Copyright 0 1994 by the Society of America

The mec-8 of Caenorhabditis elegans Affects Muscle and Sensory Neuron Function and Interacts With Three Other : unc-52, smu-1 and smu-2

Erik A. Lundquist and Robert K. Herman

Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108 Manuscript received March 14, 1994 Accepted for publication May 23, 1994

ABSTRACT in the Caenorhabditis elegans gene mec-8 were previously shown to cause defects in mech- anosensation and in the structure and dye filling of certain chemosensory neurons. Using noncomple- mentation screens, we have identified eight new mec-8 alleles and a deficiency that uncovers the locus. Strong mec-8 mutants exhibit an incompletely penetrant cold-sensitiveembryonic and larval arrest, which we have correlated with defects in the attachmentof body muscleto the hypodermis and cuticle. Mutations in mec-8 strongly enhance the mutant phenotypeof unc-52(uiable) mutations; double mutants exhibit an unconditional arrestand paralysis at thetwofold stage ofembryonic elongation, a characteristic of lethal alleles of unc-52, a gene previously shown to encode a homolog of the core protein of heparan sulfate proteogylcan, found in basement membrane, and to be involved in the anchorageof myofilament lattice to the muscle cell membrane. We have identified and characterized four extragenic recessive suppressors of a mec-8; unc-52(uiable) synthetic lethality. The suppressors, which define the genes smu-1 and smu-2, can weakly suppress all mec-8 mutant phenes. They also suppress the muscular dystrophy conferred by an unc-52(uiable) .

UTANT analysishas proved to bevery useful in the 1991). It is presumed that mec-8 mutants are defective M study of the development and function of both in touch cell function, although no morphological de- the nervous system and the musculature of the nema- fects in the touch cells of mec-8 animals have been dis- tode Caenorhabditis elegans. Many genes identified by cerned by electron microscopy (CHALFIEand SULSTON mutations that affect animal movement, mechanosen- 1981; CHALFIEand Au 1989).The results of temperature- sation, egg-laying, chemosensation,thermosensation, shift experiments with mec-8(~218ts), a temperature- feeding or defecation have been shown to be important sensitive allele, suggest that only a small amount of mec-8 in nervous system development (forreviews, see CHALFIE product is required fortouch cell function; theMec phe- and WHITE 1988; BARGMANN1993), and among theclass notype can be rescued if the mutants aregrown at per- of mutants affected in movement, manyhave been missive temperature either duringembryogenesis or for shown to have defects in muscle development, structure a time during larval development (CHALFIEand Au or function (WATERSTON 1988; WILLIAMSand WATERSTON 1989). 1994). Mutations in mec-8 also cause defects in chemosen- Mutations in the mec-8 gene were originally identified sory neurons (PERKINSet al. 1986). Neurons required in a screen for mechanosensory-defective (Mec) mu- for various chemosensory behaviors have been identi- tants (CHALFIEand SULSTON 1981; CHALFIEand Au 1989). fied by laser ablation studies (BARGMANNet al. 1990, When a fine hair is drawn gently across the body of a 1993; BARGMANNand HORVITZ1991a,b). All of the im- wild-type worm in the region of the head or tail, the plicated neurons belongto a pairof bilaterally symmet- animal moves quickly backwardor forward, respectively. ric sensilla in the head called amphids. Each wild-type Mec mutants do not respond to this gentle touch, al- amphid consists of the ciliated dendrites of 12 neurons though they do recoil to a prod with a wire and move plus two non-neuronal support cells, a sheath cell and with apparently wild-type coordination. Laser ablation a socket cell (WARDet al. 1975; WAREet al. 1975; WHITE studies have shown that the wild-type response to light et al. 1986; PERKINSet al. 1986). PERKINSet al. (1986) touch is mediated by a set of six sensory neurons, the showed by serial section electron microscopy that mu- touch cells, which havelong, anteriorly directed, longi- tation in mec-8 disrupts the fasciculation of the amphid tudinal processes (CHALFIEand SULSTON1981). The pro- cilia. A convenient method for scoring chemosensory cesses of the touch cells are embedded in the animal’s defects in mec-8 mutants (as well as many other chemo- hypodermis and lie near thecuticle; associatedwith each sensory mutants) has been described (PEFUUNSet al. process is extracellular material called the mantle,which (1986). When living wild-type,animals are bathed in a appears to be connected to the cuticle by fibrous bodies solution of fluorescein isothiocyanate (FITC), the cell (CHALFIEand SULSTON1981; FRANCISand WATERSTON bodies and processes of six neurons of each amphid,

Genetics 138: 83-101 (September, 1994) 84 E. A. Lundquist and R. K. Herman which are exposed to the external environment via a ning at the third or fourth larval stage; the disruption pore, fill with dyeand can be readily visualized by fluo- of muscle is correlated with a progressiveparalysis rescence microscopy (HEDGECOCKet al. 1985). Two neu- (MACKENZIE et al. 1978; WATERSTON 1988; GILCHIUST and rons belongingto each of a bilaterally symmetric pair of MOERMAN1992). Other unc-52 alleles cause a paralysis sensilla in the tail, called phasmids (SULSTONet al. 1980; and arrest at thetwofold stage of embryonic elongation WHITEet al. 1986; HALL and RUSSELL1991), arealso ex- (ROGALSKIet al. 1993; WILLIAMSand WATERSTON1994), posed to the outside and fill with dye. PERKINS et al. which is a phenotype characteristic of mutations in other (1986) reported that each of two different mec-8 muta- muscle-affecting genes (WATERSTON1989; VENOLIAand tions invariably block FITC filling of both phasmid neu- WATERSTON1990; BARSTEADand WATERSTON1991; rons and all but two amphid neurons, a phenotype we WILLIAMSand WATERSTON1994). We have found that refer to as Dyf, for abnormal dye filling. when viable unc-52 mutations are combinedwith anyof In this report we further characterize mec-8 mutants. several mec-8 mutations, the phenotype of the double We have identified eight new mec-8 alleles and a defi- mutant is very similar to that of an embryonic lethal ciency of the locus by noncomplementation screens, unc-52 mutant. and we have analyzed the Mec and Dyf phenes caused Suppression of a mutant phenotype by mutation in by all alleles to try to determine the mec-8 null pheno- another geneprovides a second method for identifylng type. We have also discovered that strong mec-8 muta- interactions between genes. We have identified extra- tions cause a cold-sensitive embryonic and larval lethality genic suppressors of mec-8; unc-52 synthetic lethality at low penetrance and cold-sensitive disruptions in the that define two loci, smu-1 and smu-2. We show that structure of body wallmuscle. C. elegam body wall mutations in these two loci are capable of suppressing muscle cells are located in four longitudinal strips that three distinct mec-8 phenes as well as the paralysis con- run the length of the animal, subventrally and subdor- ferred by certain viable unc-52 alleles. sally. To generate animal movement, the contractile force of the body wall muscles must be transmitted to the MATERIALS AND METHODS external cuticle (WATERSTON1988). Interposedbetween General genetic methods: Nematodes were cultured as de- the body muscle cells and the cuticle are a basement scribed by BRENNER(1974). The wild-type strain was N2. We membrane and a thin layer of hypodermis, which is re- havefollowed standard C. elegans genetic nomenclature sponsible for secreting the cuticle. The muscle cells are (HORVITZet al. 1979). Unless otherwise noted, all genetic ex- attached to the adjoining basement membrane, which periments were done at 20". Genes, mutations and chromo- some rearrangements used in this work werethe following (for in turn is anchored to thecuticle via filament-associated references, see HODCKINet al. 1988): LG (linkage group) I: structures called fibrous organelles that bridge the thin dpy-5(e61),unc-40(e271), mec-6(e1342), unc-l3(e51), unc- hypodermal layer and thus complete the mechanical 55(e402),unc-29(eI93), mec-8fe398, u303, 1174, u391, coupling of muscle to cuticle (FRANCISand WATERSTON u218 ts, u456, u314) (CHALFIEand Au 1989), mec-8(rh170) 1991; HRESKOet al. 1994). We have found that mec-8 (E. HEDGECOCK,personal communication), dpy-24(s 71), unc- 75(e950),unc-lOl(ml), unc-59(e261), unc-95(su33), unc- animals grown at 16" frequently exhibit defects in at- 54(e190), nDf23, nDf24, nDf25, nDp4.LGII: sqt-2(sc3), mup- tachment of the myofilament lattice to the hypodermis 1(e2346),unc-85(e1414), dpy-lO(e128), unc-4(e120), and cuticle. In sum, mec-8 mutations cause defects in the sqt-1 (scl3), unc-52(e669, e669su250, e444, e998, e1421, functions of at least three distinct types of cells: mech- su54, su200) and mnC1. LGIII: dpy-l8(e364). LGW. dpy- 13(mn278),emb-9(hc70), bli-6(sc16), mec-3(e1338), him- anosensory and chemosensory neuronsand body 8(e1491). LGV unc-60(e667), mec-1 (el 066), unc-23(e324), muscle. mec-9(el494), him-5(e1467).LGX: unc-l(e538), lon-2(e678), One of the virtues of C. elegans genetics is its suit- mec-2(e75),unc-97(su110), unc-6(ev400), mec-7(e1343), ability for identifylng interacting genesthrough the mec-lO(e1515),him-4(e1267), unc-3(el51), daf-6(e1377), analysis of the of double mutants. We report unc-7(bx5),mec-S(e1340), let-2(6246), osm-l(p808), mec-4(e1497). here two types of genetic interaction involving mec-8. Genetic mapping of mec-8 We have refined the map po- The first is a synthetic enhancement of an unc-52 mu- sition of mec-8, which earlier was mapped to the rightof dpy-5 tantphenotype by mec-8 mutation.Products of the Z (CHALFIEand SULSTON1981). For mapping purposes, mec-8 unc-52 gene arehomologous to the core protein of the mutants were scored either with respect to their defect in mammalian basement membrane heparan sulfate pro- mechanosensation (Mec), by gently stroking the animals with an eyebrow hair (CHALFIEand SULSTON1981), or with respect teoglycan (ROCALSKIet al. 1993); and an epitope en- to their defect in dye filling (Dyf, see below). The results of coded by unc-52 sequence (ROGALSKIet al. 1993) is con- three-factor crosses, whichare given in Table 1, put mec-8 be- centrated in those regions of the basement membrane tween unc-29 and dpy-24 and closer to unc-29 (see Figure l). that contribute to the muscle-hypodermal attachments The following two-factordata yield an unc-29-dpy-24 map dis (FRANCISand WATERSTON1991; HRESKOet al. 1994). Dif- tance of 1.2 map units: 8 of 250 Unc self progeny of unc-29/ dpy-24 hermaphrodites segregated Dpy (Unc) progeny, and 6 ferent classes of unc-5.2 mutation have been character- of 328 Dpy selfprogeny from the same parents segregated Unc ized. Viable unc-52 alleles cause a gradual disruptionof (Dpy) progeny. Among 210 Unc self progeny of unc-29/mec- the myofilament lattice posterior to the pharynx begin- 8(e398) hermaphrodites, one segregated Mec self progeny, mec-8 and85 Interacting Genes

