Interference of human and Drosophila APP and APP-like proteins with PNS development in Drosophila: Dab enhances the phenotype induced by APP, a reduction of the endogenous protein level by RNAi suppresses the phenotype

The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, transgenic Drosophila was used as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch (Merdes, 2004).

These studies show that the ectopic expression of human APP-family members induces Notch gain-of-function phenotypes during the development of the adult PNS. The severity of the induced phenotypes not only depends on the dosage and the particular APP-family member, but also on particular domains of the molecules. This led to the identification of the NPTY motif as the only critical motif within the ICD for the interference with PNS development and for the interaction of APP with Numb/Pon and Dab in vitro and in vivo (Merdes, 2004).

An interaction between APP and Numb has been demonstrated by Roncarati (2002). In mouse brain lysates as well as in cell culture, APP or APP.ICD bind to all four isoforms of Numb and to Numb-like. Surprisingly, in this study, the processing of APP and the release of the ICD of APP resulted in an inhibition of Notch signaling. Numb is a negative regulator of Notch signaling and binds directly via its PTB domain to Notch. Therefore, a direct interaction between APP and the PTB domain of Numb should result in an increase rather than in a decrease of Notch activation. From the known crystal structure of PTB-NPTY interactions, a trimeric complex between Notch, APP and Numb seems unlikely. In this study, the induced Notch gain-of-function phenotypes, the strong genetic interaction, the dependence of the asymmetric localization of APPL on Numb and the direct binding between APP and Numb support a crosstalk between Notch signaling and APP-family members. One explanation for the APP induced Notch gain-of-function phenotypes during mechano-sensory organ (MSO) development would indeed be the sequestration and inactivation of Numb by APP-family members. However, several lines of evidence are provided that (if APP competes with Notch for the binding to Numb) suggest this binding and competition must be highly regulated and requires factors which have not previously been known to be involved in MSO development (Merdes, 2004).

(1) Expression of the human APP-family proteins induces cell fate transformations during MSO development in a dosage- and construct-dependent manner, but the potency in phenotype induction of the different proteins does not correlate with their in vitro and in vivo binding affinity to Numb. Nevertheless, the NPTY motif proves to be essential both for binding to Numb and phenotype induction, suggesting that the binding to Numb might be necessary but not sufficient for phenotype induction. This implies that there is at least one additional factor which plays an important role and which must have different affinities to the APP-family members than Numb, for example, strong binding to APLP2 but weak binding to APP.
(2) Deletion of the ECD of APP results in an inactive molecule, which can no longer induce any phenotypes. This stands in contrast to all in vitro binding studies that have been performed between the NPTY motif of APP and PTB-containing proteins in cell culture. In these assays, the affinity of such a molecule to Fe65, Dab-1/2, X11L, Numb and Numb-like did not change significantly.
(3) APP molecules with a deletion of the NPTY motif could suppress the phenotypes induced by wt APP and induce the loss of macrochaete in wt flies. Such a dominant-negative effect can only be explained if APP-family members have a receptor-like function. In this scenario, APP.DeltaNPTY would compete with wt APP or APPL for ligand binding, but could not relay the 'signal', for example, crosstalk to Notch and/or inactivating Numb. Another possibility would be the necessity of homodimer formation. Such a dimer formation has been postulated, but so far no in vivo data are available. Furthermore, structural data do not provide any evidence for a dimerization of APP molecules prior to the binding of PTB-containing proteins.
(4) Overexpression of Drosophila APPL induces only very weak phenotypes, whereas the overexpression of APPL.sd induces very strong phenotypes. The difference in phenotype induction could not be correlated with significant differences in expression levels, metabolism or processing. This was surprising, since APPL.sd had been generated to impair secretion and therefore processing. As a consequence, it is postulated that the 33 aa deletion in APPL.sd changes the conformation of the ECD, confirming again the important role the ECD plays in determining the potency of the APP-family members for interference with PNS development.
(5) Overexpression of APLP2 results in bold patches, suggesting that presumptive SOPs are transformed into epidermal cells by the induction of a Notch gain-of-function phenotype very early during MSO development. This step during PNS development is known to be independent of Numb and functions via the lateral inhibition mechanism, indicating that APP-family members can also interact with Numb-independent Notch signaling processes. During these processes, so far unknown factors might take over the role of Numb as a negative regulator of Notch to add an additional level of control to the system. From the literature, it seems to be clear that endocytosis is important for inhibition and for the promotion of Notch signaling, but almost nothing is known about the factors directly involved in these events.
(6) Ectopically expressed APPL and APPL.sd as well as APP and APP/APLP2 are asymmetrically localized during MSO development and co-localization and co-immunoprecipitation with Pon has been be demonstrated in vivo. This is an interesting result since APPL and APP induce only weak phenotypes, but APPL.sd and APP/APLP2 induce very strong phenotypes. Nevertheless, both types of proteins are recognized with the same efficiency by the Numb-dependent machinery responsible for the asymmetric distribution of factors during MSO development, thus completely uncoupling this event from phenotype induction. This implies that the phenotype induction occurs after completion of the separation of the SOP siblings and that APP, even if it binds to Numb, does not compete with other binding partners of Numb for asymmetric segregation (Merdes, 2004).

