rhomboid

Effects of Mutation or Deletion

Table of contents

Rhomboid and eye morphogenesis

Regulation of Drosophila EGF receptor (Egfr) activity plays a central role in propagating the evenly spaced array of ommatidia across the developing Drosophila retina. Egfr activity is essential for establishing the first ommatidial cell fate, the R8 photoreceptor neuron. In turn, R8s appear to signal through Rhomboid and Vein to create a patterned array of ‘proneural clusters’ that contain high levels of phosphorylated ERKA and the bHLH protein Atonal. Secretion by the proneural clusters of Argos represses Egfr activity in less mature regions to create a new pattern of R8s. Propagation of this process anteriorly results in a retina with a precise array of maturing ommatidia (Spencer, 1998).

Rhomboid is a seven membrane-spanning protein that enhances Egfr signaling and activation of ERKA. It can activate Egfr signaling several cell diameters from the source of its expression, apparently by regulating release or activity of the DER ligand Spitz. An antibody specific for Rhomboid shows it to be expressed in cells near the posterior edge of each proneural cluster; a Rhomboid enhancer trap line confirms that expression begins in the 1- to 3-cell R8 equivalence group, based on the positioning of the group within the larger ‘proneural cluster’ and the apical position of its nuclei. Expression then quickly resolves to a single cell that can be unambiguously identified as R8. The previously demonstrated ability of Rhomboid to activate Egfr signaling at a distance suggests that cells of the R8 equivalence group could use Rhomboid to set the pattern of Egfr/Dras1 activation across the proneural cluster. To test this possibility, hs-rhomboid flies were used to express Rhomboid throughout the MF. The result was similar to the effect of expressing Dras1^Val12: within 30 minutes of the initiation of ectopic Rhomboid expression, an unpatterned stripe of phosphorylated dpERKA emerges throughout the MF. This suggests that, indeed, expression of Rhomboid is sufficient to activate ras pathway signaling within the MF. The ‘rebound’ effect seen with ectopic Dras1^Val12 is also observed with ectopic Rhomboid, but with a more rapid time course. 1-2 hours after initiating ectopic Rhomboid expression, all detectable dpERKA as well as Atonal expression in the proneural groups is lost; this is observed even if Rhomboid is expressed continuously during this period. In addition, the upregulation of Argos expression observed with ectopic activation of Dras1^Val12 is also observed with ectopic expression of Rhomboid. Unlike experiments with Dras1^Val12, only a minor expansion of Atonal is observed with Rhomboid overexpression, presumably due to the brevity of ras pathway activation and its rapid subsequent down-regulation; Ras^Val12 is able to produce activation for longer periods presumably because it acts intracellularly and downstream of Argos inhibition (Spencer, 1998).

If Rhomboid signaling alone were responsible for directing Egfr activation within the MF, one would expect loss of Rhomboid function to result in a loss of ERKA phosphorylation, Atonal expression and R8 specification. To test this hypothesis, Rhomboid activity was blocked in two ways: by creating patches of mutant rho- homozygous tissue and by expressing an antisense construct. Loss of Rhomboid results in a diminution, but not complete loss, of phosphorylated MAP kinase within the MF. Consistent with this observation, proneural clusters retain high levels of Atonal expression, and Boss expression is normal. These results are consistent with data indicating that loss of the Rhomboid target Spitz has little effect on R8 specification. Similar conclusions can be drawn when Rhomboid is eliminated through expression of a rhomboid antisense construct. Ubiquitous expression of rhomboid antisense eliminates detectable Rhomboid protein. Down-regulation of Rhomboid for 90 minutes results in transient loss of Atonal expression and dpERKA in proneural clusters. However, these losses are short-lived: even when antisense Rhomboid is expressed continuously, Atonal expression and dpERKA return within 2-3 hours; R8 specification, as assessed by Boss expression, remains unaffected. Therefore Rhomboid, as with Spitz, is not sufficient to account for Egfr-mediated induction of the R8 fate. Together these results suggest that another ligand for Egfr may be present in the MF, and that this ligand may function redundantly with Rhomboid to activate ras pathway signaling, Atonal expression and R8 specification (Spencer, 1998).

