Src oncogene at 42A
DEVELOPMENTAL BIOLOGY

Embryonic

For assessment of Src42A distribution, ovaries and embryos were stained for Src42A and E-cadherin. As expected from RNA staining (Takahashi, 1996), Src42A signals appeared over the entire plasma membrane of all cells, but strong Src42A signals can often be found at sites of either cell-cell or cell-matrix adhesion (Takahashi, 2005).

At the start of oogenesis, the cystoblast undergoes four rounds of mitotic divisions with incomplete cytokinesis to generate 16 cystocytes interconnected via ring canals. Follicle cells subsequently separate off individual cysts to form egg chambers. Transient but very strong Src42A signals, not associated with strong E-cad signals, have been found in cystocytes in germarium region 2a/b. E-cad signals become evident at slightly later stages. In stage 1-7 egg chambers, relatively strong Src42A signals are apparent along the nurse/follicle cell boundary as noted for E-cad; Src42A signals on the basal follicle-cell surface are very weak. In the middle of oogenesis, relatively strong Src42A signals are evident in polar and invading border cells. Middle-stage ring-canals are marked by Src42A enclosed by weak E-cad. By stage 7, cytoplasmic Src42A becomes evident in oocytes. At stage 8, Src42A unassociated with E-cad starts being deposited on the oocyte surface and is conspicuous by stage 10b, at which time strong Src42A and E-cad signals can be seen in centripetal cells (Takahashi, 2005).

Embryogenesis starts with cleavage (stages 1-4), in which the nucleus undergoes 13 divisions and the nuclei thus produced become arranged in a single layer beneath the egg surface. Membranous Src42A signals are evident. During cellularization, not only Src42A but also E-cad signals are apparent on the surface of eggs and the membrane extending inwardly. The leading edges of invading membranes are always marked by Src42A but not E-cad. At stage 6, mesoderm generation starts by invagination. In stage 7 dorsal cells lying anterior to the cephalic furrow, Src42A distribution is virtually the same as at stage 5, but in more posterior dorsal cells involved in transient furrow formation or posterior midgut invagination, strong Src42A expression associated with strong E-cad signals were confirmed only in the apical region. Src42A unassociated with E-cad expression persists in invaginated mesodermal cells and are evident on the ectoderm/mesoderm interface at stage 9. Apical tips of mesectodermal cells, situated along the ventral midline, show strong E-cad and Src42A signals (Takahashi, 2005).

In late developmental stage embryos, there are strong signals of E-cad and Src42A in some tubular structures. In all cases, E-cad is present only in apical regions, while Src42A varies in location according to tube type. In hindguts covered with thin visceral mesodermal cells, Src42A signals are evident in both apical and basal regions, whereas Malpighian tubules protruding from hindguts, salivary glands and stomodeum opening, none of which having any mesoderm association, all display Src42A signals only in apical regions. Basal strong Src42A signals are eliminated when hindgut cells acquired Malpighian-tubule fate. Similarly, strong basal Src42A signals, separating the ectoderm from mesoderm at the basal clypeus cortex, varnish with stomodeum formation (Takahashi, 2005).

During stage 12, tracheal branches develop from invaginated tracheal pits. At stage 13, the dorsal trunk anterior has fused with the dorsal trunk posterior of the anterior neighbor to form a long tubular structure. Src42A co-localizes with juxtaposition E-cad signals. Strong Src42A signals unassociated with E-cad are present in tendon cells to which the muscle system is attached. Dorsal closure is a major morphogenic process in which two epithelial sheets converge to enclose the embryo. At the leading edge, moderate Src42A signals colocalize with strong E-cad (Takahashi, 2005).

Strong Src42A signals are evident in the CNS. Longitudinal connectives and commissures stain strongly with anti-Src42A antibody. E-cad signals can be seen only in midline glial cells, mesectodermal derivatives. In the CNS, nervous system-specific N-cadherin appears co-expressed with Src42A (Iwai, 1997). Strong Src42A signals are present in the brain and the axon linking the larval eye (Bolwig's organ) to the optic lobe. Strong Src42A signals, occasionally associated with E-cad signals, are seen in the gonad (Jenkins, 2003; Takahashi, 2005).

Differing Src signaling levels have distinct outcomes in Drosophila

High levels of Src activity are found in a broad spectrum of cancers. The roles of Src and its negative regulator Csk have been extensively studied, although results have often proved contradictory or the relevance to whole organisms is unclear. In Drosophila, overexpression of either Src orthologue resulted in apoptotic cell death, but paradoxically, reducing dCsk activity led to over-proliferation and tissue overgrowth. This study showed that in Drosophila epithelia in situ, the levels of Src signaling determine the cellular outcome of Src activation. Apoptotic cell death was triggered specifically at high Src signaling levels; lower levels directed antiapoptotic signals while promoting proliferation. Furthermore, the data indicate that expression of kinase-dead Src isoforms do not necessarily act as dominant-negative factors, but can instead increase Src pathway activity, most likely by titrating Csk activity away from endogenous Src. The importance of Src activity levels was emphasized when oncogenic cooperation between Src and Ras was examined: malignant overgrowth was observed specifically when high Src signaling levels were achieved. A model is proposed in which low levels of Src signaling promote survival and proliferation during early stages of tumorigenesis, whereas strong Src signaling, coupled with antiapoptotic signals, directs invasive migration and metastasis during advanced tumor stages (Vidal, 2007).

This study provides evidence that different levels of Src signaling lead to different biological outcomes. It is speculated that Src activity plays two important but separable roles during tumor maturation: early low levels of Src contribute to tumor overgrowth, whereas later high levels of Src, coupled with other oncogenes such as Ras, lead to invasive migration. Previous work emphasized the importance of Csk/Src-dependent signals present at tumor boundaries that can provoke metastatic-like behavior. In addition, the current data reconcile the contrasting phenotypes observed between partial reduction of Csk activity versus a strong increase in Src activity by demonstrating the importance of low versus high levels of Src pathway activation, respectively (Vidal, 2007).

It was also considered what precisely is being modeled with the use of altered Src isoforms, a common approach in the study of signaling pathways including Src. Unexpectedly, it was observed that kinase-dead versions of the two Drosophila SFKs did not behave as expected for dominant-negative isoforms. Furthermore, ectopic expression of dSrc42A, including as a kinase-dead isoform, led to the activation of its sole paralogue, dSrc64B. These data suggest an alternative explanation to paradoxical and controversial observations reported for vertebrate Src. The primary phenotype of src−/− knock-out mice, osteopetrosis caused by defective osteoclasts, was rescued by introducing a kinase-dead Src isoform (SrcKD). In addition, SrcKD rescued the reduced levels of phosphorylated tyrosine observed in src−/− osteoclasts. This led to the suggestion that the essential activities of Src are mediated exclusively through protein-protein interactions and not through kinase activity. The work suggests an alternative model in which endogenous SFKs are ectopically activated in SrcKD-rescued osteoclasts through the titration of Csk activity; this ectopic activation can then compensate for the loss of any individual SFK. In this view, the ability of SrcKD to rescue Src still invokes kinase activity, albeit indirectly (Vidal, 2007).

The results indicate that high levels of Src signaling can show oncogenic cooperation with Ras to direct tumoral growth and organismal lethality. Similarly to Ras/Scrib, Ras/dCsk cells displayed overgrowth, loss of epithelial polarity, migration, and invasion into the ECM. The data indicate that, although cells did not metastasize into distant tissues, they were capable of invading nearby tissues within the cephalic complex such as the brain. In mammals, the ability to invade a local blood or lymph vessel could provide a malignant cell the ability to reach and colonize most tissues of the organism. Therefore, current Drosophila models could model some (albeit not all) fundamental aspects of the metastatic process (Vidal, 2007).

Increasing levels of Src activation correlate with advancing tumorigenic stages in a variety of human cancers, including lung, breast, pancreatic, ovarian, and colorectal cancer. Strikingly, this study observed a similar correlation with Src-driven oncogenesis in Drosophila: high (but not low) levels of Src signaling cooperated with oncogenic Ras to produce invasive overgrowth and organismal lethality. One question in the cancer field is whether ectopic expression studies truly model aspects of human tumors. The current results indicate that they can; however, attention must be paid to levels of activity and the specific aspects of tumorigenesis that each level emulates. Emerging models for tumorigenesis should consider not only the activation of multiple and specific pathways, but should also calibrate these levels to explore, e.g., benign overgrowth versus metastasis versus cell death (Vidal, 2007).

Although Src activity can affect cell proliferation and survival, a growing consensus in the field is that a major role for Src is to promote metastasis by stimulating migratory behavior in transformed cells. This study suggests that the proliferative and survival signals from low Src activation contributes to tumor growth in early stages. In line with this, a recent study shows that mice with keratinocyte-restricted deletion of Csk have mild SFK activation and develop epidermal hyperplasia, but without malignant transformation (Vidal, 2007).

In advanced stages, increased Src signaling, together with the acquisition of strong antiapoptotic signals that protect cells from Src-induced apoptosis such as oncogenic Ras, may reveal the ability of Src activation to drive invasive migration and, potentially, metastasis. For example, it is noted that pancreatic ductal adenocarcinoma involves the invasion of exocrine cells through intrapancreatic nerves, leading to severe damage and pain, promoting cancer spread, and precluding resection. Src, expressed at high levels in the majority of these tumors, is likely required for tumor progression, and cell culture studies suggest that invasion requires Ras signaling for pancreatic tumor targeting. Finally, Src-specific kinase inhibitors have entered clinical trials. The current results with dominant-negative Src isoforms suggest caution, however, as the potential of a Src:drug complex to titrate Csk/Chk activity may yield unpredictable results in the context of Src-overexpressing tumors (Vidal, 2007).

Effects of Mutation or Deletion

Since Src possesses multiple functional domains, the isolation of protein-null mutants may be required to clarify the roles of Src in development. Short Src42A deletion mutants were generated through imprecise P-element excision of Src42Ak10108 (enhancer trap line) and a protein-null lethal mutant, Src42A26-1, was identified using anti-Src42A antibody. In Src42A26-1, a 1.9 kb region containing the putative TATA box, RNA start, the first exon of Src42A and the entire P-lacZ sequence were deleted. Src42A26-1 embryos showed mild dorsal closure defects. Close inspection of mutant embryos stained for Engrailed revealed occasional segmental misalignment. Lethality and morphological defects in Src42A26-1 were eliminated by introducing the wild-type Src42A transgene driven by arm-GAL4 (Takahashi, 2005).

Using Src64P1 and Src42AE1, Src42A and Src64 have been shown to be functionally redundant to each other with respect to the dorsal closure (Tateno, 2000). Using a newly isolated protein-null Src42A mutant, it has been demonstrated that these two Src genes are functionally redundant not only in dorsal closure but in many other development contexts as well (Takahashi, 2005).

The dorsal open phenotype associated with head involution defects was exhibited by 34% of Src42A26-1;Src64P1/+ embryos. Src42A26-1;Src64P1 embryos show much severer phenotypes with no apparent germ band retraction. No defects could be found in Src64P1. CNS morphology is extensively affected by the simultaneous elimination of Src42A and Src64 activity. In Src double mutant embryos, longitudinal tracts and commissures are frequently broken without significant loss of Elav-positive neuronal cells. In Src double mutants, optic lobe/Bolwig's organ and trachea formation is significantly disrupted, while no apparent defect is detected in Src42A26-1 (Takahashi, 2005).

Normal nurse cell formation requires maternal Src64 activity (Dodson, 1998). However, no nurse cell fusion occurs in ovaries doubly heterozygous for Src42A26-1 and Src64P1 and there is hardly any enhancement of Src64 nurse cell phenotypes such as nurse cell fusion and ring-canal defects with elimination of one copy of Src42A. Src42A thus plays only a minor, if any, role in ovary development (Takahashi, 2005).

Src may thus be considered to exercise central roles in many normal developmental processes of oogenesis and embryogenesis. Src42A and Src64 contribution to total Src activity would depends on particular aspects of development (Takahashi, 2005).

To identify genes that may interact with Src42A genetically, a search was made for fly mutants that enhance the eye phenotype induced by misexpression of the dominant-negative form of Src42A (Src42A[KR]) (Takahashi, 1996). As previously noted, eyes of flies heterozygous for a P[Src42A[KR]] insertion are almost entirely normal. Seven putative enhancer lines were obtained and E(7A-1), a line with the strongest enhancing activity, was selected for subsequent experiments (Takahashi, 2005).

Flies heterozygous for E(7A-1) are viable and not associated with any apparent morphological eye defects, whereas E(7A-1) homozygotes are embryonic lethal. Complementation tests indicate the E(7A-1) lethal lesion is present in 57B5-14 on the second chromosome, which contains shotgun (shg), a gene encoding E-cad. shgR64a (a null allele) fails to complement E(7A-1). As with E(7A-1), shgR64a enhances the eye phenotype of flies heterozygous for P[Src42A[KR]] insertion. Virtually no E-cad signals could be found in E(7A-1) homozygous stage 13 embryos. Thus, it is concluded that E(7A-1) harbors a lethal mutation in shg (shgE(7A-1)) and that shg activity reduction enhances Src42A eye phenotypes (Takahashi, 2005).

Subsequent experiments indicate shg is also capable of enhancing Src42A mutant phenotypes in various developmental contexts, other than eye morphogenesis. shgR6 and Src42A6-1 are hypomorphic alleles of shg and Src42A, respectively and dorsal closure of either Src42A6-1 or shgR6 embryos appears essentially normal. But most shgR6; Src42A6-1 embryos are associated with the dorsal open phenotype, indicating that shg-Src42A interactions are required for normal dorsal closure (Takahashi, 2005).

Src42A-shg interactions may also be involved in normal thorax closure in pupal stages. Defects are apparent in thorax closure in escapers and pharate adults of Src42A6-1. Similar defects have been reported for Src42Ajp45 and classified into three classes based on severity (Tateno, 2000). Src42A6-1 notum phenotypes are found considerably enhanced in the genetic background of shgg317/+. Two thirds of class 1 were converted to severer classes, while a fraction of class 3 was doubled. In some double mutant flies, right and left halves of the notum appeared completely separated from each other (class 4) (Takahashi, 2005).

E-cad regulates cell-cell adhesion via homophilic association. Arm interacts directly with the cytoplasmic domain of E-cad and alpha-catenin. The latter is thought to associate with the actin network. Strong hypomorphic alleles of arm have defects in the dorsal closure, so attempts were made to determine whether Src42A interacts genetically with arm in dorsal closure and eye morphogenesis. As with embryos homozygous for Src42A6-1, virtually all embryos heterozygous for armYD35 (null allele) and those homozygous for armH8.6 (hypomorph) are normal in dorsal closure. By contrast, most Src42A26-1 embryos heterozygous for armYD35 are associated with the dorsal open phenotype. armH8.6/+ eyes are normal in appearance, but Src42A[KR]/armH8.6 flies possess rough eyes, as also noted for Src42A[KR]/shg. It thus follows that arm-Src interactions are essential for normal dorsal closure and eye morphogenesis (Takahashi, 2005).

During early-mid stages of the dorsal closure, dorsal-most epidermal cells and epidermal cells located more ventrally elongate along the dorsoventral axis and F-actin thickly accumulates at the leading edge. In the zippering stage, actin-based processes are essential for zippering epithelial sheets together. Dorsal-most epidermal cell elongation is associated with the redistribution of many proteins such as those involved in planar polarity and cytoskeleton. Genetic experiments show interactions between Src, shg and arm to be involved in the dorsal closure and thus examination was made of temporal change in the locations of E-cad, Arm, Fas3 and F-actin during dorsal closure in Src and shg mutants as well as wild type (Takahashi, 2005).

In wild type, not only F-actin but also E-cad and Arm signals increase at the leading edge from 9 hours after egg laying. Polarized Fas3 expression and tubulin bundling occur with dorsoventral elongation of dorsal epidermal cells. No germ band retraction occurs in Src42A26-1;Src64P1 embryos and so examination was made of the effects of reduction in Src-activity on protein distribution at the leading edge of Src42A26-1;Src64P1/+ and Src42A26-1 embryos. In Src42A26-1;Src64P1/+ embryos, dorsal-most epidermal cell elongation and polarized deposition of Fas3 and tubulin bundling appear to proceed normally. But, unlike wild-type embryos, Src42A26-1;Src64P1/+ embryos exhibit significant reduction in E-cad and F-actin deposition at the leading edge at 11-12 hours AEL. Arm signals appear reduced throughout the entire membrane region, including the leading edge and accumulate in the cytoplasm. The leading edge of Src42A26-1;Src64P1/+ embryos, initially smooth in appearance, frequently kinked with partial dorsal-most epidermal cell deformation from 10 hours AEL onwards. Kinking of the actin cable at the zipper front is thought most likely due to lamellae traction, and, accordingly, Src activity reduction in Src42A26-1;Src64P1/+ embryos may possibly give rise to defects in the cytoskeletal machinery that are essential for driving the dorsal closure. In Src42A26-1 single mutant embryos, dorsal-most epidermal cell elongation and the leading-edge structure appears virtually normal but morphological defects could sometimes be seen along the zippered midline. F-actin signals are also significantly reduced in shgR64a mutant embryos (Takahashi, 2005).

It has been shown that there is no expression of puckered (puc) or decapentaplegic (dpp), positively regulated by JNK signaling at the leading edge on Tec29 Src42A double mutants, suggesting that Src42A may act upstream of JNK signaling (Tateno, 2000). Puc is a negative regulator of JNK signaling and the absence of puc activity causes ventral expansion of the area of dpp expression normally restricted to DME cells. Examination was thus made of dpp expression in Src mutants. dpp expression was monitored using nuclear dpp-lacZ signals. Unlike Tec29 Src42A mutants, ventrally expanded dpp expression is found in all Src mutants, indicating that JNK signaling is not completely suppressed in Src42A26-1;Src64P1/+ and Src42A26-1 mutant embryos (Takahashi, 2005).

In epithelial cells, membranous Src42A is colocalized with E-cad and, consequently, shg activity may be required for proper plasma membrane localization of Src42A. To confirm this point, shgR69 clones were generated in pupal wing discs and Src42A localization was examined for any change by anti-Src42A antibody staining. Membranous Src42A signals in shg mutant clones were found to be reduced significantly in a cell-autonomous fashion, indicating that shg is essential for proper plasma membrane localization of a certain region of Src42A (Takahashi, 2005).

Taken together, these results indicate that the leading-edge adherens junction containing E-cad, Arm and actin may serve as a cytoskeletal and/or regulatory machinery for properly driving the dorsal closure, and that interactions between Src42A, shg and arm would be essential for membrane localization of their own protein products (Takahashi, 2005).

To further clarify Src function, activated (Src42A[YF]), dominant-negative (Src42A[KR]) and wild-type (Src42A[WT]) forms of Src42A were driven by pnr-GAL4 to determine any change in Arm, E-cad or Src42A signals. Immunostaining of embryos collected at 10-14 hours AEL showed that, as with wild type, nearly all Arm and E-cad signals localize in the plasma membrane when kinase-inactive Src42A[KR] is driven, while considerable cytoplasmic E-cad and Arm signals are evident in cells overexpressing Src42A[WT] or [YF]. It may thus follow that activated Src stimulates cytosolic Arm stabilization and/or arm expression. Alternatively, Src42A may be involved in regulating possible cadherin endocytosis (Takahashi, 2005).

Overexpression of activated Src42A may also cause change in cell morphology. Forced expression of UAS-Src42A[WT] and [YF] prevents dorsal epithelial cells from elongating normally. Occasionally, cells that strongly expressed Src42A and were separated from the amnioserosa or dorsal epidermis plane could be found in embryos transformed with Src42A[YF]. These cells were frequently associated with the expression of Clawless (see Kojima, 2005) or Fas3, which are maker proteins for amnioserosa and epidermis, respectively, but not with TUNEL signals at least up to the end of stage 13, suggesting that they are live cells dissociated from amnioserosa or dorsal epidermis because of elevated Src activity. Most released cells degenerated at stage 16 via apoptosis. It is concluded that Src42A is essential for proper cell migration and cell-shape regulation (Takahashi, 2005).

In Drosophila, Src64 was considered a unique ortholog of the vertebrate c-src; however, more recent evidence has been shown to the contrary. The closest relative of vertebrate c-src found to date in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time in this paper. In contrast to Src64, overexpression of wild-type Src41 causes little or no appreciable phenotypic change in Drosophila. Both gain-of-function and dominant-negative mutations of Src41 cause the formation of supernumerary R7-type neurons, suppressible by one-dose reduction of boss, sevenless, Ras1, or other genes involved in the Sev pathway. Dominant-negative mutant phenotypes are suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type Src41. The colocalization of Src41 protein, actin fibers and DE-cadherin, as well as the Src41-dependent disorganization of actin fibers and putative adherens junctions in precluster cells, suggest that Src41 may be involved in the regulation of cytoskeleton organization and cell-cell contacts in developing ommatidia (Takahashi, 1996).

