BIOLOGICAL OVERVIEW

Different classes of Drosophila photoreceptor neurons (R cells) in the Drosophila compound eye form layer-specific connections in the optic lobe, the highly structured portion of the brain dedicated to integration of visual information from the retina. R1-R6 axons (axons generated by photoreceptors 1-6) project to the lamina, the outer layer of the optic lobe; R7 and R8 axons project to separate layers of the medulla. A receptor tyrosine phosphatase (RPTP) known as Protein tyrosine phosphatase 69D (Ptp69D) is required for lamina target specificity. In Ptp69D mutants, R1-R6 axons project inappropriately through the lamina and terminate in the medulla (Garrity, 1999).

The formation of connections by R cell axons involves a complex dialog between R cell afferents and their targets in the developing optic lobe (see Development of the lamina visual center of the brain. R cell afferents induce the production and differentiation of lamina neurons as well as the migration and differentiation of lamina glia (Huang, 1998). The dependence of lamina differentiation on R cell-derived signals plays a crucial role in matching the number of afferents to their targets. R cell axons from each facet in the eye disc form a compact, highly ordered fascicle with the R1-R7 axons surrounding the R8 axon (Meinertzhagen, 1993). Axons from each bundle appear to enter the optic lobe in a sequential fashion: R8 enters first, followed by R1-R6, and then R7. Since target specificity does not simply reflect the order of axon ingrowth, target selection likely reflects molecular recognition between R cell axons and determinants in the developing optic lobe. Based on histological studies, it has been proposed that the initial targeting to the lamina is a consequence of interactions between R1-R6 growth cones and lamina glia (Perez, 1996). R1-R6 growth cones establish synaptic connections with lamina neurons during pupal development, some 4-5 days after projecting into the lamina. This involves a complex series of steps, since R1-R6 growth cones undergo stereotyped rearrangements to establish the final patterns of their connections. While inductive signals produced by R cells have been identified, the targeting signals in the optic lobe and their receptors on R cell growth cones remain unknown (Garrity, 1999).

R1-R6 axon targeting errors occur in the dreadlocks (dock) mutant (Garrity, 1996). Since dock encodes an SH3/SH2 adapter protein, the possibility that phosphotyrosine signaling plays a crucial role in lamina targeting was pursued. A collection of mutations in genes that were previously implicated in growth cone function in the embryo and that encode proteins involved in phosphotyrosine signaling were examined for R1-R6 axon targeting defects. Ptp69D was identified in this survey. Genetic and biochemical studies support a model in which Ptp69D functions in R1-R6 growth cones to detect an extracellular signal and translate it into changes in growth cone motility via the dephosphorylation of specific substrates. In Ptp69D mutants, R1-R6 growth cones project through the lamina, terminating in the medulla. Genetic mosaics, transgene rescue, and immunolocalization indicate Ptp69D functions in the photoreceptor R1-R6 growth cones. Ptp69D overexpression in R7 and R8 growth cones does not respecify their connections, suggesting Ptp69D acts in combination with other factors to determine target specificity. Structure-function analysis indicates the extracellular fibronectin type III domains and intracellular phosphatase activity are required for targeting (Garrity, 1999).

A combination of genetic mosaic analysis and cell-specific transgene rescue experiments tested whether PTP69D is required in R cell afferents or their targets. In the genetic mosaic analysis, targeting of Ptp69D mutant axons innervating normal (i.e., Ptp69D;+) brains was examined. Mutant patches of retinal tissue were created by X-ray-induced mitotic recombination, and R1-R6 axon targeting was assessed in the adult using the R1-R6-specific marker, Rhodopsin1-LacZ. In wild-type and Ptp69D heterozygous adults, all R1-R6 axons terminate in the lamina. In contrast, R1-R6 axons from all Ptp69D mutant eye patches analyzed projected through the lamina into the medulla. To exclude the possibility that Ptp69D disrupts targeting indirectly by regulating cell fate and patterning in the eye, sections of homozygous mutant eye patches were examined. Eye patterning, R cell fate determination, and cellular morphogenesis are largely normal in Ptp69D mutants. Of 204 ommatidia scored from 21 eyes, only 3 ommatidia lacked a single R1-R6 neuron. Similar defects have been observed in other R cell axon guidance mutants and may reflect a weak requirement for normal innervation for R cell survival. Furthermore, as assessed by rhabdomere morphology, no transformations of R1-R6 neurons into R7 or R8 neurons were observed. Hence, targeting errors in Ptp69D do not result from more general defects in R cell development (Garrity, 1999).

