Dominant mutations affecting muscle structure in Caenorhabditis elegans that map near the actin gene cluster

J. Mol. Riol. (1984)

180, 473-496

Dominant Mutations Affecting Muscle Structure in Caenorhabditis elegans that Map near the Actin Gene Cluster ROBERT H. WATERSTON'?,

DAVID HIRSH'

AND T. R. LANEI

’ Department of Genetics Washington University School of Medicine 660 South Euclid, St. Louis, MO 63110, U.S.A. and ’ Department of Molecular, Cellular and Developmental Biology University of Colorado, Boulder, Colo. 80309, U.S.A. (Received 14 February 1984, and in revised form 14 August 1984) By examining Fl progeny of mutagenized Caenorhabditis elegans larvae, we recovered several dominant mutations which affect muscle structure. Five of these new mutations resulted in phenotypes unlike the previously recognized uric-54 and uric-15 dominant alleles. Mapping studies placed all five mutations in the same small region of linkage group V. Polarized light, fluorescence and electron microscopic studies showed that a prominent feature of the disorganized myofilament lattice is the abnormal placement of thin filaments within the body wall muscle cells. Pharyngeal musculature is also affected by three of the mutations when homozygous. Of the five mutations only three are homozygous viable. All three of these have unusually high intragenic reversion rates either spontaneously (- 10m6) or after ethyl methanesulfonate mutagenesis (2 x 10e5). suggesting that reversion occurs through loss of function mutations. No unlinked suppressor mutations were found. The dominance of the mutations, the effect on thin filaments and the reversion properties suggested that these new dominant mutations lie in a gene or genes specifying a structural component of the thin filament. The positioning of a set of three actin sequences in the same region (Files pt al., 1983) led us to speculate that these mutations lie in actin genes.

1. Introduction The small nematode Caenorhabditis elegans is the subject of intensive investigations into the genetic specification of muscle protein biosynthesis and assembly. About 20 genes have already been identified that affect muscle structure (Brenner, 1974; Epstein & Thomson, 1974; Epstein et al., 1974; Waterston et al., 1977,198O; Zengel & Epstein, 1980; Greenwald & Horvitz, 1980). Biochemical investigations of mutants of these genes identified uric-54 I as the structural gene for the principal myosin heavy chain of the four myosins identified t Author to whom all correspondence should be addressed. 473 0022%2836/84/350473-24$03.00/O 0 1984 Academic Press Inc. (London) Ltd.

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on acrylamide gels (Epstein et al., 1974; ,MacLeod et al., 1977a,b; Waterston et al.: 1982a). In addition, evidence suggests that uric-15 I is the structural gene for paramyosin (Waterston et al., 1977). These genes and their mutants are proving useful in a detailed molecular genetic dissection of thick filament assembly and function (Karn et al., 1983; Moerman et al.. 1982; Waterston et al., 1982h: Miller et al., 1983). To complement’ these studies of the thick filament, we have begun a similar molecular genetic study of the thin filament’. Analysis of the many homozygous viable mutations in the 20 known genes has failed to reveal any effect of these mutations on the mobility in acrylamide gels of known thin filament components. despite the fact t.hat mutants in several genes appear by electron microscopy t,o alter thin filament arrangement (Waterston et al., 1980; Zengel & Epstein. 1980). This failure to identify genes for thin filament) components despite intensive genetic searches suggested the need for the isolation of additional mutants by new approaches. Virtually all of the mutants in the init’ial set’ of 20 genes were identified by their homozygous mutant phenotypes, and most of these proved to be fully recessive. In our studies of the thick filament genes uric-54 I and mu-15 I. we identified dominant alleles, and we reasoned that dominant alleles of genes encoding thin filament structural components might also be recovered (Snustad. 1968; Greenwald & Horvitz, 1980). Dominant alleles in known genes could focus efforts on these genes and provide new hints as to their product. Beyond this, dominant mutant alleles could reveal genes not easily identified by screens for homozygous viable, recessive mutants. For example, genes which are members of multigene families might be difficult to identify since mutations which result in the absence of a product might be masked by the production of sufficient protein from other members of the family. However, the production of a defective product from a mutated member of a multigene family might interfere with the function of the normal product from other family members, and would likely have effects even when heterozygous. Such dominant gain of function mutants usually require that the polypeptide product be part of a multimeric unit. Muscle, with its many levels of interactions between proteins, should be particularly amenable to analysis through isolation of dominant mutants. Accordingly, we undertook to isolate animals with abnormal movement and muscle structure in the first generation after mutagenesis. Not surprisingly, we recovered a large number of new dominant, alleles of the uric-54 locus and a however, we have recovered five smaller number of uric-15 alleles. In addition. mutations which are closely linked to one another on linkage group V. As part of our studies of these mutants we have mapped them genetically, constructed heteroallelic combinations where possible, and recovered revertants to near wildtype phenotype. Morphological studies of t,he isolates and revertants have included examination of muscle structure with polarizing and fluorescence light microscopy and t’ransmission electron microscopy. Our results suggest these new mut,ations lie in one or more members of a multigene family for a thin filament component,. The placement of a olust,er of three actin genes in the same two centimorgan region of linkage group V by Hirsh & co-workers (Files et ai., 1983) suggested to us that, these dominant mutations might involve one or more of the

