sevenless

DEVELOPMENTAL BIOLOGY

Larval

Sevenless protein is localized on the apical surface of the developing retina. It is found associated with all cells including both photoreceptor cells and cone cells (Banerjee, 1987). It is detectable several hours before any overt differentiation of R7 cells. In addition to apical localization, it is also found deeper in the same tissue, where certain cells contact the R8 cell. This suggests that R8 expresses a ligand (BOSS) for the Sevenless protein (Tomlinson, 1987).

The exact temporal requirements of sevenless gene expression have been demonstrated, revealing the stages of ommatidial development during which the presumptive R7 cell can respond to the presence of Sevenless protein. sevenless gene function is only required during a brief, defined period for the initiation of R7 development; subsequently sevenless is dispensable for both differentiation and function of the R7 photoreceptors (Basler, 1989a).

The expression patterns of sevenless and seven-up overlap in R3 and R4, the two photoreceptors which in addition to R7 express sevenless at high levels (Mlodzik, 1990).

Use of a dominant-negative form of the EGF-R in the eye reveals that EGF-R is required for differentiation of all photoreceptor cell types (R1-R8), including R7 which is also subject to the Sevenless signal. DN-EGF-R is truncated in the 13 amino acids C-terminal to the transmembrane domain. Receptor tyrosine kinases dimerize and transphosphorylate each other upon activation. The removal of the intracellular domain produces a dominant-negative function because receptor molecules without the intracellular tyrosine kinase domain can dimerize with wild-type receptors, but the dimer is unable to signal. Expression of DN-EGF-R behind the morphogenetic furrow causes complete loss of the adult retina. As well as eight photoreceptors, each ommatidium comprises four cone cells and eight pigment cells. Expression of DN-EGF-R in the presumptive cone or pigment cells leads to them not differentiating. Overexpression of secreted Spitz, the ligand of EGF-R causes overrecruitment of all cell types in the ommatidium. Spitz has extracellular protease cleavage sites that allow a fragment with an EGF repeat to be released. Overexpression of membrane-bound full-length Spitz has no effect on eye development. In all cases the source of the extra photoreceptors is the same: transformation into photoreceptors of the "mystery cells" (early members of the cluster, later destined to leave and apparently rejoin the surrounding undetermined cells) (Freeman, 1996).

Just as with EGF-R, overexpression of activated Sevenless recruits extra cells into the ommatidium. Sevenless is also able to recruit additional cone and pigment cells when expressed in the pupal retina. Sevenless can also replace EGF-R function in the wing. Finally, overexpression of secreted Spitz can replace the need for Sevenless. It is concluded that there is no significant difference in the intracellular effects of activation of these two RTKs, even in the R7 cell, where both receptors are required (Freeman, 1996).

A model is proposed for eye development based on these and other observations. First, Spitz activation of DER can trigger all the cell types in the ommatidium, the choice of fate being dependent on when the activation occurs. Argos is an extracellular inhibitor of DER activation (Schweitzer, 1995). Third, the expression of Argos is dependent of EGF-R activation, establishing a negative feedback loop (Golembo, 1996). Fourth, Argos can diffuse further than Spitz. Fifth, the successive waves of induction of each cell type (photoreceptors, cone cells, primary pigment cells, and second/tertiary pigment cells) occur in concentric rings around the ommatidium: each cluster resembles a bullseye. In this model, Spitz is initially produced by the three central cells R8, R2 and R5 and that this recruits the immediately neighboring cells and photoreceptors. In R7, the later activation of Sevenless by its ligtand, Boss, is also required. As cells differentiate, they express Argos, which diffuses outwards, preventing more distal cells from responding to Spitz; Argos is unable to block cells that have already started to differentiate or cells that are exposed to high level of Spitz. Later, more cells start to produce Spitz, overcoming Argos inhibition in the nearest cells. This allows the next concentric ring of cells around the potoreceptors to be recruited, but now as a different cell type, cone cells. Again, Argos prevents more remote cells from responding by diffusing beyond the cone cells (now themselves producing it). Later still, the Spitz source expands again, now recruiting the pigment cells (Freeman, 1996).

The R7 photoreceptor, a unique cell type within the Drosophila ommatidium, was initially proposed to be specified by two distinct signals from neighboring cells, one from the R8 photoreceptor and another from the R1/6 photoreceptor pair. The R8-to-R7 signal is the transmembrane ligand Bride of Sevenless (Boss), which is received by the receptor tyrosine kinase Sevenless (Sev) and transduced via Ras activation within the presumptive R7 cell. However, the identity of the R1/6-to-R7 signal has remained elusive. Evidence is presented that the transmembrane ligand Delta (Dl), expressed by the R1/6 pair, activates the receptor Notch (N) in the presumptive R7 cell and constitutes the postulated R1/6-to-R7 signal required in combination with the Boss/Sev signal to specify the R7 fate (Tomlinson, 2001).

To investigate a role for Delta/Notch signaling in R7 specification, 'experimental' clones of Dl^- cells marked by the white (w) mutation were generated, as well as 'control' clones of wild-type cells similarly marked by w, and the genotypes and phenotypes of mosaic ommatidia in the adult eye were scored. Ommatidia, which are entirely Dl^-, are grossly abnormal, as are the majority of mosaic ommatidia in which many of the cells are Dl^-. However, the remaining mosaic ommatidia develop a normal complement of photoreceptors. 209 such normally composed ommatidia were examined to determine whether any particular photoreceptors or combinations thereof are underrepresented in Dl^- mosaic tissue compared with control mosaic tissue (marked only by the w^- mutation). Such underrepresentation would indicate that Delta expression is required in these cells for correct specification of the photoreceptor cell pattern. Two such examples of underrepresentation were found. In the first instance, only one of the 209 ommatidia scored contained a mutant R8 cell, and only a few contained mutant R2 (8/209) or mutant R5 (28/209) cells. The absence of mutant R8 cells probably reflects a critical role of Delta in the initial establishment of the R8 cell, and the low numbers of ommatidia with mutant R2 and R5 cells likely reflect their close lineage relationship with R8 (Tomlinson, 2001).

