InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

Shark (SH2 domain ankyrin repeat kinase, Ferrante, 1995) is a Drosophila nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain. Analysis of the phenotypes associated with a shark loss-of-function mutation demonstrates that Shark activity is essential for the migration of the dorsolateral epidermis of the embryo during dorsal closure (DC). Shark kinase functions in DC upstream of Dpp expression by leading edge (LE) cells (Fernandez, 2000).

shark is also required for dorsal-appendage (DA) morphogenesis in Drosophila oogenesis. shark function is required in follicle cells for cell migration and chorion deposition. bullwinkle (bwk) regulates, through a novel germline-to-soma signal, morphogenesis of the eggshell dorsal appendages. A screen was carried out for dominant modifiers of the bullwinkle mooseantler eggshell phenotype and shark was idenfied as a dominant modifier of bullwinkle. At the onset of dorsal-appendage formation, shark is expressed in a punctate pattern in the squamous stretch cells overlying the nurse cells. Confocal microscopy with cell-type-specific markers demonstrates that the stretch cells act as a substrate for the migrating dorsal-appendage-forming cells and extend cellular projections towards them. Mosaic analyses reveal that shark is required in follicle cells for cell migration and chorion deposition. Proper shark RNA expression in the stretch cells requires bwk activity, while restoration of shark expression in the stretch cells suppresses the bwk dorsal-appendage phenotype. These results suggest that shark plays an important downstream role in the bwk-signaling pathway. Candidate testing implicates Src42A in a similar role, suggesting conservation with a vertebrate signaling pathway involving non-receptor tyrosine kinases (Tran, 2003).

The folding and remodeling of epithelia into more complex structures is a recurrent phenomenon in metazoan development. Intercellular interactions are important regulatory components of these processes. Adjacent cells typically provide cues that direct morphogenesis or establish an extracellular milieu permissive for cell movements (Tran, 2003).

In Drosophila melanogaster, remodeling epithelia can interact with an adjacent epithelium. Two well-studied examples include the migration of the embryonic dorsal epithelium over the amnioserosa, and eversion of leg and wing primordia relative to the peripodial tissue that bounds the imaginal discs. These cell layers actively regulate the patterning and movements of neighboring epithelia. Ablation of the peripodial membrane results in growth and patterning defects in the eye and wing discs. In the embryo, the amnioserosa contributes signals and mechanical force to dorsal closure. During germband retraction, the amnioserosa also signals to and extends lamellipodia-like structures towards the retracting germband cells. This study elaborates on a novel extracellular pathway defined by bullwinkle (bwk) (Rittenhouse, 1995) that is essential for proper tubulogenesis of the follicular epithelium during synthesis of the dorsal appendages (DAs), specialized respiratory structures of the eggshell. Additionally, it is demonstrated that an adjacent squamous cell layer acts as a substrate for the migrating epithelium and expresses factors required for this morphogenetic process (Tran, 2003).

DA formation occurs within the context of the Drosophila egg chamber, which consists of ~650 somatically derived follicle cells surrounding a germline cyst composed of one oocyte and 15 nurse cells. The germ cells are interconnected via cytoplasmic bridges called ring canals, which provide access for the transfer of nurse-cell material into the developing oocyte. At stage 11, the nurse cells transport most of their cytoplasm into the oocyte, and then undergo programmed cell-death. DA morphogenesis begins at stage 11, coincident with nurse-cell apoptosis (Tran, 2003).

During DA formation, the somatic layer consists of two major populations with distinctive morphologies, the stretch cells and columnar cells. At the anterior, ~50 squamous stretch cells cover the nurse cells. These cells provide signals that pattern the anterior eggshell-forming cells and ensure proper nurse-cell cytoplasmic dumping. The columnar cells overlie the oocyte at the posterior and secrete the layers and specialized structures of the eggshell. The anterior-most columnar cells (the centripetal cells)migrate inwards, closing off the anterior end of the oocyte while synthesizing the operculum and micropyle. In addition, two subpopulations of ~65 dorsoanterior follicle cells form the two dorsal appendages through a complex reshaping and reorganization of a flat epithelium into three-dimensional tubes (Tran, 2003 and references therein).

These DA-forming cells apically constrict and evert outwards, changing from a flat layer into tubular structures that extend anteriorly. Secretion of chorion proteins into the tube lumens creates the appendages. This process occurs during the final stages of oogenesis, downstream of the events that pattern the eggshell and embryonic axes (Tran, 2003).

Although much is known about the induction and refinement of follicle-cell patterning, little is known about the factors that govern the cellular movements. One pathway that contributes to the morphogenesis is the Jun-kinase (JNK) pathway. The Drosophila Jun and Fos transcription factors are expressed highly in the stretch cells and in an anterior subset of the two DA-forming cell populations. Loss of JNK-pathway function results in two short paddleless DAs and defective nurse-cell cytoplasmic transport (Tran, 2003 and references therein).

