InteractiveFly: GeneBrief

apontic : Biological Overview Regulation | Developmental Biology | Effects of Mutation | References

Gene name - apontic

Synonyms - tracheae defective

Cytological map position - 59F3--4

Function - potential transcription factor and RNA binding protein

Keywords - oogenesis, trachea, heart, sensitivity to ethanol sedation

Symbol - apt

FlyBase ID: FBgn0015903

Genetic map position - 2-

Classification - novel bZIP transcription factor and RNA binding protein

Cellular location - nuclear and cytoplasmic

NCBI link: Entrez Gene

apt orthologs: Biolitmine
Recent literature

Monahan, A.J. and Starz-Gaiano, M. (2016). Apontic regulates somatic stem cell numbers in Drosophila testes. BMC Dev Biol 16: 5. PubMed ID: 26993259
Microenvironments called niches maintain resident stem cell populations by balancing self-renewal with differentiation, but the genetic regulation of this process is unclear. The niche of the Drosophila testis is well-characterized and genetically tractable, making it ideal for investigating the molecular regulation of stem cell biology. The JAK/STAT pathway, activated by signals from a niche component called the hub, maintains both germline and somatic stem cells. This study investigated the molecular regulation of the JAK/STAT pathway in the stem cells of the Drosophila testis. The transcriptional regulator Apontic (Apt) was found to act in the somatic (cyst) stem cells (CySCs) to balance differentiation and maintenance. Apt functions as a negative feedback inhibitor of STAT activity, which enables cyst cell maturation. Simultaneous loss of the STAT regulators apt and Socs36E, or the Stat92E-targeting microRNA miR-279, expanded the somatic stem cell-like population. Genetic analysis revealed that a conserved genetic regulatory network limits JAK/STAT activity in the somatic stem cells of Drosophila testis. In these cells, JAK/STAT signaling was found to promote apt expression. Then, Apt functions through Socs36E and miR-279 to attenuate pathway activation, which is required for timely CySC differentiation. The study proposes that Apt acts as a core component of a STAT-regulatory circuit to prevent stem cell overpopulation and allow stem cell maturation.

Wang, X. F., Shen, Y., Cheng, Q., Fu, C. L., Zhou, Z. Z., Hirose, S. and Liu, Q. X. (2017). Apontic directly activates hedgehog and cyclin E for proper organ growth and patterning. Sci Rep 7(1): 12470. PubMed ID: 28963499
Hedgehog (Hh) signaling pathway and Cyclin E are key players in cell proliferation and organ development. Hyperactivation of hh and cyclin E has been linked to several types of cancer. However, coordination of the expression of hh and cyclin E was not well understood. This study shows that an evolutionarily conserved transcription factor Apontic (Apt) directly activates hh and cyclin E through its binding site in the promoter regions of hh and cyclin E. This Apt-dependent proper expression of hh and cyclin E is required for cell proliferation and development of the Drosophila wing. Furthermore, Fibrinogen silencer-binding protein (FSBP), a mammalian homolog of Apt, also positively regulates Sonic hh (Shh), Desert hh (Dhh), Cyclin E1 (CCNE1) and Cyclin E2 (CCNE2) in cultured human cells, suggesting evolutionary conservation of the mechanism. Apt-mediated expression of hh and cyclin E can direct proliferation of Hh-expressing cells and simultaneous growth, patterning and differentiation of Hh-recipient cells. The discovery of the simultaneous expression of Hh and principal cell-cycle regulator Cyclin E by Apt implicates insight into the mechanism by which deregulated hh and cyclin E promotes tumor formation.
Shen, Y., Wang, L., Hirose, S., Zhou, Z. and Liu, Q. (2018). The transcriptional factor Apt regulates neuroblast differentiation through activating CycE expression. Biochem Biophys Res Commun 499(4): 889-894. PubMed ID: 29625112
In Drosophila, the thoracic neuroblast 6-4 (NB6-4T) divides asymmetrically into a glial precursor and a neuronal precursor, while the abdominal neuroblast 6-4 (NB6-4A) divides symmetrically to produce two glial cells. The underlying mechanism by which NB6-4T and NB6-4A undergo distinct differentiation is still elusive. This study finds that the transcription factor Apontic (Apt) exclusively expresses in NB6-4T cells and is involved in regulating NB6-4T differentiation. Loss of Apt results in neuronal precursor loss. Epistasis analysis shows that Apt controls NB6-4T differentiation through activating CycE expression. On the other hand, Gcm suppresses Apt expression in the NB6-4A cell, thus inhibiting CycE expression. Taken together, these findings reveal a Gcm-Apt-CycE axis that regulates neuroblast and glia cell differentiation.
Berez, A., Peercy, B. E. and Starz-Gaiano, M. (2020). Development and Analysis of a Quantitative Mathematical Model of Bistability in the Cross Repression System Between APT and SLBO Within the JAK/STAT Signaling Pathway. Front Physiol 11: 803. PubMed ID: 32848815
Cell migration is a key component in development, homeostasis, immune function, and pathology. It is important to understand the molecular activity that allows some cells to migrate. Drosophila melanogaster is a useful model system because its genes are largely conserved with humans and it is straightforward to study biologically. The well-conserved transcriptional regulator Signal Transducer and Activator of Transcription (STAT) promotes cell migration, but its signaling is modulated by downstream targets Apontic (APT) and Slow Border Cells (SLBO). Inhibition of STAT activity by APT and cross-repression of APT and SLBO determines whether an epithelial cell in the Drosophila egg chamber becomes motile or remains stationary. Through mathematical modeling and analysis, this study examine how the interaction of STAT, APT, and SLBO creates bistability in the Janus Kinase (JAK)/STAT signaling pathway. This paper updates and analyzes earlier models to represent mechanistically the processes of the JAK/STAT pathway. Parameter, bifurcation, and phase portrait analyses were made, and reductions were made to the system to produce a minimal three-variable quantitative model. The manifold between migratory and stationary steady states was analyzed in this minimal model, and it was shown that when the initial conditions of the model are near this manifold, cell migration can be delayed.
Berez, A., Peercy, B. E. and Starz-Gaiano, M. (2020). Development and Analysis of a Quantitative Mathematical Model of Bistability in the Cross Repression System Between APT and SLBO Within the JAK/STAT Signaling Pathway. Front Physiol 11: 803. PubMed ID: 32848815
Cell migration is a key component in development, homeostasis, immune function, and pathology. It is important to understand the molecular activity that allows some cells to migrate. Drosophila melanogaster is a useful model system because its genes are largely conserved with humans and it is straightforward to study biologically. The well-conserved transcriptional regulator Signal Transducer and Activator of Transcription (STAT) promotes cell migration, but its signaling is modulated by downstream targets Apontic (APT) and Slow Border Cells (SLBO). Inhibition of STAT activity by APT and cross-repression of APT and SLBO determines whether an epithelial cell in the Drosophila egg chamber becomes motile or remains stationary. Through mathematical modeling and analysis, this study examine how the interaction of STAT, APT, and SLBO creates bistability in the Janus Kinase (JAK)/STAT signaling pathway. This paper updates and analyzes earlier models to represent mechanistically the processes of the JAK/STAT pathway. Parameter, bifurcation, and phase portrait analyses were made, and reductions were made to the system to produce a minimal three-variable quantitative model. The manifold between migratory and stationary steady states was analyzed in this minimal model, and it was shown that when the initial conditions of the model are near this manifold, cell migration can be delayed.
Wang, X. F., Liu, J. X., Ma, Z. Y., Shen, Y., Zhang, H. R., Zhou, Z. Z., Suzuki, E., Liu, Q. X. and Hirose, S. (2020). Evolutionarily Conserved Roles for Apontic in Induction and Subsequent Decline of Cyclin E Expression. iScience 23(8): 101369. PubMed ID: 32736066
Cyclin E is a key factor for S phase entry, and deregulation of Cyclin E results in developmental defects and tumors. Therefore, proper cycling of Cyclin E is crucial for normal growth. This study found that transcription factors Apontic (Apt) and E2f1 cooperate to induce cyclin E in Drosophila. Functional binding motifs of Apt and E2f1 are clustered in the first intron of Drosophila cyclin E and directly contribute to the cyclin E transcription. Knockout of apt and e2f1 together abolished Cyclin E expression. Furthermore, Apt up-regulates Retinoblastoma family protein 1 (Rbf1) for proper chromatin compaction, which is known to repress cyclin E. Notably, Apt-dependent up-regulation of Cyclin E and Rbf1 is evolutionarily conserved in mammalian cells. These findings reveal a unique mechanism underlying the induction and subsequent decline of Cyclin E expression.
Cao, X., Rojas, M. and Pastor-Pareja, J. C. (2022). Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland. Dis Model Mech 15(1). PubMed ID: 34842272
Development involves tightly paced, reproducible sequences of events, yet it must adjust to conditions external to it, such as resource availability and organismal damage. A major mediator of damage-induced immune responses in vertebrates and insects is JAK/STAT signaling. At the same time, JAK/STAT activation by the Drosophila Upd cytokines is pleiotropically involved in normal development of multiple organs. Whether inflammatory and developmental JAK/STAT roles intersect is unknown. This study shows that JAK/STAT is active during development of the prothoracic gland (PG), which controls metamorphosis onset through ecdysone production. Reducing JAK/STAT signaling decreased PG size and advanced metamorphosis. Conversely, JAK/STAT hyperactivation by overexpression of pathway components or SUMOylation loss caused PG hypertrophy and metamorphosis delay. Tissue damage and tumors, known to secrete Upd cytokines, also activated JAK/STAT in the PG and delayed metamorphosis, at least in part by inducing expression of the JAK/STAT target Apontic. JAK/STAT damage signaling, therefore, regulates metamorphosis onset by co-opting its developmental role in the PG. These findings in Drosophila provide insights on how systemic effects of damage and cancer can interfere with hormonally controlled development and developmental transitions.