TABLE 1

Three-factor map data

Phenotype of selected of Genotype of parent recombinants Number recombinant chromosome

dpy-5 unc-13 mec-8(e398)/+++ Dpy non-Unc 10/10 dpy-5 unc-13(+) mec-8( +) Mec non-Dpy 9/17 dpy-5(+) unc-13 mec-8 8/17 dpyJ(+) unc-l3(+) mec-8 dpy-5 unc-29 +/+ + rnec-8fe398) Dpy non-Unc 19/19 dpy-5 unc-29(+) mec-8 Unc non-Dpy 20/20 dpy-5( +) unc-29 mec-8( +) unc-29 + dpy-24/+ mec-8(e398)+ Unc non-Dpy 9/12 unc-29 mec-8( +) dpy-24( +) 3/12 unc-29 mec-8 dpy-24(+) Dpy non-Unc 14/15 unc-29(+) mec-8 dpy-24 1/15 unc-29(+)mec-8(+) dpy-24 unc-29 mec-B(e398) dpy-24/+++ Unc non-Dpy 13/16 unc-29 mec-8 dpy-24(+) 3/16 unc-29 mec-8(+) dpy-24(+) Dpy non-Unc 10/10 unc-29(+)mec-8(+) dpy-24 corresponding to an unc-29-mec-8distance of about 0.2 map plates) were transferred to fresh plates daily. Non-Unc-7 F, unit. Among 150Mec selfprogeny of mec-8(e398)/dpy-24her- hermaphrodite progeny were scored for touch sensitivity by maphrodites, two segregated Dpy selfprogeny, corresponding gently stroking each worm with an eyebrow hair, and all Mec to a mec-8-dpy-24 distance of about 0.7 map unit. animals were picked. Onlyone mutant per plate was saved, to Deficiency mapping was performed by mating males het- ensure independence of mutants. The deficiency mnDfl1 I erozygous for the chromosome of interest to hermaphrodites was isolated as an allele of mec-8 in a separate F, non- heterozygous for the deficiency. F, cross progeny were exam- complementation screen: EMSmutagenized N2 males were ined for the presence of mutant animals. The mutation being mated with dpy-5 unc-13 mec-8(e398) I; unc-9 X hermaph- tested was linked to dpy-5 or unc-75. For example, dpy-5 mec- rodites, and F, progeny were scored for Mec. The X-linked 8(e398)/++ males were mated to mnDfl1 l/unc-13 lin-11 markers unc-7 and unc-9 were included in these crosses to hermaphrodites, and F, progenywere scored for Mec and Dyf. prevent male cross-progeny from matingwith hermaphrodite The deficiency mnDfll1 uncovers mec-8(e398) (and all other cross-progeny; Unc-7and Unc-9 males do not mate (HODGKIN mec-8 alleles tested; see RESULTS),unc-55 and unc-29 but not 1983). Three mec-8 mutations (mn450, mn455and mn459) unc-13, dpy-5, dpy-24or lin-11. None of the three deficiencies were identified among 9,645 F, animals scored inthe first EMS nDf23, nDf24 or nDf25 uncovers mec-a(e398) or dpy-24. For screen, a frequency of 3 X The deficiency mnDfl11 was each of the latter three deficiencies, we identified comple- the only mec-8 mutation recovered among 3,553 F, animals menting and viable mec-8/Df animals by showing that they screened. Molecular analysis has shown that the mec-8 region segregated extremely few self progeny carryingthe mec-8(+)- is entirely missing in mnDfl11 (R. ELLIS,personal communi- containing chromosome (the chromosome not tagged with cation; E. LUNDQUIST,J. SHAW and R. HERMAN,unpublished ex- dpy-5 or unc-75 or with the markers balancing the deficiency periments). FourTMP-induced mec-8 mutations (mn462, stock), presumablybecause the mec-g(+)-bearing chromo- mn463, mn464 and mn465) were identified among 18,712 F, some also carried a homozygous inviable deficiency. animals scored, a frequency of 2 X Each mutation re- A stock of genotype dpy-5 unc-13 med(e398); nDp4was covered inthe EMS and TMP screens was outcrossed five times constructed by mating dpy-5 unc-13 mec-8(e398)/++ + males to N2. with unc-13; nDp4 hermaphrodites and picking non-Unc-13 To obtain potential transposon-tagged alleles,hermaphro- progeny, some of which gave rise to many Dpy non-Unc-13 dites of the mutator strain RW7097 (MOM et al. 1988) were animals. Twenty of the latter animals were picked;they all seg- mated with N2 males; the heterozygous male crossprogeny regated Dpy Unc Mec self progeny, as expected for a dpy-5 were mated to unc-29 mec-a(e398) dpy-24 I; unc-7 X her- unc-13 mec-8fe398); nDp4 parental genotype if nDp4 carries maphrodites, as in the EMS and TMP mutagenesis experi- unc-13(+) but not dpy-5(+). The Dpy non-Unc parents were ments, and the hermaphrodite cross progeny were screened all non-Mec, which indicates that nDp4 carries me&(+). for rare Mec animals. Five mec-8 mutations were identified Identificationof new rnec-balleles: Ethyl methanesulfonate among 3,316 F, hermaphrodites scored. Because of their high (EMS) treatment was performed according to BRENNER(1974) incidence and because each of the five mutations resulted in and SULSTONand HODCIUN(1988). Trimethylpsoralen (TMP) a similar temperature-sensitive Mec and Dyf phenotype (see treatment was based on the methods Of YANDELL etal. (1994): RESULTS),we suppose that the mutations were not indepen- a mixture of N2 males and hermaphrodites was washed with dent, and only one, mn472 ts, was analyzed further, after out- M9 buffer (SULSTONand HODCIUN1988) and resuspended in crossing ten times to N2. 30 pg/ml TMP in M9 buffer. After 15 min in the dark, the Microscopy: Living animals were mounted for Nomarski animals weretransferred to an unseeded NGM plate (SULSTON differential interference contrast microscopy or epifluores- and HODCIUN1988) and exposed to longwave ultraviolet ra- cence microscopy on a 5%agar pad (sometimescontaining 5 diation, using a Blak-Ray lamp, model LJVL-21, for a dose of mM sodium azide as an anesthetic) in a drop ofM9 buffer 700 pW/cm‘ (measured with a Superdose dose meter) for 45 under a coverslip. Animals viewed by polarized light optics sec. A drop of OP50 bacteria (BRENNER1974) was added to the were mounted on a slide (without agar) in a dropof M9 plus plate, and the worms were allowed to recover for 5 hr in the 5 mM sodium azide.A rotary compensator was used toenhance dark. contrast of birefringent muscles. Samples werephotographed After exposure to either EMS or TMP, N2 males weremated with Ilford XP2 400 film. with unc-29 mec-a(e398) dpy-24I; unc-7(bx5) XL4 hermaph- Dye filling of amphid and phasmid neurons: Two fluores- rodites. The mating parents (2-5 of each on 35-mm diameter cent dyeswere used: FITC (Sigma) and 3,3’-dioctadecyl- 86 E. A. Lundquist and R. K. Herman oxacarbocyanine perchlorate (DiO; Molecular Probes). FITC These animals failed to segregate viableDpy Unc-52 self- was used for scoring mec-8 in crosses. DiO, which gives better progeny; eggs laid by them were collected and mounted on resolution and less fading than FITC, was used to quantitate agar in S medium under a coverslip (SULSTONand HODGIUN defects in dye filling in various mec-8 mutants. FITC filling was 1988) and sealed around the edges with Vaseline. The devel- performed according to HEDCECOCKet al. (1985). For DiO oping zygotes were then incubated at 20" (or other indicated filling (HERMANand HEDGECOCK1990), animals were bathed in temperature) and monitored periodically by Nomarski micros- a solution of 20 pg/ml DiO in M9 buffer for 2 hr (a dilution copy. In all cases, close to one quarter of the were of a DiO stock solution containing 2 mg/ml DiO in dimeth- paralyzed and arrested at the twofold stage ofembryogenesis, ylformamide). Excess dye wasremoved by allowing the animals as described in RESULTS. Essentially no embryos from mnCl to crawl on a clean NGM plate for a few minutes before putting dpy-10 unc-52/unc-4parents or various mec-8 mutants showed them on a slide for microscopy. Most strains were scored in at twofold embryonic arrest under comparable conditions. In least two independent experiments (most were scored three other experiments, the mec-8mutation was balanced by a dpy-5 times). Little variation in filling was seen among experiments unc-29 chromosome, which also does not lead to embryonic done ondifferent days or with different stock solutions of DiO. arrest at the twofold stage. For example, mec-8(e398)/dpy-5 Scoring developmental arrest of mec-8 mutants: L4 her- unc-29; unc-52(e669) animals were generated from mec-8/ maphrodites were picked individually to NGM plates and dpy-5 unc-29; unc-52/+ parents, which segregated both transferred daily. Self progeny were scored as follows: embry- Unc-52 and Mec selfprogeny; the homozygous unc-52 animals onic arrest (any unhatched embryos remaining 1 day after exhibited the progressive paralysis conferred by unc-52,were hermaphrodite removal at 20" or 25", 2 days after hermaph- non-Mec and non-Dyf, and segregated Dpy Unc-29 self prog- rodite removal at 16") ; L1 and L2 arrest (any L1 or L2 larvae eny but no Mec Dyf self progeny. Eggs laid by these animals remaining 2 days after hermaphrodite removal, 3 days after were collected and scored, as already described, and in every hermaphrodite removal at 16"); all other animals. Scored ani- case about onequarter of the embryos were paralyzed and mals wereremoved from the plates so they wouldnot interfere arrested at the twofold stage of elongation. Finally, in a few with subsequent counts. instances, neither the mer-8 nor the unc-52 mutation was bal- Fmtion and antibody staining: A drop of 1 mg/ml polyly- anced. For example, mec-8(e398)/+; unc-52(e444)/+ ani- sine was placed on a clean slide and allowed to dry. Animals mals were generated and recognized by the fact that they seg- were washed from NGM plates with M9 buffer or water and regated both Unc-52 and Mec Dyf self progeny. No Unc-52 placed on the polylysine spot. A coverslip was placed over the Mec Dyfselfprogenywere produced, however. A large number sample; liquid was drawn from underneath the coverslip using of Mec Dyf progeny (two-thirds of which were expected to be the edgeof a papertowel until the animals appeared to flatten heterozygous for unc-52) were picked and allowed to lay eggs slightly; and the slide was placed in liquid nitrogen for at least for a few hours. The eggs were collected and inspected peri- 5 min. Immediately after removal from liquid nitrogen, the odically by Nomarski microscopy;about one-sixth showedthe coverslip was flipped off with a razor blade, and the slide was characteristic arrest at the twofold stage. put in methanol at -20" for 10 min, acetone at -20" for 5-10 Identification of genetic suppressors of mec-8(u218 ts); min and then allowed to dry. unc-52(e669su250 ts) synthetic lethality: The double mutant A drop of undiluted goat serum was placed on the sample mec-8(u218 ts) I; unc-52(e669su250 ts) I1 is viable at 16" and and incubated at 37" for 1 hr, room temperature for 2-3 hr, inviable at 25". Hermaphrodites of this genotype grown at 16" or overnight at 4" in ahumidified chamber. Excess goat serum were mutagenized with EMS and cultured on plates at 16" for was removed, and a drop (20-70 pl) of primary antibody di- two generations. The plates were then shifted to 25", and viable luted in phosphate-buffered saline (PBS) (SULSTONand and coordinated