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DEVELOPMENTAL BIOLOGY

Embryonic

Expression of ENA protein is first detected during germ-band extension at stage 10 and by stage 11 is detected in the endoderm and ectoderm of the extended germ band. During germ-band retraction [Images], ENA protein becomes restricted to the CNS and by stage 15 is observed primarily in the commissural and longitudinal axons of the CNS. During late embryogenesis (Stage 17), ENA protein is localized primarily to the longitudinal exons (Gertler, 1995).

Expression of ABL protein

ABL protein, present in all cells of the early embryo as the product of maternally contributed mRNA, transiently localizes to the region below the plasma membrane cleavage furrows as cellularization initiates. The function of this expression is not yet known. Zygotic expression of ABL is first detected in the post-mitotic cells of the developing muscles and nervous system midway through embryogenesis. In later larval and pupal stages, ABL protein levels are also highest in differentiating muscle and neural tissue including the photoreceptor cells of the eye. ABL protein is localized subcellularly to the axons of the central nervous system, the embryonic somatic muscle attachment sites and the apical cell junctions of the imaginal disk epithelium (Bennett, 1992).

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila; Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help in the understanding of animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. This study reports the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, these data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction (Fox, 2007).

Regulation of apical constriction during Drosophila VF formation is a paradigm for how signal transduction directs morphogenesis. This study identified Abl as a novel regulator of this process. The results suggest that Abl acts in parallel to the known signaling pathway that promotes apical myosin activation by helping to organize a continuous apical actin network. Furthermore, the results help to explain the greater severity of the RhoGEF2-mutant phenotype relative to other VF mutants by suggesting that RhoGEF2 plays crucial roles in both myosin and actin regulation (Fox, 2007).

Previous work established myosin as a key output of RhoGEF2 signaling during mesoderm internalization. However, ambiguities remained regarding the circuitry of this pathway, since the RhoGEF2 phenotype is much more severe than that of cta or fog mutants, suggesting that a simple linear pathway is unlikely. The data suggest that RhoGEF2 plays dual roles in actin and myosin regulation, and thus its inactivation has more severe effects (Fox, 2007).

From these data, a mechanistic model was developed for the regulation of apical constriction during VF formation. The regulation of actin localization by Abl and RhoGEF2 promotes organization of the apical actin network in constricting cells. It is suggested that Abl regulates actin by actively downregulating cortical Ena in mesoderm, thus leading to polarized actin accumulation, similar to the role that it was shown to play in follicle cells. RhoGEF2 plays a distinct, Cta-independent role in the effective assembly of organized apical actin. While RhoGEF2 and Abl are modulating actin assembly, the mesodermal transcription machinery activates Fog-Cta signaling, apically stabilizing RhoGEF2. This allows the efficient activation of apical myosin. Coupling of these two cues -- an organized apical actin ring at AJs and stable apical myosin activation -- cooperate to ensure highly coordinated actomyosin constriction throughout the sheet of mesodermal cells in a short timeframe (Fox, 2007).

This model helps explain the mutant phenotypes observed in this and previous studies. In abl mutants, Fog-Cta allow RhoGEF2 stabilization and myosin contraction, but the lack of organized mesodermal actin in these mutants, which results from inappropriate Ena regulation, prevents the uniform assembly of actin-based contractile rings. cta mutants lack a stabilizing signal for RhoGEF2, preventing uniform apical myosin activation and uniform constriction. However, some cells can constrict without Fog-Cta, accumulating apical myosin levels comparable to those in wild type. In RhoGEF2 mutants, the combined failure to stabilize/activate myosin and a lack of organized apical actin severely compromises apical constriction. The similarity between RhoGEF2 and cta;abl mutants supports this model, as both processes should be compromised (Fox, 2007).

The model suggests that organized apical actin is an essential prerequisite for cell constriction. Although both Abl and RhoGEF2 regulate actin localization, the data argue that each acts independently. First, actin defects arise during cellularization, when Abl and RhoGEF2 have non-overlapping localizations. Second, whereas Abl clearly acts through Ena, loss of RhoGEF2 disrupts actin without altering Ena localization. Finally, Abl is not a Rho effector in S2 cells (Fox, 2007).

Several unanswered questions remain. With respect to abl, a major question is why do some cells apically constrict while others fail? This phenotype resembles the cellularization defects of abl mutants, in which only some cells fail to reorganize actin into furrows. However, all cells exhibit excess apical Ena and thus form abnormally long, apical microvilli. Perhaps, in some cells, furrow actin assembly drops below a crucial threshold and furrows fail. In the VF, the absence of Abl may have similar effects. VF defects could result from both competition for cellular actin and recruitment of other regulators (e.g. the formin Diaphanous) to ectopic locations, preventing their action in VF formation. This may reduce actin assembly into contractile rings. When constriction initiates, stochastic variations in ring strength may lead some rings to fail, leading to unconstricted cells. Future work is needed to identify the full set of actin regulators involved, and to assess how they work. Interestingly, recent work implicates Abl in epithelial-mesenchymal transitions. Whereas Abl disrupts VF formation, Twist is normally localized in abl mutants, suggesting that this major regulator of such transitions is not an Abl target in flies (Fox, 2007).

The data also reveal the importance of mesodermal Ena downregulation. This may result from increased mesodermal Abl activity, suggested by elevated levels of mesodermal Abl relative to non-mesoderm; however, this remains to be tested. It is also necessary to identify the mechanism by which Abl regulates Ena. In some places, such as the syncytial blastoderm, Abl localizes to sites where Ena is normally absent and, in the absence of Abl, ectopic Ena is found at these sites. This suggests that Abl actively antagonizes Ena localization. At other times and regions, however, such as the leading-edge during dorsal closure, Abl co-localizes with Ena, and thus may hold it in an inactive state. In VFs, Abl localizes to the apical-lateral cortex, and Ena localizes to this site in its absence. Further studies of Abl action will be needed to clarify the mechanisms by which it downregulates Ena (Fox, 2007).

Interestingly, manipulating mammalian Ena/VASP can affect cell contractility Thus, Ena-downregulation may permit proper VF cell contractility. Testing this hypothesis will be important (Fox, 2007).

The results also raise questions regarding RhoGEF2. The model suggests that RhoGEF2 acts via two mechanisms, only one of which is Cta-dependent. Perhaps another upstream cue acts on RhoGEF2 to promote actin organization. Because RhoGEF2 mutants have actin-organization defects in all cells, this regulator may act in all cells prior to gastrulation. However, the data do not rule out a second mesoderm-specific RhoGEF2 regulator acting in parallel to Cta. Although Rho-Kinase is a potential Rho effector with respect to myosin, another effector may regulate actin organization. Attractive candidates are the Formins, which reorganize actin in many processes (Fox, 2007).

The data strengthen the idea that different cytoskeletal regulators direct distinct morphogenetic processes. Both Abl and Fog regulate mesodermal apical constriction but are dispensable for germband cell-cell intercalation. Thus, although both processes require dynamic myosin reorganization, distinct regulators act in each (Fox, 2007).

The picture becomes more complex when considering other roles of Fog, Cta and RhoGEF2. All are required for internalization of the posterior midgut and salivary glands, but these cells internalize in abl mutants. Thus, different types of apical constriction may be regulated differently. It will be interesting to explore the roles of Fog, Cta and RhoGEF2 during dorsal closure, which requires Abl (Fox, 2007).

This work supports mechanistic connections between VF formation and neural tube closure. Both involve actin-based apical constriction to internalize a sheet of cells into a tube. Mice lacking Abl and Arg kinases have neural tube defects, and actin organization in neuroepithelial cells appears altered; interestingly, these cells have ectopic actin that is less polarized than normal, similar to what was observed in abl-mutant VFs. Furthermore, double-mutant analysis suggests that mammalian Ena plays a role in neural tube closure in conjunction with Profilin. Thus, Abl-Ena signaling may represent a conserved mechanism of actin regulation during apical constriction. New mechanistic insights can now be pursued in mammals (Fox, 2007).

Rho also regulates neural tube closure. Mice lacking p190RhoGAP have neural tube defects. Interestingly, p190RhoGAP is an Arg substrate in the brain, suggesting possible direct links between Abl and Rho in apical constriction. The role of Drosophila p190RhoGAP in the VF has yet to be examined, but RhoGAP68F is implicated in VF formation. Future work in both flies and mice will provide further mechanistic insights into conserved mechanisms of apical cell constriction (Fox, 2007).

Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

The golgi apparatus is optimized separately in different tissues for efficient protein trafficking, little is known of how cell signaling shapes this organelle. This study finds that the Abl tyrosine kinase signaling pathway controls the architecture of the golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-golgi in developing PRs. Overexpression or loss-of-function of Ena increases the number of cis and trans-golgi cisternae per cell, and Ena overexpression also redistributes golgi to the most basal portion of the cell soma. Loss of Abl, or of its upstream regulator, the adaptor protein Disabled, lead to the same alterations of golgi as does overexpression of Ena. The increase in golgi number in Abl mutants arises in part from increased frequency of golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, it was demonstrated that the effects of Abl signaling on golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion (Kannan, 2014).

The Abl tyrosine kinase signaling pathway controls golgi morphology and localization in Drosophila photoreceptors through its regulation of the actin cytoskeleton. Ena, the main effector of Abl in morphogenesis, is associated with the cis-golgi compartment, and it regulates golgi localization and dynamics under the control of Abl and its interacting adaptor protein, Dab. Reducing the levels of Abl or Dab, or overexpressing Ena, led to similar defects in golgi fragmentation state and subcellular distribution. During golgi biogenesis, Abl increases the frequency of fusion of golgi cisternae, and decreases fission events. Abl evidently controls golgi organization through its regulation of actin structure, as the effect of Abl signaling on golgi could be blocked by modulating actin structure genetically or pharmacologically. Collectively, these data reveal an unexpected link between a fundamental tyrosine kinase signaling pathway in neuronal cells and the structure of the golgi compartment (Kannan, 2014).

The data reported here suggest that the Abl signaling pathway controls golgi morphology and localization through its control of actin structure. This is consistent with previous reports that altering the levels of actin modulators perturbs the structure and function of the golgi apparatus. A variety of proteins that modulate actin dynamics have been localized to golgi. Ultra-structural studies established the association of actin filaments with golgi membranes and the association of β and γ actin with the golgi. In cultured cell models, including neurons, actin depolymerization leads to golgi compactness, fragmentation and altered subcellular distribution. It is noted, moreover, that the reported golgi-associated signaling proteins include several that have been linked to Abl signaling, including the Abl target Abi, the Abi binding partner WAVE, and various effectors of Rac GTPase including ADF/cofilin, WASH and Arp2/3. Thus, for example, Abi and WAVE have been implicated in actin dependent golgi stack reorganization and in scission of the golgi at cell division to allow faithful inheritance of golgi complex to daughter cells in Drosophila S2 cell cycles (Kondylis, 2007). These data reinforce the importance of actin-regulating signaling pathways for controlling golgi biogenesis (Kannan, 2014).

Two lines of evidence suggest that the increase observed in golgi number in Abl pathway mutants is due primarily to net fragmentation of pre-existing golgi cisternae and not to de novo synthesis of golgi. First, live imaging of golgi dynamics in neurons of the Drosophila eye disc reveals that the steady-state number of golgi cisternae reflects an ongoing balance of fusion and fission events, much as observed previously in yeast. Quantification of these events in wildtype vs Abl mutant tissue demonstrated directly that loss of Abl significantly increased the frequency of fission events, and reduced the frequency of fusions. Second, the absolute volume of cis-golgi in Abl mutant photoreceptors was not substantially greater than that in wildtype, as judged by direct measurement of the volume of GM130- immunoreactive material in deconvoluted image stacks of photoreceptor clusters. While a small apparent increase was observed in golgi volume in the mutants (~55%, based on pixel counts), it is noted that golgi cisternae are small on the length scale of the point spread function of visible light, such that the fluorescent signal from a single cisterna extends into the surrounding cytoplasm. The increase in apparent golgi volume is therefore within the range expected due simply to fluorescence 'spillover' from the three-fold greater number of separate golgi cisternae in the mutants (Kannan, 2014).

It is striking that both increase and decrease of Ena led to net fragmentation of golgi. Why might this be? It is known that both fission and fusion of membranes requires actin dynamics: at scission, polymerization provides force for separating membranes, while in fusion, actin polymerization is essential for bringing membranes together and for supplying membrane vesicles, among other things. As a result, altering actin dynamics is apt to change the probabilities of multiple aspects of both fission and fusion events, making it impossible to predict a priori how the balance will be altered by a given manipulation, just as either increase or decrease of Ena can inhibit cell or axon motility, depending on the details of the experiment, due to the non-linear nature of actin dynamics. Indeed, this study also observed net golgi fragmentation when actin was stabilized with jasplakinolide, just as was done from depolymerization with cytochalasin or latrunculin. More direct experiments will be necessary to fully understand this dynamic, however. deficits selectively disrupt dendritic morphogenesis but not axogenesis, and perhaps consistent with this, Abl/Ena function is essential for dendrite arborization in these cells but has not been reported to affect their axon patterning. Finally, in some contexts, neuronal development requires local translation of guidance molecules in the growth cone rather than translation in the cell soma. It is likely that the need for actin dynamics to target different subcellular compartments in different cell types will be reflected in different patterns of Abl/Ena protein localization (Kannan, 2014).

This study reports the role of Abl/Ena-dependent regulation of actin structure on overall golgi structure and localization but there may be more subtle effects on golgi function as well. For example, recent evidence supports a role for actin-dependent regulation of the specificity of protein sorting in the golgi complex. Preferential sorting of cargos is achieved by nucleation of distinct actin filaments at the golgi complex. In Hela cells, for example, Arp2/3 mediated nucleation of actin branches at cis-golgi regulates retrograde trafficking of the acid hydroxylase receptor CI-MPR, while Formin family mediated nucleation of linear actin filaments at golgi regulates selective trafficking of the lysosomal enzyme cathepsin D. Similarly, the actin-severing protein ADF/cofilin, the mammalian ortholog of Drosophila twinstar, sculpts an actin-based sorting domain at the trans-golgi network for selective cargo sorting. It will be important to investigate whether the effects of Abl/Ena on golgi morphology have functional consequences on bulk secretion or protein sorting (Kannan, 2014).

Protein trafficking and membrane addition in neurons need to be coordinated with the growth requirements of the axonal and dendritic plasma membranes, but the mechanisms that do so have been obscure. Abl pathway proteins associate with many of the ubiquitous guidance receptors that direct axon growth and guidance throughout phylogeny, including Netrin, Roundabout, the receptor tyrosine phosphatase DLAR, Notch and others. The data therefore suggest a potential link between the regulatory machinery that senses guidance information and the secretory machinery that helps execute those patterning choices. Indeed, preliminary experiments suggest that some of the axonal defects of Abl pathway mutants may arise from alterations in golgi function. Beyond this, Abl signaling is essential in neuronal migration, epithelial polarity and integrity, cell adhesion, hematopoiesis and oncogenesis, among other processes The data reported in this study now compel a reexamination of the many functions of Abl to ascertain whether some of these effects arise, at least in part, from regulation of secretory function (Kannan, 2014).

Effects of Mutation or Deletion

Although Abl> mutant flies die at the end of pupation or as young adults, flies that lack Abl function and have only one copy of ena survive to adulthood with no obvious defects. The fact that reduction in the level of ena compensates for the absence of Abl activity suggests that these two molecules normally function in opposition to one another in a precise balance. The most frequent CNS defect in enb mutants is a reduction in the integrity of axon bundles. Mutated axons have diffuse and loosely bundled longitudinal and commissural axon tracts. Some homozygous embryos exhibit less obvious phenotypes such as thinning of longitudinal connectives, increased number of axons exciting the CNS from the longitudinal axons or failure of commissural axons to separate into anterior and posterior axon bundles. The overall organization of the PNS is disrupted, spacing and organization of the neurons is irregular and some clusters of neurons are mislocalised. Some defects in axon guidance are apparent, with axons crossing between PNS elements in adjacent segments (Gertler, 1995).

During Drosophila embryogenesis, the Abelson tyrosine kinase (ABL) 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 ENA participate in establishing axonal connections in the embryonic CNS of Drosophila (Gertler, 1989).

Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. Methods have been developed to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, several genes have been identified that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis (Gao, 1999).

