anterior open/yan

DEVELOPMENTAL BIOLOGY

Embryonic

At state 10, low levels of yan are expressed uniformly in most regions of the epidermis. During germband shortening, yan becomes restricted to a small number of cells that correspond to the tracheal system (Lai, 1992). Overexpressing yan in the mesoderm blocks embryonic mesodermal differentiation (Rebay, 1995).

Larval

In third instar larvae, Yan is seen in the eye imaginal disc in and near the morphogenetic furrow. Posterior to the furrow (i.e., after it passes), only basally located nuclei of uncommitted cells express high levels of Yan protein (Lai, 1992). Overexpressing yan in the eye blocks differentiation of non-neuronal cells (Rebay, 1995).

A comparative study of Pointed and Yan expression reveals new complexity to the transcriptional networks downstream of receptor tyrosine kinase signaling

The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal this study used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt-Yan network, numerous instances were found where Pnt-GFP and Yan adopt a mutually exclusive pattern of expression. However, many examples were also found of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response (Boisclair Lachance, 2014).

Oogenesis

Function of the ETS transcription factor Yan in border cell migration

Invasive cell migration in both normal development and metastatic cancer is regulated by various signaling pathways, transcription factors and cell-adhesion molecules. The coordination between these activities in the context of cell migration is poorly understood. During Drosophila oogenesis, a small group of cells called border cells (BCs) exit the follicular epithelium to perform a stereotypic, invasive migration. The ETS transcription factor Yan is required for border cell migration and Yan expression is spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor tyrosine kinase pathways (Egfr and Pvr) in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. These results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells (Schober, 2005).

To distinguish whether PVR and EGFR regulate Yan expression at the transcriptional or post-transcriptional level, activated PVR was expressed using slbo-Gal4 in a yan^P(lacZ) background. BCs that do not express activated PVR are clearly distinguishable from activated PVR-expressing BCs by their migration defects in stage 10 egg chambers. Yan has been described as suppressing its own transcription, and loss of Yan protein might thus result in activation of the yan^P(lacZ) reporter due to an autoregulatory feedback loop. Interestingly, BCs that express activated PVR strongly express ß-Gal, suggesting that PVR activation controls Yan expression post-transcriptionally, which is further supported by RNA in situ hybridization data using a yan-specific probe (Schober, 2005).

The data support a model where JAK/STAT and Notch signaling specify anterior terminal cells including BCs, resulting in a strong expression of Yan in BCs; increasing RTK activity can decrease Yan expression as BCs approach their destination (Schober, 2005).

The Notch and RTK signaling pathways function to control AP axis specification at early stages of oogenesis, resulting in expression of the ETS transcription factor pointed (pnt) at the posterior pole. In photoreceptor cells, RTK activation induces the downregulation of Yan, which subsequently allows pnt expression and a switch in cell fate. Thus, tests were performed to see whether Yan expression at the initiation of BC migration might suppress pnt expression, and Yan downregulation at the nurse cell-oocyte boundary might lead to pnt expression, and therefore, potentially, induce BC differentiation. RNA in situ hybridization data and analysis of pnt^P(lacZ) expression in ovaries revealed that pnt is not expressed in BCs at any stage of oogenesis, and ectopic expression of slbo-Gal4::UAS-pntP2 does not alter BC motility. Furthermore, ectopic activation of PVR in BCs downregulates Yan expression and delays BC migration without induction of pnt^P(lacZ) in BCs. Thus it is concluded that although the Notch and RTK signaling pathways modulate Yan expression levels in both photoreceptor cells and BCs, the mechanisms used are not identical, and the transcriptional responses and downstream mechanisms depend, at least in part, on the developmental context (Schober, 2005).

This study reveals that during oogenesis yan mutant BCs are defective in their invasive migratory behavior. In addition, Yan is upregulated as BCs exit the epithelium to become migratory, and that subsequently Yan protein levels decay as BCs approach the nurse cell-oocyte boundary. Because Yan functions as a transcriptional repressor and an inhibitor of neuronal differentiation, whether it regulates BC identity was examined. Although this possibility cannot be completely excluded, BC markers are properly expressed in the absence of Yan. Thus, it is proposed that Yan promotes BC motility, an hypothesis which is supported by the observations that: (1) Yan is upregulated prior to the BCs exiting the follicular epithelium to become migratory; (2) Yan protein levels decrease progressively as BCs approach their final destination; and (3) yan mutant BCs exhibit a delay in migration. Interestingly, ectopic expression of constitutively activated Yan in BCs also delays their migration, suggesting that the spatiotemporal activity of Yan protein needs to be precisely controlled during the migratory process (Schober, 2005).

The dynamic expression of Yan is crucial for BC migration, as indicated by the migratory defects associated with both gain- and loss-of-function alleles of yan. Analysis of mutations in the JAK/STAT and Notch signaling pathways reveals that they are required for the expression of at least two transcription factors that are crucial for BC migration and which themselves influence DE-Cad activity. Slbo is specifically expressed in BCs and enhances shg transcription. Yan, by contrast, is expressed in anterior terminal cells, but becomes upregulated in BCs at the time they exit from the epithelium to become migratory. Yan might enhance DE-Cad turnover to facilitate the transition from an immobile epithelial state to a migratory one. Enhanced BC migration defects of hypomorphic slbo mutant egg chambers overexpressing Yan further underscore their interaction to regulate DE-Cad expression and BC migration (Schober, 2005).

Interestingly, Yan expression levels gradually decrease as BCs move along an increasing PVR/EGFR activity gradient. Yan has been shown to be phosphorylated by the EGFR-MAPK pathway, which triggers its nuclear export and protein degradation. Consistent with these previous studies, expression of dominant-active PVR and EGFR in BCs blocks BC migration and abrogates Yan protein expression, whereas yan transcript or enhancer trap expression is still detectable. Expression of activated Ras and Raf similarly induced Yan downregulation, consistent with an involvement of the canonical Ras/MAPK pathway in mediating PVR/EGFR signaling. It is noted, however, that although BC migration is significantly delayed upon ectopic expression of activated Ras, activated Raf hardly affects their ability to migrate. The basis of this difference, which might be due to complex feedback loops between the implicated signaling pathways, is unclear at the present time and will need to be investigated further (Schober, 2005).

