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 three of the newly identified genes (Wasserman, 2000), while three have emerged only from the annotated genome sequence 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 (referred to here as Rhomboid-1) and Roughoid/Rhomboid-3 act together to control cell recruitment (by triggering Egfr activation) in the developing eye. Genetic mosaics show that the pair of proteins acts only in the signal-emitting cell, not the cells that receive the signal via the 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 of roughoid/rhomboid-3 alone are viable, although the rough eye is more extreme than that of the classical ru¹ mutation, implying that ru¹ is an hypomorphic allele (a conclusion also supported by the observation that the eye phenotype of ru¹/Df(ru) is more extreme than ru¹/ru¹). There is a variable loss of photoreceptors (typically one or two) in many ommatidia of the roughoid/rhomboid-3 adult eye. However, this disruption is not sufficient to account for the extent of roughness seen externally: the principle cellular phenotype can be seen in the developing pupal retina -- the loss of cone and pigment cells. Examining the earliest stages of ommatidial development in larval imaginal discs from these mutants confirms 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 the determination of the founding R8 photoreceptor, as determined by the expression of the atonal gene, is also normal in roughoid/rhomboid-3 mutant discs. Therefore, the loss of photoreceptors seen in adult eyes appears to be caused by later loss of the cells rather than initial recruitment defects. However, cone cells are dramatically under-recruited, as evidenced by the substantial loss of staining by the cone cell marker Cut. The same phenotype is seen in ru¹ discs and in ru^PLLb (a deficiency mutation) discs, although the latter have a greater loss of cone cell precursors. Using TUNEL labeling, slightly elevated apoptotic cell death was observed in the imaginal disc as compared to wild-type; however, the amount of cell death was insufficient to account for the observed loss of cone cell staining. Furthermore, ubiquitous expression (under the control of the GMR enhancer) of the baculovirus p35 gene in the eye, which prevents apoptotic cell death, did not significantly suppress the roughoid/rhomboid-3 phenotype. Therefore, it has been concluded that the cone cell deficiency results primarily from a failure of recruitment, rather than increased cell death (Wasserman, 2000).

By examining imaginal discs, the requirement for rhomboid-1 roughoid/rhomboid-3 in the R8 photoreceptor can be defined more precisely. Within rhomboid-1 roughoid/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, the isolated Elav-positive cells all express the R8-specific marker, Boss. Consistent with this, the transcription factor that specifies R8, Atonal, is expressed within rhomboid-1 roughoid/rhomboid-3 double mutant clones. In wild-type discs, Atonal first appears in all cells just ahead of the morphogenetic furrow and is gradually refined to evenly-spaced single cells that become the R8s. In rhomboid-1 roughoid/rhomboid-3 clones there are excess Atonal-positive cells and these cells are disorganized, suggesting that the refinement and/or spacing mechanisms are disrupted. In addition to the absence of non-R8 photoreceptors, there are no Cut expressing cells in the body of the double mutant clones, indicating that cone cell determination does not occur. From these results it is concluded that the only cells to initiate differentiation in the absence of Rhomboid-1 and Roughoid/Rhomboid-3 are the R8 cells: no subsequent recruitment occurs. Importantly, discs with clones mutant for rhomboid-1 alone are completely wild-type but the rhomboid-1 roughoid/rhomboid-3 double mutant phenotypes 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 regulator of Egfr signaling (Wasserman, 2000).

This implication was directly tested by examining MAP kinase activation in clones lacking both Rhomboids. In wild-type imaginal discs, the activated form of MAP kinase (as detected by an antibody specific for the diphosphorylated form of MAP kinase) is seen in regularly-spaced clusters of cells along the morphogenetic furrow. This MAP kinase activation is abolished upon removal of the Egfr. Loss of 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 combination of rhomboid-1 and roughoid/rhomboid-3 disrupts Egfr activation of MAP kinase, and that the contribution of the ru¹ mutation to this loss is critical (Wasserman, 2000).

