InteractiveFly: GeneBrief

Protein tyrosine phosphatase 4E and Protein tyrosine phosphatase 10D: Biological Overview | References

BIOLOGICAL OVERVIEW

Drosophila has six receptor protein tyrosine phosphatases (RPTPs), five of which are expressed primarily in neurons. Mutations in all five affect axon guidance, either alone or in combination. Highly penetrant central nervous system (CNS) and motor axon guidance alterations are usually observed only when specific combinations of two or more RPTPs are removed. This study examined the sixth RPTP, Ptp4E, which is broadly expressed. Ptp4E and Ptp10D are closely related type III RPTPs. Non-drosophilid insect species have only one type III RPTP, which is closest to Ptp10D. This study found that Ptp4E mutants are viable and fertile. Ptp4E Ptp10D double mutants were found to die before the larval stage and have a mild CNS phenotype in which the outer longitudinal 1D4 bundle is frayed. Ptp10D Ptp69D double mutants have a strong CNS phenotype in which 1D4 axons abnormally cross the midline and the outer and middle longitudinal bundles are fused to the inner bundle. To examine if Ptp4E also exhibits synthetic phenotypes in combination with Ptp69D, Ptp4E Ptp69D double mutants and Ptp4E Ptp10D Ptp69D triple mutants were made. No phenotype was observed in the double mutant. The triple mutant phenotype differs from the Ptp10D Ptp69D phenotype in two ways. (1) The longitudinal tracts appear more normal than in the double mutant; two or three bundles are observed, although they are disorganized and fused. (2) Axons labelled by the SemaIIB-τ Myc marker often cross in the wrong commissure. Motor axon guidance was examined, and no phenotypes were observed in any Ptp4E double mutant combination. However, triple mutants in which Ptp4E Ptp10D was combined with Ptp69D or Ptp52F exhibited stronger phenotypes than the corresponding Ptp10D double mutants. It is concluded that type III RPTPs are required for viability in Drosophila, since Ptp4E Ptp10D double mutants die before the larval stage. Unlike Ptp10D, Ptp4E appears to be a relatively minor player in the control of axon guidance. Strong phenotypes are only observed in triple mutants in which both type III RPTPs are eliminated together with Ptp69D or Ptp52F. These results allow the construction of a complete genetic interaction matrix for all six of the RPTPs (Jeon, 2008).

Signalling via tyrosine phosphorylation is essential for axon guidance in many systems. Target proteins involved in signal transduction and cytoskeletal dynamics in growth cones are phosphorylated by tyrosine kinases (TKs) and dephosphorylated by tyrosine phosphatases (PTPs). In a simplified view of phosphotyrosine pathways controlling cell growth and differentiation, signaling is triggered by engagement of receptor tyrosine kinases (RTKs) by ligands. Ligand binding induces receptor dimerization and phosphorylation of downstream targets. RTK signalling is downregulated by dephosphorylation of autophosphorylated RTKs and other signalling molecules by cytoplasmic PTPs. In this scenario, the PTPs are passive modulators of a process in which the 'informational' event that initiates signalling is ligand binding to the RTK (Jeon, 2008).

In contrast, phosphotyrosine signalling pathways involved in growth cone guidance in the Drosophila embryonic central nervous system (CNS) involve receptor tyrosine phosphatases (RPTPs) and cytoplasmic TKs. Like RTKs, RPTPs are modular signalling receptors. They have cell adhesion molecule-like extracellular (XC) domains, linked via a single transmembrane region to one or two cytoplasmic PTP domains. Five of the six fly Rptp genes are selectively expressed in CNS neurons, and all of these genes have loss-of-function phenotypes that affect axon guidance (Jeon, 2008).

The TK that is central to many growth cone guidance events in the Drosophila >embryo is Abl, a cytoplasmic kinase. Drosophila has many RTKs, but no functional RTK has been implicated in embryonic axon guidance (the kinase-related axon guidance receptors Derailed and Off-track are thought to lack enzymatic activity). These facts suggest that phosphotyrosine signalling in growth cones could be controlled in a manner opposite to that used in RTK pathways. In this scheme, the growth cone would use a cytoplasmic TK to constitutively phosphorylate targets, and the 'information' that alters signalling strength would be transmitted via engagement of RPTPs by ligands located on the surfaces over which the growth cone travels (Jeon, 2008).

Of course, this is a greatly oversimplified picture, because there are many other receptors that can influence phosphotyrosine signalling in embryonic growth cones. For example, the Roundabout 1 (Robo1) receptor is an essential regulator of axon guidance across the midline. Phosphorylation of Robo1 by Abl may be regulated by Robo1's engagement of its ligand Slit, and in this case the 'information' that triggers signalling would be delivered via Slit binding to Robo1. Also, it is unlikely that phosphorylation by Abl is an unregulated, constitutive process. Nevertheless, it is striking that the receptors are kinases and the cytoplasmic modulators are phosphatases in pathways that regulate cell growth, while the reverse seems to be true for pathways that control neuronal growth cone guidance (Jeon, 2008).

RPTP pathways are poorly understood relative to RTK pathways, partially because in vivo ligands that regulate axon guidance and synaptogenesis have been identified only for the Drosophila Lar RPTP. These are the heparan sulfate proteoglycans Syndecan and Dallylike. However, Lar also has non-heparan sulfate proteoglycan ligands, and ligands for the other five fly RPTPs have not yet been defined. It has also been difficult to identify substrates that are important for RPTP signalling in vivo (Jeon, 2008).

Five Drosophila RPTPs have been genetically characterized in published papers. Four of these (Ptp10D, Lar, Ptp69D, Ptp99A) are expressed only on CNS axons in late embryos, and the fifth, Ptp52F, is CNS-specific but is expressed on both axons and cell bodies. All of the zygotic phenotypes for these genes are alterations in axon guidance, suggesting that this is the major function of this gene family in Drosophila. In contrast, many mammalian RPTPs are expressed in non-neural tissues and have functions unrelated to axon guidance (Jeon, 2008).

The RPTPs regulate both CNS and motor axon guidance. There is extensive redundancy among the five genes, so that highly penetrant guidance phenotypes are usually observed only when two or more RPTPs are genetically removed. Studies of motor axon guidance indicate that each guidance decision made by motoneuron growth cones requires a specific subset of the RPTPs. For example, axons in the ISNb nerve are unable to defasciculate from the common ISN pathway in Lar Ptp69D Ptp99A triple mutants. The later decision by ISNb axons to enter their target muscle field fails in Lar single mutants, so that the axons bypass the muscle field, but the bypass phenotype is suppressed and muscle field entry restored in Lar Ptp99A double mutants. This example illustrates that RPTPs can exhibit either functional redundancy, in which the absence of one RPTP is compensated for by another RPTP, or competition, in which removal of a second RPTP suppresses the guidance errors caused by the absence of the first RPTP. Similar genetic interactions among RPTPs may also occur in vertebrates, as two recent papers (Stepanek, 2005; Uetani, 2006) show that double mutant combinations and RNA interference perturbations involving the vertebrate Lar and type III (Ptp10D-like) RPTP subfamilies produce complex alterations in motor axon guidance (Jeon, 2008).

This paper examines the functions of the sixth and last Drosophila RPTP, Ptp4E. This protein is closely related to Ptp10D, and is the product of a recent gene duplication. Unlike the other RPTPs, Ptp4E is widely expressed in late embryos. When these studies started, it was thought that Ptp4E mutations might have phenotypes affecting many non-neural tissues, since loss of Ptp4E could not be compensated for by neural-specific RPTPs. However, the findings show that Ptp4E single mutants have no detectable phenotypes, because Ptp4E is redundant with the closely related Ptp10D. Double mutant embryos lacking both of these RPTPs die at hatching, but they have specific phenotypes affecting only CNS axons and tracheal cells (Jeon, 2008).

This study describes the axon guidance phenotypes produced by Ptp4E mutant combinations. Tracheal phenotypes will be described elsewhere. The data in this paper, together with those in earlier papers (Desai, 1997; Schindelholz, 2001; Sun, 2001), allow construction of complete pairwise interaction matrices that define how all six Drosophila RPTPs regulate CNS and motor axon guidance (Jeon, 2008).

Ptp10D and Ptp4E are clearly the result of a gene duplication that occurred much more recently than the split between the other Drosophila Rptp genes. The amino acid sequences of their catalytic PTP domains share 89% identity, versus 36%-40% identity for pairwise comparisons of Ptp4E with other Drosophila RPTPs. Their XC domains have a very similar organization, containing chains of 11 FN3 repeats in Ptp4E and 12 FN3 repeats in Ptp10D, and are 58% identical in amino acid sequence. The Ptp4E gene encodes two predicted preproteins, of 1,767 and 1,607 amino acids, while the Ptp10D gene encodes preproteins of 1,931 and 1,631 amino acids. The sequences that differ between the alternative gene products are at the carboxyl terminus in both cases, but there is no sequence similarity between the Ptp4E and Ptp10D proteins within this region. Both genes reside on the X chromosome, and the nine Ptp4E introns within conserved coding sequence all correspond exactly in position to Ptp10D introns. Ptp10D has one additional intron not found in Ptp4E (Jeon, 2008).

The Caenorhabditis elegans gene dep-1 is the ortholog of both Ptp10D and Ptp4E. Humans and mice have five genes encoding type III RPTPs, defined as proteins with XC domains composed of long chains of FN3 domains and a single PTP domain. Among these, the product of the PTPRB gene (PTPβ, not to be confused with RPTPβ, which is a different protein also known as PTPζ) has a somewhat higher alignment score to Ptp10D and Ptp4E than the other four mammalian type III proteins. These are: DEP-1/CD148, encoded by the Ptprj gene; PTPRO; SAP-1, encoded by the Ptprh gene; and PTPRQ. Since the radiation into the five mammalian genes seen today occurred after the split between arthropods and mammals, one cannot define any of the type III mammalian genes as an ortholog of one of the fly genes. Schindelholz (2001) presents more complete description of the relationships among all the Drosophila, C. elegans, and mammalian RPTPs (Jeon, 2008).

