InteractiveFly: GeneBrief

sprouty : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Of necessity, development demands an appropriate balance between inductive and inhibitory activity. A secreted ligand of a receptor tyrosine kinase cannot be allowed to exercise an inductive capacity on adjacent tissue without a mechanism in place to oppose this induction. Without opposition, such induction could result in uncontrolled growth in the target tissue. Thus, inhibitory activities serve to focus inductive interactions to a narrow range, resulting in a refinement of the inductive effect. Such inhibitory activities assure the containment of cell growth to the limits required for the establishment of a refined and elaborate tissue patterning.

One of the best examples of the balance of inductive and inhibitory activity is to be found in eye development: the stimulation of the Epidermal growth factor receptor (Egf-r) by its ligand Spitz. This stimulation is opposed by the inhibitory action of Argos. Argos is an extracellular inhibitor of Egf-r activation (Schweitzer, 1995). The expression of Argos is dependent on Egf-r activation, establishing a negative feedback loop (Golembo, 1996) delimiting the inductive effects of Spitz.

The FGF receptor is also subject to inductive interactions by Branchless (Bnl) (Sutherland, 1996), the FGF ligand, and is opposed by Sprouty (Sty), an antagonist of FGF. Fibroblast growth factor sends signals for the patterning required to form the apical branching essential to normal Drosophila airways. In wild-type embryos, Branchless induces secondary tracheal branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sty mutants, tracheal stalk cells adopt a branching fate. These extra branching cells do not arise from extra cell divisions nor do they arise from the suppression of cell death. The extra branching cells in sty mutants appear to arise by a change in tracheal cell fate, with normally non-branching stalk cells recruited to the branching fate (Hacohen, 1998).

During normal tracheal development, secondary and terminal branching genes are induced at the ends of growing primary branches by localized expression of Branchless. Because the ectopic branches in sty mutants are formed by the prestalk cells located near the cells that are normally induced to branch, the extra branches could arise from overactivity of the Bnl pathway. To test whether sty functions by limiting the Bnl pathway or by preventing branching in some other way, an examination was made of downstream effectors in the Bnl pathway that regulate the later branching events (Hacohen, 1998).

One such effector is pointed (pnt), a downstream target of several receptor tyrosine kinase pathways. pnt expression is induced by Bnl at the ends of primary branches and promotes secondary and terminal branching. Similarly, the DSRF gene and three other marker genes (Terminal -2,-3, and -4) are induced at the ends of growing primary branches; all promote terminal branching. In sty mutants, all five downstream effectors are expressed in expanded domains that include the prestalk cells, which later form ectopic branches. The DSRF marker is activated at the same time as in the normal branching cells (Hacohen, 1998).

The transcriptional repressor Yan is another critical target of Bnl signaling. As in other RTK pathways, activation of the Btl receptor leads to MAPK-dependent phosphorylation and degradation of Yan, which is necessary to activate the later programs of tracheal branching. Normally, Yan is degraded only in the tip cells of the outgrowing primary branches. In sty mutants, Yan is degraded in an expanded domain that coincides with the expanded domains of pnt and DSRF expression. A yan-lacZ transcriptional reporter continues to be expressed normally, implying that down-regulation of Yan occurs posttranscriptionally as in other RTK pathways. The results show that sty loss of function mutations enhance all known downstream effects in this Bnl pathway. An engineered gain of function condition, in which the sty gene product is overexpressed during embryonic stages 10-12, severely blocks induction of downstream effectors and branching by Bnl. The reciprocal is also true: overexpression of Bnl can overcome the opposition of sty and induce secondary and terminal branching throughout the tracheal system. Thus, sty behaves genetically as a competitive inhibitor of the Bnl pathway (Hacohen, 1998).

To determine which cells require sty+ function, a genetic mosaic analysis was carried out using the FLP recombination system (Xu and Rubin, 1993 ). Thirty-three mosaic ganglionic branches (GBs) composed of sty- and sty+ cells were examined. In the 29 clones in which just the tracheal tip cell GB1 is sty-, an ectopic branch is always present in the neighboring sty+ stalk cell, GB2. In contrast, in three of the four clones of the opposite type, in which a subset of the other stalk cells (GB2-7) were sty-, the GB appears normal with no extra branches. It is presumed that the single exceptional clone represents the rare situation in which a second GB cell can take on the branching fate even in the presence of wild-type sty function. One particularly informative sty- clone includes all of the GB cells except the tip cell; this forms a normal GB without ectopic branches. It is concluded that sty+ is required in the tip cell and acts nonautonomously to inhibit branching by nearby stalk cells. There are two unexpected characteristics of the clones in which the GB1 cell is sty-: (1) outgrowth of ectopic branches from GB2 and GB3 usually turn away from the ventral nerve cord (VNC), whereas in sty- embryos the extra branches normally enter the VNC. This indicates that the remaining sty+ cells in the mosaic individuals somehow influence tracheal pathfinding. (2) Growth of the normal branch from a sty- GB1 cell sometimes appears to be slowed when nearby cells (GB2-7) are sty+. This suggests that sty+ might also have a cell-autonomous function that protects it from the inhibitory effects of its sty+ neighbors (Hacohen, 1998).

Given the competitive genetic interactions between Sty and Bnl, and that both appear to be membrane-associated or secreted proteins, the simplest biochemical model is that Sty competes with Bnl for binding the receptor Breathless or that Sty restricts receptor activation in some other way, such as by binding Bnl and limiting its diffusion. Alternatively Sty could bind another receptor. The short-range inhibitory action of Sty is reminiscent of lateral inhibitory effects mediated by the Notch pathway: conceivably, Sty could activate Notch or another receptor in the prestalk cells and block signaling downstream of the Btl receptor. If so, the block must occur at or upstream of MAPK and Yan, because it has been shown that Sty counteracts the MAP kinase-dependent degradation of Yan (Hacohen, 1998).

Any model of Sty action must account for the paradoxical observation that Bnl signaling is highest in the cells that express the highest levels of the inhibitor. Although this is a consequence of the induction of sty expression by the Bnl pathway, it is unclear how expression of sty and other effectors is maintained once Sty protein is present. Perhaps high levels of Bnl simply outcompete high levels of Sty. Alternatively, there may be special mechanisms that allow sty-expressing cells to escape their own inhibitory effect (Hacohen, 1998)

Sprouty, an intracellular inhibitor of Ras signaling

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty minus eyes have excess photoreceptors, cone cells, and pigment cells. Tests provide evidence that Sprouty interacts specifically with the Egfr pathway. Halving the dose of sprouty (1) strongly enhances the rough eye caused by the misexpression of rhomboid, a specific activator of Egfr signaling; suppresses the rough eye caused by underrecruitment of photoreceptors in a hypomorphic allele of spitz, the TGF-like ligand of the Egfr; (3) suppresses the phenotypes of Egfr hypomorphic mutations both in the eye and the wing and (4) flies heterozygous for both sprouty and argos have mildly rough eyes, caused by a slight overrecruitment of all types of cell, although heterozygosity for either mutation alone causes no phenotype. Other genetic interactions between sprouty and the Egfr pathway are also detailed. All point to the same conclusion: Sprouty inhibits Egfr signaling (Casci, 1999).

The expression of sprouty in the eye imaginal disc was documented. In eye imaginal discs, sprouty is detected only behind the morphogenetic furrow, in the region where Egfr-mediated recruitment occurs. It appears successively in each of the newly recruited cells, both in photoreceptors and cone cells, a pattern that strongly suggests that sprouty expression is dependent on Egfr signaling itself. This is confirmed by loss of sprouty staining in many cells when Egfr signaling is blocked (with a dominant-negative form of the receptor) and an increased number of cells expressing sprouty in the presence of an activated form of the receptor. These results in the eye are consistent with observations that sprouty expression also follows, and is dependent upon, Egfr activation in the follicle cells of the egg. Similarly, sprouty expression has been shown to be dependent on Fgfr signaling in the trachea. Although the widespread dependence of sprouty expression on RTK signaling could be indirect, it has important developmental consequences, as it implies that Sprouty participates in negative feedback control of signaling (Casci, 1999)

Hacohen (1998) found sprouty to behave nonautonomously in the trachea. In contrast, in the current study sprouty is shown to act strictly cell autonomously in R7 cells. In sevenless mutant ommatidia mosaic for sprouty, all R7s are sprouty minus. Ommatidia are found where the only sprouty minus cell is the R7, implying that absence of Sprouty in a cell can be sufficient to transform it into an R7, even when all its neighbors are sprouty plus (Casci, 1999).

