sprouty

Transcriptional Regulation

The tracheal cells that express sty are located very close to the small clusters of epidermal and mesodermal cells that express Branchless, the sole known Drosophila FGF ligand; the sty expression pattern is very similar to that of pointed and other genes induced by Bnl. These observations suggest that sty might also be induced by the Bnl pathway. Consistent with this, in bnl and breathless mutants, sty is not expressed or is expressed only weakly. When Bnl is ubiquitously expressed, sty turns on at high levels throughout the tracheal system. The downstream effector pnt is also required for sty expression, and sty expression is activated outside its normal expression domain when pnt P1 protein is ubiquitously expressed. Consistent with this, pnt sty double mutants display the same tracheal phenotype as the pnt mutant alone. These results show that sty expression is induced by the Branchless activated signaling pathway that Sprouty inhibits. Sty limits induction of its own gene, just as it limits induction of other genes by Bnl, as shown by the broadened expression domain of a sty-lacZ marker in a sty- background. This experiment is a wonderful example of how expression of a gene can be examined in cells that are mutant for that gene. By examining an expression vector (sty-lacZ) regulated by the promoter of sty in cells mutant for sty, it is see that sty is overexpressed in such cells, showing that sty functions to limit induction of its own gene (Hacohen, 1998).

During Drosophila oogenesis Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the MAPK cascade via the Drosophila EGF receptor (Egfr). Activation of this pathway induces different cell fates in the overlying follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially restricted and of constant intensity during mid-oogenesis. Gurken internalization can first be observed in all posterior follicle cells, abutting the oocyte from stage 4 to 6 of oogenesis. At the same time MAPK activation evolves in a spatially and temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms generated by target genes of the Egfr pathway (Peri, 1999).

Gurken signaling also induces the expression of a negative element in the Egfr pathway. Argos expression comes too late to explain the dynamic evolution of the MAPK and rhomboid patterns during stage 10 of oogenesis, but sprouty is expressed early enough to be part of the regulatory network controlling the Egfr pathway activation at stage 10. sprouty is induced in all posterior follicle cells abutting the oocyte at stage 4 to 6 of oogenesis. sty is absent from egg chambers lacking Gurken function, and thus sty may be one factor required to counteract the potential rho-dependent autoactivation of the Egfr pathway (Peri, 1999).

Protein Interactions

Sprouty (Spry) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogates an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. These results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis (Wong, 2001).

In examining the sequence of Drosophila Sprouty, a region was found between amino acids 179 and 199 that shows a low homology to residues 36-53 in hSPRY2. Residues 36-53 lie within region 11-53, which is important for c-Cbl binding. It was therefore investigated if the binding domain could be further refined to a smaller region. FLAG-tagged hSPRY2 truncation and deletion constructs were expressed in 293T cells, and cell lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-c-Cbl antibody. No binding is seen between c-Cbl and the 53C (without residues 1-53) and DeltaN36 (lacking amino acids 36-53) mutants, whereas binding is apparent between c-Cbl and the 30C (without residues 1-30) and full-length constructs. A reciprocal precipitation experiment was performed in which cell lysates from the same transfections were subjected to immunoprecipitation with anti-c-Cbl antibody and immunoblotted with anti-FLAG antibody. The result is in agreement with the above data. Thus, the c-Cbl-binding region of hSPRY2 is contained within sequence 36-53 (Wong, 2001).

