Gene name - argos
Synonyms - giant lens (gil)
Cytological map position - 73A3-A4
Function - ligand - secreted protein
Symbol - argos
Genetic map position - 3-
Classification - Egf-receptor ligand
Cellular location - extracellular
|Recent literature||Koca, Y., Housden, B. E., Gault, W. J., Bray, S. J. and Mlodzik, M. (2019). Notch signaling coordinates ommatidial rotation in the Drosophila eye via transcriptional regulation of the EGF-Receptor ligand Argos. Sci Rep 9(1): 18628. PubMed ID: 31819141
In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. This study shows that hypomorphic alleles of Notch, belonging to the N(facet) class, reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated cell motility process. During OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the epidermal growth factor receptor (EGFR), to fine-tune the activity of EGFR signaling. Consistently, the loss-of-function defects of N(facet) alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)-Mam-dependent transcription.
Many signaling events are non-cell autonomous. This means that inductive signals are passed from cell to cell, in contrast to cell autonomous signals, where one signaling molecule affects another within the cell, without recourse to events outside the cell. The proteins Argos and Spitz are ligands for the epidermal growth factor receptor (EGF-R). As such, they function to trigger signaling through the EGF receptor, that is, a non-cell autonomous signal. Argos is active in differentiation of several tissues including eye and wing. Argos is involved in an inhibitory loop (Brunner, 1994) whereby it restricts the duration and level of EGF-R signaling (Golembo, 1996).
The optic lobes of the adult brain originate from a population of cells derived from invaginating ectoderm that attach to the surface of the developing brain hemispheres during stages 12 and 13. During the larval stage the optic lobe primordia are organized into two parts: the inner optic anlagen and the outer optic anlagen. The outer anlagen gives rise to the lamina and medulla, the target neuropiles for rhabdomere axons. The inner optic anlagen generates the remaining neuropiles, lobula, and lobula plates. Genes involved in this process include neuralized, Notch, l(1)ogre, optomotor blind, argos, anachronism and sine oculis (Sawamoto, 1996a).
Argos plays a role in photoreceptor determination, and helps regulate optic lobe development. argos mutations cause a failure in the morphogenesis of optic lobes at the earliest stages, when the lobes are formed from invaginating ectoderm in the embryo (Sawamoto, 1996a).
Loss-of-function mutations of argos also cause an aberrant axon projection pattern of rhabdomere axons (R-axons). The role of glia in R-axon guidance has been studied using mutations in repo, a glial-specific homeodomain protein. Expression of repo in glial cells is impaired in argos mutants. Glial development is likely to be disturbed in argos mutants prior to the retinal innervation. Thus the misrouting of R-axons is likely to be due to a failure in glial development. What is most important here is the interaction between argos and two other genes, Star and rhomboid. Star and rhomboid activate signal transduction through the EGF-R -ras signaling cascade, while argos counteracts these signals. Thus glia are involved in proper R-axon projection, and argos effects are felt through its role in glial development (Sawamoto, 1996a).
Argos stimulates ommatidial rotation during eye development. The ommatidia of the Drosophila eye initiate development by stepwise recruitment of photoreceptors into symmetric ommatidial clusters. As they mature, the clusters become asymmetric, adopting opposite chirality on either side of the dorsoventral midline and rotating exactly 90°. The choice of chirality is governed by higher activity of the frizzled (fz) gene in one cell of the R3/R4 photoreceptor pair and by Notch-Delta (N-Dl) signaling. The 90° rotation also requires activity of planar polarity genes such as fz as well as the roulette (rlt) locus. Two regulators of EGF signaling, argos and sprouty (sty), and a gain-of-function Ras85D allele, interact genetically with fz in ommatidial polarity. Furthermore, argos is required for ommatidial rotation, but not chirality, and rlt is a novel allele of argos. Evidence is presented that there are two pathways by which EGF signaling affects ommatidial rotation. In the first, typified by the rlt phenotype, there is partial transformation of the 'mystery cells' toward a neuronal fate. Although most of these mystery cells subsequently fail to develop as neurons, their partial transformation results in inappropriate subcellular localization of the Fz receptor, a likely cue for regulating ommatidial rotation. In the second, reducing EGF signaling can specifically affect ommatidial rotation without showing transformation of the mystery cells or defects in polarity protein localization (Strutt, 2003).
Mutations in fz result in defects in planar polarity of the eye, characterized by ommatidia taking on random chirality, or no chirality, and rotating randomly. A hypomorphic combination of fz alleles fz19/fz20 results in a weak eye phenotype in which only 9% of ommatidia show polarity defects. This phenotype is strongly enhanced by removing one copy of the dishevelled (dsh) gene, which acts downstream of Fz in polarity signaling (Strutt, 2003).
