Ras oncogene at 85D
A good example of the function of Epidermal growth factor receptor in regulating cell development is found by examining the role of EGF-R in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the EGF-Receptor and subsequent Ras1/Raf-mediated signal transduction. In the first step six midline cells in each segment, originating from the anterior-most three of a total of eight midline progenitor cells, are determined as the midline glia equivalence group. The process of generation of the midline glia equivalence group involves Notch function and segmentation genes. It might also depend on the function of single minded, the master regulatory gene of midline development. The single minded transcript accumulates in the midline glia and, depending on the context, can act either as a transcriptional activator or repressor. By the end of embryogenesis the final number of midline glial cells is about 3 to 4. Thus, the final number of cells has to be selected from the initially defined equivalence group in a second step (Scholz, 1997).
Egf-r mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egf-r in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egf-r signaling and are destined for cell death. Both Rhomboid and Argos control activation of the EGF-R during midline glia development. It is thought that a graded activation of EGF-R is brought about by the activity of Rhomboid, which is thought to promote EGF receptor signaling, possibly by cell autonomous activation of the EGF-R ligand Spitz. Ectopic rhomboid leads to extra midline glial cells. EGF-R activates PointedP2 through phosphorylation; Pointed in turn activates the transcription of argos. Argos negatively regulates EGF-R signaling non-cell autonomously and competes with Spitz function. pointed mutants form extra glial cells. Yan antagonizes PointedP2A in midline glial cells, just as it does in the developing photoreceptor cells (Scholz, 1997).
Another factor appears to promote midline glial cell survival: a signal appears to be conveyed via contact with commissural axons. In mutants that lack commissural axons, the midline glial cells die. One can bypass the requirement of axonal contact for midline glia survival by the expression of activated Drosophila jun. Expression of activated Drosophila jun results in missing commissural axon tracts. In summary it appears that Egf-r signaling is required during at least two steps of midline development. First, it is required for the generation of the correct number of midline glial cells, and second, it controls the subsequent differentiation of these cells (Scholz, 1997).
Neuron-glia interactions are necessary for the formation of the longitudinal axon trajectories in the Drosophila central nervous system. Longitudinal glial (LG) cells are required for axon guidance and fasciculation, and pioneer neurons for trophic support of the glia. Neuregulin is a neuronal molecule that controls glial survival in the vertebrate nervous system. The Drosophila protein Vein has structural similarities with Neuregulin. Vein functions like a Neuregulin to maintain glial cell survival. Direct in vivo evidence is presented at single-cell resolution that Vein is produced by pioneer neurons and maintains the survival of neighboring LG. This mechanism links axon guidance to control of glial cell number and may contribute to plasticity during the establishment of normal axonal trajectories (Hidalgo, 2001).
Interestingly, dominant-negative expression of Ras has a stronger effect than dominant-negative expression of DER. This suggests that other signaling pathways function in parallel to control glial survival. Ras functions also downstream of the FGFR, which is also expressed in the LG. Hence, Ras may integrate signaling from both DER and FGFR. Nevertheless, the MAPKinase pathway is activated only in subsets of LG at a given time. Perhaps it is active in different cells at different times, or other signaling pathways are involved in the control of glial survival (Hidalgo, 2001).
Ras pathway and mesoderm development
Fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene is involved in early
directional migration of the Drosophila mesoderm. New data is provided that
(1) demonstrate a second role for Htl in promoting the specification of the precursors to
certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its
cell migration function; (2) suggest that Ras and at least one other signal transduction
pathway act downstream of Htl, and (3) establish a functional relationship between the Ras
pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some
of the same dorsal mesodermal cells that are dependent on Htl (Michelson, 1998a).
The involvement of Htl in mesodermal founder cell fate specification was tested by reducing its activity under conditions where earlier cell migration is not compromised. This was accomplished by ectopic expression of a dominant negative of the Htl Fgf receptor. Dominant negative Htl induces numerous defects in mesodermal structures at multiple positions along the dorsoventral axis and at different stages of development. In late stage embryos, somatic muscles are missing from ventral, lateral and dorsal groups, and gaps occur in the rows of cardial and pericardial cells. These defects can be traced to an earlier stage where the corresponding precursor cells are found to be lacking. For example, dominant negative Htl prevents the formation of progenitors of the Eve-expressing pericardial and somatic muscle cells. Small gaps in the normally continuous rows of visceral mesodermal precursors are also observed (Michelson, 1998a).
Ectopic mesodermal expression of a constitutively active form of Ras1 is capable of partially rescuing a strong hypomorphic htl mutant. Partial rescue of a null htl mutation by activated Ras1 also is manifest in the expression of Eve in the dorsal mesoderm. No Eve-positive cells are found in the complete absence of htl function, whereas a hypomorphic mutant contains a markedly reduced number of Eve-expressing segments. Although the Epidermal growth factor, a second receptor tyrosine kinase, is involved in development of Eve muscle founders, all of the Eve-positive cells generated by activated Ras1 in htl mutant embryos are confined to the dorsal mesoderm in their usual segmental pattern, consistent with the involvement of Ras1 in both the migration and cell fate specification functions of Htl (Michelson, 1998a).
Interestingly, loss-of-function mutations in tinman and htl have identical affects on the development of Eve pericardial and somatic muscle cells. Similarities are also seen between the cardial and dorsal somatic muscle phenotypes of these two genes. However, tinman differs significantly from htl in the mechanism of action since mesoderm migration is completely normal in tinman mutants. This implies that tinman is involved in only one of the processes affected by htl, namely the determination of dorsal mesodermal cell fates. Since Ras1 functions in the Htl signaling pathway and activation of this signal transduction has the opposite effect on Eve progenitor development as tin loss-of-function, an epistasis experiment could be performed. Expression of activated Ras1 in a tinman mutant background results in an Eve expression phenotype corresponding to that of tinman. That is, tinman loss-of-function completely blocks the ability of activated Ras1 to promote the formation of Eve pericardial cell and somatic muscle progenitors (Michelson, 1998a).
The following model is proposed for the role of Tinman and Htl in the formation of Dorsal mesoderm. When mesodermal cells reach the dorsal-most region of the ectoderm, they are induced by Dpp to express Tinman, thereby acquiring the competence to differentiate into visceral, cardiac, or dorsal somatic muscle derivatives. Superimposed on this process is the activation of the Ras1 pathway in a small subset of dorsal mesodermal cells. Ras1 activation is mediated by Htl in those cells destined to form the Eve pericardial progenitiors, whereas both Htl and Egf receptor function together to generate a Ras1 signal specific to Eve-positive somatic muscle fate. In this sense, Htl/Egfr/Ras1 signaling serves to distinguish a fate characterized by Eve expression from additional dorsal mesodermal fates that are also dependent on tinman. It should be noted that Tin and Ras1 regulation are not required to function in any particular order; one may precede the other or they may act simultaneously. The essential point is that both are absolutely required for the specification of Eve cardiac and somatic muscle fates in the dorsal mesoderm (Michelson, 1998a).
Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute
(l'sc)-expressing cells. Individual progenitors are specified by the sequential deployment of unique
combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and
Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on
the adjacent mesoderm in the form of L'sc preclusters. One precluster, preC1, is found in the
ventral mesoderm, and the other, preC2, is localized to the dorsal mesoderm. PreC2 encompasses the territory in which dorsal L'sc clusters C2 and C14-C17, the subject of this paper, subsequently
develop. All mesodermal cells within preC2 precluster are
competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases
(RTKs), the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast
growth factor receptor. By monitoring the expression of the diphosphorylated form of
mitogen-associated protein kinase (MAPK), these RTKs are seen to be activated in small clusters
of cells within the original competence domain (precluster). Each cluster represents an equivalence group because
all members initially resemble progenitors in their expression of both L'sc and mesodermal identity
genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The
RTKs remain active in the single progenitor that emerges from each cluster under the subsequent
inhibitory influence of the neurogenic genes. The singling out of progenitors from mesodermal equivalence groups depends on lateral inhibition
mediated by the neurogenic genes. Moreover, Egfr and Htl are differentially involved in the
specification of particular progenitors (Carmena, 1998).
Of the two clusters of dorsal mesodermal cells that express the pair-rule gene even-skipped that arise sequentially from a single precluster, one cluster gives rise to a single Eve-positive progenitor that divides to give rise to a pair of pericardial cells (P2). The second cluster (P15, arising from the same C2 precluster that gives rise to P2) gives rise to at least one dorsal
somatic muscle and forms from a second Eve progenitor. The two L'sc clusters from which these progenitors arise are
prefigured by a broader domain of L'sc expression (the precluster) that is dependent on the combined activities of Wg
and Dpp. The corresponding equivalence groups (clusters) are formed within this prepatterned mesodermal
region via localized activation of the Ras1 pathway by two receptor tyrosine kinases (RTKs), Htl, and
Egfr. Whereas Htl is required for the Eve cardiac equivalence group, both Egfr and Htl are involved
in the Eve muscle cell cluster. These findings demonstrate how positional information initially
establishes a mesodermal prepattern, and establish that individual progenitors are progressively
determined by unique combinations of intercellular signals. It is concluded that distinct cellular identity codes are generated by
the combinatorial activities of Wg, Dpp, Egf, and Fgf signals in the progressive determination of
embryonic mesodermal cells (Carmena, 1998).
Transduction of RTK signals occurs, at least in part, via the Ras/MAPK cascade. Constitutive activity of Egf receptor, Htl, or Ras1 is associated with the
development of supernumerary Eve-expressing mesodermal cells. However, it has not been established whether this response is due to activated Ras1-induced proliferation of
normal Eve cells or is due to the recruitment of additional cells to an Eve-positive fate. Activated Ras1
does not increase the sizes of L'sc clusters, suggesting that Ras1 is not simply
stimulating the division of mesodermal cells. However, to definitively address this question, the effects of constitutive Ras1 activity were examined in string (stg) mutant embryos. Because strong
alleles of stg prevent all post-blastoderm cell divisions, a potential
cell-proliferation effect of activated Ras1 should be blocked in this genetic background. However, stg should not inhibit the overproduction of Eve-expressing cells if Ras1 promotes their
determination. In a stg mutant, only one to two Eve-positive cells are present in each hemisegment. These cells correspond to the Eve progenitors that form in wild-type embryos. The presence of only one Eve progenitor in some segments of stg mutant
embryos presumably is due to the smaller number of mesodermal cells that contribute to L'sc clusters
when zygotic cell divisions do not occur. Significantly, activated Ras1 generates more Eve progenitors
in a stg mutant than are seen in the mutant alone. It is concluded that Ras1 promotes the
formation of additional Eve progenitors by inducing more mesodermal cells to assume this fate and not
by stimulating the normal progenitors to divide.
Next, an assessment was carried out to determine if the Ras1 pathway is sufficient to induce progenitor differentiation by
examining the myogenic effects of Ras1 at later developmental stages in both wild-type and myoblast
city (mbc) mutant embryos. In the absence of mbc function, muscle fusion does not occur and
differentiated muscle founders appear as spindle-shaped myoblasts that express myosin and founder
cell markers, such as Eve. In contrast to the mbc mutant in which
one Eve-expressing muscle DA1 founder is present in each hemisegment, multiple occurrences of such cells form in
mbc embryos under the influence of activated Ras1. All of the Eve plus myosin-positive
myoblasts seen in these embryos have the elongated morphology of muscle founders, as opposed to the
round shape of neighboring Eve-negative myoblasts. Large syncytia containing many Eve-positive
nuclei are present when Ras1 is activated in a wild-type background. Although extra Eve
pericardial cells initially appear under the influence of activated Ras1, Eve expression
in such cells is lost by later stages. It is concluded that ectopic activation of the Ras1
pathway not only stimulates Eve expression in additional progenitors, but also promotes the formation
and differentiation of supernumerary muscle founders (Carmena, 1998).
A new gene, heartbroken, has been identified that participates in the signaling pathways of both FGF
receptors. heartbroken has been cloned and although it appears to be a novel protein, it possesses several sequences characteristic of a signal transduction protein (Vincent, 1998). Mutations in heartbroken are associated with defects in the migration and later specification of mesodermal and
tracheal cells. Genetic interaction and epistasis experiments indicate that heartbroken acts downstream of the two FGF receptors,
but either upstream of, or parallel to, Ras1. Furthermore, heartbroken is involved in both the Heartless- and
Breathless-dependent activation of Mapk. It has been concluded that heartbroken may contribute to the specificity of developmental responses
elicited by FGF receptor signaling (Michelson, 1998b, and Vincent, 1998).
Ras1 is a key signal transducer acting downstream of all (receptor tyryosine kinases) Rtks, including Htl. Since htl and hbr mutants have similar mesodermal phenotypes and genetic interaction studies suggest a functional relationship between the products of these genes, it became interesting to see whether hbr could also be related to Ras1 function. It is known that targeted mesodermal expression of a constitutively activated form of Ras1 can partially rescue the htl mutant phenotype. This conclusion was reached by examining both the activated Ras1-induced migration of Twi-expressing cells and the recovery of dorsally restricted Eve-positive muscle and cardiac progenitors in htl embryos. Using these same assays, it has been found that activated Ras1 is capable of partially rescuing the strong hbrYY202 mutant.
The above results suggest that hbr acts either upstream of Ras1 or on a parallel pathway involved in either initiating or transducing the Htl signal. It was next asked where hbr functions in relation to the receptor by determining if a constitutively activated form of Htl can rescue the hbr phenotype. When expressed in the mesoderm of wild-type embryos, activated Htl induces the formation of additional Eve founder cells but has no effect on mesoderm migration. In a htl mutant background, activated Htl partially corrects the mesoderm migration defect and contributes to the specification of significant numbers of Eve progenitors. Quantitation of the latter effect reveals that activated Htl is significantly more efficient at rescuing loss of htl function than is activated Ras1. In contrast, the influence of activated Htl is completely blocked by a homozygous hbr mutation. These results, as well as the dominant suppression of activated Htl by hbr, argue that hbr acts either downstream of, or parallel to, this mesodermal Fgf receptor (Michelson, 1998b).
