roughoid/rhomboid-3 mRNA expression is first detectable at the morphogenetic furrow, where ommatidial development begins. The transcript is present all the way to the posterior of the eye imaginal disc, implying that roughoid/rhomboid-3 is expressed throughout the period when photoreceptors and cone cells are recruited. The transcript appears to be restricted to the developing eye: no tissue-specific expression was observed in other imaginal discs or in the embryo by RNA in situ hybridization. Furthermore, tissue-specific expression of rhomboid-2 or rhomboid-4 could not be detected in either the embryo or imaginal discs (Wasserman, 2000).

EGF receptor signalling protects smooth-cuticle cells from apoptosis during Drosophila ventral epidermis development: Rhomboid functions in cell survival

Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. This phenotype has been studied and a new function has been discovered for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning (Urban, 2004).

The denticle belt fusion phenotype is one of the distinguishing features of the spitz group genes (Mayer, 1988), yet its developmental basis has remained mysterious, since no function has been known for EGFR signalling in the smooth cuticle, which is the affected tissue. Analysis of this phenotype has revealed its cause and uncovered a previously unrecognized function for EGFR signalling in Drosophila epidermal development. Spitz is the primary EGFR ligand in epidermal patterning, and is activated by proteolysis in three rows of rhomboid-1-expressing cells in the future denticle region. High EGFR signalling is required for cells to adopt the denticle fate, and other signalling pathways are used to elaborate the different denticle morphologies. The Wingless signal emanates from one posterior row of each parasegment and spreads anteriorly, suppressing the denticle fate and thus allowing cells to secrete a smooth cuticle. These future smooth-cuticle cells also require signalling through the EGFR for viability, and its absence results in apoptosis of future smooth-cuticle cells and thus denticle belt fusions. This survival signalling is mediated by low-level stimulation of the EGFR by cooperation between the ligands Vein and Spitz, which is activated by Rhomboid-1, Rhomboid-3 and Star (Urban, 2004).

The ventral epidermis is patterned in multiple stages during development, with cell fate specification occurring late, through antagonism between EGFR and Wingless signalling around stages 12-14. Direct phenotypic analysis indicates that EGFR signalling is required for smooth-cuticle cell survival during these fate specification stages and not earlier or later: epidermal cell apoptosis is greatly elevated in mutant embryos at stages 12-14, and the fusion phenotype first becomes apparent around stage 15 as curvature of Engrailed stripes (Urban, 2004).

This direct phenotypic analysis is also supported by several independent genetic observations. EGFR signalling is not required for survival in future smooth-cuticle cells early, when the ventrolateral fates are being specified (stage 10/11) since removing Rhomboid-1 expression at only this stage using the single-minded mutation never results in denticle belt fusions (Mayer, 1988). Defects at this early stage also cause ventral narrowing in spitz group genes (Mayer, 1988), and since rhomboid-3 does not enhance this phenotype, this suggests that rhomboid-3 cooperates with rhomboid-1 only later in development. Vein acts independently of spitz group genes to suppress denticle belt fusions, and this cannot occur at stage 10/11 since at this early stage Vein expression is dependent on EGFR signalling through a positive feedback loop. Finally, the fusion phenotype itself suggests that it forms late since denticle cells are being pulled into smooth cuticle regions and, as such, their denticle fate must have already been determined and cannot be altered by receiving signals from these smooth domains (Urban, 2004).

Thus, two thresholds with different outcomes exist for EGFR signalling in patterning the ventral epidermis. The level of EGFR signalling that a cell receives is presumably dependent on its distance from the Spitz-processing cells; activated MAPK staining indicates that these rows of cells receive high levels of EGFR signalling. High levels of EGFR signalling are required to induce the denticle fate, while lower levels that reach smooth-cuticle cells are sufficient to suppress apoptosis. All ventral epidermal cells therefore require EGFR signalling, but the exact level, together with antagonism of shavenbaby transcription by Wingless signalling, determines the biological outcome. Importantly, these functions may be separate, since Wingless signalling is known to antagonize shavenbaby transcription to repress the denticle fate, but may not repress EGFR signalling itself in smooth-cuticle cells: activated MAPK staining suggests that some smooth-cuticle cells in the midline may also receive higher levels of EGFR signalling (Urban, 2004).

These results indicate that cells require EGFR signalling for their survival only when they are starting to differentiate. A similar pattern was also observed in the developing eye imaginal disc where removing the EGFR resulted in cell death only once the morphogenetic furrow had passed. These observations raise the intriguing possibility that establishing a requirement for survival signals may be inherent in the differentiation program itself, perhaps for protecting against developmental errors. However, the observation that the requirement for survival signalling is restricted to the central region of the ventral epidermis implies that either this requirement is not ubiquitous, or that another signal is also involved (Urban, 2004).

