Protein tyrosine phosphatase 69D

DEVELOPMENTAL BIOLOGY

Embryonic and Larva

In embryos, the fasciclins are localized to axonal subsets, while the RPTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, a biochemical approach has been taken. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted 'HRP proteins') apparently bear the HRP carbohydrate epitope. Polyclonal anti-HRP antibodies have been used to purify these proteins from Drosophila embryos, and protein sequences have been obtained from seven HRP protein bands. These data define three major HRP proteins as Neurotactin, Fasciclin I, and an RPTP, Ptp69d. Fasciclin II, Neuroglian, Ptp10D, and Ptp99A are also HRP proteins (Desai, 1994).

Ptp69D, like the previously characterized RPTPs, is localized to CNS axons in the embryo. Pdp69D is first present in the germ band extended embryos (stages 9-10). Axonal staining is first observed at the onset of germ band retraction. After this time, staining is primarily localized to CNS axons. In third instar larvae, Ptp69D expression is restricted to subsets of neuronal processes in the brain, ventral nerve cord, and eye disc. In each of the three thoracic ganglia, Pdp69D is expressed at high levels in the neuropil. Pdp69D is also localized to the A8 abdominal ganglion at the posterior extremity of the ventral nerve cord. Nerves connecting the leg and wing imaginal discs to the ventral cord are stained. Pdp69D is also expressed on portions of the basal surface of imaginal disc epithelia, especially near attachment stalks. In the optic lobes, Ptp69d is localized to the neuropils of the lamina and medulla, and to an array of parallel thick bundles that may be the transmedullary fibers of the developing lobula complex. In the eye-antennal disc, Pdp69D is localized to photoreceptor axons in the optic stalk (Desai, 1994).

Structure-function analyses of tyrosine phosphatase PTP69D in giant fiber synapse formation of Drosophila

PTP69D> is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2) and has been shown to play a role in axon guidanceof embryonic motoneurons as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. This study characterized the developmental role of PTP69D in the giant fiber (GF) neurons, two interneurons in the central nervous system (CNS) that control the escape response of the fly. These studies revealed that PTP69D has a function in synaptic terminal growth in the CNS. Missense mutations in the first immunoglobulin (Ig) domain and in the Cat1 domain, present in Ptp69D10 and Ptp69D20 mutants, respectively, did not affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell autonomous rescue experiments demonstrated a function for the Cat1 and the first Ig domain of PTP69D in the GFs but not in its postsynaptic target neurons. In addition, complementation studies and structure-function analyses revealed that for GF terminal growth Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domains, but not the fibronectin III or the membrane proximal region domains. In contrast, the fibronectin III but not the immunoglobulin domains were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, these studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function in photoreceptor targeting (Lee, 2014).

Adult

The spatial and temporal expression of seven Drosophila protein tyrosine phosphatase genes during oogenesis was examined by whole mount in-situ hybridization of antisense RNA probes to ovaries. Diverse expression patterns exist consistent with multiple roles for protein tyrosine phosphatases in the ovary. Ptp99A and corkscrew transcripts are expressed in follicle cells, consistent with possible roles in the EGF receptor signaling pathway. Transcripts from corkscrew and DPTP10D are detected in the germline during oogenesis and localized to the oocyte during egg chamber development. Localization of the two transcripts is disrupted by mutations in egalitarian and Bicaudal D. Dlar and DPTP4E transcripts are found in the germline during the same developmental stages as DPTP10D transcripts, but their transcripts are not localized to the oocyte. DPTP61F transcription is detected only after stage 6 of oogenesis. After stage 10B these transcripts are transported to the oocyte; thus ovarian transcription of DPTP61F may reflect a maternal contribution of the mRNA for later use during embryogenesis. Ptp69D transcripts are sequestered in the nucleus from stage 7 to stage 10, and then released to the cytoplasm. These observations suggest that the export of Ptp69d mRNA from the nucleus is temporally regulated during oogenesis (Fitzpatrick, 1999).

Effects of Mutation or Deletion

The receptor tyrosine phosphatases Ptp69D and Ptp99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking Ptp69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking Ptp99A are indistinguishable from wild type. However, motor axon defects in ptp69D;ptp99A double mutant embryos are much more severe than in embryos lacking only Ptp69D. These results demonstrate that Ptp69D and Ptp99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system (Desai, 1996).

Ptp69D mutant embryos display a variety of abnormal SNb morphologies. Some of these are similar bypass, detour, and stall SNb phenotypes described in embryos that misexpress Connectin or Fasciclin II. In the bypass phenotype, some or all of the SNb axons fail to defasciculate from the intersegmental nerve (ISN) and turning into the ventrolateral muscle field. Instead, they continue to extend dorsally within the ISN, which often appears thicker than normal owing to the addition of the misrouted SNb axons. In complete bypass segments, the ventrolateral muscles are apparently uninnervated (Desai, 1996).

Some SNb axons that bypass the ventrolateral muscles later leave the ISN distal to the muscle field and grow backward on the internal side of the muscles (U-turn phenotype), forming synaptic specializations at positions normally used by wild-type branches. These axons apparently retain the ability to recognize their target field, even though they bypassed it during the initial phase of their outgrowth (Desai, 1996).