Is the function of Yan to facilitate the transition of BCs from an epithelial to a migratory state, or to promote their motility? Although E-Cadherin is often downregulated as cells transit from an epithelial to a mesenchymal-like migratory state, this may not be the case in BCs, since DE-Cad is strongly expressed in BCs and shg mutant BCs fail to migrate. However, BCs mutant for yan or Ecdysone hormone co-receptor taiman (tai) accumulate ectopic DE-Cad-containing adhesive complexes. Consistent with these observations, ectopic stimulation of PVR in BCs, which enhances tai mutant BC migration defects, also results in elevated, cortical DE-Cad staining. Even though the observed BC migration defects in these mutants might not be due to altered surface levels of DE-Cad only, it was found that overexpression of DE-Cad alone can cause migration impaired BCs. E-cadherin not only mediates homophilic cell-cell adhesion but also functions together with its binding partners as a key regulator of the cortical actin cytoskeleton. It is therefore interesting to note that follicle cells overexpressing DE-Cad show severely enhanced filamentous actin staining (Schober, 2005).

The experiments revealed that DE-Cad was elevated in yan mutant BCs and suppressed upon expression of UAS-yan^ACT, suggesting that Yan controls, at least in part, DE-Cad expression in BCs. These observations find further support in the partial rescue of slbo-Gal4::UAS-yan^ACT-induced BC migration defects upon co-expression of UAS-DE-Cad. How does Yan affect DE-Cad expression in BCs? Although the function of Yan as a transcriptional repressor in various tissues suggests that it may act as a transcriptional regulator of shg, no change was detected in shg transcription in yan mutant follicle cells. However, increased membrane dye FM1-43 incorporation in Drosophila SL2 cells overexpressing Yan^ACT, and a decrease in incorporation after yan^RNAi, suggests a change in endocytic activity. E-Cadherin has been found in endocytic compartments and endocytosis has been speculated to modulate E-Cadherin activity regulation during morphogenetic movements. Interestingly, blocking endocytosis by the expression of dominant-negative Rab5 leads to severe BC migration defects and increased DE-Cad staining. Consistent with these observations, expression of shg under a heterologous promoter has been shown to rescue shg mutant BC migration defects, suggesting that the dynamic expression of DE-Cad in BCs might depend on both transcriptional and post-transcriptional mechanisms. Based on these results, a model is favored whereby Yan might, at least in part, function to regulate DE-Cad turnover, possibly through the transcriptional regulation of as-yet-unidentified components of the endocytic machinery (Schober, 2005).

ETS transcription factors are not only regulators of morphogenetic processes but have also been identified as oncogenes. Indeed, several ETS factors are upregulated in invasive cancers and are currently used as molecular markers to grade their invasiveness. The molecular function of ETS factors in tumorigenesis is not clear, as they can act as both oncogenes and tumor suppressors. The observations that yan is associated with similar gain- and loss-of-function phenotypes support both a positive and negative function on invasive migration, dependent on activity levels and possibly on available cofactors. Furthermore, the complexity of invasive tumors makes it difficult to assess what function ETS factors have, as they are upregulated not only in the cancerous tissue but also, for example, in forming blood vessels during tumor angiogenesis. Finally, the finding that Yan levels are regulated by JAK/STAT, Notch and RTK signaling pathways, which have been implicated in metastatic cancer, is another strong connection between Yan-like ETS factors and tumorigenesis (Schober, 2005).

Effects of Mutation or Deletion

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called ‘head midline’) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

Mutations in yan are recessive and result in a decrease in viability and fertility. The only morphological defect is a roughening of the exterior of the eye, resulting from extra photoreceptor cells. The extra cells are R7, the last of eight photoreceptors to form in each ommatidium. A decrease in yan function enhances the extra R7 cell phenotype produced by activated Gap1 and Ras1. This suggests that wild type yan acts antagonistically to Ras1 and Gap1 activation in the process of R7 cell-fate determination (Lai, 1992).

A collection of transposable-element-induced mutations have been screened for those which are dominant modifiers of the extra R7 phenotype of a hypomorphic yan mutation. The members of one of the identified complementation groups correspond to disruptions of the tramtrack gene. As heterozygotes, ttk alleles increase the percentage of R7 cells in yan mutant eyes. Just as yan mutations increase ectopic R7 cell formation, homozygous ttk mutant eye clones also contain supernumerary R7 cells. However, in contrast to yan, the formation of these cells in ttk mutant eye tissue is not necessarily dependent on the activity of the sina gene. Furthermore, although yan mutations are dominant in interactions with mutations in the Ras1, Draf, Dsor1, and rolled genes to influence R7 cell development, ttk mutations only interact with yan and rl gene mutations to affect this signaling pathway. These data suggest that yan and ttk both function to repress inappropriate R7 cell development but that their mechanisms of action differ. In particular, TTK activity appears to be autonomously required to regulate a sina-independent mechanism of R7 determination (Lai, 1996).

Genetic interactions were studied of yan with downstream components of the Sevenless pathway. yan mutation suppresses dominantly mutant raf and rolled eye phenotypes allowing single R7 cells to develop in ommatidia. raf mutation improves adult viability of mutant yan homozygotes. A reduced activity of tramtrack results in enhancement of the mutant yan phenotype. ttk mutations produce extra R7 cells even in sina homozygotes while the yan mutation does not. This results indicates thet TTK represses R7 induction downstream of the sites were YAN and SINA function (Yamamoto, 1996).

The Drosophila yan gene encodes an ETS domain nuclear protein with a transcription repressor activity that can be downregulated through phosphorylation by mitogen-activated protein kinase (MAPK). Before photoreceptor precursor cells commit to a particular cell fate, Yan is required to maintain them in an undifferentiated state. tramtrack (ttk) mutations have been identified that act as dominant enhancers of yan. ttk synergistically interacts with yan to inhibit the R7 photoreceptor cell fate. Since ttk products are nuclear proteins with zinc-finger DNA-binding motifs, yan and ttk represent two nuclear regulators essential for the control of cellular competence for neural differentiation. Reduction of either yan or ttk activity suppresses eye phenotypes of the kinase suppressor of ras (ksr) gene mutation, which is consistent with the involvement of yan and ttk in the Ras/MAPK pathway. Based on the fact that yan acts upstream of sina and ttk acts downstream of sina, it is expected that interaction between yan and ttk does not occur through direct protein associations. A more likely scenario is that yan controls expression of downstream genes that are critical for regulating ttk expression or function. A strong candidate target gene is phyllopod, which acts downstream of yan and upstream of sina (Lai, Z.-C., 1997).

An allele of the yan locus has been isolated as an enhancer of the Ellipse mutation of the Drosophila Egf-r gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations. Phenotypic and complementation analysis reveals that aop is allelic to yan; genetically the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones shows that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. These studies suggest that the role of Yan is defined by the developmental context of the cells in which it functions. An important role of this gene is in allowing a cell to choose between cell division and differentiation (Rogge, 1995).

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. faf also interacts with pointed: the extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism (Isaksson, 1997).

