1 Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
2 Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
3 Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
4 Department of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
5 Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
*Author for correspondence (e-mail: gmardon{at}bcm.tmc.edu)
Accepted 19 December 2001
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SUMMARY |
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Key words: Drosophila, eye, R8 photoreceptors
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INTRODUCTION |
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The chronology of ommatidial development is well characterized. First, differentiation of developmentally equipotent cells of the eye imaginal disc begins when movement of the morphogenetic furrow (MF) initiates at the posterior margin of the disc and progresses anteriorly. With its passage, the MF leaves in its wake clusters of differentiating neurons that will ultimately become the photoreceptors of the adult eye (Fig. 1A) (Ready et al., 1976; Tomlinson and Ready, 1987
). The order in which photoreceptors differentiate within an ommatidium is invariant and begins with the R8 photoreceptor (Jarman et al., 1994
; Tomlinson and Ready, 1987
). The R2/R5, R3/R4 and R1/R6 photoreceptors are then sequentially recruited in a pairwise fashion. Last, the R7 photoreceptor is recruited. In Drosophila, each ommatidium is not clonally derived (Lawrence and Green, 1979
). Instead, the R8 photoreceptor orchestrates ommatidial construction via induction of surrounding uncommitted cells to become photoreceptors in a process that is dependent on Epidermal Growth Factor Receptor (EGFR) signaling (Freeman, 1996
; Freeman, 1994
; Jarman et al., 1994
; Tio et al., 1994
; Tio and Moses, 1997
; Wasserman et al., 2000
).
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Despite the temporal overlap of the many processes that underlie R8 maturation, the roles of individual transcription factors and signaling pathways are specific and genetically separable. Thus, it is both feasible and instructive to subdivide R8 maturation into distinct processes that are defined by the genetic pathways that govern them. This article is therefore subdivided into four sections: first, positively and negatively acting factors required for proper R8 selection are reviewed; second, the roles of the Notch and EGFR signaling pathways, which coordinate the spacing of the selected R8 cells, are discussed; third, the genes involved in differentiation of the selected R8 cell are analyzed; and fourth, the organizing properties of R8 are reviewed. As several recent discoveries have called into question some of the basic assumptions of R8 development and function, these data, their impact on the field of R8 development, and possible controversies that surround them are highlighted throughout the review. Next, some of the unique and important developmental characteristics of R8 specification are discussed by comparing it with another paradigm for neural differentiation: Drosophila sensory organ precursor (SOP) development. Last, perhaps the most direct relevance of R8 development to neural specification is addressed by discussing the striking parallels between R8 differentiation and ganglion cell development during mammalian retinal morphogenesis.
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Positive selection of R8: proneural genes |
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The protein encoded by da is a requisite binding partner for Ato, as well as for other basic helix-loop-helix proteins (Cabrera and Alonso, 1991; Jarman et al., 1993
; Murre et al., 1989
; Van Doren et al., 1991
). Clonal analysis of a null mutation in da reveals a phenotype that is similar to that of the ato1 mutation failure of all photoreceptor development. Moreover, there is an absolute cell-autonomous requirement for da in R8, as well as a partial requirement for da in R2 and R5 (Brown et al., 1996
). Consistent with its predicted function, Da is expressed in a pattern that is very similar to that of Ato, beginning with a broad dorsoventral stripe within the MF. Posterior to the MF, Da is expressed in most cells at a low level, but in R8 at a high level. Da and Ato also appear to crossregulate each other. Only early Da expression is detected in ato1 mutant eye discs, and da function is required for proper spatiotemporal expression of Ato (Brown et al., 1996
). Moreover, analysis of loss-of-function clones of da and ato have similar effects on Ato expression both lead to expansion of Ato to all cells of the clone in a manner similar to the initial pattern of Ato expression (Chen and Chien, 1999
). This finding suggests that while not required for initiation of Ato expression, both Ato and Da are required for proper autoregulation of late Ato expression. However, a detailed analysis of the mechanisms underlying Ato/Da crossregulation has not been reported.
