Strains were maintained at 19–23°C on NGM plates spotted with E. coli
strain OP50. In all age-effect experiments, strains were strictly maintained at 20°C. For all transgenes described in this publication, only one array and/or one insertion is given in the strain list, although unless otherwise specified in the Results
section, multiple independent arrays or insertions were examined.
CB4088: him-5(e1490) V.
CB4856: Hawaii natural isolate carrying niDf9 I. niDf9 designates the 19 kb deficiency spanning peel-1 and zeel-1.
EG1285: oxIs12[Punc-47::GFP; lin-15(+)] lin-15B(n765) X.
EG4322: ttTi5605 II; unc-119(ed3) III.
EG4348: Utah natural isolate carrying peel-1(qq99) I. EG4348 was collected by M. Ailion from Salt Lake City, Utah (this publication). qq99 designates the naturally occurring nonsense mutation in peel-1.
EG5389: qqIr7[peel-1(qq99)] I; oxIs494[Ppeel-1::GFP, Cbr-unc-119(+)] II; unc-119(ed3) III.
EG5655: qqIr7[peel-1(qq99)] I; oxSi19[peel-1(+), Cbr-unc-119(+)] II; unc-119(ed3) III.
EG5766: qqIr7[peel-1(qq99)] I; oxSi77[Ppeel-1::peel-1::GFP, Cbr-unc-119(+)] II; unc-119(ed3) III.
EG5801: oxSi87[Ppeel-1::peel-112a.a.::GFP, Cbr-unc-119(+)] II; unc-119(ed3) III.
EG5955: qqIr7[peel-1(qq99)] I; ttTi5605 II; unc-119(ed3) III; oxEx1462[Phsp-16.41::peel-1, Cbr-unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
EG5958: qqIr7[peel-1(qq99)] I; oxSi186[Phsp-16.41::peel-1, Cbr-unc-119(+)] II; unc-119(ed3) III.
EG5960: qqIr7[peel-1(qq99)] I; oxSi188[Phsp-16.2::peel-1, Cbr-unc-119(+)] II; unc-119(ed3) III.
EG5961: qqIr7[peel-1(qq99)] I; ttTi5605 II; unc-119(ed3) III; oxEx1464[Phsp-16.2::peel-1, Cbr-unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
EG6297: qqIr5[niDf9] I; oxSi298[Phsp-16.41::zeel-1::tagRFP, Cbr-unc-119(+)] II; unc-119(ed3) III.
EG6298: qqIr5[niDf9] I; ttTi5605 II; unc-119(ed3) III; oxEx1501[Phsp-16.41::zeel-1::tagRFP, Cbr-unc-119(+), Pmyo-2::GFP].
EG6301: qqIr5[niDf9] I; ttTi5605 II; unc-119(ed3) III; oxEx1504[Pexp-3::peel-1, Cbr-unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
EG6306: qqIr5[niDf9] I; ttTi5605 II; unc-119(ed3) III; oxEx1509[Punc-47::peel-1, Cbr-unc-119(+), Prab-3::mCherry].
MT1344: bli-3(e767) lin-17(n677) I.
MT3301: fem-1(hc17) IV; him-5(e1490) V.
MY19: German natural isolate carrying peel-1(qq98)
I. MY19 was collected from Roxel, Germany 
designates the naturally occurring nonsense mutation in peel-1
N2: Laboratory reference strain, Bristol.
PD4790: mIs12[myo-2::GFP, pes-10::GFP and gut::GFP].
I; qqIr8[N2=>CB4856, unc-119(ed3)]
QX1197: qqIr5[CB4856 =>N2, niDf9]
is an 140–370 kb introgression from CB4856 into N2. This strain was used in some experiments instead of CB4856, in order to reduce the genetic variation segregating in the background.
QX1257: niDf9 I; qqIr8[unc-119(ed3)] III; qqIs2[zeel-1genomic::GFP, unc-119(+)].
QX1264: niDf9 I; qqIr8[unc-119(ed3)] III; qqEx2[zeel-1genomic::GFP, unc-119(+)].
QX1319: zeel-1(tm3419)/hT2[qIs48] I; +/hT2[qIs48] III.
QX1320: qqIr6[EG4348=>N2, peel-1(qq99)]
QX1384: niDf9 I; qqIr8[unc-119(ed3)] III; qqEx6[Pzeel-1:: zeel-1SOL, unc-119(+)].
QX1392: qqIr6[peel-1(qq99)] I; unc-119(ed3) III; qqEx3[peel-1(+), unc-119(+)].