The DA-forming cells require additional extracellular cues for normal tubulogenesis. Mosaic analyses demonstrate that bwk is required in the germline to regulate formation of the dorsal appendages (Rittenhouse, 1995). bwk encodes several SOX/TCF transcription factors with pleiotropic functions, regulating dorsal follicle-cell migration, anteroposterior (AP) patterning in the embryo, and transport of nurse-cell cytoplasm into the oocyte. In bwk mutants, the DA-forming cells not only fail to migrate anteriorly, but instead extend much more laterally (Dorman, 2004), as indicated by the wide DA paddle (Tran, 2003 and references therein).

To elucidate the role of bwk in DA formation, other components of this germline-to-soma signaling pathway were sought. Second-chromosome deficiencies were screened for regions that genetically interact with bwk. Tests of candidate mutations identified shark as a strong Enhancer of bwk. shark encodes an SH2-ankyrin-repeat, tyrosine-kinase protein (Ferrante, 1995) that functions upstream of the JNK pathway (Fernandez, 2000) during dorsal closure of the embryo (Tran, 2003 and references therein).

shark is shown to act downstream of bwk in the squamous stretch cells, and shark mediates the regulation of DA formation by bwk. Furthermore, detailed cellular analyses with stretch-cell markers show that the stretch cells provide a substrate for the DA-forming cells and appear morphogenetically active (Tran, 2003).

Shark non-receptor kinase is conserved, with homologs in Hydra (Chan, 1994) and sponge (Suga, 1999). The mammalian counterparts contain homologous SH2 and tyrosine-kinase domains but lack the ankyrin repeats (Chan, 1991; Taniguchi, 1991). These mammalian proteins, Zap70 and Syk, are recruited to immunoreceptor complexes upon ligand binding and regulate immune-cell activation and differentiation, functioning alongside Src kinases (reviewed by Chu, 1998). In T-cells, Zap70 also mediates signaling downstream of integrin-receptor complexes that feature in T-cell motility (Bearz, 1999; Soede, 1998: Tran, 2003 and references therein).

Mosaic analyses with loss-of-function shark alleles have established two somatic functions in DA formation. First, shark is required in the DA cells for proper DA-chorion deposition, a complex process regulated at many levels. Mutations that disrupt chorion-gene amplification or chorion-protein synthesis result in thin, collapsed DAs and main-body eggshell. Unlike those mutations, loss of shark in the main-body follicle cells does not cause defects in follicular imprints, alter the appearance of the eggshell under darkfield optics, or produce thin chorion and collapsed eggs. Although the methods used in this study may miss subtle defects in main-body chorion, shark may play a DA-cell-specific role in the production/formation of chorion. Although regulatory sequences and a putative binding protein drive specific spatial expression of chorion-reporter constructs, no reported mutants disrupt DA-specific chorion expression (Tran, 2003).

The second function of shark lies in the stretch cells and affects the migration of the DA cells. Large stretch-cell clones resulted in shortened DAs that vary in their morphology and penetrance. This variability could result from residual activity of these mutant alleles, non-cell autonomy, or functional redundancy. Although no Shark paralogs are encoded in the genome, several non-receptor tyrosine kinases share homology in the SH2 and kinase domains, including Src42A (Tran, 2003).

In addition, stretch-cell expression of shark strongly suppresses the bwk-mutant DA phenotype, in concurrence with a direct role for bwk in regulating shark expression in this tissue. These results indicate that shark is key in regulating DA migration downstream of bwk. Full rescue was not likely achieved because of insufficient expression levels, the need to localize shark RNA, or the existence of shark-independent branches downstream of bwk (Tran, 2003).

These data suggest a model in which Bwk regulates factors in the germline that are required for proper shark expression in the stretch cells. Shark then regulates the activity of targets required for DA-cell movement across the stretch-cell layer. Another factor that could be regulated by bwk is the Src42A kinase, which behaves similarly to shark. Loss of Src42A enhances bwk mutants, while stretch-cell expression of activated Src42A suppresses bwk. Mammalian homologs of Shark function together with Src kinases, suggesting a conserved signaling cascade (Tran, 2003).

Two other stretch-cell signaling pathways, JNK and DPP, regulate DA morphogenesis. Tests with bwk and shark, however, fail to reveal strong or definitive interactions. Loss of JNK activity in oogenesis results in shortened and paddleless DAs, yet expression of UAS-basket⁺ and reduction of bsk dose do not alter the morphology of bwk eggshells. Furthermore, expression of the AP-1 components is unaffected in bwk mutants and shark clones. These data support the hypothesis that the bwk/shark pathway does not primarily act through JNK signaling (Tran, 2003).

