1 Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
2 Department of Biology, University of Southern California, 835 W 37th Street, Los Angeles, CA 90089, USA
* Present address: The Genetics Company, Winterthurstr. 190, 8057 Zürich, Switzerland
Present address: Friedrich-Miescher-Labor der Max-Planck-Gesellschaft, Spemannstr. 37, D-72070 Tübingen, Germany
Author for correspondence (e-mail: zinnk{at}its.caltech.edu)
Accepted August 10, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Receptor tyrosine phosphatase, PTP, Drosophila, Neural development, Axon guidance, Neuromuscular system, Genomics, Motor axon, RNAi
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The four neural RPTPs DPTP10D, DLAR, DPTP69D and DPTP99A have been studied using genetics. All four regulate axon guidance decisions in embryos (Desai et al., 1996; Desai et al., 1997; Krueger et al., 1996; Sun et al., 2000; Sun et al., 2001; Wills et al., 1999). Ptp69D, Dlar (Lar FlyBase) and Ptp10D mutations also cause optic lobe innervation defects in larvae and pupae (Garrity et al., 1999; Newsome et al., 2000) (T. Suzuki et al., and Q. Sun and K. Z., unpublished).
Single mutants that lack DLAR or DPTP69D have guidance phenotypes affecting specific pathway decisions made by embryonic motor axons (Desai et al., 1996; Krueger et al., 1996). However, most guidance decisions within the neuromuscular system are altered only when specific combinations of two or more RPTPs are eliminated, indicating that RPTPs can have partially redundant functions at growth cone choice points (Desai et al., 1996; Desai et al., 1997; Sun et al., 2000; Sun et al., 2001).
RPTPs can also have competitive activities, in a formal genetic sense. DLAR and DPTP99A function in opposition to each other in regulating entry of the ISNb motor nerve into its target ventrolateral muscle (VLM) field (Desai et al., 1997). The Abl tyrosine kinase and its substrate Ena are also involved in DLAR signaling at this decision point (Wills et al., 1999).
Little is known about the biochemical mechanisms involved in control of motor axon guidance by RPTPs. The ligands and/or co-receptors that might interact with their extracellular (XC) domains have not been identified. The in vivo substrates for fly RPTPs are also unknown, although several proteins that they can dephosphorylate in vitro or in transfected cells have been described (Fashena and Zinn, 1997; Wills et al., 1999).
None of the previously characterized Rptp single mutants has strong phenotypes that affect the array of axons within the CNS, as assayed by staining with several different antibody markers. Removal of both DPTP10D and DPTP69D, however, generates a unique phenotype in which many longitudinal axons abnormally cross the midline (Sun et al., 2000). The Ptp10D Ptp69D double mutation interacts with roundabout, commissureless and slit, a set of mutations defining a pathway that repels longitudinal axons from the midline and prevents commissural axons from recrossing it (Zinn and Sun, 1999).
We describe DPTP52F, which is probably the last remaining RPTP encoded in the Drosophila genome. Ptp52F mutations cause specific CNS and motor axon guidance phenotypes, and exhibit genetic interactions with mutations in the other Rptp genes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Molecular biology
PCR amplification of DPTP52F, DPTP26C and DPTP36E fragments was performed on phage stocks of a 9-12 hr gt11 library (Zinn et al., 1988) using primers designed from genomic sequences. cDNA clones were isolated from this and other libraries, including a random-primed 9-12 hour
ZAPII library (K. Z., unpublished), by hybridization to these PCR fragments. We were only able to obtain one cDNA clone encoding a portion of the DPTP52F extracellular (XC) domain. The sequence of this cDNA could encode an open reading frame of 881 amino acids, corresponding to the C-terminal section of the predicted protein CG18243 (1419 amino acids) in Release 2 of GadFly. A number of in-frame stop codons are located upstream of the ATG for the reading frame, which is at nucleotide 580 of our cDNA. However, a comparison of the sequence of our cDNA to the genome sequence revealed that an intron upstream of this ATG had not been completely removed. In the cDNA, the 5' splice site of this intron is fused to sequences within the intron, and the fusion joins the 5' splice site to a site near the branchpoint, 14 nucleotides 5' to the acceptor site. This cDNA is thus a copy of an aberrantly spliced RNA. All other introns within the cDNA sequence were removed in a normal manner. To define the complete coding region for DPTP52F, we then performed reverse transcription-PCR (RT-PCR) experiments on primary embryonic first-strand cDNA preparations. We used a 3' primer within the cDNA, downstream of the aberrantly spliced intron, and a series of 5' primers, the most 5' of which was upstream of the ATG of CG18243 (in the presumed 5' UTR of the CG18243 mRNA; primer sequences available on request). (CG18243 has no ESTs, so its predicted mRNA has no 5' UTR; it begins with the ATG of the predicted protein.) Analysis of the cloned PCR products revealed that the DPTP52F protein sequence begins with the initiating methionine of CG18243. There are several in-frame stop codons immediately upstream of this ATG, so the protein must start here. The authentic DPTP52F sequence is very similar to CG18243, except that the intron that was incompletely removed from our cDNA (intron 3) does not use the predicted CG18243 5' splice site, but rather uses a 5' splice site 42 nucleotides downstream. This is the same 5' splice site that was fused to the branchpoint sequence in our clone. The actual 3' splice site of this intron is as predicted for CG18243. Joining these splice sites preserves the CG18243 reading frame, so the DPTP52F protein contains an extra 14 amino acids, making the entire preprotein 1433 amino acids in length.