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actin sequences. The analysis of revertants of three of the mutants, presented in the accompanying paper (Landel et al., 1984) is consistent with the hypothesis that these mutations are in the actin genes. 2. Materials

and methods

(a) General genetic methods and strains The general genetic methods and handling of nematodes were as previously described (Brenner, 1974; Riddle & Brenner, 1978). N2 is the wild type strain of C. eEegans var. Bristol used in these studies and all other strains were derived from this strain. Some of the strains used in this study were obtained from the Caenorhabditis Genetics Center. Strains used in the present study and their genotypes are (unless indicated otherwise all genes are on linkage group V): CB224[dpy-Il(&24)], CB25[unc-23(e25)], CB268[unc-4l(e268)], CB1407[sup3(el407)], RW15[act-?(stl5)], RWlS[smn-Z(e30)], RW22[uct-?(stZZ)], RW94[act-?(st94)] (this strain uric-51(e369)], RW1162[dpy-ll(e224) sma-l(e30)], has been lost). RW1114[dpy-Il(e224) RW1174[dpy-ll(s224) uric-41(e268)], R#W1184[dpy-ll(e224) uric-23(e25)], RW1185[dpy-ll(e224) act-?(stl5)], RW1260[unc-42(e270) sma-l(edO)], RW207O[dpy-18(e364) III; sup-7(&5) X], RW2316[ une-54(e190) I; sup-3(e1407 &go)], RW3290[+sma-l/eDj’l+], RW3296[dpy-II+sma-l/+mDfl+], RW3308[ + sup-3(e1407 st90) act-?(st120)leDfl+], RW3309[ + sup-3(e1407 st90) act-?(Ytll9)leDfl+], RW2524[dpy-18(e364) III; a&2(&15): sup-T(st5) X] and RW5018[unc-42(~270)]. Spontaneous revertants obtained from RW15 include RW2241, RW2253 through RW2255. RW2262, RW2521 and RW2452 through RW2456. EMSt-induced revertants obtained from RW15 include RW2244 through RW2250 and RW2252. Additional EMSinduced revertants obtained for st15 from RW2524 were RW2627 through RW2633. Revertants obtained spontaneously from RW22 include RW2522, RW2575 through RW2590 and RW2613 through RW2624. EMS-induced revertants of RW22 include RW2556 through RW2574 and RW2592 through RW2612. Revertants obtained spontaneously from RW94 include RW2457 through RW2460. EMS-induced revertants of RW94 include RW2461 through RW2487. Screens for dominant mutations were carried out. by treating a mixed population of worms with 0.05 M-ethyl methanesulfonate for 4 h, recovering the animals and allowing them to grow overnight before picking 5 to 10 L4 or young adult animals per plate. This results in taking animals that are early larvae at the time of mutagenesis, which allows for DNA replication and mutation fixation before progeny are produced. Slow moving animals were picked from among the Fl progeny, and after reproduction, the animals were examined by polarized light microscopy to find those with abnormal muscle structure. Dominant mutations were either isolated from N2 (these were st15, st22 and st94) or from RW2316 (these were St119 and st120). This second strain contains the uric-54(e190) deficiency allele which thereby eliminates the recovery of the more frequent une-54 dominant mutations. The RW2316 strain also contains sup-3(e1407&90), which is a than is st.ronger suppressor of the motility defect associated with uric-54(e190) sup-3(e1407) alone. This allows the recovery of additional mutations affecting muscle structure in the uric-54(e190) background. The stronger sup-3 suppressor was recovered spontaneously in an uric-54(e190); sup-3(el407) stock. We assigned t.he new suppressor activity to sup-3 on the basis of the following two sets of observations. We failed to recover the weaker e1407 suppressor activity after outcrossing and thus the st90 mutation is closely linked to e1407. Spontaneous mutants of a strain of the genotype uric-54(e190); sup-3 (e1407 st90 st92), t Abbreviations used: EMS, ethyl methanesulfonatr; cM. centimoryan.

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where st92 is an EMS-induced mutation linked to e1407 and st90 which confers yet stronger suppression, have been obtained which have lost all suppressor activity. Assuming that all 3 suppressor mutations did not revert independently, either the secondary suppressors st90 and st92 are ineffective in the absence of sup-3(el407) or a single event eliminated all 3 activities. The latter hypothesis would be consistent with the conclusion that all 3 mutations alter a single locus (Waterston, unpublished results). (See Discussion for possible involvement of actin genes in the suppressor activity.) Reversion experiments were carried out by inspecting either untreated populations or populations mutagenized with EMS in the standard manner (Brenner. 1974) for animals of greater size and improved motility as adults. In general reversion experiments used homozygous animals, as it was possible to recognize the heterozygous revertant by its greater size and improved motility as an adult. In the majority of cases the revertant was initially recovered as a heterozygote, although many were found as homozygotes as well. Screens attempting to pick out revertants in a heterozygous background (Greenwald & Horvitz, 1980) proved to be very difficult, as animals of wild-type movement and size are hard to discern in a background of heterozygotes, which are of wild-type size and of a variable motility approaching that of wild-type. Frequencies of mutagen-induced reversion were calculated from the number of revertants obtained divided by the number of treated Fl chromosomes as estimated from the number of P, animals multiplied by twice the average brood size. In experiments where more than half the plates yielded revertants the Poisson distribution was used to correct for the likelihood of obtaining multiple independent events per plate. Frequencies of spontaneous reversion were estimated from the number of independent revertants obtained divided by twice the known number of animals per plate at starvation (the 10 cm plates used support the growth of approximately 100.000 animals). (b) Microscopy Polarized light microscopy was performed on unfixed specimens mounted uuder a coverslip in M9 buffer and viewed with a Zeiss WL microscope equipped with polarizer. analyzer and strain-free objectives and condenser. Animals were photographed with Ilford HP5 film processed with Ilford Percept01 or Microphen developer. Electron microscopy was done as previously described (Moerman et al.. 1982; Waterston et al.. 1980). (c) Fluorescence microscopy Asynchronous nematode populations were rinsed from NGM-agar plates with M9 buffer and allowed to settle at unit gravity. After several changes of M9 buffer the animals were fixed in 3% (v/v) formaldehyde in 0.1 M-Na,HPO, for 3 h at 22°C with occasional agitation. After fixation, animals were gently sedimented through 2 to 3 changes of PBS 8.1 miw-Na,HPO,, pH 7.3), extracted with (0.14 M-NaCl, 2.7 mM-ECl, 15 m&r-KH,PO,, 100% acetone at -20°C for 2 min and again sedimented through 3 changes of PBS. The fixed and acetone-extracted animals were incubated for 2 to 3 h at 22°C with rhodaminephalloidin (Molecular Probes) diluted 1 : 20 to 1 : 50 in PBS from the manufacturers stock solution (100 units/ml). After 4 washes in PBS, animals were mounted on glass slides in a small amount of PBS and viewed by epifluorescent illumination with a Zeiss Universal and barrier filters selective for rhodamine microscope equipped with excitation fluorescence. Exposures (3 to 10 s) of HP-5 or TRI-X film were developed in Ilford Microphen developer for ASA400.

