InteractiveFly: GeneBrief

Activating transcription factor 3: Biological Overview | References


Gene name - Activating transcription factor 3

Synonyms - ATF3, A3-3

Cytological map position - 1E5-1F1

Function - transcription factor

Keywords - epithelial cell replacement during abdominal morphogenesis, functional partner of Jun during development

Symbol - Atf3

FlyBase ID: FBgn0028550

Genetic map position - X:1,136,773..1,168,682 [-]

Classification - bZIP transcription factor

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Bhat, S. and Jones, W. D. (2016). An accelerated miRNA-based screen implicates Atf-3 in Drosophila odorant receptor expression. Sci Rep 6: 20109. PubMed ID: 26848073
Summary:
The Drosophila olfactory system is highly stereotyped in form and function; olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) always appear in the same antennal location and the axons of OSNs expressing the same OR converge on the same antennal lobe glomeruli. Although some transcription factors have been implicated in a combinatorial code specifying OR expression and OSN identity, it is clear other players remain unidentified. In hopes of mitigating the challenges of genome-wide screening, this study examined the feasibility of a two-tiered approach comprising a primary "pooling" screen for miRNAs whose tissue-specific over-expression causes a phenotype of interest followed by a focused secondary screen using gene-specific RNAi. Since miRNAs down-regulate their targets, miRNA over-expression phenotypes should be attributable to target loss-of-function. It is the sequence-dependence of miRNA-target pairing that suggests candidates for the secondary screen. Since miRNAs are short, however, miRNA misexpression will likely uncover non-biological miRNA-target relationships. Rather than focusing on miRNA function itself where these non-biological relationships could be misleading, it is proposed using miRNAs as tools to focus a more traditional RNAi-based screen. This study describes such a screen that uncovers a role for Atf3 in the expression of the odorant receptor Or47b.
Zhou, J., Edgar, B. A. and Boutros, M. (2017). ATF3 acts as a rheostat to control JNK signalling during intestinal regeneration. Nat Commun 8: 14289. PubMed ID: 28272390
Summary:
Epithelial barrier function is maintained by coordination of cell proliferation and cell loss, whereas barrier dysfunction can lead to disease and organismal death. JNK signalling is a conserved stress signalling pathway activated by bacterial infection and tissue damage, often leading to apoptotic cell death and compensatory cell proliferation. This study shows that the stress inducible transcription factor ATF3 restricts JNK activity in the Drosophila midgut. ATF3 regulates JNK-dependent apoptosis and regeneration through the transcriptional regulation of the JNK antagonist, Raw. Enterocyte-specific ATF3 inactivation increases JNK activity and sensitivity to infection, a phenotype that can be rescued by Raw overexpression or JNK suppression. ATF3 depletion enhances intestinal regeneration triggered by infection, but does not compensate for the loss of enterocytes and ATF3-depleted flies succumb to infection due to intestinal barrier dysfunction. In sum, this study has provided a mechanism to explain how an ATF3-Raw module controls JNK signalling to maintain normal intestinal barrier function during acute infection.
BIOLOGICAL OVERVIEW

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. This study implicates Drosophila Activating transcription factor 3 (Atf3 or A3-3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. These data thus identify Atf3 as a new functional partner of Drosophila Jun during development (Sekyrova, 2010).

Metamorphosis of Drosophila larvae into pupae and adult flies provides remarkable examples of morphogenetic changes that involve replacement of entire cell populations. Epithelia that had served larval function undergo programmed cell death while imaginal cells proliferate and differentiate to take their position. The Drosophila abdomen is an attractive system for studying the developmental replacement of one epithelial cell population with another. Unlike the adult head and thorax with appendages, all forming from pre-patterned imaginal discs, the adult abdomen derives from histoblasts that reside in each abdominal segment. Soon after the onset of metamorphosis, the diploid histoblasts undergo an initial phase of synchronized cell divisions; later the histoblasts expand while proliferating and replace the old polyploid larval epidermal cells (LECs) that cover the surface of the abdomen (Ninov, 2007; Bischoff, 2009). To free space for the histoblasts, LECs are extruded from the epithelial monolayer. In order to maintain integrity of the epithelia, changes in cell adhesion and cell migration must be precisely orchestrated during this tissue remodeling (Sekyrova, 2010).