Vein is a Neuregulin ortholog postulated to bind to and activate Egfr. Consistent with this view, removal of a single copy of vein in a DER^Elp mutant background strongly enhances the rough eye phenotype observed with DER^Elp /+ alone. Vein mRNA is present at high levels throughout the anterior of second instar eye discs where Egfr is thought to play a role in cell proliferation. By the third larval instar, however, vein is restricted in the MF to single cells within the R8 equivalence group. Thus, at least one cell of the R8 equivalence group contains two potential activators of Egfr: Vein and Rhomboid. To assess the role of Vein in R8 formation, early clonal patches homozygous for a vein null mutation were created. Few such patches are observed, although commonly observed are ‘twin spots’ (groups of cells containing two copies of the GFP marker and homozygous wild type for Vein, which are formed when mitotic recombination occurs). This suggests that Vein may be required early for cell proliferation or survival, similar to the requirement previously observed for Egfr. Within the small mutant patches that do survive, Boss expression is normal; thus loss of Vein alone, as with loss of Rhomboid, does not prevent R8 formation. These results suggest that neither Rhomboid nor Vein alone is essential for R8 differentiation. This is similar to what has been observed in the embryonic CNS, where neuroblast formation requires Egfr activity, but is only strongly affected if both rhomboid and vein activity are removed together. To determine if Rhomboid and Vein also act in parallel to specify R8 in the retina, rho-;vn- double mutant clonal patches were created by mitotic recombination. Patches were created later in second and third instar larvae to circumvent the requirement for Vein in early cell survival, and many of the resulting clonal patches (and their corresponding ‘twin spots’) contained only 4-8 cells. R8 specification is never observed in the interior of these patches, although R8 cells are able to form along the periphery. In addition, often the pattern of ommatidia surrounding and anterior to the patch is altered. In rare rho-;vn- patches that cross the MF, Atonal expression in the proneural clusters also appears to be reduced; these large clones do not distinguish whether this loss is due to a direct requirement for rho vn function in proneural clusters or is a secondary consequence of a loss of more posterior, differentiated R8s. These experiments suggest that Rhomboid/Vein-mediated Egfr activation has two roles: specification of the R8 fate, and setting the pattern of proneural clusters (Spencer, 1998).

The observation that rho-;vn- mutant clones produce disturbances in the spacing of more anterior ommatidia is reminiscent of defects observed in ommatidia surrounding Egfr minus clones and suggests that the R8 neuron in one ommatidium might influence the positioning of R8s in neighboring and anterior ommatidia. By what mechanism might this influence arise? Above are presented experiments indicating that Egfr/Dras1 (through Rhomboid and presumably Vein) can activate expression of the secreted protein Argos. Therefore, the potential for Argos to direct the pattern of emerging R8s through repression of Egfr was examined. Argos is a secreted factor that can act several cell diameters from its source. It acts as a negative regulator of the Egfr pathway in vivo and can prevent autophosphorylation and activation of Egfr in tissue culture cells, leading to the suggestion that Argos directly binds Egfr. Evidence for the presence of such an Egfr repressor in the MF is provided by a chimeric Egfr protein. l-DER is a constitutively activated chimeric receptor in which the extracellular domain of Egfr has been replaced by the l-repressor dimerization domain. As described above, activation of Egfr through Dras1^Val12 or Rhomboid results in an eventual ‘rebound’ loss of dpERKA and Atonal. By contrast, ectopic expression of l-DER leads to elevation of Atonal expression, which persists for at least 3 hours, even though (as with ectopic Rhomboid and Dras1^Val12) Argos expression is also elevated in this time frame. This result suggests that the rapid ‘rebound’ effect observed with Rhomboid requires a normal Egfr extracellular domain, and supports the view that it is mediated through a repressive ligand such as Argos. Previous work in the embryo has found an upregulation of Argos transcription in response to Egfr signaling. Consistent with this observation, the highest levels of Argos expression in the MF are found in the regions of highest Egfr activity, the proneural clusters. Lower levels of the protein are observed between and anterior to these clusters, presumably due to diffusion from the proneural clusters into the surrounding tissue. Argos overexpression in the MF results in elimination of Egfr activity (as measured by ERKA phosphorylation) and Atonal expression in the proneural clusters. Overexpression of Argos eliminates expression of Rhomboid and Vein: the factors that localize Egfr activity to the cell destined to become R8. A 90 minute heat-shock leading to overexpression of Argos eliminates Rhomboid protein from cells in the MF. This is consistent with findings that down-regulation of the transcription factor CF2, a negative regulator of Rhomboid transcription, is induced by Egfr signaling. Ectopic expression of Argos eliminates most or all Vein mRNA from cells in the MF. Thus, Argos-mediated repression of Egfr pathway activity may normally contribute to the pattern of Rhomboid and Vein expression necessary for correct R8 specification. To determine if Argos is necessary for setting the normal pattern of R8s, Boss expression was examined in the hypomorphic, partial loss-of-function mutant argos^styP1. Homozygous escapers of this line have rough eyes, due in part to the formation of ectopic ommatidia. Consistent with this, Boss-staining reveals that the pattern of R8 specification in these animals is disturbed: the spacing between R8s is variable and, most tellingly, R8s form aberrantly in positions between the normal ommatidial rows. These ectopic R8s are found in every eye disc of this genotype examined. This suggests that Argos produced by proneural clusters may normally diffuse anteriorly to repress Egfr activity (and Rhomboid and Vein expression), as well as the formation of R8s directly anterior to the cluster. In this model, R8s in the next row of ommatidia will be set at positions farthest from the site of Argos release, giving rise to the ‘out-of-register’ pattern of R8s found in wild type animals. Argos expression, in turn, is controlled by Rhomboid and Vein expressed in R8, indicating that each R8 has a role in patterning succeeding rows. It should be noted, however, that the disruption of ommatidial pattern observed when argos function is reduced is not very severe, and suggests that one or more additional factors are likely to contribute to the regulation of Rhomboid and Vein transcription (Spencer, 1998).