3. Results (a) Recovery and description The five dominant recovered by subjecting

mutations, st15, C. elegans larvae

of mutants

st22, st94, St119 and st120, were all to ethyl methanesulfonate mutagenesis

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and inspecting the Fl progeny for slow or paralyzed progeny. The slow animals which reproduced and yielded both wild-type and slow or paralyzed progeny were further screened with polarized light microscopy for abnormalities in muscle structure. The majority of mutations recovered from wild-type at a frequency of about 5 x low4 are alleles of the uric-54 gene. The five mutations that are the subject of this paper occurred about 0.1 as frequently, and were readily distinguished phenotypically and by polarized light microscopy from uric-54 alleles. All five mutations, when heterozygous, result in animals slower than wildtype. but unlike the uric-54 dominant alleles which become progressively slower with age, these five mutations have their most pronounced effect on L4 larvae. Adults move better and are more difficult to distinguish from wild-type. Animals homozygous for the mutations st15, st22 and st94 are viable and fertile. The movement of the st15, st22 and st94 homozygotes is not markedly different from heterozygous forms. However, the homozygote L4 is never as slow as the heterozygous I,4 animals, and the homozygous adults are slower than their heterozygous counterparts. These homozygous adults are smaller than wild-type or heterozygous adults with the difference particularly marked for st15/&15 animals. Also, the generation time is increased and brood size is reduced compared to wild-type or heterozygotes. For example st15/st15 animals have a generation time of six days and a brood size of about 25 at 20°C compared to 3.5 days and nearly 300, respectively, for wild-type. The mutations St119 and st120, when homozygous, result in inviable animals. Approximately O-25 of the progeny of either sup-3(e1407 st90) stll9/+ or sup-3(e1407 st90) st120/+ animals arrest as first stage larvae. Whether the lethality is due to the St119 and st120 mutations per se or is a synthetic effect of the coupled sup-3(e1407 st90) mutation is uncertain, for st119 and st120 as far as we know have not been separated from the sup-3 mutations. In any case, the presumptive homozygous larva is motile after hatching and the pharynx pumps. By several hours after hatching pharyngeal pumping becomes irregular and incomplete, even though the larva is still moving relatively well, better in fact than homozygous viable alleles of other genes such as uric-54(e1273). Paralysis of the body wall musculature does not seem to be the immediate cause of inviability. (b) The mutations st,l5, st22, st94, st119, and st120 are all located in the same region of linkage group V Because the dominance of these five mutations makes the interpretation of complementation tests ambiguous, the genetic map position of each of the mutations has been determined in order to evaluate possible relatedness. The results of these mapping experiments are given in Table 1. Two-factor crosses with Dpy marker mutations on each of the six linkage groups showed that st15 is unlinked to Dpy mutations on linkage group I, II, III, IV and X, but is 4.1 CM from dpy-11. In a second cross st119 was found to be 0.5 CM from une-41. With this general position established from two-factor crosses, additional three-factor crosses were carried out for four available mutations, as shown in Table 1. We conclude that the four mutations st15, st22, St119 and st120 map to a position

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TABLE 1

Map data for st15, st22, st94, St119 and st120 Parental genotype A. Tm-factor

Progeny?

Map distance (CM)

crmaea: dpy-11 st15/+ f

st94/eDfl sup-3 stl2O/eDfl+ uric-41 + / + sup-3 at119

614 slow and wild-type 15 Dpy slow ’ DPY 700 slow 0 wild-type N 8000 slow 0 wild-type - 19,400 slow or Uric 67 wild-type

4-l

< 0.5
B. Three-factor crosaes:$ dpy-11 (12/41) at15 (29141) uric-51 dpy-11 (14/19) uric-23 (5/19) at15 dpy-11 dpy-11 dpy-11 dpy-11

(15/18) 8t15 (3/18) 8ma-l (36/39) at22 (3/39) sma-l (8/10) at119 (2/10) sma-l (17120) st120 (3/20) MU-1

dpy-11 (g/10) uric-41 (l/10) 8tlj dpy-11 (18/20) uric-41 (z/20) 8t28 dpy-11 (21/22) uric-41 (l/22) at119 dpyll (717) [~a41 8t120] urn-42 uric-42 une-42 uric-42

(l/4) (Z/7) (3/6) (2/5)

at15 (3/4) sma-1 at22 (5/7) sma-1 St119 (3/6) sma-I stl20 (3/5) sma-1

uric-41 (14/14) (sup-3 st119] t Because the st15/st15 and st94/st94 homozygotes grow appreciably more slowly than either heterozygous or wild-type strains this class was not included in counts of the first 2 crosses. Similarly the sup-3 St119 and sup-3 st120 homozygotes are inviable and were ignored. 1 Genes are indicated in their derived positions. Numbers in parentheses represent the number of crossover events in the interval between the 2 genes as a fraction of the total crossover events recovered.

nearly midway between uric-42 on the left and sma-1 on the right (Fig. 1). The information available for st94, which was lost through reversion, shows that it, too, is closely linked to sma-1 (data not shown). We have also examined the various mutations in trans to two deficiencies in the region: eDf1 (formerly e1405; Riddle & Brenner, 1978) and mDf1 (Brown & Riddle, personal communication). Both extend through we-41 and une-42, but eDf1 is believed to extend further to the right as it possesses sup-3 activity, a gene identified by mutant alleles which phenotypicaliy suppress uric-54 null alleles and uric-15 missense alleles (Riddle & Brenner, 1978). The sup-3 gene has been placed to the right of uric-42 (Riddle & Brenner, 1978). Both deficiencies behave as the

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FIG. 1, Partial map of the right half of linkage group V showing on the line the relative positions of the genes used in this study. The extent of eDf1 is shown below the line. The cluster of 3 actin sequences lies between me-23 on the left and mm-1 on the right, as indicated above the line (Files d al., 1983).

wild-type fifth chromosome in trans to all five mutations but this result is ambigious because the presumptive null alleles (see later sections) of the gene containing st15, St22 and st94 also behave as wild-type in trans to the dominant mutations. Recombinants between st15, st22, St119 and St120 and the mDf1 deficiency were recovered readily (data not shown), but wild-type animals were recovered only at frequencies of < 10V3 among progeny of any of the five mutations in trans with eDf1 (Table 1). Whether these are in fact recombinants or revertants is currently being checked with appropriately marked strains. Thus these experiments show that the five mutations lie to the right of mDf1 but do not) distinguish whether or not the mutations are outside the deficiency eDf1. To test directly for linkage of the four available mutations to one another, we attempted to construct heteroallelic strains, using appropriate markers. Neither st15 nor St22 in trans with either St119 or St120 produced viable animals. However. the st15/st22 double heterozygote is viable and resembles the St22 homozygote. In one experiment with the st15 mutation in cis with dpy-11 and St22 in cis with uric-42, no recombinant animals were found in more than 10’ progeny of heterozygotes. In summary, these mapping experiments indicate that st15, st22, St119 and st120. and probably st94, are very closely linked to one another, between uric-42 and sma-1, near the right end of eDf1. (c) The mutations

st15, st22, st94, St119 and St120 alter muscle structure with particular

effects on thin jilament

arrangement

The muscle structure of all the mutant alleles either as heterozyotes or where possible as homozygous animals has been examined with polarized light microscopy. In addition, the st15/ + and st15/st15 animals have been evaluated by fluorescence microscopy using fluorescently labeled phalloidin and by electron microscopy. (i) Polarized

light microscopy

Using polarized light microscopy, the entire body wall and pharyngeal musculatures can be rapidly evaluated in unfixed specimens. As described

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previously, the adult wild-type animal has brightly birefringent A bands running at a 6” angle to the long axis of the animals (Waterston et al., 1980; Mackenzie & Epstein, 1980). The A bands, about 1 pm in the transverse dimension, are int,erspersed with darker I bands, with a sharp regular boundary between the bands. In the center of the I band are punctate birefringent dense bodies, the functional equivalent in the nematode of the vertebrate Z line (Fig. 2(a)). Tn contrast to this regular organization of the wild-type muscle, the muscle structure of all five mutants is quite disorganized. Since the mutations are similar in their effect on body wall musculatures the description will focus on the structure of the st15 mutants. The heterozygous animal, st15/ + , has irregular and indistinct A bands, and

dense bodies are recognizable

only occasionally

in t,he I bands (Fig. 2(b)). In

addition, large patches of birefringent material are present, usually at the ends of cells or in cell processes. No comparable structures are present in wild-type. All body wall muscle cells exhibit these abnormal features. All four other mutations show similar qualitative changes. The sarcomere structure of the homozygote, st15/st15, is even more abnormal, as A and I bands are blurred and difficult to recognize in many areas where the birefringence is almost uniform across the muscle cell (Fig. 2(c)). The large patches of birefringence are numerous and often possess an angle of extinction different from the residual muscle lattice (Fig. 2(d)), suggesting that the components of the patch are not oriented uniformly parallel to the long axis of the animal. Both st22 and st94 are similar, but may be marginally less severely disorganized. The muscle structure of the stll9/stll9 and stl20/st120 Ll larvae is also abnormal with changes similar to those seen in st15/st15 larvae. (ii) Fluorescence microscopy The fungal toxin phalloidin binds to F-actin specifically and when labeled with the fluorophor NBD or rhodamine it marks F-actin filaments in fluorescence (Fig. 3(a) and (c)) phalloidin binds microscopy (Barak et al., 1980). In wild-type to the thin filaments which comprise the I band of the polarized light images and extend into the A band, stopping at the unlabeled H-zone. The dense bodies are unstained and can be detected in outline lying in a row within each thin filament band (arrows). In contrast, the thin filament organization of the mutants st15 and st22 is markedly altered (Figs 3(b) and (d) and 8(b)). There is weak staining over most, of the cell, with only traces of band organization, and patches of increased fluorescence are irregularly scattered in the cells. These areas correspond in location and shape to the patches of increased birefringence seen in polarized light microscopy. (iii) Electron

microscopy

The obliquely one to view in different levels the associated the A band is

striated organization of C. elegans body wall musculat’ure allows single transverse sections not only the thin and thick filaments at within the sarcomere, but also the alteration of A and I bands and M line and dense bodies. The overlap of thin and thick filaments in extensive and usually 11 to 12 thin filaments surround each thick

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Fro. 2. Polarized light micrographs of the body wall musculature of at15 mutants and wild-type (N2) for comparison. Animals are oriented so that their long axis is parallel to the long axis of the photograph and were rotated about their long axis under the coverslip to bring the muscle quadrant! shown onto the upper surface, parallel to the plane of the optical section. The photographs are of musculature about l/3 the body length from the anterior tip, and show the central portion of one muscle cell on the left portion of the photograph, along with the ends of 2 other cells at the right. (a) 52. Wild-type body wall musculature is characterized by sharply defined oblique striations. The thick filament containing A bands are bright and a fainter central H-zone (lower arrow) is seen in those bands in best focus. The dark I-band contains in addition to the isotropic thin filaments the bright. periodic structures called dense bodies (upper arrow). These latter are sites of thin filament insertion and a.re analogs of the vertebrate Z line. The bar represents 5 pm. (b) s115/+. The arrows mark 2 A bands with an intervening I band. These A bands are diffuse and of a non-uniform width along the length of the band. Several faint dense bodies are discernible in the I band to the left of the arrows. A bands are even less distinct in other parts of the st15/+ musculature. Magnification, same as in (a). (c) st15. No distinct A bands are present, although variations in the intensity of birefringence do suggest irregular striations. The arrows mark patches of increased birefringence. The patch marked b>the upper arrow is extinguished at this angle. Magnification as in (a). (d) st15. This shows the area marked with arrows in (c), but rotated 45” to extinguish the muscle birefringence. The patches markrtl 1)~ t.hts arrows an= not extinguished at this angle. MagnificaGon as in (a).

filament (Fig. 4: see Waterston et al., 1980; Mackenzie details). In both st15/+ and stZ5/stZ5 this organization is Tn the &15/-t animals thick filaments are abundant irregular A bands by I bands with central dense bodies bands are quite incomplete and many thick filaments do complement of surrounding thin filaments. In many

8: Epstein. 1980. for markedly altered. and are divided into (Fig. 5). Often these T not have their normal sections, clumps of

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microfilaments are seen outside the normal lattice position adjacent to the hypodermis (examples of such clumps are shown in Fig. 5(b) and Fig. 6). Muscle structure is more normal in the anterior region of the &15/t animal, with A bands clearly defined, and with more normal appearing dense bodies; however, even in this region the lattice is not as regular as in the wild-type, and clumps of thin filaments are seen (Fig. 5(b)). As expected from light microscopic studies, filament disorganization is more rxtensive in the st15 homozygotes. The thick filaments are still abundant, but show little evidence of organization into A bands (Fig. 6). Dense bodies are poorly formed, protruding only a short distance into the sarcomere. Thin filaments are only sparsely distributed in the normal lattice region. Instead bundles of microfilaments (average diameter 6.2 nm; range 56 to 71, n = 20) are found outside the normal lattice. These filaments are often oriented obliquely to transverse sections. The groups of filaments are of a size, position and number

(0)

(b)

FIG. 3. l’halloidin staining of st15 homozygotes and wild-type. In order t.o visualize the distribution of actin filaments in the body wall muscle cell animals were treated with the fungal toxin phalloidin labeled with rhodamine, which binds to F-actin. (a) N2. Low magnification views of wild-type show that the majority of fluorescence is associated with the body wall muscle cells, although the other muscles such as the pharyngeal, anal and uterine muscles can also be seen. In addition, staining is present along the lumen of t,he intestine. presumably associated with the microvilli which line the lumen. The body wall staining falls into the four quadrants where muscle cells are. The distribution of stain is nearly uniform within the quadrant at this resolution. The box indicated by arrows outlines the region typically photographed in the higher magnification photomicrographs. The bar represents 50 pm. (b) stZ5. Similar low magnification views of sfl5 show that the staining of the body wall

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(d)

musculature is non-uniform indicating that the thin filaments in these cells are abnormally placed and aggregated. Magnification, as in (a). (c) N2. A high magnification view of wild-type shows the striated pattern expected for thin filaments based on polarized light and electron microscope images. The non stained striations correspond to the H-zone of polarized light. Within each stained thin filament band is a linearly arrayed set of less intensely stained spots (arrows). These correspond in position and size to dense bodies. The bar represent.s 1Opm. (d) st1.j. The thin tilaments as drtect.eti by phallmdln show almost no sign of normal wild-type organization. Faint staining is seen in much of the cell whereas a few irregular areas have increased staining. These latter areas are apparently collections of thin filaments and correspond to the irregular areas of anisotropir material seen in polarized light microscopy and to the bundles of thin filaments seen in electron micrographs. (See Fig. 8(b) for anofhvr example.) Magnification. as in (c).

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Frc:. 4. Electron micrograph of wild-type (N2) body musculature in transverse section, for comparison with Figs 5 and 6. This Figure illustrates the normal arrangement of the myofilament lat,tice in X2. with bands of thick filaments alternating with thin filaments and dense bodies along the hypodermal margin of’ the muscle cell. (a) A complete quadrant of the body wall musculature at a lower magnification. mrb. muscle cell hodies: cut.. cuticle. The bar represents 1.0 pm. (1)) A high magnification view, comparable to Figs 5 and 6, to illustrate the details of filament distribution. A. A band: db. dense body; cut’, cuticle; I. I band: M. 111line analog. The bar represents 0.5 pm.

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similar to the bright patches of material seen in polarized light and with phalloidin. Because of the filament diameter and staining with phalloidin we conclude that these filaments represent thin filaments, displaced from their normal lattice position.

(iv) Pharyngeal

muscle structure

The second largest muscular organ in C. elegans is the pharynx, which consists of four parts: the procorpus, first bulb, isthmus and posterior bulb. In wild-type, the myofilaments are oriented radially from the membrane adjacent to the lumen of the pharynx to the outer membrane of the muscle cells (Albertson & Thomson, 1976; Waterston et al., 1980). The filament organization of these muscles can also be evaluated by polarized light microscopy. Inspection of all five heterozygous animals revealed no differences from wild-type. No abnormalities are seen in st22 or st94 homozygotes, but in st15/st15 animals circumferentially oriented birefringent material can be seen in the posterior bulb of some adults (not shown). Electron micrographs of transverse sections of the posterior bulb show that this birefringence derives from thick and thin filaments oriented perpendicular to the plane of section, i.e. longitudinally in the animal (Fig. 7). Perhaps as a functional correlate of this morphological abnormality, apparently intact bacteria are present in the distended intestinal lumen of st15/st15 adults. In wild-type, mostly bacterial ghosts are present in the intestine as the bacteria are broken upon passage through the grinder apparatus of the posterior bulb. The pharyngeal musculature of sup-3 stllglsup-3 St119 and sup-3 stlZO/sup-3 st120 Ll homozygotes is even more markedly abnormal as determined by polarized light microscopy. Both animals show little normal organization of birefringement material in the posterior bulb, and in the procorpus, birefringent material is oriented longitudinally rather than radially. The intestine appears almost empty by light microscopy suggesting these animals feed poorly. Again, because of the presumed presence of the sup-3 mutations in cis with St119 and st120, it is uncertain if this effect on the pharynges is the manifestation of St119 or St120 alone, or is a synthetic effect of these mutations in combination with sup-3. The sup-3(e1407 st90) homozygotes have a pharyngeal structure similar to wild type at all ages.

