BIOLOGICAL OVERVIEW

As a result of the Drosophila genome project, six new rhomboid-like genes have been identified, in addition to the previously characterized rhomboid (CG1004). Full-length cDNAs have been isolated from threeof the newly identified genes (Wasserman, 2000), while three have emerged only from the annotated genomesequence of Drosophila. Rhomboids belong to a large family of related proteins. Mutations have been isolated in rhomboid-3 and they correspond to one of the first described Drosophila mutations, roughoid (Strong, 1920). Rhomboid (referredto here as Rhomboid-1) and Roughoid/Rhomboid-3 act together to control cell recruitment (by triggering Egfr activation) in the developingeye. Genetic mosaics show that the pair of proteins acts only in the signal-emitting cell, not the cells that receive the signal viathe Egfr. This analysis leads to a prediction that there is a missing Egfr ligand that regulates cell death and survival in the developing eye, and a newly identified protein related to Spitz is thought to be a candidate for the missing ligand (Wasserman, 2000).

Flies with a complete deletion ofroughoid/rhomboid-3 alone are viable, althoughthe rough eye is more extreme than that of the classical ru¹ mutation, implying that ru¹ is anhypomorphic allele (a conclusion also supported by the observation thatthe eye phenotype of ru¹/Df(ru) ismore extreme than ru¹/ru¹).There is a variable loss of photoreceptors (typically one or two) inmany ommatidia of the roughoid/rhomboid-3 adulteye. However, this disruption is not sufficient to accountfor the extent of roughness seen externally: the principle cellular phenotype can be seen in the developing pupal retina -- the loss of coneand pigment cells. Examining the earliest stages ofommatidial development in larval imaginal discs from these mutantsconfirms this phenotype. Using Elav, a ubiquitous neural marker, few (if any) defects in the recruitment of photoreceptors could be seen. Consistent with this, ommatidial initiation and thedetermination of the founding R8 photoreceptor, as determined by theexpression of the atonal gene, is also normal inroughoid/rhomboid-3 mutant discs.Therefore, the loss of photoreceptors seen in adult eyes appears to becaused by later loss of the cells rather than initial recruitmentdefects. However, cone cells are dramatically under-recruited, asevidenced by the substantial loss of staining by the cone cell markerCut. The same phenotype is seen in ru¹ discsand in ru^PLLb (a deficiency mutation) discs, although the latter have a greaterloss of cone cell precursors. Using TUNEL labeling, slightlyelevated apoptotic cell death was observed in the imaginal disc as compared towild-type; however, the amount of cell death was insufficient toaccount for the observed loss of cone cell staining. Furthermore,ubiquitous expression (under the control of the GMR enhancer) of thebaculovirus p35 gene in the eye, which prevents apoptotic celldeath, did not significantly suppress theroughoid/rhomboid-3 phenotype. Therefore, it has been concluded that the cone cell deficiency results primarily from afailure of recruitment, rather than increased cell death (Wasserman, 2000).

By examining imaginal discs, the requirement for rhomboid-1roughoid/rhomboid-3 in the R8 photoreceptor can be defined more precisely. Within rhomboid-1roughoid/rhomboid-3 double mutant clones isolated cells are seem that express the neuronal marker Elav. These clones look very similar to Egfr minus clones and, as in the latter, theisolated Elav-positive cells all express the R8-specific marker, Boss. Consistent with this, the transcription factor thatspecifies R8, Atonal, is expressed within rhomboid-1roughoid/rhomboid-3 double mutant clones.In wild-type discs, Atonal first appears in all cells just ahead of themorphogenetic furrow and is gradually refined to evenly-spaced singlecells that become the R8s. In rhomboid-1roughoid/rhomboid-3 clones there are excess Atonal-positive cells and these cells are disorganized, suggesting that the refinement and/or spacing mechanismsare disrupted. In addition to the absence of non-R8 photoreceptors,there are no Cut expressing cells in the body of the double mutantclones, indicating that cone cell determination does notoccur. From these results it is concluded that the only cells to initiate differentiation in the absence of Rhomboid-1 andRoughoid/Rhomboid-3 are the R8 cells: no subsequentrecruitment occurs. Importantly, discs with clones mutant forrhomboid-1 alone are completely wild-type butthe rhomboid-1 roughoid/rhomboid-3 double mutantphenotypes closely resemble those caused by loss of the Egfr. This implies that in the eye the rhomboid-1 roughoid/rhomboid-3 pair combine to take on the role of Rhomboid-1 in other tissues; that of a positive regulatorof Egfr signaling (Wasserman, 2000).