These results suggest a model for the patterning of ommatidia within the retina. It is proposed that patterning and R8 specification is set as cells respond regionally to regulation of Egfr activity. Beginning at the anterior edge of the MF, Egfr expression is upregulated and is expressed at levels that may be high enough to allow for low-level spontaneous activity. These results indicate that within the MF some cells become competent to respond to Egfr/Dras1 signaling by differentiating as R8 photoreceptors; the nature of this change in competence is not yet understood but may involve delayed expression or activation of a novel factor. Once competent, these cells respond to Egfr signaling by establishing a row of R8 equivalence groups. Cells of this group express Rhomboid and Vein, a required step in maintaining the R8 fate. Once the R8 equivalence group is established, other factors including Notch signaling and Rough are required to select a single R8 from the group. In addition to their role in R8 differentiation, the production of Vein and Rhomboid/Spitz in the proneural clusters suggests that these diffusible factors may play a role in patterning. Based on the evidence it is proposed that R8’s release of Vein and Spitz (via Rhomboid) activates Egfr in surrounding cells. This local activation of Egfr has two effects: upregulation of Atonal and upregulation of Argos. Upregulation of Argos, in turn, blocks expression of Rhomboid and Vein in other cells within and directly anterior to the proneural group, thereby creating an ‘R8 exclusion zone’. It is proposed that creation of these exclusion zones is necessary to prevent ectopic R8s. As R8 competence progresses anteriorly to cells beyond the R8 exclusion zone, new R8 equivalence groups would be permitted to form in the niches between the exclusion zones. This localized Argos signaling should result in the arrays of R8s in neighboring rows being formed ‘out-of-register’ to each other, and this is indeed the case. In addition, loss of Argos should result in the emergence of ectopic ommatidia, and this has been observed as well. Therefore, the spacing between ommatidia and their overall pattern appears to depend on the number of cell diameters across which Argos normally diffuses. An analogous role for Argos in embryonic ectoderm and subsequent steps of ommatidial maturation have been proposed. It has been estimated that Argos can exert its effects up to five cell diameters from its source; neighboring proneural clusters, representing two sources of Argos, are typically separated by less than eight cell diameters (Spencer, 1998 and references).

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 has been identified as a candidate for the missing ligand (Wasserman, 2000).

By examining imaginal discs, the requirement for rhomboid-1 roughoid/rhomboid-3 in the R8 photoreceptor can be defined. 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).

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-1^7M43, 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-1^7M43 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-1^7M43 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-1^7M43. 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-1^7M43 (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 (Elp^B1); spitz (spi^scp1 and spi^scp2); 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-1^7M43) 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).

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).

Genetic link between Cabeza, a Drosophila homologue of fused-in-sarcoma (FUS), and the EGFR signaling pathway

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila Cabeza (Caz) (Caz) is a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror, suppresses the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescues the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila (Shimamura, 2014).

This study found that Caz knockdown in eye imaginal discs induces a rough eye phenotype associated with apoptosis, abnormal differentiation of cone cells and pigment cells, and defects in ommatidia rotation in pupal retinae. However, apoptosis and differentiation of photoreceptor cells were not affected in larval eye imaginal discs expressing Caz dsRNA. Why did Caz knockdown in eye imaginal discs affect pupal retinae but not third instar larval eye discs? In situ hybridization and immunohistochemistrical analyses demonstrated that Caz mRNA and protein are enriched in the brain and CNS during embryogenesis, and Caz protein was detected in the nuclei of several larval tissues and in imaginal discs. However, the expression level of Caz is higher in adult eyes than in larval eye discs (Flybase). Thus, it is possible that Caz plays a more important role in eye development in the pupal stage (Shimamura, 2014).