(d) Reversion

of the mutants

st15, st22 and st94

During the course of maintaining the st15/st15 stocks we noted the appearance of occasional spontaneous revertants, which were easily recognized by their larger size, improved mobility, and more normal internal structure. We have gone on to study these reversion events in greater detail for not only the st15 animals but also stZZ/stZZ and st94/st94 strains. We have not yet carried out a similar analysis of st119 and st120, because these mutations are homozygous inviable. The three mutations stZ5, st22 and st94 give rise to spontaneous revertants at similar frequencies. For st15, five independent, revertant events were found among 4 x lo6

486

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FIG. 6.

AND

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l)OMINANT

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IN C. clrgans

487

Fro. 6. Electron micrograph of st15 body wall musculature. The transverse section is at a level similar to Fig. 5(a), and shows musculature with features similar to those depicted in the polarized light micrograph (Fig. Z(c)). Bundles of thin filaments (Tn) are present away from the cuticle (cut). and the myofilament lattice itself shows few signs of organization into distinct bands. The arrow lies in a region in which few thin filaments are found. An enlargement of this area is shown in the inset. The arrow points to the same thick filament in each photograph. Tn, a large bundle of thin filaments; db. a residual dense body: cut, cuticle. The bar represents 0.5 pm.

Fro. 5. Electron micrographs of st15/+ animals, showing the abnormal organization of the nryofilaments. (a) A transverse section taken from behind the posterior pharyngeal bulb shows an area similar to that depicted in Fig. 2(b). An I band with a central dense body (db) lies near the right, but the filaments at the left are not segregated into distinct A and I bands. (b) A transverse section at the level of the pharyngeal procorpus showing more organized, but still abnormal musculature. See the text for additional description. cut, cuticle; db, dense body, the Z-line equivalent: I, I band; A. A band: Tn. a cluster of what are likely displaced thin filaments. The bar represenm 0.5 pm.

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FIG. 7. Electron micrograph of a transverse section of the at15 posterior pharyngeal bulb at the level of the grinder. The basement membrane (bm) lies at the outside of the spherical bulb. Thin filaments normally attach at a dense plaque at the cell membrane, and thin and thick filaments extend in toward the grinder at the central lumen. Some normally orientated filaments are present in this micrograph (nl). However, filaments are also running circumferentially, cut in cross-section on either side of the normally oriented filaments. No similarly oriented filaments are found in the wild-type. The bar represents 0.5 pm.

chromosomes screened, giving a reversion frequency of I.2 x 10m6. For st22, 28 revertant events were recovered among 3 x lo7 chromosomes screened, for a frequency of 1.2 x 10 -6. With st94, the numbers were four revertants from 6 x lo6 events for a frequency again of = 10m6. We have also examined the effects of EMS mutagenesis on the reversion frequency. In one experiment with st94, parental animals were divided into two groups, one treated with EMS and the other not treated, and revertants recovered from both groups. Four spontaneous revertants were recovered from 3 x IO6 animals screened, whereas 27 revertants were recovered among 0.6 x lo6 progeny of mutagenized animals (overall frequency 2 x lo-’ to 4 x 10m5). Estimated frequencies of EMS-induced revertants of st15 and st22 are in general agreement with the results with st94. Not’ all of the revertants have been outcrossed to check for linkage but of 47 events that have been examined, the original mutation was not recovered in 400 to 800 F2 progeny of crossed hermaphrodites. This includes three EMS and eight sponta.neous revertants of st15; 26 EMS-induced and four spontaneous revertants of st94; and four EMS and two spontaneous revertants of st22. Several revertant strains derived from mutagenesis did yield slow progeny but in general these had

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4x9

normal muscle. In one case, an incidentally induced allele of uric-95 Z was found, but the original dominant thin filament mutation was not recovered. All the revertants recovered in these experiments whether spontaneous or mutagen-induced are semidominant with respect to the original mutation. All are also homozygous viable although the screening procedure resulted in the recovery of the majority of revertants as heterozygotes. Revertant/mutant heterozygotes have greater size and better growth rates than the homozygous mutant strains, but the heterozygotes are slower than wild-type, particularly as L4 animals. These properties of the revertant chromosome are similar to wild-type and in fact in these simple genetic tests the revertant chromosome is indistinguishable from wild-type. To evaluate the extent of reversion, we have examined all the revertants with polarized light microscopy. The homozygous revertants have body wall and pharyngeal musculature that cannot reliably be distinguished from wild-type. The muscle structure of mutantfrevertant heterozygotes resembles that of mutant/ wild-type combinations. To look at thin filament organization directly we stained revertant animals with rhodamine-labeled phalloidin (Fig. 8(a)). The revertants, e.g. RW2458, have a pattern of thin filament organization comparable to wildtype as can be appreciated by comparing Figure 8(a) with Figure 3(a). Based on muscle organization, again the revertant chromosome cannot be distinguished from wild-type. 4. Discussion Tn the present study we have identified five new dominant mutations, and characterized these mutations revertants and their genetically and morphologically. Results of mapping experiments place all five mutations within a small region in the right arm of linkage group V. The phenotypes of the five mutations when heterozygous with wild-type are similar, but only the three mutations st15, st22 and st94 are homozygous viable, whereas at119 and at120 are recessive lethal mutations. All five result in reduced motility, but not severe paralysis, when either heterozygous or homozygous. This slowed movement correlates with, and presumably results from, a disorganized body wall myofilament lattice. A principal feature of the mutant muscle is the abnormal placement of thin filaments, a feature shared by mutants of only two other genes, uric-60 and uric-78. The three homozygous viable mutations revert at unusually high frequencies, either spontaneously or after mutagenesis, via semidominant closelg linked mutations. This combination of features, dominance, thin filament. abnormalities, and high internal reversion rates, is unique among the mutants affecting muscle structure in C. eleqans and makes this group of mutations of special interest. To carry these studies further, we wished to know the products of the gene(s) affected by these mutations. Because of the implications we drew from the present results, and because a cluster of three actin sequences had been mapped in the same general region (Files et al., 1983) we hypothesized that these mutations were in one or more of the actin genes.

