Abl tyrosine kinase


Effects of Mutation or Deletion (part 1/2)

During Drosophila embryogenesis, the Abelson tyrosine kinase is localized in the axons of the central nervous system (CNS). Mutations in Abl have no detectable effect on the morphology of the embryonic CNS, and the mutant animals survive to the pupal and adult stages. In the absence of Abl function, however, heterozygous mutations or deletions of Disabled (Dab) exert dominant effects, disrupting axonal organization and shifting the lethal phase of the animals to embryonic and early larval stages. Embryos that are homozygous mutant for both Abl and Dab fail to develop any axon bundles in the CNS, although the peripheral nervous system and the larval cuticle appear normal. The genetic interaction between these two genes begins to define a process in which both the Abl tyrosine kinase and the Dab gene product participate in establishing axonal connections in the embryonic CNS of Drosophila (Gertler, 1989).

Drosophila Armadillo and its vertebrate homolog beta-catenin have dual roles in epithelial cells: transducing signals from the Wingless/Wnt family of proteins and working with cadherins to mediate cell adhesion. Wingless/Wnt signaling also directs certain cell fates in the central nervous system (CNS), and cadherins and catenins are thought to function together during neural development. An analysis of the biochemical properties of a second Armadillo isoform, with a truncated carboxyl terminus generated by alternative splicing, was carried out. This isoform accumulates in differentiating neurons. Using armadillo alleles that selectively inactivate the cell adhesion or the Wingless signaling functions of Armadillo, Armadillo was found to have two sequential roles in neural development. Armadillo function in Wingless signal transduction is required early in development for determination of neuroblast fate. Later in development, disruption of the cell-cell adhesion function of Armadillo results in subtle defects in the construction of the axonal scaffold. Mutations in the gene encoding the Drosophila tyrosine kinase Abelson substantially enhance the severity of the CNS phenotype of armadillo mutations, consistent with these proteins functioning co-operatively at adherens junctions in both the CNS and the epidermis. This is one of the first demonstrations of a role for the cadherin-catenin system in the normal development of the CNS. The genetic interactions between armadillo and abelson point to a possible role for the tyrosine kinase Abelson in cell-cell adhesive junctions in both the CNS and the epidermis (Loureiro, 1998).

Three second-site mutations that suppress the lethality caused by the absence of Abl oncogene function have been isolated, and all three map to the gene enabled. The mutations are recessive embryonic lethal mutations but act as dominant mutations to compensate for the neural defects of Abl mutants. Thus, mutations in a specific gene can compensate for the absence of a tyrosine kinase (Gertler, 1990).

Mutations in the Drosophila Abelson tyrosine kinase have pleiotropic effects late in development that lead to pupal lethality or adults with a reduced life span, reduced fecundity and rough eyes. Evidence for ABL function was obtained by analysis of mutant phenotypes in the embryonic somatic muscles and the eye imaginal disk. The defects in musculature are apparent by late stage 16; at this point many muscle fibers are absent, and the remainder are often thin and disorganized. In mutant eyes there is a slightly reduced number of facets, missing and supernumerary bristles, and facets irregular in shape and size. Sections reveal defects in photoreceptor cells, cone cells and pigment cells. The expression patterns and mutant phenotypes indicate a role for ABL in establishing and maintaining cell-cell interactions (Bennett, 1992).

Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identify alleles of only one gene, enabled . The Ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains. Ena is also a substrate for the Abl kinase; tyrosine phosphorylation of Ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous Ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, Ena is expressed at highest levels in the axons of the embryonic nervous system. ena mutant embryos have defects in axonal architecture. It is concluded that a critical function of Drosophila Abl is to phosphorylate and negatively regulate Ena protein during neural development (Gertler, 1995).

The axonal localization of the Drosophila Abl protein and its genetic interactions with Dab and fasciclin I (Abl has a synergistic effect with fas I in axon guidance) implicate Abl in axonal pathfinding. Several changes at the amino terminus of Abl permit proper function and localization of the altered protein. In contrast, the presence of human c-Abl type 1a amino-terminal sequences or the murine c-Abl carboxy-terminal domain interfers with function and axonal localization. Rescue of phenotypes caused by mutations in Abl alone does not require tyrosine kinase activity, indicating a novel kinase-independent function for the properly localized Abl protein. However, Abl kinase activity is required to rescue the mutant phenotypes in genetic backgrounds also mutant for disabled (Henkemeyer, 1990).

Drosophila Fasciclin I is a homophilic cell adhesion molecule expressed in the developing embryo on the surface of a subset of fasciculating CNS axons, all PNS axons, and some nonneuronal cells. Protein-null mutations in the fasciclin I gene are viable and do not display gross defects in nervous system morphogenesis. The Drosophila Abl oncogene homolog encodes a cytoplasmic tyrosine kinase that is expressed during embryogenesis primarily in developing CNS axons; Abl mutants show no gross defects in CNS morphogenesis. However, embryos doubly mutant for fas I and Abl display major defects in CNS axon pathways, particularly in the commissural tracts where expression of these two proteins normally overlaps. The double mutant shows a clear defect in growth cone guidance; for example, the RP1 growth cone (normally fas I positive) does not follow its normal path across the commissure (Elkins, 1990).

Fasciclin II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In fasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and as a result, the corresponding sensory precursors fail to develop. In fasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in Abl oncogene show dominant interactions with fasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression (Garcia-Alonso, 1995).

Given the mild phenotypes of Abl mutant animals, it is possible to design genetic screens to identify mutations in genes that enhance or suppress the Abl mutant phenotypes. It has been hypothesized that in a genetic background sensitized by Abl mutations, a 50% reduction in the level of a protein that is regulated by Abl might be sufficiently detrimental to shift the lethal phase from the pharate adult stage to an embryonic or early larval stage. This effect is called haploinsufficiency dependent on an Abl mutant background (HDA). The genes identified are not haploinsufficient themselves but manifest their effects when the fly is also mutant for Abl. disabled and prospero are two of the genes identified by this strategy. Although Abl mutants exhibit no visible defects in the embryonic central nervous system (CNS), animals that are doubly mutant for Abl die as embryos and fail to form proper axonal connections in the CNS. Heterozygous deletions of pros in the absence of Abl cause embryonic and larval lethality. Examination of these embryos reveals, for the most part, normal axonal architecture in the CNS and peripheral nervous system, with variably penetrant subtle defects in the CNS, including fusion of the anterior and posterior commissural axon bundles. prosM4 homozygous mutant embryos display a segmentally repeated pattern of disrupted axon bundles in each neuromere. The longitudinal axons, which extend to the anterior and posterior between segments, are absent. The midline space between the two halves of the nervous system is wider than normal, with a loss of some midline cells of unknown identity. The anterior and posterior commissural axon bundles that cross the midline in each segment are replaced by a single axon bundle (Gertler, 1993).

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, Disabled, reveals that the two proteins are likely to function together in a process either downstream of the Abl protein tyrosine kinase or in parallel with it (Hill, 1995).