Pointed is an Ets domain-containing transcription factor that is responsible for transducing most known instances of EGFR signalling. Although it was previously clear that pointed mutant embryos rarely display denticle belt fusions (Mayer, 1988), analysis of a more recent null allele that removes both P1 and P2 transcripts demonstrates that even complete loss of pointed leads only to a very low frequency of denticle belt fusions. This is also consistent with the milder effects of pointed clones in the developing eye, and in particular the late onset of their apoptosis. These observations raise the possibility that EGFR-mediated survival signalling in general occurs primarily at a non-transcriptional level. Consistent with this model, EGFR signalling has been shown to reduce Hid protein stability, thus directly inhibiting apoptosis (Urban, 2004).

Rhomboid exists as a seven-member family in Drosophila, and at least four of these members are intramembrane serine proteases that can cleave all Drosophila membrane-tethered EGFR ligands and specifically activate EGFR signalling in vivo. Although the precise role of the rhomboid protease family in EGFR signalling and in other biological contexts has been unclear, mutations have now been isolated for both Rhomboid-2 and -3. Genetic analysis with null alleles has revealed that both act as tissue-specific activators of EGFR signalling much like Rhomboid-1. Rhomboid-2 is the only rhomboid known to be expressed early in gametogenesis, and is involved in sending EGFR signals from the germline to the soma to guide its encapsidation by somatic cells. In this context, Rhomboid-2 appears to act alone. Rhomboid-3 displays strong expression in the developing eye imaginal disc, and is allelic to roughoid, one of the first Drosophila mutants described. Rhomboid-3 is the dominant rhomboid protease during eye development, but does not act alone: Rhomboid-3 cooperates with Rhomboid-1 in the developing eye (Urban, 2004).

Despite the power of these genetic approaches, it should be noted that rhomboid-1, -2 and -3 exist as a gene cluster on chromosome 3L and, as such, combined mutations are difficult to generate by recombination. Analysis of epidermal patterning using RNAi to overcome this limitation is the first implication of a rhomboid homolog function in embryogenesis. Interestingly, the rhomboid involved is Rhomboid-3, the rhomboid that was previously thought to be eye-specific. However, unlike in the developing eye where Rhomboid-3 has the dominant role, and removing Rhomboid-1 by itself has no effect, the exact opposite is true in embryogenesis: Rhomboid-1 is the main protease in epidermal patterning while removing Rhomboid-3 alone does not result in detectable defects. This analysis suggests that different rhomboid proteases function predominantly to activate EGFR signalling in distinct tissues, but often act cooperatively or with a degree of redundancy (Urban, 2004).

The requirement for high levels of signalling for fate specification and lower levels for viability in developing tissues may not be limited to the EGFR pathway. Intriguingly, analysis of cell death in wingless mutant embryos suggests that a reciprocal signalling function may also be required to maintain cell viability in denticle regions of the ventral epidermis: in conditions of reduced Wingless signalling, specifically during the stage of epidermal fate specification (but not earlier), cells corresponding to two denticle rows were observed to undergo apoptosis. Therefore, as with EGFR signalling, high levels of Wingless signalling induce the smooth-cuticle cell fate, while lower levels may be required for survival of a subset of denticle cells. Thus, the Wingless and EGFR signalling pathways may act antagonistically in specifying cell fate, while having complementary and reciprocal functions in maintaining cell viability in the developing epidermis of Drosophila. These survival functions may be conserved since EGFR signalling also has multiple roles in mammalian epidermal development, while some mammalian epidermal tumors are also specifically dependent on EGFR signalling for cell survival. Wnt signalling has also been linked to maintaining cell viability in certain developmental contexts (Urban, 2004).

Effects of Mutation or Deletion

To understand the specific function of each of the Drosophila rhomboid genes, as well as the underlying core function of these proteins, mutations in rhomboid genes were sought. rhomboid-3 is located distal to rhomboid-1 on the third chromosome, in a region that includes the roughoid (ru) mutation (Strong 1920). Since a genetic interaction between roughoid and rhomboid-1 has been found, coupled with the approximate genetic colocalization of rhomboid-3 and roughoid, it was asked whether roughoid is in fact a mutation in rhomboid-3. The only allele of roughoid currently available, ru1, is a spontaneous mutation first reported in 1920 (Strong, 1920). A P-transposon-based mutagenesis was carried out to isolate new alleles, using as a starting stock a P-element that is inserted ~6-kb proximal to rhomboid-3. The screen allowed the isolation of either new P insertions, or of deletions flanking the P-element, as a result of P-induced recombination (Wasserman, 2000).

