atf3 is a cell-polarity response gene activated by aPKC but not JNK signaling

Previous transcriptome profiling by our group and others [11–13] has suggested that disturbed cell polarity leads to upregulation of atf3 expression. Consistently, qRT-PCR from scrib¹ homozygous mutant larvae and adult heads bearing dlg1^G0342 homozygous mutant clones showed increased levels of atf3 mRNA (Fig 1A), thus confirming induction of atf3 transcription upon depletion of the Scrib polarity module. To extend this evidence, we tested whether Scrib or Dlg1 deficiency impacts the levels of an Atf3::GFP fusion protein expressed from a recombineered BAC (atf3^gBAC) that is sufficient to rescue the lethality of the atf3⁷⁶ null mutants [21]. In control eye/antennal imaginal disc (EAD), Atf3 was enriched in differentiated photoreceptors of the eye primordium and a subset of peripodial cells of the antenna (Fig 1B). In wing imaginal discs, Atf3::GFP protein labeled the squamous cells of the peripodial epithelium and columnar cells of the outer ring that encircles the wing pouch (Fig 1C). Knockdown of dlg1 (en>dlg1^RNAi) or scrib (en>scrib^RNAi) in the posterior compartment of the wing disc resulted in a marked increase of the Atf3::GFP signal in the columnar epithelium of the wing pouch (Fig 1D and 1E). Importantly, Atf3 was also upregulated in dlg1^G0342 loss-of-function mutant clones induced in the larval EAD (Fig 1F and 1G and S1A–S1C Fig).

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Fig 1. atf3 is a polarity response gene.

(A) Transcript levels of atf3 increased in scrib¹ mutant larvae and in adult heads bearing dlg1^G0342 clones relative to control. qRT-PCR data are means of 5 and 3 biological replicates (FRT82B and FRT82B scrib¹, respectively) and 5 and 4 biological replicates (FRT19 and FRT19 dlg1^G0342, respectively). Error bars depict 95% confidence interval; Unpaired Student’s t-tests assuming unequal variance were used to calculate p-values: *p = 0.014, **p = 0.0051. (B-C) Atf3::GFP fusion protein expressed from a recombineered BAC (atf3^gBAC) labeled differentiated photoreceptors and a subset of peripodial cells in the control eye/antennal imaginal disc (B). In wing imaginal disc, peripodial cells (C) and columnar cells forming a ring surrounding the wing pouch (C’, arrows) showed Atf3::GFP enrichment. (D-E) Increase of Atf3::GFP signal appeared in the posterior compartment of the wing imaginal disc where Dlg1 (D-D’) or Scrib (E-E’) was depleted. (F-I) Clones in the EAD (myr-mRFP) deficient for Dlg1 expressed ectopic Atf3 within the eye (F’) and antenna (G’) part, which persisted when JNK signaling was blocked (dlg1^G0342 bsk^DN) (H’, I’). (J) Mild activation of JNK in the posterior compartment (J) of the wing imaginal disc (en>hep^wt) induced TRE-DsRed (J’) but did not trigger expression of GFP-tagged Atf3 (J”). (K-L) Atf3 protein levels increased following expression of a membrane tethered aPKC in the posterior compartment of the wing imaginal disc (en>aPKC^CAAX) (K’). This induction was independent of JNK signaling (en>aPKC^CAAX bsk^DN) (L’). (M-P) Driving Atf3 (M) or aPKC^CAAX (O) with dpp-GAL4 (mCherry) induces an Atf3-responsive ATRE-GFP reporter (M’,O’), which is insensitive to Hep (N’) or activated Yki (P’). The wing discs (C-D, M-P) or the posterior wing disc compartments (E,J) are outlined with dotted lines. Micrographs B and F-I are projections of multiple confocal sections, while micrographs C-E and J-P are single slices. To allow for comparisons, micrographs M-P were acquired with the same microscope settings. Scale bars: 50 μm (C-I,K-L) and 100 μm (B,J,M-P).

https://doi.org/10.1371/journal.pgen.1007241.g001

Previous studies have shown that disruption of the Scrib module leads to activation of Jun-N-terminal kinase (JNK) signaling and to deregulation of the atypical protein kinase C (aPKC) [22–25]. To determine if either one of these two pathways is sufficient to induce Atf3 expression, we assessed both Atf3::GFP protein levels and the activity of a synthetic Atf3-responsive element (ATRE) reporter. The ATRE construct expresses GFP or RFP under the control of four concatenated genomic segments, each of which is 22 bp long and contains an Atf3 binding site identified through ChIP-seq in this study (S1 Table, Materials and Methods). Both in vivo and in cultured Drosophila S2 cells, Atf3 activated the ATRE reporter (Fig 1M’ and S2A, S2B and S2E Fig) but not its mutated version (mATRE) bearing three base pair substitutions in the Atf3 recognition sequence (S2C and S2D Fig and S1 Table). While activation of JNK signaling by expressing a wild type form of Drosophila JNKK (Hemipterous; Hep) induced a JNK-responsive TRE-DsRed reporter [26] (Fig 1J and S2G Fig), it failed to upregulate Atf3 expression when targeted to the posterior compartment of the wing imaginal disc (en>hep^wt) (Fig 1J”). Similarly, the ATRE-GFP reporter was insensitive to or only minimally activated by JNK signaling in the wing imaginal disc (dpp>hep^wt) and S2 cells, respectively (Fig 1N and S2E Fig). Moreover, JNK signaling appeared dispensable for Atf3 induction caused by loss of polarity, as elevated Atf3::GFP signal persisted in dlg1 deficient EAD clones expressing a dominant negative form of the Drosophila JNK Basket (dlg1^G0342 bsk^DN) (Fig 1H and 1I). In contrast, expression of a membrane tethered aPKC (aPKC^CAAX) that causes mild overgrowth was sufficient to induce both Atf3::GFP and the ATRE reporter in specified wing disc compartments (en>aPKC^CAAX and dpp>aPKC^CAAX) (Fig 1K and 1O). Although inhibiting JNK signaling suppressed the aPKC^CAAX–mediated overgrowth, it did not prevent Atf3 induction (en>aPKC^CAAX bsk^DN) (Fig 1L). Signaling via aPKC has been shown to engage Yorkie (Yki), a transcriptional co-activator in the Hippo pathway [10,24,27,28]. However, expression of a constitutively active form of Yorkie (dpp>yki^act) did not induce the ATRE reporter (Fig 1P). Together, these findings highlight a role for Atf3 as a polarity response gene that is activated by loss of key polarity determinants downstream of aPKC but not JNK.