In the second example, and of particular relevance to this study, none of the 209 ommatidia scored were mutant for both the R1 and R6 cells, in contrast to the control experiment in which both the R1 and R6 are marked in 34% of the ommatidia. However, all other possible Dl^- mosaic combinations of the R1/6/7 trio occurred at frequencies similar to those of control (wild-type) clones. It is concluded that at least one of the two members of the R1/6 pair must express Delta for the ommatidium to form a normal pattern. Because the R1/6 photoreceptors are specified after all of the other photoreceptors except the R7, this result suggests that Delta activity in these cells is specifically required for the correct specification of the R7 (Tomlinson, 2001).

In wild-type ommatidia, all of the outer photoreceptors (R1-R6) have characteristically large rhabdomeres that extend the depth of the retina, whereas R7 has a small rhabdomere restricted to the more apical regions of the retina. Among the abnormally composed ommatidia that are mosaic for Dl^-, a distinct class can be identified that is identical to phenotypically normal ommatidia except that all three cells in the normal positions of R1, R6, and R7 have large rhabdomeres that extend the depth of the retina. The individual identities of the cells in these ommatidia were inferred from the relative positions of the photoreceptors compared with the surrounding ommatidia; the position of the R8 cell (normally situated basally and between R1 and R2) was used to corroborate the accuracy of this assessment. In such ommatidia, both R1 and R6 positions were invariably found to be Dl^-. Thus, it appears that the absence of Delta function in both R1 and R6 prevents the normal specification of R7 and that the cell in the R7 position develops instead as an outer photoreceptor. The R8 cell was wild-type in all 30 of these ommatidia, confirming the finding that the requirement for Delta signaling for the correct specification of R7 cannot be satisfied by Delta derived from R8 (Tomlinson, 2001).

Delta therefore has the behavior expected for an R1/6-derived signal that is required to specify the R7 fate. Moreover, its role is distinct from that of the R8-to-R7 signal because in the absence of Delta signaling, the presumptive R7 cell chooses an outer photoreceptor fate rather than the cone cell fate chosen when Boss/Sev signaling is compromised. It is likely that the particular outer photoreceptor fate chosen is that of R1/6 (Tomlinson, 2001).

Specification of the Drosophila R7 photoreceptor has emerged as a central paradigm for how a single cell chooses its fate based on signals received from neighboring cells. However, even in this apparently simple case, considerable uncertainty persists about the number of signals involved as well as the roles of these signals. Two signals were initially proposed, based in large part on the observation that the presumptive R7 cell makes a unique set of contacts with the R8 cell and both members of the R1/6 pair. However, only one signal, the Boss/Sev signal sent from R8 to R7, has been identified, and in the absence of any evidence for a second signal, the consensus view in the field has shifted toward one-signal rather than two-signal models. The finding that Notch activation is a necessary signal for R7 specification does not concur with reports that ectopic activation of the Notch pathway in the presumptive R7 cell causes it to develop inappropriately as a cone cell. However, these reports describe experiments using entirely mutant sev-N^intra ommatidia in which the Notch pathway is constitutively activated in several cells within each ommatidium. Under these conditions, many cells choose inappropriate fates, and the ommatidia undergo aberrant rotations and exhibit other disorders, precluding an accurate assessment of the fate of the presumptive R7 cell (Tomlinson, 2001).

One-signal models for R7 specification have been supported in large part by experiments that show that the forced activation of the Ras pathway in the presumptive cone cells appears sufficient to direct them to the R7 fate. However, Delta is expressed in the presumptive R3/4, R1/6, and R7 photoreceptors, which directly contact the presumptive cone cells. Hence, the presumptive cone cells normally receive a Notch input. Accordingly, forced activation of the Ras pathway in these cells would provide the second of the two inputs predicted by the two-signal model to specify the R7 fate. Thus, the experimental finding that forced Ras activation causes presumptive cone cells to choose the R7 fate is compatible with the two-signal model (Tomlinson, 2001).

Another challenge to the two-signal model is the evidence that Ras activity is necessary for the normal specification of cone cells. It has been reported that two genes, sparkling and prospero, show differential expression in the cone cells relative to the R7 cells and are subject to direct regulation by the Ras pathway in cone cells. Taken together with previous observations suggesting that cone cell specification depends on activation of the Drosophila EGF receptor, these results present a paradox. If the choice of the cone cells fate normally requires Ras activation, why does forced activation of the Ras pathway in these cells cause them to develop as ectopic R7s? It is suggested that the R7 and cone cell precursor choose distinct cell fates because they receive different levels of Ras input, higher for the R7 fate (via Boss/Sev signaling) and lower for the cone cell fate (Tomlinson, 2001).