In hemisegments exhibiting detour and stall phenotypes, SNb axons innervate some or all of the ventrolateral muscles, but can follow incorrect routes to reach their targets. For example, some axons leave the ISN at the normal choice points forming a synapse at the muscles 6/7 cleft. They fail to extend beyond this point or reach muscles 13 and 12 (stall phenotype). Other SNb axons in the same hemisegment do not defasciculate from the ISN at the normal choice (as in bypass) but instead leave it at muscle 30 and follow the abnormal route to muscle 12 (detour phenotype). Although muscle twelve is a normal synaptic site, the large accumulation of axonal material that is observed there suggests that inappropriate axons may have been routed to the target (Desai, 1996).

In some hemisegments, a different type of split SNb phenotype is observed. In this case SNb axons leave the ISN at the normal point but some stall at the proximal edge of muscle 14, apparently unable to defasciculate or extend further. Others, however, extend across the wrong (internal) face of muscles 7 and 6. They do not form a synaptic specialization at the muscle 6/7 cleft. The characterization of the bypass, detour and stall phenotypes indicates that SNb growth cones in Ptp69D mutants are impaired in their ability to execute pathfinding decisions correctly, and retain the capacity to recognize muscle targets (Desai, 1996).

To determine whether the incomplete penetrance of the SNb defects in Ptp69D null embryos could be due to compensation by other axonal RPTPs, such as Ptp99A, Ptp69D:Ptp99A double mutant combinations were analyzed. Penetrance and severity of SNb defects are enhanced in Ptp69D:Ptp99A embryos, even though the loss of only Ptp99A does not affect motor axons. In Ptp69D:Ptp99A embryos, the peripheral motor pathways, with the exception of the ISN, are disorganized or irregular in almost all segments. Double mutant embryos also exhibit CNS defects, including breaks and bundle fusions in the longitudinal track and variable widening of the CNS. The pattern of body wall muscle fibers appears normal in Ptp69D:Ptp99A embryos, however. The cell bodies and axons of the PNS are also normal (Desai, 1996).

The axons of the SNa exit the CNS in the SN root and follow a distinct pathway to the distal edge of the ventrolateral muscle field. The SNa then bifurcates and extends one branch to lateral muscles 21-24 and another to muscles 5 and 8. Ptp69D and Ptp69D/Ptp99A double mutants display a variety of SNa guidance defects. The SNa axons in one example leave the ISN at an abnormal exit point and then stall before reaching their targets. In another example SNa splits into three fascicles instead of two. The penetrance of such SNa phenotypes in embryos homozygous for complete deletion or for the extracellular domain deletion of Ptp69D is about 9%. As with the SNb, Ptp69D embryos have a low penetrance of SNa defects. Ptp99A embryos do not display any SNa defects. The loss of Ptp99A function, however, potentiates the effects of Ptp69D. About one in three of the SNa nerves are phenotypically abnormal in double mutants. These SNa phenotypes are also rescued by a single copy of the Ptp69D transgene. The loss of Ptp99A in combination with the weaker Ptp69D alleles has effects on SNa phenotypes similar to those described for SNb. Removing Ptp99A function increases the penetrance of SNa defects produced by each Ptp69D mutation by 3-to 4-fold (Desai, 1996).

The SNb bypass, detour and stall phenotypes observed in Ptp99A and Ptp69D mutants strikingly resemble those produced by overexpression of the homophilic adhesion molecule Fasciclin II on motor axons or by ectopic expression of the Connectin glycoprotein on ventrolateral muscles. Fasciclin II overexpression also affects the SNa nerve, again producing phenotypes similar to those observed in the RPTP mutants. Raising the level of Fasciclin II or Connectin misexpression increases the penetrance of defects just as does reducing Ptp69D or Ptp99A function (Desai, 1996).

Similar phenotypes produced by Ptp69D and Ptp99A loss-of-function mutations and fasciclin II and connectin gain-of-function constructs suggest that RPTPs and these adhesion/repulsion molecules affect the same signal transfection pathway or use parallel pathways that converge on to the same effectors. It has been reported that engagement of the homophilic adhesion molecules N-CAM, L1 and N-cadherin potentiates adhesion and neurite outgrowth via elevation of fibroblast growth factor receptor tyrosine kinase activity. Since Fasciclin II is likely to be the Drosophila homolog of N-CAM, it is possible that a similar signaling pathway could be present in flies. If so, homophilic interactions among SNb axons mediated by overexpressed Fasciclin II might elevate tyrosine kinase activity. Likewise, binding of Connectin on ventrolateral muscles to a heterophilic receptor on SNb growth cones might also elevate tyrosine kinase activity. This tyrosine kinase signal would have the net effect of repelling growth cones from the target by keeping them within the fascicle. This is consistent with results obtained in vertebrate systems, where Eph family tyrosine kinases can promote fasciculation via their activation of repulsive ligands (Desai, 1996).

In the Drosophila neuromuscular system, RPTP activity could oppose Fasciclin II-mediated adhesion and regulate the influence of repulsive ligands by decreasing signaling through tyrosine kinase pathways. Elevation of RPTP activity at specific choice points may allow growth cones to defasciculate from axon bundles. This would enable SNb growth cones to leave the ISN at the first choice point and to defasciculate and form synapses on their targets at subsequent choice points within the muscle field. Elimination or reduction of RPTP activity by mutation would interfere with transmission of defasciculation signals, producing abberant pathway decisions (Desai, 1996).