To extend the binding investigation to Drosophila Spry and Drosophila Cbl, various constructs were made. (1) To investigate whether the Ring finger domain of Cbl is involved in binding to Spry, 293T cells were transiently transfected with Cbl, dCblDeltaRF (Drosophila Cbl lacking the ring finger domain), or vector alone. Cell lysates were subjected to pull-down assays with GST-Spry or GST alone. Whereas full-length Cbl binds to Spry, dCblDeltaRF does not. This result is indicative of the Ring finger domain of Cbl being involved in its binding to Spry. (2) Binding domain analyses were performed to ascertain the site in Spry that binds to the Ring finger domain of Cbl. The possible involvement of residues 179-199 in Spry was directly addressed. FLAG-tagged full-length Spry and the dSPRYN210 (amino acids 1-210), dSPRY202C (amino acids 202-592), and dSPRYDeltaN179 (mutant with a deletion of amino acids 179-199) constructs were transiently expressed in 293T cells, and lysates were incubated with either GST alone or GST-Cbl. Spry-derived proteins that contain residues 179-199 bind to Cbl, whereas those that lack the sequence do not. The region comprising residues 179-199 of Spry is therefore responsible for its interaction with Cbl; deletion of this region of Spry can similarly abolish its binding to c-Cbl (Wong, 2001).

Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases

Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. This study shows that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. It is proposed that this double-negative feedback circuit shapes the output profile of RTK signaling events (Jarvis, 2006).

Four lines of evidence support the conclusion that Csw/SHP-2 inactivate Spry proteins by direct binding and dephosphorylation. First, genetic experiments in developing Drosophila eye and trachea and HEK293 cells demonstrated that Csw/SHP-2 and Spry act in the same RTK signaling events but in opposite directions. Indeed, manipulating their activity in opposite directions caused similar Drosophila phenotypes and similar effects on MAPK activation in HEK293 cells, and reducing spry dose suppressed the csw loss-of-function phenotype in the eye and enhanced the gain-of-function phenotype, supporting the idea that they regulate the same step in signaling. Second, molecular epistasis experiments in HEK293 cells demonstrated that SHP-2 functions upstream of, and negatively regulates, phosphorylation of the critical tyrosine residue (Y53) of Spry1. Third, biochemical studies of extracts of HEK293 cells, Drosophila S2 cells, and eye discs demonstrated that Csw/SHP-2 proteins associate in complexes with Spry proteins. Interaction was enhanced in S2 cells and eye discs when a substrate-trapping Csw was used. Interaction involves more than just binding of Csw/SHP-2 to the crucial tyrosine, because complex formation was observed with SHP-2 mutants lacking the phosphatase domain and with a Spry mutant lacking the tyrosine. Finally, purified SHP-2 selectively dephosphorylated Spry1 in vitro. These data support the conclusion that Spry proteins are direct targets of Csw/SHP-2 in all three systems examined (Jarvis, 2006).

One genetic result did not readily fit with the model that Csw functions by inactivating Spry by dephosphorylation. Whereas reduction of spry dose suppressed the eye phenotype of a hypomorphic csw allele and dominant-negative Csw^G547E, consistent with the model, it did not suppress the milder phenotype of dominant-negative Csw^C583S. This catalytically inactive, substrate trapping form of Csw has unusual properties: it behaves in a dominant-negative fashion, interfering with wild-type Csw function, but also retains some wild-type Csw function because it partially rescues other dominant-negative and hypomorphic csw alleles. This residual activity of Csw^C583S is proposed to result from its ability to partially mimic the effect of dephosphorylating a substrate by binding to it tightly. Spry binds Csw^C583S and could be such a substrate. If so, this could explain the lack of suppression of Csw^C583S phenotype by reduction in spry dose: decreasing spry levels would not reduce spry function under conditions in which it is already trapped in an inactive or partially inactive form by Csw^C583S (Jarvis, 2006).

Csw/SHP-2 binding and dephosphorylation of Spry creates an interesting regulatory circuit downstream of RTKs. Both components of the circuit are induced and activated following receptor activation, Csw/SHP-2 by SH2 domain interactions with phosphotyrosines, and Spry proteins by transcriptional induction of their genes and tyrosine phosphorylation of the proteins. One induced component (Spry) is a signaling inhibitor, the other (Csw/SHP-2) is a signaling promoter that acts by inactivating the inhibitor (Jarvis, 2006).