The recent availability of genome sequences from twelve different Drosophila species, three mosquito species, two hymenopterans, a beetle, and the silkmoth allowed tracing of the evolution of the Ptp10D/Ptp4E gene pair within insect lineages. Surprisingly, it was found that the Ptp4E gene is found only in drosophilid species. Mosquitoes, which are also dipterans, and all other sequenced insects have only a single Rptp gene corresponding to this gene pair. This gene is always much more closely related to Ptp10D than to Ptp4E. In addition, the Ptp4E sequence exhibits more sequence diversity among the drosophilid species than does the Ptp10D sequence. These data indicate that Ptp10D is the ancestral gene and that its sequence has been constrained more by evolution than the Ptp4E sequence since the time of the duplication. Ptp4E has evolved much more rapidly, suggesting that it may have acquired new function(s) since its emergence or was less essential for fitness than Ptp10D (Jeon, 2008).

The Ptp10D ortholog found in all insect species always contains the Ptp10D-specific intron, and all Ptp4E orthologs in drosophilids lack this intron. This suggests that the intron may have been lost at the time of the duplication from the copy that evolved into Ptp4E. This would have been between 235 million years ago (the estimated time at which the mosquito and fly lineages diverged from each other) and 40 million years ago (the estimated time at which the radiation among the 12 sequenced drosophilid species occurred) (Jeon, 2008).

Attempted were made to trace the history of the duplication by examining the genes adjacent to Ptp10D and Ptp4E, but the organizations of the Ptp10D and Ptp4E regions in D. melanogaster were found to have arisen long after the Ptp10D and Ptp4E genes diverged from each other. Ptp10D is flanked by the Rst(1)JH and bifocal genes. Rst(1)JH is found upstream of the Ptp10D ortholog in both the obscura and melanogaster groups, but is separated from it in D. willistoni and all other drosophilids. bifocal orthologs are adjacent to the Ptp10D gene only in the melanogaster group. Similarly, the two genes flanking Ptp4E, SIP3 and CG4068, are located next to the Ptp4E ortholog only within the obscura and melanogaster groups. There are no significant sequence similarities between the genes that flank Ptp10D and Ptp4E (Jeon, 2008).

The published in situ hybridization data suggest that Ptp4E mRNA is ubiquitously expressed in late embryos, although there are some level differences between tissues (Oon, 1993). To further analyze expression, and to ensure that the observed pattern was not affected by cross-hybridization between the closely related Ptp4E and Ptp10D phosphatase domain sequences, this analysis was repeated using a probe from the first four Ptp4E exons, which are not closely related to Ptp10D (Jeon, 2008).

In gastrulating embryos, Ptp4E mRNA is enriched in the invaginating mesoderm. In germ band extended embryos (stage 10-11), the strongest expression is observed in the posterior midgut primordium. There is also an interesting 'scalloped' pattern of expression observed at the ectodermal border. This has an intriguing correspondence to visceral mesoderm dpERK staining, suggesting that Ptp4E may be enriched at sites of RTK activation. Interestingly, thisbe, which encodes a ligand for the fibroblast growth factor receptor Heartless, is expressed in a similar pattern. Examination of a germ-band extended embryo expressing a UAS-linked Ptp4E-green fluorescent protein (GFP) fusion from the engrailed-GAL4 driver shows the expected striped pattern of expression, confirming that the probe recognizes Ptp4E and allowing an estimate of the relative levels of driven versus endogenous Ptp4E mRNA (Jeon, 2008).

At stage 14, Ptp4E is widely expressed, with highest levels observed in the midgut. Expression in the CNS can also be seen. Finally, at stage 17 expression is much higher in the gut than elsewhere, with particularly strong expression observed in the hindgut and at the anterior end of the midgut (Jeon, 2008).

To examine Ptp4E protein expression and localization, a variety of mouse monoclonal and polyclonal antibodies were generated against Ptp4E. Although the ubiquitous staining observed with the antibody is consistent with the in situ hybridization data, it is uncertain whether that antibody staining in wild-type embryos is due to Ptp4E protein, because it is not significantly reduced in Ptp4E mutant embryos (Ptp4E¹ or Df(1)ovo4). This finding could be explained in two ways. First, Ptp4E¹ mutants might continue to make an abnormal Ptp4E protein(s) due to initiation of translation at methionine residues encoded in exons not removed by the excision mutation (the second methionine residue in Ptp4E is at amino acid 377, within an undeleted exon). This protein, if it exists, would lack a signal sequence and may be nonfunctional, because the CNS phenotypes of Ptp4E¹ Ptp10D¹ and Df(1)ovo4 Ptp10D¹/Y embryos are identical. Df(1)ovo4 deletes the entire Ptp4E gene. The presence of antibody staining in Df(1)ovo4 mutants could be due to persistence of Ptp4E protein synthesized from maternal mRNA, since early embryos contain large amounts of Ptp4E mRNA. Second, it is possible that the Ptp4E antibodies cross-react with another ubiquitously expressed protein. They do not cross-react with Ptp10D, because the signal does not decrease in Ptp10D¹ mutant embryos (Jeon, 2008).

Although the antibody could not be used to define where Ptp4E is expressed in the embryonic CNS in wild-type embryos, its ability to recognize overexpressed Ptp4E protein allowed analysis of whether Ptp4E can localize to axons. To do this, Ptp4E-GFP wis drived with the pan-neuronal elav-GAL4 driver. In these embryos, bright staining of both CNS and peripheral nervous system (PNS) axons is observed with anti-Ptp4E and anti-GFP antibodies, and the two patterns are superimposable. Interestingly, Ptp4E-GFP also appears to localize to neuronal cell bodies in the PNS and CNS. In contrast, Ptp10D, Ptp69D, Lar, and Ptp99A, which are restricted to axons in wild-type embryos, are also axon-specific when overexpressed (Jeon, 2008).

Expression of Ptp10D protein was detected only in the nervous system in published work. It is selectively expressed on embryonic CNS axons (Tian, 1991), and in the neuropil of the larval and adult brain (Qian, 2007). Recent data, however, show that Ptp10D is also expressed by embryonic tracheal cells. These findings suggest that the embryonic/larval lethality of Ptp4E¹ Ptp10D¹ animals might be due to either nervous system or tracheal phenotypes. In fact, it was found that these embryos have severe tracheal defects. Their nervous system defects, however, are relatively mild, and would not be expected to produce early lethality. Consistent with this, it was found that GAL4-driven pan-neural expression of a UAS-Ptp4E-GFP fusion, which is capable of rescuing the tracheal phenotype when driven in tracheal cells by breathless-GAL4, does not rescue lethality in the Ptp4E¹ Ptp10D¹ background (Jeon, 2008).

Driving Ptp10D in tracheae with breathless-GAL4 in a Ptp4E¹ Ptp10D^EP1172 background (the EP1172 line is a UAS-containing P element insertion upstream of the gene, so it allows rescue by crossing in GAL4 drivers) rescues lethality, allowing some adults to emerge. These data confirm that lethality in the double mutant is rescuable by Ptp10D expression in tracheae (or in other cells that express breathless-GAL4). Attempts were made to rescue lethality by ubiquitous expression of Ptp4E, but pancellular overexpression of Ptp4E-GFP driven by tubulin-GAL4 was found to be lethal (Jeon, 2008).

In Drosophila, five of the six RPTPs have been reported to be neural-specific in late embryos, and all the zygotic Rptp phenotypes that have been published are axon guidance alterations. In contrast, many of the 17 mammalian RPTPs are expressed in non-neural cell types and have a variety of functions unrelated to axon guidance. Since Ptp4E is the only widely expressed Rptp gene, it is speculated that studying its mutant phenotype might reveal new functions for Drosophila RPTPs outside the nervous system, and that these might provide information about functions of mammalian non-neural RPTPs. One might have expected that Ptp4E mutations would cause lethality and produce strong phenotypes, since no other RPTPs would be able to compensate for the loss of Ptp4E in non-neural cells. This, however, is not the case. Ptp4E mutants are viable, fertile, and apparently healthy, and have no detectable phenotypes in the nervous system or elsewhere. Furthermore, evolutionary analysis indicates that Ptp4E is a relatively recent invention; it is present in drosophilids but not in mosquitoes or non-dipteran arthropods. Within the drosophilids, its sequence also changes more rapidly than that of Ptp10D, suggesting that it has been less constrained by evolution. All of these considerations indicate that Ptp4E is not essential for development of non-neural cell types in Drosophila (Jeon, 2008).

Perhaps in Drosophila the functions executed by mammalian RPTPs in non-neural cell types are carried out by one or more of the eight nonreceptor PTPs. Some of these are ubiquitously expressed. Only three have been genetically characterized. Csw and PTP-ER are involved in cell fate determination. Mutations in ptpmeg produce axonal defects in the adult brain. Ptpmeg, however, does not act in the neurons that exhibit the axonal phenotypes, but is required in surrounding cells. Thus, it is unlikely to participate in growth cone signal transduction in the same manner as the RPTPs (Jeon, 2008).

Ptp10D and Ptp4E are the only Drosophila RPTPs that are members of the same subfamily; the other four are each the sole fly representative of their subfamily. Mutations in three of the other four Rptp genes (Lar, Ptp52F, Ptp69D) cause lethality. This suggests that the viability of Ptp10D and Ptp4E single mutants might be due to compensation by the other member of the subfamily, and that a Ptp4E Ptp10D double mutation would cause lethality. This is in fact observed; the double mutant dies at hatching. However, it does not have generalized defects. Rather, the defects are all within the nervous system and the tracheal network. Ptp10D is also selectively expressed in tracheal cells. It is suggested that Ptp4E Ptp10D double mutant phenotypes are observed only where Ptp10D is expressed (Jeon, 2008).

The analysis described in this paper, together with that in several other papers allowed assembly of a complete genetic interaction matrices for pairwise combinations of mutations in all six of the Rptp genes. A matrix is presented depicting the functions of the RPTPs in regulation of longitudinal axon guidance in the CNS, as assayed by 1D4 staining. The lines represent different types of genetic interactions. Synthetic phenotypes, where neither of the single mutants exhibits a detectable phenotype but the double mutant has a phenotype, are seen for Ptp10D Ptp69D and Ptp4E Ptp10D. Enhancement of a single mutant phenotype by removal of a second RPTP are observed only for Ptp52F, since this is the only single mutant that has a CNS phenotype detectable with 1D4. Finally, suppression, where removal of a second RPTP suppresses the single mutant phenotype, is observed for Lar Ptp52F mutants, and may indicate that these two RPTPs function in a competitive manner (in a formal genetic sense) to regulate a CNS signalling pathway (Jeon, 2008).