Drosophila Fasciclin I is the prototype of a family of vertebrate and invertebrate proteins that mediate cell adhesion and signaling. The midline fasciclin gene (FlyBase ID: FBgn0024211)encodes a second Drosophila member of the Fasciclin I family. Midline fasciclin largely consists of four 150 amino acid repeats characteristic of the Fasciclin I family of proteins. Hydrophobicity plot analysis of the protein sequence suggests that Mfas is secreted and/or associated with the cell surface. The N-terminus of Mfas has a positively charged arginine followed by a stretch of 16 predominantly hydrophobic residues, a feature that resembles a membrane signal sequence. There are 9 potential glycosylation sites scattered throughout the second half of the protein. Immunostaining and biochemical analysis using antibodies to Midline fasciclin indicates that the protein is membrane-associated, although the sequence does not reveal a transmembrane domain. The gene is expressed in a dynamic fashion during embryogenesis in the blastoderm, central nervous system midline cells, and trachea, suggesting it plays multiple developmental roles. Protein localization studies indicate that Midline fasciclin is found within cell bodies of midline neurons and glia, and on midline axons. Initial cellular analysis of a midline fasciclin loss-of-function mutation reveals only weak defects in axonogenesis. However, embryos mutant for both midline fasciclin and the abelson nonreceptor tyrosine kinase, show more severe defects in axonogenesis that resemble fasciclin I abelson double mutant phenotypes (Hu, 1998).

Abelson kinase regulates epithelial morphogenesis in Drosophila

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. This study reports a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. The cellular defects are described that underlie these problems, focusing on dorsal closure as an example. Further, it is shown that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. Ena localizes to adherens junctions of most epithelial cells, and it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. Possible models for Abl function during epithelial morphogenesis are discussed in light of these data (Grevengoed, 2001).

It is useful to compare what was observed in the epidermis with Abl's role in axon outgrowth, where it is thought to modulate communication between at least two transmembrane axon guidance receptors, Robo and dLAR, and the actin cytoskeleton. Abl is thought to antagonize Ena in this process. The ena and abl phenotypes were surprising in one respect. Ena/VASP proteins had been thought to enhance actin polymerization based on promotion of intracellular motility of the bacteria Listeria. However, while promoting actin polymerization might be expected to drive growth cone extension and axon outgrowth, Ena promotes growth cone repulsion and axon stalling, whereas Abl has opposite effects. Work in cultured fibroblasts led to similar conclusions: Ena/VASP proteins inhibit cell migration (Grevengoed, 2001).

Building on this model, Abl and Ena might play analogous roles in epithelial cells, translating extracellular signals into changes in the actin cytoskeleton. This sort of cytoskeletal modulation plays a key role in cell migration and cell shape changes during epithelial morphogenesis. One model consistent with these data is that Abl acts at adherens junctions during morphogenesis. Cadherins and catenins play important roles in morphogenesis in all animals. Severe reduction in Drosophila Arm or DE-cadherin function leads to early loss of epithelial integrity. Less severe reduction in cadherin/catenin function affects head involution, dorsal closure, and other morphogenetic processes. In fact, many epithelial defects of DE-cadherin mutants are blocked by blocking morphogenetic movements, suggesting that modulating adhesion is critical to morphogenesis (Grevengoed, 2001).

Several lines of evidence support the possibility that the morphogenetic defects of ablMZ mutants result, at least in part, from Abl action at adherens junctions. (1) The effects on dorsal closure, germband retraction, and head involution are strongly enhanced by reducing the dose of DE-cadherin. (2) The defects in cell shape during dorsal closure resemble, in part, those of arm mutants. (3) The defects in morphogenesis are suppressed by mutations in ena, which is primarily found at adherens junctions. (4) A reduction in junctional Arm and alpha-catenin is seen in ablMZ mutants. It is important to note, however, that any role for Abl at adherens junctions would be a modulatory one. It is not absolutely essential for adherens junction assembly or function. Of course, it remains possible that other tyrosine kinases may act redundantly with Abl. The relationship between the cadherin-catenin system, Abl, and Ena that may occur in epithelial cells could also exist in the CNS. Arm and DN-cadherin play roles in axon outgrowth in Drosophila, and in this role arm interacts genetically with abl (Grevengoed, 2001).

One target of Abl might be Ena, which could regulate actin dynamics in the actin belt underlying the adherens junction. Just as local modulation of actin dynamics likely regulates growth cone extension or stalling, the cell shape changes and cell migration characteristic of morphogenesis will require modulation of actin dynamics and junctional linkage. The idea that Ena may regulate cell-cell adhesion recently received strong support from work in cultured mammalian keratinocytes, where inhibiting Ena/VASP function prevented actin rearrangement upon cell-cell adhesion. This model is further supported by the demonstration that both Ab1 and Ena regulate actin polymerization at the adherens junctions of ovarian follicle cells in Drosophila (Grevengoed, 2001).

Other models are also consistent with these data. Abl may act directly on the actin cytoskeleton, with its effects on junctions a more indirect consequence. Junctional linkage to actin is critical for effective cell adhesion and alterations in actin polymerization could affect the ability to assemble stable cadherin-catenin complexes, as was observed in cultured mammalian cells, resulting in the observed loss of Arm from junctions. Abl could also play a more general role in the establishment and maintenance of cell polarity. Finally, studies of cultured mammalian cells also suggest that Abl acts at cell-matrix junctions to modulate responses to integrin-mediated adhesion by associating with and phosphorylating focal adhesion proteins like paxillin and Crkl. In doing so, it may influence both tethering to actin and signal transduction. Drosophila integrins play important roles in morphogenetic processes such as dorsal closure and germband retraction. No genetic interactions were detected between abl and scab, the integrin alpha-chain that plays a role in dorsal closure. However, this does not rule out interplay between integrins and Abl in morphogenesis. It is now important to test these different models by investigating the mechanism by which Abl and Ena act during morphogenesis (Grevengoed, 2001).

Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Genetics and cell biology have been combined to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. Profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, Capulet (Capt), the Drosophila homologue of Adenylyl Cyclase Associated Proteins, functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. It is therefore proposed that Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila (Baum, 2001).

Given that the Abl tyrosine kinase binds mammalian Capt and antagonizes the function of Ena in Drosophila, whether Abl, like Capt and Ena, might have a role in the control of F-actin organization in the follicular epithelium was tested. Clonal analysis reveals that loss of Abl causes subtle defects in F-actin organization. In abl4 mutant cells, apical actin filaments are often mislocalized, appearing at elevated levels at lateral cell cortices. In addition, abl clones exhibit severe defects in epithelial architecture, with mutant tissue forming a multilayered epithelium close to the posterior pole of the egg chamber and, to a smaller extent, at the anterior pole. A similar phenotype has been described in dlg/l(2)gl/scrib mosaic egg chambers. Because these genes are required for proper epithelial cell polarity, Abl might also regulate the polarity of follicle cells. As a further perturbation of Abl function the heat-shock Gal4 driver was used to express the protein at high levels within the follicular epithelium. Like the abl loss of function, high-level overexpression of Abl also perturbs epithelial architecture, leading to the formation of multiple layers of cells at the posterior pole of the egg chamber. Thus Abl functions to modulate follicle-cell F-actin organization and cell polarity and must be tightly regulated in follicle cells to maintain proper epithelial character (Baum, 2001).

To test genetically for an interaction between Abl and Capt, Abl was expressed at more moderate levels (using the T155 Gal4 driver) in egg chambers containing capt mutant clones. Although a modest overexpression of Abl has little visible effect on wild-type follicle cells, an increase in Abl expression in capt mutant follicle cells has profound effects. Increased levels of Abl protein alter both the level and distribution of actin filaments in capt mutant cells. The formation of large localized F-actin aggregates seems to be suppressed and actin filaments often become more widely distributed around the cell cortex, as observed in abl mutant clones. In addition, in a minority of egg chambers, the combination of Abl overexpression and loss of Capt causes a profound disruption of epithelial morphology. This genetic interaction between capt and Abl implies that the two genes have related functions in the control of epithelial F-actin organization (Baum, 2001).

Finally, having found genetic evidence to suggest that Ena and Abl cooperate with Capt in the control of epithelial F-actin, the distribution was examined of Ena and Abl proteins in capt mutant follicle cells. In the capt mutant, Ena's distribution is altered so that the majority of the protein becomes localized with apical actin filaments. Thus Ena is found tightly associated with apical F-actin both in the wild type and in capt mutant cells. In contrast, significant amounts of Ena are not observed at the apical surface of capt chic double-mutant cells, in which apical F-actin aggregates are not formed. Therefore, both in the wild type and in various mutants, the amount of Ena present at adherens junctions closely parallels the level of apical F-actin. The localization of Abl was also examined in the wild type and in capt mutant tissue. Abl, like Capt, seems to have a diffuse staining pattern within wild-type follicle cells. However, in capt clones, Abl protein becomes concentrated at the apical cell surface, partly localizing with Ena. Thus, a loss of Capt leads to a change in localization of both Ena and Abl. Because these proteins act together with Capt to control the spatial organization of the follicular actin cytoskeleton, their altered distribution is likely to contribute to the generation of the marked capt mutant phenotype (Baum, 2001).

This study has used Drosophila genetics and the follicular epithelium to characterize how various actin-binding proteins act to regulate the spatial organization of F-actin. The results show that actin dynamics are regulated by distinct mechanisms within different domains of a polarized epithelial cell. Capt, Ena and Abl seem to modulate apical actin-filament formation, whereas cofilin and profilin seem to have a more global function, regulating cortical actin-filament dynamics throughout the cell. Moreover, the accumulation of F-actin at apical, basal and lateral sites in tsr mutant follicle cells and the loss of cortical actin filaments in chic mutant cells indicates that cortical actin filaments are turned over continuously throughout the cell. This being so, it is striking that F-actin becomes so highly polarized in the capt mutant (Baum, 2001).

In vitro, Capt has been shown to inhibit actin polymerization, which is consistent with its role in limiting epithelial actin-filament formation. In such assays, Capt seems to block actin polymerization by sequestering actin monomers. However, Capt protein and monomeric actin seem evenly distributed within the follicular epithelium. Because the loss of a uniformly distributed, actin-monomer sequestering protein would not be expected to result in the polarized accumulation of F-actin, it is considered possible that Capt might function by inhibiting the activity of a protein at apical adherens junctions that promote local actin-filament formation. Given Ena's ability to promote F-actin-filament assembly in mammalian cells, it is considered that Ena is a potential target of Capt activity. Cell-biological and genetic analysis of Ena within the Drosophila follicular epithelium supports this idea. Ena is concentrated together with F-actin at apical adherens junctions, and the level of Ena protein closely parallels the extent of local actin-filament formation. These results indicate that Ena has an important role in dictating the spatial organization of the actin cytoskeleton in Drosophila follicle cells. Moreover, Ena is required for the synthesis of apical actin aggregates in the capt mutant, which adds strength to the hypothesis that Capt might inhibit apical actin-filament formation catalysed by Ena. However, it is important to note that Ena is also present in cytoplasmic aggregates that lack concomitant F-actin and that in the wild type, Ena localizes with F-actin primarily at the apical cell cortex. The presence of Ena is therefore not sufficient to induce local actin polymerization, and its ability to catalyse actin-filament formation might be augmented at adherens junctions. Finally, homologues of Ena have been shown to localize to focal contacts and adherens junctions in mammalian epithelial cells in culture, suggesting that Ena might have an evolutionarily conserved function to control actin-filament formation at these sites (Baum, 2001).

Although profilin binds Ena and is required for F-actin formation within the wild type and in capt mutant follicle-cell clones, profilin seems to differ from Ena in two respects: (1) it is found that the overexpression of profilin has little effect on the level or distribution of F-actin; (2) profilin protein is not localized within follicle cells. Therefore profilin might have a general function within cells, facilitating actin-filament formation throughout, whereas Ena catalyses actin-filament formation at specific subcellular sites (Baum, 2001).

Like Capt and Ena, Abl is also required for proper F-actin organization within the follicle-cell epithelium. Interestingly, Abl also exhibits a cell-polarity phenotype reminiscent of that seen in l(2)gl/dlg/scrib mutants. Furthermore, the activity of Abl has a potent effect on the organization of F-actin in follicle cells lacking Capt. This is different from that observed in capt l(2)gl mutant cells because excess Abl generates more diffusely localized ectopic F-actin. The synergistic interaction between Capt and Abl suggests that these two proteins act in the same pathway. However, because Abl alters the organization of actin within capt mutant cells, Abl and Capt are unlikely to be components in a simple linear signalling cascade. One possibility is that Abl modulates the integrity of adherens junctions, where Ena and Capt seem to act. In support of the idea that Capt and Abl have related functions, mammalian homologues of Capt and Abl have been shown to interact physically, indicating that this relationship might be conserved in mammals. Moreover, actin aggregates reminiscent of those seen in the fly capt mutant cells are formed in the neurons of abl mutant mice. Thus Abl might modulate actin-filament formation in multiple organisms and cell types, perhaps by cooperating with Ena and Capt (Baum, 2001).

Bringing these data together, a model is envisaged in an effort to explain the aetiology of the pronounced capt mutant phenotype. Normally, in the columnar epithelium, Ena protein is concentrated at adherens junctions, where it promotes local F-actin synthesis. This activity of Ena is counterbalanced by Capt, which limits the amount of apical F-actin. Excess F-actin therefore forms at apical junctions in capt mutant cells. This newly formed apical F-actin is able to recruit additional molecules of Ena from the cytoplasm, because Ena binds microfilaments, which leads to further actin-filament formation. This explains why, in the absence of apical F-actin aggregates, Ena does not become concentrated at the apical cortex of cells in the capt chic mutant. Thus, the loss of Capt initiates an explosive cycle of local actin polymerization and Ena recruitment at adherens junctions, culminating in the striking polar actin aggregates observed in capt mutant cells. It is speculated that within wild-type epithelial cells, controlled autocatalytic cycles of actin-filament formation of this type might help to limit the accumulation of actin filaments to a single site within a cell. For instance, during the formation of a Drosophila wing hair, a similar process might be required to generate a single bundle of actin filaments at the apical cortex of the epithelium (Baum, 2001).

In Drosophila, Ena and Abl are thought to be part of a signalling pathway that changes local actin polymerization within the growth cones of neurons to guide axonal pathfinding. However, it has not been possible so far to analyse the cytoskeletal consequences of perturbations in Ena and Abl function directly within Drosophila neurons. The easily visible, asymmetric actin cytoskeleton in follicle cells has allowed the definition of cell-biological roles for these actin-regulatory genes. In these cells, Capt, Ena and Abl modulate actin-filament assembly at specific subcellular sites, probably by altering local actin dynamics. Thus, by analogy, these proteins might alter the site of actin-filament formation in response to signalling in neurons. If so, it will be interesting to determine whether similar signals impinge on this putative Capt/Ena/Abl pathway in neurons and in epithelia. Finally, given the fact that Capt, Ena and Abl control actin cytoskeletal organization in multiple tissues and in different organisms, these genes might have a conserved function, acting together to control the distribution of actin filaments in many other types of polarized animal cell (Baum, 2001).

Interactions between Amalgam, its transmembrane receptor Neurotactin, and the Abelson tyrosine kinase affect axon pathfinding

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase (Abl) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. An unusual missense ama allele, amaM109, has been identified that dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the AmaM109 mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, all five mutations originally attributed to disabled are in fact alleles of nrt. These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone (Liebl, 2003).

Genetic screens for second-site modifiers are useful tools for identifying components of signaling networks. Over the past decade, work in Drosophila has identified multiple modifiers of the Abl mutant phenotype. With the exception of the transcription factor prospero, all of the dominant modifiers identified have been cytoplasmic and co-expressed with Abl in axons. The biochemical characterization of some of the proteins encoded by these dominant enhancers has lead to an emerging model whereby the Abl tyrosine kinase supplies multiple inputs into actin cytoskeleton dynamics in the growth cone (Liebl, 2003).

The dosage-sensitive genetic interactions of ama and nrt with Abl provide unique information regarding Abl signaling networks. Five independent nrt alleles have been identified that remove Nrt function. Three are null alleles (nrtM2, nrtM29, nrtM54), while two (nrtM100 and nrtM221) are missense alleles that behave as protein nulls. Thus, simply reducing wild-type Nrt activity in an Abl-null background impairs viability, suggesting Abl and Nrt lie within one or more common signaling networks. The fact that these genetic combinations have clear effects on axon pathfinding, strongly suggests that at least one of these common signaling networks has its in vivo output in the growth cone. This is confirmed by the severe axon guidance phenotype produced by disruption of Abl and Nrt function through RNAi or homozygous zygotic mutation. Disruption of Abl and Nrt by zygotic mutation results in strong, but less severe CNS phenotypes than RNAi, probably as a result of elimination of maternally loaded Abl mRNA (Liebl, 2003).

Ama and Nrt have been shown to functionally interact to mediate cell:cell adhesion. Heterozygous null alleles of ama have no detectable dominant effects on axon pathfinding in an Abl-mutant background, presumably because the biochemical activity of secreted Ama is not directly associated with the cytoplasmic tyrosine kinase activity of Abl. However, disruption of Abl and Ama by homozygous zygotic mutation or by RNAi techniques does show clear synergistic disruptions of the CNS architecture. As with Abl and Nrt, the RNAi-induced phenotype is the more severe of the two, presumably because of the elimination of maternally supplied Abl mRNA (Liebl, 2003).

The identification of the unusual missense ama allele amaM109 as a strong dominant enhancer of the Abl mutant phenotype, affecting both viability and axon pathfinding, strengthens the conclusion that Ama, Nrt and Abl are functionally intertwined in the growth cone. AmaM109, which alters a cysteine residue needed to stabilize the first Ig domain of Ama, eliminates Ama homophilic adhesion but not the ability of AmaM109 to bind Nrt, and this is probably responsible for its unique character. The biochemical activity of this protein is clearly not wild type, since its ability to support aggregation of Nrt-expressing S2 cells is impaired (Liebl, 2003).

Genetically, the amaM109 allele phenocopies heterozygosity for nrt in the Abl1/Abl4 mutant background. Both genotypes result in 100% pre-pupal lethality, and both result in approximately one-third of embryo segments having defective commissures. Thus, it seems likely that, whatever its biochemical mode of action, the AmaM109 protein disables Nrt activity in a way that simply reducing the dose of wild-type Ama (by heterozygous null mutation) does not (Liebl, 2003).

To better understand the function of Nrt in the CNS, Speicher (1998) carried out an extensive genetic analysis, looking for cell adhesion molecules (CAMs) that are functionally redundant to Nrt. This was achieved by generating animals null for nrt and null for a variety of other CAM-encoding genes in pair-wise combinations. Removal of Nrt does not result in a strong CNS phenotype, Three different genetic combinations showed synergistic interactions in the CNS: nrt and neuroglian (nrg), nrt and derailed (drl), and nrt and kekkon1 (kek1), with the nrt, nrg combination showing the most profound synergy. This work suggests the role of Nrt in CNS cell adhesion is at least partially redundant to Nrg, Drl and Kek1. Interestingly, it has been reported that nrg and Abl have no genetic interaction when the morphology of the CNS is assayed by mAb BP102 staining (Liebl, 2003).

Whether Nrt-mediated adhesion provides novel inputs into Abl-mediated signaling networks in the growth cone or whether Nrt-mediated adhesion represents a novel output of the role of Abl in cytoskeleton dynamics can be determined by the genetic experiments that have been carried out. Intriguingly, deletion of the cytoplasmic region of Nrt eliminates its ability to promote cell:cell adhesion. Since many transmembrane cell adhesion molecules require functional interactions with the actin-based cytoskeleton, it is plausible that Ama:Nrt-mediated adhesion requires interaction of the cytoplasmic region of Nrt with actin-based cytoskeleton components. To clarify this issue molecular genetic screens are currently being conducted to identify protein:protein interactions involving the cytoplasmic domain of Nrt (Liebl, 2003).

Molecular and genetic characterization of nrt as a dominant enhancer of the Abl mutant phenotype has shown that all five mutations previously attributed to dab are nrt alleles. How were these mutations initially attributed to dab? The answer lies in incomplete characterization of proximal and distal breakpoints of Abl deletions, and mistaking the effects of dab near the proximal breakpoint with the effects of fax near the distal breakpoint. In retrospect, the difference in genetic activity between different deletions can be accounted for by the difference in the distal breakpoints of these chromosomes. Null mutations in fax dominantly enhance the Abl mutant phenotype (Liebl, 2003 and references therein).

Tyrosine phosphorylation-dependent signaling cascades play key roles in determining the formation of an axon pathway. The cytoplasmic Abelson tyrosine kinase participate in several signaling pathways that orchestrate both growth cone advance and steering in response to guidance cues. A genetic approach was used to evaluate the role for Abelson in growth cones during a decision to cross or not to cross the Drosophila embryonic midline. The data indicate that both loss- and gain-of-function conditions for Abl cause neurons within the pCC/MP2 pathway to project across the midline incorrectly. The frequency of abnormal crossovers is enhanced by mutations in the genes encoding the midline repellent, Slit, or its receptor, Roundabout. In comm mutants, where repulsive signals remain elevated, increasing or decreasing Abl activity partially rescues commissure formation. Thus, both too much and too little Abl activity causes axons to cross the midline inappropriately, indicating that Abl plays a critical role in transducing midline repulsive cues. How Abl functions in this role is not yet clear, but it is suggested that Abl may help regulate cytoskeletal dynamics underlying a growth cone's response to midline cues (Hsouna, 2003).

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, the microtubule-associated protein Orbit/MAST (proper FlyBase designation Chromosome bows) has been identified as a partner of the Abelson (Abl) tyrosine kinase. Identical axon guidance phenotypes are found in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors (Lee, 2004).

Orbit/MAST was identified as a candidate partner of Abl in a post-embyonic screem. In a retinal screen, overexpression of orbit/MAST enhanced the AblGOF phenotype, suggesting that these two proteins cooperate in vivo. However, validation of the screen required analysis of mutations in orbit/MAST (Lee, 2004).

Orbit/MAST was initially identified as a maternal effect lethal locus with defects in mitotic spindle and chromosome morphology; however, zygotic mutants display no defects in cell division, presumably due to maternal stores of the protein required for oogenesis. Independent LOF alleles were examined for zygotic phenotypes. Axon fascicles that are restricted to either side of the central nervous system (CNS) midline by Slit signaling can be visualized at stage 17 with anti-Fasciclin II (FasII, Mab1D4). In late-stage wild-type embryos (stage 17), FasII is excluded from the midline. However, in orbit/MAST mutants, ectopic midline crossing was detected, primarily by the midline-proximal MP1 axon pathway. This phenotype is qualitatively identical to that seen in Abl zygotic mutants (note that loss of maternal and zygotic Abl generates catastrophic axonal defects, underlining Abl's central role in axonal development). Since the exclusion of FasII from axon commissures reflects a redistribution of protein that could be dependent on Orbit/MAST, it was important to confirm the guidance defects with an alternative marker. Using a Tau-LacZ fusion protein under control of an Apterous promotor expressed in two medial ipsilateral axons that never cross the midline, frequent ectopic crossing of these axons was found in orbit/MAST mutants (Lee, 2004).

In order to rule out the possibility that axonal defects in orbit/MAST alleles result from some early failure in cell division or fate acquisition in the CNS, these homozygous mutants were stained with markers of neuronal cell fate. The number and position of neurons appeared to be normal even in the strongest orbit/MAST alleles. The fate of the midline glia that secrete Slit was examined, but no abnormalities were detected. To prove that the orbit/MAST axon defects represent a late, CNS-specific function of the gene, UAS-orbit(+) was expressed in mutant backgrounds under the control of postmitotic, neuron-specific GAL4 (elav-GAL4 and 1407-GAL4). Quantification of ectopic midline crossing in independent orbit/MAST mutants revealed an allelic series of guidance defects whose penetrance was consistent with the perdurance of some maternal protein. However, two independent transgenes successfully rescued the axon guidance defects of null orbit/MAST alleles. Thus, Orbit/MAST is required cell autonomously during neuronal differentiation for accurate axon guidance decisions (Lee, 2004).

The late-stage axon pathway defects in orbit/MAST mutants suggest a failure in the repellent effects of Slit on growth cone orientation. To be certain that the orbit/MAST phenotype reflects a loss of growth cone orientation and not simply a change in patterns of axon fasciculation, axon trajectories of pioneer neurons was inspected before other axons were available to serve as a substrate for fasciculation. At late stage 12, the posterior corner cell (pCC) helps to pioneer the MP1 pathway proximal to the midline; pCC neurites extend anteriorly and slightly away from the midline in wild-type. In orbit/MAST homozygotes, the pCC often orients toward the midline, sometimes crossing to meet its contralateral homolog. This shows that Orbit/MAST is required for accurate directional specificity of axon growth (Lee, 2004).

In addition to controlling midline crossing of axons, Slit repulsion determines the lateral position of longitudinal axon fascicles within the CNS neuropil. A marker for a mediolateral axon fascicle (Sema2b-Tau-myc) was used to examine this later function of Slit and its Robo receptors. In wild-type, Sema2b-positive axons cross the midline, turn, and extend along a straight longitudinal trajectory. In orbit/MAST mutants, a few Sema2b-positive axons meandered toward the midline from lateral positions. However, measurement of the lateral separation of these axon tracts reveals a significant inward shift in orbit/MAST mutants. Together, these genetic data demonstrate that Orbit/MAST performs a cell-autonomous postmitotic function during growth cone navigation (Lee, 2004).

The interaction between Orbit/MAST and Abl in the retina predicted that these proteins might cooperate to mediate axon guidance choices. However, since Abl plays both positive and negative roles in Slit signaling, it was important to test the polarity of genetic interactions in the context of embryonic development. Abl and Orbit/MAST levels were elevated, alone or in combination in postmitotic neurons. A mild synergy between the two genes during midline guidance was found that is consistent with cooperation. Interestingly, overexpression of Orbit/MAST alone induces a low but significant number of guidance errors at the midline. Stronger interactions were observed through LOF analysis. Double homozygous LOF mutants showed substantially increased ectopic midline crossing compared to single mutant controls, reminiscent of mutations in robo itself. Due to large maternal contributions of Abl and Orbit/MAST, even amorphic alleles are not zygotic null. Thus, it is not possible to use the double LOF mutant to conclude that both proteins act in a common pathway; however, the observed synergy does show that Abl and Orbit/MAST cooperate during midline axon guidance (Lee, 2004).

Since Abl is also required for motor axon pathfinding in the periphery, intersegmental nerve b (ISNb) morphology was compared in double and single mutants. Overexpression of Abl generates an ISNb bypass phenotype where this group of axons fail to enter their target domain. Coexpression of Abl and Orbit/MAST does enhance the expressivity of phenotype slightly, but the effect is subtle. Once having entered the ventral target domain, wild-type ISNb axons innervate the clefts between muscles 6, 7, 12, and 13. In Abl LOF mutants, ISNb stops short of its final targets, often terminating at muscle 13. A similar ISNb growth cone arrest phenotype is observed at very low penetrance in orbit/MAST LOF alleles. However, comparison of these phenotypes to orbit,Abl recombinant homozygotes revealed a strong enhancement of ISNb arrest in double LOF mutants, increasing the frequency of defects and shifting arrest to a more proximal position at the muscle 6/7 cleft. Thus, Abl and Orbit/MAST cooperate during axon guidance decisions in multiple contexts (Lee, 2004).