Why does a signaling pathway induce both a feedback inhibitor and a protein that inactivates it? One possibility is that this double-negative circuit provides a mechanism for rapidly resetting the signaling system: the inhibitor terminates signaling and the deactivator restores the inhibitor to its original (inactive) state, readying the cell for another round of signaling. This may be important when cells experience successive waves of signaling, such as the waves of EGFR and Sevenless signaling in eye development (Jarvis, 2006).

Another possibility is that the double-negative circuit allows precise control of the signal output profile. In the absence of feedback, the response to a signal is simple and sustained, increasing monotonically until reaching saturation. If a basic negative-feedback system is operative, the magnitude and duration of the response are limited, generating a parabolic response profile. However, if the pathway contains both a feedback inhibitor (Spry) and an inducible component (Csw/SHP-2) that deactivates it, this creates more complex output profiles, such as the irregularly shaped curve observed for MAPK activation following FGFR activation in HEK293 cells. By altering activity of individual feedback components, other complex profiles can be generated. If cells can distinguish different profiles, as some cells distinguish different calcium oscillations, this could lead to different outcomes. The shape of the RTK response profile could be as important to outcome as the magnitude and duration of the response. In a similar way, differential induction of individual components of a double-negative feedback circuit can transform simple signaling gradients into complex spatial patterns of signal output (Jarvis, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

The embryonic expression pattern of sprouty was determined by whole-mount in situ hybridization and by analysis of three sty enhancer trap markers that closely follow mRNA expression and provided cellular resolution of the pattern. sty is expressed specifically in the tracheal system and a few other developing tissues, including midline glia, where bnl and btl also function, and the dorsal vessel, where the other known Drosophila FGF pathway is operative (see Heartless). sty is also expressed in a small subset of central nervous system neurons, oenocytes, and subsequently, in the eye imaginal disc (Hacohen, 1998).

Effects of mutation or deletion

sty mutations were generated by imprecise excision of a P[lacZ] transposon at band 63D1,2, which expresses the fl-galactosidase marker in tracheal cells as they are forming new branches. The original P[lacZ] insert was a silent mutation. Three P excision alleles, designated sty5, sty64, and sty55, show excessive tracheal branching and comprise a lethal complementation group. In sty5 homozygotes, 30%-120% more branches than normal bud from the DB (dorsal branch), LTa (lateral trunk anterior), LTp (lateral trunk posterior), GB (ganglionic branch), and VB (visceral branch) primary branches. The extra branches are not distributed randomly; rather, they arise from the stalks of the primary branches, close to the positions where secondary and terminal branches normally bud. Five EMS-induced sty alleles have been recovered in an unrelated screen for suppressors of a dominant eye phenotype. All eight sty mutations have a similar tracheal phenotype and are pupal lethal in trans to deficiencies in the 63D region. sty5 homozygotes display as severe a tracheal phenotype as sty5 hemizygotes, implying that sty5 is a genetic null allele (Hacohen 1998).

The cellular basis of the extra branching phenotype has been investigated by staining sty mutants with tracheal cell and nuclear markers. There are extra branch-forming cells in the mutants, each forming a single branch at stage 16, just as do normal secondary branch cells. The extra branching cells do not arise from extra cell divisions. Cell counts show that the total number of tracheal cells is not increased in the sty5 mutant, even though the number of cells forming fine branches is substantially increased. No additional dividing cells are detected in sty5 embryos labeled with BrdU or a string-lacZ marker that expresses lacZ in dividing tracheal cells. It is also unlikely that the extra branching cells arise by suppression of cell death, because cell death does not occur in normal tracheal development, and no extra branches are found in mutant embryos, which lack all normal apoptosis. Rather, the extra branching cells appear to arise by a change in tracheal cell fate, with normally nonbranching stalk cells recruited to the branching fate. This interpretation is supported by the finding that at the same time in development as normal branching cells are activated, there is an inappropriate activation of the secondary and terminal branch markers in prestalk cells. These cells also follow the same developmental program as normal secondary branch cells, forming extensive arrays of terminal branches in the larva (Hacohen, 1998).

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. The role of Sprouty in eye development is described. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. In all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal (Kramer, 1999). sprouty is required to prevent neuronal induction of non-neuronal cells in the retina. Animals homozygous for any of the EMS-induced alleles of spry die as pharate adults. The rare escapers have eyes that are similar in size to wild-type eyes but have a disorganized exterior. A majority of the ommatidia in these animals contained supernumerary photoreceptor neurons, which by their morphology are R7 cells. In addition, some of the extra photoreceptors resemble outer photoreceptor neurons. Examination of the early stages of neuronal development in the eye imaginal disc with molecular markers reveals that the supernumerary photoreceptors originate from non-neuronal cone cells and mystery cells that have assumed R7 and R3/R4 fates, respectively. Neuronal markers are inappropriately activated in cone and mystery cells at the same time in development as in the normal photoreceptors, implying that the defect in the mutant occurs at the normal time of photoreceptor induction. Thus, spry functions in the eye imaginal disc to prevent neuronal induction of these non-neuronal cells (Kramer, 1999).

Spry is found to act in parallel to Argos in antagonizing the Epidermal growth factor receptor pathway. Many of the processes that are controlled by Egfr signaling are antagonized by the extracellular factor Argos; therefore, the genetic relationship between spry and argos was tested. Overexpression of Argos using a GMR-argos transgene causes apoptosis of photoreceptor neurons and a reduced eye structure. By contrast, GMR-argos;spry- animals have normal-sized eyes that contain excess photoreceptor neurons, similar to spry mutants. Since the effects of overexpression of Argos are ameliorated in the absence of spry function, Spry acts either downstream or parallel to Argos. If Argos activity were mediated solely by Spry, one would expect that removal of Argos would not affect the spry mutant phenotype. However, spry;argos double mutant ommatidia exhibit massive neuronal differentiation, with each ommatidium containing more than a dozen neurons. Thus argos and spry are unlikely to act in series, but have parallel and partially redundant functions in antagonizing EGFR signaling (Kramer, 1999).

spry can function mainly autonomously to prevent cone cells from becoming R7 cells. Since Spry appears to function as an intercellular signal in the developing tracheal system, it was asked whether SPRY could act at a distance in the developing eye. Initially, ommatidia located at the boundary of spry minus clones in the eye were examined. The ommatidial phenotype changes abruptly at the boundary, indicating that Spry does not act over long distances. To determine which cells require spry+ function for the construction of phenotypically wild-type ommatidia, a mosaic analysis was carried out on spry minus clones, using a white+ transgene to mark spry+ photoreceptor neurons. Since there are no lineage restrictions during ommatidial assembly, some ommatidia at the clone border are composed of both spry+ and spry- cells. Any photoreceptor cell can be genotypically spry- in normally constructed ommatidia, indicating that there is no absolute requirement for spry+ function in any of the photoreceptor neurons. However, all photoreceptor cells except R3 have a decreased probability of being spry minus. This suggests that there is redundancy among photoreceptor cells in the requirement for spry+ function, and/or that there is a requirement for spry in non-neuronal cells that are related to the photoreceptor cells, such as the cone cells. To determine whether spry is required in the cone cells that are transformed into R7 neurons, a mosaic analysis was carried out on spry- clones in a sev- background. Since sev- ommatidia lack the endogenous R7 cell, all R7 cells developing in the mutant clone must arise as a consequence of the absence of spry function. In 15 retinae containing sev;spry mutant clones, 99 mosaic ommatidia with six outer photoreceptor cells and one or more R7 cells were scored. A vast majority of the R7 cells (97.9%) were spry-, indicating that the requirement for spry+ function is mainly cell autonomous in the cells that differentiate as R7. However, three of the 140 R7 cells (2.1%) were spry+ in genotype. This percentage is significantly higher than the 0.2% expected from the dominant effect of spry in the same heterozygous genotype but in which homozygous clones were not induced. Thus, spry can also function non-autonomously to prevent cone cells from differentiating as R7 photoreceptor neurons (Kramer, 1999).

