Abl tyrosine kinase

DEVELOPMENTAL BIOLOGY

Embryonic

The expression of the Abl protein has been examined throughout embryonic and pupal development; mutant phenotypes in some of the tissues expressing abl have been analyzed. In contrast to the ABL mRNA, the Abl protein is not maternally supplied to the oocyte. As cellularization initiates, 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 and apical cell junctions. The function of this expression is not yet known. Abl is localized in cytoplasmic islands that form around the nuclei in the anterior end of the embryo. Zygotic expression of abl is first detected in the post-mitotic cells of the developing muscles and central nervous system midway through embryogenesis. Zygotic Abl in the CNS is limited to neurons; immunostaining is not detected in neuroblasts or ganglion mother cells. The Abl protein is concentrated in axons as they extend from neurons. The highest level of Abl throughout embryogenesis is observed in the axon scaffold of the CNS. At stage 14, Abl protein is detected in the visceral mesoderm and at a lower level in the somatic mesoderm. In the somatic muscle, immunostaining is concentrated at the muscle attachment sites (Bennett, 1992).

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

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).

Larval

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 and the apical cell junctions of the imaginal disk epithelium. Abl protein is detected in the epithelial cells of leg, wing and eye-antennal discs. The Abl protein is present throughout the cytoplasm, but is concentrated within the apical cortical region of the cells, a region containing actin-rich adherens-type junctions. Abl protein is found in adepithelial cells that give rise to much of the musculature of the adult thorax. In the leg disc, only the adepithelial cells that have migrated into the more distal folds of the leg pouch, and have begun morphological changes, show an increased level of Abl protein. In pupal leg and wing discs there is a higher level of staining in the proximal region where the leg and wing imaginal discs have fused to form the notum. The Abl protein is present at high levels in developing muscle (Bennett, 1992).

Low levels of Abl protein are detected in undifferentiated cells ahead of the morphogenetic furrow in the developing eye disc. Higher levels of protein are detected in developing photoreceptor cells. The higher levels of Abl protein are detected simultaneously in photoreceptor cells R2, R5 and R8. Later Abl is detected in cells R3 and R4, followed by R1 and R6, and finally R7, coinciding with the order of photoreceptor differentiation. The Abl protein is present throughout the photoreceptor cell bodies and axons, and is concentrated in the apical portions of the cells. Abl protein is detected in neurons of the developing adult CNS, but is not found in neuroblasts. Abl protein is present in the developing neuropil of the brain (Bennett, 1992).

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