The observation that the rough eye phenotype of Caz knockdown flies was significantly suppressed by co-expression of P35 and that apoptotic cells detected by immunostaining with anti-cleaved Caspase-3 antibody were significantly increased in pupal retinae of flies expressing Caz dsRNA suggests that induction of apoptosis at least partially contributes to the rough eye phenotype. It is reported that the number of dying cells increases dramatically if interactions between cells are disrupted, for instance upon cell ablation. Therefore, one possible explanation is that Caz knockdown disrupts interactions between cells in pupal retinae, as evidenced with anti-Cut immunostaining, that results in induction of apoptosis. In addition, it is well known that apoptosis is induced by JNK or p38 signaling. It is also reported that persistent activation of the JNK or p38 signaling pathways mediates neuronal apoptosis in ALS, and that TDP-43 is related to JNK signaling. Thus, another possible explanation is that Caz knockdown induces JNK or p38 signaling, resulting in increase of apoptosis in pupal retinae (Shimamura, 2014).

This study found a genetic interaction between Caz and Rhomboid, a rate-limiting component of the EGFR signaling pathway. Appropriate levels of EGFR signaling are required for cone cell-fate and ommatidial rotation. Knockdown of Caz in eye imaginal discs and pupal retinae induced abnormal differentiation of cone cells and defects in ommatidia rotation that eventually resulted in the rough eye phenotype in adults. The rhomboid-1 mutant rescued the fusion of cone cells and mutations of rhomboid-3 and mirror significantly suppressed the rough eye phenotype of Caz knockdown flies. In contrast, mutations of EGFR did not suppress the rough eye phenotype induced by knockdown of Caz. These apparently contradictory results might be explained as follows. Once activated, the signaling cascade could be amplified progressively, so that only a half reduction of some components of pathway such as EGFR may not be sufficient to suppress the effects of over-activation of the initiator such as Rhomboid. In any event, the present study suggests that Caz negatively regulates EGFR signaling. Since the expression level of Caz is much higher in adult eyes than larval eye discs, negative regulation of EGFR signaling by Caz may play a role in controlling EGFR signaling less reactive to oxidative stress during adulthood. It should be noted that a hallmark of ALS is chronic neuronal exposure to oxidative stress and inflammation (Shimamura, 2014).

In summary, this study has shown that knockdown of Caz in the Drosophila retina induces a rough eye phenotype associated with increased apoptosis, abnormal differentiation of cone cells and pigment cells, and defects in ommatidia rotation. This study provides the first definitive evidence that Caz plays an important role in regulation of the EGFR signaling pathway. It should be noted that the neurodegeneration occurring in ALS can be accounted for deviation from strict control of MAPK signaling. Thus, the Caz knockdown flies used in the present study should provide a useful tool for elucidating functions of FUS and pathological mechanisms of associated ALS (Shimamura, 2014).

Rhomboid in the brain and optic lobe

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

A large number of neural and glial cell cell types differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and its signal array is referred to as the 'proneural wave', since it is the marker of the EGFR/Ras signaling front. This study shows that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. It was also shown that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system (Kawamori, 2011).

The Fat/Hippo pathway has been known as a tumor suppressor pathway. This study and in the report of Reddy (2010), it was shown that the loss of Fat/Hippo signaling causes a delay of NB differentiation in the optic lobe. In contrast, dachs;ft double mutation, which is expected to stabilize the Fat/Hippo pathway, causes an advance of NB differentiation. This led to the question of how the Fat/Hippo pathway controls NB differentiation (Kawamori, 2011).

It has been reported that EGFR/Ras signaling is necessary and sufficient for NB induction, and its transduction is the driving force of the progress of the proneural wave. EGFR/Ras signaling sweeps the NE field through the gradual activation of Ras and its downstream EGF secretion by Rho. It was reasoned that this signal transduction is the target of the Fat/Hippo pathway in the control of NB differentiation. Indeed, ectopic expression of the EGFR/Ras signaling components Ras^V12 and rho was sufficient to induce NBs in the ft mutant background (Kawamori, 2011).

