EGF receptor


Table of contents

Egfr and eye morphogenesis (part 2/2)

Regulation of EGF receptor signaling establishes pattern across the developing Drosophila retina

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

DERElp is a mutation that has been demonstrated to alter the spacing of ommatidia. It is a hypermorphic (gain-of-function) allele of Egfr. The eyes of DERElp /+ flies are rough and irregular: spacing between ommatidia is uneven and somewhat fewer ommatidia overall are present when compared to wild type. This loss of ommatidia appears to be due to repression of Atonal expression within the MF: the initial stripe of Atonal (Stage 0) is unaffected, but expression is lost in the region where proneural clusters normally form. Remarkably, Atonal expression reappears in a 1-3 cell group (Stages 2, 3), and the majority of R8s still form. In DERElp homozygotes, nearly all ommatidia fail to form, presumably due to a more extensive loss of Atonal. These results suggest that high levels of DER activity can repress Atonal expression and thereby alter the spacing of R8 photoreceptors and developing ommatidia. They also suggest that emergence of the R8 equivalence group may not depend on prior formation of a proneural cluster or Atonal expression within it. To further explore the role of Egfr signaling on R8 specification, an activated form of the downstream target Dras1 was employed. Flies containing Dras1Val12 fused to an inducible heat shock promoter were subjected to a 1 hour heat shock followed by a rest period at room temperature to assess the effects of transient, ubiquitous expression. Within 2 hours after the initiation of Dras1Val12 expression, Atonal expression was strongly upregulated throughout the MF, leaving a broad unpatterned band of Atonal in the region where it is normally partitioned into proneural clusters. This expansion in Atonal results in the production of ectopic R8s, as assessed by Boss expression 10 hours after heat-shock. Boss is an R8- specific protein that begins expression 6-8 hours after cells have left the MF; its ectopic expression 8-10 hours after heat-shock indicates the additional R8s are derived from cells within the MF at the time of heat-shock. Based on incorporation of the nucleotide analog BrdU, the presence of ectopic R8s is not due to cell proliferation within the MF, nor is any alteration in Atonal or Boss expression observed in wild-type flies receiving a similar heat shock regimen. Interestingly, not all cells prove sensitive to R8 induction, suggesting that not all cells within the MF are competent to respond to Ras pathway signaling in this manner. These results indicate that strong Dras1 signaling can upregulate Atonal expression; however, only cells within a restricted zone are competent to respond to increased Atonal and ras pathway signaling by differentiating as R8s. The upregulation of Atonal expression is followed by a ‘rebound’ downregulation. Loss of Atonal expression is observed 4-6 hours after transient expression of Dras1Val12, leaving only a few cells near the posterior edge of the MF that still retained Atonal. Loss of Atonal is accompanied by an expansion in the expression of two inhibitors of Atonal function, Rough and E(spl), and a stable loss of R8 cells as assessed by Boss expression (see above). The arrest in the addition of new R8s persists for approximately 20 hours before reinitiating. This diminished Atonal staining is similar to that seen in DERElp and argues that strong or chronic ras pathway signaling may induce factors that feed back to shut down endogenous DER/Dras1 activity (Spencer, 1998).

Ubiquitous activation of Dras1 signaling eliminates pattern within the MF, yet endogenous Dras1 is expressed at high unpatterned levels throughout the MF. This raises the question as to which cells display active Dras1 signaling. One useful indicator of ras pathway activation is phosphorylation of the downstream target ERK: activation of the EGF receptor or Ras leads to phosphorylation of ERK on two closely spaced residues. This activation can be assessed with an antibody specific for the phosphorylated form, and an alpha-dpERK antibody recognizes doubly phosphorylated Drosophila ERKA (dpERKA). Remarkably, although ERKA is expressed in an unpatterned fashion throughout the eye disc, presence of the activated form, dpERKA, is restricted to a repeating linear pattern within the MF. Based on co-localization with Atonal expression, ERKA is activated specifically within the proneural clusters (Stage 2) and is complementary to the expression patterns of Rough and E(spl). DpERKA is found to localize primarily to the cytoplasm, though occasional nuclear localization is also observed. Ubiquitous expression of Dras1Val12 results in broad expansion of the region of cells containing dpERKA within 30 minutes, a time course significantly faster than expansion of the Atonal region. All dpERKA is lost in a ‘rebound’ 2-3 hours later. No alteration in dpERKA pattern or intensity is observed in wild type control discs that receive the same regimen of heat shocks. Therefore, although Dras1 is normally present throughout the MF and is capable of activating ERKA, the ras pathway is only highly active within the proneural clusters. Use of constitutively active and dominant negative Egfr shows that Egfr is the receptor-tyrosine kinase responsible for activating the ras pathway in the proneural clusters. Blocking Egfr activity results in a loss of proneural clusters as assessed by loss of dpERKA, a loss of Atonal expression in the proneural clusters, and a block in R8 formation (Spencer, 1998).

Rhomboid is a seven membrane-spanning protein that enhances Egfr signaling and activation of ERKA. It can activate Egfr signaling several cell diameters from the source of its expression, apparently by regulating release or activity of the DER ligand Spitz. An antibody specific for Rhomboid shows it to be expressed in cells near the posterior edge of each proneural cluster; a Rhomboid enhancer trap line confirms that expression begins in the 1- to 3-cell R8 equivalence group, based on the positioning of the group within the larger ‘proneural cluster’ and the apical position of its nuclei. Expression then quickly resolves to a single cell that can be unambiguously identified as R8. The previously demonstrated ability of Rhomboid to activate Egfr signaling at a distance suggests that cells of the R8 equivalence group could use Rhomboid to set the pattern of Egfr/Dras1 activation across the proneural cluster. To test this possibility, hs-rhomboid flies were used to express Rhomboid throughout the MF. The result was similar to the effect of expressing Dras1Val12: within 30 minutes of the initiation of ectopic Rhomboid expression, an unpatterned stripe of phosphorylated dpERKA emerges throughout the MF. This suggests that, indeed, expression of Rhomboid is sufficient to activate ras pathway signaling within the MF. The ‘rebound’ effect seen with ectopic Dras1Val12 is also observed with ectopic Rhomboid, but with a more rapid time course. 1-2 hours after initiating ectopic Rhomboid expression, all detectable dpERKA as well as Atonal expression in the proneural groups is lost; this is observed even if Rhomboid is expressed continuously during this period. In addition, the upregulation of Argos expression observed with ectopic activation of Dras1Val12 is also observed with ectopic expression of Rhomboid. Unlike experiments with Dras1Val12, only a minor expansion of Atonal is observed with Rhomboid overexpression, presumably due to the brevity of ras pathway activation and its rapid subsequent down-regulation; RasVal12 is able to produce activation for longer periods presumably because it acts intracellularly and downstream of Argos inhibition (Spencer, 1998).

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

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

The observation that rho-;vn- mutant clones produce disturbances in the spacing of more anterior ommatidia is reminiscent of defects observed in ommatidia surrounding Egfr minus clones and suggests that the R8 neuron in one ommatidium might influence the positioning of R8s in neighboring and anterior ommatidia. By what mechanism might this influence arise? Above are presented experiments indicating that Egfr/Dras1 (through Rhomboid and presumably Vein) can activate expression of the secreted protein Argos. Therefore, the potential for Argos to direct the pattern of emerging R8s through repression of Egfr was examined. Argos is a secreted factor that can act several cell diameters from its source. It acts as a negative regulator of the Egfr pathway in vivo and can prevent autophosphorylation and activation of Egfr in tissue culture cells, leading to the suggestion that Argos directly binds Egfr. Evidence for the presence of such an Egfr repressor in the MF is provided by a chimeric Egfr protein. l-DER is a constitutively activated chimeric receptor in which the extracellular domain of Egfr has been replaced by the l-repressor dimerization domain. As described above, activation of Egfr through Dras1Val12 or Rhomboid results in an eventual ‘rebound’ loss of dpERKA and Atonal. By contrast, ectopic expression of l-DER leads to elevation of Atonal expression, which persists for at least 3 hours, even though (as with ectopic Rhomboid and Dras1Val12) Argos expression is also elevated in this time frame. This result suggests that the rapid ‘rebound’ effect observed with Rhomboid requires a normal Egfr extracellular domain, and supports the view that it is mediated through a repressive ligand such as Argos. Previous work in the embryo has found an upregulation of Argos transcription in response to Egfr signaling. Consistent with this observation, the highest levels of Argos expression in the MF are found in the regions of highest Egfr activity, the proneural clusters. Lower levels of the protein are observed between and anterior to these clusters, presumably due to diffusion from the proneural clusters into the surrounding tissue. Argos overexpression in the MF results in elimination of Egfr activity (as measured by ERKA phosphorylation) and Atonal expression in the proneural clusters. Overexpression of Argos eliminates expression of Rhomboid and Vein: the factors that localize Egfr activity to the cell destined to become R8. A 90 minute heat-shock leading to overexpression of Argos eliminates Rhomboid protein from cells in the MF. This is consistent with findings that down-regulation of the transcription factor CF2, a negative regulator of Rhomboid transcription, is induced by Egfr signaling. Ectopic expression of Argos eliminates most or all Vein mRNA from cells in the MF. Thus, Argos-mediated repression of Egfr pathway activity may normally contribute to the pattern of Rhomboid and Vein expression necessary for correct R8 specification. To determine if Argos is necessary for setting the normal pattern of R8s, Boss expression was examined in the hypomorphic, partial loss-of-function mutant argosstyP1. Homozygous escapers of this line have rough eyes, due in part to the formation of ectopic ommatidia. Consistent with this, Boss-staining reveals that the pattern of R8 specification in these animals is disturbed: the spacing between R8s is variable and, most tellingly, R8s form aberrantly in positions between the normal ommatidial rows. These ectopic R8s are found in every eye disc of this genotype examined. This suggests that Argos produced by proneural clusters may normally diffuse anteriorly to repress Egfr activity (and Rhomboid and Vein expression), as well as the formation of R8s directly anterior to the cluster. In this model, R8s in the next row of ommatidia will be set at positions farthest from the site of Argos release, giving rise to the ‘out-of-register’ pattern of R8s found in wild type animals. Argos expression, in turn, is controlled by Rhomboid and Vein expressed in R8, indicating that each R8 has a role in patterning succeeding rows. It should be noted, however, that the disruption of ommatidial pattern observed when argos function is reduced is not very severe, and suggests that one or more additional factors are likely to contribute to the regulation of Rhomboid and Vein transcription (Spencer, 1998).