Rho kinase signaling, which stimulates constriction of the apical actomyosin cytoskeleton through myosin phosphorylation, is necessary for the extrusion and the ensuing apoptosis of LECs (Ninov, 2007). Perturbed myosin phosphorylation leaves the process of the epithelial exchange incomplete, with residual LECs obstructing closure of the adult abdominal epidermis at the dorsal midline. A similar defect results from compromised function of the ecdysone receptor (EcR), which is required for both the initial phase of histoblast proliferation (Ninov, 2009) and for the removal of LECs (Ninov, 2007). Other factors besides Rho signaling and EcR that regulate the epithelial cell replacement are unknown (Sekyrova, 2010).

This study implicates Atf3 (A3-3 -- FlyBase), the single Drosophila ortholog of the vertebrate Activating transcription factor 3 (ATF3) and Jun dimerization protein 2 (JDP2) in abdominal development. ATF3 and JDP2 belong among basic region-leucine zipper (bZIP) proteins, some of which play important roles in epithelial morphogenesis. Particularly the functions of Jun and Fos bZIP proteins in epithelial closure events during development are well understood owing to genetic studies in Drosophila. By contrast, no morphogenetic function has yet been reported for Atf3 in Drosophila (Sekyrova, 2010).

Mammalian ATF3 and JDP2 form homodimers but preferentially dimerize with members of the Jun subfamily (Aronheim, 1997; Hai, 1989; Hsu, 1991), functioning either as transcriptional activators (ATF3-Jun) or repressors (JDP2-Jun). Based mainly on cell-culture studies, multiple roles in cell proliferation, differentiation and apoptosis have been ascribed to ATF3 and JDP2. Atf3-/- mice are viable (Hartman, 2004) but suffer from altered glucose and immune homeostasis (Gilchrist, 2006; Qi, 2009; Rosenberger, 2008). Also Jdp2-/- mice survive but produce extra fat in their brown adipose tissue (Nakade, 2007). In vivo significance of the interaction between the ATF3 or JDP2 proteins and Jun remains unclear (Sekyrova, 2010).

This study shows that Atf3 interacts biochemically and genetically with Jun in Drosophila. Temporal downregulation of atf3 transcription during metamorphosis is crucial, since sustained atf3 expression alters adhesive properties of LECs, thus preventing their extrusion and replacement by the adult epidermis. This effect of Atf3 requires the presence of Jun (Sekyrova, 2010).

Among Drosophila bZIP proteins, the predicted product of the CG11405 gene (also referred to as a3-3), located on the X chromosome, shows the closest similarity to the mammalian ATF3 and JDP2 proteins. The DNA-binding/dimerization bZIP domains of the human ATF3 and Drosophila Atf3 proteins are identical in 60% of their amino acids; there is 58% identity between Atf3 and JDP2 in this region (Sekyrova, 2010).

Dimerization between Atf3 and Jun in Drosophila has been theoretically predicted (Fassler, 2002) and confirmed by a yeast two-hybrid screen. To demonstrate direct binding, co-immunoprecipitation experiments were conducted. The endogenous Jun protein from Drosophila S2 cells co-precipitates with a transiently expressed Atf3 whereas Fos did not. A DNA mobility-shift assay with recombinant bZIP domains of Atf3, Jun and Fos was conducted to test for their DNA-binding properties. Atf3 specifically bound an ATF/CRE consensus element but not the AP-1 site, which was recognized by the Jun-Fos (AP-1) complex. Although Atf3 bound DNA by itself, presumably as a homodimer, the binding was enhanced in the presence of Jun. Fos did not synergize with Atf3 in DNA binding. Excess unlabeled DNA bearing the ATF/CRE binding site competed for the Atf3 bandshift activity whereas the AP-1 binding element did not. These results have shown that, like ATF3 or JDP2 in mammals, Atf3 in Drosophila selectively dimerizes with Jun, with which it cooperatively and specifically binds the ATF/CRE DNA element (Sekyrova, 2010).