R. H. WATERSTON,

D. HIRSH

T. R. LANE

(b)

(a)

PIG. 8. Phalloidin-stained

4ND

of the revertant RW2458 and the mutant stl3. The similar to wild-type with broad longitudinal bands of stain alternating with the narrower unstained H-zone. This is in marked contrast to the at15 animal (b) which shows only faint signs of organization of fluorescence into bands and instead contains a few irregular regions of fluorescently stained material. The bar represents 10 pm. rev&ant

musculature

photomicrographs

(a) has a muscle structure

(a) The jive dominant

mutations

lie in a single gene or in a tightly related genes

Einlced cluster of

Most mutations in C. elegans have been assigned to specific genes by allelism testing. The dominance of the five mutations considered here prevents meaningful interpretation of allelism tests and consequently we have relied primarily on linkage data to determine the relatedness of the five mutations. All five mutations are located on the right half of linkage group V and none have been observed to

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recombine with the half map unit deficiency eDf1. Additional mapping of st15, st22, St119 and St120 place each of these mutations about O-4 to 0.6 map units to the right of we-42 outside mDf1, and left of sma-1, close to the suspected right endpoint of eDf1. Direct tests for linkage between the various mutations were only possible for st15 and St22 as the st94 mutation was lost through reversion, and other combinations are lethal. The recombination distance between the st15 and St22 mutations (
mutations

are gain

of function

mutations

The dominance of the five independently isolated mutations could be explained in at least two ways, which are not mutually exclusive. In one case, the gene(s) in which the mutations lie encode a polypeptide that is required in stoichiometric amounts. A loss of function mutation (e.g. a nonsense or deletion mutation) in the gene reduces the level of normal product in the heterozygote to one-half wild-type levels, which is below the amount required for normal movement and muscle structure. A second hypothesis is that the mutations result in a dominant gain of function for the gene product (e.g. inappropriate expression, or altered function from a missense mutation). Such gain of function mutants might be found in a gene(s) encoding a polypeptide that is part of a multimeric unit, where the assembly of an altered product into the multimer can interfere with the function

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of normal product. Muscle proteins obviously can fit either hypothesis: they are structural proteins required stoichiometrically and are assembled into a complex multimeric unit. A comparison of the phenotypes of the heterozygous and homozygous mutants suggests that the present set of mutations represent a dominant gain of function from the following reasoning. In the case of loss of function mutations, we would expect the homozygous mutant animal to be significantly more affected than the heterozygote. This is in fact true for loss of function mutations in uric-54 and uric-15, where the homozygous animal is severely paralyzed, and the heterozygote is only slightly slower than wild-type. In the case of an abnormal product interfering with normal product function, the relationship of the heterozygous to homozygous phenotype would be less predictable, and the severity of the heterozygote might approach that of the homozygote phenotype. As the motility of st15, st22 and st94 heterozygotes is not, markedly different from the motility of their respective homozygotes, we suggest that the mutations are dominant gain of function mutations. From the forward frequency of mutation after EMS mutagenesis of about 0.1 the frequency of similar gain of function missense mutations in the uric-54 gene (a large target), these mutations are not extremely rare and could represent missense mutations. However: one or more of these mutations could result in inappropriate expression. The results of the reversion analysis as discussed in the next section and in the accompanying paper (Landel et al., 1984) are consistent with either interpretation. (c) The st15, st22 and st94 mutations probably function mutations

revert to wild-type

by loss of

The high spontaneous and mutagen-induced reversion frequency of all three testable isolates is unusual for mutations in C. elegans. The reversion events are closely linked to the original mutation in all cases tested (
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M'C'TATIONS