This implication was directly tested by examining MAP kinase activationin clones lacking both Rhomboids. In wild-type imaginal discs, theactivated form of MAP kinase (as detected by an antibody specific forthe diphosphorylated form of MAP kinase) is seenin regularly-spaced clusters of cells along the morphogenetic furrow.This MAP kinase activation is abolished upon removal of the Egfr. Lossof rhomboid-1 and roughoid/rhomboid-3 together also removes MAP kinase activation completely, whereas the loss of rhomboid-1 alone does not disrupt MAP kinase activation at all. This directly demonstrates that the loss of the combinationof rhomboid-1 and roughoid/rhomboid-3disrupts Egfr activation of MAP kinase, and that the contribution ofthe ru¹ mutation to this loss is critical (Wasserman, 2000).

To understand how Rhomboid-1 and Roughoid/Rhomboid-3control Egfr signaling in the eye it is important to determine whether they act in the signal-emitting or signal-receiving cell. The Egfritself is the principle receptor of recruiting signals in theommatidium and, as such, is required in the cells being recruited. Theobservation that Rhomboid-1 and Roughoid/Rhomboid-3 areonly required in the founding R8, but that in their absence R8 forms normally without subsequent recruitment of other cells, implies thatthe proteins are not needed for reception of the signal, but insteadfor its generation. This is directly confirmed by examining geneticallymosaic ommatidia at the border of the clones in imaginal discs. Incontrast to the absence of cell recruitment in the central part ofclones, at the borders many examples of cells that are mutantfor the two Rhomboids but are nevertheless recruited normally as non-R8photoreceptors are found. This is direct proof that acell can be recruited normally even if it has no Rhomboid-1 orRoughoid/Rhomboid-3, as long as it is adjacent to awild-type cell. Similar non-autonomy is seen for cone cell recruitment: no cone cells are recruited in the center of a clone butmutant cells that are adjacent to wild-type cells can adopt a cone cellfate. This result is also confirmed when the loss of activated MAPkinase is examined closely: MAP kinase activationcan be seen in cells that are themselves mutant, when they are adjacentto wild-type cells. These results demonstrate that the rhomboid-1roughoid/rhomboid-3 combination controls thegeneration of the recruiting signal, not its reception by recruitedcells. As expected, spitz mutant clones also show the samenon-autonomy and the distance from wild-type tissue atwhich mutant cells can be recruited is a direct indication of the rangeat which Spitz can function: this is estimated to be no more than two orthree cells. The range of non-autonomy in the rhomboid-1roughoid/rhomboid-3 double mutant clones is indistinguishable, which is consistent with the idea thatRhomboid-1 and Roughoid/Rhomboid-3 control the activation of Spitz (Wasserman, 2000).

Egfr has a role inregulating cell survival in the developing eye. Intriguingly, the only known Egfr ligand to act in the eye,Spitz, does not control this survival signaling, since spitz minusclones have little excess cell death. Thisposes the question of whether the Egfr survival function is due toligand-independent, constitutive signaling by the receptor or istriggered by another as yet unknown ligand. rhomboid-1 roughoid/rhomboid-3 clones have asubstantial increase in cell death, like Egfr minusclones but distinct from spitz minus clones. Moreover, they alsohave a characteristic tapered shape (a consequence of the apoptoticloss of cells toward the posterior of the clone), again likeEgfr minus clones but not spitz minus clones. Clonesmutant for rhomboid-1 alone have no excess cell death.Therefore, loss of Rhomboid-1 and Roughoid/Rhomboid-3permits cell death, but not by virtue of controlling Spitz activation (because loss of Spitz does not induce death). In conjunction with thenon-autonomous behaviour of the Rhomboids, this is taken as a strongsuggestion that there is an unidentified Egfr ligand that controls cellsurvival in the eye (Wasserman, 2000).

The ectopic expression of rhomboid-1 activates Egfrsignaling in all tissues examined. The effects of similarly expressingroughoid/rhomboid-3 were assessed to determinewhether the redundancy between the two proteins in the eye reflects acommon molecular mechanism. Overexpression of either gene in thedeveloping eye causes severe disruptions.At the cellular level, excess cone and pigment cell recruitment is theprimary phenotype, although some excessphotoreceptors are also seen. A similar phenotype is also caused byectopic expression of a constitutive form of the Egfr or of its ligand,Spitz. In the wing, the Egfr pathway promotes theformation of veins, and ectopic expression ofeither rhomboid-1 orroughoid/rhomboid-3 again produces a similarphenotype: all cells in the wing are converted into vein cells, causingthe wing to be small, excessively pigmented and blistered. Finally the consequence of ectopic expression ofroughoid/rhomboid-3 in the anterior folliclecells of the egg was also examined. These eggs have an expansion of dorsal tissue,including the respiratory appendages, yet againcharacteristic of Egfr hyperactivity and ectopic expression ofrhomboid-1. Therefore, in threedifferent developmental contexts ectopic expression ofroughoid/rhomboid-3 leads to a specificphenotype indistinguishable from that caused by rhomboid-1 andthe ectopic activation of the Egfr pathway. Althoughrhomboid-3 function appears largely confined to the eye, theseexperiments all point to the conclusion that ectopic expression ofRoughoid/Rhomboid-3 is sufficient to activate Egfr signaling in many tissues (Wasserman, 2000).