To test whether Atf3 and Jun interact in vivo, experiments were conducted in the Drosophila compound eye, the precise structure of which sensitively reflects genetic interactions. Overexpression of atf3 under the GMR-Gal4 driver disrupted the ommatidial arrangement, resulting in smaller eyes with a glossy appearance. This atf3 misexpression phenotype could be completely suppressed by simultaneous RNAi-mediated knockdown of jun but not of fos. Conversely, the phenotype was exacerbated when jun was overexpressed in the eye together with atf3, suggesting that it is Atf3 in a complex with Jun that derails the normal eye development. Neither RNAi nor overexpression of jun alone had any effect on eye morphology. Interestingly, like depletion of Jun, co-expression of fos under the GMR-Gal4 driver completely averted the atf3 misexpression phenotype, restoring the normal appearance of the eye. Expression of fos or its mutant versions alone had no effect. These data can be explained by the ability of the surplus Fos to bind Jun and thus reduce its availability for interaction with the Atf3 protein. This interpretation is further supported by experiments showing that expression of the truncated bZIP domain of Fos is sufficient to suppress the Atf3 gain-of-function phenotype, whereas its transcription activation domain or phosphorylation sites are dispensable (Sekyrova, 2010).

Taken together, the results show that Atf3 cooperates with Jun, as Jun is specifically required for an effect caused by overexpression of Atf3 in the developing eye. Given the capacity of both Atf3 and Fos to bind Jun, and based on the ability of Jun to enhance and of Fos to suppress the Atf3 gain-of-function phenotype, it is suggested that Atf3 and Fos compete for their common partner Jun in vivo (Sekyrova, 2010).

To find out whether Atf3 is required for Drosophila development and whether its absence might resemble a phenotype caused by loss of its partner Jun, atf3 mutant flies were generated. The longest deletion (line atf376) obtained by imprecise excision of a P element, removed the entire bZIP domain of Atf3, and atf376 hemizygous (male) larvae lacked detectable atf3 mRNA. Thus, atf376 probably represents a null allele. Most atf376 larvae die soon after hatching and during all three larval stages. Only a few (approximately 2%) reach the third instar but die before metamorphosis as defective pseudopuparia. Expression of atf3 cDNA under the ubiquitous armadillo (arm-Gal4) driver rescued some atf376 hemizygotes to adults, confirming that loss of atf3 was the cause of the lethal phenotype. Interestingly, the moribund atf376 larvae abnormally enlarged lipid droplets in their fat body, thus displaying a phenotype reminiscent of that in mice lacking one of the Atf3 orthologs, JDP2 (Nakade, 2007). However, in contrast to viable Jdp2 or Atf3 (Hartman, 2004) knockout mice, atf3 is an essential gene in Drosophila (Sekyrova, 2010).

Fly embryos lacking the function of Jun or Fos die because of the failed dorsal closure. However, atf376 embryos develop normally, without the dorsal open defect, even when derived from atf3-deficient germline clones induced in atf376/ovoD1 mothers. Thus, unlike its partner Jun, Atf3 is not required for dorsal closure, suggesting that dorsal closure is regulated by Jun-Fos dimers and that the Atf3-Jun complex has another function later in development (Sekyrova, 2010).