These results have important implications for the mechanism of Spry action. Three possible models have been presented for how Spry might antagonize the Branchless FGF pathway in the developing tracheal system. One is by direct binding or blockage of the FGF ligand. Another posits binding or blockage of the FGF receptor Breathless. A third model postulates a separate Spry receptor on the receiving cells that antagonized the FGF pathway downstream of the FGF receptor in the receiving cells. The data indicating that Spry antagonizes both FGF and EGF pathways supports this third model. The structures of the two types of ligands (EGF and FGF) and the extracellular portions of their receptors do not show any striking sequence similarities, this argues against the first two models, which invoke direct interaction between Spry protein with FGF or EGF ligands or receptors. However, the intracellular portions of the EGF and FGF receptors and the downstream signal transduction pathways show significant similarities. Thus, it is easy to imagine how Spry interaction with its own receptor on a receiving cell could lead to inhibition of a common downstream step in the FGF and EGF signaling cascades. If Spry acts through its own receptor, rather than by directly antagonizing the FGF or EGF receptors, then it is also easy to see how differences in autocrine versus paracrine activity of Spry in different tissues could arise by differences in expression or activity of its receptor (Kramer, 1999).

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. Late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open (Yan), a repressor for late btl expression. Biochemical and genetic analysis has indicated that the minimum btl enhancer includes binding sites of Anterior-open (Ohshiro, 2002).

The minimum btl enhancer consists of B2 and B3 regions, the latter, a late enhancer. lacZ expression driven by B3 enhancer mimics btl late expression. Bnl/Btl signaling in developing trachea not only facilitates cell migration during primary branch formation but also induces the expression of genes required for secondary and terminal branch formation in a subset of tracheal cells. The present study indicates that the btl gene itself is a target of Bnl/Btl signaling and accordingly, Bnl/Btl signaling is regulated by a positive feedback mechanism. The results also indicated that the positive feedback of Bnl/Btl signaling includes MAPK activation. This is the first clear demonstration of the presence of a positive feedback loop in FGF signaling (Ohshiro, 2002).

The positive feedback mechanism may be an important finding, since a negative feedback mechanism has already been shown to be involved in Bnl/Btl signaling. Spry is expressed in many tissues including trachea and functions as an intracellular inhibitor of Ras pathway signal transduction through its binding to Drk and Gapl. In embryos mutant for spry, downstream target genes of Bnl/Btl signaling are misexpressed and extra secondary branch cells are generated. At the onset of secondary branch formation in wild type trachea, only one or a few cells exposed to the highest levels of Bnl overcome the inhibitory action of Spry to acquire secondary-branch-forming cell fate. Receptor molecules activated by ligands are generally considered eliminated from the cell membrane through endocytosis and the mechanism of Bnl/Btl-signaling-activity-dependent btl transcription should thus be indispensable to a constant supply of fresh Btl receptors to membranes of cells receiving Bnl. A proper balance between positive effects from Btl supply and negative effects of Spry on Bnl/Btl signaling may accordingly be essential for properly selecting secondary budding cells in the vicinity of the FGF signaling center and inducing properly terminal branch formation (Ohshiro, 2002).

Calcineurin is a Ca²⁺-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca²⁺ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca²⁺-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin is activated by a sustained increase in intracellular Ca²⁺ levels that can result from the opening of intracellular Ca²⁺ channels in response to phosphoinositide (PI) signaling. PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP₂) to yield inositol 1,4,5-trisphosphate (InsP₃), which then activates the InsP₃ receptor Ca²⁺ channel (Sullivan, 2002).

An activated form of Pp2B-14D, canA^act, was made by deleting the autoinhibitory and calmodulin-binding domains. The canA^act construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the canA^act.gl transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

The canA^act.gl screen yielded 11 complementation groups, 9 of which failed to modify rough eyes caused by other glass-induced transgenes. This demonstrates that the majority of the modifier groups do not act through the glass enhancer. The nine specific modifiers were then divided into class I genes, which act downstream of calcineurin, and class II genes, which act at the level of CanB (Sullivan, 2002).

