Ras oncogene at 85D


Transcriptional Regulation

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene's function when other approaches are not illuminating. An eye-specific, enhancer-promoter present in the P element expression vector pGMR is able to drive high level expression in the eye of genes near the site of P element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea two overexpression screens were carried out for genes that function to regulate cell death. A screen was carried out for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a reaper overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5' to hid, a potent inducer of apoptosis, and insertions 5' to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the P element insertion, new misexpression vectors were created. A screen with one of these vectors identified eagle as a suppressor of a rough eye phenotype associated with overexpression of an activated Ras1 gene. This suggests that eg may be involved in the Ras1 signal transduction pathway (Hay, 1997).

Targets of Activity

In the differentiation of photoreceptors in eye imaginal discs, activated Ras1 up-regulates the transcriptional activity of P2, but not the P1 form of Pointed. Pointed P2 may be a direct target of a Drosophila MAPK, encoded by the rolled locus at the same time that Ras 1 and Rolled negatively regulate the ability of yan to repress transcription. (O'Neill, 1994). Pointed P2 is phosphorylated by MAPK at a single site that is required for its in vivo function as a transcriptional activator. This site is located within the so-called "pointed" or RII domain, which is shared by a subset of ETS proteins (Brunner, 1994).

The expression of the transcription factor DJun in the eye imaginal disc correlates temporally and spatially with the determination of neuronal photoreceptor fate. Expression of dominant negative forms of DJun in photoreceptor precursor cells results in dose-dependent loss of photoreceptors in the adult fly. Conversely, localized overexpression of DJun in the eye imaginal disc can induce the differentiation of additional photoreceptor cells. The transformation of nonneuronal cone cells into R7 neurons elicited by constitutively active forms of sevenless, Ras1, Raf, and MAP kinase is relieved in the presence of DJun mutants. These results demonstrate a requirement of DJun downstream of the sevenless/ras signaling pathway for neuronal development in the Drosophila eye (Bohmann, 1994).

phyllopod (phyl) encodes a novel protein required for fate determination of photoreceptors R1, R6, and R7, the last three photoreceptors to be recruited into the ommatidia of the developing Drosophila eye. Genetic data suggests that phyl acts downstream of Ras1, raf, and yan to promote neuronal differentiation in this subset of photoreceptors. Ectopic expression of phyl in the cone cell precursors mimics the effect of ectopic activation of Ras1, suggesting that phyl expression is regulated by Ras1. phyl is also required for embryonic nervous system and sensory bristle development (Chang, 1995).

PACAP-like activity has been detected in larvae and neuromuscular junctions that function in the adenylyl cyclase second messenger system. The vertebrate PACAP38 triggers two muscular responses in Drosophila: an immediate depolarization and a late enhancement (Zhong, 1995b). Antibody to vertebrate PACAP-38 stains segmentally repeated larval CNS neurons as well as motor nerve terminals (Zhong, 1996). It has long been thought that the neuromuscular synapse is a good model for the synaptic basis of learning. Binding of a PACAP-like peptide to its receptors leads to activation of Rutabaga-adenylyl cyclase by the Galpha subunit and of Ras1/Raf by the Gbeta-gamma complex: the pathways then converge to modulate potassium ion-channel activity (Zhong, 1995a and Zhong, 1996).

In addition to a post-translational regulation of Head involution defective (Hid), the Ras/MAPK pathway promotes cell survival in Drosophila by downregulating the expression of hid. Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid expression. hid transcript levels are downregulated in dominantly active Dras1- (Dras1Q13) expressing embryos when assayed 3 hr after heat shock. In wild-type embryos, total HID mRNA levels do not change dramatically between stage 11, when Ras expression was ectopically induced, and stage 14, when HID mRNA levels were assayed. This eliminates the concern that developmental arrest might account for the observed difference in HID mRNA levels. It was observed that hid levels return to normal in Dras1Q13 embryos by 5 hr after heat shock. Cell death also resumes in these embryos several hours later. This indicates that a transient increase in Ras activity leads to a transient suppression of hid expression, accompanied by a transient protection from naturally occurring cell death. HID mRNA levels were also assayed through an alternative procedure: whole mount in situ analysis. These results confirm that hid transcript levels decline in dominantly active Dras1- (Dras1Q13) expressing embryos. This is particularly apparent in the midline glia, which strongly express hid. The survival of midline glia is known to depend on the activity of the Epidermal growth factor receptor pathway. To confirm that Ras regulation of hid utilizes the Raf/MAPK pathway, the effect of a constitutively active form of Draf (phlF22) on hid expression has been investigated. In situ analyses were performed on embryos expressing activated Draf under the control of the heat shock promoter. Heat-induced expression of phlF22 results in downregulation of hid transcript levels, suggesting that Ras functions through the Raf/MAPK pathway to downregulate hid expression (Kurada, 1998).

Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. The pnt gene encodes two related proteins, pnt1 and pnt2. pnt2 operates downstream of the MAPK rolled in the Ras pathway. Therefore, the consequences of ectopic expression of pnt2 were examined. Embryos were generated that carry UAS-Pnt2 and a midline glia-specific Gal4 driver (52A-Gal4), resulting in the expression of pnt2 in the midline glia cells. Such embryos were tested for hid levels by whole-mount in situ analysis. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).

Since upregulation of the Ras/MAPK pathway promotes cell survival and downregulates hid expression, it was predicted that increased hid expression is the cause of the increased apoptosis observed when Ras activity is decreased. Ubiquitous expression of the negative regulator yan is able to induce massive embryonic apoptosis. In these same embryos HID mRNA levels are increased within 2 hr of yanAct induction and continue to rise for many more hours. Thus, downregulation of Ras activity in the embryo results in increased hid transcription and apoptosis, and this transcription is regulated either directly or indirectly by yan. These results imply that Ras activation of MAPK and inactivation of yan is an important cell survival pathway in embryos (Kurada, 1998).

Blocking Epidermal growth factor receptor activity in the developing eye also enhances apoptosis. If hid is a target of Egfr/Ras/MAPK activity in this tissue, then hid levels should increase when Egfr activity is blocked. Expression of a dominant negative Egfr in the developing eye results in a band of increased hid transcription in the eye disc. This band lies several rows posterior to the furrow and corresponds well with the first developmental defects seen in these eye discs. In sum, these data implicate the downregulation of hid transcription as an important component of Egfr antiapoptotic activity. The post-transcriptional modification of Hid appears to be equally important (Kurada, 1998).

According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus, indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).

In contrast to ato broad expression just anterior to the furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential for uncommitted cells to acquire ommatidial cell fate or the ability to express ommatidial marker genes. Consistent with this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).

In the eye developmental field posterior to the morphogenetic furrow, three ommatidial cell marker genes, ro, svp, and pros, were found to be misexpressed zonally in many uncommitted cells along the A/P axis when Ras1 was transiently but ubiquitously activated. The Ras1-dependent misexpression regions appear to correspond to those regions where expression of these genes is normally initiated, indicating that Ras1-dependent zonal marker gene misexpression in presumptive uncommitted cells may reflect directly their developmental potentials. Subsequent to Ras1 activation, posterior ro misexpression is restricted only to the vicinity of the furrow. Wild type cells just posterior to the morphogenetic furrow may thus possess developmental potential for ro expression. Misexpression of svp and pros, subsequent to Ras1 activation, is strongly present from row 2 to row 6 and from row 4 to row 9, respectively, causing the region strongly misexpressing svp partially to overlap that of pros. The posterior half of the svp expression region overlaps the anterior half of the pros expression region, but this does not necessarily mean that cells in the overlapped region (cells in or near rows 4-6) possess two different developmental potentials at the same time for svp and pros expression. Indeed, optical section analysis has indicated that svp and pros expression occurs mutually exclusively (Hayashi, 2001).

In contrast to Ras1 activation in the present study, differentiating wild type cells may receive Ras1 activation signals reiteratively or for a relatively long period, this possibly being essential in order to exclude any ambiguities in developmental potential. Preliminary inspection indicates that in the overlapping region, svp- but not pros-misexpressing cells strongly tend to be localized near differentiating ommatidia, which may secrete short-ranged Spitz or Ras1 activation signals. This may suggest that the threshold of Ras1 signal for svp expression is higher than that for pros expression. In addition to the Ras1 signal, the involvement of the Notch (N) signal also has to be considered. N has been shown to play important roles in ommatidial cell fate determination. Therefore, although developmental potential appears to be essential for cell fate specification, it is not the only mechanism used and the strength or duration of Ras1 signal and the pattern of N signal activation may also be important for making a perfect ommatidium (Hayashi, 2001).

This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow. Uncommitted cells just posterior to the morphogenetic furrow are presumed to acquire ro expression potential at the earliest stage of the model (stage 1). The regulation of ro expression appears highly complicated. ro expression is abolished in all cells in smo mutant clones situated just posterior to the morphogenetic furrow but fundamentally normal in more posterior clones in which ro is expressed in putative R2/R5 and R3/R4 precursors. ro expression appears to be regulated by at least two enhancers, one for expression at or near the morphogenetic furrow and the other for expression in developing R2-R5 photoreceptor precursors situated more posteriorly. Hh signals may thus be involved only in the former, possibly through the regulation of prepattern gene expression, and ro autoregulation maintains the activity of the latter. Egfr/Ras1 signals may be involved in both of them (Hayashi, 2001).