IN C. elegans

493

5 x 10V5 is lower than EMS-induced loss of function mutations in other genes: 2 x low4 for uric-54 (Anderson, personal communication) 5 x 10e4 for uric-58 and 4 x 10e4 for uric-93 (Greenwald $ Horvitz, 1980). The lower mutagen-induced frequency as opposed to similar if not higher spontaneous frequencies of these mutants compared to other genes could be accounted for by several factors, The mutagen-induced frequency could be low perhaps because the effective target’ is small. This might reflect a smaller gene (the uric-54, where the product is known, is in fact about 5 times the size of an actin gene) or that some event not easily stimulated by EMS is involved. The latter might be true if reversion via the null state did not occur. The peculiar growth properties and small brood sizes of the st15, st22 and st94 animals might also affect the efficiency of EMS mutagenesis. Alternatively, the spontaneous frequency may be high, perhaps because events other than loss of function mutations can result in reversion. These events might include unequal recombination or gene conversion, both possible mechanisms if the mutations lie in the actin cluster. In a genetic test for reversion via null alleles, we have also tested more than 20 of the revertants with the tRNAF& nonsense suppressor, sup-7 X (Wills et al., 1983; Waterston et al., 1984), to see if the suppressor can restore the mutant phenotype and thereby identify null revertants, but this has so far been unsuccessful (Waterston, unpublished results). Greenwald & Horvitz (1980) found only one of 24 isolates tested to be suppressible for uric-93 revertants and we are testing additional revertants in an attempt to recover suppressible null revertants among the present set of revertants. A comparison of the revertants obtained here with loss of function mutants in uric-93 and uric-54 is instructive. Null alleles of uric-54, when homozygous, result in animals which are severely paralyzed, demonstrating the essential nature of the uric-54 product. On the other hand uric-93(O) null homozygotes are wild-type, showing that the uric-93 product is not required to produce a normal animal. In this regard, the null revertants of st15, st22 and st94, as RW2246 and RW2580 (Landel et al., 1984), resemble null revertants of uric-93, in that the homozygous revertant animal, in which the mutant product has been eliminated, resembles wild-type. Thus the product affected by st15, st22 or st94 is not required by the normal animal, either because the product serves no required function or that function can be supplied by the products of other similar genes. The st15, st22 and st95 revertants of nuil alleles, differ from either uric-54 or uric-93 null alleles in combination with the original mutations. For both uric-54 and me-93, null alleles are fully recessive to the dominant alleles, i.e. uric-93(d)/unc-93(O) or uric-54(d)/ uric-54(O) resemble the uric-93(d) or uric-54(d) homozygous animals. This contrasts with the phenotype of me-93(d)/+ or uric-54(d)/+ animals, which is intermediate between the phenotypes resulting from homozygous wild-type or dominant alleles. In contrast, the stl5, st22 and st94 null revertants are semidominant with the original mutant allele, so that, for example, st22/(0) has a phenotype similar to &?2/+. We interpret these observations as follows. In the case of the uric-93 and uric-54 genes, the product from the wild-type allele in uric-93(d)/ + or uric-54(d)/+ animals counteracts significantly the effects of the dominant mutation, The null allele in

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uric-93(d)/(O) or une-54(d)/(O) animals does not yield any wild-type product to balance the effects of the mutant product and is thus fully recessive to the dominant allele. For st15, st22 and st94 mutations, the similarity of the phenotypes of st22/(0) to st22/+ animals argues that the presence of the wildt,ype allele has little effect on the expression of the mutant phenotype. Rather it’ is the number of mutant genes, and thus the amount of the mutant product, that, determines the phenotype. These results are consistent with the hypothesis that the gene(s) altered by stl5: st22 and st94 are members of a multigene family, in which the presence of other identical or very similar genes makes any one gene non-essential. In this view, the dominant mutations interfere with the function of the product of one or more members of the family. The phenotype observed represents a balance between the amount of mutant and normal products from all genes. The actin genes represent such a multigene family (Files et al., 1983; Martha Wild, unpublished results). (d) Do these jive mutations identify a new gene? The dominance of the present set of isolates and the wild-type null phenotype associated with the gene(s) affected by the five mutations (see below) prevents allelism testing of these mutations with other genes in the region. Although these five mutations are clearly distinct in phenotype from other identified mutations in the region (and elsewhere in the genome), they could represent an unusual class of mutation in a gene already identified by mutations producing a different phenotype. Presuming that this hypothetical gene must involve muscle in some way, only one gene in this region, sup-3, has been shown to have effects on muscle structure. This gene was identified as a dose-dependent, dominant suppressor of uric-15 missense alleles and uric-54 null alleles. The suppressor alleles of this locus, in the wild-type although without apparent effect on muscle structure background, result in improved thick filament placement in uric-54 null animals, and thus the gene likely encodes some muscle cell product, possibly even a structural component of the muscle lattice. The map position previously determined for sup-3 is very close to the position determined for the present five mutations and the mapping experiment with sup-3 act-l(stll9) failed to separate su,p-3 from st129. Close linkage alone is of course insufficient to establish allelism. Complementation tests are complicated by the dominance of both sup-3 suppressor alleles and the thin filament mutations, as well as the fact that both classes of mutat,ions must be evaluated by their opposing effects on muscle. The small deficiency, eDf1, which has sup-3 suppressor activity, does resemble the wild-type chromosome when placed in tram with st15, st22, st94, St119 and st120. But, interpretation of this result is complicated. Revertant null alleles of st15, st22, or st94 also behave in such trans tests as the wild-type chromosome. Further recent experiments (Riddle & Brown, personal communication: Waterston, unpublished results) suggest that sup-3 suppression does not result from sup-3 loss of function mutations. If so, eDf1 does not completely inactivate sup-3 as was originally proposed (Riddle & Brenner, 1978). Thus the relationship between sup-3 and the dominant mutations described here remains an enigma.

DOMINANT (e) What is the product

MUSCLE

of the gene(s)

MUTATIONS

IN C. elegant

495

altered by st15, st22, st94, St119 and st120?

The analysis of the muscle structure of the mutant animals demonstrates that these mutations alter muscle structure, especially thin filament placement within the muscle cell. Morphological studies are insufficient to determine the altered gene product, but in studies with uric-59 and uric-15 the structural abnormalities were predictive of, and correlate well with, the known molecular defect. A straight-forward interpretation of the morphological studies taken together with the genetic studies is that the mutations affect some structural component of the t,hin filament itself or an associated component which determines thin filament. placement. The coincidence of the map position of the five dominant alleles with that of the cluster of three actin genes (Files et al., 1983) led us to the molecular analysis of the actin genes of the mutants paper (Landel et al., 1984).

and their

revertants

reported

in the following

We thank Santiago Plurad for the electron micrographs; Ross Francis for the fluorescent micrographs of the wild-type, mutants and revertants; and Kay Webb for secretarial assistance. This work was supported by a grant from the Public Health Service (GM23883).

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Edited by S. Brenner

T. R. LANE S. & Waterston,

R. H. (1983).