The interaction matrix for motor axon guidance is different from the CNS interaction matrix, it can be concluded that the relationships between the RPTP signalling pathways differ in some cases between motor neurons and CNS interneurons. However, Ptp10D Ptp69D double mutants have a synthetic SNa phenotype, so these two RPTPs interact strongly in regulating both CNS and motor axon guidance. Loss of Ptp10D also enhances both the CNS and motor axon defects of Ptp52F mutants (Jeon, 2008).

As in the CNS, there are competitive relationships between RPTPs, but they are seen for a different RPTP pair. In motor axons, removal of Ptp99A completely suppresses the Lar ISNb parallel bypass phenotype. Lar mutations enhance the Ptp52F motor axon phenotypes rather than suppressing them as they do in the CNS (Jeon, 2008).

This paper has defined the phenotypes associated with simultaneous elimination of the functions of two RPTP subfamilies, by examining triple mutants removing both Ptp4E and Ptp10D together with each of the other three RPTPs whose absence produces lethality. This analysis shows that the Ptp10D/Ptp4E subfamily is redundant with Ptp69D in controlling guidance decisions made by three neuronal types, but Ptp4E mutants have relatively minor effects relative to Ptp10D mutants. For guidance of 1D4 and SemaIIB axons within the CNS, removal of both members of the Ptp10D/Ptp4E subfamily together with Ptp69D modulates the phenotype observed in Ptp10D Ptp69D mutants. For SNa axons, the triple mutant has an enhanced phenotype, in that 10% of SNa nerves now fail to extend altogether; this is almost never observed in double mutants. Enhancement of a Ptp10D Ptp52F ISN truncation phenotype was seen by removal of Ptp4E, but no strong interactions between Ptp4E Ptp10D and Lar are observed in the CNS or neuromuscular system (Jeon, 2008).

These results suggest that there is a special relationship between the Ptp10D/Ptp4E subfamily and Ptp69D. Perhaps these two types of RPTPs have similar substrates in both CNS interneurons and motor neurons. In CNS neurons, some critical substrate(s) dephosphorylated by Ptp69D might also be dephosphorylated by either Ptp10D or Ptp4E, so that certain phenotypes, such as crossing of all the SemaIIB axons in the wrong commissure, are observed only when all three RPTPs are eliminated. However, in CNS neurons such as the neuroblast 2-5 lineage, whose axons ectopically cross the midline in Ptp10D Ptp69D double mutants, Ptp4E cannot compensate for the loss of Ptp10D. Perhaps in these cells the relevant Ptp69D substrate(s) can be dephosphorylated by Ptp10D but not by Ptp4E; however, this seems unlikely given that Ptp4E and Ptp10D have PTP domains that are much more similar to each other than are those of Ptp69D and Ptp10D. Alternatively, perhaps the Ptp4E concentration is too low in these neurons for efficient dephosphorylation to occur. Another possibility is that growth cones of these neurons contact Ptp10D ligands, but not Ptp4E ligands, and that ligand contact is required for signalling. An understanding of the biochemical origins of these genetic interactions will require identification and characterization of RPTP ligands, substrates and downstream signalling proteins, as well as localization of these proteins to specific neuronal types (Jeon, 2008).

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission

Many neurons influence their targets through co-release of neuropeptides and small molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release, as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both STED super-resolution and transmission electron microscopy. Ptp4E also controls presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content (Tao, 2019).

Synaptic complexity is enhanced by co-transmission with small molecules and bioactive peptides. The two transmitter classes differ in their postrelease distances traveled and durations of action, thus providing mechanisms for rapid point-to-point control and slow neuromodulation of circuits, development and behavior. Furthermore, from a cell biology perspective, transmission by small molecules and neuropeptides is distinguished by different vesicular loading mechanisms. The genetic results presented here are remarkable because (a) they reveal increased transmitter packaging, when past genetic screens have only yielded mutants that reduce vesicular packaging; (b) control of vesicular packaging varied between neuron subtypes based on differential Rptp expression, which represents a new mechanism for generating variation in co-transmission in the nervous system. Furthermore, this result is intriguing in the context of monoaminergic neurons because Ptp4E interacts genetically with α-synuclein toxicity in Drosophila. Given that synuclein is implicated in Parkinson's disease, DCV fusion pore dynamics and the early secretory pathway, the results here suggest that the mechanistic relationship between Rptps and synuclein may be broader than previously recognized; and (c) presynaptic abundance of a small-molecule vesicular membrane transporter and luminal neuropeptides are regulated in parallel. This shows that regulation of co-transmission is not limited to control of activity-induced vesicle exocytosis. Instead, an Rptp regulates vesicular packaging of both small-molecule and peptide neurotransmitters that underlies co-release (Tao, 2019).

How can a single Rptp simultaneously modify vesicular loading of both monoamines and neuropeptides? Peptidergic neurotransmission relies on packaging of neuropeptides in the soma, where they condense in the TGN and are sorted into DCVs. There is little DCV circulation in octopamine terminals because of their extensive axonal arbors and numerous boutons. Therefore, Rptp regulation of neuropeptide content of individual DCVs likely originates prior to axonal transport. VMAT is also processed in the TGN to be sorted into DCVs and small synaptic vesicles, rather than proceeding through the constitutive secretory pathway. A recent study found that knockdown of the TGN protein HID-1 reduces DCV luminal cargo and VMAT in DCVs by affecting sorting and DCV production. The coordinated effects on neuropeptides and VMAT are reminiscent of the results presented in this study, but the Ptp4E effect on packaging was not associated with a change in DCV number or transport. Therefore, the uncoupling of DCV number from packaging is indicative of a novel cell biological mechanism. With this in mind, a possible explanation for the effect of inhibiting Ptp4E is that the tyrosine phosphorylation stimulates TGN sorting of luminal and vesicle membrane content without changing DCV number or size. By this mechanism, Rptp regulation of vesicular packaging in the soma could scale co-release at the distal synaptic ending (Tao, 2019).

These results pose the question of the site of Ptp4E function. Rptps often mediate signaling triggered by cell-cell contacts. For example, the presynaptic Rptp Lar is activated by muscle Syndecan during development of the NMJ. By analogy, it is possible that presynaptic Ptp4E governs retrograde signaling (e.g., by interrupting tyrosine kinase-dependent mechanisms). In favor of this hypothesis, the closely related Rptp Ptp10D is found on axons in the embryo, where it is positioned to regulate axonal guidance during development. However, the potential involvement of vesicle biogenesis and the unknown localization of Ptp4E raise the possibility that somatic Ptp4E is responsible for synaptic effects. Novel tools to differentially control of Rptp activity by compartment (i.e. soma versus terminal) will be needed to distinguish between these possibilities. Regardless of the cellular location of Ptp4E signaling, the mechanism discovered here (i.e. coincident control of packaging of neuropeptides and small-molecule transmitters) represents a previously unknown cell biological strategy for regulating synaptic co-transmission (Tao, 2019).

Previous experiments have shown that increased VMAT leads to enlargement of vesicles and greater vesicular monoamine storage, but this effect was not seen in this study. Notably, the mechanistic basis of the VMAT expression effect on vesicle size is not understood because thermodynamics with a simple system suggests that maximal vesicular monoamine concentration should be reached even with one VMAT per vesicle. Therefore, to explain the previously observed effect on vesicle size, some other factor, such as monoamine leakage or membrane flexibility, must come into play. It is suggested that these parameters might differ in the synaptic terminals examined in this study. Alternatively, in contrast to the spherical vesicles studied previously, the DCVs in octopamine neurons are ovoid. Therefore, the current analysis of largest dimension cannot exclude that the narrow axis of these DCVs increased. According to the latter scenario, increases in vesicular volume and membrane surface area could have been undetected with the methodology used in the current study (Tao, 2019).

What would the expected consequences be of upregulating VMAT and neuropeptides in vesicles? Upregulating VMAT will increase the speed of vesicle loading when there is exocytosis-endocytosis cycling or kiss-and-run release. Hence, increased VMAT will affect release more when vesicle emptying by release is most marked. In Drosophila, the effect of increased VMAT on behavior has not been examined. However, the dopamine precursor L-DOPA increases vesicular monoamine content and ameliorates Parkinsonian symptoms in humans. Thus, by analogy, it is suggested that octopaminergic signaling would be boosted by increased vesicular octopamine packaging induced by synaptic VMAT upregulation. Of course, increased co-transmission by neuropeptides could further alter octopamine action. Therefore, it would be interesting to explore how Rptps in the brain affect octopamine-dependent fly behaviors such as feeding and egg laying (Tao, 2019).

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 (Sun, 2000), while all longitudinal axons are rerouted into midline-crossing commissural pathways in Lar Ptp10D Ptp69D Ptp99A quadruple mutants (Sun, 2001). 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 (Qian, 2007). 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 (Sun, 2000). 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).

Receptor tyrosine phosphatases control tracheal tube geometries through negative regulation of Egfr signaling

The formation of epithelial tubes with defined shapes and sizes is essential for organ development. This study describes a unique tracheal tubulogenesis phenotype caused by loss of both Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E is the only widely expressed Drosophila RPTP, and is the last of the six fly RPTPs to be genetically characterized. Mutations have been isolated in in Ptp4E; although Ptp4E null mutants have no detectable phenotypes, double mutants lacking both Ptp4E and Ptp10D display synthetic lethality at hatching owing to respiratory failure. In these double mutants, unicellular and terminal tracheal branches develop large bubble-like cysts that selectively incorporate apical cell surface markers. Cysts in unicellular branches are enlargements of the lumen that are sealed by adherens junctions, whereas cysts in terminal branches are cytoplasmic vacuoles. Cyst size and number are increased by tracheal expression of activated Egfr tyrosine kinase, and decreased by reducing Egfr levels. Ptp10D forms a complex with Egfr in transfected cells. Downregulation of Egfr signaling by the RPTPs is required for the construction of tubular lumens, whether extracellular or intracellular, by cells that undergo remodeling during branch morphogenesis. The Ptp4E Ptp10D phenotype represents the first evidence of an essential role for RPTPs in epithelial organ development. These findings might be relevant to organ development and disease in mammals, because PTPRJ (DEP-1), an ortholog of Ptp4E/Ptp10D, interacts with the hepatocyte growth factor receptor tyrosine kinase. PTPRJ corresponds to the murine Scc1 (suppressor of colon cancer) gene (Jeon, 2009).

This paper analyzes the phenotypes of Drosophila embryos lacking both type III RPTPs, Ptp10D and Ptp4E. Ptp10D regulates axon guidance in embryos and larvae. It is selectively expressed on CNS axons and tracheal branches, and is apically localized in tracheal cells (Jeon, 2009).

Ptp4E is broadly expressed, and Ptp4E single mutants have no detectable phenotypes. The Ptp10D/Ptp4E gene pair was generated by a relatively recent duplication event, and the two proteins are closely related to each other. Although Ptp10D and Ptp4E single mutants are viable and fertile, double mutants die at the end of embryogenesis. These data show that Ptp10D and Ptp4E have partially redundant functions during development. The lethality of the double mutants is due to respiratory failure that results from the malformation of tracheal branches (Jeon, 2009).

The Drosophila tracheal system consists of a network of tubes with different sizes and architectures. The larger tubes are multicellular, and have lumens surrounded by several cells that are separated by intercellular junctions. Smaller tracheal branches are composed of unicellular tubes with autocellular AJs. The lumen of a unicellular tube is surrounded by the apical surface of a single cell, and is sealed by a longitudinal AJ. The smallest tubes, called tracheoles, are intracellular, seamless structures that form within the cytoplasm of terminal tracheal cells (Jeon, 2009).

Loss of both type III RPTPs alters the shape of tracheal lumens, so that unicellular and seamless tubes develop large bubble-like cysts. Multicellular tubes, however, are unaffected. This is a unique phenotype that has never been seen in any single mutant. The cysts are modified apical compartments, because they selectively incorporate apical but not basolateral cell surface markers (Jeon, 2009).

Cysts in seamless tubes are necessarily intracellular compartments. Cysts in unicellular tubes, however, are expanded segments of lumen, because they are associated with single AJs. Accumulation of luminal matrix markers in unicellular tubes is reduced in double mutants, suggesting that they have a selective defect in lumen formation in certain tube types (Jeon, 2009).

RPTPs are regulators of tyrosine kinase signaling pathways. By examining the effects of manipulating tyrosine kinase activity on the cyst phenotype, this study showed that the Egfr receptor tyrosine kinase is central to cyst formation. Increasing Egfr activity enhances the cyst phenotype, while reducing Egfr suppresses it. The Btl (FGF receptor) tyrosine kinase also contributes to the phenotype. Egfr physically associates with Ptp10D in cultured Drosophila cells (Jeon, 2009).

The data suggest that the phosphatases are negative regulators of Egfr signaling, and that the loss of this negative regulation is partially responsible for the cystic phenotype of Ptp4E Ptp10D double mutants. Cysts are not observed when activated Egfr (Egfr^Elp) is expressed in tracheae in a wild-type background, but it is not known if Egfr is activated to the same extent by the Elp mutation as it is by loss of the RPTPs. If it is, then excess tyrosine phosphorylation of RPTP substrates in other signaling pathways, perhaps including the FGF receptor pathway, might be required together with Egfr upregulation in order to produce the cyst phenotype (Jeon, 2009).

Ptp10D and Ptp4E, which have catalytic domain sequences that are 89% identical, are likely to dephosphorylate the same targets, explaining why the phenotype is observed only when both RPTPs are absent. Ptp10D forms a complex with Egfr, and it is likely that the closely related Ptp4E would do so as well. Studies in mammalian cell culture have shown that the growth factor receptor tyrosine kinase Met can be dephosphorylated by the type III RPTP DEP-1, which is 50% identical to Ptp10D and Ptp4E within the catalytic domain (Palka, 2003). These findings, together with genetic and biochemical analysis, suggest that autophosphorylated Drosophila Egfr might be a direct target for the enzymatic activities of Ptp10D and Ptp4E (Jeon, 2009).

The fact that apical and luminal cell surface markers accumulate in cysts in unicellular and terminal branches, but not in multicellular branches, suggests that lumen formation in unicellular and terminal branches involves a common mechanism that is not required in multicellular tubes. This is odd, because the apical/luminal surface appears to be generated in a very different manner in unicellular and terminal branches. Lumen in unicellular branches is an extracellular compartment created by remodeling of the cell surface, whereas lumen in terminal branches is an intracellular compartment created by vesicular trafficking (Jeon, 2009).

A possible solution to this conundrum is suggested by the observation that cells in multicellular branches increase in size during development but do not radically change their shapes. Cells in unicellular and terminal branches, however, undergo dramatic shape changes during branch outgrowth. These changes involve reorganization of the apical cell surface in unicellular branches, and the de novo generation of an apical compartment in terminal branches. In unicellular branches with autocellular AJs, the apical/luminal surface coordinates its growth with the remodeling of the cell as a whole, wrapping around the luminal matrix and forming a junction with itself. During elongation of terminal branches, apical surface and lumen markers are localized to growing lines within cells, indicating that lumen and its enclosing apical membrane form in synchrony with the extension of the rest of the cell (Jeon, 2009).

It is speculated that negative regulation of Egfr signaling by Ptp10D and Ptp4E is important for synchronizing apical membrane expansion with the remodeling of the rest of the cell. This could be mediated by phosphotyrosine-regulated interactions between the cytoskeleton and the apical membrane. In this context, it is interesting that loss of the C. elegans ERM (ezrin-radixin-moesin) homolog, which links the apical membrane to the underlying cortical actin network, causes cystic phenotypes in the intestine and the excretory canal (Jeon, 2009).

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. A unique embryonic tracheal tubulogenesis phenotype has been described caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. This study demonstrates that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, it was shown that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. It was also shown that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways (Jeon, 2012).

The Drosophila tracheal system is an intricate branched network of air-filled tubes that delivers oxygen to tissues. Tube formation in the tracheal system involves complex morphogenetic events that differ between tube types. Multicellular tubes have lumens that are surrounded by the apical surfaces of several cells. Unicellular tubes are formed by rolling up of single cells to form junctions with themselves. Seamless tubes are intracellular structures within terminal cells. Many genes have been identified that affect the formation and morphology of tracheal tubes (Jeon, 2012).

The absence of the two Type III RPTPs, Ptp4E and Ptp10D, changes the geometries of the tubes in unicellular and terminal branches, so that they form spherical cysts in place of continuous tubular lumen. The phenotype involves a loss of negative regulation of the Egfr RTK, and Ptp10D physically associates with Egfr (Jeon, 2009; Jeon, 2012).

One of the mammalian Ptp4E/Ptp10D orthologs, PTPRJ (DEP-1), is a direct regulator of multiple growth factor receptor TKs (Arora, 2011; Berset, 2005; Chabot, 2009; Lampugnani, 2003; Kappert, 2007; Palka, 2003; Tarcic, 2009). This led to a test of the hypothesis that Btl and Pvr, the other two Drosophila growth factor receptor TK orthologs that are expressed in embryonic tracheae, are also required for the Ptp4E Ptp10D phenotype (Jeon, 2012).

A quantitative analysis of cyst size at the TC/LT junction showed that tracheally expressed DN mutants of Egfr, Btl, and Pvr all suppress the Ptp4E Ptp10D phenotype almost to wild-type, suggesting that dysregulation of all three RTKs is required for the replacement of linear tubes by spherical cysts. Also, CA mutants of each RTK enhance the phenotype, producing enlarged cysts. These effects cannot be produced by all RTKs, since expression of a CA mutant of InR did not enhance the phenotype (Jeon, 2012).

If the Ptp4E Ptp10D cyst phenotype is the consequence of simultaneous deregulation of all three RTKs, it might be possible to generate the phenotype in a wild-type background by expressing multiple RTK CA mutants. This did not work, even when all three CA mutants were expressed at once. Possible explanations include: 1) because PTP activity normally dominates over RTK activity, the effect on PTyr levels of removing negative regulation by the RPTPs is much greater than that produced by expressing CA RTK mutants in the presence of the RPTPs; 2) other TKs are important for the phenotype, and their activities must also be increased; 3) the RPTPs have PTP-independent activities as adhesion molecules, and generation of the phenotype requires both elevation of RTK activity and the absence of the PTP-independent functions of the RPTPs (Jeon, 2012).

Whether Ptp4E and Ptp10D both regulate all three RTKs was investigated. If they have specificity for particular RTKs, one might be able to generate the cyst phenotype by removing only one RPTP in the presence of a CA RTK mutant. Such combinations were made for Egfr (Ptp4E, Btl>Egfr-CA and Ptp10D, Btl>Egfr-CA), but neither of them had cysts. Since the PTP domains of Ptp4E and Ptp10D are 89% identical, it is likely that they have the same enzymatic targets. The idea that the RPTPs have redundant functions is also consistent with the observation that Ptp4E and Ptp10D single mutants have no detectable phenotypes, while the double mutant is lethal and has both tracheal (Jeon, 2009) and nervous system defects (Jeon, 2008: Jeon, 2012).

When each of the DN RTK mutants was expressed together with a CA mutant of one of the other RTKs in the Ptp4E Ptp10D background, the DN mutant was now unable to suppress the phenotype back to wild-type. Instead the phenotype returned to the strength of unmodified Ptp4E Ptp10D mutants. This shows that if the activity of one RTK is sufficiently elevated, it can replace the requirement for another RTK. Thus, none of the three RTKs is uniquely required to generate the phenotype. Rather, the formation of tubes vs. cysts is controlled by the total amount of activity of certain RTKs in tracheal cells. This implies that the downstream pathways whose increased activity causes cyst formation use SH2-domain effectors that can bind to PTyr sites on any of the three RTKs (Jeon, 2012).

It is interesting that the three RTKs can substitute for each other in regulating the tube vs. cyst decision when they are deregulated by loss of the RPTPs, since the RTKs do not seem to have interchangeable activities in wild-type tracheal cells (or in other tissues). Loss or gain of Btl function produces defects in primary, secondary, and terminal tracheal branching. Loss of Egfr function produces a much more subtle tracheal phenotype affecting tissue integrity. Maintenance of tissue integrity requires signaling through the MAP kinase pathway downstream of Egfr, but is unaffected by reduction of MAP kinase signaling downstream of Btl (Jeon, 2012).

