Copyright 0 1995 by the Genetics Society of America
Viable Maternal-Effect Mutations ThatAffect the Development of the Nematode Caenorhabditis eZegam
Siegfried Hekimi, Paula Boutis and Bernard Lakowski
Department of Biology, McGill University, Montreal, Quibec, Canada H3A IBI Manuscript received June 27, 1995 Accepted for publication September 12, 1995
ABSTRACT We carried out a genetic screen for viable maternal-effect mutants to identify genes with a critical function relatively early in development. This type of mutation would not have been identified readily in previous screens for viable mutants and therefore could define previously unidentified genes. We screened 30,000 genomes and identified 41 mutations falling into 24 complementation groups. We genetically mapped these 24 loci; only two of them appear to correspond to previously identified genes. We present a partial phenotypic characterizationof the mutants anda quantitative analysis of the degree to which they can be maternally or zygotically rescued.
ENETIC screens inthe nematode Caenorhabditis matically found in previous screens for viable mutants, G elegans have identified numerous genes required as in those screens in general only the first generation for normal development.Most of these genes have been of homozygotes was examined. identified through screens for viable mutants with ab- Indeed, relatively few maternal-effect viable muta- normal morphology or behavior (e.g., BRENNER1974; tions have been characterized to date. However, some HODGKINand BRENNER1977; HODGKIN1980, 1983; particularly interesting genesdisplay this type ofherita- CHALFIEand SULSTON1981; RIDDLE et al. 1981; TRENT bility, among them, genesin the dosage compensation et al. 1983; FERGUSONand HORVITZ1985; PARKand and sex-determination pathways, such as sdc-1 (VILLE- HORVITZ1986; DESAIet al. 1988; MANSER and WOOD NEW and MEYER1987), tra-3 (HODGKINand BRENNER 1990; AWRY1993; STARICHet al. 1995). However, work 1977), and dpy-27 ( MEYERand CASSON1986; CHUANG in other organisms, in particular, Drosophila, has amply et al. 1994). Other such genes include mes-1, which is demonstratedthat full loss-of-function mutations in involved in germ line formation (CAPOWSKIet al. 1991); genes controlling the earliest aspects of development eat-3, which affects pharyngeal pumping (AVERY1993); have suchprofound consequences on later develop- and genes involved in dauer larva formation, such as ment that they almost invariably lead to embryonic le- duf-1 (SWANSON and RIDDLE 1981), THOMAS MAS et thality. To identify such genes in C. elegans, genetic al. 1993) and daf-23 (GOTTLIEBand RUVKUN 1994). screens formaternal-effect and zygotic embryonic lethal To identify new genes involved in the development mutations have been carriedout anda number of genes of the worm, we carried out a screen formaternal-effect required for specific aspects of early development have viable mutants and identified 41 mutations leading to been identified (e.g., ISNENGHIet al. 1983; PRIES et al. morphological or behavioral defects. 1987; KEMPHUES et ul. 1988a,b;SCHNABEL and SCHNABEL 1990a,b; MAINS et al. 1990; BUCHERand GREENWALD MATERIALSAND METHODS 1991; MELLO et al. 1992; BOWERMANet al. 1993; WIL- General methods and strains: C.elegans strains were cul- LIAMS and WATERSTON1994). However, essential genes tured as described by BRENNER(1974). Standard genetic can also be identified through the isolation of viable methods for C. elegans were used (BRENNER1974; SULSTON hypomorphic mutations, that is, mutations that do not and HODGKIN1988). Animals were cultured at20" unless oth- fully abolish the activity of the gene but produce avisi- erwise stated. Wild type was the N2 Bristol strain. Mutations ble phenotype (e.g,, FERGUSONand HORVITZ1985). and rearrangements used were as follows: One class of genes that has not been systematically LGI: dpy-5(e61), unc-40(e271), bli-4(e937), dpyl4(eISSts), che- pursued to date is that for which mutational disruption 3(el124), unc-29(e1072), daf-l6(m26),dPy-24(~71), unc- does not necessarily lead to lethality but for which the 75(e950), unc-54(e190), dj24, nDJ23, eDf3. LGII: dpy-lO(e128), rol-6(eI87), unc-4(e120), unc-53(e404), rol- requirement is sufficiently early and/or for sufficiently l(e91), eat-2(ad465), unc-52(e669su250ts). small amounts that there would be a maternal effect. LGIII: dpy-l(el), dafZ(el37ots), emb32(g58), embZ(hc58), emb Such genes and mutations would not have been auto- I (hc57), unc-79(el030), emb7(hc66), dpy-l7(el64), gml (e24OO), ml-31(s2438), ml(jb7), h-1(e185), sma4(e729), emb16(gl9), Corresponding author: Siegfried Hekimi, 1205 Doctor Penfield Ave., lin-39(nI 760), lin-l3(n387), ncl-l(e1865), emb25(g45), mab Montreal, Quebec, Canada H3A 1B1. 5(e1239), unc-36(e251), unc-32(e189), emb34(&2), &24(g40), E-mail: [email protected] dpy-I8(e364), Dfl.