R1-R6 axons from small mutant patches exhibit R7- and R8-like axon trajectories into topographically appropriate positions in the medulla. R1-R6 axons from larger mutant patches lead to massive hyperinnervation of the medulla, disrupting its structure. Thus, Ptp69D is required in the eye for R1-R6 growth cones to terminate in the appropriate layer in the optic lobe. Furthermore, this defect is not corrected later in development, since R1-R6 axons persist in the medulla neuropil into the adult. To assess whether Ptp69D is required only in the retina, and not in both the retina and optic lobe, a test was performed to see whether expressing Ptp69D specifically in the retina rescues the targeting defect. A Ptp69D cDNA under the control of the eye-specific promoter GMR was introduced into a Ptp69D mutant background. Eye-specific expression of Ptp69D fully rescues the R1-R6 axon targeting defect. Since GMR drives expression in both neuronal and nonneuronal cells, it remains formally possible that Ptp69D is required in nonneuronal retinal cells. However, expression of Ptp69D via the neuron-specific Elav promoter rescues the R1-R6 axon targeting defect, thereby eliminating this possibility. Taken together, these results demonstrate that Ptp69D functions in R cells (Garrity, 1999).

The requirement of Ptp69D in R cell afferents is consistent with Ptp69D detecting specific targeting signals in the lamina. Alternatively, Ptp69D may be required in R cell growth cones to induce lamina target development and only indirectly disrupt R1-R6 targeting. To distinguish between these possibilities, lamina development was assessed using specific markers. Two signals released from R cell growth cones act sequentially to induce lamina precursor cells (LPCs) to produce lamina neurons (Huang, 1998). Hedgehog drives G1-arrested LPCs into S phase of their final division. The epidermal growth factor receptor ligand Spitz then induces lamina neuron differentiation. In Ptp69D mutants, LPC proliferation and lamina neuron differentiation are normal, as assessed using anti-BrdU, anti-Dachshund, and anti-Elav staining. The organization of lamina neurons into columns is also largely normal. Thus, Ptp69D is not required for lamina neuron induction. R cell growth cones terminate between two rows of glial cells. These cells depend on signals from R cell axons to migrate and to differentiate (Winberg, 1992, Perez, 1996). Glial differentiation was assessed using an antibody recognizing Repo, a glial-specific nuclear protein. In Ptp69D, as in wild type, glia migrate into the lamina and expressed Repo. In contrast to wild type, however, they formed irregular rows. It seems most likely that disruption in glial layering is a consequence of the failure of R1-R6 growth cones to terminate in the lamina, giving rise to an uneven lamina plexus of varying thickness separating glial layers. Thus, Ptp69D is probably not required for the differentiation of lamina glia (Garrity, 1999).

To assess whether Ptp69D could act as a receptor in R1-R6 growth cones to control targeting directly, its distribution was examined more precisely. Double-staining experiments using mAb24B10 and a nuclear glial marker show the dense layer of R cell growth cones between the epithelial and marginal glial cells. Single-cell labeling experiments confirm that R1-R6 growth cones terminate in this region. Electron microscopy reveals that at this stage in development, expanded R cell growth cones and their filopodia contribute the vast majority of processes to the lamina plexus and are in close juxtaposition to the epithelial and marginal glial cells. Ptp69D protein distribution within the lamina was assessed in preparations double stained with both anti-Ptp69D antibody and a glial marker. Anti-Ptp69D staining is observed in the lamina plexus between the epithelial and marginal glial cells. Staining is observed at the anterior edge of the lamina plexus where newly arriving R1-R6 axons terminate. Thus, Ptp69D protein colocalizes with R1-R6 growth cones in the lamina, indicating that it is expressed at the appropriate time and place to play a direct role in determining R1-R6 target specificity. Ptp69D immunoreactivity is also detected in the medulla neuropil. Since medulla neurons projecting into this neuropil express Ptp69D at very high levels, it could not be determined whether the R7 and R8 axons in the medulla also have Ptp69D on their surfaces (Garrity, 1999).

