Ras oncogene at 85D


Table of contents

Raf, a serine/threonine kinase (part 2/2)

A genetic screen for modifiers of Drosophila Src42A identifies mutations in Egfr, rolled and a novel signaling gene

To investigate a Ras-independent means of activating the Mapk cascade, mutations have been isolated that suppress the lethality of a Drosophila Raf mutation [also referred to as l(1) pole hole], RafC110, which cannot interact with Ras1. RafC110 contains a point mutation (Arginine217 to Leucine) in the Ras1 interaction domain. This mutation completely abolishes the ability of the RafC110 mutant protein to bind with Ras1 as tested using in vitro binding and yeast two hybrid assays. Interestingly, the phenotypes of RafC110 mutants are substantially weaker than those of Raf null mutants. This could be partially attributed to the regulation of RafC110 by Ras-independent factor(s). It has been shown that the Torso (Tor) RTK can activate wild-type Raf in the complete absence of Ras1. Raf transduces signals from several RTKs throughout Drosophila development. Because RafC110 is a partial loss-of-function mutation, the mutant animals live beyond the embryonic stage and die at the pupal stage as pharate adults. This provides a sensitized genetic background whereby suppressor mutations, which allow RafC110 mutants to develop further into fertile adults, have been isolated. Interestingly, some Suppressors of RafC110, Su(Raf), appear to function without restoring the binding between RafC110 and Ras1. For example, Su(Raf)3 is an intragenic suppressor containing a compensatory amino acid change next to the Ras1 interaction domain on RafC110. Su(Raf)3 does not restore the interaction with Ras1 even though it is the strongest suppressor and it is not an activating mutation. In this case, it is possible that a decreased affinity for Ras1 may be compensated for by an increased affinity for a member of the Ras1-independent pathway (Zhang, 1999 and references).

Six extragenic Su(Raf) loci have also been identified. These mutations not only suppress RafC110 but also other partial loss-of-function Raf alleles that do not impair Ras-Raf binding. This suggests that the suppression of RafC110 by the extragenic Su(Raf) mutations does not necessarily involve the restoration of Ras-Raf binding. Developmental analyses have shown that all six extragenic Su(Raf) mutations promote signaling in the Sevenless (Sev) and Egfr RTK pathways. Su(Raf)34B is a gain-of-function mutation in the Dsor1 locus that encodes the fly Mek. Recently, Su(Raf)1 has been shown to encode Src42A. The isolation of mutations that suppress the suppressor activity of Su(Raf)1 is reported in this paper. These mutations define two known genes, Egfr and rolled (rl; also referred to as Mapk) and two previously uncharacterized loci. In addition, two alleles of Src42A were also isolated in the screen, although these mutations are not true suppressors of Su(Raf)1 (Zhang, 1999).

One of the novel suppressor loci was named semang (sag). sag is required during both embryonic and imaginal disc development. Mutations in sag cause zygotic lethality. To identify developmental pathways where sag functions, the phenotypes associated with sag mutations were examined with particular attention to those processes controlled by known Drosophila RTKs. The results of these analyses show that sag participates in the Torso (Tor) and Drosophila DFGF-R1 RTK (Breathless) pathways during embryonic development. sag also disrupts the embryonic peripheral nervous system. During imaginal disc development, sag mutations affect two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions broadly in several RTK-mediated processes. This role of sag in RTK signaling is further supported by the genetic interaction between sag and other known RTK signaling genes. sag dominantly enhances the phenotypes caused by reductions of RTK signaling in loss-of-function Raf or rl mutants. Consistent with this, sag dominantly suppresses the formation of supernumerary R7 cells caused by the activated sev-Ras1V12 mutation. The sag mutations analyzed are likely to be loss-of-function mutations. These results suggest that sag may have a positive role in RTK signaling (Zhang, 1999).

Focus was placed on processes known to be controlled by RTKs. At the beginning of embryogenesis, the Tor RTK pathway specifies the embryonic terminal cell fates. Activation of Tor at the embryonic poles triggers the Ras-Mapk signaling cascade, resulting in the expression of two transcription factors: tailless (tll) and huckebein (hkb). tll and hkb in turn activate genes required for terminal development. Whenever there are reductions of tll and hkb expression due to reduced levels of Tor signaling, deletions of terminal structures occur. In the absence of Tor, the mutant embryos lack the anterior acron and all structures posterior to abdominal segment seven (A7). In sag homozygote mutant embryos derived from sag GLC eggs crossed to sag heterozygote males, terminal defects similar to those of the RafPB26 allele are observed. The head skeletal structure is collapsed; the tail region contains a partial deletion of the abdominal segment eight; the size of the anal pads is reduced and associated structures appear abnormal. The expressions of tll and hkb at the posterior embryonic pole are solely activated by tor signaling, whereas the anterior expressions are also activated by the bicoid morphogene. In wild-type cellular blastoderm embryos, tll is expressed posteriorly from 0% to 15% egg length (EL; 0% EL is at the posterior pole); hkb is expressed posteriorly from 0% to 9% EL. In sag mutant embryos, posterior tll expression is reduced to 10% EL; posterior hkb expression is reduced to 6% EL. In other words, there is an ~30% reduction of the posterior expressions of both tll and hkb in sag mutants. The anterior expression of these two genes appears grossly normal, with perhaps a slight broadening of tll and a slight reduction of hkb. Consistent with this, the anterior head defect appears variable and is only observed in 50% of the embryos. These results suggest that sag is involved in tor signaling, although the mutation blocks tor signaling to a lesser extent than a Ras1 gene deletion mutation. In embryos lacking maternal Ras1+, the posterior tll expression domain is reduced to 5% EL and hkb is not expressed at the posterior. The residual tll expression in the Ras1 mutant embryos reflects the functioning of the Ras1-independent pathway that activates the Mapk cascade (Zhang, 1999).