It therefore appears that many components of the canonical proneural paradigm are present during R8 selection. First, a specific proneural gene (ato) controls the development of a specific organ or cell type (R8). Second, the expression of this proneural gene is highly regulated. Third, this essential proneural gene requires binding to Da for its proper function and regulation. However, many questions remain. For example, the details of ato autoregulation in the eye, including the factors that physically interact with the crucial ato enhancers are not known. Moreover, no obvious potential direct downstream targets of Ato and Da have been identified in the eye thus far. Finally, the mechanism by which putative eye-specific factors control the transcriptional environment in the developing eye to ensure that an R8 cell, and not a chordotonal organ, develops from an Ato-expressing cell are not known.
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Negative selection of R8: hairy, extramachrochaete, rough and hedgehog |
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rough (ro) encodes a homeodomain-containing protein that helps to ensure that only one cell of each R8 equivalence group differentiates as R8, and loss-of-function mutations in ro often result in ommatidia with two or three R8 cells each (Dokucu et al., 1996; Heberlein et al., 1991
). However, the mechanism by which this occurs is not known. Furthermore, loss of ro function results in an expansion of stage 1 Ato expression, and misexpression of ro is capable of both repressing Ato prior to final R8 selection and preventing photoreceptor differentiation (Dokucu et al., 1996
; Kimmel et al., 1990
). Thus, Ro acts as a repressor of both Ato expression and R8 selection. Consistent with this role, Ro is expressed in a pattern that is mutually exclusive with that of Ato. Moreover, Ro expression begins in the MF at about the same time Ato is expressed in the R8 equivalence group (stage 3), the precise time at which Ro is believed to exercise its function in R8 selection. Establishment of the mutually exclusive expression patterns of Ro and Ato does not require Notch signaling (Dokucu et al., 1996
). This observation suggests the existence of some unidentified intermediate that relays a signal from Ro-expressing cells to the R8 equivalence group in order to repress all but one of the Ato expressing cells from being selected as R8. There are currently no stellar candidates for this relay signal, but the results of a recent genetic screen for modifiers of a dominant ro mutant phenotype has already uncovered genes that regulate Ato and may yet unveil other missing links in this process (Chanut et al., 2000
).
The Hedgehog signaling pathway also plays a role in the regulation of Ato expression (Borod and Heberlein, 1998; Dominguez, 1999
; Dominguez and Hafen, 1997
; Greenwood and Struhl, 1999
; Heberlein et al., 1995
). As Hedgehog signaling is required for early events involving the MF, it is not surprising that Hedgehog signaling is required for early expression of Ato and that ectopic Hedgehog signaling leads to increased Ato or precocious Ato expression in the eye disc (Borod and Heberlein, 1998
; Dominguez, 1999
; Greenwood and Struhl, 1999
; Heberlein et al., 1995
; Heberlein et al., 1993b
; Ma et al., 1993
). However, Hedgehog signaling also exerts a powerful repressive effect during later stages of Ato expression, and loss of Hedgehog signaling during stage 4 results in expansion of Ato expression to many cells, though at an expression level lower than endogenous Ato at this stage. These alternate effects are thought to result from a presumed Hh gradient high levels of Hh at the posterior boundary of Ato expression inhibits Ato, whereas low levels further anteriorly induce Ato (Dominguez, 1999
). It is possible that the inhibitory function of Hedgehog signaling is mediated by Ro; in addition to the increased Ato levels observed in loss-of-function clones of smoothened (smo), which prevent all Hedgehog signaling, levels of Ro in the MF are reduced. However, interior smo clones in adult eyes present relatively normal ommatidial organization (Dominguez, 1999
; Dominguez and Hafen, 1997
). As the Notch signaling target Enhancer of split [E(spl)] is expressed appropriately and Sca protein is detected in single cells within smo clones, it is possible that Notch-mediated selection of single R8 cells occurs despite the failure of Ato resolution to single cells (Dominguez, 1999
). This potential uncoupling of Ato expression and R8 selection is striking and unexpected because Ato expression and R8 selection were thought to be inextricably linked. However, a possible explanation is suggested by other data involving the aforementioned ato2 regulatory mutation. In these mutants with a compromised 5' ato enhancer, single R8 cells are selected but have disrupted Ato expression, beginning with the stage 2 intermediate groups and lack all Ato expression by stage 4 (White and Jarman, 2000
). Thus, Ato expression and R8 selection are also uncoupled in this mutant. Taken together, these data demonstrate that late Ato expression (stages 3 and 4) is necessary for normal R8 function but not sufficient for R8 selection.