Expression levels of CI (activator form; CIact) and Dac in smo mutant clones vary considerably, depending on clone position, thus making the situation much more complicated. The expression of CIact is down-regulated in smo mutant clones at or near the morphogenetic furrow and up-regulated in more posterior clones. Dac expression is up-regulated only in the latter. Although normal ro expression is observed in ci or dac mutant clones, the fact that enhanced expression of CIact and Dac occurs in smo mutant clones distant from the morphogenetic furrow may suggest that CIact and Dac have some role in expressing ro in these clones. Consistent with this, misexpression experiments indicate either misexpressed CIact or Dac to be capable of bringing about ro up-regulation in a fraction of ommatidial cells. It is thus likely that ro expression is positively regulated by CIact and Dac (Hayashi, 2001).

In the absence of Hh signals, ci may serve as a gene to encode a repressor for ro expression, since ro expression near the furrow is repressed by the repressor form of CI protein (Hayashi, 2001).

In stage 2, R3/R4 precursors expressing ro acquire svp expression potential. svp expression in wild type R3/R4 precursors along with Ras1 activation-dependent svp misexpression in uncommitted cells is assumed to be not only positively regulated by the concerted action of Ras1 signaling and Dac and Eya but also negatively regulated by the protein product of the prepattern gene, lz. R1/R6 photoreceptors are recruited into ommatidia between stages 2 and 3. R1/R6 fate is previously shown specified by dual Bar homeobox genes, BarH1 and BarH2, whose expression is positively regulated by the cell-autonomous function of lz and svp. Consistent with this, in the putative R1/R6 arising area (around row 6), considerable svp expression occurs even in the presence of Lz. svp expression is regulated by Dac and Eya, so that normal Bar expression or R1/R6 fate eventually comes under the control of putative eye prepattern genes Lz, Dac, and Eya (Hayashi, 2001).

In stage 3, which may correspond to R7 and cone cell formation stages, pros is positively regulated through the concerted action of Ras1 signaling and prepattern gene lz. svp expression in this region is negatively regulated by Lz. Lz and Pnt both been shown to directly bind to the pros promoter/enhancer region and pros expression occurs only when Pnt and Lz have bound simultaneously to the pros enhancer/promoter. In wild type, Lz is expressed prior to pros expression in rows 4-7; subsequent to Ras1 ubiquitous activation, pros expression takes place in this region. Thus, in all wild-type progenitors situated in rows 4-7, Lz may bind to the pros enhancer/promoter so as to impart progenitor cells with pros expression potential. In wild type, pros expression first becomes apparent in R7 precursors at row 8. The absence of pros expression in rows 4-7 in wild type may then be accounted for by the possible absence of Ras1 signal activity. This possibly may be an oversimplification since, for instance, this does not explain why pros is repressed in R1/R6 photoreceptors which also arise from Lz-positive progenitor cells, or why pros is not induced efficiently on Ras1 activation in row 10 and more posterior regions (Hayashi, 2001).

The former might be caused by the absence of the strong N signal in R1/R6 precursors, but another unknown mechanism may be required to explain the latter. Therefore, more remains to be discovered about pros regulation, but what is known is nonetheless an excellent model for understanding the manner in which cooperative action of prepattern genes and differentiation signals give rise to specific cell fates from common progenitors (Hayashi, 2001).

dac and eya are expressed and may serve as eye prepattern genes in the region anterior to the morphogenetic furrow. This is supported by the finding that ommatidial marker gene misexpression subsequent to Ras1 activation occurs only within the Dac/Eya expression domain. pros expression is considerably restricted but ro and svp misexpression is evident throughout the entire Dac/Eya expression domain. Considerably strong misexpression of ELAV, a neuron-specific antigen, is also apparent anterior to the furrow. Thus, as with posterior cells, those anterior to the morphogenetic furrow are capable of developing into photoreceptors or ommatidial cells with receipt of Ras1 signals. However, no apparent regularity in ommatidial marker gene expression can be detected in the region anterior to the furrow, indicating that developmental potential of anterior cells is necessarily reset to some extent before the onset of normal eye development at the morphogenetic furrow or before first receiving Spitz signals from nascent R8 (Hayashi, 2001).

ro expressed along the morphogenetic furrow may be involved in this process, in that svp expression near the morphogenetic furrow is significantly repressed by ro expression along the morphogenetic furrow. Furthermore, ro has also been shown to repress ato expression near the furrow (Hayashi, 2001).

In the developing Drosophila eye, differentiation of undetermined cells is triggered by Ras1 activation but their ultimate fate is determined by individual developmental potential. Presently available data suggest that developmental potential is important in the neurogenesis of vertebrates and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along the dorso-ventral axis in response to graded Sonic Hedgehog signals and this presages their future fates. Subdivision of originally equivalent neural progenitors through the action of prepattern genes may accordingly be a general strategy by which diversified cell types are produced through neurogenesis (Hayashi, 2001).

hibris is regulated by Notch and Ras in a Toll10b mutant background. This regulation was confirmed in vivo in wild-type embryos. hbs expression was examined in Notch and Ras loss-of-function embryos and embryos overexpressing activated forms of Notch and Ras in the mesoderm. A dominant negative Ras construct activates hbs expression in the somatic mesoderm. Zygotic null Notch embryos show lower hbs transcription. Conversely, an activated form of Notch upregulates hbs in the mesoderm, while an activated form of Ras almost completely inhibits hbs expression. These results argue that, upon stimulation, Notch activates hbs, while Ras acts as a negative signal, and predicts that hbs expression in the somatic mesoderm would be restricted to fusion-competent cells (Notch dependent) and excluded from founder cells (Ras dependent). It is not known whether this regulation is direct, that is, Notch or Ras effectors act directly on the hbs promoter, or indirect, that is, Notch/Ras converts cell fate, which in turn would lead to hbs upregulation/downregulation by some other effector (Artero, 2001).

In Drosophila, the Ras signal transduction pathway is the primary effector of receptor tyrosine kinases, which govern diverse developmental programs. During oogenesis, epidermal growth factor receptor signaling through the Ras pathway patterns the somatic follicular epithelium, establishing the dorsoventral asymmetry of eggshell and embryo. Analysis of follicle cell clones homozygous for a null allele of Ras demonstrates that Ras is required cell-autonomously to repress pipe transcription, the critical first step in embryonic dorsoventral patterning. The effects of aberrant pipe expression in Ras mosaic egg chambers can be ameliorated, however, by post-pipe patterning events, which salvage normal dorsoventral polarity in most embryos derived from egg chambers with dorsal Ras clones. The patterned follicular epithelium also determines the final shape of the eggshell, including the dorsal respiratory appendages, which are formed by the migration of two dorsolateral follicle cell populations. Confocal analyses of mosaic egg chambers demonstrate that Ras is required both cell- and non cell-autonomously for morphogenetic behaviors characteristic of dorsal follicle cell migration, and reveal a novel, Ras-dependent pattern of basal E-cadherin localization in dorsal midline follicle cells (James, 2002).

The mosaic analyses described here both clarify and contribute new information to the general understanding of dorsoventral patterning in Drosophila. In a simple model for embryonic dorsoventral patterning, Gurken/Egfr/Ras signaling in the egg chamber represses pipe transcription dorsally. pipe function in the ventral follicle cells is necessary to activate the serine protease cascade in the perivitelline space, which leads to activation of the Toll receptor in the embryonic plasma membrane by cleaved Spätzle (Spz) and, ultimately, a dorsoventral gradient of Dorsal nuclear translocation. Consistent with this hypothesis, loss of pipe function in the follicle cells leads to the absence of zygotic Twist and embryonic dorsalization, and ectopic expression of pipe throughout the follicular epithelium results in embryonic ventralization. In this work, however, no direct spatial relationship was found between dorsal Ras clones (and, therefore, ectopic pipe) in the egg chamber and ectopic Twist in the embryo (James, 2002).

These data are consistent with the hypothesis that additional patterning events modify the initial asymmetry determined by Egfr signaling and thereby establish the final Dorsal gradient. Additional patterning downstream of Egfr has also been suggested to explain another observation, that the embryonic phenotype resulting from maternal mutations in gurken or Egfr is not simply an expansion of ventral embryonic fates, as detected by Twist expression and formation of a larger ventral furrow. Rather, many embryos exhibit a pattern duplication characterized by two ventrolateral regions of maximum nuclear Dorsal and Twist, separated by a ventral minimum, which leads to the eventual invagination of two furrows. The result that Ras clones cell-autonomously derepress pipe suggests that such a refinement process occurs downstream of pipe (James, 2002 and references therein).

Consistent with these findings, the additional patterning events that govern the shape of the Dorsal gradient in wild-type embryos (and that can create two furrows in ventralizing mutants) do not occur in the follicular epithelium. Rather, the refinement process occurs at the level of Spätzle processing or Toll activation in the embryo. Furthermore, elegant mosaic analyses reveal that, while loss-of-function pipe or windbeutel (wind) clones on the ventral side of the egg chamber result in a local loss of ventral Twist expression in the embryo, ventral clones of pipe or wind also exert a non-autonomous effect on lateral embryonic fates. It has been hypothesized that an 8-12 cell wide ventral region in the egg chamber (defined by the spatial requirement for pipe) generates ventral information, perhaps imbedded in the vitelline membrane, which is then competent to establish the Dorsal gradient along the entire dorsoventral axis, presumably via outward diffusion of a ventral morphogen (James, 2002 and references therein).

In addition, other patterning mechanisms that influence the Dorsal gradient have been discovered recently. Nuclear translocation of Dorsal can be modified by maternal Sog and Dpp in a pathway parallel to Toll signaling. Also, the serine protease cascade upstream of Toll is subject to feedback inhibition, providing an additional level of regulation (James, 2002 and references therein).