Consistent with the vital requirement for Atf3 during larval stages, atf3 mRNA was expressed in embryos and larvae. Expression then sharply declines by the late-third larval instar, and no atf3 mRNA was detected by northern blot hybridization in wandering larvae and during metamorphosis from the time of puparium formation until the second day of pupal development. Detailed RT-PCR analysis showed that atf3 downregulation coincided with the cessation of feeding and the onset of metamorphosis [0 hours after puparium formation (APF)]. A pulse of expression occurred at 6 hours APF. RT-PCR from isolated fat body and abdominal integuments, together with in situ hybridization performed on puparia at this stage, showed that atf3 mRNA was primarily present in the larval epidermis (LECs) during the expression peak at 6 hours APF. From the time of head eversion (12 hours APF) the mRNA level remained low until the second day of pupal development, and then it grew steadily during morphogenesis of the adult. Quantitative RT-PCR revealed a 4.3-fold difference in atf3 mRNA abundance between 0 and 72 hours APF. In contrast to the tight regulation of atf3, the mRNAs of fos and jun fluctuated little during the examined period. Therefore, unlike Jun or Fos, Atf3 was dynamically regulated during metamorphosis at the level of transcription (Sekyrova, 2010).

The precise temporal control of atf3 expression suggested that the rise and subsequent fall of Atf3 during metamorphosis might be critical for the complex morphogenesis occurring in fly pupae. This possibility was tested by means of sustained expression of the full-length Atf3 protein using the UAS-Gal4 system with various drivers. A striking, fully penetrant metamorphic defect was observed with the pumpless (ppl) Gal4 driver. Although ppl>atf3 animals developed normally until the pupal stage, they failed to complete fusion of the adult abdominal epidermis. A dorsal cleft in the abdomen remained that could not be covered with the adult cuticle, and consequently 86% of the flies died inside the puparium. All of the ppl>atf3 adults that did eclose showed abdominal lesions filled with the old pupal cuticle lacking adult pigmentation and bristles, often with a clot covering a bleeding wound. Adults with the same abdominal cleft (but otherwise normal) also emerged when atf3 was moderately and ubiquitously misexpressed under the arm-Gal4 driver, suggesting that abdominal morphogenesis was the process most sensitive to ectopic Atf3 (Sekyrova, 2010).

The adult fly abdomen derives from histoblasts that proliferate, replace LECs and finally differentiate, giving rise to the adult cuticle. Therefore, the observed abdominal defect suggested a compromised function of the epidermis, either LECs, histoblasts or both cell types. To distinguish between these possibilities, expression of the ppl-Gal4 driver was first examined in the epidermis. It was found that ppl-Gal4 was active in LECs but not in histoblasts. Second, another driver, Eip71CD-Gal4, which was inactive in histoblasts but strongly expressed in LECs, was examined. Eip71CD-Gal4-driven misexpression of atf3 mostly produced lethal pupae lacking adult cuticle, but it occasionally yielded adults with a dorsal abdominal cleft. In addition to being active in LECs, both ppl-Gal4 and Eip71CD-Gal4 (data not shown) were also expressed in the fat body. However, no abdominal defects occurred when atf3 was misexpressed under either of three fat-body-specific Gal4 drivers, Lsp2, Cg or C7. Third, to rule out the possibility that ectopic Atf3 affected the imaginal epidermis, its expression was directed to histoblasts by using the escargot (esg) and T155 Gal4 drivers; in neither case the fusion of the adult abdominal epidermis was affected (Sekyrova, 2010).

To finally confirm that abdominal morphogenesis was disrupted by sustained atf3 activity in LECs, atf3 was induced by using the flp-out technique. Owing to the timing of heat-shock induction to the mid-third instar, this method triggers expression in the polyploid larval cells but not in the diploid histoblasts (Ninov, 2007) . Misexpression of atf3 under the actin promoter following the flp-out event invariantly led to an abdominal cleft. The lesions were often more severe than those observed in ppl>atf3 animals, affecting also lateral and ventral parts of the abdomen. Together, the above data demonstrate that the sustained expression of atf3 prevents fusion of the adult abdominal epidermis by acting upon LECs, suggesting that the replacement of these obsolete larval cells by adult histoblasts requires the developmental downregulation of atf3 expression (Sekyrova, 2010).