In the developing Drosophila eye, BarH1 and BarH2, paired homeobox genes expressed in R1/R6 outer photoreceptors and primary pigment cells, are essential for normal eye morphogenesis. BarH1 was ectopically expressed under the control of the sevenless enhancer (sev-BarH1). The sev enhancer drives gene expression strongly, not only in R7 precursors but also in R3/R5 and cone cell precursors. Evidence is presented that sev-BarH1 causes two types of cone cell transformation: transformation of anterior/posterior cone cells into outer photoreceptors and transformation of equatorial/polar cone cells into primary pigment cells. The ectopic primary pigment cells are partially similar in morphology to cone cells. sev-BarH1 represses the endogenous expression of the rough homeobox gene in R3/R4 photoreceptors, while the BarH2 homeobox gene is activated by sev-BarH1 in an appreciable fraction of extra outer photoreceptors. In primary pigment cells generated by cone cell transformation, the expression of cut, a homeobox gene specific to cone cells, is completely replaced with that of Bar homeobox genes. Extra outer photoreceptor formation is either suppressed or enhanced, respectively, by reducing the activity of Ras/MAPK signaling or by dosage reduction of yan, a negative regulator of the pathway, suggesting interactions between Bar homeobox genes (cell fate determinants) and Ras/MAPK signaling in eye development. It is concluded that cone cell precursors may adopt four different cell fates: an outer photoreceptor fate, a primary pigment cell fate, a cone cell fate, or the fate of disappearance f from ommatidia (X-cell fate). Cone cell precursors appear to be divided into two subgroups with respect to sensitivity to sev-BarH1: either anterior/posterior or equatorial/polar cone cell precursors. sev-BarH1 causes transformation of a fraction of anterior/posterior cone cells into outer photoreceptors partially expressing R1/R6-specific genes and also causes the transformation of a fraction of equatorial/polar cone cells into primary pigment cells; this suggests that BarH1 serves as a determinant of R1/R6 and primary pigment cell fates in normal eye development (Hayashi, 1998).

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Ectopic expression of a constitutively active protein (yan^ACT) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, a genetic screen was performed to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan^ACT. Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes recovered are four RTK pathway components [rolled (MAPK), son-of-sevenless, Star, and pointed], and two genes (eyes absent and string) that have not been implicated previously in RTK signaling events. Mutations in five previously uncharacterized genes were also recovered. One of these, split ends, has been characterized molecularly and is shown to encode a member of the RRM family of RNA-binding proteins (Rebay, 2000).

spen encodes a predicted protein of 5476 amino acids. Database searches indicate Spen belongs to a family of RRM proteins. The RRM is a loosely conserved RNA binding domain of ~22 conserved amino acids spread over an 80 to 100-amino-acid-long region. The most highly conserved sequences within the RRM motif are two ribonucleoprotein (RNP) domains designated RNP2 (a hexapeptide) and RNP1 (an octapeptide). Other conserved residues are scattered throughout the domain and include primarily hydrophobic amino acids. Spen contains three RRM motifs in tandem toward the N terminus of the protein (amino acids ~500-750). The Spen RRMs are most similar to RRMs in several novel proteins of unknown function. These include proteins predicted from conceptual translation of the BDGP Drosophila genomic sequence and the Caenorhabditis elegans genomic sequence, and proteins predicted from conceptual translation of human and mouse EST sequences. The first RRM of Spen is most divergent from the RRM consensus, and is also more loosely conserved among other spen-like proteins. Homology between Spen and other known RRM proteins in Drosophila or other species is less striking and strictly limited to residues defining the RRM consensus sequence. Thus, Spen may define a new subclass of RRM proteins. Other motifs in Spen include a predicted coiled coil region over amino acids 1857-1922 and amino acids 1979-2014 that could be suggestive of protein-protein interactions and a highly conserved C-terminal domain of unknown function that is found in proteins from worms to humans. Otherwise, the Spen protein sequence appears novel. Because some of the sequences identified by the database searches correspond to short EST sequences, it will be necessary to isolate full-length cDNA clones in order to determine whether these proteins contain both the RRM and the C-terminal domain. However, both motifs are found in a second Drosophila protein and in a human protein, suggesting that Drosophila Spen is a member of a novel family of proteins defined by both the RRM and C-terminal motifs (Rebay, 2000).

The isolation of spen as an enhancer of yan^ACT suggests it may play a role as a positive regulator of the RTK/Ras pathway. Preliminary results indicate Spen is a nuclear protein broadly expressed in most tissues and enriched in neuronal lineages. It is not known whether spen functions upstream or downstream of yan. One possibility is that spen might regulate the stability of the yan transcript. It has been postulated that the mechanism for downregulating yan activity involves post-translational modifications of the protein, namely phosphorylation by activated MAPK, that subsequently targets yan for degradation. Such post-translational regulation of yan would presumably need to be reinforced at the transcriptional and/or translational level. Thus, spen might play a role in destabilizing yan mRNA in response to Ras signaling. This would be consistent with the isolation of mutations in spen as enhancers of yan^ACT. Alternatively, Spen could be transcriptionally regulated by yan, and could play a role in splicing, stability, or transport of other downstream effector genes. Future phenotypic, genetic, and biochemical characterization of spen will be necessary to understand its role in Ras/yan signaling events (Rebay, 2000).

Among the remaining seven pathway relevant candidates are two genes of known function that have not been implicated previously in RTK/Ras-mediated signaling events. One of these groups, SY3-4, is allelic to the phosphatase string, a Drosophila homolog of the yeast cell cycle gene cdc25. string regulates the G2-M transition in dividing cells by dephosphorylating and activating the cdc2 kinase, thereby allowing formation of cyclin/cdc2 complexes that promote S phase. On the basis of the direction of interaction with yan^ACT, string would be postulated to be an antagonist of Ras signaling. Previous suggestions of a possible antagonistic relationship between string and Ras signaling come from a screen for modifiers of roughex, a negative regulator of G1 progression in the developing eye, which has identified string as a suppressor and ras1 as an enhancer (Rebay, 2000).

yan itself has been implicated in cell cycle control. Whereas hypomorphic yan mutations are semi-viable and have an extra photoreceptor phenotype, null mutations in yan are embryonic lethal, with the embryos dying as a result of overproliferation of cells in the dorsal neuroectoderm. Thus, depending on the developmental context, yan regulates not only the transition between undifferentiated and differentiated cell types, but also the choice between differentiation and cell division. Recovery of string alleles in this screen could reflect cross-talk between cell cycle and differentiation pathways that occurs in part at the level of transcriptional regulation. Thus, it is possible that the downstream targets of yan will include cell cycle regulators, such as string, or that yan expression and stability may be linked to cell cycle controls. Alternatively, String could have postmitotic functions essential to differentiation (Rebay, 2000).

