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First published online 8 April 2004
doi: 10.1242/dev.01063

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Conversion of cell movement responses to Semaphorin-1 and Plexin-1 from attraction to repulsion by lowered levels of specific RAC GTPases in C. elegans

¹ Samuel Lunenfeld Research Institute of Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada
² Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada

View larger version (32K): [in a new window]   Fig. 1. The isolation of a deletion allele within the C. elegans plexin 1 locus. (A) A deletion allele of Ce-plx-1 was isolated from a mutagenized C. elegans N2 strain frozen library screened using a PCR based method (see Materials and methods). The genomic DNA deletion removes all of exon 19 (red dotted rectangle), which encodes the transmembrane domain. (B) The plx-1 genomic DNA sequence was used to design primers to PCR amplify multiple cDNAs that were sequenced and assembled into a full-length clone. The cDNA from plx-1(ev724) was sequenced and revealed abnormal splicing between exons 18 and 20 resulting in a frame-shift mutation. (C) The intracellular portion of C. elegans PLX-1 is highly conserved with human and Drosophila plexins. In particular, the seven residue region (black underline) responsible for RACGTP binding in Drosophila Plexin B is well conserved in Drosophila Plexin A, C. elegans PLX-1 and PLX-2, and human plexins A3, B1 and C1. The RHO-binding region defined for Drosphila Plexin B (red underline) is less well conserved, as a large portion of it is missing in plexins from other species. However, the amino acids bordering this region are well aligned in plexins from C. elegans, Drosophila and human. The alignment includes Hm-PLX-A3 (X87852), Hm-PLX-B1 (X87904), Hm-PLX-C1 (AF030339), Dm-PLX-A (T13937), Dm-PLX-B (T13164), Ce-PLX-1 (NP_500018) and Ce-PLX-2 (NP_497001).

View larger version (97K): [in a new window]   Fig. 2. Ray 1 cells and the adult ray 1 are displaced anteriorly in plx-1(ev724). The position of ray 1 cells was determined by fluorescence microscopy using the ajm-1::GFP reporter in L3 males (Baird et al., 1991; Koppen et al., 2001). For all panels, anterior is left and ventral is bottom. (A) Male rays develop from two bilaterally symmetric ray/SET precursor cells (Rn cells, where n=1-9). In the third larval stage (L3), ray 1-4 cell clusters (pink arrow) lie ventral to their corresponding R1-4.p sister cells. The developing hook (A; white arrow) is located ventral to the ray 1-2 cells. (B-D) The position of adult male rays was determined by DIC microscopy. Ray 1 is observed at an abnormal anterior position in plx-1(ev724) adult males (C,D) when compared with wild-type males (B). In wild-type males, ray 1 (B; white arrow) is observed in close opposition to ray 2 (B; black arrow). A mild ray 1 anterior phenotype (ray 1 class 2) is scored when ray 1 (C; white arrow) is observed just anterior to it normal position (C; black arrow), but is still within the fan structure. A severe ray 1 anterior displacement phenotype (ray 1 class 1) is defined as a ray 1 located outside the fan area (D; white arrow) even further anterior to ray 2 (D; black arrow). A ray 1 that is shorter than in wild-type males is also characteristic of both types of anterior ray 1 displacement (B-D). Ray 1 cells (white arrow) are displaced anterior in plx-1(ev724) L3 males (F) when compared with wild-type animals (E) of the same stage. Other ray cells, including ray 2 cells (E,F; pink arrow), are not affected in plx-1(ev724) L3 males. (G) A detachment of all rays [ray 1 (R1) shown by large arrow] from the male tail syncitium (SET) is always observed in adult wild-type males. (H) Anterior ray 1 (large arrow) displacements in adult plx-1(ev724) males is usually accompanied by a persistent adhesion to the SET. Scale bar: 25 µm.

View larger version (82K): [in a new window]   Fig. 5. GFP reporter expression (schematics show constructs used) for smp-1 and smp-2 relative to ray 1 cells in wild-type and lin-1(e1275) animals. For all panels, anterior is left. (A-E,H) Ventral views; (F,G,I-L) lateral views. (A-E) Reporter genes for smp-1 and smp-2 are expressed in the male tail hook. The translational reporter gene smp-1::GFP is expressed at the cell membrane of the developing hook (A, L3 stage male (smp-1::GFP); B, DIC, arrowheads) and in adult hook cells (E, smp-1::GFP, arrowhead) in wild-type males. The smp-2p::gfp transcriptional reporter is also expressed in the hook (C, smp-2p::gfp; D, smp-2p::gfp/DIC overlay; arrowhead) in close proximity to ray 1 (D, arrows show ray 1) in wild-type adult males. No expression is detected in rays 1-6 for any reporters analyzed (smp-1p::gfp, smp-2p::gfp and smp-1::GFP). (F,G) Both smp-2p::gfp and smp-1::GFP express in rays 7, 8 and 9 (arrows). (H) Expression of smp-2p::gfp is observed in both the normal and ectopic hook (arrowheads) of lin-1 (e1275) mutant males, and in ray 7, 8 and 9 (arrows) and in the ray tail bursa (above arrows in G). (I-L) The ajm-1::GFP reporter was used to determine the position of both the ectopic hook and ray 1 in lin-1(e1275) adult males [the hook focal plane (red) and the ray focal plane (green) are shown in overlays I-L]. In developing lin-1(e1275) males, the ectopic hook (arrowhead) is located anterior to the developing ray 1 cells (I, early L3 stage; J, late L3 stage; K, L4 stage). Ray lineages are normal in lin-1(e1275) males [I, all ray cells are present (green), ray 1 cells shown by arrows]. By the late L3 stage, ray 1 cells are already found anterior to their normal position (J, arrow) and by the L4 stage, ray 1 is still further anterior (K, arrow). In adult lin-1(e1275) males, ray 1 is frequently observed anterior (arrow, class I phenotype) to its normal position relative to other rays (L, numbering), and in closer proximity to the ectopic hook position (L, arrowhead). Scale bars: 20 µm; bar in B applies to A-G, bar in K applies to H-L.