DERElp represents one of the first examples of a mutation that alters patterning in the retina: the eyes of DERElp /+ heterozygotes are rough and mispatterned and DERElp /DERElp homozygotes have greatly reduced eyes, which lack most ommatidia. In addition, proneural clusters appear to be absent, and Boss-expressing cells form less frequently and show aberrant patterning. Genetic evidence indicates that the DERElp mutation is a hypermorphic (gain-of-function) allele. The DERElp phenotype, then, is at odds with the data presented above, which indicates that Egfr activation results in specification of more R8s (and ommatidia) rather than fewer. However, high levels of Egfr activity also lead to induction of the Egfr inhibitor, Argos. In fact, the data presented here suggest that the patterning defects observed in DERElp retinae could result from the induction of ubiquitous and unpatterned Argos expression: DERElp heterozygotes show lower, rather than higher levels of dpERKA within the MF and Argos lacks the localization to proneural clusters present in wild-type animals. These data indicate that upregulation of Argos should result in loss of R8s, and this is observed in DERElp mutants (Spencer, 1998).

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

Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina

The Drosophila Egfr receptor is required for differentiation of many cell types during eye development. Mosaic analyses with definitive null mutations were used to analyze the effects of complete absence of Egfr, Ras or Raf proteins during eye development. The Egfr, ras and raf genes are each found to be essential for recruitment of R1-R7 cells. In addition Egfr is autonomously required for MAP kinase activation. Egfr is not essential for R8 cell specification, either alone or redundantly with any other receptor that acts through Ras or Raf, or by activating MAP kinase. As with Egfr, loss of ras or raf perturbs the spacing and arrangement of R8 precursor cells. R8 cell spacing is not affected by loss of argos in posteriorly juxtaposed cells, which rules out a model in which Egfr acts through argos expression to position R8 specification in register between adjacent columns of ommatidia. The R8 spacing role of the Egfr is partially affected by simultaneous deletion of spitz and vein, two ligand genes, but the data suggest that Egfr activation independent of spitz and vein is also involved. The results prove that R8 photoreceptors are specified and positioned by distinct mechanisms from photoreceptors R1-R7 (Yang, 2001).

It is thought that EGFR activity is required for recruiting R1- R7 photoreceptor cells to ommatidia, probably through Ras, Raf and MAPK but the role of this pathway in R8 specification has been less clear. Loss-of-function studies with putative Egfr null clones or temperature sensitivity have suggested that Egfr is dispensable for R8 specification (although involved in R8 spacing); studies with dominant negative approaches have suggested that Egfr is essential for R8 specification. There is also a particular class of Egfr mutants, the Elp alleles, that prevent R8 specification, and there is evidence that R8 specification might depend on Egfr-independent Raf activation. A study of null mutations in the Egfr/Ras/Raf pathway has been undertaken to resolve some of these issues. Two prior studies of Egfr mutant clones used the genetically amorphic point mutations flb1K35 and topCO. For topCO the molecular defect is unknown; flb1K35 corresponds to Gln267 in Ochre, which truncates the Egfr early in the extracellular domain. Although it is a reasonable assumption that these are both null alleles, it is worth noting that another mutation encoding Gln430 in Amber (top38) retains significant function, so the possibility of residual function in topCO or flb1K35 caused by readthrough, translational reinitiation or other mechanisms cannot be completely excluded. However, these possibilities can be excluded for the allele top18A, which deletes all Egfr-coding sequences from the genome. The phenotype of top18A clones is similar to flb1K35 and topCO. ato is expressed in top18A clones. It is concluded that cells completely lacking Egfr-coding capacity can still differentiate R8 photoreceptor cells, although their patterning is abnormal and they later die. Cells that completely lack Egfr are not recruited as any other photoreceptor type (Yang, 2001).

By the late third instar, cells in mutant clones have lacked Egfr gene function for approximately 120 hours. It is possible that cells might have a homeostatic mechanism (such as upregulation of another receptor) that compensates for sustained absence of Egfr function, and that some processes that would be Egfr-dependent in normal eye cells have been rescued in the clones. There is experimental evidence for such homeostasis from studies of the Egfrts2 allele. When Egfr function is interrupted, MAP kinase activation is lost from eye discs within 30 minutes, but levels of activated MAP kinase rebound within a few hours, even in the continued absence of Egfr function. MAP kinase activation was examined within clones of Egfr mutant cells. MAP kinase activation is undetectable. Thus, specification of R8 cells in Egfr mutant clones is not associated with MAP kinase reactivation via an alternative pathway. This finding indicates that the restored dpERK staining seen in the Egfrts2 allele must depend nonautonomously on loss of Egfr function in other cells. For example, loss of Egfr function from the whole animal may lead to changes in humoral signals that nonautonomously affect MAPK by some mechanism (Yang, 2001).

Genetic studies suggest that specification of most ommatidial cells depends on activation of Ras and Raf by Egfr (or by Egfr and Sevenless in the case of R7). R8 cell specification in the absence of Egfr might indicate activation of Ras and Raf by another receptor. Clones of cells null for Ras or Raf have been examined to test this. The null phenotype of Ras closely resembles that of Egfr. Ato expression initiated normally but patterning is affected and more cells than normal retain atonal expression posterior to the furrow. R8 cells are specified and express the R8 protein Senseless. No other Elav-expressing photoreceptor cells are recruited (Yang, 2001).

The phenotype of clones mutant for raf is similar. R8 cell specification begins relatively normally, as indicated by onset of Ato and Senseless expression. R8 cell precursors are improperly spaced, however. More posteriorly, raf mutant R8 cells express the neural protein Elav only transiently. These results also confirm directly that Ras and Raf are required for R1-R7 recruitment, and show that after these clones are induced in the first larval instar, Ras and Raf play no essential roles in the proliferation, survival or maintenance of eye disc identity of most eye disc cells (Yang, 2001).

Since null clones for Egfr, ras, and raf each permit R8 specification, although they affect R8 spacing, it is concluded that R8 specification can occur independently of Egfr, and is also independent of any other receptor that acts through Ras and Raf. Although the requirement for MAP kinase has not been tested directly (since the MAP kinase gene rolled maps proximal to all extant flip recombination target [FRT] sites), it was found that MAP kinase activation is undetectable in Egfr-null clones (Yang, 2001).

For both Egfr and ras, there is a nonautonomous delay of morphogenetic furrow movement and loss of ato, especially in large clones with substantial areas of mutant cells posterior to the furrow. This suggests Egfr and ras are required for expression of factors that push the morphogenetic furrow across the eye disc. Two such factors are Hh and Dpp. Hh is reported to be expressed by photoreceptor cells; therefore, fewer cells are expected to express Hh in ras or Egfr clones. There were some differences between clones mutant for raf and clones mutant for ras or Egfr. Less Elav is detected in raf mutant cells. In Egfr or ras mutant clones, Elav protein is detected in the mutant R8 cells, although at lower levels than in nearby wild type cells. In Egfr mutant clones, normal levels of Elav protein are restored by expression of baculovirus p35, indicating that low Elav levels reflect commitment of Egfr mutant cells to apoptosis. It is possible that Elav is lost more rapidly in raf mutant cells because of more rapid apoptosis than Egfr or ras mutant clones. Delayed furrow progression was not seen in raf mutant clones, but this may be because they were too small (Yang, 2001).

The differences between raf clones and Egfr or ras clones could indicate ras-independent signaling to raf, as has been proposed to occur during the determination of the embryonic termini. Such signaling to permit Elav expression in more R8 precursor cells (or preserve R8 precursor cells from apoptosis for longer) would have to be independent of Egfr as well, whereas all raf activity in the embryonic termini is dependent on torso, the relevant receptor. An alternative explanation is that these apparent differences relate to the much smaller size of raf clones compared with Egfr and ras clones. For the autosomal Egfr and ras mutations, the Minute technique was used to compensate for the growth disadvantage of the homozygous cells. This is not readily possible for the X-linked raf mutation. As a consequence, the raf clones examined were much smaller than the Egfr and ras clones, and grew at a reduced rate relative to neighboring wild-type cells. In the similar situation of Minute heterozygous clones growing slowly in wild-type backgrounds, nonautonomous interactions have been demonstrated, prolonging the cell doubling time of the slow-growing M/+ cells, and accelerating the doubling time of neighboring wild-type cells. If changes in cellular properties are also induced by the differential growth of neighboring homozygous raf mutant and wild-type cells, it is possible that faster loss of Elav might not indicate additional roles for raf in differentiation or survival, but an indirect effect of competition by the nearby wild-type cells on the raf minus cells. At present, experimental evidence to distinguish these models is not available (Yang, 2001).

The common requirements for Egfr, ras and raf in R8 spacing are not shown by null mutations in spi, which codes for an Egfr ligand required for recruitment of R1-R7. It is possible that spi is required redundantly with vn, another ligand with no essential role in ommatidium development. It was found that R8 precursor specification occurs in clones doubly mutant for both spi and vn. R8 spacing occurs almost normally, although there are rare cases of multiple R8 cells like those that occur more frequently in Egfr mutant clones. This raises the possibility that spi and vn do have redundant roles in R8 precursor spacing, but if this is so, there must be another ligand, or ligand-independent process, that is also involved. It has been found that the Drosophila genome sequence predicts another Spi-like protein. Cells doubly mutant for two putative ligand processing molecules encoded by rhomboid and roughoid resemble cells mutant for the Egfr. This suggests that rhomboid and roughoid redundantly process spi and spi-like, which act redundantly on Egfr in R8 spacing. The spi, spi-like double- and spi, spi-like, vn triple-mutant combinations that would directly test the relative contributions of all three ligands have yet to be examined (Yang, 2001).

The inhibitory ligand Argos is also required nonautonomously for R8 spacing. It had been suggested that Argos could diffuse from proneural intermediate groups, where it is expressed in response to Egfr activation, creating an 'exclusion zone' for further Egfr activation that will position future intermediate groups precisely out of phase. It was found, however, that Argos function can be performed by protein secreted several ommatidia away, which questions whether Argos conveys precise spatial information. Crucially, proneural intermediate groups are positioned normally even if immediately posterior regions are null mutant for argos, refuting the 'exclusion zone' model for argos action. Larger argos clones do affect R8 spacing distant from the clone boundary, suggesting that argos may be globally necessary in an unpatterned way to keep Egfr activity in check. An alternative is that argos is required indirectly through its effect on photoreceptor differentiation. Accordingly, ectopic photoreceptor cells in argos mutant territories might alter the expression of furrow progression signals such as Dpp and Hh (Yang, 2001).

The main result of this study is that R8 precursor specification occurs in cells null for Egfr, ras or raf. This is consistent with the proposed Egfr/Ras/Raf pathway of recruitment for photoreceptors R1-R7. These results appear definitively to exclude essential roles for Egfr, ras, raf, spi or vn, in R8 specification (although they support roles in R8 spacing), and show that argos is dispensable for the proposed signaling by each pair of proneural intermediate groups; each pair positions R8 specification in the next most anterior column. It is thought that R8 specification instead relies on autoregulatory transcription of the proneural ato gene promoted by two other DNA-binding proteins, daughterless and senseless that can occur without Egfr signaling. Defects in arrangement of R8 cell precursors show that the Egfr/Ras/Raf pathway nevertheless plays a role in the patterning of R8 cells. The increased number of R8 cells in mutants indicates that Egfr normally activates Ras and Raf to suppress R8 specification in certain locations. The Egfr pathway might modulate Notch. However, the Egfr requirement for R8 spacing was found to be more autonomous than the Egfr requirement for E(spl) expression, raising the possibility of another target. One candidate is the homeobox gene rough (Yang, 2001).

A primary role for the epidermal growth factor receptor in ommatidial spacing in the Drosophila eye

The differentiation of regularly spaced structures within an epithelium is a common feature of developmental pattern formation. The regular spacing of ommatidia in the Drosophila eye imaginal disc provides a good model for this phenomenon. The correct spacing of ommatidia is a central event in establishing the precise hexagonal pattern of ommatidia in the Drosophila compound eye. The R8 photoreceptors are the founder cells of each of the ommatidia that comprise the adult eye and are specified by a bHLH transcription factor, Atonal. The epidermal growth factor receptor (Egfr) has a primary function in regulating R8 spacing. The receptor's activation within nascent ommatidia induces the expression of a secreted inhibitor that blocks atonal expression, and therefore ommatidial initiation, in nearby cells. The identity of the secreted inhibitor remains elusive but, contrary to previous suggestions, it has been shown that this inhibitor is not Argos. This Egfr-dependent inhibition acts in parallel to the inhibition of atonal by the secreted protein Scabrous. The activation of the Egfr pathway is dependent on Atonal function via the expression of Rhomboid-1. Therefore, it was concluded that Egfr's role in promoting cell survival is largely independent of its role in photoreceptor recruitment; even when cell death is blocked, most photoreceptors fail to form. Based on these data and those of others, a model is proposed for R8 spacing that comprises a self-organizing network of signaling molecules. This model describes how successive rows of ommatidia form out of phase with each other, leading to the hexagonal array of facets in the compound eye (Baonza, 2001).

R8 cell spacing is disrupted in clones of cells mutant for Egfr. However, cell death is substantially elevated in these clones, and therefore it is not possible to tell whether the spacing defects are a direct consequence of Egfr loss or are secondary to cell death. To examine this, Egfr- clones were generated in a genetic background in which cell death was blocked in the eye by expressing the baculovirus p35 gene under the control of the eye-specific GMR enhancer. In these clones, R8 cells differentiate but their spacing is still disrupted. This result implies that the Egfr function in spacing is not secondary to cell death. The abnormal spacing is seen first as a failure of Atonal to become modulated into proneural clusters in the furrow; a broad band of fairly uniform Atonal is expressed until just posterior to the furrow. This eventually resolves into isolated Atonal-expressing cells that form a disorganized array. Since atonal expression does ultimately resolve to single cells despite the lack of proneural clusters, it is imagined that lateral inhibition mediated by Notch still occurs in the Egfr- clones (Baonza, 2001).

The Drosophila Egfr signals principally through the Ras/MAPK signal transduction pathway. The observation that Egfr signaling has a direct role in spacing the proneural clusters in the eye imaginal disc is therefore consistent with the fact that MAPK activity within proneural clusters is necessary for the repression of atonal expression in cells between proneural clusters. The MAPK signal transduction pathway is activated by a wide range of receptor tyrosine kinases; therefore, tests were made to see whether Egfr is responsible for the observed MAPK activation in the furrow. In clones of cells carrying an Egfr null mutation the receptor is autonomously required for all MAPK activation. From this it is concluded that the Egfr is the only RTK that detectably activates MAPK in the morphogenetic furrow (Baonza, 2001).

Interestingly, clones lacking the Egfr ligand, Spitz, have normal R8 cell spacing and MAPK activation. This finding suggests that another ligand for the receptor may be responsible for this function. A single novel Spitz-like ligand has recently been discovered in the completed Drosophila genome sequence (FlyBase ID FBgn0036744), and it is speculated that this could provide the missing function in R8 cell spacing. Testing this prediction awaits the identification of loss-of-function mutations in the spitz-2 gene (Baonza, 2001).

The results described above imply that Egfr has a primary function in ommatidial spacing and suggest that it is the only RTK that activates MAPK in the proneural clusters. Since it has been shown that the transcription factor Atonal is also required for this MAPK activation, how Atonal and Egfr activation are related was investigated. One possibility is that Atonal directly activates the expression of rhomboid-1, a principal activator of Egfr signaling, as it does in the embryonic chordotonal organs. Therefore whether ectopic Atonal can activate rhomboid-1 expression was investigated. In wild-type cells, rhomboid-1-lacZ is expressed only in photoreceptors R8, R2 and R5. UAS-atonal was expressed under the control of sevenless-Gal4, which is expressed in all ommatidial cells except R8, R2 and R5. When atonal is thus misexpressed, it was found that rhomboid-1 expression (as detected by rhomboid-1-lacZ) is activated in ectopic photoreceptor cells. Thus, consistent with the model in which the activation of MAPK via Atonal depends on the activation of rhomboid-1 expression, atonal expression can induce rhomboid-1 expression, which in turn activates Egfr (Baonza, 2001).

In some tissues, the expression of the Egfr activator, Rhomboid-1, is dependent on Egfr signaling itself. This dependency thereby constitutes a positive-feedback loop. If this were the case in the furrow, the Atonal-triggered expression of rhomboid-1 could not initiate Egfr signaling (as it would itself depend on prior signaling). Therefore, the expression of rhomboid-1-lacZ in Egfr- clones was examined. Since both the clone marker and the detector for rhomboid-1 expression are the lacZ gene, both are labeled in the same color. However, the ß-galactosidase that marks the clone is cytoplasmic, whereas the one that indicates rhomboid-1 expression is confined to the nucleus. In this way the rhomboid-1-expressing cells can be distinguished from the Egfr-positive cells; this is particularly obvious in transverse optical sections of the eye disc. Egfr- cells can initiate rhomboid-1 expression, and it is concluded that the initiation of rhomboid-1 expression in the furrow does not require Egfr activity (Baonza, 2001).

The results described so far indicate that within proneural clusters, Atonal activates the expression of rhomboid-1, which in turn leads to the activation of the Egfr/Ras/MAPK pathway. This MAPK activity in proneural cells leads to a nonautonomous inhibition of atonal expression in the cells between proneural clusters. Scabrous is a secreted protein, expressed within clusters, that is also required for the inhibition of atonal expression between clusters. A possible prediction of the model is that scabrous expression in proneural cluster cells would be activated by Egfr/Ras/MAPK signaling -- in other words, that Scabrous is the inhibitory signal secreted by cluster cells in response to Egfr signaling. To test this, Scabrous expression was analyzed in Egfr- clones by using a monoclonal antibody against the Scabrous protein. Normal levels of Scabrous were observed in Egfr- clones, and this finding implies that Scabrous expression is not dependent on the Egfr pathway. The pattern of Scabrous expression was nevertheless altered, which reflects the abnormal spacing of cells in the furrow in Egfr- clones (Baonza, 2001).

This result suggests that the inhibitory factor regulated by Egfr signaling works in parallel to Scabrous in repressing atonal between proneural clusters. A consequent prediction is that when both Scabrous and Egfr signaling are removed, the spacing defects in the furrow should be worse than those caused by either mutation alone. Conversely, if the Egfr-dependent inhibition is mediated by Scabrous, the double mutants should have the same phenotype as the single mutants. Complete loss of Scabrous alone causes a relatively mild defect in spacing. Clones doubly mutant for Egfr and scabrous were examined and they have reproducibly more severe spacing defects than do Egfr mutant clones alone. When the cells were stained with anti-Boss to label the R8 cells, this was particularly clear within the morphogenetic furrow; in Egfr- clones the spacing is irregular, but the overall number or R8s is not substantially increased over that of the wild type, while in Egfr-;sca- double-mutant clones, more R8s form in the furrow and typically produce a very closely spaced row of cells that is not seen in the single-mutant clones. This additive effect of removing Egfr and Scabrous supports the notion that they mediate two parallel pathways: each contributes to the inhibition of atonal expression between proneural clusters (Baonza, 2001).

It has been proposed that the secreted Egfr antagonist, Argos, could be the inhibitor of R8 determination between preclusters. This suggestion has been directly tested in argos loss-of-function clones. The arrangement and spacing of the developing ommatidia are completely normal, even in very large clones induced in a minute background; some examples cover more than half of the eye disc. This result implies that Argos cannot be significantly involved in regulating ommatidial spacing. Consistent with this result, it has previously been shown that whole eyes mutant for eye-specific argos alleles do not have substantially disrupted precluster spacing (Baonza, 2001).

A fairly simple model can be proposed for how R8 spacing is controlled; this model synthesizes the work of several groups. A key feature is that it is a self-organizing system; once atonal expression is initiated at the posterior of the disc, the pattern spreads across the whole retinal primordium without further input from signals other than those generated by the spacing mechanism itself. The first stage of ommatidial determination is the activation of a broad, uniform band of atonal expression anterior to the morphogenetic furrow. This is initiated at least in part by the secreted protein Hedgehog, which emanates from the more posterior, already differentiating, ommatidia. In the model, this band of Atonal expression becomes modulated by the combined action of two diffusible inhibitors, Scabrous and an unidentified inhibitory factor dependent on Egfr-induced signaling through the MAPK pathway, both of which are dependent on Atonal (Baonza, 2001 and references therein).

atonal expression is upregulated by an autoregulatory loop, just as the proneural clusters become apparent. It is at this point that it is proposed that Scabrous and the Egfr-dependent inhibitory factor act. They diffuse toward the anterior and inhibit atonal expression in those cells closest to the inhibitory source. By this mechanism, only the cells farthest from clusters in the previous row retain atonal expression. This produces the characteristic staggered arrangement of R8s in successive rows. Therefore, the central patterning event in establishing the overall arrangement of the ommatidia is the transformation of uniform Atonal expression into modulated expression, as controlled by a combination of Scabrous and the Egfr-dependent inhibitory signal (Baonza, 2001).

Once Atonal expression is initially modulated by these inhibitory factors, well-defined proneural clusters are formed by a combination of the same inhibitory signals and the autoregulatory positive feedback loop that maintains and increases atonal expression within the clusters. It is suggested that this autoregulation makes the proneural cluster cells refractory to the inhibitory signals they themselves are producing (Baonza, 2001).

There is no obvious candidate for either the Egfr-dependent inhibitory factor or for the signaling pathway it uses. It can be inferred, however, that it triggers the expression of the homeodomain protein Rough, since Rough expression is lost in Egfr- clones. Rough is a transcription factor that represses atonal expression and Rough is normally expressed in a complementary pattern to Atonal within the furrow. Rough expression is not affected by the loss of Scabrous, which is consistent with the idea that the Rough-mediated inhibition of atonal expression is instead controlled by the Egfr-dependent inhibitory factor. It was originally suspected that Scabrous, which regulates Notch signaling, could be the inhibitory factor. As described above, coupled with the phenotype of scabrous mutants alone, the results on Scabrous expression and the scabrous, Egfr double mutations imply that this is not the case. Nevertheless the possibility that Notch activity has a role in regulating precluster spacing has not been ruled out; it is clearly involved in the later process of lateral inhibition that inhibits atonal expression in all but one of the proneural clusters, so its function in nonautonomous atonal inhibition is well established. Despite this possibility, current evidence does not provide a convincing link between the Egfr-dependent inhibition and the Notch pathway (Baonza, 2001).

An alternative Egfr-centered model of R8 spacing has been proposed. In this model, Argos would be the Egfr-dependent diffusible molecule that prevents atonal expression and R8 specification between the proneural clusters. However, R8 specification (as opposed to spacing) occurs normally in the absence of Egfr; an Egfr inhibitor such as Argos therefore would not be expected to prevent R8 determination. This is confirmed by results demonstrating that argos null mutant clones have normal ommatidial spacing, even when they are very large, as well as by data showing that eyes from viable, eye-specific argos mutants (i.e., in which the whole eye is mutant for argos) have reasonably normal ommatidial spacing. Another model attributes the crucial uniformity-breaking step to the secreted protein Hedgehog, which can inhibit atonal expression at high levels while activating it at lower levels. This view is conceptually distinct from that presented here, since the source of the diffusible inhibitor (Hedgehog) is not the proneural clusters but the much farther posterior differentiating ommatidia. According to this model, rough expression would be activated by inhibitory levels of Hedgehog. It is worth noting that this view of ommatidial spacing is not mutually inconsistent with the one presented here; several different pathways may contribute to the patterning of the ommatidial array (Baonza, 2001).

Atonal influences the final differentiation of an R8 cell as well as its earlier selection. Reducing the amount of Atonal in already selected R8 cells leads to the reduced recruitment of subsequent ommatidial photoreceptors; conversely, overexpression of Atonal in R8 leads to excess recruitment. This result fits well with the proposed model and the observation that Atonal can activate the expression of Rhomboid-1 in R8 cells. During the recruitment phase of ommatidial development (which occurs posterior to the furrow after R8s are selected), Egfr signaling is required for triggering the determination of the non-R8 photoreceptors. This signaling is initiated by Rhomboid-1 and Rhomboid-3, which together allow the release of Spitz, the Egfr-activating ligand. A simple explanation is that the level of Atonal in R8 influences the level of Rhomboid-1 which, in turn, controls photoreceptor recruitment (Baonza, 2001).

Rhomboid-1 is expressed not only in the R8 cell but also in the next two photoreceptors to be recruited, R2 and R5. The control of this expression can now be fully explained. The evidence in this paper suggests that in R8, rhomboid-1 is regulated by Atonal. In R2 and R5, but not in R8, rhomboid-1 expression has been shown to be under the control of Rough (which has a later role in photoreceptor specification as well as the function in spacing described here) (Baonza, 2001).

These results emphasize the extraordinary diversity of functions that many proteins carry out in eye development. At least five distinct roles for Egfr have been discovered; Atonal is used first for specifying R8 cells and later for regulating their differentiation; Rough is an inhibitor of atonal in the furrow but in a later incarnation activates Rhomboid-1 expression in R2 and R5 and thereby triggers photoreceptor recruitment; Notch, too, has many functions, not all of which are fully understood. One implication of the repeated use of signaling molecules is that the signals themselves do not specify the outcome of signaling. Instead, the fate of a cell is largely determined by the 'state' of the cell that receives the signal, which can broadly be translated into the complement of transcription factors that a cell is expressing (Baonza, 2001).

There are aspects of this concept, however, that remain unclear. For example, it has previously been shown that constitutively active Egfr is sufficient to trigger the differentiation of photoreceptors anterior to the morphogenetic furrow, even in the absence of Atonal. It is therefore not understood why Egfr signaling in the proneural clusters leads to the production of a diffusible inhibitor of atonal expression rather than photoreceptor determination. A possible explanation is that cells cannot become specified as photoreceptors by Egfr signaling while they are expressing Atonal. Alternative explanations include different effects caused by different Egfr ligands or by different levels of MAPK activation. More generally, a major goal will now be to understand how the successive Egfr signaling events in the eye fit together; for example, how are the transitions from furrow initiation to proneural cluster spacing to cell recruitment controlled? In the Drosophila oocyte, integrated regulation of multiple Egfr signaling events is a key aspect of developmental progression and coordinated patterning, and it is suspected that such linking of successive signaling events may be a general feature of complex developmental systems (Baonza, 2001).

A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody (CM1) raised against a peptide from human caspase 3 was used to investigate how extracellular signals control spatial patterning of cell death. The antibody crossreacts specifically with dying Drosophila cells and labels the activated effector caspase Drice. The initiator caspase Dronc and the proapoptotic gene head involution defective are important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered the EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affects caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appear to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway (Yu, 2002).

A particularly useful feature of the CM1 antiserum is the detection of cells that would otherwise be marked for death but which are protected by baculovirus p35 expression. The morphological protection provided by p35 may permit better investigation of the location and autonomy of death and survival signals. On Western blots the CM1 antibody detects activated Drice but not the Drice zymogen; Drice is the Drosophila sequence most similar to the immunizing peptide. Definitive evidence that CM1-stained cells are apoptotic comes from the dependence of embryonic CM1-staining on the 75C1,2 chromosome region, and from the p35-sensitivity of larval and pupal CM1-stained cells. The morphology of all CM1-stained cells is altered by p35 expression. In the presence of p35, CM1-stained cells become indistinguishable from normal cells by morphological criteria, and are not distinguishable except by CM1 staining. Since baculovirus p35 blocks cell death by inhibiting caspase activity, p35-dependent morphology of CM1-stained cells shows that such morphology depends on caspase activity in the cells, which are therefore apoptotic. These results show that only apoptotic cells are labelled by the CM1 antiserum. Apoptotic cells that are unlabelled by CM1 might also exist, although none have been noticed (Yu, 2002).

Experiments using the egfrts1a allele have confirmed that Egfr is required for survival of pupal retinal cells, as suggested by misexpression experiments. Egfr is also required for survival of eye imaginal disc cells. Consistent with the model that Egfr prevents cell death by inactivating hid, hid is absolutely required for caspase activation in egfr mutant clones. Similar results have been obtained using TUNEL experiments to assess Egfr-DN-induced cell death (Yu, 2002).

Survival in pupal retina is regulated by two further extracellular signals that are not involved in eye imaginal discs. In principle, such signals might act to modulate Egfr signaling, to regulate Hid or DIAP activity in parallel to Egfr, or to activate initiator caspases. Notch (N) is required for caspase activation in the pupal retina. Epistasis experiments show that N is not required for pupal cell death in the absence of Egfr function, and therefore that the normal function of N is to inhibit the Egfr survival signaling pathway in pupae. Such results place N upstream of Egfr and indicate that N acts ultimately through hid and the anti-apoptotic DIAP proteins that prevent caspase activation, rather than through N-mediated caspase activation. Survival in pupal retinas also depends on signals from primary pigment cells and/or cone cells. Such signals must antagonize proapoptotic N activity, since N is epistatic to the primary pigment cell/cone cell signal. The data now imply a pathway in which primary pigment cells and/or cone cells promote survival by inhibiting activation of N, thus preventing N antagonism of Egfr activity in the interommatidial cells (Yu, 2002).

The essential role of Egfr now seems to be downstream of N, whereas the cone cell/primary pigment cell signal must act upstream. Downstream Egfr function raises anew the question of identity of the primary pigment cell/cone cell signal. Primary pigment cells or cone cells do not seem essential for Egfr activation, because N is still required for apoptosis after ablation of these cells. Pupal photoreceptor cells express the Egfr ligand SPI and its processing/presenting factor Rhomboid, and are one possible source of Egfr activation. One model suggests that primary pigment cells and/or cone cells are the source of an unidentified signal or mechanism that prevents N activation (in particular interommatidial cells) so that Egfr survival signaling can continue (Yu, 2002).

According to one view, survival signals are the critical extracellular regulators of developmental cell death. By contrast, results from C. elegans and mammals indicate that cell death depends on activation of initiator caspases to trigger the apoptotic cascade. Homologs of the activatory components exist in Drosophila. Studies of eye development place three extracellular signals in a pathway acting through Egfr and hid to regulate survival, in part through IAPs. The only evidence consistent with positive regulation of apoptosis is that in eye imaginal discs, hid appears to promote cell death through an unidentified mechanism independent of DIAPs, and, in this case, the role of EGF receptor signaling is still to promote survival by inhibiting Hid (Yu, 2002).

These findings do not rule out other pathways that activate initiator caspases during eye development, or that such activation might be required for cell death. Since hid is essential for cell death, however, pathways that activate initiator caspases independently of hid cannot be sufficient for any of the cell death that normally occurs during eye development. Because loss of Egfr survival signaling is sufficient for cell death, and Egfr survival signaling is only important to inhibit Hid, these data imply that release of hid is sufficient as well as necessary for normally occurring cell death. The data do not rule out any parallel Egfr-dependent signal to suppress caspase activation independently of hid, but such a pathway cannot be sufficient for cell death in the absence of hid. These findings suggest that positive activators of caspase processing may not be the direct targets of extracellular regulation. However, it will be important to investigate survival and death signals in other organs, including cell deaths that occur independently of reaper, grim and Hid in ovarian nurse cells and during autophagy, the mechanisms of which have yet to be determined (Yu, 2002).

Notch activation of yan expression is antagonized by RTK/Pointed signaling in the Drosophila eye

Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation, is a target of Notch signaling during Drosophila eye development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification (Rohrbaugh, 2002).

A role for RTK signaling in regulating yan transcription was investigated. When the RTK pathway is constitutively activated by torD-DER or Ras1V12, the yan enhancer activity is greatly reduced. Thus, RTK signaling appears to negatively regulate yan transcription, in addition to its effect on Yan protein stability. Evidence supports a view that the inhibitory effect of RTK/Ras1 signaling on yan expression is mediated through the pointed (pnt) gene. Taken together, the results demonstrated that Pnt negatively regulates yan expression, and it is likely that Pnt is directly involved in repressing yan transcription. Although a role for Pnt as a transcriptional repressor has not been extensively investigated, pnt has been shown to negatively regulate hid transcription in embryos. Interestingly, a P-DLS motif is present in the Pnt protein (amino acids 356–360 in PntP1), which might mediate interaction with the transcriptional corepressor dCtBP. At this point, the data does not exclude the possibility that Pnt might also activate expression of a repressor, which in turn switches off yan transcription (Rohrbaugh, 2002).

rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

The Drosophila homolog of Egfr is an RTK that activates a highly conserved signal transduction cascade in a variety of tissue and cell types during Drosophila development. The activation of the Egfr is dependent on the tissue/cell type-specific ligands at the specific developmental stage. In the developing eye, Egfr function is required for the determination of all retinal cell types. A single copy of the mutations in Egfr acts as a mild enhancer of the rugose eye phenotype. In addition, Ellipse, a dominant mutation in Egfr (EgfrE), acts as a suppressor suggesting that rugose interacts with the Egfr-mediated signal cascade (Shamloula, 2002).

Phospholipid membrane composition affects EGF Receptor and Notch signaling through effects on endocytosis during Drosophila development

The role of phospholipids in the regulation of membrane trafficking and signaling is largely unknown. Phosphatidylcholine (PC) is a main component of the plasma membrane. Mutants in the Drosophila CTP-phosphocholine cytidylyltransferase 1 (Cct1), the rate-limiting enzyme in PC biosynthesis, show an altered phospholipid composition with reduced PC and increased phosphatidylinositol (PI) levels. Phenotypic features of Cct1 indicate that the enzyme is not required for cell survival, but serves a role in endocytic regulation. Cct1- cells show an increase in endocytosis and enlarged endosomal compartments, whereas lysosomal delivery is unchanged. As a consequence, an increase in endocytic localization of EGF receptor (Egfr) and Notch is observed, and this correlates with a reduction in signaling strength and leads to patterning defects. A further link between PC/PI content, endocytosis, and signaling is supported by genetic interactions of Cct1 with Egfr, Notch, and genes affecting endosomal traffic (Weber, 2003).

Drosophila Cct1 is highly homologous to fly Cct2 and to CCT genes from other species ranging from yeast to human. Cct2, is located immediately downstream of Cct1. CCT catalyzes the formation of CDP-choline during PC biogenesis. Thus, it was surprising to find specific phenotypes associated with Cct1. To confirm that the Cct1 mutations indeed affect PC biogenesis, the membrane composition of wild-type and Cct1 mutant larvae were compared. Strikingly, PC levels were reduced in membrane preparations from CCT116919/Df(3L)emc5 and dCCT1EP0831/Df(3L)emc5 animals, whereas an increase in PI levels was detected. These data confirm that Cct1 is required for PC biogenesis and demonstrate that in Cct1 mutants the ratio of PC, PI, and PE is changed (Weber, 2003).

To determine the requirements of Cct1 during cellular differentiation and development and because Cct1 null mutants are lethal, Cct1 mutant tissue was generated during eye development. Surprisingly, CCT1- cells initially develop normally with no obvious defects in cellular architectureand give rise to tissue comparable in size to wild-type, indicating that Cct1 is not required for cell growth or survival. Cct1- eye disc clones revealed patterning defects similar to those observed in adult eyes of the hypomorphic P alleles. Mutant ommatidial clusters often showed typical polarity defects. Chirality defects are apparent using the (largely) R4-specific mδ-lacZ reporter, which reflects cell fate selection in the R3/R4 pair and thus ommatidial chirality. Mutant or mosaic clusters often show aberrant mδ-lacZ expression, reflecting defects in Fz/Notch signaling in the R3/R4 pair. The photoreceptor R1/R6-specific marker Bar reveals the general orientation of developing ommatidial clusters and their degree of rotation. In Cct1 clones, many clusters have an aberrant orientation, displaying defects in ommatidial rotation. These data suggest that Cct1 can affect specific processes during eye development, in particular those related to planar cell polarization (PCP; Frizzled or Notch signaling) and ommatidial rotation (Egfr signaling) (Weber, 2003).

Cct1 is additionally required during terminal photoreceptor differentiation. Although Cct1 null mutant photoreceptor cells initially appear normal, they do not form the light-harvesting organelle, the rhabdomere. The rhabdomeres, containing the rhodopsin proteins, are large stacks of membrane microvilli that are formed at the apical membrane domain of each photoreceptor during their terminal differentiation. Transmission electron microscopy (TEM) analysis of Cct1 mutant photoreceptors has revealed that the rhabdomeres failed to properly form (with only remnants of rhabdomeres sometimes to be found. This defect is not light or age dependentas is the case in many phototransduction mutants, indicating that Cct1 is autonomously required during the terminal differentiation of the rhabdomeric membrane stacks. Rhabdomere morphogenesis is thought to be a 'burst' of apical membrane synthesis, and it is also affected by blocking dynamin function (via photoreceptor-specific shits expression. Thus, this phenotype can be attributed to membrane trafficking defects (Weber, 2003).

The phenotypic features of Cct1 during eye development suggest a requirement in one or several signaling pathways. Thus, genetic interactions were tested between Cct1 and components of most of the common signal transduction cascades. Strikingly, Cct1 interacts with Egfr signaling. Cct1 alleles dominantly suppress the gain-of-function (GOF) Ellipse allele of Egfr (EgfrElpB1). The EgfrElpB1 phenotype is apparent in reduced eye size and loss of photoreceptors and ectopic veins in the wing. All aspects of the EgfrElpB1 phenotype arer suppressed by Cct1 alleles. The degree and quality of suppression was comparable to that of loss-of-function (LOF) alleles of components of the Egfr/Ras pathway, suggesting a positive Cct1 requirement in Egfr signaling. This observation was confirmed by the inverse interaction. The homozygous CCT116919 eye phenotype is dominantly enhanced by Egfr. The interactions between Cct1 and Egfr are consistent with the Cct1 eye phenotypes, as Egfr is required for photoreceptor induction, survival, and ommatidial rotation (Weber, 2003).

The ommatidial chirality defects in Cct1 mutants suggest a role in PCP establishment, which is governed by an interplay of Fz-Notch signaling. This is supported by the observation that Cct1 alleles suppress the PCP GOF phenotypes of sev-Fz and sev-Dsh, which display PCP defects with chirality inversions and symmetrical clusters. Cct1 dominantly suppress these aspects in both backgrounds, with many more ommatidia displaying a chiral arrangement. Since PCP GOF eye phenotypes are modified by components of Fz or Notch pathways, this was addressed further and an interaction between Cct1 and Notch signaling in general was tested (Weber, 2003).

Notch is haploinsufficient, showing a dominant LOF phenotype in strong alleles in the notching of the wing margin. Genetic modifications of this phenotype have been generally used to dissect the requirements of other genes in the context of Notch signaling. In the N null allele, N55e11/+, the wing notching phenotype is enhanced by removing one copy of Cct1, both with respect to the extent and frequency of the notch. In addition, although N55e11/+ flies have no dominant eye phenotype in a wild-type background, the simultaneous removal of one copy of Cct1 and aos gives rise to rough eyes with many chirality defects and a partial loss of the ventral eye. Such triple-heterozygous eyes resemble the stronger CCT116919/Df(emc5) phenotype. Thus, although neither N nor Egfr signaling show dominant LOF eye phenotypes, the simultaneous removal of one copy of Cct1 results in a haploinsufficiency of this genotype. This not only supports a requirement for CCT1 in N signaling, but also suggests a link between N and Egfr signaling, possibly mediated through Cct1. Moreover, the homozygous CCT116919 phenotype is strongly enhanced by a Notch-/+ background, with severe wing notching and dramatically reduced eyes in rare escapers. These observations support the notion that Cct1 is required for Notch signaling in several developmental contexts (Weber, 2003).

Surprisingly, the CCT enzymes are not required for cellular survival, as even clones of the double null allele (CCT299) show the same phenotypic features as the CCT1179 null, indicating that a cell can function with reduced PC and increased PI levels. This suggests that the alternate pathway of PE methylation can synthesize enough PC or that cells can function in many aspects with low PC levels. However, a decrease in PC (and an increase in PI) levels affects regulatory aspects of membrane dynamics and endocytosis. In addition, Cct1 is the major gene responsible for the observed defects, since CCT116919 and CCT1179 show the same phenotypes as the double null CCT299 allele (Weber, 2003).

The primary Cct1 defect is an increase in the rate of endocytosis. In particular, more clathrin-coated pits (CCPs) and vesicles are observed, leading also to a size increase of the endosomal compartment. However, membrane traffic progression through the endosomal compartment to the lysosome is not affected. This is in contrast to dynamin mutants (shi), where CCPs fail to pinch off and thus endocytosis is blocked (Weber, 2003).

An explanation for the increase of endocytosis in Cct1 mutants might lie in the fact that decreased PC and increased PI levels affect the dynamics of the assembly and/or removal of the clathrin coat. PIP2 (derived from PI) has been suggested to serve as a 'docking site' for clathrin coat-promoting proteins, such as Epsin. It has been proposed that several factors, including dynamin, amphyphysin, and endophilin, can also initiate vesicle budding. An increase in PI and change in the PC/PI ratio is also interesting in the context of the proposal that Sec14p could monitor the PC/PI ratio and influence endocytosis. An alternative cause for the phenotypes observed might be the fact that the plasma membrane is normally asymmetric, with a difference in phospholipid composition between the outer and inner plasma membrane layers. Mammalian plasma membranes have higher PC content in the outer layer as compared to the inside face, suggesting that PC levels in the outer membrane layer serve a specific function (Weber, 2003).

Cell surface receptor internalization and subsequent sorting to multivesicular bodies and lysosomal delivery are important mechanisms of signaling modulation. The phospholipid composition of the plasma membrane affects this process. The enlarged endosomal compartment is likely a consequence of the increase in endocytosis. Genetic and histological analyses suggest that Egfr and N are sensitive to Cct1 function and possibly PC/PI levels. The Garland cell analysis and genetic interactions indicate that Cct1- cells have an increased rate of endocytosis, suggesting that plasma membrane (PM) proteins spend a shorter time at the plasma membrane with a reduced chance to interact with their ligands. As membrane trafficking to the lysosome is functioning in Cct1- cells, the overall levels of PM receptors might be reduced. Although the N and Egfr pathways are affected the most, it is likely that other receptors are also affected. Not all RTKs are equally sensitive to Cct1 activity, however; for example, Sevenless (Sev) does not interact with Cct1. Furthermore, internalization of the Sev/Boss (Sev-ligand) complex is affected by hk mutants but is not manifest in an R7 loss phenotype. This suggests that signaling levels require tight regulation for some RTKs, such as Egfr, with multiple distinct responses at different signaling levels, as compared to others such as Sev, with a simple on/off situation (Weber, 2003).

Both Egfr and N signaling levels are reduced in Cct1 mutants, as deduced from the genetic interactions. The peak signaling levels for both pathways are affected in Cct1, whereas other aspects of their readout are less affected, supporting the notion that Cct1 function is important for signaling level output in certain contexts only. Consistently for both pathways, regulated ligand and receptor processing and trafficking are critical for correct signaling levels. Moreover, since Cct1 expression can be regulated by signaling input, a given cell population could regulate aspects of endocytosis through its membrane PC content (Weber, 2003).

It has recently been shown that a regulatory crosstalk between Egfr and Notch signaling is mediated through the transcriptional upregulation of a hypothetical factor by Egfr/Ras, leading to endocytosis and downregulation of Notch/LIN-12 in C. elegans. Strikingly, a heterozygous condition for both signaling pathways (that has no phenotypic consequence by itself) shows dramatic defects in a Cct1-/+ background, also suggesting crosstalk between Egfr and Notch signaling. Thus, some aspects of the crosstalk between these and other pathways might be generally regulated or mediated through membrane phospholipid contents (Weber, 2003).

EGF receptor/Rolled MAP kinase signalling protects cells against activated Armadillo in the Drosophila eye

ß-catenin/Armadillo are transcriptional co-activators that mediate Wnt signalling in normal development. Activated forms of ß-catenin are oncogenic. Mutant forms of Drosophila Armadillo were constructed that correspond to common human oncogenic mutations; they were found to activate Armadillo constitutively. When expressed in the Drosophila eye, these eventually induce apoptosis in all cell types. Intriguingly, cells in the eye are resistant to the effects of activated Armadillo for a long period prior to the onset of cell death at the mid-pupal stage. This latency is conferred by EGF receptor (EGFR)/MAP kinase signalling, which prevents activated Armadillo from inducing apoptosis; when EGFR signalling naturally ceases, the cells rapidly die. Nemo, the Drosophila homologue of NLK in mice and LIT-1 in Caenorhabditis elegans, does not antagonize activated Armadillo, suggesting that the Nemo-like MAP kinases may not generally interact with Armadillo/ß-catenin. Thus, the results show that activated Armadillo is subject to a specific negative control by EGFR/Rolled MAP kinase signalling (Freeman, 2001).

Expression of activated forms of Armadillo causes a late onset of apoptosis in the developing Drosophila eye. These effects are similar to those of overexpressing dTCF, and genetic interactions indicate that they are mediated by endogenous dTCF. The disruptive effects of these conditions closely mimic those of Drosophila APC mutations except that, since APC expression is restricted to neuronal cells, cell death in that case is confined to photoreceptors. Therefore, in flies as in humans, the consequences of activated Armadillo/ß-catenin are essentially equivalent to those caused by APC loss. Importantly, these new forms of activated Armadillo provide powerful tools for genetic screens which might identify ancillary signalling proteins conserved between humans and Drosophila (Freeman, 2001).

A precedent for an antagonistic effect between Wingless and EGFR signalling is found in the embryonic epidermis. In this case, signalling by these pathways appears to be integrated at the level of a common target gene, svb. Given that, in the eye disc, overexpression of dTCF causes the same delayed cell death as Arm*, and that the Arm* rough eye phenotype is suppressed by dTCF heterozygosity, the antagonism between Arm* and EGFR signalling also occurs at the level of target gene transcription. As in the embryonic epidermis, antagonistic inputs from the two pathways could be integrated at a transcriptional enhancer of a common target gene. However, svb is not a good candidate in this case since it is not expressed in the eye and no genetic interaction between svb and Arm* has been found (Freeman, 2001).

Although it has been argued that the delayed apoptotic effect of Arm* is based primarily on dTCF-mediated transcription, there may also be a post-transcriptional contribution. This is suggested by the observation that Armadillo levels were only moderately elevated in larval eye discs overexpressing Arm*, but strongly increased in 40-48-h-old pupal discs that showed severely disrupted ommatidia. As was observed for Armadillo S10, both cytoplasmic and nuclear levels were increased simultaneously, indicating that both types of activating mutations cause stabilization of free protein rather than altering its relative distribution between cytoplasm and nucleus. However, since this increase in Armadillo level correlates in time with the onset of apoptosis, it is unclear whether the accumulation is a cause or an effect of the putative transcriptional changes induced by Arm* (Freeman, 2001).

Most interestingly, in contrast to other tissues where activated Armadillo has immediate effects, Arm* is expressed for 2-4 days in eye disc cells before causing a detectable phenotype; onset of apoptosis occurs only at the mid-pupal stage. This delayed manifestation of the effects of Arm* could not have been inferred from the phenotype of dAPC mutants. These results imply that this latency is a consequence of protection conferred by EGFR signalling. The EGFR has multiple functions in the developing eye, including a protective effect against cell death in normal development. Furthermore, a strong genetic interaction has been observed between the cell death-inducing factor Hid and Ras1 signalling in the eye, and the latter apparently promotes cell survival by downregulating Hid directly at the transcriptional and post-transcriptional level (Freeman, 2001).

In contrast, no suppression is seen of the Arm* rough eye phenotype by heterozygosity for the H99 deficiency, which uncovers Hid and two further cell-death-inducing factors. This argues that the antagonism observed between Arm* and EGFR signalling is specific, and distinct from the more general survival-promoting function of the receptor, which is mediated directly by Hid. In support of this, a large number of genes including dE2F, p53 and presenilins have been found to induce cell death when expressed in the larval eye disc; in all cases, the apoptosis occurs in the third instar disc, despite the fact that normal EGFR signalling is very active at this stage. In the case of dE2F, this early-onset apoptosis is antagonized by EGFR signalling, reflecting the ongoing survival-promoting role of the EGFR throughout disc development. The distinction between this and the phenomenon report in this study is demonstrated by the fact that the rough eye phenotype caused by simultaneous overexpression of dE2F, dDP and p35 is not suppressed by dTCF or arm heterozygosity. Also, there is no known requirement for arm in the larval disc. Taken together, these observations suggest that the dramatic EGFR-mediated delay of the Arm*-induced apoptotic phenotype is unique, and that the mechanism on which it is based is distinct from that employed by the receptor in generally promoting cell survival in the disc (Freeman, 2001).

