Ras oncogene at 85D


EFFECT OF MUTATION OR DELETION (part 2/3)

Ras and glia

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).

Phosphatidylinositol 3-kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral nerves

Drosophila peripheral nerves, structured similarly to their mammalian counterparts, comprise a layer of motor and sensory axons wrapped by an inner peripheral glia (analogous to the mammalian Schwann cell) and an outer perineurial glia (analogous to the mammalian perineurium). Growth and proliferation within mammalian peripheral nerves are increased by Ras pathway activation: loss-of-function mutations in Nf1, which encodes the Ras inhibitor neurofibromin, cause the human genetic disorder neurofibromatosis, which is characterized by formation of neurofibromas (tumors of peripheral nerves). However, the signaling pathways that control nerve growth downstream of Ras remain incompletely characterized. This study shows that expression specifically within the Drosophila peripheral glia of the constitutively active RasV12 increases perineurial glial thickness. Using chromosomal loss-of-function mutations and transgenes encoding dominant-negative and constitutively active proteins, it was shown that this nonautonomous effect of RasV12 is mediated by the Ras effector phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. The nonautonomous, growth-promoting effects of activated PI3K are suppressed by coexpression within the peripheral glia of FOXO, a transcription factor inhibited by Akt-dependent phosphorylation. It is suggested that Ras-PI3K-Akt activity in the peripheral glia promotes growth of the perineurial glia by inhibiting FOXO. In mammalian peripheral nerves, the Schwann cell releases several growth factors that affect the proliferative properties of neighbors. Some of these factors are oversecreted in Nf1 mutants. These results raise the possibility that neurofibroma formation in individuals with neurofibromatosis might result in part from a Ras-PI3K-Akt-dependent inhibition of FOXO within Schwann cells (Lavery, 2007).

Activating Ras specifically within the peripheral glia was sufficient to increase growth of the perineurial glia. In addition, activating the Ras effector PI3K, but not Raf or Ral, within the peripheral glia was sufficient to increase perineurial glial growth, and inhibiting PI3K activity in the peripheral glia, but not perineurial glia, suppressed the growth-promoting effects of activated Ras. It was also found that activity within the peripheral glia of the PI3K-activated kinase Akt was both necessary and sufficient to mediate the growth-promoting effects of PI3K. Finally, it was found that overexpression within the peripheral glia of FOXO, the forkhead-box transcription factor that is phosphorylated and inactivated by Akt-dependent phosphorylation, was sufficient to suppress the growth-promoting effects of PI3K. Together, these results suggest that Ras activity in the peripheral glia activates nonautonomous growth via the PI3K and Akt-dependent inhibition of FOXO. This observation is consistent with the previous observations that Nf1 mouse Schwann cells oversecrete growth factor(s) that cause increased recruitment of mast cells into the peripheral nerve and is consistent in part with the observation that the proliferation defects of Nf1 mutant mouse or human cells requires hyperactivation of Tor in a PI3K- and Akt-dependent manner (Lavery, 2007).

Perineurial glial growth in Drosophila peripheral nerves is regulated by several genes. These genes include Nf1, which is the Drosophila ortholog of human Nf1, push, which is thought to encode an E3 ubiquitin ligase and two genes implicated in neurotransmitter signaling: amnesiac, which is thought to encode a neuropeptide similar to vertebrate pituitary adenylate cyclase-activating polypeptide, and inebriated (ine), which encodes a member of the Na+/Cl-dependent neurotransmitter transporter family. Some of these genes might regulate perineurial glial growth via the activity of Ras or PI3K within peripheral glia. For example, mutations in push, but not ine, enhance the perineurial glial growth phenotype of RasV12 expressed in peripheral glia. These observations are consistent with the possibility that the activity of ine regulates Ras-GTP levels within peripheral glia. In contrast, push might regulate PI3K in a Ras-independent manner or act in the perineurial glia to regulate sensitivity to peripheral glial growth factors. Additional experiments will be required to distinguish between these possibilities (Lavery, 2007).