View larger version (21K): [in a new window]   Fig. 6. Model of Semaphorin 1 and Plexin 1 signaling in male ray 1 positioning. (A) In a wild-type genetic background, we find that MIG-2 and CED-10 (RAC GTPAses) are probably redundant in preventing anterior displacement of ray 1 cells (however, see Discussion). There is a requirement for UNC-73 (RAC GEF) in MIG-2 and CED-10 function. Some UNC-73 functions are required in parallel with PLX-1 for preventing this phenotype. RHO-1 GTPases, and the RHO-kinases LET-502 and K08B12.5, appear to be required in parallel with the PLX-1 and UNC-73/MIG-2/CED-10 pathways to prevent anterior ray 1 displacement, but the analyses do not rule out a possible direct feed-forward from PLX-1 signaling to RHO-family GTPase signaling (dashed arrow on left). Debilitation of UNC-73, MIG-2 and CED-10 displaces ray 1 anterior to normal, whereas debilitation of UNC-33 prevents anterior ray 1 displacement. At high (normal) levels of MIG-2 and CED-10, SMP-1 and SMP-2 signaling through PLX-1 helps to prevent anterior displacements of ray 1 (pathway in blue). However, a conversion of PLX-1 function occurs at low levels of both MIG-2 and CED-10 [genotype mig-2(mu28); ced-10(n1993)/+], as a stimulation of the ray 1 anterior positioning function occurs (pathway in red). This implies that high (normal) levels of RAC GTPases (MIG-2 and CED-10) prevent the switch in the polarity of PLX-1 output. Ray anterior displacements require CRMP/UNC-33, which could act as an effector of PLX-1 at low RAC levels (dashed arrow on right), or could act independently. (B) A cell migration model for positioning of ray 1 cells during male development. In a wild-type background [normal mig-2(+) and ced-10(+) (rac) levels], expression of Semaphorin 1 proteins from the hook primordium (green) attracts PLX-1-expressing ray 1 cells (purple) toward the posterior side. At low mig-2(+) and ced-10(+) (rac) levels [genotype mig-2(mu28); ced-10(n1993)/+], ray 1 cells are repulsed away from sources of Semaphorin 1 proteins.

View larger version (49K): [in a new window]   Fig. 3. Transcriptional and translational reporters for plexin 1 are expressed in developing ray cells. For all panels anterior is left and ventral is bottom. The expression pattern of GFP reporters for plx-1 (see Materials and methods, schematics show constructs used) was determined in transgenic males using fluorescence microscopy. A similar expression pattern was observed in transgenic animals with an extra-chromosomal translational reporter array (evEx168) or an integrated transcriptional reporter array (evIs140). (A) Expression of evIs140 is observed in the dividing Rn.a and Rn.p cells, but predominantly in R1.a and R1.p cells (white arrows) in L3 males. (B) Expression of evEx168 encoding the entire plx-1(+) coding sequence (minus four C-terminal amino acids, see Material and methods) fused to a GFP reporter. Expression is observed at the cell membrane of developing ray cells, predominantly in the 3-cell clusters for rays 1 and 2 during the early L3 stage (white arrows). (C,D) Low expression is detected in all ray precursor clusters at the late L3 stage (white arrows). No expression is detected in adult rays for either the transcriptional or the translational reporter (data not shown). Scale bars: A, 15 µm; B-D, 25 µm.

View larger version (55K): [in a new window]   Fig. 4. Localization of C. elegans PLX-1 in Cos7 cells. Cos7 cells were transfected (see Materials and methods) with a DNA construct encoding the C. elegans PLX-1 fused to a MYC epitope (Fujii et al., 2002). Cells were fixed in 4% paraformaldehyde and stained with a mouse monoclonal anti-MYC antibody (9E10) and an Alexa 488-conjugated anti-mouse antibody. Actin microfilaments were revealed using phalloidin-rhodamine. Cell morphology was observed using DIC filters on a Leica DMRA2 microscope. Arrows indicate co-localization of PLX-1 with actin microfilaments in membrane ruffles. Scale bar in A: 20 µm for A-C.

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