Nematode culture and crossings
C. elegans strains were maintained on nematode growth medium (NGM) seeded with OP50 bacteria at 15°C as described previously. This temperature was chosen in preference to 20°C, since for unc-83 and unc-84 mutants, elevated temperatures were reported to increase a p-cell nuclei migration defect as well as an egg laying defect, which we thought best to avoid, since our observed phenotype is temperature independent. To create BAT1099 and BAT1488, PMW200 was crossed with BAT197 and BAT661 (Strains list see Suppl. Table 1).
Synchronized young L4 larvae (BAT60) were harvested and resuspended in 1 ml M9 buffer (approx. from three 6 cm dishes). 7 µl of EMS (Ethyl methanesulfonate, Sigma #M0880) was dissolved in 1 ml of M9 and combined with the 1 ml of worms. EMS-treated worms were incubated on a rotary shaker for 4 h at RT, washed 3 times with M9 and finally plated on NGM-plates. After a 30 min recovery, 20 healthy late L4s were transferred to fresh plates (5 P0 animals per plate). F2 generations were screened for an aberrant number of GFP-positive nuclei using a Biosorter (Union Biometrica) and candidates subsequently verified with a fluorescent dissecting microscope. The resulting positive candidate BAT173 was crossed with N2 worms to generate BAT197.
Identification of the mutated bar18 locus
To identify the mutated bar18 locus, we applied a Hawaiian SNP crossing strategy and combined it with whole-genome sequencing (WGS) as described by Doitsidou et al.. Using this strategy we introduced Hawaiian SNPs through crossing and homologous recombination into the genome of the mutant and then narrowed down the location of the mutation following WGS. After each cross, only animals that still show the mutant phenotype are analyzed and, as a result, fewer or no Hawaiian SNPs will be found around the bar18 locus, making it possible to identify it.
Briefly, we crossed bar18 mutant (BAT173) and WT (BAT60) animals with Hawaiian CB4856 males and singled F1 cross progeny. After they self-fertilized and propagated, we singled 41 independent F2s of bar18 worms that showed the nuclei displacement phenotype and 36 independent F2s of WT worms to fresh plates and allowed them to self-fertilize. After their progeny populated 6 cm NGM plates, worms were washed off with M9 and their DNA was isolated using the Gentra Puregene Tissue Kit (Qiagen, #158689) according to the manufacturer’s instructions. Pooled DNA samples of bar18 and WT worms were used to prepare libraries with the Paired-End Sample Prep Kit (Illumina #PE-102-1002) according to the manufacturer’s protocol. Libraries were subjected to WGS on an Illumina HiSeq 2500 System, using single 100 nucleotide reads. Bioinformatic analysis was done using the Galaxy-based CloudMap tool according Minewich et al..
Generation of transgenic rescue lines
To generate BAT1298 (myo-3p rescue) and BAT1300 (eft-3p rescue), the plasmids dBT599 (myo-3p::unc-83_cDNA::SL2::NLS::tagRFP::tbb-2_3'UTR) and dBT573 (eft-3p::unc-83_cDNA::SL2::NLS::tagRFP::tbb-2_3'UTR) were linearized (ScaI) and injected into hermaphrodites as complex arrays at a final concentration of 25 ng/µl or 10 ng/µl respectively, together with 4 ng/µl of a linearized (ScaI) hygromycin resistance plasmid (IR98, a gift from Sebastian Greiss) and 2.5 ng/µl of linearized (FspI) myo-2p::mCherry co-injection marker (pCFJ90, addgene plasmid #19327). Digested (PvuII) bacterial genomic DNA was added to a final DNA concentration of 150 ng/µl.
To generate BAT1906, BAT1907 (both myo-3p recue), BAT1908, BAT1909 (both eft-3p rescue), the plasmids dBT742 (myo-3p::3xFLAG::unc-83_cDNA) and dBT573 (eft-3p::3xFLAG::unc-83_cDNA) were linearized (ScaI) and injected into hermaphrodites as complex arrays at a final concentration of 5 ng/µl, together with 4 ng/µl of a linearized (ScaI) hygromycin resistance plasmid (IR98, a gift from Sebastian Greiss) and 5 ng/µl of linearized (ApaI) ttx-3p::mCherry co-injection marker (dBT158). Digested (PvuII) bacterial genomic DNA was added to a final DNA concentration of 150 ng/µl.
Sequences, plasmids and worm strains are available upon request.
To quantify the nuclei dispositioning phenotype penetrance (Fig. 1B), living worms were assessed under a fluorescent dissecting microscope. To count total numbers of GFP-positive nuclei, worms were immobilized with polystyrene microspheres as described by Fang-Yen et al.. We took image stacks of whole worms using a Leica DM6B-Z microscope. Stacks were opened using Fiji (http://fiji.sc) and nuclei quantified with the help of the multi-selection tool to mark counted nuclei. Sex muscles (vulva and uterine muscles) were excluded when counting.
List of worm strains used
(See Suppl. Table 1.)