There are several lines of evidence from mice and humans suggesting that cell nonautonomous growth regulation, as a consequence of intercellular signaling, underlies neurofibroma formation. First, although neurofibromas arise in individuals heterozygous for Nf1 after loss of Nf1+ from cell(s) within peripheral nerves, neurofibromas are heterogeneous and contain cells that are not clonally related, such as Schwann cells, perineurial cells, and fibroblasts. These observations suggest that neurofibromas arise when a core of Nf1 cells cause overproliferation of their heterozygous neighbors via nonautonomous means. Second, neurofibroma formation in mice and humans requires a homozygous Nf1 mutant genotype in Schwann cells but not other cells within the tumor. Third, Ras-GTP levels in Schwann cells from the mouse Nf1 knock-out mutant are uniformly elevated. In contrast, only a subset of Schwann cells from human neurofibromas exhibit elevated Ras- GTP levels; the possibility has been raised that this subset, but not other Schwann cells from the tumor, is homozygous for Nf1. In this view, these Nf1 cells recruited neighboring Schwann cells that were heterozygous for Nf1 into the tumor by nonautonomous means, such as by the excessive release of one or more growth factors. Fourth, it has been demonstrated that Nf1 Schwann cells oversecrete the ligand for the c-Kit receptor. This oversecretion increased migration of mast cells into peripheral nerves and might be an essential step in neurofibroma formation. These Schwann cells also oversecrete additional factors whose physiological role remains unclear. The molecular mechanisms by which neurofibromin regulates the synthesis or release of these molecules remain incompletely understood. The current observations that Ras activity in the peripheral glia promotes growth nonautonomously via the PI3K- and Akt-dependent inhibition of FOXO might provide insights into the mechanisms by which peripheral nerve growth is regulated nonautonomously by the mammalian Schwann cell (Lavery, 2007).

By hyperactivating Ras, Nf1 mutations could in principle cause tumors via any of several Ras effector pathways. In addition, the diverse types of tumors observed in individuals with neurofibromatosis could result from hyperactivation of distinct Ras effector pathways. The Raf pathway has been viewed as a more relevant effector pathway than the PI3K pathway, mostly because the importance of Ras in the activation of PI3K under physiological conditions remains controversial. In particular, although it is clear that the oncogenic RasV12 mutant is sufficient to activate PI3K, it has sometimes been difficult to demonstrate that wild-type Ras is necessary for PI3K activation. Presumably, this difficulty reflects the fact that PI3K can be activated by Ras-independent as well as Ras-dependent mechanisms, such as direct activation by activated receptor tyrosine kinases or by PIKE-L (phosphatidylinositol kinase enhancer). However, more recently, it has been demonstrated that PI3K and Akt are hyperactivated in several Nf1 mutant cell types and that this hyperactivation is Ras dependent. Furthermore, PI3K activation plays an essential functional role in Nf1-mediated growth defects, as is demonstrated by the observation that PI3K- and Akt-dependent Tor activation is necessary for the proliferation defects of Nf1 mutants to occur: application of rapamycin, a Tor inhibitor, attenuates the ability of Nf1 mutant cells to proliferate. These observations demonstrate that PI3K and Akt play key roles in at least some aspects of Nf1-induced tumor growth (Lavery, 2007).

The results are consistent with these observations. By comparing the effects on perineurial glial growth of peripheral–glial expression of activated Raf, PI3K, or Ral, it was possible to demonstrate that activation of PI3K, not Raf or Ral, is sufficient to promote perineurial glial growth and that PI3K activity in the peripheral glia is necessary to observe the nonautonomous effect of activated Ras on perineurial glial growth. It was similarly shown that Akt activity os necessary and sufficient to mediate the growth-promoting effects of PI3K. However, previous studies have observed that Tor activation is critical for the PI3K- and Akt-dependent growth regulation of Nf1 mutant cells, this study observed a critical role for the PI3K- and Akt-dependent inhibition of the transcription factor FOXO. It is possible that the phenotype observed in previous studies reflects the well characterized ability of PI3K–Tor to activate growth cell autonomously, whereas the phenotype reported in this study reflects nonautonomous growth regulation. In this view, PI3K and Akt regulate autonomous and nonautonomous growth via the Tor and FOXO pathways, respectively (Lavery, 2007).

FOXO presumably inhibits the growth-promoting effects of PI3K and Akt by transcriptional regulation of target genes. Candidate FOXO target genes include those encoding the molecules oversecreted by Nf1 Schwann cells, whereas other targets might be represented in the distinct transcript profiles exhibited by Nf1 Schwann cells or malignant peripheral nerve sheath tumors compared with wild-type Schwann cells. For example, Schwann cells from neurofibromas, but not normal Schwann cells, express the epidermal growth factor (EGF) receptor. Other potential targets include genes encoding growth factors, although ectopic expression within the peripheral glia of two candidate genes, Hedgehog and the EGF ligands spitz and gurken, failed to induce perineurial glial growth. Additional experiments will be required to identify the FOXO targets that regulate nonautonomous growth in peripheral nerves (Lavery, 2007).

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).

Notch and Ras signaling pathway effector genes expressed in fusion competent and founder cells during Drosophila myogenesis

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).

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. It is proposed that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active RasV12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. Evidence is also presented that the DVM FCs may arise from the leg imaginal disc (Atreya, 2008).

Founder cells (FCs) play an important role during myogenesis as they usually represent a pre-pattern prior to the onset of myoblast fusion. In the Drosophila embryo, each of the 30 muscle fibers in a hemisegment is seeded by a single founder cell. A group of adult flight muscles in Drosophila, the dorso-ventral muscles (DVMs) is patterned by founder cells which unlike the embryo are much larger than surrounding myoblasts. These cells have been referred to as imaginal pioneers and express the embryonic founder cell marker Dumbfounded, as detected by reporter activity. Duf is known to serve as an attractant for myoblasts and to thereby promote fusion. This study examined a role for the DVM FCs in organizing fiber formation. First the FCs were eliminated prior to the onset of fusion through targeted expression of the cell death gene reaper. Removal of these cells does not abolish fiber formation, but rather, excessive numbers of thinner fibers are formed. This outcome resembles what is observed when larval scaffolds for a related group of muscles, the dorsal longitudinal muscles (DLMs), are ablated. These scaffolds also express Duf, and the outcomes of their genetic ablation served as a useful comparison of muscle patterning in a set of functionally related fibers. In examining DVM myogenesis closely, it was found that in addition to the large Duf-expressing FCs, some (smaller) cells in the myoblast pool also express Duf, and it is proposed that these are the alternate/replacement founder cells that can seed fiber formation in absence of the large FCs. Interestingly, supernumerary fibers do not develop when reaper expression is maintained through the period of myogenesis, indicating that Duf expression is necessary in the alternate/replacement founder cells for fiber formation. In a second set of experiments, the Ras-signaling pathway was constitutively activated in using the rP298-Gal4 driver, and this manipulation also resulted in an increase in fiber number. The increase was not due to proliferation of pre-existing founder cells, and the supernumerary fibers develop in presence of the existing FCs. These outcomes for ablations of DVM organizers suggest that (1) DVM fibers can arise in the absence of the large elongate FCs; (2) a subset of cells in the myoblast pool have the potential to initiate fiber formation; (3) signals from the FC normally suppress this capability, so that the correct pattern of muscle fibers can be generated; and (4) interfering with the signaling results in the formation of supernumerary fibers. Thus, the most critical role of the DVM FCs is to regulate fiber number (Atreya, 2008).