Until now, the observation that there is no requirement forrhomboid-1 in cell recruitment in the eye has left asignificant gap in understanding of Egfr signaling in development.The eye has been one of the key model systems for analyzing the Egfr pathway and has provided countervailing evidence to the model thatRhomboid is an essential element in Spitz processing. The discoverythat Roughoid/Rhomboid-3 is an eye-specific Rhomboid, andthat the loss of both Rhomboid-1 and Roughoid/Rhomboid-3mimics the phenotype of Egfr loss, now resolves this apparentinconsistency (Wasserman, 2000).

There has been much uncertainty about how Rhomboid-1 controls Egfractivation. Recently there has been growing evidence for the idea thatit acts in the cell from which the signal emanates (perhaps bycontrolling Spitz processing). The principal evidence for this comes from experiments in which ectopic expression of rhomboid inthe embryonic midline causes lateral cells to alter their fates(Golembo,1996). The simplest explanation for these results wasthat Rhomboid controls the production of a diffusible ligand,although it has not been possible to rule out more indirect causes for thisnon-autonomy. The results in the eye imaginal discs provide direct evidence thatcells can be recruited as photoreceptors or cone cells in the absenceof either Rhomboid-1 or Roughoid/Rhomboid-3 -- as long asthey are adjacent to wild-type cells. Therefore, the evidence nowoverwhelmingly supports a model for Rhomboid function in which at leastRhomboid-1 and Roughoid/Rhomboid-3 act in thesignal-emitting cell, presumably by regulating the activation of Spitz.The molecular nature of this Spitz activation remains uncertain. It hasbeen proposed that Rhomboid-1 regulates the proteolytic release of Spitz from the cell surface, but other mechanisms are also consistent with the current evidence (Wasserman, 2000).

The discovery of Rhomboid-3 and its role in eye development allows the model of Egfr signaling in ommatidial cell recruitment to be refined further.Each ommatidium is initiated by an R8 cell, which is determined by anEgfr-independent mechanism involving the expression of the bHLHtranscription factor Atonal. The newly founded R8s then start to expressrhomboid-1 androughoid/rhomboid-3, which between them causeSpitz in the R8 to be activated, thereby recruiting neighboring cells as photoreceptors. Later in development, all photoreceptors express rhomboid-1 and roughoid/rhomboid-3, andthese then become the source of the Spitz that recruits cone cells;pigment cells are later recruited by a further iteration of the sameprocess. It is notable that rhomboid-1 mutantsalone have no phenotype, but loss ofroughoid/rhomboid-3 is sufficient to disruptcone and pigment cell determination. This presumably reflects a more substantial role for Roughoid/Rhomboid-3 than Rhomboid-1,at least in the later stages of eye development. Nevertheless, only the mutation of both can reproduce the complete absence of recruitmentcaused by loss of the Egfr, indicating that they act in cooperation at all stages of ommatidial recruitment (Wasserman, 2000).

Genetic evidence largely points to this straightforward model of cellrecruitment. However, there is a subtle distinction, detected by mosaicanalysis, between the requirement for Spitz and that for Rhomboid-1 andRoughoid/Rhomboid-3. Although the only photoreceptor toabsolutely require Spitz is the R8 -- exactly as seen for rhomboid-1roughoid/rhomboid-3 double mutants -- there is also apartial requirement for Spitz in the next two photoreceptors to berecruited, R2 and R5. No suchrequirement for rhomboid-1 androughoid/rhomboid-3 is seen. Although this may indicatethat the rhomboid-1 roughoid/rhomboid-3 pair isnot required in R2 and R5, it is also possible that this result is anartifact based on the absence of a chromosome completely null forrhomboid-1 and roughoid/rhomboid-3 (Wasserman, 2000).