Drosophila muscles originate from the fusion of two types of
myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To
better understand muscle diversity and morphogenesis, a
large-scale gene expression analysis was performed to identify genes differentially
expressed in FCs and FCMs. Embryos derived from
Toll10b mutants were employed to obtain primarily muscle-forming
mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM
fate, respectively. The transcripts present in embryos of each genotype were
compared by hybridization to cDNA microarrays. Among the 83 genes
differentially expressed, genes known to be enriched in FCs or FCMs,
such as heartless or hibris, previously characterized genes
with unknown roles in muscle development, and predicted genes of unknown
function, were found. These studies of newly identified genes revealed new patterns of gene
expression restricted to one of the two types of myoblasts, and also striking
muscle phenotypes. Whereas genes such as phyllopod play a crucial
role during specification of particular muscles, others such as
tartan are necessary for normal muscle morphogenesis (Artero, 2003).
The Toll10b mutation gives rise to embryos
composed primarily of somatic mesoderm. In these embryos FCs and FCMs are
readily detected, and they respond to the Ras and Notch signaling pathways
in the same way as their wild-type counterparts. Advantage was taken of this
fact to enrich Toll10b mutant embryos for FCs or FCMs,
which allowed a concentration on the transcription in these two specific
cell types within the context of the entire embryo. Genes known to be
expressed and regulated in FCs or FCMs emerged from the screen in the proper
categories. Not all known FC/FCM genes were detected in the screen for several
reasons: the high stringency set for interpretation of the array data; the
presence of only about one-third of the genome on the arrays; the loss of Dpp
in the Toll10b background, and the specific window of
myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora
of potential new muscle regulators were uncovered, including known genes with
no previously recognized function in the mesoderm (such as phyl and
asteroid), and genes predicted from the Drosophila genome
sequence but not previously analyzed (Artero, 2003).
Various tests were applied to ascertain the validity of the results.
Available databases were analyzed to find evidence
that the known and predicted genes were expressed at the correct time and place. In
addition, Northern analysis with eleven genes tested the reliability of the
microarray detection and selection criteria; the results from all
genes tested agreed with the array data (Artero, 2003).
A Toll10b sample on the Northern blots allowed
ascertainment of why a gene is enriched in a particular condition. For example, in
the case of FC enriched genes, the signal in the Ras and
Notch lanes can be compared with Toll10b alone to
determine whether the Ras/Notch ratio for a gene is due to activation by Ras
or repression by Notch. Those genes that are 'enriched
under Notch conditions', for example, could reflect a variety of transcription
mechanisms that would result in a ratio of less than 0.6. By Northern
analysis, many of the 'Notch-regulated' genes, and hence the predicted
FCM genes, were found to be repressed by Ras signaling and slightly activated by Notch. As
a case in point, hibris is induced by Notch (2-fold)
and repressed by Ras (10-fold), both by Northern analysis and by in situ
hybridization in embryos (Artero, 2003).
A combination of in situ hybridization, immunostaining and confocal
microscopy was used to verify that the differential expression changes that were observed
in these overexpression embryos reflected true differential expression in the
wild-type situation. The expression of nine genes from different
functional categories was analyzed. For
seven of these, expression was detected in the predicted type of myoblast. For
two, asteroid (ast) and cadmus, no specific staining in embryos was detected by in situ hybridization. For
those genes that fell into the category of 'specific role in muscle
development uncertain', in situ hybridization of several (28%) showed
expression in tissues other than somatic mesoderm that are present in the
Toll10b background. These genes changed their expression
levels in response to Ras or Notch, and may be Ras and Notch targets in
non-mesodermal tissues (Artero, 2003).
The most stringent test, mutational analysis, was applied to a set of genes
for which mutations are available. Preliminary analyses of another four FCM-enriched genes was carried out: Elongation factor Tu mitochondrial (EfTuM), Glutamine synthetase 1, cadmus and parcas. All
four mutants have muscle defects, including muscle losses and aberrant muscle
morphologies. Thus all the genes tested show some muscle defect, supporting the usefulness of the genetic and genomic approach (Artero, 2003).
Taken together, these data suggest that the majority of genes detected play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or
complete genetic redundancy. The data complement traditional forward genetic
approaches for finding genes crucial for muscle morphogenesis (Artero, 2003).
Each of the thirty FCs per abdominal hemisegment is hypothesized to produce
its own unique combination of transcriptional regulators, though the evidence
for this is limited. In turn the combination of regulators would control the
morphology of the final muscle. Although several transcriptional regulators
have been linked to FC identity, the molecular description is far from
complete. This screen contributed two more FC-specific genes. Previously known
markers, such as slouch or eve, once induced in the muscle
FC, are maintained throughout the remainder of development. Ubx,
which emerged from this screen, is a similarly simple case, as its transcripts
are steadily present in most FCs. By contrast, more
complex patterns of gene expression have been identified in FCs, such as the transient transcription of asense in a subset of FCs. The subsequent
transcriptional inactivation of asense may underlie temporal changes in
cell properties (Artero, 2003).
Even less is known about transcriptional regulators controlling FCM
differentiation. Only one gene, lame duck, has been shown to have a
role in FCMs. This screen has uncovered three more potential players: delilah,
E(spl)mß and CG4136, confirming that FCMs follow their own,
distinct, myogenic program. Discovering what aspects of FCM biology are
controlled by these transcriptional regulators awaits analysis of the
loss-of-function phenotypes (Artero, 2003).
Notch and Ras signaling pathways interact during muscle progenitor
segregation. The results suggest that phyl and
polychaetoid (pyd) may be additional links between the two
signaling pathways in FCs. phyl and pyd both interact
genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during
specification of SOPs and their progeny. This study
shows a similar regulation in muscle cells, where Notch signaling represses
phyl expression and Ras signaling increases phyl expression. Likewise, in the
nervous system, the segregation of SOPs requires pyd, a Ras target
gene, to negatively regulate ac-sc complex expression. Similarly, Pyd
may restrict the muscle progenitor fate to a single cell, perhaps by
regulating lethal of scute transcription. Thus, Pyd would collaborate
with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).
FCMs appear to integrate Ras and Notch signaling differently. Two genes
whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).
Embryos that lack or ectopically express phyl have morphological
defects in specific muscles, for example, in LL1 and DO4 in response to
diminished phyl function, and in DT1 and LT4 in response to increased
phyl function. The morphological defects in the loss-of-function
embryos appear to be due to a failure to specify particular FCs, a conclusion
that is based upon missing or abnormal production of the FC marker Kr. In eye
development and SOP specification, Phyl directs degradation of the
transcriptional repressor Tramtrack. In a subset
of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack
for degradation. The presence of Tramtrack would contribute to the specific
identity program of the muscle. Since Tramtrack is expressed in the mesoderm,
this possibility is likely. Alternatively, Phyl may be required for targeted
degradation of some other protein in a subset of FCs. The molecular partner
for Phyl during muscle differentiation is unknown, although preliminary data
suggest that sina is also expressed in somatic mesoderm and thus may
be its partner. These studies have
identified a new role for Phyl in muscle progenitor specification and suggest
the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).
A role for ubiquitination in muscle differentiation is further reinforced
by the identification of the RING finger-containing protein Goliath (Gol), induced by
activated Notch conditions, and CG17492, induced by activated Ras conditions.
Several RING-containing proteins function as E3 ubiquitin ligases, with the
ligase activity mapping to the RING motif itself. Ligase function has been experimentally confirmed for the Gol ortholog GREUL1
in Xenopus. Thus, targeted protein degradation during muscle morphogenesis could serve a host of crucial functions, such as protein turnover, vesicle sorting, transcription factor activation and signal degradation (Artero, 2003).
