Picture from Hajdu-Cronin Y et al. (2017) MicroPubl Biol "Copulation defective mutants of C. elegans"

To identify genes involved in male copulatory behavior, we carried out an F2 clonal screen in a him-5 mutant background. We identified 20 mutations that affect male mating behavior without causing gross defects in morphology.Male mating in C. elegans comprises at least five steps (Liu and Sternberg, 1995). (l) The male responds to the hermaphrodite by backing his tail along the length of the hermaphrodite, (2) he turns over or under her body before reaching the head or tail, (3) he locates the vulva with his tail, at which point he stops backing, (4) he inserts his spicules into the vulva, and (5) he transfers sperm. To study the genetic basis for male mating behavior, we are isolating and characterizing Copulation Defective (Cod) mutations. We screened for mutant strains defective in this behavior using the screen described by Hodgkin (1983). him-5(e1490) worms are mutagenized with ethyl methane sulfonate (EMS); 20 P0 L4 hermaphrodites are picked singly to Petri plates; ten F1 worms are picked per mutagenized P0; and ten F2 L4 hermaphrodites are singled per P0 and their male progeny tested for mating efficiency via a qualitative mating test (six males crossed with six unc-52(e444) hermaphrodites, which are paralyzed at adulthood (Brenner, 1974). Mutants with phenotypes that are likely to reduce mating efficiency in a non-specific manner (such as those causing an Unc, Dpy, or Lon phenotype) were discarded. Those strains that appear morphologically normal under the dissecting microscope yet fail to mate or mate at a very low efficiency (1-5% cross progeny compared to wild type) were screened under Nomarski optics for defects in male reproductive structures. We screened over 3000 haploid genomes, and picked over 100 strains with reproduction defects. Nineteen strains were successfully backcrossed, which represents about 25% of the total strains attempted. This result suggests that most strains harbor two or more mutations that contribute to the mating-deficiency defect. Preliminary analysis of behavior suggests that every major step in the wild-type mating pathway has at least one corresponding Cod mutation blocking the behavior, with several mutations blocking at the spicule insertion step. The screen also yielded morphological mutants, whose phenotypes include crumpled spicules, abnormal rays, and a gonad migration defect; some of these will be described elsewhere (see Chamberlin & Sternberg; MICROPUB in prep).

Picture from Schwarzbauer JE et al. (1993) Mol Biol Cell "The Caenorhabditis elegans homologue of the extracellular calcium binding ...."

Figure5. Expression of SPARC-lacZ in transgenic animals. L4 larvae were injected with he SPARC-lacZ plasmid plus the dominant marker, rol-6. Transgenic lines were established and stained for f1-galactosidase activity. Staining is shown in an unhatched larva (A, t) and in cells along the sides of an L1 larva (B). (C) Composite of one stained adult hermaphrodite is shown. The head is to the left. Note the symmetrical pattern in the head region and at some positions along the body. Staining extends the length of the animal.

Picture from Tu H et al. (2015) BMC Evol Biol "Membrane-associated collagens with interrupted triple-helices (MACITs): ...."

Fig. 8 Localization of COL-99::EGFP::FLAG at NMJs and in other tissues. a) Detection of COL-99::EGFP::FLAG expression in the head part of an adult C. elegans. Whole mount immunofluorescence staining was performed with a rabbit anti-GFP antibody and AlexaFluor 488-conjugated anti-rabbitIgG. c) Detection of COL-99::EGFP::FLAG in the middle body part. The specimen was prepared by a freeze-crack treatment and a mild PFA fixation.The detection antibody was rabbit anti-GFP. e Staining of methanol/acetone fixed tail (TA) section of a col-99::egfp::flag worm with rabbit anti-GFP(white) and DAPI (blue). The green fluorescence detected with AlexaFluor 488-conjugated secondary antibody was converted to white to enhance the sensitivity. g Double staining of an L1 larva with rabbit anti-GFP (green) and mouse anti-myosin (magenta). The image was converted from a3D Z-scan and signal was adjusted by Image J software. I) Whole-mount immunofluorescent staining of a col-99::egfp::flag adult worm with rabbitanti-GFP (green) and mouse anti-myosin (magenta). k Immunofluorescent staining of a freeze-crack section of worm muscle after mild PFA fixationwith rabbit anti-GFP (green) and mouse anti-myosin (magenta). m-o Immunofluorescent staining of a col-99::egfp::flag worm after freezecracktreatment with rabbit anti-GFP (green in m and o) and alpha-bungarotoxin (magenta in n and o) showing NMJ localization. o Mergedfrom (m) and (n). q-s Immunofluorescent staining of col-99 embryos with rabbit anti-GFP (green in q and s) and alpha -bungarotoxin (magentain r and s). b, d, f, h, j, l, p, and t are negative control staining of unc-119 worms for (a), (c), (e), (g), (i), (k), (o), and (s) respectively. Bars, 5 um in a-h, k-l and m-p, 10 um in i-j, and 20 um in q-t. HE, head; MB, middle body; TA, tail; BM, body muscle; NMJ, neuromuscular junction

Picture from Seetharaman A et al. (2011) Dev Cell "MADD-4 is a secreted cue required for midline-oriented guidance in ...."

Figure S3. madd-4's Post-Embryonic Expression Pattern. A. Shown is the madd-4 genomic locus (with intron 9 highlighed in blue) along with the four constructs used to investigate madd-4's expression pattern. The expression pattern from chromosomally-integrated versions the transcriptional reporter pPR721(madd-4Bp::YFP) (i.e. trIs79) and pPR731(madd-4Bp::RFP) (i.e. trIs80) is presented in Figure 3B and 3A, respectively, of the main text. In (B) through (D), the expression pattern from extra-chromosomal arrays of the transcriptional reporters pPR681(madd-4Ap::RFP) (on the left) and an integrated version of pPR731(madd-4Bp::RFP) (i.e. trIs80) (on the right) is presented. The expression pattern of an extra-chromosomal array of pPR725, which is a fosmid clone carrying a functional (see Figure 1K) bi-cistronically-tagged madd-4 locus within the madd-4 fosmid WMR0626cA02 is similar to that generated by pPR731(madd-4Bp::RFP). On the pPR725 schematic, the small purple box to the left of the red box (that represents coding sequence for mCherry) indicates a nuclear localization signal. See Tursun et al. (2009) for more details on bi-cistronic reporters. B-D. An analysis of the expression pattern of madd-4Ap::RFP (on the left) and madd-4Bp::RFP (on the right). The animals on the left habour an extrachromosomal transgenic array that contains pPR681(madd-4Ap::RFP) and a YFP marker for the array that is expressed in the pharynx (black asterisks). The animals on the right harbour the trIs80 integrated transgenic array that contains the pPR731(madd-4Bp::RFP) construct. In addition, all of the animals shown have the trIs8 chromosomally integrated transgenic array. trIs8 drives expression of YFP in all 26 gabaergic neurons from the unc-25 promoter, including the 19 motor neurons in adults (and the six in young larvae) (Jin et al., 1999), and CFP in only select cholinergic motor neurons (from the unc-129 neural specific promoter) (Colavita et al., 1998), and RFP in the excretory canal (marked with white asterisks). These are markers that allow us to unambiguously identify many of the neurons that express RFP from the madd-4 promoters. 'B' shows typical L1 hatchlings. The RFP channel on the left in B (i.e. madd-4Ap::RFP) is increased in relative intensity in order to visualize the RFP-expressing cells. 'C' shows typical L2 larvae. 'D' shows typical adults. The white arrowheads indicate overlap between RFP-expressing cells, and CFP or YFP-expressing cells. In contrast to madd-4Bp::RFP expression, which is consistently high in all classes of commissural motor neurons in all larval stages and adults, animals carrying madd-4Ap::RFP express low levels of RFP in the first larval stage in both gabaergic and cholinergic motor neurons. madd-4Ap::RFP expression then increases dramatically after the L1 stage, but only in cholinergic cells. The scale bar represents 25 μm. This figure is related to Figure 3.