dsRNA-mediated genetic interference
This was performed essentially as described in the Carthew laboratory protocol (http://www.pitt.edu/~carthew/manual/RNAi_Protocol.html) (Kennerdell and Carthew, 1998; Schmid et al., 2001). Sequences to be transcribed into dsRNAs were cloned into Bluescript KS+, linearized with the appropriate restriction enzymes, and transcribed in vitro with Ambion T3 and T7 Megascript kits following the manufacturers instructions. Transcripts were annealed in injection buffer (0.1 mM NaPO4, pH 7.8/5 mM KCl) after heating to 95°C for 1 minute and cooled gradually to room temperature by placing the water bath in a foam box over a 18 hour period. We typically injected 0.1 nl of 0.5-2.0 µM solutions of dsRNAs. The Ptp52F dsRNAs corresponded to Ptp52F sequences spanning nucleotides 1279-2444 and nucleotides 2105-3998 (counting from the initiating ATG). Cages were set up using 2- to 4-day-old Oregon R flies. Eggs were collected over a 15- to 30-minute period on grape juice agar plates for subsequent injection. The eggs were lined up lengthwise on double stick tape on a glass slide and immersed in halocarbon oil. Syncitial blastoderm embryos were injected at
50% egg length (EL) using an Eppendorf transjector. After injection, embryos were allowed to develop to stage 16-17 under oil in a moist chamber.
Antibody production
A fragment of the extracellular domain of DPTP52F (amino acids 572-1037) tagged with His6 was expressed in E. coli at the Caltech Protein Expression Facility. Monoclonal antibodies (mAbs) against this fusion protein were generated at the Caltech Monoclonal Antibody Facility and screened based on their ability to recognize the fusion protein on dot blots and by staining of whole mount embryos. mAb 13B8, which worked well for immunostaining of whole mount embryos (Fig. 2), was used at a 1:3 dilution.
|
Database searches
Celera scaffold sequence (Venter et al., 2001) was searched with blastn using cDNA sequences encoding each human PTP named in a publication. PTP protein sequences were used to search the same database using tblastn, and correlations were made between scaffolds and known RPTPs. Unknown PTP-related sequences were examined further to determine if they are pseudogenes. The names of all 19 human RPTP-like proteins, with an Accession Number for a reference sequence for each, are HPTPß=PTPß (X54131), HPTP=DEP-1=CD148 (D37781), GLEPP-1=PTP-U2 (U20489), Sap-1 (D15049), PTP-RQ=PTP-GMC1 (human fragment (134 amino acids) is AF169351, rat rPTP-GMC1 (full sequence) is AF063249), LAR (Y00815), PTP
(L38929), PTP
(U35234), PTPµ (X58288), PTP
(L77886), RPTP
=PTPRT (AF043644), PTPo=PTPRO=hPTP-J=PCP-2=R-PTP-
=PTP-
,=PTPRU=PTP
(U71075), PTP
=RPTPß (M93426), PTP
(L09247), PTP
(M34668), PTP
(X54134), CD45=LCA (Y00062), IA-2=512 antigen=PTP35 (L18983), and phogrin=IA-2ß=PTP-NP=PTP-IAR=ICAAR=PTP-X (AB002385) (the last two are catalytically inactive). The reference sequences and scaffolds of the 16 cytoplasmic PTPs and the nine new PTP pseudogenes we defined are available on request. Our conclusions regarding orthologous relationships between fly, worm and human RPTPs differ from those in a recent review (Walchli et al., 2000). These authors defined orthologs based solely on E values of blastp searches with PTP domains, and did not consider whether homology extends over the entire protein. As these E values are very similar for many pairs of PTP domains, and such values are highly sensitive to gap length, this is not a good way to determine orthologs. For example, they assigned DPTP69D as the CD45 ortholog, and DPTP99A as the PTP