Genetics 141: 1351-1364 (December, 1995) 1352 S. Hekimi, P. Boutis and B. Lakowski
LGIV: dpy-9(e12),lin-1 (el 777), dpr-l3(e184sd), blid(scl6), eat- 20" or 15", and only strains in which at least 90% of the FB 12(ad695), unc-24(e138, el 172), unc-43(e408), kt-657(~1254), shared a phenotype were kept. We also did not keep strains d&20(e1282ts), unc-31(e169, e928), unc-26(e205),tra-3(el767), that were so sick they would have required special measures dpy-4(e1166sd), nDf41, eDfl9, eDfl8, df7, sDf2. or were difficult to propagate. These conditions probably ex- LGV dpy-I 1 (e224), unc-42(e270), rol-4(sc8), lin-25(n545), him- cluded or made unlikely the recovery of alleles of dpr genes 5(e1467), unc-76(e91I), dpy-21 (e428), unc-51(e369), sDf32, involved in dosage compensation. Alleles of these genes are sDf28, ctDfl, arDf1. mostly near lethal and sometimes cold sensitive and would LGX: unc-7(e5), lin-l5(n309), sdc-1 (n485), sup-IV(n983) probably have been discarded for that reason. The daf-1, daf- Balancers: mnCl II, nT1 (W;v) 21 and duf-23 maternaleffect mutations are temperature sen- Strains used: CB4622 unc-26(e205) tra-3(el767) dpy-4(el166)/ sitive, as isdauer formation itself (GOLDEN andRIDDLE 1984). nT1 W; +/nT1 (v) (BARNES1991). A dufallele producing substantially <90% dauer larvae at 15" Isolation of mutants: Wild-type animals were mutagenized or 20" would not have been kept or possibly not even noticed, with ethyl methane sulfonate (EMS) following the standard as F3 plate carrying a substantial number of wild-type looking protocol (SULSTONand HOUCKIN 1988). Groupsof five muta- worms would have been immediately discarded. Finally, mu- genized hermaphrodites (PO) were plated on 9-cm Petri tants in eat-?, a gene defined by a single mutation, are very dishes and allowed to self-fertilize. They were removed after sick, and would not have been kept for that reason. Further- having laid -200 eggs (40 eggs each). The resulting F, ani- more, eat-j(ud426) might itself be a hypomorphic mutation, mals were left on the plate until they had laid a total of 6000 and possibly, more usual alleles are stronger and thus lethal. to 10,000 F, eggs and were then removed. When most of the Maternal rescue: To score for maternal rescue, wild-type resulting F2 animals had grown to adulthood, 50 adults with males were mated to homozygous mutant hermaphrodites no detectable morphological or behavioral mutant phenotype and 10 of the resulting wild-type F, hermaphrodites were were picked and transferred onto individual small plates. pooled and allowed to lay eggs for a24hr period. All resulting Those animals, which produced an entire broodof morpho- F2 animals were scored. When maternal rescue was incom- logically, behaviorally or developmentally (slow growth) mu- plete, it was noted which aspect of the phenotype was not tant worms, were analyzed further. Thirty thousand F2 animals rescued or how the animal's phenotype deviated from the were screened as described above, and 41 independent muta- wild type. All progeny found on a given plate were scored to tions were isolated, tested for complementation and mapped. identify possible dead eggs or larvae, but no dead animals Complementationtests: Complementation tests between were ever observed. newly isolated mutations were performed when they appeared Zygotic rescue: Wild-type males were mated to homozygous to produce similar phenotypes. Most commonly, heterozy- mutant hermaphrodites marked with a recessive mutation, gous males (m,/+) of one mutation were mated to homozy- and the anatomical and behavioral phenotypes of the cross- gous hermaphrodites of the other strain (m2/m2)and their progeny (m/+) were scored. All mutations that showed in- progeny scored formutant males. Complementation to alleles complete zygotic rescue also displayed some embryonic and of known genes was tested in the same way. This type of larval lethality in the absence of maternal and zygotic rescue. complementation test checks for the failure of zygotic rescue Lethality in these heterozygotes, however, was not scored be- of m2 by m,. However, in the case of mutations for which cause it would have been too difficult to distinguish such zygotic rescue by a wild-type allele either failed entirely or was animals fromdying self-progeny. Therefore, only animals that incomplete, failure of zygotic rescue would not have been a had survived embryogenesis and the earliest stages of larval sufficiently discriminating criterion to establish noncomple- development were scored. mentation. Inthese cases, the mutations were first individually Paternalrescue: The possibility of a paternal effect was mapped and then complementation tests were carried out investigated by testing whether sperm carrying a mutant copy using strains with linked markers. Three mutations of this of the gene but produced by a heterozygous male carrying a type (yml6, qm?I and qm35) were found to map to the same wild-type copy of a gene could contribute enough wild-type genetic location, and their allelism was then established in gene activity to the developing zygote to result in a wild-type the following way: 10 or more hermaphrodite cross-progeny phenotype. Heterozygous males (m/+) were mated to homo- from a cross of m,/+ males to marked m2/m2hermaphrodites zygous mutanthermaphrodites (m/m) and cross-progeny were individually picked and their self-progeny scored. Her- males were scored. In the absence of paternal rescue, half of maphrodite cross-progeny producing only mutant self-prog- the males should be phenotypically mutant and half should eny were found; because maternal rescue by the wild-type be wild type. When marked strains were used as a source of allele is complete for all three mutations, we concluded that hermaphrodites, both male and hermaphrodite cross-prog- they were allelic. eny were scored. In no case did we see paternal rescue leading Three mutations with a similar Mau phenotype (qm18, qm19 to a detectable amelioration of the phenotype at the level of and qm45) are X-linked. Their allelism was established by tak- the dissection microscope. ing advantage of the ability of tru-2(q276) to transform XX Penetrance: For the reference allele of each novel gene, animals into phenotypic males that areable to mate efficiently thedegree of embryonic lethality and larval lethality was (L. AVERY,personal communication). q276/y276; qm45/qm45 scored, as wellas the penetrance of visible phenotypes in males were constructed and used in complementation tests surviving adults. To determine lethality, 10-20 animals of a to qm18 and qm19. given genotype were pooled and allowed to self-fertilize for a Identification of alleles of known genes: Alleles of two 12-hr period. Approximately 200 of the eggs laid during this known genes were identified in the screen: three alleles of time were then transferred to afresh plate andcounted. sdc-l and two alleles of tru-3. The two mutations (qm44 and Twenty-four hours later, the number of unhatched (assumed qm52) with a Tra-3phenotype were tested for noncomplemen- to be dead) eggs was counted. Several days later, the number tation with tru-?