Several other RTKs function following the activation of Tor at the blastoderm stages. At the late embryonic stages examined, sag/sag embryos derived from sag GLC eggs lack gut constrictions. This feature was used in addition to the lacZ marker to confirm the genotype of sag/sag embryos. The DFGF-R1 RTK (breathless) is involved in directing the migration of tracheal cells to form tracheal branches. Monoclonal antibody 2A12, which reacts with an unknown tracheal luminal antigen, was used to visualize the branches. In contrast to those of wild-type embryos, the mutant tracheal branches are fragmented with incomplete connections. This truncation of tracheal branches could be explained by reduced Fgfr signaling in the sag mutants (Zhanga, 1999).

The Egfr pathway is involved in specifying ventral ectodermal cell fates. Reduction of Egfr signaling causes deletion of the ventral-most cell types. Consequently, the width of ventral dentical bands is shortened and the central nervous system (CNS) is disrupted. In sag/sag embryos derived from sag GLC eggs, the width of ventral dentical bands as well as the distance between Keilin's organs appeared grossly normal. Anti-HRP antibody staining, which labels all neurons and their processes, shows that the CNS axon tracks are also grossly normal. However, severe defects are observed in the peripheral nervous system (PNS). The PNS axons appear to be reduced in number; most if not all chordotonal neurons (CH) are missing; and neurons of the external sensory organs (ES) are also abnormal in number and in their arrangement in clusters (Zhang, 1999).

RTK signaling mediates cell proliferation of imaginal discs. In strong sag zygotic mutants (sag13L or sag32-3) that die at pupal stage, eyes are rough and contain only ~50% of the normal number of ommatidia. Third instar larval eye discs dissected from these mutants are also smaller in size, suggesting that sag may have a role in cell proliferation of the eye disc. The development of all cells in the eye requires a normal level of RTK signaling. Several pulses of Egfr RTK signaling are involved in the sequential recruitment of all photoreceptor cells (except R8) and subsequently the cone cells and pigment cells. In addition, activation of the Sev RTK in the R7 precursors is required for the formation of the R7 cells. In partial loss-of-function signaling mutants, such as RafHM7 or Dsor1XS520, reduced RTK signaling levels result in the reduction of both the R7 and outer photoreceptors. The eyes derived from sag zygotic mutants (pharate adults) were examined in cross section. All sag mutant ommatidia lack the normal complement of photoreceptor cells and these cells are often in the state of degeneration. To observe the eye phenotypes in the absence of cellular degeneration, mitotic clones of sag homozygous cells were generated in a heterozygotic sag genetic background. Inside the unpigmented patches of sag13L mutant clones, no wild-type ommatidia are found; 75% of the ommatidia lack the R7 cell and 94% of the ommatidia lack from one to three outer photoreceptors. No degeneration is observed for the photoreceptor cells inside the clones in mosaic eyes after aging the flies for several weeks. A similar result has been obtained for the sag32-3 allele. Thus the eye defect associated with sag mutations is similar to that observed in partial loss-of-function signaling mutants, such as RafHM7 or Dsor1XS520 (Zhang, 1999).

To examine how sag affects the development of photoreceptor cells, anti-Elav antibody was used to visualize all photoreceptor neurons as they were recruited into the preommatidial cell clusters. In third instar sag mutant eye discs, the initial formation of the R8 cell appears normal. Subsequently, more mature clusters contain reduced numbers of Elav-positive cells. This defect is more easily seen in the mutant pupal eye disc where ommatidial spacing becomes more irregular, but no further loss of photoreceptor cells is observed. These results indicate that some of the photoreceptor precursor cells fail to be recruited into the clusters at the third instar stage. Since preommatidial cluster formation requires several rounds of Egfr-mediated inductive recruitment, the most likely explanation for the observed phenotype is that sag mutations impair Egfr signaling, resulting in fewer photoreceptor precursors being recruited into the ommatidia. In heterozygotic sag/+ flies carrying clones of sag mutant cells in somatic tissues, defects are found in only two tissues, the eye and wing. sag clones in the wing cause a deletion of the L4 vein. This vein phenotype is similar to that observed in viable loss-of-function Egfr mutants, suggesting that sag may participate in Egfr-mediated vein formation (Zhang, 1999).

To provide further evidence that sag has a role in RTK signaling, sag was tested for genetic interaction with other known signaling genes. Four results are described: (1) sag dominantly enhances the eye phenotype of weak rl mutants. In the eyes derived from rl1;rl13R flies, 65% of the ommatidia are normal and the remaining 35% of the ommatidia lack the R7 cell, while outer photoreceptors are all normal. In contrast, in the eyes derived from rl1+/rl13L sag13L flies, all ommatidia lack the R7 cell and 77% of these also lack from one to three outer photoreceptor cells. (2) Although eyes from rl41-1/+ or sag13L/+ flies are normal, the eyes derived from rl41-1+/+ sag13L flies exhibit disorganized ommatidia. Sections show that only 41% of the ommatidia are normal and the remaining 59% of the ommatidia lack the R7 cells and/or R1–6 cells. (3) The expression of a constitutively activated form of Ras1 under the control of the sev promoter/enhancer sequences (sev-Ras1V12) mimics the effects of sev RTK activation. Because the promoter sequence also directs transcription in cone cell precursors and the mystery cells, these cells are transformed into R7-like cells in flies carrying the activated construct. Similar to mutations in many known RTK signaling genes, sag dominantly suppressed the formation of these supernumerary R7 cells caused by sev-Ras1V12. The number of R7 cells was reduced from an average of 2.4 ± 0.12 cells per ommatidium in sev-Ras1V12/+ flies to 1.48 ± 0.14 cells per ommatidium in sag13L/+; sev-Ras1V12/+ flies. (4) RafHM7 mutants survive at 18°-22°, but die as pupae at 29°. sag dominantly enhances the lethality of RafHM7 and causes the complete death of RafHM7/Y; sag/+ flies at the permissive temperatures. This result suggests that sag interacts genetically with RTK signaling mutations not only in the eye, but also in tissues required for survival. As expected for a gene involved in RTK signaling, sag enhances loss-of-function Raf and rl, but suppresses the activated sev-Ras1V12 mutation. These genetic interactions provide further evidence that sag plays an important role in RTK signaling (Zhang, 1999).