In order to identify additional factors involved in regulating ommatidial polarity, a large-scale genetic screen was carried out for loci interacting with fz. Unexpectedly, the principle factors identified were components of the EGF signaling pathway: three complementation groups corresponded to the genes argos, sty, and Ras85D. argos encodes an inhibitory ligand for the Drosophila EGF receptor. The new allele isolated in this screen (argos5F4) and two independent alleles enhanced the fz19/fz20 phenotype, such that about 20% of ommatidia had polarity defects. Similarly, the fz19/fz20 phenotype was also enhanced by two novel alleles and three known alleles of sty, which encodes a cytoplasmic protein that inhibits the Ras signaling pathway. Finally, the 2F4 enhancer mutation had an unusual dominant phenotype, in which a small number of ommatidia had extra R7 cells and very rare defects in specification of outer photoreceptors; also, extra vein tissue was seen in the wing. This phenotype is reminiscent of dominant mutations in the MAPK gene rl (rlSem, and the extra R7 cell phenotype is increased by removing one copy of the negative Ras pathway components sty, Gap1, and yan. Transheterozygotes of 2F4 and loss-of-function Ras85D mutations result in a weak Ras85D phenotype, in which outer photoreceptors were lost from many ommatidia. This phenotype suggests that 2F4 might be a Ras85D allele. This was confirmed by sequencing of the Ras85D gene in 2F4 mutants, which revealed a mutation of Ala59 to Thr. Interestingly, this mutation is a weak activating mutation found in viral oncogenes. Hence, mutations in three EGF pathway components, each of which are predicted to increase levels of pathway activity, are dominant enhancers of an fz ommatidial polarity phenotype (Strutt, 2003).
In wild-type eyes, 1-2 so-called 'mystery cells' are seen associated with the ommatidial cluster at the 5-cell stage of development, but these fail to differentiate as neural cells and are lost from the ommatidium by row 4. In strong argos mutants, most ommatidia in the adult have one or two extra photoreceptor neurons, as a result of mystery cells being transformed into photoreceptors. The phenotype of transheterozygotes of a null argos allele argosΔ7 and argos5F4 (from the screen) was less severe, with only 45% of adult ommatidia having extra photoreceptors. Interestingly, many of the ommatidia with a normal complement of photoreceptor cells had polarity defects; up to 50% of the ommatidia were misrotated, while only about 5% appeared achiral and typically less than 1% had wrong chirality. Therefore, in addition to photoreceptor recruitment defects, argos mutations can be characterized as particularly affecting ommatidial rotation, but not R3/R4 fate (Strutt, 2003).
The phenotype of argos mutations is in fact similar to that of rlt. The most striking defect in rlt mutants is the failure of ommatidia to rotate exactly 90°; however, some ommatidia also have an additional photoreceptor near the R3/R4 pair. rlt maps close to argos, and these loci fail to complement each other. Notably, the phenotypes of argosrlt/argosΔ7 or argosrlt/argos5F4 are identical in strength to that of argosrlt. Sequencing of the argos gene in rlt mutants did not reveal any amino acid changes, suggesting that rlt is a regulatory allele of argos, with only a weak photoreceptor recruitment defect but a strong misrotation phenotype (Strutt, 2003).
sty mutants have a severe rough eye phenotype characterized by transformation of cone cells to R7 photoreceptors and, less frequently, of mystery cells into outer photoreceptors. This phenotype is sufficiently strong that it is not possible to deduce from adult eye sections whether the ommatidia are also misrotated. However, examination of eye imaginal discs from sty homozygotes shows that the developing ommatidial clusters are not uniformly rotated relative to each other (Strutt, 2003).
Further evidence that EGF signaling was important in regulating rotation came from examination of animals carrying the dominant Ras85D allele, Ras85D2F4. In homozygotes, the extra R7 cell phenotype was not increased above that seen in heterozygotes; however, up to 20% of ommatidia were misrotated, and misrotations were also occasionally seen in heterozygotes (1%-5% of ommatidia). The dominant rotation defects seen in Ras85D2F4 heterozygotes were suppressed when placed in trans to a loss-of-function Ras85D allele; this finding is consistent with the defect being caused by inappropriate activation of Ras85D signaling (Strutt, 2003).