The neural receptor tyrosine phosphatases Ptp69d, Ptp99A and Dlar are involved in motor axon guidance in the Drosophila embryo. The requirements for these three phosphatases in growth cone guidance decisions along the ISN and SNb motor pathways have been analyzed. Any one of the three suffices for the progression of ISN pioneer growth cones beyond their first intermediate target in the dorsal muscle field. Dlar or Ptp69d can facilitate outgrowth beyond a second intermediate target, and Dlar is uniquely required for formation of a normal terminal arbor. A different pattern of partial redundancy among the three phosphatases is observed for the SNb pathway. Any one of the three suffices to allow SNb axons to leave the common ISN pathway at the exit junction. When Dlar is not expressed, however, SNb axons sometimes bypass their ventrolateral muscle targets after leaving the common pathway, instead growing out as a separate bundle adjacent to the ISN. This abnormal guidance decision can be completely suppressed by also removing Ptp99A, suggesting that Dlar turns off or counteracts a Ptp99A signal that favors the bypass axon trajectory. These results show that the relationships among the tyrosine phosphatases are complex and dependent on cellular context. At growth cone choice points along one nerve, two phosphatases cooperate, while along another nerve these same phosphatases can act in opposition to one another (Desai, 1997).

The growth cone of the aCC neuron pioneers the ISN pathway, exiting the CNS during stage 13 and then growing dorsally past the ventrolateral muscles (VLMs) and lateral muscle 4. During stage 15, ISN growth cones contact one of the three dorsal 'persistent Twist' (PT) cells, PT2, and also interact with the peripheral nervous system (PNS) and muscle fibers. The PT cells are precursors of adult muscles and express both Twist (a mesodermal nuclear marker) and Fasciclin II. Another PT cell, PT3, is initially located posterior and lateral to PT2 and does not appear to be contacted by the pioneer axons during their outgrowth. Later, however, PT3 is contacted by a posteriorly directed side branch of the ISN, and it subsequently migrates toward the main nerve. After passing PT2, the pioneer growth cones extend under the main tracheal trunk and contact a third PT cell, PT1, as well as the muscle fibers adjacent to it. By the end of stage 16, the ISN has acquired a highly stereotyped morphology, with lateral branches at the proximal edges of muscles 3 (first branch) and 2 (second branch) and a terminal arbor at the proximal edge of muscle 1, just beyond PT. PT3 is always at the first branchpoint. ISN axons form synapses on the dorsal muscles during stage 16 and early stage 17, with aCC innervating muscle 1 and RP2 innervating muscle 2 (Desai, 1997).

In wild-type, Ptp99A, or Ptp69D late stage 16 embryos, the ISN has reached PT1 and begun to form a terminal arbor in 99%-100% of abdominal hemisegments (A2-A7). Dlar embryos, however, display truncation phenotypes in which 9%-19% of ISNs terminate at the second lateral branchpoint (SB phenotype), and 22%-34% stop between the second branch and PT1 (SB+ phenotype). The distal portion of the ISN is often abnormally thin in SB+ hemisegments, suggesting that some axons failed to extend past the second branchpoint. ISNs that do reach PT1 usually form terminal arbors that are smaller and simpler than in wild-type, suggesting that growth cone exploration of the muscle fibers near PT1 is reduced in Dlar mutants. Combining Ptp69D with Dlar increases the penetrance and severity of the Dlar ISN defects. In Dlar:Ptp69D double mutant embryos, only 15-19% of ISNs reach PT1, while 43%-49% stop at the position of the second branch-point. Dlar:Ptp99A double mutants also display SB (10%-21%) and SB+ (29%-39%) phenotypes. About 5% of ISNs terminate at the first branch-point in both types of double mutants, a phenotype not observed in any single mutant embryo. Ptp69DPtp99A embryos occasionally have abnormal ISNs, but much less frequently than in the other double mutant genotypes (5% SB; 11% SB+). Triple mutants lacking all three RPTPs exhibit much stronger ISN phenotypes than any single or double mutant. 57%-70% of ISNs now terminate at the first lateral branchpoint near PT3 (FB phenotype, while 12%-17% of ISNs fail to even reach PT3 (1- phenotype) (Desai, 1997).

The RPTP mutant phenotypes described here suggest that intermediate targets for ISN growth cones may be located at the two branchpoint positions where ISN truncations are observed. The first branchpoint position is at the intersection between muscle 19 and the proximal edge of muscle 3. The second branchpoint position is at the intersection of the proximal edges of muscles 10 and 2. There may also be a target site at the intersection of the proximal edges of muscles 1 and 9 that defines the position of the terminal arbor. PT1 is also located here. Although processes branching from the ISN will later envelop it, PT3 does not appear to be directly contacted by the pioneer growth cones, so it is unlikely to define an intermediate target for the main ISN. PT2 is dorsal to the first branchpoint by stage 16. ISN pioneer growth cones reproducibly contact PT2, however, and the first lateral branch forms shortly after this contact is made, so recognition of PT2 could be involved in defining the first branchpoint position (Desai, 1997).