Loss of atf3 suppresses the effects of dlg1 deficiency on eye development

Imaginal disc clones lacking either Scrib or Dlg1 surrounded by normal epithelium are severely disorganized, suffer from disturbed vesicular transport, lose the ability to terminally differentiate and are eliminated through cell competition involving JNK signaling [22,25,29]. While JNK and its downstream transcription factor Fos are required for apoptosis and suppression of Yki-mediated hyperproliferation of scrib and dlg1 mutant cells [23,25,30–33], aPKC is responsible for the aberrant morphology and differentiation of the clonal cells [22].

Because Atf3 upregulation results from depletion of the Scrib complex components as well as from ectopic aPKC activation (Fig 1D, 1E and 1K), we investigated whether this excess Atf3 might contribute to the differentiation and morphological defects of EAD clones lacking Dlg1. While EADs bearing dlg1 deficient clones (S1B and S1C Fig) produced significantly smaller, malformed adult eyes with patches of undifferentiated tissue (Fig 2A and 2C and S3A, S3F and S3G Fig), simultaneous removal of atf3 (atf3⁷⁶dlg1^G0342) almost completely restored normal eye size and morphology (Fig 2D and S3B, S3F and S3G Fig), with only minor irregularities to the orderly hexagonal lattice (Fig 2D’). An equivalent genetic interaction was observed between atf3 and the dlg1^m52 allele [34,35] (S3D–S3G Fig). Importantly, adding a single copy of the atf3^gBAC transgene to animals with the double mutant mosaic EADs (atf3⁷⁶dlg1^G0342;; atf3^gBAC/+) was sufficient to reinstate the aberrations to the adult eye morphology (S3C Fig), thus clearly showing that Atf3 is required for this dlg1 deficiency phenotype to develop. Abnormalities were further exacerbated in adult eyes bearing dlg1 deficient clones in which Atf3 was overexpressed (dlg1^G0342 atf3^wt) (Fig 2E). It is important to note that mosaic overexpression of Atf3 alone disturbed the normal ommatidial arrangement in the adult eye (Fig 2F). However, immunostaining of third instar EADs against a pan-neuronal marker Elav showed that, unlike cells lacking Dlg1, Atf3-expressing clones (atf3^wt) differentiated (S4 Fig). In contrast, clonal loss of atf3 alone (atf3⁷⁶) did not markedly impact adult eye morphology (Fig 2B). These data provide causal evidence for the role of Atf3 downstream of disturbed epithelial polarity. They also indicate that while Atf3 is required for phenotypes caused by loss of dlg1, the gain of Atf3 alone does not fully recapitulate these defects.

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Fig 2. atf3 genetically interacts with dlg1.

(A-R) eyFLP-mediated mitotic recombination was used to generate clones (GFP) in EADs of the indicated genotypes. Lack of dlg1 (C) produced smaller adult eyes containing patches of undifferentiated tissue (C’) relative to control eyes (A,A’) and those bearing atf3⁷⁶ mutant clones (B,B’). Simultaneous clonal loss of atf3 and dlg1 (D) resulted in normally sized eyes with few to no differentiation defects (D’). In contrast, overexpression of atf3 in dlg1^G0342 clones (E) exacerbated the differentiation defects (E’). Clonal overexpression of atf3 alone disturbed the orderly ommatidia array (F) generating glossy patches lacking bristles (F’). The dlg1^G0342 (I) and atf3⁷⁶dlg1^G0342 (J) EAD clones were less abundant and smaller relative to control (G) and atf3⁷⁶ mutant clones (H). Overexpression of atf3 in dlg1^G0342 cells generated clones with smooth boundaries composed of large, round cells (K). Overexpression of atf3 alone did not impact clone shape or abundance (L). While no extrusion was observed either in case of atf3⁷⁶ mutant (N) or atf3 overexpressing (R) EAD clones compared to control EADs (M), the clonal loss of dlg1 induced both apical and basal extrusion (O). Clones that lack dlg1 and overexpress atf3 are extruded apically (Q), while simultaneous loss of dlg1 and atf3 normalizes the epithelial integrity (P). EADs M-R were stained against the p120-catenin to aid the assessment of tissue architecture. Discs were counterstained with DAPI. Micrographs A-F are scanning electron micrographs. Micrographs G-R are projections of multiple confocal sections and show EADs 7 days after egg laying. Dotted lines indicate cross sections, which appear below the corresponding panels and are oriented apical side up. Scale bars: 100 μm (A-F), 10 μm (A’-F’), 100 μm (G-L), 50 μm (M-R).

https://doi.org/10.1371/journal.pgen.1007241.g002

Loss of atf3 restores polarity and differentiation to dlg1 deficient cells independent of JNK activity

Closer examination of third instar larval EADs revealed the presence of fewer dlg1^G0342 clones relative to control and atf3⁷⁶ mosaic EADs (Fig 2G–2I). Many of these mutant cells showed increased activity of the JNK-responsive TRE-DsRed reporter (S5B Fig). Interestingly, simultaneous loss of atf3 neither restored the size or abundance of dlg1^G0342 mutant cells nor prevented JNK activation (Fig 2J and S5A and S5C Fig). The exacerbated phenotype of dlg1^G0342atf3^wt mosaic adult eyes correlated with the presence of contiguous clonal patches in third instar EAD that were markedly rounder compared to clones lacking dlg1 alone (Fig 2I and 2K). Although the individual clones appeared larger, the overall amount of GFP positive tissue was not increased when compared to dlg1 mutant clones (S5A Fig). The cross sections of the eye primordia further revealed that in contrast to dlg1 deficient cells which accumulated basally, the majority of dlg1^G0342atf3⁷⁶ clones remained in the disc proper similar to control and atf3 mutant cells (Fig 2M–2P). Strikingly, overexpression of Atf3 in cells lacking dlg1 promoted their apical extrusion (Fig 2Q). To rule out that clone elimination contributed to the observed rescue phenotype in the atf3⁷⁶dlg1^G0342 adult eyes, we took two alternative approaches. To inhibit death of clonal cells, we expressed the baculoviral caspase inhibitor p35 in dlg1^G0342 and atf3⁷⁶dlg1^G0342 EAD clones. To reduce competition elicited by neighboring cells, we utilized the EGUF/hid technique [36], which facilitates expression of a pro-apoptotic protein Hid in the non-clonal cells within the Glass multiple reporter (GMR) domain causing their elimination during pupal stages [37]. As expected, blocking apoptosis by p35 increased the abundance of mutant clonal cells to a similar extent in both genotypes (S5A Fig). Importantly, nearly four times more adults bearing atf3⁷⁶dlg1^G0342p35 mosaic EADs emerged compared to dlg1^G0342p35 animals (S5D Fig). Adult eyes derived from dlg1^G0342p35 EAD contained only small patches of differentiated photoreceptors compared to controls (S5E and S5F Fig), whereas atf3⁷⁶dlg1^G0342p35 eyes were either similar to those of atf3⁷⁶dlg1^G0342 animals (Fig 2D and S5G Fig) or exhibited modest differentiation defects (S5H Fig). Consistently, the overall morphology and differentiation pattern was markedly improved in adult eyes composed entirely of atf3⁷⁶dlg1^G0342 mutant tissue generated by the EGUF/hid method. In contrast to only remnants of differentiated tissue in dlg1^G0342 mutant eyes, clear ommatidial arrays were observed in control and atf3⁷⁶dlg1^G0342 adult eyes, (S5I–S5K Fig). Taken together, these data strongly argue against apoptosis being the basis of the genetic rescue conferred by atf3 deficiency in dlg1 mutant clones, and instead suggest that Atf3 contributes to cell extrusion and differentiation defects caused by loss of Dlg1.

To better characterize how loss of atf3 impacts dlg1 mutant phenotypes, we stained third instar larval EADs for markers of differentiation and cell adhesion. Unlike control clones, dlg1^G0342 mutant cells located posterior to the morphogenetic furrow of the eye primordium frequently lacked the pan-neuronal marker Elav. Many of them flattened and delaminated, pushing some of the non-clonal Elav-positive cells to the basal side of the epithelium (Fig 3A and 3B). In contrast, atf3⁷⁶dlg1^G0342 clones remained columnar, contributing to photoreceptor and interommatidial cell differentiation (Fig 3C). Importantly, immunostaining for the apical determinant Crumbs, the adherens junction protein DE-cadherin (DE-cad) and the lateral membrane marker Fasciclin III (FasIII) revealed a clear rescue of the apicobasal organization of the ommatidia in atf3⁷⁶dlg1^G0342 clones compared to the disturbed architecture in clones mutant for dlg1^G0342 alone (Fig 3A–3F). These findings uncover novel genetic interaction between Atf3 and the central Scrib polarity module component Dlg1 and establish Atf3 as a driver of the structural and differentiation defects stemming from the loss of dlg1.

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Fig 3. Loss of atf3 restores differentiation and polarity to dlg1 mutant clones.

(A-F) eyFLP-mediated mitotic recombination was used to generate clones (GFP) in the EADs of the indicated genotypes. (A-C) Immunostaining shows the stereotypical pattern of differentiated photoreceptors (Elav) and the lateral membrane component Fasciclin III (FasIII) in control discs (A’- A”). Patterns of these markers are disturbed in dlg1^G0342 clones (B’- B”), and restored in atf3⁷⁶dlg1^G0342 clones (C’- C”). Similar to control (A”), atf3⁷⁶dlg1^G0342 cells (C”) show columnar organization and maintain contact with the apical surface while dlg1^G0342 clones delaminate (B”). (D-F) The normal patterns of the adherens junction component DE-cadherin (DE-cad) (D’) and the apical determinant Crumbs (D”) are disturbed in dlg1^G0342 clones (E’-E”) but normalized in atf3⁷⁶dlg1^G0342 clones (F’-F”). Discs were counterstained with DAPI. White arrows indicate cross sections, which appear below the corresponding panels and are oriented apical side up. Clones are outlined with white dotted lines. Micrographs are otherwise single confocal slices. All images show EADs 7 days after egg laying. Scale bars: 20 μm (A-F).

https://doi.org/10.1371/journal.pgen.1007241.g003

Disturbances to the endosomal trafficking machinery in dlg1 mutant epithelia require Atf3

Besides altered differentiation and mislocalization of polarity proteins, disturbed cellular trafficking [29,38–40] is a hallmark feature of epithelial cells lacking the Scrib module components. Therefore, we tested if the distress of the trafficking machinery upon dlg1 loss was Atf3-dependent. Interestingly, neither dlg1^G0342 nor atf3⁷⁶dlg1^G0342 EAD clones enlarged by p35 expression showed changes in the uptake of fluorescently labeled dextran compared to surrounding tissue (S6A–S6C Fig), indicating that endocytic activity in cells lacking dlg1 was not affected. However, the staining of intracellular vesicle components revealed marked changes to the amount and distribution of early and recycling endosomes. While levels of the Rab5-positive vesicles were reduced in dlg1^G0342 and dlg1^G0342p35 mutant cells located in front or behind the morphogenetic furrow (Fig 4A’ and 4B’ and S7B and S7E Fig), the pool of Rab11 recycling endosomes increased (Fig 4A” and 4B”). In line with these findings, Dlg1-deficient cells also displayed an abnormal distribution of the Syntaxin7/Avalanche (Avl)-positive vesicles (S6E Fig). In contrast, the Rab5, Avl and Rab11 staining patterns in atf3⁷⁶dlg1^G0342 double mutant EAD clones were comparable to those seen in control clones or surrounding normal tissue (Fig 4C and 4D and S6D, S6F, S7A, S7C, S7D and S7F Figs). Strikingly, the changes to the endosomal compartment observed in dlg1 mutant tissue could be recapitulated by the Atf3 overexpression. While apical levels of Rab5 were decreased, Rab11-marked vesicles were enriched on the basal side of the wing imaginal discs in cells expressing Atf3 when compared to surrounding tissue (S8A and S8B Fig). In addition, immunostaining of wing discs bearing Atf3-expressing clones revealed changes in levels and localization of polarity proteins whose proper membrane placement requires a functional trafficking machinery [41,42]. While the apical levels of Crumbs were markedly lower, the integrin subunit Myospheroid (Mys), which normally is restricted to the basal cell surface, was detected along the entire lateral membrane in Atf3-expressing clones relative to surrounding tissue (S8C and S8D Fig). These data demonstrate that Atf3 contributes to the alterations of the endosomal machinery upon loss of polarity and its overexpression is sufficient to mimic some of the hallmark features of dlg1-deficient epithelial cells. Interestingly, despite the shift in apicobasal polarity markers Atf3-expressing cells, unlike those lacking dlg1, maintained their columnar shape and were not extruded from the epithelium (Fig 2L and 2R and S8 Fig).

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Fig 4. Impairment of Rab5 and Rab11 abundance and localization in dlg1 mutant clones is Atf3-dependent.

(A-D) eyFLP-mediated mitotic recombination combined with a co-expression of p35 was used to generate larger clones (GFP) in the EADs of the indicated genotypes. The levels and localization of early Rab5-positive endosomes are disturbed upon loss of dlg1 both posterior (A’) and anterior (B’) to the morphogenetic furrow, while recycling Rab11 vesicles are enriched in dlg1 mutant clones relative to surrounding tissue (A”,B”). Both Rab5 and Rab11 vesicle amounts and distribution are normalized in atf3⁷⁶dlg1^G0342 double mutant clones (C-D). On the cross sections, blue arrowheads indicate the regular Rab5 enrichment along the photoreceptors (A’,C’), yellow arrowheads indicate the apical localization of Rab5 in epithelial cells anterior of the MF (B’,D’). Note that both patterns are lost in dlg1 deficient cells. Discs were counterstained with DAPI. White arrows indicate cross sections, which appear below the corresponding panels and are oriented apical side up. Clones are outlined by white dotted lines. All images show EAD 7 days after egg laying. Scale bars: 10 μm (A,C), 20 μm (B,D).

https://doi.org/10.1371/journal.pgen.1007241.g004

Atf3 binds a 12-nucleotide motif in genes fundamental to cytoarchitecture

As a bZIP transcription factor, Atf3 is expected to regulate gene expression through binding to specific DNA sequences. To capture a snapshot of genomic regions bound by Atf3, we employed the Atf3::GFP fusion protein expressed from the recombineered atf3^gBAC to perform chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). The ChIP-seq using an anti-GFP antibody identified 152 genomic locations significantly enriched in samples prepared from atf3⁷⁶;; atf3^gBAC/+ mated adult males compared to yw controls (Fig 5A and S1 Dataset). Using the Multiple Em for Motif Elicitation (MEME) tool [43], we derived a 12-column position weight matrix (PWM) yielding a palindromic binding site which we call the Drosophila Atf3 motif (Fig 5B and S2 Dataset). This motif occurred in 112 of the 152 significantly enriched peaks in Atf3 samples, which correspond to 121 genes whose cis-regulatory regions contained an Atf3 motif (Fig 5A and S1 Dataset). Submission of this PWM to Tool for Motif to Motif comparison (TOMTOM) [44] determined that Atf3 recognizes DNA that is most similar to the Atf2 binding motif in Drosophila and ATF3/JDP2 motif in humans (Fig 5B). Closer examination of the Atf3-response elements revealed an 8-nucleotide core that closely resembles the cyclic AMP response element (CRE) (TGACGTCA) (Fig 5B). We have previously reported that the bZIP domain of the Drosophila Atf3 protein indeed directly binds this DNA element [5]. Our present experiments using the ATRE and mATRE reporters further confirm that Atf3 can control expression from this site in cultured cells and in vivo (Fig 1M’ and S2 Fig).

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Fig 5. Atf3 targets genes fundamental to epithelial cytoarchitecture.

(A) ChIP-seq identified 152 genomic positions bound by Atf3 in samples from atf3⁷⁶/Y;;atf3^gBAC/+ adult flies. (B) 112 genomic regions, proximal to 121 genes, contained the Drosophila Atf3 motif. The core of the Atf3 motif closely matches the CRE element but not the AP-1 consensus site. The Atf3 motif most closely resembles the Drosophila Atf2 motif (p = 5E-7) and human JDP2/ATF3 motif (p = 3E-9). (C) Of the 121 genes targeted by Atf3, 40 have roles in cytoskeleton organization and dynamics. (D) A schematic shows Atf3 targets (orange) belonging to the cytoskeleton GO cluster broken into subgroups and placed within representative spatial contexts in an epithelial cell.

https://doi.org/10.1371/journal.pgen.1007241.g005

The gene ontology (GO) analysis with FlyMine [45] revealed that structural and regulatory components of the cytoskeleton were significantly enriched among Atf3-bound regions. In total, 40 of the identified 121 genes containing Atf3 motifs belong to at least one out of five GO terms corresponding to cytoskeleton regulation (Fig 5A and 5C and S1 Dataset). Fig 5D depicts a summary of putative Atf3 targets engaged in their corresponding functions in a model epithelial cell. Importantly, an independent Atf3 ChIP experiment from third instar larval EADs determined that several of these targets including microtubule subunits (αTub84B and βTub56D) and nuclear Lamin C (LamC) were also bound by the Atf3 in imaginal epithelia (S9A and S9B Fig) suggesting a potential mechanistic basis for the Atf3 driven phenotypes in epithelial cells with compromised apicobasal axis.

Atf3 overexpression has a broad effect on gene transcription in imaginal disc epithelia

To complement the ChIP-seq approach and characterize the transcriptional response to excess Atf3, we compared the transcriptomes of mosaic EADs overexpressing Atf3 with control expressing only GFP. Strikingly, RNA-seq revealed a widespread shift in the expression of 4957 genes (S3 Dataset), with nearly three times as many transcripts being downregulated (n = 3666) than enriched (n = 1291) by fold change ≥1.5 relative to control (Fig 6A and S3 Dataset).

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Fig 6. Gain of Atf3 induces a broad transcriptional response in EAD epithelium.

(A) Transcriptome analysis identified a total of 4957 genes that were differentially regulated by a factor ≥1.5 in mosaic EADs expressing Atf3 (eyFLP^MARCM atf3^wt). Three times as many genes were transcriptionally repressed (n = 3666) than activated (n = 1291). (B-C) Tables list the Flybase ID, gene symbol (Symbol), and relative fold change in gene expression (Fold Change FRT82B atf3^wt/FRT82B) of the Atf3 targets identified by ChIP-seq that are part of the cytoskeleton GO cluster (B) and the 80 Atf3 ChIP-seq targets which do not belong to the cytoskeleton GO cluster (C). (D) List summarizing the regulation of all tubulin genes in mosaic EADs overexpressing Atf3 sorted in descending order of transcript abundance.

https://doi.org/10.1371/journal.pgen.1007241.g006

Of the 121 genes identified as Atf3 targets by ChIP-seq in the adults, 32 were differentially expressed at the mRNA level (Fig 6B and 6C) in atf3^wt mosaic EADs, most of which (23) showed transcriptional upregulation including six genes (Arp1, Chd64, DCTN6-p27, Ilk, LamC, Rep) that belonged to the cytoskeleton GO cluster (Fig 6B). In accordance with the mRNA-seq, an independent qRT-PCR of selected Atf3 ChIP-seq targets detected enrichment of LamC, ude, DCTN6-p27 and Arp1 transcripts while αTub84B and βTub56D remained unchanged (S9A Fig) despite being occupied by Atf3 in both the adults and EADs (S1 Dataset and S9B Fig). To identify additional putative Atf3 targets among genes misregulated in the RNA-seq dataset, we scanned the Drosophila genome with our experimentally derived Atf3 PWM using the Find Individual Motif Occurrences (FIMO) [46] and Peak Annotation and Visualization (PAVIS) [47] utilities. The Atf3 motif (Fig 5B) was found within 5 kb upstream and 1 kb downstream of 1252 genes differentially regulated in atf3^wt mosaic EADs (S3 Dataset). Importantly, a significant proportion of transcripts altered by Atf3 expression in the mosaic EADs overlapped with genes that had been found deregulated in either scrib or dlg1 mutant wing imaginal discs [10] (S10 Fig and S3 Dataset). This intersection shows that the gain of Atf3 and the loss of the Scrib polarity module elicited a partly overlapping genetic response.

Atf3 controls the microtubule network and Lamin C downstream of loss of polarity

The requirement of Atf3 for the manifestation of cytoarchitecture and polarity defects in dlg1 deficient cells prompted us to further focus on its regulation of microtubules and LamC, the primary building blocks of the cytoskeleton and nucleoskeleton, respectively. Microtubules are classically associated with controlling cell shape and division, but they are also central to polarity by serving as railroad tracks for directed vesicular trafficking [48]. Nuclear lamins on the other hand provide structure and stiffness to the nuclear envelope [49,50]. The coupling between the cytoskeleton and the nuclear lamina via the LINC complex is essential for maintaining the mechanical properties of the cell and signal transduction [51]. Immunostaining for LamC showed that the increase in LamC transcription in response to excess Atf3 (Fig 6B and S9A Fig and S3 Dataset) translated into a dramatic enrichment of the LamC protein in the nuclear envelope of EAD or wing disc cells overexpressing Atf3 (Fig 7A and 7D). Importantly, LamC protein levels also increased in the wing disc epithelium deficient for Scrib (en>scrib^RNAi) and in dlg1 mutant clones of the EAD (Fig 7B and 7E), whereas they were markedly reduced in atf3⁷⁶dlg1^G0342 double mutant EAD clones compared to the surrounding tissue and control clones (Fig 7C and 7F). In contrast to LamC enrichment, expression of at least two microtubule genes (βTub85D and γTub37C) was downregulated in atf3^wt mosaic EADs (Fig 6D, S3 Dataset). Interestingly, immunostaining for α- and β-Tubulin revealed a reduced microtubule network and markedly lowered levels of non-centrosomal γ-Tubulin in Atf3-overexpressing clones of the wing imaginal disc relative to surrounding epithelial tissue (Fig 8A–8C). In addition, eye primordia bearing dlg1 mutant clones showed abnormal microtubule organization that was partially normalized by simultaneous clonal loss of atf3 (Fig 8D–8F). Taken together, our molecular and genetic approaches identified microtubule encoding genes and LamC as new targets of Atf3. We demonstrate that the upregulation of LamC and disturbances to microtubule network are the common hallmarks of epithelial cells in which Atf3 activity is enhanced either by transgenic overexpression or as a result of disturbed polarity. While LamC is a bona fide target of Atf3, changes to microtubule network likely arise indirectly as a part of the secondary response to ectopic Atf3 activity.

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Fig 7. LamC is a bona fide target of Atf3.

(A-B) Both Atf3 overexpression (en>atf3^wt) and Scrib knockdown (en>scrib^RNAi) increased LamC levels in the posterior compartment of the wing imaginal disc (mCherry) (A’,B’). (C-D) eyFLP-mediated clonal overexpression of Atf3 (GFP) markedly upregulated the LamC protein (D’) compared to levels detected in surrounding tissue and control clones (C’). (E-F) LamC was also enriched in dlg1^G0342 clones (E’), but was reduced below control levels in atf3⁷⁶dlg1^G0342 cells (F’). Discs were counterstained with DAPI. Images represent single slices from confocal stacks. The wing disc posterior compartments (A-B) and EAD clones (C-F) are outlined with white dotted lines. On images C’-F’, the arrows indicate cross sections shown below the corresponding images, with the apical side facing up. Scale bars: 50 μm (A-B), 10 μm (C-F).

https://doi.org/10.1371/journal.pgen.1007241.g007

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Fig 8. Increased Atf3 levels disturb the microtubule network.

(A-B) Columnar cells in the wing disc expressing Atf3 (hsFLPout>>atf3^wt) (GFP) contained less apical, subcortical α- and β-Tubulin compared to surrounding tissue (A’,B’) and non-clonal cells of the peripodial epithelium (P) directly above (A’). (C) Wing disc clones expressing Atf3 display lower levels of non-centrosomal γ-Tubulin (C’). (D-F) eyFLP-mediated mitotic recombination was used to generate clones (GFP) in the EADs of the indicated genotypes. Clones lacking dlg1 (E) show disturbed α-Tubulin localization (E’) compared to control clones (D-D’) in the photoreceptor region of the EADs. Clones lacking both dlg1 and atf3 (F) show normalized microtubule distribution along the apicobasal axis (F’). Discs were counterstained with DAPI. The arrows indicate cross sections shown below the corresponding images, with the apical side facing up. Clones are outlined with white dotted lines. Scale bars: 10 μm (A-F).

https://doi.org/10.1371/journal.pgen.1007241.g008

Outlook

A growing body of evidence points to striking similarities in the cellular and molecular events underlying loss of polarity and wounding [10,66]. In this context, the induction of Atf3 upon loss of polarity is in line with the early discovery of ATF3 as a gene rapidly induced in the regenerating rat liver [67] and recent studies showing Atf3 induction during epithelial wounding [14,15]. Here we demonstrate that Atf3 levels remain low as long as epithelial polarity is intact, whereas loss of polarity due to deficiency in tumor suppressors of the Scrib complex triggers Atf3 expression. Future investigations into the link between Drosophila Atf3 and cell polarity are likely to unravel the impact of ATF3 expression on epithelial homeostasis and on human pathologies arising from polarity breakdown.

Flies

The following fly strains were used: (a) y w¹¹¹⁸,(b) w¹¹¹⁸,(c) y atf3⁷⁶ w/FM7i, P{ActGFP}JMR3 [5], (d) atf3^gBAC [21], (e) en-GAL4 (RRID: BDSC_30564), (f) dpp-GAL4 (RRID: BDSC_7007), (g) UAS-atf3^V (this study as atf3^wt) [5], (h) hsFLP; act>y⁺>GAL4, UAS-GFP, (i) hsFLP; act>y⁺>GAL4, UAS-GFP, UAS-atf3^wt (this study), (j) FRT19A (RRID: BDSC_1744), (k) FRT82B (RRID: BDSC_5619), (l) UAS-p35/CyO (RRID: BDSC_5072), (m) GMR-hid y w FRT19; ey-Gal4, UAS-FLP (RRID: BDSC_5248), (n) FRT19A dlg1^G0342 (111872, DGRC), (o) dlg1^m52 (a generous gift from F. Papagiannouli), (p) FRT82B scrib¹ [25], (q) eyFLP FRT19A tubGAL80; act>y⁺>GAL4, UAS-GFP (this study), (r) eyFLP FRT19A, tubGAL80; act>y⁺>GAL4, UAS-myrRFP (this study), (s) eyFLP; act>y⁺>GAL4, UAS-GFP; FRT82B, tubGAL80 [68], (t) UAS-dlg1^RNAi (41134, VDRC), (u) UAS-scrib^RNAi (105412, VDRC), (v) TRE-DsRed (attP16) [26], (w) UAS-hep^wt, (x) UAS-yki^{S111A.S168A.S250A.V5} (RRID: BDSC_28817, this study as yki^act), (y) UAS-mCD8-ChRFP (RRID:BDSC_27391, this study as UAS-mCherry), (z) UAS-aPKC^CAAX [69], (aa) UAS-LamC [70], (bb) UAS-bsk^DN. A transgenic ATRE-GFP line was obtained using the attP16 landing site [71]. The atf3^gBAC fly line was used for immunostaining of Atf3::GFP. For detailed descriptions of fly crosses, see S2 Table. All crosses were carried out at 25°C unless stated otherwise.

Quantitative reverse transcription-PCR (qRT-PCR)

For each biological replicate, total RNA was isolated from 10 larvae or 15 adult heads with Isol-RNA Lysis Reagent (5 Prime). After DNase I treatment (Ambion, Foster City, CA), cDNA was synthesized from 1μg of RNA using oligo(dT) primers and Superscript III (Life Technologies). PCR was performed in triplicate with BioRad 2x SYBR Green mix in the CFX96 real-time PCR system (Bio-Rad, Hercules, CA). All primers were designed to anneal at 62°C (see S1 Table for oligonucleotide sequences). All data were normalized to rp49 transcript levels, and fold changes in gene expression were calculated using the ∆∆CT method [72].

Genetic mosaic analysis

Clones expressing atf3 in the wing imaginal disc were generated using a hsFLPout method described in [5]. Crosses were kept at 22°C. Four days after egg lay, progeny were heat shocked for 30 min in a 37°C water bath. Imaginal discs were dissected from wandering third instar larvae. Generation of mosaics in EADs using Mosaic Analysis with a Repressible Cell Marker method (MARCM) [73] was carried out as described in [31].

ChIP-seq

For each genotype (see S2 Table), three biological ChIP replicates and one input replicate were generated. For each ChIP replicate, 1.2 g of adult males were collected and processed using a modified version of the protocol described in [74]. Anesthetized males were split into two batches of 600 mg and flash frozen using liquid nitrogen, pulverized with a mortar and pestle, and disrupted further via 20 strokes of a loose fitting pestle in a Dounce homogenizer containing 10 ml of Crosslinking Solution (1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 50 mM HEPES, pH 8.0) supplemented with proteases inhibitors (11873580001, Roche). The remainder of the protocol describes the processing of a single batch. The suspension was centrifuged for 1 min at 400 g in a swing bucket rotor and then passed through two layers of 64 μm Nitex membrane into a 15 ml Falcon tube. To crosslink Atf3 to chromatin, formaldehyde was added to a final concentration of 1.8% to the Falcon tube containing 10 ml of cleared fly homogenate. The Falcon tube was then incubated on an orbital shaker at room temperature for 10 min. Crosslinking was stopped by adding glycine to a final concentration of 225 mM and shaking for a further 5 min. After centrifugation for 10 min at 1,100 g in a swing bucket rotor, cells were resuspended in 10 ml of Cell Lysis Buffer (85 mM KCl, 0.5% IGEPAL CA-630 (v/v), 5 mM HEPES, pH 8.0) supplemented with protease inhibitors and lysed in a Dounce homogenizer via twenty strokes with a tight fitting pestle. Nuclei were pelleted at 2000 g for 4 min and washed in 5 ml of Cell Lysis Buffer three times. Nuclei were resuspended in 2 ml of TBS Lysis Buffer (50 mM Tris-Cl pH 7.8, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton-X100, 0.01% IGEPAL CA-630) supplemented with protease inhibitors and incubated for 10 min on an orbital shaker. While submerged in an ice water bath, chromatin was sheared to 300–500 bp fragments by applying fifty 30 seconds on/30 seconds off cycles of a Branson 250 microtip sonifier with Output set to 3.5 and Duty Cycle set to 50%. The 2 ml of chromatin was split equally into two 1.5 ml low binding microcentrifuge tubes (710176, Biozym). Debris was cleared from the chromatin by centrifugation at 20,000 g for 10 min. Chromatin was cleared overnight in two tubes at 4°C by the addition of 40 μl of sepharose-IgG beads (17-0969-01,GE) equilibrated in TBS Lysis Buffer. 100 μl of cleared chromatin was retained as an INPUT sample for sequencing. Remaining cleared chromatin was transferred to two new low binding tubes and precipitated overnight at 4°C with 40 μl GFP Trap beads (gta-20, Chromotek) per tube. Beads were washed 5 times with cold TBS Lysis Buffer supplemented with protease inhibitors. From this point on, the beads and INPUT sample retained earlier were processed in the same fashion. Samples were resuspended in 100 μl TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) supplemented with 50 μg/ml RNase A, transferred to PCR tubes, and incubated in a thermal cycler with heated lid at 37°C for 30 min. SDS was added to a final concentration of 0.5% (w/v) from a 10% stock, proteinase K to a final concentration of 0.5 mg/ml. Tubes were then incubated at 37°C for 12 hours followed by 65°C for 6 h in a thermal cycler with heated lid to partially reverse the crosslinks. H₂O was added to a final volume of 200 μl, and DNA was purified by phenol:chloroform extraction. DNA was ethanol precipitated in a low binding 1.5 ml microcentrifuge tube with sodium acetate and glycogen. DNA was washed once with 70% ethanol. DNA pellets derived from the original 1.2 g of starting adult fly material (4 microcentrifuge tubes in the end) were resuspended in 15 μl of TE.

Sequencing libraries were generated from input and ChIP samples according to the Illumina protocol for total ChIP-seq library preparation. All samples were loaded onto a single Illumina flow cell (Illumina, San Diego, CA). Using the Illumina HiSeq 2000 instrument, 50 bp of sequence were read from one DNA end. Image analysis and base calling were done with the Illumina RTA software (RRID: SCR_014332) at run time. Sequences were mapped to the Drosophila genome assembly (version BDGP R5/dm3, April 2006) and delivered as BAM files.

