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Semaphorin 1a and semaphorin 1b are required for correct epidermal cell positioning and adhesion during morphogenesis in C. elegans

Val E. Ginzburg1,2, Peter J. Roy1,2,* and Joseph G. Culotti1,2,{dagger}

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada
2 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
* Present address: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5427, USA



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  		Fig. 1. Multiple sequence alignment of invertebrate semaphorins. Multiple sequence alignment of members of the class I semaphorin family using the ClustalX program. The alignment includes Tribolium semaphorin 1a (T-Sema-1, Accession Number L26080), Grasshopper semaphorin 1a (G-Sema-1, Accession Number L00709), Drosophila melanogaster semaphorin 1a and semaphorin 1b (D-Sema-1a and D-Sema-1b, Accession Numbers AAF52696 and AAF57816, respectively), C. elegans semaphorin 1a and semaphorin 1b (Ce-Sema-1a and Ce-Sema-1b, Accession Number Q17330 and Accession Number unpublished, respectively). Completely conserved residues are highlighted in black, similar residues in gray. C. elegans Ce-Sema-1a and Ce-Sema-1b predicted domains and motifs are as indicated: signal sequence for secretion (purple), semaphorin domain (yellow), Furin protease consensus cleavage site (green), Met related sequence (MRS) (blue), transmembrane domain (black) and a predicted PDZ binding domain (red).

 


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  		Fig. 2. Molecular structure of smp-1 and smp-2 genes and their predicted protein products. (A) The K14B11 cosmid carries smp-1. Regions deleted in ev708 and ev715 are indicated under the summary of exon/intron structure of the appropriate gene. (B) The smp-1 cDNA is aligned with the predicted protein Ce-Sema-1a. The D1037 cosmid carries smp-2. The region deleted in ev709 is indicated. The smp-2 cDNA is aligned with the predicted protein Ce-Sema-1b. Predicted domains within the proteins are as indicated: signal sequence (purple), semaphorin domain (yellow), Furin protease consensus cleavage site (green), Met related sequence (MRS) (blue), transmembrane domain (black) and a predicted PDZ binding domain (red).

 


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  		Fig. 3. Male tail defect in smp-1 and smp-2 mutants. Posterior is upwards in each panel. The arrows indicate ray 1 or its expected position. (A) The wild-type male tail has nine rays on each side (ventral view). (B-F) A common defect in ray 1 is its position, which ranges from anterior (B,C, lateral views) or anterior and dorsal (D,E, lateral and ventral views, respectively), to a complete absence of an external ray 1 (F, right side).

 


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  		Fig. 4. Perdurant SET cell attachment to ray 1 in the sema-1 mutant males. Male tail epidermal cells and ray precursors clusters are visualized using jam-1::gfp to mark adherens junctions (A-E). Male tail ray 1 and 2 precursor clusters are indicated (arrows). In the wild type (A), the clusters are closer together than in the smp-1 and smp-2 mutants (B). In the smp-1 and smp-2 mutants, the displaced ray 1 always remains connected to the seam syncytium (Set cell) in the adult male tail (D,E), which differs from the wild type (C).

 


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  		Fig. 5. Morphological defect in hyp4 of smp-1 mutant animals results in a displaced mouth phenotype. DIC microscopy reveals a mouth displaced to the ventral side in the mutant (B, arrow) compared with the wild type (A, arrow). (C,D) jam-1::gfp is used to visualize adherens junctions between hypodermal cells. Arrows indicate buccal opening (mouth). (E,F) Analysis of contacts between the mutant (F) and wild-type (E) head epidermal cells are summarized. Instead of forming a cylindrical shaped hyp 4 cell and cylindrical head as in the wild type (A,C,E), in smp-1 (ev715) (B,D,F) hyp 4 forms an abnormal contact with itself (failure to fuse) and probably with hyp 3 and hyp 5 that results in torrid-shaped cells that are thinner on the ventral side. The oddly shaped hyp 4 presumably causes tilting of the pharynx toward the ventral side and consequent formation of a displaced mouth.

 


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  		Fig. 6. Body and tail tip shape defects in L1 stage smp-1 and smp-2 mutant animals. Hyp 10 split tail defects are found in mutants of smp-2 and smp-1 (compare arrows in A,B) The wild-type tail tip (A) and the morphological tail tip defect of smp-1(ev715) and smp-2(ev709) mutants (B) are shown. Wild-type body shape (C) is associated with a normal queue of lateral epidermal seam cells (E). Body shape defects in smp-1(ev715) and smp-2(ev709) (D, arrows) are associated with abnormal orientation or positioning of seam cells (F, arrows).

 


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  		Fig. 7. Touch cell axon guidance defects. Typical PLM axon guidance defects of smp-1 (ev715) and smp-2 (ev709) visualized with a mec-7::gfp integrated transcriptional reporter. Wild-type PLML and PLMR axons migrate longitudinally in a ventrolateral position (A,B), whereas in smp-1 and smp-2 mutants, these axons exhibit abnormal migratory paths (C,D) and branching defects (E,F).

 


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  		Fig. 8. smp-1 and smp-2 expression patterns visualized with gfp transcriptional reporters during embryogenesis. DIC (left) and GFP (right) images of the same embryo. (A,B) The expression pattern of smp-1::gfp can be initially observed at approximately 150 minutes in four cells of the E lineage and later at about 250 minutes of development spread to eight cell of the same lineage. (C,D) At the time of ventral enclosure, the leading edge epidermal cells and the muscle lineage start to express smp-1::gfp. (E,F) In the comma stage, muscle precursors and other cells in the head express smp-1::gfp. (G,H) Initial embryonic expression of smp-2::gfp is observed at the twofold stage in pharyngeal (arrows) and intestinal (arrowheads) cells. These cells were identified using SIMI BioCell CD ROMTM (Schnabel et al., 1997Go).

 


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  		Fig. 9. Post-embryonic stage expression patterns of smp-1 and smp-2 visualized by gfp transcriptional reporters. smp-1::gfp expression was observed in all four quadrants of body wall muscle, all four enteric muscles, all sex-specific muscles of hermaphrodite and male, in distal tip cells throughout their migration and in cells of the head tentatively identified as sensory neuron support cells (not shown). We also observed smp-1::gfp expression in epidermal cells hyp 4, in the head (A, arrowhead), and hyp 10, in the tail whip (B, arrowhead), gonadal sheath cell, sperm and excretory system (data not shown). (D) We have also tentatively identified inner labial neurons (data not shown) and the AVL neuron cell body (arrowhead) and axon (arrow), which is located in the nerve ring and sends its axon along the ventral nerve cord into the tail. (C) smp-2::gfp expression was observed in a mononuclear pharyngeal cell (m6) (open arrow), in tentatively identified URAV/D motorneurons and inner labial neurons, in intestinal cells and in the sphincter muscle (data not shown). smp-2::gfp is also expressed in the body wall and head muscle quadrants (C, white arrow and white arrowhead, respectively).

 

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