Drosophila has two other Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. Src64 does not affect viability when mutated. The isolation of Su(Raf)1 as a mutation in Src42A that restores the viability of Raf mutants and the isolation of Egfr, rl, and sag as extragenic suppressors of Su(Raf)1 provides the first in vivo evidence that both Src42A and sag are modulators of RTK signaling. At this moment, it is not known where Src42A and sag fit into the known RTK signaling cascade. An Src42A cDNA driven by a ubiquitously expressing promoter rescues the lethality of both Su(Raf)1 homozygotes and Su(Raf)1/Df hemizygotes. Based on this, Su(Raf)1 has loss-of-function characteristics, suggesting that Src42A is, unexpectedly, a negative modulator of RTK signaling. However, the genetics of Su(Raf)1 suggest that the suppression of RafC110 may be attributed to a dominant-interfering effect because the RafC110 lethality is not suppressed in Src42A hemizygotes of genotype Df(2R)nap9/+. Because of this, the role of Src42A in RTK signaling is still being investigated. However, the genetic interaction as revealed by the modifying screen suggests that Egfr and other RTKs may possibly regulate Src42A and sag, which in turn modulate the Mapk cascade (Zhang, 1999).

DER signaling restricts the boundaries of the wing field during Drosophila development

Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the first element of the hierarchy of regulatory genes promoting wing formation. Drosophila epidermal growth factor receptor (Egfr) signaling has many functions in fly development. Antagonizing Egfr signaling during the second larval instar leads to notum to wing transformations and wing mirror-image duplications. Egfr signaling is necessary for confining the wing subregion in the developing wing disk and for the specification of posterior identity. To do so, Egfr signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells (Baonza, 2000).

To study Egfr function during early wing development, Egfr signaling was reduced at different times by using thermosensitive alleles of Egfr or by overexpression of dominant negative Raf (DNRaf). Hypomorphic vein (vn) and connector enhancer of ksr (cnk) [a regulatory member of the Ras signaling cascade] alleles were also analyzed. Under these conditions, posterior to anterior transformations, proximal (notum) to distal (wing) transformations, and a reduction (or absence) of the notum region were observed with high frequency. When DNRaf is expressed in clones induced during the second instar, different kinds of phenotypes are found. Large clones in the posterior notum/hinge anlage lead to notum to wing transformations, whereas large clones covering the posterior of the wing give rise to posterior to anterior transformations. These phenotypes were found only after inducing a large amount of confluent clones. Clones of cells overexpressing DNRaf in other regions at this age, or anywhere at later stages, give rise to different defects, such as those previously described on cell proliferation and vein cell fates (Baonza, 2000).

Posterior to anterior transformations are associated with mirror-image duplications that are reminiscent of those observed after reducing the expression of engrailed, a gene that confers posterior identity in posterior wing cells. En represses ci and limits the expression of dpp to anterior compartment cells adjacent to En-expressing cells. Dpp acts as a long-range morphogen emanating from the compartment border and it directs the growth and patterning of the wing. Local loss of en function is sufficient to generate a complete transformation of posterior cells to anterior, and as a consequence, to induce the ectopic expression of dpp and an ectopic anterior compartment (Baonza, 2000).

The mirror-image wing duplication resulting from the reduction of Egfr signaling in the posterior compartment correlates with a down-regulation of En protein expression and the up-regulation of Ci. Accordingly, ectopic dpp expression is activated and a new A/P border is implemented. It should be mentioned that dpp is activated within the posterior compartment, suggesting that the posterior En-nonexpressing cells are not recruited from anterior regions of the wing disk (Baonza, 2000).

Cells that neighbor those that express DNRaf in clones are recruited to generate the new A/P border. This is a nonautonomous effect that also has been described for en clones, leading to mirror-image duplications. Moreover, when DNRaf is expressed in clones, not all cells within the clone down-regulate En. Egfr signaling is therefore important for the maintenance of En expression in the posterior cells of the wing pouch and the enactment of posterior cell fates, although its effects appear to be nonautonomous (Baonza, 2000).

Wg is expressed in an anterior-ventral area roughly complementary to vn expression, near the interface between the A/P and D/V compartment boundaries. Reducing Egfr signaling during the second larval instar results in the generation of ectopic wings out from peripheral notal tissue in posterior territories. It is worth pointing out that this ectopic wing tissue does not develop at the expense of the notum, which, in many cases, is not affected. These phenotypes are similar to those observed after early Wg overexpression. They are never observed in the absence of en, which only promotes posterior to anterior notum and wing transformations (Baonza, 2000).

This leads to the proposition that Egfr signaling would restrict the domain of expression of wg and define the boundaries of the field of cells that is going to undergo a wing developmental program. To examine this possibility, the expression of Wg was analyzed in clones ectopically expressing DNRaf. In large clones covering most of the notum/hinge region, Wg expression is up-regulated from the early second larval instar in the posterior of the wing disk, extending progressively. The expansion of Wg expression drives the enlargement of the D/V axis toward posterior territories (manifested in the expanded field of ap-expressing cells in the duplicated areas) giving rise to a fully developed duplicated wing (Baonza, 2000).

To test whether the ectopic expression of Wg, induced by the down-regulation of Egfr activity, is sufficient to promote notum to wing transformations, Wg was interferred with by inducing the expression, in clones, of a dominant-negative form of Wg (DNWg) along with DNRaf. In most cases, double mutant clones covering the whole posterior compartment show a partial reduction in the size of the wing pouch and notum and a suppression of notum to wing transformations or ectopic induction of wing markers (vg expression). It is concluded that the repression of wg expression by Egfr signaling in the notum, under wild-type conditions, is necessary to set a limit for the wing field. This relationship partly resembles the antagonism between the Egfr and wg signaling pathways for the specification of the ventral larval cuticle, although in this case wg and Egfr do not counteract one another, but control epidermal differentiation through the opposite transcriptional regulation of downstream genes (Baonza, 2000).