Evidence that Egfr contributes to cryptic genetic variation for photoreceptor determination in natural populations of Drosophila

One objective of quantitative genetics is to identify the nucleotide variants within genes that contribute to phenotypic variation and susceptibility. In an evolutionary context, this means characterizing the molecular polymorphisms that modify the penetrance and expressivity of perturbed traits. A survey of association (between 267 SNPs in almost 11 kb of the D. melanogaster Egfr and the degree of eye roughening due to a gain-of-function EgfrE1 allele crossed into 210 isogenic wild-type lines) provides evidence that a handful of synonymous substitutions supply cryptic variation for photoreceptor determination. Ten sites exceed Bonferroni threshold for association in two sets of crosses to different EgfrE1 backgrounds including a particularly significant cluster of sites in tight linkage disequilibrium toward the 3' end of the coding region. Epistatic interaction of this cluster with one other site enhances the expressivity of this haplotype. Replication of the strongest associations with an independent sample of 302 phenotypically extreme individuals derived from 1000 crosses of EgfrE1 to freshly trapped males was achieved using modified case-control and transmission-disequilibrium tests. A tendency for the rarer alleles to have more disrupted eye development suggests that mutation-selection balance is a possible mechanism contributing to maintaining cryptic variation for Egfr (Dworkin, 2003).

Induction and autoregulation of Bar during retinal neurogenesis

Neurogenesis in the Drosophila eye imaginal disc is controlled by interactions of positive and negative regulatory genes. The basic helix-loop-helix (bHLH) transcription factor Atonal (Ato) plays an essential proneural function in the morphogenetic furrow to induce the formation of R8 founder neurons. Bar homeodomain proteins are required for transcriptional repression of ato in the basal undifferentiated retinal precursor cells to prevent ectopic neurogenesis posterior to the furrow of the eye disc. Thus, precise regulation of Bar expression in the basal undifferentiated cells is crucial for neural patterning in the eye. Evidence is shown that Bar expression in the basal undifferentiated cells is regulated by at least three different pathways, depending on the developmental time and the position in the eye disc. (1) At the time of furrow initiation, Bar expression is induced independent of Ato by Hedgehog (Hh) signaling from the posterior margin of the disc. (2) During furrow progression, Bar expression is also induced by Ato-dependent EGFR (epidermal growth factor receptor) signaling from the migrating furrow. (3) Once initiated, Bar expression can be maintained by positive autoregulation. Therefore, it is proposed that the domain of Bar expression for Ato repression is established and maintained by a combination of non autonomous Hh/EGFR signaling pathways and autoregulation of Bar (Lim, 2004).

To identify activators of Bar expression in the basal undifferentiated cells, focus was placed on two different transcription factors, Lozenge (Lz) and Glass (Gl), as candidates. Both proteins are known to be required for normal Bar expression in R1/6 photoreceptor cells, but it has not been demonstrated whether they are also required for Bar expression in the basal undifferentiated cells. Lz is expressed in R1, 6 and 7 photoreceptor cells and is required for normal level of Bar expression in R1/6 cells. In the basal undifferentiated cells, Lz is co-expressed with Bar in a majority of Bar-expressing cells, except in a group of cells just posterior to the furrow. To test whether Lz is also required for Bar expression in the basal undifferentiated cells, Bar expression was examined in homozygous lzr15 mutants and loss-of-function (LOF) clones of lzr15, a null allele of lz. It was found that the expression level of Bar is strongly decreased but not completely eliminated in R1/6 photoreceptor cells within lzr15 mutant clones. However, Bar expression in the basal undifferentiated cells is little changed compared with its expression level in adjacent wild-type cells. These results suggest that Lz is necessary to activate Bar expression in R1/6 cells, but not in the basal undifferentiated cells behind the furrow (Lim, 2004).

Next, the requirement for Gl was examined in undifferentiated cells. Gl is a zinc-finger protein expressed in all cells posterior to the furrow. Gl is not necessary for Bar expression in the basal undifferentiated cells although it is essential for Bar expression in R1/6 photoreceptor cells. Taken together, these results suggest that Bar expression requires other activators in the basal undifferentiated cells (Lim, 2004).

Based on the evidence presented in this study, a model is proposed for the regulation of Bar expression in the basal undifferentiated cells. Prior to photoreceptor differentiation at the time of furrow initiation, Bar expression in the basal undifferentiated cells near the posterior region of the disc is induced by secreted signaling factors from the posterior margin. Hh signaling from the posterior margin is required for the initial induction of Bar expression. During furrow progression, a narrow region of Bar expression immediately posterior to the furrow depends on Ato from the furrow. EGFR signaling may partially mediate Ato effects on Bar expression. Hh produced by photoreceptor cells generated behind the furrow may also be required in part for Bar expression near the furrow during furrow progression. Finally, Bar is autoregulated to maintain its expression. The properly expressed Bar proteins repress ato transcription in the basal undifferentiated cells, thereby preventing ectopic photoreceptor differentiation posterior to the furrow (Lim, 2004).

Hh expression is dynamic, depending on the time and the position in the developing eye disc. In the early third instar eye disc, Hh is expressed in the posterior margin and is required for the furrow initiation. During furrow progression, Hh is also produced in the differentiating photoreceptor cells generated posterior to the furrow and secreted anteriorly to promote furrow progression. During this process, Bar is specifically expressed in the basal undifferentiated cells posterior to the furrow and inhibits ectopic retinal neurogenesis by repressing proneural gene ato expression (Lim, 2004).

Hh signaling is required for Bar expression in the basal undifferentiated cells during initial eye development because Bar expression is strongly reduced or absent within smo LOF clones generated near the furrow or close to the posterior margin of the disc. Prior to the photoreceptor differentiation, Hh expressed in the posterior margin of the disc is responsible for Bar expression at specific distances from the posterior region of the eye disc proper. A graded expression of Bar near the posterior region in ato1 mutant eye disc might be the effects of Hh secreted by the posterior margin (Lim, 2004).

During furrow progression, Hh signaling is required for Ato expression in the furrow, and Ato-mediated EGFR signaling is required for Bar activation. Therefore, it is possible that the loss of Bar expression near the furrow in smo LOF clones might be caused by indirect effects of reduced Ato expression rather than by direct effects of Hh signaling on Bar expression. Hh may partially contribute to Bar expression by activating normal levels of Ato expression in the furrow. Thus, the Hh-Ato-EGFR cascade activates Bar expression just posterior to the furrow. Alternatively, since Hh signaling may also affect furrow progression, it is possible that the loss of Bar expression near the furrow in smo LOF clones might be caused by indirect effects of slow furrow migration rather than by direct effects of Hh signaling on Bar expression (Lim, 2004).

The results suggest that Ato is required nonautonomously for the induction of Bar expression just posterior to the migrating furrow. Although Ato acts as an activator for Bar expression, expression of these proteins always show a juxtaposed complementary pattern along the furrow. This suggests that some mediator(s) is required for transducing Ato effects on Bar expression. EGFR activated by Ato in the furrow is required for Bar expression, suggesting that nonautonomous effects of Ato on Bar expression may be partially mediated by EGFR, as revealed by analysis of Egfr negative clones and temperature senstive mutants. Furthermore, EGFR is required for Bar expression not only in the eye disc but also in the antenna and leg discs in Drosophila, suggesting that EGFR signaling may be a common activator for Bar expression in different tissues or even in higher organisms (Lim, 2004).

Notch (N) signaling is also known to contribute to neuronal differentiation together with Hh and Dpp pathways. Thus, N signaling may play a role for Bar expression in the basal undifferentiated cells during furrow progression. Bar expression is strongly downregulated when N function is removed with a temperature-sensitive mutation (Nts) or using the Enhancer-of-split [E(spl)] mutant clones in the eye disc. This suggests that N signaling may be required for Bar expression in the basal undifferentiated cells. However, it is equally possible that loss of Bar expression in the E(spl) LOF clones or in the Nts eye disc may be an indirect secondary effect of the lack of the basal undifferentiated cells because nearly all cells in the basal region of the eye disc differentiate into photoreceptor cells without N function. Further analysis of Bar regulation at the molecular level will be helpful to identify direct regulators of Bar expression in the undifferentiated cells of the eye disc (Lim, 2004).

Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye

In the developing eye of Drosophila, the EGFR and Notch pathways integrate in a sequential, followed by a combinatorial, manner in the specification of cone-cell fate. This study demonstrates that the specification of primary pigment cells requires the reiterative use of the sequential integration between the EGFR and Notch pathways to regulate the spatiotemporal expression of Delta in pupal cone cells. The Notch signal from the cone cells then functions in the direct specification of primary pigment-cell fate. EGFR requirement in this process occurs indirectly through the regulation of Delta expression. Combined with previous work, these data show that unique combinations of only two pathways -- Notch and EGFR -- can specify at least five different cell types within the Drosophila eye (Nagaraj, 2007).

Unlike photoreceptor R cells, cone cells do not express Delta at the third instar stage of development. However, these same cone cells express Delta at the pupal stage. In addition, correlated with this Delta expression, the upregulation of phosphorylated MAPK was observed in these cells. This is very similar to the earlier events seen in R cells during larval development, in which the activation of MAPK causes the expression of Delta. Also, as in the larval R cells, the pupal upregulation of Delta in cone cells is transcriptional. A Delta-lacZ reporter construct, off in the larval cone cell, is detected in the corresponding pupal cone cells. To determine whether EGFR is required for the activation of Delta in the pupal cone cells, the temperature-sensitive allele EGFRts1 was used. Marked clones of this allele were generated in the eye disc using ey-flp at permissive conditions and later, in the mid-pupal stages, shifted the larvae to a non-permissive temperature. Cells mutant for EGFR, but not their adjacent wild-type cells, showed a loss of Delta expression. However, both mutant and wild-type tissues showed normal cone-cell development, as judged by Cut (a cone-cell marker) expression. As supporting evidence, ectopic expression of a dominant-negative version of EGFR (EGFRDN) in cone cells using spa-Gal4 after the cells have already undergone initial fate specification also causes a complete loss of Delta expression without compromising the expression of the cone-cell-fate-specification marker (Nagaraj, 2007).

Gain-of-function studies further support the role of EGFR signaling in the regulation of Delta expression in cone cells. Although weak EGFR activation is required for cone-cell fate, activated MAPK is not detectable in cone-cell precursors of the third instar larval eye disc. When spa-Gal4 (prepared by cloning the 7.1 kb EcoRI genomic fragment of D-Pax2) is used to express an activated version of EGFR in larval cone cells, detectable levels of MAPK activation in these cells were found and the consequent ectopic activation of Delta in the larval cone cells occurred. Taken together, these gain- and loss-of-function studies show that, during pupal stages, EGFR is required for the activation of Delta. However, this Delta expression is not essential for the maintenance of cone-cell fate (Nagaraj, 2007).

In larval R cells, the activation of Delta transcription in response to EGFR signaling is mediated by two novel nuclear proteins, Ebi and Sno. To determine the role of these genes in wild-type pupal-cone-cell Delta expression, sno and ebi function were selectively blocked in the pupal eye disc. A heteroallellic combination of the temperature-sensitive allele snoE1 and the null allele sno93i exposed to a non-permissive temperature for 12 hours caused a significant reduction in Delta expression. Similarly, a dominant-negative version of ebi also caused the loss of Delta expression. Importantly, pupal eye discs of neither spa-Gal4, UAS-ebiDN nor snoE1/sno93i showed any perturbation in cone-cell fate, as judged by the expression of Cut. Thus, as in the case of larval R cells, the loss of ebi and sno in the pupal cone cells causes the loss of Delta expression without causing a change in cone-cell fate (Nagaraj, 2007).

To test whether the expression of Delta in pupal cone cells is required for the specification of primary pigment cells, Nts pupae were incubated at a non-permissive temperature for 10 hours during pupal development and pigment-cell differentiation was monitored using BarH1 (also known as Bar) expression as a marker. Loss of Notch signaling during the mid-pupal stages caused a loss of Bar, further demonstrating the requirement of Notch signaling in the specification of primary pigment-cell fate. Similarly, when the 54CGal4 driver line, which is activated in pigment cells, was used to drive the expression of a dominant-negative version of Notch, pupal eye discs lost primary pigment-cell differentiation, again suggesting an autonomous role for Notch in pigment-cell precursors. In neither the Nts nor the 54C-Gal4, UAS-NDN genetic background was perturbation observed in cone-cell fate specification. It is concluded that Delta activation mediated by EGFR-Sno-Ebi in pupal cone cells is essential for neighboring pigment-cell fate specification (Nagaraj, 2007).

Delta-protein expression in pupal cone cells is initiated at 12 hours and is downregulated by 24 hours of pupal development. To determine the functional significance of this downregulation, the genetic combination spa-Gal4/UAS-Delta was used, in which Delta is expressed in the same cells as in wild type, but is not temporally downregulated. Whereas, in wild type, a single hexagonal array of pigment cells surrounded the ommatidium, in the pupal eye disc of spa-Gal4, UAS-Delta flies, multiple rows of pigment cells were observed surrounding each cluster. Furthermore, in wild type, only two primary pigment cells were positive for Bar expression in each cluster, whereas, in spa-Gal4, UAS-Delta pupal eye discs, ectopic expression of Bar was evident in the interommatidial cells. Therefore, the temporal regulation of Notch signaling and its activation, as well as its precise downregulation, are essential for the proper specification of primary pigment-cell fate (Nagaraj, 2007).

By contrast to the autonomous requirement for Notch signaling in primary pigment cells, the function of the EGFR signal appears to be required only indirectly in the establishment of primary pigment-cell fate through the regulation of Delta expression in the pupal cone cells. When a dominant-negative version of EGFR was expressed using hsp70-Gal4 at 10-20 hours after pupation, no perturbation was observed in the specification of primary pigment cells, as monitored by the expression of the homeodomain protein Bar. By contrast, the expression of dominant-negative Notch under the same condition resulted in the loss of Bar-expressing cells. Thus, in contrast to Notch, blocking EGFR function at the time of primary pigment-cell specification does not block the differentiation of these cells. Importantly, blocking EGFR function in earlier pupal stages caused the loss of Delta expression in cone cells and the consequent loss of pigment cells. Based on these observations, it is concluded that, in the specification of primary pigment-cell fate, the Notch signal is required directly in primary pigment cells, whereas EGFR function is required only indirectly (through the regulation of Delta) in cone cells (Nagaraj, 2007).

The Runt-domain protein Lz functions in the fate specification of all cells in the developing eye disc arising from the second wave of morphogenesis. At a permissive temperature (25°C), lzTS114 pupal eye discs showed normal differentiation of primary pigment cells. lzTS114 is a sensitized background in which the Lz protein is functional at a threshold level. When combined with a single-copy loss of Delta, a dosage sensitive interaction caused the loss of primary pigment cells. By contrast, under identical conditions, a single-copy loss of EGFR function had no effect on the proper specification of primary pigment-cell fate. This once again supports the notion that the specification of primary pigment cells directly requires Lz and Notch, whereas EGFR is required only indirectly to activate Delta expression in cone cells (Nagaraj, 2007).

This study highlights two temporally distinct aspects of EGFR function in cone cells. First, this pathway is required for the specification of cone-cell fate at the larval stage, and EGFR is then required later in the pupal cone cell for the transcriptional activation of Delta, converting the cone cell into a Notch-signaling cell. Delta that was expressed in the cone cell through the activation of the Notch pathway functioned in combination with Lz in a cell autonomous fashion and promoted the specification of the primary pigment-cell fate (Nagaraj, 2007).

Studies using overexpressed secreted Spitz have shown that ectopic activation of the EGFR signal in all cells of the pupal eye disc results in excess primary pigment cells. This study shows that EGFR activation in the pupal eye disc is required for the transcriptional activation of Delta in cone cells, but that the loss of EGFR function at the time when primary pigment cells are specified does not perturb their differentiation. It is concluded that the ectopic primary pigment cells seen in an activated-EGFR background result from the ectopic activation of Delta, which then signals adjacent cells and promotes their differentiation into primary pigment cells. Indeed, it has been shown that excessive Delta activity results in the over specification of primary pigment cells. The results are also consistent with the previous observation that the EGFR target gene Argos is not expressed in primary pigment cells in pupal eye discs. Additionally, loss of EGFR function in pupal eye discs does not perturb the normal patterning of interommatidial bristle development, which develop even later than the primary pigment cells (Nagaraj, 2007).

The elucidation of the Sevenless pathway for the specification of R7 led to the suggestion that different cell types within the developing eye in Drosophila will require combinations of dedicated signaling pathways for their specification. However, studies from several laboratories have suggested that the Sevenless pathway seems to be an exception, in that cell-fate-specification events usually require reiterative combinations of a very small number of non-specific signals. Cone-cell fate is determined by the sequential integration of the EGFR and Notch pathways in R cells followed by the parallel integration of the EGFR and Notch pathways in cone-cell precursors. This study shows that the most important function of EGFR in the specification of primary pigment cells is to promote the transcriptional activation of Delta in cone cells through the EGFR-Ebi-Sno-dependent pathway. The sequential integration of the EGFR and Notch pathways, first used in the larval stage for Delta activation in R cells, is then reused a second time in cone cells to regulate the spatiotemporal expression of Delta, converting the cone cells at this late developmental stage to Notch-signaling cells. Delta present in the cone cell then signals the adjacent undifferentiated cells for the specification of primary pigment cells. For this process, the Notch pathway functions directly with Lz but indirectly with EGFR. Through extensive studies of this system it now seems conclusive that different spatial and temporal combinations of Notch and EGFR applied at different levels can generate all the signaling combinations needed to specify the neuronal (R1, R6, R7) and nonneuronal (cone, pigment) cells in the second wave of morphogenesis in the developing eye disc (Nagaraj, 2007).

The EGFR and Notch pathways are sequentially integrated, in a manner similar to that described here, in multiple locations during Drosophila development. In the development of wing veins, EGFR that is activated in the pro-vein cells causes the expression of Delta, which then promotes the specification of inter-vein cells. Similarly, these two pathways are sequentially integrated in the patterning of embryonic and larval PNS, and during muscle development. Indeed, there are striking similarities between the manner in which the EGFR and Notch pathways are integrated in the developmental program in the C. elegans vulva and the Drosophila eye. During vulval fate specification in the C. elegans hermaphrodite gonad, anchor cells are a source of EGFR signal (Lin3), which induces the specification of the nearest (P6) cell to the primary cell fate from within a group of six equipotent vulval precursor cells (VPC). This high level of EGFR activation induces the transcriptional activation of Notch ligands in the primary cells in what can be considered sequential integration of the two pathways - the Notch signal from the primary cell both inhibits EGFR activity in the VPCS on either side of P6.p and also promotes the secondary cell fate. Thus, the reiterative integration of these two signals, in series and in parallel, can be used successfully to specify multiple cell fates in different animal species. Given that the RTK and Notch pathways function together in many vertebrate developmental systems, it is likely that similar networks will be used to generate diverse cell fates using only a small repertoire of signaling pathways (Nagaraj, 2007).

Egfr and eye morphogenesis (part 1/2)

Table of contents

EGF receptor : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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