Ten mutations were identified that failed to complement the ru1 allele; the mutational events that caused five of them were studied in detail. A molecular analysis of these mutations has revealed that all the new roughoid alleles are deletions that remove DNA distal to the starting P-element and extend at least as far as the rhomboid-3 gene; they all remove the rhomboid-3 coding sequence. In all cases, the original P-element (l(3)j6B4) remains at the proximal end of the deletion and the proximal flanking sequence is unaffected. The new mutant stocks still only have a single P-element in them (detected by Southern blot). Excision of this P-element fails to revert the rough eye phenotype, suggesting that the phenotype is generated by the genomic deletions, and not the P-element insertion itself. These are all hallmarks of deletions caused by P-element-induced male recombination, which it is presumed was responsible for producing the nested set of distal deletions from the original P-element. The deletions that produced the new roughoid alleles fall into two classes: one viable with rough eyes, and the other lethal. Molecular mapping of the deletions shows that the viable alleles remove only rhomboid-3, whereas the lethal alleles extend to, and disrupt, the next most distal known gene, a genetically uncharacterized tyrosine phosphatase (Ptp61F). All of the lethal alleles (i.e., those that remove rhomboid-3 and Ptp61F) are viable in trans to ru1 and have the characteristic rough eye (Wasserman, 2000).

Phenotypic analysis focused on two deletions (rho-3PLLb and rho-3PLJc) that break in the first intron of rhomboid-3 and thus remove the whole rhomboid-3 coding sequence but nothing more distally. It can be inferred from these two alleles that removal of rhomboid-3 alone causes a phenotype similar (although more extreme) to ru1, and that loss of rhomboid-3 fails to complement ru1. On the basis of the analysis with the gene prediction programs Genewise and Genie, as well as the annotated Drosophila genome sequence, there is no predicted gene between the starting P-element and rhomboid-3. In summary, the new alleles of roughoid represent deletions of rhomboid-3; deletion of rhomboid-3 alone is sufficient to cause the roughoid phenotype: no other mutagenic events have been detected in the new roughoid alleles. Therefore, it is concluded that roughoid is an allele of rhomboid-3 (Wasserman, 2000).

In an attempt to define a role for rhomboid-1 in the developing eye, mutant clones of several different rhomboid-1 alleles were made. Null mutations cause no defects in cell recruitment, leading to the conclusion that Rhomboid-1 is not required in this process. Indeed, in clones generated using the Minute technique, entire rhomboid-1 minus eyes were found to be phenotypically wild-type. In an apparently contradictory result, one EMS-induced allele, rho-17M43, was found to cause a complete failure of cell recruitment -- exactly the phenotype that had initially been predicted for rhomboid-1. Although the molecular lesion in rho-17M43 is not known, it behaves genetically like known rhomboid nulls in other tissues and has been extensively used in previous work. One distinction between rho-17M43 and the other alleles examined is that it was induced on a multiply-marked chromosome, and it still carries the ru1 mutation that is often present on such chromosomes. It has since been discovered that roughoid is a mutation in rhomboid-3. The description of rhomboid-1 roughoid/rhomboid-3 double mutants is therefore based on the phenotype of ru1 rho-17M43. Due to the very close proximity of the two genes, it is difficult to recombine ru1 with other rhomboid-1 alleles; instead new rhomboid-1 alleles have been induced on a ru1 chromosome and they confirm the interaction seen with rho-17M43 (Wasserman, 2000).

Clones of cells doubly mutant for rhomboid-1 and roughoid/rhomboid-3 do not survive into adult eyes, but rather leave a visible scar, at the edge of which there are genetically mosaic ommatidia that comprise a mixture of wild-type and mutant cells. By examining mosaic ommatidia that have formed normally, it can be concuded that only the R8 photoreceptor, the founding cell of each ommatidium, requires rhomboid-1 and roughoid/rhomboid-3 for normal photoreceptor recruitment to occur. Since neither gene alone is required for normal photoreceptor recruitment, this requirement for the pair of Rhomboids in R8 represents the only need for either gene in the formation of photoreceptors. However, note that this mosaic analysis technique cannot address which cells must express the pair of Rhomboids for normal cone cell development (Wasserman, 2000).

It is proposed that Roughoid/Rhomboid-3 is an important activator of the Egfr in eye development. A clear prediction of this proposal is that mutations in the gene will interact with mutations in known components of this pathway. Genetic interaction tests confirm this prediction. Null alleles of roughoid/rhomboid-3 interact dominantly with mutations in the Egfr itself (ElpB1); spitz (spiscp1 and spiscp2); Star (S218), and overexpressed argos (GMR-argos); the hypomorph ru1 also interacts in some of these tests but less strongly than the null mutants. Whereas rhomboid-1 mutations alone do not interact, the combination of loss of rhomboid-1 and roughoid/rhomboid-3 (ru1 rho-17M43) interacts most strongly of all. The model places the Rhomboids genetically upstream of the Egfr. Consistent with this, it has been found that overexpression of either rhomboid-1 or rhomboid-3 (both of which give strong phenotypes on their own) is unable to rescue the phenotype caused by overexpression of a dominant negative form of the Egfr (Wasserman, 2000).