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R8 spacing: Notch, scabrous and EGFR |
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The Notch signaling pathway has been reviewed extensively (Baker, 2000; Greenwald, 1998
). In brief, Notch (N) encodes a receptor that can be bound by two distinct membrane-bound ligands, Delta (Dl) and Serrate (Ser). Receptor/ligand interactions activate Notch signaling which leads to changes in activity or expression of downstream members of the pathway, including Suppressor of Hairless [Su(H)] and members of the E(spl) complex. Transduction of the Notch signal can lead to a number of outcomes. In some cases, cell fate determination and differentiation are delayed, often via the repression of proneural genes in during a process known as lateral inhibition. In other cases, inductive cues are generated (Baker and Yu, 1997
; Fortini et al., 1993
; Jennings et al., 1994
). Examples of both types of effects are seen in the Drosophila eye.
The combined actions of the Notch pathway and Sca are required for establishing the dynamic Ato expression pattern and R8 spacing. Loss-of-function clones of either N or Dl display low initial levels of Ato expression during stage 1 that do not increase or resolve into later stages of expression, as well as a complete failure of R8 differentiation (Fig. 2A). Moreover, expression of either constitutively active N or Dl ligand induces high levels of Ato anterior to the MF (Baker and Yu, 1997; Baonza and Freeman, 2001
). These findings suggest that Notch signaling is not required for initiation of Ato expression, but for achieving high levels of Ato expression or proneural enhancement. Furthermore, as both proneural gene expression and differentiation are rescued non-autonomously near the borders of Dl clones, it seems that proneural enhancement is a specific function of cells in which Notch signaling is activated (Baker and Yu, 1997
). Mechanistically, proneural enhancement requires alleviation of Su(H) repressor function by Notch signaling but does not rely on conversion of Su(H) to its active form or on E(spl) function (Li and Baker, 2001
; Ligoxygakis et al., 1998
). Moreover, recent data suggests that proneural enhancement requires Decapentaplegic (Dpp) signaling for full efficiency and that Notch signaling represses H and Emc proteins to derepress Ato expression (Baonza and Freeman, 2001
; Greenwood and Struhl, 1999
). Thus, both activation of Ato expression and alleviation of Ato repression appear to contribute to proneural enhancement.
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The expression of N and Dl are not mutually exclusive in the developing eye, and loss of either protein has similar effects on R8 selection and spacing (Baker and Yu, 1998). When N function is removed using a temperature sensitive allele (Fig. 2B), a neurogenic phenotype that leads to premature photoreceptor differentiation of most cells in the MF is observed (Cagan and Ready, 1989
). Specifically, photoreceptors are present in evenly spaced clusters of approximately 10 cells expressing R8 markers, including Ato, while Dlts mutations result in slightly smaller clusters of cells (Baker et al., 1996
; Baker and Zitron, 1995
). Thus, when N signaling is removed, Ato stage 2 intermediate clusters fail to resolve into single R8 cells. This implies that N signaling represses Ato during stage 2 and is required for establishing ommatidia with only one R8 cell. This is an example of classic lateral inhibition. Consistent with this interpretation, the pattern of E(spl) expression is mutually exclusive with that of Ato beginning with stage 2 and misexpression of activated N is sufficient to repress Ato and induce E(spl) in intermediate groups (Baker et al., 1996
; Dokucu et al., 1996
). Furthermore, unlike the process of proneural enhancement, lateral inhibition is mediated by traditional N signaling molecules as loss-of-function clones of Su(H) and E(spl) both lead to neurogenic phenotypes (Li and Baker, 2001
; Ligoxygakis et al., 1998
).
The diametrically opposed effects of Notch signaling in R8 specification raise the following question: what mediates the abrupt transition in Notch function from promoting Ato elevation during proneural enhancement to repressing Ato during lateral inhibition? One explanation is that elevated levels of Ato following proneural enhancement cause a change in sensitivity to Notch signaling. Possible mechanisms for such a change include Ato autoregulation (see above) and induction of potential target genes such as sens, which interacts strongly with the Notch pathway in the developing wing (Nolo et al., 2000). Other explanations are certainly possible, and only future experiments can resolve this conundrum.