These findings can be explained by the fact that growth factor receptor TKs are usually in an inactive state, due to insufficient levels of ligands and to negative regulation by PTPs. They become active only when they come into contact with elevated levels of their ligands at specific times and places. The activities of Type III RPTPs that dephosphorylate the RTKs might also be transiently reduced at some of these times and places, possibly through interactions of their XC domains with as yet unidentified ligands. As a consequence of the tight control of RTK activity, only those downstream signaling pathways that are most responsive to a particular RTK are likely to be activated by that RTK at any time in wild-type embryos, and the outcomes of signaling through these pathways may also be controlled by the subcellular distributions of ligands, RTKs, RPTPs, and downstream effectors. By contrast, in the absence of Ptp4E and Ptp10D, basal levels of RTK ligands may be able to drive all of the growth factor RTKs to a high level of activity, resulting in strong signaling through all of the downstream pathways they can control. Loss of negative regulation might also cause delocalization of signaling, so that effectors whose activity is normally restricted to particular parts of the cell become activated in a cell-wide manner. Under these conditions, a reduction in the activity of any one of the RTKs by a DN mutant will decrease signaling through multiple downstream pathways. Adding a CA mutant of another RTK can then turn signaling through all of these pathways back up, compensating for the effects of the DN mutant (Jeon, 2012).

The ability of RTKs to substitute for each other in control of cyst formation is conceptually similar to cell transformation through elevation of RTK signaling. RTK activity in cultured cells is tightly controlled, and only a few endogenous RTKs are normally involved in cellular responses to the mitogenic growth factors in their culture medium. Many RTKs can signal through the Ras/MAP kinase pathway, however, and elevated Ras/MAP kinase transduction is sufficient to cause transformation of established cell lines. Thus, oncogenic (CA) mutants of a variety of RTKs are able to transform fibroblastic cell lines when expressed at high levels, regardless of whether the endogenous versions of the RTKs are normally used to regulate proliferation in those cell lines (or are even expressed there) (Jeon, 2012).

The observations on the interchangeability of the RTKs suggests that the decision to form cysts rather than tubes in Ptp4E Ptp10D mutants is very sensitive to the levels of PTyr on the effector binding sites on autophosphorylated RTKs, and that cysts appear when total PTyr rises above a critical threshold. This conclusion is based on the complete suppression of the Ptp4E Ptp10D cyst phenotype that is produced by expression of any of the three DN mutants, even though each DN would eliminate only about 1/3 of total PTyr on the RTKs, if they have roughly equal activities. In wild-type embryos, negative regulation by the RPTPs keeps RTK signaling well below this threshold, so the system is insulated against random fluctuations in phosphorylation or downstream signaling (Jeon, 2012).

The Ptp4E Ptp10D GB cyst number phenotype is completely suppressed by Btl-DN, but not suppressed at all by Egfr-DN and only slightly by Pvr-DN. This might be taken as evidence that elevation of Btl activity is uniquely required to replace GB tubes with cysts. However, when Btl-DN is competed against Pvr-CA, it is only able to suppress the Ptp4E Ptp10D, Btl>Pvr-CA phenotype back to that of unmodified Ptp4E Ptp10D, indicating that Btl can be replaced by Pvr if its activity is sufficiently elevated. These findings can be explained if Btl activity is much higher than Egfr or Pvr activity in GB cells, so that a DN mutant that knocks down endogenous Btl activity by a DN mutant has a greater effect on phosphorylation of effector binding sites than mutants that reduce Egfr or Pvr activities. The activity of CA RTK mutants is independent of the endogenous levels of RTKs, so the Pvr CA mutants could still reverse the effect of Btl-DN even if Pvr-DN has no effect on its own (Jeon, 2012).

To evaluate whether the MAP kinase signaling pathway is involved in the determination of tube geometry, a CA mutant of Phl, the Drosophila Raf kinase, was expressed in the Ptp4E Ptp10D background. Phl-CA enhances the TC/LT phenotype almost as strongly as Egfr-CA. When Phl-CA is combined with Egfr or Btl DN mutants, the phenotype is suppressed back to that of unmodified Ptp4E Ptp10D. Since these RTKs are upstream of Raf, the fact that suppression occurs suggests that pathways other than the MAP kinase pathway are required to generate the phenotype. However, these other pathways may be stimulated by elevation of MAP kinase signaling, since one would expect suppression back to a near-wild-type phenotype if they were completely independent of the MAP kinase pathway (Jeon, 2012).

The involvement of Rho GTPases was tested by expressing DN mutants of Rho1, Rac1, and Cdc42 in the Ptp4E Ptp10D background. Rho1-DN and Rac1-DN completely suppress the TC/LT phenotype, and Cdc42-DN and a tracheally expressed Rho1 RNAi construct produces partial suppression. The DN mutant data do not necessarily show that Rho and Rac are both required for generation of the phenotype. DN mutants may occlude binding of wild-type Rho family GTPases to their GEFs, some of which can act on both Rho and Rac. Therefore high-level expression of Rac-DN might inhibit Rho activation, and vice versa. However, the suppression of the TC/LT phenotype by Rho1 RNAi, which is a specific inhibitor, implicates Rho1 itself in the phenotype. Rho1-CA enhances the Ptp4E Ptp10D phenotype, producing cysts on most DBs (Jeon, 2012).

The differences in the abilities of the three RTK DN mutants to suppress the effects of Phl-CA and Rho-CA may provide clues to the pathways that act downstream of these kinases. The MAP kinase and Rho pathways are not necessarily independent, since Ras/MAP kinase pathway activation can increase Rho-GTP in some cell lines. Also, each of the three RTKs is likely to activate both pathways to some extent, as well as many other downstream pathways. Defining the specific outputs of the MAP kinase and Rho pathways that control tracheal tube geometry and identifying other RTK pathways that regulate tube formation is likely to require genome-wide screens for suppressors and enhancers of the Ptp4E Ptp10D phenotype (Jeon, 2012).

Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication

This study developed a screening method for orphan receptor ligands, in which cell-surface proteins are expressed in Drosophila embryos from GAL4-dependent insertion lines and ligand candidates identified by the presence of ectopic staining with receptor fusion proteins. Stranded at second (Sas) binds to the receptor tyrosine phosphatase Ptp10D in embryos and in vitro. Sas and Ptp10D can interact in trans when expressed in cultured cells. Interactions between Sas and Ptp10D on longitudinal axons are required to prevent them from abnormally crossing the midline. Sas is expressed on both neurons and glia, whereas Ptp10D is restricted to CNS axons. Epistasis experiments were conducted by overexpressing Sas in glia and examining how the resulting phenotypes are changed by removal of Ptp10D from neurons. Neuronal Ptp10D was found to restrain signaling by overexpressed glial Sas, which would otherwise produce strong glial and axonal phenotypes (Lee, 2013).

A screen was devised for orphan receptor ligands in which CSS proteins are ectopically expressed in embryos and ligand candidates are identified by the presence of ectopic staining with receptor-AP fusion proteins. A screen of GAL4-dependent insertion lines was performed for 311 CSS protein genes with the XC domain of the Ptp10D RPTP, and a gene encoding the cell-surface protein Sas was identified. A modified ELISA assay was used to show that Sas and Ptp10D selectively bind to each other in the absence of other cofactors, and demonstrated that Sas and Ptp10D-expressing cells can aggregate with each other (Lee, 2013).

Ptp10D Ptp69D and sas Ptp69D double mutants both have strong ectopic midline crossing phenotypes, suggesting that Sas is required for Ptp10D signaling in the context of longitudinal axon guidance. Sas appears to be expressed on all cells in the CNS, including glia. Genetic epistasis experiments were conducted by overexpressing Sas in glia and asking whether loss of the neuron-specific Ptp10D modifies the resulting phenotype. The results suggest that overexpressed Sas can produce a signal that alters glia and affects their communication with neurons. Interactions between Ptp10D and Sas suppress the production of this signal (Lee, 2013).

RPTP signaling controls the decisions by axonal growth cones to choose longitudinal versus commissural pathways, because in a quadruple Rptp mutant (Ptp10D Lar Ptp69D Ptp99A), all 1D4 (FasII)-positive longitudinal axons are diverted into the commissures and the longitudinal bundles are absent. Ptp10D and Ptp69D are key to these guidance decisions, because triple Rptp mutants in which either Ptp10D or Ptp69D is wild-type have a relatively normal 1D4 pattern, but any mutant combination that includes both Ptp10D and Ptp69D mutations has thick 1D4-positive commissures. This suggests that Ptp10D and Ptp69D share some critical substrate(s) or interacting protein(s) that controls these decisions (Lee, 2013).

sas Ptp69D double mutants also have strong ectopic midline crossing phenotypes that are rescued by selective expression of Sas in FasII neurons. The simplest model to explain these findings is that Ptp10D forms a complex with Sas in FasII-expressing longitudinal tract neurons in order to activate the downstream signaling pathway(s) that it shares with Ptp69D. However, the axons of FasII neurons bundle together, so Sas on one axon could contact Ptp10D on another axon. The sas Ptp69D phenotype can also be rescued by expression of Sas in glia, and Sas protein(s) appear to be deposited in the ECM. Thus, signaling interactions relevant to midline crossing might also be mediated by binding of soluble Sas to Ptp10D on axons (Lee, 2013).

Longitudinal axon guidance and interface glial development are intertwined processes. Perturbation of interface glia can cause longitudinal axons to cross the midline. Conversely, the fates of longitudinal glia, which are a subset of the interface glia, are controlled by signals from neurons (Lee, 2013).

The analysis of glial-neuronal interactions provides an excellent system in which to examine whether signaling through Sas can be regulated by interaction with Ptp10D. Ptp10D is only on axons, whereas Sas is expressed on glia. Driving Sas overexpression in glia with Repo-GAL4 produces only subtle phenotypes. However, genetic removal of Ptp10D from Repo > Sas embryos generates strong ectopic midline crossing phenotypes. These phenotypes are accompanied by disorganization of interface glia. Glial mispositioning might be sufficient to affect axon guidance. However, given the severity of the axonal phenotype, it is thought more likely that the disruption of the glial lattice is reflective of changes in gene expression that cause the glia to send abnormal axon guidance signals to the neurons (Lee, 2013).

The 37 aa Sas cytoplasmic domain interacts with Numb in the yeast two-hybrid assay. Numb is an inhibitor of Notch signaling, and both elevation and loss of Notch signaling affect longitudinal glia. It is suggested that when Sas is overexpressed in glia and is not restrained by Ptp10D binding, it might sequester Numb, thereby increasing Notch signaling (Lee, 2013).

Binding of Sas to Ptp10D on longitudinal axons facilitates Ptp10D's functions in regulation of CNS axon guidance. In glia, overexpressed Sas produces a signal that is suppressed by interactions with neuronal Ptp10D. Other receptors involved in axon guidance exhibit interactions with ligands that produce different signaling outcomes depending on whether the ligands and receptors are expressed on the same or on different cells. In retinal ganglion cells and spinal motor neurons, Eph RTKs interact with Ephrin ligands both on other cells (in trans) and on the same cells (in cis), and cis interactions attenuate the responses of the RTKs to ligand presented in trans (Lee, 2013).

Like Sas, Ephrins and type III neuregulin 1 (a ligand for ErbB RTKs) also generate 'reverse' signals in the cells that express them. These signals are important for axon pathfinding. However, Ephrin and neuregulin signals are produced upon engagement of the ligands with their receptors, not blocked by receptor engagement as in the case of Sas and Ptp10D (Lee, 2013).

The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition

Normal epithelial cells often exert anti-tumour effects against nearby oncogenic cells. In the Drosophila imaginal epithelium, clones of oncogenic cells with loss-of-function mutations in the apico-basal polarity genes scribble or discs large are actively eliminated by cell competition when surrounded by wild-type cells. Although c-Jun N-terminal kinase (JNK) signalling plays a crucial role in this cell elimination, the initial event, which occurs at the interface between normal cells and polarity-deficient cells, has not previously been identified. Through a genetic screen in Drosophila, this study identifies the ligand Sas and the receptor-type tyrosine phosphatase PTP10D as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition. At the interface between the wild-type 'winner' and the polarity-deficient 'loser' clones, winner cells relocalize Sas to the lateral cell surface, whereas loser cells relocalize PTP10D there. This leads to the trans-activation of Sas-PTP10D signalling in loser cells, which restrains EGFR signalling and thereby enables elevated JNK signalling in loser cells, triggering cell elimination. In the absence of Sas-PTP10D, elevated EGFR signalling in loser cells switches the role of JNK from pro-apoptotic to pro-proliferative by inactivating the Hippo pathway, thereby driving the overgrowth of polarity-deficient cells. These findings uncover the mechanism by which normal epithelial cells recognize oncogenic polarity-deficient neighbours to drive cell competition (Yamamoto, 2017).

Normal epithelial cells possess an intrinsic tumour-suppression mechanism against oncogenic neighbours. For instance, in canine kidney cell cultures and zebrafish embryos, oncogenic cells that activate Ras or Src are eliminated from an epithelial monolayer when surrounded by normal cells. Similarly, in the Drosophila imaginal epithelium, oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg1; hereafter dlg) are eliminated from the tissue when surrounded by wild-type cells. The removal of these surrounding wild-type cells abolishes cell elimination and allows scrib^- loss-of-function mutant cells to overproliferate; this context-dependent cell elimination is therefore considered to be cell competition. Genetic studies in Drosophila have revealed that this tumour-suppressive cell competition is driven by JNK-dependent cell death, triggered by the Drosophila tumour necrosis factor (TNF) Eiger. However, the initial mechanism by which normal epithelial cells recognize nearby polarity-deficient cells to drive cell competition have remained unknown (Yamamoto, 2017).

To explore the initial event, which occurs at the interface between normal cells and oncogenic polarity-deficient cells, an ethyl methanesulfonate (EMS)-based genetic screen was conducted in Drosophila for genes required for wild-type 'winners' to eliminate neighbouring polarity-deficient 'losers'. In the eye imaginal epithelium, clones of homozygous mutant scrib^-/- are eliminated when surrounded by wild-type tissue. The elimination of scrib^-/- clones is also evident in adult eyes. Using the FLP/FRT-mediated genetic mosaic technique, EMS-induced homozygous mutations were induced only in wild-type winners and screened for mutations that caused an elimination-defective (eld) phenotype in neighbouring scrib^- loosers. Among 7,490 mutant strains generated, four elimination-defective mutants (eld-4, eld-6, eld-7, and eld-8) that fell into the same lethal complementation group were generated. Clones of scrib^- cells surrounded by eld-4 clones were no longer eliminated but instead grew robustly in the eye disc and survived into adult tissue, causing a characteristic melanization phenotype. Notably, clones of eld-4, eld-6, eld-7, or eld-8 cells showed neither a growth disadvantage of their own nor a suppressive effect on the growth of neighbouring wild-type tissue. Thus, the complementation group eld-4/6/7/8 possesses mutations in a gene required for elimination of neighbouring scrib^- clones (Yamamoto, 2017).

Using a series of chromosomal-deficiency lines and subsequent cDNA sequencing, a nonsense mutation in the coding region of the gene stranded at second (sas) was identified in the eld-4 mutant strain. Encoded by sas is a cell-surface ligand protein that has two extracellular domains-von Willebrand factor type C (VWC) and fibronectin type 3 (FN3) domains-as well as a transmembrane domain. Sas is required for proper axon guidance in the nervous system, but its physiological role in epithelia is unknown. Expression of Sas was indeed lost in eld-4 clones, but ectopic expression of Sas within eld-4 clones surrounding scrib^-/- clones reversed the elimination-defective phenotype. Moreover, the knockdown of Sas in cells surrounding scrib^-/- clones phenocopied the elimination-defective phenotype; a similar elimination-defective phenotype also occurred upon Sas knockdown in cells surrounding dlg^-/- mutant eye-disc clones. These data reveal that the cell-surface ligand Sas is required for normal epithelial cells to eliminate neighbouring polarity-deficient cells (Yamamoto, 2017).

Next, attempts were made to understand the mechanism by which Sas drives the elimination of nearby cells. Sas is normally localized at the apical surface of epithelial cells. Notably, however, this study found that Sas relocalized to the lateral cell surface specifically at the interface between wild-type and scrib^-/- or dlg^-/- clones. This relocalization of Sas at the clone interface was also observed between wild-type and scrib^-/- sas^-/- double-mutant clones, indicating that the Sas protein that accumulates at the clone interface is derived from surrounding wild-type cells (Yamamoto, 2017).

The fact that normal epithelial cells relocalize Sas laterally to eliminate neighbouring oncogenic cells suggests that normal cells transmit a signal to these cells through a cell-surface receptor for Sas. Attempts were made to identify the Sas receptor expressed in polarity-deficient cells. It has been reported that PTP10D, a receptor-type tyrosine phosphatase (RPTP), interacts and functions with Sas during longitudinal axon guidance in the Drosophila nervous system and that Sas-PTP10D trans-signalling occurs through glial-neuronal communication. It was therefore assumed that PTP10D and/or other RPTPs were strong candidates for the Sas receptor in the imaginal epithelium. Given that two extracellular domains of Sas, VWC and FN3, can form homophilic interactions with the same domains of other proteins and that FN3 is a domain commonly shared by RPTPs, Thirty-two RNA interference (RNAi) fly strains were screened that target expression of Drosophila transmembrane proteins bearing either VWC or FN3 domains. Only one RNAi line targeting PTP10D phenocopied the severe elimination-defective and melanization phenotypes when expressed within scrib^-/- or dlg^-/- mutant clones. Like Sas, PTP10D was relocalized to the interface between scrib^-/- and wild-type clones, whereas it normally localized at the apical surface of epithelial cells. This lateral accumulation of PTP10D was almost eliminated when PTP10D-RNAi was expressed within scrib^-/- clones, indicating that the PTP10D accumulating at the clone interface derives from scrib^-/- mutant cells. Furthermore, immunostaining analysis of scrib^-/-sas^-/- double-mutant clones indicated that Sas and PTP10D are localized adjacent to each other in neighbouring cells. Notably, the lateral relocalization of Sas and PTP10D at the clone interface was also observed for the neoplastic non-functional tumour-suppressor mutants vps25^-/-, erupted^-/-, or Rab5DN-expressing cells, all of which are eliminated as losers of cell competition when surrounded by wild-type cells; however, such relocalization was not observed for non-neoplastic polarity stardust^-/- or crumbs^-/- mutants. These data suggest that in response to the emergence of neoplastic polarity-deficient cells, adjacent normal cells relocalize Sas laterally whereas nearby polarity-deficient cells relocalize PTP10D laterally, thereby driving elimination of polarity-deficient cells through trans-activated Sas-PTP10D signalling (Yamamoto, 2017).

Next the mechanism by which Sas-PTP10D signalling drives elimination of polarity-deficient cells was investigated. It has previously been shown that the activation of Eiger-JNK signalling in polarity-deficient cells is essential for their elimination. Therefore, a possible mechanism by which PTP10D knockdown in scrib^-/- clones results in an elimination-defective phenotype is through inhibition of JNK signalling. However, JNK signalling was still strongly activated in scrib^-/- clones expressing PTP10D-RNAi, as assessed by the JNK target MMP1. This indicates that loss of PTP10D drives one or more intracellular signalling events that cause an elimination-defective phenotype in the presence of JNK activation. A strong candidate for this signalling event is activation of Ras signalling, as JNK is converted from pro-apoptotic to pro-growth in the presence of Ras activation. Notably, it has been reported that PTP10D and its mammalian orthologue PTPRJ (also known as DEP1/CD148/SCC1/RPTPeta) negatively regulate epidermal growth factor receptor (EGFR) signalling by directly dephosphorylating the intracellular tyrosine kinase domain of EGFR. This study found that EGFR normally localizes apically in wild-type cells but relocalizes to the lateral surface together with PTP10D at the boundaries between scrib^-/- and wild-type clones. More pertinently, EGFR-Ras signalling was strongly elevated in scrib^-/- clones expressing PTP10D-RNAi, as assessed by downregulation of the transcription factor Capicua. Moreover, co-knockdown of EGFR and PTP10D in scrib^-/- clones completely reversed the elimination-defective phenotype, with EGFR-RNAi alone having only a slight effect on the growth of normal tissue. Furthermore, expression of a constitutively active form of EGFR or Ras caused overgrowth of scrib^-/- clones, while expression of dominant-negative form of Ras in scrib^-/-PTP10D-RNAi clones strongly suppressed their growth. Thus, scrib clones in the absence of PTP10D signalling activate both JNK and Ras signalling and overgrow in a manner dependent on EGFR signalling. The co-activation of EGFR-Ras and Eiger-JNK signalling causes hyper-accumulation of intracellular F-actin, thereby inactivating the tumour-suppressor Hippo pathway. Inactivation of the Hippo pathway triggers nuclear translocation and activation of the downstream transcriptional co-activator Yorkie (Yki), which induces upregulation of various pro-growth and anti-apoptotic genes. Indeed, scrib^-/- clones expressing PTP10D-RNAi strongly accumulated intracellular F-actin and showed strong upregulation of the Yki target gene expanded (ex), as well as an increased nuclear signal of Yki protein; however, scrib mutation alone only slightly upregulated F-actin and ex expression. Furthermore, inhibition of Yki activity by the Yki kinase Warts (Wts) or Yki-RNAi significantly suppressed growth of scrib^-/- clones in the absence of PTP10D, while Wts-overexpression or Yki-RNAi alone had little effect on tissue growth. Similar upregulations of EGFR signalling and Yki activity were observed in scrib^-/- clones when surrounded by sas^-/- eld-4 clones. Finally, he number of dying cells at the boundaries between scrib^-/- and wild-type clones was found to be significantly reduced by PTP10D-knockdown, whereas cell proliferation was significantly increased in scrib^-/- clones expressing PTP10D-RNAi. Together, these data indicate that when neoplastic polarity-deficient cells emerge in the epithelium, neighbouring non-neoplastic cells restrain EGFR signalling of nearby polarity-deficient cells through a Sas-PTP10D trans-interaction, which enables JNK signalling activated in polarity-deficient cells to drive cell elimination. In the absence of Sas-PTP10D, elevated EGFR-Ras signalling in polarity-deficient cells cooperates with JNK signalling to cause Yki activation, thereby leading to an elimination defect and overgrowth of polarity-deficient cells (Yamamoto, 2017).

These data indicate that in response to the emergence of oncogenic polarity-deficient cells, Sas and PTP10D relocalize specifically at the clone interface to the respective lateral surfaces of normal or polarity-deficient cells, enabling the ligand and receptor to interact with each other in trans. Thus, Sas-PTP10D acts as a fail-safe system for epithelial tissue, a system that protects against neoplastic development and is normally latent but activates upon oncogenic cell emergence. Notably, the Sas-PTP10D system was not required for other types of cell competition triggered by Minute, Mahjong, Myc or Yki. Although the mechanism by which Sas and PTP10D relocalize to the clone interface is currently unknown, this study found that the apical proteins Bazooka, Patj, and aPKC and the sub-apical protein E-cadherin also relocalize to the lateral surface of the clone boundary. This suggests that the apical cell surface expands to the lateral region at the clone boundary, meaning that Sas and PTP10D meet each other in trans at the clone interface (Yamamoto, 2017).

The genetic data reveal that Sas and PTP10D act together as tumour suppressors during cell competition. Previous studies have reported that PTPRJ, the mammalian homologue of PTP10D, also acts as a tumour suppressor and negatively regulates EGFR signalling. Although no obvious homologues of Sas have been identified in mammals, thrombospondin-1 and syndecan-2 have been reported to act as ligands for PTPRJ. Given that elimination of scrib-deficient cells by cell competition also occurs in mammalian systems, and that the signalling mechanisms identified in Drosophila are evolutionarily conserved, similar cell-cell recognition mechanisms may help to safeguard human tissues against tumorigenesis (Yamamoto, 2017).

Receptor-like tyrosine phosphatase PTP10D is required for long-term memory in Drosophila

Tyrosine phosphorylation mediates multiple signal transduction pathways that play key roles in developmental processes and behavioral plasticity. The level of tyrosine phosphorylation is regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Extensive studies have investigated the roles of tyrosine kinases in memory formation. However, there were few studies on PTPs. To date, learning has been shown to be defective only for mouse knock-outs of PTPα, leukocyte common antigen-related, or PTPδ. A major limitation of these studies arises from their inability to distinguish an acute (biochemical) impairment of memory formation from a more chronic abnormality in neurodevelopment. From a behavioral screen for defective long-term memory, chi mutants were found to disrupt expression of the PTP10D protein tyrosine phosphatase gene. chi mutants are normal for learning, early memory, and anesthesia-resistant memory, whereas long-term memory specifically is abolished. Significantly, induction of a heat shock-PTP10D⁺ transgene before training fully rescues the memory defect of chi mutants, thereby demonstrating an acute role for PTP10D in behavioral plasticity. PTP10D was found to be widely expressed in the embryonic CNS and in the adult brain. Transgenic expression of upstream activating sequence-PTP10D⁺ in mushroom bodies is sufficient to rescue the memory defect of chi mutants. These data clearly demonstrate that signaling through PTP10D in mushroom bodies is critical for the formation of long-term memory (Qian, 2007).

A P element insertional mutagenesis to screen was performed for X-linked mutants that disrupt 1 d memory after spaced training. Plasmid rescue of genomic DNA flanking the P element (PlacW) insertion in one identified mutant, which was named chi ('chi' is a Chinese word that can be translated as 'foolish'), revealed the transposon to be inserted in the first intron of PTP10D, 345 bp downstream of the splice donor site of exon 1 and 943 bp upstream of the start codon in exon 2. RT-PCR analyses revealed that expression of the PTP10D transcript is greatly reduced in chi mutants. Western blot analysis identified a single protein band of 190 kDa in control flies, which is consistent with a previous observation (Tian, 1991), and this band was undetected in chi/chi mutants. These molecular results suggest that PTP10D is disrupted in chi mutants (Qian, 2007).

These PTP10D mutants were found to be defective in 1 d memory after spaced training. Both chi/chi and EP(X)1172/EP(X)1172 homozygous mutants showed significantly lower 1 d memory after spaced training than control flies. More importantly, two mutant alleles failed to complement; 1 d memory after spaced training in chi/EP(X)1172 heteroallelic mutants was significantly lower than control flies. This finding demonstrates that loss of function of the PTP10D gene per se is responsible for the defect in 1 d memory after spaced training (Qian, 2007).

Therefore, from a behavioral screen for long-term memory mutants identified chi, which carries a molecular lesion of the PTP10D gene. Homozygotes of another, independently isolated mutation in PTP10D, EP(X)1172, also are defective for long-term memory. Assays for different memory phases reveal the memory defect of chi mutants to be specific for long-term memory; STM, MTM, and ARM all were statistically indistinguishable from controls, as were basic sensorimotor responses to odors and footshock. These observations confirm that mutations in the chi locus lead to specific LTM defects (Qian, 2007).

This defect is not resulted from developmental abnormality for the LTM phenotype of chi mutants and can be rescued by acutely induced expression of an hs-PTP10D⁺ transgene in the adult fly, a conclusion also supported by the lack of abnormalities in gross brain morphology in chi mutants. More specifically, the Chi protein PTP10D plays an essential acute role in formation of LTM but is not required for maintenance of LTM. This rescue occurred only when training took place within 3 h of heat shock when induced expression of PTP10D was abundant. However, the heat shock induction failed to rescue LTM when training was conducted 27 h later at which induced expression of PTP10D was becoming undetectable. Moreover, at testing of memory (27 h after heat shock), although induced expression of PTP10D was undetectable, LTM remained to be rescued if training happened within an interval during which induced expression of PTP10D was abundant. Such observations provide a strong demonstration of an acute role for PTP10D during LTM formation (Qian, 2007).

Finally, function of PTP10D for memory formation is localized in the MB. Transgenic expression of a UAS-PTP10D⁺ transgene only in MBs is sufficient to rescue the LTM defect of chi mutants, a result consistent with many other studies that support a role for MBs during olfactory memory formation. In particular, it has been shown that the absence of the vertical lobes of the mushroom body disrupts LTM, and mutations in Crammer peptides that are expressed in the glial cells around the mushroom body also lead to defective LTM (Comas, 2004). This study presents for the first time that the function of a protein in the mushroom body is required for formation of LTM. All of these lines of evidence lead to the conclusion that this receptor-like tyrosine phosphatase plays a critical role in biochemical cascades that mediate long-term memory formation in Drosophila (Qian, 2007).

Several PTPs have been examined in Drosophila. PTP69D plays a role in axon guidance, whereas Drosophila leukocyte common antigen-related Dlar appears to be required for maturation of growth cones in synapses. The data show that PTP10D is expressed widely in embryonic and adult CNS. Thus, one might expect PTP10D also to play a role in neurodevelopment. To that end, the axon guidance defect of PTP69D mutants is exacerbated in PTP69D;PTP10D double mutants, but PTP10D single mutants display no observable defects in development of the CNS (Desai, 1996; Sun, 2000). The data support these observations and suggest, instead, a more important role for PTP10D in the biochemistry of memory formation (Qian, 2007).

The study provides the following insights. Primarily, this is a novel report in invertebrates that a receptor-like tyrosine phosphatase is involved in memory formation. In vertebrates, there are a number of reports that suggest an involvement of receptor-like tyrosine phosphatases in learning and memory, although it remains to be determined whether such effects are specific to long-term memory formation (Uetani, 2000; Skelton, 2003; Kolkman, 2004). In any case, these observations indicate that signaling via receptor-like tyrosine phosphatases may be a conserved biochemical pathway for learning and memory, as for the cAMP pathway. Also, the extracellular structure of PTP10D is similar to other cell adhesion molecules, suggesting that cell-cell interaction might be a critical signal transduction mechanism during memory formation. Consistent with this notion, mutants for Drosophila integrin and Fas II have been reported to disrupt STM. More generally, cell adhesion molecules have been implicated widely for synaptic plasticity in vertebrates and invertebrates (Qian, 2007).

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).

Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo

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).

β-Integrin de-phosphorylation by the Density-Enhanced Phosphatase DEP-1 attenuates EGFR signaling in C. elegans

Density-Enhanced Phosphatase-1 (DEP-1) (see Drosophila Ptp4E) de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover, dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling (see Drosophila EGFR signaling). Using a substrate-trapping DEP-1 mutant in a proteomics approach, this study identified the C. elegans β-integrin subunit PAT-3 (see Drosophila mys) as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin (see Drosophila rhea) recruitment. The non-phosphorylatable β-integrin mutant pat-3(Y792F) partially suppresses the hyperactive EGFR signaling phenotype caused by loss of dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the β-integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the αβ-integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. The study proposes that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through αβ-integrin activation (Walser, 2017).