The peripheral neurons in each hemisegment of the Drosophila embryo are grouped into dorsal, lateral, and ventral clusters. The neurons within each cluster can be further classified on the basis of their dendritic morphology; these categories are external sensory (es) neurons and chordotonal (ch) neurons, each containing a single dendrite; bipolar dendrite (bd) neurons, each with two simple unbranched dendritic projections; and multiple dendrite (md) neurons with extensive dendritic arborizations. The md neurons are thought to function as touch receptors or proprioceptors to sense body surface tension or deformation. The dendritic branching of md neurons does not begin until 16 hr after egg laying (AEL) and continues until and beyond hatching. Because impermeable cuticle already forms at 16 hr AEL, md neuron dendrites can not be visualized by standard antibody staining of whole mount embryos. It is possible to manually dissect individual embryos to allow antibody access; however, this technique is too laborious to be useful for a large-scale mutant screen. To circumvent these technical problems, an assay system was developed on the basis of expression of GFP in living embryos. First, a panel of Gal4 enhancer trap lines was screened to identify those that allow high levels of UAS-driven GFP expression in a subset of PNS neurons at the appropriate developmental stages. Of these, the Gal4 line 109(2)80 was chosen. Recombination was performed to create a second chromosome harboring both the Gal4 109(2) 80 transgene and a UAS-GFP transgene, but no background lethal mutations. A fly line homozygous for the Gal4 109(2) 80/GFP chromosome (denoted as Gal4 80/GFP) was then introduced. In the dorsal clusters of abdominal segments A1-A7, GFP expression labels both axons and dendrites of all six md neurons, one bd neuron, and one tracheal innervating neuron, but not the es neurons. In addition, high levels of GFP expression are detected in the lateral and ventral clusters, and in the antennomaxillary complex. Low levels of GFP fluorescence are also observed in a small subset of neurons in the central nervous system (CNS). The dendrites of dorsal cluster md neurons elaborate just underneath the epidermal layer. In larvae, these dendrites as revealed by Gal4 80/GFP are in tight association with a layer of epidermal cells labeled by Kruppel-Gal4/GFP. It is thus possible to visualize the md neuron dendrites in the dorsal cluster in living animals. A focus was placed on the development of these md neuron dendrites in wild-type as well as mutant embryos. To simplify this description, two types of easily detectable dendrites were defined: dorsal branches grow toward the dorsal midline and lateral branches grow along the approximate anterior-posterior axis toward segment boundaries (Gao, 1999).

The projection pattern of md neuron dendrites in a specific hemisegment is largely invariant from embryo to embryo, on the basis of observations on over thousands of embryos. The major characteristics of dendritic morphogenesis are summarized here. By 12-13 hr AEL, ch and es neurons have already sent out their initial dendritic projections. At this stage, bd neurons have also extended their dendrites. The primary dendrites of md neurons emerge at 13-14 hr AEL, 2 hr after the axons of PNS neurons have reached the CNS. The location of initial dendritic outgrowth and the orientation of this outgrowth are fairly invariant for md neurons. At 13 hr AEL, a dorsal dendrite first emerges from one md neuron in the anterior of the dorsal cluster; shortly after, a second dorsal dendrite emerges from a posterior md neuron of the same cluster. Both dorsal dendrites extend perpendicular to the anterior-posterior axis towards the dorsal midline. Each md neuron (in the dorsal cluster only) sends out one dorsally oriented primary dendrite; however, some md neurons have additional primary lateral dendrites. The dorsal extension essentially stops between 15 and 16 hr AEL, before the lateral branches start to develop. Between 15 and 17 hr AEL, numerous transient lateral branches extend and retract. These branches undergo constant remodeling. Only a subset is eventually stabilized between 18 and 20 hr AEL to become the final lateral branches. At this stage, dorsal and lateral branches are clearly distinguishable. The number of lateral branches in a particular segment is similar from embryo to embryo. In addition, the anterior and posterior dorsal branches within a hemisegment are clearly separated by an area devoid of dendrites. Before hatching (23-24 hr AEL), most lateral branches further elaborate tertiary branches before and after they reach the segment boundary, but only a small number of branches cross over into neighboring segments. At hatching, the dorsal branches have not yet reached the dorsal midline so there is a clear dendrite-free zone near the dorsal midline. After hatching, the dorsal branches resume elongation and reach the dorsal midline by the second instar stage. The length and the thickness of dendritic processes continue to increase with increasing larval body size (Gao, 1999).

Before hatching, the lateral branches are regularly spaced and project toward the segment boundaries. This pattern is relatively invariant from embryo to embryo for a specific hemisegment. To investigate how the dendritic patterning develops, dendrite formation was monitored in living embryos from 15 to 16 hr AEL and time-lapse analysis was carried out. Numerous lateral growth buds emerge anterior or posterior to the dorsal branches and then retract. Only a subset of these lateral branches elongates toward the segment boundaries and becomes stabilized. During this process, the length and orientation of dorsal branches remains largely unchanged. Numerous thin processes at the tips of the lateral branches undergo rapid extension and retraction. These thin processes are not labeled by a Tau-GFP fusion protein, indicating that they might not contain microtubules. This analysis reveals that dendritic development is a dynamic process (Gao, 1999).

Two approaches were used to identify genes involved in dendritic morphogenesis: (1) an investigation of the effects of previously isolated mutations, and (2) a systematic mutant screen. It was reasoned that dendrite development might share some common molecular mechanisms with axon and tracheal development, because all of these processes exhibit subcellular outgrowth and branching. Among the mutants that were examined, prospero mutants and embryos expressing a constitutively active form of Dcdc42 showed detectable dendrite phenotypes. A mutant shows a dendritic phenotype that is somewhat analogous to that in a kakapo mutant. The early budding and extension of lateral branches appear to be normal; however, at a late stage, some lateral branches turn dorsally instead of extending toward segment boundaries, resulting in an apparent reduction in the number of lateral branches. Approximately 60% of the mutant embryos exhibit the phenotype. The mutation that causes lethality and the dendritic phenotype is not complemented by a chromosomal deletion of the region 55F to 56C. Of the available lines carrying lethal P insertions in this region, l(2)02029 does not complement this mutant line. L(2)02029 carries a P-element insertion in the 5' region of the gene enabled. ena46, isolated in this study, does not complement four different enabled alleles: ena210, enaGC5, enaGC8, and ena23. enabled encodes an Abl tyrosine kinase substrate. enabled is known to cooperate with Dlar to control motor axon guidance. These results indicate that enabled affects dendrite as well as axon development (Gao, 1999).

The ena gene was identified through its ability to suppress the Abl mutant phenotype. Since reductions in ena compensate for the absence of Abl, it has been hypothesized that a precise balance between Abl and Enabled activity is required for viability. Similar to the genetic interaction between Abl and ena, heterozygous mutations in ena can partially alleviate the trio mutant phenotype. One interpretation of this interaction is that a balance between trio and ena is required, and Trio may possess a biochemical function that is antagonistic to Enabled's. Since neural enriched isoforms of Mena, the murine homolog of Enabled, are believed to be involved in filopodia formation to extend the growth cone, a potential antagonistic role for Trio is the retraction of growth cones. Drosophila Trio's second DH domain stimulates the formation of stress fibers in REF-52 cells. In neurons, the formation of similar actin-myosin contractile filaments leads to neurite retraction. Therefore, a balance between the biochemical activities of Enabled and Drosophila Trio may be required for a proper balance between extension and retraction of the growth cone in response to attractive and repulsive pathfinding cues (Liebl, 2000).

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).

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).

Actin and microtubules (MTs) are tightly coordinated during neuronal growth cone navigation and are dynamically regulated in response to guidance cues; however, little is known about the underlying molecular mechanisms. Drosophila Pod-1 can crosslink both actin and MTs. In cultured S2 cells, Pod-1 colocalizes with lamellar actin and MTs, and overexpression remodels the cytoskeleton to promote dynamic neurite-like actin-dependent projections. Consistent with these observations, Pod-1 localizes to the tips of growing axons, regions where actin and MTs interact, and is especially abundant at navigational choice points. In either the absence or overabundance of Pod-1, growth cone targeting but not outgrowth is disrupted. Taken together, these results reveal novel activities for pod-1 and show that proper levels of Pod-1, an actin/MT crosslinker, must be maintained in the growth cone for correct axon guidance (Rothenberg, 2003).

Preliminary genetic interaction data suggest that Pod-1 may function in part to transmit guidance signals to the cytoskeleton. For example, several observations were suggestive of a relationship between Pod-1 and Enabled. (1) Axon defects in embryos devoid of Pod-1 resemble defects in embryos mutant for enabled (ena). (2) Pod-1 overexpression recruits Ena to the ends of the neurite-like projections in S2 cells. (3) Extensive colocalization is observed between Pod-1 and Ena in S2 cells as well as in embryos. Therefore genetic interactions between pod-1, ena, and the robo receptor (one of several axon guidance receptors that directly binds to Ena) were tested to ask whether the genes might function together in midline repulsion, a specific axon guidance decision that involves Robo and Ena. Indeed, it was found that while pod-1 zygotic mutants, ena heterozygotes, or robo heterozygotes do not exhibit midline crossing errors, when gene dosages of ena or robo are reduced simultaneously with pod-1, frequent (i.e., in approximately 30% of abdominal segments) midline crossing errors are observed (Rothenberg, 2003).

Effect of mutations in Abl

The Drosophila Abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, abl/v-abl-chimeric Abelson murine leukemia viruses were constructed. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of Drosophila ABL protein. The chimeric Abelson murine leukemia viruses are able to mediate morphological and oncogenic transformation of NIH 3T3 cells and are able to abrogate the interleukin-3 dependence of a lymphoid cell line. A virus that contains both TPK and carboxy-terminal Drosophila ABL regions has no in vitro transforming activity for primary bone marrow cells and lacks the ability to induce tumors in susceptible mice. A virus that replaces only a portion of the v-abl TPK region with that of Drosophila ABL has low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of ABL TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity (Holland, 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 expression patterns and mutant phenotypes indicate a role for ABL in establishing and maintaining cell-cell interactions (Bennett, 1992).

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) implicates 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).

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, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase (Hill, 1995).

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).

Initial analysis of Capt in the Drosophila germline shows that this protein is required for proper oocyte polarity. To determine whether Capt is also required for the establishment and/or maintenance of follicle-cell apical-basal polarity, the localization of microtubules, Crumbs, alpha-spectrin and ß-spectrin was examined in capt clones. With these markers, it is found that capt mutant follicle cells retain many aspects of their wild-type epithelial polarity despite the profound change in actin organization. Thus, it is possible that apical actin-filament formation in capt mutant clones is localized by the action of the cell's apical-basal targeting machinery. Alternatively, proximity to the germline could define the site of preferential apical actin-filament formation in a capt mutant cell. To distinguish between these two possibilities clones were generated within the columnar epithelium that lacked both capt and lethal(2)giant larvae [l(2)gl] gene functions (because l(2)gl is required cell-autonomously for the maintenance of epithelial polarity). In capt l(2)gl double-mutant cells, F-actin is frequently seen accumulating at a single, randomly positioned site within the cell. Thus, although components of the apical-basal targeting machinery are not required to limit actin-filament formation to a single site, they are necessary to target actin-filament formation to the apical surface of capt mutant cells. These data led to a postulate of the existence of a protein that is targeted to apical junctions and that promotes local actin-filament formation after the loss of Capt. Ena was considered a potential candidate (Baum, 2001).

Ena family members are thought to be key regulators of actin-filament dynamics. In mammalian cells they promote actin-filament formation and are localized at adherens junctions and focal contacts. To determine whether Drosophila Ena shares similar properties, the distribution and function of Ena within follicle cells was analysed. In early egg chambers, Ena protein is concentrated at the apical cell cortex of wild-type follicle cells. Subsequently, as the epithelium begins to migrate, the level of Ena increases, particularly within posterior follicle cells and in motile border cells. At this stage the protein remains localized together with F-actin at the apical surface of the epithelium. However, Ena is also observed in punctate cytoplasmic structures that lack coincident actin filaments. These structures are present in follicle cells throughout oogenesis but become more striking as the egg chamber develops. Finally, at late stages of oogenesis, Ena protein becomes concentrated at the basal surface of the follicular epithelium, flanking stress-fibre-like actin filaments. To locate Ena more precisely, the distribution of Ena was compared with that of Armadillo (Arm). Arm is the Drosophila ß-catenin homologue and is localized at apical adherens junctions interconnecting cells within the epithelium. It was found that Ena and Arm localize together apically in wild-type follicle cells. Thus, through much of oogenesis, Ena is concentrated together with F-actin and Arm at follicle-cell adherens junctions, the site of ectopic actin-filament formation in capt mutant cells (Baum, 2001).

As a test of Ena function, follicular ena clones were generated. Cells homozygous for hypomorphic ena alleles (ena210, ena23) lose cortical actin filaments from apical, basal and lateral sites. However, whereas chic clones preferentially lose basal actin filaments, ena mutant cells also exhibit a marked decrease in the amounts of apical F-actin. This might reflect the fact that Ena is concentrated at apical junctions in the wild type, whereas profilin has a broader cellular distribution. It is concluded that Ena facilitates actin-filament formation in Drosophila, much as it does in mammalian cells (Baum, 2001).

In mammalian cells, the overexpression of Ena homologues is able to induce ectopic actin-filament formation. To determine whether Ena can promote excessive actin-filament formation in Drosophila, Ena was overexpressed in follicle cells and, in a separate experiment, in the germline. In both cell types Ena is able to induce the formation of novel F-actin structures at sites where Ena aggregates accumulate. Thus, Ena is likely to be a critical determinant of the subcellular distribution of actin filaments within a cell (Baum, 2001).

Given Ena's important role in the control of epithelial actin organization, ena capt double-mutant clones were generated to test whether Ena is also required for the formation of apical actin aggregates in the capt mutant. Because ena and capt genes are located on opposite arms of chromosome II, this is an experimental challenge. However, the mutants were recombined onto a double FRT chromosome to generate double-mutant cells that were identified by the absence of green fluorescent protein (GFP) and the loss of Capt. F-actin aggregates do not form in the double mutant lacking both Ena and Capt functions, and ena capt clones frequently lose actin filaments. This result places Ena genetically downstream of Capt. Thus, like profilin, Ena is required for the synthesis or nucleation of actin filaments at adherens junctions in the capt mutant.

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).

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).

Enabled plays key roles in embryonic epithelial morphogenesis in Drosophila

Studies in cultured cells and in vitro have identified many actin regulators and begun to define their mechanisms of action. Among these are Enabled (Ena)/VASP proteins, anti-Capping proteins that influence fibroblast migration, growth cone motility, and keratinocyte cell adhesion in vitro. However, partially redundant family members in mammals and maternal Ena contribution in Drosophila previously prevented assessment of the roles of Ena/VASP proteins in embryonic morphogenesis in flies or mammals. This study used several approaches to remove maternal and zygotic Ena function, allowing this question to be addressed. Inactivating Ena does not disrupt cell adhesion or epithelial organization, suggesting its role in these processes is cell type-specific. However, Ena plays an important role in many morphogenetic events, including germband retraction, segmental groove retraction and head involution, whereas it is dispensable for other morphogenetic movements. This study focused on dorsal closure, analyzing mechanisms by which Ena acts. Ena modulates filopodial number and length, thus influencing the speed of epithelial zippering and the ability of cells to match with correct neighbors. Filopodial regulation was explored in cultured Drosophila cells and embryos. These data provide new insights into developmental and mechanistic roles of this important actin regulator (Gates, 2007).

Drosophila Ena localizes to AJs and regulates cortical actin assembly in follicle cells, and Ena/VASP proteins help establish cell adhesion in keratinocytes and mammary epithelial cells (Scott, 2006). The hypothesis was tested that Ena/VASP proteins play key roles in cell-cell adhesion. Surprisingly Ena inactivation or mutation does not disrupt AJs or epithelial integrity in embryos or imaginal discs, suggesting that they do not play general roles in these processes. Of course, Ena/VASP proteins may play cell type-specific roles; e.g., filopodial zippers initiating keratinocyte adhesion may require Ena, whereas other mechanisms of establishing epithelial architecture may not. These data also reveal morphogenetic events in which Ena is nonessential; e.g. there were no apparent defects in ventral furrows or germband extension. Consistent with this, Mena/VASP/Evl triple mutant mice complete many morphogenetic processes without major defects (Gates, 2007).