QX1409: qqIr7[EG4348=>N2, peel-1(qq99)]
QX1577: qqIr5[niDf9] I; qqEx1[Pzeel-1::zeel-1cDNA::GFP, Pmyo-2::RFP].
QX1589: qqIr5[niDf9] I; qqEx4[Pzeel-1::Y71A12B.17TM:: zeel-1SOL, Pmyo-2::RFP].
QX1605: qqIr5[niDf9] I; ttTi5605 II; unc-119(ed3) III.
QX1607: qqIr5[niDf9] I; qqEx5[Pzeel-1:: zeel-1TM, Pmyo-2::RFP].
QX1618: qqIr5[niDf9] I; qqEx7[Plin-26::zeel-1, Pmyo-2::RFP].
QX1619: qqIr5[niDf9] I; qqEx8[Phlh-1::zeel-1, Pmyo-2::RFP].
QX1624: qqIr5[niDf9] I; oxSi186[Phsp-16.41::peel-1, Cbr-unc-119(+)] II; unc-119(ed3) III.
QX1650: oxSi19[peel-1(+), Cbr-unc-119(+)] II.
QX1772: qqIr5[niDf9] I; ttTi5605 II; unc-119(ed3) III; oxEx1462[Phsp-16.41::peel-1, Cbr-unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
SJ4157: zcIs21[Phsp-16::clpp-1(WT)::3xmyc-His tag+Pmyo-3::GFP] V.
Scoring Embryo Lethality
In all experiments except the age-effect experiment, embryo lethality from self-fertilizing hermaphrodites was scored by isolating hermaphrodites at the L4 stage and singling them to fresh plates the following day. After laying eggs for 8–10 h, the hermaphrodites were removed and embryos were counted. Unhatched embryos were counted ~24 h later.
To score embryo lethality from mated hermaphrodites, three or four L4 hermaphrodites were mated to six to ten L4 or young adult males for 24–36 h. Hermaphrodites were then singled to fresh plates and embryo lethality was scored as above. Broods were examined for the presence of males 2–3 d later, and any broods lacking males were excluded. To allow male sperm to age within the reproductive tract of the hermaphrodite, mated hermaphrodites were removed from males, and lethality was scored among embryos laid 3 d after removal.
In the age-effect experiment, 91 zeel-1(tm3419)peel-1(+)/niDf9 hermaphrodites were singled at the L4 stage and transferred every 12 h to fresh plates. Hermaphrodites were discarded after the first 12-h period in which they failed to lay fertilized embryos. Total embryos were counted at the end of each laying period, and unhatched embryos were counted ~24 h after each laying period had ended.
To score embryo lethality from partially mated hermaphrodites, 130 zeel-1(tm3419)peel-1(+)/niDf9 hermaphrodites were mated at the L4 stage to an equal number of PD4790 males, which carry an insertion of the fluorescent marker, Pmyo-2::GFP. After 24 h, hermaphrodites were singled and transferred every 12 h to fresh plates until day five. Embryo lethality was scored as above, except that after unhatched embryos were counted, hatched and unhatched progeny were classified as self- or cross-progeny according to presence of pharyngeal GFP. Hermaphrodites laying 100% self-progeny or more than 95% cross-progeny were excluded. The remaining hermaphrodites, which we define as “partially mated,” laid ~10%–50% self-progeny. In these broods, we calculated the portion of self-progeny, laid during days 3 to 5, that failed to hatch.
Mapping peel-1 Mutations in MY19 and EG4348
Absence of the paternal-effect in EG4348 was mapped relative to bli-3(e767)
, a visible marker located ~10 cM from the peel-1
interval. Mapping was performed as described 
. Briefly, EG4348 males were crossed to MT1344 hermaphrodites, and F1 hermaphrodites were mated to CB4856 males. The resulting hermaphrodite progeny were allowed to self-fertilize, and their broods were scored for embryo lethality (i.e., presence of peel-1
activity) and presence of Bli animals. Directionality with respect to bli-3
could be inferred because bli-3
is located at the left-hand tip of chromosome I.
Preliminary sequence analysis of the peel-1
interval in EG4348 was performed by genotyping EG4348 with a subset of the markers listed in Table S2 of 
. These markers tile across the peel-1
interval, and they distinguish all haplotypes carrying an intact copy of the peel-1/zeel-1
element from all haplotypes lacking it 
. In other words, the Bristol-like alleles of these markers are in perfect linkage disequilibrium with presence of the peel-1/zeel-1
element. At all markers we assayed, EG4348 carried the Bristol-like allele.