Moderate overexpression of dpp and loss of the type I receptors, tkv and sax, can lead to shortened and somewhat broadened DAs, resembling bwk mutants. The expression of dpp RNA and a dpp enhancer trap, however, are unaffected in bwk mutants. Both hypomorphic dpp alleles and loss of type I receptors fail to interact with a strong bwk mutant. These data suggest that bwk does not directly regulate dpp4 expression or activity but rather may modulate downstream targets (Tran, 2003).

DA-cell movement over the stretch cells may require expression of stretch-cell factors that guide or facilitate migration. Mammalian proteins that share homology with Shark can bind to and regulate integrin complexes. Shark may bind these and/or other adhesion receptors to regulate cell migration either through signaling cues or by modulating the extracellular matrix (Tran, 2003).

Shark could also regulate stretch cell behaviors, controlling the small cellular projections that extend toward the DA cells during their anterior movement. These extensions may guide or signal the DA-forming cells, as occurs in imaginal discs (Tran, 2003).

Extracellular signals and interactions are key components of morphogenetic processes. Two downstream components of the bwk pathway have been identified that act in the stretch-cell layer to relay a novel germline signal required for the movement of a third tissue, the remodeling epithelium of the dorsal appendage cells (Tran, 2003).

Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe

The molecular and cellular mechanism for clearance of dead neurons was explored in the developing Drosophila optic lobe. During development of the optic lobe, many neural cells die through apoptosis, and corpses are immediately removed in the early pupal stage. Most of the cells that die in the optic lobe are young neurons that have not extended neurites. This study shows that clearance was carried out by cortex glia via a phagocytosis receptor, Draper (Drpr). drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Drpr that was expressed in other subtypes of glia did not mediate clearance. Shark and Ced-6 mediated clearance of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe (Nakano, 2019).

Many studies have explored the cellular and molecular mechanisms for clearance of dead neurons in the developing Drosophila CNS. During embryonic development, dead neurons are phagocytosed by subperineurial glia. Draper (Drpr) acts as a phagocytosis receptor on the glial membrane to clear dead neurons in the embryo. Another receptor, Six-microns-under (SIMU), works in cortex glia to allow recognition and engulfment of apoptotic cells, whereas Drpr works to degrade apoptotic cells in the embryonic CNS. During metamorphosis, dead neurons are engulfed by glia in the CNS. Elimination of neurites of vCrz neurons during metamorphosis is performed by astrocyte-like glia via the Crk/Mbc/dCed-12 signaling pathway but not the Drpr pathway. In contrast, elimination of cell bodies of vCrz neurons, a group of neurons that express neuropeptide Corazonin, requires Drpr, but its expression is not required in astrocyte-like glia. However, recent studies have reported inconsistent results on the requirement of Drpr for dead cell clearance and the glia subtypes that work for clearance in the brain during metamorphosis. It has been reported that dead neurons that died in the central brain before the beginning of the third larval instar and in the optic lobe before the late third larval instar are cleared by cortex glia via the Drpr pathway, but neurons that die thereafter are efficiently cleared without Drpr. Drpr has been shown to be required for apoptotic cell clearance during metamorphosis and its expression is required in ensheathing glia and astrocyte-like glia, but not in cortex glia (Nakano, 2019).

One of the causes of inconsistency among previous studies may be differences in the cellular materials to be phagocytosed, and different mechanisms could work for phagocytosis of different materials in the CNS during metamorphosis. Three types of neurons need to be phagocytosed during metamorphosis. Obsolete larval neurons die, and their cell bodies and neurites are removed by phagocytosis. Larval neurons of another type are respecified from larval to adult neurons via pruning of larval neurites and extension of new adult neurites. Pruned neurites are removed by phagocytosis. Adult-specific neurons are produced by precursor cells during post-embryonic development and differentiate during metamorphosis. A number of these young neurons die during development before extending neurites. Therefore, studies on a single type of neuron or specifically defined neurons are needed to define the molecular and cellular mechanisms for clearance of dead neurons. Moreover, clearance of neurites and cell bodies of dead neurons should be studied independently (Nakano, 2019).

In this study, clearance of dead neurons in the developing optic lobe was examined. The Drosophila optic lobe is a unique center in which a large number of dying cells are observed during its development. Most dying neurons in the optic lobe are young neurons that had just started to differentiate into adult neurons. One of paired neurons derived from intermediate precursors (GMCs) is eliminated by apoptosis under the control of Notch signaling. Neurons that die in the developing optic lobe have not yet extended neurites at the time they die. Therefore, cellular materials to be cleared after the cell death include nuclei and general cytoplasm, but not neurites in the developing optic lobe (Nakano, 2019).

