Fly stocks and molecular genetics

Flies were maintained at 25°C on a standard diet. The ftz mutant was ftz^9H34/TM3Ser, hb-lacZ, with expression of β-galactosidase used to identify mutant embryos. Enhancer-reporter constructs were constructed by PCR isolation of ~ 1kb regions of genomic DNA inserted into the following sites of attBlacZ, upstream of a basal promoter and lacZ: ken—EcoRI/ XbaI, aay—EcoRI/XbaI, mid–HindIII/XbaI, tal—HindIII/XbaI, 5-HT2A –EcoRI/XbaI, trn–HindIII/XbaI, hh–XbaI/HindIII, Antp–HindIII, XbaI, and blot–XbaI/NotI. The PhiC31 integration system was used to insert transgenes into the genomic attP site VK00022 in chromosome II. Transgenic fly lines were generated by Rainbow Transgenic Flies, CA and BestGene, CA and were maintained as homozygotes. One transgenic line, containing the blot enhancer construct, was homozygous lethal but, when crossed into a ftz mutant background, was homozygous viable. In this ftz background, a rare phenotype was observed in which part of the right dorsal thorax was missing and only the left wing was present. To examine transgene expression in ftz embryos, virgin females w; ftz^9H34/Tm3Sb were crossed with w; P[enhancer-lacZ]/P[enhancer-lacZ]; Dr/Tm3Sb males. From this cross, w; P[enhancer-lacZ]/+; ftz^9H34/Tm3Sb males and females were crossed to generate w; P[enhancer-lacZ]/P[enhancer-lacZ]; ftz^9H34/Tm3Sb. Offspring were self-crossed to analyze expression in a ftz background. Embryos derived from ftz-f1 germline clones (referred to as ftz-f1mutants) were generated with the autosomal FLP-DFS technique [27–29] using ftz-f1¹⁹ [30, 31]. Briefly, yw hsFLP;FRT^2Aftz-f1¹⁹/TM3Sb virgin females were crossed with w; FRT^2A ovo^D/TM3Sb males. Females were allowed to lay eggs for 1 day in vials and their progeny were heat-shocked for 2 hours at 37°C in a circulating water bath on the third and fourth days after egg laying. Subsequently, embryos derived from the females of genotype yw hsFLP;FRT^2Aftz-f1¹⁹/ FRT^2A ovo^D (identified as non-Sb females) were analyzed. All of the FRT^2A ovo^D recombinant chromosomes were associated with a fully penetrant DFS phenotype such that all eggs laid by these females are derived from germline recombination events. For the control for the microarray experiment, yw hsFLP;FRT^2A/ FRT^2A virgins were crossed to w; FRT^2A ovo^D/TM3Sb males and subjected to the same heat shock and selection protocol in parallel.

For in situ hybridization, standard protocols were followed [32, 33] with one modification—in place of Proteinase K treatment, embryos were heated at 95°C for 5 minutes. Primer sequences for probes available upon request. Standard protocols were followed for antibody staining [34]. For reporter constructs, anti-β-galactosidase antibody (Cappel, 1:2000) was used. Stained embryos were visualized using DIC on a Leica DMRB microscope, a Zeiss Discovery V12, or a Leica SP5X Confocal microscope.

Microarray

ftz-f1 or control females (see above) were mated to w¹¹¹⁸ males in collection cages at 25°C and allowed to lay eggs for up to 2 hours. Eggs were dechorionated in 3% sodium hypochlorite for 3 minutes and then covered with halocarbon oil and aged. Embryos were visualized under phase contrast optics at 100-200x magnification. Individual embryos were pooled into groups of roughly equivalent developmental stages and kept out of the light path of the microscope as much as possible. To generate pools of embryos at specific stages, each pool was monitored closely and selected by visual inspection at late cellularization, stage 5, at the onset of gastrulation, stage 6, or at mid germband extension, stage 8. Embryos were manipulated using a small needle. As each embryo reached the desired stage of development, it was transferred to 100 μl of TRIZOL on ice. Approximately 100 embryos were collected per time point. Individual collections were stored at -80°C. Each experiment was done in triplicate. Total RNA was extracted using the Qiagen RNEasy kit according to standard protocols. Samples were processed using the Affymetrix one-cycle cDNA synthesis protocol prior to hybridization to Affymetrix Drosophila 2.0 expression arrays.

Affymetrix drosophila2 genechip CEL files were imported into BioConductor/R [35] using the ReadAffy function of the affy package [36] and assigned to developmental stage (5,6,8), wildtype or ftz-f1 mutant condition (0,1) and batch number (processing and hybridization batch). Using 3 replicates of 3 stages and 2 states of ftz-f1 gave a total of 18 genechip arrays. All arrays were normalized by the expresso function using quantiles normalization, only perfect match, and median polish summary method. This generated the normalized expression set used for all further data analysis. The normalization procedure produced log₂ expression results, and the fold change between the average of any two data sets s1 and s2 was calculated as . To identify differentially expressed genes showing a response to the presence or absence of functional Ftz-F1, the microarray analysis of variance package, maanova [37], was run in oneColor mode fitting a mixed effect ANOVA model using the formula ŷ = Stage+Ftz.F1+Batch. Batch represents the groups of RNA that were processed and hybridized on the same day and was treated as a random or non-repeatable term in the model. The function matest was run with a permutation count of 100 to compute the p-value for the Ftz.F1 model term. p-values were further controlled for the N discovery rate using the Q-value method of the adjPval function [38]. Probesets having an FDR adjusted p-value < 0.05 were considered potential target genes.

Motif Analysis

401bp windows surrounding the peak Ftz binding position for all intervals [39] were merged and the underlying genomic sequences were extracted from a repeat masked copy of the Drosophila melanogaster genome. This dataset was processed with the meme application fasta-get-markov, to generate a 5^th order hidden markov model of the available background enhancers found in the Drosophila blastoderm [40]. This model or its associated fasta file were used as the background model for all relevant processes of the meme suite of applications. A subset of the 401bp sequences, defined by the ftz_3_032707-sym-1 dataset, was used as the positive set for Ftz binding. This set contains 403 intervals identified as bound by Ftz in stage 5 Drosophila melanogaster embryos, along with the location of maximal binding within each interval. Genomic sequence for each of these regions was extracted from the repeat masked genome. For both MEME and DREME, a q-value threshold of 0.05 was used with the previously computed background file searching both forward and reverse strands. MEME was restricted to a maximum motif width of 10, while DREME’s default of 8 was used [41]. To search for additional motifs within the enhancer regions, Ftz/Ftz-F1 enhancer regions were analyzed using MelinaII (which runs 4 de novo algorithms, Consensus, MEME, MDScan, and Gibbs) and the Drosophila JASPAR database [42]. To search for motifs using the Jaspar database, a Gaussian distribution was constructed from random sequences in the Drosophila genome to determine the background likelihood of the occurrence of any given sequence. Enhancer sequences were scanned using pwms from the Jaspar database and compared to this background to determine the chance of a motif occurring randomly at each position. Motifs with a probability score of 0.9 or higher were considered candidates.

Microarray identification of Ftz-F1-responsive genes

To identify candidate targets of Ftz-F1, gene expression was compared between wildtype and ftz-f1 germline clones embryos (referred to throughout as ftz-f1 mutants) at three time points during development: stage 5, the blastoderm forms, Ftz 7 stripes are established; stage 6, gastrulation, 7 strong Ftz stripes; and stage 8, germband extension, Ftz stripes begin to fade (S1 Fig). Gene expression was compared in whole embryos. As Ftz+ cells represent a maximum of 25% of the cells in the whole embryo, this could potentially dilute the overall fold change observed for genes regulated by Ftz, but this method also allowed for identification of Ftz-F1 targets that are not co-regulated by Ftz.

Of the 18952 probesets on the genechips, 735 (4%) showed detectable alterations in response to absence of ftz-f1. To avoid genes with solely maternal expression masking zygotic responses to Ftz-F1, analysis was restricted to genes showing at least a 1.1 fold increase between stage 5 and stage 6 in wildtype microarrays. 3944 (21%) of the probesets showed a 1.1 fold increase in expression level between stage 5 and stage 6. The intersection of these two data sets produced a list of 379 potential targets for validation, fewer than 2% of all genes examined on the microarray. 314 (83%) of these potential targets showed upregulation in response to absence of Ftz-F1 but, since Ftz-F1 has been shown to activate transcription (reviewed in [31], many of these are likely regulated indirectly and were not further examined in this study. Potential direct Ftz-F1 targets were the 65 (17%) probesets that showed at least a 1.1 fold increase in expression level between stages 5 and 6 and a lower expression in ftz-f1 mutants. These 65 probesets map to 63 unique genes. Fig 1 shows a heatmap of the changes in expression level for potential target genes and S1 Table shows the top 11 candidate target genes, ordered by their fold change in expression level between control and ftz-f1 mutants. Each showed an average of at least -1.49 fold change in expression level between ftz-f1 mutants and control embryos. Pairwise comparison of Pearson correlation coefficients showed that individual genechips varied more by developmental stage than by the presence or absence of a functional Ftz-F1 protein (S2 Table). This, along with the fact that fewer than 2% of genes that increase in early zygotic expression show changes in ftz-f1 mutants, suggests that only a small percentage of genes in the genome are regulated, directly or indirectly, by any given pair-rule transcription factor.