Following the establishment of the ommatidial precluster composed of the R8 cell and adjacent R2, R3, R4, and R5 cells, the surrounding undifferentiated cells undergo a wave of division, giving rise to a distinct population from which the R1/6, R7, and cone cells will be recruited. Unlike the precursor cells that give rise to the precluster, all of the cells within this second population express the transcription factor Lozenge, which appears to distinguish their response to Notch and Ras activation from that of the first round of precursor cells. Two mechanisms are suggested by which Notch and Ras signaling may function combinatorially to direct cells within this Lozenge-expressing precursor population to choose the R1/6, R7, and cone cell fates. In the first model, Notch and Ras activation are viewed as distinct and independent inputs and a simple combinatorial code is proposed for determining the fates of the Lozenge-expressing cells, which are recruited to the ommatidium after the five-cell precluster is established: (1) if a cell receives Ras activation alone, it becomes an R1/6 type; (2) if a cell receives both Ras and Notch activation, it is directed to the R7 pathway; (3) if a cell receives only Notch activation, it becomes a cone cell. It is suggested that during normal development, only the R7 precursor receives both Ras and Notch activation (from the R8 and R1/6 cell, respectively), whereas the R1/6 precursors receive only the Ras input (probably via Spitz/EGF receptor signaling from the neighboring R2/5 cells), and the cone cell precursors receive only the Notch input (presumably via Delta/Notch signaling from neighboring photoreceptors). This combinatorial code is overly simplistic, as there is evidence that cone cell specification depends on Ras activation. Accordingly, the R7 and cone cell precursors are viewed as equivalent in terms of Notch activation, but distinct in terms of Ras activation, with only the presumptive R7 receiving an extra boost in Ras activity via Boss/Sev signaling. A more accurate description of the combinatorial code might therefore be as follows: (1) high Ras alone specifies the R1/6 fate; (2) high Ras plus Notch specifies the R7 fate, and (3) moderate Ras plus Notch specifies the cone cell fate. This model accounts for all of the changes in cell fates observed when Notch or Ras inputs are abolished or ectopically provided to the R1/6, R7, or cone cell precursors (Tomlinson, 2001).

In the second model, the possibility is considered that the Notch and Ras signals are not independent but, rather, that the action of one may facilitate the action of the other. For example, Sev is normally expressed at a low level in the R1/6 precursors but at a high level in the R7 precursor. Hence, one function of Delta/Notch signaling may be to upregulate Sev expression in the R7 precursor, predisposing this cell to receive a high level of Ras activation via its contact with the Boss-expressing R8 cell. When Notch is ectopically activated in the R1/6 precursors, these cells would be induced to express high levels of Sev, and because they also contact the R8 cell, they too would receive high levels of Ras activation. In an extreme version of this model, the R1/6 and R7 fates would be distinguished solely by different levels of Ras activation, and the only role of the Notch/Delta signal from R1/6 would be to boost the level of Ras activation in the R7 precursor cell to reach the threshold necessary to specify the R7 fate. However, less extreme versions of this model are equally tenable. For example, Delta-Notch signaling might serve both to provide a distinct Notch input to the R7 precursor as well as to predispose it to receive a higher level of Ras activation than the R1/6 precursors. In this scenario, the cell-type code would be that (1) moderate Ras alone specifies the R1/6 fate, (2) high Ras with Notch specifies the R7 fate, and (3) moderate Ras with Notch specifies the cone cell fate (Tomlinson, 2001).

Slingshot cofilin phosphatase localization is regulated by Sevenless tyrosine kinase and regulates cytoskeletal structure in the developing Drosophila eye

Animal development requires that positional information act on the genome to control cell fate and cell shape. The primary determinant of animal cell shape is the cytoskeleton and thus the mechanisms by which extracellular signals influence the cytoskeleton are crucial for morphogenesis. In the developing Drosophila compound eye, localized polymerization of actin functions to constrict the apical surface of epithelial cells, both at the morphogenetic furrow and later to maintain the coherence of the nascent ommatidia. As elsewhere, actin polymerization in the developing eye is regulated by ADF/cofilin (Twinstar in Drosophila), which is activated by Slingshot (Ssh), a cofilin phosphatase. Ssh acts in the developing eye to limit actin polymerization in the assembling ommatidia, but not in the morphogenetic furrow. While Ssh controls cell shape, surprisingly there are no direct or immediate consequences for cell type. Ssh protein becomes apically concentrated in cells that express elevated levels of the Sevenless (Sev) receptor-tyrosine kinase (RTK), even those that receive no ligand. This is interpreted as a non-signal driven, RTK-dependent localization of Ssh to allow for locally increased actin filament turnover. It is suggested that there are two modes of actin remodeling in the developing eye: a non-RTK, non-Ssh mediated mechanism in the morphogenetic furrow, and an RTK and Ssh-dependent mode during ommatidial assembly (Rogers, 2005).

Despite some sequence similarities between Drosophila Ssh and MAP Kinase phosphatases, no such activity, could be detected in vivo or in vitro. Consistent with the results of others, it was found that Ssh normally limits actin polymerization in the developing eye. However, this activity is found to be regionally specific: it is limited to the assembling ommatidia, and does not appear to function in the morphogenetic furrow, despite the intense regulation of F-actin there. Recently, a second, structurally unrelated cofilin phosphatase, chronophin (CIN) has been found in vertebrates. A CIN ortholog or some other cofilin phosphatase may control F-actin dynamics in the morphogenetic furrow (Rogers, 2005).

It is suggested that the colocalization of Sev and Ssh in later eye development may be functionally significant. It is interesting to note that the elevated apical deposits of Ssh antigen are seen at a time and place where elevated Sev expression is known to be occurring and also where there is elevated F-actin. In other systems, Ssh has been found to be associated with multi-protein complexes that include signaling receptors or scaffolds. Stimulation of human cells with neuregulin-1beta (an Egfr ligand), triggers lamellipodium formation, results in the dephosphorylation of Ssh, its release from the scaffolding protein 14-3-3 and its translocation to the F-actin rich lamellipodium. LIM Kinase is also activated by neuregulin-1beta and SSH1L. LIMK, actin and 14-3-3 can form a multiprotein complex in vivo. Therefore, it has been suggested that the local activation of SSH1L and LIM Kinase may be coordinately regulated as part of a complex, increasing cofilin-dependent actin filament turnover in areas of intense actin remodeling such as lamellipodia. Also, in cultured rat mammary adenocarcinoma cells engineered to overexpress the RTK EGFR, the application of liganded beads elicits locally elevated actin polymerization, with locally elevated cofilin (Rogers, 2005).