In summary, these results show that all three RPTPs are involved in ISN outgrowth and guidance. In Dlar single mutants, most ISNs reach PT1 but have small terminal arbors. ISNs with any abnormal phenotype are uncommon (<17% penetrance) in any single or double mutant genotype in which Dlar is wild-type, suggesting that Dlar is central to ISN guidance. Removing Ptp69D and/or Ptp99A function from Dlar mutants generates phenotypes in which the ISN pathway is truncated at specific branchpoint positions. Thus, while Ptp69d or Ptp99A are not essential for ISN development, they do participate in guidance processes involving Dlar. To investigate the basis of the requirement for Dlar, the ISN phenotypes of Dlar were examined in embryos in which Ptp69d was overexpressed. The frequency of the SB truncation phenotype in these embryos (10%) is similar to that observed in Dlar embryos, and they still have small terminal arbors. These data suggest that Ptp69d cannot substitute effectively for Dlar along the ISN pathway even when it is present at much higher than normal levels (Desai, 1997).

The cell bodies of the RP neurons, four of which contribute axons to the SNb are located in paired clusters between the commissural tracks of each segment. RP1, RP3, and RP4 express the adhesion molecule Fasciclin III at higher levels. Their axons grow across the midline and over the contralateral RP cluster. The RP neurons leave the CNS as a single fascicle of the ISN and enter the SNb pathway at the exit junction. The RP clusters in the Ptp69D mutants have the correct number of cell bodies and are located at normal positions, although they are less regularly arranged than in the wild-type. Mutant RP axons, however, sometimes exhibit guidance errors within the CNS. For example, in some cases, the RP fascicle does not turn along the ISN pathway, but instead continues along the longitudinal connnective into the next posterior segment and joins its RP fascicle. RP axon outgrowth was also studied in double mutants. Ptp69D/Ptp99A mutants exhibit the same spectrum of axon guidance as single mutants, but at a higher frequency. Since only five of 100 hemisegments examined lack RP fascicles, while greater than 50% of hemisegments exhibit SNb bypass phenotypes in double mutant embryos, it is included that the bypass phenotype is usually due to errors in axon guidance rather than to the absence of SNb axons (Desai, 1996).

The SNb motor nerve innervates the ventrolateral muscles (VLMs) and contains the axons of the identified RP1, RP3, RP4 and RP5 motoneurons. RP growth cones leave the common ISN pathway at the exit junction, enter the VLM field, and then navigate among the muscle fibers. Synapses form at highly stereotyped positions by late stage 16. Previous results have demonstrated that loss of Ptp69D function produces SNb phenotypes in which the nerve follows abnormal pathways among the muscle fibers or stalls prior to reaching synaptic targets. Although Ptp99A mutations on their own cause no SNb phenotypes, removal of Ptp69d uncovers a role for Ptp99A in SNb axon guidance. SNb axons in Ptp69D single mutants and Ptp69D:Ptp99A double mutants display similar guidance defects. The penetrance of these defects, however, is increased about 7-fold by removal of Ptp99A function. Dlar mutations also affect SNb guidance and synaptogenesis within the VLM field. In 62-74% of hemisegments in Dlar-null embryos, the entire SNb navigates the exit junction and successfully enters the VLMs. Most of these SNbs fail to form the normal pattern of synaptic branches. The morphologies of the abnormal SNbs in Dlar mutants, however, are quite different from those in Ptp69D:Ptp99A mutants. Dlar SNbs in late stage 16/early stage 17 embryos have the overall appearance of wild-type SNbs at early to mid-stage 16, suggesting that their development is delayed. They are thick and terminate in large growth cones at the distal edge of muscle 6. The prominent synapse in the cleft between muscles 7 and 6 is usually absent, as is the synapse at muscle 12 (Desai, 1997).

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. The isolation of mutations in the fourth neural RPTP, DPTP10D, is reported. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. The Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. Elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals (Sun, 2000).

To visualize individual axons and growth cones that are affected in Ptp10D;Ptp69D double mutants, lineage tracing experiments were performed in which the fluorescent dye DiI was used to label all of the progeny of single neuroblasts (NBs) in vivo. Individual neuroectodermal cells were randomly labeled at stage 8, and the embryos allowed to develop until stage 17, after which DiI-labeled NBs arising from the injected cells were identified based on their positions, and the axons and cell bodies of the NB progeny were visualized by confocal microscopy. Analysis of a large number of NB lineages in the double mutants revealed that many CNS axonal pathways are altered in complex ways by the absence of DPTP10D and DPTP69D. The projection patterns of three sets of neurons, the progeny of NBs 3-1, 4-2, and 2-5, are described that illustrate essential aspects of the phenotype. No alterations in numbers or positions of cell bodies are observed for these lineages in Ptp10D;Ptp69D embryos. The NB 2-5 lineage generates 15-22 cells by stage 17, of which 8-16 are intersegmental interneurons. Some of these (4 to 8 neurons) extend axons across the midline in the anterior commissure; these axons then turn anteriorly in the contralateral longitudinal tract and grow all the way to the brain (up to 10 segments). The remaining intersegmental interneurons (4 to 8 neurons) extend axons anteriorly (in the ipsilateral longitudinal tract) that stop after projecting about half as far. These contralateral and ipsilateral axons form the most substantial fibers in the longitudinal connectives. There is also a single motoneuron that extends an axon in the ipsilateral ISNd pathway and innervates muscles 15-17. In Ptp10D;Ptp69D mutants, the contralaterally projecting interneuronal axons cross the midline and turn anteriorly in a normal manner, but then double back across the midline after about two segments and grow posteriorly in the ipsilateral longitudinal tract. The axons of the ipsilateral intersegmental neurons grow anteriorly for a short distance and stop. The ISNd motoneuron extends an axon toward the midline that stalls and never enters the ISN root. This lineage illustrates that interneuronal axons abnormally cross the midline in the Rptp double mutant, and that a motor axon is deflected toward the midline (Sun, 2000).