(el767) and balanced over nT1 simultane- of animals that had reached adulthood was scored. ously, by crossing qm44/+ or qm52/+ to the strain CB4622 Phenotypicanalysis: Threemutant strains suspected by and recovering the appropriate progeny classes.Alleles of overall phenotype to have muscle defects were stained with other genes for which maternal-effect viable mutations are fluorescein isothiocyanate-labeled phalloidin to visualize F- known were not isolated, probably because of the experimen- actin in muscle cells. 4',&Diamidino-2-phenylindole (DAPI) tal conditions of the screen. The screen was carried out at was also used to stain nuclei. Mutant and control N2 worms Viable Maternal-Effect Mutations 1353 Mutations Maternal-Effect Viable were harvested in M9 buffer and pelleted. The animals were the mother is sufficient to produce a wild-type pheno- frozen for 10 min at -80" and thawed to permeabilize the type in the offspring. However, it neither selects for, worms. For fixation, theworms were immersed inacetone for nor against, mutations forwhich the presence of a wild- 10 min and pelleted again. The acetone was aspirated, and the animals were airdried. The concentrationsused for phal- type copy in the zygote is insufficient for a wild-type loidinand DAPIwere 0.1and 1 pg/ml, respectively.The phenotype (strict maternal effect). animals were stained in the solutions for 3 hr, rinsed three Summary of the mutant phenotypes:The 24 comple- to four times in M9 buffer, and then mounted and examined mentation groups fall into seven phenotypic classes: (1) by fluorescence microscopy. All three strains were also ob- mau (maternal-effect uncoordinated), eight genes; served by polarized light and differential interference contrast (2) microscopy. mum (gaternal-effect gncoordinated and Eorphologi- Animals carrying mau mutations were also examined with cally abnormal), three genes; (3) mal (maternal-effect the carbocyanidedye DiI to stain thesensory neurons morphologically abnormal), four genes; (4) mad (ma- (HEDGECOCKel al. 1985; E.M. HEDGECOCK,personal commu- ternal-effect dumpy), three genes; (5) mud (Eaternal- nication). effect gncoordinated and dumpy), one gene; (6) clk (abnormal function of biological cJo&s), three genes; RESULTS and (7) genes involved in sex determination and/or A geneticscreen for viablematernal-effect muta- dosage compensation, two genes (whose phenotypes tions: Wild-type (N2) worms were mutagenized with are not discussed further here). EMS and allowed to self-fertilize for two generations. Table 1 gives short summaries of the phenotype, ex- Animals resulting from the second round of self-fertil- pressivity and penetranceof the reference mutation for ization (F2) are likely to be homozygous carriers of a each gene. In the text below, we describe the mutant number of newmutations with or without visible pheno- phenotypes in more detail. The phenotypic descrip- typic effects. Such animals were examined, and those tions given in Table 1 and in the main text refer to the that displayed wild-type overall morphology and behav- adult phenotypes, unless explicitly stated. Most of the ior were picked onto individual fresh plates and allowed phenotypic defects of all the mutants can be observed toself-fertilize. Those strains that then produced an in L1 larvae, indicating thatthe genes are required entirely mutant brood (F,) were analyzed further in during embryogenesis as suggested by the fact that their the following way. After the initial isolation, all mutants phenotype can be rescued by a maternal effect. How- were crossed to wild-type (N2) males and three to five ever, at this stage of the characterization of these genes, phenotypically wild-type hermaphrodites from the re- we have not attempted to determine whether the gene sultant progeny (F, ) were picked onto individual plates. products were also required during postembryonic de- The self-progeny (F2) of these individuals were then velopment. examined for the presence of phenotypically wild-type Desniption of mau genes: mau genes can be subdivided animals. When -25% of these F2s showed the mutant in three groups: (1) mau-1, mau-2 and mau-4, mutations phenotype, the strain was discarded because it sug- in which result in neuroanatomical defects; (2) mau-3, gested that theanimal picked originally was an escaper, mau-5 and mau-6, mutations inwhich result in defective that is, an animal carrying a fullyzygotic mutation, muscle function; and (3) mau-7 and mau-8, mutations which was not strongly expressed. When substantially that result in generalized locomotory defects that we fewer than 25% of the animals displayed a mutant phe- ascribe to defects in synaptogenesis. Among the mau notype, 24 phenotypically wild-type F2s were picked mutants, only mau-7 and mau-8 show significant embry- onto individual plates and their F3 progeny examined. onic lethality. Dying embryos, however, appear fully dif- At this stage, in most cases, some of the FS broods were ferentiated and not deformed. composed almost entirely of animal displaying a mutant mau-1 (two alleles): For both mau-1 alleles, the mutant phenotype. When none of the F3 broods displayed a phenotype is extremely variable. The most affected ani- fully mutant phenotype, the procedure was repeated. mals are almost totally paralyzed at hatching, grow very When the repeated procedure was still unsuccessful at slowly and many die before reaching adulthood. The obtaining fully mutant F3 broods, we considered that most mild phenotype is shown by a small proportion the phenotype of the original mutant strain was syn- of animals that grow at a normal rate butkink severely thetic in origin, that is, due to more than one indepen- when attempting backward movement. Between these dently segregating mutation, andthe strain was dis- extremes, individual mau-1 mutants display qualitatively carded. distinct phenotypes. For example, althoughmost of the Strains thatappeared generally sick without dis- animals appear to lay their eggs normally, a proportion playing specificmovement defects or anomalies of body (1/5) of the mutants are strongly egg-laying defective shape were not kept. We analyzed 30,000 FP animals (Egl). Similarly, a subset of the mutants resembles unc- and their progeny inthis way and isolated 41 mutations 6 and unc-5 mutants in their locomotory pattern, sug- representing 24 complementation groups. gesting abnormalfunction of their dorsal cord The screening procedure selects for mutations for (HEDGECOCKet al. 1985). A differentsubset of the ani- which the presence of a wild-type copy of the gene in mals tends to lie witha stiff ventral bend butwith mobile 1354 S. Hekimi, P. Boutis and B. Lakowski
