argos

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

argos mutant embryos show expansion of ventral cell fates suggesting hyperactivation of the EGF-R pathway (Golembo, 1996).

A good example of the function of EGF-R in regulating cell development is found by examining the role of EGF-R in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the EGF-Receptor and subsequent Ras1/Raf-mediated signal transduction (See Drosophila Ras1). In the first step six midline cells in each segment, originating from the anterior-most three of a total of eight midline progenitor cells, are determined as the midline glia equivalence group. The process of generation of the midline glia equivalence group involves Notch function and segmentation genes. It might also depend on the function of single minded, the master regulatory gene of midline development. The single minded transcript accumulates in the midline glia and, depending on the context, can act either as a transcriptional activator or repressor. By the end of embryogenesis the final number of midline glial cells is about 3 to 4. Thus, the final number of cells has to be selected from the initially defined equivalence group in a second step (Scholz, 1997).

Egf-r mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egf-r in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egf-r signaling and are destined for cell death. Both Rhomboid and Argos control activation of the EGF-R during midline glia development. It is thought that a graded activation of EGF-R is brought about by the activity of Rhomboid, which is thought to promote EGF receptor signaling, possibly by cell autonomous activation of the EGF-R ligand Spitz. Ectopic rhomboid leads to extra midline glial cells. EGF-R activates PointedP2 through phosphorylation; Pointed in turn activates the transcription of argos. Argos negatively regulates EGF-R signaling non-cell autonomously and competes with Spitz function. pointed mutants form extra glial cells. Yan antagonizes PointedP2A in midline glial cells, just as it does in the developing photoreceptor cells (Scholz, 1997).

The midline glia of the Drosophila embryonic nerve cord undergo a reduction in cell number after facilitating commissural tract morphogenesis. The numbers of midline glia entering apoptosis at this stage can be increased by a loss or reduction of function in genes of the spitz group or the Drosophila EGF receptor pathway. Argos, a secreted molecule with an atypical EGF motif, is postulated to function as a Egf-r antagonist. argos function reflects or is involved in the process that restricts midline glia numbers developmentally. In this study, the role of argos is assessed in the determination of midline glia cell numbers. Fewer midline glia enter apoptosis in embryos lacking argos function. Ectopic expression of argos is sufficient to remove all Egf-r-expressing midline glia from the nerve cord, even those that already express argos. Egf-r expression is not terminated in the midline glia after spitz group signaling triggers changes in gene expression. Paradoxically, although all midline glia express Egf-r, argos expression is restricted to the midline glia that do not enter apoptosis. It is therefore likely that an attenuation of Egf-r signaling by Argos is integrated with the augmentation of Egf-r signaling by Spitz throughout the period of reduction of midline glia numbers, and argos-expressing midline glia depend upon continued Spitz activation of the EGF-R at levels higher than adjacent non-argos expressing midline glia to overcome possible autocrine inhibition by released Argos. It is suggested that signaling by Spitz (but not Argos) is restricted to adhesive junctions. In this manner, midline glia not forming signaling junctions remain sensitive to juxtacrine Argos signaling, while an autocrine Argos signal is excluded by the adhesive junction (Stemerdink, 1997).

The argos null mutation causes an increase in chordotonal (Ch) organs in both the thoracic and the abdominal segments, whereas overexpression of the argos gene results in a decrease in these organs. Argos transcripts are expressed transiently in the cells surrounding the Ch organ precursor, and rhomboid, which is involved in the regulation of the number of Ch organs, acts epistatically to argos in this event. These findings suggest that argos plays a role in Ch organ precursor formation and regulates the final number of Ch organs (Okabe, 1996).

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called ‘head midline’) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

Signaling by the epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. These denticles derive from a segmental zone of embryonic cells in which EGFR signaling activity is maximal (Szuts, 1997).

Both Egfr and spitz are expressed fairly uniformly throughout the embryonic epidermis. However, Spi appears to be incapable of activating Egfr unless it is processed into a secreted form; there is genetic evidence that the membrane-spanning products of the rhomboid and Star genes may be mediating this process. In young embryos (before the germ band is fully extended) rhomboid is expressed in distinct segmental stripes. These stripes remain visible until stage 16 by which time they fade away. They become circumferential, and one cell wide in the dorsal half of the embryo. In the ventral half, they are also one cell wide in the thoracic segments, a bit wider in the first abdominal segment, and at least two cells wide in all other segments. These stripes correspond roughly to the cells giving rise to denticle rows 2 and 3. argos is observed in, and adjacent to, cells expressing rhomboid, and indeed is expressed in circumferential stripes after completion of germ-band retraction. The segmental stripes of argos are fairly similar to the rhomboid stripes but a bit wider. Argos is an inhibitor of Egfr function, so Argos reduction is expected to result in overactivation of Egfr signaling. argos loss of function mutants result in an entire additional row of denticles anterior to row 1 (Szuts, 1997).

High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of Argos. These homeotic genes account for the main difference in shape between abdominal and thoracic denticle belts (Szuts, 1997). argos mutants show reduced viability and a rough eye phenotype, including blistering in the posterior region. There is abnormal rhabdomere morphology and extra outer photoreceptors occur as well. In argos mutants, mystery cells are transformed into extra photoreceptors, at the expense of pigment cells (Freeman 1992 and Kretzschmar, 1992). Loss-of-function mutations in the argos gene cause impaired retinal projections to the optic lobe (Kretzschmar, 1992), and the formation of extra veins (Sawamoto, 1994).

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homolog (Egfr) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. This phenotype suggests a possible role for Egfr in specifying the founder R8 photoreceptor cells for each ommatidium. Ellipse mutations have been used to probe the role of Egfr in eye development: Elp mutations result from a single amino acid substitution in the kinase domain, which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirm that the mutation is hypermorphic in vivo, but the Egfr function is elevated less than by ectopic expression of the ligand Spitz. Ectopic Spi promotes photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of Egfr activation were assessed to explore the basis of these distinct outcomes. Elp mutations cause overexpression of the Notch target gene E(spl) mdelta and require the function of Notch to suppress ommatidium formation. E(spl) mdelta is known to be expressed in cell clusters in the morphogenetic furrow of wild type eyes. The Elp phenotype also depends on the secreted protein Argos and therefore, in Elp;aos double mutants, the phenotype is reverted. Complete loss of Egfr function in clones of null mutant cells leads to delay in R8 specification and subsequently to the loss of mutant cells. The Egfr null phenotype is distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands is required for aspects of Egfr function. No delay in ato refinement is found for spi mutant eyes, and vn mutants have vestigial eye discs that usually fail to differentiate any type of eye cell. In normal development, Egfr protein is expressed in most retinal cells, but at distinct levels. Antibody specific for diphospho-ERK as well as expression of the Egfr target gene argos was used to assess the pattern of Egfr activity; highest activity was found in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggest that this activity may not be required for normal ommatidium development. Since distinct phenotypic effects for four different levels of Egfr activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function are seen, it is proposed that multiple thresholds separate several aspects of Egfr function. These include activation of N signaling to repress R8 specification; turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation. It is suggested that Egfr activation by Spi is the only signal that the R8 cell needs to provide for most photoreceptor cells and that ubiquitous targeted Spi expression converts every retinal cell to an R1-R7 photoreceptor, at the expense of R8 and other cell types. Argos is required for loss of ommatidia in Elp. Nonautonomy of Elp mutations suggests that aos acts nonautonomously to inhibit ommatidium formation (Lesokhin, 1999).

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, a genetic screen was performed for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, the eye and wing phenotypes of the clown mutants were examined in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation is impaired, showing a phenotype similar to that caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There is also an increased number of pigment cells in the clown eyes. Epistatic analysis places clown between argos and Ras1. In addition, clown negatively regulates the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, six mutations of novel genes have been identified. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway (Taguchia, 2000).

The effects of argos overexpression on eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and are enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutation. A loss-of-function argos mutation enhances the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversley, overexpression of argos inhibits formation of the extra R7 cells that is caused by high-level MAPK/ERK-A activity. A phenotype of the sevenless-argos double mutants reveals that sevenless is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade (Sawamoto, 1996b).

There are several partially redundant gene functions in the immediate vicinity of argos at the chromosomal position 73A. One of them bulge suppresses the rough eye phenotype associated with overexpression of argos; conversely, amorphic argos mutations suppress the eye phenotype seen in flies bearing a single dominant bulge allele. bulge is 0.15 cM distal to argos. A second gene, suppressor of bulge and argos (soba) suppresses the eye phenotypes seen in flies expressing either the dominant bulge allele or the hs-argos construct. soba resides 120 kb proximal to argos. In addition, one allele of a new gene, clown, suppresses the eye phenotypes associated with hs-argos and bulgeDominant. clown maps on chromosome 3 at the cytological position 68CD (Wemmer, 1995).

What are the biological roles of the related EGF domains of the Drosophila EGF ligands? The EGF domain contains a series of six cysteines, which form three disulfide bonds to generate a looped structure, and a number of other highly conserved residues that are known to be required for binding and activating members of the vertebrate ErbB receptor family. The EGF domains of Vein and Spitz are not highly related (38% conserved) but have more sequence conservation with each other than with Aos. Additionally, the length of the predicted B loop that forms from the region between cysteines 3 and 4 is significantly longer in Aos than in the activating ligands (Schnepp, 1998).

Chimeric molecules were created by exchanging the EGF domain of Vn for those of either Spi or Aos. The activity of these chimeras was compared with the native factors in vitro and in vivo. Secreted Spi (sSpi, the active form of Spi) and Aos increase or decrease, respectively, the level of Egfr tyrosine phosphorylation in Drosophila S2-DER tissue-culture cells. The Vn:Vn EGF chimera, which serves as a control for the effect of the additional residues introduced during construction of the chimeras, behaves like native Vn. In contrast, possession of the Spi-EGF domain converts Vn into a stronger Egfr activator. The Vn:Aos EGF chimera behaves as an inhibitor, rather than an activator and caused a reduction in Egfr activation resulting from the ligand-independent activation of Egfr. These results show that the properties of Vn are changed when its EGF domain is swapped with that of Spi or Aos so that the chimeras behave like the factors from which the EGF domain is derived (Schnepp, 1998).

In the embryo, ectopic activation of the DER pathway by sSpi, using the Gal4-UAS system, causes an expansion of ventral cell fates that can be monitored by expression of the ventral cell marker orthodenticle (otd). Ectopic expression of native Vn causes no change in the expression of otd. The Vn:Vn EGF chimera causes a very mild expansion of otd expression. This slight effect could be the result of higher expression of the transgene. In contrast, ectopic expression of the Vn:Spi EGF chimera causes a dramatic expansion of otd expression that is similar to that seen with ectopic expression of sSpi. In the wing, ectopic activation of the DER pathway is characterized by the appearance of extra veins. Ectopic expression of native Vn in pupal interveins produces a mild or moderate extra-vein phenotype, whereas ectopic expression of sSpi causes a strong extra-vein phenotype. A direct role for Vn in normal vein development has been demonstrated; such a role has not been demonstrated for sSpi but is likely to take place. Ectopic expression of the Vn:Vn EGF chimera gives extra-vein phenotypes similar to those seen after ectopic expression of native Vn. In contrast, ectopic expression of the Vn:Spi EGF chimera produces a strong extra-vein phenotype like that seen following ectopic expression of sSpi. In the eye, ectopic activation of the Egfr pathway is characterized by loss of ommatidia, over-recruitment of cell types, and blistering. Ectopic expression of native Vn posterior to the morphogenetic furrow in the eye disc has no effect on the adult eye phenotype; in contrast, ectopic expression of sSpi and the Vn:Spi EGF chimera produces small disorganized eyes with blisters. Surprisingly, ectopic expression of the Vn:Vn EGF chimera also showed a strong eye phenotype. This result suggests that regions outside the EGF domain can affect the activity of a factor because the manipulation used to create the chimeras changed 4 residues flanking the EGF domain. These in vivo data corroborate the biochemical data that Vn is a less potent activator of Egfr than sSpi. The EGF domain is a key feature that differentiates Vn and sSpi because Vn can be converted into a more potent Egfr activator if its EGF domain is swapped with that of Spi. The ability to differentially regulate signaling, depending on whether Vn or sSpi is utilized, may be one mechanism by which DER elicits specific cell responses during development (Schnepp, 1998).

To test whether Vn can be converted into an inhibitor by swapping its EGF domain with that of Aos, the effects of ectopic expression of Vn, Aos, and the Vn:Aos EGF chimera were compared in larval wing and eye discs. Native Vn produces an extra-vein phenotype when expressed ectopically in larval wing discs, as expected for an activator of Egfr signaling. In the wing, ectopic suppression of the Egfr pathway is characterized by vein loss; ectopic expression of native Aos or the Vn:Aos EGF chimera results in vein loss. The vein loss phenotype associated with ectopic expression of Vn:Aos EGF is not as severe as that caused by native Aos. In the eye, reduction in activity of the Egfr pathway is characterized by loss of cell types and fusion of ommatidia. There is no observable effect on adult eye phenotype following ectopic expression of native Vn in eye discs, but ectopic expression of the Vn:Aos EGF chimera produces a rough eye phenotype with fused lenses similar to, but not as severe as, that produced by ectopic expression of native Aos. These results show that the EGF domain is a key determinant responsible for the difference between Vn and Aos and that the EGF swap is sufficient to convert an Egfr activator into an inhibitor. The Vn:Aos EGF chimera is apparently not as potent an inhibitor as native Aos in the eye or the wing, suggesting that other regions of the proteins (Vn and/or Aos) may play modulating roles (Schnepp, 1998).

The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, since it is a secreted protein with a single epidermal growth factor (EGF) domain. An investigation was carried out to determine which regions of the Argos protein are essential for inhibition. A series of deletions were made and tested in vivo; analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-alpha-like factor), would disclose what makes one protein inhibitory and the other activating (Howes, 1998).

In one set of chimeras, the entire EGF domains of Spitz and Argos were swapped. The SAS (Spitz N-terminal; Argos EGF domain; Spitz C-terminal) and ASA pair (in which only the EGF domains themselves were swapped) were designed to test the possibility that the EGF domain is solely an EGFR binding domain, with no role in activation or inhibition. If this were the case, the EGF domains would be interchangeable and SAS might behave like Spitz, and ASA like Argos. In the SA and AS pair, the complete C termini of Spitz and Argos were swapped, including the EGF domains. If the Argos EGF domain and the adjacent C terminus were sufficient to confer the Argos protein's inhibitory function, the SA chimera (Spitz N terminus with the Argos EGF domain and C terminus) might be expected to behave like Argos. Similarly, since the N terminus of Argos appears not to contain sufficient information to confer inhibitory function, the AS chimera (Argos N terminus with the Spitz EGF domain and C terminus of the secreted form) might retain Spitz activity. However, neither SAS nor ASA has any activity; they neither suppress nor enhance the argos mutant phenotype. This suggests that the EGF domain is more than simply an EGFR binding domain; instead, it participates in specifying whether the receptor is activated or inhibited. SA also has no Argos activity (nor, as expected, is it Spitz-like). This implies that sequences N-terminal to the Argos EGF domain are necessary for its function. The AS chimera activates the EGF receptor, suggesting its function is similar to Spitz, not Argos. When overexpressed in the wing, it produces the characteristic extra vein phenotype of EGFR activation. In this same assay, overexpression of Argos causes the opposite phenotype, namely loss of wing veins. These results indicate that the large N-terminal region of Argos is not sufficient to convert Spitz into an inhibitor. AS, however, rescues spitz eye mutants only partially, suggesting that Spitz's potency is reduced by the addition of 355 amino acids of the Argos N terminus (Howes, 1998).

The spacing between the six essential cysteines in EGF domains varies only slightly. In particular, between C3 and C4 (the B-loop), there are usually 10 amino acids in EGF-like ligands; the range is 8-13 in all known EGF repeats. In Argos, there are 20 residues between C3 and C4, and this extension might account for Argos's unusual inhibitory function. Indeed, although the function of this B-loop remains uncertain, many studies have implicated it in EGF binding and activation of the receptor. The C-loop of EGF domains, between cysteines 5 and 6, is also critical for the function of EGF-like ligands. In Argos, the C-loop is also atypical. It is only five residues long, compared with eight in Spitz and all the mammalian activating ligands, and it has two extra positive charges. To examine the functional importance of the Argos B- and C-loops, four chimeras were constructed between Argos and Spitz in which the B- and C-loops were exchanged, and tested for whether they activate or inhibit the EGFR. The extended B-loop is necessary for Argos function, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting substantially affecting this inhibitory function. Comparison of the argos genes from Drosophila and the housefly, Musca domestica, supports this structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors (Howes, 1998).

A screen was carried out in order to identify genes interacting with Armadillo, the Drosophila homolog of ß-catenin. Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Arm^over) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Arm^under). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).

Mutations in 17 genes (26 deficiencies) were characterized that interact with Arm^over and/or Arm^under. The 17 genes were sorted into four groups. Group 3 consisted of EGF pathway genes: Interactions have been observed with some, but not all, members of the EGF pathway. Those identified were Egfr, veinlet/rhomboid (ve/rho), and argos (aos). All enhance Arm^over, increasing the number of ectopic bristles in the wing blade, with ve^M4 being the strongest interactor. None show an interaction with the Arm^under stocks.

The interactions with genes encoding components of the Egf pathway were initially dismissed because argos and Egfr, which have opposite effects on the Egf pathway, interact similarly with Arm^under and Arm^over. However, recent work has demonstrated an antagonism between the Wg and Egf pathways in the embryonic epidermis. This antagonism is probably not universal since another embryonic function of Wg, the maintenance of Engrailed expression, is not affected by Egf signaling. However, the interactions that were uncovered in the wing suggest that the Egf-Wg antagonism may not be limited to cuticle patterning. It is noteworthy that, in the wing, only an interaction with Arm^over (which involves ectopic bristles) was seen. It may thus be that Wg and Egf only compete at places where specialized cuticular structure are formed, although, while denticles are negatively regulated by Wg, bristles are made in response to Wg signaling. No explanation is available as to why argos, a negative regulator of Egf signaling, should interact in the same manner as Egfr (Greaves, 1999).

The role of Ras signaling was studied in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, causes excessive cell death in developing eyes at pupal stages. The Argos-induced cell death is suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppresses the Argos-induced cell death. Thus, Argos-induced cell death seems to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid is suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death (Sawamoto, 1998).

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).

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).

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).

Argos (aos) is a secreted protein having an EGF motif. Partial loss-of-function mutations in argos result in rough eyes with supernumerary cone and R cells and extra-wing-vein phenotypes. Complete loss-of-function mutants of argos are embryonic lethals. While loss-of-function argos mutants have increased numbers of cone cells, heat-shock promoter-driven overexpression of Argos leads to a reduction in the number of cone cells. Cell culture experiments have shown that Argos is a negative regulator of the Egfr. argos mutations act as strong suppressors of the rugose mutation. The rough eye phenotype of rg is completely suppressed by a single copy of the argos loss-of-function mutation. rg/Y; argos/+ double mutants have smooth eyes and normal complement of R cells as well as cone cells. Consistent with this result is the finding that heat-shock promoter-driven overexpression of Argos acts as a dominant enhancer of the rg mutant phenotype. In a genetic screen for second-site modifiers of the argos phenotype two interacting genes have been identified and it has been suggested that they may function in the Argos-mediated signaling pathway. Mutations in bulge and soba act as dominant suppressors of the rough eye phenotype of an argos amorphic allele as well as the rough eye phenotype caused by the heat-shock-induced overexpression of the Argos protein. A single copy of bulge and soba dominantly enhance the rough eye phenotype of the rg mutants. These results are consistent with the finding that argos mutations act as strong suppressors of rugose (Shamloula, 2002).

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90°, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Epidermal growth factor receptor (Egfr) signalling is required for rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. It is proposed that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, there is an error-correction mechanism operating during pupal stages to reposition inappropriately oriented ommatidia. These results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling (Brown, 2003).

The rotational phenotypes caused by perturbation of Egfr signalling are very similar to the published phenotype of the roulette mutation, one of the few mutations reported to specifically disrupt rotation and not chirality. Interestingly, roulette turns out to be allelic to argos. The roulette mutation is now referred to as argos^rlt (Brown, 2003).

Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).

Since ommatidial rotation is a cell biological event, it is probable that among the main read-outs affected are cell-adhesion properties of the precluster cells and effects on cytoskeletal elements. This is further supported by observations that (1) Raf/MAPK-independent and thus transcription-independent Egfr/Ras signaling pathways are important, and (2) that canoe is required in this context. To address this further, two sets of experiments were performed. First, tests were performed for genetic interactions between the dosage-sensitive Star^–/+ rotation phenotype and selected factors required in cell adhesion and cytoskeletal regulation; and second, whether cell-adhesion components such as cadherins and integrins are normally localized in aos^rlt and cno^Mis1 mutant backgrounds was directly analyzed (Gaengel, 2003).

To specifically test the involvement of cytoskeletal elements and adhesion as well as junctional components, candidate genes were tested for dominant interaction of the mild Star rotation phenotype. These genetic data argue for an involvement of E-Cadherin/shotgun, the atypical cadherin Flamingo (Fmi), the adherens junction protein canoe, non-muscle myosin II (zipper), the septin peanut, and capulet, a protein with actin and adenylate cyclase-binding ability (Gaengel, 2003).

Next, the expression of Fmi and Shotgun in ommatidial preclusters was examined during rotation. Strong LOF alleles of Egfr and its signaling components also affect cell proliferation, fate specification and survival, making the analysis of cell adhesion and junctional components in the context of rotation rather difficult. Thus localization of the cadherins and Arm/ß-catenin was examined in imaginal discs of the rotation-specific aos^rlt allele (Gaengel, 2003).

Although the overall expression and localization of Shotgun and Arm/ß-catenin are largely unaffected, the localization of Fmi is changed in aos^rlt discs. In WT, Fmi is initially present apically in all cells of the morphogenetic furrow and subsequently becomes asymmetrically enriched in the R3/R4 precursor pair. In and posterior to column 6, Fmi is expressed at the membrane of R4, and largely depleted from R3 membranes that do not touch R4, forming a horseshoe-like R4-specific pattern. In contrast, in aos^rlt discs, Fmi restriction to the R4 precursor is generally delayed, and often not established even in columns 8-12, where high levels of Fmi are still seen around the apical membrane cortex of R3 and R4. Since Fmi is thought to act as a homophyllic cell-adhesion molecule, its increased presence on R3 membranes should have a direct effect on Fmi localization in neighboring cells and thus possibly the adhesive properties of the precluster. It is worth noting that although Fmi is required during PCP establishment and R3/R4 cell-fate specification, the delay in Fmi restriction to R4 has no significant effect on the R3/R4 cell-fate decision. Although Fmi interacts with Fz and Notch in this context, the R4-specific mDelta-lacZ marker does not differ significantly from WT and adult aos^rlt eyes also display no defects in R3/R4 specification. Thus, it appears that the delay in Fmi localization specifically affects ommatidial rotation, probably through adhesion, and possibly explains the broad range of rotation angles in aos^rlt and other Egfr pathway mutants (Gaengel, 2003).

A gradient of epidermal growth factor receptor signaling determines the sensitivity of rbf1 mutant cells to E2F-dependent apoptosis

Retinoblastoma (Rb) family proteins control E2F-dependent transcription and restrict cell proliferation. In the early G₁ phase of the cell cycle, Rb family proteins bind to E2F family members, inhibiting their ability to activate transcription and recruiting repressor complexes to DNA. In late G₁ to S phase, cyclin-dependent kinases (CDK) phosphorylate Rb family proteins, liberating E2F and activating E2F-dependent transcription. One of the least-well-understood aspects of in vivo studies of Rb function is the fact that the inactivation of Rb often sensitizes cells to apoptosis. The extent of apoptosis caused by the inactivation of Rb is highly cell type and tissue specific, but the underlying reasons for this variation are poorly understood. This study characterizes specific time and place during Drosophila development where rbf1 mutant cells are exquisitely sensitive to apoptosis. During the third larval instar, many rbf1 mutant cells undergo E2F-dependent cell death in the morphogenetic furrow. Surprisingly, this pattern of apoptosis is not caused by inappropriate cell cycle progression but instead involves the action of Argos, a secreted protein that negatively regulates Drosophila epidermal growth factor receptor (EGFR [DER]) activity. Apoptosis of rbf1 mutant cells is suppressed by the activation of DER, ras, or raf or by the inactivation of argos, sprouty, or gap1, and inhibition of DER strongly enhances apoptosis in rbf1 mutant discs. RBF1 and a DER/ras/raf signaling pathway cooperate in vivo to suppress E2F-dependent apoptosis and the loss of RBF1 alters a normal program of cell death that is controlled by Argos and DER. These results demonstrate that a gradient of DER/ras/raf signaling that occurs naturally during development provides the contextual signals that determine when and where the inactivation of rbf1 results in dE2F1-dependent apoptosis (Moon, 2006).

This study takes advantage of the observation that the inactivation of rbf1 in the Drosophila eye results in a distinctive pattern of apoptosis that is tightly linked to eye development, and this model system was used to define a cellular context in which RBF1 is needed to protect cells against dE2F1-dependent cell death. The results show that the cellular response to the inactivation of rbf1 involves a combination of signals. Deregulated dE2F1 provides one function that is required for apoptosis. However, in most situations, deregulation of the endogenous dE2F1 is not sufficient to induce apoptosis. In addition, a second condition, the down-regulation of an EGFR/Ras/Raf signaling pathway, is also necessary. In the eye imaginal disc, the EGFR/Ras/Raf signaling pathway is down-regulated at the region immediately anterior to the 'intermediate group' (IG) of cells, from which the R8 founder cell will be selected. When rbf1 mutant cells pass through this gradient, they become highly sensitive to dE2F1-dependent apoptosis. Elevation of the level of DER/Ras/Raf signaling by a variety of means suppresses apoptosis in rbf1 mutant cells. Conversely, expression of a dominant-negative mutant of DER strongly synergized with mutation of rbf1 to induce apoptosis (Moon, 2006).

Before starting this work, several different ways were considered in which the inactivation of RBF1 might result in apoptosis. If differentiated/differentiating cells try to reenter the cell cycle following the inactivation of RBF1, then an abnormal or inappropriate S-phase entry might cause apoptosis. Alternatively, one could argue that rapidly proliferating cells contain the highest levels of E2F transcriptional activity, and hence these cells ought to be most sensitive to E2F-induced apoptosis when RBF is removed. Although both models were plausible, in fact, neither explanation fits the data. rbf1 mutant eye discs display little apoptosis in either the population of differentiated cells or in actively cycling cells. Instead, rbf1 mutant cells are sensitive to apoptosis in the MF, at a time when some cells exit the cell cycle and initiate a differentiation program. This apoptosis was not accompanied by inappropriate cell cycle progression. Indeed, when rbf1 mutant cells were rescued from apoptosis, they showed no indication of S-phase entry. Hence rbf1 mutant cells were not dying because they were inappropriately progressing through the cell cycle. Instead, apoptosis was dependent on a specific developmental context. This need for the correct context may be particularly significant when designing cell culture-based screens for treatments that are synthetic lethal with the inactivation of Rb (Moon, 2006).

Several studies have shown that E2F complexes regulate the expression of proapoptotic genes, but why would the effects of losing RBF1 be sensitive to EGFR signaling? While it seems likely that deregulated dE2F1 activates transcription of several proapoptotic targets, the results indicate that an important part of the explanation lies in the regulation of the proapoptotic gene hid. hid transcripts are up-regulated in rbf1 mutant eye discs and halving the gene dosage of hid dramatically reduced apoptosis. Previous studies have shown that HID-induced apoptosis is highly sensitive to EGFR/Ras/Raf signaling. Signaling through this pathway suppresses transcription of hid and is thought to induce an inhibitory phosphorylation on the HID protein. This provides a simple model, in which the loss of RBF1 results in the elevated expression of a proapoptotic protein, which is then held in check by EGFR/Ras/Raf-mediated signaling. Apoptosis would then occur when EGFR signals are reduced. Consistent with this model, it was found that the region of the eye disc that is most sensitive to loss of RBF1 is also highly sensitive to low levels of ectopic hid expression (Moon, 2006).