To determine where in the Ras signal transduction pathway Sprouty acts, it was asked whether altering its amount could modify the phenotypes caused by expressing constitutive forms of the Egfr, Ras1, and Raf in the eye. Halving the dose of sprouty enhances the phenotype of constitutive Egfr but has no effect on the constitutive Ras1 or Raf phenotypes. Overexpression of sprouty is able to rescue the phenotype caused by expression of the constitutive Egfr. These interactions indicate that Sprouty acts downstream of (or parallel to) the Egfr, but upstream of Ras1 and Raf (Casci, 1999).

Both full-length Sprouty and a truncated Sprouty containing residues 1-369 (i.e., without the cys-rich domain and C-terminal residues) were assayed for their ability to bind in vitro translated members of the Ras pathway. Strong interactions are detected between Sprouty and Drk, an SH2-SH3 containing adaptor protein homologous to mammalian Grb2, and between Sprouty and GTPase-activating protein 1 (Gap1), a Ras GTPase-activating protein. No interactions were seen between Sprouty and several other proteins involved in the Ras pathway: Sos, Dos, Csw, Ras1, Raf, and Leo (14-3-3). The interactions with Drk and Gap1 did not require the presence of the C-terminal cysteine-rich domain, the region of Sprouty most conserved between flies and humans. Since the well-conserved cysteine-rich domain of Sprouty is not required for binding to Drk or Gap1, it might instead target the protein to the plasma membrane. To test this, two truncated forms of Sprouty were expressed in cultured cells. One form lacks the conserved cysteine-rich domain, whereas a second exclusively comprises the cysteine-rich domain. The form with the cysteine-rich domain is membrane associated and is indistinguishable from the wild-type protein. In sharp contrast, the form lacking the cysteine-rich domain is distributed uniformly throughout the cell, with no specific localization to membranes. Cell fractionation confirms these results. It is concluded that the 147-residue cysteine-rich domain in Sprouty, which corresponds to the most conserved region in the published human ESTs, is responsible for the specific localization of Sprouty to the plasma membrane (Casci, 1999).

Sprouty's function is, however, more widespread. It also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, biochemical analysis suggests that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. These indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction (Casci, 1999).

Sprouty is a general inhibitor of receptor tyrosine kinase signaling

Sprouty was originally identified as an inhibitor of Drosophila FGF receptor signaling during tracheal development. By following the capacity of ectopic Sprouty to abolish the pattern of activated MAP kinase in embryos, it has been shown that Sprouty can inhibit other receptor tyrosine kinase (RTK) signaling pathways, namely the Heartless FGF receptor and the Egf receptor. Similarly, in wing imaginal discs, ectopic Sprouty abolishes activated MAP kinase induced by the Egfr pathway. Sprouty expression is induced by the Egfr pathway in some, but not all, tissues in which Egfr is activated, most notably in follicle cells of the ovary, the wing imaginal disc and the eye disc. In the ovary, induction of sprouty expression follows the pattern of Egfr activation in the follicle cells. Generation of homozygous sprouty mutant follicle-cell clones demonstrates an essential role for Sprouty in restricting EGFR activation throughout oogenesis. At the stage when dorso-ventral polarity of the follicle cells is established, Sprouty limits the ventral expansion of the activating Gurken signal. Later, when dorsal appendage fates are determined, reduction of signaling by Sprouty facilitates the induction of inter-appendage cell fates. The capacity of Sprouty to reduce or eliminate accumulation of activated MAP kinase indicates that in vivo it intersects with the pathway upstream to MAP kinase. The ability of ectopic Sprouty to rescue lethality caused by activated Raf suggests that it may impinge upon the pathway by interacting with Raf or downstream to it (Reich, 1999).

Identification of Sprouty as an inhibitor of both FGF receptor and EGF receptor pathways raises the issue of the mechanism by which Sprouty exerts its effects. Since Sprouty is a novel molecule, its biological properties are still obscure. sprouty mutant clones have been shown to exert a non-autonomous phenotype in tracheal development. It is not known, however, if Sprouty itself is the signal transmitted from one cell to the other, or if the activity of Sprouty in the leading tip cells affects their capacity to signal to the adjacent cell through a different signaling module. If Sprouty is a secreted protein, one would expect its inhibitory signal to be relayed to the cytoplasm, by a hitherto unknown pathway. Conversely, if Sprouty functions in the cells in which it is produced, it may impinge directly on the cytoplasmic signaling pathways of RTKs. Recent evidence suggests that Sprouty may be an intracellular protein. Sprouty interacts in vitro with Drk/Grb2 and GAP proteins, but the possible functional significance of this interaction has not been determined. The capacity of Sprouty to suppress activated lambdaTop and lambdaHtl constructs demonstrates that it intersects with the pathway downstream to the receptor. Ectopic Sprouty eliminates the normal accumulation of dpERK in the embryo and wing discs, demonstrating that the intersection point is at the MAP kinase kinase (MEK) stage, or upstream to it. Due to lethality resulting from ectopic expression of activated Ras or Raf, it was difficult to determine the intersection point more precisely. However, the capacity of ectopic Sprouty to rescue lethality induced by activated Raf suggests that it can inhibit Raf itself or components downstream of Raf. Similarly, in the ovary, Sprouty expression is also capable of eliminating phenotypes induced by activated Raf (Reich, 1999).

Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation

During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).

During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).

Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).

Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).

Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several Ras^GTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of Ras^GTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).

The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active Pointed^P1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).

This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).

In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of Ras^GTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).

Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development

The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. This study profiled the Drosophila larval brain by single cell RNA-sequencing and identified Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved (Ariss, 2020).

The RTK signaling pathway is critical in a plethora of glial cell functions, such as migration, differentiation, proliferation, neurodegeneration and locomotion. This study identified Ama as a regulator of RTK signaling in glial cells and uncover an essential function of Ama in the maintenance of the BBB. This study underscores the power of scRNA-seq profiling to explore a knockdown phenotype and led to the identification of Sty as a major Ama target during regulation of RTK signaling (Ariss, 2020).

Profiling cells by scRNA-seq identified distinct changes in gene expression profiles across multiple glial cell types in Ama-depleted brain that otherwise would have not been possible using conventional approaches. First, it was found that Ama depletion affects gene expression in surface glia, which in turn leads to a disruption in the BBB. Second, the increase in hemocyte numbers in repo>Ama^RNAi scRNA-seq dataset enabled the discovery of infiltrating blood cells in Ama-depleted brains. Third, the increase in Sty and decrease in PntP1 levels following Ama knockdown in glia encouraged exploration of the impact of Ama in RTK signaling (Ariss, 2020).

scRNA-seq identifies and clusters cells based on similarity in gene expression profile to uncover the cellular heterogeneity in a tissue. In this study, scRNA-seq of the normal fly brains identified all the glial cell types in addition to a Fas cluster that appeared to encompass multiple glial cell types. This cell cluster displayed high expression levels of cell adhesion proteins, which is a hallmark of glial cells, while also supporting the observation that scRNA-seq not only clusters cells by type but also by similar biological features. This underlies the robustness of scRNA-seq, since it uncovers complex cellular dynamics related to certain stimuli and continuous temporal differentiation processes, as well as the spatial arrangement of cells. Notably, the cellular perturbations in clustering that were observed following Ama depletion were validated experimentally by genetic analysis, immunofluorescence and other assays. This type of single-cell data validation is essential as it addresses the concern of a batch effect between scRNA-seq samples. Thus, this work highlights the power of scRNA-seq to profile a knockdown or mutant phenotype (Ariss, 2020).

The results revealed that Ama is critical for maintaining the BBB, as its depletion results in discontinuous surface glia (SG) membranes, suggesting a lack of tight junctions or organization that leads to disruption of the barrier. This in turn exposes the larval brains to the high potassium content of the hemolymph, which damages neurons. Ama knockdown decreases overall glial cell proliferation, which can affect SG cells and the BBB in two additional ways. First, lack of proliferation in PG cells can potentially affect the secretion of metabolites, which is important to prevent neurodegeneration. Second, Ama depletion can alter subperineurial glia (SPG) growth by reducing endoreplication and endomitosis, as evident by reduced expression of cell cycle genes in repo>Ama^RNAi, and, therefore, hinder the ability of SPG to accommodate the growing brain during late larval stages (Ariss, 2020).

