QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harrington, R. J. Articles by Chisholm, A. D. Search for Related Content PubMed PubMed Citation Articles by Harrington, R. J. Articles by Chisholm, A. D. Social Bookmarking                         What's this?

The C. elegans LAR-like receptor tyrosine phosphatase PTP-3 and the VAB-1 Eph receptor tyrosine kinase have partly redundant functions in morphogenesis

Robert J. Harrington¹, Michael J. Gutch^2,*, Michael O. Hengartner², Nicholas K. Tonks² and Andrew D. Chisholm^1,

¹ Department of Molecular, Cell, and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA
² Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
^* Present address: Electronic Publishing Services Inc., 880 Third Avenue, 14th Floor, New York, NY 10022, USA

View larger version (23K): [in a new window]   Fig. 1. Structure of the ptp-3 locus. (A) Genomic structure of the ptp-3 locus and location of op147::Tc1. The long transcript, encoding PTP-3A, is generated from 29 coding exons that span 36.2 kb of genomic DNA; this transcription unit is denoted C09D8.1 in the C. elegans genome database (also previously known as clar, ypp-1, and ptp-1). Four large introns are not shown to scale (dotted lines). Exons 1-13 are specific to the long transcript. Exon 14 contains the ATG for the short transcript and is transcribed from an internal promoter. Exon 13 (PTP-3A) or exon 14 (PTP-3B) both splice to exon 15. Exons 15-30 are common to both transcripts. The transmembrane domain (tm) and the D1 phosphatase domain are encoded by exon 26. The Tc1 insertion of op147 is in exon 26, disrupting the D1 phosphatase domain. The PstI site used to insert GFP is indicated. The genomic region contained in cosmid clone F38A3 is indicated. (B) Northern blot of total C. elegans mRNA extracted from mixed-stage populations, probed with the PTP-3B cDNA (see Materials and Methods). Messages of approx. 8 kb and approx. 5 kb are detected. (C) Partial genetic map of the center of linkage group II, showing map location of ptp-3, vab-1, and markers used in strain constructions.

View larger version (71K): [in a new window]   Fig. 2. PTP-3 is the C. elegans ortholog of the LAR subfamily. (A) Percentage identity and similarity of PTP-3A Immunoglobulin-like (Ig-like) domains, Fibronectin Type III (FNIII) repeats, and phosphatase domains to those of its closest relative (DLAR), and a representative vertebrate LAR family member (rat; RnLAR). The PTP-3B isoform is also shown for comparison; cartoons are not to scale. DLAR contains a ninth FNIII repeat; the FNIII domains of PTP-3 align with the first eight repeats of DLAR. PTP-3A is the largest member of the LAR family; the extra size is mostly due to a larger ‘spacer’ region between the last FNIII repeat and the predicted transmembrane domain. Percentages are calculated from alignments using ClustalW. (B) Alignment of the N-terminal (D1) phosphatase domain of PTP-3 with those of other LAR family members (DLAR, HmLAR2, and rat LAR), using ClustalW. Within the phosphatase domains, DLAR is the most similar protein to PTP-3; within the vertebrate LAR family, PTP proteins are slightly more similar to PTP-3 than are LAR or PTP. The op147 insertion disrupts codon Y1705 in the first phosphatase domain, between the conserved residues YINAN and FWRM. The predicted catalytic cysteine residue C1833 is marked (asterisk). Accession numbers are M27700 (DLAR), AF017083 (HmLAR2), and S46216 (rat LAR). The sequences of PTP-3 cDNAs have been deposited in GenGank, with accession numbers AF316539 (PTP-3A) and AF316540 (PTP-3B).

View larger version (89K): [in a new window]   Fig. 3. Embryonic and neuronal expression of PTP-3. (A-F) Expression of PTP-3B-GFP in enclosure stage embryos. Animals are of genotype juEx189; GFP is visualized by anti-GFP immunostaining (green), animals are also immunostained with MH27 to visualize adherens junctions (red). (A-C) A lateral confocal projection of an embryo prior to epidermal enclosure. The dorsal sheet of epidermal cells is visualised by MH27 staining; PTP-3B::GFP expression is widespread in surface cells; expression in ventral neuroblasts is marked in A (arrowhead). (D-F) A post-gastrulation embryo; medial confocal section showing widespread expression of PTP-3B::GFP at cell surfaces in epidermal, neuronal, pharyngeal, muscle, and endodermal tissue layers. (G) Wild-type L1 larva stained with anti-PTP-3 antibodies and MH27. Nerve ring staining is marked by the arrowhead. (H,I) PTP-3B-GFP expression in L1 (H) and adult (I) (juEx189), stained with anti-GFP antibodies; note intense nerve ring expression (arrowhead in I) and staining in neuronal processes. Scale bar, 10 µm (A-F); 30 µm (G, H); 100 µm (I).