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Fig 1. Microarray identification of Ftz-F1-responsive genes in Drosophila embryos.

RNA was extracted from control or ftz-f1 mutant embryos at stages 5, 6, and 8, as indicated, and used to synthesize cDNA for hybridization to Affymetrix Drosophila 2.0 expression arrays. The dendrogram shows Pearson correlation coefficients of mean expression levels across probesets under assayed conditions. The heatmap shows expression levels of probesets that increased between stages 5 and 6 in wildtype embryos and were expressed at lower levels in ftz-f1 mutant embryos than control. Genes were sorted from highest to lowest average fold change in expression between control and ftz-f1 mutants after cellularization.

https://doi.org/10.1371/journal.pone.0163128.g001

Expression of Ftz-F1-responsive genes overlaps with Ftz

All previously identified Ftz-F1 target genes are co-regulated by Ftz. In order to determine whether this is the case for the microarray identified Ftz-F1-responsive genes, expression patterns were examined for ten of the top eleven genes from the microarray and compared to ftz expression (Fig 2, Table 1): ken and Barbie (ken), engrailed (en), astray (aay), midline (mid), tarsal-less (tal), 5-hydroxytryptamine (serotonin) receptor 2A (5-HT2A), tartan (trn), hedgehog (hh), Antennapedia (Antp), and bloated tubules (blot). tektin-C was not included because it does not show a detectable level of expression in early embryos by in situ hybridization. For 9 of the 10 remaining candidate target genes, expression of the target gene overlapped with ftz expression. ken is expressed in two stripes, which overlap with ftz stripes 1 and 7 (Fig 2A–2A”). en is expressed in 14 stripes and every other stripe overlaps with a ftz stripe (Fig 2B–2B”). The 7 stripes of aay and ftz overlap (Fig 2C–2C”). mid is expressed in 14 stripes, seven of which overlap with ftz stripes (Fig 2D–2D”). Of the six (of a total seven) tal stripes visible in blastoderm stage embryos, stripes 2 and 3 are more strongly expressed than the other stripes, but none overlap with ftz (Fig 2E–2E”). Thus, tal could not be a direct target of Ftz. 5-HT2A expression is broader than ftz expression, but all 7 5-HT2A stripes overlap the 7 ftz stripes (Fig 2F–2F”). trn is expressed in 8 stripes; the 7 posterior stripes of trn overlap the 7 stripes of ftz, while the most anterior trn stripe does not (Fig 2G–2G”). Of the 14 hh stripes, 7 alternating stripes overlap with ftz (Fig 2H–2H”). The single Antp stripe overlaps with the second ftz stripe (Fig 2I–2I”). blot expression, while in a striped pattern, is not as sharp as other targets. The stripes are blurred and broad, with soft edges. However, this expression does overlap with ftz stripes (Fig 2J–2J”). In sum, nine of the eleven top candidate target genes identified in the microarray experiment overlap with ftz, tektin-C and tal being the exceptions. The former is not detectably expressed and is thus a false positive from the microarray. tal may be an indirect target or a gene regulated by Ftz-F1 without Ftz. Overlap for the remaining nine genes was seen in blastoderm or very early gastrulation stages, consistent with the possibility that these genes are regulated directly by Ftz.

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Fig 2. Top Ftz-F1-responsive genes are co-expressed with ftz.

Fluorescent double in situ hybridization was performed against ftz and each target gene, as indicated. ftz stripes (A-J, red); target gene stripes (A’–J’, green); overlay (A”–J”, yellow). Some or all of the target genes’ expression patterns overlap ftz expression during the blastoderm stage for all candidate targets except tal. (A’) ken, (B’) en, (C’) aay, (D’) mid, (E’) tal, (F’) 5-HT2A, (G’) trn, (H’) hh, (I’) Antp, (J’) blot. Photographs of embryos from confocal microscopy are shown; anterior, left.

https://doi.org/10.1371/journal.pone.0163128.g002

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Table 1. Summary of Ftz/Ftz-F1 dependent target genes and CREs.