The adult optic lobe develops from the primordium during metamorphosis. Optic lobe neurons are produced by two proliferation centers, the outer proliferation center (OPC) and inner proliferation center (IPC). Neurons differentiate, extend neurites, and produce four types of neuropil, the lamina, medulla, lobula plate, and lobula. Then, the optic lobe consists of four types of neuropil and surrounding cortices of neuronal cell bodies. According to previous studies, many neurons and a small number of precursor cells undergo cell death during optic lobe development. This cell death does not occur randomly in the optic lobe but occurs in clusters in a specific temporal and spatial pattern. The number of dead cells in the optic lobe starts to increase at the puparium formation, reaches a peak at 24 h after puparium formation (24 h APF), and decreases to almost zero by 48 h APF. Two types of cell death are involved in this process: ecdysone dependent and independent. Both types of cell death are apoptosis and involve the Drosophila effector caspases, DrIce and Dcp-1. DrIce plays an important role in dead cell clearance as well. The role of cell death is to prevent the emergence of abnormal neural structures in the optic lobe (Nakano, 2019).

This study explored the cellular and molecular mechanisms for clearance of dead young neurons in the developing optic lobe. The results showed that clearance was carried out by cortex glia via a phagocytosis receptor, Drpr. Drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Signaling molecules, Shark and Ced-6 mediated clearance downstream of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe (Nakano, 2019).

This study revealed that Drpr expressed in cortex glia were required for dead cell clearance in the MLpL region of the developing optic lobe, and that Drpr in other subtypes of glia did not mediate clearance. This is the first study that showed clearance of dead young neurons in the developing optic lobe required Drpr expression in the cortex glia. In the lamina region, lamina distal cortex glia work for dead cell clearance (Nakano, 2019).

The expression pattern of Drpr agreed with the alteration in the activity of dead cell clearance in the optic lobe during metamorphosis. At early pupal stages, Drpr is expressed weakly in a mesh-like pattern and strongly in a centripetal pattern in cortex glia in the MLpL region. This expression weakened thereafter, and only weak expression was seen in a mesh-like pattern during the last half of the pupal period. Moreover, no protrusion was seen of Drpr expressing glial cytoplasm into the neuropil from neuropil glia (NG) at early pupal stages. This agrees with the fact that cell death in the developing optic lobe occurs mainly in young neurons before they extend neurites or in abnormal neurons with no neurites (Nakano, 2019).

After 48 h APF, cell death was rarely observed and thus activity of dead cell clearance was low. However, this does not mean that glia lost potential ability to clear corpses at late pupal stages. Forced expression of wild-type drpr on the drpr mutant background at 48 or 72 h APF resulted in clearance of accumulated TUNEL-positive cells. This indicates that the components of the mechanism for dead cell clearance except Drpr are retained until late pupal stages. Therefore, if some cells died at late pupal stages and Drpr expression was induced in cortex glia, the dead cells would be cleared via Drpr pathway. Moreover, the fact that accumulated TUNEL-positive cells were removed when wild-type drpr was forcibly expressed in late pupal stages on the drpr mutant background suggests that 'eat me' signals were secreted or displayed by accumulated TUNEL-positive cells in drpr mutants not only at early pupal stages, when the cells died, but also at late pupal stages long after cell death (Nakano, 2019).

At late pupal stages, strong Drpr expression appeared in the cytoplasmic protrusions from the neuropil glia (NG), and astrocyte-like glia simultaneously started expressing molecular markers (specific GAL4s). This Drpr was not utilized for clearance of dead neurons, as almost no cell death arises at this stage in control conditions. In the neuropil of the optic lobe at late pupal stages, neurites extend and form synapses to make and complete neural networks. When new synapses are formed during development of the Drosophila larval neuromuscular junction, significant amounts of presynaptic membranes and a subset of immature synapses are removed from the junction by surrounding glia and postsynaptic muscle via the Drpr/dCed-6 pathway (Fuentes-Medel, 2009). Thus, the same process may arise at developing synapses in the developing optic lobe, and astrocyte-like glia expressing Drpr in the cytoplasmic protrusions may function to remove unnecessary presynaptic membranes and immature synapses (Nakano, 2019).