The NB 4-2 lineage produces about 22 cells, including the well-characterized RP2 motoneuron that extends its axon along the ISN pathway and innervates the dorsal muscle 2. The NB 4-2 also generates the CoR motoneurons, whose axons constitute all of the SNc motor nerve. All of the interneurons are local; two or three of them extend axons across the anterior commissure that bifurcate in the contralateral connective. In Ptp10D;Ptp69D double mutants, the RP2 axon stalls before reaching its target, and the CoR axons do not branch onto all of their target muscles. An ipsilateral longitudinal projection is formed that extends anteriorly from the clone and crosses the segment border; this is never observed in wild type. Finally, the local interneuronal projection splits after crossing the midline, so that two pathways form instead of one; this was observed in all lineages examined. In summary, this lineage illustrates that abnormal longitudinal pathways are formed in mutant embryos and that pathway selection in the commissures is altered (Sun, 2000).

NB 3-1 produces the RP1, RP3, RP4 and RP5 motoneurons, which extend axons across the anterior commissure and into the ISNb nerve, eventually innervating the ventrolateral muscles. It also generates a variable number of interneurons, which cross the midline and project both posteriorly (intersegmental interneurons) and anteriorly (local interneurons) in the contralateral connective. In Ptp10D;Ptp69D mutants, the RP neurons extend axons normally across the commissure and into the ISNb nerve, although they do not form normal synapses. The interneuronal projections, however, are radically altered. They still cross the midline, but do not form defined anterior and posterior projections in the contralateral connective. Instead, they grow anteriorly in a circular path around the neuropil, contacting the midline at the end of their trajectory. Like the other lineages, 3-1 illustrates that longitudinal pathways cannot form normally. Both the anterior and posterior interneuronal projections are missing, and are replaced by a swirl of axons that grow to the midline. These kinds of pathway alterations could give rise to the connective breaks that are observed in mutant embryos (Sun, 2000).

The fact that longitudinal axons can be changed into commissural axons by elimination of RPTP activity suggests that tyrosine phosphorylation controls the manner in which growth cones respond to midline repulsive signals. This is consistent with the observation that pharmacological inhibition of tyrosine kinase activity in grasshopper embryos causes a robo-like phenotype in which the longitudinal axon of the pCC neuron crosses the midline and circles back to the ipsilateral side. Further evidence that the effects of the inhibitor may actually be due to blockage of Robo signaling is provided by the recent observation that the Drosophila pCC axon in robo embryos has a unique branched morphology that is identical in appearance to that of the grasshopper pCC in inhibitor-treated embryos (Sun, 2000 and references therein).

The repulsive response to midline signals is encoded within the Robo cytoplasmic domain. The cytoplasmic domains of fly, nematode and mammalian Robo family proteins (Robos) contain conserved tyrosine-containing PYATT sequence motifs, suggesting that these domains could be direct targets for tyrosine kinases. Phosphorylated tyrosine motifs usually function by binding to SH2 and PTB-domain adapter proteins that mediate downstream signaling events. Robo also contains two proline-rich sequences that could interact with SH3-domain adapters. Robo2 has the tyrosine-containing motif, but lacks the proline-rich sequences (Sun, 2000).

How are Robo signaling pathways regulated by RPTPs? There is no evidence at present that the RPTPs directly alter signaling by the Robo protein. It is possible that the RPTPs and Robo feed into separate pathways that only intersect after several signaling steps. There is, however, a known mechanism for RPTP-mediated positive regulation of tyrosine kinase pathways that suggests how DPTP10D and DPTP69D could facilitate Robo signaling. During T cell receptor (TCR) signal transduction, the RPTP CD45 removes an inhibitory C-terminal phosphate group from the Src-family tyrosine kinase Lck, thereby activating it and allowing it to phosphorylate the z chain of the TCR. The phosphorylated z chain in turn binds to an SH2-domain containing tyrosine kinase (ZAP-70), which mediates downstream signaling events. CD45 is required for TCR signaling because in its absence Lck is not activated and thus cannot efficiently phosphorylate the z chain. (Interestingly, CD45 may also be involved in the termination of the TCR signaling response, since it can dephosphorylate the z chain and prevent it from binding to ZAP-70) Another mammalian receptor phosphatase, RPTPa, also dephosphorylates and activates Src-family kinases. Fibroblasts derived from RPTPa knockout mice have reduced Src and Fyn activities, suggesting that RPTPa is an in vivo regulator of Src family kinase function (Sun, 2000 and references therein) .

