BIOLOGICAL OVERVIEW

Most homeodomain proteins act early in embryonic development. Rough is an exception. It is expressed in the eye imaginal disc behind the morphogenic furrow, well after the actions of upstream genes like eyeless and hedgehog.

Rough plays a critical role in restriction of the number of photoreceptor precursors in the specification of R8 photoreceptors, the first to develop during the initial specification of rhombdomeres. In the developing retina, the proneural gene for photoreceptor neurons is atonal, a basic helix-loop-helix transcription factor. Using atonal as a marker for proneural maturation, the stepwise resolution of proneural clusters was examined during the initiation of ommatidial differentiation in the developing eye disc. atonal is negatively regulated by rough. This interaction leads to the refinement of proneural clusters to specify R8, the first neuron to emerge in the retinal neuroepithelium. Either ectopic expression of atonal or the removal of rough results in the transformation of a discrete 'equivalence group' of cells into R8s. In addition, ectopic expression of rough blocks atonal expression and proneural cluster formation within the morphogenetic furrow. Thus, rough provides retina-specific regulation to the more general atonal-mediated proneural differentiation pathway. Expression of Rough and Atonal is mutually exclusive: as atonal expression resolves from an initial ubiquitous stripe to individual proneural clusters, rough expression emerges in the intervening cells. The opposing roles of atonal and rough are not mediated through the Notch pathway, as their expression remains complementary when Notch activity is reduced. These observations suggest that homeobox-containing genes can serve a function of tissue-specific repression for bHLH factors. Rough is not likely to be a direct negative regulator of Enhancer of split expression since their expression patterns show extensive overlap. Instead, Rough-induced loss of E(spl) expression may be due to loss of atonal expression in a manner analogous to E(spl) requirement for achaete and scute activity. Notch signaling is also presumably required for E(spl) expression in this system (Dokucu, 1997).

Other functions have been ascribed to Rough. It appears that one function of rough in R2 and R5 cells represses seven up, a member of the steroid receptor superfamily (Heberlein, 1991). Another function of rough may be the induction of soluble signals from the R2/R5 pair for the production of Bar H1 and Bar H2 in R1/R6 (Higashijima, 1992), the third set of cells to contribute to ommatidial development. An additional function, the possible induction of Star in R2/R5 photoreceptors, is suggested by genetic interactions between Star and rough in photoreceptor determination (Heberlein, 1991).

ro^Dom is a dominant allele of rough (ro) that results in reduced eye size due to premature arrest in morphogenetic furrow (MF) progression. The ro^Dom stop-furrow phenotype is sensitive to the dosage of genes known to affect retinal differentiation, in particular members of the hedgehog (hh) signaling cascade. ro^Dom interferes with Hh's ability to induce the retina-specific proneural gene atonal (ato) in the MF and normal eye size can be restored by providing excess Ato protein. ro^Dom was used as a sensitive genetic background in which to identify mutations that affect hh signal transduction or regulation of ato expression. In addition to mutations in several unknown loci, multiple alleles of groucho (gro) and Hairless (H) were recovered. Analysis of their phenotypes in somatic clones suggests that both normally act to restrict neuronal cell fate in the retina, although they control different aspects of ato's complex expression pattern (Chanut, 2000).

Loss-of-function ro mutations cause eye roughness, due to mis-specification of photoreceptors R2 and R5, and the formation of ommatidia with more than one R8 photoreceptor. Repression of R8 cell fate has been attributed to inhibition of ato expression by the Ro homeodomain protein. In support of this proposal, Rough and Atonal proteins appear in complementary sets of cells behind the MF, and ato expression is expanded behind the MF in ro mutants. Generalized expression of ro under a heat-shock promoter (hs-ro) leads to loss of ato expression in the MF and eventually results in furrow arrest. Furrow arrest in ro^Dom is also accompanied by loss of ato expression in the MF. By analogy to the hs-ro phenotype, it is proposed that ro^Dom leads to excess Ro production, although that excess is not detectable by antibody staining (Chanut, 2000).

ro expression at the posterior edge of the MF is under the control of hh signaling. The position of ro-expressing cells, adjacent to hh-expressing cells, suggests that high levels of hh signaling are required for ro expression. By comparison, ato, also a target of hh signaling in the MF, is expressed further away from the hh source, suggesting a requirement for lower levels of hh signaling. It is proposed that the ro^Dom rearrangement sensitizes the ro gene to hh signaling, either by removing a negative regulatory cis-element or by bringing in an additional hh-responsive enhancer element. The resulting anterior expansion of the ro expression domain would prevent ato expression and ultimately cause differentiation arrest (Chanut, 2000).

While this model cannot be proven at this point, it provides a simple explanation for the surprising genetic interactions between ro^Dom and hh: if the rearranged ro gene is more sensitive to Hh, then increasing hh gene dosage will cause more Ro production and accelerate the differentiation arrest. However, reducing Hh signaling, by removing one copy of hh or by providing the inhibitor Ptc in excess, will diminish the amount of Ro protein made and restore Ato accumulation. Modifiers recovered in this screen should therefore include, among other things, components of hh signaling that affect ro or ato expression or partners of Ro in the inhibition of ato transcription (Chanut, 2000).