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

A distal-to-proximal gradient of EGFR activity predicts a source of ligand(s) at the presumptive tip. Potential ligands are the TGF-alpha family members Spitz and Keren, and the neuregulin, Vn; the former require activation by the membrane protein Rhomboid (Rho), or the homolog Roughoid (Ru). Both vn and rho are expressed in the center of the leg disc in early third instars. Genetic studies show that they are redundant so that loss of either gene alone has no effect on tarsus development, but loss of both together along with ru, which shows partial redundancy with rho even though no expression can be detected, has marked effects on leg patterning and growth. Large ru rho vn triple mutant clones can result in truncations of the tarsus, although these are never as extreme as in Egfrts mutants, possibly because of the difficulty of removing all ligand-expressing cells at the center of early leg discs using this technique, or because the ru mutant used is not null. Wild-type tissue located at the tip of adult legs always correlates with rescue of tarsal development. In addition, misexpression of a secreted form of Spitz results in non-autonomous activation of al. Verification of high levels of EGFR signalling in the distal leg is revealed by expression of sprouty in this location; this is upregulated in many tissues by EGFR signalling (Campbell, 2002).

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90°, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Epidermal growth factor receptor (Egfr) signalling is required for rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. It is proposed that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, there is an error-correction mechanism operating during pupal stages to reposition inappropriately oriented ommatidia. These results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling (Brown, 2003).

The Egfr ligand Keren was misexpressed in developing photoreceptors and cone cells under the control of sev-Gal4. Surprisingly, this caused a disruption in the orientation of ommatidia relative to WT, a phenotype not previously associated with excess Egfr signalling. In the WT adult eye, all ommatidia are oriented at 90° relative to the equator. By contrast, when Keren is misexpressed, many ommatidia are abnormally oriented, with some ommatidia having rotated more than 90° and some less than 90°. In general, excess Egfr signalling leads to over-recruitment of cells in the eye, but photoreceptor recruitment is not affected when Keren is expressed at these levels. However, analysis of the pupal retina shows that Keren misexpression causes over-recruitment of cone cells, consistent with it acting through the Egfr. Previous work has shown that recruitment of cone cells is more sensitive than photoreceptors to Egfr overactivation; these results support this view, and also suggest that rotation is more sensitive than photoreceptor recruitment to perturbation of Egfr signalling (Brown, 2003).

Further examination of the adult phenotype indicates that it is rotation specifically that is disrupted on overexpressing Keren; the chirality (i.e. the correct specification of R3 and R4) of the ommatidia remains unaffected. This distinguishes the UAS-Keren phenotype from disruption of PCP components, which can cause both rotational and chiral defects (Brown, 2003).

Is Egfr activity normally required for correct rotation? Several conditions were examined that decrease Egfr signalling, including a haploinsufficient Star allele (which has slight rotational defects), rho3/roughoid mutants, and expression of dominant-negative Egfr under the control of heatshock HS-Gal4. In all these cases, rotational defects are clearly seen in correctly specified ommatidia. In order to quantify and compare the rotational defects further, the rotation angles of approximately 600 ommatidia each were measured in WT, UAS-keren and ru1 eyes. Strikingly, defects caused by too little or too much Egfr activity are very similar -- ommatidia are over- or under-rotated, although in both cases there is a bias towards rotation angles of greater than 90°. The similarity of the rotational defects caused by increasing and decreasing pathway activity is reminiscent of some PCP mutations (Brown, 2003).

The rotational phenotypes caused by perturbation of Egfr signalling are very similar to the published phenotype of the roulette mutation, one of the few mutations reported to specifically disrupt rotation and not chirality. Interestingly, roulette turns out to be allelic to argos. The roulette mutation is now referred to as argosrlt (Brown, 2003).

There are four ligands that activate the Drosophila Egfr: Spitz, Gurken and Keren (which resemble mammalian TGFalpha), and Vein, a neuregulin-like molecule. Spitz is thought to mediate most of the Egfr functions in eye development, although spitz clones do not phenocopy Egfr clones in all respects. Specifically, spitz clones do not show defects in cell survival or ommatidial spacing, which are seen in Egfr loss-of-function clones. spitz hypomorphic eyes were examined to determine whether these show rotational defects. Under-recruited ommatidia are very common in the spiscp1 hypomorph, indicating that Egfr activity is substantially impaired – to beneath the threshold for photoreceptor recruitment. Despite this, very few misrotated ommatidia are seen. In comparison, ru1 eyes show only minor recruitment defects, indicating a less dramatic reduction of Egfr activity than spiscp1. ru1 eyes, however, show severe rotational defects. These data suggest that Spitz is not essential for normal rotation. They do not, however, rule out the possibility that Spitz acts redundantly with another ligand. To test this, a genetic interaction between Star and a spitz hypomorph was tested. As expected, heterozygosity for spitz enhances the recruitment defects in the S/+ eye. A significant enhancement of rotational defects is observed, implying that Spitz does function in ommatidial orientation. Together, these results suggest that Spitz acts redundantly with another Egfr ligand to control rotation. The fact that loss of Rho3/ru, a protease that activates Egfr ligands, results in rotational defects, whereas spitz mutants do not, implies the involvement of another cleaved ligand. Gurken is restricted to the germline. By elimination, it is therefore tentatively concluded that Keren also acts in the Egfr-dependent regulation of ommatidial rotation. Note, however, that keren expression is too low to detect by in situ hybridisation in any tissue so it is not possible to tell whether keren is transcribed appropriately. Confirmation of this hypothesis awaits the identification of a keren mutant (Brown, 2003).