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Hekimi,and P. Boutis B. Lakowski heads and tails. This phenotype resembles that of unc- strongly suggest that these genes are required for nor- 4 (WHITEet al. 1992), suggesting abnormal function of mal neuroanatomy and that theirmovement defects (in ventral cord motor neurons. particular for mau-1 and mau-2) probably arise from mau-1 mutants were examined by staining with the abnormal or missing neuronal connections. carbocyanide dye DiI, which stains sensory neurons in mau-3, mau-5, mau-6 (one allele each): Mutants in each the head and in the tail. In most animals, the projec- of these genes appear to have defective muscles. The tions of amphid(those in thehead) and phasmid phenotype of all three mutantsworsens throughout de- (those in the tail) neurons appeared normal. In a few velopment. mau-5 mutants are Egl and only become cases, however, the processes that the phasmids send fully paralyzed after filling up with eggs. The progres- into the ventral cord appeared twice as long as in the sion of the paralysis of mau-3 mutants is very gradual wild type. during postembryonic life, becoming fullyparalyzed mau-2 (three alleles): All three alleles of this gene lead only during adult life. By contrast, the phenotype of to a similar phenotype. When attempting backward mau-6mutants worsens to full paralysis very rapidlyafter movement, these animals kinkseverely. In addition, reaching adulthood, leading rapidly to death. mau-2 mutants are strongly Egl. The egg-laying defect When worms are examined under polarized light, is not maternally rescued: a homozygous mau-2 mutant muscles can be visualized by their birefringent proper- originating from a mother carrying a wild-type allele ties. The muscle bands in all three mutants appearvery of the gene displays fully wild-type locomotion but is narrow and ragged compared with wild-type worms,and severely Egl (see Table 1).Approximately 15% of mau- some muscle cells appear to be degenerating. In some 2 mutants die during larval development. Examination mau-6 mutants, muscle cells are found in isolation and of these worms by DIC (differential interference con- not aligned into a bandnor attached to each other. All trast) microscopy showed that the excretory canal was other aspects of anatomy appear normal at thelevel of invariably very short and strongly convoluted in places DIC microscopy. To date, it is unclear which aspect of (Figure 1B) . muscle development or function is affected in these Examination of mau-2mutants with DiI did not reveal mutants; however, the maternal effect makes it unlikely any defects in the amphid neurons, but dye filling of that a structural molecule directly involved in muscle the phasmid neurons failed entirely in >90% of the contraction is affected. animals. In most of the remaining cases, they stained mau-7, mau-8 (one allele each): Mutants in these two on only one side and only very rarely on both sides. genes display the same pattern of phenotypes. They are mau-4 (three alleles): All mutant animals are severely lethargic and, when undisturbed, tend to lie on the Unc when attempting backward movement. These mu- plate with a fully extended body posture. However, they tants have a partially wasted posterior body similar to are also jerky; even when undisturbed, they display fre- the phenotype of some mutants with abnormal migra- quent very rapid forward or backward movements over tions of the canal associated neuron (MANSER and very short distances that are not followed by a more WOOD,1990). Wasting is generally confined to the most posterior one-fourth of the body. This wasted part of continuous movement. In addition, they display a num- the posterior body can appear stiff or flaccid, but it ber of distinct movement defects. For example, when is always unable to propagate normal waves of muscle moving forward, the head and the anterior half of the contraction. Two observations suggest that the normal body propagate only very shallow waves. Atthe extreme substrates necessary for proper cell and axonal migra- of the phenotypic continuum, the anteriorone-third of tions are altered in the wasted part of the body. First, the body is fully stiff and pushed forward by the rest of the posterior gonad armgenerally reflexes back prema- the body in a manner that is very reminiscent of the turely toward the anterior of the animal instead of en- locomotory defects of levamisole-resistant uncoordi- tering the wasted region. Second, all amphid and phas- nated mutants like unc-29 (LEWISet al. 1980). Further- mid neurons could be normally stained with DiI and more, when induced to move backward, these mutants the amphidprojections always appeared wild type. How- frequently kink but also displaya discontinuous ratchet- ever, the anteriorly directed processes of the phasmids like movement reminiscent of mutants of unc-11, a gene into the ventral nerve cord were always very short in shown to be required for normal cholinergic neuro- these mutants, whereas the posterior projection into transmission (CULO~TIet al. 1981; HOSONOand KAMIYA the tail spike appeared normal. 1991; NGUYEN et al. 1995). Finally,all animals are Two alleles of the gene (qml8ts and qml9ts) are tem- strongly constipated and show defects in the defecation perature sensitivewith a steep temperaturedepen- cycle, including very long periods and the frequent ab- dence: most animals are almost fully wild type when sence of expulsion contractions, a behavior known to raised at 20", when raised at 22" are fully mutant, and require the normalfunction of GABAergic neurotrans- die aslarvae at 25". One allele (qm45) is Unc at all mission (MCINTIREet al. 1993a,b). These observations temperatures, although it is also subviable at 25". suggest that some general aspect of synaptic function The phenotypes seen in mau-1, mau-2 and mau-4 is affected. As these mutants display a maternal effect, Viable Maternal-Efkct Mutations 1.757
F 1