ortholog, but there are no relationships between the XC domain sequences within these pairs.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We performed searches of the Berkeley Drosophila Genome Project (BDGP) genomic sequence database for additional PTP sequences in 1998 and 1999. When these searches were conducted, about 25% of the sequence was available, but much of it was in unordered pieces. We found PTP sequence fragments at three cytological locations: 26C, 36E and 52F. These fragments all encoded a sequence closely related to VHCSAGRGV, the canonical PTP active-site motif (Fauman and Saper, 1996). We isolated and sequenced cDNA clones encoding these three PTPs. Analysis of these sequences showed that we had identified two cytoplasmic PTPs (DPTP26C and DPTP36E) and an RPTP (DPTP52F).
Our analysis of cDNA clones and PCR products showed that the DPTP52F preprotein is 1433 amino acids in length (see Materials and Methods for details). The amino acid sequence immediately following the initiating methionine has a 23 amino acid uncharged region (amino acids 15-38) and resembles a signal peptide. A single predicted transmembrane domain is located at residues 1038-1062. The XC domain has 17 potential N-linked glycosylation sites. Its C-terminal region (amino acids 370-1037) contains five fibronectin type III (FN3) repeats like those found in other Drosophila RPTPs. FN3 repeats are also found in many adhesion and extracellular matrix molecules (Fig. 1D). The N-terminal 370 amino acids of the XC domain is not significantly related to any other sequences in the database.
Among published RPTP sequences, DPTP52F is most similar overall to DPTP4E (42% similarity), although they differ in the number of FN3 repeats in their XC domains (five versus 13). DPTP52F has a single cytoplasmic phosphatase domain that is 32-36% identical in sequence to the PTP domains of the other known Drosophila RPTPs.
We were unable to detect DPTP52F mRNA by in situ hybridization to embryos, suggesting that it is expressed at very low levels. We generated mAbs against the XC domain of DPTP52F, and used these to perform immunohistochemical staining of whole-mount embryos. Expression of DPTP52F is observed at the cellular blastoderm stage. During formation of the ventral furrow, DPTP52F can be detected on cells flanking the invaginating region (Fig. 2A).
Later in development (stages 13-17), DPTP52F is selectively expressed in the CNS (Fig. 2B). This CNS specificity resembles that seen for the four neural RPTPs DPTP10D, DLAR, DPTP69D and DPTP99A (Desai et al., 1994; Hariharan et al., 1991; Sun et al., 2000; Tian et al., 1991; Yang et al., 1991). These RPTPs, however, are localized to axons and are present only at very low levels on neuronal cell bodies. By contrast, DPTP52F appears to be expressed primarily on neuronal cell bodies (Fig. 2C).
By amplifying the antibody signal, we can sometimes detect CNS axon bundles that appear to be positive for DPTP52F staining (data not shown). We cannot state with certainty that these axons express DPTP52F, however, as they are on top of positive cell bodies, and some or all of the brown color of the axon bundles could be due to these underlying cells.
The absence of motor axon staining with anti-DPTP52F is not surprising, because these axon bundles are thin and therefore do not stain strongly. We also cannot visualize expression of DLAR or DPTP99A on motor axons in wild-type embryos, and DPTP10D can only be detected on the SNa nerve (Sun et al., 2000; Sun et al., 2001). Determining that these other RPTPs are axonally localized was not a problem, however, because visualization of positive axon bundles within the CNS was not impaired by staining of underlying cell bodies.
Df(2R)JP4/Df(2R)JP8 embryos, which have a complete deletion of the Ptp52F gene (Fig. 1A), do not stain with anti-DPTP52F mAbs. Embryos homozygous for point mutations in Ptp52F (see below) also do not stain (Fig. 2D). These results show that the mAbs selectively recognize DPTP52F.