Mutations in Ptp69D cause different types of guidance defects in different neurons. For instance, while Ptp69D is necessary for R1-R6 neurons to stop in the lamina, it is required with other RPTPs for motoneuron growth cones within the intersegmental nerve to advance beyond specific landmarks toward their targets (Desai, 1997). Similarly, Ptp69D mutants exhibit diverse axon bundling defects. In double mutants of Ptp69D and the related RPTPs Dlar or Ptp99A, motor axons within the SNb fail to defasciculate upon entering their target region (Desai, 1997). Conversely, the normally fasciculated axons within Bolwig's nerve defasciculate in Ptp69D mutants. The fact that specific guidance molecules may function differently in different contexts is emerging as a common theme in neurodevelopment. Indeed, the netrin receptors (i.e., Dcc, Unc40) are required for both chemorepellent and chemoattractant function, as well as for fasciculation of specific axon bundles. Hence, the same protein can subserve different functions in the context of different combinations of other guidance molecules. Combinatorial mechanisms provide the flexibility and complexity of interactions needed to establish large networks of connections between neurons with a limited number of signaling components. Future progress in dissecting the role of Ptp69D in R1-R6 targeting will require identification of extracellular ligands and phosphatase substrates (Garrity, 1999 and references).

As in the fly visual system, the projection of neurons to specific layers of the brain is a prominent feature of neuronal organization in vertebrates. Studies in the cerebral cortex and the optic tectum argue for the importance of molecular targeting cues in establishing layer-specific connections. As in flies, cell adhesion molecule-like RPTPs are expressed on axons and growth cones in the developing vertebrate CNS. Given the conservation in function of other developmentally important molecules, it is tempting to speculate that CAM-like RPTPs will play a role in regulating layer specificity in the vertebrate brain (Garrity, 1999 and references).

Receptor tyrosine phosphatases regulate birth order-dependent axonal fasciculation and midline repulsion during development of the Drosophila mushroom body

Receptor tyrosine phosphatases (RPTPs) are required for axon guidance during embryonic development in Drosophila. This study examined the roles of four RPTPs during development of the larval mushroom body (MB). MB neurons extend axons into parallel tracts known as the peduncle and lobes. The temporal order of neuronal birth is reflected in the organization of axons within these tracts. Axons of the youngest neurons, known as core fibers, extend within a single bundle at the center, while those of older neurons fill the outer layers. RPTPs are selectively expressed on the core fibers of the MB. Ptp10D and Ptp69D regulate segregation of the young axons into a single core bundle. Ptp69D signaling is required for axonal extension beyond the peduncle. Lar and Ptp69D are necessary for the axonal branching decisions that create the lobes. Avoidance of the brain midline by extending medial lobe axons involves signaling through Lar (Kurusu, 2008).

The mushroom bodies (MBs) are highly conserved paired structures in the insect brain that are essential for olfactory learning and other higher-order functions. MBs vary greatly in size between insect species, but their overall organization is very similar in all insects. In Drosophila, an adult MB contains about 2500 principal neurons, known as Kenyon cells. All Kenyon cells are generated from four neuroblasts (NBs) in each brain hemisphere. Each NB produces three types of Kenyon cells (γ, α′/β′, and α/β) in a strict temporal order, and the four lineages are indistinguishable. An MB is thus a fourfold-symmetric structure. γ neurons are generated by the mid-third instar stage, α′/β′ neurons between mid-third instar and puparium formation, and α/β neurons after puparium formation (Kurusu, 2008).

Kenyon cell dendrites extend into a glomerular structure called the calyx, which receives olfactory input from the projection neurons of the antennal lobe (AL). The axons of Kenyon cells from each lineage group fasciculate into a bundle, and the four bundles merge to form the peduncle, a massive parallel tract that extends ventrally and then splits into two branches, each composed of intertwined lobes (Kurusu, 2008).