Song, F., Zhang, W., Li, X., Chen, X., Yuan, X., Jiang, M., Zhao, Y., Liu, Q. and Zhou, Z. (2023). FSBP suppresses tumor cell migration by inhibiting the JNK pathway. iScience 26(4): 106440. PubMed ID: 37035004
The main cause of high mortality in cancer patients is tumor metastasis. Exploring the underlying mechanism of tumor metastasis is of great significance for clinical treatments. This study has identified the transcription factor Apt/FSBP is a suppressor for tumor metastasis. In Drosophila wing disc, knockdown of apt is able to trigger cell migration, whereas overexpression of apt hampers scrib-RNAi-induced tumor cell migration. Further studies show that loss of apt promotes cell migration JNK pathway . To investigate the role of FSBP, the homolog of Apt in mammals, Fsbp liver-specific knockout mice were constructed. Knockout of Fsbp in liver does not cause any detectable physiological defects, but predisposes to tumorigenesis on DEN and CCl(4) treatment. In addition, loss of Fsbp accelerates tumor metastasis from liver to diaphragm. Taken together, this study uncovers FSBP is a novel tumor suppressor, and provides it as a considerable drug target for tumor treatment.


Apontic was identified in four different studies, each of which discovered different facets to the complex biological functions of this protein. Having first been identified as a modifier of Hox gene function in gnathal development, apontic may serve in a parallel pathway to Deformed and Sex combs reduced in the development of sclerite (cuticular) structures in the ventral portion of the gnathal region derived from maxillary and mandibular segments, with the exception of mouth hooks (Gellon, 1997). In the guise of tracheae defective, apontic has been shown shown to be required for the formation of the tracheal system during Drosophila embryogenesis. apt is not necessary for determining tracheal cell identity but for subsequent morphogenetic cell movements (Eulenberg, 1997). A third study involved a screen for enhancer trap lines that exhibit an early pattern of gene expression in cardiac precursor cells. apt proved to be adjacent to one plasmid insert showing expression in heart precursors, and apt mutant embryos show distinct abnormalities in heart morphology. A fourth study showed that Apt acts in concert with Bruno (also termed Arrest), a known translational repressor of Oskar, to repress the translation of OSK mRNA. Apt physically interacts with Bruno, and reducing the amount or activity of both Bru and Apt proteins appears to lead to a modest derepression of OSK translation (Lie, 1999).

While the observations made in the first three studies are more or less consistent in concluding that apontic might code for a transcription factor, the fourth study, showing that Apt binds to Bru and may regulate OSK mRNA translation rather convincingly leads to the conclusion that Apt is a cytoplasmic protein involved in translational control. This overview will deal with the fourth study and consider how these results impinge on the conclusions of the first three studies.

Proper control of OSK mRNA translation is essential and requires strict coordination with localization of the transcript to the posterior pole of the oocyte. Not surprisingly, translational regulation of OSK mRNA appears to be complex, and several factors involved in repression and activation have already been identified. The evidence for a direct role in OSK translation is strongest for Bru, a protein that binds specifically to regulatory sequences in the OSK mRNA 3' untranslated region (UTR). Another protein suggested to act on OSK in repression is p50, which was identified by virtue of its binding to OSK mRNA, but for which genetic confirmation of such a role has not been obtained. apt can be added to this roster of proteins and genes implicated in negative regulation of translation (Lie, 1999 and references).

It has been suggested that Apt functions as a transcription factor during embryogenesis, perhaps acting as a cofactor for certain Hox genes (Gellon, 1997 and Eulenberg, 1997). Two types of evidence have been presented to support this conclusion. (1) The Apt protein is highly concentrated in nuclei during most of embryogenesis, which strongly implicates a nuclear function. (2) The predicted structure of the Apt protein includes domains similar to those found in certain transcription factors. One is a short region enriched in glutamine residues, which may serve as a transcriptional activation domain. This by itself does not strongly support a role as a transcription factor, since similar glutamine-rich regions are found in a wide variety of Drosophila proteins, some of which are not involved in transcriptional regulation. The other domain is a potential bZIP motif. One part of this motif, the leucine zipper, is clearly present in Apt and may imply that the protein homodimerizes or forms a heterodimer with another protein in vivo. The second part of the bZIP motif, a flanking basic region, appears in an unusual form: certain amino acids known to be involved in DNA binding are present, but these are positioned much closer to the leucine zipper than in any other characterized bZIP domain. In addition, there are few basic amino acids. Consequently, Apt is either a rather unusual example of a bZIP transcription factor, or it may be a related protein whose function in the nucleus is less certain (Lie, 1999).

Apt is not always nuclear. Apt protein is persistently retained in the cytoplasm of early stage embryos even after other maternally provided proteins have shifted to the nuclei. This evidence for programmed control of the subcellular location of Apt suggests a requirement for Apt in the cytoplasm of early embryos. In the nurse cells of the ovary Apt protein is partitioned primarily to the cytoplasm. One possibility for how the subcellular distribution of Apt may be controlled is suggested by differences in APT mRNAs. The use of alternate 5' exons leads to variation at the amino terminus of the protein. Exon choice appears to vary during development, with one form of the mRNA found primarily among maternal transcripts while other forms are ubiquitous or most prevalent among zygotic transcripts. This pattern correlates well with the changing distribution of Apt protein: cytoplasmic Apt protein is synthesized largely or entirely from maternal mRNAs, while nuclear protein is synthesized from both maternal and zygotic mRNAs. Thus one form of the protein could be targeted to the nucleus and the other form to the cytoplasm (Lie, 1999).