FIGURI:.I.-Morphological phenotypes of uncoordinated mutants. mltm-l and v~~u-2mutants. (,.I) A wton-1 mutant with an anteriorly mispositioned vulva. (B) Part of a mnu-2 mutant at the LS stage (between the posterior of the pharynx and the AIAI (anterior lateral microtuble) newon cell body; anterior to the left). On the left, scvcr~lnuclri 01' head neurons can lxx seen (round structures), as well as a cell with a complex shape (indicated by an arrow) that is the amphid shcath cell. In this animal, the excretory canal stops prematurely in a cluhshaped structure indicated hy a large arrowhead. In the wild type. the excretory canal extends normally to the anus of the animal, which would be well to the right of his picturc. The cell lying on thr canal. just left of the point of termination, is probablv the BDU neuron. (C) A mum1 mutant displaying a normally tlr\rlopcd hr;d and an undifferentiated posterior body:
it suggests that their altered synapticfunction could Animals that survive to adulthood display a nlunber result from a defect in synaptogenesis. of distinct phenotypes. A variably deformed head is frr- DPsmpions of mum gm,~.s:Each of thethree mum quentlyobserved, ranging from a relatively rounded genes is defined by a single mutation, which results in rather than a pointed head, to hypertrophic dorsal and similar phenotypes, and they are discussed as a single ventral bulges. The pharynx is often highly abnormal, class. Mutants are variably uncoordinated, ranging from ranging from abnormal relative sizrs of' its parts to ap- classic kinking to total uncoordination. The most se- parent total absence of parts (r.~.,no anterior bulb or verely affected animalsare unableto propagate contrac- no isthmus). Deformations of other parts of the body tion waves along their body; instead different regions were also observed, albeit more rarely. of the body appear to contract independently.All three The most reproducible and interprrtable abnormali- mum mutations result in a high level of lethality (see ties we observed in these mutants are those affecting Table 1). In all three mutants, severedefects in em- thereproductive and excretory systems. In the wild bryogenesis can be observed. In almost all cases, the type, the posterior gonad arm is on the left of the gut dying embryos are severely deformed, but with no sim- and the anterior arm is on the right. In all three mu- ple or obvious pattern to the abnormalities. However, tants, one gonad arm is frequently on the wrong side in some of the dying embryos, the head developed nor- of the gut, resulting in animals that have both gonad mally, whereas the rest of the body remains a relatively armson the same side of thegut. More rarely, the undifferentiated ball of cells (Figure 1C). This pheno- gonad arms are fully inverted relative to wild type (Fig- type resembles that of known mutants [(P.R., nohl) ure 2). These defects arc highly penetrant; in mw3, (K. L. VANAUKEN, L. G. EDGARand W. R. Woon, per- for example, 80% of worms that reach adulthood haw sonal communication)]. their posterior gonad on the right rather than on the 13.58 S. I-lckinli,1'. I3owis ;mtl 13. 1;lkowski
rior c;ln;ds. suggc.sting that the normally ;Interiorly tli- rcctcd c;~llillsarc directed posteriorly in thcsc animals. Tnkcw togcthcr. our ohscnations suggcst that in thcsc mut;mts some fw~damcntal ;~spcctof' ccll movc- ment is impairccl, resulting in misplaccmcnt of cclls throughout tlcvlopmcnt. including those cclls that do not u~~tlc~golong tlistancc migriltions. 1Ir.sniptiom of n~olgrnrs: 1no1-I (ow nllrlr): This muta- tion produces extensive cmh~~onicand l:~nallethality. Ahout half of the srln-iving mutants sl~owone or more of a few stcrcotypic;d dcl'c.cts. Thcsc include ;1 clors;\l anteriorly directed protrusion on the head and ;I postc- riorly dircctctl tlors;d protrusion ovcr thc anus (Figurc
e- ;" w 3). Thcsame clcfects. htmuch mort. sevcrc, can frc- " qucntly he ohsc~-\ctlin dying c;lrly-stagc mutant lanae and embryos. Tllc dcfccts in thcsc mrltants do not rc- I:KA.KI:. ?.--I\ ,,rrc,rr-2II1I1t;I11t hnving IWO ol'tl~c.pl~c.~~otypcs semble those of any prc*viorlslyknown mtmnt. frc.qucntly found in mon mItxnIs: tlw posterior go~~;ltlis psi- mol-2 (thm/dlrlr.s): Thc thrc~illlc'lcs of this gene :UT rioncd on thc right of 01c* gut, rcwlting in an ;lnilllal wit11 VCI? similar ;Ind protlucc most frquently a sevw". ven- I)oth gonad ;mns on tllr same sitlc. and tl~cright ~*xcrct(m g~illl~~cc.~~is ;llsO pos~l~onct~ilntc.riOrly ill,(l t~,<.c.scrcrol? cc.il tral hypertrophy loc;1tctl untlcr the phiIry11gcill isthmus is positioned too posrrriorly. I\lthough rhe nlispositioning of antl postcrior h~dh(Figure 4h). Other lesscircum- rhrsr ~worcll is intlcpcntlcnt, in this particular;minwI. tl~rsr scril>ctl &:fccts ;ire illso ol)sclyctl, prcscq1til1g ;1 general IWO cc4s II~~c-almost cx;~crlyinvcwrtl tlwir rcspcctivc psi- anti not oln,iollsly stc,rcorypc,~tlcformation (,fthe hody. lions. Thcsc tlcfccts often lcatl to cwly lethalit\, and can also he ohsc~~cdin dying embryos. The defects seen in mnl- left. Abnormally positioned and shaped gonad primor- 2 mutants are reminiscent of those ohsewed in rmb-3 dia can hc ohscnctl in 1.1 and later land stages. More- mutants (LIWIS and HOIX;KIS1977). ova-, wt lrcqucntly ohscnc an abnorm;d placement of mol-3 (ow nllrlr): The- main defect in this mutant the \TIIW (cither postc8rior or anterior of its normal ap pcars to hc that most of the time the buccal cavity does position)(Figurc I A). Together,these ohsewations not open at the tip of the head. Instead, it appears to suggest that in thcsc m\lt;llltS, the cells formingthe open at random positions more posteriorly, in the head, gonild primordium migrate ahnormally. including on the dorsal, ventral or lateral sides (Figure U'c ;\Is0 ohscnc~tlhighly sterc.otypiciIl anomalies in 4C). Embryonic and I;un.;d Ictll;dity is high in this mu- thc positions of the cclls of the cxcrctorv system, in tant because many dying embryosand Ialxw appear to prticul;Ir, the CXCWIOI'\' CCII i111tl tllc cxcrctory gl;rds have the pharynx and the buccal c;i\.iv dctxhetl from (Figurc 2). In the wild typc. thc cxcrctory cell is on the left, ventral and anterior to the terminal hulh of the the hvporlcrmis. The few animals that swvive to adult- hood generally show only a relatively mild pharynx, whereas the excretory glands, :I hiliit<:rdly syn- phenotype metric pair of cells, arc locatcd ventrally and posterior (Table 1). to the terminal hulh and scntl thick proccsscs anteriorly md4 (onr nll~l~):In this mutant, there is a conspicu- where they mcct and IIISC. In the mutants, individual ous deformation of the head resulting from a severe cxcrctory glands arc frcq~~c*ntlylocated morc anteriorly, hypertrophy of the left side of the head only. The hypcr- mitlwlv between the pharyngeal hulhs, and send i1 pro- trophy is variable in size and anteroposterior extenthut cess postcriorly to mcct the proccssof the contr;d:ltcd mostly is located in the anterior partof the head (Figure gland ccll. In all thrcc mutants, thc right gland cc4 is 4B). The lineages that form analogous bilaterally sym- iIlli.ctcd morc frequently than the left. For example, in metrical p;\irs of hypodermal cells in the head :Ire not mum-I. 60% of thc right itnd 15% of tho left gland cells homologous by lineage (SrFr-5~osrt (11. 