Expression of dpp is also sharply decreased in ro^Dom. Like ato, dpp expression could be inhibited by ro directly. This may explain its sharp downregulation behind the MF in wild type at the location where ro begins to be expressed. Alternatively, its decrease in ro^Dom could be a secondary consequence of decreased ato transcription. In support of the latter, dpp transcription is sharply reduced in the MF of ato¹ homozygous larvae. Surprisingly, Hairy protein levels remain elevated ahead of the MF in ro^Dom, although h has been shown to be a target of Dpp signaling. The same is true in ato¹ mutants. This suggests that h is under the control of other, as yet unidentified, mechanisms that are not dramatically impaired by the ro^Dom mutation and the accompanying loss of dpp transcription. In any case, effects of ro^Dom on dpp and h expression are unlikely to explain the furrow arrest since the ro^Dom phenotype is not detectably affected by changes of h and dpp gene dosage (Chanut, 2000).

In contrast, ro^Dom is very sensitive to alterations of ato gene dosage, since it is enhanced by loss-of-function ato alleles and almost completely rescued when high levels of ato expression are restored ahead of the MF. The ro^Dom phenotype therefore appears to result primarily from inhibition of ato expression due to excess Ro protein. On the basis of this understanding, the role of two of the strongest suppressors isolated in this screen, new alleles of gro and H, were analyzed on ato regulation and furrow progression (Chanut, 2000).

gro encodes a transcription inhibitor that combines with b-HLH genes of the E(spl) complex to inhibit expression of proneural genes such as achaete and scute. In gro mutant clones, expression of ato persists behind the MF longer than in wild-type tissue. This is consistent with a role for Gro in the N signaling events that lead to the refinement of ato expression behind the MF (Chanut, 2000).

However, Gro is also known to form inhibitory complexes with other transcription factors of the b-HLH class, such as Hairy, or of other classes, such as the c-Rel homolog Dorsal or the homeodomain, segment polarity regulator Engrailed. Association of Gro with Hairy deserves to be envisaged here, since Hairy has been implicated in inhibition of ato as well. It is found, however, that gro mutant clones expand ato expression posterior to the MF, whereas h inhibits ato expression anterior to the MF. Another intriguing possibility is that Gro associates with Ro to mediate inhibition of ato expression behind the MF. Although this hypothesis cannot be completely eliminated, it is unlikely, because complete loss-of-function phenotypes of ro and gro are different. While both of them lead to increased ato expression and imperfect R8 resolution, this effect is much more extensive and long lasting in gro mutant tissue than in ro mutant tissue. In addition, neuronal hyperplasia is not observed in ro mutant tissue, which suggests that at least this gro function must involve factors other than Ro. However, removal of E(spl) function results in neuronal hyperplasia and excess R8 development very similar to removal of gro function. Therefore, the hypothesis is favored that Gro restricts ato expression by combining with proteins of the E(spl) complex whose expression is induced by N signaling (Chanut, 2000).

Even in the complete absence of gro [or E(spl)] function, some refinement of ato expression still occurs, which indicates that factors independent of N and Gro play important roles in patterning Ato behind the MF. Candidates include Ro, the Egfr inhibitor Argos, and Hh. Moreover, outer photoreceptors differentiate in large excess between the R8 precursors and are the main cause of neuronal hyperplasia. Neuronal hyperplasia could occur as a direct consequence of the excess of R8 precursors in gro [and E(spl)] mutant tissue, which would, through the normal serial induction process, recruit an excess of neighboring cells into ommatidial clusters. However, differentiation of extra outer photoreceptors was observed with a hypomorphic gro allele in the absence of excess R8 differentiation. The excess of all photoreceptor types observed with a stronger gro allele may therefore reflect an involvement of gro in the restriction of cell fates at each step of ommatidial formation (Chanut, 2000).

gro mutant clones can also induce extensive overgrowth of head capsule and retinal tissues. In the wing, gro clones have been found to cause overgrowth via the induction of ectopic hh expression. Hh is also a powerful inducer of overgrowth in eye discs, when provided in excess or ectopically. However, overgrowth due to ectopic hh expression is accompanied by ectopic and premature photoreceptor differentiation, a phenotype not observed in overgrown gro mutant tissue. It is therefore unlikely that gro mutant clones cause ectopic hh expression in the eye. Besides, if gro mutations allowed increased Hh production, one would expect enhancement, rather than suppression, of the ro^Dom phenotype. While scenarios cannot be eliminated for ro^Dom in which a slight increase in cell proliferation allows the MF to progress further, it is more likely that gro mutations suppress ro^Dom by allowing Ato protein to persist longer in the MF (Chanut, 2000).

Hairless inhibits N signaling by preventing Su(H), a transcription factor, from translocating to the nucleus and activating transcription of N targets such as the E(spl) complex genes. In the absence of H, Su(H) is free to enter the nucleus upon activation of N. Su(H) mutant clones lead to expanded Ato expression behind the MF, consistent with a role for Su(H) in the N-mediated lateral inhibition that leads to the refinement of ato expression. In H clones are found in which the refinement of ato expression to single cells appears accelerated. This is consistent with a role for H as an inhibitor of N and Su(H) in lateral inhibition. Surprisingly, however, individual clusters of Ato-expressing cells often persist in H mutant tissue behind the MF, instead of resolving to single R8 precursors; in adults as well, mutant ommatidia often contain more than one R8. This would suggest that at later stages H is required to resolve ato expression to single R8 precursors, a role that is not expected for an inhibitor of the N pathway. Anterior H mutant clones show precocious ato expression anterior to the MF. This might explain the patterning defects behind the MF, if precocious and excessive accumulation of Ato protein in the MF interferes with the proper execution of lateral inhibition via N or with downregulation by Ro. In this regard, it is noted that excess Ato protein, as provided under heat-shock control, is found to perturb the resolution of ato expression to single R8 precursors (Chanut, 2000).