The sca locus encodes a fibrinogen-like secreted peptide that is expressed in a subset of Ato-expressing cells beginning in the stage 2 intermediate groups and is maintained in R8 at high levels until just after Ato expression in R8 ceases (Baker et al., 1990; Baker et al., 1996
; Lee et al., 1996
; Mlodzik et al., 1990
). sca mutant eye discs contain more R8 cells than expected. Moreover, these R8 photoreceptors are located too close together and Ato intermediate groups do not form correctly (Fig. 2C) (Baker and Zitron, 1995
; Lee et al., 1996
). Strikingly, misexpression of high levels of Sca has a similar phenotype (Ellis et al., 1994
). The similarity in gain- and loss-of-function phenotypes suggest that differential levels of Sca establish the R8 spacing pattern. In one model for sca function, secreted Sca protein diffuses away from its source in the intermediate groups to surrounding cells where it represses Ato anteriorly. This establishes both the phase and spacing of the developing eye field (Baker and Zitron, 1995
; Baonza et al., 2001
). The mechanism for this function of sca is not known, but it is likely to be independent of Notch signaling (as Nts mutants show proper phase and spacing of R8) and require an unidentified receptor.
Analysis of developing eye tissue mutant for both sca and members of the Notch pathway firmly established their roles in R8 spacing. Tissue mutant for sca and either N or Dl have similar phenotypes: a worsening of either single mutant phenotype such that a nearly continuous field of R8 cells that lack any trace of clustering or organization is observed (Fig. 2D) (Baker and Zitron, 1995). These data, in conjunction with sca and Nts phenotypes, suggest that Sca establishes the intermediate groups and that Notch signaling restricts the intermediate groups to single R8 cells. As sca and N genetically interact, and their encoded proteins bind one another, it is possible that sca provides a bias for Notch signaling in particular regions (Baker et al., 1990
; Baker and Zitron, 1995
; Ellis et al., 1994
; Powell et al., 2001
). Establishment of such a bias by Sca is distinct from its role in determining intermediate groups and would occur within the intermediate groups themselves to potentiate Notch activity and generate single R8 cells through lateral inhibition. Consistent with this model, removal of only the Notch pathway results in large, evenly spaced clusters of R8 cells in which the initial function of sca is preserved, whereas removal of Sca or increased levels of Sca causes a lack of intermediate groups and unbiased Notch activity which results in multiple, randomly spaced, single R8 cells (Baker and Zitron, 1995
; Ellis et al., 1994
).
The role of the EGFR in ommatidial spacing has long been debated, but despite earlier conclusions to the contrary, recent evidence suggests that EGFR signaling via the Ras pathway does participate in R8 spacing (Baonza et al., 2001; Kumar et al., 1998
; Spencer et al., 1998
; Yang and Baker, 2001
). In fact, clones of null mutations in EGFR, other members of the Ras signal transduction pathway, and members of the Rhomboid (Rho) family, which are required for EGFR ligand processing, all result in R8 cells that are spaced too close together (Fig. 2E) (Baonza et al., 2001
; Wasserman et al., 2000
; Yang and Baker, 2001
). Indeed, in both EGFR and rho1, rho3 double mutants, Ato expression is disrupted such that stage 1 expression is expanded and intermediate group formation is disrupted (Baonza et al., 2001
; Wasserman et al., 2000
).
Interestingly, ato function is absolutely required for activation of MAP kinase in the eye (Chen and Chien, 1999). This activation begins in a pattern that overlaps the stage 2 intermediate groups, and MAP kinase signaling then leads to repression of Ato non-autonomously (Chen and Chien, 1999
). As loss- and gain-of-function data suggest that non-autonomous Ato repression via EGFR signaling is crucial in establishing intermediate groups (Lesokhin et al., 1999
); it therefore appears that Ato induces a negative feedback loop to regulate both its own expression and R8 spacing via EGFR-mediated (and Sca-mediated) non-autonomous repression. The mechanism governing EGFR control over R8 spacing is not entirely clear, but does appear to rely on an EGFR ligand that is not Spitz (Spi). The identity of this ligand is unknown, but data suggest that it probably is not a known EGFR ligand, is almost certainly processed by Rho family members and may be encoded by the uncharacterized spitz2 locus (Baonza et al., 2001
; Kumar et al., 1998
; Wasserman et al., 2000
; Yang and Baker, 2001
). As Rough expression is dependent on EGFR signaling and Rough plays a role in both R8 selection and the regulation of Ato, it has been hypothesized that MAP kinase activation in the intermediate groups leads to non-autonomous induction of Rough in the surrounding areas to repress Ato. In existing models, this non-autonomous induction of Rough would be accomplished by a proposed secreted factor that diffuses from the intermediate groups (Baonza et al., 2001
; Dominguez et al., 1998
).