An important consequence of eliminating the DVM FCs is that multiple fibers are formed, suggesting that cells from the myoblast pool are recruited to initiate fusion. Formation of supernumerary fibers is also observed when larval scaffolds that pattern the functionally opposing muscle group, the DLMs are ablated, or eliminated using reaper. Thus, although the two muscle groups develop using different modes of development, the larval scaffolds and the single celled FCs serve a similar function-specification of the number of adult fibers. These organizers serve as a pre-pattern, partitioning myoblasts through attractive cues, and subsequently when fusion begins, the final muscle pattern begins to emerge. The size of the myoblast pool remains unaffected by the manipulation (targeted reaper expression) and this is borne out by the fact that muscle volume in the adult thorax is not altered. These outcomes are similar to what is observed after DLM scaffolds are genetically ablated or laser ablated (Fernandes and Keshishian, 1996).

If a role for the large DVM founder cells is to initiate fusion, how does it occur in their absence? Two possibilities are suggested: that myoblasts randomly fuse with each other, or that 'replacement' founder cells begin to seed fiber formation. If the first scenario were true, many more than the 6-7 supernumerary fibers would be expected. Also, it would be a departure from the well understood separation of 'founder' and 'fusion-competent' fates (reviewed in Taylor, 2003) and the normal tendency of myoblasts to only fuse with a founder/scaffold. The current results do suggest the recruitment of 'alternate/replacement' founder cells. The rP298 promoter is active in a small subset of cells in the myoblast pool as early as 6 h APF, as detected by using the rP298-Gal4/UAS mCD8GFP. These cells are the same size as other cells in the myoblast pool and thus much smaller than the DVM FCs. It is proposed that these smaller Duf-expressing cells are recruited to initiate fusion when FCs are eliminated. They are capable of sustaining fusion, as suggested by multiple EWG-positive nuclei within a supernumerary fiber. It was also observed that fiber formation is disrupted upon prolonged exposure to reaper, which is most likely due to elimination of the smaller Duf-expressing cells as well. Thus, it appears that Duf expression is necessary in these cells to generate supernumerary fibers (Atreya, 2008).

It is useful to compare another manipulation which also eliminates Duf-lacZ expressing FCs, but which has different outcomes with respect to fiber formation. When the mesothorax is denervated at the third larval instar, the expression of Dumbfounded in DVM FCs is gradually abolished during the period of fusion, 12-24 h APF. Although the size of the myoblast pool is unaffected by the denervation, and Duf-expressing FCs are present prior to fusion (0-12 h APF), DVM fibers fail to form. In light of the reaper studies, it is reasonable to propose that during the 0-12 h period, DVM FCs engage in a 'lateral inhibition' process that is sufficient to prevent cells of the myoblast pool from seeding fibers during the subsequent fusion phase. Moreover, just as innervation is needed to maintain Duf expression in the FCs during the fusion phase, it may also be necessary to maintain expression in the smaller 'replacement/alternate' founder cells (Atreya, 2008).

In considering a role for the smaller Duf-positive cells during normal development, it is proposed that a subset of cells in the myoblast pool have the capability to initiate fiber formation, and that interactions with the founder cell prior to fusion are responsible for active suppression of this 'organizer' competency. When the FCs are eliminated, it is conceivable that the suppression is incomplete and they can go on to seed fibers. Thus, the smaller Duf-expressing cells serve as a reserve pool of founder cells to initiate fusion when necessary. The suppression could involve signaling mechanisms such as those that have been described for the embryonic mesoderm, wherein an equivalence group is first defined, from which a single founder cell is then selected, and the potential of the remaining cells is inhibited (Atreya, 2008).