Fine-mapping in MY19 and EG4348 was performed by crossing each strain to MT1344 and collecting Lin Non-Bli and Bli Non-Lin recombinants in the F2 generation. Recombinant animals were genotyped (via a portion of their F3 broods) at each of two markers flanking the peel-1 interval. The right-hand marker for the MY19 cross was a BstCI snip-SNP amplified with primers 5′-GTA TTC CGA CGA TTC GGA TG-3′ and 5′-CAT TGA GAA CAC AAA AAC AAA CG-3′. The right-hand marker for EG4348 cross was an AfeI snip-SNP amplified with primers 5′- GAC ATA TTT CCC GCA ACC TG-3′ and 5′- GTG ACG AGG CTT GAG GAT TC-3′. The left-hand marker for both crosses was a BanI snip-SNP amplified with primers 5′-CGC CAA ATA TGT TGT GCA GT-3′ and 5′-CAC CAC GTG TCC TTT CTC ATT-3′.
Recombinants breaking within the peel-1
interval were homozygosed for the recombinant chromosome, and the resulting homozygotes were phenotyped for peel-1
activity. Phenotyping was performed by crossing each line to CB4856 and scoring embryo lethality from self-fertilizing, F1 hermaphrodites, and from F1 males backcrossed to CB4856 hermaphrodites. Recombinants were classified as having peel-1
activity if these crosses produced ~25% and ~50% embryo lethality, respectively. Next, the locations of recombination breakpoints were mapped more finely by sequencing six to ten sequence polymorphisms, located throughout the peel-1
interval, that distinguish MY19 or EG4348 from Bristol. The MY19 polymorphisms were determined from the MY19 sequence described in 
, and the EG4348 polymorphisms were determined by amplifying and sequencing arbitrary fragments from this strain. Some polymorphisms are shared between MY19 and EG4348, and these were used in both crosses. For the most informative recombinants, we later sequenced across the entire breakpoint region in order to map these breakpoints to the level of adjacent polymorphisms. This approach mapped the peel-1
-disrupting mutations to regions of 5 kb in MY19 and 8 kb in EG4348. These intervals were then sequenced in the corresponding strains, and all sequence polymorphisms were identified. Finally, we genotyped these polymorphisms in a panel of 38 wild strains previously identified as having intact peel-1
. These strains, as well as the primers used for genotyping them, are given in Figure S2
. The MY19 sequence was deposited in GenBank previously 
, and the EG4348 sequence was deposited under accession number HQ291558.
Identification of peel-1 Transcript
RNA was collected from mixed-staged Bristol animals by freeze-cracking and extracting in Trizol (Invitrogen) according to the manufacturer's protocol. Reverse transcription-PCR (RT-PCR) was performed using pairs of primers flanking each candidate mutation in MY19 and EG4348. For each pair of primers, the forward and reverse primers were located ~100 bp apart, and two reactions were performed, one using each of the two primers as the RT primer. Product was observed for only one pair of primers, and for that pair, only in one direction. These primers were 5′-ACA TGT ATC TTG ATC TGC CTG A-3′ (forward) and 5′-AAA AAT TAA CCA CAA TGA AGC AA-3′ (reverse), and product was only observed using the reverse primer as the RT primer. To recover the remainder of this putative transcript, 3′ and 5′ RACE were performed using standard methods 
. For 3′ RACE, the RT reaction was performed using 5′-GTT TTC CCA GTC ACG ACT TTT TTT TTT TTT TTT TT-3′, and PCR was performed using the gene-specific primer, 5′-ACA TGT ATC TTG ATC TGC CTG A-3′ (forward), and the adaptor primer, 5′-GTT TTC CCA GTC ACG AC-3′ (reverse). For 5′ RACE, the RT reaction was performed using a gene-specific primer that spanned the putative stop codon, 5′-TCA ATT TCA TGG ATT TTC AAC A-3′, and PCR was performed using 5′-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3′ (forward) and a nested, gene-specific primer, 5′-AAA AAT TAA CCA CAA TGA AGC AA-3′ (reverse). Then, a second round of PCR was performed using the adaptor primer, 5′-GGC CAC GCG TCG ACT AGT AC-3′ (forward), and another nested, gene-specific primer, 5′-AGA GCA ATA ACA TGC GCA AA-3′ (reverse). SuperScript III (Invitrogen) was used in all RT reactions, and PlatinumTaq (Invitrogen) was used for all PCR reactions. The peel-1
transcript did not contain a splice leader sequence and was deposited in GenBank under accession number HQ291556. More recently, the peel-1
transcript was identified independently by WormBase curators and assigned the identification number, Y39G10AR.25
To search for transcripts carrying both peel-1 and zeel-1, an RT reaction was performed using the peel-1-specific primer, 5′-AAA AAT TAA CCA CAA TGA AGC AA-3′, and PCR was performed using a forward primer located in the 3′ end of zeel-1 (5′-CCA TCC GAG ATA ACC GAA AA-3′) and a reverse primer located in the 5′ end of peel-1 (5′-AGA GCA ATA ACA TGC GCA AA-3′). No product was observed.
CB4088 and MT3301 animals were grow at 15°C and synchronized at the L1 stage by bleaching and hatching overnight in M9. L1s were split into two populations, and one population was shifted to 25°C. When animals had reached young adulthood, hermaphrodites and males were separated by hand, and RNA was collected as above. Real-time PCR of peel-1, spe-9, and rpl-26 was performed in triplicate, for 40 cycles, on an ABI 7900HT using the QuantiTect SYBR Green Kit (Qiagen). Relative expression levels of peel-1 and spe-9 were calculated separately for males and hermaphrodites, using the 2−ΔΔCt method, with rpl-26 as the endogenous control and the 15°C MT3301 sample as the reference sample. Primers used to amplify peel-1 were 5′-TAC ACC CGT CAC ACC AAC TG-3′ and 5′-TCC GAC TAT GAT GTT CCA CAA-3′; primers for spe-9 were 5′-CGG CTT GCA TAC ACA ATG AG-3′ and 5′-ACG CCA TGA CTC TTG CTC TT-3′; and primers for rpl-26 were 5′-TCC AAT CAG AAC CGA TGA TG-3′ and 5′-GTG CAC AGT GGA TCC GTT AG-3′.
Among the hermaphrodite samples, relative expression levels of peel-1 and spe-9 were roughly equivalent, except for the 25°C MT3301 sample, where expression of peel-1 and spe-9 was undetectable. That is, in this sample, signal for peel-1 and spe-9 failed to rise above the detection threshold, even after 40 cycles, despite rpl-26 amplifying normally.
Single molecule FISH of was performed as in 
, with the embryos and hermaphrodites squashed down to ~9 µm thickness for imaging. Automated counting of nuclei in embryos was performed using software developed in 
Rescue of peel-1
Transgenic animals carrying peel-1(+)
were generated by two methods: bombardment 
and Mos1-mediated, single-copy insertion 
. For bombardment, a fragment containing the Bristol allele of peel-1
, along with ~2.8 kb of upstream sequence and ~1 kb of downstream sequence, was excised from fosmid WRM0633bE09 (Bioscience LifeSciences, Nottingham, UK) using AhdI and NgoMIV. This fragment was cloned into the yeast shuttle vector, pRS246 (ATCC, Manassas, VA), via yeast-mediated ligation 
of the fragment's ends. The resulting plasmid, pHS11, was bombarded into QX1320, along with the unc-119(+)
rescue vector, pDP#MM016B 
. Bombardment was performed as in 
, although only extra-chromosomal arrays were recovered. Nine independent transgenic lines were tested for peel-1
activity by crossing them to CB4856 and scoring embryo lethality from self-fertilizing, F1 hermaphrodites (self-cross) and F1 males backcrossed to CB4856 hermaphrodites (backcross).
For Mos1-mediated insertion, the peel-1
fragment from pHS11 was amplified by PCR, using primers having NheI cut sites, and this amplicon was cut with NheI and ligated into pCFJ151 
linearized with AvrII. The resulting plasmid, pHS26, was injected into QX1409 along with the vectors needed to generate single-copy insertions 
. Insertion-carrying animals were recovered by the direct insertion method 
, and five independent insertion-carrying lines were tested for peel-1
activity as above. For one of the two insertions that did exhibit peel-1
activity, the self-cross and backcross were repeated, and hatched progeny were collected and genotyped for a PCR-length polymorphism located less than 1 kb from niDf9
. The primers used to amplify this polymorphism were 5′-TGG ATA CGA TTC GAG CTT CC-3′ (forward) and 5′-CCC CCT AAT TTC CAA GTG GT-3′ (reverse).