To understand how Rhomboid-1 and Roughoid/Rhomboid-3 control Egfr signaling in the eye it is important to determine whether they act in the signal-emitting or signal-receiving cell. The Egfr itself is the principle receptor of recruiting signals in the ommatidium and, as such, is required in the cells being recruited. The observation that Rhomboid-1 and Roughoid/Rhomboid-3 are only required in the founding R8, but that in their absence R8 forms normally without subsequent recruitment of other cells, implies that the proteins are not needed for reception of the signal, but instead for its generation. This is directly confirmed by examining genetically mosaic ommatidia at the border of the clones in imaginal discs. In contrast to the absence of cell recruitment in the central part of clones, at the borders many examples of cells that are mutant for the two Rhomboids but are nevertheless recruited normally as non-R8 photoreceptors are found. This is direct proof that a cell can be recruited normally even if it has no Rhomboid-1 or Roughoid/Rhomboid-3, as long as it is adjacent to a wild-type cell. Similar non-autonomy is seen for cone cell recruitment: no cone cells are recruited in the center of a clone but mutant cells that are adjacent to wild-type cells can adopt a cone cell fate. This result is also confirmed when the loss of activated MAP kinase is examined closely: MAP kinase activation can be seen in cells that are themselves mutant, when they are adjacent to wild-type cells. These results demonstrate that the rhomboid-1 roughoid/rhomboid-3 combination controls the generation of the recruiting signal, not its reception by recruited cells. As expected, spitz mutant clones also show the same non-autonomy and the distance from wild-type tissue at which mutant cells can be recruited is a direct indication of the range at which Spitz can function: this is estimated to be no more than two or three cells. The range of non-autonomy in the rhomboid-1 roughoid/rhomboid-3 double mutant clones is indistinguishable, which is consistent with the idea that Rhomboid-1 and Roughoid/Rhomboid-3 control the activation of Spitz (Wasserman, 2000).

Egfr has a role in regulating 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 minus clones have little excess cell death. This poses the question of whether the Egfr survival function is due to ligand-independent, constitutive signaling by the receptor or is triggered by another as yet unknown ligand. rhomboid-1 roughoid/rhomboid-3 clones have a substantial increase in cell death, like Egfr minus clones but distinct from spitz minus clones. Moreover, they also have a characteristic tapered shape (a consequence of the apoptotic loss of cells toward the posterior of the clone), again like Egfr minus clones but not spitz minus clones. Clones mutant for rhomboid-1 alone have no excess cell death. Therefore, loss of Rhomboid-1 and Roughoid/Rhomboid-3 permits cell death, but not by virtue of controlling Spitz activation (because loss of Spitz does not induce death). In conjunction with the non-autonomous behaviour of the Rhomboids, this is taken as a strong suggestion that there is an unidentified Egfr ligand that controls cell survival in the eye (Wasserman, 2000).

The ectopic expression of rhomboid-1 activates Egfr signaling in all tissues examined. The effects of similarly expressing roughoid/rhomboid-3 were assessed to determine whether the redundancy between the two proteins in the eye reflects a common molecular mechanism. Overexpression of either gene in the developing eye causes severe disruptions. At the cellular level, excess cone and pigment cell recruitment is the primary phenotype, although some excess photoreceptors are also seen. A similar phenotype is also caused by ectopic expression of a constitutive form of the Egfr or of its ligand, Spitz. In the wing, the Egfr pathway promotes the formation of veins, and ectopic expression of either rhomboid-1 or roughoid/rhomboid-3 again produces a similar phenotype: all cells in the wing are converted into vein cells, causing the wing to be small, excessively pigmented and blistered. Finally the consequence of ectopic expression of roughoid/rhomboid-3 in the anterior follicle cells of the egg was also examined. These eggs have an expansion of dorsal tissue, including the respiratory appendages, yet again characteristic of Egfr hyperactivity and ectopic expression of rhomboid-1. Therefore, in three different developmental contexts ectopic expression of roughoid/rhomboid-3 leads to a specific phenotype indistinguishable from that caused by rhomboid-1 and the ectopic activation of the Egfr pathway. Although rhomboid-3 function appears largely confined to the eye, these experiments all point to the conclusion that ectopic expression of Roughoid/Rhomboid-3 is sufficient to activate Egfr signaling in many tissues (Wasserman, 2000).

Until now, the observation that there is no requirement for rhomboid-1 in cell recruitment in the eye has left a significant 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 that Rhomboid is an essential element in Spitz processing. The discovery that Roughoid/Rhomboid-3 is an eye-specific Rhomboid, and that the loss of both Rhomboid-1 and Roughoid/Rhomboid-3 mimics the phenotype of Egfr loss, now resolves this apparent inconsistency (Wasserman, 2000).

