of Flippase (Flp) in the early eye disc, leading to FRT recombination and the formation of homozygous goi and cl clones [17]. The cl homozygous cells die due to the recessive cell lethal (cl) allele, leaving the eye disc composed mainly of homozygous goi cells. We scored the external eye phenotype of ey-FLP/w; FRT goi/FRT cl flies. If we saw defects, such as reduced or rough eye, we performed adult eye sections as previously described [18]. Table 1 lists stocks that were found to cause adult eye defects.

Table 1

Mutant alleles associated with novel eye phenotypes.

Gene	Allele	Nature of allele	Eye colora	Reference
blot	01658	P-element in intron near 5′UTR	w−	[82]
CG12007	LL03248	Piggybac in ORF	Strong w+	[83]
CG13192	EY07746	P-element in ORF	Weak w+	FBrf0132177b
CG2747	KG09552	P-element in 5′UTR	Strong w+	FBrf0132177b
CG8108	EY14316	P-element in ORF	Weak w+	FBrf0132177b
l(3)j2D3	j2D3	P-element in ORF	Strong w+	[84]
omd	EY04837	P-element in 5′UTR	Weak w+	FBrf0132177b
Syp	03806	P-element in intronc	w−	[84]

Eye color of homozygous mutant tissue in the adult. Weak w and w tissue can be distinguished from heterozygous tissue by color; strong w tissue cannot.

Personal communication to FlyBase; related publication: [85]

The allele is in the 5′UTR of CG31195, which maps to an intron of Syp, and hence the phenotype may potentially be due to loss of expression of CG31195.

A few transposon insertions of interest had been recombined onto an FRT chromosome previously by the UCLA Undergraduate Research Consortium in Functional Genomics (URCFG; http://www.bruinfly.ucla.edu/). Where possible, we ordered these flies, performed complementation tests, and proceeded directly to assaying the eye phenotype with ey-FLP; FRT cl/Bal.

Results

So ChIP-seq binding profile

Given the importance of so in Drosophila eye development, we used the genome-wide ChIP-seq approach to identify genes that may be directly regulated by So. Mid-third instar eye discs were chosen for the analysis as they exhibit retinal progenitors in different stages of eye development. Thus, performing So ChIP-seq at this stage is expected to identify targets of So in retinal progenitors ahead of the MF as well as in cells undergoing differentiation posterior to the MF. We performed two biological replicates of So ChIP-seq that yielded ∼4.74 million 35 bp reads, of which ∼3.4 million reads are unique. The combined unique reads from the two biological replicates were mapped to the Drosophila melanogaster FlyBase genome release 05, resulting in 7,566 regions enriched for So binding. These So-bound regions are henceforth referred to as ‘So-ChIP-seq peaks’. All ChIP-seq peaks are listed in Dataset S1.

The So-ChIP-seq peaks were associated with estimated 5,952 genes with a mean peak width of ∼1 kb. The majority of the So-ChIP-seq peaks (84.7%) overlap one or more genes at least partially, and these peaks were assigned to the gene(s) they overlap. 15.3% of So-binding peaks are intergenic peaks, which were assigned to the nearest gene in either direction. Most peaks are less than 5 kb from an annotated transcription start site (TSS), with 52.4% of the peaks being <1 kb from a TSS, and 16.7% of peaks between 1 and 5 kb from the nearest annotated TSS. Only 10.8% of the peaks are more than 20 kb from an annotated TSS (Figure 1).

Figure 1

Peak distribution and motif enrichment of the So ChIP-seq data set.

(A) Genome distribution of So-enriched regions (peaks) with respect to annotated transcription start sites (TSS). 52.4% of So peaks are <1 kb from the nearest TSS, and 78.9% of peaks are no more than 10 kb from the nearest TSS. (B) Transcription factor binding motifs enriched in the So ChIP-seq peak set. The name of the TF and the percentage of peaks that have at least one occurrence of the TF motif are listed under each motif. Abd-B, Abdominal-B; Cad, Caudal; Dref, DNA Replication Element factor; Hb, Hunchback; Hr78, Hormone receptor 78; Iro-C, Iroquois Complex; Trl, Trithorax-like; Usp, Ultraspiracle.