Interestingly, Wg overexpression induces notum to wing transformations, but never mirror-image duplications, such as those obtained after reducing Egfr activity. This suggests that wg function mediates notum to wing conversion, but its overexpression is not sufficient for posterior to anterior transformation. Indeed, the overexpression of DNWg is not able to rescue the down-regulation of En, the up-regulation of Ci, or wing mirror-image duplications, which result from interfering with Egfr signaling. These data suggest that the effects of Egfr signaling on en and wg functions are independent, although the possibility of regulatory interactions between wg and en cannot be disregarded at this stage (Baonza, 2000).

The reiterative use of Egfr has been demonstrated in the generation of multiple fates in the developing fly eye. A fundamental early function for Egfr in the underlying patterning system of the wing subfield, controlling the activity of two genes, en and wg has been documented. It is significant that fibroblast growth factor [Ras-mitogen-activated protein kinase (MAPK)] activities are implicated in the initiation of the whole program of limb development in vertebrates, a program remarkably similar to that of the Drosophila wing. It remains to be seen whether a similar strategy applies to the activities of Ras-MAPK cascades during vertebrate limb bud development (Baonza, 2000).

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila

The heat shock protein Hsp90 has been shown to associate with various cellular signaling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. Mutations in the hsp83 gene, the Drosophila homolog of hsp90, have been identified in a search for dominant mutations that attenuate signaling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. Both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Hsp83 is essential for Raf function in vivo (van der Straten, 1997).

How does Hsp83 facilitate Raf function? One possibility is that Hsp83 is needed merely for the maturation of the Raf protein. Another possibility is that Hsp83 may help to assembly complexes consisting of both Raf and other signaling components, such as Ras and MEK. Both of these proteins associate with the Raf-Hsp90 complex; it has been suggested that the interaction with Ras is required for the Raf-Hsp90 complex to be translocated to the membrane, while the interaction with MEK is a prerequisite for this kinase to be a substrate of Raf. The model favored is that Hsp90 facilitates Raf signaling in a manner similar to that proposed for Hsp90's function in steroid receptor signaling, allowing Raf to switch rapidly to its active conformation once it reaches the plasma membrane (van der Straten, 1997).

Dominant mutations of Drosophila MAP kinase kinase and their activities in Drosophila and yeast MAP kinase cascades

Eight alleles of Dsor1 encoding a Drosophila homolog of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppress mutations for receptor tyrosine kinases (RTKs) torso, sevenless, and to a lesser extent, Drosophila EGF receptor. Furthermore, the Dsor1 alleles show no significant interaction with gain-of-function mutations of Egfr. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrates that Dsor1 can activate yeast MAP kinase homologs if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes reveal that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors rather than constitutive activity (Lim, 1997).

Transcriptional regulation of the Drosophila-raf proto-oncogene by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system

The DRE/DREF system plays an important role in transcription of DNA replication genes, such as those encoding the 180 and 73 kDa subunits of DNA polymerase alpha as well as the gene that encodes PCNA. Two sequences were found homologous to DNA replication-related element (DRE; 5'-TATCGATA) in the 5'-flanking region (-370 to -357 with respect to the transcription initiation site) of the D-raf gene. Transcriptional activity was confirmed through gel mobility shift assays, transient CAT assays, and spatial patterns of lacZ expression in transgenic larval tissues carrying D-raf and lacZ fusion genes. The D-raf gene was found to be another target of the Zerknullt (Zen) protein with the observation of D-raf repression by Zen protein in cultured cells and its ectopic expression in the dorsal region of the homozygous zen mutant embryo. The evidence of DRE/DREF involvement in regulation of the D-raf gene strongly supports the idea that the DRE/DREF system is responsible for the coordinated regulation of cell proliferation-related genes in Drosophila (Ryu, 1997).

Lilliputian: an AF4/FMR2-related protein that controls cell identity and cell growth

lilliputian has a partially redundant function downstream of Ras/MAPK signaling in cell fate specification in the Drosophila eye. Loss-of-function mutations in lilli were identified as dominant suppressors of the specification of supernumerary R7 photoreceptor cells in response to constitutive activation of Raf in the developing eye. However, without constitutive activity of the Ras/MAPK pathway, the normal number of photoreceptor cells is specified in each ommatidium in the complete absence of Lilli function. It is concluded that Lilli has a specific function in regulating the efficiency of signal transduction downstream of Raf. As a putative transcription factor, Lilli may regulate the expression levels of one or multiple components of the Ras/MAPK signaling pathway that become rate-limiting when Ras/MAPK signaling is too high, in cells where it is normally low (as in the case of ectopic activation of Raf in the eye), or when signaling is reduced (Wittwer, 2001 and references therein).

Raf acts to elaborate dorsoventral pattern in the ectoderm of developing embryos

In the early Drosophila embryo the activity of the EGF-receptor (Egfr) is required to instruct cells to adopt a ventral neuroectodermal fate. Using a gain-of-function mutation it has been shown that D-raf acts to transmit this and other late-acting embryonic Egfr signals. A novel role for D-raf was also identified in lateral cell development using partial loss-of-function D-raf mutations. Thus, evidence is provided that zygotic D-raf acts to specify cell fates in two distinct pathways that generate dorsoventral pattern within the ectoderm. These functional requirements for D-raf activity occur subsequent to its maternal role in organizing the anterioposterior axis. The consequences of eliminating key D-raf regulatory domains and specific serine residues in the transmission of Egfr and lateral epidermal signals were also addressed in this study (Radke, 2001).

In the Drosophila embryo, Egfr activity is required to instruct a field of cells that lie on either side of the ventral furrow to adopt a ventral ectodermal fate. It is from this neuroectodermal cell population that the ventral nervous system and epidermis arise. At later times, Egfr functions in germband retraction and cuticle formation. Embryos that develop without Egfr activity fail to form ventral cuticular structures and show the 'faint little ball' phenotype. A constitutively active form of the D-raf protein, D-raftor4021, was used to bypass the requirements for Egfr function in embryos that lacked Egfr gene activity. For the generation of hyperactive D-raftor4021-proteins, the extracellular and transmembrane domains of the torso RTK gene were fused to the D-raf kinase domain. Chimera D-raftor4021proteins were shown to act independently of sevenless RTK gene function in developing photoreceptor cells: the chimeric proteins exhibited gain-of-function effects in the Tor signaling pathway (Radke, 2001).