1983). Ow view, arc positioned anteriorly. In atltlition, tho cxcrctory ccll thcrcforo, is that the defect in mol4 mutants is in the is also frcqwntly positioned too posteriorly and/or 011 spccification of only the lineages on the left side. the right sitlc (Figurc 2). For cximplc, in IIIIIVI-~, 3.5% 1)r.srriptiom of mod nnd mtrd pnr.s: The phenotype of of thc excretory cc-lls is in the ;h"m;d posterior posi- nwdantl mud mutants have not been extensively studied tion antl (X)%, is on thc right. to data. and the description we can give does not go The canals protlucc~lby thcc-xcrctory cc-lls i~rc;dmost beyond the descriptive summary given in Table 1. In invariably abnormal in thcsc animals. M'c obsc!nrc very all cases, males and hermaphrodites are an'ected, sug- frcqrlcntlv one or both postcrior c;mals in the ventral gcsting that thcsc genes arc not involved in dosage com- cord or both posterior c;In;ds on thc same sidc of the pensation in any simple way. mnd-2, ~nnd-3and mid-I animal. In raw cases, we ohsc~~cclmorc than two postc- mutants display significant embryonic lethality. Dying \'i;lld(- \I;~~~~~~1i~I-I~I~i.~~\llll;lliolls 1 :<.5!b
~ ""f"-7 - r;;"*=7" r-" .y: \ i iI' 2 L
FI(;I'KI.3.- tnd/ n1uI;InIs. (;\) I Ic;d of';III ;lnil11;d showing ;I typicxI tlors;~lprolrllsion. (1%) :\nothcr ;Inim;ll showing ;I lvpi(xl tlorsal protrusion in ~hc.poswrior p~rtof' rhr I)otly. ((:) ;\II cn1;wgt-d vicw ol' ~IICprotrusion of' ~hr;lnin1id shown in I\. sl~o\vi~lg thr lunlrtl of the. g111~III~ IIIC locarion ol'vc-n~r;~Inclyc cord nllc1c.i. Thc. ilnin1;ds sllo\\*n ;IW1.4 IilI~I~~ cI11hlyos ZIIT frcqtlt'lltly ~00rly<*lo1lgatctl ~III iIpl)cilr ol'thc aninwls. 111 T;~l,lc1, the fr;Iction given in I~xkcts Idly tlifli.rc~ntiatctland arc not highly tlcformcd. I,csitlcs the statcmcnt on maternal rcsc-~~cc-orrc*sponds Ilm-ri/)tinn.s nf rlk pms: rlk-1 CJIW ~~llt~lt~.s):Thrw of thc 10 thc fraction of the self-progeny from ;I scIf-fC*rtilizing itllcl DlSCUSSIOS We identified a number of mutations whose phcntr types and mode of transmission suggest that they are involved in the development of the worm. \'en few viable maternaleffectmutations had been identified before our screen (see Introduction),probably because only a screen specifically designed to find them would allow their systematic iclentification. It is possible that some of the mutations we isolated are not null mutations and that null mutations in these genes would lead to lethality or abolish maternal rescue. However, whatever the effects of nnll mutations, the phenotypes we identified point to at least some of the processes for which these genes are required. Indeed, the identification and study of hypomorphic mutations to study the processes they affect has heen a very SIIC- cessfd approach in C. t+gans (~.g.,HILI. and STERSIWKG 1993). As several of the phenotypic gene classes wc identified in this screen show novel phenotypes, we ex- pect they will be useful in the study of the genetic basis of development. Is there a class of genes defined only by maternal- effect viable mutations? None of our genes mapto the vicinity of other viable genes with a similar phenotype.It is likely therefore that most of them have not previously been defined by only strictly zygotic viable mutations. In particular, the ttnr gene class is believed to be close .- to saturation,reinforcing this argument for the mot1 and mttm genes (which exhibit uncoordination). Fur- thermore, for several of our genes, we recovered muta- tions at the same rate as for sdr-1 (three alleles) and Ira-3 (twoalleles). As all known alleles of these two genes show a maternal effect and are not lethal (albeit hn-3 mutants are sterile),they were probably recovered near the normal loss-of-function frequency. This sug- gests that for all genes for which we have obtained at least two alleles (maximum is five for rlk-l), we might have obtained these alleles at the normal loss-of-func- tion frequency. This would imply that at least a propor- tion of our genes can he defined only by viable mater- Fl mu-2 ml-31 mad-2 md-1 II I I 1 I I I Unc-35 unc-29 Unc-75 Unc-54 bli4 - nM23 eM3 - nDf24 I. rol--6 wIc-4 clk- 1 mud mud-1 I dk-2 mum-3 111 IV mi-2 c V - sDf28 SM32 mu4 c X bn-2 urn7 Iln-15 sup10 -10 m.u. FIGURE.i.-Genetic map positions of the new genes descrihed in this study. This diagram is a summa?; for the ultimate mapping data consult Table 2. The full data are available from the Caenorhahditis (knetics Center. Each horizontal line represents an entire linkage group (scale bar is given).Genes defined in this study are drawnahove the line.positioned hetween the reference markers against which they were mapped (drawn helow the line). Deficiencies are shown only where complementation data formally narrows the interval for a gene. The inset for linkage group IV shows details not present on the full-scale map. "here a gene was not separated from a marker, its position is marked hy a dot ahow that marker with a har representing its likelv position. genes would be lethal as well. This impression should, previously known genes with viable alleles: sdr-I and /ru- however, be tempered by the observation that we have 3. For both of these genes,all previously known alleles obtained mnu-I,mnu-2 and mol-2 alleles at relatively exhibitmaternal rescue (HODGKIN198.5; VII.I.EsEr\'F. high frequencv (two to three alleles), and all alleles of and MEYER 1987, 1990). The frequencvat which we thcse genes also produce very high lethality. Further- recovered mutations in these genes therefore provides more, the phenotypes of mnu-1, mnu-2, and mnd-1 over a baseline to evaluate the significance of the frequencies deficiencies are notworse than that of mutant homozy- we observe with other genes. From 30.000 mutagenizcd gotes. genomes screened, we obtained thrcc dr-I allc*les and Mutation frequency: We isolated alleles of only two two /m-3allclcs. This frequency appears to hold for all 1362 S. Hekimi, P. Boutis and B. Lakowski TABLE 2 Summary of genetic mapping for 22 genes G ene Genetic mapping data"mapping Genetic Gene data"mapping Genetic Gene mau-1 V Inside sDj28, sDf32 mal-2 V [ dpy-11 mal-2/unc-51] mau-2 I [ dpy-14 