Thus, in the developing eye, local and transiently elevated RTK levels may lead, over a period of hours, to the stabilization of apical actin through the recruitment of cofilin and the molecules that regulate it, such as Ssh. Thus, the coincident Sev and Ssh apical pattern may represent a transient state of elevated RTK expression, which then serves to elicit the apical constrictions that regulate the Armadillo-positive junctional structures, which then stabilize the ommatidial cell clusters. Later in life, this apical constriction is remodeled to form the light sensing rhabdomere: the site of opsin function. It is further suggested that this concentration of Ssh must remain localized to the apical tip of the cell, otherwise (as in mutant clones), the polymerization of actin over-runs and the differentiation and ultimately the viability of the cell is affected. Thus the local, limited, but long-lasting concentration of Ssh at the tips of developing photoreceptor cells in response to RTK levels may serve to stabilize the ommatidial cell cluster. Thus, while no direct biochemical evidence is available, the tight apical co-localization of Sev and Ssh is consistent with a direct interaction between Ssh and a complex that includes RTKs (Rogers, 2005).

Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads

Drosophila germline stem cells are regulated by the somatic microenvironment, or 'niche,' which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. A receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, it is proposed that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads (Kitadate, 2007).

These data show that the posterior somatic gonadal cells (SGCs), as well as the anterior SGCs, have the capacity to contribute to the germline-stem-cell niche within the male embryonic gonads. However, during development, niche differentiation is normally repressed in the posterior SGCs by Sev. In the absence of Sev activity, posterior SGCs are recruited to form an expanded niche. Sev is activated in the SGCs by the Boss ligand emanating from pole cells. This implies that varying the number of pole cells will alter the niche size. A model predicts that a decrease in the number of pole cells should induce ectopic niche formation within the gonads, which consequently increase their chance to recruit pole cells as the stem cells. Thus, it is speculated that the interaction between SGCs and PGCs via the Boss/Sev pathway acts as a key component of a negative-feedback loop to maintain an optimal number of germline stem cells in male gonads. A similar feedback mechanism has been reported in the stem-cell system of plant meristem and in Drosophila larval ovaries (Kitadate, 2007).

However, it was found that overexpression of a constitutively active Sev results in neither hyperactivation of Rl nor repression of hub-cell fate in the anterior SGCs, suggesting that activation of signaling components downstream of Sev is suppressed in the anterior SGCs. It has been widely accepted that the Notch transmembrane receptor and the receptor tyrosine kinase (RTK)/RAS/MAPK pathways antagonize each other in various developmental contexts, and that Notch signaling is involved in stem cell maintenance and differentiation in several stem-cell systems. In Drosophila ovaries, Notch signaling is required for the formation and maintenance of the germline-stem-cell niche. Overexpression of activated Notch induces expansion of the niche, while a reduction of Notch activity results in loss of the niche. In addition, germline cells express ligands for Notch to induce Notch-receptor activity and thereby to promote their own maintenance and function within the niche. Since the Notch receptor is also expressed predominantly in SGCs of male embryonic gonads, it is likely that Notch may antagonize Boss/Sev signaling in the anterior region of the gonads. It is speculated that the negative- and positive-feedback loops between germline and soma through Sev and Notch signaling act antagonistically to regulate proper niche formation during gonadal development. It will be interesting to test this hypothesis in future experiments. This study provides an important step toward understanding the regulatory mechanisms of niche formation in germline development (Kitadate, 2007).

beta amyloid protein precursor-like (Appl) is a Ras1/MAPK-regulated gene required for axonal targeting in Drosophila photoreceptor neurons

beta amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease, was found in a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development. Appl expression was found in the eye imaginal disc and it is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, Appl null mutants were analyzed and reduced preference for UV light was found, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, whether Ras1/MAPK is responsible for the high Appl expression was examined. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. Appl was shown to be directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism (Mora, 2013).

Two main conclusions can be drawn from this work. First, Drosophila Appl is involved in R7 axonal targeting. Moreover, the finding that the Appl loss-of-function defects are enhanced when combined with Nrt heterozygous mutant suggest that Appl acts at the membrane of R7, where it interacts with other proteins such as Nrt. Second, Appl activation downstream of the RTK/Ras1 is independent of neural specification, occurs in vivo, and is mediated by direct binding of PntP2 to ETS sequences in the Appl regulatory region (Mora, 2013).

Together, these findings may provide insights into the pathogenesis of neurological disorders such as Alzheimer's disease. The β-amyloid peptides, which accumulate in the amyloid plaques found in the brain of Alzheimer's disease patients, are produced after APP proteolysis. However, Alzheimer's disease has not only been associated to the production of the primary component Aβ by proteolysis of APP, but also by transcriptional regulation. Increased APP transcription underlies the phenotype in some cases of familial Alzheimer's disease. In addition, overexpression of APP appears to be responsible for the early onset of Alzheimer's disease in individuals with Down syndrome. Thus, the current results open the possibility to explore whether in some cases of Alzheimer's disease a burst of RTK/Ras1/MAPK occurs and whether this signaling activity ends with high APP accumulation (Mora, 2013).

Amyloid β peptides are known to be involved in vision dysfunction caused by age-related retinal degeneration in mouse models. Thus, the current in vivo observations could be the basis for further research in mammalian models for neurodegenerative retinal disorders that share several pathological features with Alzheimer's disease (Mora, 2013).