Analysis of CNS axons suggested that Orbit/MAST is an effector in the Slit/Robo repellent pathway. To test the hypothesis, the same genetic assay was used that was used to identify Slit as the ligand for the Robo receptor family. While heterozygotes lacking one copy of Slit or its receptors show very few guidance errors at the midline choice point, transheterozygotes that also remove one copy of a second gene in the pathway often reveal strong, synergistic phenotypes. Indeed, while orbit/MAST heterozygotes show no significant midline defects, very strong synergy is observed with mutations in slit (roughly 10-fold). As a control for the specificity of the interaction, embryos were examined lacking different alleles of orbit/MAST and an allele of capulet (capt), an actin binding protein that shows strong interactions with both slit and Abl (Wills, 2002). No synergy was observed between capt and orbit/MAST. The same transheterozygote analysis was performed with single mutations in the repellent receptors; orbit/MAST was found to enhance robo. Additional crosses revealed that orbit/MAST interacts with robo and robo2 but not with robo3, consistent with the specialization of Robo and Robo2 for midline crossing. To be certain that Orbit/MAST is not required simply for the expression or delivery of Slit and/or Robo protein, staining in wild-type and orbit/MAST embryos was compared, but no obvious differences were seen (Lee, 2004).

While all the data supported the model that Orbit/MAST is necessary for Abl function during axon guidance, a more rigorous test was desired. If Orbit/MAST acts as an effector of Abl, orbit/MAST mutations would be expected to be epistatic to an Abl GOF phenotype. The fact that Abl acts in both positive and negative capacities during midline guidance complicates the interpretation of such an experiment within the CNS; however, Abl plays a less complex role for ISNb motor axons. When overexpressed under a strong postmitotic neural GAL4 source, Abl generates an ISNb bypass phenotype; neuronal expression of GAL4 alone has no effect. However, when Abl is overexpressed in an orbit/MAST homozygous background, the frequency of ISNb bypass drops approximately 2-fold. This indicates that Orbit/MAST acts genetically downstream of Abl in embryonic growth cones (Lee, 2004).

Abelson, enabled, and p120 catenin exert distinct effects on dendritic morphogenesis in Drosophila

Neurons exhibit diverse dendritic branching patterns that are important for their function. However, the signaling pathways that control the formation of different dendritic structures remain largely unknown. To address this issue in vivo, the peripheral nervous system (PNS) of Drosophila was used as a model system. Through both loss-of-function and gain-of-function analyses in vivo, it has been shown that the nonreceptor tyrosine kinase Abelson (Abl), an important regulator of cytoskeleton dynamics, inhibits dendritic branching of dendritic arborization (DA) sensory neurons in Drosophila. Enabled (Ena), a substrate for Abl, promotes the formation of both dendritic branches and actin-rich spine-like protrusions of DA neurons, an effect opposite that of Abl. In contrast, p120 catenin (p120 ctn) primarily enhances the development of spine-like protrusions. These results suggest that Ena is a key regulator of dendritic branching and that different regulators of the actin cytoskeleton exert distinct effects on dendritic morphogenesis (Li, 2005).

Previous studies implicated Ena in the actin-dependent process of axon guidance. Ena, one of the founding members of Ena/VASP family proteins, is present at the leading edge of lamellipodia and at the tips of filopodia and directly binds to the actin monomer-binding protein profilin (Krause, 2003). In cultured hippocampal neurons, Ena/VASP activity is required for the normal formation of filopodia on growth cones and neurite shafts. Using both loss-of-function and gain-of-function approaches, it has been demonstrated that Ena regulates the dendritic branching of different subtypes of DA sensory neurons in Drosophila. Dendritic filopodia may be involved in dendritic growth and branching. As shown by time-lapse analysis, numerous filopodia-like thin processes at the tips of lateral dendrites of DA neurons undergo rapid extension and retraction during Drosophila embryogenesis. Some are stabilized and eventually become lateral branches. These immunostaining studies showed that Ena was localized in dendrites of DA sensory neurons and that the number of dendritic filopodia is significantly reduced in ena mutant embryos. Therefore, it is highly likely that Ena plays analogous roles in dendrites to control the formation of filopodia, precursors of stable dendritic branches (Li, 2005).

Abl, a nonreceptor tyrosine kinase, plays a key role in several developmental processes including axon guidance and epithelial morphogenesis and has a cell-autonomous function in limiting dendritic branching, opposite that of Ena. Loss of Abl activity results in an increased number of dendritic branches and spine-like protrusions, while overexpression of Abl inhibits the formation of dendritic branches and spine-like protrusions. A mutant Abl construct that abolishes its kinase activity has no effect on dendritic development, suggesting that phosphorylation of its substrates is required for its activity in this process. Thus, the extent of dendritic branching appears to be controlled, in part, by the balance between the activities of Ena and Abl (Li, 2005).

Several lines of evidence indicate that Abl also interacts with cadherin complexes. Abl mutations in Drosophila significantly enhance the axonal defects found in armadillo (Arm, the ß-catenin homologue) mutants. Drosophila E-cadherin interacts with Abl in controlling epithelial morphogenesis. Furthermore, Abl forms a complex with the axon guidance receptor Robo and N-cadherin, and the kinase activity of Abl is essential for phosphorylation of ß-catenin. Abl also interacts with and phosphorylates ß-catenin, a member of the p120ctn subfamily in mammals. Several components in the N-cadherin complex regulate various aspects of neuronal morphogenesis (Li, 2005 and references therein).

The potential interaction between Abl and p120ctn prompted an examination of the role of p120ctn in dendritic branching. Drosophila p120ctn mutant embryos do not show defects in adherens junctions, and p120ctn is not required for DE-cadherin function in vivo. However, studies in mammalian systems indicate that p120ctn plays a key role in maintaining normal levels of cadherin. In this study, morphological and molecular differences between dendritic branches and spine-like protrusions on some DA neurons are described. Although those spine-like protrusions do not make synaptic connections with other neurons, they do share some similarities with mammalian spines. Most notably, these processes are more or less perpendicular to dendritic shafts and are highly enriched in actin. Evidence is provided that p120ctn has a function in neural development. It was found that loss of p120ctn activity reduced the number of spine-like protrusions on dendrites of some DA sensory neurons, while overexpression of p120ctn promotes the formation of these fine dendritic structures. Since overexpression of ß-catenin, another cadherin-associated protein, increases spine density in cultured mammalian neurons, it remains possible that p120ctn regulates actin cytoskeleton dynamics through modulating the cadherin complex. Interestingly, ena and p120ctn interacted genetically, revealing a supporting role for p120ctn in modulating dendritic branching of a subset of DA neurons, which was not obvious when p120ctn was mutated alone. Taken together, these genetic analyses suggest that different regulators of the actin cytoskeleton exert their specific effects on dendritic morphogenesis through interactive molecular pathways (Li, 2005).

The receptor protein tyrosine phosphatase PTP69D antagonizes Abl tyrosine kinase to guide axons in Drosophila

During Drosophila embryogenesis, both the cytoplasmic Abelson tyrosine kinase (Abl) and the membrane bound tyrosine phosphatase PTP69D are required for proper guidance of CNS and motor axons. Evidence is provided that PTP69D modulates signaling by Abl and its antagonist, Ena. An Abl loss-of function mutation dominantly suppresses most Ptp69D mutant phenotypes including larval/pupal lethality and CNS and motor axon defects, while increased Abl and decreased Ena expression dramatically increase the expressivity of Ptp69D axonal defects. In contrast, Ptp69D mutations do not affect Abl mutant phenotypes. These results support the hypothesis that PTP69D antagonizes the Abl/Ena genetic pathway, perhaps as an upstream regulator. It was also found that mutation of the gene encoding the cytoplasmic Src64B tyrosine kinase exacerbates Ptp69D phenotypes, suggesting that two different cytoplasmic tyrosine kinases, Abl and Src64B, modify PTP69D-mediated axon patterning in quite different ways (Song, 2008).

Two enzyme classes, tyrosine kinases and tyrosine phosphatases, dynamically maintain protein phosphotyrosine modifications that are critical for axon guidance. Studies that revealed physical interactions between members of these families have led to an investigation of the relationship between the membrane bound tyrosine phosphatase, PTP69D and cytoplasmic tyrosine kinases. Evidence is provided that PTP69D modulates signaling by the tyrosine kinase, Abl, and its substrate Ena. (1) Ptp69D mutant phenotypes, including adult lethality, embryonic CNS and ISNb motor axon defects, are significantly suppressed by loss of Abl function, and dramatically enhanced by gain of Abl function. (2) Ptp69D does not suppress Abl, suggesting that their interaction is asymmetric. (3) Ena, a strong suppressor and a downstream substrate of Abl, dominantly exacerbates the defects of Ptp69D.

Abl mutants display evident phenotypes such as adult lethality and ISNb arrest defects. Bi-directional suppression was expected between Ptp69D and Abl by analogy to Dlar mutations, which interact symmetrically with Abl for such phenotypes as midline crossing defects in the CNS. However, although Abl mutants modify Ptp69D phenotypes, no evidence was found of reciprocal suppression by introducing Ptp69D mutations into Abl embryos despite trying various combinations of alleles. While several models might explain this, one simple interpretation is that Abl is epistatic to Ptp69D, i.e., Ptp69D acts through Abl (Song, 2008).

What could be downstream targets of PTP69D and Abl? As a substrate of Abl and antagonistic genetic component of the Abl pathway, Ena is an excellent candidate, and indeed, ena mutations enhanced the lethality and axonal defects of Ptp69D mutants. Ena is known to play a role in cell motility, and likely supports F-actin assembly within cells by antagonizing capping protein at the barbed ends of actin and reducing filament branching. In Drosophila, Ena associates with the PTP69D D2 domain and is phosphorylated by Abl in vitro, and its specific cellular localization is regulated by Abl. The consistent pattern of interactions of Ptp69D with Abl and ena (suppressed by Abl mutations and enhanced by the Abl antagonist, ena) supports the idea that the Abl effector, Ena is also a key to signaling by PTP69D (Song, 2008).

The data define a functional relationship among PTP69D, Abl and Ena, but what could be their physical relationship? Extrapolating from genetic interactions to molecular mechanism is not straightforward, however, an attractive speculation that could provide one framework for further thinking is the idea that PTP69D, Abl and Ena may coexist in a complex, where PTP69D inhibits Abl, which in turn inhibits Ena. Such a model would be consistent with the available biochemical evidence, as well as with the genetic interactions observed. Many other models are equally possible, however, and a great deal of additional experimentation would be required to establish this hypothesis. For example, whether these three proteins co-immunoprecipitate has not been investigated, nor have it been demonstrated that they act simultaneously in the same cell. Moreover, although the kinase activity of Abl is required for its axonal function, it is thought that tyrosine phosphorylation of Ena is not the sole function of Abl. Axon guidance by Abl seems also, for example, to be associated with the action of small Rho family GTPases, particularly Rac, and any model for the mechanism of axon guidance by Abl and its partners will have to take these data into account (Song, 2008).

The patterns of genetic interactions of Ptp69D with Abl and ena described in this paper bear some similarities to those of Dlar. Does either RPTP substitute for each other? Previous studies demonstrated that Ptp69D and DLAR cooperate at growth cone choice points along one nerve, ISNb, while along another nerve, ISN, they do not act together. In the adult eye, moreover, a Dlar transgene rescues Ptp69D R7 axon phenotype, but not vice versa. Thus, the relationship between PTP69D and DLAR is complex and depends on cellular context (Song, 2008).

This analysis of PTP69D was extended by investigating the functional interaction of PTP69D with a Drosophila Src gene, Src64B. Mammalian Src protein (c-src) has been shown to regulate the stability and remodeling of actin structures. In Drosophila, Src64B has been shown to function in nervous system development in the embryo, the mushroom body of the adult brain and in the adult eye, making it a plausible candidate for interacting with PTP69D in axon guidance. Moreover, in mouse the RPTP CD45 functions to positively regulate SFK in T cells. Indeed, the data show that Ptp69D does interact with Src64B, but in a sense opposite to that with Abl: Ptp69D and Src64B interact synergistically rather than antagonistically (Song, 2008).

Hints as to a possible mechanism that could underlie the interaction of PTP69D with Src64B are suggested by experiments in mammals. The SFK Fyn binds to LAR and phosphorylates the LAR D2 domain. In turn, LAR dephosphorylates a C-terminal inhibitory motif of Fyn, increasing Fyn activity. The current data as well could potentially be explained by an analogous model whereby PTP69D derepresses Src activity by removing an inhibitory phosphate, though other models are clearly possible and more study is required to test this speculation. It is interesting that the biochemical association of LAR with Fyn in mammals is reminiscent of that observed for DLAR with Abl in Drosophila, but the biological consequences in the two cases are quite different, and in fact opposite: activation of Fyn activity, but suppression of Abl (Song, 2008).

Superficially, it seemed surprising that two cytoplasmic kinases had opposite interactions with PTP69D, antagonizing Abl but cooperating with Src64B. To test this further, the genetic interaction between Src64B and Abl was examined. Although Src64BΔ17 did not show any significant effect on Abl lethality, it dramatically suppressed the ISNb stall defect of Abl mutants, further supporting the hypothesis that Src64B and Abl kinases may have opposing functions in axon guidance (Song, 2008).

In summary, the receptor protein tyrosine phosphatase PTP69D interacts both with the Abl-Ena tyrosine kinase pathway and with Src64B to control axon patterning in Drosophila. PTP69D antagonizes Abl, perhaps as an upstream regulator, but functions synergistically with Src64B, thus revealing previously unrecognized specificity in the action of these tyrosine kinase pathways (Song, 2008).

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision (Hsouna, 2008).

The developmental defects observed in the formation of the CNS axon scaffold clearly point to individual and co-operative roles for CaM- and Abl-dependent signaling pathways during axon guidance at the midline. Moreover, the range of defects suggest that both CaM and Abl have multiple roles during the transduction of midline attractive and repulsive cues, and probably converge to regulate key aspects of the cytoskeletal dynamics underlying axon outgrowth and steering (Hsouna, 2008).

When the signaling strength of either pathway is individually decreased, the predominant phenotype is ectopic midline crossing errors of pCC/MP2 axons. Ectopic midline crossing errors is also the primary defect in embryos experiencing a mild, but simultaneous, reduction of both CaM and Abl signaling. CaM and Abl appear to work together in the same neurons to transduce guidance cues because ectopic crossovers are replicated if a kinase inactive Abl transgene (ftzng-AblKN) is co-expressed with the CaM inhibitor in the same neurons. It also appears that the level of Abl activity is particularly important. When both copies of the endogenous abl gene are mutated, expression of even one copy of the CaM inhibitor is sufficient to induce major defects in the axon scaffold. The converse, one copy of abl4 in homozygous iCaMKA (inhibitor of CaM-KA were KA stands for a novel competitive inhibitor called Kinesin-antagonis) embryos, does not significantly affect the frequency of ectopic crossovers. Together, these data are consistent with the earlier hypothesis that both signaling pathways function to transduce midline repulsive cues (Hsouna, 2008).

Robo, the receptor for the midline repellent Slit, is expressed in pCC/MP2 neurons from the onset of axonogenesis and its activity is required to prevent them from crossing the midline. Loss-of-function robo mutations enhance the frequency of ectopic crossovers induced by either iCaMKA expression or abl mutations. Thus, the observation that a simultaneous decrease in CaM and Abl synergistically increases ectopic crossovers was anticipated. Mechanistically, Abl is known to bind to, and phosphorylate, Robo to potentially inhibit its activity. In addition, Enabled, a known substrate for Abl, binds to Robo and is required to signal midline repulsive activity. While little is known about how CaM contributes to Robo signaling, the meandering crossovers observed in robo mutants are replicated when iCaMKA is combined with loss-of-function mutations in sos and Sos is now known to bind to Robo. A direct role for Sos in Robo signaling is also supported by the observation that ectopic midline crossovers occur in sos abl double mutants (Hsouna, 2008).

However, it is unlikely that CaM and Abl are operating solely downstream of Robo during midline guidance. When the level of CaM and Abl activity is substantially reduced in embryos using multiple copies of iCaMKA and abl alleles, the frequency of crossovers increase but, in addition, commissures begin to fuse and gaps in the longitudinal connectives form. These latter defects are difficult to explain solely on the basis of a disruption in Robo signaling, since they are not generally evident in robo null embryos. Thus, the efficacy of other guidance mechanisms functioning at the midline must also be affected in the CaM and Abl mutants. One obvious candidate is Netrin-dependent midline attraction (Hsouna, 2008).

Netrin is a major midline attractant required for commissure formation in the Drosophila CNS, and Frazzled is the Netrin receptor guiding many commissure axons across the midline. In the absence of Fra, most posterior commissures do not form correctly. However, it is suspected that an alteration in Fra activity is not responsible for the fused commissures and longitudinal gaps observed in iCaMKA and abl mutants. First, in abl and fra double mutants, a loss of Abl activity in fra mutants exacerbates commissure loss, and second, expression of iCaMKA still induces crossovers in the absence of Fra. Most vertebrate literature also predicts that CaM-dependent enzyme activity increase during Netrin-dependent attraction, not decrease, as occurs with iCaMKA expression (Hsouna, 2008).

There is, however, growing evidence that at least one other Netrin-dependent receptor is functioning during the midline guidance of pCC/MP2 neurons. While pioneering the longitudinal connective, pCC/MP2 neurons follow an axon trajectory delineated by Netrin localized along commissure axons by Fra. To project past these Netrin-rich commissures, midline attraction must be briefly inhibited by Robo activity. If this inhibitory signal fails, pCC/MP2 axons cross the midline using the newly emerging commissures and leave gaps in the longitudinal connective. This is, in fact, similar to the defects observed in iCaMKA and abl mutants. Importantly, while they are responding to Netrin, most pCC/MP2 neurons do not appear to express Fra. Thus, in addition to inhibiting Robo-dependent midline repulsion, a combined loss of CaM and Abl activity may be preventing Robo from blocking this Netrin-dependent attraction at the segmental boundary. Testing this hypothesis awaits characterization of the Netrin-dependent receptor that is functioning at the segmental boundary. The absence of Fra expression in pCC/MP2 neurons may also explain 1) why iCaMKA and abl induce crossovers of pCC/MP2 neurons even though fra and abl interact to reduce commissure formation, and 2) why iCaMKA induces pCC/MP2 axons to cross the midline even in the absence of Fra (Hsouna, 2008).

Interestingly, this Netrin-dependent, but Fra-independent attraction near the segmental boundary is known to be sensitive to IP3 levels, which are presumably leading to an increase in intracellular calcium. Thus, the ability of these neurons to remain on the correct side of the midline is quite sensitive to the level of calcium, and now CaM, signaling. Calcium and/or CaM may function downstream of specific receptors or more generally as second messengers governing basic cell processes, such as motility. Certainly, inhibiting even a small amount of CaM activity (using one copy of iCaMKA) sensitizes these neurons to over-expression of Fra. Moreover, the frequency of crossovers (57%) observed in this experiment is approximately the same as observed when wild type Fra is over-expressed in a heterozygous robo mutant (55%). This implies that expression of a single copy of iCaMKA is reducing Robo activity by half, a conclusion difficult to reconcile with previous data. Therefore, it is suspected that expression of iCaMKA is altering the spatial and temporal regulation of calcium-dependent activity underlying growth cone movement and steering. In the case of pCC/MP2 neurons, this appears to preferentially result in ectopic midline crossovers, a defect which is further enhanced when the levels of receptor for midline attraction (Fra) or repulsion (Robo) are genetically manipulated (Hsouna, 2008).

An alteration in key calcium-dependent regulatory events would also be further exacerbated by a simultaneous loss in Abl activity, especially since Abl is a key regulator of the actin and myosin dynamics underlying growth cone advance. While not previously appreciated, there is some evidence linking CaM and Abl activity during axon guidance in the embryonic axon scaffold. For example, heterozygous abl mutations suppress the ectopic midline crossing errors induced by expression of an activated Myosin Light Chain Kinase (a CaM-dependent enzyme) even when Fra is co-expressed. In addition, actin dynamics are likely to be important since both iCaMKA and abl mutations interact with Profilin loss-of-function mutations to alter axon path finding. This study specifically demonstrates a strong, synergistic interaction between CaM- and Abl-dependent signaling during in vivo development of the embryonic CNS. Clearly, it will be important to identify where these key signaling pathways converge to regulate actin and myosin dynamics and how these regulatory events contribute to axon guidance decisions at the midline (Hsouna, 2008).

Distinct effects of abelson kinase mutations on myocytes and neurons in dissociated Drosophila embryonic cultures: mimicking of high temperature

Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. This study demonstrates that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl1 and Abl4) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. The results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of Abl cultures. Despite the extensive alterations by Abl mutations, myocyte fusion events and nerve-muscle contact formation were observed between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos (Liu, 2014).

Abl tyrosine kinase Effects of mutation part 2/2

Abl tyrosine kinase: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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