Which step of this cycling process does Fat/Hippo pathway mutation affect? Based on ectopic expression experiments, the Fat/Hippo pathway lies upstream of Ras and Rho, and it is expected to control the process from EGF transmission to Ras activation. The phenotypic difference produced by Ras^V12 and rho overexpression should be noted. When rho was expressed in the ft mutant background, several NE clones with abnormal morphology remained. This phenotype was not observed when Ras^V12 was expressed. In this model, Ras^V12 drives NB differentiation in Ras^V12-expressing cells in a cell-autonomous manner. In contrast, Rho activates Ras signaling in neighboring cells through the secretion of an EGF ligand. Based on these phenotypic differences, the Fat/Hippo pathway is expected to control the cell-to-cell EGF transmission, including its secretion, distribution or reception at the cell surface. This hypothesis is supported by the fact that several signaling components of the EGFR/Ras pathway, including Rho and EGFR, are localized to the apical side of epithelial tissues, and it is thought that this signal is transmitted along the apical side in epithelial tissues. It has also been reported that Fat/Hippo pathway mutations enhance the expression level of several apically localized molecules, such as aPKC, PatJ, Crumbs and E-cadherin. Thus, Fat/Hippo signaling targets could include unknown apical components that are involved in EGF transmission and this could account for the incomplete NB induction by rho overexpression. rho-expressing cells secret the EGF ligand, which diffuses in the NE surface, but Fat/Hippo pathway mutation would prevent its cell-to-cell transmission and subsequent EGFR/Ras pathway activation in the receiving cells. In this hypothesis, EGF transmission would be disturbed in the NE mutant for the Fat/Hippo pathway, causing the delay of proneural wave progress (Kawamori, 2011).

As an alternative hypothesis, the Fat/Hippo pathway could regulate signal transduction from the EGFR to Ras activation. If this is the case, the Fat/Hippo pathway regulates the intracellular signal transduction of the EGFR pathway. Many of the known targets of the Fat/Hippo pathway are components of growth regulatory, cell survival and cell adhesion molecules. There could be unknown targets that modulate other signaling pathways, including the EGFR pathway, and the NB differentiation defect would thus be caused by a failure in the activation of differentiation signals in the absence of Fat/Hippo signaling (Kawamori, 2011).

This study shows that the Fat/Hippo pathway mutation also affected the morphological character of the NE. Fat/Hippo pathway mutant clones were induced, and they often included NE tissue with a folded morphology and disrupted the medulla structure. The results showed that the Fat/Hippo pathway functions in the regulation of NB differentiation and in NE morphology are distinct, but the two functions could affect each other. The morphological defect of the NE could affect EGFR/Ras signal transduction. The possibility is discussed that the EGF ligand could be distributed along the apical membrane of the NE. The invagination of the apical membrane of the folded NE into the inner region could prevent EGF ligand signaling. There were clones with a normal NE morphology in which NB differentiation was delayed and, thus, morphological defects are not determinate, but they could promote the delay of NB induction (Kawamori, 2011).

How is the activity of the Fat/Hippo pathway regulated throughout the development of the optic lobe? Ft is a member of the cadherin family, and an extracellular molecule is expected to regulate its activity. Ds is a candidate for the Ft ligand that regulates planar cell polarity and Fat/Hippo signaling activity in other epithelial tissues. The expression of ds with a posterior-specific pattern in the developing optic lobe (Reddy, 2010) was confirmed. In the rescue experiments for the ds mutation, the expression of either ds lacking its intracellular domain (ds^ΔICD) or ft lacking its extracellular domain (ft^ΔECD) was sufficient to compensate for ds function, suggesting that Ds functions as a ligand and that Ft lies downstream of Ds in this context (Kawamori, 2011).

The phenotypes of ds and ft mutants were compared to assess whether the mutation of ds by itself accounts for the phenotype of the ft mutants. In contrast to the ft mutants that exhibited altered NB differentiation in the entire outer optic anlagen, the ds mutant phenotype was regionally specific; NB differentiation was severely delayed in the posterior region, and the development of the anterior region was not significantly affected. These differences suggest that there might be some regulatory mechanisms that control Ft activity independently of Ds in the anterior region of the optic lobe (Kawamori, 2011).

The Fat/Hippo pathway is known as a tumor suppressor pathway, and many studies related to this pathway have focused on tissue growth or cell survival. This study has reported a new function of the Fat/Hippo pathway in the regulation of neural differentiation. The Fat/Hippo pathway regulates the progress of neural differentiation signaling, and the EGFR/Ras pathway is a candidate target of this pathway. The data suggest that the Fat/Hippo pathway includes unknown targets involved in EGFR/Ras signal transduction. Further studies are required to identify the targets of the Fat/Hippo pathway and determine the interplay between Fat/Hippo and EGFR/Ras pathways, specifically in NB differentiation (Kawamori, 2011).

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