Supernumerary fibers can arise despite the presence of pre-existing rP298-lacZ positive FCs Overexpression of RasV12 with the rP298-Gal4 driver prior to the onset of fusion also results in the development of supernumerary fibers. Two additional phenotypes are seen compared to what is seen under conditions of reaper expression (1) the large rP298-lacZ expressing FCs are still present and (2) many more rP298-lacZ expressing nuclei are seen in the myoblast pool than in controls. The formation of additional rP298-lacZ expressing cells is considered first. The BrDU studies demonstrate that they do not arise by proliferation. Thus the cells are being generated from the myoblast pool due to a change in cell fate. Two possibilities are suggested: one that activation of the Ras/Map kinase pathway in FCs signals cells in the myoblast pool to turn on rP298, and the second and more intriguing possibility considers the small subset of cells that express the rP298-reporter at low levels. There may be additional cells that are below the level of detection with the lacZ reporter; if the Ras/Map kinase pathway is activated in several of these cells, it would similarly result in activation of the rP298 promoter, giving rise to the increased number of rP298-positive cells. The levels of RasV12 activated in these unfused myoblasts may be just enough to alter cell fate, conferring founder cell like properties. In the Drosophila embryo, when RasV12 is targeted to the embryonic mesoderm an overproduction of progenitor cells is seen, which is brought about as a result of a change in cell fate of mesodermal cells. Although RasV12 is also being targeted to the large DVM FC, it does not alter cell fate there since that cell is already expressing high levels of rP298 and it continues to seed fiber formation, as in controls (Atreya, 2008).

Ras regulates intracellular signaling through the ERK/MAP kinase pathway, and RasV12 is known to increase levels of activated MAPK within cells. A recent report on founder cell specification during adult abdominal myogenesis in Drosophila has shown that restricted activation of the Ras/Map kinase pathway by the FGF receptor Htl, in a few myoblasts reinforces founder cell properties in them, leading to the upregulation of rP298-lacZ levels, and the eventual loss of reporter expression in the other myoblasts. While expression of Htl has not been reported in IFM myoblasts, a similar mechanism that uses the ERK/MAPK pathway might be at play. It is also considered that overexpression of RasV12 causes death of the founder cells. But this is clearly not the case, since TUNEL labeling does not show any cell death, and the original founder cells are present and seed fiber formation (Atreya, 2008).

Expressing RasV12 in DLM scaffolds has two prominent outcomes. First, there is an increase in the number of rows of nuclei within the developing fibers. The increase in number of nuclei is suggestive of rapid fusion, and an outcome of enhanced Ras signaling. Another interesting aspect is in regard to muscle splitting. The six DLM fibers are generated as a collective outcome of the longitudinal splitting of the three larval scaffolds and fusion of myoblasts with these scaffolds. It is thought that as myoblasts fuse with the scaffolds there is an increase in surface area, which is manifested as splitting. Under conditions of RasV12 expression, there is an increase in the number of nuclei within each DLM fiber, indicative of rapid fusion. However, in 45% of animals, splitting does not proceed normally. It follows therefore, that fusion occurs too rapidly, and becomes uncoupled from a sustained incorporation of new membrane, which is then manifested as a lag in splitting. Under conditions of nerve ablation, a lag in muscle splitting is observed, but in that case, a reduced pool of myoblasts and slower fusions are responsible (Atreya, 2008).

Which of the known organizer cells do they resemble — grasshopper pioneer, larval scaffolds, or embryonic FCs? The cells that organize the DVM myoblasts share features that are common to embryonic founder cells and to grasshopper pioneers. Like the embryonic FCs and grasshopper pioneer cells, they serve as fusion targets and prefigure a muscle fiber. They bear a closer resemblance to grasshopper pioneers with respect to their large size including cellular expanse. However, they are unique with respect to the manner in which they interact with surrounding myoblasts, a feature that has been revealed as a result of manipulations carried out in the present study. They are not necessary for fiber formation, per se, as in their absence, a reserve pool of 'alternate/replacement' FCs is recruited. When these cells are removed as well, fiber formation fails and this scenario is the most similar to the outcome of targeting reaper to embryonic founder cells (Atreya, 2008).