These data reveal key roles for Ena in many morphogenetic events: germband retraction, head involution, segmental groove retraction and dorsal closure. These build upon previously documented roles for Ena in axon guidance and actin regulation in follicle cells, providing a view of the spectrum of biological events requiring Ena. It is hypothesized that Ena acts in a mechanistically similar manner in each process, as an actin anti-Capping protein promoting filament elongation. Flies use this tool in many different ways, promoting distinct cell behaviors. Some events can be linked fairly directly to known Ena/VASP functions. The role of Ena in axon guidance fits its actin regulatory role well. During dorsal closure, Ena inactivation reduces filopodia, consistent with its biochemical function. The data reveal that this slows epithelial zippering and disrupts precise alignment between the two sheets. Leading-edge filopodia have been proposed to function both as sensors directing proper cell matching, and to facilitate adhesion of cells from opposing edges in Drosophila and C. elegans. Although the data support a sensory role for filopodia, disrupted epithelial adhesion/fusion was not observed. The data support and contrast with work on embryos expressing dominant-negative Cdc42 (Cdc42DN), which produce rudimentary protrusions. Like ena mutants, Cdc42DN embryos display mismatching of cells from opposing edges, but, unlike ena mutants, Cdc42DN embryos have gaps between cells along the midline. Although Cdc42DN-expressing leading-edge cells produce only rudimentary protrusions, FP4mito-expressing cells produce robust lamellipodia. Because AJ formation in cultured mammalian cells can be mediated either by filopodia or lamellipodia, lamellipodia may mediate adhesion in the absence of filopodia (Gates, 2007).

Ena is also essential for morphogenetic events in which its cell biological role is more speculative. Germband retraction requires integrin-mediated adhesion of amnioserosa and epidermis to couple cell-shape changes in the two tissues. Ena inactivation mimics integrin loss. Ena/VASP proteins localize to focal adhesions, and Drosophila Ena colocalizes with integrins at ends of planar-polarized actin bundles in follicle cells. Ena/VASP inactivation does not disrupt focal adhesions, but may modulate their size and stress fiber robustness under mechanical stress. Drosophila Ena may strengthen the cytoskeleton during germband retraction, promote amnioserosal lamellipodia or regulate extracellular matrix (ECM) adhesion more directly, as VASP does in platelets and as Ena/VASP proteins may do in Xenopus somitogenesis. Less is known about mechanisms by which segmental grooves form and retract. However, Ena is planar-polarized to dorsal-ventral cell boundaries in these cells; perhaps it stabilizes actin attachment at borders where it is enriched. Defects in head involution result from alterations in dorsal-fold cell shape change, which may share mechanistic similarities with segmental groove formation (Gates, 2007).

One key challenge is to identify machinery required to generate filopodia and lamellipodia. a striking correlation was found between Ena activity and filopodial length and number. Inactivating Ena significantly decreased filopodial length and number, whereas increasing Ena activity increased filopodial length. Interestingly, maximum filopodial length was not substantially altered, and thus is probably not limited by Ena levels. However, Ena can be rate-limiting in filopodial formation as Ena overexpression generated filopodia on lateral epithelial cells that normally do not produce them. Together, these data suggest that Ena promotes both initiation and elongation of leading-edge filopodia (Gates, 2007).

Ena is concentrated at the tips of elongating filopodia, consistent with its influence on filopodial length and its biochemical function. Interestingly, GFP-Ena particles move rearward prior to retraction, presumably by retrograde flow, and some GFP-Ena is retained at filopodial tips as they retract. This was unexpected, since the anti-capping function of Ena/VASP suggested that Ena at filopodial tips would promote extension. Although this could be an artifact of GFP-Ena, it may indicate complexity in the control of filopodial dynamics. For example, whether filopodia continue extending or retract may be determined not only by actin polymerization rates at the tip, but also by depolymerization and/or retrograde flow rates at its base. In addition, filopodial dynamics may be regulated at individual filaments within filopodia rather than the structure as a whole. GFP-Ena particles may be locally inactivated Ena on individual filaments moving away from the tip by retrograde flow (Gates, 2007).

One unanswered question is whether different filopodial regulators act additively or in series. Mammalian Ena/VASP can act downstream of Cdc42 together with IRSp53, but IRSp53 can promote Ena/VASP-independent filopodia (Nakagawa, 2003). Formins also promote filopodia, but whereas Dictyostelium dDia2 and VASP directly interact, Ena/VASP:formin relationships remain unclear. The reduced number of short filopodia formed when Ena is inactivated is consistent with multiple mechanisms acting additively/synergistically to produce the appropriate filopodial number/length (Gates, 2007).

These data also test in vivo one aspect of the convergent elongation model. This proposes that tip complex proteins bind filaments and protect them from capping, allowing continued elongation, and then interact laterally, bundling filaments and forming filopodia. Ena/VASP proteins may supply anti-capping activity, and could also help mediate lateral association via tetramerization. GFP-Ena overexpression promoted large lamellipodia containing numerous actin microspikes; these often converged at their distal ends to form filopodia, supporting the convergent elongation model (Gates, 2007).

Filopodial regulation was also examined. The data demonstrate that Abl is a key negative regulator of filopodial extension in cultured cells and in vivo, inhibiting Ena accumulation at nascent filopodial tips. This idea is further supported by a parallel analysis of embryos expressing activated Bcr-Abl or excess wild-type Abl; both reduce filopodia on leading-edge and amnioserosal cells. This provides a means for signal transduction pathways to regulate cell behavior (Gates, 2007).

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).

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. Null alleles of the single Drosophila ZO protein Polychaetoid (Pyd), have been generated. Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure (Choi, 2011).

ZO family proteins localize to mammalian TJs and also to AJs in mammals, flies, and nematodes. Elegant work in cell culture revealed important roles for mammalian ZO family proteins in properly localizing TJ strands into a functional, apically-localized barrier. Furthermore, whereas cultured mammalian cells lacking ZO family function can assemble AJs, their maturation into smooth belt junctions, a phenotype thought to involve remodeling the linkage to the actin cytoskeleton, is impaired (Choi, 2011).

It was thus hypothesized that ZO family proteins would be essential for AJ maturation and/or maintenance during normal development. However, assembly of spot AJs into more continuous belt AJs occurred normally in pydMZ mutants, and there were no apparent defects in DE-cad levels or localization, even late in embryonic morphogenesis. Furthermore, loss of Pyd did not perturb tracheal trunk fusion, an event that requires AJ function. Finally, loss of Pyd did not perturb the junctional localization of its AJ binding partner Cno. The data also suggest that Pyd is dispensable for assembly of tracheal septate junctions -- although this is perhaps not surprising, as fly Pyd does not localize to septate junctions. The data are consistent with analysis of the nematode ZO-1 orthologue ZOO-1 (Lockwood, 2008), which is also dispensable for AJ assembly. It will be interesting to examine mouse ZO family double and triple mutants to determine the full role of these proteins in both AJs and TJs during mammalian development (Choi, 2011).

Subtle changes in levels of AJ proteins in the absence of Pyd cannot be ruled out. Djiane (2011) recently reported that although AJs remain in pyd mutant cells, cells lacking Pyd accumulate higher levels of membrane-associated DE-cad than neighboring wild-type cells. Djiane's data provides support for a model in which Pyd binds and may regulate the E3 ubiquitin ligase Su(dx), which regulates the endocytic trafficking of Notch. Perhaps Pyd plays a similar role in regulating the trafficking of AJ proteins (Choi, 2011).

Pyd's role in Notch signaling during postembryonic development was not explored in this study, since that was the subject of parallel of Djiane (2011). However, the current data do not support an essential role for Pyd in embryonic Notch signaling, as Notch mutant embryos lose ventral epidermal cells and gain excess neurons, phenotypes not observed int this study. Subtler roles for Pyd in Notch or other signaling pathways in the embryo cannot be ruled out. In fact, the presence of extra terminal cells in the tracheal system may be indicative of Notch signaling defects in that tissue (Choi, 2011).