For three of the peel-1 array lines, a small number of severely deformed L1s were observed in the backcross, similar to the “escapers” typically observed among peel-1-affected embryos sired by hermaphrodites. We suspected that these L1s had “escaped” the paternal-effect due to partial germline silencing of the peel-1 arrays. Consistent with this hypothesis, we genotyped 13 of these animals, using the PCR-length polymorphism described above, and all were zeel-1(niDf9) homozygotes. We then calculated the frequency of these escapers relative to the total number of peel-1-affected progeny (i.e., relative to the total number of dead embryos and deformed L1s).
ZEEL-1::GFP Fusion and Domain Swapping
was generated by amplifying GFP
from PD95.75 and inserting it into pHS4.1, a genomic subclone of zeel-1(+)
described previously 
. pHS4.1 was linearized with AhdI, and yeast-mediated ligation 
was used to insert GFP
just upstream of the zeel-1
stop codon. Later, a second ZEEL-1::GFP
construct was generated using the cDNA of zeel-1
, instead of the genomic locus. This construct was generated by first cutting pHS4.1 with EcoNI and BglII, in order to remove the entire coding region of zeel-1
, and then inserting a full cDNA of zeel-1
, followed by GFP
. The cDNA of zeel-1
was cloned previously 
, and this replacement was performed using yeast-mediated ligation 
. Both constructs showed full rescue of peel-1
-affected embryos, and data from the two constructs were combined.
To generate ZEEL-1SOL
, pHS4.1 was cut with EcoNI and KpnI, and the fragment containing zeel-1
codons 5 to 205 was removed. The remaining fragment was then re-circularized, using yeast-mediated ligation 
, to fuse codon 4 to codon 206. To generate ZEEL-1TM
, the entire coding region of zeel-1
was excised from pHS4.1 using EcoNI and BglII, and this fragment was replaced with a partial cDNA of zeel-1
encoding the first 205 amino acids of the protein. This replacement was performed using yeast-mediated ligation 
To generate Y71A12B.17TM::ZEEL-1SOL
, the coding region of zeel-1
was excised from pHS4.1, as above, and yeast mediated ligation 
was used to replace this fragment with a partial cDNA of Y71A12B.17
, followed by a partial cDNA of zeel-1
. The resulting construct contained the N
-terminal 207 codons of Y71A12B.17
fused to the C
-terminal 712 codons of zeel-1
. The junction of this fusion was chosen to overlap a string of seven amino acids (KNERKEG) that are perfectly conserved between the two proteins. The Y71A12B.17
cDNA was cloned by reverse transcribing RNA from the Bristol strain using primer 5′-TTG AAC AAA AAC AAT GGA TAT GTA A-3′, and then performing PCR using primers 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GTC GGA TTT CGA CTC AGA-3′
(forward) and 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC ATT TAT TAA CTC CAA CAA TGA TTC G-3′ (reverse). This PCR product was then cloned into the vector, pDONR221 (Invitrogen), using the Gateway cloning kit (Invitrogen). The Y71A12B.17
cDNA differs slightly from the WormBase gene prediction and was deposited in GenBank under accession number HQ291557.
All constructs were bombarded 
into QX1015 along with the unc-119(+)
rescue vector, pDP#MM016B 
, or they were injected 
into QX1197 at ~80ng/µl, along with the fluorescent marker, Pmyo-2::RFP
at 3 ng/µl. To test each transgene for its ability to rescue peel-1
-affected embryos, transgenic animals were crossed to the Bristol strain, and lethality was scored among embryos derived from two crosses: self-fertilizing F1 hermaphrodites (self-cross) and F1 males backcrossed to hermaphrodites of the original transgenic line. To calculate hatch rates among peel-1
-affected embryos inheriting ZEEL-1::GFP
, transgenic animals were crossed to QX1319, and embryos were collected from (i) transgenic, self-fertilizing, F1, zeel-1(tm3419)peel-1(+)/niDf9
hermaphrodites and (ii) transgenic, F1, zeel-1(tm3419)peel-1(+)/niDf9
males backcrossed to non-transgenic, niDf9/niDf9
hermaphrodites. Inheritance of ZEEL-1::GFP
was inferred by expression of the co-injection marker, Pmyo-2::RFP
, which can be scored even in arrested embryos.
All other transgenes were generated using the three-site Gateway system from Invitrogen. This method allows three separate DNA fragments to be joined together and inserted into pCFJ150, which contains Cbr-unc-119(+)
and the sequences needed for Mos1-mediated insertion at the ttTi5605
Mos site on chromosome II 
. In most cases, this method was used to join together a promoter of interest, a coding sequence, and a 3′ UTR.