Would this activated D-raf protein act independently of Egfr to rescue the embryonic lethality associated with homozygous mutations in the Egfr gene? In the case of noninjected control, 25% of the embryos derived from heterozygous Egfr parents (Egfr-/+) failed to hatch, showed the faint little ball phenotype, and were homozygous for the Egfr mutation. D-rafWT mRNA was used as a control for the injection procedure, and it was found that after injection 27% of the embryos from heterozygous Egfr parents failed to hatch. These embryos showed the Egfr mutant phenotype at 24 hr. When D-raftor4021 mRNA was injected into the central region of embryos collected from heterozygous Egfr parents, all aspects of defective Egfr signaling were rescued for some of the mutant Egfr embryos. Of the 258 embryos that received injection, 217 (84%) hatched out of their egg cases as larvae, while 41 (16%) remained within their eggshells. Thus, an increase in embryonic hatching and suppression of Egfr-induced lethality was observed after injection of D-raftor4021 mRNA. Partial rescue of the Egfr phenotype was found in unhatched embryos that had received D-raftor4021 mRNAs with ventral cuticular structures observed. It was concluded that constitutively active D-raftor4021 molecules can bypass the requirement for Egfr activity in the embryo and direct cells of the embryonic ectoderm to adopt a ventral fate. These results show that D-raf participates downstream of Egfr in developing embryos (Radke, 2001).

Once it had been found that an activated form of the D-raf protein could suppress the effects of a loss-of-function Egfr allele, it was reasoned that embryos lacking maternal and zygotic D-raf activity would exhibit an Egfr-like phenotype. These embryos would also be expected to show defects associated with the loss of maternal D-raf function in Tor signaling. To determine whether the identities of cells in the ventral ectoderm were dependent on D-raf activity, marker gene expression patterns and cuticles produced by D-raf embryos were compared to those of wild-type and Egfr embryos. To generate these D-raf embryos, mosaic D-raf females were produced whose eggs lacked maternal D-raf proteins. Once fertilized, these eggs gave rise to two classes of embryos: the first class was composed of the paternally rescued D-raf torso embryos (D-raf-/+) that had inherited a wild-type D-raf gene from their fathers: they were defective in Tor RTK signaling and were missing head and tail structures at 24 hr. These D-raf torso embryos lacked maternal but not zygotic D-raf activity. The second phenotypic class was composed of the D-raf null embryos (D-raf-/Y) whose exoskeletons consisted of what appeared to be a small patch of dorsal cuticle. These embryos lacked maternal and zygotic D-raf activity throughout development. It was anticipated that this D-raf null embryonic class would exhibit the phenotypic characteristics consistent with defective Egfr signaling, a consequence of defective D-raf protein activity (Radke, 2001).

Initially, to determine whether the establishment of ventral cell identity by the maternal dorsal gene system occurred normally in D-raf embryos, the accumulation of rhomboid (rho) mRNAs between 4 and 6 hr (stages 9-12) of development was assayed. As visualized by in situ hybridization, a column of cells ~2-3 wide on either side of the ventral midline showed the accumulation of rho mRNAs. This temporal and spatial pattern of rho expression was observed in all embryos in the D-raf collections, with each embryo a member of either the D-raf torso (lacking maternal but not zygotic D-raf activity) or null class. An equivalent rho expression pattern was observed in wild-type and Egfr embryos. Thus, the initial step in the establishment of ventral cell identity, by dorsal and other maternal genes that act to define the dorsoventral embryonic axis, is not perturbed when these events take place in the absence of maternal or zygotic D-raf activity (Radke, 2001).

To determine whether EGR-receptor signaling occurs normally in D-raf embryos, expression of the orthodenticle (otd) gene was monitored. In wild-type control embryos, at 6 hr (stage 11) otd mRNAs accumulate in cells adjacent to the ventral midline and in the head. In embryos lacking Egfr activity, otd expression occurred only in those cells within the embryonic head. In D-raf embryo collections, two patterns of embryo staining were observed with approximately one-half of the embryos showing otd expression in cells along the ventral midline and in the head. For the remaining D-raf embryos, the accumulation of otd mRNAs was observed only in the head, similar to Egfr embryos (Radke, 2001).

To distinguish between torso and null embryos in D-raf collections, a ftz-ß-gal marker gene located on the paternal X chromosome was used. Males with the ftz-ß-gal gene were allowed to fertilize eggs from mosaic females that lacked D-raf activity. In this double-labeling experiment, embryos that showed a ftz pattern of ß-gal expression were assigned to the D-raf torso class. These embryos also displayed a wild-type pattern of otd expression. In those D-raf null embryos lacking ß-gal expression, otd mRNAs were detected only in cells of the head, similar to Egfr embryos (Radke, 2001).

Between 4 and 7 hr (stages 9-11) of development, wild-type and Egfr embryos accumulated decapentaplegic (dpp) mRNAs in cells that formed two lateral stripes, when embryos were viewed ventrally. A similar pattern of dpp mRNA accumulation is seen in D-raf mutant embryos at this developmental stage. However, the ventral distance between dpp stripes becomes smaller in Egfr embryos as they develop. The distance between lateral dpp stripes was recorded and compared in wild-type, Egfr, and D-raf embryos at 10 hr (stage 13) of development. For wild-type embryos the average stripe distance was 0.111 units. In the collection of Egfr embryos, ~75% showed an average dpp lateral stripe distance of 0.118 units, similar to wild type. This phenotypic class contained embryos that were heterozygous mutant (Egfr-/+) or wild type with respect to the Egfr gene. In the remaining 25% of the embryos the average dpp stripe distance was reduced to 0.075 units as anticipated for homozygous mutant Egfr embryos (Radke, 2001).