Previous studies have reported that astrocyte-like glia are responsible for clearance of degenerating axons of dying obsolete larval neurons in the ventral nerve cord (Tasdemir-Yilmaz, 2014) and of pruned axons of γ neurons in the mushroom body. In contrast, degenerating axons are removed by ensheathing glia in the olfactory lobe following Wallerian degeneration of the olfactory nerve. Therefore, different subtypes of glia work to clear degenerating axons in different contexts. Astrocyte-like glia may specifically function for clearance of 'programmed' degenerating axons and ensheathing glia for clearance of 'accidently' degenerating axons. In addition, another subtype of glia, cortex glia, functions to remove dead young neurons. These young neurons had just started to differentiate into adult neurons in the developing optic lobe and have not yet extended neurites at the time they die. Tasdemir-Yilmaz (2014) reported that elimination of cell bodies of obsolete vCrz neurons requires Drpr, but its expression is not required in astrocyte-like glia. Young neurons in the optic lobe and cell bodies of obsolete vCrz neurons in the ventral nerve cord both locate in the cortex and almost the same cellular materials are cleared after the cell death, including nucleus and general cytoplasm, but not neurites. Therefore, as with dead young neurons in the optic lobe, the expression of Drpr in cortex glia would be required for clearance of cell bodies of dead vCrz neurons. Comparative studies are expected in the future on the mechanisms for clearance of degenerating axons of dead neurons, degenerating axons of cut nerves, dead young neurons, and cell bodies of dead obsolete neurons. Moreover, considering that 'accidently' degenerating axons are cleared by a different subtype of glia from 'programmed' degenerating axons, a possibility should be tested that cell bodies of neurons that died 'accidently' are cleared by a distinct subtype of glia (Nakano, 2019).

This is the first study to reveal that Shark mediates Drpr-dependent clearance of dead neurons in the CNS. Moreover, this study suggests that Ced-6, Crk/Mbc/dCed-12, and Rac1 are partially involved in clearance of dead young neurons. Therefore, both Drpr/Shark/dCed-6 and Crk/Mbc/dCed-12 pathways work for dead cell clearance in the developing optic lobe. As cortex glia function for clearance of dead neurons in the developing optic lobe, these pathways must work in cortex glia (Nakano, 2019).

A previous study reported that these pathways function to mediate removal of degenerating axons in ensheathing glia in the adult olfactory lobe when the olfactory nerve is cut (Ziegenfuss, 2012). The same pathways work in astrocyte-like glia around the mushroom body when axons of γ neurons are pruned, and in the ventral nerve cord when neurites of dead vCrz neurons are cleared during metamorphosis (Tasdemir-Yilmaz, 2014). Therefore, Drpr/Shark/dCed-6 and Crk/Mbc/dCed-12 pathways generally function to clear corpses in different glia in different contexts. However, the relative role played by each pathway depends on the situation. Although drpr mutation strikingly inhibited dead cell clearance in the optic lobe, knockdown of the Crk/Mbc/dCed-12 pathway had only a moderate effect. In contrast, removal of olfactory nerve axons that have undergone Wallerian degeneration is strongly affected by both drpr mutation and Crk/Mbc/dCed-12 knockdown (Ziegenfuss, 2012). In the pruning of mushroom body γ axons, mutation of drpr and knockdown of Crk/Mbc/dCed-12 additively affect clearance, although mutation of drpr has a stronger effect (Tasdemir-Yilmaz, 2014). In the removal of axons from dead vCrz neurons during metamorphosis, knockdown of dCed-12 causes a moderate defect, whereas drpr mutation causes no defect by itself but only enhances the defect caused by dCed-12 knockdown (Tasdemir-Yilmaz\, 2014). How the relative role of these pathways is regulated and why remain to be defined (Nakano, 2019).

Previous studies reported that Pretaporter, CaBP1 and phosphatidylserine act as Drpr ligands when dead embryonic cells are phagocytosed in the Drosophila embryo and cultured cells. The present study suggested a possibility that these molecules mediate signaling for dead cell clearance as a Drpr ligand in the developing optic lobe. However, Pretaporter and CaBP1 are not essential for clearance and their role would be minor. Therefore, relative role of molecules that work for dead cell clearance as ligands for Drpr may be different depending on the context. As described above, different subtypes of glia work to clear corpse in the CNS: ensheathing glia for Wallerian's degenerating axons, astrocyto-like glia for pruned axons and degenerating axons of dead vCrz neurons, and cortex glia for dead young neurons in the optic lobe. Therefore, it is to be defined whether difference in ligand molecules is involved in activating different subtypes of glia (Nakano, 2019).

The present study agrees with results described by Tasdemir-Yilmaz (2014), who reported that Drpr is required for elimination of cell bodies of vCrz neurons that die at 3-7 h APF. However, the current study disagrees with other studies (Nakano, 2019 and references therein).

