the-utility-stage-specific-mid-to-late-drosophila-follicle

The Utility of Stage-specific Mid-to-late <em>Drosophila</em> Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

University of Iowa Carver College of Medicine

ETS family transcription factors serve as downstream effectors of signal transduction pathways, mediating cellular proliferation, differentiation and, when misregulated, tumorigenesis. The transcriptional repressor YAN prevents inappropriate responses to Receptor Tyrosine Kinase signaling by outcompeting POINTED for access to target gene promoters. We demonstrate that the molecular mechanism underlying downregulation of YAN involves CRM1-mediated nuclear export and define a novel role in this context for MAE, a co-factor previously implicated in facilitating MAPK phosphorylation of YAN. In addition to promoting YAN downregulation, MAE also participates in an inhibitory feedback loop that attenuates POINTED-P2 activation. Thus, we propose that MAE plays multiple independent roles in fine-tuning the levels of POINTED and YAN activity in accordance with changing RTK signaling conditions.

CRM1-mediated nuclear export and regulated activity of the Receptor Tyrosine Kinase antagonist YAN require specific interactions with MAE.

Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.

The transcription factor Eyes absent is a protein tyrosine phosphatase.

Ets transcription factors play crucial roles in regulating diverse cellular processes including cell proliferation, differentiation and survival. Coordinated regulation of the Drosophila Ets transcription factors YAN and POINTED is required for eliciting appropriate responses to Receptor Tyrosine Kinase (RTK) signaling. YAN, a transcriptional repressor, and POINTED, a transcriptional activator, compete for regulatory regions of common target genes, with the ultimate outcome likely influenced by context-specific interactions with binding partners such as MAE. Previous work in cultured cells has led us to propose that MAE attenuates the transcriptional activity of both YAN and POINTED, although its effects on POINTED remain controversial. Here we describe a new layer of complexity to this regulatory hierarchy whereby mae expression is itself directly regulated by the opposing action of YAN and POINTED. In addition, we report that MAE can antagonize POINTED function during eye development; a finding that suggests MAE operates as a dual positive and negative regulator of RTK-mediated signaling in vivo. Together our results lead us to propose that a combination of protein-protein and transcriptional interactions between MAE, YAN and POINTED establishes a complex regulatory circuit that ensures that both down-regulation and activation of the RTK pathway occur appropriately according to specific developmental context.

MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN.

Transcription factors provide nodes of information integration by serving as nuclear effectors of multiple signaling cascades, and thus elaborate layers of regulation, often involving post-translational modifications, modulating and coordinate activities. Such modifications can rapidly and reversibly regulate virtually all transcription factor functions, including subcellular localization, stability, interactions with cofactors, other post-translational modifications and transcriptional activities. Aside from analyses of the effects of serine/threonine phosphorylation, studies on post-translational modifications of transcription factors are only in the initial stages. In particular, the regulatory possibilities afforded by combinatorial usage of and competition between distinct modifications on an individual protein are immense, and with respect to large families of closely related transcription factors, offer the potential of conferring critical specificity. Here we will review the post-translational modifications known to regulate ETS transcriptional effectors and will discuss specific examples of how such modifications influence their activities to highlight emerging paradigms in transcriptional regulation.

Post-translational modifications influence transcription factor activity: a view from the ETS superfamily.

Post-translational modifications such as phosphorylation provide versatile context-specific strategies for modulating transcription factor activity. In the prevailing view of this process, modifying enzymes indirectly influence gene expression by shuttling to and from the nucleus where they alter the activity of their target transcription factors. However, a new paradigm has recently been suggested from studies of Eyes absent (EYA), a member of a conserved network of transcriptional regulators implicated in the development of numerous tissues and organs including the eye, ear, muscle and kidney. These findings indicate that EYA operates both as a transcriptional coactivator and as the prototype of a novel class of protein tyrosine phosphatases. The regulatory potential of such a bifunctional transcription factor is enormous and suggests a new layer of dynamic regulation in which transcription factors themselves might provide intrinsic enzymatic activities to fine-tune nuclear output.

New vision from Eyes absent: transcription factors as enzymes.

Eyes absent (EYA) proteins are defined by a conserved C-terminal EYA domain (ED) that both contributes to its function as a transcriptional coactivator by mediating protein-protein interactions and possesses intrinsic protein tyrosine phosphatase activity. Mutations in human EYA1 result in an autosomal dominant disorder called branchio-oto-renal (BOR) syndrome as well as congenital cataracts and ocular defects (OD). Both BOR- and OD-associated missense mutations alter residues in the conserved ED as do three missense mutations identified from Drosophila eya alleles. To investigate the molecular mechanisms whereby these mutations disrupt EYA function, we tested their activity in a series of assays that measured in vivo function, phosphatase activity, transcriptional capability, and protein-protein interactions. We find that the OD-associated mutations retain significant in vivo activity whereas those derived from BOR patients show a striking decrease or loss of in vivo functionality. Protein-protein interactions, either with its partner transcription factor Sine oculis or with EYA itself, were not significantly compromised. Finally, the results of the biochemical assays suggest that both loss of protein tyrosine phosphatase activity and reduced transcriptional capability contribute to the impaired EYA function associated with BOR/OD syndrome, thus shedding new light into the molecular mechanisms underlying this disease.

Using Drosophila to decipher how mutations associated with human branchio-oto-renal syndrome and optical defects compromise the protein tyrosine phosphatase and transcriptional functions of eyes absent.

Prostaglandins are local transient hormones that mediate a wide variety of biological events, including reproduction. The study of prostaglandin biology in a genetically tractable invertebrate model organism has been limited by the lack of clearly identified prostaglandin-mediated biological processes and prostaglandin metabolic genes, particularly analogs of cyclooxygenase (COX), the rate-limiting step in vertebrate prostaglandin synthesis. Here, we present pharmacological data that Drosophila ovarian follicle maturation requires COX-like activity and genetic evidence that this activity is supplied in vivo by the Drosophila peroxidase Pxt. pxt mutant females are sterile, and maturing follicles show defects in actin filament formation, nurse cell membrane stability and border cell migration. Maturation of pxt follicles in vitro is stimulated by prostaglandin treatment and fertility is restored in vivo to pxt mutants by expressing mammalian Cox1 protein. Our experiments suggest that prostaglandins promote Drosophila follicle maturation, in part by modulating the actin cytoskeleton, and establish Drosophila oogenesis as a model for understanding these critical biological regulators.

Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation.

Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals.

Drosophila eggshell production: identification of new genes and coordination by Pxt.

Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling.

Prostaglandins (PGs) are lipid signals that are produced at their sites of action by cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs), and PG-type specific synthases. Active PGs serve as ligands for G protein-coupled receptors (GPCRs). The functions of PGs have largely been elucidated using pharmacologic, expression-based (synthesis and signaling components), and genetic studies. In this review, we discuss the in vivo roles of PGs in cancer, development, and reproduction that have been characterized using genetic knockout/knockdown and overexpression approaches in mice, zebrafish, and invertebrate model systems, and how pharmacologic inhibition of PG synthesis affects cardiovascular health/disease and cancer incidence and progression.

Genetic insights into the in vivo functions of prostaglandin signaling.

While Prostaglandins (PGs), lipid signals produced downstream of cyclooxygenase (COX) enzymes, regulate actin dynamics in cell culture and platelets, their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton-temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). Additionally, loss of Pxt results in early actin remodeling, including extensive actin filaments and aggregates, within the posterior nurse cells of stage 9 (S9) follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts), the homolog of Adducin, and Villin (Quail), an actin bundler, localize to all early actin structures, while Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to the model that PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, while at S10B, it promotes Ena-dependent actin remodeling.

Article	Year
CRM1-mediated nuclear export and regulated activity of the Receptor Tyrosine Kinase antagonist YAN require specific interactions with MAE. Development (Cambridge, England)\| PubMed ID: 12538513	2003
The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature\| PubMed ID: 14628053	2003
MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN. Mechanisms of development\| PubMed ID: 15511639	2004
Post-translational modifications influence transcription factor activity: a view from the ETS superfamily. BioEssays : news and reviews in molecular, cellular and developmental biology\| PubMed ID: 15714552	2005
New vision from Eyes absent: transcription factors as enzymes. Trends in genetics : TIG\| PubMed ID: 15734575	2005
Using Drosophila to decipher how mutations associated with human branchio-oto-renal syndrome and optical defects compromise the protein tyrosine phosphatase and transcriptional functions of ... Genetics\| PubMed ID: 15802522	2005
Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation. Development (Cambridge, England)\| PubMed ID: 18216169	2008
Drosophila eggshell production: identification of new genes and coordination by Pxt. PloS one\| PubMed ID: 21637834	2011
Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling. Molecular biology of the cell\| PubMed ID: 23051736	2012
Genetic insights into the in vivo functions of prostaglandin signaling. The international journal of biochemistry & cell biology\| PubMed ID: 23685076	2013
Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis. Molecular biology of the cell\| PubMed ID: 24284900	2013

Tina L. Tootle

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