The simplest view of the 'founder cell' hypothesis is that each FC contains
all the information for the development of a particular muscle. By contrast,
FCMs have been seen as a naïve group of myoblasts, entrained to a
particular muscle program upon fusion to the FC. This work indicates that these
two groups of myoblasts have distinct transcriptional profiles. These data
raise the possibility of a greater role for FCMs in determining the final
morphology of the muscle and emphasize a need to characterize fully those FCM
genes. For
example, this screen identified a protein kinase of the SR splice site selector
factors (SRPK) whose transcripts are enriched in FCMs, suggesting that
regulation of the splicing machinery is important for muscle morphogenesis.
The Mhc gene undergoes spatially and temporally regulated alternative
splicing in body wall muscles conferring different physiological properties on
these muscles. This FCM-specific expression of SRPK may indicate that the
production of a particular Mhc isoform is regulated by the FCMs that
contribute to that muscle, rather than by the particular FC that seeds the
muscle. In addition, a number of observations suggest that FCMs may be a
diverse population of myoblasts, with different subsets having different
potential to contribute to the final muscle pattern. For example, hbs
expression suggests that only a subset of FCMs express the gene, and
twist expression in lame duck mutant embryos persists in a
subset of FCMs. This study provides additional genes
for exploring whether FCMs are a heterogeneous population of myoblasts as well
as determining the nature of FCM contribution to the final muscle (Artero, 2003).
The molecular events underlying complex morphological changes, such as
migration, cell fusion, cell shape changes or changes in the physiology of a
cell, require a rich and dynamic program of transcription changes. This study has described approximately one-third of this transcriptional profile. The FC- or
FCM-specific transcription of seven genes, and the mutant phenotype of four
selected genes, allowed the definition of new muscle mutations that
specifically affect the morphological traits of a subset of muscles (Artero, 2003).
Ras proteins are small GTPases with well known functions in cell proliferation and differentiation. In these processes, they play
key roles as molecular switches that can trigger distinct signal transduction pathways, such as the mitogen-activated protein kinase
(MAPK) pathway, the phosphoinositide-3 kinase pathway, and the Ral-guanine nucleotide dissociation stimulator pathway.
Several studies have implicated Ras proteins in the development and function of synapses, but the molecular mechanisms for this
regulation are poorly understood. The Ras-MAPK pathway is involved in synaptic plasticity at the Drosophila larval neuromuscular junction. Both Ras1 and MAPK are expressed at the neuromuscular junction, and modification of their activity levels results in an altered number of synaptic boutons. Gain- or loss-of-function mutations in Ras1 and MAPK reveal that regulation of synapse structure by this signal transduction pathway is dependent on Fasciclin II localization at synaptic boutons. These results provide evidence for a Ras-dependent signaling cascade that regulates Fasciclin II-mediated cell adhesion at synaptic terminals during synapse growth (Koh, 2002).
Synapse stability and synapse expansion during muscle growth are regulated by changes in FasII expression at presynaptic and postsynaptic membranes and FasII expression is in part controlled by electrical activity. One mechanism through which electrical
activity alters FasII levels is by regulating its synaptic clustering
via CaMKII-dependent phosphorylation of Discs large. An additional mechanism by
which the levels of FasII at the presynaptic terminal are modified has been documented in this study: the
activation of the Ras-MAPK pathway. This redundant mechanism may serve
the differential regulation of FasII localization at the presynaptic
and postsynaptic site or may represent FasII regulation in response to
different signals. Whereas activation of CaMKII is elicited by an
increase in electrical activity, activation of the MAPK pathway may be
triggered by activity or by an as yet unknown but different signaling mechanism (Koh, 2002).
Studies in Aplysia indicate that activity-dependent endocytosis of ApCAM results in an increase in the number of synaptic contacts during long-term facilitation. ApMAPK is likely to induce ApCAM internalization in a process that depends on ApMAPK activity in dissociated neurons. However, its involvement in the intact organism has not been tested (Koh, 2002 and references therein).
In this study, Drosophila larval neuromuscular synapses have been used to determine the involvement of the Ras-MAPK pathway in the regulation of synaptic FasII levels and in morphological synaptic plasticity. Both Ras and MAPK are expressed at the NMJ, where they regulate presynaptic expansion. This regulation is accomplished by altering FasII levels at synaptic boutons. A ras hypomorph mutant and anti-Ras antibodies have been used to determine that Ras1 is specifically expressed at the larval NMJ. Although Ras1 immunoreactivity at synapses and muscles is severely reduced in ras1 hypomorphic mutants, nuclear staining persists (Koh, 2002).
Two antibodies were used to demonstrate the synaptic localization of MAPK at the NMJ, an antibody that recognizes all forms of the MAPK Rolled (DmERK-A) and an antibody that exclusively labels active, double-phosphorylated MAPK (DpMAPK).
Interestingly, although both antibodies labeled synaptic boutons, their
distribution was not identical. In particular, the antibody against
active MAPK-labeled hot spots was more restricted in its localization
than general MAPK staining. This suggests that active MAPK is recruited
to specific domains within the synaptic bouton or that MAPK activation
occurs at discrete regions within the boutons. Interestingly, the same
domain that is occupied by active MAPK has lower levels of FasII,
consistent with the idea that MAPK activation might be involved in the
downregulation of FasII. It has been suggested that the regions of low FasII concentration correspond to the active zone, suggesting that active MAPK is localized to the active zone. The localization pattern of Ras1 and MAPK at synapses is also consistent with the localization protein 14-3-3, another protein that has been involved in the Ras1-Drosophila Raf-MAPK signal transduction pathway (Koh, 2002).
Expression of constitutively active
Ras (Ras1V12) drastically increases the number of synaptic boutons.
This change is indistinguishable from the increase in boutons observed
in the Ras1V12S35 variant and the constitutively activated
RafF179, suggesting that these changes
are induced by activation of the MAPK pathway. Consistent with these results is the observation that a hypomorphic mutation in ras1, ras15703, has the opposite phenotype, a decrease in bouton number, and that a gain-of-function mutation in rl leads to an increase in bouton number. The finding that Ras1V12 and Ras1V12S35 elicit identical phenotypes at the NMJ is consistent with findings in other tissues, such as in the retina, in which the epidermal growth factor receptor-Ras1 pathway is involved in photoreceptor survival, or in the wing discs, where the Ras pathway is involved in hyperplastic growth (Koh, 2002).
Notably, expression of Ras variants that activate the PI3-K and Ral
signal transduction pathways and a constitutively active RalA also
induce an increase in bouton number that is similar in extent to
RasWT and considerably lower than Ras1V12. These results raise the
possibility that Ras1V12G37 and Ras1V12C40 may still retain some degree
of affinity for Raf or, alternatively, that other Ras-mediated pathways
might also influence NMJ development. All known ras genes
encode a protein region, the effector loop, that is highly conserved in
all species. Mutations in this loop interfere with the ability of Ras
to bind to specific effectors without altering its catalytic activity.
A series of mutations in the effector loop that allow almost exclusive
activation of a single effector havs been isolated in mammals. The
specificity of these mutants has been tested by in vitro
binding assays as well as by genetic and biochemical approaches in cell
culture. In Drosophila, a genetic
approach has been used to demonstrate specificity. These studies
suggest that Ras1V12 and RasV12S35 phenotypes are emulated by a
hyperactivated form of Raf and suppressed by Raf, MEK, and MAPK mutants (Koh, 2002).
Studies in vertebrate cells and in Drosophila suggest that
although Ras activation by receptor tyrosine kinases is blocked by the
putative dominant-negative RasN17, Ras activation by PKC and the
Ras1V12C40/PI3-K effect on cytoskeletal reorganization in fibroblasts
are not. At the NMJ, Ras1N17 does not behave as a dominant negative. Thus,
taken together, this analysis of NMJ structure in the different Ras
strains suggests that Ras1 regulates the number of type I glutamatergic
synapses in Drosophila and this regulation depends to a
considerable extent on the activation of the MAPK pathway. Although
activation of PI3-K and Ral-GDS-Ral by presumably PKC activation also
points to a role for these pathways, their effect on NMJ growth is
less prominent than the MAPK pathway (Koh, 2002).
Immunocytochemical studies of FasII immunoreactivity at synaptic
terminals of MAPK gain- and loss-of-function mutants suggest that MAPK
regulates levels of synaptic FasII, a cell-adhesion molecule that plays
a key role in the maintenance and expansion of NMJs in
Drosophila. This model was supported by experiments in which only type I synaptic FasII was immunoprecipitated. This was accomplished by using anti-DLG antibodies, because DLG binds directly to FasII at type I boutons but not at other
bouton types. The immunoprecipitation experiments
demonstrate that enhancing the levels of MAPK activity at synaptic
terminals results in a reduction of type I synaptic FasII. Conversely,
decreasing levels of MAPK activity results in an increase in type I
synaptic FasII levels. These results are in agreement with the studies
in Aplysia dissociated neurons, which show that ApMAPK is
involved in the internalization of ApCAM (Koh, 2002).
Additional support for the idea that the changes in bouton number
elicited by alterations in Ras1 and MAPK activity are mediated by
alterations in FasII levels was demonstrated by examining the overall
expression of FasII in MAPK gain- or loss-of-function alleles,
examining the distribution of FasII within single synaptic boutons in
relation to active MAPK, and using hypomorphic fasII mutants. The studies with rl mutants demonstrate that there is an inverse relationship between levels of synaptic FasII and MAPK activity. Furthermore, active MAPK localization coincides with regions of the bouton that have no or low FasII levels (Koh, 2002).
Two main functions of FasII in the regulation of synapse number have been demonstrated. (1) FasII is critically required for synapse maintenance: below threshold FasII levels, synaptic boutons are not maintained. (2) FasII operates by
constraining synaptic growth, similar to the Aplysia system. Therefore, a decrease in FasII to a level still sufficient for maintenance results in an increase in synaptic arbor size. On the basis of this model,
the following interpretation of the results is proposed. The dramatic decrease in
FasII levels in the homozygous fasII mutant does not allow
any influence of MAPK activity changes on NMJ structure. Similarly,
when FasII levels are decreased to approximately one-half the
wild-type levels (fasIIe76/+), an
increase in MAPK activity does not induce an additional increase in
bouton number, probably because an additional decrease in FasII
compromises synaptic maintenance, thus preventing NMJ growth. However,
the increase in FasII levels induced by a reduction of MAPK activity
(rl10a/+) in a fasIIe76/+ background suppresses the increase in boutons observed in fasIIe76/+ alone. This result suggests that MAPK regulates FasII levels and exists upstream of FasII at signal transduction pathways that regulate the number of type I synaptic boutons (Koh, 2002).
Notably, the hypomorph rl10a/+
has no significant decrease in bouton number, although these mutants
have a striking increase in FasII levels compared with wild-type
controls. An explanation for this result is that FasII is a homophilic
cell-adhesion molecule that is required both in the presynaptic and in
the postsynaptic cell for function. If the Ras-MAPK pathway functions to regulate FasII at the presynaptic cell, as suggested by studies with cell-specific Gal4
drivers, then an asymmetric increase in FasII levels in the presynaptic cell alone may not have much of an effect. Previous studies also show
that although the NMJ is very sensitive to a decrease in FasII levels, an increase in FasII over wild-type levels does not have much of an effect (Koh, 2002).
Although the results are consistent with a regulation of FasII-mediated
synapse growth by the Ras-MAPK pathway, it is important to note that
several other molecules in addition to FasII are involved in the
regulation of synapse growth. Moreover, several studies suggest that many
changes at the fly NMJ are compensated by yet unknown homeostatic
mechanisms. Therefore, further understanding of these regulatory and compensatory signals will be necessary to fully explain these observations (Koh, 2002).
In conclusion, a signaling pathway intimately involved in the regulation of synaptic growth at the NMJ has been identified. Identification of the mechanisms involved in the activation of this pathway may provide valuable clues toward understanding the plasticity of this synapse (Koh, 2002).
The Drosophila Ras1 gene is required for proper cell fate specification throughout development; the loss-of-function phenotype of Ras1 suggests an additional role in cell proliferation or survival. However, direct role for Ras1 in promoting cell proliferation has not been established. Expression of an activated form of Ras1 (Ras1[V12]) during Drosophila wing imaginal disc development is sufficient to drive ectopic cell proliferation and hyperplastic tissue growth. Expression of Ras1(V12) induces widespread cell death in the imaginal discs, including cells not expressing the transgene, which results in ablation of adult structures. It is thought that the non-autonomous cell death induced by ectopic Ras could be a manifestation of cell competition Increased cell death may represent a mechanism to compensate for excessive proliferation and regulate the overall disc size. Loss-of-function mutations in the genes encoding RAF, MEK, MAPK and KSR dominantly suppress Ras1(V12)-induced cell proliferation. Two Ras effector loop mutations (E37G and Y40C) that block the Ras-RAF interaction, also suppress Ras1(V12)-induced proliferation, consistent with a requirement for the MAPK cascade during the Ras1 mitogenic response. These two Ras effector loop mutants, however, retain some activity and can act synergistically with a MAPK gain-of-function mutation, suggesting that Ras1 may also act through signaling pathway(s) distinct from the MAPK cascade, but which act in parallel with the MAPK pathway (Karim, 1998).
Growth and patterning of the Drosophila wing imaginal disc depends on its subdivision into dorsoventral (DV)
compartments and limb (wing) and body wall (notum) primordia. Evidence is presented that both the DV and
wing-notum subdivisions are specified by activation of the Drosophila Epidermal growth factor receptor (Egfr). Egfr signaling is necessary and sufficient to activate apterous (ap) expression, thereby segregating
the wing disc into D (ap-ON) and V (ap-OFF) compartments. Similarly, Egfr signaling directs
the expression of Iroquois Complex (Iro-C) genes in prospective notum cells, rendering them distinct from, and immiscible with, neighboring wing
cells. However, Egfr signaling acts only early in development to heritably activate ap, whereas it is required persistently during subsequent
development to maintain Iro-C gene expression. Hence, as the disc grows, the DV compartment boundary can shift ventrally, beyond the range of the
instructive Egfr signal(s), in contrast to the notum-wing boundary, which continues to be defined by Egfr input (Zecca, 2002a).
