Copyright 0 1995 by the Genetics Society of America

Isolation of Dominant XO-Feminizing Mutations in Caenorhabditis elegans: New Regulatory tra Alleles and an X Chromosome Duplication With Implications for Primary Sex Determination

Jonathan Hodgkin and Donna G. Albertson

Medical Research Council Laboratoly of Molecular Biology, Cambridge GB2 24H, England Manuscript received April 12, 1995 Accepted for publication July 7, 1995

ABSTRACT A strain of Caenmhabditis elegans was constructed that permits selection of dominant or sex-linked mutations that transformX0 animals (normally male) into fertile females,using a feminizing mutation, tra-2(e204m, which by itself doesnot sexually transformX0 males. Twenty-three mutations were isolated after chemical mutagenesis and found tofall into both expected classes (four dominanttra-l mutations andeight recessive xol-1 mutations)and novelclasses. The novel mutations include 10 secondsite mutations of tra-2, which are called eg mutations, for enhanced gain-offunction. The tra-2(d eg) alleles lead to complete dominant transformation of X0 animals from fertile male into fertile female. Also isolated was a duplication of the left endof the Xchromosome,eDp26, which has dominant X0 lethal and feminizing properties, unlikeall previously isolated duplications of the Xchromosome. The properties of eDp26indicate thatit carries copies of oneor more numerator elements, which act as partof the primary sexdetermination signal, the XA ratio. The eDp26 duplication is attached tothe left tip of the X chromosome in inverted orientation and consequently can be used to generate unstable attached-X chromosomes.

EX determination in the nematode Caenorhabditis (2X3A and 2X4A) develop into males, while tetra- S elegans has been the subject of extensive genetic, ploids with three Xchromosomes (3X4A) develop into molecular and developmental investigations (for re- hermaphrodites (-L and HERMAN1979). The nature views see HODGKIN1988, 1990; VILLENEWEand MEYER of what is measured in assessing XA ratio has hitherto 1990; KWABARA and KIMBLE 1992; PARKHUMT and been theleast understood partof the sex determination MENEELY1994). Itoffers an opportunity to trace a major process in C. elegans. developmental process in its entirety, from its initiating This enigmatic primary signal acts on a set of at least point, which is the primary sex determination signal, four regulatory genes (xol-1, sdc-1, sdc-2, sdc-?), which through to specific differentiative and morphogenetic have a dual role (VILLENEUVEand MEXER 1987; MILLER events involved in sexual maturation and reproduction. et al. 1988; NUSBAUMand MEYER1989; KLEIN and MEXER Much progress to this end has been achieved. This pa- 1993). On the onehand they regulate subordinate per describes the selection and analysis oftwo new kinds genes that are involved in somatic sex determination of feminizing mutation, which shed light on primary sex and germ line sex determination, and on theother determination and on regulatory interactions among hand they regulate subordinate genes required for X downstream control genes. chromosome dosage compensation. These two sets of The two natural sexes of C. elegans are the male and subordinate genes appear to function independently: the self-fertilizinghermaphrodite, which is essentially a mutations affecting sex determination do not usually female that has a limited capacity for spermatogenesis affect dosage compensation and vice versa.Figure 1 pro- inaddition to oogenesis. Primary sex determination vides an overview of the major genes involved in these depends on X chromosome dosage: diploid animals processes and the predicted interactions between them. with two Xchromosomes (XX) develop into hermaph- C. elegans, like most other organisms with dimorphic rodites, while diploids with one X chromosome (XO) sex chromosomes, actively compensates for the differ- develop into males. Experiments with triploid and tetra- ence in X chromosome dosage between the two sexes ploid strains have indicated that X dosage is assessed (MEYERand CASSON1986; MENEELYand WOOD 1987; by the ratio of X chromosome to autosomes, rather HSUand MEYER1993). A set of at least five genes, called than by the absolute number of Xchromosomes. Thus, the dosage compensation dumpy genes, is specifically re- triploids and tetraploids with X chromosomes two quired for X chromosome dosage compensation (dpy- 21, dpy-26, dpy-27, dpy-28 and dpy-30). These all appear Corresponding author:Jonathan Hodgkin,MRC Laboratory of Molec- ular Biology, Hills Road, Cambridge CB2 2QH, England. to be required for downregulating the Xchromosome E-mail: [email protected] activity in XXanimals, so as to equalize expression levels

Genetics 141: 527-542 (October, 1995) 528 J. Hodgkin and D. G. Albertson

dm-21,dpy-26 - - - genes on X dpy-27,dpy-28, 1 chromosome dpy-30 u X CHROMOSOME DOSAGE Sdc-1 COMPENSATION A / X: RATIO 1 x01-1 "-I S~C-2 S~C-3 U /;: PRIMARY SIGNAL - COORDINATE CONTROL OF fern-1 SEX DETERMINATION AND\ her-I + 21; "I fern-2 + tra-1 DOSAGE COMPENSATION fern-3 , \ I I I INTERMEDIATE REGULATORS: COORDINATION BY CELL-CELL TERMINAL INTERACTION SOMATIC REGULATOR

PREDICTED ACTIVITY STATES :