DNA motif analyses

To identify regions bound by Atf3, BEDTools [75] (RRID: SCR_006646) was used to convert BAM files from Atf3 and control samples (3 biological ChIP replicates and one input sample each) into BED files, which were subsequently submitted to a Comparative Profile Analyzer (CoPrA) workflow to call and test the statistical significance of differentially enriched genomic locations in Atf3 ChIP-seq samples relative to control. The differential analysis depends on base-resolution coverage profiles of the Atf3 and control ChIP samples as well as the input data from both samples. They were generated from uniquely mapped reads that were extended to the original fragment length of 200 bp resulting in 6 ChIP and 2 input coverage profiles. All profiles are normalized through division of the base-wise coverage values by the total number of respective sample reads multiplied by one Million. Thereby normalized input profiles from Atf3 and control samples are subtracted from normalized Atf3 and control ChIP-profile replicates, respectively. Every processed ChIP profile replicate is then scaled separately to transform its values into the range [0,1]. They were then discretized by calculating the coverage mean of a defined sliding window. Since transcription factor binding peaks cover a small region on the genome, its size was adjusted to 50 bp. The shift is set by default to half of the window size. In the next step the discretized replicate profiles of the same sample type are combined to one resulting discretized profile by taking the mean of the window values at the same genomic position respectively. The thus processed sample profiles of Atf3 and control are finally utilized to generate a difference profile by taking the Euclidean difference between Atf3 and control values at the same genomic position. The obtained values of the difference profile are normally distributed and are located between -1 and 1 with a mean very close to 0. To filter out non-informative difference regions the profile is cleaned by removing all values within the range of 4 times the standard deviation of the mean. Remaining difference regions which are adjacent to each other and that possess the same sign are merged. They either show enrichment for Atf3 or control. They are size filtered by taking the discretization window size as a minimal threshold. The resulting difference regions are finally tested for significance by a two-sided Kolmogorov-Smirnov test of the discretized region values of Atf3 and control and multiple testing corrected by Benjamini-Hochberg. We accepted differential regions with a q-value < 0.05 that are enriched in the Atf3 sample and extracted their DNA sequences with BEDTools. Peaks were assigned to genes when located within a gene or 1 kb upstream of the transcriptional start site. Multiple genes were manually associated with a peak in the case of nested genes or dense gene regions. Enriched regions mapping to positions within the CH321-51N24 BAC (source of the atf3^gBAC) were discarded (n = 59), except for two peaks corresponding to the genes atf3 and CG11403, which contained the Drosophila Atf3 motif.

Sequences of regions bound by Atf3 were uploaded to MEME (http://meme-suite.org/) for motif identification [43]. The positional weight matrix returned by MEME was submitted to TOMTOM (http://meme-suite.org/tools/tomtom) for interspecies motif comparison [44]. FlyBase gene ontology (GO) terms identified with FlyMine (http://www.flymine.org, RRID: SCR_002694) were used for functional annotation [45].

ChIP from eye/antennal imaginal discs

A custom protocol combining the methods described in [74,76] was used to perform ChIP against Atf3 in the EADs. For each replicate, 100 EADs with mouth hooks attached were dissected from third instar larvae in ice-cold PBS. Discs were fixed at room temperature by gently mixing in 1 ml cross-linking solution (1.8% formaldehyde, 50 mM Hepes pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl), which was changed 3–4 times during fixation. Fixation was stopped by washing for 3 min in 1 ml PBS/0.01% Triton X-100/125 mM glycine 3–4 times. Fixed discs were washed for 10 min in 1 ml wash A (10 mM Hepes pH 7.6, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and subsequently for 10 min in 1 ml wash B (10 mM Hepes pH 7.6, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100), with the wash solutions being changed 3–4 times over the 10 min. Eye discs were separated from the brain and mouth hooks and transferred into 550 μl of TBS Lysis Buffer (see ChIP-seq protocol). Chromatin was sheared using an Active Motif EpiShear sonicator (Active Motif, 53052) equipped with a 1.5 ml EpiShear Cooled Sonication Platform (Active Motif, 53080) and 1.5 ml Benchtop Cooler (Active Motif, 53076). With Amplitude set to 50%, sonication was performed in cycles of 20 seconds on/30 seconds off for 20 minutes. Chromatin was centrifuged at 14,000 RPM for 10 min to clear debris, transferred into clean 1.5 ml tubes, and cleared overnight at 4°C with 20 μl sepharose-IgG beads equilibrated in TBS Lysis Buffer. Cleared chromatin was transferred to new 1.5 ml tubes and precipitated overnight at 4°C with 25 μl GFP Trap beads per tube. Beads were washed the indicated number of times with 1 ml of each of the following buffers at 4°C on a rotating wheel for 10 min: 1 × TBS Lysis Buffer, 4 × TBS 500 Lysis Buffer (50 mM Tris-Cl pH 7.8, 500 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton-X100, 0.01% IGEPAL CA-630), 1 × LiCl buffer (250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630 (v/v), 10 mM Tris-HCl, pH 8.0). Beads were transferred into clean 1.5 ml tubes and washed two times with TE for 10 min at 4°C on a rotating wheel. Beads were subsequently transferred into PCR tubes. Chromatin was decrosslinked and precipitated as described in the ChIP-seq protocol. Purified DNA was raised in 500 μl water.

qPCR was performed in triplicate with BioRad 2x SYBR Green mix in the CFX96 real-time PCR system (Bio-Rad, Hercules, CA). All primers were designed to anneal at 62°C (see S1 Table 1 for oligonucleotide sequences). Data were normalized to amplification values for ecd. Fold enrichment was calculated using the ∆∆CT method [72].

RNA-seq

RNA was isolated from FRT82B atf3^wt mosaic EADs of third instar larvae as described [77]. Total RNA libraries were generated according to the Illumina protocol and single-end sequenced on an Illumina NextSeq 500 instrument at 75 bp read length. Image analysis and base calling were done with the Illumina RTA software at run time. Published sequence data from FRT82B mosaic EAD samples [11] were used as control. Data were processed using a high-throughput Next-Generation Sequencing analysis pipeline [78]. Basic read quality check was performed with FastQC (v0.10.1) (RRID: SCR_014583) and read statistics were acquired with SAMtools v0.1.19 (RRID: SCR_002105) [79]. Reads were mapped to the Drosophila reference assembly (version BDGP R5/dm3, April 2006) using Tophat v2.0.10 (RRID: SCR_013035) [80], and gene quantification was carried out using a combination of Cufflinks v2.1.1 (RRID: SCR_014597) [81], and the DESeq2 package v1.10.1 (RRID: SCR_000154) [82], with genomic annotation from the Ensembl database (RRID: SCR_002344), version 84. In all samples, the number of total reads exceeded 50 million, from which an average 83.6 percent could be mapped, and on average 97.5% of these mapped reads fulfilled the MAPQ≥30 criterion. The results were uploaded into an in-house MySQL database and joined with BiomaRt (RRID: SCR_002987) v2.26.1 [83] annotations from Ensembl, version 84. Lists of differentially expressed genes were defined by a final database export using 5 and 0.01 as cutoffs for DESeq2-based FCs and p-values, respectively. To identify genes differentially expressed under the respective conditions, the average of at least three biological replicates was calculated. The S3 Dataset shows all transcripts whose expression differed ≥ 1.5-fold in FRT82B atf3^wt compared to FRT82B control.

Atf3 motif search

The experimentally derived Drosophila Atf3 PWM was submitted to the online FIMO utility (http://meme-suite.org/tools/fimo) (RRID: SCR_001783) to identify Atf3 motifs in Drosophila melanogaster genome (UCSC, dm3), with a p-value threshold set at 0.0001. FIMO results, in the form of a BED file, were subsequently submitted to the online PAVIS tool (https://manticore.niehs.nih.gov/pavis2), using default settings.