Although Pyd is not essential for assembly or maintenance of AJs, this study found that it does play important roles in embryonic morphogenesis in both the epidermis and trachea. From 40 to 70% of embryos lacking maternal and zygotic Pyd die as embryos, with characteristic defects in head involution. This was true in embryos mutant for three different deletion alleles, two of which did not remove any other coding sequences. Even for events that usually go to completion in the absence of Pyd, like dorsal closure, execution does not proceed normally. For example, loss of Pyd disrupts coordinated cell shape changes in the epidermis during dorsal closure and significantly slows this process. Pyd also plays an important role in effective zippering together of the two epidermal sheets at the canthi and in maintaining a straight leading edge. Furthermore, the tracheal defects observed are consistent with defects in intercalation, as were previously documented in weaker alleles, along with possible defects in cell fate. Thus fly Pyd, like nematode ZOO-1 (Lockwood, 2008), is an important regulator of morphogenesis. Because Pyd is a complex, multidomain protein with many binding partners, in the future, it will be of interest to explore how the different domains of ZO-1 contribute to its functions in vivo (Choi, 2011).

Of interest, zygotic cno mutants share all of the cell shape and morphogenesis defects of pydMZ mutants. This is consistent with early data demonstrating both physical and genetic interactions, thus strongly suggesting that Cno and Pyd work together in regulating coordination of adhesion and the cytoskeleton. Recent work suggests that during apical constriction and invagination of mesoderm cells, Cno is one of the linkers anchoring the actomyosin cytoskeleton at AJs (Sawyer, 2009). Consistent with this idea, an apparent rupture of the LE actomyosin cable was shown in both pydMZ and cno mutants, leading to splayed open and hyperconstricted LE cells. During dorsal closure, these data would be consistent with a model in which Cno and Pyd specifically reinforce AJ-actomyosin connections at points where tension is the greatest. It will be interesting to examine whether mammalian ZO-1/ZO-2 and afadin functionally interact in a similar way (Choi, 2011).

Another player in dorsal closure is the fly nectin-like protein Echinoid (Ed). Like the mammalian nectins, Ed is an immunoglobulin-superfamily cell adhesion molecule. Both nectins and Ed associate with afadin/Canoe. Ed plays an important role during dorsal closure (LaPlante, 2011), and Ed, like Pyd, plays a role in tracheal development (Laplante, 2010). During dorsal closure, Ed expression is lost from the amnioserosa but maintained in the epidermis (Laplante, 2011, and juxtaposition of adjacent cells that express and those that do not express Ed can lead to actin cable assembly. However, ed maternal and zygotic mutants differ from both pydMZ and cno zygotic mutants: in ed mutants the actomyosin cable fails to assemble. Furthermore, unlike Ed, Cno and Pyd continue to be expressed in the amnioserosa. The mechanistic role of Ed remains somewhat controversial, with suggestions that it works through fly myosin VI to regulate myosin contractility and suggestions that it sets up a tissue boundary, allowing proper polarization of junctional and cytoskeletal proteins in the leading edge (Laplante, 2011). It will be interesting to explore whether Ed, Cno and Pyd work together during dorsal closure (Choi, 2011).

The suite of defects during dorsal closure shared by pydMZ and cno mutants is complex, including defects in LE cell shapes, a wavy leading edge, defects in zippering at the canthi, persistent deep segmental grooves, and simultaneous disruption of head involution. This entire suite of defects was strikingly reminiscent of those previously observed in embryos in which the function of the actin regulator Ena was disrupted by genetic inactivation, sequestration to mitochondria, or expression of a constitutively active form of its negative regulator Abelson (Abl) kinase. This led to an exploration of the hypothesis that Pyd and Cno worked together with or regulated Ena (Choi, 2011).

During dorsal closure, Ena has an interesting localization pattern in epidermal cells. It localizes to AJs and is particularly enriched at tricellular junctions. It also localizes to ends of filopodia produced by LE cells. However, the most striking feature of Ena localization during this stage is its dramatic accumulation in 'LE dots', which form at the dorsal ends of the AJs between LE cells, where they overlay the amnioserosa. These overlap locations where the actomyosin cable is anchored. It was initially hypothesized that LE dots might play a role in cadherin-based cell adhesion, but this is not disrupted in ena mutants. Reducing Ena function does reduce filopodia, which is suspected to underlie defects in zippering of the epidermal sheets. The role of Ena in LE dots is less clear. It is speculated that LE dots are Ena storage places, from which it is released to modulate cell protrusions at the leading edge. Consistent with this, activation of the formin Diaphanous leads to loss of Ena from LE dots and dramatic alterations in protrusive behavior. However, the defects seen in LE cell shape in ena mutants are also consistent with the idea that Ena plays a role in anchoring or maintaining the actin cable at the leading edge (Choi, 2011).

Clear alterations were seen in Ena localization in LE cells in pydMZ or cno mutants. Enrichment of Ena in LE dots was reduced overall and became very uneven. It is tempting to speculate that the failure to effectively recruit Ena to LE dots leads to the defects in LE cell shape observed in pydMZ or cno mutants. If failure to deliver Ena to LE dots also interfered with subsequent release to the leading edge, this might alter protrusive behavior and slow zippering of the epidermal sheets at the canthi—this remains to be tested. To test the hypothesis that regulating Ena is an important part of the roles of Pyd and Cno during dorsal closure, genetic interactions were examined. Loss of zygotic Ena has only a subtle effect on epidermal morphogenesis, as the maternal Ena suffices for most events. However, reduction of maternal/zygotic Ena significantly enhanced the epidermal phenotype of zygotic cno mutants, and reduction of maternal/zygotic Cno enhanced the epidermal phenotype of zygotic ena mutants, consistent with them working together during this process; it is important to note that in both zygotic mutants maternal Ena or Cno remains, so enhancement is a plausible prediction for double mutants of genes in the same pathway. It will be interesting to further explore this mechanistic connection, probing whether Ena physically interacts with either Cno or Pyd and how they regulate Ena localization and/or activity (Choi, 2011).

Dissecting regulatory networks of filopodia formation in a Drosophila growth cone model

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. This study has focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, embryonic Drosophila primary neurons were as one consistent cellular model to study filopodia regulation. The data for loss-of-function of capping proteins, Enabled, different Arp2/3 complex components, the formin DAAM, and profilin, reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of the Drosophila model system. Using combinatorial genetics, this study demonstrated that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM, filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. These genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps (Goncalves-Pimentel, 2011).

The Drosophila actin regulator Enabled regulates cell shape and orientation during gonad morphogenesis

Organs develop distinctive morphologies to fulfill their unique functions. Drosophila embryonic gonads were used as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. A membrane GFP marker was developed to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. It is proposed that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction (Sano, 2012).

SGPs and PGCs display dramatic changes as they progress from cell aggregates to the well-compacted embryonic gonads. In the mature gonad, PGCs are embedded in a mesh network of SGP cytoplasmic extensions, and the interaction between these cells is enhanced by SGP-driven gonad compaction. The compaction process is important not only in maintaining the structural integrity of the gonad but also in ensuring proper differentiation of germline stem cells, which requires the intimate association of PGCs and the niche derived from anterior SGPs. This study found that Ena, an actin regulator, is required for gonad compaction. Ena acts in the soma, and loss of ena alters SGP cell shape and orientation, and intracellular distribution of DE-cad. Mathematical simulation suggests that Ena has an important role in regulating the cadherin-dependent cell adhesive forces necessary for proper organ morphogenesis (Sano, 2012).

Detailed analysis of SGP morphology during gonad formation revealed that gonad compaction relies on coordinated changes in SGP cell shape and orientation. Anterior SGPs turn less than those located in the middle or posterior pole, which may contribute to the overall anterior movement of the gonad primordium from its original position at PS10-12 to its final location at PS10-11. Consistent with their failure to form a compact gonad, the angle of ena mutant SGPs is smaller with respect to the gonad AP axis (Sano, 2012).

It was noticed that the ena mutation affects SGP cell shape and axis orientation differently in different regions of the gonad. Defects in cell shape changes were more severe at the anterior than in the posterior, while cell orientation was disturbed more severely in the middle and posterior SGPs. Since ena is detected uniformly in all SGPs, these phenotypic differences are likely due to internal differences in the SGPs. In the early stages of development, SGPs are specified as three clusters in PS10, 11, and 12 in the mesoderm. The homeobox gene, abd-A, is expressed in PS10 through 12, while abdominal-B (abd-B) is only expressed in PS11 and 12 at the coalescence stage. Although the ultimate fate of individual SGPs from each parasegment remains unknown, differential expression of abd-A and abd-B could account for the different cellular behaviors (Sano, 2012).

