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

Gene name - sprouty

Synonyms -

Cytological map position - 63D1--63D2

Function - antagonist of FGF signaling

Keywords - trachea, FGF signaling

Symbol - sty

FlyBase ID: FBgn0014388

Genetic map position -

Classification - novel protein

Cellular location - intracellular (Casci, 1999)



NCBI links: Precomputed BLAST | Entrez Gene
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 RasGTP 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 RasGTP. 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 PointedP1, 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 RasGTP, 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).


GENE STRUCTURE

cDNA clone length - 3255 - longest of three cDNA classes

Bases in 5' UTR - 1323

Exons - 2

Bases in 3' UTR - 159


PROTEIN STRUCTURE

Amino Acids - 591

Structural Domains

sprouty encodes a novel cysteine-rich protein. Its most striking feature is a 124-residue cysteine-rich region (22 cysteines), which is flanked by N- and C-terminal cysteine-free regions that contain many stretches of repeated or alternating amino acids. There are eight potential N-linked glycosylation sites. There is a predicted signal peptide near the N terminus but no clear transmembrane domains, suggesting that Sty protein may be secreted (Hacohen, 1998).


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-rasG12D 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-rasG12D-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).


sprouty: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
date revised: 10 July 99

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