Effects of Mutation or Deletion

Temperature-sensitive alleles of sev show that SEV activity is required for several hours during the development of each R7 cell to specify R7 cell differentiation. This suggests that the presumptive R7 cell remains for approximately 5 hr in an undetermined state in the absence of the SEV-mediated signal before committing to an alternative fate. One mutation disrupts the Gly-Xaa-Gly-Xaa-Xaa-Gly consensus in the ATP-binding site of the Sevenless kinase domain (Mullins, 1991).

The use of ectopic expression of sevenless and rough has provided insight into the mechanisms of positional signaling 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, like other homeobox genes, acts as a selector gene that determines the fate of single cells (Hafen, 1990).

A gain-of-function sevenless mutation (SevS11), consisting of a truncated Sevenless protein, is overexpressed in the cells where sevenless is normally expressed. In SevS11 mutant flies, all sevenless-expressing cells initiate neural development. This results in the formation of multiple R7-like photoreceptors per ommatidium. Therefore, sevenless activity appears to be necessary and sufficient for the determination of R7 cell fate (Basler, 1991).

The Sevenless protein is expressed transiently in 8 of the 20 precursors of an ommatidium. Activation of the Sevenless kinase in these eight cells indicates that six of them are competent to become R7 cells. To test the competence of all 20 ommatidial precursors in a temporally unrestricted manner, a constitutively activated Sevenless kinase has been created and expressed in all 20 precursors. Competence to develop as neuronal cells in response to Sevenless activity is spatially and temporally limited to the cells expressing sevenless. Therefore, the expression of sevenless marks a preexisting pattern of developmental potential in the disc epithelium (Dickson, 1992).

Some aspect of R7 differentiation is independent of the genetic pathway(s) involving sevenless, BOSS and sina. An enhancer trap line, H214, has been developed in which beta-galactosidase is primarily expressed in the R7 cell throughout its development. In mutations of sevenless, boss and sina, expression in H214 is initially reduced although still present in the R7 precursor and persists in the Equatorial cone cell (the alternative fate of R7 cells in mutants) into which R7 cells develop. The EQC in wild type never expresses lacZ in H214. Thus the presumptive R7 cell receives positional information independent of sevenless (Mlodzik, 1992).

A Drosophila gene with similarity to the mammalian Ras GTPase activating protein has been isolated in screens for mutations that affect eye development. Inactivation of the locus, GTPase-activating protein 1 (Gap1), mimics constitutive activation of the Sevenless receptor tyrosine kinase and eliminates the need for a functional Sevenless protein in the R7 cell. These results suggest that Gap1 acts as a negative regulator of Sevenless signaling by down-regulating the activity of the Ras1 protein, which has been shown to be a key element in signaling by Sevenless (Gaul, 1992).

An Enhancer of sevenless mutation acts as a dominantly inhibiting allele of corkscrew. csw function is essential for Sevenless signaling. Expression of a membrane-targeted form of CSW can drive R7 photoreceptor development in the absence of sevenless function. The dominantly inhibiting CSW shows a substitution of glutamate for glycine at codon 547. In mutant eye discs, prepared by transducing the dominantly inhibiting CSW, were examined, elav expression appeared to be normal at the stage when R8, R2 and R5 express elav. However, subsequent staining for Elav was abnormal. Staining of cells occupying the normal position of R3 and R4 was only rarely observed. Transduction of normal csw suppresses the dominant negative mutant. The dominantly inhibiting csw allele was used to examine the role of CSW during signaling by activated forms of Ras1 and Raf. csw function is still required during signaling by activated Ras1 and Raf proteins. These results define a function for CSW that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development (Allard, 1996).

Eight alleles of Dsor1 encoding a Drosophila homolog of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppress mutations for receptor tyrosine kinases (RTKs) torso, sevenless, and to a lesser extent, Drosophila EGF receptor. Furthermore, the Dsor1 alleles show no significant interaction with gain-of-function mutations of Egfr. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrates that Dsor1 can activate yeast MAP kinase homologs if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes reveal that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors rather than constitutive activity (Lim, 1997).

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. faf also interacts with pointed: the extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism (Isaksson, 1997).

Evidence for information flow from R7 to R8 photoreceptors, that is in a direction that is the reverse of that usually thought to occur, comes from a study of rhodopsin expression in sevenless mutants. The function of the compound eye is dependent on a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant flies, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes (Chou, 1996).

Additional evidence for information flow from R7 to R8 photoreceptors comes from a subsequent study of rhodopsin expression in sevenless mutants. The photoreceptor cells of the Drosophila compound eye are precisely organized into elementary units called ommatidia. The outer (R1-R6) and inner (R7, R8) photoreceptors represent two physiologically distinct systems with two different projection targets in the brain. All cells of the primary system, R1-R6, express the same rhodopsin and are functionally identical. In contrast, the R7 and R8 photoreceptors are different from each other. They occupy anatomically precise positions, with R7 on top of R8. In fact, there are several classes of R7/R8 pairs, which differ morphologically and functionally and are characterized by the expression of one of two R7-specific opsins: rh3 or rh4. A new opsin gene has been identified, rhodopsin 5, expressed in one subclass of R8 cells. Interestingly, this subclass represents R8 cells that are directly underneath the R7 photoreceptors expressing rh3, but are never under those expressing rh4. These results confirm the existence of two subpopulations of R7 and R8 cells, which coordinate the expression of their respective rh genes. Thus, developmental signaling pathways between R7 and R8 lead to the exclusive expression of a single rhodopsin gene per cell and to the coordinate expression of another one in the neighboring cell. Consistent with this, rh5 expression in R8 disappears when R7 cells are absent (in sevenless mutant) (Papatsenko, 1997).