Ppeel-1::GFP, Ppeel-1::PEEL-112a.a.::GFP, and the PEEL-1::GFP Fusion
For Ppeel-1::GFP, we joined together the peel-1 promoter, GFP, and the peel-1 3′UTR. For the peel-1 promoter, we used all intergenic sequences between the peel-1 start codon and last coding segment of zeel-1 (i.e., 2,473 bp of sequence). The peel-1 3′ UTR was determined empirically and extended 86 bp downstream of the peel-1 stop codon. For GFP, we used a variant containing S65C and three internal introns (identical to the variant in pPD95.75).
For Ppeel-1::PEEL-112a.a.::GFP, the PEEL-1 leader peptide was added by extending the promoter fragment to include the first 12 amino acids of PEEL-1. This signal peptide was discovered while we were investigating a possible regulatory role of the first intron of peel-1. We had generated a GFP reporter driven by the peel-1 promoter and the first intron of peel-1, and this construct also happened to carry the first 12 amino acids of PEEL-1. GFP driven by this construct was packaged into sperm (unpublished data), and in order to confirm that sperm packaging was caused by the leader peptide, rather than the intron, we generated Ppeel-1::PEEL-112a.a.::GFP (which excludes the first intron). Conversely, we also generated a reporter carrying the first intron and a randomized leader peptide, and for this construct, no sperm packaging was observed (unpublished data).
To tag PEEL-1 with GFP, the promoter fragment was extended even further to include the entire peel-1 gene, up to (but excluding) the stop codon.
All three constructs were injected into EG4322 or QX1409, and single-copy insertions were obtained using the direct insertion MosSCI method 
. Three to six independent insertions were analyzed for each construct, and no differences were observed among insertions of the same construct. We note that although PEEL-1::GFP appears to localize normally, it failed to exhibit peel-1
activity (unpublished data), presumably because the GFP tag inhibited function.
Plin-26::zeel-1 and Phlh-1::zeel-1
For Plin-26::zeel-1 and Phlh-1::zeel-1, the promoters of lin-26 or hlh-1 were joined to the cDNA of zeel-1 and the 3′ UTR of let-858. For the promoters of lin-26 and hlh-1, 7,122 bp of sequence and 3,037 bp of sequence upstream of the respective start codons were used. For the let-858 3′ UTR, 434 bp of sequence downstream of the stop codon was used. Transgenic animals were generated by injecting Plin-26::zeel-1 and Phlh-1::zeel-1 into QX1197 at ~80 ng/µl, along with the fluorescent marker, Pmyo-2::RFP at 3 ng/µl.
To evaluate rescue among male-sired embryos, transgenic animals were crossed to QX1319, and transgenic, F1, zeel-1(tm3419)peel-1(+)/niDf9 males were backcrossed to non-transgenic, niDf9/niDf9 hermaphrodites. Embryos were dissected from these hermaphrodites and imaged every 10–20 min, starting before the 2-fold stage and ending at least 6 h after the 2-fold stage. To evaluate rescue among hermaphrodite-sired embryos, transgenic lines were crossed to QX1319, and embryo viability was scored among embryos collected from transgenic, self-fertilizing, F1, zeel-1(tm3419)peel-1(+)/niDf9 hermaphrodites. Among both male- and hermaphrodite-sired embryos, inheritance of the transgene was inferred by expression of Pmyo-2::RFP.
Heat-Shock Constructs and Constructs for Cell-Specific Expression of peel-1
, and Punc-47::peel-1
were generated by joining the peel-1
cDNA downstream of the appropriate promoter and upstream of the tbb-2
3′ UTR. We describe the promoter and 3′UTR fragments in terms of length of sequence upstream or downstream of the appropriate start or stop codons: Phsp-16.41
(501 bp), Phsp-16.2
(493 bp), Pexp-3
(2,877 bp), Punc-47
(1,251 bp), and tbb-2
3′ UTR (331 bp). Phsp-16.41::peel-1
were injected into QX1409 at 25 ng/µl, and arrays and MosSCI insertions were recovered as in 
. The arrays carry co-injection markers Pmyo-2::mCherry
, and Prab-3::mCherry
were injected into QX1605 at 25 and 10 ng/µl, respectively, along with co-injection markers Prab-3::mCherry
, and Pmyo-3::mCherry
) and marker Prab-3::mCherry
). In all cases, Pmyo-3::mCherry
were injected at 10 ng/µl, and Pmyo-2::mCherry
was injected at 5 ng/µl.