These results demonstrate that Egfr signalling is required for the maintenance through eye development of the correct orientation of ommatidia. It was speculated that rotation may rely at least partly on the adhesive properties of the cells. In an initial attempt to examine this hypothesis, genetic interactions between components of the Egfr pathway and various adhesion molecules were sought. A Star heterozygote, in which Egfr signalling is slightly reduced, was used as a background in which to look for interactions, because this phenotype is very weak, allowing any enhancement of rotational defects to be easily recognized. Halving the dose of alpha-laminin (wing blister) and the integrin ß subunit (myospheroid) does not modify the Star/+ phenotype. In contrast, alleles of E-cadherin (shotgun) shows a significant interaction with Star, with many more misrotated ommatidia. Under the strongest condition, there is also an enhancement of the rare misrecruitment defects seen in Star/+ eyes, but the enhancement of the rotational defect is independent of this by two criteria. First, the rotational defects were only measured in correctly specified ommatidia; and second, the weaker alleles of shotgun affected rotation without enhancing recruitment. On the basis of these results, it is concluded that the control of rotation by Egfr signalling is linked to cadherin-based adhesion (Brown, 2003).

A model that might account for these results is proposed that suggests that the role of Egfr signalling is to establish a 'locking' mechanism that ensures that ommatidia remain in their final orientation. Such a mechanism might be necessary to protect the ommatidia against positional disruption during later events in eye development. Signalling would therefore be required during or at the end of normal rotation in order to set in place this hypothetical 'lock', although defects might not arise until significantly later than this, when processes occur that would cause ommatidia to reorient in the absence of such a lock (Brown, 2003).

The Epidermal growth factor receptor (Egfr) pathway controls cell fate decisions throughout phylogeny. Typically, binding of secreted ligands to Egfr on the cell surface initiates a well-described cascade of events that ultimately invokes transcriptional changes in the nucleus. In contrast, the mechanisms by which autocrine effects are regulated in the ligand-producing cell are unclear. In the Drosophila eye, Egfr signaling, induced by the Spitz ligand, is required for differentiation of all photoreceptors except for R8, the primary source of Spitz. R8 differentiation is instead under the control of the transcription factor Senseless. High levels of Egfr activation are incompatible with R8 differentiation; the mechanism by which Egfr signaling is actively prevented in R8 is described. Specifically, Senseless does not affect cytoplasmic transduction of Egfr activation, but does block nuclear transduction of Egfr activation through transcriptional repression of pointed, which encodes the nuclear effector of the pathway. Thus, Senseless promotes normal R8 differentiation by preventing the effects of autocrine stimulation by Spitz. An analogous relationship exists between Senseless and Egfr pathway orthologs in T-lymphocytes, suggesting that this mode of repression of Egfr signaling is conserved (Frankfort, 2004).

In this analysis of sens function in R8 differentiation, it was found that the extra R2/R5 cell that develops from the pre-R8 in sens mutants expresses Ro, which is normally expressed in R2/R5 but not R8. Ro is expressed downstream of Egfr pathway activation, and both ro function and high levels of Egfr pathway activation are required for R2/R5 differentiation. Since the pre-R8 cell consistently expresses Ro and differentiates as an R2/R5 cell in sens mutants, it was hypothesized that this transformation occurs as a consequence of high levels of Egfr activation in the pre-R8 cell (Frankfort, 2004).

This hypothesis was tested by simultaneously removing sens function and blocking Egfr activation in the developing Drosophila eye. Egfr activation was blocked by removing function of both rhomboid-1 (rho-1) and rhomboid-3 (rho-3; FlyBase: roughoid, ru). Loss of both rho-1 and rho-3 function prevents processing of secreted Egfr ligands, including Spi, and results in the loss of all ERK (MAP kinase) activation. Furthermore, loss of rho-1 and rho-3 phenocopies Egfr loss-of-function in that only R8 cells differentiate. Loss of sens function results in pre-R8 differentiation as a founder R2/R5 cell which is sufficient to recruit a reduced number of photoreceptors. However, the absence of rho-1, rho-3 and sens together causes total photoreceptor loss, except for a few photoreceptors near the clonal boundary that are rescued non-autonomously by neighboring wild-type cells that produce and process Spi appropriately. A similar phenotype is detected in tissue mutant for both spi and sens. This loss of photoreceptors seen in rho-1 rho-3 sens and spi sens mutants is not due to cell death because apoptosis was prevented in these experiments by expression of GMR-p35. Furthermore, pre-R8 selection still occurs in both rho-1 rho-3 and rho-1 rho-3 sens mutant tissue, suggesting that a potential founding photoreceptor is present. Therefore, these results are interpreted to mean that, in the absence of sens function, pre-R8 differentiation as a founder R2/R5 photoreceptor requires activation of the Egfr signaling pathway via the Spi ligand. In other words, in sens mutants, the pre-R8 switches from a Spi/Egfr-independent R8 differentiation pathway to a Spi/Egfr-dependent R2/R5 differentiation pathway (Frankfort, 2004).

Rhomboid 3 orchestrates Slit-independent repulsion of tracheal branches at the CNS midline

EGF-receptor ligands act as chemoattractants for migrating epithelial cells during organogenesis and wound healing. Evidence suggests that Rhomboid 3/EGF signalling, which originates from the midline of the Drosophila ventral nerve cord, repels tracheal ganglionic branches and prevents them from crossing the midline. rho3 acts independently from the main midline repellent Slit, and originates from a different sub-population of midline cells: the VUM neurons. Expression of dominant-negative Egfr or Ras induces midline crosses, whereas activation of the Egfr or Ras in the leading cell of the ganglionic branch can induce premature turns away from the midline. This suggests that the level of Egfr intracellular signalling, rather than the asymmetric activation of the receptor on the cell surface, is an important determinant in ganglionic branch repulsion. It is proposed that Egfr activation provides a necessary switch for the interpretation of a yet unknown repellent function of the midline (Gallio, 2004).

The morphogenesis of the embryonic tracheal network depends on the charted migration of ~2000 epithelial cells deriving from 20 epidermal invaginations. These cells undergo three successive rounds of branching to generate a tubular network that extends along stereotyped paths towards specific target tissues. The last branching event produces thin, unicellular terminal branches that associate with distinct organs. The ventral nerve cord (VNC) is invaded by 20 ganglionic branches (GBs), which sprout from the lateral trunk of the trachea. GB migration towards and inside the CNS is highly stereotyped. Each GB initially tracks along the inter-segmental nerve and toward the CNS. GB1, the leading cell of the ganglionic branch, enters the nerve cord and changes substrate to track along the segmental nerve, proceeding ventrally on top of the longitudinal fascicles and towards the CNS midline. Finally, after reaching the midline, GB1 takes a sharp turn and migrates dorsally through the dorsoventral channel and then turns posteriorly on the dorsal side of the VNC. At the end of embryogenesis, GB1 will have trailed a remarkable 50 ┬Ám inside the CNS. Genetic analysis has uncovered a number of factors that are necessary for this fixed migratory path: the FGF homolog Branchless is required to guide the GBs towards the CNS and to induce them to enter it, in part by inducing the expression of the nuclear protein Adrift. Once inside the CNS, Slit, the main repulsive cue for axons at the midline, becomes a key guiding cue for the migrating GBs. Slit controls several, distinct aspects of ganglionic branch pathfinding into the CNS: it is first required to attract GBs toward the CNS, an effect mediated by Slit's receptor Robo2, and then to prevent GBs from crossing the midline once they reach it: this is also mediated by Robo (Gallio, 2004 and references therein).

A collection of 2640 P-element insertions was screened for mutants affecting the pathfinding of the ganglionic branch (GB) into the CNS. One of the recovered mutants, named inga (from ingen återvändo, meaning 'no turning back' in Swedish) was characterised by a specific midline-cross phenotype: at stage 16.3-4, upon approaching the CNS midline, a significant number of inga GBs failed to turn posteriorly and dorsally at the midline and crossed to the other side, or remained lingering on it. No other defect was detected in the tracheal system of inga embryos. Sequence analysis of the genomic region surrounding the transposon in inga mutants showed that the P-element was inserted in roughoid/rho3, and all available ru/rho3 alleles as well as inga/Df(3L)Ar14-8 embryos (a chromosomal deficiency removing the 61-62 region). These showed the same tracheal phenotype as inga. Therefore, it was concluded that inga is an allele of rho3 and subsequent analysis focused on a previously characterised null allele (Gallio, 2004),

The essential components of the Egfr signalling pathway were associated with ventral nerve cord development soon after their discovery. rhomboid, spitz and pointed mutants were originally identified for their effect on the ventral ectodermal region. Egfr signalling also plays a central role in the development of the VNC midline, where it is first required for cell differentiation and positioning of midline glia and later for their survival during the late stages of embryogenesis (Gallio, 2004)

The expression of rho3 in VUMs and its function in GB1 guidance away from the midline identifies a new role for Egfr signalling in the VNC. Unlike rho1, rho3 mutants have a normal VNC pattern in which longitudinal connectives and glial populations appear normal, suggesting that rho3 is specifically required for GB1 guidance. Expression of dominant-negative forms of the EGF receptor or Ras in GB1 phenocopies the rho3 guidance phenotype. In addition, overactivation of Egfr signalling in the trachea is sufficient to redirect GB1 and induce early turn phenotypes. Finally, rho3 is required in parallel to slit, the main repulsive cue deriving from midline glia. Taken together, these results suggest that rho3 mutant GB1s are misrouted because of reduced levels of Egfr/Ras signalling in GB1 cells, rather than to indirect, subtle defects of midline patterning or signalling capacity in rho3 mutants. This led to the proposal of a simple model in which Rho3 activates one or more Egfr ligands secreted by the midline cells. Reception of this signal by migrating GBs is mediated by Egfr and Ras, and promotes turning away from the midline (Gallio, 2004)

Three Drosophila Egfr ligands are activated by Rhomboid proteases: Gurken (which is only present in oocytes), Spitz and Keren, the latter expressed in embryos below the detection level of in situ hybridisation or antibody staining. Thus, the ligand activated by Rho3 to guide GB1 migration is very likely Spitz; it is expressed and is functional at the midline, but a contribution by Keren cannot be firmly excluded (Gallio, 2004)

The mammalian EGF receptors regulate migration in a variety of contexts, but in all known examples they appear to promote responses to chemoattractants. They do so by directly affecting cytoskeletal organisation, mainly through the PI3K, PKC or PLC pathways. The proper activation of the fly Egfr is also necessary for the migration of border cells toward the source of Gurken in the dorsal part of the oocyte. During this migration Egfr activation is coordinated with the activation of the fly PDGF/VEGF receptor homologue and requires the conserved adaptor protein Mbc (Dock 180/CED-5). Mbc provides a link to activated Rac and actin re-arrangements, which leads to the stereotyped attraction of the border cells towards the oocyte. It is, however, unclear whether Egfr provides the necessary spatial information for border cells during their pathfinding, or if it is required for the interpretation of positional cues provided by Pdgf/Vegfr or other receptors (Gallio, 2004 and references therein)

Egfr signalling is mediated by a number of downstream effectors in different cell types. In order to determine which one is used in GB1 pathfinding, a panel of mutants and dominant-negative constructs of known downstream effectors were analysed for their effect on GB migration. myoblast city (mbc) is a conserved adaptor necessary for the chemo-attractant function of Gurken during border cell migration in the ovary. mbc alleles had no defects in GB pathfinding. Since mbc has negligible maternal contribution and is not readily detected in tracheal tissues, it is concluded that it is unlikely to have a role in Egfr-mediated GB repulsion from the midline. Two additional effectors were tested that have been implicated in Egfr-elicited migratory responses in other systems: PLCgamma and PI3K. The fly PLCgamma is encoded by the small wing (sl) locus. small wing embryos had extra terminal sprouts emanating from the primary tracheal branches but show no specific defects in GB migration inside the VNC. Deltap60 is a deletion variant of the adaptor p60, which has dominant-negative effects on PI3K activity in vivo and in vitro. SRF-Gal4 driven expression of Deltap60 resulted in a stalling phenotype of 19% of the GBs but not midline crosses. This may reflect a requirement of PI3-K in the early extension of the GBs toward the midline, which is also impaired by the expression of the dominant-negative form of Egfr in GB1 (Gallio, 2004).

The activation of Ras is a necessary step in many of the cellular responses induced by Egfr signalling in Drosophila. It leads to the activation of Raf, and culminates with activation of the Ets-transcription factor Pointed and the nuclear export of Yan, another Ets protein that antagonises Pnt in the activation of target genes. SRF-Gal4-directed expression of a dominant-negative form of Ras results in stalled branches inside or outside the VNC. Importantly, a significant number of GBs was grossly misrouted (8%) or crossed the midline (4%) suggesting that Ras is required in the GB1 cells for their turn away from the midline. The large proportion of arrests in cell migration observed in these experiments might reflect a broader requirement for these common effectors in tracheal cell migration and sprouting (Gallio, 2004).

To analyse whether Egfr mediated repulsion of GB1 from the midline requires Raf or downstream pathway components, a dominant-negative form of Raf and an activated form of Yan were expressed under the control of SRF-Gal4. These constructs caused many of the branches to stall or misroute but in neither case could any branches that crossed the VNC midline be found. As an example, expression of the activated Yan construct stalled the migration of 45% of the GBs, and misrouted an additional 7%, but not a single midline cross was observed (Gallio, 2004).

