Axonal heparan sulfate proteoglycans regulate the distribution and efficiency of the repellent slit during midline axon guidance

The presentation of secreted axon guidance factors plays a major role in shaping central nervous system (CNS) connectivity. Recent work suggests that heparan sulfate (HS) regulates guidance factor activity; however, the in vivo axon guidance roles of its carrier proteins (heparan sulfate proteoglycans, or HSPGs) are largely unknown. Here we demonstrate through genetic analysis in vivo that the HSPG Syndecan (Sdc) is critical for the fidelity of Slit repellent signaling at the midline of the Drosophila CNS, consistent with the localization of Sdc to CNS axons. sdc mutants exhibit consistent defects in midline axon guidance, plus potent and specific genetic interactions supporting a model in which HSPGs improve the efficiency of Slit localization and/or signaling. To test this hypothesis, we show that Slit distribution is altered in sdc mutants and that Slit and its receptor bind to Sdc. However, when we compare the function of the transmembrane Sdc to a different class of HSPG that localizes to CNS axons (Dallylike), we find functional redundancy, suggesting that these proteoglycans act as spatially specific carriers of common HS structures that enable growth cones to interact with and perceive Slit as it diffuses away from its source at the CNS midline.     

Receptor protein tyrosine phosphatases regulate retinal ganglion cell axon outgrowth in the developing Xenopus visual system

Receptor protein tyrosine phosphatases (RPTPs) are regulators of axon outgrowth and guidance in a variety of different vertebrate and invertebrate systems. Three RPTPs, CRYP-alpha, PTP-delta, and LAR, are expressed in overlapping but distinct patterns in the developing Xenopus retina, including expression in retinal ganglion cells (RGCs) as they send axons to the tectum (Johnson KG, Holt CE. 2000. Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system. Mech Dev 92:291-294). In order to examine the role of these RPTPs in visual system development, putative dominant negative RPTP mutants (CS-CRYP-alpha, CS-PTP-delta, and CS-LAR) were expressed either singly or in combination in retinal cells. No effect was found on either retinal cell fate determination or on gross RGC axon guidance to the tectum. However, expression of these CS-RPTP constructs differentially affected the rate of RGC axon outgrowth. In vivo, expression of all three CS-RPTPs or CS-PTP-delta alone inhibited RGC axon outgrowth, while CS-LAR and CS-CRYP-alpha had no significant effect. In vitro, expression of CS-CRYP-alpha enhanced neurite outgrowth, while CS-PTP-delta inhibited neurite outgrowth in a substrate-dependent manner. This study provides the first in vivo evidence that RPTPs regulate retinal axon outgrowth.     

The C. elegans LAR-like receptor tyrosine phosphatase PTP-3 and the VAB-1 Eph receptor tyrosine kinase have partly redundant functions in morphogenesis

Receptor-like protein-tyrosine phosphatases (RPTPs) form a diverse family of cell surface molecules whose functions remain poorly understood. The LAR subfamily of RPTPs has been implicated in axon guidance and neural development. Here we report the molecular and genetic analysis of the C. elegans LAR subfamily member PTP-3. PTP-3 isoforms are expressed in many tissues in early embryogenesis, and later become localized to neuronal processes and to epithelial adherens junctions. Loss of function in ptp-3 causes low-penetrance defects in gastrulation and epidermal development similar to those of VAB-1 Eph receptor tyrosine kinase mutants. Loss of function in ptp-3 synergistically enhances phenotypes of mutations in the C. elegans Eph receptor VAB-1 and a subset of its ephrin ligands, but does not show specific interactions with several other RTKs or morphogenetic mutants. The genetic interaction of vab-1 and ptp-3 suggests that LAR-like RPTPs and Eph receptors have related and partly redundant functions in C. elegans morphogenesis.     

The Cell Surface Receptor Tartan Is a Potential In Vivo Substrate for the Receptor Tyrosine Phosphatase Ptp52F

Receptor-linked protein-tyrosine phosphatases (RPTPs) are essential regulators of axon guidance and synaptogenesis in Drosophila, but the signaling pathways in which they function are poorly defined. We identified the cell surface receptor Tartan (Trn) as a candidate substrate for the neuronal RPTP Ptp52F by using a modified two-hybrid screen with a substrate-trapping mutant of Ptp52F as “bait.” Trn can bind to the Ptp52F substrate-trapping mutant in transfected Drosophila S2 cells if v-Src kinase, which phosphorylates Trn, is also expressed. Coexpression of wild-type Ptp52F causes dephosphorylation of v-Src-phosphorylated Trn. To examine the specificity of the interaction in vitro, we incubated Ptp52F-glutathione S-transferase (GST) fusion proteins with pervanadate-treated S2 cell lysates. Wild-type Ptp52F dephosphorylated Trn, as well as most other bands in the lysate. GST “pulldown” experiments demonstrated that the Ptp52F substrate-trapping mutant binds exclusively to phospho-Trn. Wild-type Ptp52F pulled down dephosphorylated Trn, suggesting that it forms a stable Ptp52F-Trn complex that persists after substrate dephosphorylation. To evaluate whether Trn and Ptp52F are part of the same pathway in vivo, we examined motor axon guidance in mutant embryos. trn and Ptp52F mutations produce identical phenotypes affecting the SNa motor nerve. The genes also display dosage-dependent interactions, suggesting that Ptp52F regulates Trn signaling in SNa motor neurons.Receptor-linked protein-tyrosine phosphatases (RPTPs) are enzymes with extracellular (XC) domains, a single transmembrane domain, and one or two cytoplasmic protein tyrosine phosphatase (PTP) homology domains. Many RPTPs have XC sequences that resemble those of cell adhesion molecules (for a review, see reference 33). This sequence organization suggests that RPTPs can couple cell-cell recognition events to dephosphorylation of cytoplasmic substrates. Interestingly, while phosphotyrosine (PY) pathways involved in cell growth and differentiation typically involve receptor tyrosine kinases that bind to growth factors and are regulated by nontransmembrane PTPs, those that control axon guidance often use RPTPs and nontransmembrane TKs. This implies that the cues that affect PY signaling in axonal growth cones may interact with RPTPs rather than with receptor tyrosine kinases (reviewed in reference 14).There are 17 active RPTPs encoded in the human genome, while Drosophila has six. Most of the mammalian RPTPs are expressed in nonneural tissues, but four of the six fly RPTPs are expressed only by central nervous system (CNS) neurons in late embryos. All published zygotic phenotypes produced by Rptp mutations are alterations in axon guidance or synaptogenesis. These results suggest that the major functions of the Drosophila RPTPs are in neural development (for a review, see reference 16). Analysis of axon guidance phenotypes in embryos bearing single or multiple Rptp mutations is consistent with the idea that RPTP interactions with ligands at growth cone choice points convey “information,” in the form of changes in substrate phosphorylation within growth cones, that is used to determine pathway decisions.In the Drosophila neuromuscular system, 36 motor axons grow out within six nerve bundles in each abdominal hemisegment, and each axonal growth cone makes a series of genetically determined guidance decisions that direct it to the appropriate muscle fiber (for a review, see reference 27). Our work on Rptp mutant combinations suggests that each pathway decision uses a specific subset of the six RPTPs. RPTPs can exhibit functional redundancy, so that the loss of one does not produce a defect unless another RPTP is also absent, or competition, in which loss of one RPTP suppresses the phenotype produced by loss of another (5, 6, 31). Examination of RPTP expression patterns suggests that the RPTPs are expressed by most (or possibly all) CNS neurons, including motor neurons. If so, the requirements for individual RPTPs for execution of particular guidance decisions cannot be due to selective expression of these RPTPs on specific motor axons. These requirements might instead be determined by the expression patterns of RPTP ligands, so that only RPTPs whose ligands were localized to the vicinity of a growth cone choice point would participate in that pathway decision. Alternatively (or in addition), the necessity of a particular RPTP for a pathway decision might arise from selective expression of RPTP substrates, so that an RPTP would be important for guidance decisions made by a growth cone of a specific motor neuron only if that neuron expressed the relevant substrate(s).Evaluation of such models requires identification of specific XC ligands and intracellular substrates for the Drosophila RPTPs. Only one set of ligands has been identified thus far. These are the heparan sulfate proteoglycans Syndecan (Sdc) and Dallylike (Dlp), which bind to the Lar RPTP with nanomolar affinity and contribute to its functions in axon guidance and synapse growth (9, 15). Similarly, little is known about substrate specificity in vivo. Lar can dephosphorylate the Enabled (Ena) protein, which regulates the growth cone cytoskeleton, and genetic interaction studies suggest that Ena may be an in vivo substrate for Lar (35). The transmembrane protein gp150 can be dephosphorylated by Ptp10D in cell culture and intact fly larvae, but genetics has not provided evidence that Ptp10D and gp150 are in the same signaling pathway in vivo (7).The identification of in vivo substrates for RPTPs has been hampered by the fact that purified RPTP cytoplasmic domains often do not exhibit high selectivity in vitro when tested for dephosphorylation activity on peptides or proteins. The most fruitful method for finding substrates for both RPTPs and cytoplasmic PTPs has been the use of “substrate-trapping” mutants. The most effective substrate traps were devised by Tonks and coworkers, and are created by changing an invariant Asp (D) residue within the PTP active site to Ala (A) (8). The D residue has an abnormal pK and is thus able to donate a proton to the phosphorus-oxygen bond, facilitating displacement of the tyrosine (Y) OH by the invariant Cys (C) nucleophile of the enzyme. This creates a phosphoenzyme intermediate. The dephosphorylated substrate then dissociates, and water attacks the Cys-phosphate bond, releasing the phosphate and reconstituting the enzyme. In D→A mutants, the polarization of the phosphorus-oxygen bond by protonation cannot take place, and the PY substrate remains bound to the enzyme. Substrate-trapping mutants expressed in cells often bind to only a few phosphoproteins, suggesting that PTPs exhibit high specificity in vivo (see, for example, reference 11).We conducted a modified yeast two-hybrid screen to find Drosophila phosphoproteins that bind selectively to RPTP substrate-trapping mutants. We identified the cell surface receptor Tartan (Trn) in this screen and showed that it is a substrate for the Ptp52F RPTP in Drosophila Schneider 2 (S2) cells. Axon guidance phenotypes in trn mutants are identical to those seen in Ptp52F mutants, and trn and Ptp52F exhibit dosage-dependent genetic interactions. These results suggest that Ptp52F is a regulator of Trn signaling in motor neurons in vivo.     