A distinction between adult and embryonic myogenesis is exemplified by the manner in which founder cells are selected during abdominal myogenesis. rP298-lacZ expressing founder cells in the adult abdomen are specified by signaling through the FGF receptor, Heartless, which is under positive as well as negative regulation through Sprouty and Heartbroken. This is different from founder cell specification in the embryo which uses Notch-mediated lateral inhibition. This difference between embryonic and adult abdominal muscles is particularly striking since the two muscle groups are more similar to each other with respect to the occurrence of segmentally repeated patterns, a feature that is not present in the adult thorax. The manner in which founder cells of the adult thoracic muscles are specified is not known, but it does not include a Notch-mediated lateral inhibition (Atreya, 2008).

In considering how a myoblast pool is patterned, these studies thus far have shown that an important property of organizer cells that regulate DVM patterning, is the regulation of fiber number. This property is related to the expression of Duf, which is known to serve as an attractant for myoblasts. Thus, the pre-pattern of large, elongate rP298-lacZ positive cells enables myoblast segregation in a manner that the fidelity of fiber number is ensured. There is a scattering of smaller rP298-lacZ expressing adult myoblasts in the pool, and these have the potential to seed fiber formation. The manipulations have shown that the DVM FCs suppress these smaller myoblasts from seeding fibers, and that interfering with this capacity can result in the formation of supernumerary fibers being generated. The interference can take the form of eliminating FCs or by activating the Ras/Map kinase pathway. Interestingly, cues from the epidermis are able to guide the nascent supernumerary fibers to attach appropriately. The organizer cells are contacted by neurons prior to the onset of fusion and this is the basis for another property that is distinct from embryonic founder cells -- the nerve dependence of rP298-lacZ expression. The innervating neurons continue to exert an influence on myogenesis during the stages of fusion and proliferation as well (Atreya, 2008).

Future work will be aimed at examining the mechanisms by which the correct number of FCs is established, the signaling mechanisms underlying founder cell-myoblast interactions, and how supernumerary founder cells are normally prevented from arising in the myoblast pool (Atreya, 2008).

Ras regulates synaptic plasticity at the neuromuscular junction

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).

Ras and wing development

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).

A dual function of Drosophila capping protein on DE-cadherin maintains epithelial integrity and prevents JNK-mediated apoptosis

E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. This study shows that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down Capping Protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, these findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours (Jezowska, 2011).

Actin filament (F-actin) turnover and organization is a critical regulator of AJs assembly, maintenance, and remodelling. F-actin growth, stability, disassembly and also their organization into functional higher-order networks are controlled by a plethora of actin-binding proteins (ABPs), strongly conserved between species. Capping protein (CP), composed of an α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer by restricting accessibility of the filament barbed end, inhibiting addition or loss of actin monomers. In Drosophila, removing either cpa or cpb, promotes accumulation of F-actin within the cell and gives rise to identical developmental phenotypes. In the whole larval wing disc epithelium, loss of CP activity reduces Hpo pathway activity and leads to ectopic expression of several Yki target genes that promote cell survival and proliferation. However, inappropriate growth can only be observed in the proximal wing domain. In the distal wing primordium, cpa or cpb mutant cells mislocalize the AJs components DE-Cad and Arm, upregulate puc expression, extrude and die. This indicates that while loss of CP can under certain conditions result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue (Jezowska, 2011).

This study investigated the role of the actin-CP heterodimer in survival of cells in the distal wing disc epithelium. CP has a dual function in regulating DE-Cad: it stabilizes DE-Cad at cell-cell junctions, thereby preventing loss of epithelial integrity and inhibits upregulation of the DE-cad gene. DE-Cad would otherwise provide an active signal, which affects Wg signalling and promotes JNK-mediated apoptosis. However, when cells lacking CP are kept alive, JNK is converted into a potent inducer of proliferation (Jezowska, 2011).