In summary, activation of Ras appears to be required for repulsion of GB1 from the midline, whereas the remaining components of the pathway are required for tracheal cell extension inside the VNC but not for the decision to cross the midline barrier (Gallio, 2004).

There are substantial differences in the ways by which Egfr controls migration in GB1 and in border cells. This analysis indicates that Egfr signalling is not a chemotactic cue for tracheal pathfinding -- rather, it reveals a surprising role in mediating repulsion from the signalling source. In addition, mbc mutants do not show any midline crossing phenotypes that would resemble the phenotypes of rho3 or the ones generated by inactivation of the receptor. Furthermore, the increase of signalling levels in GB1, either by the expression of Rho1, activated receptor or activated Ras, results in a significant phenotype opposite that of the rho3 mutants: such treatments induced GBs to turn early before reaching the midline. This suggests that at the appropriate distance from the midline, Egfr activation becomes a switch to initiate the turn of GB1 away from it. Hence, an experimental increase of signalling levels can shift the crucial switch further away from the midline, while decreased signalling causes midline crosses. In essence, migrating GBs use Egfr activation to efficiently compute their relative distance from the midline, fine-tuning their response to the repulsive and attractive cues originating from it (Gallio, 2004)

Migration in general, and axonal pathfinding at the midline in particular, is known to rely on a number of guidance signals, at times redundant ones. The major midline repulsive signal for GB1 is Slit, yet a genetic test shows that rho3 acts in parallel to Slit. It is hypothesized that Egfr works in an analogous manner by activating a second, yet undiscovered, signalling system for GB repulsion. Such a guidance cue may be specific for GB1 migration, since axonal fascicles are not affected in rho3 mutants. Alternatively, the activation of Egfr in GB1 provides an epithelial specific regulation of a common repulsive signal used by both axons and GB1 (Gallio, 2004)

What could this repulsive signal be? Likely candidates fall in the short list of conserved signals repelling axons and non-neural cells in different systems: Netrins, Semaphorins and Ephrins. Netrins are involved in the repulsion of motor axons in both vertebrates and invertebrates and both Drosophila Netrins are expressed at the CNS midline, where they mediate attraction of commissural axons. Semaphorins and Ephrins are also capable of repelling axons and non-neural cells in different contexts, and they therefore represent possible guiding cues for GBs. Intriguingly, each family uses receptor tyrosine kinases as receptors (in the case of Ephrin) or co-receptors (in the case of Semaphorins). Most of these signals are bi-functional, they can elicit both attractive and repulsive responses on the receiving cells depending on context. Egfr activation in GB1 may lead to the post-translational modifications that activate a repellent receptor or inactivate an attractant one and may represent a general 'switch' mechanism for changing the orientation of cell migration depending on the strength of RTK signalling (Gallio, 2004)

Affecting Rhomboid-3 function causes a dilated heart in adult Drosophila

Drosophila is a well recognized model of several human diseases, and recent investigations have demonstrated that Drosophila can be used as a model of human heart failure. Previously, Optical coherence tomography (OCT) can be used to rapidly examine the cardiac function in adult, awake flies. This technique provides images that are similar to echocardiography in humans, and therefore it is postulated that this approach could be combined with the vast resources that are available in the fly community to identify new mutants that have abnormal heart function, a hallmark of certain cardiovascular diseases. Using OCT to examine the cardiac function in adult Drosophila from a set of molecularly-defined genomic deficiencies from the DrosDel and Exelixis collections, an abnormally enlarged cardiac chamber was detected in a series of deficiency mutants spanning the rhomboid 3 locus. Rhomboid 3 is a member of a highly conserved family of intramembrane serine proteases and processes Spitz, an epidermal growth factor (EGF)-like ligand. Using multiple approaches based on the examination of deficiency stocks, a series of mutants in the rhomboid-Spitz-EGF receptor pathway, and cardiac-specific transgenic rescue or dominant-negative repression of EGFR, it was demonstrate that rhomboid 3 mediated activation of the EGF receptor pathway is necessary for proper adult cardiac function. The importance of EGF receptor signaling in the adult Drosophila heart underscores the concept that evolutionarily conserved signaling mechanisms are required to maintain normal myocardial function. Interestingly, prior work showing the inhibition of ErbB2, a member of the EGF receptor family, in transgenic knock-out mice or individuals that received herceptin chemotherapy is associated with the development of dilated cardiomyopathy. These results, in conjunction with the demonstration that altered ErbB2 signaling underlies certain forms of mammalian cardiomyopathy, suggest that an evolutionarily conserved signaling mechanism may be necessary to maintain post-developmental cardiac function (Yu, 2010; Full text of article).


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roughoid: Biological Overview | Developmental Biology | Effects of Mutation

date revised: 5 August 2011

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