The class I modifier group CS3-3 failed to complement the hypomorphic sprouty alleles sty^Delta5 and sty^Delta64; both sty^Delta5 and sty^Delta64 also suppressed canA^act.gl, and the sty gene from CS3-3⁵¹⁸ harbored a nonsense mutation (Q250Stop). Therefore, it is concluded that the CS3-3 complementation group is sprouty. The fact that sty falls into the class II group suggests that sprouty functions downstream of calcineurin and/or in a parallel pathway (Sullivan, 2002).

Two lines of evidence suggest that calcineurin is a negative regulator of Egf receptor/Ras signaling. First, a negative regulator of RTK signaling, sprouty, was isolated as a suppressor of the canA^act.gl rough eye phenotype in the dominant modifier screen. Both sprouty and canA^act suppress wing vein formation and reduce the number of photoreceptor cells per ommatidium. Egf receptor/Ras signaling is essential for both wing vein and R-cell formation (Sullivan, 2002).

A thorough examination of genetic interactions between canA^act and components of RTK and other signaling pathways has confirmed that canA^act specifically represses the Egf receptor/Ras pathway and that it acts upstream in the pathway. The lack of convincing genetic interactions with other signaling pathways in the imaginal eye disc does not rule out a role for calcineurin in these pathways in other developmental contexts. With the exception of pnt, activated calcineurin was not modified by components downstream of Ras and was modified only by a subset of genes that act between the Egf receptor and Ras. While Gap1 and sty alleles modify the effects of activated calcineurin, drk and cbl do not. Thus calcineurin may act downstream of, or parallel to, drk and cbl. The more downstream components of the Ras/MAP kinase pathway may not interact with activated calcineurin because they are too far removed from the point(s) of intersection between calcineurin and the pathway. Alternatively, these components may not be limiting, so that reduction of gene dose, which is the basis of a dominant modifier screen, would have no appreciable effect (Sullivan, 2002).

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 (argos^5F4) 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 (rl^Sem, 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).

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).

It is concluded that EGF signaling is required for correct ommatidial rotation. A fz ommatidial polarity phenotype is dominantly enhanced by argos, Ras85D^2F4, 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 argos^rlt, 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).

Basson, H. R., et al. (2005). Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction. Dev. Cell 8: 229-239. 15691764

Casci, T., Vinos, J. and Freeman, M. (1999). Sprouty, an intracellular inhibitor of Ras signaling. Cell 96(5): 655-65.

Chambers, D., et al. (2000a). Differential display of genes expressed at the midbrain - hindbrain junction identifies sprouty2: an FGF8-inducible member of a family of intracellular FGF antagonists. Mol. Cell. Neurosci. 15(1): 22-35.

Chambers, D. and Mason, I. (2000b). Expression of sprouty2 during early development of the chick embryo is coincident with known sites of FGF signalling. Mech. Dev. 91(1-2): 361-4.

Chi, L., et al. (2004). Sprouty proteins regulate ureteric branching by coordinating reciprocal epithelial Wnt11, mesenchymal Gdnf and stromal Fgf7 signalling during kidney development. Development 131: 3345-3356. 15201220

Davidson, B. P., et al. (2000). Exogenous FGF-4 can suppress anterior development in the mouse embryo during neurulation and early organogenesis. Dev. Biol. 221: 41-52.

de Maximy, A. A., et al. (1999). Cloning and expression pattern of a mouse homologue of Drosophila sprouty in the mouse embryo. Mech. Dev. 81(1-2): 213-6.

Furthauer, M., et al. (2001). sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128: 2175-2186

Glienke, J., et al. (2000). Human SPRY2 inhibits FGF2 signalling by a secreted factor. Mech. Dev. 91-99. 10940627

Golembo, M., Schweitzer, Ronen, Freeman, M. and Shilo, B.Z. (1996). argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop. Development 122: 223-230

Hacohen, N., et al. (1998). sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92(2): 253-263.