Two phenotypic classes of D-raf embryos were also distinguished on the basis of a statistically relevant difference in dpp stripe distance. In approximately one-half of the embryos the average dpp lateral stripe distance was 0.120 units, with the remaining embryos showing an average separation of 0.064 units. It was speculated that this second phenotypic class contained the D-raf null embryos. To test this idea, the marker ftz-ß-gal X chromosome was again employed in a double-labeling experiment to distinguish between D-raf torso and null embryos. As anticipated, it was the male D-raf null embryonic class that showed the decrease in distance between lateral dpp stripes, indicative of a loss in ventral cell fates (Radke, 2001).

On the basis of this analysis of rho, otd, and dpp gene expression patterns in D-raf null embryos, it has been concluded that ventral ectoderm cells are specified incorrectly in the absence of D-raf activity. This loss results in the production of a mature D-raf null exoskeleton that is severely reduced in size and devoid of ventral structures, consistent with the Egfr embryonic phenotype. However, the distance between lateral dpp stripes in Egfr (0.075 units) and D-raf null (0.064 units) embryos was compared: it was smaller in D-raf null embryos. In addition, after cursory inspection, the size of the exoskeleton patch produced by D-raf null embryos appeared smaller than that from Egfr embryos. These differences could be biologically significant and the analysis was expanded to address this potentially interesting finding (Radke, 2001).

To better understand the role that D-raf plays in the ectoderm and to access its regulation in various developmental pathways partial loss-of-function alleles of D-raf generated in vitro were used. D-raf shares homology with family members in CR1 that contain (1) D-ras binding motifs; (2) CR2, a region rich in serine and threonine residues, and (3)the CR3 kinase domain. CR1 is thought to exhibit positive control in the regulation of the D-raf protein via its interaction with D-Ras, while CR2 appears to be involved in the negative regulation of the molecule. Whether conserved subdomains (CR1 and CR2) or putative phosphorylation sites (serine 388 or 743) are essential for the activity of D-raf in the embryo or involved in its positive or negative regulation was tested. These modifications of D-raf often result in decreased D-raf activity. Thus, by expressing partial loss-of-function D-raf alleles in D-raf null embryos the role D-raf plays in developing embryos could be deciphered (Radke, 2001).

Using a structure-function strategy, several modified forms of the D-raf protein were generated. The D-rafWT and D-rafK497M genes were constructed as positive and negative controls, respectively, with the D-rafWT allele a full-length copy of a D-raf cDNA. D-rafK497M lysine 497, which was shown to be critical for D-raf protein kinase activity and likely involved in ATP binding, was replaced with a methionine. The N-terminal and CR1 deletion mutation, D-rafDelta315, was likely to show a partial loss-of-function in D-raf null embryos. For the D-rafDelta445 mutation both positive (CR1) and negative (CR2) control elements were lost, and it was predicted that this form of D-raf would act in a manner similar to wild type or, on the basis of its structural similarity to oncogenic forms of Raf-1, and show a gain-of-function effect in the embryo. Of the five phosphorylation sites identified for the human Raf-1 kinase, two are conserved in the D-raf protein. Serine to alanine substitutions at these sites were generated and it has been shown that S388 (CR2) plays a negative role while S743 (CR3) is involved in the positive control of D-raf in the Tor pathway. It was predicted that the D-rafS388A and D-rafS743A proteins would show similar phenotypic consequences for developing cells in the embryo (Radke, 2001).

Using P-element-mediated transformation, Drosophila lines were generated that contained an insertion of the D-rafWT, D-rafK497M, D-rafDelta315, D-rafDelta445, D-rafS388A, or D-rafS743A gene on either the second or third chromosome. Each of these modified D-raf genes were paternally introduced into D-raf embryos lacking maternal D-raf protein. The level and stability of D-raf proteins produced by expression of each paternally inherited D-rafmodified gene was tested. In this assay 100 embryos were collected for each sample and processed for Western analysis. Since the expression of each D-rafmodified gene was under the control of the hsp70 promoter, samples were processed from non-heat-shocked or heat-shocked embryos at 5 and 10 hr of development. These D-rafmodified proteins are variably stable and in D-raf null embryos show differences in the rescue of dorsoventral cuticular defects caused by the loss of D-raf maternal and zygotic function. The degree of phenotypic rescue observed in D-raf null embryos was as follows: D-rafWT > D-rafS388A > D-rafDelta445 > D-rafS743A > D-rafDelta315 > D-rafK497M (Radke, 2001).

The accumulation of D-raf protein was assayed in D-raf embryos that had inherited the D-rafWT gene. For these embryos the accumulation of D-raf proteins after heat induction was approximately twofold greater than that found in wild-type embryos at 5 hr. At 10 hr, the level of the D-rafWT protein was unchanged. The effect of D-rafWT proteins on otd and dpp gene expression patterns was determined in D-raf embryos. As anticipated, induction of the D-rafWT gene results in 100% of the D-raf null class showing wild-type ventral otd stripe expression and a normal pattern of dpp expression. Embryonic cuticles were examined at 24 hr to assess the ability of the D-rafWT gene to promote signaling in the late-stage Egfr pathway responsible for epidermal differentiation and the final cuticular pattern. Of these D-raf null embryos that had inherited the D-rafWT gene, 99% developed cuticles indistinguishable from their D-raf torso sisters. Thus, all ectodermal signaling pathways dependent on D-raf activity could be fully restored in null embryos by expression of the D-rafWT gene (Radke, 2001).

In the phenotypic analysis, 84% of D-rafS388A expressing D-raf null embryos showed rescue of Egfr-induced otd expression in ventral cells and the distance between dpp stripes appeared normal. By the completion of embryonic development, 97% of the D-raf null embryos showed the torso phenotype, while the remaining 3% showed a composite 'imperfect torso' phenotype. In addition to showing head and tail defects associated with the torso phenotype, embryos of the 'imperfect torso' class were twisted and had denticle bands of reduced width, indicative of partial loss of signaling in ventral cells that depend on the Egfr pathway for development. Since all of the D-raf null embryos showed some phenotypic rescue by D-rafS388A, it was concluded that serine 388 is not essential for the function of D-raf in the ectoderm. Instead, it was thought likely that S388 plays a negative role in the regulation of D-raf similar to its function in Tor signaling (Radke, 2001).