So ChIP-seq identifies previously known direct targets of So

To date, seven direct targets of So during eye development have been identified, namely ey, lz, so, hh, dac, ato, and pros. Enhancer analysis for these genes has suggested that So regulates their expression during eye development [6], [7], [8], [9], [10]. We mapped the known So-dependent enhancers of the seven known So targets to our ChIP-seq data set to see how many of these So dependent enhancers are bound by So in the developing eye disc. Our So ChIP-seq analysis showed that six out of seven genes are enriched for So-binding at the published So-dependent enhancers. For example, the ato enhancer that harbors a So-binding site is bound by So in the third instar eye disc (Figure 2, [10]). Our data accurately identified six out of seven published So-dependent enhancers, suggesting that our ChIP-seq data can predict biologically relevant targets. Although we do observe an So peak within the ey gene, it does not overlap the previously reported eye enhancer [8].

Figure 2

So ChIP-seq peak in the So target gene atonal maps to a previously known So-regulated enhancer.

The top panel (“So”) shows the So binding profile, which is significantly enriched compared with the negative control (“Control” panel). The bottom panel shows the atonal (ato) protein coding region in green. The yellow bar in the bottom panel marks the 3′ ato enhancer, which is necessary for the onset of ato in the eye and is directly activated by So [10]. The So ChIP-seq peak overlaps this previously identified 3′ ato enhancer.

So peaks are enriched for transcription factor binding motifs

Previous studies suggest that So acts with other transcription factors to regulate the expression of its target genes. For example, So and Ey bind to adjacent motifs in an ato enhancer, and both are necessary for the onset of ato expression in the eye [10]. So also cooperates with PntP2, the downstream effector of Egfr signaling, in activating hh and pros expression during eye development [6], [19], [20]. Therefore, we tested whether the So bound regions are enriched for binding motifs of other transcriptional regulator(s) in order to identify synergistic interactions between So and other DNA binding TFs that play a role during eye development. Toward this end we used the RSAT program [15] to identify binding motifs enriched within the So ChIP-seq peaks.

Analysis of the entire So ChIP-seq peak set with RSAT software revealed enrichment for several motifs (Figure 1B). One of the top hits among these is AGATAC, which closely matches the consensus motif for So, YGATAY (Y = C/T) [11]. A second enriched motif, STTWTCA (S = C/G, W = A/T), matches the consensus motif for the So paralog and RD network member Optix, which is expressed along with So in the anterior portion of the eye disc [21]. A third enriched motif is AACAYAA, the motif for homeodomain transcription factors of the Iroquois Complex, which establish dorsal-ventral polarity in the eye disc – Mirror (Mirr), Caupolican (Caup), and Araucan (Ara) [22], [23]. A fourth enriched motif is TATCGATA, which corresponds to the DNA Replication Element (DRE), the binding site for the zinc finger protein DRE factor (Dref) [24], while a fifth motif, AGAGMGMG (M = A/C), resembles the consensus for Trl (Trithorax-like), a zinc finger chromatin remodeling protein. A sixth motif, CGGTCACACTG, corresponds to the nuclear hormone receptor Hr76 and to Ultraspiracle (Usp), which mediates the transcriptional response to the hormone ecdysone [25]. ATTTKTA (K = G/T), a seventh enriched motif, resembles the motifs for several transcription factors that are important for determining the embryonic body plan and specification of embryonic as well as larval retinal fields, including the homeodomain factors Abdominal-B (Abd-B), Caudal (Cad), and Zerknüllt (Zen); the zinc finger transcription factors Hunchback (Hb) and Broad-Z1 (Br-Z1); and the helix-loop-helix factor Bric a brac 1 (Bab1).

So binds to or near genes predicted to function in eye development

To identify major biological processes associated with genes enriched for So binding, we performed Gene Ontology (GO) analysis. We ranked the ChIP-seq peaks based on their P-values and selected the top 10% most significant ChIP-seq peaks (757 peaks corresponding to 782 annotated protein-coding genes, P≤10−64). The GO analysis of genes that correspond to the top 10% most significant ChIP-seq peaks was done using the program DAVID [26], [27], to obtain enrichment scores for different GO terms. Highly enriched GO terms (enrichment score >1.3, corresponding to P<0.05) include terms predicted to be associated with putative So target genes, such as Imaginal disc development, Sensory organ development, Neuron differentiation, and others (Table S1).