Plasmid constructs

To create the Atf3 expression reporter, oligonucleotides containing four intact or mutated Atf3 sites (see S1 Table for sequences) were cloned via Mlu1 and Not1 sites into the pRedRabbit and pGreenRabbit vectors [84]. To express N-terminally tagged GFP- or FLAG-Atf3 proteins from the UAST promoter, atf3 cDNA (see S1 Table for oligonucleotide sequences) was cloned into pENTR4 and subsequently recombined using LR Clonase II (11791–020, Life Technologies) into pTGW and pTFW vectors, respectively (T. Murphy, Drosophila Genomic Resource Center).

S2 cell culture

Schneider 2 (S2) cells were cultured at 25°C in Shields and Sang M3 insect medium (S8398-1L, Sigma-Aldrich) containing 8% fetal bovine serum (Gibco, Life Technologies) without antibiotics. Cells were transfected using X-tremeGENE (Roche Applied Science). Expression of UAS-driven genes was induced by co-transfection with a pWA-GAL4 plasmid expressing GAL4 under an actin5C promoter. Cells were fed 24 hours after transfection. Cells were imaged or lysed 72 hours after transfection.

Tissue staining

Tissues from third instar larvae were processed as described previously [5]. The following primary and secondary antibodies were used at the indicated dilutions: anti-Dlg1 (RRID: AB_528203; 1:200), anti-Fasciclin III (RRID: AB_528238; 1:200), anti-Elav (RRID: AB_528217 and AB_528217; 1:200), anti-DE-cad (RRID: AB_528120; 1:200), anti-Crumbs (RRID: AB_528181; 1:200), anti-p120-catenin (RRID: AB_2088073; 1:200), anti-α-Tubulin (RRID: AB_579793; 1:200), anti-β-Tubulin (RRID: AB_2315513; 1:200), anti-LaminC (RRID: AB_528339; 1:500) and anti-βPS integrin (RRID: AB_528310; 1:200) from the Developmental Studies Hybridoma Bank (DSHB) (Iowa City, Iowa), anti-γ-Tubulin (T6557, Sigma Aldrich; 1:50), anti-GFP (G10362, Invitrogen; 1:500), anti-Rab5 (ab31261, Abcam; 1:200), anti-Rab11 (BD610656, BD Biosciences; 1:200), chicken-anti-Avl (a gift from D. Bilder, 1:500) and Cy2, Cy3- and Cy5-conjugated secondary antibodies (Jackson Immunoresearch). Tissues were counterstained with DAPI (1 μg/ml) and mounted in DABCO-Mowiol medium (Sigma-Aldrich).

Dextran uptake assay

Third instar imaginal discs were incubated in M3 media containing 0.5 mM MW3000 Texas Red dextran (D3328; ThermoFisher Scientifc) for 15 minutes (pulse) at 25°C. Discs were briefly washed 2 times in M3 media followed by incubation in M3 media for 60 minutes (chase). Discs were subsequently fixed in 1% formaldehyde in PBS for 20 min, counterstained with DAPI, and mounted in DABCO-Mowiol medium (Sigma-Aldrich).

Image acquisition and processing

S2 cells were imaged using a DP72 camera mounted on an Olympus CK4X41 miscroscope, in conjunction with cellSens 1.1 Software (RRID: SCR_014551). Confocal images were acquired at room temperature using an Olympus FV1000 confocal microscope equipped with 20x UPlan S-Apo (NA 0.85), 40x UPlan FL (NA 1.30) and 60x UPlanApo (NA 1.35) objectives. Transversal sections were generated using Imaris 7.0.0 (Bitplane) (RRID: SCR_007370). Figure assembly and image brightness and contrast adjustments were done in Photoshop CS5.1 (Adobe Systems, Inc.) (RRID: SCR_014199). Z-stacks of adult eyes were taken using a motorized Leica M165 FC fluorescent stereomicroscope equipped with the DFC490 CCD camera. Images were processed using the Multifocus module of LAS 3.7.0 software (Leica).

Flow cytometry of eye/antenna imaginal discs

Third instar larvae were collected and washed two times with PBS. EADs were dissected in PBS (on average sixty EADs /replicate/genotype) and transferred to 1.5 ml low binding microcentrifuge tubes (no more than one hour before dissociation). PBS was removed from EADs and was replaced with 100 μl dissociation solution containing 1 mg/ml collagenase I (Sigma, C2674), and 1 mg/ml papain (Sigma P4762). Samples were incubated for 60 min at room temperature and gently swirled every 15 min. After the dissociation solution was removed, discs were carefully rinsed with 500 μl PBS, which was then replaced with 100 μl of PBS. The final dissociation was performed by passing the discs through a 27G insulin syringe (Terumo) five times. Additional 200 μl of PBS was added for a final volume of 300 μl (5 μl PBS per disc). Following sample filtration through a Filcone filter, propidium iodide was added to measure cell viability and samples were stored on ice until sorting. For each sample (n≥3 per genotype), 30,000 events were counted on a BD LSRFortessa Cell Analyzer in combination with BD FACSDiva software v8.0 (both BD Bioscience) using gates set to distinguish GFP+, GFP-, and PI- cells.

Scanning electron microscopy

Adult heads were fixed in 80% ethanol, postfixed with 1% osmium tetroxide, dehydrated in ethanol, critical point dried, gold coated, and observed under a JEOL JSM-7401F (Tokyo, Japan) 6300 scanning electron microscope.

Immunoblotting

S2 cells were lysed in 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.01% Igepal, and protease inhibitors (Roche Applied Science). Protein concentration was quantified using Pierce 660 reagent (22660, Thermo Scientific) according to manufacturer’s instructions. Following SDS-PAGE, proteins were detected by immunoblotting with anti-Flag M2 (1:1000, Sigma Aldrich), anti-GFP (1:5000, TP401, Acris) and anti-α-Spectrin (1:1000, RRID: AB_528473) antibodies, followed by incubation with corresponding HRP-conjugated secondary antibodies (Jackson Immuno Research). Chemiluminescent signal was captured using ImageQuant LAS4000 reader (GE Healthcare).

Sample size criteria

For sample size criteria, post hoc analysis of results presented in Figs 1, S1, S3, S5 and S9 using G*Power 3.1 (RRID: SCR_013726) [85] determined that the statistical power of all statistically significant differences (1-β)exceeded 0.94, given sample size, mean, and standard deviation for each condition. Raw data are available in S4 Dataset.

Accession numbers

All raw next generation sequencing data from FRT82B and FRT82B atf3^wt mosaic EAD samples and the Atf3 ChIP-seq data are available from the Gene Expression Omnibus (GEO) database (RRID: SCR_007303) under accession numbers GSE65261, GSE89845, GSE71206.