During MSO development, the asymmetric distribution of Numb ensures that the siblings arising from one mother cell show a difference in response to the activation of the Notch receptor. Numb is responsible for the asymmetric segregation of α-adaptin and binds both the ICD of Notch and α-adaptin, suggesting that Numb may regulate Notch by controlled endocytosis. The difference in response to Notch signaling is further amplified by the asymmetric localization of the E3 ubiquitin ligase Neuralized, which upregulates the endocytosis of the Notch ligand Delta. However, one has to take into account that it has also been reported that Numb can (1) bind the ICD of Notch after release, (2) inhibit the ability of this ICD to cause nuclear translocation of Su(H) and (3) can inhibit Notch signaling during wing development by ectopic misexpression. Therefore, even if it is very tempting to suggest that Numb solely regulates Notch by endocytotic mechanisms, there might still be other Numb functions (Merdes, 2004).

Nevertheless, more and more evidence is emerging that regulated endocytosis is an important general feature for the modulation of developmental signals. In this respect, it is especially intriguing that Drosophila Dab has been identified as an essential factor for the interaction of APP with Notch signaling. Whereas the overexpression of Dab enhances the phenotype induced by APP, a reduction of the endogenous protein level by RNAi suppresses the phenotype. Notch gain-of-function phenotypes during MSO development can be induced by expression of high levels of Dab alone. This is remarkable since it has been proposed that the mammalian Dab-2 homologs belong to a family of cargo-specific adaptor proteins, which, like Numb and β-Arrestin, regulate cargo selection and pit formation. Accordingly, APP molecules could induce the observed phenotypes during PNS development, influencing endocytosis and processing of Notch with the help of Dab. A function for APP as endocytotic receptor is supported by the finding that full-length APP is internalized via clathrin-coated vesicles. Furthermore, a direct interaction between Drosophila Dab and Notch has been demonstated previously (Giniger, 1998). These binding studies have been reproduced, but the binding of Dab to Notch in vitro was shown to be very weak in comparison to the binding affinity of Su(H) or Numb. However, additional studies suggest not only the presence of a second Notch-binding motif within the C-terminal domain of Dab, but also reveal the presence in vivo of a complex which contains Notch and Dab in Drosophila embryos (LeGall and Giniger, personal communication to Merdes, 2004). The second binding motif could allow a direct interaction between the Notch receptor and APP mediated by Dab, and it will be of great interest to elucidate the role of Dab with respect to Notch and APP signaling in the future. A crosstalk between the APP family and Notch receptors has also been shown to take place in the mammalian system (Merdes, 2004).

Originally, mutations in the dab gene were isolated by genetic interactions with the Drosophila Abl homolog. It has recently been reported that these mutations have been erroneously attributed and that all mutations isolated as dab alleles in fact affect the nrt locus (Liebl, 2003). Nrt is a single-pass type-II transmembrane protein and belongs to the family of neuronal cell adhesion molecules (N-CAMs). Nrt mutants are viable and fertile, but its function in growth cone guidance can be revealed in combination with other N-CAM mutants. Since the originally described dab alleles were used for the first genetic studies, mutations were identified in nrt as dominant suppressors of the APP-induced phenotype and also the overexpression of Nrt itself induces very strong and very specific Notch gain-of-function phenotypes. However, genetic studies ruled out an involvement of Abl in the APP-induced phenotype. Preliminary genetic data suggest a genetic interaction between appl and nrt mutations resulting in lethality of the otherwise viable alleles. Additional experiments will be necessary both in Drosophila and vertebrates to further explore this interaction. Especially, the isolation of new mutants for Drosophila dab and appl generated in a clearly defined genetic background, and their use for genetic interactions with Notch, numb and nrt, should provide insights into the mechanisms underlying the potential functions of APP-family members in endocytosis, Notch signaling and PNS development. However, the identification of appl as a quantitative trait locus already provides evidence for a function of appl during PNS developmen (Merdes, 2004).

Although it has not been established that the binding interactions between APP, Numb and Dab are functionally important in AD, signaling pathways emanating from aberrant APP function, as it occurs in AD, may influence Dab/Numb and thus Notch activity. Also, the use of drugs to lower APP processing and Aβ production could result in altered APP functions and an interference with Notch signaling in the adult brain. As already mentioned, an interaction between APP and Numb and Numb-like in the mouse brain has been demonstrated and there is accumulating evidence for a role of the Notch signaling pathway not only in early events during cell fate specifications but also in stem cells, in already differentiated neuronal cells and in neurodegeneration in the adult vertebrate nervous system. Furthermore, the view that only the production and deposition of Aβ plays a decisive role in AD has been challenged by recent evidence from different model systems that attribute numerous functions to APP and derivatives thereof. These findings together with the current data make it likely that alterations in the processing of APP either during the onset and progression of AD or by the use of therapeutics may result in loss- as well as in gain-of-function phenotypes contributing to the disease or side effects (Merdes, 2004).

Mouse disabled 1 regulates the nuclear position of neurons in a Drosophila eye model

Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Disabled 1, which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhances the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduces the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling that regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons (Pramatarova, 2006).

How Notch establishes longitudinal axon connections between successive segments of the Drosophila CNS

Development of the segmented central nerve cords of vertebrates and invertebrates requires connecting successive neuromeres. This study shows both how a pathway is constructed to guide pioneer axons between segments of the Drosophila CNS, and how motility of the pioneers along that pathway is promoted. First, canonical Notch signaling in specialized glial cells causes nearby differentiating neurons to extrude a mesh of fine projections, and shapes that mesh into a continuous carpet that bridges from segment to segment, hugging the glial surface. This is the direct substratum that pioneer axons follow as they grow. Simultaneously, Notch uses an alternate, non-canonical signaling pathway in the pioneer growth cones themselves, promoting their motility by suppressing Abl signaling to stimulate filopodial growth while presumably reducing substratum adhesion. This propels the axons as they establish the connection between successive segments (Kuzina, 2011).

The axons of the longitudinal pioneer interneurons of the Drosophila ventral nerve cord establish the initial connection between successive segments of the animal. The receptor Notch is crucial for making those first connections, performing two parallel, partially redundant but completely separate functions. Canonical Notch signaling in the interface glia constructs an unbroken track for longitudinal pioneer axons to follow by shaping a continuous band of neuronal membrane that bridges from segment to segment. Simultaneously, non-canonical Notch/Abl signaling in the pioneer neurons themselves promotes the motility of their growth cones, suppressing the activity of the Abl tyrosine kinase signaling module to stimulate filopodial development. Either signaling mechanism provides substantial rescue of a Notch mutant, but both are required for full activity in formation of longitudinal connections of the CNS (Kuzina, 2011).

The mechanism that guides the very first axon to establish the path of a nascent nerve is one of the most fundamental problems in neural development. For longitudinal pioneers of the Drosophila CNS, it is now seen that constructing their path requires coordinated contributions from four interacting cell types. First, the axons of commissural interneurons bearing the Notch ligand Delta contact interface glia. The glia respond by activating canonical Notch signaling, enhancing expression of Notch target genes, including prospero. The genetic program stimulated by Notch directly or indirectly allows the glial cells to attract a 'cap' of fine filopodial processes from nearby differentiating neurons, and shapes that cap into a continuous longitudinal band that bridges between segments. The neuronal cap atop the glia bears the Netrin receptor Frazzled (DCC), which in turn recruits soluble Netrin, thus constructing a domain of accumulation of Netrin protein that hugs the surface of the associated glia. Finally, the pioneer growth cones advance along the edge of that domain of immobilized Netrin until they meet and fasciculate with their partners pioneering from the next segment. The consequence of this choreography is a nerve trajectory that follows, indirectly, the shape of the row of interface glia (Kuzina, 2011).

This view suggests plausible explanations for many aspects of longitudinal axon development that have, up to now, been confusing. Previous investigators have documented that the pioneer growth cones extend amidst a thicket of filopodia that cap the interface glia. The provenance and significance of those filopodia were unknown, though it was clear that they did not derive from other axons. It is now seen that these filopodia come from surrounding, differentiating neurons, and that their function is to hold Frazzled, and therefore Netrin, in a pattern dictated by the positions of the overlying glia, creating the Netrin domain along which the pioneers extend. This explains why the positions of the interface glia correlate so closely with the axon trajectory even though the glia are not the direct substratum. The data, along with other recent results, also suggest why previous experiments investigating the guidance function of interface glia might have given such confusing results. Transformation of the glia into neurons in a gcm mutant would be predicted to place a row of DCC-expressing neurons in precisely the position of the wild-type filopodial carpet. Moreover, genetic experiments ablating or displacing the glia have relied on reagents targeting the progeny of the longitudinal glioblast, but it is now known that only nine of the ten interface glia come from this precursor; the tenth, M-ISNG, is from a different lineage. M-ISNG is appropriately positioned to anchor the filopodial carpet in the absence of the other interface glia and preliminary experiments suggest that it is sufficient for this. Moreover, it has been argued that in those rare segments where pioneer axons stall owing solely to manipulations of the interface glia, all ten of them, including M-ISNG, tend to be absent or displaced. Finally, as in previous studies, this stud found that the Netrin zygotic mutant has a mild, and genetically enhanceable phenotype, showing that the null for the gene is not null for the genetic pathway. It might be that there is a maternal contribution to Netrin, as there is for frazzled. Alternatively, because the receptor on longitudinal pioneers presumably recognizes a Netrin-Frazzled complex, it might be that this receptor has some affinity for Frazzled even in the absence of Netrin. Identification of the missing Netrin receptor will be necessary to clarify this point. It also seems likely that other neuronal components cooperate with the Netrin-Frazzled complex on the meshwork to provide substratum function, as expression of Fra in interface glia is not sufficient to rescue the Notch axonal phenotype (Kuzina, 2011).

Once the pathway for an axon has been constructed, there remains the problem of driving the motility of the growth cone along that pathway. Somehow, the information encoded in a pattern of occupancy of cell surface receptors must be transformed into a pattern of cytoskeletal dynamics that drives growth cone motion. At the level of the axon, this is the bedrock problem in axon guidance, and it, too, has resisted analysis. The current data reveal how Notch modulates an elementary property of the actin cytoskeleton to promote motility of longitudinal pioneer growth cones. Through its antagonism of the Abl signaling network, Notch de-represses the Abl antagonist Enabled and suppresses the Rac GEF Trio. Enabled directly promotes filopodial growth; suppressing Rac indirectly promotes filopodia, probably by redirecting various factors away from lamellipodia. Stimulating filopodial development probably promotes longitudinal axon growth in at least two ways. First, converging pioneer growth cones from successive segments need to encounter one another and fasciculate to establish the connection between segments. Extension of filopodia increases the area searched by an advancing growth cone, increasing the probability that it will encounter its partners advancing from the adjacent segment. Second, filopodia promote neurite growth by promoting microtubule invasion of the leading edge (Kuzina, 2011).

In parallel with stimulating filopodia, suppression of Trio, and thus of Rac, is expected to reduce substratum adhesion. When the pioneers are growing towards the segment border, small gaps in the Frazzled-Netrin pattern are not uncommon, so release of the advancing growth cone from the substratum is likely to aid its forward motion. Moreover, initially there is more Frazzled-bound Netrin within the neuromeres than there is at the segment border, requiring advancing pioneers to go down a gradient of Netrin, towards a region with less Netrin. It is possible that both of these properties make it helpful to limit substratum adhesion of the growth cone via reduction of Rac signaling by Notch (Kuzina, 2011).

It might seem paradoxical that Notch promotes axon growth by suppressing Abl signaling when Abl has been the archetype of a motility-promoting signaling pathway. Indeed, genetic studies of Abl in axon guidance have often appeared to be confusing and contradictory. In part, this reflects pleiotropy. Abl appears to act in the glia, and in the cells providing the filopodial carpet, in addition to the pioneers. As the phenotype in a whole-animal mutant reflects the sum of unrelated functions in different cells, seemingly similar experiments can produce contradictory results if different cellular processes become limiting. For longitudinal axons, for example, if pathway establishment is limiting (in Abl- or fra- animals), reduction of Notch interferes with axon growth synergistically; if growth cone function is limiting (in Notch- animals), reduction of the Abl pathway restores axon growth. It was therefore essential in the current work to control gene activity, and analyze phenotypes, in single, identified cells (Kuzina, 2011).

Beyond pleiotropy, however, complexity arises because the effect of signaling molecules in motility is profoundly context dependent. Ena promotes actin polymerization but often restricts cell motility; cofilin severs actin filaments but can promote net actin polymerization and cell migration. As axon growth is achieved by throughput through a cycle of actomyosin dynamics, it requires a balance among the steps of that cycle. Excessive activity or inactivity of any single step in the process inhibits motility by impairing progression through the cycle. The data now reveal that, for Drosophila longitudinal pioneers, an essential aspect of growth cone movement is restraint of Abl activity to allow filopodial development, and perhaps also to limit substratum adhesion (Kuzina, 2011).

The data reported in this study reveal that Notch promotes CNS longitudinal axon growth in two very different ways, constructing a pathway using its canonical signaling mechanism and promoting motility via the Notch/Abl interaction. This dual role bears striking parallels to the dual role of Notch in radial migration of neurons in the mammalian cortex. There, as in the fly, canonical signaling by Notch is essential for the development of glial cells that define a migration pathway, whereas interaction with the Abl pathway protein Disabled controls neuronal motility and adhesion. Further study will be required to assess whether these parallels between Notch function in the fly and vertebrate nervous systems reflect a deeper mechanistic similarity. Similarly, it will be interesting to see whether formation of longitudinal nerve tracts in the spinal cord uses machinery homologous to that which this study has described in the fly (Kuzina, 2011).

Protein Interactions

Drk, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the Sevenless receptor tyrosine kinase (Sev). One role of Drk is to provide a link between activated Sev and the Ras1 activator Sos. Drk-binding proteins other than Sos may play important roles in signaling by Sev and other RTKs. Biochemical studies performed with mammalian systems have provided evidence that such Grb2-binding partners do exist. These include Cbl, a proto-oncogene product, and GAB1, a downstream component of the insulin and epidermal growth factor receptors. The possibility that Drk performs functions other than binding to Sos has been been investigated by identification of additional Drk-binding proteins. The phosphotyrosine-binding (PTB) domain-containing protein Disabled (Dab) binds to the Drk SH3 domains (Le, 1998).

To characterize the nature of the in vitro Dab-Drk interaction, it was necessary to determine which domains of Drk are required for binding to Dab. To answer this question, well-characterized mutations were used that had been shown to inactivate the function of either the SH2 or SH3 domain of Grb2. For example, changing the proline 49 residue to leucine (P49L) inactivates the N-terminal SH3 domain, while the arginine 86-to-lysine (R86K) mutation disrupts the SH2 domain and the glycine 203-to-arginine (G203R) mutation affects the C-terminal SH3 domain. The corresponding mutations were introduced, individually or in combination (P49L, R85K, G199R, P49L/G199R), into the [32P]GTK-DRK fusion protein and the ability of the mutant proteins to interact with the lambda gt11-encoded beta-galactosidase-DAB fusion protein was tested. Mutation of the SH2 domain does not affect binding, indicating that the in vitro Dab-Drk interaction does not require a functional Drk SH2 domain. However, the Dab-Drk interaction is dependent on the function of the SH3 domains because simultaneous mutations of both SH3 domains abolish binding. Moreover, while Dab binds to both SH3 domains, it appears to interact more strongly with the C-terminal domain. In addition, in vitro interaction between Drk and Dab requires the presence of the proline-rich region of Dav and suggests that the SH3 domains of Drk bind directly to sequences within the Dab proline-rich core (Le, 1998).