There are also GO clusters unrelated to eye development, including Gland development, Ovarian follicle cell development, Leg disc development, and Muscle attachment, among others (Table S1). Some of these terms may be expected since some putative So targets are known to function in multiple developing organs and tissues. For example, Leg disc development, an enriched GO term, requires dac, a direct target of So in the eye [28], [29]. In addition to eye development, So/SIX transcription factors, along with other members of the RD network, orchestrate multiple developmental processes. For example, enrichment for GO terms associated with muscle may reflect the requirement for the So cofactor Eya in the developing muscle [30]. The Notch and Epidermal growth factor receptor (Egfr) signaling pathways (reviewed by [31]) are also employed in the development of multiple organs and tissues, and several components of each pathway are present among genes corresponding to the top 10% of the So peaks.

Several enriched GO terms refer to processes that are not currently known to be regulated by So during eye development. It is possible that the presence of genes associated with these developmental process may reflect a previously undescribed role for So. These GO terms include Protein kinase cascade, Cell-cell junction organization, and Asymmetric protein localization (Table S1). Thus GO analysis of the top putative targets of So based on So-binding suggests that our So ChIP-seq analysis contains genes involved in diverse processes during eye development including those that were not previously associated with So function.

Identification of novel eye genes

Over half of the genes that harbor So peaks that rank among the top 10% based on P-value (≥642.39, corresponding to P≤10−64) have no previously described role in eye development. Some of these genes have been shown to play roles in the development of other organs such as gonad or brain, some cause early lethality, and the mutant phenotypes for many have not been reported. To expand our understanding of how So regulates eye development, we investigated if these genes with highly enriched So ChIP-seq peaks are required for eye development. To this end, we compiled a list of genes that met the following criteria: 1) the role of the gene in the eye had not been reported; 2) a So peak with P<10−20 maps to the gene; and 3) the gene is expressed (expression level ≥10) in wild-type third instar eye discs based on previously published microarray data [32]. We then obtained available alleles, including P-element insertions, in these genes from the Drosophila stock centers (see Materials and Methods). Recessive lethal alleles that failed to complement a deficiency covering the gene of interest were recombined onto an FRT chromosome in order to assay the phenotype in the eye using the ey-FLP/FRT technique [17], [20]. Out of 26 alleles tested with ey-FLP, loss-of-function mutations in fifteen genes had no effect on adult eye morphology (4EHP, akirin, CG43658, CG6767, CG7675, CG8223, CG9932, cnc, Eaf6, ems, Imp, l(3)L1231, Mpcp, vkg, and wde), and loss of two genes caused very subtle disorganization of the adult eye (CG1965 and att-ORFA); these 17 genes were not analyzed further. Eight showed severe defects in adult eye morphology and a ninth gene, krotzkopf verkehrt (kkv/CG2666), resulted in pharate lethal flies with severe reduction of the head (data not shown). Eight mutant lines resulted in viable flies with reduced and misshapen eyes (Figure 3). These mutants lead to a range of defects in ommatidial architecture from minor disorganization to complete loss of ommatidia as compared to the control (Figure 4): bloated tubules (blot/CG3897), CG12007, CG13192, CG2747, CG8108, l(3)j2D3/CG6801, oocyte maintenance defects (omd/CG9591), and Syncrip (Syp/CG17838) (Table 1).

Figure 3

Novel eye phenotypes of putative So target genes.

Adult eye phenotypes were analyzed using ey-FLP; FRT goi/FRT cl. Homozygous mutant tissue has less pigmentation than heterozygous tissue in omd, CG13192, blot, CG8108, and Syp. In CG2747, CG12007, and l(3)j2D3, homozygous vs. heterozygous tissue cannot be distinguished by color. omd, CG2747, CG12007, and Syp are on chromosome 3R (FRT82B); blot, l(3)j2D3, and CG8108 are on 3L (FRT80B); and CG13192 is on 2R (FRT42D). Bottom panel shows control external eye phenotypes of ey-FLP; FRT/FRT cl flies.

Figure 4

Ommatidial defects in putative So target gene mutants.