Perturbation of DPTP52F expression using RNAi produces axon guidance phenotypes
Injected double-stranded RNA (dsRNA) is a potent and specific inhibitor of gene expression in Drosophila; this method of gene expression blockage is called RNA interference (RNAi) [(Kennerdell and Carthew, 1998; Misquitta and Paterson, 1999); see also (Schmid et al., 2001)]. We initially used RNAi to evaluate the functions of DPTP52F during embryonic development. We examined motor axon and CNS phenotypes in these embryos by staining them with mAb 1D4 (Van Vactor et al., 1993), which recognizes motor axons in the periphery and three paired longitudinal bundles within the CNS. In abdominal segments A2-A7, 32 motor axons exit the CNS within the ISN (intersegmental nerve) and SN (segmental nerve) roots; these then split into five pathways that innervate 30 muscle fibers (Keshishian et al., 1996). The SNa and SNc pathways emerge from the SN root, and the ISN, ISNb, and ISNd pathways arise from the ISN root. ISNb and ISNd are also known as SNb and SNd.
To evaluate the efficacy of RNAi for perturbation of Rptp gene expression, we first injected Dlar dsRNA into embryos and allowed them to develop to stage 16-17. Injection of buffer produced no phenotypes (Fig. 3B,F,J), whereas injection of Dlar dsRNA phenocopied the characteristic Dlar mutant ISNb phenotype, known as parallel bypass (Fig. 3G). In parallel bypass hemisegments, ISNb leaves the ISN tract in an apparently normal manner and continues to extend dorsally, but fails to innervate its VLM targets (Krueger et al., 1996). In Dlar RNAi embryos, 15% of hemisegments had parallel bypass phenotypes. This penetrance is very similar to that observed for zygotic Dlar null mutants (18%) (Desai et al., 1997). SNa and CNS pathways are not affected in Dlar mutants (Krueger et al., 1996), and these also appeared normal in Dlar dsRNA-injected embryos (Fig. 3C,K).
|
The ISNb, ISNd and SNc motor nerves appeared identical to wild type in Ptp52F RNAi embryos (Fig. 3H; data not shown). The SNa nerve, however, displayed a clear and diagnostic phenotype. SNa has a characteristic bifurcated morphology. The bifurcation occurs at a choice point located between muscles 22 and 23. The posterior (or lateral) branch of the SNa innervates muscles 5 and 8, and the anterior (or dorsal) branch innervates muscles 21-24 (Fig. 3I). In embryos injected with Ptp52F dsRNA, either the posterior or anterior branch was sometimes missing (Fig. 3L). The penetrance of this bifurcation phenotype was 18%. In summary, our RNAi experiments suggested that DPTP52F is involved in guidance of longitudinal axons within the CNS and in bifurcation of the SNa motor nerve.
Identification of Ptp52F mutants
We examined a collection of EMS-induced mutations that are lethal over Df(2R)JP4, Df(2R)JP6 and Df(2R)JP8 (Fig. 1A) for the Ptp52F CNS and SNa phenotypes that we had defined using RNAi. There are 13 lethal complementation groups in this interval. We collected embryos from lines representing each of these complementation groups and stained them with mAb 1D4. Mutations within one complementation group produced phenotypes identical to those we had observed with RNAi. This group consists of eight alleles. We sequenced the Ptp52F gene from three of these alleles, and found the following mis-sense mutations (Fig. 1C): Ptp52F18.3, Ser to Pro in the fifth FN3 repeat (S941P), Ptp52F7.8.1, Pro to Ser in the second FN3 repeat (P633S), and Ptp52F8.10.3, Tyr to Asn in the PTP domain (Y1348N).
Transheterozygous Ptp52F18.3/Df(2R)JP4 flies can survive until the first instar larval phase. Ptp52F embryos appear morphologically normal during all phases of development. The peripheral nervous system (PNS) displays no defects, and the pattern of body wall muscle fibers is also normal. There are also no abnormalities in the patterns of expression of the Even-skipped (Eve) and Engrailed (En) homeodomain proteins during segmentation or later within the CNS, suggesting that the fates of Eve- and En-expressing cells are not altered by these mutations (data not shown).
Embryos homozygous for any of the three Ptp52F alleles express no detectable DPTP52F protein, as assayed by staining with anti-DPTP52F mAbs (Fig. 2D). Embryos homozygous for Ptp52F18.3 or transheterozygous for Ptp52F18.3 over Df(2R)JP4 have approximately the same phenotypic penetrances. Thus, Ptp52F18.3 may be a null allele. Ptp52F7.8.1 or Ptp52F8.10.3 produce the same phenotypes as Ptp52F18.3, but with somewhat lower penetrances, suggesting that they may be hypomorphic alleles (see Table 1, Table 2, Table 3). All of these data indicate that the phenotypes described in more detail below are due to the absence of the DPTP52F protein.
|
|
|
|
|
SNa pathways are altered in Ptp52F and double mutants
Ptp52F mutants display a variety of SNa guidance defects. The most common defect, as in Ptp52F RNAi embryos, is a failure to bifurcate (Fig. 6C). In other hemisegments, the SNa has extra branches (Fig. 6B), or stalls near the bifurcation point (Fig. 6D). The penetrances of such SNa phenotypes in Ptp52F18.3 homozygotes or Ptp52F18.3/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%; Table 1).
|
Ptp10D Ptp52F and Ptp52F Ptp69D embryos display synergistic ISNb phenotypes
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 (Fig. 7A). Ptp52F mutations produce any detectable ISNb phenotypes only at low frequencies (<13%; Table 2).
|
Dlar Ptp52F double mutants have parallel bypass phenotypes identical to those of Dlar single mutants (Krueger et al., 1996). Ptp99A mutations cause no ISNb phenotypes on their own or in combination with Ptp52F (Table 2).
DPTP52F regulates ISN outgrowth
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 (Fig. 7C). In Ptp52F mutants, most ISNs are normal (Fig. 7C), but 5-17% terminate at SB (Table 3). Dlar mutations produce SB phenotypes with a similar penetrance (19% for null alleles) (Desai et al., 1997). When Dlar and Ptp52F mutations are combined, the frequency of the SB termination phenotype is similar to those of the single mutants. Ptp99A mutations have no effects on ISN on their own, and also cause no enhancement of the Ptp52F phenotype (Table 3).
Ptp10D and Ptp69D single and double mutants have no ISN phenotypes (Fig. 7D) (Sun et al., 2001). 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 (35%; Fig. 7E) that is less frequently observed in Ptp52F single mutants (<10%). Removal of DPTP69D does not affect the T phenotype, but produces an increase in the SB phenotype (5%40% for Ptp52F8.10.3; Table 3; Fig. 7F). In summary, our 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.
Removal of DLAR suppresses the Ptp52F CNS phenotype
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 (Fig. 4E,F). We do not, however, observe a new synergistic phenotype like that produced by removal of DPTP10D and DPTP69D together (Sun et al., 2000). Removal of DPTP99A does not affect the Ptp52F CNS phenotype (data not shown).
In contrast to these results, we find that when a Dlar mutation is introduced into a Ptp52F mutant background, the morphology of the 1D4-positive bundles reverts to wild type (Fig. 4D). 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.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RPTPs in flies, worms and humans
The existence of complete genome sequences for humans, Drosophila and the nematode C. elegans allows the determination of how many members of each gene family are required for development and function of these metazoans. We find that Drosophila has 14 genes encoding tyrosine-specific phosphatases: six Rptp genes and eight genes encoding potentially active PTPs without transmembrane domains (cytoplasmic PTPs). The two new cytoplasmic PTPs we identified are now called PEZ/CG9493 (DPTP26C) and CG7180 (DPTP36E). The completed genome sequence allowed identification of three additional PTP genes at 8F (CG3101), 61C (CG1228) and 86F (CG14714); none of these is likely to encode RPTPs (Table 4).
|
C. elegans has many more RPTPs (15) and cytoplasmic PTPs (>57) than does the fly (Table 4) (Hutter et al., 2000; Plowman et al., 1999). To determine how many PTPs are encoded in the human genome, we examined the Celera human genome sequence database (Venter et al., 2001). Surprisingly, our analysis showed that all of the genes that are likely to encode functional human PTPs have already been identified by PCR techniques or by ESTs. The human genome appears to encode 17 active RPTPs and 16 cytoplasmic PTPs. We also found nine PTP pseudogenes that had not been previously identified.
Two human PTP-like genes (IA-2 and phogrin) encode orthologs of CG4355 in Drosophila and ida-1 in C. elegans (Zahn et al., 2001). All of these proteins contain sequence changes at crucial positions, indicating that they do not function as active phosphatases (Cai et al., 2001; Magistrelli et al., 1996).
Three of the six Drosophila RPTPs have human and C. elegans counterparts, as follows:
(1) The DPTP10D/DPTP4E subfamily (Type III) is characterized by a large number of FN3 repeats (8) and a single PTP domain. It has a single member in C. elegans (F44G4.8), two members in Drosophila (DPTP10D and DPTP4E), and five members in humans (HPTPß, HPTP
, GLEPP-1, Sap-1 and PTP-RQ). PTP-RQ is only defined in humans by a 134 amino acid fragment, but our analysis indicates that the complete protein is >2000 amino acids and is the ortholog of rat rPTP-GMC1.
(2) The LAR subfamily (Type IIa) has three Ig domains and eight or nine FN3 repeats, and two PTP domains. It has a single member in C. elegans (C09D8.1) and Drosophila (DLAR) and three members in humans (LAR, PTP, and PTP
).
Two other fly RPTPs have counterparts in C. elegans but no obvious human orthologs:
(1) DPTP69D has two Ig domains and three FN3 repeats, and is probably orthologous to C. elegans CLR-1.
(2) DPTP99A corresponds to worm K04D7.4. It has three FN3 repeats and two PTP domains, but the second domain lacks the essential cysteine residue. DPTP69D and DPTP99A have been classified as Type IIa (LAR-like) and Type III (DPTP10D-like), respectively, but this may be incorrect, as their domain organizations differ from the other members of these subfamilies.
DPTP52F does not have clear nematode or human orthologs. It is equally related to a number of different PTPs. DPTP52F might be classified as a member of the DPTP10D/DPTP4E (Type III) subfamily, however, as it has an XC domain with FN3 repeats and only one cytoplasmic PTP domain.
The 11 C. elegans RPTPs that are not orthologous to Drosophila or human RPTPs all have a nematode-specific extracellular domain denoted as FE (Hutter et al., 2000). This domain has no close relatives in Drosophila or other sequenced species.
The human genome encodes nine RPTPs in four subfamilies not found in worms or flies: (1) MAM domain RPTPs (Type IIb), some of which can function as homophilic adhesion molecules (PTPµ, PTP, PTP
and PTPo); (2) Carbonic anhydrase domain-containing (Type V) RPTPs (PTP
and PTP
); (3) Type IV RPTPs, which have short glycosylated extracellular domains (PTP
and PTP
); and (4) CD45 (Type I). (Most human RPTPs have several names, and one name was arbitrarily selected here; see Materials and Methods for listings of all names).
Regulation of axon guidance by DPTP52F
To evaluate the functions of DPTP52F during development, we first used RNAi to inhibit its expression. Ptp52F RNAi embryos have distinctive motor axon and CNS phenotypes (Fig. 3). We used these phenotypes as a tool for analysis of a set of lethal mutations within the interval containing the Ptp52F gene. Mutations in one complementation group produced phenotypes identical to those observed with RNAi. We sequenced the Ptp52F gene from three mutant lines and found nonconservative missense substitution mutations (Fig. 1). Embryos from all three mutant lines fail to stain with anti-DPTP52F mAb, suggesting that the mutant proteins do not fold correctly or are unstable (Fig. 2).
We characterized motor axon phenotypes produced by these mutations. In Ptp52F mutants, the SNa motor nerve fails to bifurcate in a normal manner in about 40% of hemisegments (Table 1). SNa normally splits into anterior and posterior branches at a choice point just distal to muscle 12. Mutant SNa nerves lack either the anterior or posterior branches with approximately equal frequency. SNa nerves can also have ectopic branches or stall near the normal bifurcation point (Fig. 6).
To further examine the roles of DPTP52F in regulating motor axon guidance, we made double mutants lacking DPTP52F together with each of the other four neural RPTPs. Our earlier work has defined three modes of genetic interaction among Rptp genes: partial redundancy (a guidance decision can be facilitated by any one of a set of RPTPs), collaboration (expression of two specific RPTPs is required to allow a decision to take place) and competition (loss-of-function mutations in one Rptp gene suppress a phenotype produced by mutations in the other gene). Some of these genetic interactions could be explained by formation of RPTP heteromultimers (Sun et al., 2001).