Why does this pattern of apoptosis occur? It is likely that several different factors are needed to establish the gradient of DER activity. Important regulators of DER activity in the eye include Gap1, Sprouty, and Argos. In this particular context, the ability of Argos to diffuse and act at a distance from the p-Erk-positive cells appears to be important. It is suggested that the pattern of Argos expression in the developing eye disc generates a zone in which cells that have failed to exit the cell cycle and inappropriately inactivate RBF1 become prone to undergo apoptosis. In essence, this could be viewed as a developmental failsafe mechanism against inappropriate proliferation. In support of this, it is noted that E2F1 levels are transiently elevated in G₁ phase cells in the MF, even though E2F regulation is not needed for S phase entry in the second mitotic wave. Consistent with the idea that this region of the disc may be more sensitive to apoptosis, it was found that a transient pulse of cyclin E expression, which drives ectopic S phases throughout much of the disc, generates a similar stripe of apoptosis in the MF. It is curious that this sensitivity occurs at the time when the role of EGFR is apparently changing from being needed for cell proliferation in the anterior part of the disc to being required for differentiation in the posterior part of the disc. It will be interesting to discover whether similarly sensitive regions exist in other discs (Moon, 2006).

As seen with rbf1, the effects of mutating Rb in the mouse are most evident at points in development when cells attempt to exit the cell cycle and differentiate. Rb-null mouse retinas show increased cell death during the transition from proliferation to differentiation. Whether this is due to an analogous interaction between Rb/E2F and EGFR/Ras signaling has not been tested but is an interesting possibility. It is also tempting to speculate that some of the different cellular responses to the inactivation of Rb in the mouse retina may be caused by differences in EGFR/Ras-mediated differentiation signals (Moon, 2006).

There are several indications that the general phenomenon described in this study is likely to be conserved in mammalian cells. For example, recent studies have shown that apoptosis in cultured fibroblasts lacking Rb family proteins (TKO) can be suppressed by activation of Ras/Raf. Interestingly, a functional homologue of Hid, Smac/Diablo, was recently shown to be a direct target of E2F1 in mammalian cells, raising the possibility that mammalian cells may contain a regulatory loop that directly parallels the regulation of Hid. However, a connection between the proapoptotic function of Smac/Diablo and EGFR pathway has yet to be described (Moon, 2006).

The molecular events underlying the convergence of EGFR signaling and Rb/E2F may be different between flies and humans. It is noted that Akt activation suppresses E2F1-induced apoptosis in mammalian tissue culture cells, while neither the overexpression of dAKT1 nor the mutation of dPTEN is sufficient to prevent cell death in rbf1 mutant eye discs. This may reflect a difference between an in vivo analysis and tissue culture conditions, or it may reflect species-specific differences in the regulation of apoptosis. It is known, for example, that caspase activation is regulated differently between species. In vertebrates, cytochrome c release from mitochondria is a key step in the promotion of caspase activation, while in Drosophila, this step is largely dispensable. It is possible that EGFR activity converges on E2F-dependent cell death through a previously identified E2F target whose activity is regulated by Raf/Erk- and/or AKT-mediated signals, such as Bim. In order to define this circuitry, it is first necessary to identify the appropriate in vivo context in mice or humans in which Rb/E2F and EGFR activity cooperate to regulate cell survival. Once the appropriate context is found, then it may be possible to identify the molecular mechanism linking E2F-dependent cell death to survival signals (Moon, 2006).

Both EGFR family and Rb pathways are often altered in cancer. Given that developmentally controlled fluctuations in EGFR signaling have dramatic effects on the sensitivity of rbf1 mutant cells to apoptosis, it is speculated that therapeutic cancer drugs that target EGFR family proteins may induce cell death most efficiently in tumor cells that have the highest levels of E2F1 activity. One of the curious features of human retinoblastoma is that, unlike many other cancers, these tumors rarely contain mutations in p53, suggesting that either these cells do not need to mutate p53 or that they find a more effective way to suppress apoptosis. Identification of the critical components that protect premaligant Rb mutant cells from apoptosis may lead to new ways to target these cells for treatment (Moon, 2006).

gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary

In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. This study shows that a novel germline factor, Gone early (Goe; CG9634), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases (see Neprilysin4). At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment (Matsuoka, 2014: PubMed).