Top panel: tangential sections through adult eyes of ey-FLP; FRT goi/FRT cl flies reveal defects ranging from loss of the inner photoreceptor (CG13192) to complete loss of rhabdomeres (blot). Bottom panel: Control sections through the eyes of ey-FLP; FRT/FRT cl flies.

One of the novel genes, omd, is of particular interest due to its proposed role as a negative regulator of Decapentaplegic (Dpp) signaling [33], which is necessary for normal eye development [34]. omd has a So ChIP-seq peak (P = 10−56) that maps to the first intron and first coding exon of omd, as well as to the TSS of an overlapping gene, falafel (flfl/CG9351). Given the overlap between omd and flfl it is not possible to determine whether the So-ChIP-seq peak reflects So-mediated regulation of omd, flfl, or both. We have used a lethal P-element insertion in the 5′ UTR of omd (omd) to test if the omd/flfl locus plays a role during eye development (it is unknown whether the P-element lethality is due to disruption of omd or flfl). Clonal analysis using omd results in reduced and disorganized adult eyes, with some homozygous mutant tissue surviving in the adult (Figure 3 and 4). omd encodes a subunit of the Integrator complex, which processes small nuclear RNAs (snRNA) [35]. Knockdown of other Integrator complex subunits causes multiple developmental defects in Drosophila, including in the embryo, wings, and bristles [36]. In addition, an RNAi screen has identified omd as a putative regulator of neural stem cell self-renewal in the larval brain [37]. The flfl gene also has a role in neurogenesis: it encodes a protein phosphatase regulatory subunit that regulates asymmetric protein localization in dividing neuroblasts [38].

Transcriptional profiling to detect putative So targets

So ChIP-seq detects So binding to genes in the third instar eye disc, but not necessarily the regulation of a target gene by So. If a gene is a putative target of So, we predict that its expression would be affected by changes in So levels. Therefore, we reasoned that putative direct So targets can be identified as the genes that are bound by So in the eye disc and that are differentially regulated in response to changes in so expression. However, observing transcriptional changes in the so mutant eye disc is challenging, because so homozygous mutant eye discs undergo massive apoptosis in the early third instar stage, around the time when wild type eye discs initiate differentiation [3]. Therefore, we used an alternative approach by monitoring changes in gene expression during ectopic eye formation in Drosophila imaginal discs. The changes in gene expression were analyzed by microarray analysis [32]. Previous studies have shown that overexpression of transgenes encoding the RD network members Ey, So, and Eya can lead to eye formation in ectopic sites such as legs [39], [40], [41]. We have used a combination of microarray data sets analyzing the gene expression changes due to loss and gain of so during ectopic eye induction ([32] and our unpublished data). In the current study, we used the microarray data in two ways to identify candidate direct targets of So.

The first approach was based on the observation that the RD gene eyeless (ey) is a potent inducer of ectopic eyes in multiple imaginal discs, but it cannot induce ectopic eye formation in so mutant discs [40], [42]. This observation suggests that similar to normal eye development, ectopic eye development requires so. Therefore, we compared the gene expression profiles of leg imaginal discs expressing a UAS-ey (Uey) transgene in presence and absence of endogenous so (wild-type and so mutant background, respectively) to identify genes whose expression is affected by loss of so function.

Second, overexpression of so alone is a weak inducer of ectopic eyes and, consistent with this observation, few genes are differentially regulated between wild type and so-overexpressing leg discs ([41] and our unpublished data). However, co-overexpression of so and eya, which encodes a transcriptional coactivator that binds So, results in robust induction of ectopic eyes [5], [43]. Therefore, we compared gene expression profiles of wild-type leg discs to those that co-overexpress so and eya (Ueya+so) transgenes in order to identify genes that respond to elevated levels of so and/or eya (we note that this approach does not distinguish genes that respond to the So/Eya complex from genes that respond to Eya alone).

Putative direct targets of So were identified as genes that show either altered expression in ey-overexpressing leg discs in the presence vs. absence of so (abbreviated Uey and Uey; so, respectively) or a change in expression in leg discs co-overexpressing eya and so compared to wild type leg discs (abbreviated Ueya+so). Genes meeting these criteria were then intersected with the list of genes that have So ChIP-seq peaks to identify putative direct targets of So. Intersection of these data sets identifies a total of 810 genes that show differential expression between Uey and Uey; so leg discs, between wild-type and Ueya+so leg discs, or both (Table S2).