To assess the requirement for signals transduced by the Egfr during normal wing disc development, the behavior was examined of clones of cells that are homozygous for null or temperature-sensitive mutations of the Egfr gene (referred to subsequently as Egfr- or Egfrts), or for a loss of function mutation of the ras gene (ras-), which encodes the Ras GTPase, a conserved downstream effector of the Egfr signal transduction pathway. Clones of mutant cells were generated during different stages of larval development and their size, shape and distribution assayed in each of the four distinct primordia that make up the mature wing disc: the prospective wing blade, wing hinge, lateral notum and medial notum. In general, loss of Egfr activity caused more penetrant and severe effects than the loss of Ras activity, possibly reflecting a shorter perdurance of Egfr function relative to that of Ras following loss of the wild-type gene, or a restricted requirement for Ras in mediating some, but not all, downstream outputs of Egfr activation. ras- clones, in particular, were more viable than Egfr mutant clones, allowing use of the twin spot method of clonal analysis and allowing the generation of mutant clones of large size using the Minute technique. However, aside from this difference, the effects of Egfr and ras mutant clones on Iro-C gene expression were the same. In these experiments mutant clones were marked either by the presence or absence of the reporter proteins GFP or CD2 (Zecca, 2002a).
Unlike Egfr- clones, ras- clones induced during the first or second larval instar can survive without the benefit of the Minute technique. Under these conditions, mitotic recombination generates 'twin spots' composed of genetically marked ras- and ras+ sister clones, which descend from the same mother cell. Twin spots could be recovered in the prospective wing blade domain, wing hinge and medial notum domains. However, only single ras+ spots were generally observed in the prospective lateral notum domain, indicating that their ras- sister spots failed to survive in this domain; the few ras- sister spots obtained in this domain appeared abnormal. Similar results were obtained when ras- cells were generated during the first larval instar using the Minute technique. Such ras- clones could form large, and apparently normal, regions of the prospective wing blade and wing hinge. Nevertheless, they appeared to be excluded from the presumptive notum territory. Strikingly, some of the discs obtained under these conditions appeared to lack most or all prospective notal tissue and to consist predominantly of prospective wing blade and hinge tissue (Zecca, 2002a).
In summary, Egfr-, Egfrts and ras- clones can contribute to the prospective wing blade, wing hinge and medial notum. However, all three classes of mutant clones generally failed to populate the prospective lateral notum, indicating that Egfr signaling is essential for the normal development of this region of the wing disc (Zecca, 2002a).
Prospective notum cells are distinguished from wing cells by the activity of the Iroquois Complex (Iro-C) genes. These results demonstrate (1) that activation of Egfr/Ras pathway is both necessary and sufficient to drive Iro-C gene expression in wing disc cells, and (2) that wing disc cells persistently monitor their level of Egfr/Ras input and are allocated to the wing or notum primordium on an ongoing basis, depending on the level of Egfr/Ras input they receive. This means that the wing-notum subdivision is not a stable compartmental partition between differently committed cell types, but rather a labile demarcation that reflects the current distribution of an instructive Egfr ligand (Zecca, 2002a).
Despite the provisional nature of the wing-notum segregation, the boundary between the two primordia is relatively straight and sharp. By manipulating Egfr/Ras signaling, it was shown that presumptive notum cells that lose the capacity to maintain Iro-C gene expression sort out of the notum primordium. Conversely, presumptive wing cells that ectopically activate the Iro-C genes sort out of the wing primordium. Similar results have been obtained by altering Iro-C gene function directly, rather than through the manipulation of Egfr/Ras signaling. Taken together, these results suggest that Iro-C gene activity, under Egfr control, programs prospective notum cells to have a different affinity from prospective wing cells, thereby straightening and sharpening the boundary between the two primordia. Further support for such a mechanism comes from experiments in which clones of cells were generated that ectopically express an activated form of Spi, an Egfr ligand, in the prospective wing hinge. All of the cells within these clones express the Iro-C genes and interdigitate freely with neighboring wild-type cells that are also induced to express the Iro-C genes. However, cells located further away do not receive sufficient Spi to activate Iro-C gene expression and these form a smooth boundary encircling the ectopic Iro-C-expressing cells (Zecca, 2002a).
As in the case of the Iro-C genes, Egfr/Ras signaling is both necessary and sufficient to activate ap expression in early wing disc cells. Furthermore, evidence is provided that each wing disc cell chooses to express, or not to express, ap at this time, depending on its level of Egfr/Ras activation. However, in contrast to the Iro-C genes, the descendents of each cell then inherit this initial choice without further reference to Egfr/Ras signaling. The results of eliminating Egfr/Ras activity before the establishment of the DV compartments are particularly striking. Early loss of Egfr activity causes dorsally positioned cells within the disc to choose, incorrectly, to become V compartment founders. These cells and their descendents generally sort into the existing V compartment or out of the disc epithelium. In rare cases, they can form an ectopic V compartment within the D compartment. By contrast, later loss of Egfr activity has no effect on the DV compartmental segregation. These findings establish that Egfr signaling is responsible for establishing the D and V compartments through the heritable activation of ap (Zecca, 2002a).
The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal growth factor receptor (Egfr) signaling, which heritably activates
apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. The source, identity and mode of action of the Egfr ligand(s) that specify these subdivisions has been examined. Of the three known ligands for the Drosophila Egfr, only Vein (Vn), but not Spitz or Gurken, is required for wing disc
development. Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression.
However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather
than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where
Vn-Egfr signaling is effective. After ap is heritably activated, the level of Egfr activity declines in D compartment cells as they proliferate and
move ventrally, away from the source of the instructive ligand. Evidence is presented that this reduction is necessary for D and V compartment cells to
interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing
primordium (Zecca, 2002b).
All cells within the wing imaginal disc require a minimum level of Egfr/Ras activity to sustain a normal rate of proliferation. It is not known whether this activity reflects the basal activity of the Egfr/Ras transduction pathway, or the response of the receptor to a specific ligand. However, it is clear that this low level of Egfr/Ras activity does not require Vn dependent Egfr signaling, since it has been shown that ectopic expression of Ap in vn mutant discs can rescue growth and differentiation of the wing primordium. This result demonstrates that the absence of wing development in vn mutant discs is an indirect consequence of the failure to establish an apON-apOFF interface (Zecca, 2002b).
During normal development, the ap and Iro-C genes are initially activated in overlapping dorsoproximal domains in response to Egfr signaling, and hence, at this early stage, it appears that most or all D compartment cells are exposed to relatively high levels of Egfr/Ras signaling. Thereafter, as the wing disc grows, ventrally situated D compartment cells inherit the 'on' state of ap expression, even as they populate areas of the disc progressively farther from the domain of high Egfr/Ras signaling and sustained Iro-C expression. It is suggested that the progressive reduction of Egfr/Ras activity in these ventrally situated D cells enables them to interact with neighboring V compartment cells to induce Wg and Vg expression and stimulate growth of the wing primordium. By contrast, early induced clones of RasV12-expressing cells autonomously express ap and experience persistent high levels of Ras activation, as indicated by sustained expression of the Iro-C genes. As a consequence, the ectopic DV boundary cannot shift outside of the domain of high Egfr/Ras signaling. Cells flanking this ectopic DV boundary fail to engage in the reciprocal induction of Wg and Vg expression or to stimulate growth. Hence, the apON-apOFF interface may normally have to shift to a region of relatively low Egfr activity for the DV boundary to acquire wing organizer activity (Zecca, 2002b).