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. We show that the Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function.     

Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system

Receptor protein tyrosine phosphatases (RPTPs), are involved in axon outgrowth and guidance not only in the Drosophila visual system (Garrity et al., 1999. Neuron 22, 707-717) but also in the developing vertebrate retina (Ledig et al., 1999a. J. Cell Biol. 147, 375-388). We have cloned a variety of Xenopus RPTPs, including four RPTPs expressed in the developing visual system (LAR, PTP-delta, CRYP-alpha and PTP-rho). These four RPTPs are transcribed in the developing optic vesicle during differentiation and in overlapping but distinct patterns in the developing retina during retinal layer formation. LAR, PTP-delta, and CRYP-alpha are also expressed in retinal ganglion cells during axonogenesis and during axon guidance from the retina to the optic tectum.     

Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F.

Receptor-linked protein tyrosine phosphatases (RPTPs) regulate axon guidance and synaptogenesis in Drosophila embryos and larvae. We describe DPTP52F, the sixth RPTP to be discovered in Drosophila. Our genomic analysis indicates that there are likely to be no additional RPTPs encoded in the fly genome. Five of the six Drosophila RPTPs have C. elegans counterparts, and three of the six are also orthologous to human RPTP subfamilies. DPTP52F, however, has no clear orthologs in other organisms. The DPTP52F extracellular domain contains five fibronectin type III repeats and it has a single phosphatase domain. DPTP52F is selectively expressed in the CNS of late embryos, as are DPTP10D, DLAR, DPTP69D and DPTP99A. To define developmental roles of DPTP52F, we used RNA interference (RNAi)-induced phenotypes as a guide to identify Ptp52F alleles among a collection of EMS-induced lethal mutations. Ptp52F single mutant embryos have axon guidance phenotypes that affect CNS longitudinal tracts. This phenotype is suppressed in Dlar Ptp52F double mutants, indicating that DPTP52F and DLAR interact competitively in regulating CNS axon guidance decisions. Ptp52F single mutations also cause motor axon phenotypes that selectively affect the SNa nerve. DPTP52F, DPTP10D and DPTP69D have partially redundant roles in regulation of guidance decisions made by axons within the ISN and ISNb motor nerves.     

Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance

Axon guidance receptors guide neuronal growth cones by binding in trans to axon guidance ligands in the developing nervous system. Some ligands are coexpressed in cis with their receptors, raising the question of the relative contribution of cis and trans interactions to axon guidance. Spinal motor axons use Eph receptors to select a limb trajectory in response to trans ephrins, while expressing ephrins in cis. We show that changes in motor neuron ephrin expression result in trajectory selection defects mirrored by changes in growth cone sensitivity to ephrins in vitro, arguing for ephrin cis-attenuation of Eph function. Furthermore, the relative contribution of trans-signaling and cis-attenuation is influenced by the subcellular distribution of ephrins to membrane patches containing Eph receptors. Thus, growth cone ephrins are essential for axon guidance in vivo and the balance between cis and trans modes of axon guidance ligand-receptor interaction contributes to the diversity of axon guidance signaling responses.     

The metalloprotease tolloid-related and its TGF-beta-like substrate Dawdle regulate Drosophila motoneuron axon guidance

Proper axon pathfinding requires that growth cones execute appropriate turns and branching at particular choice points en route to their synaptic targets. Here we demonstrate that the Drosophila metalloprotease tolloid-related (tlr) is required for proper fasciculation/defasciculation of motor axons in the CNS and for normal guidance of many motor axons enroute to their muscle targets. Tlr belongs to a family of developmentally important proteases that process various extracellular matrix components, as well as several TGF-beta inhibitory proteins and pro-peptides. We show that Tlr is a circulating enzyme that processes the pro-domains of three Drosophila TGF-beta-type ligands, and, in the case of the Activin-like protein Dawdle (Daw), this processing enhances the signaling activity of the ligand in vitro and in vivo. Null mutants of daw, as well as mutations in its receptor babo and its downstream mediator Smad2, all exhibit axon guidance defects that are similar to but less severe than tlr. We suggest that by activating Daw and perhaps other TGF-beta ligands, Tlr provides a permissive signal for axon guidance.     

Dimerization of protein tyrosine phosphatase sigma governs both ligand binding and isoform specificity

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase sigma (PTPsigma) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTPsigma forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTPsigma ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTPsigma can alter the ligand binding specificities of PTPsigma ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.     

The Extracellular and Cytoplasmic Domains of Syndecan Cooperate Postsynaptically to Promote Synapse Growth at the Drosophila Neuromuscular Junction

The heparan sulfate proteoglycan (HSPG) Syndecan (Sdc) is a crucial regulator of synapse development and growth in both vertebrates and invertebrates. In Drosophila, Sdc binds via its extracellular heparan sulfate (HS) sidechains to the receptor protein tyrosine phosphatase LAR to promote the morphological growth of the neuromuscular junction (NMJ). To date, however, little else is known about the molecular mechanisms by which Sdc functions to promote synapse growth. Here we show that all detectable Sdc found at the NMJ is provided by the muscle, strongly suggesting a post-synaptic role for Sdc. We also show that both the cytoplasmic and extracellular domains of Sdc are required to promote synapse growth or to rescue Sdc loss of function. We report the results of a yeast two-hybrid screen using the cytoplasmic domains of Sdc as bait, and identify several novel candidate binding partners for the cytoplasmic domains of Sdc. Together, these studies provide new insight into the mechanism of Sdc function at the NMJ, and provide enticing future directions for further exploring how Sdc promotes synapse growth.     

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. We demonstrate here that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies revealed that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also showed axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes were able to rescue some sdc visual-system phenotypes, but sdc+ transgenes were ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential.     

Syndecan contributes to heart cell specification and lumen formation during Drosophila cardiogenesis

The transmembrane proteoglycan Syndecan contributes to cell surface signaling of diverse ligands in mammals, yet in Drosophila, genetic evidence links Syndecan only to the Slit receptor Roundabout and to the receptor tyrosine phosphatase LAR. Here we characterize the requirement for syndecan in the determination and morphogenesis of the Drosophila heart, and reveal two phases of activity, indicating that Syndecan is a co-factor in at least two signaling events in this tissue. There is a stochastic failure to determine heart cell progenitors in a subset of abdominal hemisegments in embryos mutant for syndecan, and subsequent to Syndecan depletion by RNA interference. This phenotype is sensitive to gene dosage in the FGF receptor (Heartless), its ligand, Pyramus, as well as BMP-ligand Decapentaplegic (Dpp) and co-factor Sara. Syndecan is also required for lumen formation during assembly of the heart vessel, a phenotype shared with mutations in the Slit and Integrin signaling pathways. Phenotypic interactions of syndecan with slit and Integrin mutants suggest intersecting function, consistent with Syndecan acting as a co-receptor for Slit in the Drosophila heart.     