It has been suggested that early ato expression, ahead of the MF, is in part the result of an as yet unsuspected 'proneural' effect of N signaling. The anterior expansion of ato expression in H mutant clones is consistent with this model, assuming that H would act as an inhibitor of N there as well. However, the proneural function of N must not be mediated by Su(H), since removal of Su(H) function does not abolish ato expression ahead of the MF. The results presented here may indicate that H antagonizes the proneural function of N via a mechanism that does not involve Su(H). Alternatively, the role of H on early ato expression may be independent of N signaling. Regardless of the exact mechanism, the enhanced expression of ato ahead of the MF in H mutants is likely to explain suppression of the ro^Dom phenotype by counteracting the effect of ectopic Ro on ato expression in the MF (Chanut, 2000).

Finding that similar levels of suppression can be achieved by loss-of-function mutations in H and gro (which act in opposite directions in the N pathway) is not unique. A similar situation was encountered in another study where mutations in gro and H were both found to enhance the wing and bristle phenotypes associated with loss-of-function mutations in Egfr. The observation that mutations in both genes elevate ato expression in the vicinity of the MF, but at different stages of the differentiation process, helps resolve this paradox. The results also indicate that the exact timing (or location) of ato expression might not be crucial to MF progression, provided adequate levels are reached. This conclusion is supported by the finding that Ato supplied anterior to its normal expression domain, in the h expression domain, restores normal eye size in a ro^Dom background. Whether proper R8 spacing and ommatidial patterning can be achieved under these conditions remains to be shown (Chanut, 2000).

PROTEIN STRUCTURE

Amino Acids - 350

Structural Domains

The Rough protein has a typical homeodomain, distantly related to Antennapedia (Saint, 1988). There are opa (CAX) repeats on either side of the homeodomain. The analysis of chimeric proteins, in which parts of the homeodomain are swapped between Rough and Antennapedia proteins reveals that the C-terminus of the rough homeodomain is critical for transcription factor activity. The same C-terminal region is found to be required for the recognition of Rough binding sites (Heberlein, 1994).

Transcriptional Regulation

According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus, indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).

In contrast to ato broad expression just anterior to the furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential for uncommitted cells to acquire ommatidial cell fate or the ability to express ommatidial marker genes. Consistent with this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).

In the eye developmental field posterior to the morphogenetic furrow, three ommatidial cell marker genes, ro, svp, and pros, were found to be misexpressed zonally in many uncommitted cells along the A/P axis when Ras1 was transiently but ubiquitously activated. The Ras1-dependent misexpression regions appear to correspond to those regions where expression of these genes is normally initiated, indicating that Ras1-dependent zonal marker gene misexpression in presumptive uncommitted cells may reflect directly their developmental potentials. Subsequent to Ras1 activation, posterior ro misexpression is restricted only to the vicinity of the furrow. Wild type cells just posterior to the morphogenetic furrow may thus possess developmental potential for ro expression (Hayashi, 2001).

This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow. Uncommitted cells just posterior to the morphogenetic furrow are presumed to acquire ro expression potential at the earliest stage of the model (stage 1). The regulation of ro expression appears highly complicated. ro expression is abolished in all cells in smo mutant clones situated just posterior to the morphogenetic furrow but fundamentally normal in more posterior clones in which ro is expressed in putative R2/R5 and R3/R4 precursors. ro expression appears to be regulated by at least two enhancers, one for expression at or near the morphogenetic furrow and the other for expression in developing R2-R5 photoreceptor precursors situated more posteriorly. Hh signals may thus be involved only in the former, possibly through the regulation of prepattern gene expression, and ro autoregulation maintains the activity of the latter. Egfr/Ras1 signals may be involved in both of them (Hayashi, 2001).

Expression levels of CI (activator form; CI^act) and Dac in smo mutant clones vary considerably, depending on clone position, thus making the situation much more complicated. The expression of CI^act is down-regulated in smo mutant clones at or near the morphogenetic furrow and up-regulated in more posterior clones. Dac expression is up-regulated only in the latter. Although normal ro expression is observed in ci or dac mutant clones, the fact that enhanced expression of CI^act and Dac occurs in smo mutant clones distant from the morphogenetic furrow may suggest that CI^act and Dac have some role in expressing ro in these clones. Consistent with this, misexpression experiments indicate either misexpressed CI^act or Dac to be capable of bringing about ro up-regulation in a fraction of ommatidial cells. It is thus likely that ro expression is positively regulated by CI^act and Dac (Hayashi, 2001).

In the absence of Hh signals, ci may serve as a gene to encode a repressor for ro expression, since ro expression near the furrow is repressed by the repressor form of CI protein (Hayashi, 2001).

dac and eya are expressed and may serve as eye prepattern genes in the region anterior to the morphogenetic furrow. This is supported by the finding that ommatidial marker gene misexpression subsequent to Ras1 activation occurs only within the Dac/Eya expression domain. pros expression is considerably restricted but ro and svp misexpression is evident throughout the entire Dac/Eya expression domain. Considerably strong misexpression of ELAV, a neuron-specific antigen, is also apparent anterior to the furrow. Thus, as with posterior cells, those anterior to the morphogenetic furrow are capable of developing into photoreceptors or ommatidial cells with receipt of Ras1 signals. However, no apparent regularity in ommatidial marker gene expression can be detected in the region anterior to the furrow, indicating that developmental potential of anterior cells is necessarily reset to some extent before the onset of normal eye development at the morphogenetic furrow or before first receiving Spitz signals from nascent R8 (Hayashi, 2001).

ro expressed along the morphogenetic furrow may be involved in this process, in that svp expression near the morphogenetic furrow is significantly repressed by ro expression along the morphogenetic furrow. Furthermore, ro has also been shown to repress ato expression near the furrow (Hayashi, 2001).

In the developing Drosophila eye, differentiation of undetermined cells is triggered by Ras1 activation but their ultimate fate is determined by individual developmental potential. Presently available data suggest that developmental potential is important in the neurogenesis of vertebrates and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along the dorso-ventral axis in response to graded Sonic Hedgehog signals and this presages their future fates. Subdivision of originally equivalent neural progenitors through the action of prepattern genes may accordingly be a general strategy by which diversified cell types are produced through neurogenesis (Hayashi, 2001).

An outstanding model to study how neurons differentiate from among a field of equipotent undifferentiated cells is the process of R8 photoreceptor differentiation during Drosophila eye development. Senseless/Lyra is a Zn finger transcription factor that is expressed and required in the sensory organ precursors (SOPs) for proper proneural gene expression. In senseless mutant tissue, R8 differentiation fails and the presumptive R8 cell adopts the R2/R5 fate. senseless repression of rough (ro) in R8 is an essential mechanism of R8 cell fate determination and misexpression of senseless in non-R8 photoreceptors results in repression of rough and induction of the R8 fate. Surprisingly, there is no loss of ommatidial clusters in senseless mutant tissue and all outer photoreceptor subtypes can be recruited, suggesting that other photoreceptors can substitute for R8 to initiate recruitment and that R8-specific signaling is not required for outer photoreceptor subtype assignment (Frankfort, 2001).

Rough (Ro), was examined to confirm and extend the hypothesis that the presumptive R8 photoreceptor becomes an R2 or R5 photoreceptor in senseless mutants. Ro is normally expressed in the R2/R5 photoreceptors and later expands to include the R3/R4 photoreceptors. Within sens mutant clones, Ro is detected abnormally in three cells per ommatidium at the time when it should only be detected in the R2/R5 photoreceptor pair. Moreover, when the presumptive R8 cell is marked with the R8-specific enhancer trap in sca, the enhancer trap is consistently expressed in one of the three Ro-expressing cells. These data suggest that sens normally represses Ro in the differentiating R8 photoreceptor (Frankfort, 2001).

Since ro is not normally expressed in R8, and misexpression of Ro induces changes in cell fate, it was hypothesized that Ro misexpression in the presumptive R8 cell is responsible for the loss of R8 observed in sens mutant clones. To test whether ro is epistatic to sens, sens mutant clones were generated in a ro mutant background. Many ommatidia mutant for both sens and ro contain photoreceptors with small rhabdomeres, suggesting the presence of either R8 or R7 photoreceptors. Furthermore, some double mutant ommatidia contain more than one photoreceptor with a small rhabdomere, similar to the ro mutant phenotype. To determine if the photoreceptors with small rhabdomeres were R8 cells, the expression of two late markers of R8 differentiation, Boss, and an R8-specific enhancer trap, BBO2, were examined in sens mutant clones generated in a ro mutant background. While Boss is never expressed in tissue mutant only for sens, Boss expression is restored in many ommatidia in the double mutant tissue. BBO2 expression is also restored in the double mutant tissue, sometimes in two photoreceptors within the same cluster, suggesting the presence of more than one R8 photoreceptor. Thus, inappropriate Ro expression in the presumptive R8 cell is likely responsible for the early abortion of R8 differentiation and adoption of the R2/R5 fate in sens mutant ommatidia (Frankfort, 2001).

Since sens-mediated repression of ro plays a critical role in R8 differentiation, whether sens misexpression is sufficient to repress ro and induce additional photoreceptors to adopt the R8 fate was tested. sens was misexpressed in clones posterior to the MF using a variation on the MARCM system. Within sens misexpression clones, Ro is repressed in those cells showing the highest levels of Sens. sens misexpression is also sufficient to induce Boss expression in multiple cells per ommatidium and at higher levels than it is normally expressed. Finally, in the adult retina, misexpression of sens causes ommatidial disruption near the center of the clone and induces the formation of ectopic small rhabdomeres within ommatidia near the clonal border. Thus, sens is capable of repressing Ro when misexpressed, and is sufficient to induce both R8-specific gene expression and rhabdomere morphology (Frankfort, 2001).

In sens mutant ommatidia, the presumptive R8 cell rapidly expresses R2/R5-specific genes and adopts the fate of an R2/R5 photoreceptor in essentially all cases. One R2/R5-specific marker that is abnormally expressed in the presumptive R8 within sens mutant ommatidia is Ro. ro acts as a repressor of R8 differentiation and Ro is not normally expressed in R8. The consistent misexpression of Ro within the presumptive R8 photoreceptor in sens mutant tissue suggests that sens represses Ro in R8. Such a relationship between sens and ro is further supported by the observation that sens misexpression causes the repression of Ro in outer photoreceptors. Moreover, it is clear that repression of Ro in R8 is of functional significance because loss of ro function is sufficient to rescue the R8 loss observed in sens mutant clones. These data imply that ro is epistatic to (downstream of) sens and that sens-mediated repression of Ro is essential for R8 differentiation. Thus, repression of a cell-fate repressor is identified as a major mechanism of R8 differentiation. These findings are also consistent with the observations that sens acts as a repressor in the Drosophila CNS and that the sens homologs Gfi-1 (murine) and pag3 (C. elegans) function as repressors as well (Frankfort, 2001).

In addition to its role as a repressor of R8 differentiation, ro is sufficient to induce changes in subtype specification, and it is thought that ro acts downstream of photoreceptor recruitment to specify photoreceptor subtype identity as an R2/R5 cell. Moreover, it is clear that in ro mutant ommatidia the presumptive R2/R5 photoreceptors adopt the fate of an R1, R3, R4, or R6 photoreceptor. Similarly, both loss- and gain-of-function experiments reveal that sens function is not required for establishment or maintenance of neural fate in the developing eye, but specifically for directing a cell to follow the R8 differentiation pathway. Thus, sens and ro seem to have analogous roles in directing the specification of specific photoreceptor cell fates. The transcriptional and genetic relationships identified between sens and ro imply that the process of R8 differentiation involves a hierarchical interaction where sens normally represses ro to prevent both ro repression of R8 and ro induction of R2/R5. When sens function is removed, ro is abnormally expressed in the presumptive R8 cell and the R2/R5 fate is adopted (Frankfort, 2001).

Thus a new model is proposed for the genetic regulation of R8 differentiation that includes the relationships among ato, sens, and ro. In this model, ato induces sens within the R8 equivalence group and R8, and sens is in turn required for maintenance of ato expression. Since R8 may transiently differentiate in sens mutant clones, ato is likely sufficient to confer specificity to R8 differentiation, whereas sens is required to 'lock-in' and maintain this program of R8 differentiation, primarily via the repression of ro. Thus, mutual antagonism of ato and ro is likely mediated by sens. sens presumably has a ro-independent role in R8 differentiation as well, because loss of ro function does not completely rescue the sens mutant phenotype (Frankfort, 2001).

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 work suggests that Sens acts to ensure that the organizing center of each ommatidium is refractory to the developmental signals it produces -- the R8 cell can secrete Spi and even activate Egfr on its own cell membrane, yet remains protected from the deleterious effects of activation of Pnt and other Egfr targets, such as Ro, in R8 (Frankfort, 2004).

The mechanism by which Sens regulates the discrepancy between levels of Egfr activation at the receptor/cytoplasmic and nuclear levels in R8 is probably through repression of pnt transcription. This is supported by the observation that pnt transcription is not induced by misexpression of an activated form of Egfr when sens is co-misexpressed. Furthermore, expression of the pnt-P1 isoform in R8 disrupts R8 differentiation. Since misexpression of pnt-P2 has no effect on R8 differentiation, this suggests that Sens negatively regulates transcription of pnt-P1, but not pnt-P2. This mode of regulation is consistent with established models for transduction of the Egfr signal to the nucleus. Specifically, ERK phosphorylates Pnt-P2, which is thought to be a transient positive regulator of pnt-P1 transcription. In this model, transduction of Egfr activation occurs all the way into the nucleus of R8, but Sens represses the pathway at the final step -- positive regulation of pnt-P1 by Pnt-P2. When sens function is removed, the block on pnt-P1 transcription is relieved, and Pnt-P1 can exert its transcriptional effects on the nucleus, including ro induction (Frankfort, 2004).

There is evidence that pnt-P1 transcription can be regulated by Egfr signaling independently of pnt-P2 during Drosophila embryogenesis. If this is the case during eye development, the model would remain essentially the same -- Sens would still act as a negative regulator of pnt-P1 in R8. However, this regulation would occur independently of pnt-P2 rather than downstream of pnt-P2 (Frankfort, 2004).

Sens is also a potent negative regulator of ro and this relationship appears to specifically affect the cell fate decision between R8 and R2/R5 differentiation. Several lines of evidence suggest that Sens-mediated repression of ro is distinct from other effects of Sens in R8: (1) loss of ro function does not rescue R8 differentiation in all ommatidia in sens mutants; (2) even those R8 cells that do differentiate in sens ro double mutants require Spi/Egfr pathway activation; (3) misexpression of ro in R8 causes a different phenotype than misexpression of pnt-P1 in R8. Specifically, even though Egfr pathway activation is necessary and sufficient for Ro expression, misexpression of pnt-P1 in R8 does not cause an obvious cell fate transformation from R8 to R2/R5, while misexpression of ro in R8 does. Indeed, R8 markers are still expressed when pnt-P1 is misexpressed in R8. However, aberrant nuclear movements and the absence of small rhabdomeres at the level of R8 in adults suggest that misexpression of pnt-P1 does perturb R8 differentiation. Together, these results suggest that Sens repression of pnt-P1 occurs independently of Sens function as a repressor of ro, and that Sens-mediated repression of pnt-P1 is probably required for normal R8 differentiation upstream or independently of cell fate determination (Frankfort, 2004).

Targets of Activity

In the absence of rough activity, seven up expression is derepressed in cells that would otherwise develop into R2 and R5. Curiously, this derepression is not observed in R3 and R4 cells, raising the possibility that rough is modulated in the two pairs (Heberlein, 1991a).

The gene rhomboid is a putative target gene of Rough (Freeman, 1992).

Additional targets are BarH1 and BarH2, which require rough for expression in R1 and R6 photoreceptors (Higashijima, 1992).

Directly or indirectly glass controls the expression of approximately 25 % of all enhance trap lines expressed in the eye disc. The phenotype of eye discs doubly mutant for glass and rough suggest that glass is required for subtype specification and for recruitmant of cells to the ommatidial cluster. rough and glass appear to act on common target genes . However the is no simple relationship between ro and gl. While RO represses both seven up and boss in R2 and R5 precursors, and GL activates both in cells which normally express them, in the absence of both ro and gl it is found that svp is expressed but boss is not. One possibility is that GL is critical for the establishment of R8-specific genes and RO for the establishment of R2/5-specific genes and for the repression of inappropriate genes in these cells (Treisman, 1996).

To identify the transcriptional targets through which rough acts to specify the R2/R5 neuronal sub-type, enhancer trap lines expressed in developing photoreceptors were screened for those whose expression patterns are altered when ro function is inactivated. In this way two potential ro targets were identified; these are also targets of the zinc finger transcription factor Glass. An enhancer trap line was identified that exhibits altered morphogenetic furrow expression in a ro mutant background. Finally, an enhancer trap line, AE33, was molecularly characterized that was identified in earlier screens as a target of both ro and gl. The transcript interrupted by AE33 shares similarity with the mammalian vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases that is associated with actin filaments, focal adhesions, and dynamic membrane regions. There is also similarity with Enabled, a substrate of the Drosophila Abl tyrosine kinase and with two human Expressed Sequence Tags (ESTs) (DeMille, 1996).

Two-step selection of a single R8 photoreceptor: a bistable loop between senseless and rough locks in R8 fate

Patterning of sensory organs requires precise regulation of neural induction and repression. The neurocrystalline pattern of the adult Drosophila compound eye is generated by ordered selection of single founder photoreceptors (R8s) for each unit eye or ommatidium. R8 selection requires mechanisms that restrict R8 potential to a single cell from within a group of cells expressing the proneural gene atonal (ato). One model of R8 selection suggests that R8 precursors are selected from a three-cell 'R8 equivalence group' through repression of ato by the homeodomain transcription factor Rough (Ro). A second model proposes that lateral inhibition is sufficient to select a single R8 from an equipotent group of cells called the intermediate group (IG). This study provides new evidence that lateral inhibition, but not ro, is required for the initial selection of a single R8 precursor. In ro mutants ectopic R8s develop from R2,5 photoreceptor precursors independently of ectopic Ato and hours after normal R8s are specified. Ro directly represses the R8 specific zinc-finger transcription factor senseless (sens) in the developing R2,5 precursors to block ectopic R8 differentiation. These results support a new model for R8 selection in which lateral inhibition establishes a transient pattern of selected R8s that is permanently reinforced by a repressive bistable loop between sens and ro. This model provides new insight into the strategies that allow successful integration of a repressive patterning signal, such as lateral inhibition, with continued developmental plasticity during retinal differentiation (Pepple, 2008).

Ro is a homeodomain-containing protein and has been shown to bind DNA at two sites in its own enhancer containing an ATTA core sequence. To explore the possibility that Ro directly represses sens, the R8 specific sens enhancer was identified and the mechanisms regulating sens expression was characterized. A 645 bp fragment within the second intron of the sens genomic locus named F2 was identified that is sufficient to drive reporter expression specifically in photoreceptors of the developing eye-antennal imaginal disc. To test whether the F2 region is necessary for R8-specific sens expression, the 645 bp region was specifically deleted from the sens-L genomic rescue construct generating DeltaF2. In sens-null mutants, one copy of DeltaF2 rescues the null phenotype in all tissues except the eye. Thus, F2 is the sens eye enhancer and is necessary and sufficient for R8-specific sens expression (Pepple, 2008).

F2 contains two potential Ro-binding sites known as H1 and H2, for homeodomain 1 and 2. To test for a direct interaction, electrophoretic mobility shift assays (EMSAs) were performed. A probe containing H1 and H2 is bound specifically by Ro protein in vitro. Complete loss of binding occurs with mutation of H2. Mutation of H1 does not prevent Ro binding, but there may be a mild decrease in binding compared with the wild-type probe. To test the in vivo significance of these interactions, each site was mutated in a reporter generated with the minimal R8-specific enhancer, B-short-GFP, and the effect on GFP was evaluated. Although H1 is not required for Ro binding in vitro, mutation of H1 in B-short (termed H1^*) leads to consistent expression of GFP in two extra cells per ommatidium. These two cells were identified as the R2,5 photoreceptor pair by co-localization of GFP with β-galactosidase from the R2,5-specific enhancer trap RM104. GFP expression is also expanded into the R2,5 pair with the H2 mutation (H2^*). Mutation of both H1 and H2 (H1,2^*) results in a GFP expression pattern indistinguishable from H2^*. To test whether the loss of ro function has the same effect on B-short-GFP expression as does mutation of the Ro-binding sites, ro^X63 clones were generated. In the absence of ro function, both Sens and B-short-GFP expression are detected in two to three cells per ommatidium. Together with the in vitro binding data, these in vivo results suggest that Ro directly represses sens expression in R2,5 photoreceptors (Pepple, 2008).

