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Volumn 288, Issue 5463, 2000, Pages 150-154

Positioning of longitudinal nerves in C. elegans by nidogen

Author keywords

[No Author keywords available]

Indexed keywords

ENTACTIN;

EID: 0034616141     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5463.150     Document Type: Article
Times cited : (90)

References (38)
  • 2
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    • note
    • 2 embryos were screened by epifluorescence microscopy for axon scaffold defects and by Nomarski microscopy for general morphological defects. The isolated mutant strains were back-crossed against wild-type (N2) five times to remove other possible mutations. The ur41 mutation was mapped relative to lon-3(e2175) and unc-76(e911). From the Lon non-Unc recombinants, 21 of 97 segregated ur41. Subsequently, ur41 was mapped relative to lin-2S(n545) and unc-76(e911). One of six Lin non-Unc recombinants segregated ur41, which placed nid-1 to right side of lin-25. By complementation test with chromosomal deficiencies, arDf1 deleted nid-1(ur41) but not odr-3. These results ordered ur41 between lin-25 and odr-3 of linkage group V, spanned by 28 genomic cosmids (C. elegans Genome Project). Pools of three to four cosmids were injected at 5 to 10 μg/ml per cosmid along with the dominant transformation marker, pRF4, which contains rol-6(su1006) (29) at 100 μg/ml into ur41 animals carrying a dorsal sublateral nerve GFP marker transgene, kyls123(zcZ1.2::GFP) (30). The nerves were examined for the rescue of the nid-1(ur41) phenotype. Germ line transformation with one cosmid F54F3 (accession number Z79696) completely rescued the ur41 phenotypes. An 11-kb PCR product that included the entire predicted coding sequence for the nidogen gene and 2.5 kb of 5′ flanking sequence also rescued the phenotypes.
  • 3
    • 0002221492 scopus 로고
    • To analyze the migrations of different classes of neurons, we created strains by standard genetic crosses that contained GFP markers for different axons: urls13(unc-119::GFP) for visualization of whole nervous system (23), evls82a(unc-129::GFP) for visualization of DA and DB neurons (26) [for these and other cell names, see J. White, E. Southgate, J. Thompson, S. Brenner, Philos. Trans. R. Soc. (London) B 314, 1 (1986)], rhls4(glr-1::CFP) for visualization of AVA, AVB, AVD, AVE, AVG, and PVC interneurons within the ventral nerve cord (37, 32), and kyls39(sra-6::GFP) for visualization of PVQ neurons (33). Images were obtained by using a Zeiss LSM 410 Invert Laser Scan microscope. For the electron microscopy analysis, animals were fixed with glutaraldehyde and osmium tetraoxide (34). Five or six L4 or young adult animals were aligned within a small agar block, embedded, and sectioned together. Sections were poststained with uranyl acetate and lead citrate.
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    • White, J.1    Southgate, E.2    Thompson, J.3    Brenner, S.4
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    • 0032545933 scopus 로고    scopus 로고
    • The nid-1 (RNAi) animals were generated as described previously [A. Fire etal., Nature 391, 806 (1998)] by using a 1-kb sequence from exon 8, which was cloned into pBLuescript (Stratagene) as template for RNA synthesis. RNA was produced by both T3 and T7 RNA polymerase, and the reactions were pooled before being injected into the intestines of edls20(unc-119::GFP) transgenic animals. Phenotypes of the nervous system were observed under epifluorescence microscopy.
    • (1998) Nature , vol.391 , pp. 806
    • Fire, A.1
  • 8
    • 0342811294 scopus 로고    scopus 로고
    • note