string has not been isolated in any other Ras pathway screens; however, it was isolated, again as a suppressor, in a screen for modifiers of activated Notch. The direction of interaction suggests string acts as a positive regulator of Notch signaling. Expression of N^ACT and yan^ACT have similar developmental consequences since both inhibit or delay differentiation of the cell types in which they are expressed. Isolation of string in both screens could indicate a point of cross-talk between the Notch and the RTK/Ras pathway. Alternatively, isolation of string as a suppressor of both Notch^ACT and yan^ACT could have more to do with the similar terminal phenotype of these two backgrounds rather than reflecting direct interactions with the two pathways. Supporting the first hypothesis, cross-talk between the Notch and RTK pathways has been reported by numerous labs. Despite all the genetic interaction data, the mechanisms whereby the Notch and RTK pathways intersect remain to be determined. Experiments designed to study signaling by both pathways in vivo have suggested an antagonistic relationship, which would be consistent with string acting as a negative regulator of Ras signal transduction and a positive regulator of Notch signal transduction (Rebay, 2000 and references therein).

The second gene with a previously defined function that may be relevant to the RTK pathway on the basis of these genetic tests is eyes absent (eya. eya encodes a novel nuclear protein of unknown function that functions in a hierarchy of 'master eye regulatory genes' that are required to specify and promote differentiation of eye tissue. However, on the basis of expression pattern and phenotypes, it is possible that eya plays additional roles in development independent of its role in determining competence to become eye tissue. One possibility is that eya could be directly complexed with yan, and could direct its transcriptional repressor activity in certain tissues. However, preliminary yeast two-hybrid experiments have failed to indicate Yan-Eya protein-protein interactions. An alternate possibility to be investigated is transcriptional regulation of eya by Yan. Given the genetic interactions observed between eya and yan^ACT, it will be interesting to investigate the possible role of eya in RTK/yan-mediated signaling events in the embryo and developing eye. It could be that in order to differentiate as eye tissue, a developing cell must receive both a 'general' differentiation signal from the RTK pathway and a more specific eye fate specification signal (Rebay, 2000 and references therein).

Signaling by Egfr, the Drosophila epidermal growth factor receptor tyrosine kinase (RTK), is essential for proper migration and survival of midline glial cells (MGCs) in the embryonic central nervous system (CNS). A gene called split ends (spen) was isolated in a screen designed to identify new components of the RTK/Ras pathway. Drosophila Spen and its orthologs are characterized by a distinct set of RNA recognition motifs (RRMs) and a SPOC domain, a highly conserved carboxy-terminal domain of unknown function. To investigate spen function in the context of RTK signaling, the consequences of spen loss-of-function mutations on embryonic CNS development were examined. spen is required for normal migration and survival of MGCs; embryos lacking spen have CNS defects strikingly reminiscent of those seen in mutants of several known components of the Egfr signaling pathway. In addition, spen interacts synergistically with the RTK effector pointed. Using MGC-targeted expression, it was found that increased Ras signaling rescues the lethality associated with expression of a dominant-negative spen transgene. Therefore, spen encodes a positively acting component of the Egfr/Ras signaling pathway (Chen, 2000).

To examine the consequences of complete loss of spen function, spen germline clones were generated using the ovo^D-FLP/FRT system. Embryos lacking both maternal and zygotic spen will be referred to as 'spen mutants'. These mutants exhibit moderate defects in CNS morphology, as visualized by anti-Elav antibody, a pan-neuronal marker. In stage 16 spen mutant embryos, the space separating the two longitudinal halves of the CNS is reduced compared with the wild type and, in some segments, the two sides are completely collapsed across the midline. Because of this collapse, the midline neurons are difficult to detect; however, labeling with the antibody 22C10 reveals that the ventral midline neurons are present and have no obvious defects in their projections. Because such collapsed CNS phenotypes might be indicative of defects in the midline, spen mutant embryos were examined for expression of the MGC-specific marker Slit. Initial determination of the MGCs appeared normal in spen mutants. The first detectable defects occur at late stage 12/early stage 13 when the MGCs normally initiate their migration. In the wild type, the glial cells migrate in a tightly packed configuration along the dorsal surface of the ventral nerve cord, whereas in spen mutants, the glia migrated aberrantly and became spread out in a more diffuse pattern. The results of these analyses differ from a recent report that excessive numbers of MGCs are initially specified in spen mutants. Using the Slit-lacZ nuclear enhancer trap marker to count the MGCs, comparable numbers of MGCs in the wild type and in spen mutants were detected up until stage 13, and a reduction in MGC number in spen mutants beginning at stage 14. Thus, in these spen mutants, which appear to be genetic and protein nulls, normal numbers of MGCs are initially specified, a phenotype consistent with what has been reported for other Egfr pathway mutants (Chen, 2000).

By stage 16, in a wild-type embryo, the Slit-positive MGCs have migrated and elongated to ensheathe the anterior commissure (AC) and posterior commissure (PC) axons, thereby maintaining proper separation and bundling. In similarly staged spen mutant embryos, the MGCs had not properly migrated or wrapped themselves around the commissure bundles. In addition, while apoptosis reduces the number of MGCs in wild type embryos from ~8 per segment at stage 13 down to only ~3 per segment by stage 16-17, in spen mutants this reduction is even more drastic, leaving only 1-2 MGCs per segment. Thus, in spen mutants, although initiation of MGC differentiation appears normal, the later aspects of glial development, including migration, wrapping, and survival or maintenance of the MGC fate, are defective. To confirm that Spen is expressed in the MGCs at stage 13 when its function is required, embryos carrying the MGC-specific enhancer trap line AA142, were double labeled with anti-beta-galactosidase and anti-Spen antibodies. The highest level of Spen expression is seen in the MGCs (Chen, 2000).

Because defects in glial cell development are likely to perturb organization of the CNS, spen mutant embryos were labeled with the antibody BP102, which highlights all axon tracts in the CNS. As predicted, the AC and PC axon bundles are not properly organized or separated and, in some segments, are completely fused. In addition, the two longitudinal connectives appear closer together than normal and are occasionally fully collapsed across the midline. Staining with the anti-Fasciclin II (FasII) antibody, which highlights a distinct set of three axon bundles in each longitudinal branch, further clarifies this phenotype. These longitudinal axon tracts never cross the midline in a wild-type embryo. In contrast, the FasII-positive axons cross and recross the midline in spen mutants, producing a fragmented and disorganized longitudinal axonal array (Chen, 2000).