F, progeny were identified and picked. No HODCKIN1988) was added to the sample. Samples were incu- more than one suppressed line from each plate was saved. Four bated in ahumidified chamber for 1 hr at 37", 2-3 hr at room independent mutants were analyzed. Each of the four muta- temperature or overnight at 4". Each slide was washed three tions suppressed, at 25", the paralysis conferred by unc- times in PBS,20 min per wash. A drop of fluorescein- 52(e669su250 ts), both the Mec and Dyfphenes conferred by conjugated secondary antibody, diluted 1:lOO to 1:150 in PBS, mec-8(u218 ts) and the synthetic lethality conferred by the was added to the sample. After incubation in a humidified unc-52 and mec-8 mutations together (see RESULTS); all four chamber at 37" for 1 hr orat room temperature for 2-3 hr, the lines bred true for these characteristics. sample was washed twice in PBS, 20 min per wash, and once All mapping and complementation experiments involving in PBS with 4 mM ascorbic acid. The sample was mounted in the suppressors were done at 25". The suppressor stocks were a drop of PBS with 4 mM ascorbic acid, and the coverslip was crossed to N2 males, and F, cross progeny were picked and sealed with fingernail polish. allowed to self. Among the F, progeny in all four cases were The following antibodies were used, each at afinal dilution Mec and Unc-52 animals that were indistinguishable from the of 1:500: DM 5.8 (MILLERet al. 1983), which recognizes original rned(u218 ts) and unc-52(e669su250 ts) strains, MHC-B, the major body wall muscle myosin heavychain; MH2 which indicates that all four of the suppressor mutations are (FRANCISand WATERSTON1991), which recognizes UNC-52 extragenic to both mec-8 and unc-52. Many of Unc-52progeny (ROCALSIUet al. 1993) in the basement membrane adjacent to of the triply heterozygous strains segregated about onequarter muscle strips ;and MH27 (WATERSTON1988), which recognizes non-Unc-52 (suppressed) animals, which indicates that the an antigen present in the junctions of hypodermal cells and suppressors are recessive for suppression of the paralysis con- therefore outlines hypodermal cells. ferred by unc-52(e669su250 ts). Analogous results indicated Construction and analysisof mec-8; unc-52 double mutants. that the suppressors are also recessive for suppression of the Several mec-8; unc-52(e444) strains were constructed and Mec phene conferred by med(u218ts). The suppressor mu- maintained using the chromosome balancer mnCl dpy-10 tationswere assignedallele names mn415, mn416, mn417and unc-52(e444)II. Males of genotype mec-8/+; unc-4/+ were mn433, and the loci defined by the suppressor mutations are mated to homozygous mnCl dpy-10 unc-52 hermaphrodites. called smu (suppressor of mec-8 and gnc-52). F, cross-progeny that segregated both Mec and Unc-4 progeny Complementationtests between suppressor mutations: were selected, and Mec non-Unc-4 F, hermaphrodites, geno- The characteristic of suppressing the paralysis conferred by type mec-8; mnCl dpy-10 unc-52(e444)/unc-4,were picked. unc-52(e669su250 ts) was used for complementation testing mec-8 and87 Interacting Genes between pairs of suppressors. In generating new strains, the mn417 and mn433 were 2/55 and 1/56, respectively. Taken presence of the suppressor was ascertained by first identifylng together, these results confirm that the three smu-1 alleles Unc-52 animals,which must be homozygous for unc- map close together and that smu-1 maps about 0.7 map unit- 52(e669su250 ts), and then identifylng non-Unc-52 (homozy- correcting for growth at 25" (ROSEand BAILLIE1979)-to the gous suppressor) self progeny. Complementation tests were rightof unc-101. We have shownthat smu-l(mn415)and unc- performed by crossing unc-52(e669su250ts); smu(u); him-5 101 complement: mec-8(~218ts) smu-l(mn415) I; unc- males to dpy-5; unc-52(e669su250ts); smu(6) hermaphro- 52(e669su250 ts) II; him-5 Vmales were crossedto dpy-5 unc- dites (for these tests, we made no special effort to follow the 101 I; unc-52(e669su250 ts) IIhermaphrodites; the non-Dpy fate of the original mec-8(u218 ts) mutation; the construction progeny were allUnc-52 (not suppressed). Furthermore, unc- of suppressor stocks in which mec-$(u218 ts) was clearly absent lOl/smu-1 animals (see above) were non-Unc-101. is described below). Non-Dpy hermaphrodite progeny were Mapping smu-2 ZI: The progeny ratios from hermaphro- then scored for suppression of the paralysis conferred by unc- dites heterozygous for smu-2(mn416), mec-8(~218ts) and 52(e669su250 ts). The F, progeny were alsochecked for sup- unc-52(e669su250ts) suggested that smu-2 is loosely linked pression; in all casesof noncomplementation, no nonsup- to unc-52 II (Figure 1). This surmise was confirmed by pressed segregantswere found in the F, generation, suggesting subsequent mapping experiments. For example, smu-2 that noncomplementing alleles were closely linked. The sup- was shown to be very near or to the left of dpy-10 ZI by the pressor mutations fell into two complementation groups: following three-factor cross: mec-8(u218 ts); smu-2 unc- mn415,mn417 and mn433 wereassigned to smu-1, and 52(e669su250 ts); him-5 males weremated to mec-8(u218 ts); mn416 was assigned to smu-2. dpy-10 unc-4 hermaphrodites, and Mec hermaphrodite prog- Mapping smu-1 I: For each of the smu-1 alleles, N2 males eny were picked. Dpy non-Unc4 and Unc4 non-Dpy F, re- were mated to mec-$(u218 ts) smu-1; unc-52(e669su250ts) combinant progeny were picked, and their self progeny were hermaphrodites. From the triply heterozygous F, hermaph- screened for animalsshowing suppression ofMec: 10/10 rodites, manynon-Unc-52 progeny hermaphrodites were Unc4 non-Dpy recombinants and 0/8 Dpy non-Unc recom- picked individually, and the proportion of animals that seg- binants carried smu-2. The following three-factor cross placed regated about threequarters Mecself progeny was deter- smu-2 near or to the left of unc-85 0/6 Unc-85 non-Dpy re- mined. The ratios obtained-5/100 for mn415, 1/20 for combinants and 4/4 Dpy non-Unc-85 recombinants among mn417 and 2/87 for mn333, for an average of 0.04-indicate the self progeny of + unc-85 dpy-IO +/smu-2 + + unc- that smu-1 is situated on LGI, about 9 map units from mec-8. 52(e669su250 ts) hermaphrodites carried smu-2, asascer- The location of smu-1 on LGI (Figure 1) was determined more tained by segregants showing suppressionof paralysis in unc- precisely bytwo- and three-factor crosses. dpy-5 unc-75/+ 52(e669su250 ts) homozygotes. The following cross placed maleswere used to generate dpy-5 + unc-75 +/+ mec- smu-2 between sgt-2 and unc-85: 6/8 Sqtnon-Unc-85 re- 8(u218 ts) + smu-l(mn415); unc-52(e669su250 a)/+ her- combinant and 0/2 Unc-85 non-Sqt recombinants among maphrodites. The progeny of these animals were screened for the self progeny of sqt-2 + unc-85 +/+ smu-2 + unc- Dpy non-Unc-75 non-Unc-52and Unc-75 non-Dpy non-Unc-52 52(e669su250 ts) hermaphrodites carried smu-2. This posi- recombinants. Only recombinants that segregated Unc-52 tion is also supported by the following results: 0/29 Sup non- animals among their self progeny were saved. Ten Unc-52 Sqt recombinants and 5/5 Sqt non-Suprecombinants among progeny from each selected recombinant were picked and the self progeny of sqt-2 smu-2 + unc-52(e669su250 ts)/+ + scored for the segregation of non-Unc-52 (suppressed) prog- unc-85 unc-52(e669su250 ts) hermaphrodites carried unc- eny. Amongthe Dpy non-Unc recombinants, 13/13 harbored 85. Finally, the following two-factor cross givesa smu-2-unc-85 smu-1 (mn415);only 1/10 Unc-75 non-Dpyrecombinants har- map distance of 3.8 map units, corrected to about 2.5 map bored smu-l(mn415).These data suggest that smu-1 is either units because of the growth at 25" (ROSEand BAILLIE1979): to the right of unc-75 (in which case one of the recombinant 9/120 non-Unc-52 (suppressed) segregantsfrom smu-2 + unc- chromosomes was generated by two crossovers) or very close 52(e669su250 a)/+ unc-85 unc-52(e669su250 ts) hermaph- and to the left. A similar cross involvinga dpy-5 unc-59 chro- rodites carried unc-85. mosome gave the following results: 8/10 Dpy non-Unc-59 re- Wehave shown that smu-2 and the closely linked gene combinants and 1/7 Unc-59 non-Dpy recombinants carried mup-1 (GOHand BOCAERT1991) complement, a cross between smu-l(mn415),which positions smu-1 nearer but to the left of smu-2 unc-4 unc-52(e669su250ts)/+ + + males and mup- unc-59. A similar cross involvingdpy-5 unc-I01 gave the fol- 1 (e2436) dpy-10 unc-52(e669su250 ts) hermaphrodites, lowing results: 9/9 Dpy non-Unc-101 recombinants and 0/8 yielded equal proportions ofUnc-52 (not suppressed) and Unc-101 non-Dpyrecombinants carried smu-1 (mn415),which wild-type cross progeny. indicates that smu-1 is near or to the right of unc-101. That Isolation of smu-1 alleles away from mec-8(u218ts) and it is to the right was determined by the following cross: dpy-5 unc-52(e669su250 ts): Three strains, each carrying a differ- mec-8(~218ts)/+ + males were mated to mec-8(u218 ts) unc- ent smu-1 allele alone, were constructed by the following pro- 101smu-l(mn415) hermaphrodites. F,Mec animalswere cedure. unc-52(e669su250ts)/+ males were mated to dpy-5 picked, and non-Unc-101 non-Mec recombinants (recombi- smu-1; unc-52(e669su250 ts) hermaphrodites (derived from nant between unc-I01 and smu-1) were selected. Four of five three-factor mapping crosses) and Unc-52 progeny were such recombinants segregated Dpy self progeny. The one re- picked. Non-Dpynon-Unc-52 (suppressed) recombinants combinant chromosome without dpy-5 could have been in- were picked among the progeny and allowed to self. Several volved in two crossovers. Finally, two-factor crosses were used non-Dpy hermaphrodite progeny werepicked, and a line that to measure the unc-101-smu-1 map distance: mec-8(~218ts) segregated no Dpy progeny was kept. The smu-1; unc- unc-lO1/+ + males were mated with mec-8(~218ts) smu-1; 52(e669su250 ts)stock so generated was crossed to unc- unc-52(e669su250 ts) hermaphrodites, and Mec progeny 52(e669su250ts)/ + males five times, always retaining the (mec-8 unc-101 +/mec-8 + smu-1; unc-52/+) were picked. unc-52 allele in homozygousform to score for the presence of Among the self progeny of these animals, Unc-101 hermaph- smu-1. Next, unc-l01/+ males were mated to smu-1; unc- rodites were picked;their self progenywere then scored for the 52(e669su250 ts) hermaphrodites, and progeny were picked. presence of non-Mec (suppressed) segregants. For mn415, Non-Unc-101 progeny were pickedfrom broods that con- 3/189 Unc-101 parents segregated recombinants. The datafor tained Unc-101 animals. Non-Unc-101 progeny were then 88 E. A. Lundquist and R. IC Herman