To understand the cellular events underlying the incomplete epithelial closure in ppl>atf3 animals, cell membranes were visualized by antibody staining of the septate junction component, Discs large 1 (Dlg1), or used a transgenic DE-cadherin::GFP fusion protein (shg::gfp). In wild-type animals 24 hours APF, LECs covering the surface of the abdomen gave way to the rapidly expanding nests of histoblasts that began to fuse laterally and ventrally (Bischoff, 2009; Ninov, 2007). In ppl>atf3 pupae the histoblast nests also spread, and at least at 16 hours APF, before their fusion, they comprised normal numbers of histoblasts. By 48 hours APF a control abdomen was fully covered with adult epidermis consisting exclusively of histoblasts, now forming sensory bristles. Histoblasts in ppl>atf3 abdomens also differentiated the adult cuticle with sensory bristles, although polarity of the bristles near the dorsal cleft was altered. However, in contrast to the control, a large population of LECs remained in the dorsal abdomen of ppl>atf3 animals at 48 hours APF. The membranes of the persisting LECs accumulated the Dlg protein, and although these cells became severely deformed they survived throughout metamorphosis to the adult stage. When visualized in live ppl>atf3 pupae, the apical junctions of the remaining LECs displayed interdigitation and accumulation of DE-cadherin::GFP. Another adherens junction component, the Drosophila β-catenin Armadillo, was also enriched in atf3-expressing LECs (Sekyrova, 2010).

Cooperation between adherens junctions and the apical ring of actomyosin cytoskeleton is required for basal extrusion of LECs (Ninov, 2007). The altered pattern of DE-cadherin and β-catenin therefore suggests that excessive Atf3 might prevent LEC extrusion through stabilization of the cell-cell adhesion complex. To examine the effect of Atf3 on LECs in further detail, the flp-out technique, which allows comparisons of atf3-misexpressing and control LECs within one tissue, was employed. Membrane interdigitation occurred between atf3-positive LECs already at 18 and 24 hours APF, even in areas where the LECs had no contact with histoblasts. At 48 hours APF only LECs expressing atf3 persisted, apparently being squeezed by the expanding histoblasts. The membrane-associated DE-cadherin::GFP signal was stronger in adjacent atf3-positive LECs compared with non-induced LECs, and quantitative analysis of confocal images acquired at 18 hours APF and at 24 hours APF both revealed a statistically significant 1.4-fold increase of the DE-cadherin::GFP signal intensity upon atf3 induction. Enrichment of DE-cadherin on apical membranes of atf3-expressing LECs was further confirmed on confocal cross sections (Sekyrova, 2010).

Although some atf3-positive LECs began the extrusion process, they could not detach from the apical surface even when entirely surrounded by histoblasts, possibly being tethered to it by the excessive adhesion protein. By contrast, control LECs did completely separate from the epithelium. In addition, LECs overexpressing atf3 displayed apical enrichment of moesin, an actin-binding protein of the ERM (ezrin, radixin, moesin) family, which links transmembrane proteins to cortical actin filaments. Interestingly, prominent accumulation of DE-cadherin was also observed in atf3-expressing clones of epithelial cells within the hinge region of wing discs that form the adult thorax, indicating that the effect of Atf3 on cell adhesion components may not be limited to larval epithelia (Sekyrova, 2010).

In summary, these results show that deregulation of atf3 expression causes marked changes of cell membranes, including interdigitation and accumulation of cell adhesion molecules, suggesting that LEC adhesiveness might be increased. Although some of the affected LECs initiate extrusion, this process stays incomplete. Consequently, the adhering LECs present a physical barrier for the migrating histoblasts (Sekyrova, 2010).

