Ras oncogene at 85D
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).
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).
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 pathwaysónamely 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).
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).
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).
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).
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