There has been much uncertainty about how Rhomboid-1 controls Egfr activation. Recently there has been growing evidence for the idea that it acts in the cell from which the signal emanates (perhaps by controlling Spitz processing). The principal evidence for this comes from experiments in which ectopic expression of rhomboid in the embryonic midline causes lateral cells to alter their fates (Golembo,1996). The simplest explanation for these results was that Rhomboid controls the production of a diffusible ligand, although it has not been possible to rule out more indirect causes for this non-autonomy. The results in the eye imaginal discs provide direct evidence that cells can be recruited as photoreceptors or cone cells in the absence of either Rhomboid-1 or Roughoid/Rhomboid-3 -- as long as they are adjacent to wild-type cells. Therefore, the evidence now overwhelmingly supports a model for Rhomboid function in which at least Rhomboid-1 and Roughoid/Rhomboid-3 act in the signal-emitting cell, presumably by regulating the activation of Spitz. The molecular nature of this Spitz activation remains uncertain. It has been 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 an Egfr-independent mechanism involving the expression of the bHLH transcription factor Atonal. The newly founded R8s then start to express rhomboid-1 and roughoid/rhomboid-3, which between them cause Spitz in the R8 to be activated, thereby recruiting neighboring cells as photoreceptors. Later in development, all photoreceptors express rhomboid-1 and roughoid/rhomboid-3, and these then become the source of the Spitz that recruits cone cells; pigment cells are later recruited by a further iteration of the same process. It is notable that rhomboid-1 mutants alone have no phenotype, but loss of roughoid/rhomboid-3 is sufficient to disrupt cone 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 recruitment caused 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 cell recruitment. However, there is a subtle distinction, detected by mosaic analysis, between the requirement for Spitz and that for Rhomboid-1 and Roughoid/Rhomboid-3. Although the only photoreceptor to absolutely require Spitz is the R8 -- exactly as seen for rhomboid-1 roughoid/rhomboid-3 double mutants -- there is also a partial requirement for Spitz in the next two photoreceptors to be recruited, R2 and R5. No such requirement for rhomboid-1 and roughoid/rhomboid-3 is seen. Although this may indicate that the rhomboid-1 roughoid/rhomboid-3 pair is not required in R2 and R5, it is also possible that this result is an artifact based on the absence of a chromosome completely null for rhomboid-1 and roughoid/rhomboid-3 (Wasserman, 2000).

The loss of the Egfr in clones in the developing eye induces ectopic cell death and this cell death occurs at about the time that differentiation in the eye begins. Surprisingly, spitz minus clones do not trigger this excess apoptosis. There are two possibilities to explain this discrepancy and they can now be resolve. Either the cell survival signaling by the Egfr is constitutive (that is ligand-independent) or it may be controlled by a ligand other than Spitz. The observation that, like loss of the receptor, loss of Rhomboid-1 and Roughoid/Rhomboid-3 triggers cell death, implies that cell survival signaling in the eye must be mediated by another ligand, also controlled by Rhomboid-1 and Roughoid/Rhomboid-3. Gurken and Vein, the other known Egfr activating ligands in Drosophila, can be ruled out based on their phenotypes. Therefore, it is proposed that there is a novel ligand, 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 been sequenced 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 remains enigmatic. The only function deduced for Drosophila Rhomboid-1 and Roughoid/Rhomboid-3 is the activation of Spitz. But there is no experimental evidence, nor anything in the sequence of the proteins, to hint that they are proteases that catalyse the cleavage. Moreover, the enzymes that release similar ligands have been discovered in mammals and they are a recognizable family of ADAM metalloproteases; 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 Rhomboids available 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 some invariant charged residues that suggest the presence of a hydrophilic pocket that might constitute an enzymatic active site or a channel. It is striking that by comparison, the hydrophilic amino-terminal domains show little if any conservation, suggesting that they do not form part of the core function of the Rhomboid family. Indeed, Rhomboid-1 that has had its amino-terminal artificially removed, retains its ability to activate Egfr signaling (M. Sohrmann and M. Freeman, unpubl., cited in Wasserman, 2000). All Rhomboid-like proteins detected in the database do have a hydrophilic amino terminus, so it is imagined that this may be an important regulatory region. In this regard, it is interesting that Rhomboid-4 of Drosophila, as well as human Rhomboid-like protein, have sequences 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 Project database 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 cytologically located very close to the rhomboid-1 (rhomboid) gene on the third chromosome, whereas rhomboid-4 (CG1697) has been mapped to position 10C on the X chromosome by polytene chromosome in situ hybridization. Full length cDNAs were isolated for each of the new genes and their sequences were compared. The most highly conserved region spans the seven transmembrane domains; the hydrophilic amino terminus is strikingly divergent. This pattern of similarity is very like that between Drosophila rhomboid-1 and its recently identified mammalian homologs (Pascall, 1998), and suggests that the transmembrane domains provide a core function for Rhomboid-like proteins. 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 arranged EF-hand motifs that are putative calcium-binding domains. There are three further rhomboid-like genes predicted (rhomboid-5, rhomboid-6, and rhomboid-7). Rhomboid-5 (CG5364) is located at 31C; Rhomboid-6 (CG17212) at 33C, and Rhomboid-7 (CG8972) at 48E. The most conserved region encompasses the transmembrane domains, while diverging in the hydrophilic amino termini. This striking conservation of rhomboid-like genes suggests that the primordial function of these proteins is a fundamental cellular process. The restriction of Drosophila 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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