In situ hybridization experiments were carried out to compare the expression patterns of Dock and dPTP61F, a receptor tyrosine phosphatase that interacts with Dock. Drosophila embryos were hybridized with antisense digoxigenin-incorporated RNA probes. The dPTP61F RNA probe was generated from sequences common to both splice variants. Dock mRNA expression localizes to the developing brain and ventral nerve cord of these embryos. dPTP61F mRNA also localizes to the central nervous system but requires increased staining times due to the lower abundance of message. The signal observed in the central nervous system is contributed by expression of the membrane-associated splice variant (dPTP61Fm) while staining outside of the central nervous system is due to background and low level staining of dPTP61Fn in the gut. Comparison of the expression patterns of dPTP61F and Dock transcripts by Northern analysis indicates that these messages are expressed in a similar manner throughout development. The overlapping expression patterns of the membrane associated splice variant, dPTP61Fm, and Dock in the Drosophila embryo and the similarly regulated mRNA expression patterns during development are in complete agreement with the in vivo association experiments (Clemens, 1996).

Dock protein is expressed in most or all central nervous system (CNS) axons and cell bodies. It can be visualized in motor axons, which exit the CNS via two nerve roots and then branch into five nerves that innervate the body wall muscles. All five motor nerves are labeled with anti-Dock antisera. The entire length of the intersegmental nerve (ISN) can be visualized using anti-Dock, but expression levels are highest in growth cones. The SNb (also known as ISNb) nerve is also stained with anti-Dock, with highest expression in growth cones, including the RP3 growth cone, which forms a synapse along the cleft between ventrolateral muscle fibers 7 (also known as VL4) and 6 (VL3). The darkest anti-Dock staining in the embryo is in body wall muscles where the muscles attach to the epidermis. The muscle attachment sites appear as lines, marking the insertion points of longitudinal muscles such as the dorsal acute (2), lateral longitudinal (4) and ventrolateral muscles (7, 6, 13 and 12; muscles 13 and 12 are also known as VL2 and VL1, respectively), or as spots, marking the insertion point of transverse muscle fibers such as the dorsal (18) and lateral transverse muscles (21, 22, 23 and 24). All of the body wall muscle attachment sites appear to express Dock, but those belonging to longitudinally oriented fibers are particularly prominent. Most sensory neurons in the peripheral nervous system (PNS) express Dock, including chordotonal organ neurons, multiple dendrite neurons, and external sensory and dendritic arborization neurons in the dorsal cluster. PNS axons also express Dock (Desai, 1999).


Dock expression is detected in photoreceptor R cells but not in glia. In R cell growth cones Dock is expressed in the lamina neuropil sandwiched between layers of glial cells. Immunoreactivity is also observed in R cell bodies and medulla neurons, making it likely that, in addition to R7 and R8, medulla neurons contribute to staining in the medulla neuropil (Garrity, 1996).

Effects of Mutation or Deletion

Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. It is proposed that Dock transmits signals in the growth cone in response to guidance and targeting cues. dock mutants were examined by electron microscopy. A cross sectional view of a wild-type optic stalk reveals a regular array of axon bundles separated by fine glial processes. Each fascicle contains eight R cell axons, with a central axon fiber surrounded by the remaining seven. The youngest ingrowing axons found near the perimeter of the optic stalk are not yet sorted into fascicles of eight fibers. In dock, the sorting process occurs normally; nearly all fascicles contain eight axons surrounded by glial processes as in wild type. Furthermore, the proportion of axons segregated into fascicles in dock and wild-type optic stalks is the same. However, fascicles are less densely packed, with glial cells showing larger cellular profiles in all dock animals. This loose packing may explain the gaps between axon bundles observed in the light microscope and could reflect disruptions in neuron-glia interactions. In mosaics, examination of the R cell projection pattern reveals defects in the medulla terminal field innervated by mutant R cells. Defects include gaps in the array, hyperinnervation, and crossing of fibers from adjacent columns. In all cases, the positions of mutant R cell projection defects in the medulla are consistent with the location of the mutant patch in the retina; for instance, anterior patches result in defects in the posterior medulla neuropil. In large part, then, the gross retinotipic order of the fibers is maintained in mosaic animals. These results provide strong evidence that the dock gene is required in the eye for normal connectivity. Lamina neurons and glia are disorganized in preparations from dock mutants. This is most likely a consequence of defects in R cell innervation rather than an intrinsic defect in these neurons or glia. The requirement of dock in R cells was verified by expressing dock in mutant R cells. Expressing dock using a glass promoter restores axon pathfinding in dock mutants (Garrity, 1996).

Dock protein is expressed in most or all CNS axons and cell bodies and is also expressed in body wall muscles where they attach to the epidermis. The observed pattern of Dock expression suggests that Dock may be important for the establishment of neuronal connections and the attachment of muscles to the epidermis. Three mutations that eliminate or reduce Dock expression have been isolated and previously described (Garrity, 1996). All three disrupt photoreceptor axon guidance and targeting and result in pupal lethality. A study was performed to examine the effect of these mutations on the development of the embryonic nervous system. In contrast to the severe defects in optic lobe innervation observed in dock mutant larvae, embryos that lack zygotic Dock have very subtle nervous system defects. In the ventral nerve cord, anti-Fasciclin II (FasII) monoclonal antibody stains three pairs of longitudinal axon bundles. These bundles are always present in dock embryos, but they appear somewhat thicker and wavier than in wild-type embryos, and the outermost bundle is occasionally discontinuous. The thickened, wavy appearance of the longitudinal axon bundles could be caused by intermittent defasciculation. There are also low-penetrance defects in muscle organization (less than 10% of mutant hemisegments have one or more muscle fibers that are missing or abnormally cross over one another), which may be caused by loss of Dock at muscle attachment sites. The penetrance of these muscle phenotypes is not increased in embryos lacking both maternal and zygotic Dock. Dock is thus not necessary for the attachment of muscle fibers (Desai, 1999).