Drosophila Src42A is a negative regulator of RTK signaling

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. An extragenic suppressor of Raf, Su(Raf)1, has been isolated that encodes a Drosophila Src family gene (Src42A) identical to the previously cloned DSrc41. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Src42A negatively regulates Egfr signaling during oogenesis and negatively regulates receptor tyrosine kinase signaling in the eye. For example Src42A suppresses the rough eye phenotype caused by expression of hyperactive Ras or Raf. Src42A mutation also leads to defects in head and tail morphology, tracheal development and wing morphogenesis. This study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed (Lu, 1999).

The functional status of Ras, Raf, Mek, or Mapk proteins does not appear to alter the ability of wild-type Src42A to repress receptor tyrosine kinase signaling: this would favor a model in which Src42A defines a branch pathway parallel to the main Ras/Mapk cascade with an integration point downstream of Mapk. This model is consistent with the reduction of maternal Src42A activity, which can still enhance Torso receptor tyrosine kinase signaling in the absence of Ras1 protein. The manner in which Src42A acts to modulate receptor tyrosine kinase signaling is similar in two ways to another branch pathway component, Kinase suppress of Ras-1 (ksr-1: see Drosophila Kinase suppressor of ras), of C. elegans. (1) Src42A does not appear to dramatically alter RTK-mediated processes when mutated alone. For example, mitotic clones of Src42A mutant cells in the eye do not produce extra photoreceptor cells. (2) The negative role of Src42A is only revealed when the Ras/Mapk cascade is compromised or hyperactived. Recently, Therrien (1998) reported the isolation of Src42A as a suppressor of a dominant negative form of fly ksr in the eye. In attempts to understand more about Src42A, a genetic screen was performed that isolated loss-of-function mutation in Egfr, rolled, and a new gene, semang, as suppressors of Src42A mutants (Zhang, 1999). It is suggested that Src42A works together with other branch pathway modulators such as Ksr to regulate signal transduction downstream of Egfr and other RTKs. If each branch pathway modulator takes over only a part of the total regulatory power, it would explain why Src42A or ksr-1 shows mild phenotypes when mutated alone. However, the phenotypes of Src42A do not overlap with two other Drosophila Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. It is tempting to speculate that Src42A is activated following the activation of Egfr RTK via a Ras-1 independent mechanism. The signal from the activated Src42A would then integrate, in a negative fashion, with that from the main Ras/MAPK pathway to determine the final readout of an RTK pathway (Lu, 1999).

A genetic screen for modifiers of a Kinase suppressor of ras-dependent rough eye phenotype reveals genetic interactions with Src42A

kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of Ksr is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of Ksr remains unresolved. To further elucidate the role of Ksr and to identify proteins that may be required for Ksr function, a dominant modifier screen was conducted in Drosophila based on a Ksr-dependent phenotype. Overexpression of the Ksr kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with Ksr might modify the Ksr-dependent phenotype. Approximately 185,000 mutagenized progeny were screened for dominant modifiers of the Ksr-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for Ksr in Ras1 signaling. Mutations in genes encoding known components of the Ras pathway were isolated in a screen for the 14-3-3epsilon, Dsor1/mek, rolled/mapk, pointed, yan, and ksr loci. Furthermore, due to the ability of dominant-negative KSR (KDN) to block RAS/MAPK-mediated signaling, mutations in genes expected to function upstream of ksr were also isolated. These included mutations in the Egfr, Star, Sos, and Ras1 loci. In addition, 4 additional complementation groups were identfied. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor (Therrien, 2000).

Complementation test results reveal that SK2-4 is allelic to Src42A, the closest Drosophila homolog of the Src kinase family. Intriguingly, genetic data (suppression of sE-KDN, enhancement of sev-Ras1V12, and suppression of RafHM7) suggest an inhibitory role for Src42A in Ras1 signaling, which is the opposite of the described function of Src-like kinases in vertebrates. During the course of this work, Src42A was also identified by another laboratory as being a dominant suppressor of a hypomorphic allele of Raf (RafC110). Genetic and molecular characterization of different Src42A alleles clearly supports an inhibitory function for Src42A in different RTK-dependent signaling pathways. Therefore, the further elucidation of the molecular function of Src42A in Drosophila may unveil a novel mechanism of action for this family of nonreceptor tyrosine kinases (Therrien, 2000).

Regulation of JNK by Src during Drosophila development

In Drosophila, the Jun amino-terminal kinase (JNK) homolog Basket (Bsk) is required for epidermal closure. Mutants for Src42A, a Drosophila c-src protooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depends on the amount of Bsk activity, and the amount of Bsk activity depends on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrate a physiological link between Src and JNK (Tateno, 2000).

Genes that regulate cell shape changes in Drosophila are required for dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis. Mutations in genes such as hemipterous (hep) and basket (bsk, also known as DJNK) result in abnormal embryos with a dorsal hole or abnormal adults with a dorsal midline cleft. Hep and Bsk are homologous to the mammalian MKK7 (MAPK kinase 7) and JNK, and they are components of a MAPK (mitogen-activated protein kinase) cascade. Although the role of the Hep-Bsk cascade during dorsal closure has been extensively studied, the upstream trigger of this cascade is poorly understood. To identify the trigger, a screen was carried out for mutants showing the dorsal midline cleft phenotype, like a mild hep mutant. The mutant for Src42A shows this phenotype and Src42A regulates Bsk during Drosophila development (Tateno, 2000).

Furthermore, the Tec29 Src42A double mutant shows complete embryonic lethality, and a certain fraction of the dead embryos show the dorsal open phenotype. Activated DJun, a transcription factor downstream of Bsk, partially rescues the dorsal open phenotype in the Tec29 Src42A double mutant. Thus, Src42A appears to regulate Bsk in the fusion of epithelial sheets during embryogenesis and metamorphosis, and Tec29 is involved in this regulation. The double mutant for Src64 and Src42A manifests a mild but clear dorsal open phenotype, which suggests a functional redundancy between Src64 and Src42A (Tateno, 2000).

Expression of puc is known to be induced by the Bsk signal. In the wing disc of the wild-type third-instar larva, puc is expressed in the dorsal midline of the adult notum. In the wing disc of the Src42AJp45 mutant, puc expression is reduced. In contrast, larvae with a constitutively activated form of Src42A (Src42ACA) shows ectopic expression of puc. Further, introduction of a hep null mutation reduces the amount of ectopic puc expression. It is known that Bsk induces expression of puc and decapentaplegic (dpp) during embryonic dorsal closure. The embryos of the Tec29 Src42A double mutant do not show any puc or dpp expression in the leading edge cells. These results indicate that Src42A, Tec29, Hep, and Bsk regulate dpp and puc expression during embryonic dorsal closure (Tateno, 2000).

During embryonic dorsal closure, the Hep-Bsk signal is required for elongation of the leading edge cells. In the absence of the Bsk signal, these cells do not fully elongate. The accumulation of F-actin and phosphotyrosine (P-Tyr) in leading edge cells is associated with the elongation of these cells. Accumulation of these substances is disturbed in the DJun and the puc mutants. In the double mutant for Tec29 and Src42A, the leading edge cells contain reduced quantities of F-actin and P-Tyr, and these cells are only partially elongated. Thus, the defect in embryonic dorsal closure in the Tec29 Src42A double mutant is caused by this failure in cell shape change, as is the case in the DJun mutant (Tateno, 2000).

A model is proposed in which Src42A, upon receiving an unidentified signal, activates the Hep-Bsk pathway to regulate cell shape change and epidermal layer movement. This is consistent with the observation in mammals that c-Src regulates the cell morphogenetic and migratory processes and is known to activate JNK. As in Drosophila, c-Src definitely affects F-actin organization and P-Tyr localization during cell morphogenesis. Therefore, Src regulation of JNK activity toward a change in cell shape may be conserved (Tateno, 2000).

It can be also interpreted that Src42A acts upstream of DFos, a dimerization partner of DJun. Although the Src42A, Tec29, and Src64 single mutants do not show a dorsal open phenotype, the DFos mutant clearly exhibits it. This relationship is also analogous to that in mammals. Both c-src and c-fos knockout mice have a similar defect, osteopetrosis caused by reduced osteoclast function. But the phenotypic severity is milder in c-src than in c-fos knockouts; this can be explained by the functional overlap in multiple Src-family tyrosine kinases. Accordingly, in both Drosophila and mammals, multiple nonreceptor tyrosine kinases may cooperate to regulate the function of the Jun/Fos complex (Tateno, 2000).

Drosophila Src-family kinases function with Csk to regulate cell proliferation and apoptosis

Elevated Src protein levels and activity are associated with the development and progression of a variety of cancers. The consequences of deregulated Src activity have been studied extensively in cell culture; however, the effects of this deregulation in vivo, as well as the mechanisms of Src-induced tumorigenesis, remain poorly understood. In this study, the effect of expressing wild-type and constitutively active Drosophila Src-family kinases (SFKs) in the developing eye was examined. Overexpression of either wild-type Drosophila SFK (Src64 and Src42) is sufficient to induce ectopic proliferation in G1/G0-arrested, uncommitted cells in eye imaginal discs. In addition, both kinases trigger apoptosis in vivo, in a dosage-dependent manner. Constitutively active mutants are hypermorphic; they trigger proliferation and death more potently than their wild-type counterparts. Moreover, SFK-induced proliferation and apoptosis are largely independent events, since blocking ectopic proliferation does not block cell death. Further, Csk (the Drosophila C-terminal Src kinase) phosphorylates and interacts genetically with the wild-type SFKs, but not with the constitutively active mutants in which a conserved C-terminal tyrosine was mutated to phenylalanine, providing the first in vivo evidence that Csk regulates SFKs during development through phosphorylation of their C-terminal tyrosine (Pedraza, 2004).

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. The Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk–/– overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. The data provide in vivo evidence that Src activity requires JNK and STAT function (Read, 2004).

Partial reduction of Src64B, Src42A, or Btk29A activity suppresses the dCsk–/– phenotype, providing functional data to support the view that the imaginal disc overgrowth, defective larval and pupal development, and lethality of dCsk–/– mutants results from inappropriate activation of the Src-Btk signal transduction pathways. Mutations in Btk29A more strongly suppress dCsk phenotypes than either Src42A or Src64B mutations, perhaps reflecting that (1) Src paralogs act redundantly to each other in Drosophila as in mammals and (2) Btk29A has been shown to act downstream of Src family kinases (SFK) in flies and in mammals. In vivo evidence is provided that loss of Csk function hyperactivates Btk to drive cell cycle entry in development, demonstrating that Tec-Btk family kinases are critical to SFK-mediated proliferation. The data raise the possibility that partial reduction of Tec-Btk kinase activity could reduce proliferation in other cellular contexts in which overgrowth is driven by hyperactivated SFKs, such as in colon tumors (Read, 2004).

Tissue culture models show that constitutively activated SFK signal transduction modulates the function of numerous downstream effector molecules and pathways. Using a loss-of-function approach to identify effectors that mediate the dCsk overgrowth phenotypes, some of these pathways were not implicated in dCsk function. For example, SFKs up-regulate the SOS-Ras-ERK pathway in multiple tissue culture studies and Drosophila overexpression models. However, although dRas1 signaling is active throughout retinal development, reduced dEGFR, Sos, and Jra (c-jun) gene dosage failed to affect the dCsk phenotype. dCsk mutations also failed to modify a hypermorphic allele of dEGFR. Levels of doubly phosphorylated and activated ERK appeared unaltered in dCsk–/– tissue. Moreover, the dCsk phenotype failed to phenocopy defects caused by Ras pathway hyperactivation. For example, constitutively active dRas1 causes increased cell size and patterning defects in the developing imaginal discs, defects that were not observed in dCsk mutant eye tissues. These data argue that not every signal transduction pathway implicated in SFK tissue culture models necessarily functions as predicted within a developing epithelial tissue (Read, 2004).

These studies emphasize the importance of two signaling pathways in dCsk and SFK function. Since certain defects in dCsk–/– animals, such as a split notum, resembled those of hep (JNKK) mutants, it is suspected that JNK pathway activity is involved in dCsk function. Phenotypic and FACS analysis established that reduced JNK (bsk) function suppresses the phenotypes and cell cycle defects caused by loss of dCsk. These results confirm studies indicating that JNK functions downstream of the Src-Btk pathway in Drosophila and mammalian tissue culture cells. Components of the JNK pathway are required for Src-dependent cellular transformation, but the exact role of JNK in these cells is unknown. Importantly, the data show that the JNK pathway mediates proliferative responses to Src signaling in vivo. Further work will be needed to precisely understand its role in proliferation (Read, 2004).

Genetic studies also highlight the importance of the Jak/Stat signal transduction pathway. dCsk proves a negative regulator of Jak/Stat signaling; for example, dCsk mutant tissues show up-regulation of Stat92E protein, a hallmark of Jak/Stat activation in Drosophila. Stat92E, the sole Drosophila STAT ortholog, is most similar to mammalian STAT3. In mammalian cells, Src directly phosphorylates and activates STAT3 and STAT3 function and activation are required for Src transforming activity. Conversely, overexpression of Csk blocks STAT3 activation in v-Src transformed fibroblasts. Activating mutations in STAT3 can also promote oncogenesis in mice. However, the physiological significance of these interactions within developing epithelia remains unclear (Read, 2004).

dCsk; Stat92E double mutant clones reveal that blockade of STAT function in dCsk mutants severely reduces Src-dependent overgrowth and promoted apoptosis of mutant tissue. dCsk–/–; Stat92E–/– EGUF adult eyes (the EGUF method produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation) are nearly identical to phenotypes caused by overexpression of Dacapo, the fly ortholog of the cdk inhibitor p21, and PTEN, a negative regulator of cell proliferation and growth. Importantly, removing Stat92E function in dCsk mutant tissue led to a synthetic small eye phenotype and did not simply rescue the dCsk–/– proliferative phenotype. This outcome distinguishes Stat92E from mutations in Src64B, Btk29A, or bsk, which rescue dCsk-mediated defects toward a normal phenotype. The loss of tissue in dCsk–/–; Stat92E–/– clones indicates that Src-Btk signaling provokes apoptosis in the absence of Stat92E function. Consistent with this interpretation, reduced Btk29A function rescued the dCsk–/–; Stat92E–/– EGUF phenotype to a more normal phenotype, demonstrating that the reduced growth and increased apoptosis observed in the dCsk–/–; Stat92E–/– tissues is indeed Src-Btk pathway dependent (Read, 2004).

The data suggest the existence of a Src-dependent proapoptotic pathway that is normally suppressed by STAT. One possible component of this pathway is JNK, given that JNK signaling is an important activator of apoptosis in both flies and mammals. Perhaps Src-dependent hyperactivation of Bsk (JNK) in dCsk–/–; Stat92E–/– tissue contributes to cell death in the absence of proliferative and/or survival signals provided by Stat92E. However, a number of other candidate pathways may also mediate this response. The further characterization and identification of these pathways may have important implications for interceding in Src-mediated oncogenesis (Read, 2004).

Together, these observations indicate that, in tissue that contains hyperactive Src or reduced Csk, blocking STAT function is sufficient to trigger apoptosis and decrease proliferation in the absence of any further mutations or interventions. Reduced STAT3 function can promote apoptosis within breast and prostate cancer cells that show elevated SFK activity, but the molecular pathways driving apoptosis in these cells are unknown. These cells may require survival signals provided by STAT3 to counteract apoptosis due to chromosomal abnormalities or other defects. Alternatively, these cells may die because of proapoptotic signals provided by hyperactive SFKs in the absence of STAT3 function. The data argue that the latter may be true, which suggests the intriguing possibility that therapeutic blockade of STAT function in tumors with activated Src may actively provoke Src-dependent apoptosis and growth arrest in tumor tissues (Read, 2004).

Drosophila Src42 binding activity regulates RAF by a novel CNK-dependent derepression mechanism

Connector enhancer of KSR (CNK), an essential component of Drosophila receptor tyrosine kinase/mitogen-activated protein kinase pathways, negatively regulates RAF function. This bimodal property depends on the N-terminal region of CNK, which integrates RAS activity to stimulate RAF and a bipartite element, called the RAF-inhibitory region (RIR), which binds and inhibits RAF catalytic activity. The repressive effect of the RIR is counteracted by the ability of Src42 to associate, in an RTK-dependent manner, with a conserved region located immediately C-terminal to the RIR. Strikingly, several cnk loss-of-function alleles have mutations clustered in this area and provide evidence that these mutations impair Src42 binding. Surprisingly, the derepressing effect of Src42 does not appear to involve its catalytic function, but critically depends on the ability of its SH3 and SH2 domains to associate with CNK. Together, these findings suggest that the integration of RTK-induced RAS and Src42 signals by CNK as a two-component input is essential for RAF activation in Drosophila (Laberge, 2005).

RTK-induced activation of the small GTPase RAS was recognized early on as a critical event in RAF activation. RAS triggers plasma membrane anchoring of RAF through a direct contact between GTP-loaded RAS and RAF. However, this step is insufficient to induce RAF activation, but is a prerequisite for a complex series of regulatory events. For example, Ste20-like kinases and Src family kinases (SFKs) have been shown to collaborate with RAS in RTK-induced Raf-1 activation, owing to their ability to directly phosphorylate Raf-1 serine 338 (S338) and tyrosine 341 (Y341), respectively. However, these particular events are probably specific to Raf-1 as the equivalent S338 residue in B-RAF is constitutively phosphorylated, whereas the Y341-like residue is not conserved in B-RAF or in Drosophila and C. elegans RAF. Nonetheless, it remains possible that these kinases use different means to regulate RAF members. This would be consistent with genetic findings in Drosophila, which suggest that RAF is also regulated by an RTK-induced but RAS-independent pathway linked to SFKs (Laberge, 2005).

In addition to kinases and phosphatases regulating RAF activity, a number of apparently nonenzymatic proteins also modulate RAF function. One of these is Connector eNhancer of KSR (CNK), an evolutionarily conserved multidomain-containing protein originally identified in a KSR-dependent genetic screen in Drosophila. Genetic experiments in flies have indicated that CNK activity is required downstream of RAS, but upstream of RAF, thus suggesting that CNK regulates RAF activity. In agreement with this interpretation, CNK was found to interact directly with the catalytic domain of RAF and to modulate its function. The role of CNK, however, is probably not restricted to the MAPK pathway. Indeed, although mammalian CNK proteins have also been found to modulate the RAS/MAPK pathway, recent studies have indicated that they also control other events, including membrane/cytoskeletal rearrangement, Rho-mediated SRF transcriptional activity and RASSF1A-induced cell death. Given their ability to influence distinct signaling events, it is possible that CNK proteins act as signal integrators to orchestrate crosstalks between pathways (Laberge, 2005).

Intriguingly, although CNK activity is vital for RAS/MAPK signaling in Drosophila, it has opposite effects on RAF function. A structure/function analysis revealed that two domains (SAM and CRIC) located in the N-terminal region of CNK are integrating RAS signals, enabling RAF to phosphorylate MEK. However, the ability of CNK to associate with the RAF catalytic domain was mapped to a short bipartite element, named the RAF-inhibitory region (RIR), that strongly antagonizes MEK phosphorylation by RAF. Surprisingly, the RIR exerts its effect even in the presence of RAS signals, hence resulting in lower RAS-induced MAPK signaling output (Laberge, 2005).

The inhibitory effect of the RIR is relieved by an RTK-induced SFK signal. Specifically, a region located immediately C-terminal to the RIR including tyrosine 1163 (Y1163) is essential for CNK's positive function in vivo and for Sevenless (Sev) RTK-dependent MAPK activation. Upon SEV expression, one of the two SFKs found in Drosophila, Src42, associates and mediates (through the Y1163 region of CNK) RTK positive effects on the MAPK module. Remarkably, cnk loss-of-function mutations affecting the Y1163 region are fully compensated by inactivation of the RIR, thereby arguing that the Y1163 region is integrating the RTK-induced Src42 signal to counteract the RIR inhibitory function. Unexpectedly, genetic and molecular evidence has revealed that it is not Src42 catalytic function per se, but rather its binding capacity that is the key event in this process. Taken together, these results provide compelling evidence that CNK regulates RAF function by integrating both RAS and Src42 signals elicited by an RTK (Laberge, 2005).

This study shows that CNK integrates RAS and Src42 signals as a binary input, thereby allowing RAF to send signals to MEK. The RAS signal is received by the SAM and CRIC domains of CNK, which appears to enhance RAF catalytic function, whereas Src42 activity is integrated by the Y1163 region of CNK and seems to relieve the inhibitory effect that the RIR imposes on RAF's ability to phosphorylate MEK. Why would RAF activation depend on two distinct but corequired signals emitted by the same RTK? One possibility is that this requirement generates specificity downstream of an RTK. For example, only receptors that activate both RAS and Src42 would lead to activation of the MAPK module within discretely localized CNK complexes. Consequently, the combinatorial use of multifunctional signals might be a means to produce a specific output from generic signals (Laberge, 2005).

Intriguingly, despite the fact that the second Drosophila SFK, Src64, is naturally expressed in S2 cells, it does not act like Src42 in response to Sev, Egfr and InsR activation. Although the reason for this difference is not immediately clear, it was found that, unlike Tec29, overexpression of an Src64YF variant is nonetheless capable of associating with CNK and inducing its tyrosine phosphorylation. It is thus possible that Src64 fulfills a similar role to Src42, but downstream of other RTKs or in response to other types of stimuli and that difference in either their subcellular localization, requirement for specific cofactors or additional regulatory events account for their distinct behavior (Laberge, 2005).

The mechanism by which the binding of Src42 to CNK deactivates the RIR is currently unknown and a number of scenarios can be envisioned. For example, it might induce a conformational change that suppresses the inhibitory effect that the RIR imposes on RAF catalytic activity. Alternatively, it is possible that Src42 binding displaces an inhibitory protein interacting with CNK or facilitates the relocalization of a CNK/RAF complex to a subcellular compartment that is required for RAS-dependent RAF activation. However, it is not believed that this mechanism involves displacing CNK away from RAF since neither Sev expression nor Src42 depletion alters the CNK/RAF interaction (Laberge, 2005).

Although several questions are left unanswered regarding the Src42/CNK association, collectively, the data suggest a subtle binding mode reminiscent of the mammalian Src/FAK interaction. Indeed, it appears that CNK is phosphorylated on the Y1163 residue not by Src42 itself, but either by the receptor or by another kinase and that this step generates a high-affinity binding site for the SH2 domain of Src42 thereby triggering its recruitment. This event is presumably not sufficient for a stable association and/or derepression of the RIR, but also requires the binding of the SH3 domain to an unidentified sequence element within CNK. The engagement of the SH3 and SH2 domains of Src42 on CNK would not only relieve the RIR's inhibitory effect, but would also derepress Src42 autoinhibited configuration and possibly orient favorably Src42 to phosphorylate one or a few specific tyrosine residues on CNK. This scenario is certainly plausible considering that CNK has a total of 39 tyrosine residues. This would explain why depletion of endogenous Src42 led to a reduction, but not a complete elimination, of SEV-induced CNK tyrosine phosphorylation or why the Y1163F mutation impairs CNK phosphorylation mediated by Src42Y511F, since a disruption of the Src42/CNK association would prevent Src42 from phosphorylating the other sites. Although these Src42-dependent phosphorylated residues do not appear to play a role in activating the MAPK module, their concerted regulation suggests that CNK is coordinating signaling between the MAPK module and at least one other pathway (Laberge, 2005).

The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila

Fes/Fer non-receptor tyrosine kinases regulate cell adhesion and cytoskeletal reorganisation through the modification of adherens junctions. Unregulated mammalian Fes/Fer kinase activity has been shown to lead to tumours in vivo. Drosophila Fer localises to adherens junctions in the dorsal epidermis and regulates a major morphological event, dorsal closure. Mutations in Src42A cause defects in dorsal closure similar to those seen in dfer mutant embryos. Furthermore, Src42A mutations enhance the dfer mutant phenotype, suggesting that Src42A and DFer act in the same cellular process. DFer is required for the formation of the actin cable in leading edge cells and for normal rates of dorsal closure. A gain-of-function mutation in dfer (dfergof) expresses an N-terminally fused form of the protein, similar to oncogenic forms of vertebrate Fer. dfergof blocks dorsal closure and causes axon misrouting. In dfer loss-of-function mutants ß-catenin is hypophosphorylated, whereas in dfergof ß-catenin is hyperphosphorylated. Phosphorylated ß-catenin is removed from adherens junctions and degraded, thus implicating DFer in the regulation of adherens junctions (Murray, 2006),

DFer localises to adherens junctions during Drosophila embryogenesis, where it regulates leading edge morphology and dorsal closure. In dfer null mutants, P-Tyr staining is reduced at the leading edge, in particular at the actin-nucleating centers. Two potential substrates of DFer, p120ctn and ß-catenin, are localised to adherens junctions. It was found that in dferΔ1 mutants ß-catenin phosphorylation is reduced. Conversely, ß-catenin is more highly phosphorylated in dfergof mutants, demonstrating that the role of Fer in the phosphorylation of ß-catenin is conserved in Drosophila. Interestingly, the overall level of ß-catenin at cell-cell junctions is lower in dfergof mutants, suggesting that phosphorylated ß-catenin is lost from AJs and subsequently degraded (Murray, 2006),

dfer mutants also exhibit a disorganised and reduced F-actin cable at the leading edge. Formation of the F-actin cable appears to depend on adherens junctions, as F-actin nucleation begins at the level of the AJs and the F-actin cable is disrupted in DE-Cadherin mutants. It has been suggested that elevated levels of cytoplasmic α-catenin near stable AJs could favour the formation of F-actin bundles. DFer may contribute to the formation of the F-actin cable by phosphorylating ß-catenin, reducing its affinity for α-catenin, and thereby increasing the local levels of cytoplasmic α-catenin. If DFer promotes stable F-actin bundles then the regulated loss of DFer from the leading edge at stage 14 may enable the more motile Arp2/3 regulated F-actin filopodia to form and complete dorsal closure by 'zipping up' (Murray, 2006),

DFer and Src42A cooperate during dorsal closure. DFer localises to AJs and regulates ß-catenin phosphorylation. In Drosophila, Src42A binds and phosphorylates ß-catenin, although this may not be direct. Consequently, the more severe phenotypes seen in the dfer;Src42A loss-of-function mutants are most likely due to a combined loss of phosphorylation on at least two different tyrosine residues of ß-catenin (Murray, 2006),

dfer mRNA is upregulated in leading edge cells. This, together with reports that vertebrate v-Fps and Fes mediate JNK pathway activation (Li, 1998), suggested that dfer might activate the JNK pathway during dorsal closure. Although DFer itself cannot induce dpp expression, it does play a supporting role in the maintenance of dpp levels, as revealed in the Src42A mutant background. A similar failure in the maintenance of dpp, as opposed to its induction, is seen in mutants of the Wnt pathway. Given the comparable phenotypes, and the fact that phosphorylated ß-catenin is reduced in dfer mutants, it is possible that DFer contributes to the maintenance of dpp via the Wnt pathway (Murray, 2006),

This study isolated a novel, gain-of-function mutation, dfergof, in which a fragment of the White protein is fused to the N terminus of Dfer. This protein, Wex1-DFerRB, is analogous to oncogenic forms of Fps in which part of the viral GAG protein is fused to the N terminus of the endogenous proto-oncogene, generating an activated kinase. Although dfergof mutants express DFer at higher levels, this alone seems unlikely to account for the observed defects, as overexpression of DFerRB gives no obvious embryonic phenotype (Murray, 2006),

In dfergof mutants, the leading edge cells fail to elongate and the F-actin-rich filopodia are greatly reduced. The overall levels of the AJ junction components DE-Cadherin and ß-catenin are reduced, and ß-catenin is hyperphosphorylated. This suggests that AJs are disrupted in dfergof mutants. By contrast, it is interesting that the morphology of amnioserosal cells is shifted to a more motile appearance: F-actin is reduced at the cortex and there is an increase in the number of filopodia, perhaps because of a disruption of cell-cell junctions. In vertebrates, Fer has the capacity to both positively and negatively regulate cadherin-complex stability. This dual function may reflect a difference in binding partners present at AJs in different tissues (Murray, 2006),

Although loss of dfer does not appear to affect axon guidance, dfergof mutants have a clear CNS phenotype in which axons aberrantly cross the midline. A similar phenotype is seen with overexpression of the Abelson tyrosine kinase, which antagonises the receptor Robo. dfergof mutants also disrupt axon guidance in the PNS, with some general misrouting of motor nerves and some overly large synapses. In vertebrates, Fer associates with N-Cadherin in elongating neurites, where it can coordinately regulate N-Cadherin and integrin adhesion (Arregui, 2000). Fer has been shown to be concentrated in growth cones of stage 2 hippocampal neurons and is required for neuronal polarisation and neurite development (Lee, 2005). Similar to the leading edge, DFer may be required at growth cones to regulate filopodial extensions. In chick retinal cells, the phosphatase PTP1B when phosphorylated by Fer, localises to the catenin-binding domain of N-Cadherin (Xu, 2004). Interestingly, the Drosophila homologue of PTP1B, DPTP61F, is expressed in the CNS and binds to the axon guidance molecule Dock (Murray, 2006),

Strikingly, all of the phenotypes associated with dfergof mutants are rescued by expression of the Puckered tyrosine phosphatase. Given that JNK pathway activity appears normal in dfergof mutants, Puckered may target DFer itself, or its substrates, at least one of which is hyperphosphorylated in dfergof mutants. A role for DFer during Drosophila embryonic development in the regulation of AJ stability, in the formation of the contractile leading edge during dorsal closure, and in axon guidance. It cooperates with Src42A to regulate ß-catenin phosphorylation at AJs. A gain-of-function mutant with structural similarity to oncogenic forms of vertebrate Fer was isolated. Unregulated Fer activity leads to oncogenesis, possibly through unregulated epidermal to mesenchymal transition. This study has shown that activated DFer, or loss of DFer together with Src42A, disrupts AJs. This may provide a model for studying oncogenesis in the whole organism (Murray, 2006),

The Drosophila formin DAAM regulates acts upstream or parallel to Src42A and Tec29 to regulate the tracheal cuticle pattern through organizing the actin cytoskeleton

Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. This study analyzes a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, it is shown that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. The results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system (Matusek, 2006).

Drosophila DAAM is required to organize an array of parallel running actin cables beneath the apical surface of the tracheal cells that define the taenidial fold pattern of the cuticle. The actin ring pattern corresponds exactly to that of the taenidial fold pattern, and it is proposed that the actin rings organized by DAAM define the position of taenidial fold formation. The genetic interaction and epistasis data are consistent with a model that DAAM activity is regulated by RhoA. In addition, DAAM works together with the non-receptor tyrosine kinases Src42A and Tec29 to regulate the actin cytoskeleton of the Drosophila tracheal system (Matusek, 2006).

The basic structure of the insect tracheal system is a highly conserved tubular network in every species. The most important function of this network is to allow oxygen flow to target cells. Thus, tracheal tubes need to be both rigid enough, to ensure continuous air transport, and flexible enough along the axis of the tubes, to prevent the break down of the tube system when body parts or segments move relative to each other. These requirements are mainly ensured by the tracheal cuticle, which covers the luminal surface of the tubes and displays cuticle ridges (making the overall structure similar to the corrugated hose of a vacuum cleaner). Analysis of DAAM mutants provides the first direct evidence that this hypothesis is correct. The data demonstrate that in the absence of DAAM the taenidial fold pattern is severely disrupted, often leading to the collapse of the tubes and to discontinuities in the tubular network. In addition, the analysis revealed that the remarkably ordered cuticle pattern, displayed in the wild-type trachea tubes, depends on DAAM-mediated apical actin organization. Apical actin is organized into parallel-running actin cables, much the same way teanidial folds run in the cuticle. Significantly, the formation of these actin bundles precedes the onset of cuticle secretion, and the number and phasing of the actin rings correspond exactly to that of the taenidial folds in the cuticle. Thus these studies revealed a novel aspect of apical actin organization in the tracheal cells that has not been appreciated before (Matusek, 2006).

The DAAM gene encodes a novel member of the formin family of proteins, involved in actin nucleation and polymerization. Consistent with this, DAAM is colocalized with apical actin in the tracheal cells, and the activated form of DAAM is able to induce actin assembly when expressed in tracheal cells and in other cell types (unpublished). In DAAM mutant tracheal cells, apical actin is still detected, albeit at a somewhat lower level than in wild type, but the bundles formed in the mutant are much shorter and thinner than in wild type, and often appear to be crosslinked to each other. Most strikingly, global actin organization is almost completely lost, although some local order can still be detected. Remarkably, the cuticle pattern in mutant tracheal cells still follows the underlying aberrant actin pattern. Overall, in DAAM mutants, both the tracheal cuticle and the apical actin pattern resemble a mosaic of locally ordered patches that failed to be coordinated and aligned with each other and the axis of the tracheal tubes. It is thus proposed that the apical actin bundles play a key role in patterning the tracheal cuticle by defining the place of taenidial fold formation. Regarding the function of DAAM, the results suggest that the major role of this formin in the tracheal cells is to organize the actin filaments into parallel running actin rings or spirals instead of simply executing the well characterized formin function related to actin assembly. However, whether this is a direct effect on actin organization, and thus represents a novel formin function, needs to be further elucidated. An alternative model could be that DAAM is primarily required for actin polymerization but tightly coupled to an actin 'organizing' protein. In such scenario, the polymerization activity should be a redundant requirement, whereas the link to the organizing protein would be a DAAM-specific function, thereby explaining the presence of unorganized actin bundles in DAAM mutant tracheal cells (Matusek, 2006).

In the case of the main tracheal airways, which are multicellular along their periphery, it is striking that in wild type the run of the actin bundles is perfectly coordinated across cell boundaries. In addition, the run is always perpendicular to that of the tube axis. How does DAAM ensure the coordination of these two aspects of actin organization? Because the DAAM protein and the apical actin cables are both found at the level of the adherens junctions, it is possible that DAAM regulates the coordination of the actin cables at the cell boundaries through a direct interaction with junctional protein complexes. However, other explanations are also possible, and further experiments will be required to elucidate the molecular mechanism of this regulatory function. The fact that actin cables normally run perpendicular to the tube axis seems to suggest that tracheal cells are able to sense a global orientation cue and align their actin bundles accordingly. The nature and source of this cue is unknown, as is the mechanism by which DAAM is involved in the read-out of this signal. Nevertheless, it is interesting that in DAAM and btl-Gal4/UAS-C-DAAM mutant trachea, the main pattern of the cuticle phenotype is often changing from one segment to the other, suggesting that the effect of the 'global' orientation cue is limited to metameric units (Matusek, 2006).

Sequence comparisons of FH2 proteins suggest a close phylogenetic relationship between the DRF, FRL and DAAM subfamilies. Members of these three subfamilies have a high level of conservation in the FH2 domain, and importantly, also in the region of the GBD and DAD domains, suggesting that the FRL and DAAM family formins are also regulated by autoinhibition and RhoGTPases, like the DRFs. Further evidence is presented in support of this view. First, DAAM and RhoA display a strong genetic interaction. Second, C-DAAM (an N-terminally truncated form of DAAM) behaves like an activated form much the same way DRF family formins behave. Third, epistasis experiments with C-DAAM and RhoA suggest that RhoA acts upstream of DAAM. Thus, the data support the model in which DAAM, at least in the Drosophila tracheal system, is regulated by autoinhibition that can be relieved by RhoGTPases (Matusek, 2006).

This conclusion, however, contradicts the observation that human DAAM1 is required for Wnt/Fz/Dvl dependent RhoA activation in cultured cells and that xDaam1 appears to mediate Wnt-11 dependent RhoA activation in Xenopus embryos. These results suggested that DAAM functions upstream of RhoA in non-canonical Wnt/Fz-PCP signaling. An explanation for these distinct conclusions might be related to the fact that DAAM, in contrast to xDaam1, does not appear to be required for Fz/Dsh-PCP signaling. Hence, it is possible that the Drosophila ortholog is regulated in the same way as the DRF formins, while the vertebrate family members can be regulated in a different way, once bound by Dsh/Dvl and recruited into PCP signaling complexes (Matusek, 2006 and references therein).

Genetic interactions with the hypomorphic DAAMEx1 allele identified two non-receptor tyrosine kinases, Src42A and Tec29, as strong interacting partners. Although both of these kinases play multiple roles during embryogenesis, single mutants for both affect the tracheal cuticle pattern in a similar way to DAAM. These results suggest that DAAM and the Src family kinases work together to regulate the actin cytoskeleton and cuticle pattern in tracheal cells. Although it is not known whether DAAM physically binds Src42A and/or Tec29, it has been established that the FH1 region of DRFs and other formins can bind SH3 domains, including those of the Src family kinases. In agreement with these data that DAAM acts upstream of Src42A and Tec29 in tracheal cells, cytoskeleton remodeling and SRF activation mediated by mouse Dia1 and mouse Dia2 requires Src activity. Moreover, a recent report suggests that RhoD and human DIA2C regulate endosome dynamics through Src activation, proposing that Src activity is stimulated via human DIA2C dependent recruitment to early endosomes. Similarly, the Limb deformity protein (a formin) interacts with Src on the perinuclear membranes of primary mouse fibroblasts. Based on these examples, it is speculated that in Drosophila tracheal cells the RhoA/DAAM/Src module may not only be required to organize apical actin bundles, but additionally it might represent a link to secretory vesicles and to the regulation of exocytosis. Future studies will be required to test this hypothesis, and to unravel the mechanisms whereby DAAM family formins and Src family kinases contribute to cytoskeletal remodeling in the Drosophila tracheal system and in other tissues (Matusek, 2006).

Dcas supports cell polarization and cell-cell adhesion complexes in development

Mammalian Cas proteins regulate cell migration, division and survival, and are often deregulated in cancer. However, the presence of four paralogous Cas family members in mammals (BCAR1/p130Cas, EFS/Sin1, NEDD9/HEF1/Cas-L, and CASS4/HEPL) has limited their analysis in development. The single Drosophila Cas gene, Dcas, was deleted to probe the developmental function of Dcas. Loss of Dcas had limited effect on embryonal development. However, Dcas is an important modulator of the severity of the developmental phenotypes of mutations affecting integrins (If and mew) and their downstream effectors Fak56D or Src42A. Strikingly, embryonic lethal Fak56D-Dcas double mutant embryos had extensive cell polarity defects, including mislocalization and reduced expression of E-cadherin. Further genetic analysis established that loss of Dcas modified the embryonal lethal phenotypes of embryos with mutations in E-cadherin (Shg) or its signaling partners p120- and beta-catenin (Arm). These results support an important role for Cas proteins in cell-cell adhesion signaling in development (Tikhmyanova, 2010).

This work identifies a strong interaction between the Dcas, and integrin pathway genes, including integrins and their effector kinases Fak56D and Src42A, during early embryonal development in Drosophila. The synthetic lethal phenotypes found in double mutants of Dcas and Src or FAK56D were marked by defects in dorsal closure and in some cases by the appearance of anterior cuticle holes that suggested head involution defects. These defects were commonly accompanied by abnormalities in epithelial function, including failure to appropriately localize shg/E-cadherin to cell junctions, and reduced shg expression. The data are compatible with the idea that either Fak56D or Dcas is sufficient to support shg/E-cadherin localization and cell polarization during morphogenetic movements in Drosophila embryos, but the absence of both cannot be sustained (Tikhmyanova, 2010).

Building from these observations, a novel synthetic lethal relationship was established between DCas, shg, and arm. As with crosses to alleles of Fak56D and Src42A, the point of lethality was at the time of dorsal closure, at embryonal stages 15-16, and associated with defective cuticle formation. One way to integrate these observations is to hypothesize that the DCas, Fak56D, and Shg protein products are normally in dynamic balance, with Dcas regulating Shg cycling. The fact that Crb and Dlg1, a mammalian homolog of Dlg, have been reported to support Shg localization to adherens junctions, suggests that Dcas/Fak56/Src42A specifically interact to support this cell polarity/cell junctional control system. In this context, it is suggestive that the Crb family protein CRB3 has been described as part of a complex including CRB3, Pals1, and PatJ that becomes tightly associated with Src kinase during reorganization of cell polarity. In the absence of DCas and Fak56D, Shg cannot localize properly; the moderately elevated levels of Shg proteins found in these embryos most likely arises as part of a cellular compensatory mechanism in response to decreased functional Shg signaling complexes. In further indirect support of the idea that this is a specific Dcas action, the fact that genetic interactions were not observed between Dcas1 and Aur or Dock indicates that Dcas does not promiscuously interact with other genetic lesions to reduce viability (Tikhmyanova, 2010).

A previous study demonstrated a role for Dcas in axonal guidance in the development of the nervous system of adult flies (Huang, 2007). That work analyzed the hypomorphic Dcas mutant allele DcasP1, and the small deficiency Df(3L)Exel6083, including Dcas and five adjacent genes, which were also used in this study. The earlier study focused exclusively on analyzing the contribution of Dcas to axonal guidance in late (stage 16/17) embryos: in that analysis, Dcas functioned similarly to integrins, and genetically interacted with integrins (if, mew, and mys) in regulating axon guidance and axonemal defasciculation. In this context, it is intriguing that the mammalian Cas family NEDD9 gene is abundant during neuronal development, has been proposed as a candidate locus for oral cleft defects in humans based on its chromosomal location near the OFC-1 locus. Together these findings raise the possibility that this specific Dcas paralog has a specific role in human neuronal migration and morphogenesis of the head. As with the current data using the new Dcas1 allele, homozygous deletion of Dcas in conjunction with integrins had moderate effect on viability of adult flies, although this work for the first time demonstrates an interaction between Dcas and if and mew, and also between Dcas and Src, in regulation of wing development (Tikhmyanova, 2010).

Generation of the first null allele of Dcas provides a useful new tool to study the role of this protein in Drosophila development. This work illuminates the evolutionary conservation of Dcas function within the integrin and receptor tyrosine kinase network, including FAK, Src, and integrins genes. The finding that a low percentage of embryos with mutant Dcas and all embryos with double mutations in Dcas and Fak56D, have perturbed localization of polarity markers, including Shg, indicates a novel function for Cas family in regulation of cell polarity. To date, the evidence directly connecting Cas proteins to a known mechanism for control of cell polarity is sparse. Although NEDD9 was in fact discovered in a functional genomics screen for cell cycle and polarity modifiers in budding yeast (leading to its designation as HEF1, Human Enhancer of Filamentation 1), the mechanism involved was not established, and given the great evolutionary distance involved, may not be relevant to a role in metazoans. Both BCAR1 and NEDD9 interact physically with proteins that influence cell polarity controls during pseudopod extension and other actin polarization processes: these include the GTP exchange factor AND-34 (Tikhmyanova, 2010).

The data in the present study indicating genetic interactions with cell-cell junction regulatory proteins Shg, Arm and p120-catenin may have considerable significance in the sphere of cancer research, as it implies that overexpression of Cas proteins may promote cancer progression by influencing the polarized movement of cells and influencing lateral attachments. The fact that one report has indicated interactions between BCAR1 and nephrocystins at cell-cell junctions in polarized epithelial cells implies that a potentially direct interaction of Cas proteins in these structures is conserved through mammals. However, given the known interactions of Cas proteins with FAK and SRC at focal adhesions, another possibility is that Cas may additionally or alternatively impact Shg function through indirect signaling emanating from these structures. Notably, it has been reported that NEDD9 overexpression induced by dioxin caused downregulation of E-cadherin, and it will be of great interest to study the consequences of overexpressing Dcas on Drosophila development. Consequences for loss of NEDD9 expression on E-cadherin expression or localization are not yet known. Resolving these questions will provide intriguing directions for future studies (Tikhmyanova, 2010).

Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. A variety of fluorescent reporters were developed that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. Four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-β-cyclodextrin, and hydrogen peroxide) were found to be sufficient to globally activate epidermal wound response genes in Drosophila embryos. The epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals were examined. The results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration (Juarez, 2011).

Drosophila wound healing is an example of a regenerative process, which requires localized epidermal cytoskeletal changes, and localized wound-induced changes in epidermal transcriptional activity. This genetic screen with wound-dependent reporters has allowed identification of novel components that regulate the localized transcriptional response to wounding in epidermal cells. This research identifies seven genes that are required to either activate (Duox and ghost/stenosis) or localize (Flo-2, Src42A, wurst, varicose, and Drosophila homolog of yeast Mak3) the expression patterns of epidermal wound reporters. The number of new functions involved in the delimitation of epidermal wound response near wound sites was unexpected, but indicates that considerable genetic effort is devoted to localizing the activity of transcriptional wound responses during regeneration (Juarez, 2011).

One of the genes that limits the spread of epidermal wound reporters after clean epidermal punctures is Flo-2, as mutants of this gene show a broad expansion of epidermal wound gene activation. Drosophila Flo-2 is itself transcriptionally activated around epidermal wound sites, consistent with an evolutionarily conserved role in regeneration after wounding. In vertebrates, reggie-1/Flo-2 gene expression is activated in wounded fish optic neurons, and reggie-1/Flo-2 and reggie-2/Flo-1 morpholino knockdowns in wounded zebrafish retinal explants reduced axon outgrowth compared to controls. Flo-2 transcriptional activation around Drosophila epidermal wound sites is dependent on the grh genetic function, which is required to activate at least a few other epidermal wound response genes. Flo-2 thus appears to act in the same pathway as grh, although it may act both downstream and upstream of grh, since overexpression of Flo-2 can inhibit the activation of other grh-dependent wound response genes. In this respect, Flo-2 resembles stit receptor tyrosine kinase gene (Wang, 2009), which is both transcriptionally activated by Grh, as well as required for grh-dependent activation of other downstream wound genes. Amazingly, overexpression of Flo-2 can even inhibit the global activation of the Ddc and ple-WE1 wound reporters that are induced by the serine protease trypsin, or by hydrogen peroxide. The inhibitory function of overexpression of Flo-2 on wound induced transcription is cell non-autonomous, at least over the range of a few cell diameters, as shown by the ability of striped overexpression of Flo-2 to silence puncture or trypsin-induced gene activation throughout the epidermis (Juarez, 2011).

The only animal where Flo-2 null mutants have so far been characterized is Drosophila, where Flo-2 has been shown to regulate the spread of Wingless (Wg) and Hedgehog (Hh) signals in the wing imaginal discs. In the wing discs, both the secretion rate and the diffusion rate of these two lipid-modified morphogens were increased when Flo-2 was overexpressed, and decreased when Flo-2 and Flo-1 proteins were not expressed. Despite the reduced spread of Wg and Hh morphogen proteins in Flo-2 mutant imaginal discs, adult morphology of mutants was normal, presumably because of compensatory mechanisms that occur later in development. Whereas a reduced range of activation of wg and hh long range transcription target genes was observed in Flo-2 mutant imaginal discs, a greatly increased range of wound-induced gene activation was observed in Flo-2 mutant embryos. This apparent discrepancy could be explained if one invokes of a long-range wound-induced inhibitory signal that in wild type embryos diffuses faster and farther than a wound activating signal, and thereby functions to limit the wound response to nearby epidermal cells, and that in Flo-2 mutants this potential inhibitory signal has reduced secretion, concentration, and/or diffusion range. This notion is consistent with the cell non-autonomous effect of overexpressed Flo-2 on inhibiting wound- or trypsin-induced gene activation. A similar scheme of controlling signal spreading has been seen in the way that Mmp2 acts cell non-autonomously to limit FGF signaling during Drosophila tracheal development and branch morphogenesis. It's also possible that Flo-2 normally is required to set a global threshold that wound-induced signals must overcome in order to activate wound transcription, for example via Flo-2-dependent endocytosis/degradation of a diffusible wound signal and its receptor (perhaps the Stit RTK), and that signal strength normally surpasses the Flo-2 threshold only in the vicinity of a wound. In this model, loss of Flo-2 would result in all epidermal cells being able to exceed the wound signal threshold, and overexpression of Flo-2 would prevent any cells from exceeding the wound signal threshold. The cell non-autonomous effects of Flo-2 overexpression under this model might be explained by an increase in Flo-2-dependent endocytosis/degradation that rapidly depletes an activating signal from the extracellular space (Juarez, 2011).

Many previous studies have documented biochemical, molecular biological, and cell biological interactions between Src family kinases and Flotillins. In Drosophila, lack of Src42A and Flo-2 leads to expanded spread of wound gene activation, and overexpression of Flo-2 or activated Src42A can inhibit wound gene activation, which is consistent with an interaction between the two functions during the process of wound gene regulation. In cultured mammalian cells, Flo-2 can be phosphorylated by Src family kinases in an extracellular signal-dependent fashion. This phosphorylation is associated with changes in the normal intracellular trafficking of Flotillin-containing membrane microdomains and vesicles. Since overexpressed Flo-2 in Drosophila can act in a cell non-autonomous fashion to inhibit wound gene activation, and overexpressed Src42A acts in a cell autonomous fashion to inhibit wound gene activation, one interpretation is that Flo-2 lies genetically upstream of Src42A in the epidermal wound response. This hypothesis appears to be inconsistent with the vertebrate biochemical data indicating that Src kinases phosphorylate Flotillins to activate their diverse functions. However, an observation that is consistent with Src42A activating Flo-2 protein function, is that even when Flo-2 is overexpressed, addition of chemical inhibitors of Src family kinases to wounded embryos, results in widespread Ddc .47 or ple-WE1 wound reporter activation. One interpretation of this suggests Flo-2 protein, no matter the level of expression, is inactive in the absence of Src42A function. Complex feedback loops involving signaling proteins being regulated by a transcription factor, while the activity of the same transcription factors is regulated by the same signaling pathway, have been observed in the control of Drosophila epidermal wound gene expression and reepithelialization, so there may be similar dynamic cross-regulatory interactions between Flo-2 and Src42A in the localization of the epidermal wound response, interactions not easily captured in linear genetic pathway diagrams (Juarez, 2011).

The inhibitory effect of Src42A on wound gene activation suggests that it might antagonize a signaling cascade that leads to the epidermal wound response. A good candidate for such a signaling cascade is the RTK pathway involving the Stit kinase. Stit is a RET-family RTK that is required for robust activation of the Ddc and stit wound reporter genes in wounded embryos (Wang, 2009). Other evidence consistent with RTK pathway importance in wound gene activation is that phosphotyrosine accumulates persistently around wound sites, and that ERK kinase function is required for robust activation of the Ddc wound reporter gene. Interestingly, Src42A has been shown to act as an inhibitor of some Drosophila RTK proteins (those encoded by the torso, Egfr, and sevenless genes) in a few different tissues during Drosophila development. The Flo-2 and Src42A functions in epidermal wound localization after clean wounding are reminiscent of the role of Drosophila WntD during infectious wounding. WntD mutants show higher levels of some antimicrobial peptide genes after septic injury of adults (Juarez, 2011).

Previous evidence suggested that H2O2 and Duox could provide wound-induced inflammatory signals and antimicrobial activities. The current studies show that Duox is required to activate wound reporter genes after epidermal wounding, and that injected exogenous H2O2 is sufficient to activate widespread epidermal wound gene expression. Overexpression of either Flo-2 or Src42A.CA can inhibit the H2O2 -dependent wound reporter expression, suggesting that all of these components are in a common pathway controlling the activation of epidermal wound reporters. However, the ability of trypsin injection to activate the Ddc .47 and ple-WE1 wound reporters in Duox mutants suggests that a serine protease might act downstream of, or in parallel to, H2O2-dependent wound signals. A recent report showed that in cultured mammalian cells, a Src kinase phosphorylates and inhibits a Flo-2-associated enzyme, peroxiredoxin-1, which results in increased stability of H2O2. This is consistent with the results placing Flo-2, Src42A, and H2O2 in a common wound signaling pathway (Juarez, 2011).

Like H2O2, the injection of methyl-β-cyclodextrin (MβCD) into wounded embryos triggers a global wound response in the epidermis. MβCD strongly depletes cholesterol and other sterols from membranes and disrupt lipid rafts, but was also shown to remove sphingolipid-associated proteins such as Src-Family Kinases. The effects of MβCD, in combination with the effects of loss of Flo-2, suggests that the integrity of lipid rafts and associated proteins are required to inhibit epidermal wound signals. In cultured cells, MβCD treatments trigger a release of EGF receptors from membrane microdomains, which increases EGFR, and perhaps other RTK, signaling in a ligand-independent manner. Interestingly, in cultured keratinocytes, MβCD treatment can induce the expression of involucrin, which encodes a protein, analogous to Drosophila Ple/tyrosine hydroxylase, which is required for the formation of an epidermal barrier. Similarly, MβCD injections into Drosophila embryos might also cause an increase the levels of a wound signal produced or released from cells adjacent to the wound site, allowing more widespread transcriptional activation of wound reporter genes. The observations that overexpression of Src42A or Flo-2 can inhibit the MβCD -triggered activation of epidermal wound reporter genes suggest that high levels of these proteins might overcome lipid raft-inhibitory effects on wound signaling pathways (Juarez, 2011).

Other genes (wurst and varicose) identified in the screen have phenotypes similar to Flo-2 and Src42A mutants. wurst encodes an evolutionarily conserved trans-membrane protein, containing a heat shock cognate protein 70 binding domain and a clathrin binding motif. wurst is ubiquitously expressed in embryonic epithelial cells, strongly up-regulated during endocytosis-dependent luminal clearance, and mislocalized in mutants with endocytosis defects. wurst mutant embryos have tortuous tracheal tubes, due to a failure to properly endocytose matrix material from the tracheal lumen. varicose encodes an evolutionarily-conserved septate junction scaffolding protein, in the Membrane Associated GUanylate Kinase (MAGUK) family. varicose is expressed in epidermally-derived cells (including the hindgut and trachea) and co-localizes with the septate junction proteins, Coracle and Neurexin4. varicose mutant embryos develop permeable tracheal tubes and paracellular barrier defects in epithelia. Like wurst mutants, varicose mutants also have abnormal matrix composition in the tracheal lumen, and may also have abnormal extracellular matrix composition produced by other epidermal cells (Juarez, 2011).

Another gene (ghost), also known as stenosis) identified in this screen is required for wound reporter activation like Duox or grh. ghost encodes the Drosophila Sec24CD homolog, a coat protein of COPII vesicles in the ER/Golgi trafficking pathway. Transport of cargo from the ER to the Golgi via COPII vesicles is required to achieve normal amounts of secretion of extracellular matrix proteins into the developing Drosophila tracheae and normal apical-basal localization of membrane proteins. Presumably, similar secretion and membrane localization defects occur in non-tracheal epidermal cells, which account for the severe cuticle deposition defects in ghost (Sec24CD) mutants. It is fascinating to note that the finding that ghost (Sec24CD) is required for transcriptional activation of epidermal wound reporter genes is consistent with the finding that RNAi knockdowns of Sec24C in a planaria (Schmidtea mediterranea) interfered with normal regeneration after amputation wounds. It is possible that the ghost mutants do not secrete enough wound signals, or the protein matrix necessary for the propagation of a wound signal (Juarez, 2011).

Another gene required for the activation of wound reporters is shroud (sro). It is believed sro to be an allele in the Drosophila Fos-D isoform, and it was hypothesized that one of the Drosophila kayak/Fos transcription factors was required for the activation of some epidermal wound gene reporters. However, has been recently discovered that sro[1] and other sro point mutant alleles do not map in the kayak/Fos gene, but in an immediately adjacent transcription unit (Nm-g/sro) that encodes an enzyme in the sterol metabolic pathway that is necessary for production of ecdysone hormone. At first glance, the requirement of sro to activate some wound reporters suggested that these reporters rely on ecdysone signaling. This is possible, although deletions were tested that eliminate zygotic functions of the ecdysone receptor gene, as well as of the phantom gene (which encodes another enzyme in the ecdysone synthesis pathway), and embryos that are zygotic mutants in either gene show normal activation of the ple-WE1 wound reporter after puncture wounding (Juarez, 2011).

In summary, though this large unbiased screen, several genes were identified that add to the understanding of the complex pathways that control the signals that activate wound response transcription near puncture wounds. At the cellular level, there appears to be a correlation between genetic functions required to localize wound-induced gene activation, and cellular functions required for endocytosis and/or apical-basal polarity. For example, one function of Flo-2 is in signal-dependent endocytosis, although Flo-2 also plays other roles in vesicular trafficking. There have been many studies showing that endocytosis can regulate extracellular signaling strength and duration. For example, one study found that tagged-FGF8 showed increased accumulation, spread, and target gene activation when Rab-5-mediated endocytosis was reduced in zebrafish embryos. It is believed that further studies on wound response signaling may provide new insights into how membrane microdomains, endocytosis of membrane receptors, and the composition and organization of the extracellular matrix, regulates the transmission of wound signals (Juarez, 2011).

Drosophila Src regulates anisotropic apical surface growth to control epithelial tube size.

Networks of epithelial and endothelial tubes are essential for the function of organs such as the lung, kidney and vascular system. The sizes and shapes of these tubes are highly regulated to match their individual functions. Defects in tube size can cause debilitating diseases such as polycystic kidney disease and ischaemia. It is therefore critical to understand how tube dimensions are regulated. This study identified the tyrosine kinase Src as an instructive regulator of epithelial-tube length in the Drosophila tracheal system. Loss-of-function Src42 mutations shorten tracheal tubes, whereas Src42 overexpression elongates them. Surprisingly, Src42 acts distinctly from known tube-size pathways and regulates both the amount of apical surface growth and, with the conserved formin dDaam, the direction of growth. Quantitative three-dimensional image analysis reveals that Src42- and dDaam-mutant tracheal cells expand more in the circumferential than the axial dimension, resulting in tubes that are shorter in length-but larger in diameter-than wild-type tubes. Thus, Src42 and dDaam control tube dimensions by regulating the direction of anisotropic growth, a mechanism that has not previously been described (Nelson, 2012).

To define the molecular mechanisms by which Src42 acts, loss-of-function mutations were tested in candidate Src interactors for a short-tracheal phenotype. dDaam (also known as DAAM, Dishevelled associated activator of morphogenesis), a conserved Diaphanous-related formin that has been shown to bind vertebrate Src, showed a mild, but significant, short-tracheal phenotype near the end of embryogenesis (stage 16). Shortening was more apparent after hatching, raising the possibility that the weaker embryonic phenotype was due to maternal dDaam (Matusek, 2006; Matusek, 2008); failure of dDaam maternal/zygotic embryos to cellularize (Matusek, 2008) precludes a direct test of this possibility). Similarly to Src42, dDaam acts autonomously in the tracheal system, because tracheal expression of Flag-tagged dDaam fully rescued the shortened dorsal trunk of dDaam mutants. Remarkably, despite only causing modest reductions in dorsal-trunk length at embryonic stage 16, zygotic dDaam mutations were still able to completely suppress the overelongation of septate-junction, aECM, apico-basal and PCP mutants. Further, whereas Src42; scrib mutants had gross embryonic defects, dDaam; scrib mutants underwent largely normal development and had trachea with the dDaam short-tracheal phenotype. Thus, dDaam seems to act downstream of or in parallel to all characterized control pathways for tracheal-tube size (Nelson, 2012).

Src42A-dependent polarized cell shape changes mediate epithelial tube elongation in Drosophila

Although many organ functions rely on epithelial tubes with correct dimensions, mechanisms underlying tube size control are poorly understood. This study analysed the cellular mechanism of tracheal tube elongation in Drosophila, and describes an essential role of the conserved tyrosine kinase Src42A in this process. Src42A was shown to be required for polarized cell shape changes and cell rearrangements that mediate tube elongation. In contrast, diametric expansion is controlled by apical secretion independently of Src42A. Constitutive activation of Src42A induces axial cell stretching and tracheal overelongation, indicating that Src42A acts instructively in this process. It is proposed that Src42A-dependent recycling of E-Cadherin at adherens junctions is limiting for cell shape changes and rearrangements in the axial dimension of the tube. Thus, distinct cellular processes are defined that independently control axial and diametric expansion of a cylindrical epithelium in a developing organ. Whereas exocytosis-dependent membrane growth drives circumferential tube expansion, Src42A is required to orient membrane growth in the axial dimension of the tube (Forster, 2012).

These findings establish a critical role of Src in epithelial tube morphogenesis. Src42A is necessary and sufficient for cell shape changes that specifically mediate tracheal tube elongation, whereas diametric tube expansion is independent of Src42A function. Consistent with previous work, this study found that Src42A regulates E-Cad recycling at adherens junctions. On the basis of these data it is proposed that Src42A-induced adherens-junction remodelling is limiting for polarized cell shape changes that mediate axial, but not diametric, dorsal-trunk expansion. In this model, secretion drives apical membrane growth independently of Src42A function, which is required to direct apical membrane growth in the axial dimension of the tube. However, the signals activating Src42A in tracheal cells and the Src42A substrates mediating polarized cell behaviours remain elusive. No evidence was found for planar-polarized Src activity in tracheal cells or for a role of Src42A in PCP signalling, indicating that potential PCP cues that might direct axial expansion may be downstream or parallel to Src42A activity. In principle, even subtle asymmetries in the distribution of adherens-junction components may result in large-scale tissue transformations. Alternatively, polarized cell behaviour may result from the inherent anisotropy imposed by cylindrical geometry of tubular epithelia. On the basis of Laplace's law, circumferential tension on a pressurized cylinder's surface is larger than longitudinal tension. Anisotropic tissue tension might orient cell behaviour in tubular epithelia. Normal epithelial cells cultured on cylindrical substrates orient circumferentially, whereas Ras-transformed cells orient axially, accompanied by re-orientation of actin filaments, indicating that cells may be able to sense and respond to tissue geometry. It is proposed that Src42A is involved in sensing cylindrical tissue geometry, possibly as a mechanical force sensor, or by translating this information into polarized cell behaviour. The specific effect of Src42A mutations on axial, but not circumferential, cell shape changes may reflect different requirements for Src42A in remodelling axial and circumferential adherens junctions, respectively. A possible explanation for Src42A affecting elongation of the dorsal trunk and transverse connective, but not of dorsal branches, whose elongation relies on adherens-junction remodelling, is that dorsal-branch cell intercalation relies on pulling forces generated by migrating tip cells, which may override the Src42A requirement. Conversely, the absence of tip-cell-mediated pulling forces in the dorsal trunk and transverse connective may imply an increased requirement for Src42A-mediated junction remodelling in these branches. This work provides a genetically tractable paradigm to analyse Src function in regulating polarized cell behaviours that control epithelial tube dimensions during normal organogenesis or under pathological conditions, such as polycystic kidney disease, in which Src has been implicated (Forster, 2012).

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, this study identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl -/-triggered tumor invasion and loss of cell polarity gene-induced cell migration. The genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, this study showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, it was found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. These data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a (Ma, 2013).


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Reference names in red indicate recommended papers.

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Src oncogene at 42A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 December 2013

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