Ptp52F exhibits both partial redundancy and competition in its interactions with the other Rptp genes. It has strong interactions with Ptp10D, Dlar and Ptp69D, but not with Ptp99A. Interactions with Ptp10D and Ptp69D are synergistic: both the CNS and motor axon phenotypes are worsened in double mutants. In some cases, there is a dramatic difference between double mutants and either single mutant. For example, the ISNb stall phenotype is almost never observed in either Ptp52F (hypomorph) or Ptp10D mutants (1% of hemisegments), while 30% of hemisegments have this phenotype in double mutants (Table 2). These kinds of interactions suggest that some process needed for ISNb extension through the VLM field can be mediated by either DPTP10D or DPTP52F, and this process only fails when both are missing.
Ptp52F embryos have longitudinal tract phenotypes in which the 1D4-positive bundles are irregular and the outer bundle is often missing. These phenotypes are absent in Dlar Ptp52F double mutants, suggesting that DLAR and DPTP52F interact in a competitive manner to regulate longitudinal tract axon guidance/outgrowth decisions (Fig. 4). Dlar does not suppress the Ptp52F SNa or ISN phenotypes, however, so these two RPTPs do not always interact competitively.
DLAR also participates in another competitive interaction: the Dlar ISNb parallel bypass phenotype is absent in Dlar Ptp99A double mutants (Desai et al., 1997). 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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cai, T., Krause, M. W., Odenwald, W. F., Toyama, R. and Notkins, A. L. (2001). The IA-2 gene family: homologs in Caenorhabditis elegans, Drosophila and zebrafish. Diabetologia 44, 81-88.[Medline]
Desai, C. J., Popova, E. and Zinn, K. (1994). A Drosophila receptor tyrosine phosphatase expressed in the embryonic cns and larval optic lobes is a member of the set of proteins bearing the HRP carbohydrate epitope. J. Neurosci. 14, 7272-7283.[Abstract]
Desai, C. J., Gindhart Jr., J. G., Goldstein, L. S. B. and Zinn, K. (1996). Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84, 599-609.[Medline]
Desai, C. J., Krueger, N. X., Saito, H. and Zinn, K. (1997). Competition and cooperation among receptor tyrosine phosphatases control motoneuron growth cone guidance in drosophila. Development 124, 1941-1952.
Fashena, S. J. and Zinn, K. (1997). The transmembrane glycoprotein gp150 is a substrate for the receptor tyrosine phosphatase DPTP10D in Drosophila cells. Mol. Cell. Biol. 17, 6859-6867.[Abstract]
Fauman, E. B. and Saper, M. A. (1996). Structure and function of the protein tyrosine phosphatases. Trends Biochem. Sci. 21, 413-417.[Medline]
Garrity, P. A., Lee, C. H., Salecker, I., Robertson, H. C., Desai, C. J., Zinn, K. and Zipursky, S. L. (1999). Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase. Neuron 22, 707-717.[Medline]
Hamilton, B. A., Ho, A. and Zinn, K. (1995). Targeted mutagenesis and genetic analysis of a Drosophila receptor-linked protein tyrosine phosphatase gene. Rouxs Arch. Dev. Biol. 204, 187-192.
Hariharan, I., Chuang, P.-T. and Rubin, G. M. (1991). Cloning and characterization of a receptor-class phosphotyrosine phosphatase gene expressed on central nervous system axons in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 88, 11266-11270.[Abstract]
Hidalgo, A. and Brand, A. H. (1997). Targeted neuronal ablation-the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila. Development 124, 3253-3262.
Hutter, H., Vogel, B. E., Plenefisch, J. D., Norris, C. R., Proenca, R. B., Spieth, J., Guo, C. B., Mastwal, S., Zhu, X. P., Scheel, J. et al. (2000). Cell biology: Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes. Science 287, 989-994.
Karim, F. D. and Rubin, G. M. (1999). PTP-ER, a novel tyrosine phosphatase, functions downstream of Ras1 to downregulate MAP kinase during Drosophila eye development. Mol. Cell 3, 741-750.[Medline]
Kennerdell, J. R. and Carthew, R. W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017-1026.[Medline]
Keshishian, H., Broadie, K., Chiba, A. and Bate, M. (1996). The Drosophila neuromuscular junction: A model for studying development and function. Annu. Rev. Neurosci. 19, 545-575.[Medline]
Krueger, N. X., Van Vactor, D., Wan, H. I., Gelbart, W. M., Goodman, C. S. and Saito, H. (1996). The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 84, 611-622.[Medline]
Magistrelli, G., Toma, S. and Isacchi, A. (1996). Substitution of two variant residues in the protein tyrosine phosphatase-like PTP35/IA-2 sequence reconstitutes catalytic activity. Biochem. Biophys. Res. Commun. 227, 581-588.[Medline]
McLaughlin, S. and Dixon, J. E. (1993). Alternative splicing gives rise to a nuclear protein tyrosine phosphatase in Drosophila. J. Biol. Chem. 268, 6839-6842.