The three types of Kenyon cells differ with respect to their dendritic and axonal projection patterns in the calyx and lobes. These anatomical subdivisions are likely to be important for the analysis of complex sensory inputs, because γ, α′/β′, and α/β neurons receive projections from different sets of AL glomeruli (Kurusu, 2008).

In larvae, the axonal structures formed by the splitting of the peduncle are called the dorsal and medial lobes. Every Kenyon cell axon bifurcates during outgrowth and sends a branch into each lobe. The γ axons extend first and may serve as pathways to guide the axons of later-born α′/β′ neurons in the larva. α/β neurons follow similar trajectories during the pupal phase. The distal portions of the γ axons degenerate during pupal development, and their medial branches later regrow to form the adult γ lobe. The adult MB has a dorsal branch composed of the intertwined α and α′ lobes, and a medial branch containing the β, β′, and γ lobes. The lobes within the medial branch extend toward the corresponding lobes of the MB in the other hemisphere, but stop at the edge of a midline region that is devoid of MB axons (Kurusu, 2008).

The temporal order of Kenyon cell birth is reflected in the organization of axons in the peduncle and lobes, with the axons of the youngest neurons (the core fibers) in the center and those of older neurons arranged as a series of concentric rings around the core. The core fibers and the layers formed by older axons can be distinguished using antibody markers, GAL4 drivers, and staining for polymerized actin using phalloidin. The actin-rich core fibers are a transient population, because the ingrowth of new axons from sequentially generated later-born neurons occurs successively at the center and displaces the old core fibers outward into the innermost ring. Axons lose bright phalloidin staining as they move outward within the peduncle. This organization is consistent with the idea that the core fibers form a pioneer pathway used to guide extension of the axons of later-born neurons, which then in turn become the new core and are used as pathways by still later axons. To understand axon guidance and lobe morphogenesis during MB development, it is thus valuable to define and genetically characterize receptors and adhesion molecules expressed on these core fibers (Kurusu, 2008).

This study shows that four receptor tyrosine phosphatases (RPTPs) are selectively expressed on core fibers within the peduncle and lobes, and that RPTP signaling regulates segregation of core fibers into a single bundle. RPTPs have cellular adhesion molecule (CAM)-like extracellular regions containing immunoglobulin (Ig) domains and/or fibronectin type III (FN3) repeats. The ligands that interact with these extracellular domains are largely unknown. However, vertebrate and Drosophila Lar RPTPs bind to heparan sulfate proteoglycans (HSPGs), and the cell-surface HSPG Syndecan (Sdc) contributes to Lar's functions during axon guidance and synaptogenesis (Kurusu, 2008).

The four RPTPs localized to core fibers are Lar, Ptp10D, Ptp69D, and Ptp99A. They are all selectively expressed on central nervous system (CNS) axons in the embryo. There are a total of six RPTPs encoded in the Drosophila genome. The other two RPTPs, Ptp52F and Ptp4E, were not studied because Ptp52F mutants die as embryos and Ptp4E mutants have not been characterized. Also, good antibodies against these two proteins are not available (Kurusu, 2008).

RPTPs have been extensively analyzed as regulators of motor axon guidance in the embryonic neuromuscular system. Each guidance decision made by motor axons can be defined by a requirement for a specific subset of the five RPTPs that have been examined. There is substantial redundancy among RPTPs, so that high-penetrance alterations in a guidance decision are usually observed only when two or more RPTPs are removed. For example, axons of the ISNb nerve fail to leave the common ISN pathway and thus remain fasciculated to ISN axons ('fusion bypass' phenotype) when Lar, Ptp69D, and Ptp99A are all absent. When only Lar is missing, ISNb axons leave the ISN but then sometimes fail to enter their target muscle field (Kurusu, 2008).