Rho kinase (Rok)-dependent phosphorylation of myosin regulatory light chain was shown to be required for LEC extrusion (Ninov, 2007). To examine a possible relationship between the Rok-dependent cytoskeletal regulation and Atf3, the function of the GTPase Rho1 (also called RhoA), which acts immediately upstream of Rok, was disrupted. RNAi silencing of Rho1 using the ppl-Gal4 driver produced a phenocopy of atf3 misexpression, causing a dorsal abdominal cleft in 100% of ppl>Rho1(RNAi) adults, of which most died in the puparium and about 12% eclosed, similar to ppl>atf3 animals. However, when Rho1 RNAi and misexpression of atf3 in LECs were combined, the abdominal defect became more severe, not allowing any pharate adults to eclose. Conversely, co-expression of a dominantly active Rho1V14 protein suppressed the otherwise fully penetrant abdominal defect in some ppl-atf3 flies. Surprisingly, it was found that the endogenous Rho1 protein was mislocalized in atf3-misexpressing LECs, showing a diffuse cytoplasmic signal, compared with membrane localization in control LECs. These results suggest a genetic interaction between Rho signaling and atf3, and support the idea that excess Atf3 prevents extrusion of LECs by altering their cell adhesion properties (Sekyrova, 2010).

Disturbed function of the ecdysone receptor (EcR) has been shown to prevent extrusion of LECs, causing a dorsal abdominal cleft that closely resembles the Atf3 gain-of-function phenotype (Ninov, 2007). Therefore whether stimulating EcR-dependent signaling by addition of the natural agonist 20E might overcome the defect caused by sustained atf3 expression was examined. Indeed, supplying third-instar ppl>atf3 larvae with dietary 20E increased the number of eclosing adults, the abdominal scars of which were in 22% of the cases partially or completely sealed with normal adult cuticle (Sekyrova, 2010).

Atf3 interacts with Jun to form a DNA-binding complex and genetically when overexpressed in the developing compound eye. To see if this interaction is biologically relevant during abdominal morphogenesis, whether Atf3 relies on the presence of Jun to cause the dorsal cleft phenotype was tested. First, it was confirmed that Jun is indeed expressed in LECs during metamorphosis. RNAi-mediated depletion of Jun in animals that misexpressed atf3 under the ppl-Gal4 driver restored viability of adults from 14% (atf3 alone) to 100%. Strikingly, 87% of the ppl>atf3, jun(RNAi) adults eclosed with a completely normal abdomen. By contrast, RNAi knockdown of Fos in ppl>atf3 background did not improve the abdominal defect. RNAi silencing of either jun or fos alone under the ppl-Gal4 driver had no effect on the abdomen. These results demonstrate that Atf3 requires its partner Jun but not Fos to disrupt abdominal morphogenesis. Similar to the situation in the compound eye, the effect of misexpressed atf3 can be neutralized by simultaneously expressing Fos or its truncated bZIP domain under the ppl-Gal4 driver. Therefore, the model in which Atf3 and Fos compete for their common partner Jun may be extended to the developing abdomen (Sekyrova, 2010).

This study has identified Atf3 as a new partner of Jun in Drosophila. Previously, Jun has only been known to dimerize with itself and with the Drosophila homolog of Fos. Functional analysis of Atf3 has not yet been reported. Biochemical data show that, similar to mammalian ATF3 and JDP2, the Atf3 protein selectively binds Jun but not Fos. Also consistent with the properties of ATF3 and JDP2 is the ability of Atf3 to bind the ATF/CRE response element alone or synergistically with Jun. In contrast to its mammalian counterparts, however, neither Atf3 alone nor in complex with Jun bound to the AP-1 element under the same conditions. The selective interactions of Atf3 point to distinct biological roles for the Atf3-Jun and the Fos-Jun dimers, respectively (Sekyrova, 2010).

This study has shown a genetic interaction between Atf3 and Jun. The evidence is based on the ability of ectopic Atf3 to disturb morphogenesis of the adult abdomen and the compound eye, which strictly depends on the availability of Jun. Importantly, none of the Atf3 gain-of-function phenotypes could be induced by misexpression of the truncated bZIP domain of Atf3, suggesting that the functional Atf3 protein in complex with Jun is required. Based on the selectivity of Atf3 in a DNA-binding assay, it is predicted that the Atf3-Jun complex regulates specific target genes distinct from those targeted by Fos-Jun dimers (Sekyrova, 2010).

The data also reflect a relationship between the AP-1 and Atf3-Jun complexes. Although Fos does not dimerize or bind DNA with Atf3, its ability to suppress the Atf3 misexpression phenotype in the eye suggests that Fos and Atf3 compete in vivo for their common partner Jun. The fact that the same suppression can be achieved by overexpressing either the truncated Fos bZIP domain or Fos lacking phosphorylation sites indicates that the suppression does not rely on a transcriptional function of Fos but probably occurs through sequestering of Jun, even by a transcriptionally inactive Fos protein. Early in vitro studies have proposed a competition model for the AP-1 and Atf3 proteins to explain a temporal regulation of gene expression in the regenerating liver. However, to date such a relationship among Fos, Jun and Atf3 has not been supported with direct genetic evidence (Sekyrova, 2010).

Removal of LECs is normally complete by 36 hours APF, at which time the sheets of histoblasts reach the dorsal midline (Bischoff, 2009; Ninov, 2007). The data strongly support the argument that the temporal downregulation of atf3 expression during abdominal morphogenesis is necessary for LECs to be replaced by the adult epidermis. When experimentally sustained, atf3 activity in LECs interfered with this exchange by blocking extrusion and death of the LECs. This was evident as the atf3-expressing LECs survived within the epithelial layer for days after their scheduled destruction (Sekyrova, 2010).

Interdigitation of cell membranes and accumulation of adherens junction proteins in LECs suggested that ectopic Atf3 caused adjacent LECs to reinforce their mutual contacts. This probably resulted from altered distribution of the proteins, as levels of the shg (DE-cadherin) mRNA remained unchanged in LECs of ppl>atf3 animals. By contrast, junctions between atf3-expressing LECs and their normal neighbors or histoblasts were smooth and presumably less rigid. DE-cadherin was similarly enriched in clones of imaginal disc cells. These observations suggested that differential adhesion of atf3-expressing cells might have led to their sorting out from the surrounding epithelium. Even modest differences in cadherin levels have been shown to cause segregation of cells within a population by altering their adhesiveness (Sekyrova, 2010).

Recent live imaging data (Bischoff, 2009) have revealed that migrating histoblasts push the LECs ahead of themselves towards the dorsal midline, where histoblasts fuse last. The atf3-expressing LECs that adhered to each other were probably moved and pressed by the expanding histoblasts to the dorsal side, whereas non-induced LECs were eliminated. This explains why the abdominal lesions primarily occurred at the dorsal midline, although flp-out experiments showed that atf3 misexpression could affect LECs in other areas as well. Strengthened contacts among persisting LECs probably blocked invasion of histoblasts in between them and inhibited LEC extrusion, eventually causing gaps in the adult epidermis (Sekyrova, 2010).

In accord with the notion that extrusion from the epithelium is a prerequisite for LECs to undergo apoptosis (Ninov, 2007), it is assumed that sustained presence of Atf3 primarily enhanced adhesiveness of LECs, which only consequently prevented their death. This view is supported by the observation that membranes of atf3-expressing LECs interdigitated and accumulated DE-cadherin as early as 18-24 hours APF, even in areas of the larval epidermis that were far from histoblasts and where control LECs did not yet extrude. In addition, the Atf3 gain-of-function phenotype was stronger than abdominal closure defects caused by caspase mutation or inhibition (Muro, 2006; Ninov, 2007). When the anti-apoptotic proteins p35 or DIAP1 (Thread — FlyBase) was misexpressed under the ppl-Gal4 driver, the resulting dorsal lesions were not lethal and were clearly milder than the broad, mostly fatal scars in ppl>atf3 animals. Compared with the large contiguous populations of persisting LECs in ppl>atf3 pupae, inhibiting apoptosis with p35 only allowed small islands of LECs to survive (Sekyrova, 2010).