was generated using the hsp-16.41
promoter described above, the zeel-1
cDNA, and the let-858
3′UTR fused downstream of tagRFP
was added to confirm expression of zeel-1
after heat-shock. Phsp-16.41::zeel-1
was injected at 10 ng/µl into QX1605 and the arrays and the MosSCI insertion were recovered as in 
, except that a GFP-based co-injection marker (Pmyo-2::GFP
injected at 2.5 ng/µl) was used in order to distinguish these arrays from the Phsp-16.41::peel-1
Microscopy and Analysis of Live Embryos
Imaging of fixed embryos and live imaging of ZEEL-1::GFP embryos was performed on a PerkinElmer RS3 spinning disk confocal. All other imaging was performed on a Nikon 90i equipped with a CoolSNAP HQ2 camera and a X-Cite 120 Series fluorescent light source. Images were acquired and background subtracted with either Volocity (PerkinElmer) or NIS Elements (Nikon), and (in some cases) multiple channels were overlaid in Adobe Photoshop. To image dissected gonads, spermatocytes, and sperm, adult males or mated hermaphrodites were dissected into sperm media containing dextrose (50 mM Hepes, 1 mM MgSO4, 25 mM KCl, 45 mM NaCl, 5 mM CaCl2, 10 mM dextrose).
To measure the onset of epidermal leakage in peel-1-affected embryos, pre-arrest embryos were dissected from the following crosses. Hermaphrodite-sired embryos were dissected from (i) self-fertilizing, zeel-1(tm3419)peel-1(+)/niDf9 hermaphrodites and (ii) self-fertilizing, zeel-1(tm3419)peel-1(+)/zeel-1(tm3419)peel-1(+) hermaphrodites. Male-sired embryos were dissected from niDf9/niDf9 hermaphrodites mated to three types of males: (i) zeel-1(tm3419)peel-1(+)/niDf9; (ii) zeel-1(tm3419)peel-1(+)/zeel-1(+)peel-1(+); and (iii) zeel-1(tm3419)peel-1(+)/zeel-1(+)peel-1(+); oxSi19[peel-1(+)]/+. The self-fertilizing hermaphrodites were aged 24 h post-L4 at the time of dissection, and mated hermaphrodites were aged 24–48 h at the time of dissection. After dissection, embryos were imaged every 10 min, starting before the 1.5-fold stage and ending 7 or more hours after the 1.5-fold stage. The onset of epidermal leakage was calculated as the time between the 1.5-fold stage and the first frame in which leakage was observed. Calculations were truncated at 7 h past the 1.5-fold stage because this represents 1 h after the average hatching time of wild-type embryos. Finally, embryos were binned into 30-min intervals in order to generate the inverted histograms shown in .
To calculate the percentage of peel-1-affected embryos elongating past 2-fold, zeel-1(tm3419)peel-1(+)/niDf9 hermaphrodites were isolated at the L4 stage and allowed to age for 24, 48, 60, and 72 h. Embryos were then dissected and imaged every 20 or 30 min for at least 10 h.
Fixation of Sperm and Embryos
Anti-PEEL-1 is a rabbit polyclonal generated against the C
-terminal 15 amino acids of PEEL-1. This antibody was generated and purified by GenScript, Piscataway, NJ. 1CB4 is a mouse monoclonal used to stain FB-MOs 
. 1CB4 was a gift from Steven L'Hernault. To stain sperm, adult males were dissected into sperm media on charged slides, freeze-cracked in liquid nitrogen, and fixed overnight in −20°C methanol. Slides were washed with PBST (PBS+0.1% Triton-X 100), blocked for 30 min with PBST+0.5% BSA, and incubated for 4 h with anti-PEEL-1 (1/100) and 1CB4 (1/2,000), diluted in PBST+0.5% BSA. Slides were then washed three times in PBST and incubated for 2 h with Alexa568-labeled anti-mouse (1/500) and Alexa488-labeled anti-rabbit (1/500) (Invitrogen), diluted in PBST+0.5% BSA. Slides were washed again three times in PBST and mounted in Vectashield mounting media with DAPI.