Misquitta, L. and Paterson, B. M. (1999). Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): A role for nautilus in embryonic somatic muscle formation Proc. Natl. Acad. Sci. USA 96, 1451-1456.
Morrison, D. K., Murakami, M. S. and Cleghon, V. (2000). Protein kinases and phosphatases in the Drosophila genome. J. Cell Biol. 150, F57-F62.
Newsome, T. P., Asling, B. and Dickson, B. J. (2000). Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics. Development 127, 851-860.
Oon, S. H., Hong, A., Yang, X. H. and Chia, W. (1993). Alternative splicing in a novel tyrosine phosphatase gene (DPTP4E) of Drosophila-melanogaster generates 2 large receptor-like proteins which differ in their carboxyl termini. J. Biol. Chem. 268, 23964-23971.
Patel, N. H. (1994). Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology. Vol. 44 (ed. E. Fyrberg and L. S. B. Goldstein), pp. 446-488. San Diego, CA: Academic Press.
Perkins, L. A., Larsen, I. and Perrimon, N. (1992). corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso. Cell 70, 225-236.[Medline]
Plowman, G. D., Sudarsanam, S., Bingham, J., Whyte, D. and Hunter, T. (1999). The protein kinases of Caenorhabditis elegans: A model for signal transduction in multicellular organisms. Proc. Natl. Acad. Sci. USA 96, 13603-13610.
Schmid, A., Schindelholz, B. and Zinn, K. (2001). Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila. Trends Neurosci. (in press).
Seeger, M., Tear, G., Ferres-Marco, D. and Goodman, C. S. (1993). Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron 10, 409-426.[Medline]
Streuli, M., Krueger, N. X., Tsai, A. Y. M. and Saito, H. (1989). A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proc. Natl. Acad. Sci. USA 86, 8698-8702.[Abstract]
Sun, Q., Bahri, S., Schmid, A., Chia, W. and Zinn, K. (2000). Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo. Development 127, 801-812.
Sun, Q., Schindelholz, B., Knirr, M., Schmid, A. and Zinn, K. (2001). Complex genetic interactions among four receptor tyrosine phosphatases regulate axon guidance in Drosophila. Mol. Cell. Neurosci. 17, 274-291.[Medline]
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.[Medline]
Van Vactor, D., Sink, H., Fambrough, D., Tsoo, R. and Goodman, C. S. (1993). Genes that control neuromuscular specificity in Drosophila. Cell 73, 1137-1153.[Medline]
Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A. et al. (2001). The sequence of the human genome. Science 291, 1304-1353.
Walchli, S., Colinge, J. and van Huijsduijnen, R. H. (2000). MetaBlasts: tracing protein tyrosine phosphatase gene family roots from Man to Drosophila melanogaster and Caenorhabditis elegans genomes. Gene 253, 137-143.[Medline]
Wills, Z., Bateman, J., Korey, C. A., Comer, A. and Van Vactor, D. (1999). The tyrosine kinase Abl and its substrate Enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance. Neuron 22, 301-312.[Medline]
Yang, X., Seow, K. T., Bahri, S. M., Oon, S. H. and Chia, W. (1991). Two Drosophila receptor-like tyrosine phosphatase genes are expressed in a subset of developing axons and pioneer neurons in the embryonic CNS. Cell 67, 661-673.[Medline]
Zahn, T. R., Macmorris, M. A., Dong, W. J., Day, R. and Hutton, J. C. (2001). IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm. J. Comp. Neurol. 429, 127-143.[Medline]
Zinn, K., McAllister, L. and Goodman, C. S. (1988). Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila. Cell 53, 577-587.[Medline]
Zinn, K. and Sun, Q. (1999). Slit branches out: a secreted protein mediates both attractive and repulsive axon guidance. Cell 97, 1-4.[Medline]