In Drosophila, the genes rhomboid, Star, pointed and spitz, all positively acting components of the Egfr pathway, share a characteristic CNS phenotype similar to that of spen mutants. Specifically, whereas the proper number of MGCs are initially specified, they later migrate abnormally and eventually degenerate and die. The phenotypic similarities between spen and the Egfr pathway genes, as well as the isolation of spen as an enhancer of an activated yan allele, are consistent with the hypothesis that spen may be a positively acting factor in the Egfr/Ras signaling pathway. To explore this possibility, whether spen and the RTK pathway effector pointed interact synergistically in the midline was investigated. The expectation is that a reduction in activity of a proven positive effector of the Egfr pathway, such as pnt, should dominantly enhance the spen phenotype. Embryos lacking maternal spen can be partially rescued by zygotic spen expression from a paternally inherited wild-type allele (this genotype is referred to as spen/+). Stage 15-17 spen/+ embryos appear phenotypically wild type, with only ~4% of the embryos exhibiting CNS defects. Embryos heterozygous for a pnt loss-of-function mutation (pnt/+) have no apparent dominant defects. Reducing the pnt dosage in the spen/+ background increases the frequency of axonal defects to ~25%. The predominant phenotype is reduced separation between the two longitudinal axon pathways and a single inappropriate crossing of the midline by one of the FasII-positive axon tracts. This dose-sensitive interaction between pnt and spen strongly supports a role for spen as either a positively acting component of the Egfr pathway or as a component of a parallel pathway synergizing with Egfr during MGC development (Chen, 2000).

To investigate further the connection between spen and Egfr/Ras signaling in the MGCs, a putative dominant-negative spen transgene was generated that truncates the carboxy-terminal ~1500 amino acids, including the highly conserved SPOC domain. When transfected into S2 cultured cells, this construct (SpenDeltaC) is expressed at high levels and localizes to the nucleus just as is found for the endogenous wild-type Spen protein. Ubiquitous expression of SpenDeltaC is unable to rescue the lethality or phenotypes associated with spen mutants, implying an essential function for the conserved carboxy-terminal SPOC domain. To determine whether SpenDeltaC might behave as a dominant-negative mutation, the Slit-Gal4 driver was used to induce high levels of expression specifically in the MGCs. MGC-specific expression of SpenDeltaC results in completely penetrant lethality. In contrast, and consistent with the lack of primary neuronal defects associated with spen mutants, pan-neural expression of SpenDeltaC using the Elav-Gal4 driver does not compromise the viability or patterning of the fly. To test the hypothesis that the Slit-Gal4/SpenDeltaC lethality might be due to compromised RTK/Ras pathway signaling, a determination was made of whether increasing the level of Egfr/Ras pathway signaling, specifically in the MGCs, could compensate for the reduction in spen function associated with expression of the dominant-negative SpenDeltaC transgene. Whereas Slit-Gal4-driven expression of either an activated Ras^V12 or the SpenDeltaC transgene results in lethality, flies expressing both Ras^V12 and SpenDeltaC in the MGCs are viable and appear normally patterned. The mutual suppression is extremely penetrant, since over 50% of the expected class of flies was recovered. Similar, but less penetrant, rescue was obtained when SpenDeltaC and a secreted form of the Egfr ligand Spitz were coexpressed in the MGCs. Together, these results strongly suggest that spen functions autonomously in the MGCs, acting either downstream of or in parallel to Ras as a positive effector or regulator of RTK signaling (Chen, 2000).

Although the molecular mechanisms underlying Spen function in the RTK/Ras pathway remain to be elucidated, given its membership of the RRM family, one possibility is that Spen might directly regulate the processing and/or stability of specific transcripts to generate functionally distinct protein isoforms in response to, or required for, Ras signaling events. Post-transcriptional regulation of gene expression allows quick responses to external or developmental signals, and RRM family members have been shown to mediate many different cellular processes including mRNA splicing, stabilization, localization and transport. Two attractive potential targets of such activity in the CNS are the Ras pathway effector pointed and the zinc finger transcription factor tramtrack. Both genes produce alternatively spliced transcripts and are required in the MGCs. The synergistic interactions detected between spen and pointed make pointed a particularly appealing candidate. A third possibility, given that spen was isolated as an enhancer of an activated yan allele, is that Spen might function to destabilize yan transcripts in response to RTK-initiated signals. In this model, spen would contribute a second level of post-translational regulation that would reinforce the transient mitogen-activated protein (MAP) kinase signal that downregulates Yan protein, thereby stabilizing release from the Yan-mediated block to differentiation. In all these scenarios, spen could either function in parallel to the Ras/MAP kinase cascade, or could itself be directly regulated or activated by the pathway (Chen, 2000).

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors, and both of them are implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, a gain-of-function (GOF) screen was performed for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Among the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of the screen. Observed GOF phenotypes have led to the identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Among them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Analysis of rac2 phenotypes clearly demonstrate that the altered mesodermal level of Rac2 can influence specification of a number of cardiac and muscular cell types, including those expressing ladybird. The finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicates a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinase (RTK) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types (Bidet, 2003).

The presented rho, yan and rac2 gain and loss-of-function phenotypes, clearly demonstrate that these genes play critical roles in the specification of lb-expressing mesodermal lineages. When over-expressed, the regulator of EGF-ligand maturation rho is able to induce specification of an increased number of lb-positive lateral adult muscle precursors (LaPs). Consistent with this observation, the GOF of a negative effector of RTKs signalling yan leads to the loss of LaPs. Interestingly, the large number of LaPs in rho GOF embryos suggests that during segregation of the LaPs progenitor, the Notch-mediated lateral inhibition is affected. Antagonistic activities of the EGFR and the Notch signalling pathways have been reported, thus indicating that the excess of EGFR signalling can overrule the lateral inhibition during specification of muscular progenitors. The highly restricted mesodermal expression of rho suggests, however, that in wild type embryos the rho-triggered EGF signals can interfere with lateral inhibition only in a subset of promuscular clusters. This indicates that other RTKs contribute to the negative interactions with Notch. Taking into consideration all the available information, it is speculated that the ectopically expressed rho induces the EGFR pathway that antagonizes Notch dependent lateral inhibition, specifically during segregation of the LaP progenitor. This results in promoting the LaP fate. Since in rho and yan mutants the segmental border muscle (SBM) is duplicated, it is proposed that during specification of SBM founder the repressive action of yan is relieved by a Rho/EGFR-independent RTK pathway (Bidet, 2003).

These data also demonstrate new roles for rho, yan and rac2 in the specification of cardiac lineages. Interestingly, mutations of rho and rac2 affect specification of pericardial cells with no major effects on cardioblast identity. yan loss and GOF leads to even more pronounced phenotypes suggesting that, in addition to EGFR, other RTKs are involved in diversification of cardiac fates. rho and Ras/MAPK pathway have been shown to influence specification of eve-expressing pericardial cells. In addition, this study shows that rho represses and yan promotes specification of lb-positive pericardial cells. Surprisingly, in rho mutants, the supernumerary lb-positive pericardial cells co-express eve, a situation never observed in wild type embryos because of mutual repressive activities of eve and lb. This suggests that cross-repression requires the co-ordinated action of identity gene products and effectors of RTK signalling pathway. The overproduction of tin/eve-positive pericardial cells observed in rho GOF and in rac2 loss of function mutants suggests that the diversification of this particular cell type involves a rac2-dependent trafficking of EGF receptor. A future challenge will be to unravel whether Drosophila rac2 indeed co-operates with cell fate specification machinery by controlling the intracellular processing of EGFR and others RTKs (Bidet, 2003).

Rhomboid belongs to a large family of intermembrane serine proteases regulating the EGF-like ligand maturation in different species from prokaryotes to Human. One of the mouse rho homologs, ventrhoid, exhibits a very dynamic expression in central nervous system and forming somites, suggesting it may regulate early cell fate specification genes in a manner similar to that in which rho regulates lb in Drosophila. Several yan-like genes have also been identified in vertebrates. Two human yan homologs, named tel1 and tel2 share similar mesodermal embryonic expression pattern restricted to hematopoietic lineages. In addition, in adult mouse, tel1 is expressed in the heart and in skeletal muscles. As in Drosophila, yan functions with its closely related partner pointed. It is important to note that the vertebrate pnt genes ets-1 and ets-2 are involved in early embryonic heart and muscle development. The numerous vertebrate homologs of the third candidate gene of this study, rac2, control a variety of cellular processes including actin polymerization, integrin complex formation, cell adhesion, membrane trafficking, cell cycle progression, and cell proliferation. The majority Rho-GTPases are ubiquitously expressed, including the developing muscular and cardiac tissues, but their myogenic functions have not yet been investigated. The vertebrate Rac2 gene is specifically required for hematopoiesis. Its mutation in mice leads to the defective neutrophil cellular functions reminiscent of human phagocyte immunodeficiency. The only described link between Rho-GTPases and muscle concerns the binding and activation of a Serine/Threonine protein kinase homologous to myotonic dystrophy kinase by a small GTP binding protein Rho. It is speculated, however, that given the involvement of RhoB in EGFR trafficking, the vertebrate Rho GTPase can contribute to RTK-controlled myogenic pathways (Bidet, 2003).

Altogether, these data suggest that the RTK signalling involving rho, yan and rac2 might play an important and at least partially conserved role in diversification of cardiac and muscular lineages (Bidet, 2003).

Atrophin genetically interacts with yan to contributes to the negative regulation of epidermal growth factor receptor signaling in Drosophila

Dentato-rubral and pallido-luysian atrophy (DRPLA) is a dominant, progressive neurodegenerative disease caused by the expansion of polyglutamine repeats within the human Atrophin-1 protein. Drosophila Atrophin and its human orthologue are thought to function as transcriptional co-repressors. Drosophila Atrophin participates in the negative regulation of Epidermal Growth Factor Receptor (EGFR) signaling both in the wing and the eye imaginal discs. In the wing pouch, Atrophin loss of function clones induces cell autonomous expression of the EGFR target gene Delta, and the formation of extra vein tissue, while overexpression of Atrophin inhibits EGFR-dependent vein formation. In the eye, Atrophin cooperates with other negative regulators of the EGFR signaling to prevent the differentiation of surplus photoreceptor cells and to repress Delta expression. Overexpression of Atrophin in the eye reduces the EGFR-dependent recruitment of cone cells. In both the eye and wing, epistasis tests show that Atrophin acts downstream or in parallel to the MAP kinase rolled to modulate EGFR signaling outputs. Atrophin genetically cooperates with the nuclear repressor Yan to inhibit the EGFR signaling activity. Finally, it was found that expression of pathogenic or normal forms of human Atrophin-1 in the wing promotes wing vein differentiation and these forms act as dominant negative proteins inhibiting endogenous fly Atrophin activity (Charroux, 2006).

Four facts are evidence that Atro contributes to the negative regulation of EGFR signaling: (1) clones mutant for Atro display phenotypes characteristic of overactive EGFR signaling and express high levels of the known EGFR target gene Dl. These effects are enhanced when negative regulators of EGFR signaling, such as Argos, are simultaneously removed in Atro⁻ clones. (2) Increased amounts of Atro reduce the activity of EGFR signaling; (3) ectopic expression of Atro enhances the effects of decreased EGFR signaling, whereas reduced Atro enhances the effects of ectopic signaling and (4) Atro genetically interacts with yan suggesting that both repressors may cooperate to block EGFR signaling output (Charroux, 2006).

The likely C. elegans orthologue of Atrophin, Egl27, has been shown to inhibit vulval development induced by the Ras signal transduction pathway. Thus, the role of Atro as a negative regulator of the RTK/EGFR pathway may have been conserved during evolution. Egl27 is a component of a repressor complex, the nucleosome remodeling and histone deacetylase (NURD) complex, which is composed of HDAC-1, HDAC-2, two proteins of the Mi-2/CHD family, and MTA1 or MTA2. During vulval induction, the NURD complex is proposed to interact with the sequence-specific transcription factors LIN-31, an Ets-related transcription factor and LIN-1, a winged-helix molecule. LIN-1 and LIN-31 are repressors of vulval development that are negatively regulated upon phosphorylation by the MAPK mpk1/sur-1 (Charroux, 2006).

MAPK-dependent phosphorylation of the ETS transcription factor Pnt is necessary for the activation of the EGFR target genes in third instar eye imaginal discs and in embryos. Yan and Atro show synergistic genetic interaction, suggesting that both are required for the repression of EGFR signaling function. Thus, by analogy with EGL-27 and LIN-31 from C. elegans, a model is proposed where Yan cooperates with Atro in order to achieve tight repression. How does EGFR signaling counteract Atro-mediated repression? Localized downregulation (such as nuclear export and/or protein degradation) of specific repressors is a common mechanism for the activation of target genes by the EGFR pathway. Two observations argue against this mechanism for the co-repressor Atro: (1) in cells with high levels of EGFR activity, such as either side of the dorso-ventral boundary in the wing pouch, or later in prospective veins of pupal wings, Atro protein is detected ubiquitously and at invariant levels in all nuclei and (2) when EGFR signaling is overactivated in clones (by expressing the constitutive form of EGFR, EGFR^ACT), the amount and/or subcellular localization of the co-expressed Atro protein is unchanged (Charroux, 2006).