Several possible causes may have led to these inconsistencies. One possibility is the difference in cellular materials to be cleared, that is, cell bodies or neurites, as mentioned earlier. The present study examined dead young neurons in the developing optic lobes. Cellular materials to be cleared include only nucleus and general cytoplasm, but not neurites. Tasdemir-Yilmaz (2014) studied vCrz neurons and found that molecular mechanisms for clearance of cell bodies and neurites are different. However, other studies examined the central brain or the whole brain, which include cell bodies of dead neurons, neurites of dead neurons and pruned neurites. Another possibility is the difference in methods to detect dead neurons. This study used the ABC TUNEL method, which detects degraded DNA in dead cell nuclei with the streptavidin-biotin-peroxidase complex (Vector Laboratories). This method is far more sensitive and reliable than the fluorescent TUNEL method (compare the number of TUNEL-positive cells between the present study and other studies). Another method to detect dead cells is anti-Dcp-1 antibody staining. This method has some problems with detection of accumulated dead cell corpses in phagocytosis-defective mutants. It detects activated Dcp-1, one of the effector caspases. However, another effector caspase, DrIce, is also expressed and is a more effective inducer of apoptosis than Dcp-1. Therefore, this method may not detect all dead cells. Another problem with this method is the unknown stability of activated Dcp-1 in dead cells. Therefore, detection of activated Dcp-1 does not show exactly how many dead cells have accumulated in phagocytosis-defective mutants. Moreover, when dendrites are pruned during remodeling of dendritic arborization sensory neurons during metamorphosis, caspase activity is detected in the dendrite. This suggests that the anti-Dcp-1 antibody may detect pruned dendrites as well as dead cells. Finally, the subtypes of glia that clear corpses are also different in the present study and previous ones. This study found that expression of GAL4 in glia subtype-specific GAL4 lines drastically changed during metamorphosis, and the expression pattern at pupal stages was different from adult stages in many GAL4 lines. However, previous studies did not examine the expression pattern of the GAL4 lines they used. Altogether, studies on a single type of dead neuron or identified neurons are required in the future. The mechanisms for clearance of dead cell bodies and degenerating neurites should be studied independently. The expression pattern of GAL4 lines in subtypes of glia should be carefully assessed before using the line as a GAL4 driver (Nakano, 2019).

REGULATION

Protein Interactions

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. This study shows that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70 (Ferrante, 1995), binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. The Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. It is proposed that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade (Ziegenfuss, 2008).

Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases. In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis. CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains. The PTB domain adaptor protein CED-6 can bind the NPXY motif of CED-1, is required for cell corpse engulfment and acts in the same genetic pathway as CED-1. CED-1 ultimately mediates actin-dependent cytoskeletal reorganization through the Rac1 GTPase, and Dynamin modulates vesicle dynamics downstream of CED-1 during engulfment, but the molecular signalling cascade that allows CED-1 to execute phagocytic events remains poorly defined (Ziegenfuss, 2008).

Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning or during Wallerian degeneration in the adult brain. In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1. Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY and YXXL motifs in its intracellular domain. Drosophila Ced-6 is also required for the clearance of pruned axons, indicating possible conservation of the interaction between CED-1 and CED-6 in flies, but additional signalling molecules acting downstream of Draper have not been identified (Ziegenfuss, 2008).

Draper was identified in a yeast two-hybrid screen for molecules interacting with the regulatory region of Shark. When LexA-Shark, constitutively active Src kinase and AD-Draper are present, Shark and Draper interact physically. In the absence of Src kinase, Shark and Draper fail to interact, indicating that phosphorylation of Draper by Src may be essential for Shark-Draper interactions. The Draper intracellular domain contains an ITAM (YXXI/L-X_6-12-YXXL), a key domain found in many mammalian immunoreceptors including Fc, T-cell and B-cell receptors. SFKs phosphorylate the tyrosines in ITAM domains, thereby allowing ITAM association with SH2-domain-containing signal transduction proteins including Syk and Zap-70. Y-->F substitutions of the tyrosine residues were generated within or near the Draper ITAM, and it was found that Tyr 949 and Tyr 934 are critical for robust Draper-Shark binding. These correspond to the consensus tyrosine residues in the predicted Draper ITAM. Plasmids were transfected with carboxy-terminally haemagglutinin-tagged Draper (Draper-HA) or with Draper-HA and Shark with an amino-terminal Myc tag (Myc-Shark) into Drosophila S2 cells, immunoprecipitated with anti-HA antibodies, and western blots were performed with anti-phosphotyrosine, anti-Myc and anti-HA antibodies. Myc-Shark was found to co-immunoprecipitate with Draper-HA, and that anti-phosphotyrosine antibodies label a band corresponding to the position of Draper-HA that is absent in empty vector controls. Further, it was found that a Y949F substitution markedly reduces Draper-Shark association. Taken together, these data indicate Draper and Shark can associate physically through the Draper ITAM domain (Ziegenfuss, 2008).

Attempts were made to determine whether Shark is required for glial phagocytic activity in vivo. Severing adult Drosophila olfactory receptor neurons (ORNs) initiates Wallerian degeneration of ORN axons. Antennal lobe glia surrounding these severed axons respond to this injury by extending membranes towards severed axons and engulfing degenerating axonal debris. These glia express high levels of Draper, and in draper^δ5 null mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the central nervous system (CNS). Thus, both the extension of glial membranes to severed axons and the phagocytosis of degenerating axonal debris require Draper signalling (Ziegenfuss, 2008).