Evidence implicating apt in the control of OSK translation is indirect. Biochemical experiments indicate that Bru and Apt proteins interact physically but provide no insight into the significance of the association. The genetic evidence -- head defects among progeny of mothers transheterozygous for apt and bruno (aret) mutations -- reveals a functionally significant interaction between the apt and aret genes but does not specify the exact nature of the interaction. Nevertheless, given the established role for Bru in repression of OSK mRNA translation, one likely explanation is that Bru and Apt both act in this process. Consequently, reducing the amount or activity of both Bru and Apt proteins could lead to a modest derepression of OSK translation. This interpretation is supported by the sensitivity of the phenotype to reduction of nanos gene dosage (Lie, 1999).

Although the genetic interaction of aret and apt supports a role for apt in repression of OSK mRNA translation, this function may not be essential. One of the apt mutants that shows a nanos-sensitive interaction with aret has only a modest phenotype in germline clonal analysis: a small fraction of the embryos from the homozygous mutant germlines display head defects. While this phenotype is consistent with a partial relaxation of the controls on OSK activity, it is inconsistent with a complete derepression of OSK mRNA translation. Could this particular mutant be a weak allele? This seems somewhat unlikely (but not impossible) since it displays a stronger genetic interaction with aret than does another allele, which has a strong arrested oogenesis phenotype in homozygous mutant germlines. Another possibility is that apt performs a redundant or partially redundant role in repression of OSK mRNA translation. An appealing feature of this model is that a candidate exists for a protein with overlapping function. Gunkel (1998) recently described a protein, p50, that also binds to the regions of the OSK mRNA bound by Apt; Apt and p50 could have similar roles in regulation of OSK expression. The gene encoding p50 has not been identified, so genetic tests of this model are not yet possible (Lie, 1999).

The demonstration that Apt is an RNA binding protein is somewhat unexpected, since none of the well-characterized RNA binding motifs appear in the predicted protein sequence. The ability of Apt to discriminate in its binding to certain regions of the OSK mRNA 3' UTR is striking, but its significance is uncertain, especially given the binding of Apt to a wide variety of other RNAs. In further characterization of apt function, it will be of interest to determine whether Apt RNA binding activity is important for proper regulation of OSK mRNA translation, or if the interaction of Apt and Bru proteins is sufficient. Notably, Apt protein does not colocalize with Bru and OSK mRNA to the posterior pole of the oocyte, raising the possibility that displacement of Apt from Bru may allow translational activation (Lie, 1999).

A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila

In the fruit fly Drosophila melanogaster, as in mammals, acute exposure to a high dose of ethanol leads to stereotypical behavioral changes beginning with increased activity, followed by incoordination, loss of postural control, and eventually, sedation. The mechanism(s) by which ethanol impacts the CNS leading to ethanol-induced sedation and the genes required for normal sedation sensitivity remain largely unknown. This study identified the gene apontic (apt), an Myb/SANT-containing transcription factor that is required in the nervous system for normal sensitivity to ethanol sedation. Using genetic and behavioral analyses, it was shown that apt mediates sensitivity to ethanol sedation by acting in a small set of neurons that express Corazonin (Crz), a neuropeptide likely involved in the physiological response to stress. The activity of Crz neurons regulates the behavioral response to ethanol, as silencing and activating these neurons affects sedation sensitivity in opposite ways. Furthermore, this effect is mediated by Crz, as flies with reduced crz expression show reduced sensitivity to ethanol sedation. Finally, both apt and crz were found to be rapidly upregulated by acute ethanol exposure. Thus, two genes and a small set of peptidergic neurons were identified that regulate sensitivity to ethanol-induced sedation. It is proposed that Apt regulates the activity of Crz neurons and/or release of the neuropeptide during ethanol exposure (McClure, 2013).

This work identifies apt and crz that mediate the fly's sensitivity to ethanol-induced sedation. Flies with reduced expression of either gene display dramatically decreased ethanol sedation sensitivity; thus, both apt and crz normally promote ethanol sedation. Normal sensitivity to ethanol sedation requires apt expression in neurons during two distinct life stages, metamorphosis and adulthood. Apt function in a subset of crz-expressing neurons (approximately 6 of the 12-16 crz-expressing cells) is necessary and sufficient for normal sensitivity to ethanol sedation. Acute manipulations of the activity of crz neurons led to altered ethanol sedation sensitivity, demonstrating that these neurons play an active role in regulating the behavioral response to ethanol-induced sedation. The neuropeptide Crz is also involved in ethanol sedation, as flies with reduced crz expression, specifically during adulthood, show dramatically decreased ethanol sedation sensitivity. Finally, in response to acute ethanol exposure the expression of both apt and crz are rapidly upregulated during ethanol exposure. It is hypothesized that the Apt-Crz system, functioning in a very small group of neurosecretory cells, may be an early target of ethanol in the fly brain whose function is crucial for normal sensitivity to ethanol (McClure, 2013).

How does Apt regulate ethanol-induced sedation? Although Apt's role could be to regulate the expression of crz, no such function was observed. It is thus postulated that Apt functions to regulate the activity of crz neurons and/or neuropeptide release. For example, Apt, acting as a transcription factor, could regulate the transcription of proteins required for synthesis, packaging, and/or release of Crz and possibly other neuropeptides. Alternatively, Apt could regulate the expression of proteins required for synapse formation. In support of these two possibilities, apt mutant embryos were observed to have defective synaptic transmission at the neuromuscular junction, as well as fewer numbers of active zones within motoneurons, indicating a presynaptic defect (McClure, 2013).

Another possible function for Apt in regulating ethanol sedation behavior may be found in its neuronal requirement during metamorphosis, a time of intense remodeling to construct the adult CNS. During embryogenesis, Apt functions in multiple morphogenetic processes, including tracheal, head, CNS, and heart morphogenesis, as well as border cell migration. It is therefore possible that during metamorphosis Apt establishes proper development and neuronal connectivity of the adult CNS, and in particular the Crz neurons. However, this possibility seems somewhat unlikely given that the adult CNS in apt13-66 flies appeared normal, as was the number and morphology of Crz-expressing neurons. Additionally, it was found that adult-specific expression of apt in neurons was necessary for normal sedation sensitivity. However, there may be subtle defects in the adult CNS of apt mutant flies, which were not detected, that could contribute to their altered sedation sensitivity (McClure, 2013).

Apt shows highest sequence conservation with the human FSBP, a negative regulator of transcription of the gamma chain of fibrinogen (see Starz-Gaiano, 2008). Sequence conservation between apt and FSBP is observed within the DNA-binding domain. Interestingly, moderate alcohol consumption in humans has been known to exert a cardioprotective effect, in part by lowering levels of circulating Fibrinogen. The mechanism for how alcohol consumption regulates Fibrinogen is currently unknown, but in light of the current findings it is speculated that it may occur at the level of transcription. Ethanol exposure in flies was found to acutely upregulate apt expression. A similar situation may occur in humans, whereby alcohol consumption could upregulate the transcription of FSBP, ultimately leading to negative regulation of the gamma chain of fibrinogen and lowered levels of circulating Fibrinogen, which in turn would provide cardioprotection (McClure, 2013).

The observations implicate crz-expressing neurons in the regulation of ethanol sedation behavior, a function not previously attributed to these neurons. This study demonstrated that adult-specific silencing of crz neurons significantly reduced ethanol sedation sensitivity, while increasing their activity resulted in the opposite phenotype, an increase in ethanol sedation sensitivity. Based on the observation that inhibiting crz expression also reduced ethanol sedation sensitivity, it is believed that the phenotypes associated with crz neuronal manipulations reflect changes in the release of the neuropeptide Crz and activation of its signaling pathway. However, a few Crz neurons also express the short Neuropeptide F (sNPF). sNPF is considered to be a multifunctional neuropeptide due to its broad expression in diverse neuronal types, and its possible role in crz-expressing neurons and in ethanol sedation sensitivity has not been excluded. However, the observation that Apt function is required in a subset of crz neurons to promote ethanol sedation behavior, firmly establishes the importance of these neurons in mediating the behavioral response to ethanol. The data also suggest that the function of both genes, crz and apt, overlaps in a small set of neurons likely located in the pars lateralis (PL) to mediate the behavioral response to ethanol-induced sedation (McClure, 2013).

It has been hypothesized that Crz is released in response to various types of stress in insects (Veenstra, 2009; Boerjan, 2010), and that this could explain its pleiotropic effects. This hypothesis was bolstered by a recent study showing that flies deficient in Crz are resistant to metabolic, osmotic, and oxidative stress, as measured by survival (Zhao, 2010). In addition, Crz plays a role in stress physiology through its association with well characterized stress hormones. For instance, crz-expressing neurons in the PL also express receptors for two diuretic hormones, DH44 and DH31. By virtue of receptor similarity, DH44 and DH31 are related to corticotrophin-releasing factor (CRF) and calcitonin-gene related peptide (CGRP), respectively, both of which mediate the mammalian physiological and behavioral responses to stress. Interestingly, both CRF and CGRP act to inhibit secretion of GnRH in the mammalian hypothalamus. This is significant because Crz is thought to be the homolog of mammalian GnRH, and suggests that analogous regulation occurs in Drosophila (Cazzamali, 2002). It is thus possible that in flies a stress signal or the animal's stress status may be relayed to Crz neurons and alters their function. Thus, based on its functional and molecular associations with stress physiology, it is tempting to speculate that the role of Crz signaling in ethanol sedation sensitivity is related to a stress response. A previous study has shown that stress, in the form of heat shock, induces tolerance to a subsequent ethanol exposure, and that ethanol tolerance relies on the gene hangover, a large nuclear zinc-finger protein, that mediates various other stress responses (Scholz, 2005). In addition, several genes related to stress responses have been shown to be upregulated by ethanol exposure in transcriptional profiling studies, including nearly half of all Drosophila heat shock protein genes, as well as genes involved in the regulation of oxidative stress and aging. Importantly, a maladaptive response to stress has been shown in humans to be a major and common element contributing to drug addiction. Finally, an increase in ethanol self-administration has been observed in animal models with physical, social, and emotional stress. In light of these findings, it will be interesting to further explore the role of Crz and its function in stress physiology and the regulation of ethanol-related behaviors (McClure, 2013).

Neuropeptides are diverse signaling molecules that mediate a broad spectrum of physiological and behavioral processes. Several studies have linked neuropeptides to behavioral responses to ethanol. For instance, one of the first ethanol sensitivity mutants described in Drosophila, amnesiac, encodes a neuropeptide homologous to the vertebrate pituitary adenylate cyclase-activating peptide. In addition, mice lacking either neuropeptide Y (NPY), a neuromodulator abundantly expressed in many regions of the CNS, or its Y1 receptor subtype, display increased ethanol consumption and resistance to ethanol sedation, whereas animals overexpressing NPY show the opposite behavioral phenotypes. Neuropeptide F (NPF), the sole member of the NPY family in Drosophila, and its receptor NPFR1, has similarly been shown to mediate the fly's sensitivity to ethanol-induced sedation. Finally, flies with neuronal perturbations in the insulin signaling pathway displayed increased ethanol sedation sensitivity. These studies, implicating the neuropeptide Crz in sensitivity to ethanol sedation, suggest that neuropeptides are important regulators of the behavioral response to ethanol, and it would therefore be interesting to survey all known Drosophila neuropeptides and their downstream signaling components for possible role(s) in ethanol-related behaviors (McClure, 2013).


Transcriptional Regulation

tracheae defective (tdf), now more properly termed apontic, is required for the formation of the tracheal system during Drosophila embryogenesis. It encodes a putative bZIP transcription factor (Tdf). Antibodies directed against Tdf detect a nuclear protein in all tracheal cells before invagination and throughout tracheal system morphogenesis. Examination of tdf mutants reveals that tdf activity is not necessary for determining tracheal cell identity but for subsequent morphogenetic cell movements. tdf activity is under the control of trachealess, the key regulator gene for tracheal development. In contrast, tdf activity is not dependent on and does not interfere with the fibroblast growth factor-(FGF) and Decapentaplegic- (Dp) mediated signaling that directs guided tracheal cell migration. These results suggest that lack of tdf activity affects tracheal cell migration in general rather than specific aspects of cell migration. tdf activity involves both maternal and zygotic components; its requirement and hence, its effects, are not limited to tracheal system formation. The complex spatiotemporal patterns of Tdf expression in the embryo suggests that defects in tdf mutants may be the result of impaired cell migration. Therefore, it is proposed that the bZIP protein Tdf functions as a co-regulator of target genes that provide cells with the ability to migrate (Eulenberg, 1997).

The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some downstream genes but not others. While tracheal expression of tkv depends on vvl, it appears to be independent of trh. The opposite appears to be the case for two other tracheal genes, apontic/tracheal defective (tdf) and pebbled (peb) [also known as hindsight (hnt)], which code for two putative transcription factors. Both genes appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes seem to be common targets of vvl and trh but others seem to depend only on one of them (Boube, 2000).

The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hntpeb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt308-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes -- apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc) -- regulate tissue autonomously and, thus, may be direct transcriptional targets of Hnt. Three of these genes -- apt, Dl, and dpp -- are also regulated nonautonomously in adjacent non-Hnt-expressing tissues. The expression of several additional interactors -- viking (vkg), Cg25, and laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).

Feedback inhibition of JAK/STAT signaling by Apontic is required to limit an invasive cell population

In both normal development and in a variety of pathological conditions, epithelial cells can acquire migratory and invasive properties. Border cells in the Drosophila ovary provide a genetically tractable model for elucidating the mechanisms controlling such behaviors. An apontic (apt) mutant has been identified in which the migratory population expands and separation from the epithelium is impeded. This phenotype resembles gain-of-function of JAK/STAT activity. Gain-of-function of APT also mimics loss of function of STAT and its key downstream target, SLBO. APT expression is induced by STAT, which binds directly to sites in the apt gene. The data suggest that a regulatory circuit between STAT, APT, and SLBO functions to convert an initially graded signal into an all-or-nothing activation of JAK/STAT and thus to proper cell specification and migration. These findings are supported by a mathematical model, which accurately simulates wild-type and mutant phenotypes (Starz-Gaiano, 2008).

In many migratory cell types, including metastatic carcinomas, motile cells must detach from an epithelium to move to their final location. However the precise mechanisms by which cells disengage from their neighbors remain poorly understood, and in most cases it is not possible to view the process directly in vivo. Border cells in the Drosophila ovary represent a model for studying epithelial cell migration in vivo that is amenable both to genetic approaches and live imaging. This study reports the identification of a mutant, apt, in which the distinction between invasive and noninvasive cells was compromised. In apt mutants, as the border cell cluster moved away from the epithelium, additional migratory cells -- the stretched border cells -- ingressed in between the nurse cells. These stretched border cells maintained connections with both the main cluster of border cells, and the outer epithelial cell layer, resulting in a defect in detachment (Starz-Gaiano, 2008).

Recent technological advances have enabled analysis of border cells throughout their six hour migration by live imaging. Time-lapse movies of wild-type egg chambers revealed that the process of border cell detachment is surprisingly slow. This indicates that the ability to extend and retract protrusions is not sufficient for the border cells to exit the epithelium, and that there is sufficient time for transcriptional events to contribute to the process. In apt mutants, the border cells rounded up and advanced in between the nurse cells normally, but cells with an apparently intermediate identity were frequently trapped in between the border cell cluster and the follicle cell epithelium, unable to detach from either one. Thus, the two cell types must be clearly distinguished in order for them to be able to disconnect from one another (Starz-Gaiano, 2008).

In a variety of contexts throughout development, a graded distribution of a signaling molecule in a field of cells can elicit discrete cellular responses. Such threshold-like behavior can be achieved by positive autoregulation. Therefore, prior to the current work, it would have been reasonable to propose that STAT autoregulation could convert initially graded activity in the follicular epithelium to 'on' and 'off' states. In wild-type, the migrating border cell cluster takes the source of JAK/STAT activation (UPD expressed by the polar cells) with it, reinforcing SLBO expression in the migratory cells and removing the source of JAK/STAT activation from the anterior follicle cells. So, one could have postulated that the physical separation of the JAK/STAT signaling center from the anterior follicle cells was sufficient to create a significant difference in levels of STAT activity between the migrating cells and those left behind, and thus to distinguish the two cell types and behaviors. However, unexpectedly this study showed that neither STAT autoregulation nor the movement of the signaling center is sufficient to convert the gradient into a step function in the absence of APT (Starz-Gaiano, 2008).

It is proposed instead that feedback inhibition of JAK/STAT combined with the mutual repression of APT and SLBO is responsible for generating the stepwise activation pattern. When two genes mutually repress each other, a slight increase in the activation of one leads to a stronger repression of the second, which, in turn, leads to a further increase of the first. Thus, together these two genes behave as an autocatalytic system. Since apt and slbo are both targets of STAT activity, a three-component regulatory circuit is proposed. The mathematical model demonstrates that this circuit is sufficient to explain what is observed in vivo. In the absence of APT, JAK/STAT activation takes place in an enlarged region and, remarkably, the 'on-off' character of the JAK/STAT activation is lost. This suggests that the threshold behavior of the system does not result from JAK/STAT autoregulation but from the mutual repression of APT and SLBO (Starz-Gaiano, 2008).

The model that most accurately simulates the wild-type and mutant phenotypes is one in which SLBO antagonizes APT activity more strongly than its expression. This is consistent with experimental observation that overexpression of SLBO completely mimics the apt loss-of-function phenotype, but only reduces and does not eliminate APT expression (Starz-Gaiano, 2008).

It is striking that different patterns of SLBO and APT are induced by the same gradient of JAK/STAT activity. An important consequence is that, at high concentrations of active STAT, more SLBO is produced than APT. One way this could be explained is through the observation that STAT binds four different sites in the slbo enhancer with differing affinities. In cells with high concentrations of STAT, more sites, including low affinity sites, should be occupied and thus a higher level of slbo expression generated than in cells with lower STAT levels. In contrast, the apt gene contains only two detectable STAT binding sites, to which STAT can bind nearly as well as it binds the optimal STAT consensus sequence. Thus, apt expression should turn on in response to lower levels of active STAT than slbo and also should saturate at relatively low concentrations of active STAT, yielding a broad and shallow expression gradient across the anterior field of follicle cells. These are precisely the expression patterns observed. Therefore, in cells adjacent to the polar cells, SLBO wins the competition whereas further away from the source of UPD, APT wins the APT/SLBO competition. Higher levels of SLBO block the repression of JAK/STAT by APT in the cells next to the polar cells, causing an even stronger JAK/STAT activation and so on (Starz-Gaiano, 2008).

In addition, evidence was found for a low level of JAK/STAT-independent APT expression, which was also incorporated into the model. This baseline APT expression depended on the transcription factor known as Eyes absent, and based on the model it is proposed that its function could be to prevent any possibility of a renewed trigger of the JAK/STAT pathway in the cells that remain in the anterior epithelium (Starz-Gaiano, 2008).

The JAK/STAT pathway is highly conserved from insects to mammals and is critically important in development, immunity, and inflammation. Intriguingly, Drosophila APT is expressed in many domains where JAK/STAT signaling occurs, including embryonic trachea and the hub of the testes. In addition, apt has been uncovered as a downstream target of STAT in microarray analysis of testis and border cells. Therefore, apt may be a downstream target of STAT signaling in a variety of cell types (Starz-Gaiano, 2008).

It is also possible that this relationship is conserved in other animals, since genes highly related to apt are found in all sequenced insect genomes. In humans, the closest gene to apt is fibrinogen silencer-binding protein (FSBP). Interestingly, two strong loss-of-function alleles of apt contain missense mutations in well-conserved residues, demonstrating the functional significance of this region. Although FSBP has not been extensively characterized, it has been reported to be a negative regulator of the gamma chain of fibrinogen transcription. Fibrinogen is highly expressed in hepatocytes in response to inflammatory cytokine-mediated activation of the JAK/STAT pathway, and there are at least three STAT3 binding sites on the human gamma-fibrinogen promoter. This suggests that APT and FSBP could fulfill similar functions as negative regulators of STAT-responsive genes (Starz-Gaiano, 2008).

All of the major growth factor and cytokine signaling pathways are subject to extensive positive and negative feedback regulation, which is crucial to generate appropriate physiological responses. The work presented here establishes APT as a feedback inhibitor of JAK/STAT signaling and cell invasion (Starz-Gaiano, 2008).

Evolutionarily conserved transcription factor Apontic controls the G1/S progression by inducing cyclin E during eye development

During Drosophila eye development, differentiation initiates in the posterior region of the eye disk and progresses anteriorly as a wave marked by the morphogenetic furrow (MF), which demarcates the boundary between anterior undifferentiated cells and posterior differentiated photoreceptors. However, the mechanism underlying the regulation of gene expression immediately before the onset of differentiation remains unclear. This study shows that Apontic (Apt), which is an evolutionarily conserved transcription factor, is expressed in the differentiating cells posterior to the MF. Moreover, it directly induces the expression of cyclin E and is also required for the G1-to-S phase transition, which is known to be essential for the initiation of cell differentiation at the MF. These observations identify a pathway crucial for eye development, governed by a mechanism in which Cyclin E promotes the G1-to-S phase transition when regulated by Apt (Liu, 2014).

Microarray analyses suggested that the bZIP transcription factor, Apontic, regulates the genes required for the neuron, trachea, oocyte, germ cell, bristle and eye development, apoptosis, and cell-cycle regulation. Among these candidates for Apt target genes, this study shows that Apt directly controls the expression of cyclin E and is required for the G1-to-S phase transition during eye development (Liu, 2014).

In the wild type, Cyclin E protein accumulates in a stripe of cells posterior to the MF. Cyclin E was not induced by N, Hh, or Dpp signal in the eye disk because Cyclin E accumulation occurred in Su(H) mutant cells or Mad1-2 Su(H) ci mutant cells. Ci, Mad, and Su(H) are the transcriptional targets of Hh, Dpp, and N signaling, respectively. A prior work suggested that the expression of cyclin E requires activation by both E2F/DP and tissue-specific activators. This study has shown that Apt and Cyclin E are coexpressed at the posterior cells to MF. The expression of Cyclin E was reduced in the apt mutant clones and induced by overexpression of Apt at the eye disk. Apt directly activated the expression of cyclin E at the posterior cells to MF. These data suggest that Apt and E2F1 function together in the activation of cyclin E in the eye disk (Liu, 2014).