By analogy to these pathways, DPTP10D and DPTP69D might regulate growth cone repulsion by activating Src-family tyrosine kinase(s) that phosphorylate Robos. This could explain the genetic data, since the loss of RPTP function would be expected to cause a decrease in the extent of Robo phosphorylation. One might also propose that positive regulation of repulsion by the RPTPs occurs through direct dephosphorylation of Robos, and that dephosphorylated Robos are more active in signaling. This would be unusual, however, since normally it is the phosphorylated form of a signaling motif that binds to downstream adapters. A variant of the direct interaction model proposes that Robos become phosphorylated on tyrosines after engagement of Slit, and that DPTP10D or DPTP69D are recruited into a Robo/Slit signaling complex by their interactions with the phosphotyrosine motifs. RPTPs might remain bound to these sites for a significant time period, because they often hydrolyze phosphate-tyrosine bonds quite slowly. The RPTPs could then function as adapters themselves, binding to downstream signaling proteins and recruiting them into Robo/Slit receptor complexes. Determining which, if any, of these models is correct will require biochemical or genetic identification of in vivo substrates for RPTPs (Sun, 2000).

Four receptor-linked protein tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Three of these (DLAR, DPTP69D, DPTP99A) regulate motor axon guidance decisions during embryonic development. The role of the fourth neural phosphatase, DPTP10D, has been examined by analyzing double-, triple-, and quadruple-mutant embryos lacking all possible combinations of the phosphatases. This analysis shows that all four phosphatases participate in guidance of interneuronal axons within the longitudinal tracts of the central nervous system. In the neuromuscular system, DPTP10D works together with the other three phosphatases to facilitate outgrowth and bifurcation of the SNa nerve, but acts in opposition to the others in regulating extension of ISN motor axons past intermediate targets. These results provide evidence for three kinds of genetic interactions among the neural tyrosine phosphatases: partial redundancy, competition, and collaboration (Sun, 2001).

Bifocal is a putative cytoskeletal regulator and a Protein phosphatase-1 (PP1) interacting protein that mediates normal photoreceptor morphology in Drosophila. Bif and PP1-87B, as well as their ability to interact with each other, are required for photoreceptor growth cone targeting in the larval visual system. Single mutants for bif or PP1-87B show defects in axonal projections in which the axons of the outer photoreceptors bypass the lamina, where they normally terminate. The functions of bif and PP1-87B in either stabilizing R-cell morphology (for Bif) or regulating the cell cycle (for PP1-87B) can be uncoupled from their function in visual axon targeting. Interestingly, the axon targeting phenotypes are observed in both PP1-87B mutants and PP1-87B overexpression studies, suggesting that an optimal PP1 activity may be required for normal axon targeting. bif mutants also display strong genetic interactions with receptor tyrosine phosphatases, dptp10d and dptp69d, and biochemical studies demonstrate that Bif interacts directly with F-actin in vitro. It is proposed that, as a downstream component of axon signaling pathways, Bif regulates PP1 activity, and both proteins influence cytoskeleton dynamics in the growth cone of R cells to allow proper axon targeting (Babu, 2005).

Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics

During development of the adult Drosophila visual system, axons of the eight photoreceptors in each ommatidium fasciculate together and project as a single bundle towards the optic lobes of the brain. Within the brain, individual photoreceptor axons from each bundle then seek specific targets in distinct layers of the optic lobes. The axons of photoreceptors R1-R6 terminate in the lamina, while R7 and R8 axons pass through the lamina to terminate in separate layers of the medulla. To identify genes required for photoreceptor axon guidance, including those with essential functions during early development, a strategy has been devised for the simple and efficient generation of genetic mosaics in which mutant photoreceptor axons innervate a predominantly wild-type brain. In a large-scale saturation mutagenesis performed using this system, new alleles of the gene encoding the receptor tyrosine phosphatase PTP69D were recovered. PTP69D functions in the correct targeting of motor axons in the embryo and R1-R6 axons in the visual system. PTP69D is also required for correct targeting of R7 axons. Whereas mutant R1-R6 axons occasionally extend beyond their normal targets in the lamina, mutant R7 axons often fail to reach their targets in the medulla, stopping instead at the same level as the R8 axon. These targeting errors are difficult to reconcile with models in which PTP69D plays an instructive role in photoreceptor axon targeting, as previously proposed. Rather, it is suggested that PTP69D plays a permissive role, perhaps reducing the adhesion of R1-R6 and R7 growth cones to the pioneer R8 axon so that they can respond independently to their specific targeting cues (Newsome, 2000).

How might an instructive model for PTP69D function explain the behaviour of R7 axons? These axons also require PTP69D for correct targeting, but appear insensitive to any 'stop' signal it might transduce in the lamina and instead interpret PTP69D activation as a 'continue' signal in the superficial layers of the medulla. Such a context-dependent interpretation of a single guidance cue is not without precedent. In tissue culture assays, the response of Xenopus spinal cord growth cones to gradients of the guidance cue Netrin-1 is mediated by the receptor DCC. Depending on the coexpression of another Netrin-1 receptor, UNC5, or intracellular cAMP levels, these growth cones may be either attracted or repelled by the Netrin-1 source. In an instructive model for PTP69D function, a similar mechanism might be invoked to account for the different responses of R1-R6 and R7 growth cones to signals transduced by PTP69D (Newsome, 2000).