Early induced clones that express Egfrlambda, the constitutively active form of the Egfr, can induce the formation of ectopic D compartments that retain organizer activity. However, the level of constitutive Egfr/Ras activity in such Egfrlambda-expressing clones appears to be significantly lower than in clones of RasV12-expressing cells. Consistent with this, it is found that ectopic expression of Egfrlambda considerably reduces but does not completely eliminate vg expression. Hence, it is inferred that the levels of Ras activation in Egfrlambda-expressing cells are not sufficiently high to prevent productive interactions between D and V compartment cells, thus allowing the ectopic DV compartment boundary to acquire organizer activity (Zecca, 2002b).
How might Egfr signaling regulate the capacity of the DV compartment boundary to function as an organizer? One possibility is that high levels of Egfr/Ras activity block the ability of cells to transduce Notch signals. During normal development, D and V cells engage in a positive auto-feedback loop of Delta/Notch and Serrate/Notch signaling that drives the reciprocal induction of Wg and Vg expression on both sides of the DV compartment boundary. Hence, if high levels of Egfr/Ras activity block Notch signal transduction, then persistent high levels of Ras activity on even one side of the DV boundary would suffice to disrupt the feedback loop and block the reciprocal induction of Wg and other 'boundary' genes. Accordingly, the DV boundary might have to be located in a region of low Egfr activity in order to allow reciprocal Notch signaling to induce the expression of these, and perhaps other, organizer genes (Zecca, 2002b).
Another possibility is that the apON-apOFF interface may only be able to function as an organizer when cells on both sides are of prospective wing type. Prior to the initial activation of ap and the Iro-C genes, the nascent wing disc appears to be subdivided into mutually antagonistic domains of Egfr and Wg signaling that at least transiently define the incipient notum and wing primordia. Because ap and the Iro-C genes are initially activated in response to a common source of Egfr signaling, most or all D cells at this stage may be notum type. It is only later, when ventrally situated D cells move out of range of Vn-dependent Egfr signaling and switch to being wing type, that inductive interactions occur across the DV boundary to create a new and stable source of Wg signaling. It is suggested that cells on both sides of the DV boundary may have to be of wing type for the boundary to have organizer activity. One possible reason for why this might be the case is that vg, the selector-like gene that defines the wing state, is itself an integral component of the reciprocal signaling mechanism that allows D and V cells to induce the expression of DV boundary genes. High levels of Egfr/Ras signaling actively maintain Iro-C gene expression (and hence the notum state) and block vg expression. Hence, the DV boundary may normally have to shift ventrally, into a domain of low Egfr/Ras signaling and high Wg signaling that defines the incipient wing state, to allow the positive feedback loop of inductive signaling to initiate across the DV compartment boundary. Once this loop is established, it would provide a stable source of Wg and other signals generated along the DV boundary that govern the subsequent growth and differentiation of the wing blade (Zecca, 2002b).
The Ras GTPase links extracellular signals to intracellular mechanisms that control cell growth, the cell cycle, and cell identity. An activated form of Drosophila Ras (RasV12) promotes these processes in the developing wing, but the effector pathways involved are unclear. Evidence is presented indicating that RasV12 promotes cell growth and G1/S progression by increasing dMyc protein levels and activating PI3K signaling, and that it does so via separate effector pathways. Endogenous Ras is required to maintain normal levels of dMyc, but not PI3K signaling during wing development. Finally, induction of dMyc and regulation of cell identity are separable effects of Raf/MAPK signaling. These results suggest that Ras may only affect PI3K signaling when mutationally activated, such as in RasV12-transformed cells, and provide a basis for understanding the synergy between Ras and other growth-promoting oncogenes in cancer (Prober, 2002).
In the developing Drosophila wing, Ras, dMyc, and PI3K regulate rates of cellular growth (i.e., mass accumulation) and progression through the G1/S transition of the cell cycle without affecting overall rates of cell division. These results concur with experiments in mice showing that Ras, Myc, and PI3K promote cell growth without affecting rates of cell
division. This study shows that an activated form of
Drosophila Ras (RasV12) is capable of increasing
dMyc protein levels as well as levels of PI3K signaling, suggesting
that RasV12 drives growth and G1/S progression via
both of these mechanisms. RasV12 effector loop
mutants were used to show that RasV12 affects dMyc and PI3K signaling via separate pathways, and that overexpressed dMyc and PI3K do
not cross-regulate each other. Thus, a hierarchy
has been established for these growth-regulatory proteins (Prober, 2002).
Wing disc cells lacking ras have reduced levels of dMyc
protein, indicating that Ras is required to maintain normal dMyc
protein levels during wing development. ras-/-
cells contain significant levels of dMyc protein, however, indicating
that Ras is not absolutely necessary for dMyc expression, and
suggesting that reduced dMyc levels may not fully explain the growth
deficit of ras-/- cells. However, dMyc antibody staining intensity was ~40% lower for
dmycP0 or dmycP1 homozygotes than
for dmycP0 heterozygotes in regions of the wing disc
that normally contain high dMyc levels (i.e., wing pouch and notum. Because dmycP0/P0 clones have severely reduced growth rates, it seems reasonable to expect that the ~20% reduction of dMyc levels in
ras-/- clones will also reduce growth rates. RasV12 increases dMyc levels post-transcriptionally, and studies in
mammalian cell culture has shown that RasV12 stabilizes Myc
protein. Therefore, it is likely that ras-/- cells still transcribe dmyc mRNA, but that following translation, dMyc protein is less stable. What other mechanisms may regulate dMyc levels? Wingless (Wg) signaling represses dmyc expression along the dorsal-ventral boundary of the developing wing. In addition, expression of an activated version of the Decapentaplegic (Dpp) receptor Thickveins (TkvQ238D) can increase levels of dMyc protein in the wing,
whereas loss of this same receptor suppresses dMyc levels. Thus, Ras signaling may be one of many inputs affecting dMyc expression in the wing. Ras may
stabilize the low levels of dMyc protein observed throughout the
developing wing and/or refine the patterned dmyc expression
regulated by other signals. The complex regulation of dMyc expression
in vivo may account for the lack of a clear correspondence between
patterns of high endogenous Ras activity and dMyc expression (Prober, 2002).
Overexpressed Drosophila RasV12
recruits the tGPH (aPH-GFP fusion protein used as an indicator of dPI3K signaling) reporter to the cell membrane, suggesting that
RasV12 activates PI3K signaling, and thereby increases
PIP3 levels, in the developing wing. It is inferred that
Drosophila RasV12 directly activates PI3K, because
mammalian studies have shown that RasV12 can directly bind
and activate PI3K. Alternatively, Drosophila RasV12 may activate PI3K signaling via other mechanisms, such as by inhibiting the lipid
phosphatase PTEN. This possibility seems less likely, however, since
direct interactions between Ras and PTEN have not been described.