For D-raf null embryos that inherited the D-rafDelta445 gene, 52% showed rescue of the Egfr-induced otd expression pattern. This was approximately one-half the percentage rescued by the D-rafWTgene, although the quantity of truncated ~38-kD D-raf protein in these embryos was equivalent to that observed for D-raf embryos expressing the D-rafWT gene at 5 hr. For the human Raf-1 protein, removal of CR1 and CR2 resulted in unregulated kinase activity. Whether the D-rafDelta445 protein acted ectopically to create a wide ventral otd stripe was tested, but all of the otd stripes were of wild-type width. When dpp mRNA patterns were analyzed in D-rafDelta445 expressing null embryos the distance between lateral stripes in the third thoracic segment at 10 hr was similar to those that had inherited the D-rafWT gene (Radke, 2001).

In the analysis of 24-hr cuticular patterns 52% of the D-rafDelta445 embryos were rescued and showed the torso phenotype. For the remaining embryos, partial rescue was observed with signaling by the D-rafDelta445 protein defective in the determination of the ventral ectoderm. Of these embryos, 18% showed the 'imperfect torso' phenotype and 30% showed the 'null with denticles' phenotype. These 'null with denticles' embryos were twisted, had faint cuticles with narrow denticle bands, and were phenotypically similar to Egfr embryos homozygous for intermediate defective alleles of Egfr. Overall, it was found that signal transmission by D-rafDelta445 was less reliable when compared with D-rafWT, although the D-rafDelta445 protein had the potential to rescue all aspects of the embryonic D-raf null phenotype (Radke, 2001).

Analysis of D-raf embryos expressing the D-rafS743A gene was somewhat complicated by the insertion of D-rafS743A on the TM2 balancer chromosome. Thus, only one-half of the D-raf null embryos fertilized by D-rafS743A transgenic males inherited the D-rafS743A gene. The amount of D-rafS743A protein that accumulated in D-raf embryos with the D-rafS743A gene was determined; the D-rafS743A protein was ~1.5-fold greater than that observed for those embryos that had inherited the D-rafWT gene. Although greater levels of this modified D-raf protein accumulated in D-raf null embryos expressing the D-rafS743A gene, otd stripe expression was not observed. Also, the distance between lateral dpp stripes in these D-rafS743A embryos was diminished when compared with wild type, but not to the degree observed for embryos expressing the D-rafDelta315 or D-rafK497M genes. Thus, the specification of ventral cell fates at the midline requires the positive regulation of the D-raf protein at serine 743 (Radke, 2001).

Accordingly, 99% of the D-raf null embryos expressing the D-rafS743A gene showed the 'imperfect torso' phenotype. To better assess the pattern deletions generated by the loss of epidermal cell fates in these D-rafS743A embryos, epidermal sensory organs that develop in ventral and lateral domains of the embryo were scored. The separation between Keilin's organs and ventral black dots on the ventral surface was measured. Also, to determine whether patterning in lateral cells was normal for these embryos the distance between ventral and dorsal black dots was recorded. When compared with wild type, D-rafS743A embryonic cuticles showed a decreased distance between Keilin's organs and ventral black dots. A decrease in the distance between ventral and dorsal black dot material was also observed. This latter finding proved very informative for it led to the hypothesis that a novel pathway, dependent upon the D-raf protein, was operating for signal transmission in cells undergoing lateral epidermal development. It appears that cell fate specification in the ventralmost ectoderm via the EGR receptor and proper development of a subpopulation of lateral cells requires an optimal level of D-raf activity that is not achieved by the D-rafS743A protein (Radke, 2001).

Rescue of epidermal patterning defects was further diminished in D-raf null embryos that expressed the D-rafDelta315 gene. Using Western analysis it was found that the D-rafDelta315 protein migrated as an ~60-kD band detected at a level equivalent to that of the 90-kD D-rafWT protein at 5 hr. Approximately 80% of this D-rafDelta315 protein was present at 10 hr. When D-raf null embryos that inherited the D-rafDelta315 gene were assayed for otd and dpp stripe expression, ventral otd expression was not observed and the distance between lateral dpp stripes was much reduced when compared with embryos expressing the D-rafWT gene. Thus, a substantial decrease in the output of the Egfr-induced signal was detected. By the completion of development, 83 (81%) of the expected 102 D-raf null embryos with D-rafDelta315 protein showed cuticles with the 'null with denticles' phenotype (Radke, 2001).

Epidermal sensory organs were scored in D-raf null embryos expressing the D-rafDelta315 gene and their relative positions noted. Significantly, an absence of Keilin's organs was recorded and a corresponding expansion in the size of ventral black dot material was observed. The distance between these enlarged ventral dots was substantially reduced when compared with wild-type embryos. A reduction in the distance between ventral and dorsal black dot sensory organs was also observed. This finding again implicates D-raf in a pathway required for the development of lateral cells. Thus, by reducing the ability of the D-raf protein to act in signaling its role in the Egfr pathway has been verified and its function in a novel pathway involved in lateral cell development has also been uncovered (Radke, 2001).

As anticipated, D-raf-dependent pathways were not rescued when D-raf null embryos expressed the kinase defective D-rafK497M gene (Radke, 2001). Thus, D-raf acts downstream of the Egfr for the specification of ventral ectodermal cell fates. D-raf also plays a second role in a novel pathway that is required for lateral cell development. In particular the D-rafS743A and D-rafDelta315 alleles generated in vitro proved useful in defining the function of D-raf in cells of the lateral epidermis. It is hypothesized that this novel pathway acts to specify cells of the lateral ectoderm subsequent to instructions received by nuclei from the dorsal maternal gene product. Thus, dorsoventral patterning in the embryo is likely dependent on the activity of three zygotic signaling pathways with Dpp that acts in dorsal cells, Egfr that directs cells in the ventral ectoderm, and a novel RTK pathway that specifies lateral cell fates (Radke, 2001).