The most striking observation reported here is that rh5 is expressed in a subset of R8 cells, which corresponds exactly to the R7 cells expressing rh3. This makes it very likely that there is a mechanism for synchronization of rh gene expression between the two cells. Two models are suggested:

(1) Two subclasses of ommatidia may be specified early, at the time of the determination of the eight photoreceptor clusters. Later on, they may express either the rh3/rh5 pair, or the rh4/putative rh6 pair. It must be noted that rh gene expression occurs at least 4 days later than photoreceptor determination. In favor of this model, enhancer trap lines have been identified that are expressed in a subset of R8 cells just posterior to the morphogenetic furrow. Unfortunately, their expression does not last long enough to overlap rh expression. This may either represent an early distinction of different R8 cells during photoreceptor differentiation, or may be simply due to variable expression of a weakly expressing line.
(2) The distinction between the two types of R7/R8 pairs appears late, when the rh genes are first transcribed. Expression of a specific rh gene (in R7 or in R8) may be a stochastic event, which is reinforced by an unknown mechanism, while expression of the other rh gene would be repressed in this cell. Communication between the R7 and R8 cells expressing specific rh pairs (at least one signal going from R7 to R8, as shown by loss of rh5 expression in sev mutants) would permit coordination of expression of the rh genes. It is possible that the Rhodopsin molecules themselves mediate this communication, in a manner that is similar to what has been observed for the olfactory system. The olfactory receptor molecules serve as guidance signals for projection to specific targets in the olfactory bulb (Papatsenko,1997).

Although the absence of rh5 in sevenless mutants does not provide evidence in favor of either model, it may reflect the existence of a positive feedback loop in the interaction between the R8 and R7 cells. Since the R8 cells do not develop properly (rh5 is not expressed) in the absence of R7, a signal from R7 must be necessary to complete retinal development and for the formation of the different subclasses of ommatidia. The sevenless-boss pathway, or some part of it, may be involved in this interaction. Alternatively, a totally distinct pathway may lead to coordination of rh gene expression in R8. It is interesting to mention that physiological data indicate that there are still functional R8 cells in sevenless mutants. Indeed, a photopigment could be detected in the retina of flies lacking both functional R1-6 cells (ninaE mutants) and R7 cells (sev mutants). Thus, an unknown rhodopsin (rh6?), which is predicted to be expressed in the remaining subset of R8 cells (R8y; in concert with rh4 in R7y), could be expressed in all R8 cells in sevenless flies. If this is true, the R7 cell controls the expression of rh5 in R8, and thus the determination of the particular subclass of ommatidia. In other words, the decision for transcriptional exclusion between rh3/5 and rh4/6 would occur in R7, downstream of the boss-sev signaling and upstream of the suggested feedback control from R7 to R8. It must be noted that expression of rh3 in R8 cells of the dorsal margin is not affected in sevenless mutants. This indicates that at least some of the R8 development is not affected by R7 cells. In conclusion, the discrimination between rh3 and rh4 (and between rh5 and putative rh6) expression, and the coordination between pairs of rh genes in neighboring photoreceptors provides a powerful paradigm for studying not only the mechanism of transcriptional exclusion often found in sensory systems, but also for studying the concerted evolution of rhodopsin genes in achieving specific functions, such as color vision (Papatsenko, 1997).

Opsin gene expression in the R7 and R8 photoreceptor cells of the Drosophila compound eye is highly coordinated. The R8 cell specific Rh5 and Rh6 opsins are expressed in non-overlapping sets of R8 cells, in a precise pairwise fashion with Rh3 and Rh4 in the R7 cells of individual ommatidia. Double labeling with antibodies against both Rh6 and Rh1 show that the Rh6 protein is restricted to the proximal retina, and that Rh6 is only expressed in a subset of ommatidia. This result was confirmed by preparing dissociated ommatidia from adult fly heads and labeling them with antibodies against Rh6 and Rh1. The R8 rhabdomere that contains Rh6 is located basally within the ommatidium and is surrounded by the rhabdomeres of the Rh1-expressing R1-6 cells. No evidence is found of Rh6 expression in other regions of the head, retina, or in the ocelli. These findings indicate that Rh6 is expressed specifically in a subset of R8 photoreceptor cells (Chou, 1999).

Removal of the R7 cells in sevenless, boss or sina mutants, disrupts Rh5 expression and dramatically increases the number of Rh6-expressing R8 cells. This suggests that the expression of Rh5 may be induced by an Rh3-expressing R7 cell, whereas Rh6 expression is most likely a default state of the R8 cell. The paired expression of opsin genes in the R7 and R8 cells occurs in a sevenless and boss independent manner. Furthermore, the generation of both Rh3- and Rh4-expressing R7 cells can occur in the absence of an R8 cell. These results suggest that the specification of opsin expression in the R7 cells may occur autonomously, whereas the R7 photoreceptor cell may be responsible for regulating a binary developmental switch between induced and default cell-fates in the R8 cell. An immediate question raised by these results is the nature of this novel signaling pathway and the identity of the molecules that mediate it. It is quite clear that neither sev nor boss are required for the establishment of the paired expression of visual pigments in the R7 and R8 cells, and that the presence of either boss or sev or both is insufficient to restore Rh5 expression in the absence of an R7 cell. These results indicate that there must be other novel signaling molecules that are responsible for patterning of the R7 and R8 cells (Chou, 1999).