So has been suggested to function as a transcriptional activator as well as transcriptional repressor during development [43], [44]. Consistent with this, the 810 genes include ones that respond positively as well as ones that respond negatively to So. 468 genes (57.8%) are putative positive targets of So – genes that are downregulated in absence of so, upregulated in presence of ectopic so, or both. In contrast, 290 genes (35.8%) are candidate negative So targets, which show higher expression in so mutant discs, lower expression in so-overexpressing discs, or both. We refer to the remaining 52 genes (6.4%) as “ambiguous” genes, because they appear positively regulated by So in the Uey vs. Uey; so assay and negatively regulated in the wt vs. Ueya+so assay, or vice versa (Table S2). The regulation of the “ambiguous” genes by So may be context-specific or indirect. Three genes (nonA, CG2225, and Gαq) are both up- and downregulated in Uey vs. Uey; so, possibly reflecting different regulation of distinct splice isoforms of each gene by So (for each of these three genes, distinct Affymetrix probes show up- and downregulation in response to loss of so).

Among the 810 candidate So targets, 460 show differential expression in response to loss of so (Uey vs. Uey; so) and 444 respond to gain of ectopic so (wt vs. Ueya+so), with a 94-gene overlap between the two sets. Among the 486 genes that only respond positively to So, 257 are more highly expressed in Uey vs. Uey; so, and 244 are upregulated in Ueya+so relative to wt, with 33 genes (7.1% of all positively regulated genes) that respond positively to So in both loss- and gain-of-function conditions. The 290 genes that act only as negative targets of So include 151 genes that show lower expression in Uey than in Uey; so, and 149 genes with lower expression in Ueya+so than in wt, with 10 genes (3.4% all negative targets) that respond negatively to So in both microarray conditions (this summary excludes the “ambiguous” genes that respond positively to So in one assay and negatively in another) (Table S2).

Putative targets of So during eye development

The 468 genes that are activated by So in the microarray datasets include many previously shown to play an important role during eye development. These eye genes include previously known direct targets of So such as ey, ato, and lz, as well as genes encoding a variety of transcription factors and co-factors, signaling pathway components, cytoskeletal components, and chromatin modifying proteins. The transcription factors and co-factors include retinal determination genes such as Optix, eya, distal antenna (dan), and distal antenna related (danr) [21], [39], [45], as well as several transcription factors needed for differentiation of retinal cells posterior to the MF such as senseless (sens) and glass (gl) [46], [47]. The putative So targets in signaling pathways include genes such as the receptor tyrosine kinase sevenless (sev, required in the R7 photoreceptor), phyllopod (EGFR pathway target and antagonist of Notch signaling), roughoid, and pointed (EGFR signaling) [48], [49], [50], [51], [52], [53], [54]. The putative So targets Arpc1 (Actin-related protein 2/3 complex subunit 1) and roughest (rest) are involved in pupal retinal development [55], [56] while putative So targets involved in chromatin structure such as Caf1 (Chromatin assembly factor 1 subunit) and lola (longitudinals lacking) have published eye phenotypes [57], [58].

Discussion

The homeodomain transcription factor So plays vital roles in Drosophila eye development, yet only a few of its direct targets are currently known. Using ChIP-seq, we have mapped So-enriched regions throughout the genome in third instar larval eye discs. We found 7,566 So-enriched regions, which map to approximately 5,952 genes, including previously characterized So targets in the eye. The genes that map to the 10% most significant So peaks are enriched in GO categories that are relevant to eye development, such as sensory organ development, neuron differentiation, imaginal disc pattern formation, and transcription regulation. The ChIP-seq data set greatly expands our list of putative So targets in the eye, and it suggests that So regulates multiple processes during eye development.