This work, together with the discoveries discussed above, points to a model in which the initial asymmetry generated by restriction of pipe to the ventral-most 40% of the egg chamber leads to the cleavage of the zymogen Spz in the ventral-most 40% of the perivitelline space. The positive ventralizing activity of C-Spz (the active form of Spz) is opposed by the activity of a diffusible inhibitor, possibly N-Spz. In wild-type patterning, this inhibitor narrows the domain of ventralizing C-Spz activity, originally determined by pipe expression in follicle cells, from 40% of the perivitelline space to approximately 20%. This narrower region of ventralizing activity then activates Toll in a graded fashion, defining the final shape of the Dorsal gradient (James, 2002).

In Ras mosaic egg chambers, pipe is expressed ectopically in dorsal Ras clones, but is prevented from activating Twist because the size or width of the clone is too small to overcome the action of the inhibitor. Theoretically, this inhibitor could be present throughout the perivitelline space but overcome only by a large enough pool of C-Spz. The inhibitor could itself be a byproduct of Spz cleavage (N-Spz), emanating only from sources of ventralizing activity. If this mechanism is correct, then the data predict that, until the pool of C-Spz reaches a certain size, the inhibitor is more potent than C-Spz. It is hypothesized that only very large dorsal Ras clones could exceed the size or width threshold needed to overcome the inhibitor and cause ectopic expression of Twist in the embryo. In the rare cases in which embryos ectopically expressed Twist, the region of ectopic Twist was always found in contact with the normal ventral domain of Twist. It is hypothesized that these embryos result from egg chambers in which Ras clones overlap the edge of the normal pipe expression domain and alter the shape of the domain by creating a local bulge in pipe expression. The inhibitor downstream of pipe may then narrow that distorted ventral shape to produce a corresponding, though smaller, bulge in the Twist domain (James, 2002).

Support for this hypothesis comes from the analysis of egg chambers with ventral wind clones surrounding small 'islands'of wild-type cells in ventral-most positions. In these instances, if the wild-type 'island' is less than 4-6 cells wide, it is not able to induce Twist expression in the embryo. This situation is similar to the result that dorsal Ras clones fail to induce Twist. Together, these data suggest that ventral information from small patches of cells expressing pipe can be 'swamped out' by neighboring dorsally-fated cells, probably through the function of a diffusible inhibitor (James, 2002 and references therein).

The ventralizing activity of ectopic pipe expression in Ras mosaic egg chambers can be overcome by post-pipe patterning events. The existence of this compensatory mechanism demonstrates that embryonic dorsoventral patterning is a robust process. This resilience is generated by the action of additional rounds of pattern refinement, which may help to buffer these important early events of embryonic development from perturbations in signaling caused by genetic defects or environmental factors. Furthermore, the zygotic genes that orchestrate dorsoventral patterning (Dpp/BMP4 and Sog/chordin) are conserved between arthropods and chordates, and, extraordinarily, their biological function in dorsoventral patterning is also conserved. However, where flies use post-pipe pattern refinement to buffer the Dorsal gradient, which determines the Dpp/Sog pattern, chordates use a completely different developmental pathway to arrive at the BMP4/chordin patterning event. Consequently, the specific mechanism of dorsoventral 'buffering' in flies cannot be shared by chordates, suggesting that each group evolved unique mechanisms to buffer the conserved aspects of dorsoventral patterning. Understanding the degree of diversity among buffering systems will be useful in determining evolutionary relationships and will facilitate studies examining the evolution of developmental mechanisms (James, 2002).

To explain the differential phenotype of eggs laid by females transheterozygous for strong hypomorphic alleles of Ras, it is hypothesized that either eggshell patterning is more sensitive than embryonic patterning to reductions in Ras, or a Ras-independent Egfr effector pathway mediates embryonic patterning. The cell-autonomous derepression of pipe in Ras null clones demonstrates that Ras is required to initiate embryonic dorsoventral patterning and suggests that the first hypothesis is correct. Conversely, the result that embryos developing from Ras mosaic egg chambers are rarely abnormal supports the second hypothesis, with the added complexity that the Ras-independent effector pathway must also bypass pipe. As described above, however, the data suggest that very large Ras clones, or an entirely mutant epithelium, would induce ectopic Twist expression in the embryo and lead to severe embryonic dorsoventral patterning defects (James, 2002).

Indeed, in five of 26 cases, Ras follicle cell clones did result in ectopic Twist in the embryo, presumably because those clones were large enough to overcome the post-pipe patterning events that dampen the ventralizing effects of smaller clones. Furthermore, ectopic expression of pipe throughout the follicular epithelium is indeed sufficient to cause embryonic ventralization. Therefore, the lack of embryonic defects resulting from most Ras mutant follicle cell clones, which are too small to overcome post-pipe patterning processes, is misleading with respect to the question of whether Ras is required generally for embryonic dorsoventral patterning. Given these considerations, the results are more consistent with the first hypothesis, that is, Ras is required for dorsoventral patterning of the embryo, and eggshell patterning is more sensitive than embryonic patterning to reductions in Ras (James, 2002).

Why would eggshell patterning be more sensitive than embryonic patterning? Furthermore, why would that sensitivity be specific to reductions in Ras and not Gurken or Egfr? Two hypotheses can explain why eggshell patterning would be acutely sensitive, specifically to reductions in Ras. (1) Dorsoventral patterning of the eggshell requires amplification and modulation of Egfr activity, achieved by autocrine signaling involving Spitz, Rhomboid, Vein, and Argos, to define two dorsolateral populations of follicle cells. These post-Gurken patterning events are eggshell-specific, while pipe repression is achieved solely by Gurken signaling. Furthermore, since the results support the hypothesis that low lateral levels of Gurken suffice to repress pipe, it is likely that pipe repression requires only low-level signaling. The hypomorphic Ras mutations primarily affect the transcription of Ras, which, theoretically, reduces the number of Ras molecules rather than the effectiveness of each molecule. Therefore, the hypomorphic mutant may produce enough Ras molecules to transduce the initial Gurken signal, repress pipe and pattern the embryo, but too few Ras molecules to transduce the high levels of Egfr signaling required for eggshell patterning. Thus, the number of Ras molecules may be limiting for eggshell patterning, which requires an intense surge of Ras signaling, but not limiting for pipe repression, which may be accomplished with only a trickle of signaling that is easily transduced by a small number of Ras molecules (James, 2002).

(2) A second hypothesis can also explain why eggshell patterning would be more sensitive than embryonic patterning specifically to reductions in Ras. The data demonstrate that Ras mutant cells fail to initiate or accomplish dorsal follicle cell morphogenesis. These phenotypes, along with the pipe-lacZ results, suggest that Ras is required to establish dorsal follicle cell fate, a prerequisite for morphogenesis. The data are also consistent with an additional requirement for Ras specifically regulating morphogenesis. Gurken and Egfr, however, may be directly required only for patterning and may therefore influence morphogenesis only indirectly. In support of this hypothesis, the Nrk receptor tyrosine kinase, identified as an Enhancer of Ras in eggshell development, may provide an independent Ras pathway input specific for morphogenesis. Thus, a Ras mutation that produces weak embryonic defects may dramatically disrupt eggshell structures by affecting both patterning and morphogenesis. This synergism may compromise dorsal appendage morphology enough to resemble the eggshell phenotype of a severe gurken or Egfr allele, which affects eggshell and embryo equally (James, 2002).

In addition to extending understanding of embryonic dorsoventral patterning and defining the relative contribution of Ras signaling toward establishing eggshell and embryonic fates, mosaic analyses have revealed the requirement for Ras during dorsal appendage morphogenesis. How might Ras regulate dorsal follicle cell migration? By transducing signals for dorsal follicle cell fate, Ras may affect transcription of genes involved in morphogenesis. Notably, Broad-Complex (BR-C), Mirror, Bunched, and Fos/Kayak respond to Egfr signaling and likely affect transcription of genes involved in dorsal appendage formation. Alternatively, Ras activity may directly affect key cytoskeletal or adhesion molecules that play active roles during the morphogenetic process. Since many of the events of dorsal follicle cell morphogenesis occur quite rapidly -- stage 11, for example, encompasses profound morphogenetic changes, but is completed in less than 30 minutes, and since MAP kinase activity is dynamic during the early stages of morphogenesis, it is suspected that Ras signaling directly modulates the activity of migration molecules. Most likely, Ras functions in dorsal follicle cell migration through some combination of direct cytoplasmic effects and transcriptional regulation (James, 2002).

To understand the role of Ras in cell migration, other migration events controlled by Ras signaling have to be taken into consideration. In Drosophila, disruptions in Ras signaling can hinder the migration of a subset of follicle cells called border cells. Significantly, border cell fate remains properly specified in these experiments, revealing migration-specific functions for Ras. Importantly, the movement of dorsal follicle cells differs significantly from that of border cells, which navigate through germline cells as a small epithelial patch. Dorsal appendage formation involves the coordinated morphogenetic movements of an epithelial sheet. These differences demonstrate that each migration event offers a unique opportunity to study the role of Ras during developmentally regulated cell migration in Drosophila (James, 2002).

What are the effectors of Ras during dorsal follicle cell migration? One possibility is E-cadherin, since Ras is required for the basal localization of this molecule in midline follicle cells. Importantly, the levels of apicolaterally-localized E-Cad appear normal in Ras clones. This result suggests that Ras signaling does not regulate the canonical adherens-junction function of E-Cad, which provides integrity to the epithelium during morphogenesis. Instead, Ras affects the basal localization of E-Cad on the dorsal midline, which may anchor the midline cells or otherwise influence the mechanical movements of the dorsal appendage primordia (James, 2002).