Whether Shark function in glia is essential for glial responses to axon injury was explored by driving a UAS-regulated double-stranded RNAi construct designed to target shark (shark^RNAi) with the glial-specific repo-Gal4 driver, severing ORN axons, and assaying the recruitment of Draper and green fluorescent protein (GFP)-labelled glial membranes to severed axons. Maxillary palp-derived ORN axons project to 6 of the roughly 50 glomeruli in the antennal lobe. Within hours after maxillary palps have been ablated in control animals, Draper immunoreactivity decorates severed axons projecting to and within maxillary palp ORN-innervated glomeruli, and GFP-labelled glial membranes are recruited to these severed axons. Strikingly, knocking down Shark in glia completely suppresses these events. Next, antennal ORN axons were severed; these axons project to about 44 of the 50 antennal lobe glomeruli. Antennal ablation therefore injures nearly all glomeruli in the antennal lobe and results in the majority of antennal lobe glia in control animals upregulating Draper and undergoing hypertrophy. It was found that knocking down Shark in glia also blocks this glial response to axon injury. Thus, Shark is essential for all axon-injury-induced changes in glial morphology and Draper expression (Ziegenfuss, 2008).

To determine whether Shark is required for glial phagocytosis of severed axons, a subset of maxillary palp ORN axons were labelled with mCD8::GFP, Shark function was knocked down in glia, and the clearance of severed axons was assayed. In control animals severed GFP-labelled ORN axonal debris was cleared from the CNS within 5 days. In contrast, glial-specific shark^RNAi potently suppresses the clearance of degenerating axons, with severed axons lingering in the CNS for at least 5 days. Then whether mutations in the shark gene affected the glial clearance of degenerating axons was examined. The null allele of shark, shark¹, is pupal lethal (Fernandez, 2000). Therefore glial responses to axon injury were assayed in shark¹ heterozygous mutants, and dominant genetic interactions between draper^δ5 and shark¹ were tested. It was found that both draper^δ5/+ and shark¹/+ animals showed defects in glial phagocytic function: 5 days after injury, significant amounts of axonal debris remained within OR85e-innervated glomeruli. Moreover, shark¹/+; draper^δ5/+ animals showed a striking suppression of glial clearance of severed axons almost equivalent to that of draper^δ5 mutants. Thus, shark mutations dominantly suppress the glial clearance of degenerating ORN axons, and this phenotype is strongly enhanced by removing one copy of draper. These data, taken together with shark^RNAi data, show that Shark is essential for the clearance of degenerating axons by glia (Ziegenfuss, 2008).

Is Shark required for the glial clearance of neuronal cell corpses? In embryonic stage 14-15 control animals, 24.4 cell corpses were found per hemisegment. In contrast, it was found that shark¹ null mutants showed a marked increase in CNS cell corpses, with null mutants containing almost twice as many corpses per hemisegment. shark¹/Df(2R)6063 mutants accumulate cell corpses at levels similar to those in shark¹, indicating that this phenotype maps to shark. These cell corpse engulfment phenotypes are indistinguishable from that of draper^δ5 mutants. It is concluded that Shark, like Draper, is also essential for the efficient clearance of embryonic neuronal cell corpses by glia (Ziegenfuss, 2008).

Because it was found that Shark binds Draper only in the presence of an active Src kinase in two-hybrid assays, Drosophila Src kinases were screened for roles in glial phagocytic activity. Interestingly, it was found that glia-specific knockdown of Src42A (src42A^RNAi) potently suppress glial phagocytic activity: in src42A^RNAi animals, Draper is not recruited to severed maxillary palp axons; glial hypertrophy and upregulation of Draper after antennal ablation was blocked; and GFP-labelled severed axons lingered in the CNS for 5 days. Knockdown of two other Drosophila Src kinases, Btk29A and Src64B, had no effect on the glial phagocytosis of severed axons. Thus, Src42A seems to be essential for all morphological responses of glia to axon injury and for the efficient clearance of degenerating axonal debris from the CNS (Ziegenfuss, 2008).

It was predicted that Draper phosphorylation status should be sensitive to the SFK inhibitor PP2. Indeed, addition of PP2 to S2 cultures led to a decrease in the phosphorylation of Draper and Draper-Shark association. Strikingly, co-transfection of Draper and Src42A led to a marked increase in Draper phosphorylation, which was PP2-sensitive and Draper-specific. Draper-Shark interactions are not dependent on Shark kinase activity because kinase-dead Shark (Shark K698R) associates with Draper. These data indicate that Src42A may phosphorylate the Draper intracellular domain, thereby increasing the association of Shark with Draper and the activation of downstream glial phagocytic signalling (Ziegenfuss, 2008).