unc-29/mau-2] dpy-11 (35/35) unc-51 (0/35) mal-2 dpy-14 (22/36) mau-2 (14/36) unc-29 [ dpy-11 qm31/+ +]' inside nDj24; outside nDf23 58/100 d@-11 m~l-2:24 CM complements che-3 mal-3 I [ unc-40 bli-4/mal-?] mau-3 IV [ bli-6 eat-12 unc-24/mau-3] unc-40 (5/6) mal-3 (1/6) blz-4 bli-6 (16/31) eat-12 (13/31) mau-? mal-4 I1 1 ) [ dpy-10 unc-53/ma1-4] (2/31) unc-24 dpy-10 (6/16) mal-4 (10/16) unc-53 outside nDf41 2) [ rol-6 unc-4/mal-4] mau-4 X [ lin-15 suplO/mau-4] rol-6 (0/23) mal-4 (23/23) unc-4 lin-15 (5/5) mau-4 (0/5) sup10 mad-1 I [dpy-24 unc-54/mad-l] mau-5 I11 [ dpy-l daf-2 unc-32/mau-~ dpy-24 (52/53) mad-1 (1/53) unc-54 dpy-1 (3/10) mau-5 (7/10) daf-2 inside eDf3 mau-6 V 1) [unc-42 dpy-2l/mau-6] mad-2 I [ dpy-5 daf-16 unc-75/mad-2] unc-42 (3/9) mau-6 (6/9) dpy-21 dpy-5 (7/12) mad-2 (5/12) unc-75 2) [ro64 lin-25 him-5 unc-76/mau-6] [ unc-29 mad-2/+ +] roc4 (0/7) mau-6 (1/7) lin-25 (2/7) him-5 111/118 unc-29 mad-2 :3.0 cM (4/7) unc-76 mad-? V [ rol-4 lin-25 h1m-5 unc-76/mad-?] outside ctDfl, arDf1 rol-4 (0/8) lin-25 (3/8) mad-3 (1/8) him-5 mau- 7 IV [ dpy-9 lin-l/mau-a (4/8) unc-76 dpy-9 (0/11) mau-7 (11/11) lin-1 outside arDf1 mau-8 IV [dpy-13 unc-24/mau-8] mud-1 111 [unc-79 lon-l/mud-1] dpy-13 (30/31) mau-8 (1/31) unc-24 unc-79 (4/10) mud-1 (6/10) lon-1 outside nDf41 elk-1 I11 [dpy-l7 elk-1 lon-l/grpl] mum-1 IV 1) [dpy-13 unc-?l/mum-1] dpy-17 (7/18) gr0-1 (0/18) elk-1 (11/18) dpy-13 (36/40) mum-1 (4/40) unc-31 Con-1 2) [ dpy-20 unc-3l/mum-l] complements mel-31, me1 (jb7) dpy-20 (0/11) mum-1 (11/11) unc-31 Clk-2 I11 [sma-4 mab5 unc-36/clk-2] inside df7; outside eDf19, sDj2 sma-4 (35/52) elk-2 (3/52) mab5 complements let-657, unc-43 (14/52) unc-36 mum-2 IV [ unc-24 dpy-2O/mum-2] complements embl, emb2, emb7, emb16, unc-24 (4/13) mum-2 (9/13) dpy-20 emb24, emb25, emb32, emb34, ncl-1, lin- inside eDfl9; outside eDfl8 13, lin-39 mum-3 111 [ unc-32 dpy-18/mum-3] elk-3 I1 [ rol-1 elk-3 unc-52/eat-2] unc-32 (8/24) mum-3 (16/24) dpy-18 elk-3 (3/4) eat-2 (1/4) unc-52 outside Dfl [ elk-3 unc-52/+ ] I + ' mal-1 IV [ unc-26 tra-3 dpy-4/mal-l] 430/439 elk-3 unc-52 : 1.0 cM unc-26 (6/17) mal-1 (10/17) tra-3 (1/17) dpY-4 " a Only the data critically determining the positionof the gene on the genetic map, as shown in Figure5, is given; the results of other mapping experimentscan be obtained from the CGC (Caenorhabditis Genetic Center) gopher. The genotypes given in square brackets are the relevant genotype of the animals whose descendants were scored to obtain multifactor or two factor data. *Two factor data; the numeratorof the fraction represents the number of self-progeny from animals of the given genotype which were homozygous for the doubly mutant chromosome. Only animals homozygous for the marker mutation were scored (the denominator). the genes in our screen, as only clk-1 has yielded more subtly different and produced alower level ofmutagen- alleles with five mutations isolated (Table 1).The stan- esis. Instead, we suggest two alternative explanations. dard EMS mutagenesis protocol (SULSTONand HODG First, the criteria of our screening method might be KIN 1988) is known to have an approximate 5 X lop4 responsible. We chose F2 animals that looked as fully probability of inactivating an average gene (BRENNER wild type as possible to avoid picking animals carrying 1974; GREENWALDand HORVITZ1980). The average fre- phenotypically weak but nonethelessfully zygoticmuta- quency of isolation in our screen is -10-fold lower (6 tions. When visible recessive mutants are selected di- X We believe it is unlikely that this low mutation rectly from a plate of mutagenized F2 animals, such frequency is due to maternal-effect genes beingon aver- discrimination is not necessary. We might thus have age smaller targets for mutagenesis or that our muta- been selecting indirectly for genomes that, for some genesis conditions, although standard, mighthave been reason, were not strongly mutagenized. The viable ma- V iable Maternal-Effect Mutations Maternal-Effect Viable 1363 ternal-effect loci would then also be hit more rarely in GOTTLIEB,S., and G. B. RUVKUN,1994 duf2, dufI6and du.23: genet- ically interacting genescontrolling dauer formation in Cuenorhub such genomes. Second, our screen allowed usto recover ditzs ekguns. Genetics 137: 107-120. only relatively severe mutations because of the type of GREENWALD,I. S., and H. R. HORVITZ,1980 unc-93(e1500): a behav- heritability of the genes in which we were interested. ioral mutant of C. ekguns that defines a gene with a wild-type null phenotype. Genetics 96: 147-164. For most of our genes, a maternal or zygotic contribu- HEDGECOCK,E. M., J. G. CULO~I,J. N. THOMSONand L. A. PERKINS, tion from a wild-type allele is sufficient for a fully wild- 1985 Axonal guidance mutants of Cuenorhubditis ekguns identi- type phenotype (Table 1).It is possible, therefore, that fied by filling sensory neurons with fluorescein dyes. Dev. Biol. 111: 158-170. when such a gene is only partially inactivated by muta- HILL,R. J., and P. W. STERNBERG,1993 Cell fate patterning during tion, the double (maternal and zygotic) contribution is C. ekguns vulval development. Development (Suppl.): 9-18. sufficient to produce a wild-type phenotype. HODGKIN, A,,J. 1980 More sex-determination mutants in C. ekguns. Genetics 96: 649-664. We thank LEONAVERY, who suggested to S.H. that he should stop HODGKIN,J. A,, 1983 Male phenotypes and mating efficiency in claiming muu genes exist and just carry out the screen instead. We Cuenmhabditis ekguns. Genetics 103: 43-64. thank JUI.IA PAK,SHIN TAKAGI, DENISE Au-LEUNG and CWREBENARD, HODGKIN,J. A,, 1985 Novel nematode amber suppressors. Genetics for some mapping data,as well asTOM BARNES for advice on mapping 111: 287-310. HODGKIN,J., and S. BRENNER,1977 Mutations causing transforma- and for Figure 5. We thank TOMBARNES and JONATHAN EWBANKfor tion of sexual phenotype in the nematode C. eleguns. Genetics critically reading themanuscript. Thiswork was supported principally 86: 275-287. by the Canadian National Science and EngineeringResearch Council HOSONO,R., and Y. KAMIYA, 1991 Additional genes which result in (NSERC Operating grant 0121351), the Medical Research Council an elevation of acetylcholine levels by mutations in Cuenmhubditis of Canada (MRC Operating grant MT 12085) and a J.W. McConnell eleguns. Neurosci. Lett. 128 243-244. Foundation fellowship to B.L. The earliest phase of this work was ISNENGHI,E., K. T. R. DENICH,K. RADNIA, G. VON EHRENSTEIN,R. C. supported by the Medical Research Council’s Laboratory of Molecu- CASSADA,et ul. 1983 Maternal effects and temperature-sensitive lar Biology in Cambridge and the Swiss National Science Fund. Some period of mutations affecting embryogenesis in C. ekguns. Dev. nematode strains used in this work were provided by the Caenorhab- Biol. 98: 465-480. ditis Genetic Center, which is funded by the National Institutes of KEMPHUES, K. J., M. KUSCHand N. WOW, 1988a Maternaleffect lethal mutations on linkage group I1 of Cuenorhubditis ekguns. Health(NIH) National Center for Research Resources (NCRR). Genetics 120: 977-986. Some other strains used in this work were provided by the C. ekguns KEMPHUES, R,J. PRIESS,D. MORTON and N. CHENG,1988b Identifica- Genetic Toolkit Project, funded by the NIH NCRR. We also thank tion of genes required for cytoplasmic localization in early C. DON MOERMAN,VICTOR AMBROS, ANDREW CHISOI.Mand LEONAVERY ekguns embryos. Cell 52: 31 1-320. for strains. LEWIS,J. A,, and J. HODGKIN,1977 Specific neuroanatomical changes in chemosensory mutants of the nematode C. ekguns. J. Comp. Neurol. 