Drosophila Plexin B is a Sema-2a receptor required for axon guidance

Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how PlexB functions during neural development and which ligands serve to activate this receptor. Here, we show that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. We observe a physical association between these two plexin receptors in vivo and find that they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema-2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance.     

Notch steers Drosophila ISNb motor axons by regulating the Abl signaling pathway

The central problem in axon guidance is to understand how guidance signals interact to determine where an axon will grow. Here we investigate a specific axon guidance decision in Drosophila embryos, the sharp inward turn taken by the ISNb motor nerve to approach its muscle targets. We find that this turn requires Notch and its ligand Delta. We show that Delta is expressed on cells adjacent to the ISNb turning point, and we know from previous work that Notch is present on axonal growth cones, suggesting that Delta and Notch might provide a guidance signal to ISNb. To induce the turning of ISNb axons, Notch interacts genetically with multiple components of a signal transduction pathway that includes the Abl tyrosine kinase and its affiliated accessory proteins. In contrast, genetic interaction experiments fail to provide evidence for a major role of the "canonical" Notch/Su(H) signaling pathway in this process. We suggest that the Notch/Abl interaction promotes the turning of ISNb axons by attenuating the Abl-dependent adhesion of ISNb axons to their substratum, thus releasing the axons to respond to attraction from target muscles.     

Motoneuronal Sema3C is essential for setting stereotyped motor tract positioning in limb-derived chemotropic semaphorins

The wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. To overcome the limited number of guidance cues, modulation of axon responsiveness is crucial for specifying accurate trajectories. We report here a novel mechanism by which ligand/receptor co-expression in neurons modulates the integration of other guidance cues by the growth cone. Class 3 semaphorins (Sema3 semaphorins) are chemotropic guidance cues for various neuronal projections, among which are spinal motor axons navigating towards their peripheral target muscles. Intriguingly, Sema3 proteins are dynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed in most of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selective manipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilin receptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenous Nrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where this modulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. We show first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration of motoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limb-derived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.     

Ephrin-As play a rhombomere-specific role in trigeminal motor axon projections in the chick embryo

In this study, we investigate the possible role of ephrin-Eph signaling in trigeminal motor axon projections. We find that EphA receptors are expressed at higher levels by rhombomere 2 (r2) trigeminal motor neurons than by r3 trigeminal motor neurons in the chick embryo. Mapping of rhombomere-specific axon projections shows that r2 and r3 trigeminal motor neurons project to different muscle targets, including the mandibular adductor and the intermandibularis muscles respectively. Ephrin-A5 is expressed in these muscles, especially in some regions of the intermandibularis muscle, and can cause growth cone collapse of both r2 and r3 motor axons in vitro. We demonstrate that in vivo overexpression of ephrin-A5 in the intermandibularis muscle, or overexpression of dominant-negative EphA receptors in trigeminal motor neurons leads to a reduction in branching of r3-derived motor axons specifically. Overexpression of full-length EphA receptors impairs the formation of r3 projections to the intermandibularis muscle. These findings indicate that ephrins and their Eph receptors play a role in trigeminal motor axon topographic mapping and in rhombomere 3-derived projections in particular.     

Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion.     

EphA3 null mutants do not demonstrate motor axon guidance defects

Motor axon projections are topographically ordered. Medial motor column axons project to axial muscles, whereas lateral motor column axons project to limb muscles and, along the rostrocaudal axis of the animal, the more rostral motor neuron pools project to more rostral muscle targets. We have shown that EphA3 is specifically expressed in the developing medial motor column and have postulated that EphA3 might be responsible for directing their axons to axial muscle targets. This hypothesis was supported by our demonstration that EphA3 can direct retinal ganglion cell axon targeting and by studies of ephrin-A5(-/-) mutants that show that EphA receptor signaling controls the topographic innervation of the acromiotrapezius. To test the role of EphA3 in motor axon guidance, we generated an EphA3 null mutant. Retrograde labeling studies in EphA3(-/-) embryos and adults indicate that, contrary to our predictions, EphA3 is not necessary to direct motor axons to axial muscle targets. Our results also demonstrate that ephrin A5's ability to direct topographic innervation of the acromiotrapezius must be mediated through EphA receptors other than, or in addition to, EphA3.     

The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance

Genetic analysis of growth cone guidance choice points in Drosophila identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. We now demonstrate that the Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. We also show that the Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton.