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Caenorhabditis elegans PlexinA, PLX-1, interacts with transmembrane semaphorins and regulates epidermal morphogenesis

Takashi Fujii1, Fumi Nakao1, Yukimasa Shibata1, Go Shioi1,2, Eiji Kodama1, Hajime Fujisawa1,2 and Shin Takagi1,*

1 Division of Biological Science, Nagoya University Graduate School of Science, Chikusa-ku, Nagoya 464-8602, Japan
2 CREST, Japan Science and Technology Corporation, 2-6-15, Shiba Park, Minato-ku, Tokyo 105-0011, Japan



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  		Fig. 1. The structure of the plx-1 gene and its product. (A) The primary structure of PLX-1 aligned with the Drosophila PlexA and the mouse plexin-A2 sequences. (B) The similarity (%) between each region of PLX-1 and that of Drosophila PlexA and mouse plexin-A2. (C) A scheme showing the structure of the plx-1 gene, deletions, the construct plx-1::egfp used in the expression analysis and the PCR products used for rescue experiments. DDBJ Accession Number for the plx-1 cDNA is AB080022.

 


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  		Fig. 2. Expression of plx-1::egfp in him-5; ncEx[plx-1::egfp, rol-6(su1006)]. The animals are shown with anterior towards the left. (A) A lateral view of the left side of a third larval-stage male tail at about 30 hours after hatching. GFP expression is observed in R(n) cells (arrows). (C) An early to mid-L4 stage male at about 35 hours after hatching. The ray precursor clusters (arrows) express GFP. (B,D) The corresponding DIC images shown in A,C, respectively. (E) A lateral view of seam cells at L4 stage. Both parental seam cells (arrows) and a daughter cell (arrowhead) express GFP. (F) A ventral view at early L3 stage. GFP expression is observed in ventral cord motoneurons (small arrows) and seam cells (an arrowhead). All vulval precursor cells (some are indicated with arrows) aligned along the ventral midline also express GFP. Scale bars: 20 µm.

 


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  		Fig. 3. Male tail defects of plx-1 mutant, mab-20 mutant and Ce-sema-1 (RNAi) animals. All animals contain him-5. Anterior is towards the left. Arrows indicate ray 1 and arrowheads indicate ray 2. (A,B) DIC photomicrographs of ventral views of (A) a control and (B) a plx-1(nc37) mutant adult tail. In B, ray 1 on both sides show displacement defects but to different extents; the right ray 1 (arrow) is located outside of a fan (class I defect) and the left ray 1 remains in a fan (class II defect). (C-G) DIC photomicrographs of lateral views of a control (C), plx-1(nc37) (D), mab-20(bx24) (E), Ce-sema-1a, Ce-sema-1b (RNAi) (F) and mab-20(bx24); plx-1(nc37) (G) animal. In the mab-20(bx24) animal (E), ray 1 fused to ray 2, and rays 3-5 fused together. The Ce-sema-1a, Ce-sema-1b (RNAi) animal (F) shows displacement of ray 1 similar to the plx-1(nc37) animal (D). In the mab-20(bx24); plx-1(nc37) animal (G), ray 1 is displaced anteriorly and ray 3 fuses to ray 4. Scale bars: 20 µm.

 


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  		Fig. 4. Displacement of ray precursors in plx-1 mutants. Cell boundaries of ray precursor clusters were visualized in wild-type (A,C) and plx-1(nc37) (B,D) animals with jam-1::GFP. All animals contain him-5. (B) At the early L4 stage, ray 1 precursor cluster, processes are localized to the junction site between R1.p, R2.p and the surrounding hypodermal syncytium (hyp7). (A,B) R1.p of a plx-1(nc37) animal is abnormally small (B) compared with that of the control animal (A). In the plx-1 mutant at the mid L4 stage (D), the position of the ray 1 precursor cluster (1) is shifted anteriodorsally from the ray 2 precursor cluster (2) to the body seam cell (s), while it is associated with the ray 2 precursor cluster in the wild-type animal (C). For reference, each ray precursor cluster, R1.p, R2.p, the seam cell (s) and the hypodermal syncythium (hyp7) are labeled. Scale bars: 20 µm.

 


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  		Fig. 5. Seam cell defects of plx-1(nc37) hermaphrodites. Lateral views with anterior towards the left. All animals contain jcIs1; him-5. (A,C,E) DIC images of adult alae (arrowheads) in a control (A) and plx-1 mutant (C,E) hermaphrodites. In plx-1 animals, alae were discontinuous (arrows, C) or bifurcated (E). (B,D,F) Fluorescent images of the jam-1::gfp transgene expression in a control animal (B) and plx-1 mutant animals (D,F) shown in A,C,E. (D) In a plx-1 animal, the seam cell boundaries closed midway (arrows). The position of the gap without seam cells corresponds with that of the gap of the alae. (F) In a plx-1 animal, extra cell boundaries formed within a seam cell where the alae made bifurcations. (G,H) Fluorescent images of the jam-1::gfp transgene expression in a control (G) and a plx-1 L4 (H) hermaphrodite. In the plx-1 animal, the arrangement of seam cells is disrupted (arrowheads). Some seam cells were separated by gaps (asterisks). Scale bars: 10 µm.

 


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  		Fig. 6. Binding of C. elegans semaphorins to PLX-1 expressed in the cultured cells. (A) A western blot of PLX-1 expressed in HEK293T cells. An immunoreactive band the size of 220 kDa was detected for Myc-PLX-1 with anti-Myc antibody. The predicted size of the peptide is 210 kDa. (B) A western blot of C. elegans semaphorins secreted in the culture medium of HEK293T cells. An immunoreactive band the size of 143 kDa (lane 1) and 110 kDa (lane 2) for Ce-Sema-1a-{Delta}C-Fc-AP and Ce-Sema-2a-Fc was detected, respectively, with anti-Fc antibody. The predicted size of each peptide is 139 kDa and 94 kDa, respectively. (C) Scatchard analysis of the binding of Ce-Sema-1a-{Delta}C-Fc-AP to PLX-1. The inset shows the binding curves of Ce-Sema-1a-{Delta}C-Fc-AP to PLX-1 expressed on HEK293T cells (the upper line) and to HEK293T cells transfected with pCAGGS as a control (the lower broken line). (D) Binding of Ce-Sema-1a-{Delta}C-Fc-AP to PLX-1. (i,ii,iii) Untransfected HEK293T cells. (iv,v,vi) HEK293T cells expressing PLX-1. (i,iv) Cells were reacted with the anti-Myc antibody. (ii,v) Cells were reacted with Ce-Sema-1a-{Delta}C-Fc-AP. (iii,vi) Cells were reacted with Ce-Sema-2a-Fc. Scale bar: 100 µm.

 

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