Recently, it was reported that removal of both EGFR and sca function results in a more severe phenotype of multiple R8 cells than is seen in either mutant alone (Fig. 2F). Given the established roles of both EGFR signaling and Sca in intermediate group formation and R8 spacing, this result indicates that the EGFR pathway and Sca probably act independently, implying parallel pathways for establishing R8 spacing and phase. Thus, an inclusive model for R8 spacing that requires both Sca and EGFR signaling has been presented (Baonza et al., 2001; Dominguez et al., 1998
). A key assumption of this model is that the self-organizing properties of the developing Drosophila eye have their roots in the intermediate groups, which produce an inhibitory gradient that represses ato transcription in all directions. New intermediate groups emerge outside of the repressive domain established by the preceding intermediate groups such that they are evenly spaced and out of phase (Fig. 3).
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R8 differentiation: senseless, rough and spalt |
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sens is genetically downstream of ato and Sens expression specifically overlaps Ato beginning in the two to three cells of the R8 equivalence group (stage 3). Expression of Sens is then maintained solely in R8 throughout and beyond larval development. Mosaic analysis of null mutations in sens reveal a cell-autonomous requirement for sens in R8, and no requirement for sens function in any other photoreceptor. Indeed, loss of sens function leads to a complete failure of R8 differentiation and loss of Ato expression in stage 4 in most (75%) cases. Consistent with the position of sens downstream of ato, this failure of R8 differentiation occurs after proper selection and spacing of the presumptive R8 cell has occurred (Fig. 4A). Finally, sens acts at or near the top of the cascade of R8 differentiation as misexpression of sens induces non-R8 photoreceptors to express R8-specific markers and to adopt specific adult morphologic characteristics of an R8 cell (Frankfort et al., 2001).
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Thus, it appears that Sens and Ro have analogous functions during eye development: to control differentiation of a specific cell-type after selection has occurred. Moreover, a mutual antagonism between Sens and Ro is suggested by their non-overlapping patterns of expression, as well as by data demonstrating that the presumptive R8 adopts the R2/R5 fate and expresses Ro when sens function is removed. This is consistent with a model where Sens represses Ro in the developing R8 cell, and is supported by the observation that misexpression of sens is sufficient to repress Ro in non-R8 photoreceptors. Furthermore, simultaneous removal of both sens and ro function results in the restoration of R8 differentiation in many ommatidia (Fig. 4C). Together, these results suggest that sens-mediated repression of Ro regulates the process of R8 differentiation (Fig. 4D) (Frankfort et al., 2001). This finding highlights that an important developmental mechanism, repression of a repressor of cell fate, is at work during a crucial stage of R8 development. A similar mechanism was recently identified during Notch-mediated repression of Hairy and Emc during proneural enhancement, and may be at the root of other aspects of R8 development.