The most specific defect observed in dock embryos is the variable absence of a single synapse in the neuromuscular system: the synapse formed by the RP3 neuron along the cleft between muscles 7 and 6. In wild-type embryos, axons from the RP1, RP3, RP4 and RP5 neurons extend together within the SNb nerve until they reach the 7/6 cleft. At this point, the RP3 growth cone defasciculates from the other RP axons and makes a sharp medial turn to grow between muscle fibers 7 and 6. Concomitantly, the RP1 and RP4 growth cones form a large presynaptic structure along the nearby edge of muscle 13. Upon reaching the internal surface of muscles 7 and 6, the RP3 growth cone again reorients to extend posteriorly along the cleft, resulting in a long branch perpendicular to the SNb nerve. Meanwhile, the RP5 axon extends distally across the breadth of muscle fiber 13 and forms synapses at the cleft between muscles 13 and 12. Although the growth cones of RP1 and RP4 arrive at their target first, RP3 usually forms a mature synapse first, while RP5 is usually last. In late-stage 16 embryos, the RP5 growth cone explores muscle fiber 13 and begins to contact the 13/12 cleft. Mutants homozygous for three different dock alleles as well as those trans-heterozygous for two different combinations of dock alleles all variably lack the RP3 synapse at the 7/6 cleft. Pan-neuronal expression of dock largely restores this innervation. Although the RP3 synapse is not present, the other synapses made by SNb motoneurons are apparently forming in a normal manner in the dock mutant. Additonal experiments show that the absence of RP3 synapses is due to a defect in terminal guidance and/or differentiation of the RP3 growth cones, rather than to alterations in their axonal outgrowth from the CNS. The innervation of muscles 7 and 6 by RP3 is apparently normal later in development, in dock mutant third-instar larvae, and no ectopic synapses are observed. This indicates that RP3 can always form synapses in these mutants and that synapse formation is delayed so that synapses have all developed by the time of hatching or shortly thereafter (Desai, 1999).

The delay in RP3 synapse formation (lack of 7/6 cleft innervation at late stage 16, but normal innervation in third instar larvae) seen in dock mutant embryos is identical to the phenotype of late bloomer (lbm) mutants (Kopczynski, 1996). lbm encodes a member of the tetraspanin family that is expressed on motoneurons. Like dock mutants, lbm embryos also have low-penetrance body wall muscle defects. The delay in synapse formation seen in dock and lbm mutants might indicate that Dock and Lbm function in partially redundant biochemical pathways necessary for RP3 synaptogenesis. If so, embryos lacking both Lbm and Dock should display more penetrant and/or more severe defects than either single mutant. To test this hypothesis, the neuromuscular system was examined in dock;lbm double mutants. Neither the delay of RP3 synapse formation nor the occasional muscle and SNb guidance defects are more penetrant in double mutants than in lbm or dock single mutants. No obvious additional defects were observed in other axon pathways. These observations indicate that Dock and Lbm are not essential components of separate, partially redundant pathways required for RP3 synaptogenesis. Nck, the mammalin homolog of Dock, associates with activated focal adhesion kinase (FAK; Schlaepfer, 1997), which is an important effector for integrins. The interaction between FAK and Nck is interesting in light of the genetic evidence suggesting that Dock and the tetraspanin Lbm participate in the same processes during RP3 synaptogenesis. Tetraspanins associate with integrins, and this interaction can increase integrin signaling. These observations are consistent with a model in which Lbm expressed on the RP3 growth cone might facilitate integrin-mediated activation of FAK and recruitment of Dock to the FAK signaling complex (Desai, 1999).

In the course of selecting homozygous dock mutant embryos for dissection, it was noticed that none of the progeny of mothers heterozygous for dock alleles completely lacked Dock protein expression. Dock is clearly detectable in CNS axons and at muscle attachment sites in mutant embryos, although the level of expression is much lower than in wild type. One explanation for the presence of Dock protein in embryos homozygous for dock mutations is that they receive a maternal contribution of Dock mRNA and/or protein that persists late in embryogenesis. Persistent maternal Dock could account for the incomplete penetrance and/or the mild nature of the RP3 defect observed in dock embryos. To determine the origin of the anti-Dock reactivity and to discover the complete loss-of-function dock phenotype, females heterozygous for dock mutations that had homozygous dock mutant ovaries were generated using the FLP/ovo D system. Such females produce oocytes devoid of Dock message and protein which, when fertilized with dock mutant sperm, develop into complete-loss-of-function dock mutant embryos. The CNS in such embryos is not stained by anti-Dock, indicating that the residual staining in zygotic loss-of-function dock mutants is indeed due to maternally derived Dock rather than to a cross-reacting epitope (Desai, 1999).