Several lines of evidence show that, in the late phases of imaginal disc patterning, Atro plays a specific role for EGFR repression. It was found that Atro does not contribute to other signaling pathways during imaginal disc development. For instance, expression of both Distal-less and the vestigial quadrant enhancer (vgQE), two known wingless (wg) target genes, is not affected in Atro⁻ clones located in the wing pouch. Plus, it was found that signaling from the Notch (N) receptor does not require Atro activity since Atro⁻ clones expressing the constitutively active, intra-cellular fragment of the N receptor (Nintra) display identical phenotypes to Nintra control clones, when located in the wing pouch (Charroux, 2006).

Other signaling pathways are known to affect vein differentiation such as Decapentaplegic (DPP), which promotes vein differentiation in late pupae, and N whose activity is necessary to restrict vein territories. However, the idea is favored that Atro contributes mainly to EGFR signaling since Atro acts in third instar larvae and is dispensable for N activity in the wing (Charroux, 2006).

Despite the strong correlation of Atro repression of EGFR target genes in the imaginal discs, Atro is required for patterning where EGFR has not been implicated. For example, Atro is required for normal segmentation of the Drosophila embryo. However, it is noted that both EGFR signaling and Atro are required for cell survival during embryogenesis. Additionally, Atro is not required for all EGFR-dependent events. For example, Atro is not involved in the function of the EGFR defining the identity of the proximal wing disc. These observations indicate that variable mechanisms of control are implicated in the negative regulation of EGFR signaling in the nucleus (Charroux, 2006).

This notion is supported even in different imaginal tissues. EGFR signals via Strawberry notch (Sno) and Ebi, to inhibit the repressor activity of a Su(H)/SMRTER complex, leading to activation of Dl expression. Clones of cells mutant for the Su(H)^SF8 hypomorphic allele cause high level expression of Dl in PR cells, but not in the wing pouch. It was found that clones of cells mutant for the Su(H)^del47 null allele similarly do not show ectopic expression of Dl in the wing pouch. As expected, Su(H)^del47 cells located at the D/V border abolish the expression of Cut. Thus, Su(H), unlike Atro, is dispensable for Dl repression in the wing pouch. The reverse is true in the eye, where Su(H) activity is absolutely required to repress Dl expression whereas Atro is less important. This is in agreement with the weak phenotype caused by the Atro⁻ clones in the eye (i.e. no ectopic PRs, few extra cone cells), and indicates a redundancy with other negative regulators of EGFR signaling. This distinction between the relative requirements in different tissues for different regulators of EGFR signaling provides an interesting insight into tissue-specific control of ubiquitous signaling pathways. Regulators such as Atro, with functions restricted to some tissues, may contribute to the diverse outcomes of signaling through these common pathways (Charroux, 2006).

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant, hereditary malady typified by the degeneration of specific neurons in the brain. Although DRPLA has been mimicked in a mouse model, the molecular and cellular mechanisms leading to the disease remain obscure. The data point to the role of Atro in the repression of EGFR signaling. It was found that expression of human N917Atrophin-1 in the wing mimics the loss of Atro activity; this raises the possibility that N917Atrophin-1 is acting as a dominant negative. Additionally, this phenotype is independent of polyQ expansion and is sensitive to the dose of EGFR signaling components. Such effects are not seen following expression of polyQ repeats alone or the exon 1 of Huntingtin with expanded polyQ (93Q) in the wing, indicating that human N917Atrophin-1 has specific effects on this pathway. This mechanistic insight into the role of the fly gene may have broader implications concerning Atrophin function in other organisms (Charroux, 2006).

Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye

yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, is an antagonist of the Wingless signalling pathway. Cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan^-/- cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. It is concluded that yan represents a new and unsuspected molecular link between the Wingless and DER pathways (Olson, 2012).

On the basis of in vivo and in vitro characterization of the antagonistic interaction between Yan and the Wingless pathway, it is proposed that yan has a dual role at the moving anterior/posterior boundary defined by the morphogenetic furrow. In addition to the previously defined function of Yan in inhibiting premature photoreceptor recruitment, it is suggested that Yan blocks Wingless signalling at the morphogenetic furrow, probably by regulating the activity of Armadillo protein. By maintaining low Wingless signalling activity, it is proposed that Yan maintains the competence of these cells to adopt a retinal fate. In the absence of Yan, Armadillo is free to either participate in adherens junctions, thereby causing the apical constriction phenotype, or interact with TCF in the nucleus to activate target genes. It is speculated that in yan^−/− clones generated far from a source of Wingless, most of the liberated Armadillo that does not localize to adherens junctions is degraded and is unable to robustly signal in the nucleus. This could explain why apical constriction is observed in all yan^−/− clones, but ectopic Fz3–RFP activation is seen only in yan^−/− clones near the lateral margins of the eye disc. Similarly, expression of Axin—which can strongly downregulate both the membrane-directed and signalling-competent pool of liberated Armadillo—within yan^−/− clones results in a stronger rescue of apical constriction than the expression of dnTCF, which only interferes with the signalling-competent pool of Armadillo. These data indicate that both the junctional and nuclear signalling functions of Armadillo probably contribute to the yan^−/− phenotype (Olson, 2012).

In conclusion, this study shows a new and unsuspected function for yan in the negative regulation of Wingless signalling. These results might be relevant to the understanding of the molecular regulation of Wnt–RTK signalling crosstalk in human disease. The closest human homologue of yan, encoded by TEL/ETV6 is known to be associated with leukaemia, which can also result from uncontrolled activation of Wnt signalling (Olson, 2012).

The Ras-Erk-ETS-signaling pathway is a drug target for longevity

Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. This study has identified a critical role for Ras-Erk-ETS signaling in aging in Drosophila. Inhibition of Ras was shown to be sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. ETS transcriptional repressor Anterior open (Aop) was identified as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, it was demonstrates that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals (Slack, 2015).

The key role of IIS in determining animal lifespan has been well appreciated for more than two decades and shows strong evolutionary conservation. Alleles of genes encoding components of this pathway have also been linked to longevity in humans. Multiple studies have demonstrated the importance of the PI3K-Akt-Foxo branch of IIS, while this study has identified an equally important role for Ras-Erk-ETS signaling in IIS-dependent lifespan extension (Slack, 2015).

Downstream of chico, preventing the activation of either Ras or PI3K is sufficient to extend lifespan. Ras can interact directly with the catalytic subunit of PI3K, which is required for maximal PI3K activation during growth. Thus, inhibition of Ras could increase lifespan via inactivation of PI3K. However, several lines of evidence indicate that the Erk-ETS pathway must also, if not solely, be involved. In this study and elsewhere, it has been demonstrated that direct inhibition of the Ras-dependent kinase, Erk, or activation of the Aop transcription factor, a negative effector of the Ras-Erk pathway, is sufficient to extend lifespan. Importantly, this study shows that Ras-Erk-ETS signaling is genetically linked to chico because activation of Aop is required for lifespan extension due to chico loss of function. Furthermore, altering the ability of Chico to activate Ras or PI3K does not result in equivalent phenotypes: it has been shown that mutation of the Grb2/Drk docking site in Chico is dispensable for multiple developmental phenotypes associated with chico mutation, while disruption of the Chico-PI3K interaction is not. Overall, the observations strongly suggest that lifespan extension downstream of chicomutation involves inhibition of the Ras-Erk-ETS-signaling pathway (Slack, 2015).

A simple model integrates the role of Ras-Erk-ETS signaling with the PI3K-Akt-Foxo branch in extension of lifespan by reduced IIS. It is proposed that, downstream of Chico, the IIS pathway bifurcates into branches delineated by Erk and Akt, with inhibition of either sufficient to extend lifespan, as is activation of either responsive TF, Aop or Foxo. The two branches are not redundant, because mutation of chico or the loss of its ability to activate either branch results in the same magnitude of lifespan extension. Furthermore, Aop and Foxo are each individually required downstream of chico mutation for lifespan extension. At the same time, the effects of the two branches are not additive, as simultaneous activation of Aop and Foxo does not extend lifespan more than activation of either TF alone. Taken together, these data suggest that the two pathways re-join for transcriptional regulation, where Aop and Foxo co-operatively regulate genes required for lifespan extension. The model is corroborated by a previous finding that, in the adult gut and fat body, some 60% of genomic locations bound by Foxo overlap with regions of activated-Aop binding (Alic, 2014; Slack, 2015).

It is proposed that functional interactions of Aop and Foxo at these sites may be such that each factor is both necessary and sufficient to achieve the beneficial changes in target gene expression upon reduced IIS. It remains to be determined how promoter-based Foxo and Aop interactions produce such physiologically relevant, transcriptional changes. It is, however, curious that activation of either TF alone promotes longevity when one is known as a transcriptional activator (Foxo) and the other as a transcriptional repressor (Aop). A subset of Foxo-bound genes, albeit a minority, has been consistently observed that are transcriptionally repressed when Foxo is activated (Alic, 2014). Furthermore, the Foxo target gene myc is downregulated in larval muscle when Foxo is active under low insulin conditions, while deletion of foxo or its binding site within the myc promoter results in de-repression of myc expression in adipose of fed larvae (Teleman, 2008). Thus, on some promoters under certain conditions, Drosophila Foxo appears to act as a transcriptional repressor. Mammalian Foxo3a may also directly repress some genes. It will therefore be important to test whether the lifespan-relevant interactions between Foxo and Aop occur on promoters where Foxo acts as a repressor with Foxo possibly acting as a cofactor for Aop or vice versa (Slack, 2015).

In mediating the effects of IIS on lifespan, the Ras-Erk-ETS- and PI3K-Akt-Foxo-signaling pathways both appear to inhibit Aop/Foxo. To understand why signaling might be so wired, it is important to consider that the two pathways are also regulated by other stimuli, such as other growth factors, stress signals, and nutritional cues. The re-joining of the two branches at the transcriptional level would therefore allow for their outputs to be integrated, producing a concerted transcriptional response, a feature that is also seen in other contexts. For example, stability of the Myc transcription factor is differentially regulated in response to Erk and PI3K signals, allowing it to integrate signals from the two kinases. Transcriptional integration in response to RTK signaling also confers specificity during cell differentiation, with combinatorial effects of multiple transcriptional modulators inducing tissue-specific responses to inductive Ras signals. Similar integrated responses of lifespan could be orchestrated by transcriptional coordination of Aop and Foxo (Slack, 2015).

Direct inhibition of Ras in Drosophila can extend lifespan, suggesting that the role of Ras in aging is evolutionarily conserved. In budding yeast, deletion of RAS1 extends replicative lifespan, and deletion of RAS2 increases chronological lifespan by altering signaling through cyclic-AMP/protein kinase A (cAMP/PKA), downregulation of which is sufficient to extend both replicative and chronological lifespan. This role of cAMP/PKA in aging may be conserved in mammals, as disruption of adenylyl cyclase 5' and PKA function extend murine lifespan. However, cAMP/PKA are not generally considered mediators of Ras function in metazoa. Instead, the data suggest that signaling through Erk and the ETS TFs mediates the longevity response to Ras. Interestingly, fibroblasts isolated from long-lived mutant strains of mice and long-lived species of mammals and birds show altered dynamics of Erk phosphorylation in response to stress, further suggesting a link between Erk activity and longevity. Importantly, the ETS TFs are conserved mediators of Ras-Erk signaling in mammals. Investigation of the effects of Ras inhibition on mammalian lifespan and the role of the mammalian Aop ortholog Etv6 are now warranted (Slack, 2015).

A role for Ras-Erk-ETS signaling in lifespan offers multiple potential targets for small-molecule inhibitors that could function as anti-aging interventions. Importantly, due to the key role of this pathway in cancer, multiple such inhibitors exist or are in development (Slack, 2015).

This study has shown that trametinib, a highly specific allosteric inhibitor of the Mek kinase, prolongs Drosophila lifespan, thus validating the Ras-Erk-ETS pathway as a pharmacological target for anti-aging therapeutics. Trametinib joins a very exclusive list of FDA-approved drugs that promote longevity in animals, the most convincing other example being rapamycin (Slack, 2015).

Rapamycin not only increases lifespan in multiple organisms, including mammals, but also improves several indices of function during aging (Ehninger, 2014; Lamming, 2013). While rapamycin can protect against tumor growth, the effects on longevity appear to be independent of cancer prevention, as rapamcyin-treated animals still develop tumors and rapamycin can increase lifespan in tumor-free species. Furthermore, increased activity of certain tumor suppressors such as lnk4a/Arf and PTEN as well as the RasGrf1 deficiency all increase lifespan independently of anti-tumor activity. The findings that trametinib can increase lifespan inDrosophila, which are mainly post-mitotic in adulthood, and that doses of trametinib that increase lifespan do not alter proliferation rates of ISCs inDrosophila suggest that the anti-aging effects of trametinib are separable from its anti-cancer activity (Slack, 2015).

Finally, due to the high degree of evolutionary conservation in the Ras-Erk-ETS pathway, this study suggests the intriguing possibility that pharmacological inhibition of Ras-Erk-ETS may also increase lifespan in mammal (Slack, 2015).

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date revised:  10 July 2015 

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