RPTPs also regulate CNS axon guidance in a redundant manner. A subset of longitudinal axons abnormally cross the midline in Ptp10D Ptp69D double mutants, while all longitudinal axons are rerouted into midline-crossing commissural pathways in Lar Ptp10D Ptp69D Ptp99A quadruple mutants. All four RPTPs thus participate in midline crossing decisions in the embryo. This paper shows that Lar regulates avoidance of the brain midline by MB axons in the larval brain, and that high-penetrance ectopic crossing phenotypes are observed when only Lar is absent (Kurusu, 2008).

RPTP function has also been characterized during later development and in adulthood. Lar mutants have reduced numbers of boutons at neuromuscular junctions (NMJs) in larvae, and Lar and Ptp69D mutants display alterations in photoreceptor axon guidance and synaptic maintenance in the optic lobes. Finally, hypomorphic Ptp10D mutants are defective in long-term memory formation, and this phenotype can be rescued by restoration of Ptp10D expression in the MB or by acute induction of Ptp10D in adults. This result indicates that RPTPs are likely to regulate synaptic function in mature animals. Consistent with this, this study shows that all four RPTPs are expressed in specific patterns within the neuropil of the adult brain (Kurusu, 2008).

This paper demonstrates that RPTPs are selectively expressed on the core fibers of the MB. At any one time during larval development, the core fiber bundle is composed of the youngest axons within the peduncle and lobes. Core fibers are likely to serve as pathways for guidance of axons of later-born neurons. These later axons then become the new core fiber bundle and displace the old core fibers outward into concentric rings (Kurusu, 2008).

Two of the RPTPs, Ptp69D and Ptp10D, are expressed only on the innermost core fibers, while Lar and Ptp99A are expressed within an expanded core region including somewhat older axons. The data indicate that Ptp10D and Ptp69D are involved in segregation of core fibers. Split-core phenotypes, in which two or more phalloidin-rich, OK107::mCD8-GFP-low bundles are observed within the peduncle, are observed in MBs lacking Ptp69D function. They were also observed in larvae hemizygous for a complete deletion of Ptp10D and the adjacent bif gene, Δ59. The split-core phenotype occurs only when both Ptp10D and Bif are absent. These two genes have been shown to genetically interact in other contexts (Kurusu, 2008).

Ptp69D is also required for outgrowth of axons from the peduncle into the lobes, because regions near the junctions of the peduncle and the lobes are expanded at the expense of the lobes in MBs lacking Ptp69D function. Another distinctive phenotype seen in these experiments is abnormal extension of medial lobe axons across the brain midline. This is seen in Lar mutant larvae and in larval and adult Lar mutant NB clones (Kurusu, 2008).

Prior to this work, only two genes had been identified as having mutant phenotypes affecting core fiber segregation. Both of these encode adhesion molecules. mRNA from the Dscam gene, which encodes homophilic Ig domain CAMs, is alternatively spliced to generate up to 38,000 different protein isoforms. Like the RPTPs, Dscam is selectively expressed on young axons that travel within the core fiber bundle. Dscam mutant MBs and NB clones exhibited multiple core fiber bundles within the peduncle. The similarities between Dscam, Ptp69D, and Ptp10D phenotypes and expression patterns suggest that these RPTPs could be involved in Dscam signaling during outgrowth of young axons within the peduncle. Alternatively, the RPTPs could regulate adhesion via other pathways that also affect core fiber segregation and are partially redundant with Dscam (Kurusu, 2008).

Core fiber segregation defects were also observed in ~25% of NB clones bearing null mutations eliminating expression of the homophilic Ig-domain CAM FasII. FasII, unlike Dscam and the RPTPs, is expressed only on older axons outside the core region, and engagement between FasII molecules on different cell surfaces is thought to trigger adhesion rather than repulsion. This suggests that core fiber segregation defects in FasII mutants arise by a different mechanism. Perhaps older axons lacking FasII fail to adhere sufficiently to each other and thus open passageways that allow young axons to pioneer multiple pathways within the peduncle (Kurusu, 2008).

Mutants lacking expression of Lar exhibit phenotypes in which one or both of the medial lobes of the bilaterally symmetric MBs extend across the midline, so that they appear fused to one another. It was also found that Lar mutant NB clones extended axons across the midline into the territory occupied by the other MB. These phenotypes suggest that a repulsive signal emanates from the brain midline region, and that Lar participates in reception of this signal by growing MB axons within the medial lobe (Kurusu, 2008).

This is formally similar to repulsion from the CNS midline in the embryo, where binding of midline Slit to Roundabout (Robo) receptors on neuronal growth cones causes them to navigate away from the midline. Axons abnormally cross the midline in Ptp10D Ptp69D double mutant embryos, and genetic interaction studies showed that these RPTPs are positive regulators of Robo signaling in the embryo. Lar is also implicated in the decisions of axons to cross the embryonic midline (Kurusu, 2008).

The MB midline crossing phenotypes seen in Lar mutants are probably not mediated through alterations in Slit-Robo signaling. Robo1, 2, and 3 mutant phenotypes in the larval MB have been described, and they do not include fusion of medial lobes across the midline. The Lar ligand Sdc is not selectively expressed at the brain midline, so binding of Lar to Sdc probably does not trigger repulsion of MB axons. Perhaps other, as yet unidentified, Lar ligands are expressed in the midline region, and interactions between these ligands and the RPTP facilitates repulsion. Studies showed that Lar binds to at least one non-HSPG ligand in the embryo (Kurusu, 2008).

A number of other genes that regulate repulsion of MB axons from the midline have been identified in other studies. Mutations in derailed (linotte), which encodes a protein related to Ryk receptor tyrosine kinases, cause overgrowth of medial lobes across the midline. The Derailed receptor responds to a Wnt5 signal, and mediates exclusion of axons from posterior commissural pathways that cross the embryonic midline. Medial lobe fusion across the midline is also observed in fmr1 mutants and in FMR1 overexpression animals. FMR1 is an RNA-binding protein that is orthologous to the human gene affected in Fragile X mental retardation syndrome (Kurusu, 2008).

Other genes potentially involved in repulsion from the brain midline were identified in a microarray screen for MB-expressed genes. ML fusion was observed in some larvae when expression of these genes was inhibited using RNAi methods. The gene with a known function for which RNAi produced ML fusion with the highest penetrance is CG6083, encoding an aldehyde reductase. RNAi for a gene encoding a cGMP phosphodiesterase also produced ML fusion. cGMP signaling has been implicated in growth cone repulsion in vertebrate neuronal cultures (Kurusu, 2008).

The work described in this paper shows that four RPTPs are localized to growing MB axons and are important for the creation of the distinctive architecture of the MB's axonal network. Examination of Rptp mutant phenotypes shows that these CAM-like signaling molecules control several different axon guidance decisions that occur during outgrowth. Ptp10D and Ptp69D regulate segregation of the growing axons into a single core bundle within the peduncle. Ptp69D and Lar are necessary for the later axonal extension and branching events that create the dorsal and medial lobes. Medial lobe axons cease outgrowth before they reach the brain midline, and their decision to stop involves Lar (Kurusu, 2008).

PROTEIN STRUCTURE

Amino Acids - 1997

Structural Domains

Ptp69D is one of three Drosophila receptor protein tyrosine phosphatases (RPTPs) that are known to play important roles in motor axon guidance (Desai, 1996, Desai, 1997 and Krueger, 1996). These RPTPs have large extracellular domains containing multiple immunoglobulin (Ig) and/or fibronectin type III (FNIII) repeats, reminiscent of cell adhesion molecules (CAMs). Similar RPTPs are expressed on vertebrate growth cones (Stoker, 1995). The cytoplasmic tails of most RPTPs contain two tandemly arranged protein tyrosine phosphatase (PTP) catalytic domains. The membrane-proximal PTP domain (PTP1) provides most or all of the catalytic activity. The C-terminal PTP domain (PTP2) is proposed to play various regulatory roles such as mediating proper subcellular localization or serving as a binding site for downstream regulatory factors or substrates (Serra-Pages, 1995; Kashio, 1998 and Wallace, 1998). Ptp69D was originally isolated by probing a Drosophila cDNA library for sequences coding for a consensus amino acid sequence VHCSAGV identified as belonging to the PTPase domain (Streuli, 1989). Ptp69D protein has a 23-aa signal peptide, a 782-aa extracellular region, an 18-aa transmembrane segment, and a 639-aa cytoplasmic domain (Streuli, 1989)