To visualize actin filaments in peel-1
-affected embryos, embryos were stained with Alexa568-labeled Phalloidin (Invitrogen) according to Protocol 7 in 
. To visualize all the other proteins, embryos were stained with monoclonal antibodies MH2 (perlecan), DM5.6 (myosin heavy chain A), MH5 (VAB-10A), and MH4 (intermediate filaments). All monoclonals were obtained from The Developmental Studies Hybridoma Bank, Iowa City, Iowa. For these experiments, embryos were fixed for 10 min in 3% paraformaldehdye, freeze cracked in liquid nitrogen, and incubated for 5–10 min in −20°C methanol. Embryos were then washed three times in PBST and incubated overnight with the primary antibody diluted in PBST+1% BSA. MH2, MH4, and MH5 were diluted 1/150, and DM5.6 was diluted 1/1,000. Embryos were washed three times in PBST and incubated overnight with Alexa488-labeled anti-mouse (1/500) (Invitrogen), diluted in PBST+1% BSA. Embryos were washed again three times in PBST and mounted in Vectashield mounting media with DAPI.
Phylogenetic Analysis of zeel-1
Separate phylogenetic trees were built for (i) zyg-11
and all zyg-11
homologs in C. elegans
and (ii) zyg-11
and all zyg-11
homologs in C. elegans
, C. briggsae
, C. remanei
, and C. japonica
homologs were defined as all genes carrying the zyg-11
-like leucine-rich repeat region. After removing the predicted transmembrane domains of ZEEL-1, Y71A12B.17, and Y55F3C.9, all protein sequences were aligned using MUSCLE 
. The alignments were performed using the BLOSUM30 substitution matrix, a gap open penalty of −10, and a gap extend penalty of −1. The C. elegans
–only alignment was trimmed to exclude residues having gaps in more than 90% of sequences, and the multi-species alignment was trimmed using the heuristic method, automated1
, from TrimAL 
, which is optimized for maximum likelihood tree construction. Finally, phylogenetic trees were constructed using PhyML 
, using the LG substitution model 
, zero invariant sites, and four substitution rate categories. Branch support was determined using bootstrap sampling with 100 replicates.
Divergence between zeel-1
was determined by aligning the two proteins with MUSCLE 
, trimming the alignment of gaps, and using PAML 
to calculate synonymous site divergence on the corresponding nucleotide sequences. (The total length of gaps was less than 0.1% of the length of the total alignment.) The summary statistics are as follows: number of synonymous sites
715.5; number of non-synonymous sites
2,002.5; synonymous substitutions per site (dS
1.0709; non-synonymous substitutions per site (dN
Adults and larvae were heat-shocked by submerging sealed agar plates in a 34°C water bath for 1 h. Embryos were heat-shocked by mounting embryos on an agar pad, incubating the slide at 19–20°C for the prescribed number of hours before placing the slide on the floor of a sealed, 1 cm×8 cm×8 cm plastic box, and submerging the box in a 34°C water bath for 20 min. After heat-shock, embryos were imaged every 20 min for at least 10 h. Initially, embryos were staged directly by collecting and mounting four-cell embryos. Later, when it became clear that the vast majority of heat-shocked embryos developed to the 2-fold stage without defects or delay, throughput was increased by collecting mixed stage embryos and mounting, incubating, and heat-shocking as above. These embryos were staged relative to the time at which they initiated elongation and by comparing their morphology before heat-shock to images of embryos that had been staged using the direct method.
To quantify peel-1-mediated killing of the egg-laying muscles and the anal depressor muscle, the Pexp-3::peel-1 arrays were crossed to SJ4157, which carries an integrated array of the muscle marker, Pmyo-3::GFP. In 1-d-old, F1 hermaphrodites, two of the four egg-laying muscles and the single anal depressor muscle were observed and classified as live or dead based on expression of GFP. Live egg-laying muscles were classified as morphologically normal or atrophied, and both live egg-laying muscles and live anal depressor muscles were then classified as mCherry+ or mCherry−, indicating that they had or had not inherited the Pexp-3::peel-1 array. For each cell type, the percent of cells killed by the arrays was calculated assuming that all dead cells had inherited the array. In addition, each F1 animal was classified as constipated if it contained bacteria in the posterior intestine and as egg-laying defective if it contained 3-fold embryos in the uterus.
To quantify peel-1-mediated killing of GABA neurons, the Punc-47::peel-1 arrays were crossed to EG1285, which carries an integrated array of the GABA-neuron marker, Punc-47::GFP. GABA-neurons were observed in F1 hermaphrodites, and peel-1-mediated killing was quantified as above. Typically, the DVB neuron and five to nine ventral cord neurons were scored per hermaphrodite. In addition, each animal was classified as mosaic or non-mosaic based on expression of the co-injection marker, Prab-3::mCherry.