It is well established that Ena family proteins promote actin filament elongation. Therefore, it is likely that Ena is involved in DE-cadherin dynamics through regulation of the local actin morphology in SGPs. Ena translocation to the inner surface of SGPs might accelerate actin filament bundling at the inner surface. DE-cad is known to associate with bundled actin filaments and this may contribute to its accumulation at the inner surface of SGPs. Mechanical force could also be involved in DE-cad localization. In vitro analysis has demonstrated the force-dependent recruitment of the actin-binding protein, Vinculin, by α-Catenin. Adhesion by E-cad to the adjacent cell stretches the membrane, with the apparent transmission of the stretching force to α-Catenin resulting in the recruitment of Vinculin and actin filaments. This mechanism could also act at the contact surface between SGPs, thus stabilizing DE-cad (Sano, 2012).

Recent studies have shown that cellular pattern formation during morphogenesis is coordinated via the localization and/or activity of force-generating molecular machinery, such as cell adhesion molecules and Myosin. Thus, the altered DE-cad distribution in ena mutants prompted an examination of whether ena controls the force balance in gonadal cells by regulating DE-cad. Since ena expression and function are necessary in the soma, and PGCs are dispensable for gonad compaction, it is reasonable to focus on SGPs to determine the parameters controlling these forces by numerical simulation. in silico analysis showed that gonad compaction is promoted by increased adhesive force on the inner surface. This is consistent with the DE-cad relocation observed in vivo. Larger adhesive forces between SGPs increase SGP-SGP adhesion surfaces leading to SGP rearrangement and incorporation to form a spherical gonad. It was also shown that a larger contraction force along the outer surface (between the SGP and surrounding non-gonadal environment) resulted in increased gonad compaction; however, no significant Myosin II accumulation at the outer membrane of SGPs was detected. One possibility is that Myosin activity, instead of localization, is increased at the outer membrane. Alternatively, Myosin II could act in the SGP cytoplasmic extensions to contract them, thereby generating the inward force required for gonad compaction. However, no overt changes were observed in the cellular protrusions in ena mutants, making this alternative less likely (Sano, 2012).

A previous study found that robo genes are required for gonad compaction and Robo2 was localized at contact sites between SGPs. The Robo receptor family is reported to be involved in homophilic and heterophilic adhesion and repulsion. Indeed, Ena has been shown to bind to Robo, suggesting that the observed effects of Robo on gonad morphogenesis could be mediated by Ena. Ena's function appears, however, primarily associated with the morphogenetic movements and cell shape changes observed during gonad formation, as ena mutants, in contrast to robo mutants, do not affect PGC ensheathment. Future quantitative measurement of force dynamics and force-generating molecular machinery, coupled with live observation in specific genetic backgrounds, will further clarify the mechanics of gonad compaction (Sano, 2012).

Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

Prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. This study defines a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton-temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling (Spracklen, 2014).

Development and adult tissue homeostasis require dramatic movements and reorganization of both cells and whole tissues. Underlying all of these processes is the actin cytoskeleton, which serves as a dynamic scaffold to facilitate cell migration, cell division, and cell shape. Tight regulation of actin cytoskeletal dynamics is mediated by the concerted activity of over one hundred known actin binding proteins. While much is known about how the activity of individual actin binding proteins are regulated, very little is known about the mechanisms by which the activity of multiple actin binding proteins is coordinated to mediate developmental processes and tissue homeostasis (Spracklen, 2014).

One possible mechanism by which such coordination may occur is through prostaglandin (PG) signaling. PGs are small, bioactive lipids that act as paracrine and autocrine signaling molecules to regulate numerous physiological processes including pain, inflammation, fertility, and cardiovascular function. PGs are synthesized downstream of cyclooxygenase enzymes (COX1 and COX2), which convert free arachidonic acid into the precursor PGH2, and are the pharmacologic targets of non-steroidal anti-inflammatory drugs. PGH2 is then processed into biologically active prostanoids (including PGD2, PGE2, PGF, PGI2, and TXA2) downstream of COX enzymes through the activity of specific synthases. Following their synthesis, PGs most commonly serve as ligands for specific G protein-coupled receptors (GPCRs), which elicit their downstream effects through activation of Gα and, in some cases, Gβγ. Additionally, PGs may induce MAPK signaling pathways, activate Rho GTPases, or serve as PPARγ nuclear hormone receptor ligands (Spracklen, 2014).

In vitro studies have provided evidence that PG signaling can regulate the actin cytoskeleton in both a cell-type and PG-type dependent manner. For example, TXA2 and PGF2α stimulate actomyosin-based contractility, whereas PGE2 and PGI2 promote relaxation in hepatic stellate cells. Subsequently, PGs were found to have opposing effects on cytoplasmic actin filaments (i.e., actin stress fibers) in multiple cell types. While PGE2 promotes actin stress fiber assembly in rat IMCD cells and stability in IEC-6 cells, it induces actin stress fiber disassembly in A431 cells, HeLa cells, rat-1 fibroblasts and human aortic smooth muscle cells. Similarly, both PGE2 and PGI2 promote actin stress fiber disassembly in human pulmonary artery endothelial cells. PGF promotes filopodia retraction and actin stress fiber assembly in 293-EBNA cells. In human umbilical vein endothelial cells, TXA2 slows αvβ3-dependent cell adhesion and inhibits cell spreading, while PGE2 accelerates cell adhesion and promotes cell spreading. Interestingly, cytoskeletal inputs (i.e., mechanical stretching) have been shown to induce COX2- dependent production of PGE2, which subsequently leads to disassembly of actin stress fibers in murine podocytes (Spracklen, 2014 and references therein).

PG signaling is also known to directly regulate platelet activation and aggregation, which requires actin cytoskeletal remodeling, including the rapid generation of filopodia that mediate protrusion and adhesion. TXA2, the major prostanoid produced in human platelets, is a potent activator of platelet aggregation, while PGI2, PGE1, and PGD2 inhibit platelet aggregation. Additionally, PGE2 has been shown to both potentiate and inhibit platelet aggregation (Spracklen, 2014 and references therein).

The above-mentioned studies have provided some insight into the mechanisms by which PGs regulate cytoplasmic actin filament remodeling. Multiple in vitro studies demonstrate that the PG-dependent morphological changes in cytoplasmic actin bundles occur via cAMPdependent mechanisms, albeit through different downstream events including: cAMP-dependent kinase (PKA) and nucleotide exchange proteins directly activated by cAMP (Epac1)/Ras-related protein 1 (Rap1)-dependent activation of Rac, PKA-dependent Rac activation and Rac-independent activities, or PKA-dependent decreases in focal adhesion kinase (FAK) phosphorylation. Other in vitro studies implicate Rho activation downstream of PGs in driving the changes in actin stress fiber assembly. Furthermore, PGI2 and PGE1 block platelet activation through cAMP/cGMPdependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of actin elongation factors. Thus, while these studies have provided some insight into the mechanisms by which PG signaling regulates cytoplasmic actin filament assembly/disassembly, much remains to be determined including how multiple PG signals are integrated to coordinate actin remodeling and the mechanisms through which particular PG signals regulate actin dynamics (Spracklen, 2014 and references therein).

Drosophila oogenesis is a well-established model system for studying actin cytoskeletal remodeling and regulation. It consists of 14 well-characterized, morphological stages of follicle development. At stage 9 (S9) of follicle development, the follicle consists of 16 germline-derived cells (15 support or nurse cells and a single oocyte), which are surrounded by ~1000 somatically-derived epithelial cells. Multiple processes occur during S9 that are critical for female fertility. A small group (6-8) of cells, termed border cells, delaminate from the anterior of the follicle and migrate between the nurse cells toward the dorsal-anterior of the oocyte, while the remaining follicle cells migrate posteriorly over the nurse cells and oocyte to form an anterior-posterior gradient of follicle cell thickness. During S9, the oocyte actively takes up yolk granules from the hemolymph, and microtubule-dependent, slow cytoplasmic streaming establishes oocyte polarity. Aside from cortical actin deposits, the cytoplasm of the nurse cells is largely devoid of actin filament bundles through the end of stage 10A (S10A). During stage 10B (S10B), the actin cytoskeleton within the nurse cells undergoes rapid remodeling resulting in increased cortical actin deposition and the formation of a cage-like network of parallel actin filament bundles extending from the nurse cell membranes inward, toward the nurse cell nuclei. This dramatic actin remodeling is required to provide the contractile force necessary for the rapid transfer of nurse cell cytoplasm (nurse cell dumping) into the growing oocyte at S11, while preventing the nurse cell nuclei from obstructing the ring canals-specialized cytoplasmic bridges-that the cytoplasm must flow through (Spracklen, 2014).