Therefore, this work shows that Ro directly represses sens in developing R2,5 cells and that de-repression of Sens is sufficient to initiate R8 cell fate in the absence of ectopic Ato. Although there are a small number of ectopic Ato-expressing cells in column 3 in ro^x63 mutants, it is not likely that the additional 'R8' cells are due to misregulation of Ato since the great majority of ectopic 'R8s' never express detectable Ato protein after the intermediate group stage. It is more likely that the extra Ato-positive cells are due to secondary Sens activation of proneural gene expression, a previously reported phenomenon (Pepple, 2008).

sens is required for R8 differentiation to occur through repression of Ro in R8, and that ectopic Sens is sufficient to repress endogenous Ro expression. Thus, in the absence of sens, three R2,5 cells develop and in the absence of ro up to three R8 cells form per ommatidium. This reciprocal phenotype supports the existence of the three cell R8 equivalence group and a mechanism of mutual repression between sens and ro that specifies opposite cell types. Although one mechanism regulating this mutual repression is the direct repression of sens by Ro, other roles for Ro may exist. The Ro-binding site mutations do not produce the same level of GFP reporter protein expression elevation in R2,5 precursors that would be predicted from the level of GFP expressed in ro mutants. This suggests that Ro may also regulate sens by repressing an activator of sens expression in R2,5 precursors (Pepple, 2008).

Regardless of the mechanism, the negative-feedback loop between sens and ro is secondary to the initial force driving R8 selection in which Ato and Sens are transiently repressed by lateral inhibition in all but one cell within an IG. Thus, lateral inhibition transiently represses neural differentiation in the eye, establishing the patterned array of precisely spaced ommatidia while retaining the potential for later recruitment of undifferentiated cells to the photoreceptor cell fate. If the effects of lateral inhibition were to repress permanently the potential for neuronal differentiation, further retinal development would be blocked. Therefore, the effects of lateral inhibition must be limited, and the data indicate that column 3 is the boundary of its influence. Since the effects of lateral inhibition diminish, the negative-feedback loop between sens and ro reinforces the pattern of selected R8s and ensures that only one Sens-expressing cell from the R8 equivalence group develops as an R8. This simple bistable loop translates the transient developmental signal of lateral inhibition into a committed irreversible fate (Pepple, 2008).

In later R8 differentiation, another bistable loop is used to specify the 'pale' or 'yellow' subtypes of R8 photoreceptors. During this late developmental step, the bias for the 'pale' R8 fate is provided by a signal from a 'pale' R7. It is proposed that the bias signal that tips the fate decision in the sens-ro loop is provided by resolution of Ato to a single cell by lateral inhibition. Ato then directly activates Sens expression and biases that cell to the R8 cell fate. It is not yet known what activates Ro expression and thereby establishes the R2,5 cell fates. However, it has been suggested that epidermal growth factor receptor (EGFR) or Hedgehog signaling may be required for Ro expression. As a result, after the R8 bias is established, a signal such as the EGFR ligand Spitz could be sent from R8 to the two neighboring cells that bias their sens-ro loop towards Ro expression and the R2,5 fate. Once Ro expression is initiated in the R2,5 pair, the pattern of a single Sens-expressing R8 per ommatidium becomes irreversible (Pepple, 2008).

Proper patterning of the Drosophila eye requires precise selection of R8 precursors in a highly ordered array. Previously, the potential to assume the R8 fate was generally believed to reside in the single cell that achieved the highest balance of proneural induction by ato and escaped repression by lateral inhibition. This concept has influenced the interpretation of mutants that exhibit multiple R8 phenotypes, such as ro, by linking the extra R8s that form to cells that inappropriately maintain Ato expression. However, the data show that the expression pattern of Ato and Sens in a ro-null mutant is not altered in a manner consistent with this model. This re-evaluation of the ro phenotype suggests the intriguing possibility that undifferentiated cells posterior to the furrow retain the developmental plasticity to develop as R8s even in the absence of ongoing Ato expression (Pepple, 2008).

The ro phenotype demonstrates that, despite initial repression of the R8 cell fate by lateral inhibition, at least two additional cells have the potential to develop as R8s starting in column 3 if Sens expression is de-repressed. One of the subfragments of the sens eye enhancer, fragment C-GFP, is expressed in nearly all cells posterior to the MF, suggesting that sens could be de-repressed in cells other than the R2,5 cell precursors and initiate R8 development. The widespread expression of fragment C-GFP suggests that it lacks an important negative regulatory region distinct from Ro repression. One potential mechanism that may explain the fragment C-GFP expression pattern is that the stripe of Ato expression in the MF confers R8 potential to all cells and that this potential is only transiently repressed by lateral inhibition during patterning. Then, as the effects of lateral inhibition fade, secondary mechanisms repress sens expression and R8 differentiation in cells posterior to the MF. This model, demonstrated by the function of Ro and suggested by fragment C-GFP expression, is distinct from the previous concept that R8 cell fate is limited to cells of the intermediate group (Pepple, 2008).

The minimal eye specific enhancer of sens, fragment B-long, contains at least four potentially discreet regulatory elements that balance the positive and negative inputs required to specify a single R8 precursor per ommatidium. The first positively acting element is under the direct control of Ato/Da heterodimers and contains E-boxes 1 and 4. This element is required for Ato-dependent sens expression in the IGs and in columns 1-3. Although ato is at the top of the genetic cascade required for eye differentiation, sens is only the third direct target identified in the eye after bearded (brd) and dacapo (dap). Ato/Da heterodimers bind to two E-boxes (E1 and E4) to drive early sens expression in R8. This is in contrast to the previously described direct regulation of sens in SOPs of the embryonic and developing adult PNS by Ato and Scute at a single E-box in their common enhancer (Pepple, 2008).

The second positively acting regulatory element resides within the boundaries of fragment E1^*, although the minimal necessary sequence was not specifically identified. This element responds to an Ato-independent mechanism that is sufficient to maintain Sens expression in selected R8 cells after column 3. Sens is known to respond to Ato-independent inductive cues much later in R8 development (48 hours after pupation) when Sens expression requires the spalt genes. However, larval expression of Sens is not disrupted in spalt mutants, suggesting the existence of yet another unidentified positive regulator (Pepple, 2008).

In addition to these two positively acting elements, there are also at least two negative regulatory elements. The Ro-binding element H2, that is responsible for repressing Sens expression in R2,5 cells, was specifically identified. The second element was not specifically identified, but its presence is suggested by the nearly ubiquitous expression of fragment C-GFP. Together these positive and negative regulatory elements outline an elegant strategy for the multi-staged selection of a single R8 per ommatidium and highlights a model where blocking R8 cell fate potential with sequential, independent, repressive mechanisms is an important strategy for patterning and cell fate development in the Drosophila eye (Pepple, 2008).

DEVELOPMENTAL BIOLOGY

Larval

The rough gene is expressed maximally in all cells in morphogenic furrow. It is involved in specification of photoreceptor cell fate in the eye-antennal disc. Behind the furrow it is expressed in a subset of cells (Heberlein, 1991b). Genetic evidence points to rough as specifying the fates of precursors of R2 and R5 photoreceptor cells (Heberlein, 1991a). In the absence of rough function, R2 and R5 fail to be correctly determined and transform into cells of the R3/4/1/6 subtypes (Heberlein, 1991a). Rough transcripts are also found in the larval brain (Saint, 1988).

Effects of Mutation or Deletion

The size of eyes in rough mutants is reduced and the facets are disorganized. rough ommatidia have a variable number of photoreceptor cells and their arrangement is abnormal (Heberlein, 1991a)

The use of ectopic expression of sevenless and rough has provided insight into the mechanisms of positional signalling and the normal function of rough. Ubiquitous expression of sevenless does not alter cell fate suggesting that the inducing signal is both spatially and temporally controlled. Conversely, ectopic expression of rough in the R7 precursor causes a transformation of R7 cells into R1-6 type cells. This indicates that rough acts, similar to other homeobox genes, as a selector gene that determines the fate of single cells (Hafen, 1990).

The planar polarity of Drosophila ommatidia is reflected in the mirror-symmetric arrangement of ommatidia relative to the dorso-ventral midline, the equator. This arrangement is generated when ommatidia rotate towards the equator and the photoreceptor R3 displaces R4, creating different chiral forms in each half. Analysis of ommatidia that are mosaic for the tissue polarity gene frizzled (fz) shows that the presence of a single Fz+ photoreceptor cell within the R3/ R4 pair is critical for the direction of rotation and chirality. By analysing clones mutant for seven-up (svp) in which R3/R4 precursors reside in their normal positions and become photoreceptor neurones but fail to adopt the normal R3/R4 fate, it has been found that the R3/R4 photoreceptor subtype specification is a prerequisite for planar polarization in the eye. In mosaic R3/R4 pairs the svp- cell always adopts the R4 position. This bias is reminiscent of what happens in fz mosaic R3/R4 pairs, where the fz- cell also almost always adopts the R4 position. A possible interpretation of the data is that the svp mutant cell is not able to receive the polarity signal or to interpret it (or to communicate with the other cell of the R3/R4 pair). Mutations in the rough gene cause the mis-specification of R2 and R5 and their transformation to an R3/4 pair as seen by their expression of svp and their dependence on svp to develop as outer photoreceptors. In genotypes where too many cells adopt the R3/R4 fate, ommatidial polarity is also disturbed. This defect could arise because, in a situation with too many R3/R4 cells within a cluster, there is crosstalk/competition for the R3 fate between more than two cells that confuses the cluster as a whole. Taken together, these data imply that correct specification of a single R3 cell per ommatidium is a prerequisite for the normal interpretation of the Fz-mediated polarity signal (Fanto, 1998).

REFERENCES

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date revised: 10 August 2009 

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