Contradicting the generally accepted idea that PI3K is normally an
effector of Ras signaling, this study found that
localization of the PI3K reporter tGPH was not detectably affected in
ras-/- cells. Although the observations using the
tGPH reporter were not quantitative, and small effects could have been
missed, these results nevertheless indicate that Ras does not normally
play a major role in regulating PI3K in the developing wing.
Consistent with this hypothesis, expression of an activated form of
Drosophila EGFR (EGFRlambdatop) had no effect on PI3K signaling. Because the ability of dEGFRlambdatop
to activate downstream pathways is limited by the amount of endogenous Ras, this result suggests that higher levels of Ras activity than can
be generated in wild-type cells are required to activate PI3K (Prober, 2002).
An alternative explanation for the discrepancy between the involvement
of Drosophila and mammalian Ras in regulating PI3K signaling
may relate to the evolution of ras genes. The
Drosophila and C. elegans Ras homologs are more
homologous to mammalian K-Ras than to H- or N-Ras, suggesting that K-Ras may have an older, more general function than the other mammalian ras
genes. In support of this idea, H- and N-Ras are dispensable, whereas
K-Ras is essential, for normal mouse development. It is also interesting to note that overexpressed K-Ras preferentially activates Raf over PI3K,
whereas the opposite is true for H-Ras. Thus, K-Ras
may play a more fundamental role in developmental processes dependent
on Raf, but independent of PI3K, whereas H- and N-Ras may have evolved to perform less critical functions in which they regulate PI3K (Prober, 2002).
Using the tGPH reporter, it was found that levels of PI3K signaling are
not patterned but rather are uniform throughout wing development. It is therefore unlikely that PI3K signaling
is regulated by localized patterning signals such as the morphogens
Vein, Dpp, and Wg, which are secreted from the notum,
anterior-posterior boundary, and dorsal-ventral boundary of the wing,
respectively, and are thought to pattern growth and cell proliferation
of the wing. Furthermore, cell-autonomous activation of Dpp signaling
using an activated form of its receptor (TkvQ253D), which is
a potent growth driver in the wing, has no effect on tGPH localization. It may be that Dpp and Wg regulate cell growth rates
by affecting the ability of cells to respond to ubiquitous PI3K-dependent growth signals. They may do so by regulating the expression or activity of signaling proteins or transcription factors
required for transducing PI3K-dependent signals (Prober, 2002).
The tGPH reporter revealed that the polarized epithelial cells of
Drosophila wing discs contain dense regions of tGPH
colocalized with Armadillo at the apical region of the cell membrane,
with lower tGPH levels present throughout the basolateral cell membrane. This does not simply reflect an apical accumulation of
membrane microdomains enriched in PIP3 in polarized cells, because inhibiting PI3K activity (by expressing Deltap60) dramatically reduces apical tGPH fluorescence. In contrast, tGPH is
uniformly localized throughout the cell membranes of unpolarized Drosophila fat body cells in vivo and Drosophila S2
cells in culture. Similarly, mammalian PI3K is
uniformly active throughout the cell membrane of cultured HEK 293 cells. Thus, the dynamics of PI3K signaling are dependent
on the cellular context, which is likely disturbed when tissues are
dissociated into single cells that are studied in culture. This process
may allow signaling interactions not normally occurring in vivo. In support of this idea, overexpression of the Drosophila Insulin receptor homolog (Inr) does not activate MAPK in the developing wing or affect Ras-mediated cell fate specification in the developing eye, whereas addition of insulin to
cultured Drosophila or mammalian cells does activate Ras/MAPK signaling. Alternatively, the failure to detect activation of Ras signaling in
response to overexpressed Inr may reflect a cell-type specificity for this interaction or insufficient sensitivity of the assays. It will
therefore be interesting to compare the subcellular localization of
PI3K signaling complexes in cultured mammalian cells with the tissues
from which they are derived (Prober, 2002).
PI3K signaling is thought to be regulated by a family of secreted
Drosophila insulin-like peptides (dilps) that bind and activate Inr. dilp2 is ubiquitously expressed in imaginal tissues, whereas dilp2 and other dilp family members are expressed in a variety of larval tissues including the gut and neurosecretory cells in the brain.
dilp2 that is expressed in imaginal tissues is likely secreted
apically into the lumen between cells of the columnar epithelium and
the overlying peripodial membrane. This would result in preferential
binding of dIlp2 to Inr at the apical region of the cell, which could
account for the high levels of apically localized tGPH that were observed. Alternatively, apical PI3K signaling may reflect a local concentration of PI3K-signaling complexes. Consistent with the latter
possibility, RasV12 recruited tGPH to only apical regions of
the cell membrane. This result suggests that RasV12 may
require other apically localized factors to activate PI3K signaling.
This possibility is supported by the finding that coexpressed Deltap60,
which prevents PI3K from interacting with upstream activators, blocks
RasV12-mediated activation of PI3K signaling, as it does in
mammals. Because mammalian Ras can
directly bind the catalytic subunit of PI3K, it is inferred that coexpressed Deltap60 should not affect the ability of Drosophila Ras to activate PI3K signaling unless Ras-dependent activation requires other apically
localized factors that bind Deltap60. These factors may include the
Insulin receptor substrate Chico, Inr itself or other receptor
tyrosine kinases, G-protein coupled receptors, or components of
signaling complexes that are recruited upon activation of these
receptors. Several receptor tyrosine kinases, including EGFR, as well
as phosphotyrosine-containing proteins, are concentrated at the apical cell surface, although Inr is distributed throughout the cell membrane. The Drosophila homolog of the
heterotrimeric G-protein subunit Galphai, which
presumably transduces signals from a large family of associated
receptors, is also concentrated apically in wing disc cells. This is consistent with the possibility that heterotrimeric G-proteins may regulate PI3K signaling in Drosophila, as they do in mammals (Prober, 2002).
Much of the current understanding of Ras function, and that of most
oncogenes, derives from studies in homogenous cell culture systems. These studies have focused primarily on cell-autonomous effects of
oncogenes rather than upon the roles of interactions among cells within
tissues in tumor development. Tissue homeostasis is maintained by a
continuous exchange of signals between cells, the extracellular matrix,
and the local environment. An important feature of tumor development is
escape from this regulation, initially allowing the autonomous growth
and proliferation of tumor cells, and eventually resulting in altered
adhesion and migration of tumor cells away from their site of origin.
The behavior of clones of cells with elevated Raf/MAPK signaling levels
in developing Drosophila epithelia is strikingly similar to
that of tumor cells within mammalian tissues. These cells have altered
adhesive properties and cell identities, and as a result minimize
contact with neighboring wild-type cells. In contrast, PI3K and dMyc
do not regulate cell identity or adhesion. Studies in
Drosophila and vertebrates have also suggested that even
though both dMyc and PI3K stimulate growth, they appear to do so via
different mechanisms. PI3K signaling promotes nutrient import and
storage, whereas dMyc promotes nucleolar growth
and protein synthesis. Thus, the ability of
RasV12 to up-regulate both of these pathways may generate a
more robust and balanced growth response than activation of either dMyc
or PI3K alone. Furthermore, the ability of RasV12 to
deregulate cell identity and adhesion may underlie the strong synergy
between Ras and other growth-promoting oncogenes in vivo (Prober, 2002).
Continued Ras85 Effects of mutation: part 3/3 | back to part 1/3
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