In the course of these experiments, a number of apparent exceptions to the predominant even or odd pairing of opsins expressed in the R7 and R8 photoreceptor cells were found. In particular, instances of expression of Rh3 and Rh6 in the R7 and R8 cells of individual ommatidia occurred in 6% of cases. Because Rh5 is not expressed in these R8 cells, and because Rh5 expression appears to be an induced state, it may be that the geometry of the R7 and R8 cells in these exceptional ommatidia is somehow abnormal at the time when this signal is required. Thus, these R8 cells may have failed to receive the inductive signal, and then assumed the Rh6-expressing default state. Consistent with this, studies of ommatidia having multiple R7 cells seem to support the idea that the signal to induce Rh5 expression must be highly spatially restricted. Additionally, ommatidia from wild type strains that show paired expression of Rh4 and Rh5 are never found. This suggests that while the inductive signal may fail in some rare cases, Rh5 can never be induced in a normal eye by an Rh4-expressing R7 cell because it does not transmit the inductive signal. Curiously, in mutant strains that lack R7 cells, Rh5 expression is not completely abolished. Many of these Rh5-expressing cells are morphologically abnormal, in that they have long rhabdomeres or rhabdomeres in an apical position. Such photoreceptor cells have been observed previously in sev mutants and shown to be R8 cells, based on their axonal projections and the timing of their terminal mitoses. Perhaps these cells are receiving or improperly processing an inductive signal, based on their abnormal position within the eye. This result does show however, that Rh5 expression is not absolutely dependent upon the presence of the R7 cell. The signal used for the induction of Rh5 could be used elsewhere within the eye for other purposes, or the signal from the R7 cell may not be direct. Identification of the genes required to establish R7 and R8 cell patterning should allow a resolution of some of these questions (Chou, 1999).

In Drosophila, Src oncogene 1 was considered a unique ortholog of the vertebrate c-src; however, more recent evidence has been shown to the contrary. The closest relative of vertebrate c-src found to date in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time in this paper. In contrast to Src64, overexpression of wild-type Src41 causes little or no appreciable phenotypic change in Drosophila. Both gain-of-function and dominant-negative mutations of Src41 cause the formation of supernumerary R7-type neurons, suppressible by one-dose reduction of boss, sevenless, Ras1, or other genes involved in the Sev pathway. Dominant-negative mutant phenotypes are suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type Src41. The colocalization of Src41 protein, actin fibers and DE-cadherin, as well as the Src41-dependent disorganization of actin fibers and putative adherens junctions in precluster cells, suggest that Src41 may be involved in the regulation of cytoskeleton organization and cell-cell contacts in developing ommatidia (Takahashi, 1996).

To determine if karst, coding for

betaHeavy Spectrin belongs in the Sevenless signaling pathway, an epistasis experiment was performed in which the constitutively activated Sevenless receptor, Sev S11 was crossed into a karst mutant background. In a wild-type background, Sev S11 produces an excess of R7 cells because it is expressed in more than one cell per precluster. Siblings with three phenotypes (karst, S11 and S11/karst) were analyzed. If karst is epistatic to Sev S11 (i.e. blocking signaling when penetrant), the mean number of R7s per cluster in S11/karst double mutant flies would be expected to be lower than the mean in S11 flies. Unfortunately, the wider effects of the karst mutation on cell position and rhabdomere morphology prevent a reliable counting of the number of R7-type photoreceptors in S11/karst flies. Nevertheless, the mean number of photoreceptors per ommatidium for the S11/karst flies is not significantly different from the S11 flies. This is consistent with the interpretation that Sev S11 is epistatic to karst, suggesting that karst is upstream of Sevenless or in a parallel pathway contributing to the ligand interaction (Thomas 1998).

Whether D-Fos mediates ERK signaling during eye morphogenesis was investigated. Defects in photoreceptor differentiation can be induced by the RTK gain-of-function alleles Elp^B1 and sev^S11. The Elp^B1 allele dominantly causes an abnormal eye phenotype that manifests itself in roughness and the occasional lack of outer photoreceptors. This phenotype can be suppressed largely by the removal of one copy of D-fos and restored subsequently by simultaneous transgenic expression of wild-type D-Fos. A gain-of-function transgene of the RTK-coding gene sevenless (sev^S11) causes the characteristic appearance of ectopic R7 photoreceptor cells in nearly all ommatidia. The sev^S11 phenotype can be suppressed by the expression of dominant-negative Fos. In flies carrying sev^S11 in a heterozygous kay² background, the ectopic R7 photoreceptor phenotype is suppressed significantly; the number of normal ommatidia increases from 5% to approximately 20%. Reintroduction of D-fos by a transgene in this double mutant background restores the percentage of ommatidia with extra photoreceptors observed in sev^S11 heterozygous animals. Taken together, these results indicate that D-Fos can act as a rate-limiting component downstream from the RTKs Sev and DER during eye development (Ciapponi, 2001).

Modulation of reactive oxygen species (ROS) plays a key role in signal transduction pathways. Selenoproteins act controlling the redox balance of the cell. This study reports how the alteration of the redox balance caused by patufet (selD^ptuf), a null mutation in the Drosophila melanogaster selenophosphate synthetase 1 (sps1) gene, which codes for the SelD enzyme of the selenoprotein biosynthesis, affects the Ras/MAPK signaling pathway. The selD^ptuf mutation dominantly suppresses the phenotypes in the eye and the wing caused by hyperactivation of the Ras/MAPK cassette and the activated forms of the Drosophila EGF receptor (Egfr) and Sevenless (Sev) receptor tyrosine kinases (RTKs), which signal in the eye and wing, respectively. No dominant interaction is observed with sensitized conditions in the Wnt, Notch, Insulin-Pi3K, and DPP signaling pathways. The current hypothesis is that selenoproteins selectively modulate the Ras/MAPK signaling pathway through their antioxidant function. This is further supported by the fact that a selenoprotein-independent increase in ROS caused by the catalase amorphic Catⁿ¹ allele also reduces Ras/MAPK signaling. This study presents the first evidence for the role of intracellular redox environment in signaling pathways in the Drosophila organism as a whole (Morey, 2001).