Dab is expressed in the ommatidial clusters; loss of Dab function disrupts ommatidial development. Intense anti-Dab staining is observed both in the morphogenetic furrow and in developing ommatidial clusters posterior to the furrow. An apical-to-basal cross section reveales that Dab is localized to a small region just below the apical surface of the retinal epithelium. To determine which cells express Dab, the discs were costained with an antibody to ELAV, a neuronal marker present in the nuclei of developing and mature photoreceptors. The results from these experiments show that Dab accumulates at the apical membrane of the developing photoreceptor cells. However, it is not possible to assign Dab expression to particular photoreceptors due to the apical constriction of these cells. The subcellular localization of Dab is similar to that of Drk, consistent with its role as a Drk-binding partner (Le, 1998).

Numerous abnormalities are observed in Dab homozygous mutant clones. The most common defects are the absence of the R7 cell and the lack of one or more outer photoreceptors (R1 to R6) in mosaic ommatidia. In addition, large Dab mutant clones show extensive ommatidial disorganization. including regions in which no photoreceptors are present. This phenotype is observed with three different alleles of Dab and resembles those observed in clones of cells homozygous for weak alleles of either Sos or Ras1. These results indicate that Dab has an important function during photoreceptor and ommatidial development. Reduction of Dab function attenuates signaling by a constitutively activated Sev. Biochemical analysis suggests that Dab binds Sev directly via its PTB domain, becomes tyrosine phosphorylated upon Sev activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that Dab is a novel component of the Sev signaling pathway (Le, 1998).

Using Bcr-Abl to examine mechanisms by which abl kinase regulates morphogenesis in Drosophila; The interaction of Abl and Ena

Signaling by the nonreceptor tyrosine kinase Abelson (Abl) plays key roles in normal development, whereas its inappropriate activation helps trigger the development of several forms of leukemia. Abl is best known for its roles in axon guidance, but Abl and its relatives also help regulate embryonic morphogenesis in epithelial tissues. This study explores the role of regulation of Abl kinase activity during development. First the subcellular localization of Abl protein and of active Abl were compared, by using a phosphospecific antibody, providing a catalog of places where Abl is activated. Next, the consequences for morphogenesis of overexpressing wild-type Abl or expressing the activated form found in leukemia, Bcr-Abl, were explored. Dose-dependent effects of elevating Abl activity were found on morphogenetic movements such as head involution and dorsal closure, on cell shape changes, on cell protrusive behavior, and on the organization of the actin cytoskeleton. Most of the effects of Abl activation parallel those caused by reduction in function of its target Enabled. Abl activation leads to changes in Enabled phosphorylation and localization, suggesting a mechanism of action. These data provide new insight into how regulated Abl activity helps direct normal development and into possible biological functions of Bcr-Abl (Stevens, 2008).

Loss-of-function mutations in abl disrupt many morphogenetic events. To further the mechanistic understanding of the roles of Abl, whether deregulated kinase activity disrupts morphogenesis waas examined. Inappropriate activation of Abl affects many of the same morphogenetic events disrupted by loss of Abl. Normally, Abl is likely to exist primarily in an inactive form. Docking with ligands for the SH2 or SH3 domains may help trigger the active conformation. Embryos are relatively resistant to overexpression of wild-type Abl. It is suspected that increasing protein levels are largely accommodated by normal regulatory mechanisms until levels become extremely high. Consistent with this, it was found that active Abl has a more restricted localization than total Abl, suggesting that Abl activation is normally restricted to the apical cell cortex and in particular to tricellular junctions, with a pool of inactive Abl in the cytoplasm. Increasing wild-type Abl levels may drive formation of more active Abl, or it may titrate negative regulators (Stevens, 2008).

Misexpression of Bcr-Abl has more drastic consequences on morphogenesis, consistent with the constitutive activation of Bcr-Abl. In some cases, effects were simply quantitatively stronger, e.g., both Abl and Bcr-Abl affected head involution and segment grooves. However, other processes such as dorsal closure were only affected by Bcr-Abl. These processes may simply be less sensitive, affected only by very high level Abl activity. Alternately, Bcr-Abl may have cell biological effects in Drosophila distinct from those of Abl (Stevens, 2008).

The best-known target of Drosophila Abl is Ena. Abl negatively regulates Ena, in part by restricting its localization. This study examined effects on embryogenesis of depleting maternal and zygotic Ena (enaM/Z). This allows evaluation of which effects of Abl activation result from negative regulation of Ena (Stevens, 2008).

Ena loss-of-function and Abl activation share striking similarities. Both disrupt head involution. In both segmentalgrooves are substantially deepened and persist long after they normally retract. Finally, both alter cell behavior during dorsal closure in similar ways: dorsal closure is significantly slowed, leading edge cells produce fewer filopodia, and epithelial cell matching and zippering are disrupted. These data are consistent with the idea that Ena is the major target of both Abl and Bcr-Abl during Drosophila morphogenesis. This mechanism of action is further supported by other data. First, reduction in Ena levels enhances effects of Bcr-Abl overexpression. Second, overexpression of GFP-Ena partially rescues the effects of Abl activation on filopodial behavior. Finally, Ena localization is regulated by Abl. In abl loss-of-function mutants, Ena accumulates inappropriately at the apical cortex of early embryos and at the leading edge during dorsal closure. In contrast, in embryos overexpressing wild-type Abl, Ena is lost from places it normally accumulates (e.g., tricellular junctions), and it localizes instead at lower levels all around the cell cortex and in the cytoplasm. These data are consistent with Ena being a key Abl target (Stevens, 2008).