Ecdysone signaling promotes replacement of the abdominal epithelia by stimulating both the early histoblast proliferation and the extrusion of LECs (Ninov, 2007; Ninov, 2009). As atf3 misexpression affected LECs but did not impair early histoblast proliferation, the latter possibility remains, that added 20E counteracted the effect of ectopic Atf3 by facilitating the extrusion process. Since normal 20E titers was detected in ppl>atf3 larvae or prepupae, the failure of LEC extrusion was not a result of steroid deficiency. Also, 20E had no effect on atf3 mRNA levels, at least in Drosophila S2 cells or third-instar larvae. Atf3 and ecdysone signaling therefore probably influence LEC extrusion by acting independently (Sekyrova, 2010).

Although the mechanism through which ecdysone contributes to LEC removal is unknown, one attractive possibility is that it might cooperate with Rho signaling, which is required for LEC extrusion as well (Ninov, 2007). It has been demonstrated that genetic interaction between the 20E-response gene broad and components of the Rho pathway including RhoGEF2, Rho1 and myosin II is important for ecdysone-dependent epithelial cell elongation during Drosophila leg morphogenesis. The current data show that Rho1 becomes mislocalized in LECs upon atf3 misexpression and that Rho1 silencing enhances the abdominal gain-of-function phenotype of atf3. The exact relationship between Atf3, Rho1 and ecdysone remains to be determined. However, Atf3 clearly represents a new intrinsic regulator of epithelial cell replacement during Drosophila metamorphosis (Sekyrova, 2010).


REFERENCES

Search PubMed for articles about Drosophila A3-3

Aronheim, A., Zandi, E., Hennemann, H., Elledge, S. J. and Karin, M. (1997). Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol. Cell. Biol. 17: 3094-3102. PubMed ID: 9154808

Bischoff, M. and Cseresnyes Z. (2009). Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila. Development 136: 2403-2411. PubMed ID: 19542353

Fassler, J., Landsman, D., Acharya, A., Moll, J. R., Bonovich, M. and Vinson, C. (2002). B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners. Genome Res. 12: 1190-1200. PubMed ID: 12176927

Gilchrist, M., et al. (2006). Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 441: 173-178. PubMed ID: 16688168

Hai, T. W., Liu, F., Coukos, W. J. and Green, M. R. (1989). Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 3: 2083-2090. PubMed ID: 2516827

Hartman, M. G., et al. (2004). Role for activating transcription factor 3 in stress-induced beta-cell apoptosis. Mol. Cell. Biol. 24: 5721-5732. PubMed ID: 15199129

Hsu, J. C., Laz, T., Mohn, K. L. and Taub, R. (1991). Identification of LRF-1, a leucine-zipper protein that is rapidly and highly induced in regenerating liver. Proc. Natl. Acad. Sci. 88: 3511-3515. PubMed ID: 1902565

Muro, I., et al. (2006). The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process. Development 133: 3305-3315. PubMed ID: 16887831

Nakade, K., et al. (2007). JDP2 suppresses adipocyte differentiation by regulating histone acetylation. Cell Death Differ. 14: 1398-1405. PubMed ID: 17464331

Ninov, N., Chiarelli, D. A., Martín-Blanco, E. (2007). Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis. Development 134(2): 367-79. PubMed ID: 17166923

Ninov, N. and Martín-Blanco, E. (2009). Changing gears in the cell cycle: histoblasts and beyond. Fly (Austin) 3(4): 286-9. PubMed ID: 19934653

Qi, L., et al. (2009). Adipocyte CREB promotes insulin resistance in obesity. Cell Metab. 9: 277-286. PubMed ID: 19254572

Rosenberger, C. M., Clark, A. E., Treuting, P. M., Johnson, C. D. and Aderem, A. (2008). ATF3 regulates MCMV infection in mice by modulating IFN-gamma expression in natural killer cells. Proc. Natl. Acad. Sci. 105: 2544-2549. PubMed ID: 18268321

Sekyrova, P., Bohmann, D., Jindra, M. and Uhlirova, M. (2010). Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis. Development 137(1): 141-50. PubMed ID: 20023169


Biological Overview

date revised: 20 February 2010

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