The most striking result is that selD function modulates the activity of Sev and Egfr RTKs, in the eye and wing respectively, suppressing in transheterozygous combination the phenotype caused by the gain-of-function mutations of these RTKs. In contrast, InR, another well-known RTK belonging to the insulin pathway, shows no modulation by selD^ptuf mutation neither at the InR RTK level nor at the level of the Pi3K. Therefore, selD somehow modulates RTKs involved in the activation of the Ras/MAPK cassette but not other RTKs. The suppression of the Raf gain-of-function phenotype has been detected both in the eye and wing. A mild but statistically significant suppression of the rl^Sem phenotype by selD^ptuf has been found in the eye but not in the wing. This is probably due to the different sensitivity of both systems. The mild suppression of rl^Sem phenotype could be due to the fact that the MAPK is the last step of the pathway prior to the transcription of target genes. At this point of the pathway, it may be difficult to block or dilute the amplified signal triggered by the rl^Sem gain-of-function mutation (Morey, 2001).

Surprisingly, no modulation of ras by selD^ptuf mutation has been detected. There are two main explanations for this result: (1) ras is not modulated by selD at all. The Sev, Egfr, Raf and MAPK molecules, which are modulated by an alteration of the redox balance, either by selD^ptuf or Catⁿ¹ mutations, are kinases, whereas Ras is a GTPase. It is possible that GPTases are not sensitive to changes in the redox balance. This is supported by the fact that rasV12 rough eye phenotype is suppressed neither by selD^ptuf nor Catⁿ¹ mutations. Similarly, the Rho1 Small-GTPase rough eye phenotype is not suppressed by selD^ptuf either. (2) ras is actually modulated by selD but beyond the detection level, and therefore, no suppression can be scored. It could also be that this modulation is towards an enhancement. It has been reported that 3T3 fibroblasts, when stably transformed with rasV12, produce large amounts of ROS superoxide (O22) by the activation of the NADPH oxidase enzyme, which is triggered by the Rac signaling pathway. There is evidence that in certain cells MAPK activation in response to growth factors is dependent on the production of ROS. Reduction of 50% of selD dose might be not enough to alter the initial ras rough eye phenotype, neither to detect a suppression nor an enhancement. Besides, it must be kept in mind that Ras is a crosstalk point for other signaling pathways (such as Pi3K) and that the Raf/MAPK pathway is also activated in a Ras independent way (Morey, 2001).

With the exception of ras, a gradient was observed in the strength of the suppression of phenotype has been observed from sev being the strongest to rl^Sem the weakest. This could be attributed to the differential sensitivity of the particular gain-of-function alleles or to the position in the pathway of the triggering gain-of-function element (Morey, 2001).

Homozygous selD^ptuf mutants lack selenoproteins and accumulate free radicals. Heterozygous selD^ptuf individuals show no sign of impaired Ras/MAPK signaling due to the loss of one dose of selD. However, when using activated elements of the Ras/MAPK pathway, the effects of the lack of one dose of selD are evident. Therefore, it is tempting to speculate that selD^ptuf in heterozygosis results in an accumulation of ROS due to lower activity of the selenoproteins biosynthesis. This accumulation would be sufficient to impair the Ras/MAPK signaling, suppressing the gain-of-function phenotypes of elements of the pathway. However, the findings are in contrast with the results obtained in tissue culture experiments. It has been observed that ligand stimulation with peptide growth factors acting through RTKs results in an increase in intracellular ROS. Moreover, ligand-stimulated ROS generation appears to have a role mediating tyrosine phosphorylation. In addition to that, it has also been shown that extracellular administration of non-lethal concentrations of H2O2 activates MAPK. Altogether, these results in tissue culture systems point to activation of the pathway by ROS (Morey, 2001).

In this study, elimination or reduction of selenoprotein function could result in prolonged activity of the signaling pathway (due to the ROS increase caused by selD^ptuf) and consequently induce apoptosis. Therefore, one could conclude that the suppression observed in these experiments could be due to cell death of the extra R7s. Loss of any cell of the sevenless equivalence group by apoptosis will result in rough eye phenotype. However, a rescue of the normal ommatidium organization, which can only be achieved if the number of cells of the ommatidium is not altered, has been demonstrated. For this reason, it is thought that apoptosis does not explain the strong suppression observed in these experiments. Rather, these results point to a downregulation of the pathway. The discrepancies found between cell culture and Drosophila as a whole organism (i.e., activation versus downregulation of the pathway by ROS respectively) may be due to the inherent differences between the two systems used. A more important difference, however, is that experiments performed in tissue culture study the effect of transient low concentration increases of ROS whereas in this system a gene-dosage dependent constant increase in ROS occurs. To reconcile these discrepancies, it is proposed that transient increases in ROS could have a physiological function on activation of phosphorylation and thus trigger the Ras/MAPK signaling pathway, whereas a constitutive pathological change in redox potential could activate a defense mechanism blocking the pathway (Morey, 2001).

This is the first example of the role of intracellular redox environment on the Ras/MAPK signaling pathway in a whole organism. The high specificity of these results (i.e., no interaction with other signaling pathways and results confirmed in two different developing tissues (wing and eye) gives strong support to the notion that signaling through the Ras/MAPK pathway is modulated by ROS. The current hypothesis is that selenoproteins, through an undisclosed subset of ROS, modulate the Ras/MAPK signaling pathway. This is consistent with the finding that this pathway is also modulated by catalase. A selenoprotein-independent increase of a subset of ROS (i.e., H2O2) is able to modulate this pathway as well. This observation favors a scenario in which modulation of the Ras/MAPK pathway by selenoproteins would be achieved by their control of the redox balance rather than one in which selenoproteins would exert their role directly, interacting with one or more elements of this signaling cassette. These results may help to shed light on the role of redox on signaling events under physiological conditions in multicellular organisms (Morey, 2001).

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