172 489-510 LITERATURE CITED LEWIS,J. A,, C. H. WU, J. H. LEVINEand H. BERG,1980 Levamisole- resistant mutants of the nematode C. ekguns appear tolack phar- AVERY,L., 1993 The genetics of feedingin Cuenurhubditis ekguns. macological acetylcholine receptors. Neuroscience 5: 967-989. Genetics 133: 897-917. MAINS, P. E., I. A. SULSTONand W. B. WOOD,1990 Dominant mater- BARNES,T. M., 1991 Molecular analysis of the tru-3 region of the naleffect mutations causing embryoniclethality in Cuenurhubditis Cuenorhubditis eleguns genome. Ph.D. Thesis, University of Cam- ekguns. Genetics 125: 351-369. bridge, England. MANSER, J., and W.B. WOOD, 1990 Mutations affecting embryonic BOWERMAN,B.,B. W. DRAPER,C. C. MELLOand J. R.PRIESS, 1993 cell migrations in Cuenurhubditis ekguns. Dev. Genet. 11: 49-64. The maternal gene skn-1 encodes a protein that is distributed unequally in early C.eleguns embryos. Cell 74 443-452. MCINTIRE,S. L., E. JORGENSEN andH. R. HORVITZ,1993a Genes BUCHER,E. A., and I. S. GREENWALD,1991 A genetic mosaic screen required for GABA function inCuenurhubditis ekguns. Nature 364 of essential zygotic genes in Cuenorhubditis ekguns. Genetics 128 334-337. 281-292. MCINTIRE,S. L., E. JORGENSEN, J. M. KAPLAN and H. R. HORVITZ, BRENNER,S., 1974 The geneticsof Cuenurhubditis eleguns. Genetics 199313 The GABAergic nervous system of Cuenurhabditis ekguns. 77: 71-94. Nature 364: 337-341. CUZP(IWSKI,E.E.,P.~~IN,C.GARVIN~~~S.STROME,~~~~Identifica- MELLO,C., B. DRAPER,M. KRAUSE, H. WEINTRAUBand J. PRIESS,1992 tion of grandchildless loci whose products are required fornor- The pie-1 and mex-I genes and maternal control of blastomere mal germ-line development in the nematode Cuenurhubditis ek- identity in early C. ekguns embryos. Cell 70: 163-176. guns. Genetics 129: 1061-1072. MEYER,B. J.,and L. P. CASSON,1986 C. ekguns compensatesfor CHAI.FIE, M., and J. E. SULSTON,1981 Developmentalgenetics of the difference in X chromosome dosage between the sexes by mechanosensory neurons in Cuenorhubditisekguns. Dev. Biol. 102: regulating transcript levels. Cell 47: 871-881. 391-410. NGUYEN, M., A.ALFONSO, C. D. JOHNSON and J. B. RAND, 1995 Cuene CHUANc;. P.-T., D. G. ALBERTSON and B. J. MEYER,1994 DPY-27: a rhubditis ekguns mutants resistant to inhibitors of acetylcholines- chromosome condensation protein homolog that regulates C. terase. Genetics 140: 527-535. ekgans dosagecompensation through association with the X PARK, E.-C., and H. R. HORVITZ,1986 Mutations with dominant ef- chromosome. Cell 79: 459-474. fects on thebehavior and morphology of :he nematode C. ekguns. Cu1.0~1.J. G., G. VON EHRENSTEIN,M. R. CULOTTI and R. RUSSELL, Genetics 113: 821-852. 1981 Asecond class of acetylcholinesterasedeficient mutants PRIESS,J. R., H. SCHNABELand R. SCHNABEI.,1987 The glpl locus in the nematode C. eleguns. Genetics 97: 291-305. and cellularinteractions in early C. eleguns embryos. Cell 51: DESAI,C., G. GARRIGA,S. L. MCINTIREand H. R.HORVITL, 1988 A 601-611. genetic pathway for the developmentof the Cuenorhubditisekguns RIDDLE, D. L., M.M. SWANSONand P. S. ALBERT. 1981 Interacting HSN motor neurons. Nature 336: 638-646. genes in nematode dauer larva formation. Nature 290: 668-671. FERGUSON,E. L., and H.R. HORVITZ,1985 Identification and charac- SCHNABEL,H., and R. SCHNABEL,1990a An organ-specific differenti- terization of 22 genes that affect the vulval cell lineages of the ation gene phu-1, from Cuenorhabditis ekguns. Science 250: 686- nematode Cmurhubditis eleguns. Genetics 110: 17-72. 688. GOLDEN,J. W., and D. L. RIDDLE, 1984 A pheromone-induced devel- SCHNABEL,R., and H. SCHNABEL,1990b Early determination in the opmental switch in C. ekguns temperature-sensitive mutants re- C. ekguns embryo: a gene, cibl, required to specify a set of stem- veal a wild-type temperaturedependent process. Proc. Natl. cell-like blastomeres. Development 108: 107- 120. Acad. Sci. USA 81:819-823. STARICH,T. A., R. K. HERMAN,C. K. KARI, W.-H. YEH, W. S. SCHACK- 1364 S. Hekimi. P. Boutis and B. Lakowski WTZ, et ul., 1995 Mutations affecting the chemosensory neurons VILLENEUVE,A. M., and B. J. MEYER,1987 sdc-1: a link between sex of Caenwhabditis ekguns. Genetics 139: 171-188. determination and dosage compensation in C. eleguns. Cell 48: SULSTON,J. E., and J. HODGKIN,1988 Methods, pp. 587-606 in The 25-37. Nematode Cuenwhubditis ekgans, edited by W. WOOD.Cold Spring VILLENEWE,A. M., and B. J. MEYER, 1990 The role of sdc-1 in the Harbor Laboratory, Cold Spring Harbor, NY. sex determination and dosage compensation decisions in Cueno- SULSTON,J. I., E. SCHIERENBERG,J. G. WHITE and J. N. THOMSON, rhubditis eleguns. Genetics 124: 91-1 14. 1983 The embryonic cell lineage of the nematode Cuenorhub WHITE,J. G., E. SOUTHGATEand J. N. THOMSON,1992 Mutations in ditis ekgans. Dev. Biol. 100: 64-119. the Cuenorhubditis eleguns unc-4 gene alter the synaptic input to SWANSON,M. M., and D. L. RIDDLE, 1981 Critical periods in the ventral cord motor neurons. Nature 355: 838-841. development of the Cuenorhubditis ekgunsdauer larva. Dev. Biol. WII.I.IAMS,B. D., and R. H. WATERSTON,1994 Genes critical for 84: 27-40. muscle development and function in Cuenorhubditiseleguns identi- THOMASJ. H., D. A. BIRNBYand J. J. VOWELS,1993 Evidence for fied through lethal mutations. J. Cell Biol. 124: 475-490. parallel processing of sensory information controlling dauer for- WON(;, A,, P. BOUTISand S. HEKIMI,1995 Mutations in the rlk-l mation in Cuenmhabditis ekguns. Genetics 134 1105- 1117. gene of Caenorhubditis eleguns affect developmental and behav- TRENT,C., N. TSIJNG,and H. R. HORVITZ,1983 Egg-laying defective ioral timing. Genetics 139: 1247-1259. mutanb of the nematode Cuenorhabditis ekguns. Genetics 104: 619-647. Communicating editor: R. K. HERMAN