This study has identified Shark and Src42A as novel components of the Draper pathway. One potential model for Draper-Shark-Src42A interactions is that Shark and Src42A drive the recruitment of Draper to engulfment targets. However, CED-1 has been shown to cluster around cell corpses even in the absence of its intracellular domain. Moreover, Zap-70 and Syk bind phosphorylated ITAM domains in mammalian immunoreceptors when ITAM domains are phosphorylated by Src after ligand-dependent receptor. Therefore a model is favoured in which the engagement of Draper with its ligand (presumably presented by engulfment targets) promotes receptor clustering, tyrosine phosphorylation of Draper by Src42A, association of Shark, and activation of downstream phagocytic signalling events. This work suggests that Draper is an ancient immunoreceptor in which the extracellular domain is tuned to recognize modified self and the intracellular domain signals through ITAM-Src-Syk-mediated mechanisms. This is the first identification of ITAM-Src-Syk signalling in invertebrates, and it suggests that a pathway similar to Draper-Ced-1 may ultimately have given rise to ITAM-based signalling cascades in mammalian myeloid and lymphoid cells, including those regulated by Fc, B-cell and T-cell receptors (Ziegenfuss, 2008).

DEVELOPMENTAL BIOLOGY

Tyrosine kinases, ankyrin repeats, and Src homology 2 domains play central roles in developmental processes. The cloning of a cDNA for Shark, a single protein that possesses all three domains, is described. During Drosophila embryogenesis, Shark is expressed exclusively by ectodermally derived epithelia and is localized preferentially to the apical surface of these cells. Initiating expression at the end of gastrulation, the most prominantly Shark positive structure is the cephalic furrow. Ectodermal structures associated with the ventral midline and the anterior and posterior gut ectoderm express Shark. Tracheal placodes and tracheal lumen express Shark, and expression in the trachea persists throughout its development. The stomatogastric nervous system precursors express Shark in a pattern that overlaps that of Crumbs. Gnathal structures including the labium, mandible and maxilla express Shark from the time they are visible as distinct protruberances. Apical localization of Shark persists, even as tissues undergo complex invaginations, moving from the external surface of embryos to form internal structures, but expression is lost when cells lose their polarity. This pattern closely mimics the expression of Crumbs, a protein necessary for proper organization of ectodermal epithelia. Shark's structure and localization pattern suggest that it functions in a signaling pathway for epithelial cell polarity, possibly transducing the Crumbs signal (Ferrante, 1995).

To localize the distribution of shark transcripts, whole-mount in situ hybridization was performed in wild-type embryos. Large amounts of shark mRNA are observed in ovarian nurse cells, unfertilized eggs, and syncitial blastoderm embryos. Owing to the abundant maternal contribution, shark message expression appears ubiquitous during early embryogenesis up until about stages 6-7. By the end of germ-band extension, shark mRNA is expressed primarily in the ectoderm. Following germ-band retraction, stages 12-15, the strongest shark mRNA expression occurs uniformly in the epidermis and other ectodermally derived structures such as the anterior foregut, hindgut, and Malphigian tubules. Most of the shark message is down-regulated by the end of embryogenesis. Protein expression was analyzed using an antiserum raised to the Shark N-SH2 domain. In general, staining with this antiserum paralleled the expression of shark mRNA. Shark protein is expressed abundantly from the onset of cellularization, resulting in ubiquitous expression until the commencement of gastrulation. From this stage on, its expression is most prominent in the ectoderm and some of the ectodermally derived tissues. At stage 10 (full germ-band extension) and thereafter, Shark protein expression increases again but is restricted to the ectoderm, foregut, hindgut, and Malphigian tubules. During germ-band extension (stages 9-10) and in migrating epidermal cells during DC, Shark protein expression is particularly strong at the LE of the epidermis. By the end of embryogenesis, epidermal expression decreases. Embryos homozygous for deficiency Df(2R)Jp7, which removes the shark locus, as well as shark¹ homozygote embryos, show a reduction in immunoreactivity only after stages 13-14, consistent with a substantial contribution of maternal mRNA. In contrast, staining is reduced in embryos derived from shark¹ germ-line clones (GLCs), consistent with the specificity of the antiserum for Shark, which could detect residual mutant protein. Also, in agreement with genetic evidence for a predominant role of Shark in the epidermis, late shark¹ GLC-derived embryos consistently lack all immunoreactivity in epidermal cells. This pattern of Shark protein expression differed significantly from the previously reported immunolocalization of Shark protein with antibodies directed to the kinase domain (Ferrante, 1995), where poor pregastrulation and epidermal staining was seen that was contributed to by the dominance of apical staining of epithelia and staining of the tracheal system by one of three antibodies in the mixture of antipeptide antibodies used. This antibody was subsequently shown to detect an unrelated epitope (Fernandez, 2000).

REFERENCES

Search PubMed for articles about Drosophila shark

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date revised: 18 February 2024

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