The permissive model, however, offers a simpler explanation for the targeting errors made by both R1-R6 and R7 axons in Ptp69D mutants. A common feature of many mistargeted R1-R6 axons, and probably all mistargeted R7 axons, is that they terminate in the medulla at the same level as the R8 axons. The R8 growth cone pioneers the pathway into the optic lobe. It is not known whether the R8 axon is an essential pioneer for this pathway, or whether the R1-R6 and R7 axons can independently navigate towards the optic lobe. The eight photoreceptor axons are however tightly fasciculated as they traverse the optic stalk and penetrate the brain, and it is likely that specific targeting cues for R1-R6 and R7 growth cones must counteract their adhesion to the R8 axon. PTP69D activation may reduce adhesion between photoreceptor axons, thus facilitating but not directly mediating their independent targeting decisions. In this model, the low penetrance of targeting errors in Ptp69D mosaics need not reflect genetic redundancy in the targeting signals, as required in the instructive model. The low penetrance may instead reflect a delicate balance between adhesive and targeting forces that is often decided in favour of targeting even without a reduction in adhesion. In the mosaic screen for visual system connectivity mutants, mutations were recovered in at least three distinct complementation groups that lead to a fully penetrant R1-R6 'passthrough' phenotype. These mutations suggest that specific non-redundant targeting mechanisms may indeed exist, at least for R1-R6 axons (Newsome, 2000).

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. DPTP52F, the sixth RPTP to be discovered in Drosophila, is described. 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, RNA interference (RNAi)-induced phenotypes were examined 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 (Schindelholz, 2001).

Ptp52F mutants display a variety of SNa guidance defects. The most common defect, as in Ptp52F RNAi embryos, is a failure to bifurcate. In other hemisegments, the SNa has extra branches, or stalls near the bifurcation point. The penetrances of such SNa phenotypes in Ptp52F^18.3 homozygotes or Ptp52F^18.3/Df(2R)JP4 transheterozygotes are 37% and 41%, respectively. The two other Ptp52F alleles and the transheterozygous combinations of the three Ptp52F alleles with Df(2R)JP8 have a lower penetrance of SNa defects (22-28%) (Schindelholz, 2001).

Single mutants that lack any of the other four neural RPTPs do not display SNa phenotypes. However, combinations of Rptp mutations do affect the SNa. To evaluate how removal of other RPTPs might affect Ptp52F SNa phenotypes, double mutants lacking both DPTP52F and each of the other RPTPs were made. The absence of DPTP10D, DPTP69D or DLAR increases the penetrance of the Ptp52F^18.3 defects, particularly those in which the SNa stalls near the bifurcation point. No new phenotypes are observed in double mutants, however. Removal of DPTP99A does not affect the overall penetrance of SNa phenotypes, but does decrease the frequency of ectopic branches (Schindelholz, 2001).

The ISNb motor nerve innervates the VLMs and contains the axons of the identified RP1, RP3, RP4 and RP5 motoneurons. RP growth cones leave the common ISN pathway at the exit junction, enter the VLM field, and then navigate among the muscle fibers. Synapses begin to form at stereotyped positions by late stage 16. Ptp52F mutations produce any detectable ISNb phenotypes only at low frequencies (Schindelholz, 2001).

Ptp10D mutations produce no ISNb phenotypes. Removal of both DPTP10D and DPTP52F, however, generates a strong phenotype in which the ISNb stalls within the VLMs, often at the proximal edge of muscle 13. This stall phenotype is observed in Ptp52F single mutants, but its frequency can be dramatically increased in double mutants for addition of a Ptp10D mutation to the hypomorphic mutation Ptp52F^8.10.3;. Removal of DPTP69D also greatly enhances the Ptp52F stall phenotype (Schindelholz, 2001).

Dlar Ptp52F double mutants have parallel bypass phenotypes identical to those of Dlar single mutants. Ptp99A mutations cause no ISNb phenotypes on their own or in combination with Ptp52F (Schindelholz, 2001).

The ISN passes its first (FB) and second (SB) lateral branchpoints before reaching the position of its terminal arbor at the proximal edge of muscle 1. In Ptp52F mutants, most ISNs are normal. Dlar mutations produce SB phenotypes with a similar penetrance (19% for null alleles). When Dlar and Ptp52F mutations are combined, the frequency of the SB termination phenotype is similar to that of the single mutants. Ptp99A mutations have no effects on ISN on their own, and also cause no enhancement of the Ptp52F phenotype (Schindelholz, 2001).

Ptp10D and Ptp69D single and double mutants have no ISN phenotypes. However, removal of either of these RPTPs from a Ptp52F mutant background enhances the penetrances of the Ptp52F ISN phenotypes. Ptp10D Ptp52F double mutants have a reduced terminal arbor (T) phenotype that is less frequently observed in Ptp52F single mutants. Removal of DPTP69D does not affect the T phenotype, but produces an increase in the SB phenotype. In summary, these results indicate that DPTP52F, DPTP10D and DPTP69D have partially redundant functions in regulation of ISN outgrowth. It is interesting that Ptp52F mutations do not produce synergistic phenotypes when combined with Dlar mutations, which are the only other Rptp mutations that generate strong ISN phenotypes on their own. Perhaps there are two separate 'functions' needed for normal ISN outgrowth, one of which involves DLAR and the other DPTP52F (Schindelholz, 2001).

DPTP52F is the only RPTP whose removal produces clear phenotypes in the 1D4-positive longitudinal bundles of the CNS. The 1D4 pathways are usually indistinguishable from wild type in single mutants lacking each of the other four RPTPs. Removal of DPTP10D or DPTP69D from a Ptp52F background strengthens the Ptp52F CNS phenotype. The longitudinal 1D4-positive bundles become more irregular, and frequent breaks and discontinuities in the middle bundle are observed. No new synergistic phenotype like that produced by removal of DPTP10D and DPTP69D together is observed. Removal of DPTP99A does not affect the Ptp52F CNS phenotype (Schindelholz, 2001).

In contrast to these results, when a Dlar mutation is introduced into a Ptp52F mutant background, the morphology of the 1D4-positive bundles reverts to wild type. In a few segments of Dlar Ptp52F double mutants, breaks in the outer 1D4-positive bundle are still seen, but defasciculation and irregularities in the inner two bundles are not observed. The suppression is specific to the CNS phenotypes detected at late stage 16, because the introduction of Dlar mutations into a Ptp52F mutant background does not correct the failure of the pCC growth cone to extend at the appropriate time. DLAR also participates in another competitive interaction: the Dlar ISNb parallel bypass phenotype is absent in Dlar Ptp99A double mutants. Here, however, it is a Dlar phenotype that is suppressed by removal of another RPTP, rather than the reverse. Ptp52F mutations do not affect Dlar parallel bypass phenotypes. Determination of the mechanisms that underlie these genetic interactions will require biochemical analysis of DPTP5F and of the signaling pathways in which it participates (Schindelholz, 2001).

The neural receptor Protein tyrosine phosphatase DPTP69D is required during periods of axon outgrowth in Drosophila

A series of 18 chemically induced alleles of Ptp69D, ranging in strength from viable to worse than null, have been isolated and characterized, that represent unique tools for probing the structure, function, and signaling pathway of DPTP69D. Three alleles are strongly temperature sensitive and were used to define the developmental periods requiring DPTP69D function; adult health requires DPTP69D during the mid- to late-pupal stage, eclosion requires DPTP69D during the early to mid-larval stage, and larval survival requires DPTP69D during embryogenesis. Mutations predicted to abolish the phosphatase activity of the membrane proximal D1 domain severely reduce but do not abolish DPTP69D function. Six alleles appear null; only 20% of null homozygotes pupate and <5% eclose, only to fall into the food and drown. One allele, Ptp69D⁷, confers axon and viability defects more severe than those of the null phenotype. Sequence analysis predicts that Ptp69D⁷ encodes a mutant protein that may bind but not release substrate. Like mutations in the protein tyrosine phosphatase gene Dlar, strong Ptp69D alleles cause the ISNb nerve to bypass its muscle targets. Genetic analysis reveals that the bypass defect in Dlar and Ptp69D mutants is dependent upon DPTP99A function, consistent with the hypothesis that DPTP69D and DLAR both counteract DPTP99A, allowing ISNb axons to enter their target muscle field (Desai, 2003).

Bypass defects arise when some or all ISNb axons fail to enter the target ventro-lateral muscle (VLM) field, but instead grow dorsally within or next to the ISN nerve. These defects occur at a rate of ~15% in Ptp69D null embryos and 30% in Dlar mutants. Strikingly, one of the new alleles, Ptp69D⁷, confers the bypass phenotype at rates approaching those seen in Dlar mutants. Previous results have demonstrated that the Dlar bypass phenotype requires a third RPTP, DPTP99A. The ISNb bypass phenotype observed in Ptp69D embryos is likewise dependent upon DPTP99A. Only one copy of Ptp99A was removed in these experiments because Ptp69D Ptp99A double-mutant embryos display severe ISNb defects that can preempt the bypass phenotype. These results suggest that DPTP69D and DLAR function together to allow ISNb axons to innervate the VLM by counteracting DPTP99A at this choice point (Desai, 2003).

To determine the molecular basis for reduced or absent function of the Ptp69D gene, selected alleles were sequenced. For the most part, alleles directing the expression of immunologically detectable DPTP69D were chosen for sequence analysis. A shared molecular defect may be responsible for the similar phenotypes exhibited by two temperature-sensitive alleles, Ptp69D¹² and Ptp69D¹⁸. Both alleles bear a missense mutation, glycine to aspartate, in the extracellular region of the protein, C-terminal to the cleavage site 47 residues from the start of the trans-membrane segment. Two of the EMS alleles, Ptp69D²⁰ and Ptp69D²¹, as well as Ptp69D^Y125, have missense mutations in the active site of the D1 phosphatase domain. The mutation in Ptp69D²¹ changes the invariant catalytic cysteine residue to tyrosine and almost certainly abrogates the activity of this domain. In Ptp69D²⁰ and Ptp69D^Y125, conserved active-site glycines are mutated to alanine and arginine, respectively. It will be interesting to determine the effects of these changes on phosphatase activity and then to correlate activity with the ability to support axon guidance. The two alleles that cause the most severe axon guidance defects have strikingly different mutations. Ptp69D⁷ has a small three-amino-acid deletion in the D1 phosphatase domain that removes a conserved aspartate residue. In other phosphatases, this residue is required to complete catalysis; mutants in which this residue is changed to alanine bind but do not release their substrates. By contrast, the change in Ptp69D¹⁰ converts a valine between the immunoglobulin domains to a glutamate. Finally, embryos homozygous for Ptp69D⁹ and Ptp69D¹⁷ express low or undetectable levels of DPTP69D. Both alleles have changes in the trans-membrane region, suggesting that the C-terminal cleavage product may stabilize the DPTP69D extracellular portion and/or anchor it to the cell surface (Desai, 2003).

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