Previous studies have identified critical roles for PG signaling in regulating actin bundle formation during S10B and gene expression during Drosophila oogenesis. Using this same model, Fascin, an actin bundling protein, has been established as a novel downstream target of PG signaling during PG-dependent actin remodeling during S10B. Thus, Drosophila oogenesis is an attractive model for identifying the likely conserved mechanisms by which PG signaling coordinates actin cytoskeletal remodeling. This study shows that PG signaling temporally regulates the onset of actin remodeling during Drosophila oogenesis. While prior studies have largely focused on the cytoskeletal events occurring during S10B, this study primarily focused on the previously undescribed role of PGs in preventing actin filament formation during S9. Wild-type S9 follicles exhibit a low level of early actin structures in the posterior nurse cells, whereas loss of Pxt, the Drosophila COX-like enzyme, results in the highly penetrant presence of extensive actin filament and aggregate formation in the posterior nurse cells at S9. This study found that two actin binding proteins, Hts-RC (Adducin) and Quail (Villin), localize to early actin structures in wild type and pxt mutant S9 follicles, while Enabled (Ena), the sole Ena/VASP family member found in Drosophila, localizes preferentially to those early actin structures found in pxt mutants. Furthermore, genetic reduction of Ena in pxt mutants suppresses this early actin remodeling. Additionally, this study found that Ena localization to the sites of parallel actin filament bundle formation at S10B is reduced in pxt mutants. Together, these data are consistent with the model that PG signaling cascades regulate Ena localization/activity to temporally regulate actin filament formation during Drosophila oogenesis, at least in part, by restricting Ena localization/activity earlier in oogenesis (S9) and promoting appropriate Ena localization/activity later in oogenesis (S10B). Further understanding the mechanisms by which PGs exert opposing effects on Ena localization/activity during Drosophila oogenesis is likely to shed light on conserved mechanisms by which PGs may generally regulate the Ena/VASP family of proteins (Spracklen, 2014).

This study provides strong evidence that PGs temporally regulate cytoplasmic F-actin rtical actin, suggest that Fascin may generate microspikes or short filopodia that are requ penetrant, early induction of filamentous and aggregated actin structures in the posterior nurse cells of S9 follicles. Importantly, overexpression of Pxt suppresses early actin remodeling, and similar structures are observed at a low frequency in wild-type follicles. Previously, it has been shown that Pxt is also required for cortical actin integrity and bundle formation during S10B. Together these data lead to the model that, during S9, Pxt-dependent PG production initiates a signaling cascade that prevents or restricts early actin remodeling, while during S10B, Pxt-dependent PG signaling induces actin remodeling events necessary for nurse cell dumping. These opposing activities could be achieved through different PGs at S9 and S10B, or the same PG may be produced at both stages, but elicit distinct signaling cascades. The first possibility is favored as exogenous PGE2 inhibits, while PGF promotes in vitro nurse cell dumping and restores dumping in the presence of COX inhibitor treatment or genetic loss of Pxt (Spracklen, 2014 and references therein).

While early actin remodeling in the posterior nurse cells is observed in response to certain stresses and in a few mutant backgrounds, current understanding of how these structures form and the consequences of such formation is severely limited. Overexpression of death-inducing factors in the follicle cells, or starvation, results in follicle death at S8/9 and the accumulation of actin filaments and aggregates. Interestingly, these actin structures colocalize with Hts-RC, similar to what was observed in both wild-type and pxt mutants. Additionally, expression of active Dcp-1 disrupts the actin cytoskeleton within the nurse cells at S10B, suggesting that limiting caspase activation may prevent the destruction of the nurse cell cytoskeleton. Furthermore, loss of Midway, a diacylglycerol acyltransferase, causes S8 checkpoint death and the dying follicles accumulate extensive actin filaments in the posterior nurse cells. Thus, early actin structures may either cause or be caused by the induction of follicle death (Spracklen, 2014).

If premature actin remodeling in pxt mutants is either driving or caused by induction of follicle death, then the prevalence of the actin structures should be similar to the levels of death. This is not what was observed, as 34% of pxtf and 74% of pxtpxtEY S9 follicles exhibit early actin structures, while there is a much higher level of follicle death in pxtf (54% death), the allele with a lower frequency of early actin structures, than in the pxtEY allele (22% death). These data suggest that early actin structures can form and not result in follicle death, and that follicles can die without forming such structures. Thus, the function of these early actin structures remains unclear (Spracklen, 2014).

These early actin structures may function to assess whether the nurse cells are capable of the dramatic S10B remodeling events. In this case, small structures form and are rapidly depolymerized, as few wild-type S9 follicles exhibit visible, early actin structures. Supporting this idea, it was found that strong germline expression of actin labeling tools, which likely stabilize actin structures, result in increased frequency and size of these structures. Alternatively, the early actin structures may regulate nuclear position. Indeed, Hts (one of the factors found to be associated with the early actin structures) localizes to a perinuclear actin meshwork that maintains nuclear position during nurse cell dumping. Defining the function of these early actin structures will require further analyses of their structure, dynamics, and regulation (Spracklen, 2014).

The data are consistent with the model that both the early actin remodeling during S9 and the inhibition of canonical actin remodeling during S10B observed in pxt mutants are due, at least in part, to misregulation of Ena, the sole Drosophila Ena/VASP family member. Supporting this model, this study found that while the actin regulators Hts and Villin localize the early actin structures in both wild-type and pxt mutant follicles, Ena preferentially localizes to the early actin structures in pxt mutants. Furthermore, a reduction in Ena level suppresses the early actin remodeling observed in pxt mutant S9 follicles, but has no effect on the prevalence of those structures in a wild-type background. Ena has been previously shown to promote actin remodeling during S10B. Interestingly, Ena localization to the sites of canonical F-actin elongation is reduced in pxt mutants during S10B. The alterations in Ena localization in pxt mutants during both S9 and S10B are not due to changes in mRNA or protein expression. These data lead to the hypothesis that Pxt-dependent production of PGs results in the activation of signaling cascades that either directly or indirectly lead to altered Ena localization/activity. Ena may be regulated by protein-protein interactions, its antagonist Capping protein, or phosphorylation. Interestingly, loss of kinases known to regulate Ena/VASP proteins, PKA and Abl also result in early actin remodeling (Spracklen, 2014).

While PG signaling is known to regulate VASP, the extent to which the other homologs, Mena and Evl, are regulated by PG signaling is unclear. As Ena exhibits a higher level of homology to Mena and Evl than to VASP, PG signaling is likely to regulate all three mammalian forms. Uncovering the means by which PG signaling regulates, either directly or indirectly, Drosophila Ena to temporally regulate actin remodeling during oogenesis is expected to reveal conserved mechanisms through which PG signaling modulates the activity of this family of actin regulators. Such mechanisms are likely to play critical roles, not only during development, but also in human diseases including heart disease and cancer (Spracklen, 2014).

The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization, and can stimulate filopodia in cultured cells. However, redundancy in mammals and the Diaphanous role in cytokinesis limit analysis of whether and how they regulate protrusions during development. This study used two tissues driving Drosophila dorsal closure, migratory leading-edge (LE) and non-migratory amnioserosal (AS) cells, as models to define how cells shape distinct protrusions during morphogenesis. Non-migratory AS cells were found to produce filopodia that are morphologically and dynamically distinct from those of LE cells. It was hypothesized that differing Enabled and/or Diaphanous activity drive these differences. Combining gain- and loss-of-function with quantitative approaches revealed Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative 'fingerprint', the protrusive profile, which the data suggest is characteristic of each actin regulator. The data suggest LE protrusiveness is primarily Enabled-driven, while Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence (Nowotarski, 2014).


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

date revised: 2 March 2016

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