The loss of basal E-Cad in a Ras clone on the midline was surprising because the dorsal midline is thought to be a region of significantly diminished Egfr activity. Why, then, would Ras be required there for the localization of an adhesion molecule? Perhaps Ras signaling is actually active on the dorsal midline between stages 10B and 12. Alternatively, a history of high and then low Egfr/Ras signaling may be required for basal E-Cad protein localization in midline cells. Further exploration of the precise regulation of E-Cad during dorsal follicle cell morphogenesis is needed to elucidate the relationship between Ras signaling and E-Cad localization, and to address whether basal localization of E-Cad in anterior and midline cells is required for the proper morphogenesis of dorsal appendages (James, 2002).

In addition to E-Cad, Ras may regulate other adhesion, signaling, or cytoskeletal molecules to permit or instruct dorsal follicle cell migration. Known cellular effectors of Ras in mammalian cells include c-Raf, RalGDS, and PI 3 kinase. To identify molecules that interact with Ras in Drosophila follicle cells, dominant enhancers of a weak Ras eggshell phenotype have been sought. Interestingly, two enhancers, dock and Tec29A, encode signaling molecules that directly regulate cytoskeletal function. Studies that separate cell fate specification from morphogenetic events are needed to determine whether Ras actively controls the morphogenesis of dorsal follicle cells (James, 2002).

In conclusion, the finding that Ras is required for pipe repression argues against the hypothesis that a Ras-independent pathway transduces Egfr signals to pattern the embryo. This result contributes to the growing body of evidence that, in Drosophila, Egfr signaling is transmitted to the nucleus primarily by the Ras pathway rather than by alternative effector molecules. Furthermore, the data demonstrate that dorsoventral patterning is buffered by post-pipe patterning events that define the final shape of the embryonic dorsoventral gradient. Additionally, it has been shown that Ras is required for dorsal appendage patterning and morphogenesis as well as for the proper subcellular localization of E-cadherin, a major epithelial adhesion protein. Ras signaling is linked to cell migration in many developmental and disease contexts, providing justification for further dissection of Ras pathway function during dorsal appendage morphogenesis in Drosophila (James, 2002).

Ras function during myogenesis

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined. Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).

This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2 divides asymmetrically to give rise to two founder cells, one specific for a pair of persistently Eve-positive heart-associated or pericardial cells (EPCs) in every hemisegment and a second of previously undetermined identity. This second founder coexpresses Eve along with the gap gene Runt, with Eve levels rapidly fading but Runt persisting as development proceeds. By the time that Eve is evident in the EPCs, Runt labels a single somatic muscle, dorsal oblique muscle 2 (DO2). Runt is also detected in the muscle DO2 precursor during germband retraction (Carmena, 2002).

The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably expressed marker for it is unavailable (Carmena, 2002).

To further substantiate the lineage relationships among these progenitors and founders, observations related to RTK signaling dependence of P2 and P15 specification were used: whereas P15 requires the activities of both Egfr and Htl, only Htl is involved in P2 formation. In this way, targeted mesodermal expression of a dominant negative form of Egfr strongly blocks formation of DA1 but not the EPCs. Also, consistent with DO2 and EPC founders being the progeny of P2, DO2 development, like that of the EPCs, is not affected by dominant negative Egfr. Additional support for the sibling relationship between the DO2 and EPC founders derived from the analysis of targeted expression of a dominant negative form of Htl. Under conditions in which early mesoderm migration is not perturbed, dominant negative Htl generates an incompletely penetrant phenotype in which different hemisegments lose derivatives of P2, P15, or both progenitors. With such partial inhibition of Htl activity, muscle DO2 and the EPCs are consistently either both present or both absent from any given hemisegment; in no cases did one of these cell types develop without the other, as expected for cells derived from a common progenitor. In contrast, muscle DA1 frequently forms in the absence of muscle DO2 and the EPCs, consistent with its derivation from an independent progenitor. Taken together, these data establish that the EPC and DO2 founders are sibling cells of the P2 division, whereas the other Eve-expressing muscle founder arises from a different progenitor (Carmena, 2002).

This model differs from one derived on the basis of clonal analysis in which it was proposed that the two Eve-positive mesodermal cell types originate from the same progenitor. This discrepancy may relate to the fact that muscles form by sequential cell fusions involving both founders and fusion-competent cells of potentially different parental cell origins, thereby confounding the interpretation of clonal analysis in which the cytoplasm of a single myotube is labeled by the lineage tracing marker (Carmena, 2002).

Autoregulation of a signal transduction cascade can cause either enhancement or attenuation of the transduced signal, depending on whether the feedback loop acts positively or negatively. Both types of feedback control occur during the Ras- and N-mediated specification of Eve mesodermal progenitors. Ras activation leads to increased expression of several proximal components of both the Fgfr and Egfr pathways that serve to amplify and/or prolong both fate-inducing RTK/Ras signals in the emerging Eve progenitors. A similar amplification of Egfr signaling occurs via induction of Rho during Drosophila oogenesis and mesothoracic bristle formation, and via upregulation of Egfr expression during C. elegans vulva development. The present analysis also uncovers a positive feedback mechanism for inductive Fgfr signaling, in this case via increased expression of not only the Htl receptor but also its specific signal transducer, Heartbroken (Hbr). Interestingly, the data suggest that the downstream components may respond to different thresholds of Ras activity since Rho exhibits a less robust response than either Htl or Hbr to Ras activation (Carmena, 2002).

A negative feedback loop occurs in the Egfr pathway through autoactivation of the inhibitory ligand, Aos. Aos cooperates with Dl to block the progenitor-inducing Ras signal in both adjacent and more remote cells of the cluster. Aos could also exert a late inhibitory effect on the progenitor by terminating the inductive Egfr signal since Spi levels decrease following the establishment of cellular identity. Consistent with this possibility, MAPK activation fades from the singled out progenitor prior to its asymmetric division, suggesting that prolonged RTK signaling does not occur (Carmena, 2002).

Positive and negative feedback also occur during N function in the mesoderm. N activation both downregulates its ligand Dl and upregulates its own expression, thereby enhancing the potential for inhibitory signaling in cells not destined for the progenitor fate. Together, these opposing changes in Dl and N expression produce a unidirectional inhibitory signal emanating from the prospective progeni\tor and directed toward the adjacent nonprogenitor cells. Similar feedback mechanisms regulate the N pathway in the Drosophila embryonic CNS, adult PNS and wing vein-forming cells, and also apply to the N receptor-ligand combinations controlling gonadal and vulval cell fates in C. elegans (Carmena, 2002 and references therein).

Competitive cross-talk between Ras and N is manifest by the ability of the latter to block the expression of proximal components of the two RTK pathways—namely Htl/Hbr and Rho -- as well as to prevent the associated activation of MAPK. An antagonistic relationship between the RTK and N pathways is also revealed by the strong genetic interaction between Dl and Egfr, in agreement with previously reported genetic studies. Collectively, these results establish that the RTK and N pathways are not simply acting in parallel to exert opposing influences on progenitor specification; rather, N must be interfering with the generation and/or transmission of the inductive RTK signal. This effect could occur at multiple levels. The ability of activated N to at least partially block MAPK activation induced by constitutive Ras argues that N functions downstream of Ras. An additional direct effect of N on expression of Ras-responsive target genes cannot be excluded, particularly since Enhancer of split repressors are involved in the specification of progenitor cell fates. Such targets could include eve itself, or, given positive autoregulation of RTK signaling, one or more RTK pathway components (Carmena, 2002).

During C. elegans vulva development, Lin-12/N inhibits Egfr activity by stimulating the expression of a MAPK phosphatase. This is an attractive explanation for the effect of N observed here. However,while stimulation of a MAPK phosphatase could contribute in part to N inhibition of Ras signaling in the Drosophila embryonic mesoderm, it cannot be the only explanation since a constitutively activated form of Pointed is completely unable to reverse the activity of constitutive N. This is in marked contrast to the substantial reversal of N exhibited by activated Ras and occurs even though Pnt is a major Ets domain activator involved in RTK-dependent eve regulation (Carmena, 2002).

To account for the differential abilities of constitutive Ras and Pnt to compete effectively with constitutive N, the idea is favored that an additional, as yet uncharacterized, Pnt-independent function of Ras may be a target of N inhibition. Hence, there exist at least four potential sites of competitive interaction between these pathways: (1) direct regulation of target gene enhancers by pathway-specific transcriptional activators and repressors; (2) regulation of MAPK phosphorylation; (3) inhibition by N of RTK pathway component expression; and (4) an additional level of Pnt-independent cross-talk between Ras and N. These mechanisms modulate the relative flux through the competing Ras- and N-dependent processes and determine which pathway predominates, thereby achieving a critical threshold for a given cell fate. The importance of relative activity thresholds is underscored by results with different combinations of activated Ras and N insertions in which different numbers of Eve-expressing cells were induced, presumably reflecting slight fluctuations in the relative strengths of each pathway in individual cells. Similar levels of control may underlie the antagonistic effects of Ras and N in other developmental contexts (Carmena, 2002 and references therein).

Although the net effect of Ras and N signaling in the present system is the result of their antagonistic relationship, several forms of cooperative cross-talk also occur. For example, Ras activation induces the expression of Dl. Since the Ras signal is amplified by a positive feedback loop, this has the effect of biasing Dl expression to the emerging progenitor, thereby generating a polarized, nonautonomous inhibitory signal that acts on adjacent cells of the cluster. Aos is also a target of Ras activation, and Aos acts synergistically with the neurogenic pathway to block inductive RTK signaling. Thus, through its effects on the two inhibitory ligands, Dl and Aos, Ras cooperates with N to ensure that only one cell segregates as a progenitor from each equivalence group (Carmena, 2002).