unim LGI mmap 012

I II

LGII PFi 024

FIGURE1.-Genetic map showing positionsof mec-8 I, unc-52 11, smu-1 I, smu-2 11 and other loci and chromosome rearrange- ments used in mapping. picked from broods that segregated Unc-101 but no Unc-52 strain is in fact homozygous for smu-1. In a few cases where self progeny. A line that segregated no Unc-101 animals was suppression was not observed, a second, independent stock assumed to be homozygous forsmu-1. The presence of smu-1 was was constructed. Patterns of suppression of a given mec-8 al- confirmed by crossing in unc-52(e669su250ts) and identlfymg lele by different suppressors were generally consistent (see non-Unc-52 (suppressed) descendantsof Unc-52segregants. The RESULTS). absence of mec-8(u218ts) was confirmed by crossing to mec- Construction of mec-8; smu-2 double mutants mec-8/+; S(u218 e)/+ males and finding no Mec cross progeny. unc-85/+ males were mated to smu-2 hermaphrodites and Isolation of smu-2 away from mec-8(u218ts) and unc- many progeny were picked. Fromthe progeny of those F, her- 522e669su250 ts): Beginning with the mec-S(u218 ts) I; smu- maphrodites that segregated Mec and Unc-85 self progeny, 2(mn416) unc-52(e669su250 ts) 11 strain, mec-S(u218 ts) several Mec non-Unc-85 animalswere picked. Those that seg- was crossed out by segregation against dpy-5, to derive the regated no Unc-85 self progeny were assumed to bethe geno- stock dpy-5; smu-2 unc-52(e669su250ts). Next, unc- type mec-8; smu-2. Because of the possibility of recombination 52(e669su250 ts)/+ males were mated to dpy-5; smu-2 unc- between smu-2 and unc-85 (which are about 4 map units apart; 52(e669su250 ts) hermaphrodites, and non-Dpy Unc-52prog- see Figure2), we cannot be positive that a given stock display- enywere picked. Non-Dpy non-Unc-52 (suppressed) self ing no suppression of mec-8 is homozygous for smu-2. How- progenywere picked, and the resulting smu-2 unc- ever, only mec-g(u74) showed no apparent suppression by 52(e669su250ts) homozygousline was then outcrossedto smu-2, and in this case, none of the smu-1 alleles showedsig- unc-52(e669su250ts)/+ males five times. Finally, unc-85/+ nificant suppression either (see RESULTS). males were crossed tosmu-2 unc-52(e669su250ts) hermaph- Construction of smu-I; unc-52 and smu-2 unc-52 double rodites, and hermaphrodite progeny were picked.Many non- mutants smu-1 and smu-2 alleles were testedfor suppression Unc-52non-Unc-85 self progeny hermaphrodites were of the paralysis conferred by several unc-52 alleles. unc-52/+ picked from a brood that contained Unc-85 animals and males were mated to smu-l or smu-2 hermaphrodites, and F, scored for segregation of Unc-52 animals. From broods con- progeny were picked. From F, broods containing Unc-52 ani- taining Unc-85 animals but noUnc-52 animals, several non- mals, many Unc-52animals werepicked, three per plate. Their Unc-85 hermaphrodites were picked. Those segregating no progeny were then scored with respect to (Unc-52) paralysis. Unc-85 animals were assumed to be smu-2 homozygotes. The Non-Unc-52 animals were presumed to be smu-1; unc-52 or presence of smu-2 was confirmed by crossing in unc- smu-2 unc-52. 52(e669su250 ts) and identifying non-Unc-52 (suppressed) descendants ofUnc-52 segregants. The absence of mec- RESULTS S(u218 ts) was confirmed by crossing to mec-S(u218ts)/+ males and finding no Mec cross progeny. mec-8 map position and identificationof new alleles: Construction of mec-8 smu-1 double mutants Each of the We have used two- and three-factor genetic crosses (data three smu-1 alleles was combined with various mec-8 alleles by given in MATERIALS AND METHODS) to refine the genetic the following procedure. mec-8 unc-lUl/+ males were mated map position of mc-8 on linkage group I. Both touch in- with smu-1 homozygotes. Many hermaphrodite progeny were picked, and Mec non-Unc-101recombinants were picked from sensitivity and dye-filling defectsconferred by rnec-8 muta- the progeny of those F, hermaphrodites that gave rise to Mec tions were used the in mapping. The resulting genetic map Unc-101 self progeny.The F, progeny were scored for the pres- in the neighborhood of mec-8 is given in Figure 1. ence of Unc-101 animals,and several non-Unc-101F, animals Eight mec-8 mutations were identified previously by were picked.Those that segregated no Unc-101 progeny were other workers: e398 (CHALFIEand SULSTON1981), u74, assumed to be mec-8 smu-1 homozygotes. There is a small u218 ts, u303, u314, u391and u456 (CHALFIEand AU chance (1-2747) that a smu-l(+) allele is present in these stocks, owing to recombination between unc-101 and smu-1. 1989) were identified in screens for mechanosensory- Suppression of the mec-8 mutation provides evidence that the defective mutants, and rhl70 was identified in a screen and Interacting Genes Interacting mec-8 and 89

FIGLKE'L.--Neuron filling ofwild-type and vfNpr-8anim;ds with the fluorescent dye DiO. Antcrior is to the left, and dorsal is up. Scale bar in each panel represents 'LO pm. (a) Six neuron cell bodies (marked by a bracket and depictedin the inset),all belonging to one amphid of a wild-type animal, have filled with dye and are visualized by fluorescence microscopy. The processes of the neurons also show staining. The fluorescence below the bracketed amphid neurons is due to two phasmid neurons of a separate but nearby worm. (b) Two neuron cell bodies (arrow) of a single phasmid in a wild-type animal are shown; the broad staining to the left of the phasmid cell bodies is due to dye in the intestine. (c) A mec-8(~456)mutant is defective in dye filling; only two neuron cell bodies of an amphid have filled with dye. (d) Only one phasmid neuron cell body (arrow) of a mec-8(u456) animal has filled with dye. (The intestine also contains dye, and the animal has expelled some dye, seen below the phasmid cell body, by defecation.) for mutants defective in dye filling (E. HEDCECOCK,per- 3 X is approximately the frequency expected for sonal communication). Thetwo alleles u456 and u391 the generation of loss-of-functionmutations in an aver- were yray-induced (M. CHALFIE,personal communica- age C. eleguns gene (BRENNER1974). Trimethylpsoralen tion); the others were identified following EMS mu- mutagenesis yielded four mec-8 mutations (mn462, tagenesis. To identify new and possibly null alleles of mn463, mn464 and mn465) out of 18,712 F, heterozy- mec-8, we conducted F, noncomplementation screens gotes scored, a mutation frequency of 2 X and identified eight new independent mec-8 mutations. The eighth new mec-8 allele was recovered from a mu- Males that were either mutagenized or derived from a tator strain,RW7097, whichexhibits enhanced germline mutator stock were mated to homozygous unc-29 mec- transposition of the transposable element Tcl (MORI 8(e398) dpy-24 I; unc-7Xhermaphrodites, and thehet- et ul. 1988). In this case, male progeny from a cross erozygous wild-type hermaphrodite cross-progeny were between N2 males and RW7097 hermaphrodites were scored for their ability to respondto light touch. Touch- employed in the same noncomplementation screen de- insensitive animals were picked; and their self progeny scribed above. Five Mec mutants were recovered among were checked for touch insensitivity and possible ho- 3,316F, heterozygotes scored, but allfive mutations mozygosity for the new mutation. Thisscreen is capable when homozygous conferreda similar temperature- of identifylng complete loss-of-function (null) muta- sensitive phenotype. We suppose that the five mutants tions because the recessive lethal deficiency mnDfl1 I all carried thesame mutation, which was already present was isolated in a similar noncomplementation screen in our RW7097 stock; we therefore restricted our focus against mec-8(e398) (see MATERIALS AND METHODS) and to one mutation in this set, mn4 72 ts. This mutationwas uncovers the mec-8 locus, i.e., mec-8(e398)/mnDfl I I outcrossed to N2 10 times. The other seven new muta- animals are touch insensitive,viable and fertile. All eight tions were outcrossed five times. new alleles, like the previous eight, are homozygous vi- As will be shownin detail below,all of the eight able and fertile at 20". new mec-8 alleles confer a stronger defect in dye fill- Three mec-8 alleles (mn450, mn455 and mn459) ingthan does mec-8(e398),with the exception of were recovered among 9,645 F, heterozygotes scored mn472 ts, which confers a weakerdye-filling defect; following EMS mutagenesis. This mutation frequency, these results indicate thatthe newalleles arenot 90 E. A. Lundquist and R. K Herman

TABLE 2 Dye-f&g defects and mechanosensory defects of mec-8 mutauts

Proportion Proportion of phasmid of phasmid neurons Touch neurons Touch Genotype (temperature) filled (n) sensitivity Genotype (temperature) filled (n) sensitivity

N2 (wild-type) 0.99 (240) ++ mec-8(rhl70) 0.01 (180) - mec-8(e398) 0.11 (176) - mec-8(rhl70)/mnDflll 0.01 (116) - mec-8(e398)/mnDflll 0.02 (404) - mec-8(u 74) 0.02 (136) - rned(mn450) 0.03 (324) - mec-8(u 74)/mnDflll 0.04 (80) - mec-8(mn450)/mnDflll 0.02 (260) - mec-8(~74)/mec-8(mn472ts) (25") 0.06 (268) - mec-8(mn455) 0.02 (328) - mec-8(~74)/mec-8(mn472ts) (16") 0.20 (280) - mec-8(mn455)/mnDflll 0.03 (260) - mec-8(u 74)/mec-8(~218ts) (25") 0.05 (192) - mec-g(mn459) 0.04 (336) - mec-8(~74)/mec-8(~2I8ts) (16") 0.39 (208) - mec-8(mn459)/mnDflll 0.02 (248) - mec-8(u218 ts) (25") 0.18 (368) - mec-8(mn462) 0.04 (380) - mec-g(u218ts)/mnDflll (25") 0.08 (336) - mec-8(mn462)/mnDflll 0.03 (288) - mec-8(u218 ts) (16") 0.94 (120) ++ mec-8(mn463) 0.03 (488) - mec-g(u218ts)/mnDflll (16")0.36 (240) - mec-8(mn463)/mnDflll 0.02 (288) - mec-8(~303) 0.05 (228) - mec-8(mn463)/mec-8(mn472ts) (25") 0.06 (164) - mec-8(u303)/mnDflll 0.01 (200) - mec-8(mn463)/mec-8(mn472 ts) (16") 0.18 (300) - mec-8(~314) ~0.01(172) - rnec-8(mn463)/mec-8(~218ts) (25") 0.07 (216) - me~-8(u314)/mnDflll 0.02 (200) - rnec-B(mn463)/mec-8(~218ts) (16") 0.34 (128) - mec-8(~391) 0.02 (184) - mec-8(mn464) 0.02 (404) - mec-8(u39l)/mnDflll 0.03 (228) - mec-8(mn464)/mnDjlll 0.04 (288) - mec-8(~391)/mec-8(mn472ts) (25") 0.05 (264) - mec-8(mn465) 0.02 (400) - rnec-8(~39l)/mec-8(mn472ts) (16") 0.18 (276) - mec-8(mn465)/mnDfllI 0.03 (288) - mec-8(u391)/mec-8(~218ts) (25") 0.05 (212) - mec-8(mn472 ts) (25") 0.10 (396) - mec-8(u391)/mec-8(u218 ts) (16") 0.44 (180) - mec-8(mn472 ts)/mnDfllI (25") 0.05 (208) - mec-8(~456) <0.01 (284) - med(mn472 ts) (16") 0.57 (132) + mec-8(u456)/mnDflll 0.02 (256) - mec-g(mn4 72 ts)/mnDfllI (16") 0.23 (208) - ++ = touch sensitive; + = partially touch sensitive; - = touch insensitive. Animals were scored at 20" unless otherwise noted. n is the total number of phasmid neurons scored; each animal has four phasmid neurons. Scored in one experiment; all other strains were scored in at least two independent experiments.

reisolates of e398, the allele used in the noncomple- type and mec-8(~456)amphids and phasmids after ex- mentation screens. posure to DiO. When neurons fill with dye, cell bodies Mec and Dyf phenes of mec-8 mutants: All 16 mec-8 as well asprocesses are stained. Only two neurons of the mutations result in an insensitivity to light touch, the mec-g(u456) amphid illustrated in Figure 2c filled with Mec phene (CHALFIEand Au 1989; Table 2). Indeed,all dye, and one neuronof the phasmid in Figure 2d filled. but rhl70 were originally identified on the basis of this All 16 mec-8 mutations caused dye-filling defects and phenotype. CHALFIEand Au (1989) showed earlier that were recessive to mec-8(+) with respect to this phene. u218 ts causes a temperature-sensitive touch response; Both amphids and phasmids were affected, but for ease we have confirmed this result and shown that mn4 72ts of scoring, we have counted the proportionof phasmid is also temperature-sensitive: mn472 ts animals are neurons thatfilled with dye for each homozygous mec-8 weakly touch sensitive at 16" and touch insensitive at 25" allele, each mec-8 allele over a deficiency, and various (Table 2). None of the other 14 alleles confers a trans-heterozygous combinations of mec-8 alleles (Table temperature-sensitive touch response. For all 16 muta- 2). When phasmid dye filling was rare, animals exhib- tions, mutant animals respond to a prod with a wire and iting staining usually had only one phasmid neuron generally move normally. All 16 alleles are recessive to filled. mec-8(+) with respect to the touch response. All but threeof the mec-8 alleles showabout 1-4% dye As noted in the introduction, when living wild-type filling of phasmid neuronswith DiO. The two alleles that animals are bathed in a solution of a fluorescent dye, were temperature-sensitive with respect to the touchre- either FITC or DiO, 16 chemosensory neurons, six in sponse were also temperature sensitive with respect to each of a pair of amphids in the head and two in dyefilling,showinglO% (mn472ts)and18%(u218ts) each of pair of phasmids in the tail, fill with dyeand can dye filling at 25" and considerably more dye filling at 16" be readily visualized by fluorescence microscopy (Table 2). Theallele e398 was also weakerthan the oth- (HEDGECOCKet al. 1985). PERKINSet al. (1986) reported ers, showing 11% dye filling at 20". The Dyf phenes that when either mec-8(e398) or mec-g(u74) animals caused by u218 ts, mn472 ts or e398 over mnDfl11, a were exposed to FITC, usually onlytwo neurons peram- deficiency that does not complement mec-8, are more phid and no phasmid neurons filled with dye, the Dyf severe than those caused by the homozygous alleles, sug- phene. Figure 2 shows fluorescence micrographs of wild- gesting that u218 ts, mn4 72ts and e398 are incomplete and Interacting Genes Interacting mec-8 and 91

TABLE 3 Cold-semitive embryonic arrest and early larval arrest of rnec-8 mutants

Proportion of Proportion of embxyonic or embxyonic or early larval early larval Genotype "C n arrest Genotype "C n arrest

N2 (wild-type) 16 731 <0.01 mec-8(mn472 ts) 16 1106 0.01 N2 (wild-type) 25 659 <0.01 mec-8(rhl70) 16 385 0.16 mec-8(e398) 16 683 0.06 mec-8(rhl70) 25 561 0.02 mec-8(e398) 25 363 0.02 mec-B(u 74) 16 597 0.31 mec-8(mn450) 16 1828 0.26 mec-8(u 74) 25 369 0.14 mec-b'(mn450)327 25 0.02 mec-8(u218 ts) 16 391 0.00 mec-8(mn455) 16 750 0.17 mec-8(u218 ts) 25 302 0.02 mec-8(mn455) 25 370 0.03 mec-8(u303) 16 459 0.03 mec-g(mn459) 16 752 0.14 mec-8(~303) 25 165 0.02 mec-8(mn459)414 25 0.02 mec-8(~314) 16 410 0.27 mec-8(mn462) 16 641 0.23 meA(u314) 25 328 0.01 mec-8(mn462) 25 506 0.01 mec-8(~391) 16 601 0.29 mec-8(mn463) 16 501 0.28 med(u391) 25 370 0.02 mec-8(mn463) 25 580 0.03 mec-8(~456) 16 425 0.28 mec-8(mn464) 16 588 0.23 mec-8(u456) 25 666 0.05 mec-8(mn464)625 25 0.01 mec-8(mn465) 16 516 0.30 <0.01 mec-8(mn465) 25 622 I loss-of-function (hypomorphic) alleles of mec-8. The Dyf Arrested mec-8 embryos and larvae aredeformed and phenes caused by all other mec-8 alleles over mnDf1 I1 display muscle defects: We have used light microscopy are similar to those caused by the homozygous alleles. to inspect arrested mec-8 embryos and larva reared at Three of the strong alleles (u74, mn463and u391) be- 16". Arrested embryos and larvae homozygous for six haved as did mnDfl l l when placed in trans to u218 ts strong alleles-mn450, mn463,u74, u314, u391 and and mn472 ts (Table 2); for example, mec-8(u74)/mec- u456-and two weaker ones-e398 and rhl70-were in- 8(u218 ts) and mnDfl1 l/mec-8(~218ts) at 25" gave5% spected by both Nomarski differential interference mi- and 8% phasmid filling, respectively. These results are croscopy and polarized light microscopy. Arrested em- consistent with the hypothesis that u74, mn463 and bryos frequently showed incomplete elongation; some u391 are null. of these embryos subsequently hatched, but others did Cold-sensitive embryonic and early larval lethalityof not. Wild-type embryos elongate to approximately 3.5 mec8 mutants: We have discovered that many mec-8 times the length of the egg before hatching (PRIESSand mutants grown at 16" display variable embryonic or early HIFLSH1986). Figure 3 shows Nomarski micrographs of larval arrest. Table 3 lists the results ofself-progeny wild-type 2- and 3.5-fold embryos (Figure 3, a and b) and counts for mec-8 mutants grown at 16" and 25". The defective mec-8(mn463) embryos (Figure 3, c and d) arrest phenotype is clearly cold-sensitive;mec-8 animals reared at16". The mec-8(mn463)embryos pictured have grown at 16" show more lethality than those grown at 20" arrested at about the twofold stage of elongation but or 25". The two alleles that conferred a temperature- show pharyngeal development characteristic of later em- sensitive touch response and a temperature-sensitive bryonic development. This is the earliest stage of mec-8 dye-filling defect, u218 ts and mn472 ts, did not display arrest we saw; other embryos arrested at later stages of the cold-sensitive arrest. The e398 allele, which was not elongation and many of these hatched. All of the ar- among thestrongest alleles with respect to its Dyfphene, rested embryos, even those that failed to elongate be- also showed little arrest at 16". u303 is a weak allele by yond the twofold stage, exhibited some movement. In the criterionof cold-sensitivearrest, and mn455, mn419 addition to showing elongation defects, virtually all ar- and rhl70 seem also to be somewhat weaker than the rested mec-8 embryos displayed hypodermal constric- other alleles (Table 3). This leaves nine alleles that gave tions and bulges; the tips of the noses, particularly, were 23-31% embryonic or larval arrest when grown at 16"; often pinched and bent (Figure 3, c and d). Arrested these include all ofthe strongalleles as defined by other mec-8 L1 and L2 larvae reared at 16" also displayed hy- criteria. We suggest that the variable cold-sensitive em- podermal kinks and bulges. Indeed, for the strong al- bryonic or earlylarval arrest is a null phene. Sixof leles, about 5-10% of mec-8 adults reared at 16" exhib- the seven weaker mec-8 alleles were EMSinduced; ited morphological deformities-bulges andkinksin the seventh, mn472 ts, was derived from the mutator the dissecting microscope, and such animals were fre- strain RW7097. Among the nine strongalleles, four were quently uncoordinated in their movement as well. induced by trimethylpsoralen and two were ?-ray- We used polarized light microscopy to inspect the bi- induced. refringent body muscle cells (WATERSTON1988) of the 92 E. A. Lundquist and R. K. Herman

FIGUREJ."M'ild-type and mu-#embryos,as observed by Nomarski differential interference microscopy. All c.mbl-\.os\\wcreared at 16". (a) Wild-type at nearly twofold stage of elongation (approximately 450 min after fertilization). The pharynx has not yet undergone extensive morphogenesis to form distinct bulbs and a grinder. (b) Wild-type embryo at about 3.5-fold stage of elongation (approximately 750 min after fertilization, just prior to hatching). Thepharynx has undergone morphogenesis and is pumping. The nose of the animal is below the plane of focus. (c) A mec-t?(rnn463) embryo arrested at nearly the twofold stage of elongation. The pharynx is well developed but the hypodermis shows constrictions and bulges. (d) A mec-8(mn463) embryo arrested at abouta 2.5-fold stage of elongation. This animalalso shows severe hypodermal constrictions and bulges. The scale bar, shown in (a), represents 5 pm; all panels are at the same magnification. same set of eight mec-8 mutants arrested as embryos or showed any disruption. The same set of mec-8 mutants young larvae. Body wallmuscle in C. elegans is arranged reared at 25" exhibited very few or no disruptions as in four longitudinal strips that run the length of the adults. animal (WATERSTON1988). All arrested embryos and lar- We have also used antibody staining to visualize the vae displayed disruptions in the patterns of birefrin- body muscle defects in mec-8 mutants. We stained wild- gence (data not shown). A bulge or kink in the hypo- type and rnec-g(mn463) mutants grown at 16" with DM dermis invariably corresponded to an area of defective 5.8 (MILLERet al. 1983), amouse monoclonal antibody muscle birefringence; usually in such regions, a muscle that recognizes MHC B, the major muscle myosin heavy strip appeared to be detached from the hypodermis, chain, and the results are illustrated in Figure 4. The as has been observed formutants affected in myo-3 muscle strips in rnec-g(mn463) arrested embryos were (WATERSTON1989), unc-45 (VENOLIAand WATERSTON often disorganized; the strips frequently appeared lo- 1990) and mup-I (GOH and BOCAERT1991). We also in- cally unattached to the hypodermis and cuticle as if they spected the two ventral strips of body muscle in adult had peeled away from the body wall and collapsed in- mec-8 animals reared at 16". Animalschosen at random ward. Many muscle cells gavea patternof staining char- were anesthetized and rolled onto their dorsal sides so acteristic of an organized myofilament lattice, but other that the complete lengthsof the ventral strips could be muscle cells showed onlya general cytoplasmic staining, inspected for possible local disruptions. For each geno- with nuclei outlined, as if an organized lattice structure type, 20-30 animals were scored. For each of the alleles had not formed.About 40% of rnec-g(mn463) embryos e398, rhl70, u74, u314, u391and u456, over halfof the and larvae reared at 16" and stained with anti-MHC B animals displayed at least one localized disruption in showed some abnormality in muscle structure. Qualita- birefringence. For the alleles u2I8 and u303, which tively similar results were obtained for mec-8(u 74) and caused little cold-sensitive embryonic arrest (Table 3), mec-g(u456) animals. 3/22 and 5/30 animals showed at least one disruption Distribution of unc-52 protein in mec-8 mutants: The in birefringence, respectively. None of 20 N2 animals C. elegans unc-52gene encodes homologa of perlecan, Interacting Genesmec-8 and Interacting 93

y MH2 de bar in (e) represents 10 pm for (e-11). Embryos'in (a-d) were stained with DM 5.8 (MIL.I.ERr/ n/. 1983), an antibody against myosin heavy chain B, the major myosin heavy chain of the body wall muscles; embryos in (e-h) were stained with MH2 (FRANCISand WATERSTON 1991); the antigen recognized by MH2 has beenlocalized to the basement membrane of contractile tissues (FRANCISand WATERSTON 1991) and has been shown to be encoded by unc-52 sequence (ROG~VSKIet al. 1993). (a) Awild-type embryo at nearly the twofold stage of elongation. One of the fourlongitudinal muscle strips canbe seen throughout its length. The myofilament lattices of some cells have not yet organized, as is indicated by the rounded appearance andoutlined nuclei of some muscle cells in the tail. The anterior portion of a second muscle strip is in the plane of focus. (b) An N2 embryo at about thethreefold stage of elongation. Two longitudinal muscle strips can be seen. (c andd) Two mec-8(mn463) embryos displaying muscle defects. Strips of body wall muscle are displaced, as if they are unattached andhave collapsed away from the body wall (arrows). (e) Wild-type embryo at twofold stage of elongation, stained with MH2. (f) Wild-type embryo at 3.5-fold stage of elongation. (g and h)Two mec-8(mn463) arrested embryos. Strips of MH2 staining are displaced from the body wall (arrows), much like defective muscle strips. The embryo in (g) shows some MH2 staining that has not organized into strips, but remains diffuse and unorganized. 94 E. A. Lundquist and R. K. Herman

TABLE 4

Synthetic lethal combinations of mec-8 and unc-52

mec-8 allele

~~ unc-52 allele u218 ts u456 e398 mn455 mn463 mn472 ts

~ ~ ~~ ~ e444a t (w,t (25") t t t t e669 t (257 t t e669su250 ts V (le"),t (25") t (257 t (16") e998 t e1421 t su54 t Unless otherwise noted, the growth temperature was 20". Where no symbols are given, the double mutantwas not made. t = synthetic lethal combination, showing paralyzed arrest at twefold stage of embryonic elongation, V = viable. aAll unc-52(e444)-bearing animals carried the mnC1 dpy-10 unc-52 chromosome (see MATERIALS AND METHODS). the core protein of the mammalian basement mem- membrane;the basement membranein turn is an- braneheparan sulfate proteoglycan (ROGALSKIet al. chored to an adjacentand very thin hypodermis and also 1993).A monoclonal antibody, MH2, identified and de- tethered through the hypodermis to external cuticle scribed by FRANCISand WATERSTON(1991), recognizes an (FRANCISand WATERSTON 1985,1991; HRESKOet al. 1994). antigen localized to basement membrane immediately Animals homozygousfor viable unc-52 alleles are fertile, adjacent to the body muscle cells; the positions in the although they do show some abnormalities in gonadal basement membrane directly adjacent to the densebod- structure (GILCHRISTand MOERMAN1992). Morere- ies and "lines of the muscle show greatly intensified cently, a zygotic lethal class ofunc-52 mutation has been staining with MH2. ROGALSIUet al. (1993) have shown discovered; embryos homozygous for lethal unc-52 mu- that a recessive lethal unc-52 mutation abolishes MH2 tations arrest at thetwofold stage of elongation and re- staining; these workers also mapped the epitope recog- main virtually paralyzed, a phenotype referredto as Pat, nized by MH2 to a region of unc-52-encoded sequence. for paralyzed and arrested at the twofold stage of elon- (There are alternative unc-52 transcripts, which pre- gation (WILLIAMSand WATERSTON1994; GILCHRISTand sumably correspond to alternate protein products, and MOERMAN1992; ROGALSIUet al. 1993). The Pat pheno- MH2 probably does not recognize them all.) We have type is characteristic of muscledefective mutants, such as stained wild-type and mec-8 embryos reared at 16" with mp? (WATERSTON1989), unc-45 (VENOLIAand WATERSTON MH2, and the results are illustrated in Figure 4, e- h. The 1990), &bl (BARSTFADand WATERSTON 1991)and many wild-type embryos show staining that corresponds to the other loci (WILLIAMSand WATERSTON1994). body muscle strips, but the arrestedmec-8 animals show We have discovered that mec-8 mutations strongly en- strips of staining that are disorganized and that appear hance the mutant phenotype of otherwise viable and to be locallyunattached to thehypodermis, patterns that fertile unc-52 alleles such that the double mutant ani- are very similar to those already described for staining malsmimic the Pat phenotypeconferred by lethal muscle myosin of arrested mec-8 animals. Furthermore, unc-52 alleles. The synthetic lethality of mec-8; unc- in some areas the staining was diffuse and notorganized 52(viable) double mutants is not specific to particular into strips. Defects visualized by MH2 staining were seen pairs of alleles, and itis not cold sensitive. We tested six as early as the 1.5-fold stage of embryonic elongation. As alleles of each gene invarious combinations (Table 4), was the case with staining myosin, the defects seen with and all resulted in a 100%penetrant Pat phenotype. An MH2 staining were cold-sensitive and variable from ani- example of these experiments is illustrated in Figure 5. mal to animal. In this case, animals of genotype mec-8(mn46?) I; mnCl Synthetic lethality of mec-8; unc-52(viable) double dpy-IO unc-52(e444)/unc-4ZZ, which are touch insen- mutants The unc-52 gene was first defined by muta- sitive and dye-filling defective, were generated; they seg- tions that lead to a progressive dystrophy of body mus- regated Mec Unc4 andMec non-Unc self progeny but culature (BRENNER1974). Mutant animals develop nor- no (Mec) Dpy Unc-52 selfprogeny. In place of the latter, mally as larvae, but a gradual paralysis posterior to the one quarter of the self progeny were Pat embryos (Fig- pharynx begins late in larval development. The progres- ure 5), which were not produced by mec-tl(rnn463)or sion of the paralysis is correlated with a gradual disor- mnCl dpy-10 unc-52/unc-4 parents. Some, but not all, ganization of muscle structure (MACKENZIE et al. 1978; Pat embryos hatched. The Pat embryos typically had hy- WATERSTONet al. 1980), the cause of which has been podermal constrictions and bumps, particularly near ascribed to a disruption of weak anchorages of the myo- the nose, which weresimilar to those seen in the arrested filament lattice (WATERSTON1988). The lattice of the mec-8 embryos reared at 16" and to those observed with muscle cells is normally attached at many points to the lethal unc-52 mutants (WILLIAMSand WATERSTON 1994; muscle cell membrane and to the adjacent basement B. WILLIAMS,personal communication). Interacting Genesmec-8 and Interacting 95

TABLE 5 mec-8(d98) or unc-Z2(e669) in double mutant combinations that are viable and fertile

Fertile with Fertile with mec-8(e398) unc-52(e669)

bli-6 sq1-I bli-6 mec-6 dPY-5 unc-1 day-6 mpc- 7 dpy-IO unc-4 dpy-5 mec-9 dpy-I3 unc-6 dpy-IO osm-1 dpy-I 8 unc-7 dpy-24 unc-1 dpy-24 unc-13 him-4 unc-3 em b- 9" unc-23 lon-2 unc-4 him-4 unc-29 mec-1 unc-23 him5 unc-40 mec-2 unc-29 him-8 unc-54 mrc-3 unc-55 lPt-2h unc-60 mec-4 unc-97 lon-2 uno75 mec-5 mec-1 unc-95 mec-2 unc-97 " emb-9(hc70) is a temperature-sensitive embryonic lethal muta- tion (WOOD et al. 1980). mec-a(e398);emb-9fhc70) doubles show temperature-sensitive embryonic arrest like emb-9(hc70) alone. 'let-2(b246) is a temperaturesensitive embryonic lethal mutation (HIRSHand VANDERSUCE1976). mec-8(e398); let-2(6246) double mutants show temperature-sensitive embryonic arrest like &2(b246) alone.

When temperature-sensitive alleles for bothgenes were combined,the resulting mec-8(u218ts);unc- 52(e669su250 ts) strain survived at 16" but was inviable at 25". The somewhat stronger temperature-sensitive mec-8 allele mn4 72ts was inviable in combination with unc-52(e669su250) even at 16". mec-8(~218ts) in com- bination with unc-52(e444) even at 16" resulted in a Pat phenotype. No semidominantinteractions between mec-8 and unc-52 mutations were detected. Forex- ample, at 20" mec-g(e398);unc-52(e444)/+ animals were Mec and fullyviable and fertile, and mec- 8(e398)/+; unc-52(e444) animals were Unc-52, viable and fertile (and neither Mec nor Dyf). The mec-8; unc-52(viable) interaction seems to be gene-specific. Table 5 lists other double mutants that were constructed involving either mec-g(e398) or unc- 52(e669).Some of the othermutations that were tested are in genes that encode componentsof the basement membrane, such as unc-6(ev400),which encodes a lami- nin B2-like molecule (ISHIIet al. 1992), and emb-9 and let-2, which encode basement membrane collagen (Guo .I et al. 1991; SIBLEYet al. 1993) or in genes that have 1 of the myofilament lattice; the muscle cells maintaina FI(;IXI.:.5.-Microgaphs showing cmbryonic sclf progeny of rounded appearance with staining outlining the nuclei. We rnrc-#(mn463) I; mnC1 dpy-10 unc-52(r444)/unc-4 I1 her- presume that the genotype of this animal is mec-8; mnCI maphrodites. Anterior is to the upperleft. The scale bar in (a) dpy-IO unc-52. (c) This 1.75-fold embryo is stained with anti- represents 10 pm for panels (a-c); the scale bar in (d) rep MHC B only. It shows the phenotype characteristic of mec-8; resents 10 pm for panel (d). All embryos were reared at 20". unc-52 double mutants. No myofilament lattice organization (a) A 1.5-fold embryodouble-stained with DM 5.8 (anti- is apparent; muscle cells are rounded, andmuscle cell nuclei MHGB) and MH27, which recognizes junctions of hypoder- are outlined by MHC B staining. (d) Nomarski micrograph mal cells. This embryo displays wild-type organization of de- showing the terminal phenotype of a mec-8(mn463); mnCl veloping myofilament lattices into longitudinal strips, so we dpy-IO unc-52(e444).The embryo is paralyzed and arrested at presume thatit is not homozygous for unc-52. (b) This 1.7-fold the twofold stage of elongation (Pat phenotype);it also shows embryo, which is doublestained as in (a), shows about the a well developed pharynx and hypodermal constrictions and same hypodermal stainingas in (a) butshows no organization lumps. 96 E. A.and Lundquist R. K. Herman been implicated in attachment of muscle cells to the mec-8(~303)and mec-8(mn455), neither of which, on hypodermis and cuticle, such as unc-2? (WATERSTON the basis of its cold-sensitive embryonic arrest phene, is 1988), unc-95 and unc-97 (ZENGELand EPSTEIN1980; likely to be null. Conversely, the Dyf phene of mec- WATERSTON1988) or in genes thataffect the response to 8(u 74),which is a candidatefor being null, was not sup light touch (CHALFIEand Au 1989). Noneof the muta- pressed by any of the suppressors. The cold-sensitive le- tions tested in combination with mec-8(e398) or unc- thality phene of two strong mec-8 alleles, u74 and 52(e669) resulted in a Pat phenotype or showed any mn463, was scored in a smu-1 and a smu-2 background other discernable interaction. (Table 7). Both mec-8 alleles were suppressed by smu-1 Mutations in smu-1 and smu-2 suppress mec-8 alleles, and smu-2 mutations. Overall, thedata suggest that unc-52 alleles and mec-8; unc-52 syntheticlethality: smu-1 and smu-2 mutations are fairly general suppres- We have identified extragenic suppressors of the sors of the mec-8 mutations. The weaker mec-8 alleles are mec-8; unc-52 synthetic lethality by making use of the suppressed better than the strongalleles for theMec and conditional lethality of the mec-8(u218 ts); unc- Dyfphenes, but the strongalleles are suppressed for the 52(e669su250ts) strain. Animalsgrown at 16" were cold-sensitive lethality. treated with EMS and allowed to propagate for at least Finally, each of the suppressors was checked for its two generations at 16" before shifting to 25". Four in- ability to suppress the paralysis conferred by each of dependent suppressed mutants were identified as sur- seven viable unc-52 alleles: e444, e669, e669su250 ts, vivors at 25". In the triple mutants at 25", each of the e998, el421, su54,and su200. As expected, all four sup- suppressor mutations suppresses, in addition to thesyn- pressor mutations suppressed unc-52(e669su250 ts). thetic lethality of the mec-8; unc-52 combination, the The e669su250 ts mutation confers atemperature- paralysis conferred by unc-52(e669su250 ts) and the sensitive paralysis, but even at 16" mutant adults move Mec and Dyf phenes conferred by mec-8(u218 ts). All slowly. The suppression by all four suppressors was four suppressors are recessive to their wild-type alleles strong at 25" but was also significant at 16" Adult unc- for suppression of the paralysis conferred by unc- 52(e669su250 ts) animals reared at 25"display dis- 52(e669su250 ts), as well asfor suppression of the Mec rupted myofilament lattice structure posterior to the phene of mec-8(u218 ts). By complementation tests for pharynx when viewed by polarized light microscopy. We suppression of unc-52(e669su250 ts) paralysis, we de- observed thatdouble mutants carrying unc- termined that one suppressor locus, smu-l, has three 52(e669su250 ts) and any one of the four suppressor alleles (mn415, mn417 and mn433) andanother, mutations showed partial restoration of myofilament lat- smu-2, has one allele (mn416),where smu stands for tice structure when grown at 25" (data notshown). None suppressor of mec-8 and gnc-52. We mapped the three of the otherunc-52 mutations was suppressed by any of smu-1 alleles to a position about 1 map unitto the right the smu-1 alleles, and only one other unc-52 mutation, of unc-101 I, and we mapped smu-2 about 2.5 map units e669, was suppressed by smu-2, albeit weakly. The left of unc-85 I1 (Figure 1). temperature-sensitive allele e669su250 ts, was derived Stocks containing eachof the homozygous suppressor as a partial revertant of e669 (MACKENZIE et al. 1978; mutations alone were constructed. The smu-1 mutants GILCHRISTand MOEFWW 1992). have no highly-penetrant visible phenotype. However, GILCHRISTand MOERMAN(1992) identified suppressors they do show a slight tendency to coil their bodies, and of unc-52(viable) mutations that define the sup-38 IV they are slower growing and have slightly smaller gene. Because their screen was for dominant suppres- brood sizes than N2 (data not shown).smu-2(mn416) sors, it is not surprising that they did not recover smu-1 mutants also show no highly penetrant visible phe- or smu-2 alleles. The fact that we did not identify sup-38 notype but, like the smu-1 mutants, have a tendency to mutations may be the consequenceof our focus on mu- tations that suppress boththe Mec phene of coil their bodies. mec- 8(u218 and the paralysis of unc-52(e669su250 Double mutants involving each of the smu-1 and ts) ts). smu-2 suppressors with 11 different mec-8 alleles were DISCUSSION constructed and scored for suppression of the Mec and Dyf phenes of mec-8. Table 6 gives the results. The data We have shownthat mutations in the mec-8 gene ofC. for the mec-8 alleles alone are repeated from Table 2. elegans cause, in addition to the previously reported The two temperature-sensitive alleles of mec-8, u218 ts mechanosensory defects and defects in the amphid and and mn472 ts, were well suppressed for both Dyf and phasmid chemosensory neurons, an incompletely pen- Mec phenes at 25". None of the other mec-8 alleles etrant, cold-sensitive embryonic and larval arrest. The was suppressed for mechanosensation, but many were strongest mec-8 alleles cause about 30% of the animals suppressed by at least one suppressor for Dyf, many by reared at16" to arrest developmentas embryos or larvae. all four suppressors. Apart from the two temperature- The arrested embryos often fail to complete the elon- sensitive alleles, the two mec-8 alleles showing the gation phase of embryonic morphogenesis and exhibit greatest suppression for all four suppressors were morphological abnormalities, including kinks and mec-8 and Interacting Genes 97

TABLE 6

Suppression of dye" defects and mechanosensory defects of mee-8 mutants by smu-1 I and smu-2 I1 Proportion of Proportion of phasmid phasmid neurons Touch neurons Touch Genotype (temperature) filled (n) sensitivity Genotype (temperature) a filled (n) sensitivity

N2 (wild-type) 0.99 (240) ++ mec-S(u74) 0.02 (136) - mec-S(mn455) 0.02 (328) - mec-S(u74) smu-l(mn415) 0.02 (372) - mec-S(mn455) smu-1 (mn415) 0.30 (192) - mec-S(u74) smu-l(mn417) 0.04 (332) - mec-S(mn455) smu-1 (mn4I 7) 0.30 (152)c - rned(u74) smu-l(mn433) 0.02 (196) ' - mec-B(mn455) smu-l(mn433) 0.31 (176) - mec-S(u74); smu-2(mn416) 0.02 (204) - mec-S(mn455); smu-2(mn416) 0.29 (196) - mec-S(u218 ts) (25") 0.18 (368) - mec-S(mn459) 0.04 (336) - mec-S(u218 ts) smu-I(mn415) (25") 0.79 (448) ++ mec-S(mn459) smu-I(mn415) 0.06 (176) - mec-S(u218 ts) smu-l(mn417) (25") 0.80 (448) ++ mec-S(mn459) smu-l(mn417) 0.02 (176) - mec-S(u218 ts) smu-l(mn433) (25") 0.73 (400) ++ mec-8(mn459)smu-I(mn433) 0.15 (198)' - mec-S(u218 ts); smu-2(mn416) (25") 0.90 (432) ++ mec-S(mn459); smu-2 (mn416) 0.13 (200) - mec-S(u303) 0.05 (228) - mec-S(e398) 0.11 (176) - mec-S(u303) smu-1 (mn415) 0.20 (152) - mec-S(e398) smu-l(mn415) 0.08 (312) - mec-S(u303) smu-1 (mn417) 0.21 (308) - mec-S(e398) smu-l(mn417) 0.09 (212) - mec-S(u303) smu-1 (mn433) 0.21 (336) - mec-S(e398) smu-1 (mn433) 0.15 (416) - mec-S(u303); smu-2(mn416) 0.43 (180)' - mec-S(e398); smu-2(mn416) 0.19 (180) - mec-S(u391) 0.02 (184) - mec-S(mn463) 0.03 (408) - mec-S(u391) smu-l(mn415) 0.08 (220) - mec-S(mn463) smu-I(mn415) 0.04 (108) - mec-S(u391) smu-1 (mn417) 0.04 (168) - mec-S(mn463) smu-1 (mn417) 0.12 (352) - mec-S(u391) smu-1 (mn433) 0.06 (204) - mec-S(mn463) smu-l (mn433) 0.23 (296) - mec-S(u391); smu-2(mn416) 0.20 (172) ' - mec-S(mn463); smu-2(mn416) 0.18 (184) - mec-S(u456) <0.01 (284) - mec-S(mn472 ts) (25") 0.10 (396) - mec-S(u456) smu-l(mn415) 0.02 (180) - mec-S(mn472 ts) smu-I(mn415) (25") 0.55 (524) + mecd(u456) smu-l(mn4I 7) 0.02 (124) ' - mec-S(mn472 ts) smu-l(mn4I 7) (25") 0.52 (132) + mec-S(u456) smu-l(mn433) 0.10 (312) - mec-S(mn472 ts) smu-l(mn433) (25") 0.54 (180) + mec-S(u456); smu-2(mn416) 0.14 (196)' - mec-8(mn472 ts); srnu-.?(m?k416) (25") 0.69 (144) + mec-S(rhl70) 0.01 (180) - mec-S(rhl70) smu-l(mn415) 0.22 (212) - mcc-S(rhl70) smu-l(mn417) 0.06 (228) - mec-S(rhl70) smu-I(mn433) 0.12 (180) - mec-S(rhl70); smu-2(mn416) 0.08 (124) -