The loss of the Egfr in clones in thedeveloping eye induces ectopic cell death and this cell deathoccurs at about the time that differentiation in the eye begins. Surprisingly, spitz minus clonesdo not trigger this excess apoptosis. There are two possibilities toexplain this discrepancy and they can now be resolve. Either the cellsurvival signaling by the Egfr is constitutive (that isligand-independent) or it may be controlled by a ligand other thanSpitz. The observation that, like loss of the receptor, loss ofRhomboid-1 and Roughoid/Rhomboid-3 triggers cell death,implies that cell survival signaling in the eye must be mediated byanother ligand, also controlled by Rhomboid-1 andRoughoid/Rhomboid-3. Gurken and Vein, the other knownEgfr activating ligands in Drosophila, can be ruled out basedon their phenotypes. Therefore, it is proposed that there is a novelligand, activated by Rhomboid-1 and Roughoid/Rhomboid-3, which acts to protect cells from apoptosis in the eye. Interestingly, there is also evidence that there is a 'missing'Rhomboid-controlled ligand in the wing. A candidate for this missing ligand has recently beensequenced by the Drosophila genome project; this gene (CG8056) has 49%identity with Spitz and all the hallmarks of a true Egfr ligand (J. R.Lee and M. Freeman, unpubl., cited in Wasserman, 2000). It is speculated that this Spitz-like gene is the ligand that is predicted to be missing in the eye and wing (Wasserman, 2000).

The molecular mechanism of the Rhomboid-like protein family remainsenigmatic. The only function deduced for Drosophila Rhomboid-1 and Roughoid/Rhomboid-3 is the activation ofSpitz. But there is no experimental evidence, nor anything in thesequence of the proteins, to hint that they are proteases that catalyse the cleavage. Moreover, the enzymes that release similar ligands havebeen discovered in mammals and they are a recognizable family of ADAMmetalloproteases;homologs exist in Drosophila, although it is not yet known which, if any, are responsible for Spitz cleavage (Wasserman, 2000).

There are some clues about the molecular functions of Rhomboidsavailable from the conservation of different regions of the proteins.Notably, the transmembrane domains are the most highly similar,particularly domains 2, 3, and 4. Within these domains there are someinvariant charged residues that suggest the presence of a hydrophilicpocket that might constitute an enzymatic active site or a channel. Itis striking that by comparison, the hydrophilic amino-terminal domainsshow little if any conservation, suggesting that they do not form partof the core function of the Rhomboid family. Indeed, Rhomboid-1 thathas had its amino-terminal artificially removed, retains its ability toactivate Egfr signaling (M. Sohrmann and M. Freeman, unpubl., cited in Wasserman, 2000). AllRhomboid-like proteins detected in the database do have ahydrophilic amino terminus, so it is imagined that this may be an importantregulatory region. In this regard, it is interesting that Rhomboid-4 of Drosophila, as well as human Rhomboid-like protein, havesequences that fit the consensus for Ca²⁺-binding EF hands (Wasserman, 2000).

PROTEIN STRUCTURE

Amino Acids - 235

Structural Domains

Three sequences from the Berkeley Drosophila Genome Projectdatabase were identified that exhibit high similarity to rhomboid. These three were named rhomboid-2, (CG12083) rhomboid-3 (CG1214)and rhomboid-4. Both rhomboid-2 and rhomboid-3 are cytologicallylocated very close to the rhomboid-1 (rhomboid) gene on the third chromosome, whereas rhomboid-4 (CG1697) has been mapped to position 10C on the Xchromosome by polytene chromosome in situ hybridization. Full length cDNAs were isolated for each of the new genes and theirsequences were compared. The most highly conserved region spans the seven transmembrane domains; the hydrophilic aminoterminus is strikingly divergent. This pattern of similarity is very like that between Drosophila rhomboid-1 and its recently identifiedmammalian homologs (Pascall, 1998), and suggests that the transmembrane domains provide a core function for Rhomboid-likeproteins. A phylogenetic tree derived from these sequences indicates that rhomboid-3 is most closely related to rhomboid-1,followed by rhomboid-2; rhomboid-4 is the least related. The amino-terminal region of Rhomboid-4 contains two tandemly arrangedEF-hand motifs that are putative calcium-binding domains. There are three further rhomboid-like genes predicted (rhomboid-5, rhomboid-6, andrhomboid-7). Rhomboid-5 (CG5364) is located at 31C; Rhomboid-6 (CG17212) at 33C, and Rhomboid-7 (CG8972) at 48E. The mostconserved region encompasses the transmembrane domains, while diverging in the hydrophilic amino termini. This striking conservationof rhomboid-like genes suggests that the primordial function of these proteins is a fundamental cellular process. The restriction ofDrosophila Rhomboid-1 and Rhomboid-3 function to Egfr signaling presumably represents a specialization of this original function (Wasserman, 2000).

date revised: 27 July 2000

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