Protein tyrosine phosphatase receptor type J (PTPRJ) regulates retinal axonal projections by inhibiting Eph and Abl kinases in mice

Eph receptors play pivotal roles in the axon guidance of retinal ganglion cells (RGCs) at the optic chiasm and the establishment of the topographic retinocollicular map. Previous work has demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) is specifically involved in the control of retinotectal projections in chicks through the dephosphorylation of EphA and EphB receptors. It was subsequently revealed that all the mouse R3 subfamily members (PTPRB, PTPRH, PTPRJ, and PTPRO) of the receptor protein tyrosine phosphatase (RPTP) family inhibited Eph receptors as their substrates in cultured mammalian cells. This study investigated the functional roles of R3 RPTPs in the projection of mouse retinal axon of both sexes. Ptpro and Ptprj were expressed in mouse RGCs; however, Ptprj expression levels were markedly higher than those of Ptpro. Consistent with their expression levels, Eph receptor activity was significantly enhanced in Ptprj-knockout (Ptprj-KO) retinas. In Ptprj-KO and Ptprj/Ptpro-double-KO (DKO) mice, the number of retinal axons that projected ipsilaterally or to the contralateral eye was significantly increased. Furthermore, retinal axons in Ptprj-KO and DKO mice formed anteriorly-shifted ectopic terminal zones in the superior colliculus. c-Abl was found to be downstream of ephrin-Eph signaling for the repulsion of retinal axons at the optic chiasm and in the superior colliculus. c-Abl was identified as a novel substrate for PTPRJ and PTPRO, and the phosphorylation of c-Abl was up-regulated in Ptprj-KO and DKO retinas. Thus, PTPRJ regulates retinocollicular projections in mice by controlling the activity of Eph and c-Abl kinases (Yu, 2018).

PTPRJ Inhibits leptin signaling, and induction of PTPRJ in the hypothalamus is a cause of the development of leptin resistance.

Leptin signaling in the hypothalamus plays a crucial role in the regulation of body weight. Leptin resistance, in which leptin signaling is disrupted, is a major obstacle to the improvement of obesity. This study has demonstrated that protein tyrosine phosphatase receptor type J (Ptprj) is expressed in hypothalamic neurons together with leptin receptors, and that PTPRJ negatively regulates leptin signaling by inhibiting the activation of JAK2, the primary tyrosine kinase in leptin signaling, through the dephosphorylation of Y813 and Y868 in JAK2 autophosphorylation sites. Leptin signaling is enhanced in Ptprj-deficient mice, and they exhibit lower weight gain than wild-type mice because of a reduced food intake. Diet-induced obesity and the leptin treatment up-regulated PTPRJ expression in the hypothalamus, while the overexpression of PTPRJ induced leptin resistance. Thus, the induction of PTPRJ is a factor contributing to the development of leptin resistance, and the inhibition of PTPRJ may be a potential strategy for improving obesity (Shintani, 2017).

Search PubMed for articles about Drosophila Ptp10D

Arora, D., Stopp, S., Bohmer, S. A., Schons, J., Godfrey, R., Masson, K., Razumovskaya, E., Ronnstrand, L., Tanzer, S., Bauer, R., Bohmer, F. D. and Muller, J. P. (2011). Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling. J Biol Chem 286: 10918-10929. PubMed ID: 21262971

Berset, T. A., Hoier, E. F. and Hajnal, A. (2005). The C. elegans homolog of the mammalian tumor suppressor Dep-1/Scc1 inhibits EGFR signaling to regulate binary cell fate decisions. Genes Dev 19: 1328-1340. PubMed ID: 15901674

Chabot, C., Spring, K., Gratton, J. P., Elchebly, M. and Royal, I. (2009). New role for the protein tyrosine phosphatase DEP-1 in Akt activation and endothelial cell survival. Mol Cell Biol 29: 241-253. PubMed ID: 18936167

Comas, D., Petit, F. and Preat, T. (2004). Drosophila long-term memory formation involves regulation of cathepsin activity. Nature 430: 460-463. PubMed ID: 15269770

Desai, C. J., et al. (1996). Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84(4): 599-609. PubMed ID: 96178472

Desai, C. J., et al. (1997). Competition and cooperation among receptor tyrosine phosphatases control motoneuron growth cone guidance in Drosophila. Development 124(10): 1941-52. PubMed ID:

Grazia Lampugnani, M., Zanetti, A., Corada, M., Takahashi, T., Balconi, G., Breviario, F., Orsenigo, F., Cattelino, A., Kemler, R., Daniel, T. O. and Dejana, E. (2003). Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J Cell Biol 161: 793-804. PubMed ID: 12771128

Jeon, M., Nguyen, H., Bahri, S. and Zinn, K. (2008). Redundancy and compensation in axon guidance: genetic analysis of the Drosophila Ptp10D/Ptp4E receptor tyrosine phosphatase subfamily. Neural Dev. 3: 3. PubMed ID: 18237413

Jeon, M. and Zinn, K. (2009). Receptor tyrosine phosphatases control tracheal tube geometries through negative regulation of Egfr signaling. Development 136(18): 3121-9. PubMed ID: 19675131

Jeon, M., Scott, M. P. and Zinn, K. (2012). Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila. Biol Open 1: 548-558. PubMed ID: 23213447

Kappert, K., Paulsson, J., Sparwel, J., Leppanen, O., Hellberg, C., Ostman, A. and Micke, P. (2007). Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation. FASEB J 21: 523-534. PubMed ID: 17158785

Kolkman, M. J., et al. (2004). Mice lacking leukocyte common antigen-related (LAR) protein tyrosine phosphatase domains demonstrate spatial learning impairment in the two-trial water maze and hyperactivity in multiple behavioural tests. Behav. Brain Res. 154: 171-182. PubMed ID: 15302123

Kurusu, M. and Zinn, K. (2008). Receptor tyrosine phosphatases regulate birth order-dependent axonal fasciculation and midline repulsion during development of the Drosophila mushroom body. Mol. Cell. Neurosci. 38(1): 53-65. PubMed ID: 18356078

Lee, H. K., Cording, A., Vielmetter, J. and Zinn, K. (2013). Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication. Neuron 78(5): 813-826. PubMed ID: 23764287

Oon, S. H., Hong, A., Yang, X. and Chia, W. (1993). Alternative splicing in a novel tyrosine phosphatase gene (DPTP4E) of Drosophila melanogaster generates two large receptor-like proteins which differ in their carboxyl termini. J. Biol. Chem. 268: 23964-23971. PubMed ID: 8226938

Palka, H. L., Park, M. and Tonks, N. K. (2003). Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1. J. Biol. Chem. 278: 5728-5735. PubMed ID: 12475979

Palka, H. L., Park, M. and Tonks, N. K. (2003). Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1. J Biol Chem 278: 5728-5735. PubMed ID: 12475979

Qian, M., et al. (2007). Receptor-like tyrosine phosphatase PTP10D is required for long-term memory in Drosophila. J. Neurosci. 27(16): 4396-402. PubMed ID: 17442824

Schindelholz, B., et al. (2001). Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F. Development 128: 4371-4382. PubMed ID: 11684671

Shintani, T., Higashi, S., Suzuki, R., Takeuchi, Y., Ikaga, R., Yamazaki, T., Kobayashi, K. and Noda, M. (2017). PTPRJ Inhibits leptin signaling, and induction of PTPRJ in the hypothalamus is a cause of the development of leptin resistance. Sci Rep 7(1): 11627. PubMed ID: 28912580

Skelton, M. R., et al. (2003). Protein tyrosine phosphatase alpha (PTP alpha) knockout mice show deficits in Morris water maze learning, decreased locomotor activity, and decreases in anxiety. Brain Res. 984: 1-10. PubMed ID: 12932834

Stepanek, L., Stoker, A. W., Stoeckli, E. and Bixby, J. L. (2005). Receptor tyrosine phosphatases guide vertebrate motor axons during development. J. Neurosci. 25: 3813-3823. PubMed ID: 15829633

Sun, Q., et al. (2000). Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo. Development 127: 801-812. PubMed ID: 10648238

Sun, Q., et al. (2001). Complex genetic interactions among four receptor tyrosine phosphatases regulate axon guidance in Drosophila. Mol. Cell. Neurosci. 17(2): 274-91. PubMed ID: 11178866

Tao, J., Bulgari, D., Berkhoudt, D. A., Calderon, M. J., Watkins, S. C., Fonseca Velez, H. J., Sabeva, N., Deitcher, D. L. and Levitan, E. S. (2019). Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission. J Cell Sci 132(7). PubMed ID: 30837287

Tarcic, G., Boguslavsky, S. K., Wakim, J., Kiuchi, T., Liu, A., Reinitz, F., Nathanson, D., Takahashi, T., Mischel, P. S., Ng, T. and Yarden, Y. (2009). An unbiased screen identifies DEP-1 tumor suppressor as a phosphatase controlling EGFR endocytosis. Curr Biol 19: 1788-1798. PubMed ID: 19836242

Tian, S. S., Tsoulfas, P. and Zinn, K. (1991). Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Cell 67: 675-685. PubMed ID: 1657402

Uetani, N., et al. (2000). Impaired learning with enhanced hippocampal long-term potentiation in PTPdelta-deficient mice. EMBO J. 19: 2775-2785. PubMed ID: 10856223

Uetani, N., et al. (2006). Mammalian motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma and delta. J. Neurosci. 26: 5872-5880. PubMed ID: 16738228

Walser, M., Umbricht, C.A., Fröhli, E., Nanni, P. and Hajnal, A. (2017). β-Integrin de-phosphorylation by the Density-Enhanced Phosphatase DEP-1 attenuates EGFR signaling in C. elegans. PLoS Genet [Epub ahead of print]. PubMed ID: 28135265

Yamamoto, M., Ohsawa, S., Kunimasa, K. and Igaki, T. (2017). The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition. Nature 542(7640): 246-250. PubMed ID: 28092921

Yu, Y., Shintani, T., Takeuchi, Y., Shirasawa, T. and Noda, M. (2018). Protein tyrosine phosphatase receptor type J (PTPRJ) regulates retinal axonal projections by inhibiting Eph and Abl kinases in mice. J Neurosci 38(39):8345-8363. PubMed ID: 30082414

date revised: 12 August 2023

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