The conventional way to determine the intactness of the BBB function is by labeling brains with a fluorescent dextran dye. If the barrier is permissible to large molecules, such as the dye, then the BBB is considered to be broken. This study presents an alternative approach to monitor a disruption in the BBB by measuring the increase in infiltrating hemocytes in larval brains both by scRNA-seq and through staining with a hemocyte-specific antibody. Although SG protect neurons from the hemolymph, penetration of hemocytes into the brain through the damaged BBB has not been previously reported. Whether infiltrating hemocytes have a role in inflicting the damage in the brain is unknown but raises the possibility that they might have a function in that context (Ariss, 2020).

Ama and Lachesin are part of the IgLON family, with Lachesin also shown to be required for the BBB maintenance. Since IgLONs are also expressed in the BBB in mammals, this suggests that the immunoglobin superfamily may indeed also have an evolutionarily conserved BBB function in humans. These findings highlight an important role of IgLONs in neurodegenerative diseases that result in BBB breakdown (Ariss, 2020).

Through scRNA-seq and measuring P-ERK and PntP1 levels, this study found a drastic reduction in the level of RTK signaling in Ama-depleted brains. It is suggested that Ama regulates the RTK pathway since its depletion increases Sty levels, a general inhibitor of the pathway, and, conversely, overexpression of Ama has an opposite effect. Sty in repo>Ama^RNAi brains predominantly localizes to the nuclear and plasma membranes. Intriguingly, in mammalian cells, SPRY2 localization to the membrane has been shown to be crucial for its phosphorylation and inhibitory effect. The results of this study therefore suggest that, while depletion of Ama increases Sty levels, there might additionally be an effect in the cellular localization or post-translational modification of Sty. Genetic experiments indicate that Sty is the key target of Ama and that activated ERK can partially rescue the repo>Ama^RNAi phenotype, whereas activated EGFR in the glioma fly model cannot. These epistatic interactions support the model that Ama, acts through Sty, and is downstream of EGFR but upstream of ERK in the RTK signaling pathway (Ariss, 2020).

Although knockdown of Sty in Ama-depleted brains partially rescues the glial cell numbers and brain size, it fails to suppress the neuronal apoptosis in repo>Ama^RNAi brains. Since Ama can act non-cell-autonomously, it cannot be excluded that Ama might also affect neurons non-cell-autonomously and, therefore, the expression of Sty in glial cells fails to rescue neurons. This is noteworthy since LSAMP, the human counterpart of Ama, was reported to control neurite growth (Akeel, 2011). The precise mechanism of how Ama affects Sty is unknown and requires further investigation. However, SPRY2 and EGFR in human cell lines have been shown to compete with c-Cbl (a ubiquitin ligase) binding. In this context, the ubiquitin ligase attenuates the inhibitory effect of SPRY2 and vice versa. Since Ama and Sty localize to cell membranes, one possible explanation is that they may interact with each other, whereby loss of Ama releases Sty to bind to RTK receptors and promotes inhibition of the signaling pathway (Ariss, 2020).

Finally, this study shows that the role of Ama in controlling Sty levels is conserved in human glioblastoma (GBM) cell lines. Additionally, GBM patients with EGFR mutations display a significant increase in survival when they have low expression levels of LSAMP, suggesting a potential role for the IgLON family member in this cancer. Previous work has shown that LSAMP either promotes growth or acts as a tumor suppressor (Kresse, 2009). Intriguingly, SPRY2 inhibits or activates RTK signaling based on the context and cell type and also acts either as an oncogene or tumor suppressor (Masoumi-Moghaddam, 2014). These results suggest a potential connection between LSAMP and SPRY2 that may help to explain the role of LSAMP in tumorigenesis and, thus, point to LSAMP as a potential therapeutic target (Ariss, 2020).

REGULATION

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

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).

Overexpression screen in Drosophila identifies neuronal roles of GSK-3 beta/shaggy as a regulator of AP-1-dependent developmental plasticity

AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. This paper outlines results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, >4000 enhancer and promoter (EP) and EPgy2 lines were used to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, a set of selection criteria were used to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, one candidate, the GSK-3α-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity (Franciscovich, 2008).

The transcription factor AP-1 is a key regulator of neuronal growth, development, and plasticity, and in addition to cAMP response element binding (CREB) protein, it controls transcriptional responses in neurons during plasticity. Acute inhibition of Fos attenuates learning in mice and in invertebrate models such as Drosophila; AP-1 positively regulates developmental plasticity of motor neurons. Essential to the understanding of AP-1 activity in neurons is the knowledge of other proteins that influence AP-1 function or are downstream transcriptional targets. This study describes a forward genetic screen for modifiers of AP-1 in Drosophila (Franciscovich, 2008).

Using a conveniently scored AP-1-dependent adult-eye phenotype, 4307 EP and EPgy2 lines were screened for genes that modified this phenotype. Several advantages of this screen include: (1) the ease and rapidity of screening as compared to the neuromuscular junction, (2) immediate gene identification, (3) the potential to analyze in vivo phenotypes that arise from overexpression/misexpression, and finally (4) the scope for rapidly generating loss-of-function mutations through imprecise excision of the same P-element. A total of 249 known genes were isolated of which 73 can be directly implicated in eye development. The selection was prioritized using several criteria, to derive a short list of 13 final candidates that were then tested at the NMJ. Future work will focus on other predicted but as yet unstudied genes that are likely to have important functions at the NMJ (Franciscovich, 2008).

The prescreening strategy using the adult eye was successful because (1) almost all the genes selected did not result in eye phenotypes when expressed on their own, but selectively modified a Fbz dependent phenotype (Fbz is a dominant-negative transgenic construct that expresses the Bzip domain of Drosophila Fos); (2) several genes were identified that are known to interact with AP-1 in regulating synaptic phenotypes (these include ras and bsk); (3) multiple alleles of some genes were recovered confirming the sensitivity of the screening technique; (4) several genes involved in eye development were isolated (including cyclinB, which has been shown to be a downstream target of Fos in the regulation of G2/M transition in the developing eye); (5) a large number of putative interactors have connections with neural physiology and/or AP-1 function in other cell types; (6) some candidates with strong phenotypes have previously been shown to play important roles in motor neurons; and finally (7) the majority of candidates (but not all) isolated as enhancers or suppressors of Fbz in the eye exerted a similar effect on AP-1 at the synapse (Franciscovich, 2008).

Although the relative success and merits of a functional screen are considerable, there are a few disadvantages. First, the use of P-element transposons naturally excludes a large fraction of genes that are refractory to P-element transposition events. Second, insertions of EP elements within or in inverse orientation to the gene make it difficult to assign phenotypes to specific genes. Even in instances where overexpression was predicted, it has to be verified that this is indeed the case and also the phenotypes derive from hypomorphic mutations that result from the insertion of the P-element close to the target gene have to be tested. Third, although recover genes that play conserved roles in AP-1 biology is to be expected, those genes that specifically affect synaptic physiology and play no role in the eye will be excluded by this scheme. Finally, this screen will not discriminate between genes that function upstream or downstream of AP-1 in neurons. In spite of these deficiencies, it is believed that candidates identified in this screen provide strong impetus for the investigation of additional factors that are involved in the regulation of synaptic plasticity and development by AP-1 (Franciscovich, 2008).

Following their identification, it was found that several candidates had synaptic functions since several of these genes resulted in significant differences in synaptic size when compared to appropriate controls. This provided the first confirmation of the screening strategy. Next, experiments to determine genetic interaction with AP-1 showed that expression of four genes (pigeon, lbm, Cnx99A, and sty) suppressed the Fbz-dependent small synapse phenotype. Of these, sty had been isolated as an enhancer while the other three similarly suppressed the Fbz-derived eye phenotype, suggesting potentially conserved functions of these genes in the two tissues (Franciscovich, 2008).

Four genes isolated as enhancers, similarly enhanced an Fbz-mediated small synapse (cnk, pde8, fkbp13, and sgg). Notably, expression of these genes also suppressed an AP-1-dependent synapse expansion at the NMJ. These two lines of evidence indicate that these genes are negative regulators of AP-1 function in these neurons. Together with the fact that all four have previously described functions in the nervous system, these observations confirm the validity of the screen and highlight the utility of genetic screens to uncover novel molecular interactions. Further studies will provide a more comprehensive understanding of the interplay between these genes and AP-1 in the regulation of neuronal development and plasticity. For instance, more careful analysis needs to be carried out to discern whether synaptic phenotypes in each of these cases are due to overexpression or potential insertional mutagenesis of specific genes (Franciscovich, 2008).

Although GSK-3β-signaling has been implicated in several neurological disorders such as Alzheimer's disease, it is only recently that neuronal roles for this important kinase have come to light. For instance, several studies have demonstrated the role of GSK-3β in the regulation of long-term potentiation (LTP) in vertebrate hippocampal synapses (Hooper, 2007; Peineau, 2007; Zhu, 2007). In particular, these reports highlight the negative regulatory role of GSK-3β in the induction of LTP or in one case, the switching of long-term depression (LTD) into LTP. Interestingly, LTP induction leads to GSK-3β-inhibition thus precluding LTD induction in the same neurons. In flies, sgg mutations have defects in olfactory habituation, circadian rhythms and synaptic growth. These observations point to a conserved and central role for GSK-3β in neuronal physiology (Franciscovich, 2008).

GSK-3β-dependent modulation of transcriptional responses is widely acknowledged. Among several transcription factors that are known to be regulated by this kinase, are AP-1, CREB, NFAT, c/EBP, and NF-kappaB. In the context of neuronal function, for instance, RNA interference-based experiments in cultured rat cortical neurons have shown that GSK-3β-activity influences CREB and NF-kappaB-dependent transcription. Additionally, two other transcription factors, early growth response 1 and Smad3/4 have been identified in DNA profiling experiments in the same study. Significantly, GSK-3β is also a primary target of lithium, a drug used extensively to treat mood disorders. Lithium treatment has been reported to result in an upregulation of AP-1-dependent transcription, though a role for GSK-3β in this phenomenon has not been tested directly (Franciscovich, 2008).

In Drosophila, recent experiments have described the negative regulation of synaptic growth by the GSK3β-homolog shaggy (Franco, 2004). These studies demonstrate that sgg controls synaptic growth through the phosphorylation of the Drosophila MAP1B homolog futsch. The current studies suggest that Sgg-dependent regulation of synapse size occurs through the immediate-early transcription factor AP-1. GSK-3β is believed to inhibit transcriptional activity of AP-1 in cultured cells by direct inhibitory phosphorylation of c-Jun. Circumstantial evidence also suggests that GSK-3β provides an inhibitory input into AP-1 function in neurons (Franciscovich, 2008).

It was intriguing to find that Sgg inhibition leads to an expanded synapse with reduced presynaptic transmitter release, similar to highwire mutants. Given that in several instances, Sgg-dependent phosphorylation targets a protein for ubiquitination, and that Highwire encodes an E3 ubiquitin ligase, it is conceivable that sgg and hiw function in the same signaling pathway. Consistent with this hypothesis, both hiw and sgg function at the synapse seem to impinge on AP-1-dependent transcription through modulation of the JNK signaling pathway. Considering previous reports of GSK-3β-involvement in multiple signaling cascades, it will be interesting to study how sgg controls multiple aspects of cellular physiology to regulate neural development and plasticity, particularly in the context of brain function and action of widely used drugs such as lithium (Franciscovich, 2008).

EVOLUTIONARY HOMOLOGS

A search of the expressed sequence tag (dbEST) database identified three human homologs designated h-Sprouty1, h-Sprouty2, and h-Sprouty3. A complete coding sequence of h-Sty2 and a partial sequence of h-Sty1 from overlapping cDNAs has been determined. The h-Sty2 sequence encodes a 315-residue (35 kD) polypeptide. It contains a cysteine-rich domain, which is highly conserved with Sty (51% identity, with 21 of the 22 Sty cysteines conserved), and two additional short stretches of similarity to Sty in the N-terminal region but no predicted signal peptide. h-Sty1 and h-Sty3 also show strong conservation of the cysteine-rich domain, with 51%-70% identity to other family members in the available sequences (Hacohen, 1998).

In Drosophila embryos, the loss of sprouty gene function enhances branching of the respiratory system. Three human sprouty homologs (h-Spry1-3) have been cloned recently, but their function has been unknown. A murine sprouty gene (mSpry-2) whose product shares 97% homology with the respective human protein, is expressed in the embryonic murine lung. Antisense oligonucleotide strategy was used to reduce expression of mSpry-2 by 96%, in E11.5 murine embryonic lungs cultured for 4 days. Morphologically, the decrease in mSpry-2 expression results in a 72% increase in embryonic murine lung branching morphogenesis as well as a significant increase in expression of the lung epithelial marker genes SP-C, SP-B and SP-A. These results support a striking conservation of function between the Drosophila and mammalian sprouty gene families to negatively modulate respiratory organogenesis. It is suggested that mSpry-2 is a negative regulator of FGF signaling and its apparent function may be to negatively modulate new bud formation (Tefft, 1999).

Signaling molecules belonging to the Fibroblast growth factor (Fgf) family are necessary for directing bud outgrowth during tracheal development in Drosophila and lung development in mouse. Sprouty, a potential inhibitor of the Fgf signaling pathway, has been identified in Drosophila. Three potential mouse homologues of sprouty have been identified. One of them, Sprouty4, exhibits a very restricted expression pattern. At 8.0 dpc (days post coitum) Sprouty4 is strongly expressed in the primitive streak region. At 9. 5 and 10.5 dpc, Sprouty4 is expressed in the nasal placode, the maxillary and mandibular processes, the otic vesicle, the second branchial arch, in the progress region of the limb buds and the presomitic mesoderm. Sprouty4 expression is also detected in the lateral region of the somites. In the developing lung, Sprouty4 is expressed broadly in the distal mesenchyme, but not in the epithelium or trachea. Strong expression is detected in the mesechyme of the accessory lobe of the lung. In contrast, Fgf10 is expressed in discrete areas of the distal mesenchyme. At 11.5 dpc, strong expression is observed in the distal mesenchyme of the median, accessory and light caudal lobes and in the lateral and distal region of the left lobe: no signal is detected in the trachea. At 12.5 dpc, Fgf10 expression is restricted to the distal mesenchyme between buds. In conclusion, Sprouty4 and Fgf10 have overlapping but not identical expression patterns (de Maximy, 1999).

A family of vertebrate Sprouty homologs is described and it is demonstrated that the regulatory relationships with FGF pathways are conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there is a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3 (Minowada, 1999).

cDNAs representing four mouse genes with sequence homology to Drosophila and human spry genes, and the chick orthologs of Spry1 and Spry2 were isolated. Sequence analysis indicates that all contain full coding regions except the mSpry3 and cSpry1 cDNA clones, which are partial cDNAs encoding only the C-terminal region of the proteins. The predicted mSPRY1, mSPRY2, and mSPRY4 proteins are similar in size (34, 34.6 and 32.6 kDa, respectively), and each contains an ~125 amino acid (aa) residue cysteine-rich C-terminal domain with at least 23 cysteines. mSPRY3 contains a similar cysteine-rich domain. Throughout the C-terminal domain the four mouse proteins share 56%-70% aa sequence identity. In the N-terminal domain mSPRY1 and mSPRY2 are more similar to one another (~37% aa identity) than mSPRY1 is to mSPRY4 (~25% aa identity) or mSPRY2 is to mSPRY4 (~25% aa identity). Comparison of the mouse, chick, and previously described human SPRY2 sequences demonstrates that the Spry2 gene has been highly conserved during vertebrate evolution; most of the differences among species are in the N-terminal domain. The similarity between the vertebrate and Drosophila sprouty (dSPRY) protein sequences is limited mostly to the cysteine-rich domain, with each vertebrate protein showing ~44%-52% aa identity to dSPRY in this region. The cysteine residues are particularly highly conserved, with 18 or more of the 22 cysteines in the Drosophila protein present in each vertebrate gene. Outside the cysteine-rich domain there are two short regions that show similarity between dSPRY and all three mouse proteins. There are also short stretches of sequence similarity between dSPRY and individual vertebrate Sprouty proteins, such as the region of the mapped mouse Sprouty genes, that co-localize with a known mouse mutation (Minowada, 1999).

Members of the fibroblast growth factor (FGF) family of peptide growth factors are widely expressed in the germ layer derivatives during gastrulation and early organogenesis of the mouse. The effect of administering recombinant FGF-4 in the late-primitive streak stage embryo was examined to test if the patterning of the body plan may be influenced by this growth factor. Shortly after FGF treatment, the embryonic tissues up-regulate the expression of Brachyury and the RTK signaling regulator Spry2, suggesting that FGF signaling is activated as an immediate response to exogenous FGF. Concomitantly, Hesx1 expression is suppressed in the prospective anterior region of the embryo. After 24 h of in vitro development, embryos display a dosage-related suppression of forebrain morphogenesis, disruption of the midbrain-hindbrain partition, and inhibition of the differentiation of the embryonic mesoderm. Overall, development of the anterior/posterior axis in the late gastrula is sensitive to the delivery of exogenous FGF-4. The early response associated with the expression of Spry2 suggests that the later phenotype observed could be primarily related to an inhibition of the FGF signaling pathway (Davidson, 2000).

The expression of mouse Spry2 was examined to determine the extent of FGFR activation following FGF-4 treatment. Expression of Spry2 is normally restricted to the primitive streak region of the gastrula embryo, a region where several FGF genes (Fgf3, Fgf4, Fgf5, Fgf8, and Fgf17) as well as Spry4 are expressed. A marked elevation in Spry2 expression is observed shortly after FGF-4 delivery. Spry2 expression is widespread in the epiblast of the embryo, a region which is also a site of Fgfr1 expression. Of particular interest is the absence of Spry2 expression in the node and head process of the embryo and in extraembryonic tissues. To determine whether Spry2 expression is sustained after FGF treatment, embryos were examined at 24 h of culture. In control embryos, Spry2 is expressed in sites of endogenous Fgf activity such as the region where the caudal midbrain adjoins the rostral hindbrain, the tail bud, and, in some embryos, the rostral part of the forebrain. Spry2 is also expressed in the posterior paraxial mesoderm and the tail bud. In contrast, FGF-4-treated embryos display, in addition to expression of Spry2 in the neuroectoderm, ectopic expression of Spry2 in the mesendoderm of the foregut and the midgut. In severely affected embryos, histological examination revealed that paraxial mesoderm is absent and Spry2 mRNA is found in the endoderm lying directly underneath the neural tube. The primitive streak of treated embryos is abnormal in tissue organization and fails to express detectable levels of the Spry2 gene, although expression is observed in the ventral neural tube and the posterior paraxial mesoderm in some embryos (Davidson, 2000).

SPRY2, a human homolog of Drosophila Spry, is involved FGF2 signaling. In primary human dermal endothelial cells (MVEC) SPRY2 mRNA is transiently upregulated in response to FGF2. Overexpression of SPRY2 in A375 cells leads to the secretion of a soluble factor that inhibits FGF2- but not VEGF-stimulated proliferation of MVEC. Direct administration of recombinant SPRY2 protein has no effect on MVEC proliferation. However, SPRY2 protein binds the intracellular adaptor protein GRB2, indicating an intracellular localization. A SPRY2/GFP fusion protein remains in the cell, further supporting the intracellular localization of SPRY2. So the intracellular protein SPRY2 is involved in the non-cell autonomous inhibitory effect indirectly, via regulating the secretion of an inhibitor of FGF2 signaling in vertebrates, the evidence of which is presented here for the first time (Glienke, 2000).

Studies in Drosophila and chick have shown that members of the Sprouty family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Direct evidence is provided that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new branches at E12.5. mSprouty2 is expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression is restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 is most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulate the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 show greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduce branching and decrease mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 does not change Shh and FgfR2 mRNA levels in the lungs. Sprouty2 function during lung development was investigated by using two different but complementary approaches. The targeted over-expression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, results in a low level of branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also results in a low level of branching. These results indicate that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth (Mailleux, 2001).

Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior-posterior axis. The Drosophila gene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. Xenopus sprouty2 is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Using Xenopus oocytes it has been shown that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis (Nutt, 2001).

In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, a zebrafish sprouty4 gene was isolated, based on its extensive similarities with the expression patterns of both fgf8 and fgf3. Through gain- and loss-of-function experiments, it has been demonstrated that Fgf8 and Fgf3 act in vivo to induce the expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses, Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, a phenotype opposite that of the dorsalization induced by overexpression of Fgf8 or Fgf3. Conversely, inhibition of Spry4 function through injection of antisense morpholino oligonucleotide leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, it has been shown that Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, this analysis reveals that signaling through FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the regulation of bone morphogenetic protein (BMP) expression (Furthauer, 2001).

The zebrafish Spry cDNA codes for a 310 amino acid protein. It is most closely related to mouse Sprouty4, the two proteins displaying 65.7% overall amino acid similarity while showing less than 50% amino acid similarity with the mouse or human Spry1, Spry2 and Spry3. Phylogenetic analysis further confirms that this clone encodes a zebrafish Sprouty4 homolog. Alignment of the peptide sequence of the sprouty genes reveals the existence of three domains of particularly extensive conservation. Most prominent among these is the C-terminal 130 amino acid cysteine-rich domain, which constitutes the distinctive feature of Spry proteins and has been shown to be sufficient for the localization of Spry at the plasma membrane. In zebrafish Spry4 this domain contains 25 cysteine residues, 17 of which are found at conserved positions in all Spry proteins (Furthauer, 2001).

To investigate at which level Spry4 interferes with FGF signaling, an assessment was made of its ability to rescue a constitutively active (CA) FGFR1-induced dorsalization. Coinjection of CA-FGFR1 with increasing doses of spry4 mRNA progressively rescues this dorsalization phenotype. For 125 pg spry4 mRNA, only 29% (32/109) embryos remain dorsalized while using 250 pg led to a complete rescue of the dorsalization phenotype. This clearly demonstrates that spry4 antagonizes the FGF signaling mediated through FGFR1. Stimulation of FGFR1 ultimately leads to the phosphorylation of the extracellular-regulated protein kinases (ERK) 1 and 2. Therefore advantage was taken of the use of an antibody recognizing the activated form of ERK to estimate the effect of Spry4 on MAPK activity. In accordance with an activation of ERK after the stimulation of FGFR1, localized misexpression of CA-FGFR1 induces ectopic activation of MAPK at blastula stage, whereas activated MAPK is barely detectable in wild-type control embryos. Conversely, localized injection of 250 pg spry4 mRNA causes a local inhibition of MAPK activation at mid-gastrula stages, when the MAPK is ubiquitously activated in wild-type embryos. These results therefore demonstrate that spry4 interferes with FGF signaling by acting downstream of FGFR1, leading to a subsequent downregulation of MAPK activity (Furthauer, 2001).

Cellular proliferation and differentiation of cells in response to extracellular signals, are both controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Described here are two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signaling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibits growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection reveals that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associates with Ras but does not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibits the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signaling (Wakioka, 2001).

Spred-1 is tyrosine phosphorylated in response to stem cell factor (SCF), platelet-derived growth factor (PDGF) and EGF, and efficient phosphorylation of Spred-1 requires the KBD region. Using immunofluorescence microscopy, endogenous Spred-2 was found to be localized to the plasma membrane. Membrane localization of Spred was confirmed by exogenously expressed Spred fused to enhanced green fluorescent protein (EGFP). The C-terminal SPR domain is essential for plasma membrane localization, since a deletion mutant lacking SPR domain (GFP-C) is localized in the cytoplasm (Wakioka, 2001).

The molecular mechanism by which Spred suppresses the Ras-MAP kinase pathway was investigated. Since one of the nuclear targets of MAP kinase is Elk-1, a transcription factor of the Ets family, EGF-induced activation of MAP kinase can be monitored by measuring the rate of Elk-1-dependent transcription. In 293 cells, forced expression of Spred-1 or -2 dose-dependently suppresses EGF-dependent Elk-1 activation. The negative effect of Spred-1 and -2 is comparable to that of Ras GTPase activating protein (rasGAP) and N17-Ras, and Spred-1 and -2 are more potent inhibitors than is murine Sprouty-4 or the Raf kinase inhibitor protein 1. Both EVH-1 and SPR domains are necessary for the suppression of Elk-1 activation. Replacement of the EVH-1 domain of Spred-1 with that of Wiskott-Aldrich syndrome protein (WASP) abolishes the inhibitory activity of Spred-1, suggesting that the EVH-1 domain of Spred-1 may interact with a specific target required for suppression of the MAP kinase pathway. In contrast, the KBD region is not essential but required for efficient suppression of the MAP kinase pathway (Wakioka, 2001).

Ras directly interacts with and activates Raf. Raf phosphorylates and activates MEK, which in turn phosphorylates and activates MAP kinases. Spred inhibits activation of Elk-1 induced by active Ras (V12-Ras), but not that induced by active MEK or active Raf (N-Raf). Therefore, the target of Spred is probably located between Ras and Raf. To test this hypothesis, the effect of Spred-1 on EGF-induced Ras and Raf activation was examined. Interestingly, Spred sustains Ras activation, whereas it inhibits Raf activation, as measured by autophosphorylation and by in vitro kinase assay. Furthermore, like rasGAP, Spred inhibits the phosphorylation of Raf on Ser 338, which is required for Raf activation, but not on Ser 259, which is not. Thus, Spred inhibits MAP kinase activity by suppressing Raf activation (Wakioka, 2001).

The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, supporting cells of the organ of Corti, there are three, resulting in the formation of an ectopic tunnel of Corti. These effects are due to a postnatal cell fate transformation of a Deiters’ cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. These results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing (Shim, 2005).

Sprouty interations with Shp2

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, Sprouty (Spry) is demonstrated to be a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 is able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry results in dissociation of Spry from Grb2. Furthermore, Shp2 can reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry (Hanafusa, 2004).

Shp2/Ptpn11 tyrosine phosphatase is a general regulator of the RTK pathways. By genetic ablation, it was demonstrated that Shp2 is required for lacrimal gland budding, lens cell proliferation, survival and differentiation. Shp2 deletion disrupts ERK signaling and cell cycle regulation, which could be partially compensated by activated Kras signaling, confirming that Ras signaling is the main downstream target of Shp2 in lens and lacrimal gland development. It was also shown that Sprouty2, a general suppressor of Ras signaling, is regulated by Shp2 positively at the transcriptional level and negatively at the post-translational level. Only in the absence of Sprouty2 can activated Kras signaling robustly rescue the lens proliferation and lacrimal-gland-budding defects in the Shp2 mutants. It is proposed that the dynamic regulation of Sprouty by Shp2 might be important not only for modulating Ras signaling in lens and lacrimal gland development, but also for RTK signaling in general (Pan, 2010).

Sprouty2-mediated inhibition of fibroblast growth factor signaling is modulated by the protein kinase DYRK1A

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. This study identifies DYRK1A (Drosophila homolog: Minibrain) as one of the protein kinases of Sprouty2. DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, Thr75 on Sprouty2 is identified as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhances the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. These results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2 (Aranda, 2008).

Sprouty and brain patterning

In a differential display analysis to identify genes involved in patterning the mid/hindbrain region of the chick neural tube, a sprouty ortholog, sprouty2, has been identified. In the developing chick embryo there is a close correlation with known sites of FGF activity but little correlation with expression patterns of members of the EGF family. Initially, transcripts are associated with the primitive streak. During the period of neural tube patterning expression is detected in the anterior neuropore, in the isthmic region and in neural plate and posterior spinal cord. Transcripts are also detected in the otic placode, tail bud, mesoderm of the branchial arches, somitic myotome, retina, limb buds and gut mesenchyme; all are known sites of FGF action (Chambers, 2000a).

Specification and polarization of the midbrain and anterior hindbrain involves planar signals originating from the isthmus. Current evidence suggests that FGF8, expressed at the isthmus, provides this patterning influence. In this study, novel genes were sought that are involved in the process by which regional identity is imparted to midbrain and anterior hindbrain (rhombomere 1). An enhanced differential display reverse transcription method was used to clone cDNAs derived from transcripts expressed specifically in either rhombomere 1 or midbrain during the period of isthmic patterning activity. This gene expression screen has identified 28 differentially expressed cDNAs. A clone upregulated in cDNA derived from rhombomere 1 tissue shows a 91% identity at the nucleotide level to the putative human receptor tyrosine kinase antagonist: sprouty2. In situ hybridization on whole chick embryos shows chick sprouty2 to be expressed initially within the isthmus and rhombomere 1, spatially and temporally coincident with Fgf8 expression. However, at later stages this domain is more extensive than that of Fgf8. Introduction of ligand-coated beads into either midbrain or hindbrain region reveal that sprouty2 can be rapidly induced by FGF8. These data suggest that sprouty2 participates in a negative feedback regulatory loop to modulate the patterning activity of FGF8 at the isthmus (Chambers, 2000b).

Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development. A chick electroporation assay and in vitro mouse brain explant experiments have been used to compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. (1) FGF8b is the only protein that can induce the r1 gene Gbx2 and strongly activate the pathway inhibitors Spry1/2, as well as repress the midbrain gene Otx2. Consistent with previous studies that indicated high level FGF signaling is required to induce these gene expression changes, electroporation of activated FGFRs produce similar gene expression changes to FGF8b. (2) FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development. By contrast, FGF17b and FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating midbrain gene expression. This result is consistent with Fgf17 and Fgf18 being expressed in the midbrain and not just in r1 as is Fgf8. (3) Analysis of gene expression in mouse brain explants with beads soaked in FGF8b or FGF17b shows that the distinct activities of FGF17b and FGF8b are not due to differences in the amount of FGF17b protein produced in vivo. Finally, brain explants were used to define a positive feedback loop involving FGF8b mediated upregulation of Fgf18, and two negative feedback loops that include repression of Fgfr2/3 and direct induction of Spry1/2. Since Fgf17 and Fgf18 are co-expressed with Fgf8 in many tissues, these studies have broad implications for how these FGFs differentially control development (Liu, 2003).

The following steps in midbrain and cerebellum development in mouse are proposed. At the four-somite stage, Fgf8 is induced in the presumptive r1 territory by an unknown factor. Pax2 is required for this induction and OTX2 inhibits Fgf8 from being induced in the midbrain. FGF8b then induces Fgf18 in the surrounding cells, producing a larger domain and gradient of Fgf mRNA that extends into the midbrain. FGF8b also maintains two negative feedback loops by inducing Spry1 and Spry2 expression and inhibiting Fgfr2 and Fgfr3. Fgf17 is then induced by an unknown mechanism that is dependent on Fgf8 in a broader domain than Fgf18, further extending the gradient of Fgf mRNA expression. FGF17 and FGF18 protein, and possibly FGF8a and a low level of FGF8b, then regulate proliferation of the midbrain and cerebellum and En expression. The narrow domain where Fgf8 is expressed becomes the isthmus because of the activity of FGF8b, and the adjacent Otx2-negative r1 cells become the cerebellum. By the 15-somite stage Gbx2 is not required in r1 for cerebellum development, but is required earlier to specify r1. Thus, once Fgf8 expression in r1 is stabilized, perhaps by a secreted factor from the midbrain, a key function of high level signaling by FGF8b is to maintain a cascade of gene expression in the midbrain/r1 that maintains an Otx2-negative domain in r1 in which the cerebellum develops (Liu, 2003).

FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. This study employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. Mathematical models were generated to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. Sprouty genes were tested as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. A model is proposed in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain (Dyer, 2014).

Sprouty and kidney development

The kidney is a classic model for studying mechanisms of inductive tissue interactions associated with the epithelial branching common to many embryonic organs, but the molecular mechanisms are still poorly known. Sprouty proteins antagonize tyrosine kinases in the Egf and Fgf receptors and are candidate components of inductive signalling in the kidney as well. The function of sprouty proteins was addressed in vivo by targeted expression of human sprouty 2 (SPRY2) in the ureteric bud, which normally expresses inductive signals and mouse sprouty 2 (Spry2). Ectopic SPRY2 expression led to postnatal death resulting from kidney failure, manifested as unilateral agenesis, lobularization of the organ or reduction in organ size because of inhibition of ureteric branching. The experimentally induced dysmorphology associated with deregulated expression of Wnt11, Gdnf and Fgf7 genes in the early stages of organogenesis indicated a crucial role for sprouty function in coordination of epithelial-mesenchymal and stromal signalling, the sites of expression of these genes. Moreover, Fgf7 induces Spry2 gene expression in vitro and leads with Gdnf to a partial rescue of the SPRY2-mediated defect in ureteric branching. Remarkably, it also leads to supernumerary epithelial bud formation from the Wolffian duct. Together, these data suggest that Spry genes contribute to reciprocal epithelial-mesenchymal and stromal signalling controlling ureteric branching, which involves the coordination of Ffg/Wnt11/Gdnf pathways (Chi, 2004).

Intercellular signaling molecules and their receptors, whose expression must be tightly regulated in time and space, coordinate organogenesis. Regulators of intracellular signaling pathways provide an additional level of control. Loss of the receptor tyrosine kinase (RTK) antagonist Sprouty1 (Spry1) causes defects in kidney development in mice. Spry1^-/- embryos have supernumerary ureteric buds, resulting in the development of multiple ureters and multiplex kidneys. These defects are due to increased sensitivity of the Wolffian duct to GDNF/RET signaling, and reducing Gdnf gene dosage correspondingly rescues the Spry1 null phenotype. It is concluded that the function of Spry1 is to modulate GDNF/RET signaling in the Wolffian duct, ensuring that kidney induction is restricted to a single site. These results demonstrate the importance of negative feedback regulation of RTK signaling during kidney induction and suggest that failures in feedback control may underlie some human congenital kidney malformations (Basson, 2004).

Sprouty interacts with c-Cbl thus attenuating epidermal growth factor receptor ubiquitylation and endocytosis

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signaling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF-mediated lung and limb morphogenesis. Evidence is presented that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl (see Drosophila Cbl), and its impact on the cellular localization and signaling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signaling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signaling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpryDelta2N11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. It is concluded that hSpry2 potentiates EGFR signaling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation (Wong, 2002).

The means by which receptor ubiquitylation influences protein trafficking remain obscure, including the role of mono- and poly-ubiquitylation. The consensus view is that monoubiquitin or short ubiquitin chains are sufficient to direct internalization of cell surface proteins, whereas the proteasomal machinery recognizes polyubiquitylated proteins in Saccharomyces cerevisiae. In mammalian cells however, the situation is not as clear because a number of plasma membrane proteins that are ubiquitylated appear to be degraded through both the proteasomal and lysosomal pathways. Other classical endocytic signals include reversible modification such as phosphorylation, damage to the protein, genetically encoded sequence motifs (e.g. YXXPhi, where Phi is a bulky hydrophobic amino acid; MPXY or di-leucine), as well as sorting events that are coupled to clathrin-dependent or -independent routes. There are currently disparate views on how and where c-Cbl ubiquitylates its target RTKs; evidence derived from studies with yeast, growth hormone receptor and inhibition of ErbB-1/EGFR (and other diverse receptors) uptake into internalized vesicles using dynamin mutants suggest that ubiquitylation may be associated with sorting at the plasma membrane. c-Cbl-mediated ubiquitylation of EGFRs has been shown to occur at the plasma membrane, which then facilitates recruitment of activated EGFRs into clathrin-coated pits and the complex remains associated throughout the endocytic route. The results of this study further support the notion that c-Cbl is likely to act on EGFR at the cell surface, and inhibition of this interaction by hSpry2 attenuates early stages of receptor internalization. The data concur with previous evidence pertaining to the endocytic events governing mCSF-1R, where internalization of the macrophage receptor is retarded in c-Cbl-defective cells and yeast membrane receptor regulation, but is seemingly at odds with reports on EGFR endocytosis where c-Cbl has been arguably implicated as an endosomal sorting protein with signaling potential (Wong, 2002 and references therein).

In recent studies involving the analysis of crystal structures, it was demonstrated that UbcH7 interacts closely with both the RING finger domain and the N-terminal 70Z linker region of c-Cbl, apparently initiated upon tyrosine phosphorylation on residue 371 on the linker sequence by activated EGFR. The present study provides additional insights into the mechanism of c-Cbl's mediatory effect on receptor ubiquitylation, in that the binding of UbcH7 can be successfully competed off by hSpry2. Much remains to be elucidated regarding the specific details of c-Cbl-dependent ubiquitylation, such as: resolving the identity of the candidate lysine residue on c-Cbl that becomes ubiquitylated, elucidating the structural conformation of phosphorylated c-Cbl (on Y371) and determining whether dimerization of c-Cbl might be important in its function as a ubiquitin ligase -- all of which will advance understanding of how hSpry2 intercepts and disrupts the functional role of its E3-binding partner (Wong, 2002).

Growth factors and their receptor tyrosine kinases play pivotal roles in development, normal physiology, and pathology. Signal transduction is regulated primarily by receptor endocytosis and degradation in lysosomes ('receptor downregulation'). c-Cbl is an adaptor that modulates this process by recruiting binding partners, such as ubiquitin-conjugating enzymes. The role of another group of adaptors, Sprouty proteins, is less understood; although, studies in insects have implicated the founder protein in the negative regulation of several receptor tyrosine kinases. By utilizing transfection of living cells, as well as reconstituted in vitro systems, a dual regulatory mechanism has been identified that combines human Sprouty2 and c-Cbl. Upon activation of the receptor for the epidermal growth factor (EGFR), Sprouty2 undergoes phosphorylation at a conserved tyrosine that recruits the Src homology 2 domain of c-Cbl. Subsequently, the flanking RING finger of c-Cbl mediates poly-ubiquitination of Sprouty2, which is followed by proteasomal degradation. Because phosphorylated Sprouty2 sequesters active c-Cbl molecules, it impedes receptor ubiquitination, downregulation, and degradation in lysosomes. This competitive interplay occurs in endosomes, and it regulates the amplitude and longevity of intracellular signals. It is concluded that Sprouty2 is an inducible antagonist of c-Cbl, and together they set a time window for receptor activation. When incorporated in signaling networks, the coupling of positive (Sprouty) to negative (Cbl) feedback loops can greatly enhance output diversification (Rubin, 2003).

Genetic screens performed in invertebrates have identified several negative regulators of RTKs, including Cbl, Sprouty, Kekkon, and Argos. However, unlike the Cbl pathway, which is present in worms, the other three pathways do not exist in C. elegans, suggesting that they were added as secondary regulators that fine-tune RTK function. Consistent with this notion, biochemical studies have identified an interlinked dual feedback loop that combines c-Cbl and Spry2. A previously unidentified stimulus-dependent phosphorylation of Spry2, on an evolutionary conserved tyrosine residue (tyrosine 55), plays a key role in these interactions. Once phosphorylated, this residue acts as the core of an inducible docking site for the SH2 domain of c-Cbl, and, subsequently, it enables the flanking RING finger of c-Cbl to covalently link ubiquitin molecules to Spry2. Similar to trans-phosphorylation of Spry2, a c-Cbl docking site is established upon autophosphorylation of RTKs (e.g., tyrosine 1045 of EGFR), and it consequently enables receptor ubiquitination. These results imply that these two analogous processes occur concurrently, most likely at the plasma membrane and in endosomes, and that they are mutually competitive. As a result, Spry2 regulates ubiquitination and subsequent degradation of EGFR, and EGFR targets Spry2 to proteasomal degradation. The uncovered interplay between c-Cbl and Spry2 fine-tunes downstream signaling, which explains the role of these adaptor molecules in balancing between activation and repression of RTKs (Rubin, 2003).

An interesting outcome of the coupling between phosphorylation and ubiquitination of Spry2 and EGFR is a predicted oscillation of the Spry2 level. Studies in insects and in mammalian cells have revealed that Sprouty proteins, along with other regulators like Kekkon and Argos, are transcriptionally induced upon activation of RTKs. Hence, transcription from the spry2 gene is expected to follow the rapid, ligand-induced degradation of Spry2. Interestingly, a similar compensatory loop enables EGF to upregulate transcription from the egfr gene. Thus, the protein degradation-based feedback loops that regulate Sprouty and RTKs are mirrored at the gene transcriptional level, and this bimodal circuit ensures receptor homeostasis. Consistent with the critical role of tyrosine 55 of Spry2 in regulating EGFR signaling, a screen of Spry2 and Spry4 mutants has identified the respective Y55F and Y53F point mutants as dominant-negative proteins capable of potentiating MAPK activation by FGF. Likewise, deletion of residues 11-53 of Spry2 abolishes c-Cbl binding, probably because the Spry2 deletion construct impairs the amino-terminal part of the Cbl's docking site. In contrast to the current results, previous studies have identified the RING finger of c-Cbl, not the SH2 domain, as the site of interaction with Spry2. While the current results do not exclude weak binding with the RING finger, the strong interaction between Spry2 and an SH2-only mutant of Cbl indicates that the phosphotyrosine-dependent interactions govern the functional outcome in living cells (Rubin, 2003).

Along with the prospect that Sprouty proteins interfere with signaling events at a point upstream to that predicted by genetic and biochemical analyses, these results imply that Sprouty acts as a positive rather than a negative regulator of EGF signaling. It is reasonable to assume that autophosphorylation of EGFR and subsequent recruitment of c-Cbl precede the indirect transphosphorylation of Spry2. Thus, delayed activation of the Spry-mediated loop is expected to occur after c-Cbl initiates receptor ubiquitination and endocytosis. In the next step, Spry2 itself is inactivated, by means of ubiquitination and degradation, which enables a new cycle of receptor activation/inactivation. At the gene expression level, EGF-induced synthesis of Spry2 is expected to replenish the cellular pool of active Spry2 molecules. This highly regulated sequence of events predicts staggering waves of active RTKs and Sprouty proteins in growth factor-stimulated tissues. In the context of a signaling network, such as ErbB, temporal regulation of the levels of RTKs and Sprouty proteins would enable repeated stimulation by growth factors, resist perturbations, and diversify the output by balancing receptor desensitization and resensitization (Rubin, 2003).