Current models of Ena function provide good insight into some of the biological and cell biological effects of Abl activation. Both Ena inactivation and Abl activation led to a reduction in filopodia produced by leading edge cells and defects in the last stages of dorsal closure. These roles fit well with the role of Ena as an anti-capping protein that may also mediate filament bundling into filopodia. Reduction in Abl function leads to the formation of excess filopodia with elevated levels of Ena at the tips, further supporting this regulatory mechanism. Abl activation and Ena loss of function also have parallel effects on head involution and segmental groove formation. In both biological events a row of cells adopts an unusual localization of Ena, with elevated Ena levels and Ena planar polarized at the dorsal-ventral cell boundaries. It seems reasonable that the substantial alterations of Ena subcellular localization caused by Abl activation could interfere with Ena function in these key subsets of cells. However, it remains unclear precisely how localized Ena activity contributes to the distinctive cell shape changes of cells of the segmental grooves or head fold (Stevens, 2008).

One key question is the mechanism(s) by which Abl regulates Ena. The data above and earlier loss-of-function experiments are consistent with a model in which Abl regulates Ena localization, restricting its activity to places it is essential. Abl may form a complex with Ena, keeping it in an inactive state. Consistent with this, Abl can bind Ena, Ena and active Abl colocalize to tricellular junctions and leading edge cell AJs, and Abl overexpression or Bcr-Abl misexpression leads to elevated Abl activity all around the cell cortex, disrupting normal Ena localization. Perhaps Abl docks inactive Ena at sites near where its activity will be needed. For example, Ena at leading edge cell AJs could be the source of Ena needed at the leading edge to make filopodia. Although this model is attractive, some data cannot be easily accommodated by it, e.g., Abl and active Abl both localize to the cortex of syncytial embryos, but Ena is not normally localized there, and Ena localizes there in the absence of Abl, suggesting that there may be alternate mechanisms by which Abl regulates Ena. Further experiments are needed to test these hypotheses (Stevens, 2008).

One way Abl may regulate Ena is by phosphorylation. The effects on embryogenesis of Abl and Bcr-Abl require kinase activity. However, mutating all the Ena phosphorylation sites does not lead to the 'activated' phenotype expected if this is the sole mechanism of negative regulation (i.e., mimicking abl loss-of-function); instead, it has a weak ena loss-of-function phenotype. Thus, Abl regulation of Ena involves more than direct phosphorylation. Abl may phosphorylate itself and other partners, creating or disrupting protein complexes. Consistent with this, although Ena phosphorylation sites are not conserved in its mouse homologues, mouse Abl promotes Mena phosphorylation and binds VASP. Given the clear ability of Bcr-Abl to alter Ena localization/activity in Drosophila, further exploration of Ena/VASP proteins as possible targets in mammalian cells seems warranted (Stevens, 2008).

Although many effects of Abl activation can be explained by negative regulation of Ena, a subset of the effects suggest alternate targets. For example, effects of Abl overexpression on leading edge cell behavior are more drastic than those seen in enaM/Z mutants, e.g., reduced lamellipodial activity was not seen after Ena was inactivated, and high-level Bcr-Abl expression during embryogenesis is more detrimental than Ena loss. Thus, both Abl and Bcr-Abl likely have Ena-independent effects on actin and cell behavior in Drosophila (Stevens, 2008).

One critical issue in interpreting these results is whether Bcr-Abl acts in Drosophila simply as a deregulated form of Abl, or whether it has additional effects on cell behavior. In most of assays, Bcr-Abl expression has effects similar to but stronger than those of Abl overexpression. In some cases, high-level Bcr-Abl expression affected processes not affected by high-level Abl overexpression (e.g., amnioserosa integrity), but sufficient levels of wild-type Abl overexpression may not have been achieved to mimic them. However, one striking effect of Bcr-Abl was not seen either with Abl overexpression or Ena loss-of-function: the explosive production of lamellipodia by amnioserosal cells, which normally only produce filopodia. Perhaps the Bcr part of the fusion recruits additional proteins that influence its abilities. Alternately Bcr-Abl may stimulate signaling pathways such as those of c-Jun NH2-terminal kinase or mitogen-activated protein kinase, targets of mammalian Bcr-Abl; both affect Drosophila epidermal cell shape or fates. Further studies of the mechanisms of action of Bcr-Abl in Drosophila may offer clues into additional targets of Abl (Stevens, 2008).

Although both Abl and Bcr-Abl modulate actin dynamics, the cytoskeletal response they program is complex. In fibroblasts, loss of Abl prevents ruffling in response to PDGF, whereas loss of Arg reduces lamellipodial dynamics. These data suggest that Abl regulates formation of branched actin filaments involved in lamellipodia, consistent with its ability to speed migration. Many of the current observations are consistent with this, including reduced filopodial number after Abl activation, and Bcr-Abl-triggered lamellipodia. Likewise, Drosophila Abl inhibits dendrite branching. However, in other contexts, Abl modulates actin differently. Mouse Abl and Arg maintain dendrite branching, and Abl promotes actin microspikes in fibroblasts plated on fibronectin. Both are consistent with promoting unbranched actin. Bcr-Abl expression also has distinct effects in different cell types, triggering ruffling and filopodial extension in BaF3 cells, while preventing spreading and polarization on fibronectin in dendritic cells (Stevens, 2008).

Bcr-Abl adds additional complexity. Distinct effects were seen of Bcr-Abl expressed at different levels. This dose sensitivity mimics that seen in myeloid cells expressing different levels of Bcr-Abl, which differ in adhesion to fibronectin and ability to induce tumors. A second complexity involves differences between p210 and p185. In Drosophila, p210 produced consistently stronger phenotypes and also triggered higher levels of tyrosine-phosphorylated proteins. p185 and p210 differ in their biochemical and biological activities in mammals as well, and p185 and p210 cause distinct diseases in patients, and induce different pathways of differentiation in primary bone marrow cells. However, in human cells, p185 is the more active kinase. Further exploration of these functional distinctions will help illuminate the different pathways activated by Abl and Bcr-Abl during normal development and oncogenesis (Stevens, 2008).

Thus, both Abl and Bcr-Abl have distinct and at times seemingly opposite cytoskeletal effects in different cells. Perhaps this is not surprising, given the array of cytoskeletal regulators Abl can target, including those promoting unbranched actin filaments, such as Ena/VASP, and those regulating Arp2/3 and branching, such as WASP and WASP family Verprolin-homologous protein. The choice of target may be dictated by upstream inputs regulating Abl, and the consequences for actin dynamics will depend on the suite of other regulators active in the same cell. Understanding how individual cells integrate these inputs and outputs is one challenge for the future (Stevens, 2008).

Notch interaction with Disabled regulates axon patterning

Notch is required for many aspects of cell fate specification and morphogenesis during development, including neurogenesis and axon guidance. Genetic and biochemical evidence is provided that Notch directs axon growth and guidance in Drosophila via a 'non-canonical', i.e. non-Su(H)-mediated, signaling pathway, characterized by association with the adaptor protein, Disabled, and Trio, an accessory factor of the Abl tyrosine kinase. Forms of Notch lacking the binding sites for its canonical effector, Su(H), are nearly inactive for the cell fate function of the receptor, but largely or fully active in axon patterning. Conversely, deletion from Notch of the binding site for Disabled impairs its action in axon patterning without disturbing cell fate control. Finally, it was showm by co-immunoprecipitation that Notch protein is physically associated in vivo with both Disabled and Trio. Together, these data provide evidence for an alternate Notch signaling pathway that mediates a postmitotic, morphogenetic function of the receptor (Le Gall, 2008).

Previous studies have led to the speculation that the Abl tyrosine kinase and its associated accessory factors might define an alternate, 'non-canonical', Su(H)-independent signaling pathway for the receptor Notch. The data reported in this study provide strong support for this hypothesis. In extracts of wild type Drosophila, Notch is associated with Disabled and Trio, two proteins that have been associated with the action of Abl tyrosine kinase. The functions of Notch in axon growth and guidance are likely to be executed by these complexes of Notch with Disabled and Trio, and not by its association with Su(H), since deletion of the Disabled binding site from Notch significantly impairs the axon patterning function of the receptor, whereas the Su(H) binding sites are largely dispensable for this process. Moreover, two other Notch derivatives are described that are still capable of executing the axon patterning functions of the receptor despite being completely inactive for specifying Notch-dependent cell fates. Taken together with previous data demonstrating that the genetic interaction of Notch with multiple Abl pathway components is required specifically for Notch-dependent axon patterning, these data provide a molecular picture of a Notch signaling machinery that is distinct from the well-established mechanism by which a proteolytic fragment of Notch enters the nucleus to directly control transcription of Su(H)-dependent target genes (Le Gall, 2008).

The key genetic data in favor of this hypothesis stem from the targeted construction of Notch derivatives that preferentially impair either the canonical, cell fate function of the receptor or its Abl-dependent axon patterning function, respectively. Deletion of the Su(H) binding sites from Notch progressively and dramatically reduces the ability of the receptor to limit neurogenesis, but has only limited effect on growth of CNS longitudinal axons, and no detectable effect on Notch-dependent defasciculation of ISNb motor axons. In contrast, deletion of the Disabled binding site substantially reduces the axon patterning activity of Notch (30%-40%, depending on the assay), while having no effect on cell fate function beyond what can be accounted for by the known Su(H) binding site within the deletion. The properties of these complementary Notch mutants argue for the action of a qualitatively different Notch mechanism in axon patterning. Further supporting this hypothesis is the observation that Notch co-precipitates from wildtype fly extracts with two cytoplasmic signaling proteins, the Abl cofactor, Trio and the adaptor protein, Disabled, potentially providing a molecular machinery to account for the phenotypic data. In principle, a good way to further test the basis of the Notch axonal phenotype would be to examine a disabled mutant, but unfortunately no such mutants are currently available. The phenotype of a trio mutant, in contrast, is consistent with the results documented in this study. The zygotic mutant phenotypes of trio are somewhat subtle, evidently because of persistence of maternally-provided trio RNA and protein, but they include defects in some of the CNS longitudinal axons that are affected in Notchts embryos, as well as defects in ISNb motor axon guidance, while trio has not been reported to produce any Notch-like defects in cell fate (Le Gall, 2008).

While deletion of the Disabled binding region of Notch clearly reduces the axonal activity of the protein, substantial residual activity still remains. In the case of Su(H), residual activity of a Notch mutant lacking all in vitro Su(H) binding sites can be traced to an association of Su(H) with the Notch ankyrin repeats that requires the cofactor, Mastermind. By analogy, perhaps Disabled can also associate with Notch via a second site that requires a cofactor present in vivo. Consistent with this idea, preliminary biochemical experiments hint that the RamΔA mutation (Notch deleted for the Disabled binding site) does not wholly ablate recruitment of Disabled and Trio in vivo, though current reagents do not allow this to be assessed rigorously. If so, the ankyrin/cdc10 repeat region would be a plausible candidate for a secondary site of association. Experiments show that the ankyrin repeats, together with the C-terminal portion of the Ram region, contribute substantially to Notch-dependent axon patterning. Since the experiments clearly show that the canonical Notch signaling pathway is dispensable for axonal function, the axonal requirement for this portion of the protein cannot be traced to its function in canonical signaling, and a contribution to formation or activity of Notch/Abl pathway complexes would offer the simplest explanation (Le Gall, 2008).

To date, the role of Disabled in the Abl signaling pathway has been difficult to establish due to the lack of loss-of-function Disabled mutant alleles. The placement of Disabled in the Abl pathway was based initially largely upon the observation that modest overexpression of Disabled suppressed both the embryonic lethality and morphological defects produced by genetic interactions of Abl with its accessory genes Nrt and Fax. The data now show that deletion of the Disabled binding region of Notch specifically impairs a Notch function, axon patterning, that depends on the interaction of Notch with multiple Abl pathway components. Moreover, GAL4-driven overexpression of Disabled modifies the Notch ISNb phenotype in the same way as do other treatment that enhance Abl signaling, such as overexpression of Abl or reduction of enabled. Thus, these data provide further support for association of Disabled with the Abl signaling pathway, though a definitive demonstration awaits the generation and characterization of a disabled mutation (Le Gall, 2008).

The presence of Trio in Notch complexes suggests that Rho family GTPases, particularly Rho and Rac, are good candidates for a downstream readout of Notch/Abl signaling. Consistent with this, dominant genetic interactions of Notch were observed with mutations in the three Drosophila Rac genes, but not, for example, with Cdc42. Such a readout would make sense in the context of the effects of Notch on growth cone guidance and would be consistent with previous studies of Drosophila Trio. It is interesting to note that identification of Rac as an effector of Notch/Abl signaling might suggest the possibility of Notch/Abl signaling having a non-Su(H) nuclear component in some developmental contexts in addition to its cytoskeletal targets. Rho family GTPases typically have multiple downstream targets, including nuclear gene regulation in addition to cytoskeletal structure and dynamics (Le Gall, 2008).

The key step in canonical Notch signaling is the proteolytic cleavage of the receptor by γ-secretase to release the active, intracellular moiety of the molecule, NICD. Does γ-secretase also play a role in Notch/Abl signaling. Since Disabled and Trio associated were found with full-length Notch prior to cleavage, and since Notch/Abl signaling in the growth cone presumably targets the cortical actin cytoskeleton, one possibility is that γ-secretase cleavage terminates the Notch/Abl signal by separating the receptor-bound complex from membrane-tethered components of the pathway such as Abl kinase and Rho GTPases. Alternatively, in contexts such as ISNb, perhaps displacement of Disabled and Trio away from the membrane is part of the mechanism by which Notch antagonizes Abl pathway activity. Moreover, while proteolytic activity is the most apparent function of γ-secretase there have been suggestions that the complex may also have a separate function in Notch trafficking, aside from cleavage. If so, this activity could modulate Notch/Abl signaling independent of any role for protease cleavage. Clearly, additional experiments will be necessary to assess the various possible models (Le Gall, 2008).

Is the interaction of Notch with Abl pathway proteins limited to just a few Drosophila growth cones, or is it of more general biological significance. The ability to detect Notch complexes with Disabled and Trio in extract of whole embryos argues for the latter, as does the strong phylogenetic conservation of all the components of the pathway. Good candidates for potential Notch/Abl-dependent processes are provided by those developmental contexts in which non-Su(H) Notch signaling has been proposed previously. In Drosophila, these include organization of actin structure at the D/V boundary of the developing wing; in mammals, they include myogenesis and B-cell development, as well as a ligand-stimulated cytoplasmic signaling process of Notch that is essential for the survival of mouse neural stem cells and human embryonic stem cells (Le Gall, 2008).

Axon guidance and classic lateral inhibition seem to represent limit cases in which the Notch signal is largely transduced selectively through either the Abl pathway or the Su(H) pathway, respectively. It seems likely, however, that in each case both pathways make some contribution to Notch function: deletion of Su(H) binding sites does have some deleterious effect on growth of CNS longitudinal axons, while mutation of Abl and Nrt cause small but reproducible decreases in the efficacy of the classic Notch function that discriminates the identities of sibling cells. Perhaps two parallel Notch signals, one through the canonical Su(H) pathway and the other mediated by the Notch/Abl interaction, can be used in concert to provide a richer nuclear readout, or to coordinate nuclear gene regulation with cortical properties such as cytoskeletal structure and cell adhesion. It will be of great interest to determine whether some classic functions of Notch, such as dendritic patterning or oncogenesis, reflect more balanced contributions both from canonical Notch signaling and from the Notch/Abl pathway (Le Gall, 2008).

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

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

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