So peaks show enrichment for transcription factor binding motifs

Many transcription factors, including So, regulate their targets by binding cooperatively with other transcriptional regulators [6], [10]. To identify putative So-interacting transcriptional regulators in the eye, we performed de novo motif analysis of the entire So peak set using the online software RSAT [15]. As predicted, we observed enrichment for AGATAC, which closely matches the consensus So motif [11]. We also found enrichment for other transcription factor consensus motifs, some of which are known to be active in the eye disc. One of the enriched motifs corresponds to Optix, a So paralog and an RD network member that is expressed anterior to the MF in the eye disc [21]. The So peak set also shows enrichment for the consensus motif of Iroquois complex (Iro-C) homeodomain transcription factors – Mirror (Mirr), Araucan (Ara), and Caupolican (Caup). These factors, expressed in the dorsal half of the early eye disc, establish dorsal/ventral (DV) polarity in the eye disc, and activate Notch signaling at the DV midline that is necessary for eye disc growth [59], [60]. Whether So and Iro-C interact functionally to regulate target genes is unclear, but this could potentially happen in the dorsal-anterior quadrant of the third instar eye disc, where So and Iro-C are coexpressed.

Our motif analysis also suggests a putative link between So and transcriptional response to the hormone ecdysone, which triggers larval molting and pupariation in Drosophila, and regulates MF progression and the cell cycle in the eye disc [61], [62]. The So ChIP-seq peak set is enriched for the consensus motif for Br-Z1, an isoform of the Broad transcription factor, which is expressed posterior to the MF and is transcriptionally activated in response to ecdysone [63], as well as for Ultraspiracle (Usp), which forms an ecdysone-responsive heterodimer with the Ecdysone Receptor (EcR) [25]. Usp and Br appear to have opposite effects on eye development: while Br promotes MF progression, Usp antagonizes it, and Usp represses br expression in the eye disc [64], [65]. The enrichment of Br-Z1 and Usp motifs in the So peak set, as well as the presence of the term Response to ecdysone in the GO analysis of genes with highly enriched So peaks, suggests a possible role for So in regulating the response to ecdysone, a possibility that will need to be tested in future studies.

Another highly enriched motif among So peaks is the DNA Replication Element (DRE), which binds the DRE factor (Dref), an activator of DNA replication and cell proliferation [66]. In the eye disc Dref is expressed predominantly in proliferating cells anterior to the MF, and DREs are enriched in the promoter regions of genes that show high expression anterior to the MF [67]. Dref can also bind a chromatin boundary element [68], and several chromatin remodeling factors interact genetically with Dref in the eye [69]. These data suggest that Dref may play a role in chromatin remodeling. Trithorax-like (Trl, a.k.a. GAGA Factor) is also a chromatin remodeling protein active in the eye that shows consensus motif enrichment in the So peak set [70]. Altogether, these data suggest that So may contribute to regulating the cell cycle and/or chromatin state in the eye disc by acting together with Dref and Trl. This is consistent with the loss-of-function phenotypes of so mutant tissue, which shows defects in cell proliferation [5]. Future studies will be needed to test the functional significance of motif enrichment in the So peak set.

So binds to genes predicted to function in eye development

The top 10% most significant So peaks map to genes that are enriched in GO categories pertaining to eye and neuronal development, such as Imaginal disc development, Sensory organ development/Compound eye development, Neuron differentiation, and Regulation of photoreceptor cell differentiation. Most of the previously known So target genes encode transcription factors that drive successive stages of eye development: ey, which is necessary for eye specification and for initial expression of So, which then regulates ey in a feedback loop [71]; dac, which is necessary for the onset of differentiation in the eye disc; ato, which is required for the specification of the R8, the first photoreceptor that forms in the eye; and lz and its target pros, both of which regulate the differentiation of specific cell types. Consistent with these previous data, highly So-enriched genes are over-represented in the GO term Regulation of transcription, as well as InterPro protein domains that occur in transcription factors, such as homeobox and zinc finger (Table S1).

Putative direct targets of So

Of the many genes enriched for So binding in eye discs, only a fraction are expected to be true So targets. In order to identify candidate direct targets of So in eye development, we have overlapped the set of genes enriched for So binding with genes that are differentially regulated in response to loss or gain of so, using genome-wide expression data sets. Ectopic overexpression of genes that encode transcriptional regulators in the RD network, such as so and ey, is sufficient to trigger ectopic eye formation in Drosophila [40], [41]. A previous study analyzed microarray gene expression changes in response to ectopic eye induction in the leg imaginal disc ([32] and our unpublished data). Based on these previous data, we assembled two lists of candidate So target genes. The first list contains genes that are regulated by ectopic ey (UAS-ey, abbreviated Uey) in a so-dependent manner (i.e., genes that show differential expression between Uey leg discs and Uey; so leg discs). The second list includes genes that respond to ectopic overexpression of so and its binding partner eya in the leg disc (differential expression between wild-type and Ueya+so leg discs).