View larger version (144K): [in a new window]   Fig. 4. Morphological phenotypes of ptp-3 mutants. (A-D) Representative ptp-3(op147) mutant L1 stage larvae (grown at 25°C) showing defects in morphogenesis. The most common defect is a bulging or pinching of the posterior body (A); however, pinched or notched heads are also occasionally seen (B). Some inviable ptp-3(op147) larvae are starved, apparently a result of defects in pharyngeal morphogenesis (arrow in C). Some ptp-3(op147) larvae are deformed along the entire body (D). A wild-type L1 larva (E) and vab-1(null) mutant larva (F) showing the head morphology defect are shown for comparison. Scale bar, 13 µm (A-D), 20 µm (E,F).

View larger version (24K): [in a new window]   Fig. 5. Transgenic rescue of vab-1 ptp-3 synthetic lethality by PTP-3B. Embryonic lethality was quantitated in transgenic strains as described in Materials and Methods. All strains are homozygous for vab-1(e2) and ptp-3(op147). (A) ptp-3(+) transgenes (4 lines) contain the cosmid F38A3; PTP-3B::GFP transgenes (3 lines) contain the PTP-3B::GFP transgene injected at low concentration (see Materials and Methods). The vab-1 ptp-3 synthetic lethality is suppressed in lines containing these transgenes, relative to control extrachromosomal arrays containing the pRF4 marker plasmid (juEx195). Data for vab-1(e2) (George et al., 1998) are shown for comparison. (B) Suppression of vab-1 ptp-3 synthetic lethality by neuronally expressed PTP-3B (juEx377-379) but not by epidermally expressed PTP-3B (juEx422-424). Only arrays juEx378 and juEx379 caused significant reduction in lethality (Student’s t-test, P<0.01).

View larger version (149K): [in a new window]   Fig. 6. Time-lapse analysis of morphogenesis in ptp-3 mutants and vab-1 ptp-3 double mutants. Time series from five different embryos are shown: the first row is wild type; the second and third rows depict two representative ptp-3(op147) embryos; the fourth row shows a vab-1(dx31) ptp-3(op147) embryo (vab-1(0) double mutant), and the fifth row shows a typical vab-1(e2) ptp-3(op147) embryo (vab-1(k) double mutant). All panels are ventral views, with anterior to the left. First column: beginning of closure of the ventral gastrulation cleft (approx. 230-250 minutes after first cleavage); note the enlarged gastrulation cleft (arrowheads) in all mutant genotypes. Second column: later closure of gastrulation cleft. Third column: early epidermal enclosure. Fourth column: mid-epidermal enclosure. Times for each series are relative to the frame in the first column. Of the two ptp-3 embryos shown, the upper series shows a severely affected embryo, with enlarged gastrulation cleft and early failure in epidermal enclosure; this animal arrested at the enclosure stage, corresponding to the Class I phenotype of vab-1 mutants (George et al., 1998). The lower ptp-3 series shows an embryo with slightly enlarged gastrulation cleft; this embryo underwent normal epidermal enclosure and hatched with normal morphology, corresponding to the Class V phenotype of vab-1 or vab-2 embryos. Both vab-1 ptp-3 embryos shown displayed defects in gastrulation cleft closure and arrested at epidermal enclosure.

View larger version (23K): [in a new window]   Fig. 7. Synergism of ptp-3 with vab-1 and vab-2 and lack of synergism with ina-1 or clr-1. (A) Lethality was quantitated as described in Materials and Methods; error bars show s.e.m. Strains were raised at 20°C unless indicated. Data for vab-1 are from George et al. (George et al., 1998). Strains doubly mutant for ptp-3(op147) and weaker vab-1 kinase alleles (the missense alleles e2 and ju63) showed partially penetrant synergistic lethality yet were viable as homozygotes; strains containing stronger kinase alleles were completely inviable as double mutants with op147 (not shown). (B) Synergism of ptp-3 with efn mutations. Data for efn-1 from Chin-Sang et al. (Chin-Sang et al., 1999). Only efn-1 displays synergistic lethality with ptp-3. Over 90% of the lethality in vab-1(kinase) ptp-3 double mutants occurred during embryogenesis, whereas vab-1(e699) and efn-1 double mutants displayed approx. 60% embryonic lethality and approx. 30% larval lethality. (C) Synergism of the RPTP clr-1 with vab-1 or ptp-3 was tested using the temperature-sensitive clr-1(e1745), which is fully viable at 15°C and 20°C and is a fully penetrant late larval lethal at 25°C. vab-1 clr-1 homozygotes were viable at 20°C; clr-1 ptp-3 strains were viable at 15°C but not at 20°C, possibly suggestive of a mild enhancement of the Clr-1 phenotype. Embryonic lethality was quantitated using balanced strains of genotype clr-1/mIn1 mIs14, vab-1 clr-1/mIn1 mIs14, and clr-1 ptp-3/mIn1 mIs14. Strains were raised at 25°C and the embryonic lethality of non-GFP-expressing animals quantitated.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter