Materials and Methods

C. elegans strains and culture conditions
C. elegans were routinely grown on NGMSR plates (Avery 1993). All animals were maintained at 20° on E. coli HB101 (Sulston and Hodgkin 1988) except for mua-3(uy19) (YJ35) mutants, which were maintained at 15°. The wild-type strain was C. elegans variant Bristol, strain N2. Mutant strains used are YJ35 mua-3(uy19) III, OT136 mua-3(rh195) III, CB164 dpy-17(e164) III, CB1072 unc-29(e1072) I, DP38 unc-119(ed3) III, CB1282 dpy-20(e1282ts) IV, CB369 unc-51(e369) V, RB1547 sta-2(ok1860) V, NU3 dbl-1(nk3) V, and YJ208 mua-3(uy19) III; dbl-1(nk3) V.

Mapping of the new mua-3 mutation

SNP mapping:
The death phenotype during the L4 molt was called drop-dead, Drd. To identify the chromosome on which the drd mutation resides, the mutant was crossed to four mutants: unc-29 I, unc-119 III, dpy-20 IV, and unc-51 V. F2s homozygous for the marker from each cross produced approximately 20% Drd except the cross to unc-119 (<3%). This mapped drd to chromosome III. Mating unc-119(ed3) with Drd homozygotes yielded wild-type F1 progeny, indicating the mutation is recessive. Thirteen out of 14 F2 Unc produced no Drd progeny (F3), validating that drd is linked to unc-119 on chromosome III. One Unc produced progeny with both Unc and Drd phenotypes by recombination. This unc-119(ed3) drd (uy19) grows normally at 15°. We used it for further SNP mapping. The unc-119(ed3) drd (uy19) double mutants mated with Hawaiian CB4856 males produced wild-type progeny (F1). We placed 48 F2 Unc singly onto plates, and then the DNA of F3 progeny was collected for further SNP mapping (Wicks et al. 2001; Davis et al. 2005). Because unc-119 is located at 10.9 Mb, SNP markers on ZK1236 (8.4 Mb), C07A9 (9.6 Mb), T21C12 (10.5 Mb), Y39A1A (10.6 Mb), and Y75B8A (12.0 Mb) were selected. One hundred percent (44/44) of Unc progeny carried only the N2 marker (0% Hawaiian) at 12.0 Mb, suggesting that the mutation is located to the left of unc-119. Forty-eight percent (21/44) of Unc progeny carried only the N2 marker at T21C12 (10.5 Mb) and 58% (26/45) of Unc progeny carried only the N2 marker at Y39A1A (10.6 Mb). Based on the recombination frequencies, we estimated the drd location at approximately 10.1–10.2 Mb.

Whole genome sequencing:
The genomes of the mutant strains after outcrossing twice against N2 were sequenced using Illumina sequencing. Sequences were aligned to the WS220 reference C. elegans genome with bowtie2 (Langmead and Salzberg 2012), variants called with the samtools/bcftools suite (Li et al. 2009), and effects on gene function predicted and variants filtered with snpEff and SnpSift (Cingolani et al. 2012). Further specific analyses used vcftools (Danecek et al. 2011), bedtools (Quinlan and Hall 2010), and custom scripts.
After whole genome sequencing, we found a single deletion in the mua-3 gene, but no other mutations in the range of 9.8 Mb to 10.5 Mb for chromosome III. The mutation was also validated because the original strain was from the knockout consortium of C. elegans, where the mutations are generated by the UV/TMP method to create deletions. The new allele of mua-3(uy19) contains a 488-bp in-frame deletion that includes parts of the 30th and 31st exons and the short intron between them. The flanking sequences are as follows: 5′ end of the deletion: 5′-TGAGGAGAATGGATA-3′ and 3′ end of the deletion: 5′-CACCACAGTCCAGTC-3′. The deletion results in deletion of the following 131 amino acids:
RCRCRNGYHD DDPAHPGHRC SFMINECDSS NLNDCDRNAN CIDTAGGYDC ACKAPYRDEG PPQSPGRICR LNECLNPNRN TCDRNADCRD LDYGYTCTCR HGFYDQSPNP QEPGRICIEF QQEEHIERVK V.

Characterization of mua-3 phenotypes

Temperature-dependent lethality:
Eggs were isolated via hypochlorite treatment (Sulston and Hodgkin 1988) and then harvested in M9 buffer to obtain a synchronous population. Synchronized populations of YJ35 mua-3(uy19) mutants and wild-type were transferred to E. coli HB101 seeded NGM plates at the L1 stage and cultivated at 15°, 20°, or 25° in triplicate. Synchronized populations of approximately 100 mua-3 and wild-type L1s were placed onto each plate. At 25°, approximately 30 hr after L1s were given food, L4s started to molt. At 20°, L4s started to molt after approximately 40 hr, and at 15° L4s started to molt after approximately 60 hr. The majority of death occurred during the molt; therefore, timeframes for the experiments were designated accordingly. The number of dead animals and the number of total animals were counted to determine percent survival of mua-3 and wild-type at each hour from 50 hr at 15°, 37 hr at 20°, and 30 hr at 25°.

Sterility:
To determine the number of offspring produced by individual animals of OT136 mua-3(rh195) mutants and wild-type, five L4s of each strain were transferred singly to E. coli-seeded NGM plates and their progeny were counted. Each worm was transferred to a new plate every day to avoid crowding and to visualize all the progeny easily. Progeny were counted 3 d later.

Suppressor screens

Primary screen:
mua-3(uy19) were collected at the L4 stage and were randomly mutagenized with ethyl methanesulfonate (EMS) (Brenner 1974). Po animals were plated onto E. coli HB101 seeded plates and moved to 15°. F1 progeny were synchronized and remained at 15°. The homozygous F2 generation was synchronized and moved to 25°. The surviving animals were isolated.

Secondary screen:
Individual suppressors of the mua-3 mutant lethality were isolated and moved to individual plates at the restrictive temperature, 25°. Sterile animals and escapers (animals that produced more than 90% of nonviable progeny) were removed. A total of 20 F2s passed the secondary screen and were used to found lines maintained at 25°.

Complementation test for Dpy suppressors:
Unidentified suppressor hermaphrodites were crossed with wild-type males. The F1 progeny males were crossed with an alternate unidentified suppressor. The F1 progeny of this cross were counted to calculate the percentage of Dpy. The percentage of males in the population was also counted to determine whether Dpy progeny were from cross-fertilization or self-fertilization. If 50% of the progeny were Dpy, then suppressors failed to complement each other.

RNAi of dbl-1 and dpy-17
The bacteria-mediated feeding RNAi was performed as described (Fraser et al. 2000), with the following modifications. The mua-3(uy19) mutants were fed with the clones of dbl-1 and dpy-17 from the Ahringer feeding library (Fraser et al. 2000; Kamath and Ahringer 2003). The plates containing NGM agar with 1 mM IPTG and 50 mg/ml carbenicillin were inoculated with bacterial cultures grown 16–18 hr for each targeted gene. L4-stage C. elegans were transferred to the plates for each gene at 15°. Forty-eight hours later, adults were removed and the plates were placed at 25°. Forty-eight hours later, the number of progeny that survived was counted and the percentage was calculated. The RNAi clones of dpy-17 and dbl-1 were sequenced and validated (data not shown).

Photography
mua-3 mutants were observed under differential interference contrast (DIC) microscopy using a Zeiss Axio A2 Imager at either 63× or 100× magnifications. Images were acquired using Zeiss Axiovision software.

Statistical analysis
Student's t tests and one-way ANOVA test were performed to determine statistical significance.

mua-3 homology comparison
The phylogenetic tree was created using the MEGA program. CLUSTALW was used for multiple sequence alignment. The p-distance method was used to calculate distance and the neighbor-joining method was used to create a tree. The tree was statistically evaluated by using the bootstrap method (1000 times), and robustness of the tree branch was indicated at the branch. The following protein sequences were used for the phylogenetic analysis: C. elegans MUA-3 (NP_001022674.1), C. elegans MUP-4 (NP_498645.1), C. elegans FBN-1 (NP_001263711.1), Drosophila melanogaster DP (Dumpy; NP001260037.1), Homo sapiens FBN1 (NP_000129.3), FBN2 (NP_001990.2), FBN3 (NP_115823.3), and EYS (Eyes shut; NP_001278938.1). H. sapiens EYS sequence was included as an outgroup member to infer the root of this unrooted tree.