https://doi.org/10.1371/journal.pone.0163128.t001

Candidate Ftz-F1 target genes require Ftz and Ftz-F1 for expression in embryos

To test whether these candidate target genes are in fact controlled by Ftz and/or Ftz-F1, their expression patterns were examined in ftz and in ftz-f1 mutant embryos (Fig 3, Table 1). en, a known target, was not re-examined here [12]. For all nine genes examined here, expression in early stage embryos (Fig 3A–3I) was altered or undetectable in ftz (Fig 3A’–3I’) and in ftz-f1 (Fig 3A”–3I”) mutants. Both ken stripes were undetectable in ftz and in ftz-f1 mutants (Fig 3A–3A”). aay was expressed in seven stripes in control embryos, which were undetectable in ftz and in ftz-f1 mutants (Fig 3B–3B”). mid was normally expressed in 14 stripes. Like en, 7 of the 14 mid stripes were lost in ftz and in ftz-f1 mutants (Fig 3C–3C”). tal expression was undetectable in ftz and decreased in ftz-f1 mutants (Fig 3D–3D”). 5-HT2A was expressed in seven stripes beginning in early gastrulation in control embryos, which were undetectable in ftz and in ftz-f1 mutants (Fig 3E–3E”). In ftz and in ftz-f1 mutant embryos, the expression of the 7 posterior stripes of trn that overlap with Ftz (Fig 2G) was lost while the most anterior stripe, that does not overlap with Ftz, was present (Fig 3F–3F”). hh was expressed in 14 stripes, 7 of which were lost in ftz and in ftz-f1 mutants (Fig 3G–3G”). Antp was expressed in one band posterior to the cephalic furrow of the embryo, which was lost in either mutant (Fig 3H–3H”). blot was expressed in seven diffuse stripes (Fig 3J). Unlike the other candidate targets, the expression pattern was not changed qualitatively but appears weaker in ftz and in ftz-f1 embryos (Fig 3I–3I”).

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Fig 3. Ftz-F1-responsive genes are regulated by Ftz and Ftz-F1.

(A-I) The expression patterns of nine of the top candidate Ftz-F1 target genes from the microarray are shown. All are expressed in stripes in early embryos. (A) ken, (B) aay, (C) mid, (D) tal, (E) 5-HT2A, (F) trn, (G) hh, (H) Antp, (I) blot. (A’–I’) Expression was examined in ftz mutant embryos. (A”–I”) Expression was examined in ftz-f1 germline clone (GLC) embryos.

https://doi.org/10.1371/journal.pone.0163128.g003

In sum, the Ftz-F1-responsive genes identified in the microarray that are expressed in early embryos responded similarly to loss of Ftz and to loss of Ftz-F1 (Table 1). This brings to fourteen the Ftz-F1-responsive genes identified to date. All require Ftz for expression in embryos. These genes are ftz itself [11], en [12], apt, Sulf1 [25], drm, noc, and 5-HT2A [26] and seven new targets found in the microarray (ken, aay, mid, tal, trn, hh, Antp). For blot, Ftz and Ftz-F1 appear to have a quantitative effect on blot expression levels but are not responsible for establishing its striped expression. Finally, the loss of tal expression in ftz-f1 mutants verifies its identification in the microarray experiment. The fact that tal does not overlap ftz expression (Fig 2E”) suggested that it could be the only Ftz-F1 target identified to date not co-regulated by Ftz. However, since tal expression was lost in ftz mutants, it is more likely that Ftz and Ftz-F1 work together to regulate tal expression indirectly in Ftz^- cells. Overall, these findings are consistent with the fact that ftz and ftz-f1 pair-rule phenotypes are indistinguishable, strengthening the conclusion that Ftz-F1 absolutely requires Ftz for its activity in early embryos.

Identification of candidate Ftz/Ftz-F1-dependent enhancers

The Ftz-F1-responsive target genes analyzed above could be either directly or indirectly regulated by Ftz and/or Ftz-F1. To address this, we made use of BDTNP published ChIP-chip data on Ftz [39] to identify candidate Ftz/Ftz-F1-responsive enhancers within 70 kb each of the top ten Ftz-F1 targets identified from the microarray (S3 Table). Overall, this ChIP-chip experiment identified 403 Ftz binding sites in the genome of blastoderm stage embryos using a 1% FDR (Fig 4A). This cut-off was effective for identifying Bcd target genes [43]. Ftz binding was found using a 1% FDR for eight candidate target genes (Table 1). Genomic regions of ~1 kb surrounding each of these Ftz binding intervals were queried for consensus Ftz-F1 binding sites (BSAAGGHYRHH). At least one candidate Ftz-F1 binding site was found in the region of every Ftz binding peak examined, except for tal, for which no Ftz-F1 binding sites were found. DREME and MEME queries of regions within all Ftz binding peaks in the genome identified the core Ftz-F1 binding sequence (AAGG) as the most over-represented sequence (Fig 4B and 4C).

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Fig 4. Ftz-F1 binding sites are overrepresented in Ftz genomic binding peaks.

(A) Genomic positions of Ftz binding based on Ftz ChIP-chip data from BDTNP is shown schematically mapped to the four Drosophila chromosomes [39]. Red lines represent strong Ftz binding; green lines represent weak Ftz binding. The genomic loci of candidate Ftz-F1 targets from the microarray are indicated in pink. ftz-responsive genes not found in this study are indicated in black. Positions of FRT[2A] and ftz-f1, used to generate ftz-f1 germline clones, are indicated in blue. (B,C) Over-represented sequences in the Ftz ChIP-chip binding data, identified by (B) DREME and (C) MEME. Ftz-F1 candidate binding sites (core, AAGG) are overrepresented in genomic regions where Ftz binds DNA.

https://doi.org/10.1371/journal.pone.0163128.g004

For each gene, the region that contained the strongest Ftz binding and at least one candidate Ftz-F1 binding site was selected to be tested functionally (Fig 5). An enhancer was not generated for en, even though it was identified in the microarray, because the Ftz/Ftz-F1- dependent enhancer identified previously was also identified by the above searches. Enhancer-reporter constructs generated were: ken-lacZ, aay-lacZ, mid-lacZ, 5-HT2A-lacZ, trn-lacZ, hh-lacZ, Antp-lacZ, and blot-lacZ. For aay, a Ftz binding peak was not found in the dataset using 1% FDR but was found at 25% FDR. For 5-HT2A, no Ftz binding was found. However, because 5-HT2A was identified as a ftz/ftz-f1 target here and in a previous study [26], a candidate enhancer was selected based on a candidate Ftz-F1 binding near the gene. In order to increase the strength of this candidate enhancer, an exception for the size of the region was made, making it 2 kb, to include both the strongest potential Ftz-F1 binding sites and Zelda binding sites, which are important in the activation of transcription of many developmental genes [44]. This provided a good test of the importance of Ftz binding in choosing an enhancer. Interestingly, based on BDTNP data, all candidate enhancers were in accessible chromatin regions at stage 5 (Fig 5).

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Fig 5. Identification of candidate Ftz/Ftz-F1 dependent enhancers.

A screen shot from Flybase GBrowse showing genomic regions around Ftz-F1-responsive genes. Published BDTNP data available at http://bdtnp.lbl.gov/Fly-Net/browseChipper.jsp was downloaded and all interval coordinates along with the peak binding positions were remapped from release 4 to release 6 of the Drosophila genome using the Coordinate Converter provided by Flybase. The Ftz binding data was then uploaded into Flybase GBrowse for visualization. Candidate enhancers (red arrows) were identified using three major criteria: 1) Location near Ftz-F1-responsive target gene (light blue); 2) Ftz ChIP-chip binding, yellow arrow (1% FDR) or orange arrow (25% FDR); 3) candidate Ftz-F1 binding sites (thin purple arrows). Open chromatin (green arrows), was also found at blastoderm stage at all enhancers. Enhancer chromosomal locations (flybase v. 6) are: (A) ken 2R:23868513..23869466, (B) en 2R:11524814..11525958, (C) aay 3L:9416225..9417230, (D) mid 2L:5473726..5474732, (E) 5-HT2A 3R:4612279..4614294, (F) trn 3L:13073977..13075024, (G) hh 3R:23138154..23139164, (H) Antp 3R:6948758..6949766, (I) blot 3L:17409206..17410251.

https://doi.org/10.1371/journal.pone.0163128.g005

Candidate enhancers are functional CREs

To determine whether the candidate enhancers actually function as CREs in vivo, expression of reporter genes was analyzed in transgenic embryos (Fig 6). The ken-lacZ reporter gene was expressed in two stripes, anterior and posterior, mimicking endogenous ken expression (Fig 6A and 6A’). aay-lacZ was the only transgene for which no β-galactosidase was detected (not shown). mid-lacZ was expressed in seven stripes (Fig 6B and 6B’), presumably those that overlap the seven ftz stripes (Fig 2D”). 5-HT2-lacZ was expressed weakly, not evident until germband extension stages when three thick evenly spaced bands were observed (Fig 6C and 6C’). These bands are located between engrailed stripes 2 and 3, 4 and 5, and 6 and 7 (data not shown). This weak expression did not correspond to wildtype expression of 5-HT2A, and the expression was spotty. trn-lacZ was expressed in seven of the eight stripes in which endogenous trn is expressed (Fig 6D and 6D’). This finding clearly indicates that a Ftz-F1/Ftz-responsive enhancer was identified for trn, as only seven of the eight endogenous trn stripes overlap with ftz [45] and this work). hh—lacZ stripes arose during gastrulation, with fourteen stripes evident by germband extension; alternating stripes were strong and weak (Fig 6E and 6E’). The endogenous hh gene is expressed in all fourteen stripes at similar levels but only alternating stripes could be regulated directly by Ftz. Antp-lacZ was expressed in one strong stripe just posterior of the cephalic furrow at the blastoderm stage, similar to the endogenous Antp gene (Fig 6F). Additional stripes appear later, during gastrulation and germband extension (Fig 6F’). blot-lacZ was expressed in a broad band in the central region of the embryo at early and late stages (Fig 6G and 6G’) and later in seven stripes, similar to the endogenous expression (Fig 6G’).

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Fig 6. Target gene CREs direct ftz-dependent striped expression patterns.

Expression of enhancer-lacZ reporter constructs, as indicated, in transgenic embryos is shown. (A-G) Early transgene expression. (A’-G’) Late transgene expression. Note that β-galactosidase is stable and accumulates in embryos such that expression appears stronger at late germband extension. Expression of ken-lacZ, mid-lacZ, trn-lacZ, hh-lacZ, Antp-lacZ and blot-lacZ was similar to the corresponding endogenous gene. 5-HT2A-lacZ was expressed weakly. (A”-G”) Expression of enhancer-lacZ reporter transgenes (brown) in ftz mutant embryos. To identify ftz mutants, in situ hybridization was used to detect en (blue), which is expressed in 14 stripes in wildtype embryos and 7 stripes in ftz embryos.

https://doi.org/10.1371/journal.pone.0163128.g006

In sum, six of the eight enhancers tested directed strong reporter gene expression in patterns virtually identical to the corresponding endogenous genes (ken, mid, trn, hh, Antp, blot), demonstrating that these are functional CREs (Table 1). The two enhancers that were tested despite not fulfilling the criteria used for enhancer identification (see above) were weak or non-functional. aay-lacZ was not detectably expressed. This was the one enhancer for which the Ftz ChIP-chip peak was chosen at a 25% FDR. 5-HT2A-lacZ was expressed but the pattern was extremely weak and differed from the endogenous gene, suggesting that partial regulatory information was present in the region isolated. The 5-HT2A region chosen was the only enhancer tested that lacked a Ftz ChIP-chip peak but the fact that expression was lost in ftz mutants (see below) suggests that there could be low levels of Ftz genomic binding not detected even at 25% FDR. Together, these results suggest that the presence of Ftz binding and the ability to bind Ftz-F1 are highly predictive of enhancer activity in vivo.

Target gene CREs are Ftz-responsive

To test whether these CREs are ftz-responsive, expression was examined in ftz^9H34 homozygotes. Embryos were double stained with anti-β-galactosidase antibody (brown) to detect enhancer expression and en (blue) to identify the ftz mutants, which express only seven en stripes. Enhancer-lacZ reporter gene expression pattern was examined during germband extension when expression was strongest (Fig 6A”–6G”). The two stripes of ken-lacZ expression were lost in the mutants (Fig 6A”). The seven strong stripes for mid-lacZ were undetectable in the ftz background (Fig 6B”). For 5-HT2A-lacZ, the one weak enhancer, expression was not detectable in a ftz background (Fig 6C”). While some bands were still visible for trn-lacZ in ftz mutants, these were much weaker and spotty (Fig 6D”), suggesting the presence of additional CRE(s) for trn stripes. All fourteen stripes of hh-lacZ were lost (Fig 6E”), which was surprising, as only the seven stripes overlapping ftz expression should be lost if the enhancer is only under Ftz control; this suggests that this enhancer contains an additional CRE(s) that responds indirectly. Note that this indirect regulation cannot be via en, as Ftz regulates only the seven alternate en stripes that overlap with Ftz. Antp-lacZ expression was much weaker in the ftz background (Fig 6F”), suggesting the presence of additional CRE(s). The seven stripes of expression driven by blot-lacZ were lost in ftz mutants but the broad, central band was still detectable, indicating that this band was not regulated by Ftz. This is consistent with decreased levels of blot expression in ftz and ftz-f1 mutants (Fig 3). Overall, seven newly identified enhancers directed expression in embryos in patterns that were weakened or undetectable in a ftz mutant background, indicating that these CREs are Ftz-responsive.

Further analysis of Ftz-dependent enhancers

These Ftz-responsive CREs were next examined for additional motifs that might be important for regulation. For this analysis, the six strong CREs found in this study (enhancers for ken, mid, trn, hh, Antp, and blot) were analyzed along with four previously confirmed Ftz/Ftz-F1-responsive CREs (ftz proximal enhancer, en, drm2, and drm34 [12, 26, 46] to determine if there were any binding sites common to all ten. These ‘confirmed enhancers’ were compared to ‘false enhancers,’ those that contain Ftz and Ftz-F1 binding sites, but did not function as CREs in reporter transgene experiments in vivo. Three false enhancers have been found to date, those for aay (this study), drm1 and drm5 [26]. These two groups of enhancers were searched independently for binding sites for additional transcription factors that may be necessary regulators of gene expression, either as co-activators/repressors themselves or as factors modulating Ftz/Ftz-F1 DNA binding. All four of the de novo algorithms used by the MelinaII program -Consensus, Meme, MDScanner, and Gibbs—identified the binding site for Ftz-F1 as the most common motif in the confirmed enhancers (S2A and S2B Fig). Note that Ftz-F1 sites were also found to be the most common motif in in all Ftz ChIP-chip genomic peaks (Fig 4) suggesting that Ftz and Ftz-F1 binding may be sufficient for target site selection. For the false enhancers, the most common binding motif found was for Forkhead (Fkh) (S2C and S2D Fig).

To determine whether other factors—activating either positively, negatively, or affecting Ftz/Ftz-F1 binding—further influence Ftz/Ftz-F1 target gene regulation, the enhancers were next analyzed to identify known transcription factor (TF) binding sites, using the JASPAR database [42]. Binding sites for 67 known TFs were found in the confirmed enhancers (Table 2). 57 of the 67 TFs contain a homeodomain, as does Ftz, and thus were likely identified by the program because their binding site is similar to the binding site of Ftz. Similarly, this analysis found many homeodomain binding sites in the false enhancers (64 out of 77) as well as 13 TFs that do not contain a homeodomain. Excluding the homeodomain proteins, binding sites for one TF (Zeste) was present in all of the confirmed enhancers but not in all of the false enhancers. Binding sites for 5 TFs (Trithorax-like (Trl, GAF), Scalloped, Dichaete (D), Forkhead, and Sloppy-paired1) were present in all of the false enhancers but not in all of the confirmed enhancers. One TF (DEAF-1) had a particularly high PWM score in both groups of enhancers.

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Table 2. Transcription factor binding motifs within the confirmed and false enhancer groups.

https://doi.org/10.1371/journal.pone.0163128.t002

Published ChIP-chip data from 4–12 hours AEL embryos for several chromatin markers, including H3K4me3 and H3K27me3, as well as GAF, PC, and Pho binding [47], was used to analyze regions surrounding the Ftz/Ftz-F1 targets and CREs. For the histone methylation marks, no clear pattern was detected, and in fact, some targets had a strong signal for H3K27me3, a marker for repression, even though they are actively expressed. This could be explained by the broad collection time of the embryos in the ChIP-chip data. While PC and Pho showed no distinct difference between confirmed and false enhancers, strong GAF binding was found only near the three false enhancers. GAF was found near only one of the confirmed enhancers (ken), but binding was weak (S3 Fig), suggesting that GAF could repress gene activation by Ftz/Ftz-F1.

Ftz binding in the Drosophila genome

The ChIP-chip data published by BDTNP identified 403 Ftz binding sites in the genome of blastoderm stage embryos using a 1% FDR (Fig 4A). An additional 3,318 sites were identified when a 25% FDR was used as a cutoff. While some of the sites identified at 25% FDR may represent stable Ftz binding, the one tested here (candidate aay enhancer) was not functional in directing reporter gene expression. Ftz binding was found in the vicinity of all of the Ftz-F1-responsive genes identified in this study and also near previously identified Ftz-responsive genes not identified in the microarray (Figs 1 and 4A). These include teashirt [48], gooseberry [48], Ubx [49–51], and the ftz upstream element [46]. Two Ftz/Ftz-F1-responsive enhancers had been identified for drumstick (drm)–drm2 and drm34 [26]. Only drm2 was bound by Ftz in the ChIP-chip analysis—drm34 was not identified at either 1% or 25% FDR. This is the only known CRE missed in the ChIP-chip analysis, suggesting that a small number of bona fide Ftz target genes were not found with the current data. Within the 403 Ftz binding peaks, the most over-represented sequence was the Ftz-F1 binding site (Fig 4B), suggesting that Ftz-F1 is the primary determinant of stable Ftz binding in the genome. This is consistent with biochemical studies showing that Ftz-F1 dramatically increases the affinity and specificity of Ftz DNA binding in vitro [11, 17].

Other factors influencing Ftz/Ftz-F1 activity in vivo

Analysis of verified Ftz/Ftz-F1-regulated enhancers (confirmed enhancers), compared to sequences containing potential Ftz and Ftz-F1 binding sites but not functioning as enhancers (false enhancers), was carried out to identify candidate co-regulators that may limit the binding and/or activity of Ftz and/or Ftz-F1 to specific genes. Binding sites for Deaf-1 and Zeste were present in all of the Ftz-dependent enhancers while false enhancers were enriched in binding sites for Dichaete and GAGA Factor. All of these proteins are likely to contribute to the regulation of many early embryonic genes, consistent with their complex phenotypes.

Deaf-1, first identified as a putative cofactor of the Hox protein Deformed, appears to function as a general factor in the early embryo, as mutants display wide-ranging effects on embryonic development including segmentation defects [52]. Zeste binding sites were identified in all confirmed enhancers, and other studies have shown that it can act as both an activator and repressor [53, 54], functioning at the chromatin level [55, 56]. Dichaete and GAF binding sites were both identified in all of the false enhancer sites. Dichaete also affects transcription at the chromatin level Interestingly, it is expressed in stripes in the early embryo [57, 58] and was previously found to interact with Ftz-F1 in a whole genome yeast two-hybrid experiment [59]. Like Zeste, GAF can also act as an activator or repressor on the chromatin level [60], and GAF binding sites have been found in Polycomb response elements (PREs) [61–63], where Polycomb Group proteins (PcG) bind to repress homeotic gene expression, along with Pleiohomeotic (Pho) [64–66]. Examining ChIP-chip binding data of GAF surrounding both the confirmed and false enhancers revealed that GAF bound only at the false enhancers, with the exception of weak binding near ken-lacZ (S3 Fig). Together these results suggest that while other TFs may play roles in the regulation of expression of Ftz/Ftz-F1-responsive genes, Ftz-F1 is the main determinant of Ftz genomic binding. In addition, GAF may act as a repressor of Ftz/Ftz-F1 binding.

Targets of pair-rule genes

This study identified more than 50 genes that are expressed at higher levels in wildtype than ftz-f1 mutants (microarray). Of the genes showing the largest difference in the microarray experiment, ten of the top eleven are co-regulated by Ftz and Ftz-F1, and identification of enhancers suggests that at least seven of these ten are directly regulated by them. Ftz/Ftz-F1 are PRGs responsible for the formation of even-numbered parasegments and are thought to be among the more downstream PRGs, that, for example, directly regulate en expression. However, this study and others suggest that they have many other direct targets.

The new Ftz/Ftz-F1 target genes identified in this study all have known roles in embryonic development. en and hh play multiple roles, the most relevant to this study being their well-known roles as segment polarity genes [67]. While En is a homeodomain-containing transcription factor, Hh is a signaling molecule. mid encodes a T-box transcription factor, classified as a segment polarity gene, but its most well-studied roles are in development in neuroblast specification, axon guidance, and heart development [68–70]. Antp is a central Hox gene and was one of the first proposed candidate targets of pair-rule genes, including Ftz [71, 72]. This study confirms the proposed direct regulation of Antp by Ftz, along with Ftz-F1. ken encodes a zinc finger transcription factor implicated in genitalia development and as a regulator of the JAK/STAT pathway [73–75]. Interestingly, a previous study identified the gap protein Kruppel (Kr) as a negative regulator of ken [76], suggesting that a combination of activation by Ftz/Ftz-F1 and repression by Kr could explain the ken 2-stripe pattern. aay was identified in screens for regulators of peripheral nervous system development [77]. It encodes a putative 3-phosphoserine phosphatase and is necessary for axon guidance in the PNS has a role in axon guidance/nervous system development While initiatilly expressed in a 7-stripe pattern ([78] and Fig 3), it is later expressed in clusters in each segment as well as in the gut [78]. trn encodes a cell surface protein that has been implicated in cell movement and migration in various cellular contexts including roles in imaginal discs, tracheal branch outgrowth, retinal epithelial integrity [79–83]. Trn, and its partner Capricious appear to regulate cell-cell interactions in all of these contacts by mediating homophilic cell adhesion. This in turn suggests that Trn may be an important regulator of segment integrity in the early Drosophila embryo (unpublished observation). Blot is a putative neurotransmitter with a role in morphogenesis of epithelium [84]. Similarly 5HT2 encodes a serotonin receptor with specific ligand binding with a documented role in convergent extension in Drosophila [85–88].

In sum, of the known Ftz/Ftz-F1targets, excluding ftz itself, for which there is strong evidence for direct Ftz/Ftz-F1 regulation, seven encode nucleic acid binding proteins that function as TFs themselves (en, apt, drm, noc, ken, mid, Antp) and one is a signaling molecule (hh). Five fall into a number of different classes that could implicate them in playing more direct roles in morphogenesis: protein phosphatase (aay), serotonin receptor (5-HT2A), cell surface protein (trn), neurotransmitter transport (blot), and sulfatase (Sulf1). This suggests that the hierarchy remains regulatory to a large extent at the level of pair-rule genes but also identifies a group of direct targets that may play direct roles in segment formation. However, we suggest that the list of known targets is biased by their strong phenotypes. All of the top microarray targets investigated here were known genes, but the next set (Fig 1) includes many genes for which phenotypes have not been analyzed (CGs). Future experiments will determine whether these genes are also direct PRG targets, contributing to segment formation in subtle ways, such that they had not been previously identified in mutant screens.