Experiments involving ectopic expression of Apt in a GMR-Gal4/UAS-apt fly demonstrate that apt expression can induce S phase.Ectopic expression of Apt at the eye disk, therefore, has a similar effect as that of ectopic expression of cyclin E from a heat-inducible transgene (hs-cycE), suggesting that Apt and CyclinE work on the same pathway. CyclinE is essential and rate limiting for the G1-to-S phase transition. The current finding indicates that Apt controls G1-to-S phase progression by inducing cyclin E expression in the eye imaginal disk (Liu, 2014).

What is the function of Apt during development? Apt is expressed at trachea, head, heart, and CNS and is required for the development of these tissues. However, how Apt controls the development of these tissues is unknown. In this study, the candidates for Apt target genes were identified using microarray analysis. Many of these genes can be assigned to specific aspects of the development of these tissues (Liu, 2014).

Apt and Cyclin E are evolutionarily conserved from Drosophila to humans. However, the function of Apt in human is unknown. In humans, the expression of cyclin E is related to many cancers. Furthermore, Apt overexpression suppresses cancer metastasis in Drosophila. The role of Apt in the regulation of cyclin E might be a conserved function because its human homolog fibrinogen silencer binding protein (FSBP) is also a cancer-related factor and is also expressed in many tissues, including heart, brain, lung, liver, skeletal muscle, kidney, and pancreas. The discovery of a direct connection between cell differentiation and Cyclin E provides insights into the mechanism by which Apt promotes tumor formation (Liu, 2014).

Protein Interactions

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the Oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3' untranslated region of the Oskar mRNA. To begin to understand how Bruno acts in translational repression, a yeast two-hybrid screen was performed to identify Bruno-interacting proteins. One interactor proves to be the product of the apontic gene (Lie, 1999).

Apt is an RNA binding protein. Remarkably, the regions of the OSK 3' UTR bound by Apt, the AB and C regions, are precisely those bound by Bru. A test of Apt binding was performed to determine if Bru and Apt have the same RNA binding specificity: a series of RNAs was used to map the Bru binding sites, called BREs, within the OSK C region. Three of these RNAs retain the BREs and are bound by Bru, while a fourth RNA, CDelta4, lacks the BREs and fails to bind Bru. Apt binds all four RNAs, including CDelta4, indicating that Apt can bind to sites other than BREs (Lie, 1999).

Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. Mutants in apt are zygotic lethal, and some alleles also cause arrested oogenesis. Several different apt alleles were used for all analyses, since the genetics of apt are complex and different alleles have different effects. Testing for a genetic interaction between the aret (bruno) and apt mutants, dosages of both genes were reduced to see if this would provide a distinct phenotype. Females heterozygous for aret, heterozygous for any of the five apt alleles, or transheterozygous for both aret and an apt allele, were crossed to wild-type males, and the progeny embryos were then examined for cuticular defects. Females transheterozygous for aret and apt produce a fraction of embryos with head defects. Head defects can result from ectopic or excessive posterior body patterning activity, because this activity interferes with expression of the anterior body patterning morphogen, Bicoid. Consequently, the observed head defects could be explained if both Bru and Apt contribute to repression of OSK mRNA translation. Given this interaction, Apontic is likely to act together with Bruno in translational repression of Oskar mRNA (Lie, 1999).

Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the Oskar mRNA 3' untranslated region. A sequence shared by all of the bound RNAs could not be identified. Thus, despite its ability to efficiently discriminate between different parts of the OSK mRNA, Apt appears to be relatively promiscuous in its binding and may recognize many sites or perhaps a structural feature common to many RNAs (Lie, 1999).

Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems

During gene activation, the effect of binding of transcription factors to cis-acting DNA sequences is transmitted to RNA polymerase by means of co-activators. Although co-activators contribute to the efficiency of transcription, their developmental roles are poorly understood. Drosophila has been used to conduct molecular and genetic dissection of an evolutionarily conserved but unique co-activator, Multiprotein Bridging Factor 1 (MBF1), in a multicellular organism. Through immunoprecipitation, Drosophila Mbf1 was found to form a ternary complex including Mbf1, TATA-binding protein (TBP) and the bZIP protein Tracheae Defective (Tdf)/Apontic. A Drosophila mutant has been isolated. This mutant lacks the mbf1 gene; no stable association between TBP and TDF is detectable, and transcription of a TDF-dependent reporter gene is reduced by 80%. Although the null mutants of mbf1 are viable, tdf becomes haploinsufficient in mbf1-deficient background, causing severe lesions in tracheae and the central nervous system, similar to those resulting from a complete loss of tdf function. These data demonstrate a crucial role of MBF1 in the development of tracheae and central nervous system (Liu, 2003).

A cDNA encoding Drosophila Mbf1 was cloned from a larval CNS library. The predicted protein of 145 amino acids has 44%, 64% and 83% identity to MBF1 from yeast, human and silkmoth, respectively. MBF1 consists of two structural domains: a well-structured C-terminal half that binds the general transcription factor TBP; and a flexible N-terminal half that participates in binding to various activators. The region conserved in Drosophila Mbf1 includes both of these functional domains. Expression of Drosophila mbf1 cDNA partially rescued the yeast mbf1 mutant phenotype upon amino acid starvation, indicating that the ability to bind partner transcription factors is also conserved between yeast and Drosophila. In situ hybridization has revealed that a large amount of maternal mbf1 mRNA is deposited to the egg. Likewise, MBF1 protein is present in preblastoderm embryos and is later expressed in many tissues, including the CNS and the trachea. Widespread expression of MBF1 is also seen in post-embryonic stages, with particularly high levels in the larval salivary glands, gonads and adult gonads (Liu, 2003).

The relationship between Drosophila Mbf1 and Tdf is similar to that between yeast MBF1 and its partner transcription factor GCN4. Just as yeast MBF1 contacts GCN4 through its bZIP domain, Drosophila Mbf1 binds the bZIP domain of Tdf. Moreover, the lack of GCN4-dependent activation in yeast mbf1 mutant can be partially restored by expressing Drosophila Mbf1. The sequence and functional conservation between yeast and Drosophila Mbf1 indicates that the interaction with bZIP proteins is a conserved feature of the bridging factor MBF1 (Liu, 2003).

Genetic studies of mbf1 in yeast and Drosophila suggest that MBF1-associated transcription factors have two pathways for activation. In addition to the MBF1-mediated recruitment of TBP via its bZIP domain, GCN4 also recruits the SAGA complex with its N-terminal activation domain and effects transcription through chromatin modification. Likewise, Drosophila Tdf has a region similar to the glutamine-rich transactivation domain and may employ an activation pathway independent of recruiting TBP through Mbf1. Such a pathway may account for the residual expression of the TDS-lacZ reporter gene in the absence of Mbf1. Although Yeast MBF1 is essential for GCN4-dependent transcription of its target gene HIS3, low level of Tdf-dependent transcription of the TDS-lacZ gene can still occur in the absence of MBF1. This suggests that the relative importance of the two pathways is different between GCN4 and Tdf. The DNA-binding domain of Drosophila FTZ-F1 carries a basic region homologous to those in bZIP proteins and binds Mbf1 through this region. However, loss of mbf1 showed no effect on FTZ-F1-dependent transcription in vivo, suggesting that the activation by FTZ-F1 relied solely on the pathway through its transactivation domain. In an in vitro transcription system, the transactivation domain does not seem to be functional because FTZ622 polypeptide bearing only the DNA-binding domain of FTZ-F1 shows the same transcriptional activity as the intact FTZ-F1. This may explain the difference in the MBF1 requirement between FTZ-F1-dependent transcription in vivo and in vitro (Liu, 2003).

It is possible that the role of Mbf1 becomes more critical under certain circumstances, when rapid induction of gene expression is demanded by environmental conditions. The expression of the TDS-lacZ reporter in mbf1- background varies considerably from embryo to embryo, suggesting that certain conditions that are uncontrolled in these experiments may render transcription particularly dependent on the Mbf1-mediated pathway. In the natural environment, there are many stimuli that alter the gene expression profile: UV radiation, poison agents, nutrient starvation and so on. Therefore, direct recruitment of TBP by Mbf1 may become essential for rapid activation of transcription under such conditions. In agreement with this idea, the yeast mbf1 disruptant is viable under normal culture conditions, but sensitive to amino acid starvation (Liu, 2003).

Studies on MBF1 homologs also support the idea that MBF1 may function when gene expression is required in response to developmental or environmental signals. Rat MBF1 has been isolated as a calmodulin-associated peptide 19 (CAP-19) and human MBF1 has been identified as endothelial differentiation-related factor 1 (EDF1). EDF1/MBF1 is downregulated when endothelial cells are induced to differentiate. Interestingly, EDF1/MBF1 binds to calmodulin in the cytoplasm under low Ca2+ conditions but the two proteins dissociate when intracellular Ca2+ is high. The released EDF1/MBF1 is then phosphorylated and shuttled into the nucleus, where it binds TBP. Nuclear translocation of MBF1 has also been observed at a specific stage of molting in the silkworm B. mori. Considering the Ca2+ elevation upon exposure to the molting hormone ecdysteroid , these data raise an intriguing possibility that MBF1 is involved in Ca2+-induced gene activation. Although in this study the developmental roles of MBF1 were studied only in association with Tdf function, Drosophila Mbf1 may also be involved in other biological processes, such as stress response, homeostasis and longevity (Liu, 2003).

Several lines of evidence suggest that Drosophila Mbf1 has partners other than Tdf. Mbf1 is expressed in a wide spatiotemporal pattern, including tissues and stages where Tdf is absent. Although Tdf is not expressed in the salivary gland, immunolocalization of Mbf1 on salivary gland chromosome revealed a large number of loci associated with Mbf1. Furthermore, FLAG-tagged Mbf1 pulled down many proteins besides Tdf. Although mbf1-null mutants are viable under laboratory conditions, tdf becomes haploinsufficient in mbf1- genetic background, clearly indicating the importance of Mbf1 in the expression of the genomic information. This finding opens a way to identify new partners of Mbf1 through genetic screening for loci that exhibit dominant phenotypes in the absence of Mbf1. Characterization of Mbf1 partners will contribute to the knowledge of how co-activators mediate specific biological events (Liu, 2003).


Maternal and Embryonic

apontic expression occurs in both the somatic follicle cells and the germline nurse cells and oocyte. APT transcripts are detected as early as stage 2A at low levels in the germarium and at higher levels in the follicle cells. The amount of APT mRNA in the soma decreases during the remainder of oogenesis, while the level in the germline increases. APT mRNA becomes concentrated in the oocyte and also accumulates in the nurse cells at about stage 6. APT transcripts continue to be found in both the oocyte and nurse cells throughout oogenesis. To determine when and where Apt protein is expressed during oogenesis, antisera directed against a recombinant Apt protein were prepared and used for protein detection by confocal microscopy of whole-mount ovaries. Apt protein appears in both the germline and somatic cells of the ovary throughout all stages of oogenesis. In the germline, Apt protein is present in both cytoplasm and nuclei. Within the nurse cells the protein is more concentrated in the cytoplasm, while in the oocyte more protein is found in the nucleus. The protein, however, is not localized to any subdomain within the cytoplasm of either the nurse cells or the oocyte. Although Apt protein is not strictly nuclear or cytoplasmic in cells of the female germline, the protein is highly concentrated in nuclei of the ovarian follicle cells and in post-cellularization-stage embryos. The developmental differences in subcellular location suggest that Apt may have functions, perhaps different, in both nuclei and cytoplasm (Lie, 1999).

Nuclear proteins expressed from maternal mRNAs are sometimes present at high levels in the cytoplasm of early embryos. Examples include the Bicoid, Caudal and Hunchback proteins, which appear in both nuclei and cytoplasm shortly after egg laying. As nuclear divisions progress and the density of nuclei increases, nuclear localization of these proteins remains strong while the fraction of protein in the cytoplasm diminishes. Thus there appears to be no early impediment to nuclear localization, simply a paucity of nuclei. In contrast, the subcellular distribution of Apt protein appears to be actively controlled in early development. Apt protein was monitored in early embryos. Even after migration of nuclei to the surface of the embryo, Apt protein remains evenly distributed between nuclei and cytoplasm, unlike any of the examples described above. This unusual persistence of Apt protein in the cytoplasm suggests the existence of a mechanism to control its distribution, reinforcing the notion of roles for Apt in both cytoplasm and nuclei (Lie, 1999).

Transcripts from apt are expressed in a complex and dynamic pattern that includes, but is not limited to, the head regions affected in apt mutants. At the syncytial blastoderm stage maternal apt transcripts are distributed throughout the embryo. At cellular blastoderm, apt transcripts can be detected at both poles of the embryos. During germ band elongation most of the posterior expression disappears and a segmentally repeated pattern of expression arises in the trunk. apt transcripts in the trunk region are detected in cells along the ventral midline, in dorsal cells abutting the amnioserosa, and in the tracheal placodes (cells that will invaginate to form the respiratory tree). In the head of stage 9-10 embryos, apt is expressed in the dorsal part of the acron, the entire clypeolabrum and the ventral gnathal region. Transcripts can also be detected in the anterior-lateral regions of the mandibular, maxillary and labial lobes. During stage 11, apt transcripts accumulate at high levels in the dorsal ridge, and at this stage, transcript levels gradually disappear from the vicinity of the tracheal pits. As the amnioserosa is absorbed and dorsal closure ensues, dorsal mesodermal cells arranged in a single row on either side of the amnioserosa accumulate apt transcripts; later, these cells, still expressing APT transcripts, will contribute to the dorsal vessel. The patterned expression of apt also persists to late stages of embryogenesis in head epidermis and CNS (Gallon, 1997).

APT mRNA is maternally supplied. After gastrulation, at the beginning of germ band extension (stage 8), apt is uniformly expressed in the mesoderm and extends into the hindgut primordium. At stage 10, expression in the mesoderm ceases, except for the mesectodermal cells of the ventral midline. Later, during stage 11, apt expression is observed in the tracheal pits, the CNS, and the head region, as well as prominently in the heart progenitor cells. Expression in the heart precursors persists during the process of heart tube formation and in the differentiated heart during embryogenesis (Su, 1999).