We identified 810 genes that have a So ChIP-seq peak and are differentially regulated in one or both of the above conditions (Uey vs. Uey; so and wild-type vs. Ueya+so). 460 genes respond to loss of so (differential expression between Uey and Uey; so), and 444 genes respond to gain of so (difference in expression between wt and Ueya+so leg discs). The overlap between the Uey; so and the Ueya+so gene lists is 94 genes (11.6%). Although both Uey and Ueya+so are capable of inducing ectopic eye formation, ectopic ey is a much more potent inducer of retinal fate than ectopic eya+so, and this may account, at least in part, for the small overlap between these two datasets [40], [72]. Hence, Uey and Ueya+so appear to regulate largely distinct sets of genes in the leg disc. Our approach likely resulted in many false negatives, as ectopic overexpression of RD genes does not fully recapitulate normal eye development. Notably, the microarray used RNA prepared from the whole leg disc, but Uey triggers ectopic retinal development in only a small fraction of the leg disc cells; hence, a gene that is only moderately upregulated in the minority of cells that take on an ectopic eye fate is unlikely to be detected as significantly upregulated in RNA prepared from whole Uey leg discs. Nonetheless, loss of so in Uey leg discs leads to reduced expression of the previously known So targets lz and ato, indicating that many gene expression changes observed in ectopic eye induction may be relevant to normal eye development. GO analysis of the genes that appear to be activated by So based on microarray data shows enrichment for terms associated with eye, imaginal disc, and neuron development. This is consistent with the ability of Ueya+so to induce ectopic eyes, and the requirement for so in Uey-mediated ectopic eye induction. The Ueya+so responsive genes in the leg disc include genes previously known to be required in eye development, such as danr, which is expressed anterior to and within the MF and is required for the onset of photoreceptor differentiation. Danr is part of the RD network; it interacts physically with the RD proteins Ey and Dac, and its overexpression can trigger ectopic eye formation in the antenna [73].

Novel targets of So in eye development

In an attempt to uncover novel targets of So that are necessary for eye development, we obtained fly stocks with mutations in novel, So-enriched genes, and tested the requirement for these genes in eye development using the FLP/FRT system. We identified eight genes that appear to be required for eye development, as their loss in the eye disc leads to reduced, misshapen, and/or disorganized eyes. A loss-of-function mutation in one additional gene, kkv, causes a pharate lethal phenotype with severe reduction in head size, suggesting that it may act early in development, upstream of so.

When choosing putative So target genes, we made the assumption that the gene nearest to or overlapping a So peak is the putative target, an assumption that may not always be correct. A transcription factor may regulate genes many kilobases away, through long-distance interactions mediated by chromatin looping [74]. If a small gene is nested in an intron of a larger gene that has a So peak, the small gene rather than the large gene may be the true So target. Moreover, if a So peak maps to an intergenic region, we automatically map it to the nearest gene in either direction, which is not necessarily the true target.

In addition to potential inaccuracies in mapping So peaks to target genes, there are several other reasons why only nine genes tested resulted in eye or head phenotypes. First, we limited ourselves to already existing alleles that were available from stock collections. Many genes that would have been interesting to test, such as novel So-enriched genes that show differential regulation during ectopic eye induction (Table S2), do not have publicly available mutant alleles. Second, most of the alleles we ordered were transposon insertions in an intron or UTR of the gene of interest that were likely to disrupt gene function only weakly or not at all. Availability of stronger alleles may have resulted in eye phenotypes with a larger percentage of the genes tested. Third, as discussed above, So does not necessarily regulate the gene to which it binds, as the So peak may be an experimental artifact, reflect nonfunctional binding, or be regulating an adjacent gene. For example, the novel gene CG7576 overlaps a large So peak, yet it has no phenotype in the eye, possibly because the So peak reflects regulation of the nearby gene glass, known to be necessary for eye differentiation [47].