It is important to note that EGFR signaling, though required for the differentiation of all non-R8 photoreceptors, is not required for R8 differentiation, but is required for both R8 cell survival and R8 spacing (Baonza et al., 2001; Kumar et al., 1998
; Yang and Baker, 2001
). However, as ascertained from both ectopic expression experiments and EGFR gain-of-function Ellipse mutations, hyperactive EGFR signaling can hinder or prevent R8 selection (Baker and Rubin, 1989
; Baker and Rubin, 1992
; Chen and Chien, 1999
; Lesokhin et al., 1999
). Furthermore, MAP kinase activation is highest in the intermediate group the future source of R8. Thus, despite exposure to EGFR ligands, expression of EGFR protein, and probably activation of MAP kinase within its cytoplasm, the presumptive R8 somehow remains refractory to the effects of EGFR signaling. How is this accomplished? One published hypothesis is that autoregulatory Ato expression makes the future R8 cell refractory to EGFR signaling (Baonza et al., 2001
). However, another plausible explanation is that Sens represses EGFR signaling in the developing R8 cell. This is suggested by the observations that in sens mutant tissue, MAP kinase activation in the intermediate group is not altered and that Ro is expressed in the presumptive R8 cell (Frankfort et al., 2001
). As EGFR signaling is required for Ro expression, one interpretation is that EGFR signaling is effective in sens mutant presumptive R8 cells, which leads to Ro expression and assumption of the R2/R5 fate. Furthermore, sens mutant clones induced in animals heterozygous for the Ellipse mutation have a phenotype that is reminiscent of Ellipse homozygotes, possibly suggesting that Sens represses EGFR signaling (B. J. F. and G. M., unpublished).
Last, while R8 specification occurs during third instar larval development, terminal R8 photoreceptor differentiation occurs during pupal stages 3 to 4 days later. These late events, which include rhabdomere morphogenesis and opsin expression, are under the control of the spalt (sal) gene complex in both R8 and the other UV-sensitive photoreceptor, R7. Sal proteins are expressed specifically in the R8 and R7 photoreceptors beginning in late-pupal stages (Mollereau et al., 2001). How is the 3 day gap between R8 fate specification and highly specific Sal expression and terminal differentiation bridged? As Sens expression is maintained in R8 into adult life and misexpression of sens is sufficient to induce R8 rhabdomere morphology (B. J. F. and G. M., unpublished) (Frankfort et al., 2001
), Sens is a good candidate for this role. Indeed, a genetic relationship between sens and the sal gene complex has been identified, but the details of this relationship are not understood at this time (B. Mollereau, personal communication).
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Photoreceptor recruitment: role of R8 as a coordinator |
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The function of R8 in the recruitment of R7 has been reviewed extensively and will not be presented in detail here (Cagan, 1993; Raabe, 2000
; Zipursky and Rubin, 1994
). In short, boss encodes a membrane-associated protein with a large extracellular domain that is produced exclusively in the R8 photoreceptor and is absolutely required for R7 development (Hart et al., 1990
; Reinke and Zipursky, 1988
). Boss is presented on the apical surface of R8 such that it is able to bind to the Sev receptor tyrosine kinase (RTK), which is also required for R7 development (Hafen et al., 1987
; Kramer et al., 1991
; Tomlinson and Ready, 1986
). This interaction leads to internalization of the Boss protein in the presumptive R7, phosphorylation of the Sev RTK, induction of MAP kinase signaling and ultimately R7 differentiation (Fortini et al., 1992
; Hart et al., 1993
; Kramer et al., 1991
; Simon et al., 1991
). The position of R8 and precise localization of Boss to the apical cell surface are crucial in restricting the inductive cue to only the presumptive R7 cell and represent a key regulatory role in R8 organizer function (Van Vactor et al., 1991
). Finally, MAP kinase signaling via Spi/EGFR interactions is concomitantly required in R7 for proper differentiation, and combinatorial mechanisms of these two signals have been proposed (Freeman, 1996
; Tio and Moses, 1997
).
The activating ligand for EGFR that is required for photoreceptor recruitment is Spi (Freeman, 1997; Freeman, 1996
; Freeman, 1994
; Lesokhin et al., 1999
; Spencer et al., 1998
; Tio et al., 1994
; Tio and Moses, 1997
; Yang and Baker, 2001
). Moreover, the initial source of this ligand is R8, and there is a cell-autonomous requirement in R8 for spi function in normal ommatidial formation (Freeman, 1994
; Tio et al., 1994
). However, R8 is not the only source of Spi, and the next two sets of photoreceptors to be recruited, R2/R5 and R3/R4, have partial requirements for spi function as well (Freeman, 1994
; Tio et al., 1994
). The spi gene is expressed in photoreceptors other than R8, R2/R5 and R3/R4, but proper processing by both Star (S) and Rhomboid family members is thought to occur only in at most the first five photoreceptors (Heberlein et al., 1993a
; Heberlein and Rubin, 1991
; Hsiung et al., 2001
; Kolodkin et al., 1994
; Pickup and Banerjee, 1999
; Wasserman et al., 2000
). This process of expression and secretion of Spi from the R8 photoreceptor begins before overt differentiation. Specifically, sens mutations that allow R8 selection but not R8 differentiation show successful activation of the EGFR pathway (Frankfort et al., 2001
). Moreover, the same study revealed that R8 differentiation is not required for photoreceptor recruitment, and that differentiation of the presumptive R8 cell as an R2 or R5 photoreceptor results in a founder cell that is nonetheless sufficient to recruit outer photoreceptors of all subtypes (Frankfort et al., 2001
). Thus, all that is required to begin photoreceptor recruitment and differentiation is an initial source of Spi from a founding photoreceptor that need not be R8.
This conclusion helps to explain a puzzling result that ectopic expression of the proneural gene scute (sc) in an ato mutant background is capable of inducing ommatidia that typically lack a discernable R8 photoreceptor (Sun et al., 2000). One explanation for this result is that Sc replaces currently undiscovered basic helix-loop-helix proteins which direct photoreceptor differentiation and that are normally induced by Ato, thereby bypassing the requirement for Ato (Sun et al., 2000
). Another plausible interpretation is that Sc is capable of priming the eye disc so that it is possible for the selection to occur, much like the presumed earliest function of Ato. Then, selection of non-R8 founding photoreceptors occurs and some recruitment of other photoreceptors follows. Future experimentation is required to distinguish these two models.
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A paradigm for neural selection and differentiation |
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Comparison of R8 development to another well-studied Drosophila neural paradigm, sensory organ precursor (SOP) specification, reveals both similarities and differences. As each presumptive R8 cell becomes a single photoreceptor, each SOP gives rise to a single sensory organ, either of the external or chordotonal class. Moreover, the overall mechanism of each is the selection of a single neural precursor from among a field of equipotent undifferentiated cells. Both rely on proneural genes as the key protagonists to establish a zone of neural competency (proneural clusters for SOPs, intermediate groups for R8) from which a single proneural-expressing cell is selected. Finally, both require proneural self-stimulation, repression by other HLH proteins (Hairy and Emc), and Notch-mediated lateral inhibition to achieve this selection (Cubas et al., 1991; Culi and Modolell, 1998
; Jarman et al., 1993
; Skeath and Carroll, 1991
; Van Doren et al., 1991
). However, there are many differences between R8 and SOP selection (Fig. 5).
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The process of R8 specification also has a direct parallel in the mammalian retina: retinal ganglion cell (RGC) development. Evidence for this parallel originally was correlative. For example, analogous to R8 in the Drosophila retina, RGCs are the first cells to be born in the mammalian retina. Moreover, RGCs are selected from among a group of competent progenitor cells that comprise much of the early embryonic mammalian retina, and their differentiation is regulated by Notch (Austin et al., 1995). Furthermore, RGCs develop as a patterned, spaced array at the leading edge of a wave that spreads from the central to peripheral retina during a process reminiscent of MF progression in Drosophila (McCabe et al., 1999
). In addition, despite the fact they are not photoreceptors, RGCs project axons directly to the brain, much as R8 cells do. Recently, a genetic basis for the relationship between R8 and RGCs was established. Two groups reported that Math5, a murine ortholog of ato, is expressed in RGCs and their progenitors coincident with the onset of RGC differentiation, and that Math5 function is required for proper RGC differentiation (Brown et al., 2001
; Wang et al., 2001
). These results imply that Math5 is a proneural gene required for RGC differentiation. Therefore, despite seemingly dissimilar function, R8s and RGCs appear to have much in common with regard to axonal projection, timing of differentiation, and genetic control of cell fate (Kumar, 2001
).
These distinctions make R8 development in Drosophila a unique and relevant paradigm for the study of neural specification. In particular, its self-organizing nature provides an opportunity for the study of relationships that may not be accessible in other systems. Furthermore, despite a common goal of neural differentiation, R8 development and SOP development are very distinct, and comparisons of gene function between the two has already identified a number of example where genetic relationships are changed between the two systems. Finally, as a system with a direct mammalian correlate in RGC differentiation, it follows that the study of R8 specification in Drosophila will probably yield important insights to the more complicated process of mammalian eye development.
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ACKNOWLEDGMENTS |
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