Hall, A. B., et al. (2003). hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl. Curr. Biol. 13: 308-314. 12593796

Hanafusa, H., Torii, S., Yasunaga, T., Matsumoto, K. and Nishida, E. (2004). Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty. J. Biol. Chem. 279(22): 22992-5. 15031289

Jarvis, L. A., et al. (2006). Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases. Development 133: 1133-1142. 16481357

Kim, H. J., Taylor, L. J. and Bar-Sagi, D. (2007). Spatial regulation of EGFR signaling by Sprouty2. Curr. Biol. 17: 455-461. Medline abstract: 17320394

Kramer, S., et al. (1999). Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development 126: 2515-2525.

Liu, A., et al. (2003). FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors. Development 130: 6175-6185. 14602678

Mailleux, A. A., et al. (2001). Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis Mech. Dev. 102: 81-94. 11287183

Mason, J. M., et al. (2004). Tyrosine phosphorylation of Sprouty proteins regulates their ability to inhibit growth factor signaling: a dual feedback loop. Mol. Biol. Cell. 15(5): 2176-88. 15004239

Minowada, G., et al. (1999). Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 126: 4465-4475.

Nutt, S. L., et al. (2001). Xenopus Sprouty2 inhibits FGF-mediated gastrulation movements but does not affect mesoderm induction and patterning. Genes Dev. 15: 1152-1166. 1133161

Ohshiro, T., Emori, Y. and Saigo, K. (2002). Ligand-dependent activation of breathless FGF receptor gene in Drosophila developing trachea. Mech. Dev. 114: 3-11. 12175485

Peri, F., Bokel, C. and Roth, S. (1999). Local gurken signaling and dynamic MAPK activation during Drosophila oogenesis. Mech. Dev. 81(1-2): 75-88.

Perl, A.-K., et al. (2003). Temporal effects of Sprouty on lung morphogenesis. Dev. Biol. 258: 154-168. 12781690

Reich, A., Sapir, A. and Shilo, B.-Z. (1999). Sprouty is a general inhibitor of receptor tyrosine kinase signaling. Development 126: 4139-4147.

Rubin, C., et al. (2003). Sprouty fine-tunes EGF signaling through interlinked positive and negative feedback loops. Curr. Biol. 13: 297-307. 12593795

Schweitzer, R., Howes, R., Smith, R., Shilo, B.Z. and Freeman, M. (1995). Inhibition of Drosophila EGF receptor activation by the secreted protein argos. Nature 376: 699-702

Shaw, A. T., et al. (2007). Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev. 21: 694-707. Medline abstract: 17369402

Shim, K., Minowada, G., Coling, D. E. and Martin, G. R. (2005). Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling. Dev. Cell 8(4): 553-64. 15809037

Sivak, J. M., Petersen, L. F. and Amaya, E. (2005). FGF signal interpretation is directed by Sprouty and Spred proteins during mesoderm formation. Dev. Cell 8(5): 689-701. 15866160

Strutt, H. and Strutt, D. (2003). EGF signaling and ommatidial rotation in the Drosophila eye. Curr. Biol. 13: 1451-1457. 12932331

Sullivan, K. M. C. and Rubin, G. M. (2002). The Ca2+-calmodulin-activated protein phosphatase calcineurin negatively regulates Egf receptor signaling in Drosophila development. Genetics 161: 183-193. 12019233

Sutherland, D., Samakovlis, C. and Krasnow, M. A. (1996). branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and pattern of branching. Cell 87: 1891-1101

Tefft, J. D., et al. (1999). Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr. Biol. 9(4): 219-22.

Wakioka, T., et al. (2001), Spred is a Sprouty-related suppressor of Ras signalling. Nature 412: 647-651. 11493923

Wong, E. S. M., et al. (2001). Evidence for direct interaction between Sprouty and Cbl. J. Biol. Chem. 276: 5866-5875. 11053437

Wong, E. S. M., et al. (2002). Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signaling. EMBO J. 21: 4796-4808. 12234920

date revised: 30 October 2007 

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.