~~ ~ ++ = touch-sensitive; + = partially touch-sensitive; - = touch-insensitive. 'Animals were scored at 20" unless otherwise noted. n is the number of phasmid neurons scored each animal has four phasmid neurons. Scored in one experiment; other strains were scored in at least two separate experiments. TABLE 7 tion of body wall muscle cellsin arrested mec-8 embryos Suppression of the coldsensitive (16")arrest of mee-8 mutants by and larvae by both polarized light microscopy and an- alleles of smu-1 and smu-2 tibody staining of body wall myosin, we found disrup tions in body wall muscle organization that suggested Proportion embryonic muscle attachment defects; strips of body wall muscle or early larval Genotype n arrest were frequently displaced asif they had been pulled away fromthe bodywall. Furthermore, an unc-52- 73 1 <0.01 N2 (wild type) et smu-l(mn415)0.01 639 encoded epitope (ROGALSKI al. 1993),which in wild- smu-l(mn417)0.02 719 type animals is localized to regions of basement mem- smu-1 (mn433) 708 <0.01 brane involved in attachments to bodywall muscle smu-2('mn416) 0.02 498 mec-S(mn463) 501 0.28 (FRANCISand WATEWTON1991), showed disruptions in mec-S(mn463) smu-I(mn415) 574 0.05 arrested animals that were very similar to those seen by mec-S(mn463) smu-l(mn417) 1197 0.06 myosin staining. We suggest that the cold-sensitive le- mec-S(mn463)0.01 smu-1 (mn433) 619 mec-S(mn463);0.04 smu-2(mn416) 416 thality and morphological abnormalities of mec-8 mu- mec-S(u 74) 597 0.31 tants may all be a direct consequence of defects in mec-S(u74) smu-l(mn415) 340 0.1 1 mec-S(u 74) smu-I0.09 (mn,417) 1030 muscle cell attachment. Embryonic elongation appears mec-S(u 74) smu-l (mn433) 649 0.18 to be driven by the squeezing of the outermost layer of mec-S(u74); smu-2(mn416) 642 0.08 cells, the hypodermis (PRIESSand HIRSH1986), but elon- gation beyond the twofold stage appears to depend, per- bulges in the hypodermis. Some, but not all, animals haps in addition to hypodermal squeezing, on func- exhibiting defects in embryonic elongation hatch;other tional body wall musculature; embryos lacking muscle arrested larvae are normally elongated. Upon inspec- function have a common (Pat) phenotype of paralysis 98 E. A.and Lundquist R. K. Herman and arrest at thetwofold stage of elongation (WATERSTON to the reinforcement of muscle attachments, particu- 1989; VENOLIAand WATERSTON1990, BARSTEADand larly at low temperature, and that the completeabsence WATERSTON 1991; WILLIAMSand WATERSTON 1994;HRESKO of mec-8(+) leads to weak muscleattachment atlow tem- et al. 1994), which is the earliest arrest we see in mec-8 perature, eitherbecause attachments arenot secured in animals. Unlike the Pat embryos, however, all of the the first place or because they are made but are fragile. mec-8 arrested embryos exhibited some movement. In either case, those animals in which the defects are We characterized all 16 mec-8 alleles with respect to particularly severe are arrested, whereas other animals, the Mec, Dyf and lethal arrest phenes,and the effectsof although frequently exhibiting minor attachment de- different alleles on the different pheneswere quite con- fects, survive and reproduce. sistent. Two alleles, u218 ts and mn472 ts, exhibited a The relationship between the muscle attachment de- weakMec phene; both also showed the weakest Dyf' fects and the Mec and Dyf phenes of mec-8 mutants is phene, were temperature-sensitive for bothMec and Dyf unclear. One possible common theme could be cell at- and exhibited no cold-sensitive lethality. A third allele, tachments or adhesion, perhaps involving extracellular e?98, was incompletely Dyf and showed very little cold- matrix. The fasciculation or bundling defects of the pro- sensitive lethality. Supporting the idea that u218 ts, cess endings of the chemosensory neurons, which un- mn472 ts and e398 are all hypomorphs were the results derlie the Dyf defects, may be the consequence of de- showing that animals carrying these mutations opposite fects in cell-cell attachments, perhaps mediated by the a deficiency were more strongly Dyf. It is worth noting extracellular matrix in which the process endings are that e398 is an amber mutation (CHALFIEand Au 1989), embedded (PERKINSet al. 1986). The touch cell pro- so apparently the truncated mec-8 protein produced by cesses are also embedded in an extracellular matrix, the the e?98 mutant has residual function. At least four mantle, which plays a role in anchoring the touchcells more alleles-u303, mn455, mn459 and rhl70-appear to the external cuticle (CHALFIEand SULSTON1981). to be non-null, since they gave only 17% or less embry- We have discovered a synthetic lethal interaction be- onic or larval lethality at 16".The other ninealleles were tween mutations in mec-8 and viable alleles of unc-52. fully Mecand Dyf and gave 23-31 % embryonic or early The interaction is not allele specific for either gene,ex- larval arrest at 16". We also showed that three of these cept that the unc-52 mutations must be partial loss-of- nine were equivalent to a deficiency when placed op- function because null alleles of unc-52 confer a phe- posite weak allelesfor the Dyfassay. We could not readily notype (ROGALSKIet al. 1993; WILLIAMSand WATERSTON gauge the effect on cold-sensitive lethality of putting our 1994) that is similar if not identical to thatof the mec-8; strongest alleles opposite a deficiency. Such animals are unc-52(uiable) double mutant,i.e., mec-8 mutations en- generally viable, however, because we easily generated hance the phenotype conferred by unc-52 viable alleles them at16", 20" and 25".We suggest that the mec-8 null to that of unc-52 null. The synthetic interaction appears phenotype isMec, Dyf, and incompletely penetrant, to be genespecific: no interactions were found involving cold-sensitive lethal. Consistent with this view is the fact either mec-8 or unc-52 with any of a variety ofmutations that five of the strong alleles arose in noncomplemen- in other genes. It seems relevant that the cold-sensitive tation screens that also identified a deficiency of the muscle defects observed in the strongmec-8 mutants ap locus. Finally, the frequency at which we recovered EMS pear to be similar to the muscle defects conferred by induced alleles was approximately that expectedfor the mutation in unc-52, except that in comparison to unc- inactivation ofan average C. elegans gene (BRENNER 52(lethal) mutants, the defects are much weaker, ex- 1974). hibiting incomplete penetrance and variable expressiv- It might be consideredsurprising that a null mutation ity, and in contrast to unc-52(uiable) mutants, there is can result in an incompletely penetrantand cold- no evidence that the mec-8 cold-sensitive defects worsen sensitive lethality. There are clear examples inyeast, during postembryonic development. The Mec and Dyf however, in which complete inactivation of a gene re- phenes of mec-8 seem completely independent of unc- sults in lethality only at low temperature. One example 52, however. All of the seven viable unc-52 mutants we is the SAC1 gene, which was first defined by mutations have studied are wild-type with respect to both of these that suppress a temperature-sensitive mutation in an ac- phenes. We therefore suggest that the mc-8 unc-52 inter- tin gene (NOVICKet al. 1989). Additional examples in- action involves defects in muscleattachment specifically. clude the ClNl, CIN2 and GIN4 genes; mutations in One possible interpretation of a synthetic enhance- each of these lead to defects in microtubule-mediated ment is that the two loci involved encode partially re- processes (Hour et al. 1990). Itmay be relevant that all dundant functions (GUARENTE1993; THOMAS1993). An of these genes are involved in cytoskeletalfunctions. If example of this phenomenon in C. elegans is provided we assume, as already suggested, that the cold-sensitive by the ace-1, ace-2 and ace-3 genes, each of which en- lethality of mec-8 mutants is a consequence of defects in codes an acetylcholinesterase; the single mutants have muscle attachment, we are led to suggest that the mec- no obvious phenotype, but the ace-1 ace-2 double mu- 8(+) product is not absolutely essential but contributes tant is severely uncoordinated (CULOTTIet al. 1981) and mec-8 and Interacting Genes 99 the ace-1 ace-2 ace-3 triple mutant arrests development defective, and as a consequence, adult muscle cell at- shortly after hatching UOHNSONet al. 1988). Another tachments would be fragile and embryonic attachments example is provided by the glp-1 and lin-12 genes, would become dependent on unc-52 products that re- which encode very similar molecules and confer a syn- quire mec-8(+) splicing function. We emphasize that thetic lethal phenotype (LAMBIE and KIMBLE 1991). this picture is speculative. An alternative possibility is FERGUSONand HORVITZ(1989) have characterized re- that mec-8(+) regulates the splicing of a transcript for a dundant genetic pathways that direct C. elegans vulva protein that interacts with unc-52 protein. In either development; a mutant phenotypeis revealed onlywhen case, we suggest that mec-8 may control the splicing of each pathway has been mutated. We do notbelieve that transcripts of one or moreadditional genes that arere- the mec-8; unc-52 synthetic lethality reveals a redun- sponsible for the Mec and Dyf phenes. dancy of function between mec-8 and unc-52 or redun- We have identified and characterized four suppres- dant pathways,however, because thedouble mutant sors of the mec-8 unc-52synthetic lethality conferred by phenotype is the same as that of the unc-52 null. Instead, weak temperature-sensitive alleles of each gene. The we suggest that mec-8(+) and unc-52(+) contribute four suppressors were assigned to two genes, smu-1 I, nonredundantly to the process of muscle attachment. and smu-2 II. The suppressor mutations by themselves This makes their interaction analogous to one seen be- seem to bemildly deleterious butconfer no easily scored tween fem-l and fem-2, which are involved in the C. el- phenotypethat we have been able to discern. We egans sex determination pathway; a double mutantcon- checked all four suppressors for their ability to suppress sisting of partial loss-of-function alleles of each gene is the Mec and Dyfphenes of 11 mec-8 alleles. Onlythe two synthetically enhanced to resemble the null phenotype weakest mec-8 alleles showedsuppression of Mec-by all for eachsingle gene (KIMBLE et al. 1984).We suggest that four suppressors. All but oneof the 11 mec-8 alleles were the absence of mec-8 functionrenders the residual suppressed at least weakly for Dyf by at least one smu-1 unc-52 activity provided by unc-52(viable) insuffkient allele and the smu-2 mutation. None was completely for establishing or maintaining the muscle cell attach- suppressed, i. e., the difference between the Mec and Dyf ments necessaryfor successful embryogenesis. (This sug- responses may simplybe a consequenceof our ability to gests that mec-8(+) functions in muscle attachment at measure a weak suppression of Dyf. The general rule higher temperatures as well as low,which is not surpris- from these results seems to be that theweakest allelesare ing because it functions to make animals non-Mec and most readily suppressed. By this criterion, u74 may be non-Dyfat all temperatures.) One possibility inline with the best candidate for a null, since it was the only one this view would be that mec-8 encodes a component of for which we were unable to detect suppression of the the muscle attachment machinery, which would both Dyf phenotype. We also looked at suppression of strengthen muscle attachment in unc-52(+) animals at the cold-sensitive lethality of two strong mec-8 alleles, low temperature and be requiredfor muscle attachment including u 74,and bothwere suppressed by smu-1 and in unc-52(viable) embryos. A second possibility would smu-2 mutations. Because the weak mec-8 alleles did not be that mec-8 product controls the formationof a com- exhibit any cold-sensitive lethality, it seems that only a ponent of the muscle attachment machinery, perhaps little mec-8 activity is needed to suppress this mutant even unc-52 protein. phene. Preliminary molecular analysis of mec-8 supports the The action of smu-1 and smu-2 suppressors on mec-8 idea that mec-8 protein controls the expression of other mutations seems to be allele-nonspecific. The suppres- genes (E. LUNDQUIST, J. SHAWand R. HERMAN,unpub- sors seem able to compensate for a small amount of lished results). The gene encodes two 80-amino acid missing mec-8 function for each of the three mutant RNA recognition domains foundin many RNA-binding phenes. The recessive nature of the mutations suggests proteins, including the70-kD protein componentof the that they may be loss-of-function. We think it very un- U1 small nuclear ribonucleoprotein (snRNP) complex likely that thesuppressors are informational because in- (QUERY et al. 1989) and various RNA splicing factors formational suppressors should be allele-specific. The (KENAN et al. 1991), which suggests that the mec-8 pro- suppressor genes do not map near any of the known tein may regulate patterns of alternative RNA splicing. amber suppressors (KONDOet al. 1990) or smg suppres- unc-52 has been shown to undergoextensive alternative sors (HODGKINet al. 1989), nor do they exhibit any ofthe splicing (ROGALSKIet al. 1993; D. MOERMAN,personal morphological features of smg mutants. We also think it communication), so it is possible that it is a target of unlikely that the suppressors encode proteins that in- mec-8 control. According to this picture, in unc-52(+) teract directly with altered mec-8 protein to compensate animals mec-8(+) directs the RNA splicing necessary for for the mec-8 defects. Here too we would expect such the synthesis ofa species of unc-52 protein thatstabilizes suppression to be allele-specific (and perhapsalso domi- muscle cell anchorage at low temperature.In uric- nant). We suggest that the suppressors result in a small 52(viable) mutants, aspecies of unc-52 protein thatcon- amount of bypassfunction. Abypass function would ex- tributes to muscle cell attachment would be missing or plain how a mec-8null mutation could be suppressed; we 100 E. A. Lundquist and R. K Herman

propose that 74u is null, and itis suppressed with respect CHALFIE,M., and J. WHITE,1988 The nervous system,pp. 337-391 in to thecold-sensitive lethality. We would explain the weak The Nematode Caenorhabditis elegans, edited byW. B. WOOD. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. suppression of Mec and Dyf phenes by supposing that CULOTTI,J. G., G.VON EHRENSTEIN, M. R. Cu~ornand R. L. RUSSELL, the amount of bypass function provided is small. What 1981 A second class of acetylcholinesterasedeficient mutants of is the natureof the bypass function? Ifwe are correct that the nematode Caenorhabditis elegans. Genetics 97: 281-305. FERGUSON,E. L., and H. R. HORVITZ,1989 The multivulva phenotype mec-8 acts by controlling alternative splicing of target of certain Caenorhabditis elegans mutants results from defects in genes, then the bypass could be generated by loss-of- two functionally redundant pathways. Genetics 123: 109-121. function mutations in genes involvedin the general FRANCIS,G. R., and R. H. WATERSTON,1985 Muscle organization in Caenorhabditis elegans:localization of proteins implicated in thin splicing machinery such that the normalsplicing restric- filament attachment and I-band organization. J. Cell Biol. 101: tions are loosened and splices that in wild-type animals 1532-1549. are mec-8dependent are now permitted, albeit at low FRANCIS,R., and R. H. WATERSTON,1991 Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114: 465-479. levels, in the absence of mec-8 protein. GILCHRIST,E. J., and D. G. MOERMAN,1992 Mutations in the sup-38 We also tested the abilityof the suppressors to gene of Caenorhabditis elegans suppress muscle-attachment de- suppress the paralysis conferred by seven unc-52 muta- fects in unc-52 mutants. Genetics 132 431-442. GOH,P. Y., and T. BOGAERT,1991 Positioning and maintenance of tions in a mec-8(+) genetic background. All four sup- embryonic body wall muscle attachments in C. elegans requires pressors suppressed the paralysis conferred by unc- the mup-1 gene. Development 111: 667-681. 52(e669su250 ts). The only other allele suppressed was GUARENTE,L., 1993 Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet. 9: 362-366. unc-52(e669), which was suppressed weakly bysmu-2. If, GUO,X. D., J. J. JOHNSON and J. M. KRAMER,1991 Embryonic lethality as we have just supposed, the suppressor mutations re- caused by mutations in basement membrane collagen of sult in loosened restrictions on alternative splicing, then C. elegans. Nature 349: 707-709. HALL,D. H., and R. L. RUSSELL,1991 The posterior nervous system novel products generated in the smu mec-8(+) unc-52 of the nematode Caenorhabditis elegans:serial reconstruction of background couldpossibly lead to the strengtheningof identified neurons and complete pattern of synaptic interactions. muscle attachments in adult animals. This kind of sup- J. Neurosci. 11: 1-22. HEDGECOCK,E. M., J. G. CULOTTI,J. N. THOMSONand L. A. PERKINS, pression might be allele-specific and in any case would 1985 Axonal guidance mutants of Caenorhabditis elegans iden- be expected to be weak. tified by filling sensoryneurons with fluorescein dyes. Dev. Biol. 111: 158-170. HERMAN,R. K., and E. M. HEDGECOCK,1990 Limitation of the size of We thank WTYCHALFIEfor generously providing mec-8 strains and the vulval primordium of Caenorhabditis elegans by Gin-15 ex- encouragement, DONMOERMAN for helpful discussion, BOB WATERSTON pression in surrounding hypodermis. Nature 348: 169-171. and MICHELLECOUTU HRESKO for strains and antibody, ED HEDGECOCK HIRSH,D., and R. VANDERSUCE,1976 Temperaturesensitive develop for mec-8(rhl70), LOIS EDGARfor her TMP mutagenesis protocol and mental mutants of caenorhabditis elegans. Dev. Biol. 49: 220-235. RONELLIS for communicating unpublished results. 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