Sprouty was originally identified in a genetic screen in Drosophila as an antagonist of fibroblast (FGF) and epidermal growth factor (EGF) signaling. Subsequently, four vertebrate homologs were discovered; among these, the human homolog Sprouty 2 (hSpry2) contains the highest degree of sequence homology to the Drosophila protein. It has been shown that hSpry2 interacts directly with c-Cbl, an E3-ubiquitin ligase, which promotes the downregulation of receptor tyrosine kinases (RTKs). In this study, the functional consequences of the association between hSpry2 and c-Cbl has been investigated. hSpry2 is found to be ubiquitinated by c-Cbl in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of hSpry2, which in turn enhances the interaction of hSpry2 with c-Cbl. The c-Cbl-mediated ubiquitination of hSpry2 targets the protein for degradation by the 26S proteasome. An enhanced proteolytic degradation of hSpry2 is also observed in response to FGF stimulation. The FGF-induced degradation of hSpry2 limits the duration of the inhibitory effect of hSpry2 on extracellular signal-regulated kinase (ERK) activation and enables the cells to recover their sensitivity to FGF stimulation. These results indicate that the interaction of hSpry2 with c-Cbl might serve as a mechanism for the downregulation of hSpry2 during receptor tyrosine kinase signaling (Hall, 2003).

Paracrine signaling mediated by FGF-10 and the FGF-R2IIIb receptor is required for formation of the lung. To determine the temporal requirements for FGF signaling during pulmonary morphogenesis, Sprouty-4 (Spry-4), an intracellular FGF receptor antagonist, was expressed in epithelial cells of the fetal lung under control of a doxycycline-inducible system. Severe defects in lobulation and severe lung hypoplasia were observed when Spry-4 was expressed throughout fetal lung development (E6.5-E18.5) or from E6.5 until E13.5. Effects of Spry-4 on branching were substantially reversed by removal of doxycycline from the dam at E12.5, but not at E13.5. In contrast, when initiated late in development (E12.5 to birth), Spry-4 caused less severe pulmonary hypoplasia. Expression of Spry-4 from E16.5 to E18.5 reduces lung growth and results in perinatal death due to respiratory failure. Expression of Spry-4 during the saccular and alveolar stages, from E18.5 to postnatal day 21, causes mild emphysema. These findings demonstrate that the embryonic-pseudoglandular stage is a critical time period during which Spry-sensitive pathways are required for branching morphogenesis, lobulation, and formation of the peripheral lung parenchyma (Perl, 2003).

Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Sprouty proteins become tyrosine phosphorylated after growth factor treatment. Tyr55 was identified as a key residue for Sprouty2 phosphorylation; phosphorylation is required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augments signaling. Tyrosine phosphorylation of Sprouty2 affects neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation is necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl is required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 is dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, it was shown that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling (Mason, 2004).

Spatial regulation of EGFR signaling by Sprouty2

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates multiple signal-transduction pathways as well as trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Although there is growing awareness that endocytic transport can play a direct role in signal specification, relatively little is known about the molecular mechanisms underlying this link. This study shows that human Sprouty 2 (hSpry2), a protein that has been implicated in the negative regulation of receptor tyrosine kinase (RTK) signaling, interferes with the trafficking of activated EGFR specifically at the step of progression from early to late endosomes. This effect is mediated by the binding of hSpry2 to the endocytic regulatory protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and leads to a block in intracellular signal propagation. These observations suggest that EGFR signaling is controlled by a novel mechanism involving trafficking-dependent alterations in receptor compartmentalization (Kim, 2007).

Sprouty homologs and mesoderm development

Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. Two genes for each family have been identified in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments it is shown that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca²⁺ and PKCδ signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca²⁺ or PKCδ signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation (Sivak, 2005).

Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis

Somatic activation of Ras occurs frequently in human cancers, including one-third of lung cancers. Activating Ras mutations also occur in the germline, leading to complex developmental syndromes. The precise mechanism by which Ras activation results in human disease is uncertain. This study describes the phenotype of a mouse engineered to harbor a germline oncogenic K-ras^G12D mutation. This mouse exhibits early embryonic lethality due to a placental trophoblast defect. Reconstitution with a wild-type placenta rescues the early lethality, but mutant embryos still succumb to cardiovascular and hematopoietic defects. In addition, mutant embryos demonstrate a profound defect in lung branching morphogenesis associated with striking up-regulation of the Ras/mitogen-activated protein kinase (MAPK) antagonist Sprouty-2 and abnormal localization of MAPK activity within the lung epithelium. This defect can be significantly suppressed by lentiviral short hairpin RNA (shRNA)-mediated knockdown of Sprouty-2 in vivo. Furthermore, in the context of K-ras^G12D-mediated lung tumorigenesis, Sprouty-2 is also up-regulated and functions as a tumor suppressor to limit tumor number and overall tumor burden. These findings indicate that in the lung, Sprouty-2 plays a critical role in the regulation of oncogenic K-ras, and implicate counter-regulatory mechanisms in the pathogenesis of Ras-based disease (Shaw, 2007).

Sprouty genes prevent excessive FGF signalling in multiple cell types throughout development of the cerebellum

Fibroblast growth factors (FGFs) and regulators of the FGF signalling pathway are expressed in several cell types within the cerebellum throughout its development. Although much is known about the function of this pathway during the establishment of the cerebellar territory during early embryogenesis, the role of this pathway during later developmental stages is still poorly understood. This study investigated the function of sprouty genes (Spry1, Spry2 and Spry4), which encode feedback antagonists of FGF signalling, during cerebellar development in the mouse. Simultaneous deletion of more than one of these genes resulted in a number of defects, including mediolateral expansion of the cerebellar vermis, reduced thickness of the granule cell layer and abnormal foliation. Analysis of cerebellar development revealed that the anterior cerebellar neuroepithelium in the early embryonic cerebellum was expanded and that granule cell proliferation during late embryogenesis and early postnatal development was reduced. The granule cell proliferation deficit correlated with reduced sonic hedgehog (SHH) expression and signalling. A reduction in Fgfr1 dosage during development rescued these defects, confirming that the abnormalities are due to excess FGF signalling. These data indicate that sprouty acts both cell autonomously in granule cell precursors and non-cell autonomously to regulate granule cell number. Taken together, these data demonstrate that FGF signalling levels have to be tightly controlled throughout cerebellar development in order to maintain the normal development of multiple cell types (Yu, 2011).

Sprouty1 and Sprouty2 limit both the size of the otic placode and hindbrain Wnt8a by antagonizing FGF signaling

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. This study reports that in Spry1, Spry2 compound mutant embryos (Spry1^-/-; Spry2^-/- embryos), the otic placode is increased in size. The otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1^-/-; Spry2^-/- embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. Both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1^-/-; Spry2^-/- embryos by reducing the gene dosage of Fgf10. These results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center (Mahoney Rogers, 2011).

Coordinated activity of Spry1 and Spry2 is required for normal development of the external genitalia

Development of the mammalian external genitalia is controlled by a network of signaling molecules and transcription factors. Because FGF signaling plays a central role in this complicated morphogenetic process, this study investigated the role of Sprouty genes, which are important intracellular modulators of FGF signaling, during embryonic development of the external genitalia in mice. Sprouty genes were found to be expressed by the urethral epithelium during embryogenesis, and they have a critical function during urethral canalization and fusion. Development of the genital tubercle (GT), the anlage of the prepuce and glans penis in males and glans clitoris in females, was severely affected in male embryos carrying null alleles of both Spry1 and Spry2. In double mutant embryos, the internal tubular urethra was absent, and urothelial morphology and organization was abnormal. These effects were due, in part, to elevated levels of epithelial cell proliferation in mutant embryos. Despite changes in overall organization, terminal differentiation of the urothelium was not significantly affected. Characterization of the molecular pathways that regulate normal GT development confirmed that deletion of Sprouty genes leads to elevated FGF signaling, whereas levels of signaling in other cascades were largely preserved. Together, these results show that levels of FGF signaling must be tightly regulated during embryonic development of the external genitalia in mice, and that this regulation is mediated in part through the activity of Sprouty gene products (Ching, 2013).

REFERENCES

Search PubMed for articles about Drosophila sprouty

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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. PubMed Citation: 10704865

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

Ching, S. T., Cunha, G. R., Baskin, L. S., Basson, M. A. and Klein, O. D. (2014). Coordinated activity of Spry1 and Spry2 is required for normal development of the external genitalia. Dev Biol 386: 1-11. PubMed ID: 24361260

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