Mutant embryos lacking both the maternal and zygotic contributions for dock were examined, and there are marked CNS axon defects. The outermost FasII-positive axon bundles are severely discontinuous. In addition, it appears that some FasII-positive axons cross the midline that do not normally do so. Although the pattern of motor nerves is still fairly normal, complete dock null embryos do display increased levels of SNb abnormalities, some of which might result from guidance errors within the ventral cord. For example, a number of embryos display segments in which the SNb on one side is abnormally thick while the contralateral SNb is abnormally thin. This phenotype could be explained if some SNb axons fail to cross the midline and contributed to the ipsilateral SNb instead. Maternal loss of Dock also enhances dock-induced lethality. Zygotic dock mutants usually survive until the pupal stage, while embryos lacking both maternal and zygotic Dock fail to hatch. By contrast, the inhibition of RP3 synapse formation is not worsened in late stage 16/early stage 17 embryos lacking both maternal and zygotic Dock. The innervation of the 7/6 cleft in embryos mutant for maternal and zygotic dock is reduced by 39%, relative to wild-type embryos of the same stage, while 44% of 7/6 clefts completely lack synapses. This penetrance is actually lower than that displayed by zygotic dock embryos, indicating that the presence of maternally derived Dock is not responsible for 7/6 cleft innervation in zygotic dock mutants (Desai, 1999).

Likewise, the other tissues that normally express Dock appear normal in complete dock null embryos. The body wall musculature appears intact despite the loss of Dock at muscle attachment sites. The PNS also appears largely normal, although there is some variability in chordotonal organ neuron numbers. 8% of hemisegments in complete null embryos have four chordotonal neurons instead of five, and 6% have six chordotonal neurons (Desai, 1999).

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. The guanine nucleotide exchange factor (GEF) Trio has been identified as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. It is proposed that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension (Newsome, 2000).

Dscam, a Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dreadlocks (Dock), an SH3/SH2 adaptor protein required for axon guidance. Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, which contains a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable immunoglobulin (Ig) and transmembrane (TM) domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity is likely to contribute to the specificity of neuronal connectivity (Schmucker, 2000).

To gain insight into the mechanisms by which growth cones integrate guidance cues, a combined biochemical and genetic analysis of the Dock signal transduction pathway has been pursued. Dock is an adaptor protein containing 3 SH3 domains and a single SH2 domain, and is closely related to mammalian Nck. dock mutants show defects in axon guidance in the adult fly visual system and in the embryonic nervous system. Based on the role of the adaptor protein Grb-2 in linking receptor tyrosine kinases to Ras, it is proposed that Dock links guidance receptors to downstream regulators of the actin cytoskeleton. Pak, a p21-activated serine/threonine kinase, acts downstream of Dock in adult photoreceptor neurons. Dock binds through its second SH3 domain to Pak and Pak binds directly to Rho family GTPases, evolutionarily conserved regulators of the actin-based cytoskeleton. Genetic studies reveal that both Pak's kinase activity and its interaction with Rho family GTPases are essential for axon guidance (Schmucker, 2000 and references therein).

Dscam binds directly to multiple domains of Dock and is widely expressed on axons in the embryonic nervous system. Dscam is required for recognition of an intermediate targeting determinant for Bolwig's nerve: Dock and Pak are required for this step, and Dscam shows dosage-sensitive interactions with both dock and Pak. Based on these studies, it is proposed that Dscam recognizes a guidance signal(s) and translates it into changes in the actin-based cytoskeleton through Dock and Pak (Schmucker, 2000).

Dscam RNA is expressed in Bolwig's organ as well as more generally within the CNS and PNS. The protein product is exclusively expressed on axon processes. To assess whether selective expression of Dscam in Bolwig's nerve is sufficient to rescue the mutant, a transgene encoding full-length Dscam driven by the GMR promoter (a strong transcriptional driver providing Bolwig's organ-specific expression) was constructed and it was introduced into the germline by P element DNA transformation. Two independent insertions were characterized. In a wild-type (or a Dscam mutant) background, 100% of the embryos carrying one or two copies of GMR-Dscam exhibit strong axon guidance phenotypes. Individual axons project in abnormal directions over the surface of the optic lobe and rarely contact P2. It is unclear whether this reflects the sensitivity of Bolwig's nerve guidance to increased levels of Dscam or misexpression in Bolwig's organ of an inappropriate isoform, or both. Due to the large size of the Dscam locus (61 kb), whether or not the wild-type gene rescues the mutant phenotype could not be assessed. In any case, the dominant phenotype precludes assessing transgene rescue of the mutant phenotype. In contrast, GMR-dock rescues 85% of dock mutant embryos (Schmucker, 2000).

Whether dock and Pak are functional components of a Dscam guidance pathway was assessed through genetic analysis. Mistargeting defects of Bolwig's nerve were observed in some 44% of the embryos heterozygous for both Dscam and dock. In contrast, only 4%-6% and 10%-13% of embryos heterozygous for either dock or Dscam, respectively, show defects. Similarly, whereas some 38% of embryos heterozygous for both Dscam and Pak have an abnormal Bolwig's nerve, only 5% were defective in embryos heterozygous for Pak. The synergistic interactions between Dscam, dock, and Pak, the similarity of complete loss-of-function phenotypes, and the physical interactions between these proteins are consistent with their acting together to mediate recognition between the Bolwig's nerve growth cones and P2 (Schmucker, 2000).

The convergence of olfactory axons expressing particular odorant receptor (Or) genes on spatially invariant glomeruli in the brain is one of the most dramatic examples of precise axon targeting in developmental neurobiology. The cellular and molecular mechanisms by which olfactory axons pathfind to their targets are poorly understood. The SH2/SH3 adapter Dock and the serine/threonine kinase Pak are necessary for the precise guidance of olfactory axons. Using antibody localization, mosaic analyses and cell-type specific rescue, it is observed that Dock and Pak are expressed in olfactory axons and function autonomously in olfactory neurons to regulate the precise wiring of the olfactory map. Detailed analyses of the mutant phenotypes in whole mutants and in small multicellular clones indicate that Dock and Pak do not control olfactory neuron (ON) differentiation, but specifically regulate multiple aspects of axon trajectories to guide them to their cognate glomeruli. Structure/function studies show that Dock and Pak form a signaling pathway that mediates the response of olfactory axons to guidance cues in the developing antennal lobe (AL). These findings therefore identify a central signaling module that is used by ONs to project to their cognate glomeruli (Ang, 2003).

ONs of the antennae and maxillary palps undergo terminal differentiation during early metamorphosis and become predestined to express particular Or genes and synapse in specific glomeruli. Between 20 and ~50 hAPF, their axons leave the nascent antenna in fascicles and enter the AL in search of their targets. Projection neurons (PNs) the targets of the incoming axons acquire their cell fates, which predetermine their glomerular choice, during larval development. During early pupal development their dendrites enter the AL and become precisely paired with ON axons in specific glomeruli. Thus, ONs expressing a given Or gene rendezvous with PNs of a particular identity within a topographically defined glomerulus in the AL (Ang, 2003).

In wild type flies, olfactory axons take stereotyped paths on the surface of the AL to converge on their cognate glomeruli. Detailed characterization of the axon trajectories, using Gal4 drivers expressed in different subclasses of ONs shows that, upon arrival at the anterolateral point of the AL, afferents project directly, with little sidetracking to their postsynaptic targets. As in the mouse and moth, these axon pathways are bilaterally symmetric and invariant from AL to AL. How is this precise wiring pattern formed during development? In one model, each ON initially sends collaterals to multiple glomeruli and then withdraws the inappropriate branches in a process requiring odorant-evoked activity. Alternatively, the invariant pattern of connections is the result of directed axon migrations in response to spatially restricted pathfinding cues in the developing AL. A definitive answer to this question will require developmental study or direct observation of the extending axons. However, at least two observations are consistent with the notion that olfactory axons navigate directly to their cognate glomeruli. (1) A temporal lag between early axon pathfinding and subsequent Or gene expression indicates that an odorant-evoked activity is unlikely to play an important role. Indeed, activity is neither required for formation nor maintenance of the olfactory map in mouse and moth. (2) Importantly, the finding that the growth cone guidance genes, dock and Pak, are needed for development of the olfactory map, provides strong evidence that directed axon migration plays a key role in the matching of ON axons with their correct glomeruli. Directed navigation of olfactory axons to their targets is also observed in zebrafish and moth (Ang, 2003).

In dock and Pak mutants, the stereotyped connectivity of AL neuropil is severely disrupted, leading to an aglomerular phenotype. Three pieces of evidence are presented indicating that dock and Pak function in ONs: (1) antibody staining shows that Dock and Pak proteins are expressed in antennal axons during the period in which they are projecting to the brain; (2) consistent with their requirements in ONs, removal of dock and Pak activities from only the antennae results in ectopic targeting of olfactory axons, and (3) expression of dock and Pak cDNAs specifically in ONs in otherwise mutant animals leads to strong rescue of the mutant AL phenotype. Although numerous glomeruli were restored upon the expression of the wild-type cDNAs, some glomeruli were not. The incomplete rescue is thought to be due to the expression of SG18.1-Gal4 in only a subset of all the ONs. However, it is also possible that the partial rescue reflects an additional requirement of dock and Pak functions in the brain. A recent study indicates that ONs may be divided into different classes based on the timing of their projections. It was not determined further whether dock and Pak are required in all ONs or in only a specific subset. Although dock and Pak are specifically required in ONs, finding of nonautonomous effects on the morphogenetic changes of the PNs and AL glia is in accord with earlier studies in which ONs were physically or genetically ablated. The data therefore show that proper termination of ON axons is also an important step in the sculpting of the AL neuropil into distinct glomeruli (Ang, 2003).

Evidence is provided that the disruption in AL development in dock and Pak mutants is not an indirect effect of aberrant cell-fate determination or axonogenesis. By contrast, the precise targeting of ON axons is severely disrupted in dock and Pak mutants. To identify the cause of the mistargeting, the axon pathways of individual ON classes (Or47a, and Or47b) were examined at the single-cell level. Although an additional short branch was observed in 9% of dock mutant neurons, the most striking defect observed in single-cell clones was the chaotic trajectories exhibited by both the ipsilateral and contralateral axons of the ONs. It is concluded that the primary function of dock and Pak in ONs is axon pathfinding, to steer ON axons precisely to their target glomeruli. In mouse, mutations in the odorant receptor genes abolish the ability of olfactory axons to pathfind in the anteroposterior axis without affecting their migration in the dorsoventral axis, leading to the proposal that odorant receptors participate in the recognition of only anteroposterior guidance cues. However, after examining several hundred ALs for each dock and Pak mutant, no consistent patterns were observed in the mistargeting of ON axons. The ON classes are affected to different degrees by the loss of dock and Pak activities. Although Or22a and Or47a axons terminate in numerous ectopic glomeruli, Or47b axons terminate in a single glomerulus, albeit mis-shapen, in the approximate position of the wild-type VA1lm. The reason for the differential sensitivity of the ON subtypes to the loss of dock and Pak functions is not known. One possibility is that Or47b axons, which are among the first axons to enter the AL, are confronted with fewer developing glomeruli and hence fewer guidance choices than Or22a and Or47a axons that enter the AL later. Alternatively, Or47b axons may have less need for dock- and Pak-mediated navigational functions because VA1lm is located near the nerve entry point. Indeed, while the Or47b ipsilateral axons frequently terminate accurately on VA1lm, the contralateral axons, which have to project across the entire AL surface, are often misrouted. In contrast to the severe projection defects in the AL, the migration of dock and Pak mutant axons through the antennal nerve takes place normally. It is possible that the lack of requirement of dock and Pak functions during this phase of axon growth reflects a different guidance mechanism in the antennal nerve (Ang, 2003).

The observation that the ON axon trajectories are severely disrupted in dock and Pak mutants suggests that the genes may mediate the detection or response of the growth cones to guidance cues in the environment. The results indicate that in these events, dock and Pak are very likely to act in a signaling pathway: (1) loss of either dock or Pak functions results in olfactory connectivity phenotypes that are indistinguishable; (2) both dock or Pak function autonomously in ONs; (3) mutations that disrupt the domains of Dock (second SH3 domain) and Pak (N-terminal PXXP domain; Pak4) that mediate interaction between the two proteins, disrupt ON axon targeting. It is therefore proposed that Dock and Pak are part of a signal transduction cascade that allows ONs to find and precisely pair with the correct postsynaptic partners. Although severely disrupted, the guidance of ON axons in dock and Pak mutants is not completely abolished, indicating that other genes function to steer ON axons to their targets as well (Ang, 2003).

The Drosophila HEM-2/NAP1 homolog KETTE interacts with Dock and Rac to control axonal pathfinding and cytoskeletal organization.

In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. The gene kette (correctly termed Hem-protein, or simply Hem) is required for the normal projection of the VUM axons and interference with kette function disrupts glial migration. In spite of the fact that glial migration is disrupted in kette mutants, both the axon guidance and glial migration phenotypes have their origin in midline neuron expression and not in midline glial expression. Axonal projection defects are found for many moto- and interneurons in kette mutants. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The Hem/Kette protein is homologous to the transmembrane protein HEM-2/NAP1 (Nck-associated protein) evolutionary conserved from worms to vertebrates. In the CNS, the membrane protein Kette could be participating directly in the neuron-glial interaction at the midline, where it could act as a signal to direct glial migration. Alternatively, Kette could serve as a receptor of possibly glial-derived signals during VUM growth cone guidance. The experimental data suggest that Kette transduces information to the neuronal cytoskeleton, which is in agreement with a receptor function (Hummel, 2000).

The vertebrate homolog of KETTE has been shown to interact with the first SH3 domain of the Nck adapter protein (Kitamura, 1996). The Drosophila homolog of Nck is encoded by dreadlocks (Garrity, 1996). dock was identified in a screen for mutations affecting axonal pathfinding and targeting of the adult photoreceptor neurons. In wild-type third instar larvae, the different photoreceptor cells stop their axonal growth in two distinct layers of the optic lobe, the lamina and the medulla. In contrast, dock mutant photoreceptor cells fail to establish this specific targeting, leading to a disruption of the lamina neuropile organization. In ~70% of the third instar larvae homozygous for the hypomorphic ketteDelta2-6 allele (n = 25), a weak disorganization was found of the lamina plexus and an abnormal bundling of R-cell axons in the medulla. The remaining larvae showed a stronger disorganization of the R-cell axons (20%) or were indistinguishable from wild type (10%). Further reduction of the kette gene function results in an enhancement of this axonal phenotype in 50% of the analyzed transheterozygous mutant kette larva. If one copy of dock is removed in the background of a hypomorphic kette mutation, a considerable enhancement of the larval projection phenotype is observed in 60% of the individuals. In addition, a significant enhancement of the homozygous dock phenotype is observed when removing one copy of kette in a dock mutant background (Hummel, 2000).

A reduction in the size of the longitudinal connectives in the embryonic CNS is observed in dock mutants. This phenotype resembles a hypomorphic kette connective phenotype. In mutant dockP2 embryos, commissure separation is also affected, comparable with the hypomorphic phenotype seen in ketteJ1-70 embryos. In correlation with the commissural phenotype, the VUM axons do not project properly in mutant dock embryos (Hummel, 2000).

In summary, these data show that both kette and dock mutants genetically interact and share a number of phenotypic traits. This suggests that these genes might be acting in the same genetic pathway during axonal pathfinding (Hummel, 2000).

The Rho family of small GTPases constitutes important regulatory factors also interacting with Nck. To analyze the functional interaction of KETTE with members of the Rho family, both the activated as well as the dominant-negative versions of Cdc42 and Rac1 were expressed in the midline cells of wild-type and kette mutant embryos. Expression of both of these mutant proteins in all midline cells using the simGAL4 driver line results in similar axonal defects. The projection of the VUM neurons resembles the phenotype observed in kette mutant embryos. In addition, the cell bodies of the VUM neurons appear sometimes displaced. In stage 16 embryos, the segmental commissures appear fused, which again indicates the importance of the midline neurons for the migration of the midline glia. Only a weak commissural disorganization is observed when the different Cdc42 or Rac1 proteins are expressed in the midline glial cells only. In all experiments, the expression of Rac1 appears to have more pronounced effects on the axonal morphology (Hummel, 2000).

To further test the interaction of kette and Rac1, activated Rac1 was expressed in all midline cells of mutant ketteJ4-48 embryos. The commissures appeared separated, indicating that the midline glial cells are able to migrate between anterior and posterior commissures. Concomitantly, the connectives are further distant from the midline), indicating that expression of activated Rac1 can partially rescue the kette phenotype (Hummel, 2000).

Among others, the Nck adapter protein transduces signals via CDC42 and Rac1 to the Actin cytoskeleton. A GFP-moesin transgene was used to analyze the Actin cytoskeleton of mutant kette embryos. This protein binds to the F-actin fibers and thus allows a determination of their subcellular distribution using confocal microscopy. In wild-type embryos, F actin is found in axonal processes that are arranged in the typical ladder-like pattern. Prominent expression is also detected in the epidermis and the somatic musculature. In similar focal planes, kette embryos appear very different. Within the CNS, the typical fused commissure phenotype of mutant kette embryos is evident. Frequent intense granular staining is observed in the CNS and in the lateral body wall. Furthermore, the regular appearance of the cytoskeleton is disrupted in both mesoderm and ectoderm. In a tangential section of the dorsal epidermis, individual cells can be seen in wild-type embryos. Some cells form hairs, characterized by thin F-actin bundles. In mutant ketteC3-20 embryos, pronounced F-actin bundles are found, which often have a wavy appearance. In addition, the cortical actin cytoskeleton appears to stain weaker compared with wild-type embryos (Hummel, 2000).

Thus, mutations in kette affect the organization of the cytoskeleton. kette is expressed in neurons and is needed for correct axonal pathfinding. The KETTE protein seems to interact with the SH2-SH3 adapter Dock and at least part of the kette function might be mediated via small GTPases such as Rac1 (Hummel, 2000).

In addition to Kette function in axonal pathfinding, defects are observed in the morphology of trichomes and bristles in flies homozygous for the weak hypomorphic ketteDelta2-6 allele. Around 10% of the bristles appear wavy or do bend sharply and wing trichomes are enlarged and sometimes split. These phenotypes resemble those observed for mutations affecting the organization of the F-actin bundles or following expression of mutated Cdc42 or Rac1. Similarly, elevated levels of GTPase function in the developing eye cause late developmental defects as observed in hypomorphic kette mutations. cdc42 mutations have been isolated, but, presumably due to maternal contribution, loss of cdc42 function does not lead to an embryonic CNS phenotype. Both Cdc42 and Rac1 are important regulators of the actin cytoskeleton. The transduction of extracellular signaling to small GTPases is believed to involve Nck-type adapter proteins. Several phenotypic traits of kette are shared by mutations in the Drosophila gene dock, which encodes a Nck homolog. Furthermore, dock and kette genetically interact. The genetic data in combination with the kette loss-of-function and kette overexpression phenotypes led to the proposal of a model relating Dhem2/NAP1 function to cytoskeleton organization (Hummel, 2000).

The vertebrate KETTE homolog is HEM-2/NAP1 with 86% amino acid identity over the entire ORF, indicating that presumed protein-protein interactions are also conserved. To date, no hem-2 mutation has been described in vertebrates. The first SH3 domain of Nck was used to isolate Nck-associated proteins (NAP) and led to the identification of HEM-2/NAP1. Binding of HEM-2/NAP1 to Nck appears to be mediated by a 140-kD protein. Interestingly, in a screen for proteins interacting with activated Rac1, a complex consisting of HEM-2/NAP1 and a 140-kD protein was isolated. Thus, the 140-kD protein might be a novel adapter linking HEM-2/NAP1 signaling along two routes to the small GTPases. It will be of interest to identify Drosophila genes interacting with kette (Hummel, 2000 and references therein).

The Drosophila Nck homolog is encoded by dock. dock function appears highly specialized for growth-cone guidance since no mutant phenotypes have been reported in the mesoderm or the ectoderm. Because kette shows more pleiotropic defects, other adapter proteins may interact with the Kette protein (Hummel, 2000).

During axonal pathfinding, coordinated cytoskeletal remodeling occurs at the tip of the extending neurites, the growth cone. The Rho family of GTPases mediates the regulation of the reorganization of the actin cytoskeleton induced by extracellular signals: Cdc42, Rac1, and RhoA. In fibroblast cells, the different GTPases induce different cellular responses. Similarly, different functions appear to be associated with the different Drosophila GTPases. Rho as well as Cdc42 function is needed for cell shape changes during gastrulation, dorsal closure, bristle, and hair formation. Bristle and hair formation are similarly affected by kette (Hummel, 2000).

These data suggest that Kette provides a novel mechanism linking extracellular signals to the neuronal cytoskeleton. Central relay proteins are SH2-SH3 adapter proteins that control the organization of the actin cytoskeleton via a number of downstream proteins. Biochemical data suggest that additional proteins (p140 kD) may bypass the function of SH2-SH3 adapter proteins, but a detailed analysis awaits its isolation. The Kette protein might interact with extracellular signals, which, in the CNS, might possibly be presented by glial cells. To gain further insight in the neuron-glial interaction at the midline, future work will be directed toward the identification of these components (Hummel, 2000).


Ang, L.-H., et al. (2003). Dock and Pak regulate olfactory axon pathfinding in Drosophila. Development 130: 1307-1316. 12588847

Anton, I. M., et al. (1997). The Wiskott-Aldrich syndrome protein-interacting protein (WIP) binds to the adaptor protein Nck. J. Biol. Chem. 273(33): 20992-5. PubMed Citation: 9694849

Bladt, F., Aippersbach, E., Gelkop, S., Strasser, G.A., Nash, P., Tafuri, A., Gertler, F.B., and Pawson, T. (2003). The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. Mol. Cell. Biol. 23: 4586-4597. Medline abstract: 12808099

Bokoch, G. M., et al. (1996). Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271(42): 25746-9. PubMed Citation: 8824201

Braverman, L. E. and Quilliam, L. A., (1999). Identification of Grb4/Nckbeta, a src homology 2 and 3 domain-containing adapter protein having similar binding and biological properties to Nck. J. Biol. Chem. 274(9): 5542-9. PubMed Citation: 10026169

Chen, M., et al. (1998). Identification of Nck family genes, chromosomal localization, expression, and signaling specificity. J. Biol. Chem. 273(39): 25171-8. PubMed Citation: 9737977

Chou, M. M., Fajardo, J. E. and Hanafusa, H. (1992). The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels. Mol. Cell. Biol. 12(12): 5834-42. PubMed Citation: 1280326

Clemens, J. C., et al. (1996). A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance. J. Biol. Chem. 271(29): 17002-5. PubMed Citation: 8663600

Desai, C. J., et al. (1999). The Drosophila SH2-SH3 adapter protein Dock is expressed in embryonic axons and facilitates synapse formation by the RP3 motoneuron. Development 126(7): 1527-1535. PubMed Citation: 10068645

Eden, S., et al. (2002). Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418(6899): 790-3. 12181570

Fan, X., Labrador, J. P., Hing, H. and Bashaw, G. J. (2003). Slit stimulation recruits Dock and Pak to the Roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline. Neuron 40: 113-127. 14527437

Galisteo, M. L., et al. (1996). The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1. J. Biol. Chem. 271(35): 20997-1000. PubMed Citation: 8798379

Garrity, P. A., et al. (1996). Drosophila photoreceptor axon guidance and targeting requires the Dreadlocks SH2/SH3 adapter protein. Cell 85(5): 639-50. PubMed Citation: 8646773

Gupta, R. W. and Mayer, B. J. (1998). Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus. Oncogene 17(17): 2155-65. PubMed Citation: 9811447

Harden, N., et al. (1996). A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures. Mol. Cell. Biol. 16(5): 1896-908. PubMed Citation: 8628256

Herrick, T. M. and Cooper, J. A. (2002). A hypomorphic allele of dab1 reveals regional differences in reelin-Dab1 signaling during brain development Development 129: 787-796. 11830577

Hing, H., et al. (1999). Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila. Cell 97(7): 853-63. PubMed Citation: 10399914

Howe, A. K. (2001). Cell adhesion regulates the interaction between Nck and p21-activated kinase. J. Biol. Chem. 276: 14541-14544. 11278241

Hu, Q., Milfay, D. and William, L. T. (1995). Binding of NCK to SOS and activation of ras-dependent gene expression, Mol. Cell. Biol. 15: 1169-1174. Medline abstract: 7862111

Hummel, T., Leifker, K. and Klämbt, C. (2000). The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization. Genes Dev. 14: 863-873. 10766742

Innocenti, M., et al. (2004). Abi1 is essential for the formation and activation of a WAVE2 signalling complex. Nat. Cell Biol. 6(4): 319-27. 15048123

Izadi, K. D., et al. (1998). Characterization of Cbl-Nck and Nck-Pak1 interactions in myeloid FcgammaRII signaling. Exp. Cell Res. 245(2): 330-42. 9906915

Jones, N. and Dumont, D. J. (1998). The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R. Oncogene 17(9): 1097-108. PubMed Citation: 9764820

Jones, N., et al. (2006). Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 440: 818-823. PubMed Citation: 16525419

Kim, H., Rogers, M. J., Richmond, J. E. and McIntire, S. L. (2004). SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans. Nature 430: 891-896. Medline abstract: 15318222

Kitamura, T., Kitamura, Y., Yonezawa, K., Totty, N. F., Gout, I., Hara, K., Waterfield, M. D., Sakaue, M., Ogawa, W. and Kasuga, M. (1996). Molecular cloning of p125Nap1, a protein that associates with an SH3 domain of Nck. Biochem. Biophys. Res. Commun. 219: 509-514. 8605018

Kitamura, Y., Kitamura, T., Sakaue, H., Maeda, T., Ueno, H., Nishio, S., Ohno, S., Osada, S., Sakaue, M., Ogawa, W. et al. (1997). Interaction of Nck associated protein 1 with activated GTP binding protein Rac. Biochem. J. 322: 873-878. 9148763

Kochhar, K. S. and Iyer, A. P. (1996). Hepatocyte growth factor induces activation of Nck and phospholipase C-gamma in lung carcinoma cells. Cancer Lett. 104(2): 163-9. PubMed Citation: 8665484

Kopczynski, C. C., Davis, G. W. and Goodman, C. S. (1996). A neural tetraspanin, encoded by late bloomer, that facilitates synapse formation. Science 271: 1867-1870. PubMed Citation: 8596956

Kremer, B. E., Adang, L. A. and Macara, I. G. (2007). Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7. Cell 130(5): 837-50. Medline abstract: 17803907

Lawe, D. C., Hahn, C. and Wong, A. J. (1997). The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68. Oncogene 14(2): 223-31. PubMed Citation: 9010224

Li, W., et al. (1992). The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors. Mol. Cell. Biol. 12(12): 5824-33. PubMed Citation: 1333047

Lu, W., et al. (1997). Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck. Curr. Biol. 7(2): 85-94. PubMed Citation: 9024622

Lu, W. and Mayer, B. J. (1999). Mechanism of activation of Pak1 kinase by membrane localization. Oncogene 18(3): 797-806. PubMed Citation: 9989831

Lussier, G. and Larose, L. (1997). A casein kinase I activity is constitutively associated with Nck. J. Biol. Chem. 272(5): 2688-94. PubMed Citation: 9006905

Meisenhelder, J. and Hunter, T. (1992). The SH2/SH3 domain-containing protein Nck is recognized by certain anti-phospholipase C-gamma 1 monoclonal antibodies, and its phosphorylation on tyrosine is stimulated by platelet-derived growth factor and epidermal growth factor treatment. Mol. Cell. Biol. 12(12): 5843-56. PubMed Citation: 1448108

Mohamed, A. M. and Chin-Sang, I. D. (2011). The C. elegans nck-1 gene encodes two isoforms and is required for neuronal guidance. Dev. Biol. 354(1): 55-66. PubMed Citation: 21443870

Montanaro, F. and Carbonetto, S. (2003). Targeting dystroglycan in the brain. Neuron 37: 193-196. Medline abstract: 12546815

Noguchi, T., et al. (1999). Tyrosine phosphorylation of p62Dok induced by cell adhesion and insulin: possible role in cell migration. EMBO J. 18: 1748-1760. PubMed Citation:

Oldenhof, J., et al. (1998). SH3 binding domains in the dopamine D4 receptor. Biochemistry 37(45): 15726-36. PubMed Citation: 9843378

Park, D. and Rhee, S. G. (1992). Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP. Mol. Cell. Biol. 12(12): 5816-23. PubMed Citation: 1333046

Qu, Q. and Smith, F. I. (2004). Alpha-dystroglycan interactions affect cerebellar granule neuron migration. J. Neurosci. Res. 76: 771-782. Medline abstract: 15160389

Quilliam, L. A., et al. (1996). Isolation of a NCK-associated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling. J. Biol. Chem. 271(46): 28772-6. PubMed Citation: 8910519

Rao, Y. and Zipursky, S. L. (1998). Domain requirements for the Dock adapter protein in growth- cone signaling. Proc. Natl. Acad. Sci. 95(5): 2077-82. PubMed Citation: 9482841

Rivero-Lezcano, O. M., et al. (1995). Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains. Mol. Cell. Biol. 15(10): 5725-31. PubMed Citation: 7565724

Rockow, S., et al. (1996). Nck inhibits NGF and basic FGF induced PC12 cell differentiation via mitogen-activated protein kinase-independent pathway. Oncogene 12(11): 2351-9. PubMed Citation: 8649775

Ruan, W., Pang, P. and Rao, Y. (1999). The SH2/SH3 adaptor protein Dock interacts with the Ste20-like kinase Misshapen in controlling growth cone motility. Neuron 24: 595-605. PubMed Citation: 10595512

Schlaepfer, D. D., Broome, M. A. and Hunter, T. (1997). Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol. Cell. Biol. 17(3): 1702-13. PubMed Citation: 9032297

Schmucker, D., et al. (2000). Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101: 671-684. PubMed Citation: 10892653

Sells, M. A., et al. (1997). Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7(3): 202-10. PubMed Citation: 9395435

Shcherbata, H. R., et al. (2007). Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 26(2): 481-93. Medline abstract: 17215867

She, H. Y., et al. (1997). Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells. Mol. Biol. Cell 8(9): 1709-21. PubMed Citation: 9307968

Sitko, J. C., Guevara, C. I. and Cacalano, N. A. (2004). Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation. J. Biol. Chem. 279(36): 37662-9. 15173187

Stein, E., et al. (1998). Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase. J. Biol. Chem. 273(3): 1303-8. PubMed Citation: 9430661

Su, Y. C., et al. (1997). NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain. EMBO J. 16(6): 1279-90. PubMed Citation: 9135144

Tanaka, M., et al. (1997). Expression of mutated Nck SH2/SH3 adaptor respecifies mesodermal cell fate in Xenopus laevis development. Proc. Natl. Acad. Sci. 94(9): 4493-8. PubMed Citation: 9114017

Tang, J., Feng, G. S. and Li, W. (1997). Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor. Oncogene 15(15): 1823-32. PubMed Citation: 9362449

Tu, Y., et al. (1999). The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. Mol. Cell. Biol. 19(3): 2425-34. PubMed Citation: 10022929

Tutor, A. S., Prieto-Sanchez, S. and Ruiz-Gomez, M. (2013). Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies. Development 141(2): 367-76. PubMed ID: 24335255

Ursuliak, Z., Clemens, J. C., Dixon, J. E. and Price, J.V. (1997). Differential accumulation of DPTP61F alternative transcripts: regulation of a protein tyrosine phosphatase by segmentation genes. Mech. Dev. 65(1-2): 19-30. PubMed Citation: 9256342

Vaynberg, J., et al. (2005). Structure of an ultraweak protein-protein complex and its crucial role in regulation of cell morphology and motility. Mol. Cell. 17(4): 513-23. 15721255

Worby, C. A., et al. (2001). The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton. J. Biol. Chem. 276(45): 41782-9. 11546816

Worby, C. A., Simonson-Leff, N., Clemens, J. C., Huddler, D., Muda, M. and Dixon, J. E. (2002). Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance. J. Biol. Chem. 277(11): 9422-8. PubMed Citation: 11773052

Wunderlich, L., Farago, A. and Buday, L. (1999). Characterization of interactions of Nck with Sos and dynamin. Cell Signal. 11(1): 25-9. PubMed Citation: 10206341

Xu, Z., et al. (2005). Molecular dissection of PINCH-1 reveals a mechanism of coupling and uncoupling of cell shape modulation and survival. J. Biol. Chem. 280(30): 27631-7. 15941716

Yablonski, D., et al. (1998). A Nck-Pak1 signaling module is required for T-cell receptor-mediated activation of NFAT, but not of JNK. EMBO J. 17(19): 5647-57. PubMed Citation: 9755165

Yang, L. and Bashaw, G. J. (2006). Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline. Neuron 52(4): 595-607. Medline abstract: 17114045

Zeng, R., Cannon, J. L., Abraham, R. T., Way, M., Billadeau, D. D., Bubeck-Wardenberg, J. and Burkhardt, J. K. (2003). SLP-76 coordinates Nck-dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site. J. Immunol. 171: 1360-1368. 12874226

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

date revised: 10 June 2014

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