This study demonstrates that in the distal wing disc epithelium, JNK signalling triggers apoptosis of cells with reduced CP expression but induces massive proliferation when apoptosis is blocked with P35. Yki activity is also required to allow overgrowth of 'undead' Cpa-depleted tissues. Induction of apoptosis has been shown to activate Yki through the JNK pathway and triggers compensatory cell proliferation. Thus, in CP-depleted cells kept alive with P35, Yki may act downstream of JNK signalling. Consistent with this, targeting Yki degradation in these tissues fully suppresses ectopic N-Cad expression but not MMP1 upregulation. Because CP also prevents Yki activity in the whole wing disc epithelium, independently of its effect on JNK signalling, (Fernandez, 2011; Sansores-Garcia, 2011), in the distal wing domain, excess Yki activity of 'undead' CP-depleted tissues may result from a dual effect, which involves a JNK-dependent and independent mechanisms (Jezowska, 2011).

JNK signalling has been reported to propagate from cell to cell in the wing disc, where it could trigger apoptosis or Yki-dependent compensatory proliferation. Neither non-autonomous apoptosis or activation of JNK signalling was observed when patches of CP mutant cells were induced or dsRNA for CP was expressed with or without P35. Therefore, the propagation of JNK activation might be impaired in tissues knocked down for CP. However, increase proliferation was observed of wild-type cells apposed to 'undead' Cpa-depleted tissues. This suggests that JNK propagation is not required to trigger compensatory cell proliferation (Jezowska, 2011).

Several observations argue that in cells lacking CP, a DE-Cad-dependent signal promotes JNK-mediated apoptosis by inhibiting Wg signalling. First, knocking down Cpa affects Wg signalling, which has been shown to prevent JNK-dependent cell death in this region. Second, removing one copy of DE-cad in Cpa-depleted cells partially suppresses apoptosis and ectopic MMP1 expression and restores Wg target genes expression. Third, loss of CP is associated with upregulation of the DE-cad gene and increased levels of the DE-Cad protein. One way by which DE-Cad may block Wg signalling is by tethering Arm. In agreement with this possibility, in the distal wing disc epithelium, overexpression of DE-cad compromises Wg signalling, while co-expression of Arm rescues the DE-cad overexpression phenotype. Moreover, in mouse, overexpression of E-Cad induces apoptosis and sequesters the transcriptionally competent pool of β-cat, effectively shutting off expression of Lef/TCF/β-cat-responsive genes. Interestingly, in Cpa-depleted tissues, the faster mobility form of Arm is enriched. Because this form was proposed to correspond to the cytoplasmic pool of Arm, following CP loss, increase DE-Cad levels might tether and stabilize Arm in the cytoplasm, preventing it to transduce Wg signalling. How a defect in Wg signalling triggers JNK-mediated cell death is not known. In cells lacking CP, JNK activation may occur in response to loss of DIAP1 since overexpressing DIAP1 strongly reduces ectopic MMP1 expression. However, it cannot be excluded that JNK signalling reduces DIAP1 levels since JNK signalling can also function upstream of DIAP1 (Jezowska, 2011).

In the distal wing domain, cells lacking CP mislocalize DE-Cad and Arm at AJs, upregulate expression of DE-cad and extrude from the epithelium (Janody, 2006). DE-cad appears to be a direct transcriptional target of the Hpo signalling pathway. CP inhibits Yki activity (Fernandez, 2011; Sansores-Garcia, 2011) and prevents shg-LacZ upregulation, even in mutant clones that maintain a polarized epithelial architecture in the proximal wing domain. Thus, increased DE-cad expression likely results from inhibition of Hpo pathway activity. However, while mutant clones for Hpo pathway components accumulate DE-Cad, mutant cells do not extrude from the wing disc epithelium. Therefore, the polarity defect of cells lacking CP is unlikely to result from increased DE-Cad levels. Different observations also argue that altered cell-cell adhesion does not result from a defect in Wg signalling or from ectopic activation of JNK signalling, as previously reported. First, reducing DE-cad levels do not restore Arm localization at AJs. Second, in Cpa-depleted tissues in which JNK signalling is blocked, dividing nuclei surrounded by dense F-actin patches are recovered on the basal surface of the distal wing disc epithelium. Third, unlike cells lacking CP, tissues expressing P35 and defective for Wg signalling or overexpressing DE-cad or in which high apoptotic levels were induced maintain a polarized epithelial architecture). Therefore, following loss of CP, the mislocalization of DE-Cad and Arm and the loss of cell-cell contacts are likely upstream or parallel events to DE-cad upregulation and JNK-mediated cell death. Because disruption of apical-basal polarity can trigger JNK activation, a model is favored by which CP prevents JNK-mediated cell death though a dual function on DE-Cad: it promotes DE-Cad-mediated cell adhesion and restricts DE-cad expression (Jezowska, 2011).

While the effect of CP loss on DE-cad transcription is not context dependent, the polarity defect is mainly observed in the distal wing domain. Different regions of the wing disc may have specific requirements in terms of AJs stability and remodelling. Because the distal wing disc is under higher mechanical stress, this epithelium may require higher dynamics of DE-Cad remobilization. CP might be critical to control this kinetic, making distal wing cells lacking CP more prone to lose cell-cell adhesion and extrude from the epithelium (Jezowska, 2011).

Interestingly, the proto-oncogene of the Src family kinases Src42A antagonizes DE-Cad-mediated cell adhesion and stimulates the transcription of DE-cad. Moreover, in the distal wing disc epithelium, the major inhibitor of Src family kinases C-terminal Src kinase (Csk), maintains AJs stability, prevents JNK-mediated apoptosis, whereas halving the genetic dose of DE-cad suppresses the apoptotic phenotype of dCsk-depleted cells. CP and mammalian c-Src both regulate F-actin. Conversely, the control of F-actin impacts on the kinase activity of c-Src. Thus, whether the main role of CP is to regulate Src activity in the distal wing disc is an exciting possibility to be tested in the future (Jezowska, 2011).

This study and others have previously shown that the CP heterodimer acts as tumour suppressor through its control of Hpo pathway activity. This study now shows that in specific epithelia, loss of CP also affects cell-cell adhesion, which is a fundamental step to an epithelial-to-mesenchymal transition (EMT), triggers MMP1 expression, which degrades the basal extracellular matrix, induces cell invasion and promotes massive proliferation of cells that fail to stably retain associations with their neighbours when cell death is blocked with P35. Moreover 'undead' CP-depleted cells show ectopic N-Cad expression, whose de novo expression promotes the transition from a benign to a malignant tumour phenotype. Finally, like other tumour suppressors, loss of CP cooperates with RasV12 in tissue overgrowth. These findings argue that in some epithelia in which CP activity is affected, the appearance of a second mutation that prevents apoptotic cell death may trigger the development of aggressive tumours in humans. However, in contrast to tumour progression, which correlates with loss of overall E-Cad expression and stimulation of canonical Wnt signalling, this study observed increase DE-Cad levels and inhibition of Wg signalling in tissues knocked down for CP. Interestingly, in flies, shg-LacZ expression is also enhanced in response to ectopic expression of the two oncogenes Src42A and Yki. This suggests the interesting hypothesis that transcriptional stimulation of DE-cad is an early mechanism of tumour suppression, which would promote the elimination of deleterious cells, possibly through inhibition of Wg signalling, rather than allowing them to proliferate and form tumours. Malignant cells that become resistant to cell death may compete successfully by losing the overall E-Cad expression and upregulating mesenchymal cadherins such as N-Cad to reinforce their fitness (Jezowska, 2011).

Ras pathway and tracheal development

Continued Ras85 Effects of mutation: part 3/3 | back to part 1/3


Ras85D: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.