Despite the caveats above, we identified eight novel genes that appear to regulate eye development downstream of So. The eight mutants show a range of phenotypes, from a disorganized but full-sized eye to almost complete loss of eye, reflecting the requirement for So at multiple stages of eye development, from cell survival and MF initiation to differentiation [3]. One putative direct So target gene is oocyte maintenance defects (omd), predicted to encode an RNA processing protein. Little is currently known about the function of omd, although previous studies suggest omd interacts with the Dpp pathway [75] and regulates larval neurogenesis [37], suggesting that it may function downstream of So in the developing photoreceptors. Detailed characterization of omd and other novel genes, as well as their regulatory interaction with So, awaits further studies. In summary, our work identifies a wealth of putative direct So targets that will expand our understanding of So-mediated transcriptional regulation in development.

ChIP-seq data suggest the presence of multiple feed-forward loops. For example, previous studies have shown that So and Ey positively regulate each other in early eye development [8], [76], and subsequently So and Ey together initiate ato expression [10]. Ato is necessary for the specification of the R8 photoreceptor, where it initiates the expression of senseless (sens), which encodes a zinc finger transcription factor necessary for R8 development [46], [77]. sens has a So peak, and sens induction by ectopic ey in the leg disc requires so (unpublished data), suggesting that So may directly regulate sens. Such a result would be a three-level feed-forward loop, whereby Ey and So activate each other, So and Ey activate ato, and So and Ato activate sens. Similarly, in differentiating cells posterior to the MF, So activates lz [9], which encodes a transcription factor that regulates multiple downstream targets, including the direct So target pros [6]. Lz also activates the transcription factors D-Pax2 in cone cell precursors and Bar-H1/2 in R1/6 photoreceptor precursors [78], [79]. D-Pax2, Bar-H1, and Bar-H2 all have So ChIP-seq peaks, suggesting the possibility of another feed-forward loop: So activates lz and then cooperates with Lz in activating downstream transcriptional regulators in different cell types posterior to the MF.

Another intriguing possibility is that So may regulate the expression of cytoskeleton and cell junction components, many of which are encoded by genes that have So peaks, and some of which appear to be regulated by So in ectopic eye formation. While many cell junction components are expressed uniformly throughout the developing eye disc, there are examples of dynamic regulation of cell junction protein expression in the eye: immediately posterior to the MF, the transcription factor Ato and the Egfr signaling pathway together promote high expression of the cell junction protein-encoding genes shotgun and armadillo in nascent ommatidial clusters [80]. Drosophila So has not been shown to control the expression of cytoskeleton component genes, but the murine So homolog Six1 directly activates Vil2, which encodes the actin cytoskeleton regulator Ezrin, thus promoting cell motility and metastasis in cancer [81]. Since So-expressing cells of the eye disc undergo dynamic shape changes associated with the passage of the MF and the formation of ommatidial clusters, and the optic lobe does not invaginate correctly in so mutant embryos [3], [4], it is possible that one of the functions of So in development is to regulate the expression of cell adhesion and cytoskeleton molecules that control morphogenesis. Our results provide the basis for many specific hypotheses concerning eye development.

Gene Ontology (GO) terms associated with genes highly enriched for So binding in the eye disc.

(DOCX)

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Putative direct targets of So based on transcriptional response to loss or gain of in ectopic eye. All genes listed have a So ChIP-seq peak, and show significant change in expression in response to either loss of so in ectopic eye (Uey vs. Uey;so) or ectopic overexpression of so and its cofactor eya (Ueya+so vs. wt). Depending on their response to So, genes are classified as POS (positively regulated, i.e. activated, by So), NEG (negatively regulated, i.e. repressed, by So), or AMB (ambiguous – respond positively to So in one assay and negatively in another).

(XLSX)

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So ChIP-seq peaks in the eye disc. Each peak has a unique index number, which reflects its P-value (lower index number corresponds to higher P-value and hence more significant peak). P-value  = 50 corresponds to P = 10−5. The coordinates of each peak are given, as is the identity of the nearest gene and the nearest transcription start site (TSS). The GENE_STATUS column indicates whether a peak is intragenic (GENE_OVERLAP) or intergenic (GENE_CLOSE). For intergenic peaks, the distance to nearest gene is given in the GENE_DIS column. The distance to the nearest TSS is in the TSS_DIS column. Note that the nearest TSS may belong to a gene different from the one listed in the GENE column.

(XLSX)

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