Protein tyrosine phosphatase 69D: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Protein tyrosine phosphatase 69D

Synonyms -

Cytological map position - 69D

Function - receptor tyrosine phosphatase

Keywords - axon guidance

Symbol - Ptp69D

FlyBase ID: FBgn0014007

Genetic map position - 3-

Classification - Immunoglobulin-C2-domain,
tyrosine specific protein phosphatase,
type III fibronectin domain protein

Cellular location - surface

NCBI links: Precomputed BLAST | Entrez Gene

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).


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).


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

Protein tyrosine phosphatase 69D: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 21 July 99

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.