The lateral epidermis consists of two narrow stripes of tissue on the left and right sides of the embryo extending from the anterior head to the posterior tail region. For the meta- and meso-thoracic regions this lateral tissue gives rise to epidermal cuticular structures that form between dorsal and ventral black dot sensilla. Along the circumference of each abdominal segment these two regions of lateral cuticle can be subdivided into dorsolateral and ventrolateral domains. Normally in late-stage embryos the dorsolateral region is characterized by numerous discontinuous rows of long slender hairs that have a pattern similar to that found for region b of the dorsal epidermis. These dorsolateral hairs are most similar in size and morphology to a subset of dorsal hairs, the 4° hairs. The ventrolateral domain is characterized by a segmental organization of naked cuticle alternating with two to three sparse rows of denticles similar to those found in the ventral belts although not as strongly pigmented (Radke, 2001).

Several findings have indicated that a novel pathway acts in the determination of lateral ectodermal cell fates and are consistent with a role for D-raf in this pathway. Embryos that developed in the absence of dpp and dorsal activity are lateralized. Mutations in the Drosophila dCREB-A gene are also important for defining lateral embryonic regions. In the absence of dCREB-A gene function, embryos show development of only lateral epidermal structures. Two consequences of lateral cell induction have also been identified: activation of the MAP kinase protein and expression of the msh gene encoding a homeodomain protein product. Using D-raf proteins with partial function it has been found that D-raf also participates in the development of the lateral epidermis most likely to specify cellular fates in the lateral ectoderm (Radke, 2001 and references therein).

Is there a receptor tyrosine kinase responsible for triggering the activation of the D-raf protein and MAP kinase in cells of the lateral ectoderm? In mammalian systems, mitogenic signaling by insulin in fetal rat, brown adipocyte, and primary cultures involves the activation of Ras and Raf-1 proteins. Insulin also triggers an increase in Raf-1 activity in several cell lines that expressed large numbers of insulin receptors (Radke, 2001 and references therein).

The Raf-MEK-MAP kinase cascade acts in a variety of cells to transmit RTK-generated signals during Drosophila development. The protein kinase activity of D-raf is required to elicit distinct ventral cell fates specified by the EGR receptor in early embryos. Using partial loss-of-function mutations in D-raf, cell fates normally specified by high levels of Egfr activity were lost while those that required lower receptor activity appeared normal or were expanded (Radke, 2001).

How is a graded pattern of cell types within a developmental field generated by a receptor tyrosine kinase? It has been hypothesized that the main function of the Raf-MEK-MAPK phosphorylation cascade is to amplify RTK-initiated signals. In this case, the quantity of activated Raf, MEK, and MAPK molecules is directly proportional to the number of receptor molecules activated, in the absence of feedback mechanisms. This information is then translated into position-dependent gene expression patterns that lead to morphological changes and cellular development. In this model, the quantity of activated RTK receptors defines the determined state of the cell. However, a number of studies in Drosophila reveal the existence of parallel signaling pathways emanating from a receptor during embryonic development. To extend the amplification hypothesis, the Raf-MEK-MAP kinase cascade may also act to integrate signals received from these parallel pathways and ultimately define precise transcriptional outcomes using a multistep mechanism. In mammalian cells, Raf-1 is regulated by a variety of inputs including the enzymatic function of PKC, Src, and Jnk kinases that upregulate activity. Autophosphorylation also plays a role in regulating Raf-1, as well as binding to Ras, 14-3-3, KSR, hsp90, and p50 proteins. In addition, PKA, Atk (PKB), and phosphatases have been implicated in the downregulation of Raf-1 function (Radke, 2001).

This study has addressed the consequences of eliminating key D-raf regulatory domains or specific serine residues that might act to integrate distinct signaling pathways in the Egfr pathway for ventral cell determination. In general, signal transmission was less reliable for D-raf proteins that lacked the negative regulatory site S388 (D-rafS388A) or the regulatory sequences CR1 and CR2 associated with the N-terminal one-half of the molecule (D-rafDelta445). However, both proteins showed the potential to transmit the highest level of ventral signal. This phenomenon was perhaps indicative of an important role played by the D-raf protein in the assembly of multiprotein complexes with components derived from parallel pathways. The full-length wild-type D-raf molecule, which contains several conserved motifs, may serve to bring parallel-signaling components together. Thus, the structural integrity of the D-raf protein may be important for the efficiency of complex assembly or its stability. In this model only complete and stable-signaling complexes achieve the highest level of signal output. It is speculated that in the case of D-rafS388A and more often for D-rafDelta445 proteins, complete signaling complexes were not built, leading to the phosphorylation of fewer D-MEK molecules, decreased signal output, and fewer cell fate choices specified within the Egfr developmental field (Radke, 2001).

In contrast, the Egfr signal was severely compromised when transmitted by either D-rafS743A or D-rafDelta315 proteins. The range of cell types specified by these mutant D-raf molecules was dramatically reduced from the wild type. In both cases, the establishment of cell fates that require the highest level of Egfr activity was consistently lost. Serine 743 may be important for the formation of D-raf dimers or oligomers as has been suggested for Raf-1. This type of complex may be essential for the generation of the highest level of ventral signal. In embryos that developed with D-rafDelta315 proteins, cell fates were generated that required substantially lower levels of Egfr activity. It is speculated that the wild-type D-raf protein undergoes release from negative regulation imparted by the CR2 domain via its N-terminal and CR1 sequences. In the case of the D-rafDelta315 protein, maintenance of the negative regulatory function of CR2 severely limited the ability of D-raf molecules to activate D-MEK. These results point to a multistep process in the generation of active D-raf molecules with multiple upstream factors acting in parallel. The highest level of D-raf signal was generated when all inputs were received. In the absence of one or several interactions the signaling potential of the D-raf protein was reduced, but not abolished (Radke, 2001).

Table of contents

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

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