    • Seven PCR fragments, including the whole coding sequence and intron region, were amplified from the genomic DNA of nid-1(ur41) animals. PCR fragments were cloned into a pBluescript vector and subsequently sequenced by automatic sequencer. The mutation was confirmed by sequencing two independent PCR fragments.
  • 10
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    • J. W. Fox et al., EMBO J. 10, 3137 (1991).
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    • Fox, J.W.1
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  • 16
    • 0343681559 scopus 로고    scopus 로고
    • note
    • Immunostaining was performed by using freeze-fracture and methanol-acetone fixation as previously described (35). Polyclonal antibodies raised against mouse nidogen and a mouse monoclonal antibody against myosin heavy chain B (UNC-54) were used. For costaining, anti-rabbit fluorescein-conjugated and anti-mouse rhodamine-conjugated secondary antibodies were used.
  • 17
    • 0342811293 scopus 로고    scopus 로고
    • Supplemental Web material is available at www. sciencemag.org/feature/data/1048303.shl
  • 24
    • 0343681557 scopus 로고    scopus 로고
    • note
    • Transgenic strains were generated by standard methods (36). pIM#194, an expression construct for nid-1, was constructed by cloning the 2.5-kb 5′ flanking region of nid-1 into pPD 95.77 vector (from A. Fire). This GFP construct was coinjected at 10 μg/ml with pRF4 at 100 μg/ml To establish a stable line, IM329 urls151 [pIM#194, pRF4]. transgenes were integrated by γray irradiation. For the ectopic expression construct of nid-7. constructs pIM#195, pIM#196, and pIM#197, were made by using the 7-kb genomic nid-1 coding region, which was amplified by high-fidelity PCR, ligated to Nhe I-Bgl II-digested vectors, pPD96.41, pPD49.83, and pPD96.52 (from A. Fire). These vectors contained 5′ flanking regulatory sequences of mec-7, hsp16-41, and myo-3, respectively (36). The unc-119 regulatory sequence was amplified by using pIM175 as template (23), and cloned into the pPD49.26 vector (from A. Fire) to construct pIM#198. These constructs were injected at 10 μg/ml, with pRF4 into nid-1(ur41); kyls123 (zc21::GFP) animals. The resulting strains are IM330 urEx153 [pIM#195]; nid-1(ur41): kyls123(zc21::GFP); IM331 urEx153 [pIM#196]; nid-1(ur41); kyls123 (zc21::GFP), IM332 urEx154 [pIM#197]; nid-1(ur41); kyls123(zc21::GFP), IM333 urEx155 [pIM#198]; nid-1(ur41); kyls123(zc21::GFP). Ectopic expression of nid-1 was checked by in situ hybridization. IM331 embryos collected 1 to 6 hours after being laid were heat-shocked at 29.5°C for 1 hour. After heat shock, embryos were placed at 20°C, grown to the L4 stage, and examined for mispositioned nerves. Of those treated, 96% (n = 54) of the animals had wild-type nerves. For IM330, IM332, and IM333 animals, 32% (n = 41), 53% (n = 72), and 97% (n = 71) have wild-type nerves. Detection of RNA in whole-mount C. elegans embryos was performed as described (37). AP-anti-Dig antibody was used for alkaline phosphatase (AP)-mediated detection. 4′,6′-Diamidino-2-phenylindole (DAPI, 1 mg/ml) was included in the staining solution to allow nuclei to be identified by epifluorescence microscopy.
  • 38
    • 0343245942 scopus 로고    scopus 로고
    • note
    • We thank C. Bargmann, J. Culotti, E. Hedgecock, H. Hutter, and D. Pilgrim for generously providing GFP marker strains, P. Yurchenco for providing the nidogen antibodies, D. Miller for providing the UNC-54 antibody, the Caenorhabditis Genetics Center for strains, A. Coulson for cosmids, M. Driscoll, G. Patterson, J. Schwarzbauer, and P. Yurchenco for comments on the manuscript, Z. Altun-Gultekin, C.-C. Huang, G. Kao, Y.-S. Lim, P. Yurchenco, and Q. Wang for helpful discussions, R. Patel for assistance with electron microscopy, and X.-C. Ren for superb technical assistance.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.