Ptp69D has a CAM-like extracellular region and an intracellular region encoding two protein tyrosine phosphatase domains, PTP1 and PTP2. To determine which regions contribute to R1-R6 targeting, mutated Ptp69D cDNAs were assessed for their ability to rescue the Ptp69D R1-R6 targeting phenotype. The requirement for PTP catalytic activity was assessed by deletion analysis and by inactivating point mutations. A construct lacking PTP2 [69DDelta(PTP2)] rescues R1-R6 axon targeting defects, demonstrating that this domain is dispensable for R1-R6 targeting. Unlike wild-type Ptp69D, however, 69DDelta(PTP2) driven by Elav-gal4 fails to restore viability beyond the pupal stage, indicating that PTP2 is required for other developmental processes. To assess the role of phosphatase catalytic activity, a point mutation (D->A) was introduced into a conserved aspartate residue within the catalytic site of PTP1. Biochemical and structural studies in other receptor tyrosine phosphatases indicate that this conserved residue is important for phosphatase activity and that an alanine for aspartate substitution inhibits catalysis. Surprisingly, this mutant rescues the R1-R6 targeting phenotype. Thus, either phosphatase activity is not required, or PTP2 provides compensatory activity. To distinguish between these models, the aspartate to alanine mutation was introduced into both PTP domains. This mutant does not rescue R1-R6 targeting. Thus, while the PTP1 domain is sufficient for rescue, both PTP1 and PTP2 can contribute to Ptp69D function. These data indicate that Ptp69D must dephosphorylate one or more substrates in R1-R6 growth cones to promote lamina targeting (Garrity, 1999).

The extracellular domain of Ptp69D is composed of two Ig and three FNIII domains and a membrane-proximal region (MPR) of some 300 amino acids with no detectable homology to other proteins. To determine which regions are critical for function, a series of deletion mutants were tested for their ability to rescue Ptp69D targeting defects. Two mutant proteins containing essentially no extracellular domain (except for the signal sequence and 20 or 40 amino acids of the MPR) did not rescue Ptp69D R1-R6 targeting defects but were expressed at very low levels. Hence, the failure to rescue may reflect the level of expression rather than a requirement for the extracellular domain for Ptp69D function (Garrity, 1999).

Constructs lacking the Ig domains fully rescued the Ptp69D targeting phenotype. While the Ig domains are not necessary for R1-R6 targeting, they are required to restore viability, indicating that they are important in other developmental contexts. Mutants lacking only the FNIII domains, or larger deletions removing the Ig domains or the MPR in addition to the FNIII domain, do not rescue R1-R6 targeting or restore viability. In summary, these data support a model in which the FNIII domains bind targeting determinants in the lamina, translating them into changes in the catalytic activity, specificity, or localization of the tyrosine phosphatase domains (Garrity, 1999).

Slit and Receptor tyrosine phosphatase 69D confer spatial specificity to axon branching via Dscam1

Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. This paper reports that cell-autonomous loss of the receptor tyrosine phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single-growth-cone imaging reveals that Slit/RPTP69D are not required for general branch initiation but instead promote the extension of specific axon collaterals. Hence, although regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth-cone compartments enable the spatial specificity of axon collateral formation (Dascenco, 2015).

This study reports on a molecular mechanism regulating Dscam1 activity in growth cones and provides insight in the regulation and spatial specificity of axon collateral formation. Biochemical and genetic results are consistent with the molecular model that the specificity of mechanosensory (ms)-axon branching arises from a spatially restricted change of Dscam1 phosphorylation in growth cone (Dascenco, 2015).

Previous studies on the function of Dscam1 have established the model that isoform-specific homophilic Dscam1-Dscam1 interactions trigger repulsion between sister dendrites. This controls for regular spacing of sister dendrites in a process termed neurite self-avoidance. In addition, cell-intrinsic and isoform-specific interactions have also been shown to be important in sensory axons for growth-cone sprouting and branching. Importantly, for both of these functions, it is thought that Dscam1 signaling is primarily dependent on and initiated by homophilic binding of matching isoforms present on sister neurites. The results reported in this study provide evidence that Dscam1-Dscam1 interactions in axonal growth cones are subject to branch-specific modulation by extrinsic cues. Binding of the ligand Slit to Dscam1 can locally enhance cis-interactions with the receptor tyrosine phosphatase RPTP69D as well as the dephosphorylation of Dscam1. Although homophilic Dscam1 interactions can be considered to play an initial permissive role in all neurite-neurite interactions in a sprouting growth cone, the spatial restriction of an extrinsic Dscam1 ligand likely initiates functional disparity of Dscam1 signaling across different growth-cone compartments (Dascenco, 2015).

The biochemical data support the notion that RPTP69D directly dephosphorylates Dscam1 at specific cytoplasmic tyrosines. Three candidate tyrosines were identified for the regulation of Dscam1 phosphorylation: Y1857, Y1890, and Y1981. Two of the tyrosine residues, Y1857 and Y1890, are part of consensus SH2-binding sites and therefore are likely involved in regulating recruitment of SH2-domain-containing adaptor molecules. Given that these mutations diminish the Dscam1 GOF effects, it seems reasonable to speculate that they are required for downstream signaling and/or receptor turn-over or trafficking. Surprisingly, the single Y1981F mutation causes strong dominant interference with axon branching where the phenotypic effects are qualitatively indistinguishable from a loss of Dscam1 isoform diversity, which is thought to increase the probability of matching isoform interactions (i.e., GOF activity). The primary amino acid sequence surrounding Y1981 does not reveal any distinct signaling motif. However, in silico 3D protein modeling based on structural predictions suggests that phosphorylation of Y1981 could directly result in structural changes of the Dscam1 cytoplasmic domain and thereby influence Dscam1 activity (Dascenco, 2015).

Biochemical results suggest that Slit can enhance Dscam1-RPTP69D complex formation and Dscam1 dephosphorylation. Furthermore, Slit-N can directly bind to the N-terminal Ig domains of Dscam1 (Ig1-4) with an affinity comparable to that of other guidance cue/receptor interactions, suggesting that Slit-N can function as a bona fide Dscam1 ligand. Numerous studies have shown that the repellent as well as the branch-promoting function of vertebrate Slit require the function of Robo receptors. The current results show that for the formation of specific axon collaterals of Drosophila ms-neurons, Slit functions via Dscam1 in a Robo1-3-independent pathway (Dascenco, 2015).

Slit is one of the best characterized 'axon-repellent' cues and also contributes to axon branching. Imaging single ms-axons and growth-cone branching, this study found that in Slit mutant animals, only filopodia or micropodia with a midline-directed growth direction are reduced, consistent with a positive role of Slit in promoting the extension of specific branches. In contrast, branch-point initiation in ms-neurons is likely independent of Slit or RPTP69D (Dascenco, 2015).

Given that high Slit protein concentrations are likely only encountered by filopodia- or micropodia-like extensions that reach the midline proximity, the Slit-Dscam1-RPTP69D interactions are likely only occurring in a spatially restricted sub-compartment of the branching growth cone. It is envisioned that the Dscam1-RPTP69D interactions in ms-axons constitute a molecular selection process, which depends on Dscam1-RPTP69D complex formation in a subset of axonal processes that encounter sufficient Slit protein. As a result, Dscam1 dephosphorylation by RPTP69D is increased locally and triggers a response by either promoting axon-branch extension or blocking repulsion (Dascenco, 2015).

The loss of only a subset of axon branches in RPTP69D/Slit mutants suggests that there are multiple molecular control pathways accounting for the selection of different axon collaterals or the extension of the main axon shaft. Although this study has focused on RPTP69D and Slit, it is most likely that other co-receptors and extracellular cues control the activity of Dscam1 in growth cones (Dascenco, 2015).

EVOLUTIONARY HOMOLOGS

Please refer to the dlar: Evolutionary Homologs section for information on receptor protein tyrosine phosphatases in other species.

date revised: 22 May 2023

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