Further cooperation is evident in the N-mediated down-regulation of Dl and Aos in prospective nonprogenitors, a combination of negative feedback and cross-talk that effectively prevents neighboring cells from sending an inhibitory signal to the emerging progenitor. Yan is yet another Ras-dependent component that reinforces the effect of N: when MAPK is suppressed in cells in which N is active, Yan is a functional repressor that blocks progenitor fate. Other examples of cooperation between Ras and N signaling include mammalian cell tumorigenesis and photoreceptor specification in the Drosophila eye (Carmena, 2002).

One seemingly paradoxical signaling interaction is N expression upregulation by Ras. Since Ras output is amplified in the progenitor, N protein might be expected to decrease in this cell, thereby restricting lateral inhibition to the appropriate direction. However, increased N in the Eve progenitor does not actually affect the polarity of lateral inhibition because the activating ligand, Dl, is downregulated by N in the adjacent nonprogenitors. Of further relevance, Dl may inhibit N activity when the two proteins are expressed in the same cell. Moreover, upregulation of N normally occurs very late in progenitor specification, as opposed to Dl which increases in one cell of the cluster at an earlier stage. Lastly, increased N has independent biological significance in progenitors since N is required for an asymmetric division that immediately follows the specification of these cells. In this respect, the response of the N receptor to Ras activation is an efficient, anticipatory 'feed forward' mechanism for insuring that this cell division is appropriately regulated (Carmena, 2002).

A model is presented that summarizes the progressive changes in Ras and N signaling and the cellular events corresponding to each stage. The model emphasizes that, while clusters of equivalent cells begin with the same signaling repertoires, they acquire distinct biochemical states which uniquely determine progenitor and nonprogenitor identities. Most important, this complex circuitry drives the reciprocal alterations in Ras and N activities toward the requisite thresholds that are essential for determining these fates. What biases one cell in an equivalence group toward the imbalance in Ras and N signaling that initiates the entire mechanism remains an open question. One possibility is that localized expression of the RTK ligands may provide the initiating event. Similarities of other developmental systems reinforce the general relevance of these conclusions (Carmena, 2002).

The reinforcing effects of Aos and Dl are essential since neither is fully capable of insuring that only one progenitor segregates from each Eve cluster. Furthermore, simultaneous loss of both inhibitory pathways leads to the formation of additional Eve cells within the competence domain but outside of the normal Eve equivalence groups. This suggests that the combined actions of both inhibitors prevent the spreading of the inductive signal beyond the normal cluster boundaries, as might occur through positive feedback of Rho expression and the associated increase in secreted Spi production. Such a remote inhibitory mechanism is particularly relevant to Aos, which is hypothesized to act at a longer range than Spi. Of note, synergistic inhibition by Aos and the N pathway has not been observed in other systems (Carmena, 2002).

These results also revealed an effect of Aos on Htl- but not Egfr-dependent C2 cluster development. Although this could be interpreted as indicating a role for Aos in the inhibition of the Htl Fgfr, the idea that Aos is actually blocking basal and/or spontaneous levels of Egfr activation in C2 cells is favored. This interpretation is supported by the finding that a dominant negative form of Egfr suppressed the effect of aos loss-of-function not only in Egfr-dependent C15, but also in C2, which does not require Egfr for its specification. In this cluster, the requisite Aos expression is dependent on Htl activity (Carmena, 2002).

Another advantage of combining Aos and Dl relates to their differing properties. Aos is a secreted inhibitor capable of acting over several cell diameters, whereas Dl -- although subject to proteolytic processing -- is generally considered a membrane-bound ligand requiring cell contact for its activity. If a progenitor emerges from the center of a cluster such that it is in close proximity to all of its initially equivalent neighbors, then Dl alone might be sufficient for the segregation of only one progenitor. However, if a progenitor forms on the periphery of a cluster, then the addition of Aos would compensate for the inability of Dl to inhibit its more distant neighbors. Thus, two distinct modes of lateral inhibition have complementary and reinforcing functions (Carmena, 2002).

The involvement of RTK/Ras and N pathways in the specification of Eve muscle and heart progenitors exemplifies the complex regulatory interactions that can occur between two antagonistic signaling pathways acting in concert. These findings demonstrate RTK/Ras and N signals do not function independently, converging only at the most distal step leading to a particular biological response. Rather, their effects are intertwined at multiple levels to form an integrated network of cross-talk nodes and feedback loops. The combination of cell autonomous and nonautonomous components of both pathways affords the high degree of regulatory versatility and specificity required to generate the polarized signaling activities that distinguish progenitors from their nonprogenitor neighbors. These interactions are especially remarkable since, once initiated, they propagate into self-sustaining cascades that differentially drive equipotent cells to their individual fates. Of further significance, the mesodermal cells produced by these mechanisms give rise to the differentiated derivatives that compose the stereotyped structures of the embryonic heart and body wall muscles. Thus, the signaling circuitry uncovered here not only establishes the finely tuned balance between the inductive and inhibitory influences which coordinately generate progenitor cell patterns, but also sets the stage for subsequent morphogenetic events (Carmena, 2002).

Downstream-of-FGFR is a fibroblast growth factor-specific scaffolding protein and recruits Corkscrew upon receptor activation - Signaling upstream of Ras

Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. A functional characterization has been performed of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, it was demonstrated that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, two regions were identified that are important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, the results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response (Petit, 2004).

Genetic epistasis experiments have shown that Dof functions downstream of the activated FGFRs and upstream or in parallel to Ras. However, the biochemical function of Dof in the interpretation of the chemotactic response to FGFR signaling has not been addressed so far. Using in vivo rescue assays, a minimal Dof protein containing the first 600 amino acids of Dof was identified that allows rescue of both mesodermal and tracheal cell migration. Although the rescue in the tracheal system is not as efficient as the rescue observed with the wild-type construct, all six branches can migrate out, demonstrating that the first 600 amino acids of Dof retain the capacity to read out the local activation state of the FGFRs and to relay the signal to the migration machinery, albeit with somewhat reduced efficiency. Deletion from the C terminus of this dof minigene, as well as internal deletions, results in loss of rescue capacity, thus identifying regions of functional importance (Petit, 2004).

All of the constructs were examined in a Drosophila S2 cell culture assay, in which either the FGFR or the Torso signaling system was activated. Both full-length Dof and Dof600 are phosphorylated on tyrosine residues upon FGF signaling, but Torso cannot use Dof as a substrate. These results are consistent with in vivo data showing that Dof is exclusively needed for FGF-mediated signal transduction and that Torso is able to activate the MAPK cascade in the absence of Dof in dof mutant embryos. Using coimmunoprecipitation experiments, it was shown that Dof forms a complex with both FGFRs and that the first 484 amino acids, although not phosphorylated upon FGF signaling, are required and sufficient for the association with the FGFRs, demonstrating that phosphorylation of Dof is not necessary for complex formation. Cell culture analysis is in line with studies showing that the N-terminal part of Dof directly interacts with the kinase domains of Btl and Htl in yeast two hybrid assays. In addition, it was observed that both the juxtamembrane and the C terminus of Btl can be deleted without affecting considerably the quality of the rescue capacity of the receptor. Thus, it appears that Dof directly docks onto the kinase domain of the FGF receptor, in contrast to the vertebrate FGFR adapter SNT/FRS2, which interacts with a sequence motif in the juxtamembrane region of the receptor (Petit, 2004).

Since Dof becomes phosphorylated upon FGFR signaling in S2 cells, it was asked whether it was possible to identify functionally important phosphorylation sites, the proteins recognizing these sites in the phosphorylated state, and confirm the results in vivo by making use of the rescue assay and genetic analysis. Two potential phosphorylation target sites were identified by sequence analysis in the essential region comprising amino acids 485 to 600. While mutation of each individual site results in reduced phosphorylation of Dof600 in S2 cells upon FGFR signaling, mutation of only one of these sites, tyrosine 515, abolished the migration rescue capacity in vivo. Since the functionally required tyrosine residue was part of a putative consensus binding site for the SH2 domain of the nonreceptor tyrosine phosphatase Csw/SHP-2, the interaction of Csw with Dof was tested using coimmunoprecipitation experiments; Csw is indeed recruited to the activated signaling complex via Dof. It was found in rescue assays that both the region 485 to 600 as well as the region from 600 to the C terminus (construct dofDelta485-600) are able to confer function to the signaling-deficient N terminus (residues 1 to 484). It is known that the C-terminal sequences also recruit the Csw phosphatase in the absence of tyrosine 515, but it is not known know whether they do so directly or indirectly. Further deletion analyses and biochemical studies will be required to address this question (Petit, 2004).

Genetic evidence supporting an interaction between Dof and Csw was provided some time ago by the finding that mutations in csw produce a phenotype identical to bnl, btl, and dof; i.e., tracheal and mesodermal cells fail to migrate. The sum of these results clearly assign a crucial role for both Dof and Csw downstream of the FGFRs in the migratory response, indicating that the ligand-dependent phosphorylation of Dof leads to the recruitment of Csw to the signaling complex, ultimately triggering cell locomotion. SHP-2, the vertebrate homologue of Csw, has been shown to be required at the initial steps of gastrulation, as mesodermal cells migrate away from the primitive streak in response to chemotactic signals initiated by fibroblast growth factors. In addition, SHP-2 has also been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAPK pathway upon induction of another chemotactic factor, the hepatocyte growth factor/scatter factor, thus placing SHP-2/Csw as a key player in branching morphogenesis induced by diverse chemotactic factors. Therefore, it appears that both in invertebrates and vertebrates, SHP-2/Csw plays a major role in RTK signaling in the control of cell migration. The similarity of the Drosophila FGF signal transduction pathway to the vertebrate FGF pathway make the fly system accessible to address future issues not resolved in vertebrates, such as the targets of SHP-2/Csw involved in Ras activation and/or cell migration (Petit, 2004).

Using the dpERK antibody as a readout for the activation of the Ras/MAPK pathway in vivo, it was found that abolishing the interaction between the Dof600 minimal protein and Csw abolishes the activation of the MAPK cascade upon FGFR signaling. The strong correlation found between migration and MAPK activation when analyzing all mutant dof constructs in this assay might indicate that local activation of the Ras/MAPK pathway in tracheal tip cells is sufficient to trigger the migratory response upon Btl signaling. However, two lines of evidence suggest that this might not be the case (Petit, 2004).

In one case, it has been observed that under conditions in which all tracheal cells sustain high levels of Ras/MAPK activity (upon RasV12 overexpression), tracheal cells migrate normally in wild-type embryos. In sharp contrast, ectopic expression of the Bnl ligand leads to a complete disruption of directed migration. Therefore, high levels of Ras/MAPK activity do not appear to produce the same migratory response as ligand-activated FGFR signaling. Indeed, and again in contrast to ectopic Bnl, overexpression of RasV12 in wild-type embryos does not produce significant filopodial activity in DT tracheal cells, confirming that the activation of Ras is not sufficient to produce cytoskeletal rearrangements by itself (Petit, 2004).

In the other case, it was also observed that while the Dof600 protein lacking the ankyrin repeats did allow FGFR-dependent activation of the Ras/MAPK pathway and downstream nuclear response genes, this protein failed to induce migration. Thus, even local Ras activation under the control of the endogenous ligand Bnl, Btl, and Dof600DeltaAR is unable to activate the migratory machinery. Interestingly, it has also been reported that Ras activation is insufficient to guide RTK-mediated border cell migration during Drosophila oogenesis (Petit, 2004).

Is Ras activation then required at all for cells to produce a cytoskeletal response and migrate directionally? Unfortunately, genetic analysis cannot be used to directly address this question in the embryo since maternal and zygotic loss of Ras activity results in embryos that do not develop far enough to analyze the tracheal system. However, when activated Ras (RasV12) is expressed in the tracheal system or in the mesoderm of dof mutant embryos, a certain rescue of migration can be obtained. This suggests that Ras signaling is essential but not sufficient for efficient FGFR-dependent cell migration; additional proteins binding to the receptor, to Dof or to Csw appear to be crucial for a chemotactic response. To analyze the role of Ras experimentally and in detail, mitotic clones lacking Ras activity should be analyzed with regard to their migration properties. Recent reports concerning the role of FGF signaling in the migration of mesodermal and tracheal cells during late larval development might provide the basis for such analyses (Petit, 2004).

Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl cyclase activation requiring neurofibromin and Ras

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in human and in other animal models. A novel growth factor stimulated adenylyl cyclase (AC) pathway has been identified in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galphas. This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galphas, can activate AC. This study also shows that Galphas is the major Galpha isoform in fly brains, and a second AC pathway is defined stimulated by serotonin and histamine requiring NF1 and Galphas. A third, classical Galphas-dependent AC pathway, is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, it is shown that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, it is demonstrated that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galphas-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies (Hannan, 2006).

This study defines three separate pathways for AC activation: (1) a novel pathway for AC activation, downstream of growth factor stimulation of EGFR that requires both Ras and NF1, but not Galphas; (2) an NF1/Galphas-dependent AC pathway operating through the Rutabaga-AC (Rut-AC) and stimulated by serotonin and histamine, as observed in the larval brain; (3) a classical G-protein coupled receptor-stimulated AC pathway operating through Galphas alone. The Rut-AC pathway may also be stimulated by PACAP38 at the larval neuromuscular junction and in adult heads as shown in previous studies. The AC activated by NF1/Ras (AC-X), or Galphas (AC-Y), has not yet been identified (Hannan, 2006).

This study shows for the first time that Ras can stimulate AC in an NF1-dependent manner in higher organisms, via an RTK-coupled pathway that is independent of the Galphas G-protein. The functionality of human NF1 in the fly system, and the high degree of identity between human and fly NF1 (60%), suggests that similar pathways for AC activation may also operate in mammals. Previous studies failed to detect stimulation of AC by Ras in cultured vertebrate cell lines, and in Xenopus oocytes, however, these cell types may not contain sufficient NF1 to support NF1/Ras-dependent AC activation. This is consistent with the observation that levels of both Ras and NF1 are critical for stimulation of AC activity in adult head membranes. The reported EGF activation of AC in cardiac myocytes and other tissues requires both Galphas, and the juxtamembrane domain of the EGFR, which is not present in the Drosophila EGFR (Hannan, 2006).

Experiments with human NF1 mutants show that the GRD domain and the RasGAP activity of NF1 are both necessary and sufficient for growth factor-stimulated NF1/Ras-dependent AC activity. It is also concluded that C-terminal residues downstream of the GRD are critical for both body size regulation and neurotransmitter-stimulated NF1/Galphas-dependent AC activity, thus defining for the first time a region outside the GRD that contributes to this pathway. Interestingly, expression of a human NF1 GRD fragment in Nf1-/- astrocytes results in only partial restoration of NF1-mediated increases in cAMP levels in response to PACAP. Thus, regions outside the GRD also seem to be necessary for activation of AC in these mammalian cells (Hannan, 2006).

Thus, NF1, while being a negative regulator of Ras, is also actively involved in stimulation of AC activity. Moreover, it regulates AC activity through at least two different mechanisms, one of which depends on the RasGAP activity of NF1. The multifunctional nature of the NF1 protein illuminates its importance in nervous system development, tumor formation and behavioral plasticity, and may also explain the wide range of clinical manifestations in neurofibromatosis type I (Hannan, 2006).

A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila

Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. This study demonstrates activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (Calcium ATPase at 60A: a SERCA mutant), and presents an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. All of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from the genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila (Freeman, 2010).

This study describes a new model for activity-dependent pre-synaptic plasticity in Drosophila. In the double mutant combination of comt and Kum, sustained elevation of neural activity (potentially including seizure-like motor neuron firing under normal rearing conditions) results in the expansion of motor synapses with a concomitant increase in transmitter release. These synaptic changes are mediated by the Ras/ERK signaling cascade and the activity of at least two key transcription factors, CREB and Fos. In vivo reporter assays also directly demonstrate Ras activation and enhanced transcription of Fos in the nervous system. CK is the only genetic model of synaptic plasticity in Drosophila in which pre-synaptic plasticity has been correlated with the Ras/ERK signaling cascade. This result is especially relevant given the wide conservation of the Ras/ERK signaling cascade in plasticity and recent demonstrations of the involvement of this signaling cascade in learning behavior in flies (Godenschwege, 2004; Moressis, 2009). Significant insights into Ras mediated regulation of both synapse growth and transmitter release are also presented (Freeman, 2010).

Non-invasive methods to manipulate neural activity in select neurons continue to be an important experimental target in plasticity research. In Drosophila, combinations of the eag and Shaker potassium channel mutants have long been used to chronically alter neural activity and study downstream cellular events. In recent years, transgenic expression of modified Shaker channels has also been generated and used to alter excitability in both neurons and muscles. However, the CK model of activity-dependent plasticity was developed since in synaptic changes in CK were consistently more robust than eag Sh and core plasticity-related signaling components were activated in a predictable manner in CK mutants. Another advantage with CK is the option of acutely inducing seizures as has been used to identify activity-regulated genes. CK thus combines advantages of both eag Sh and seizure mutants, and as is shown in this study, leads to an activity-dependent increase in synaptic size and transmitter release. It is believed that this model will prove highly beneficial to the large community of researchers who investigate synaptic plasticity in Drosophila. The utility of more recent techniques (such as the ChannelRhodopsin or the newly reported temperature sensitive TrpA1 channel transgenes) to induce neural activity-dependent synaptic plasticity at Drosophila motor synapses has not been tested yet and it will be interesting to see if these afford greater experimental flexibility in the future (Freeman, 2010).

Signal transduction through the Ras cascade has been shown to affect both dendritic and pre-synaptic plasticity in invertebrate and vertebrate model systems. In mammalian neurons, Ras signaling has been linked to hippocampal slice LTP, changes in dendritic spine architecture and plasticity of cultured neurons. In this context, Ras signaling has been shown to impinge on downstream MAP kinase signaling, thus implicating a canonical signaling module already established as a mediator of long-term plasticity in vertebrates. In Drosophila, expression of a mutant constitutively active Ras that is predicted to selectively target ERK leads to synapse expansion and increased localized phosphorylation of ERK at pre-synaptic terminals. In light of these observations, tests were performed to see if Ras signaling os necessary and sufficient for synaptic plasticity in CK. The results suggest that synaptic changes in CK are driven by stimulated Ras/ERK signaling in Drosophila motor neurons, and these can be replicated by directly enhancing Ras signaling in these cells. Furthermore Ras activation was found to be sufficient to cause stable elevation in pre-synaptic transmitter release. Finally, evidence is provided to show that synaptic effects of Ras activation require the function of both Fos and CREB in motor neurons. The consistency of signaling events in CK with those observed in mammalian preparations makes this a more useful and generally applicable genetic model of synaptic plasticity (Freeman, 2010).

In vivo reporters of neural activity have been difficult to design but offer better experimental resolution and flexibility over standard immuno-histochemical or RNA in situ methods to detect changes in gene expression in the brain. Thus, a good reporter permits increased temporal and spatial resolution, the option of live imaging (for fluorescent reporters) and in the case of transcriptional reporters, better understanding of cis-regulatory elements that control activity-dependent gene expression. This paper describes two genetically encoded reporters with utility clearly beyond the current study; a Raf based reporter to detect Ras activation in neurons and an enhancer based reporter to detect transcription of Fos (Freeman, 2010).

The Ras binding domain of Raf has been used previously to detect Ras expression in yeast, mammalian cell lines, and recently in hippocampal neuron dendrites. This study used a similar strategy to model the reporter using the conserved Ras binding domain and the cysteine-rich domain (RBD + CRD) from Drosophila Raf, under the reasonable assumption that this would provide sensitive reporter activity in neurons. This is the first time that a Ras reporter has been utilized in an intact metazoan organism to measure changes in endogenous Ras activity. In addition to confirming Ras activation in CK brains, it is expected that this reporter will find widespread use in tracing Ras activation in multiple tissues through development and in response to signaling changes in the entire organism. Since the reporter is based on the GAL4-UAS system, it can be expressed in tissues of choice, limiting reporter activity to regions of interest. Indeed, the experiments with the eye-antennal imaginal disc illustrate the utility of this reporter in identifying regions of activated Ras signaling during eye development (Freeman, 2010).

The Fos transcriptional reporter is one of the very few activity-regulated reporters in existence in Drosophila and it should find broad acceptance as a tool to map neural circuits in the fly brain that show activity-dependent plasticity. The reporter believed to be reasonably accurate since it is expressed in expected tissue domains (embryonic leading edge cells, for instance), and also co-localizes extensively with anti-Fos staining in the larval brain. There are several recognizable transcription factor binding motifs that can be detected in this 5 kb region of DNA (including binding sites for CREB, Fos, Mef2 and c/EBP). Which of these transcription factors regulate activity-dependent Fos expression from this enhancer is currently unknown. However, future experiments that dissect functional elements in this large enhancer region are expected to refine and identify these regulatory elements. Such studies are likely to lead the way in the development of a new generation of neural activity reporters in the brain (Freeman, 2010).

Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release

In Drosophila, each of the three larval instars ends with a molt, triggered by release of steroid molting hormone ecdysone from the prothoracic gland (PG). Because all growth occurs during the larval stages, final body size depends on both the larval growth rate and the duration of each larval stage, which in turn might be regulated by the timing of ecdysone release. This study shows that the expression of activated Ras, PI3 kinase (PI3K), or Raf specifically in the PG reduces body size, whereas activated Ras or PI3K, but not Raf, increases PG cell size. In contrast, expression of either dominant-negative (dn) Ras, Raf, or PI3K increases body size and prolongs the larval stages, leading to delayed pupariation, whereas expression of dn-PI3K, but not of dn-Raf or dn-Ras, reduces PG cell size. To test the possibility that altered ecdysone release is responsible for these phenotypes, larval ecdysone levels were measured indirectly, via the transcriptional activation of two ecdysone targets, E74A and E74B. It was found that the activation of Ras within the PG induces precocious ecdysone release, whereas expression of either dn-PI3K or dn-Raf in the PG greatly attenuates the [ecdysone] increase that causes growth cessation and pupariation onset. It is concluded that Ras activity in the PG regulates body size and the duration of each larval stage by regulating ecdysone release. It is also suggested that ecdysone release is regulated in two ways: a PI3K-dependent growth-promoting effect on PG cells, and a Raf-dependent step that may involve the transcriptional regulation of ecdysone biosynthetic genes (Caldwell, 2005; full text of article).

Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila

Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. This study finds that autophagy is induced following growth arrest, and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. These results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death, and that multiple degradation pathways cooperate in the efficient clearance of cells during development (Berry, 2007).

These studies indicate that arrest of PI3K-dependent growth is an important determinant of autophagic cell death of salivary glands during Drosophila development. Maintenance of growth by expression of either activated Ras, Dp110, or Akt in salivary glands is sufficient to inhibit salivary gland degradation. It is possible that the larger Dp110-, Akt- and RasV12-expressing salivary glands simply have more material to degrade, and this is why they persist. It is suspected that this is not the case, however, since Dp110-expressing glands are larger than RasV12-expressing glands, yet RasV12-expressing glands are less degraded. Although PI3K-dependent growth inhibits autophagy, growth could influence other downstream targets. However, the Atg1-induced suppression of the Dp110 persistent salivary gland phenotype and the persistence of vacuolated salivary gland cell fragments in atg loss-of-function mutants support the conclusion that growth arrest and autophagy are required for proper salivary gland degradation (Berry, 2007).

Ras and class I PI3K signaling are complex, and cross-talk occurs between these pathways. Although the data indicate that both activated Ras and PI3K have similar effects on salivary gland cell growth and inhibition of autophagy, it is observed that Ras-expressing cells were more intact 24 hours apf. Caspase activity is detected in Dp110-and Akt-expressing glands, and it is speculated that part of the degradation observed in Dp110 and Akt glands was due to caspases. Indeed, combining Dp110 expression with caspase inhibition resulted in intact salivary glands. This additive phenotype indicates that multiple degradation pathways are involved in autophagic cell death in vivo. Caspases were also active in Ras-expressing glands that were predominantly intact; thus activated Ras likely influences factors separate from caspases and the PI3K pathway. Ras regulates PI3K-independent pathways including MAPK and the cell cycle. Proliferating cells usually double in size prior to division, and because of this, cell growth and division are often considered synonymous. These studies demonstrate that although expression of either Myc or CyclinD with Cdk4 is sufficient to induce nuclear size, they do not inhibit salivary gland degradation. These data support the conclusion that growth arrest, but not cell cycle arrest, is an important determinant of salivary gland autophagic cell death. While many studies have defined relationships between cell cycle arrest and cell death, this study defines a unique relationship between cell growth arrest and cell death (Berry, 2007).

Given autophagy’s well established function in cell survival, a role for autophagy in cell death seems paradoxical. The discovery that caspases function in cells dying with a Type II autophagic morphology led to speculation that all programmed cell death is regulated by apoptosis factors. Further, the preponderance of in vitro evidence shows a role for autophagy in cell death when caspases or apoptosis factors are inhibited. This study found that reduced function of any one of seven atg genes inhibits salivary gland degradation. The incomplete degradation of salivary glands in multiple atg loss-of-function mutants provides the first in vivo evidence that autophagy and atg genes are required for proper degradation of cells during developmental cell death. Caspase activity and caspase-dependent DNA fragmentation occurs in these atg mutants, indicating that autophagy is a caspase-independent degradation pathway required for complete cell degradation in autophagic cell death during development. Further, induction of autophagy by Atg1 expression leads to premature caspase-independent salivary gland degradation. The data do not exclude a role for caspases in autophagic cell death. Either inhibition of caspases by p35 or reduced atg gene function result in delayed and incomplete degradation of salivary glands, and the combined inhibition of caspases with reduced atg function results in increased persistence of this tissue. These data suggest that autophagy and caspases function in parallel pathways during salivary gland cell death, and that both independently contribute to cell destruction. Further, the presence of both autophagy and caspases appears to be more typical of autophagic cell death that occurs under physiological conditions. Autophagic cell death models of mammary lumen formation and embryonic cavitation, as well as amphibian developmental cell death, all involve both processed caspase-3 and autophagy (Berry, 2007).

The designations of type I apoptotic death and type II autophagic death are based on morphological criteria. The current studies indicate that cell morphology likely reflects difference in the factors that are used to activate cell death and degrade the dying cell. The degradation of salivary glands in caspase mutants indicates that caspase-independent factors are involved in autophagic cell death. The presence of autophagosomes in dying salivary glands led to an investigation of cell death in atg gene mutants; stronger defects were observed in salivary gland degradation with perturbed atg gene function than with drice, ark, and dronc mutants. These data indicate that cell morphology is informative, given that it suggested autophagy is involved in the death of salivary glands. However, it is important to note that cell death classification that is based on morphology can be misleading, since salivary glands clearly use both caspases and autophagy, degradation mechanisms that had been speculated to be strictly associated with a single morphological form of cell death (Berry, 2007).

Now that it is clear that autophagy participates in cell death under some circumstances, it will be critical to determine how autophagy participates in cell killing and removal. A recent study showed that autophagy is required to generate the energy needed to promote phagocytosis signaling in an in vitro model of embryonic cavitation (Qu, 2007). This is not believed to be the same as the role of autophagy in salivary glands, since no phagocytosis is observed during salivary gland death. Alternatively, autophagy may be used to recruit and degrade factors that promote cell survival, such as the degradation of cytoplasmic catalase in mouse L929 cells. Finally, extreme levels of autophagy may be sufficient to cause a metabolic catastrophe by degrading substrates and mitochondria that are needed for energy. The latter possibility does exist in salivary glands, as expression of the Atg1 kinase is sufficient to induce the death of fat (Scott, 2007) and salivary gland cells. Unlike fat cells, elevated autophagy does not induce caspase-dependent DNA fragmentation in salivary gland cells, and expression of p35 does not inhibit Atg1-induced death (Berry, 2007).

The prevalence of apoptosis and the potent killing potential of caspases raise the question of why autophagy participates in developmental cell death. In the context of Drosophila and other insects, larval cells have a modified endoreplication cell cycle that results in the production of gigantic cells. The number and size of cells may prohibit engulfment and digestion by phagocytes, and autophagy may be necessary for self-degradation. Further, the life history of the organism may lead to an understanding of why autophagy participates in the destruction of tissues. Drosophila do not feed during the 3 day period of metamorphosis. Thus, the differentiation and morphogenesis of the entire adult occurs in the absence of food, and the resources to build the adult fly must come from reserves that are set aside during larval development. One important source of these resources is the fat that exhibits elevated levels of autophagy at the onset of metamorphosis. Several other large larval tissues are destroyed by autophagic cell death during metamorphosis including the midgut and salivary glands. It is speculated that like fat, catabolism of these tissues by autophagy provides resources that are needed to construct the adult. Similarly, it is speculated that the large number of autophagosomes observed in dying amphibian cells may serve to recycle nutrients during metamorphosis when these animals do not feed (Berry, 2007).

These studies have indicated that it is necessary to be cautious when considering autophagy to be either a cell survival or cell death process. Perhaps it is useful to consider autophagy for what it is; a catabolic process that contributes to many cellular and biological processes. This is not that different from the caspase proteases that are widely considered to be apoptosis proteases, as it is now clear that caspases also function in cell differentiation. Future studies are likely to show that autophagy functions in many cell types, and that its contribution to cell survival and cell death are dependent on the type and physiological context of the cell (Berry, 2007).

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders, and in mature tissues, it can lead to cancer. This study identified Rabex5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking and ubiquitin ligase activity. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis (Yan, 2010).

MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila

The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. This study presents the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels (Zhang, 2011).

The insulin signal transduction pathway is regulated by cross-talk from several other signaling pathways. This includes input from the amino-acid sensing TOR pathway into regulation of insulin pathway activity by way of S6 kinase regulating IRS. Signaling downstream of growth factor receptors has also been linked to regulation of insulin signaling. The active form of the small GTPase Ras can bind to the catalytic subunit of PI3K and promote its activity. Expression of a form of PI3K that cannot bind Ras allows insulin signaling, but at reduced levels. The work reported in this study provides evidence for a second mechanism through which growth factor receptor signaling through the MAPK/ERK pathway modulates insulin pathway activity. Transcriptional control of inr gene expression by EGFR signaling may provide a means to link developmental signaling to regulation of metabolism. In this context, a statistically significant correlation wass noted between EGFR target gene sprouty and inr gene expression at different stages during Drosophila development (Zhang, 2011).

Several steps of the insulin pathway can be regulated by phosphorylation. Given that the MAPK/ERK pathway is a kinase cascade, a priori, the possibility of phosphorylation-based interaction between these pathways would seem likely. However, this appears not to be the case. Acute pharmacological inhibition of the MAPK/ERK pathway proved to have no impact on insulin pathway activity. Thus short-term changes in MAPK/ERK pathway activity do not seem to be used for transient modulation of insulin pathway activity. Instead, the MAPK/ERK pathway acts through the ETS-1 type transcription factor Pointed to control expression of the inr gene. Transcriptional control of inr suggests a slower, less labile influence of the MAPK pathway. Taken together with the earlier studies, these findings suggest that growth factor signaling can regulate insulin sensitivity by both transient and long-lasting mechanisms (Zhang, 2011).

Why use both short-term and long-term mechanisms to modulate insulin responsiveness to growth factor signaling? The use of direct and indirect mechanisms that elicit a similar outcome is reminiscent of feed-forward network motifs. Although these motifs are often thought of in the context of transcriptional networks, the properties that they confer are also relevant in the context of more complex systems involving signal transduction pathways. In multicellular organisms, feed-forward motifs are often used to make cell fate decisions robust to environmental noise. The findings suggest a scenario in which a feed-forward motif is used in the context of metabolic control, linking growth factor signaling to insulin responsiveness. In this scenario, growth factor signaling acts directly via RAS to control PI3K activity and indirectly via transcription of the inr gene to elicit a common outcome: sensitization of the cell to insulin. This arrangement allows for a rapid onset of enhanced insulin sensitization, followed by a more stable long-lasting change in responsiveness. Thus a transient signal can both allow for an immediate as well as a sustained response. The transcriptional response also makes the system stable to transient decreases in steady-state growth factor activity. It is speculated that this combination of sensitivity and stability allows responsiveness while mitigating the effects of noise resulting from the intrinsically labile nature of RTK signaling. As illustrated by the data, failure of this regulation in the fat body leads to elevated circulating glucose levels, likely reflecting impaired clearance of dietary glucose from the circulation by the fat body. Maintaining circulating free glucose levels low is likely to be important due to the toxic effects of glucose. In contrast, circulating trehalose, glycogen or triglyceride levels showed no significant change in animals with reduced InR expression, suggesting that these aspects of energy metabolism can be maintained through compensatory mechanisms in conditions of moderately impaired insulin signaling (Zhang, 2011).

Earlier studies have shown that the transcription of the inr gene is under dynamic control. Activation of FOXO in the context of low insulin signaling leads to upregulation of inr transcription, thus constituting a feedback regulatory loop. Thus, InR expression appears to be under control of two receptor-activated cues, which have opposing activities: inr expression is positively regulated by the EGFR-MAPK/ERK module, but negatively regulated by its own activity on FOXO. In the setting of this study, the cross-regulatory input from the MAPK/ERK pathway was found to dominate over the autoregulatory FOXO-dependent mechanism. If conditions exist in which the FOXO-dependent mechanism was dominant, a limited potential for crossregulation by the MAPK/ERK pathway would be expected. Whether Pointed and FOXO display regulatory cooperativity at the inr promoter is an intriguing question for future study (Zhang, 2011).

beta amyloid protein precursor-like (Appl) is a Ras1/MAPK-regulated gene required for axonal targeting in Drosophila photoreceptor neurons

beta amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease, was found in a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development. Appl expression was found in the eye imaginal disc and it is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, Appl null mutants were analyzed and reduced preference for UV light was found, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, whether Ras1/MAPK is responsible for the high Appl expression was examined. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. Appl was shown to be directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism (Mora, 2013).

Two main conclusions can be drawn from this work. First, Drosophila Appl is involved in R7 axonal targeting. Moreover, the finding that the Appl loss-of-function defects are enhanced when combined with Nrt heterozygous mutant suggest that Appl acts at the membrane of R7, where it interacts with other proteins such as Nrt. Second, Appl activation downstream of the RTK/Ras1 is independent of neural specification, occurs in vivo, and is mediated by direct binding of PntP2 to ETS sequences in the Appl regulatory region (Mora, 2013).

Together, these findings may provide insights into the pathogenesis of neurological disorders such as Alzheimer's disease. The β-amyloid peptides, which accumulate in the amyloid plaques found in the brain of Alzheimer's disease patients, are produced after APP proteolysis. However, Alzheimer's disease has not only been associated to the production of the primary component Aβ by proteolysis of APP, but also by transcriptional regulation. Increased APP transcription underlies the phenotype in some cases of familial Alzheimer's disease. In addition, overexpression of APP appears to be responsible for the early onset of Alzheimer's disease in individuals with Down syndrome. Thus, the current results open the possibility to explore whether in some cases of Alzheimer's disease a burst of RTK/Ras1/MAPK occurs and whether this signaling activity ends with high APP accumulation (Mora, 2013).

Amyloid β peptides are known to be involved in vision dysfunction caused by age-related retinal degeneration in mouse models. Thus, the current in vivo observations could be the basis for further research in mammalian models for neurodegenerative retinal disorders that share several pathological features with Alzheimer's disease (Mora, 2013).

Sriskanthadevan-Pirahas, S., Lee, J. and Grewal, S. S. (2018). The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis. Dev Biol 439(1):19-29. PubMed ID: 29660312

The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis
The Ras small G-protein is a conserved regulator of cell and tissue growth during animal development. Studies in Drosophila have shown how Ras can stimulate a RAF-MEK-ERK signalling pathway to control cell growth and proliferation in response to Epidermal Growth Factor (EGF) stimulation. This work has also defined several transcription factors that can function as downstream growth effectors of the EGF/Ras/ERK pathway by stimulating mRNA transcription. This study reports on stimulation of RNA polymerase I (Pol I)-mediated ribosomal RNA (rRNA) synthesis as a growth effector of Ras/ERK signalling in Drosophila. Ras/ERK signalling promotes an increase in nucleolar size in larval wing discs, which is indicative of increased ribosome synthesis. Activation of Ras/ERK signalling promotes rRNA synthesis both in vivo and in cultured Drosophila S2 cells. Ras signalling can regulate the expression of the Pol I transcription factor TIF-IA, and that this regulation requires dMyc. Finally, TIF-IA-mediated rRNA synthesis was found to be required for Ras/ERK signalling to drive proliferation in both larval and adult Drosophila tissues. These findings indicate that Ras signalling can promote ribosome synthesis in Drosophila, and that this is one mechanism that contributes to the growth effects of the Ras signalling pathway (Sriskanthadevan-Pirahas, 2018).

Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila

The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. This study reports on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. Activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. Pol III function was also shown to be required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. The transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead, Ras signalling was shown to promote Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. It is proposed that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth (Sriskanthadevan-Pirahas, 2018).

Post-transcriptional Regulation

regulates pupal size of Drosophila melanogaster via directly targeting Ras85D

MicroRNAs play diverse roles in various physiological processes during Drosophila development. This study reports that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'UTR in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, this work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis (Li, 2016).

Ras85D: Biological Overview | Evolutionary Homologs | Protein Interactions | Effects of Mutation | Ras as Oncogene | References

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