Only a few genes have been identified that participate in the developmental pathways that modulate homeotic (HOX) protein specificity or mediate HOX morphogenetic function. To identify more HOX pathway genes, a screen was carried out for mutations in loci on the Drosophila second chromosome; a number of genes in this region interact with the homeotic gene Deformed. Genetic and molecular tests on the eight genes isolated in the screen place them in three general categories. (1) Two genes appear to encode trithorax group functions, i.e. they are general activators of Hox gene expression or function. (2) Four genes encode abundant, widely expressed proteins that may be required to mediate Dfd morphogenetic functions in certain tissues, including two genes for collagen IV protein variants. (3) two of the genes are required for the development of a subset of embryonic Dfd-dependent structures, while leaving many other segmental structures intact. One of these two was cloned and characterized. The cloned gene, apontic (apt), is required for the elaboration of dorsal and ventral head structures. The apt transcript pattern is normal in Dfd and Scr mutants, and the Dfd and Scr transcript patterns are normal in apt mutants. It is proposed that apt acts in parallel to, or as a cofactor with, HOX proteins to regulate homeotic targets in the ventral gnathal region (Gellon, 1997).

apontic is required for the formation of some but not all Deformed- and Sex combs reduced-dependent ventral gnathal structures. The lateral arms of the H-piece are missing and the lateralgraten are shortened in Dfd mutants; the hypostomal sclerites and dorsal pouch are missing in Scr mutants. Other Dfd- and Scr-dependent structures are intact in apt mutants, such as the ectostomal sclerites (Dfd-dependent), mouth hooks (Dfd-dependent) and cross bar of the H-piece (Scr-dependent). Thus, the apt phenotype suggests that apt might be contributing to the diversification of Dfd and Scr function in a specific cell population within each selector's domain. Such overlap in phenotypes could occur in principle by three different mechanisms: (1) apt could mediate Dfd and Scr functions by regulating their transcription in a particular region (i.e. apt acts upstream); (2) apt could be a target of Dfd and Scr in a discrete population of cells (apt acts downstream), or (3) apt could act in conjunction with Dfd and Scr (or with Dfd and Scr targets) to produce a distinct biological effect in a subpopulation of cells (apt acts in parallel). Whole mount in situ hybridizations of apt mutant embryos with DFD and SCR mRNA probes were performed their and patterns of transcription were found to be indistinguishable from wild type. Therefore, apt is not required to establish or maintain Dfd and Scr transcription. Conversely, apt transcription was examined in Dfd and Scr mutants by in situ hybridization and no changes were detected. Thus, neither Dfd nor Scr is required to establish or maintain apt transcription. It is concluded that apt acts in parallel with Dfd and Scr proteins to produce ventral gnathal structures (Gellon, 1997).

In an effort to isolate genes required for heart development and to further the understanding of cardiac specification at the molecular level, PlacZ enhancer trap lines were screened for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, is particularly interesting because of its early pattern of expression in cardiac precursor cells, an expression pattern dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. A gene was isolated and characterized in the vicinity of B2-2-15 that exhibits an identical expression pattern to that of the reporter gene of the enhancer trap. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles: segments of heart cells (those of myocardial and pericardial identity) are often missing. These abnormalities become obvious shortly before the assembly of the heart precursor cells at the dorsal midline. The defects in heart tube formation are seen with three markers: (1) Evenskipped, which is present in a subset of pericardial cells (EPC); (2) Mef2, which marks the cardial cells of the heart, and (3) Zfh-1, which is primarily present in the non-EPC pericardial cells. No obvious defects are observed in somatic and visceral muscle patterning, suggesting a specific requirement for apt in heart formation, as opposed to other mesodermal derivatives. Since the initial cardiac mesoderm seems to form normally in these mutants, it seems likely that apt is primarily required for a late differentiation step, such as the correct assembly of the heart tube. This would be consistent with a cell autonomous function of apt in the developing heart (Su, 1999).

apt mutant embryos or larvae develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. The heart rate of wildtype or apt heterozgous animals varies over a large range (28-146 beats per minute). The overall average heart rate is about 80 beats per minute. When moving, the apt homozygous mutant embryos and larvae have heart rates, on average, only about a quarter the heart rate in wildtype. The heart rate and cardiac defects may be the cause of death for apt mutants during late embryonic or early larval stages (Su, 1999).

Earlier genetic analyses of apt have concentrated on the zygotic phenotype. To define more completely the role of apt in the female germline, females were created with apt minus germline clones using the FLP/DFS method. Ovaries containing germline clones were dissected, stained with DAPI to highlight nuclei, and examined for phenotype. Different apt mutants display dramatically different ovarian phenotypes. One allele is indistinguishable from wild type, because females with germline clones of this allele have phenotypically wild-type ovaries and lay eggs that develop into fertile adults. Females with germline clones of another allele also have phenotypically wild-type ovaries, but a small fraction of the eggs laid develop into embryos with head defects. In contrast, ovaries from females with germline clones from two other mutants have phenotypes that are similar to one another and severe: development is arrested in early oogenesis, and the oocyte fails to differentiate, with all nuclei becoming polyploid. In addition, some of the egg chambers have an abnormal number of nuclei. It is concluded that apt is necessary for oogenesis and that loss of apt activity leads to a developmental arrest during oogenesis. Just as for arrest (bruno) mutants, the developmental arrest occurs too early to allow the ovaries to be examined for defects in OSK mRNA translation (Lie, 1999).


Search PubMed for articles about Drosophila apontic

Boerjan, B., Verleyen, P., Huybrechts, J., Schoofs, L. and De Loof, A. (2010). In search for a common denominator for the diverse functions of arthropod corazonin: a role in the physiology of stress? Gen Comp Endocrinol 166: 222-233. PubMed ID: 19748506

Boube, M., Llimargas, M. and Casanova, J. (2000). Cross-regulatory interactions among tracheal genes support a co-operative model for the induction of tracheal fates in the Drosophila embryo. Mech. Dev. 271-278. PubMed ID: 10704851

Cazzamali, G., Saxild, N. and Grimmelikhuijzen, C. (2002). Molecular cloning and functional expression of a Drosophila corazonin receptor. Biochem Biophys Res Commun 298: 31-36. PubMed ID: 12379215

Eulenberg, K. G. and Schuh, R. (1997). The tracheae defective gene encodes a bZIP protein that controls tracheal cell movement during Drosophila embryogenesis. EMBO J. 16(23): 7156-7165. PubMed ID: 9384592

Gellon, G., et al. (1997). A genetic screen for modifiers of Deformed homeotic function identifies novel genes required for head development. Development 124(17): 3321-3331. PubMed ID: 9310327

Gunkel, N., Yano, T., Markussen, F. H., Olsen, L. C. and Ephrussi, A. (1998). Localization-dependent translation requires a functional interaction between the 5' and 3' ends of Oskar mRNA. Genes Dev. 12: 1652-64. PubMed ID: 9620852

Lie, Y. S. and Macdonald, P. M. (1999). Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation. Development 126: 1129-1138. PubMed ID: 10021333

Liu, Q.-X., et al. (2003). Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems. Development 130: 719-728. 12506002

Liu, Q. X., Wang, X. F., Ikeo, K., Hirose, S., Gehring, W. J., Gojobori, T. (2014) Evolutionarily conserved transcription factor Apontic controls the G1/S progression by inducing cyclin E during eye development. Proc Natl Acad Sci U S A 111: 9497-9502. PubMed ID: 24979795

McClure, K. D. and Heberlein, U. (2013). A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila. J Neurosci 33: 4044-4054. PubMed ID: 23447613

Scholz, H., Franz, M. and Heberlein, U. (2005). The hangover gene defines a stress pathway required for ethanol tolerance development. Nature 436: 845-847. PubMed ID: 16094367

Starz-Gaiano, M., Melani, M., Wang, X., Meinhardt, H. and Montell, D. J. (2008). Feedback inhibition of Jak/STAT signaling by apontic is required to limit an invasive cell population. Dev Cell 14: 726-738. PubMed ID: 18477455

Su, M. T., et al. (1999). The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart. Mech. Dev. 80(2): 125-32. PubMed ID: 10072779

Veenstra, J. A. (2009). Does corazonin signal nutritional stress in insects? Insect Biochem Mol Biol 39: 755-762. PubMed ID: 19815069

Wilk, R., Pickup, A. T., Hamilton, J. K., Reed, B. H. and Lipshitz, H. D. (2004). Dose-sensitive autosomal modifiers identify candidate genes for tissue autonomous and tissue nonautonomous regulation by the Drosophila nuclear zinc-finger protein, Hindsight. Genetics 168(1): 281-300. 15454543

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Biological Overview

date revised: 25 September 2023

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