Ommatidial rotation occurs in the eye imaginal disc and begins at the 5-cell cluster stage of development, by row 6. Therefore, the developing ommatidial clusters were examined in argos eye discs by using specific photoreceptor markers. At this stage of development, the seven-up (svp) gene is specifically expressed in the differentiating R3/R4 photoreceptors, and later on in the R1/R6 cells, as they are recruited to the cluster. These cells can therefore be marked by using a svp-lacZ reporter gene. In the intermediate-strength argos allele combination argos5F4/argosΔ7, 65% of clusters had extra svp-lacZ-expressing cells in the R3/R4 position that were first visible in row 4 and were maintained as the clusters matured. Adult eyes of the same genotype contained extra photoreceptors in a position consistent with being R3/R4 type. Thus, the mystery cells that are transformed to a photoreceptor fate in argos mutations take on an R3/R4 fate. Extra R3/R4 cells are never seen in wild-type eye discs (Strutt, 2003).
Interestingly, in argosrlt/argos5F4 eye discs, a large number of immature clusters also have extra svp-lacZ-expressing cells. In particular, 60% of clusters in rows 4-6 have extra cells, a similar proportion to that seen for stronger alleles. However, the number of extra svp-lacZ-expressing cells decreases to about 25% in rows 7 and 8; furthermore, costaining with antibodies against the neuronal antigen Elav reveals that many of the extra cells fail to take on a neuronal fate. This is consistent with the adult phenotype in which only 15% of ommatidia have extra R3/R4 cells. Therefore, in argosrlt mutants, mystery cells are partially transformed into R3/R4 cells; but, most of them ultimately fail to develop into neurons (Strutt, 2003).
In addition, expression of mδ0.5-lacZ, a marker for high N activity and thus R4 fate, was examined. In wild-type eye discs, mδ0.5-lacZ is initially expressed at a low level in both R3 and R4, but the pattern is rapidly resolved to high-level expression in just R4. The expression of mδ0.5-lacZ is largely unperturbed in argosrlt/argos5F4, and expression fails to be resolved to a single cell in only occasional clusters. Therefore, the presence of transient, extra R3/R4 cells in the cluster does not affect signaling between the R3 and R4 cells to define high N activity in R4, as expected from the lack of chirality defects in argosrlt adults (Strutt, 2003).
Whether the transient presence of extra R3/R4 cells in argosrlt had any effect on the subcellular localization of the Fz receptor was examined. In the early stages of photoreceptor recruitment and rotation, Fz exhibits a dynamic localization pattern; in particular, it localizes differentially in the R3 and R4 photoreceptors. The planar polarity protein Flamingo (Fmi) also colocalizes with Fz in the R3 and R4 cells. In the absence of Fz activity, or its correct localization in R3/R4, ommatidial chirality and rotation is disrupted, suggesting that Fz localization in R3/R4 may provide a subcellular cue that controls both ommatidial chirality and rotation (Strutt, 2003).
Since the mystery cells are partially transformed into photoreceptors of the R3/R4 type in argosrlt, it was predicted that this might lead to aberrant Fz localization in the early ommatidium. In row 4 of wild-type eye discs, Fz-GFP is localized to the apicolateral membranes of the R3 and R4 cells, except where they contact R2/R5, and to the posterior side of R8. By row 6, Fz-GFP in the R3 cell is localized specifically at the R3/R4 boundary, whereas in the R4 cell, it is excluded from the R3/R4 boundary and the boundary with R5 but remains enriched on other apical membranes. In argosrlt mutants, a dramatically altered localization pattern is observed. In row 4 of most clusters, Fz-GFP is enriched on the apical membranes of several cells, which from their position correspond to the R3/R4 cells and a variable number of partially transformed mystery cells. By row 6, Fz-GFP is still apically localized in these additional cells in most clusters, rather than specifically in the R3/R4 pair. As expected, Fmi is also mislocalized in an identical pattern in argosrlt eye discs. Therefore, at the time when ommatidia begin to rotate, Fz-GFP distribution is abnormal, and it is asymmetrically distributed in multiple cells that are partially transformed to the R3/R4 fate (Strutt, 2003).
Extra R3/R4 cells and corresponding mislocalization of Fz-GFP are also seen in sty mutants, and these extra cells may be an underlying cause of the ommatidial rotation defect observed. Nevertheless, evidence was also sought of EGF signaling affecting rotation independently of the induction of extra photoreceptors (Strutt, 2003).
The phenotype caused by overexpression of a second Ras homolog in flies, Ras64B, was examined. Overexpression of activated Ras64BV14 under control of the sevenless enhancer or heat shock promoter causes rough eyes, in which ommatidia are improperly oriented. A similar phenotype is seen if Ras64BV14 is expressed by using the actin promoter; the predominant defect is misrotations, with occasional loss of pigment cells and fusion of ommatidia. A role for Ras64B in eye development has not yet been determined, since no mutants have been identified. However, the actin-Ras64BV14 misrotation phenotype can be suppressed by removing one copy of argos or sty, or in Ras85D2F4 heterozygotes; this finding is consistent with Ras acting in the EGF signaling pathway, but, in this context, as a negative regulator (Strutt, 2003).
Since actin-Ras64BV14 appears to act by lowering EGF signaling, it is unlikely that its rotation phenotype is due to extra photoreceptor cells or mislocalization of polarity proteins. Indeed, no extra photoreceptor cells were visible in the adult, and staining of imaginal discs from actin-Ras64BV14 males also failed to show any extra R3/R4 photoreceptor recruitment. Furthermore, Fz-GFP and Fmi localization was normal in these eye discs. Therefore, it is believed that alteration in EGF pathway activity by expression of Ras64BV14 causes misrotations without resulting in defects in cell fate determination or polarity protein localization (Strutt, 2003).
Since the exact role of Ras64B in EGF signaling is unclear, the effect on ommatidial rotation was examined of lowering the amount of a known component of the pathway, the EGF receptor, by using a temperature-sensitive allele, Egfrts1a. To generate a very weak phenotype, flies were raised at just above the restrictive temperature, at 18°C-19°C: examination of adult eyes revealed that, in addition to loss of photoreceptors in some clusters, occasional ommatidia were misrotated. While the number of misrotations was low (1-2 per eye section), the degree of misrotation was generally at least 45°, supporting a role for EGF signaling in this process (Strutt, 2003).
It is concluded that EGF signaling is required for correct ommatidial rotation. A fz ommatidial polarity phenotype is dominantly enhanced by argos, Ras85D2F4, and sty, all of which result in excess EGF pathway activation. Additionally, ommatidial rotation defects are seen in conditions in which EGF pathway activity is either increased or decreased (Strutt, 2003).
It is proposed that there are two mechanisms by which EGF signaling affects ommatidial rotation. The first is that this is a result of mystery cells inappropriately taking on an R3/R4 fate. In argosrlt, most ommatidia show partial transformation of mystery cells into R3/R4 photoreceptors. Although most of these extra R3/R4 cells do not ultimately differentiate into neurons, Fz-GFP is mislocalized in them at the time of ommatidial rotation. Since fz is required in the R3/R4 photoreceptor pair for correct ommatidial chirality and rotation, the presence of extra cells containing localized Fz-GFP could be providing the ommatidium with conflicting cues that disrupt normal rotation (Strutt, 2003).
The largely normal expression of mδ0.5-lacZ and the lack of chirality defects in argosrlt suggest that the presence of Fz-GFP in extra cells does not affect N-Dl signaling between the cells that finally take on the R3 and R4 fates. It is supposed that only cells that eventually take on neural fate are competent to participate in the N-Dl signaling event (Strutt, 2003).
Evidence was also found for a second mechanism whereby EGF signaling affects ommatidial rotation, without induction of extra R3/R4 cells. Lowering EGF signaling by using a temperature-sensitive allele of Egfr results in misrotations, even though this would be predicted to cause loss rather than gain of photoreceptors. In addition, expression of activated Ras64B causes rotation defects without inducing extra photoreceptors. While the role of Ras64B in EGF signaling has not been fully characterized, its rotation phenotype is dominantly suppressed by argos, sty, and Ras2F4, suggesting that it is acting as a negative regulator of the EGF pathway in this context. One possibility is that it acts by competing with Ras85D for binding to exchange factors or downstream effectors, thus reducing Ras85D activity. These observations support an additional, more direct role for the EGF pathway in control of ommatidial rotation, downstream of Fz localization (Strutt, 2003).
Finally, it is noted that the RhoA locus, which also controls ommatidial rotation, interacts with neither fz19/fz20 nor actin-Ras64BV14. Hence, RhoA may be required for another aspect of ommatidial rotation, perhaps via regulation of dynamic changes in actin structure needed for cell movement (Strutt, 2003).
Bases in 5' UTR - 536
Exons - two
Bases in 3' UTR - 907
argos has an epidermal growth factor repeat, and an N-terminal signal sequence, which controls protein secretion (Kratzschmar, 1992 and Freeman, 1992). The signal sequence is potentially cleaved during protein secretion.
date revised: 20 March 2004
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