m:HIGH LOW HIGH LOW HIGH LOW HIGH xo: LOW HIGH LOW HIGH LOW HIGH LOW

FIGURE1.-Simplified overview of genes controllingsexual phenotype and dosage compensation in C. ekgans, and theproposed interactions between them in the two sexes. Pointed arrows indicatepredicted positive regulation, barred arrows indicate predicted negative regulation. The two lines at the bottom show the proposed activity states for the various genes in the two sexes. Somatic sexual development is dictated by the final gene in the pathway, tru-I, that directs female development when active at a high level, and male development when active at a low or zero level. The fern genes appear to have a dual function: in the soma they promote male development indirectly, by downregulating tra-1 in X0animals, and in the germ-line they direct spermatogenesis, being essential for this process. Additional genes and interactions are involved in germ-line sex determination in both sexes and are not shown here (see SCHEDL1991 for review). Various postulated minor interactions and feedbacks have also been omitted for the sake of simplicity. For more extensive description and discussion, see the reviews cited in the text. of sex-linked genes between XX and X0 karyotypes. The her-1 gene, expressed only in X0animals, encodes Eliminating their activity has no effect on X0 animals an apparent secreted protein (TRENTet al. 1991; PERRY but is usually lethal to XX animals, presumably because et al. 1993), which probably acts as a repressive ligand of X chromosome overexpression (HODGKIN1983, for a predicted transmembrane receptor protein,TRA- 1987b; PLENEFISCHet al. 1989; Hsu and MEYER 1994). 2A, encoded by the tru-2 gene (KLJWABARAet al. 1992). Conversely, inappropriately expressing these genes in Consequently, TRA-2A is inactive in X0animals. In XX X0animals (via a xol-1 mutation) leads to lethality from animals, the absence of HER-1 means that TRA-2A is presumedunderexpression of the X chromosome active and able to inhibit or sequester one or more of (MILLERet al. 1988).Alterations in the dosage compen- the FEM proteins, encoded by fern-1, fern-2 and fern-3. sation process do notnormally affect sexual phenotype, These proteins act as direct or indirect inhibitors of only viability. TRA-1A the net result is that in XXanimals, TRA-1A is The other set of genes, those required for sex deter- not inhibited by FEMs and can direct female develop- mination but not for dosage compensation, have been ment. In X0animals, FEMs are not inhibited by TRA- extensively studied; the seven genes in Figure 1 have 2A, so they arefree to downregulate TRA-1 activity, now all been cloned and sequenced. The last gene in permitting male development. the pathway, tra-1, encodes TRA-lA, a DNA-binding The continuing investigations of this system raise new protein that directs female development in all somatic questions and provide new tools. The present work was tissues (HODGKIN1987a; ZARKOWERand HODGKIN1992; prompted in part by the molecular characterization of D. ZARKOWEKand J. HODGKIN, unpublisheddata). In tra-2 (KUWABARAet al. 1992). IfTRA-2A is a receptor the soma, all upstream regulatory genes act only to for HER-1, then one might expect to find mutations of achieve the correctactivity state of TRA-1A active(femi- tra-2 encoding a protein thatwould fail to interact with nizing) in XX animals and inactive (masculinizing) in HER-1 and consequently remain constitutively activein X0 males. Events and gene interactions in the germ both XX and X0 animals. Such mutations should be line are more complicated and will not be discussed in dominant and transform X0 animals from males into detail here (for review, see SCHEDL 1991). hermaphrodites. Molecular investigations of the genes upstream of tra- Two classes of dominant regulatory mutations of tra- 1 have revealed an apparent signal-transduction system 2 have been identified in previous work (DONIACH1986; that ultimately controls the activity of this gene. The HODGKIN1986; SCHEDLet al. 1989), but neitherof these current model suggested by these studies is as follows. has the properties expected for mutants insensitive to Nematode Feminizing Mutations 529 inhibition by HER-1. Both types have a dominant femi- et ul. 1979) with minor modifications as follows. First, part of nizing effect, primarily on thegerm line of XXanimals. this work led to the isolation of dominant enhancing muta- tions of tru-2, isolated on chromosomes that already carried One class, known as the mxalleles, leads to feminization a gfmutation of tru-2, e2046. For clarity, these additional muta- of the XXgerm line but has an opposite, weakly mascu- tions are given the descriptive suffix, or abbreviation, eg, stand- linizing effect on the XX soma, and no effect on X0 ing for enhanced guin-offunction. The resulting doubly mutant animals (hence mx, for mixed character). The other class alleles are consequently referred to as tru-2(gf; eg) alleles. (the gf; or gain-offunction alleles) appears to be hyper- Second, some of the experiments generatedanimals appar- ently carrying attached Xchromosomes, that is, two Xchromo- morphic for tru-2 activity (DONIACH1986). XX animals somes joined at theirleft ends. This attachment is symbolized carrylng such mutations are female; X0 animals are by a caret symbol ("). Thus, an XX hermaphrodite animal usually fertile males but may exhibit some signs of femi- carrying attached X chromo_somes, both marked with lon-2, nization such as limited oogenesis in the germ line and has a genotype written LonSlon-2, as distinct from a normal yolk production by the intestine.Recent molecular data diploid, lon-2/lon-2. Cytogenetics: Fluorescent in situ hybridization (FISH) was support the interpretation of the gf alleles as hyper- carried out as previously described (&.BERTSON 1993).Probes morphs (GOODWINet ul. 1994). All mutations in this derivedfromYACclonesY47C4,Y51E2,Y8Dl,Y42C9,Y29FlO, class affect one orboth copies of a 28-bp repeat in the Y46A5, Y5E3and Y23A3 all gave duplicated hybridization sig- 3' untranslated region (UTR) of tru-2 transcripts and nals on eDp26 and eDp27. Probes from eight YAC clones fur- probably result in increased synthesis ofTRA-2 proteins. ther to the right (Y48C6,Y70E6, Y40H5, etc.) gave only a single signal. This suggests that the gf mutations affect a separate Strains: The following mutations were used in this work. regulatory control, which acts normally to reduce syn- LGII: dpy-1 O(e128), tru-2(~1095),tru-2(e2046gf) , tru-2(q276), thesis of these proteins. This control may be necessary unc-4(e120), mnC1. only to ensure that tru-2 activity remains low enough to LGIII: dpy-27(rh18), dpy-28(yl),tru-l(e1099), tru-l(e1575gf). respond properly to specific masculinizing signals in LGIV: unc-l7(e245), unc-5(e53),fem-1 (hcl7ts), mm"2(el125), unc-24(~138),fem-3(q20gf), him-8(e1489), dpy-20(e1282), dpr- the XXgerm line and in the X0 soma and germ line. 26(n199), tra-j(el767). One possible explanation for the failure, in previous LGV her-l(n695gf), him-5(e1490), dpy-21(e428). screens, to obtain a strong feminizing dominant muta- LGX: egl-I 7(el313), unc-l(e719), unc-l(e1598dm), dpy-3(e27), tion of tru-2 was that any mutation affecting the pre- unc-2(e55), unc-20(el12), lon-2(e678), d@-8(e130),d@-7(e88), unc- dicted binding site for the HER-1 ligand might also 1O(e102), xol-1 (y9), dpy-6(e14),s&-2(y15), unc-7(e5), mnDp57, mnDp65, mnDp66, mnBp67, mnDp68, mnDp70, mnDp73. affect the folding or stability of the TRA-2A protein. Except where noted, these alleles and rearrangements can Therefore, elevating levels of tru-2 products by means be found listed in HODCKINet al. (1988).The tru-2 allele q276 of a tra-2(@ mutation might be necessarya prerequisite is an unusual allele isolated by T. SCHEDI.(unpublished data), for obtaining aconstitutive mutation of this type. Also, which leads to fertile male development in homozygous tru- tru-2 is known to be haploinsufficient, so strong femini- 2(q276) XX animals, in contrast to most If (loss-of-function) zation by a single dose of a constitutive mutation might tra-2 alleles, which lead to slightly abnormal, infertile male development. The Xchromosomeduplications utilized in this also depend on elevated levels of tru-2 products. paper are described by HERMANand KARI (1989). For these reasons, we selected for feminizing muta- For examiningpatroclinous X0 individuals, a standard tions in a strain homozygous for a gf allele of tru-2, strain CB40 17 = fem-1 (hcl7ts) him-8(el489); unc-1(el 598dm) e2046, which carries a deletion of one 28-bp repeat in was used (ZARKOWER and HODCKIN1992). Animals of this the 3' UTR of tru-2 transcripts. Using a tru-2(@ back- strain were raised during larval growth at 25", to eliminate self-progeny; they were then crossed, and progeny examined ground has a separate advantage in such a selection, at 20". The fem-l(hcl7)mutation is both temperature sensitive which is that strains homozygous for tra-2(e2046) are and fully recessive (NELSONet ul. 1978), so it has no effect necessarily male/female, obligate crossing strains. Con- on the phenotype of progeny from such crosses. The him-8 sequently any feminizing mutations recovered must be mutation leads to extensive production of nullo-X oocytes, dominant or sex-linked. Previous screens and selections and the unc-l(e1598) mutation is dominant, so the non-Unc progeny from such crosses will be those that have received for feminizing mutations have been biased toward the Xchromosomes only from the male parent (patroclinous recovery of recessive mutations, such as those in her-1 progeny). and the three fern genes (HODGKIN1980, 1986). To Selection of feminizing mutations: The strain CB4919 = select for X0 feminization, we combined tru-2(e2046) tru-2(e2046gf); dpy-28(ylts) was constructed using standard with a temperature-sensitive mutation in a dosage com- methods. Partly synchronized cultures of this strain,con- pensation gene, dpy28(yl), which leads to the death of taining numerous L4 individuals, were grown at 15" and muta- genized for 4 hr with 0.05 M EMS, using standard conditions. XX individuals at restrictive temperature but has no After mutagenesis, cultures were washed and plated on 10- effect on X0 animals (PLENEFISCHet ul. 1989). 20 large plates and thengrown for approximately one genera- tion at 15". Plates were examined at this time to estimate the MATERIALS AND METHODS total F1 progeny for each mutagenesis experiment. Theplates were then shifted to room temperature (20-22'). Under nor- Culture conditions: Standard conditions for growth, main- mal growth conditions, the strain rapidly becomes asynchro- tenance, mutagenesis and genetic crosses of nematodes were nous. The presence of very large numbers of males leads to used (SULSTONand HODCKIN 1988). extensive burrowing inthe cultureplates, especially after shift- Nomenclature: Standard nomenclature is used (HORVITZ up, when many thousand frustrated adult males are scouring 530 J. Hodgkin and D. G. Albertson the depthsof each plate in search of the few remaining mori- were constructed in each case and segregated Tra-2 XXmales bund females. To extract fertile females from such plates, that were competent to mate, as expected for tra-Z(elO95);xol- each 9-cm plate was soaked in 10 ml M9 buffer for several 1 XX males. These males were crossed with hermaphrodites hours, duringwhich most of the motile worms emerged from ofgenotype tra-2(el095)/rnnCl; unc-5; xol-l(y9)and fertile non- the agar and became trapped in the overlying liquid. The Unc XX male cross progeny were produced, demonstrating resulting worm suspension was then removed; worms were that each of the eight new x01 mutations fails to complement permitted to settle and were pipetted in a small volume to a the reference allele y9 with respect to tra-2(e1095) enhance- fresh culture plate, which was then incubated for additional ment. days at room temperature. This procedure facilitated recovery The other two linesdid not carry xol-1 mutations. One of viable female individuals. carried a duplication of the left end of the X chromosome, Characterization of mutants: The following strategies, or eDp26, described in the RESULTS section. The other proved to minor variants thereof, were adopted forpreliminary charac- be extremely difficult to propagate and cross, segregating only terization of the female survivors obtained by this selection. rare self-fertile hemaphrodites. Analysis of this mutation has Individual lines were established by picking sib-mated females not yet been pursued. and propagatedat 20" to confirm viability. Only one line Reversion of tr~-2(e2046e2531):The double mutant tm- was established from each original selection plate to ensure 2(e2046~25?1)exerts dominant feminization of both XX ani- independence. Females from eachline were then crossed mals and X0animals. The two aspects of the phenotype (femi- with dpy-28 males for at least two generations, to separate (if nization of XXand feminization of XO) were both reverted, possible) tm-2(e2046) from the new mutation. After this step, using a strain CB4940, genotype tra-2(e2046e2531) unr-4/ 14 out of 24 resulting lines were still male/female, indicating mnC1. This strain is barely self-fertile, because almost all ani- linkage to tra-2, or else the induction of an unlinked mutation mals of' this genotype are female, but some individuals pro- that feminizes both XXand X0 animals, such as a strong tm- duce a few self-progeny, so the strain can be propagated with l(g;r3allele. To distinguish between these possibilities, females patience.It segregates Unc animals, homozygous for trrc- were crossed with dfi-10 unc-4/++ males, and single female 2(e2046e2531), which are invariably female. When crossed progeny crossed again with dpy-10 unc-4/++ males. As ex- with wild-typemales, these females produce nomale progeny, pected, half of these crosses produced Dpy Unc progeny. only females and occasional weakly self-fertile hermaphro- Males from these crosses were counted: 10 out of 14 produced dites. The reversion was carried out as follows: an asynchro- wild-type and DpyUnc males in equal numbers, rather than nous culture of CB4940 was mutagenized with EMS and dis- the 3:l ratio expected for an unlinked dominant feminizing tributed over 12 large plates. Forty-eight P,, L4 Unc-4 females mutation. This indicated the generation of a dominant muta- were picked from these plates and crossed withwild-type tion on LGII, which causes the death orsexual transformation males in sets of four. A total of about 9400 F, progeny was of X0 animals. Further crosses established, in all 10 cases, the produced. Three ofthe12 crosses yielded one or morefertile presence of a dominant mutation tightly linked to dpy-lo(+), males, which were crossed with marked hermaphrodites. The which transforms X0 animals into phenotypic females. For resulting hermaphrodite progeny were selfed, and in each each of the 10 cases, rare Unc non-Dpy recombinants were case some of' the hermaphrodites segregated Unc-4 Tra-2 XX also picked from the test-crosses and crossed withfem-?(yZOgf) males. For two of these cases, the mutagenized chromosome dpy-20 males. The q20 mutation is a masculinizing mutation marked with unr-4 exhibited neither dominant feminization offem-3 (BARTONet al. 1987) that suppresses the feminization of X0 nor dominantfeminization of XXand instead exhibited of XXanimals caused by tm-2(e2046), and therefore permitted recessive masculinization due to new loss-of-function alleles the construction, in all 10 instances, of homozygous animals of tm-2 (designated e2046~2531e2543and r2046e2531~2545). of genotype tra-2(gf;eg)unc-4; fem-?(gf) dpy-20. These animals This indicates that both dominant mutations had been simul- grow as viable fertile Unc Dpy hermaphrodites at all culture taneously reverted in rzs. In the third case, a temperature- temperatures. The viability of these lines demonstrates the sensitive loss-of-function allele of tm-2 had beeninduced absence of any recessivedeleterious properties associated with (e2544).At 25" XXanimals of genotype tm-2(~2046e2531r2544) each new mutation. are Tra-2 pseudomales, at 20" they are variably masculinized The other four male/female lines seemed likely to carry hermaphrodites, and at 15" they are females, consistent with new tm-1 alleles, especially since test-crosses generated X0 expectation. females with truncated tail-spikes, characteristic of strong tm- XX hermaphrodites of normal self-fertility were also ob- 1(& alleles. Further crosses established very tight linkage to tained in the reversion: one of the Unc-4 female X wild-type the tra-l locus for all four by establishing tru-l(gf)/tm-l(Zf)fe- male crosses yielded such a hermaphrodite, which when male/male lines in each case (as in HODCKIN 1987).These selfed segregated Tra-2 Unc-4 pseudomale progeny. Again, segregated only female and male individuals and no hermaph- this was found to be a new tm-Z(Zf) allele, e2046e25?1e2546. rodites. These four mutations were therefore assumed to be Also, the 12 original P,, plates were examined for the appear- tra-l(gf) alleles. ance of hermaphrodites of increased fertility. Three plates For 10 out oftheoriginal 24 lines selected, the crosses with yielded such animals: two segregated Trd-2 Unc-4 pseu- dpy-28 yielded animals that were self-fertile X0 hermaphro- domales (new alleles e2046e2531c2557 and e2046e2531e2558) dites, rather than males or females. These hermaphrodites and one carried an extragenic modifier, as yet incompletely produced self-progeny broods of viable X0 animals, ranging characterized, that leads to increased spermatogenesisin in phenotype from male through intersex to hermaphrodite. e2046 XX animals. Complementation tests were carried out Eight of 10 proved to carry xol-1 alleles. This was established for some of these new tm-2 alleles using the reference allele by the following crosses: rare fertile male X0 animals were of tm-2, ~1095.These tests confirmed that ~2543,e2544 and crossed with unc-10 dpy-6 hermaphrodites, and the resulting e2558 are all loss-of-function alleles of trrc-2. hermaphrodite progeny were crossed with wild-type males. In Construction of eDp26 sdc-2: XX and X0 animals of geno- all eight cases, this cross generated no wild-type males but type eDp26 5d-2 were constructed as follows. Hermaphrodites >lo0 DpyUnc males, plus occasional Dpy or Unc recombi- of genotype un~l(e719)dpy-7/sdc-2 were selfed and Unc non- nant males. This demonstrated thepresence of a xolmutation Dpy recombinants were picked. These were selfed to confirm tightly linked to dpy-6, which is the same location as xol-1. In genotype (unc-l .sdc-2/unc-l dpy-7), then crossed with X0 addition, strains of genotype tm-2(el095)/d~-10unc-4; xol-1 males of genotype dpy-26; eDp26. Single hermaphrodite prog- Nem atode Feminizing Mutations Feminizing Nematode 531 eny were pickedand crossed with wild-type males. Five out of properties of its constituent mutations (HODGKIN1986; ten hermaphrodites gave progeny male ratioswith Unc males PLENEFISCHet al. 1989).At 15" the temperature-sensitive greatly in excess of wild-type males (total score: 137 Unc, 16 wild), consistentwith a parental genotype dpy-26/+; unc-1 sdc- mutation yl has only a slightly deleterious effect and 2/ eDp26. Fifteen of these wild-type male progeny (putative the strain grows as well as a population of 50% female, genotype eDp26 sdc-2 XO) were crossed singly with unc-l dpy- 50% maleanimals, as a resultof the XX-feminizing 7 hermaphrodites. Hermaphrodite progeny from each cross properties of tru-Z(e2046). Whenshifted to 20" or were selfed for 24 hr and then crossed with wild-type males higher, the strain becomesinviable, because of the dele- in excess to ensure complete outcrossing. Of seven hermaph- rodites that produced both abundant self-progeny and abun- terious effects of yl on all XX animals. The XX animals dant cross-progeny, allsegregated self-progeny that included all die or become moribund, but the X0 animals re- -25% Sdc dead eggs or larvae, and cross-progeny males con- main normal males in phenotype. A shift in tempera- sisting of approximately equal numbers of wild-type and Un- ture from low to high therefore constitutes a powerful cDpy males, some Unc males, and zero Dpy males (for exam- ple, one brood had 35 wild, 29 UncDpy, 8 Unc and 0 Dpy). selection for mutations that transformX0 animals into Cross-progeny included occasional recognizable Xol larvae, females or hermaphrodites,because only these animals presumably eDp26dpy-7 X0 recombinants. The conclusion will be able to produce any progeny. A selection using from these crosses is that sdc-2 is fully epistatic to eDp26, that d@-28(yl) alonehas been carried out previously by is, eDp26 sdc-2 X0 is a viable fertile male, and eDp26 sdc-2 XX PLENEFISCHet ul. (1989), which yielded only recessive is an inviable Sdc animal (sex not determined). her-1 and xoZ-1 mutations. No mutations leading to XX Construction of eDp26 xoG2: A chromosome of genotype eDp26 xol-l(y9) was constructed as follows: tru-2(e1095); xol-1 viability were obtained. The present selection was bi- XXmales were crossed withunc-l (e719) dpy-3(e27)hermaphro- asedagainst the isolation of recessive mutations, be- dites.Unc non-Dpy recombinant progeny hermaphrodites cause the strain is propagated by outcrossing, so it was were crossed againwith tru-2; xol-1 XXmales, and the resulting expected that only dominant mutations (such as tru- non-Unc males crossed with eDp26 dpy-7(e88). Wild-type her- maphrodite progeny from thiscross were genotyped by prog- I(& alleles) or hemizygous recessive mutations (such eny testing to identify those of genotype tru-2/+; eDp26 dpy- as xol-1 alleles)would be recovered.Both of these 7/unc-l xol-1. Wild-type progenyfrom these animals were classes were indeed obtained, but two additional kinds picked to separate plates and again genotyped by progeny of feminizing mutation were also isolated. testing. Six of 88 were tru-2/+; eDp26 xoL-l/eDp26 dpy-7, and A total of eight separate mutagenesis experiments 3/88 were tru-2/+; eDp26xoL-l/unc-l xol-I.Tra-2 XXmaleprog- eny from these broods were crossed with unc-1 dpy-7, to yield were carried out, involving 94 large plates and an esti- wild-type hermaphrodite progeny of presumed genotype tru- mated total F1 of 2200,000 mutagenized genomes. Ac- 2/+;eDp26 xol-l/unc-l dpy-7. Sixof these hermaphrodites curate assessment of the total is difficult because the were checked by selfing for 24 hr, then crossing with wild- large number of males in the strain lead to extensive type males. All produced self-progeny including wild-type and UncDpy hermaphrodites, and cross progeny, including nu- burrowing in the agar plate,so this estimate should be merous UncDpy males, but no Unc, Dpy, or wild-type males. regarded as a minimum. In all, 24 independent fertile This demonstrates that both eDp26 and xol-1 were present on lines were selected, all of which grew more or less well the unmarked chromosome. Individual hermaphrodite self- at 20°, as male/female strains. As described in MATERI- progeny were selfed to identify homozygous eDp26 x061 indi- ALS AND METHODS, these lines were subjected toa series viduals. One of these was crossed with dpy-26 males to obtain (from the F3 progeny of this cross) an X0 hermaphrodite of test-crosses, which defined the natureof the feminiz- line of genotype dpy-26; eDp26 xol-1. ing mutation inall but onecase. Ten of the lines carried Construction of a freederivative of eDp26: In X0 individu- enhancing mutations of tru-2, the tru-2(g$ eg) alleles. als of genotype dpy-26; eDp26, wild-type X chromosomes are Four lines carried strong dominant mutations of tru-1. generated at a significant frequency, implyingloss of the Eight lines carried mutations of One line carried attached Xchromosome and regeneration ofan intact Xchro- xol-1. mosome. Crosses were carried out to recover the detached a duplication of the X chromosome, which has domi- duplication as a free derivative, by crossing hermaphrodites nant XO-lethal and feminizing effects. These classes are of genotype him-8; dpy-3 Lon-2 unc-7 with dpy-26 eDp26 males. discussed in order. The hermaphrodite parent is expected to produce -8% tra-2(gf, eg) mutations: The first mutation analysed diplo-X oocytes, as a result of nondisjunction caused by the him-8 mutation (HODGKINet ul. 1979). If such an oocyte is was given the allele number e2531. Preliminary crosses fertilized by a nullo-X sperm carrying a free duplication de- established that theX0 sexual transformation by e2531 rived from eDp26, then a non-Dpy Lon Unc hermaphrodite cosegregated with the XX feminizationcaused by will result. In one set of crosses, progeny included 267 wild- e2046gf. This suggested that an enhancing mutation in type hermaphrodites and four non-Dpy Lon Unchermaphro- dites. Two of the latter were further propagated, and both the tru-2 gene had been induced, which (either alone genetic and cytogenetic data showed that they carried free or in combination with e2046) is able to transform an duplications of the left end of the X chromosome, covering X0 animal from male into female. The cosegregation markers from unc-1 to unc-2. Both appeared similar in proper- is consistent with this explanation but does not prove ties, so only one of these was selected for detailed analysis it, especially since both mutations are strongly domi- and designated eDp27. nant. A reversion experiment was carried out to test RESULTS this point. If both mutations lie in the tru-2 gene, then The strainCB4919, genotype tru-2(e2046);d@-28(yI), introducing a tru-Z(lf)mutation in cis should simultane- was constructedand behaves as expectedfrom the ously revert both dominant phenotypes, XX feminiza- 532 J. Hodgkin and D. G. Albertson tion and X0 feminization. As described in MATERIALS P AND METHODS, cis-revertants of both phenotypes were readily obtained, and in both cases these exhibited all I the properties of standard tru-2(w alleles, with a Tra-2 masculinized phenotype in XX homozygotes and no dominant feminizing properties in either XXor XO. In summary, reversion of the XX dominant feminization (due to e2046) also reverts the X0dominant feminiza- tion (due to e2531) and vice versa, indicating that the e2531 mutation lies in tru-2. Molecular characterization of the tru-2(eg) mutations has confirmed that they are indeed alterations in the tru-2 sequence (P. E. KWA- BARA, unpublished results). The properties of tru-2(e2046e2531)were briefly inves- tigated to compare the X0 feminization by this geno- typewith that caused by other mutations in the sex - determination pathway, such as loss-of-function muta- tions in her-1, fern-1, fern-2, or fern-3, or gain-of-function FIGUREP.”Sexual transformation caused by dominant mutations of tru-1. Effects on XXanimals appear to be feminizing mutations. Photographs show the posterior re- similar to those of tru-2(e2046) alone: XX animals car- gions of live worms, viewed by Nomarski optics. In all photo- rying either one or two doses of tru-2(e2046e2531) de- graphs anterior is to the left. Scale, 20 pm. Animals are viewed velop as fertile females. Suppression of tru-3(el767) was laterally unless otherwise indicated. (A) Wild-type XX her- also examined: the masculinizing effects of tru-3(el767) maphrodite. Arrowhead indicates vulva, white arrow indicates long spike tail. (B) A tru-2(eg, @/+ X0female, showing com- are largely suppressed by tru-2(e2046), and, in fact, tru- plete transformation. For full genotype, see text. (C) A tm- 2(e2046) was originally isolated because of this property I(@/+ X0female, showing almost complete transformation; (DONIACH1986; HODGKIN1986). The enhanced allele, truncated tail spike is indicated by smallwhite arrow. (D) tr~-2(e2046e2531),appeared to be equally effective in Wild-type X0 male. Black arrow indicates characteristic male suppressing tru-3, suggesting thatthe hypermorphic tail structure, with fullydeveloped fan, rays, spicules and other distinctive structures. properties of tru-2(e2046)have not been reduced. Inter- actions with the masculinizing mutation her-l(n695gf) (TRENT et ul. 1988) were not examined, because tru- wild-type males. Half of the Unc-7 progeny from these 2(e2046) alone is able to suppress her-I(n695) (DONIACH crosses were male, and half were completely female in 1986). somatic phenotype, indicating sexual transformation Sexual transformation of X0individuals from male (Figure 2B). In particular, the tail spike shows complete into female is implicit in the original isolation of the transformation, in contrast to the incompletefeminiza- tru-2(& eg) mutations, but it was desirable to examine tion caused by a single dose of a tru-l(& mutation, such this transformation in the absence of other sex deter- as e1575 (Figure 2C). In this respect, therefore, the tru- mination or dosage compensation mutations. X0indi- 2(& eg) alleles are the most potent feminizing muta- viduals carrying a single dose of tru-2(e2046e2531) were tions yet isolated. However, the fertility of these trans- constructed by first establishing a stable XX hermaph- formed X0 individuals was low: of 15 Unc-7 female rodite lineCB5055, of genotype tru-2(e2046e2531);fern- individuals mated with wild-type males, only eight pro- 3(q2Ogf) dB-20; unc-7. The fern-3(@ mutation sup duced eggs (range 1-9) and only one of these eggs presses XX feminization caused by tru-2(e2046), and hatched. Itis possible that this poor fertility results from the unc-7 marker is a sex-linked recessive mutation a dosage compensation effect, because the ancestral used as a standard marker foridentifying hemizygous females, of genotype tr~-2(e2046e2531)/+;dpy-28(yl), X0individuals (HODGKIN1980, 1986). When CB5055 were significantly more fertile, otherwise the selection hermaphrodites were crossed with wild-type males, all would not have worked. More direct and extensive evi- Unc non-Dpy progeny [necessarily of genotype tru- dence for such an effect has been obtained by P. E. 2(e2046e2531)/+; fern-3(q20) dpy-20 /+ + ; unc-7/0] KWABARA (unpublished results), in further work on were either female or (rarely) hermaphrodite in phe- the e2531 mutation. Also, two stable male/female notype, showing no sign of masculinization. strains have been constructed,using other dosage com- The phenotype of these animals, however, might pensation mutations d&26(nl99) and dpy-27(rh18). have been affected by the dominant fern-)(q20) muta- Each of these strains consists of tru-2(& eg)/+ ;dpy X0 tion. CB5055 hermaphrodites were therefore crossed females and dpy X0 males. These strains grow reason- twice with mor-2 males to remove the fern-3 mutation. ably well, indicating substantial fertility in the X0 fe- Some of the resulting Mor females, of genotype tru- males. 2(e2046e2531)/+; mor-2; unc-7/+, werecrossed with Interactions of tru-2(&eg) mutations with some of the Nematode Feminizing Mutations 533 other sex determination mutations were alsoexamined, + X0 tail phenotype exhibits signs of residual masculin- providing results consistent with the standardsex deter- ization, such as the absence of a tail spike (Figure 2C). mination pathway model. It was particularly important This phenotype contrasts with that seen in wild-type XX to test the interaction of tru-Z(g$ eg) with tru-l(& muta- hermaphrodites and in tru-Z(g$ eg)/+ X0 individuals, tions in view of the strongfeminization caused by these both of which haveperfect tail spikesand show no signs new alleles. The model predicts that the double mutant of male development (Figure 2, A and B). tru-Z(g$ eg); tru-l(& should nevertheless be completely The majorityof tru-l(& allelesanalyzed hitherto male in somatic phenotype. This is indeedthe case (HODGKIN1987) were isolated as mutations causing and was demonstrated by constructing a stable strain feminization of XX individuals or as suppressors of the consisting of tru-2(e2046e2531); tru-1(el 099)/+ XX fe- XX masculinization caused by tru-3. Many of these are males and tr~-2(e2046e2531);tru-1 (e1099) XX males. weak gfalleles, which cause incomplete feminization of These males are indistinguishable from tru-2(+); tru-1 X0 animals. The present selection was biased toward males, exhibiting a male soma and low male fertility. the isolation of strong gfalleles (class 1, in the classifica- This demonstrates that tru-l(e1099) is fully epistatic to tion used in HODGKIN1987 and DE BONO et ul. 1995), tru-2(e2046e2531). The presence of a dominanthomozy- because only these will cause efficient transformation gous feminizing mutation in the males of this strain of X0 animals to a fertile female phenotype. It was means that they should sire only female progeny when therefore hoped that some of these new alleles might crossedwith females or hermaphrodites from other carry novel changes in tru-1. The sequence alterations strains. This prediction was tested in two crosses: first, in 29 tru-l(gf) alleles have now been determined (DE to examine XX progeny, the males were crossed with BONOet ul. 1995), including these four. Almost all carry marked hermaphrodites (homozygous for unc-17). All single missense alterations in a small stretch of the cod- non-Unc progeny were female (33/33 tested). Second, ing region, corresponding to a 16-amino acid sequence to examine X0 progeny, recipient females of genotype close to the predicted amino-terminus of tru-1 proteins. fem-1 him-8; unc-l(dm) were used. Non-Unc progeny This is likely to be the site for an inhibitory protein- from such females are patroclinous X0 animals (see protein interaction, which may prevent tru-1 proteins MATERIALSAND METHODS), and in control crosses with from exerting afeminizing effect in X0individuals. All tru-l(e1099)XXmales, the non-Unc progeny were invari- four alleles isolated in this work have changes identical ably male (134/134). In contrast, in crosses with tru- to those seen inpreviously isolated class 1 gf alleles Z(e2046e2531); tru-1 (el099) XX males, the non-Unc (H123Y for e2535 and an identical P122S change in the progeny were invariably female or (rarely) hermaphro- other threealleles). The sequencechanges confirm the dite (238/238). interpretation of these alleles as tru-l(gf)alleles, but no In this last cross, tru-2(e2046e2531) is supplied in the novel tru-l(& changes were obtained in this selection, paternal gamete to oocytes produced by a homozygous suggesting that saturation has been reached for EMS- tru-2(+) mother, but the patroclinous tru-Z(g$ eg) allele induced changes of this class. nevertheless dictates fully female development. There- xoGl mutations: Ten of 24 lines failed to exhibit dom- fore, maternal contributionsfrom the tru-2(g$ eg) allele inant feminization of X0animals after outcrossing with are not necessary to achieve complete feminization. A dpy-28 males, suggesting that they carried xol-1 alleles single dose of this allele, supplied zygotically, is suffi- or had a morecomplex origin. As summarized in MATE- cient to transform X0 individuals from male to female WSAND METHODS, initial tests indicated that nine car- in phenotype. ried a x01 mutation and one had a weaker and more Nine other lines independently isolated from the se- variable phenotype. Eight of thenine x01 mutations lection all carried mutations that were found to be ex- mapped to the xol-1 locus on LGXand failed to comple- tremely similar to e2531 in map position and in pheno- ment xol-l(y9)with respect to enhancing the masculini- typic effect. These nine mutations (e2534, e2536, e2538, zation of tru-2(el095)XXanimals. Neither of these prop- e2540, e2551, e2552, e2555, e2581, e2582) were therefore erties alone provides unambiguous proof of allelism, assumed to be further tra-Z(eg) mutations. Preliminary but taken together they justifythe presumption that all characterization revealed no obvious differences be- eight are xol-1 alleles. All appeared superficially identi- tween any ofthese mutations and e2531, so further anal- cal in properties to the reference allele, xol-l(y9), so they ysis of these lines was deferred. are presumed to besimple loss-of-function mutations of Era-1 (gf) mutations: Four of the 24 lines carried femi- this gene. nizing mutations tightly linked to tru-1, which were as- An X0 lethal duplicationof the Xchromosome: One sumed to be tru-I(& alleles (e2535, e2537,e2554, mutation with a Xol phenotype behaved differently e2579). Each of these exhibited the dominantfeminiza- from the other nine. As explained below, this proved tion characteristic of strong tru-l(& alleles. Both XX to be a duplication of the left end of the Xchromosome, and X0animals are transformed by a single dose of tru- which exerts a dominant X0 lethal and feminizing ef- I(& to a fertile female phenotype. The two karyotypes fect. This duplication is attached in inverted orientation remain distinguishable, however, because the tru-l(&/ to the left tip of the Xchromosome andis called eDp26. 534 J. Hodgkin and D. G. Albertson

* + + + + ++"- eDp26

r I mnDp57 mnDp73 - 0- 5 10 m.u -mnDp65 -mnDp66 -mnDp70 FIGURE3.-Map of the X chromosome, showing the location of genes and rearrangements used in this study. The extent of the XO-lethal duplication, eDp26, was initially inferred from FISH analysis (see MATERIALS AND METHODS) and is indicated by candy-striping. Locations of probes inside the duplicated region are indicated by a plus sign, and probes outside by a minus sign. The @26 duplication is attached to the end of the X chromosome in inverted orientation. *, predicted location of a major numerator site or region.

For consistency, it is given this name throughout this eDp26 might be associated with an unusual mutation of paper, although the nature of the mutation was not xol-1, comparable to the xol-l(mn467) allele described immediately apparent. by WINDet ul. (1995), which also leads to X0lethality In all initial experiments, eDp26 behaved like a xol-1 without enhancing tru-Z(lf). The mappingdata de- mutation: a stable, homozygous viablemutation on the scribed below preclude this possibility. An alternative X chromosome with no recessive lethal effects on XX explanation is that eDp26may lead to some downregula- animals but complete lethality to X0 animals. Closer tion of xol-I(+) in xol-l/eDp26 XX heterozygotes. inspection of eDp26 XX homozygotes revealed minor The X0 lethality of eDp26 was mapped, indicating a differences from wild-type XX animals: these homozy- genetic location at the left end of the X chromosome. gotes are slightly shorter thanwild-type hermaphrodites A three-factor cross with the markers egl-17 and unc-1 and have somewhat smaller broods (mean brood size indicated that the X0 lethality maps left of egl-17, the for 12 individuals was 234 hermaphrodites, 0 males, 3 leftmost marker on the currentgenetic map. A location inviablezygotes, as compared to the wild-type mean clearly distinct from xol-1 was established, and wild-type of 329 hermaphrodites, 0.2 males, 0.8 inviable zygotes X chromosomes, with no Xol property, were readily (HODCKINet ul. 1979). In contrast, x061 mutations do recovered by recombination from eDp26/xol-l heterozy- not affect the phenotype or fertility of XX hermaphro- gotes. Therefore, the eDp26 chromosome is not defec- dites (MILLER et ul. 1988). The lethal effects of eDp26 tive in xol-1. wereshown to be associatedwith X0 karyotype, not The X0 lethality of eDp26 might also have been due maleness per se, by constructing animals of genotype tru- to loss or mutation of material at the left end of the X Z(q276); eDp26 XX and tru-l(el099); eDp26 XX. Both of chromosome. Therefore, attempts were made to rescue these genotypes result in a viable male phenotype, and the X0 lethality using duplications that were found to some of these males are able to sire progeny (fertility complement both egl-17 and unc-1 and are therefore of tru-2 XX or tru-1 XX males is always low). likely to include the entire left end of the X chromo- Further results indicating that eDp26 is not a simple some (Figure 3). None of the duplications mnDp65, loss-of-function allele of xol-1 were as follows: first, that mnDp66, mnDp67, mnDp68 or mnDp70were able to res- it fails to confer male mating competence on tru-Z(lf) cue the X0 lethality of eDp26. XX animals and second, anomalous mapping behavior. Two recombinational anomalies became apparent in A separate property of xol-1 mutations, distinct from the course of this mapping. First, eDp26 causes a domi- the X0 lethality, is that tru-2(lf; xol-l(lf) XX males are nant partial suppression of recombination on the left fertile mating males (MILLERet ul. 1988), unlike tru- end of the Xchromosome. For example, the dpy-3 lon- 2(@; xol-I(+) XXmales, which are slightly abnormal in 2 interval was reduced from 10.0 map units (control) male anatomy and do notmate (HODGKINand BRENNER to 3.9 map units in eDp26/+ heterozygotes (chi-squared 19'77).As noted above, all eight xol-1 mutations isolated probability < 0.01). The unc-2 dpy-7 interval was re- in this study enhance tra-2(8 in this way. In contrast, duced from 15.4 map units to 8.1 map units (chi- tra-Z(lf; eDp26 XX males do not exhibit mating behav- squared probability < 0.01). In contrast, recombination ior, and their tail anatomy resembles that of tru-2(@; elsewhere on the Xchromosome did not appear to be xoGl(+) XXmales. Confusingly, however,eDp26 fails to affected: the interval lon-2 unc-7 was measured as 30.1 complement xoZ-l(y9)with respect to this property. That map units in eDp26/+ heterozygotes, similar to control is, tru-2(1J); xol-l/eDp26 XX animals are fertile mating values. males. This result initiallyraised the possibility that The second anomaly was that no recombinants car- Nem atode Feminizing Mutations 535 Mutations Feminizing Nematode

(the probable location of unc-2) gavetwo signals on eDp26 chromosomes, while probes further to the right gave only one signal. Furthermore, the FISH patterns showed that eDp26 is attached in inverted orientation to the left tip of the X (see Figure 4). No evidence for rearrangement, deletion or duplication within eDp26 was obtained, so cytogenetically it appears to bea simple duplication of the left end of the Xchromosome, -6.5 map units or 2100 kb (estimated from correlations be- tween the genetic and physical maps of the genome) (COULSONet al. 1991; ALBERTSON 1993). The eDp26 duplication is unstable, giving rise to a wild-type X chromosome under certain conditions. However, the instability of eDp26 was not immediately apparent. The penetrance of the X0 lethal phenotype was tested by crossing dpy-IO unc-4; eDp26 homozygous hermaphrodites with wild-type males.In all, 3162 cross progeny (non-Dpy non-Unc) hermaphrodites were ob- tained, togetherwith large numbers of unhatched eggs and arrested L1 larvae, and no males. This indicates X0 lethality in excess of 99.9%.To explore X0lethality further, a strain of genotype him-8; eDp26 was con- structed. In this strain, males were occasionally seen, though vastly fewer than the 35-40% normally seen in an him-8 strain (HODGKINet al. 1979), and the males were often small or otherwise abnormal. Twelve com- plete self-progeny broods were counted forhim-8; eDp26 XX hermaphrodites, yielding a total of 1442 hermaph- rodites and five males. Each of these males was crossed with hermaphrodites carrying sex-linked markers [unc- l(e719) d@-3(e27)]. One male failed to mate, but the otherfour sired wild-type hermaphrodite progeny. These hermaphroditeswere then test-crossed with wild- FIGURE4.-FISH images: hybridization of labeled YACs to type males. In two of the four cases, equal numbers of bivalents from XX hermaphrodites carrying eDp26 (A-C) or dp27 (D). The site of hybridization of rhodamine dUTP- UncDpymales and wild-typemales (entirely normal labeled YACs (red false color) was visualized on DAPI-stained and fertile in phenotype) were produced, indicating chromosomes (blue false color). Probes shown areY51E2 (A), that a normal X chromosome had been generated in Y8D1 (B) and Y46A5 (C and D), with locations indicated by the parental male, presumably by detachment of the the first, third and sixth + symbols in Figure 3. A-C demon- duplicated tip of the X chromosome, leaving behind strate that eDp26is attachedin inverted orientation to the left end of the X chromosome, because a probe covering lin-32 an intact normal chromosome. In the other two cases, (Y46A5)shows two separated spots of hybridization, whereas UncDpy males but no wild-type males were generated a probe close to the left end (Y51E2) shows two almost coales- from the test cross, indicating that the parental male cent spots. D shows that eDp27 is a free double duplication had transmitted an unaltered eDp26 chromosome. with two spots of hybridization; a single spotof hybridization Thus, it appears that on rare occasions an eDp26 X0 to the normal X bivalents is also visible. genotype is compatible with normal male development. Further examples of this effect are described below. rying both eDp26 and markers left of unc-2 could be The reversion to a wild-type Xchromosome is signifi- recovered, although recombinants with markers further cant because it confirms that eDp26 is attached to an to the right, such as lon-2, were readily obtained. otherwise unaltered Xchromosome, and therefore that Subsequent experiments, described below,also re- its X0 lethal effects are dominant. The fact that rever- vealed that eDp26 is unstable, reverting to a wild-type X sion to wild type appears to happen at a significant chromosome under certain conditions. The instability, frequency in a him4 background but not a wild-type together with the abnormal mapping properties, sug- background can be ascribed to the effect of him4 on gested that eDp26 might be a duplication of the left meiosis in XX hermaphrodites. In the him background, end of the X chromosome. FISH analysis was therefore recombinational events on the X chromosome are ab- carried out, using a series of YAC probes covering the normal (HODGKINet al. 1979; BROVERMANand MENEELY left end of the X. Probes up to and including Y23A3 1994), so exchanges or otherinteractions between sister 536 D. andJ. Hodgkin G. Albertson

5). Feminization caused by a null allele of xol-1 is sig- nificantly stronger than that caused by eDp26, but still incomplete (Table 1). The male animals in a strain of genotype dtq-26; eDp26 X0 are of two types. Some are indistinguishable from dpy-26 X0males, and others aredistinctly smaller. Genetic evidence, obtained by crossing the males with marked recipient hermaphrodites, also indicates that

1 C there are two types of males. Some (most or all of the larger males) are revertants, which have lost eDp26 and carry only a normal X chromosome. Others (most or all of the smaller males) appear to carry and transmit anunaltered eDp26 chromosome.Thus, in a dm-26 FIGURE5.-Variable sexual phenotypes of X0 animals of background, eDp26 can lead to fertile male develop genotype dm-26; d4126. Presentation as in Figure 2. (A) Her- ment, as can xol-I. The reversion to wildtype in the maphrodite. Note spike tail (white arrow), vulva (arrowhead), progeny of X0 hermaphrodites is not surprising, given developing eggs and oocytes. (B) Intersex (ventral view). Note the previous evidence for increased reversion in a him- vulva (arrowhead), egg and oocytes, and abnormal male tail 8 XX background. (small black arrow). (C) Male. Note fully developed male tail (black arrow); compare with Figure 2D. Epistatic interactions of ~Dp26with other mutations affecting sex determination were investigated. As de- chromatids are likely to occur more often than in a scribed above, eDp26 does not interfere with the com- wild-type background. plete XX masculinization caused by tm-2(q276) or tm- In addition to X0 lethality, eDj126 has a strong femi- l(e1099). A double mutantwith a dominant masculizing nizing effect. This is implicit in the original isolation of allele of her-1 (TRENTet al. 1988) was also constructed. this mutation, in the form of dj1~7-28;eDp26 X0 her- Homozygous animals of genotype h~r-l(n695gf);eDp26 maphrodites. The viability of these animals indicates XXwere found to exhibit variable masculinization, like that 49-28 is able to rescue the X0 lethality, which can her-l(n695gf) XXalone. Finally, a double mutant of sdc- therefore be ascribed to incorrect dosage compensa- 2 and eDp26 was constructed (see MATERIALS AND METH- tion. In contrast to normal dpy-28 X0 individuals, which ODS), and both XXand X0 phenotypes were examined. are invariably male, most of the 49-28; eDp26 X0 ani- The eDp26 sdc-2 XX animals are not viable, having an mals are either self-fertile hermaphrodites or feminized Sdc phenotype, and the X0 animals are viable fertile intersexes, demonstrating that eDp26 is both lethal and males, like sdc-2 alone. Thus, both thefeminization and feminizing. Similar properties are seen in xol-I mutants the X0lethality of eDp26are fully suppressed by a muta- (MILLER et al. 1988). Double mutants of eDp26 with tion in sdc-2. This is consistent with action at a very other dosage-compensation mutants, dpy-26(nI 99) and early step in the sex determination pathway, at a level d&27(rhl8), were also constructed. In both cases the equivalent to, or preceding, xol-I. dosage compensation mutation restores viability to Interactions of eDp26with xol-I itself are notso easily eDp26 X0animals, and these surviving animals are often tested or interpreted, because both mutations have sim- transformed to a fertile hermaphroditephenotype. ilar phenotypic effects. A double mutant chromosome, One of these strains, dpy-26(nI99); eDp26 X0 was exam- eDp26 xol-I was constructed as described in MATERIALS ined in more detail and compared with a strain of geno- AND METHODS. This was found to be homozygous viable type dpy-26; xol-l(y9) XO. In both cases the phenotype in an XX strain, with a phenotype similar to eDj126, but is variable, ranging from self-fertile hermaphrodite X0 lethal, like either mutation alone. Feminization by through sterile or fertile intersex to fertile male (Figure the double mutant was examined by constructing a

TABLE 1 Sexual transformationof dpy-26 X0 animals

______~~~~ ~ ~ ~~~ ~ ~ Genotype Hermaphrodite Intersex Male % Hermaphrodites dB-26 0 0 >loo 0 lip?-26; xol-I 209 45 50 69 dj1y-26; PDp26 151 92 68 49 djq-26; t.11/126 Xol-1 378 3 4 98

Data for the first line refer to progeny of dB-26 XX hermaphrodites (HODGKIN 1983).Data for lines two, three and four were obtained by counting complete viable progeny produced by 12 separate X0 hermaphro- dites (picked as L4’s), for each genotype. Numerous unhatched eggs or dead young larvae were also produced, as is usual with dB-26 X0hermaphrodites. Nematode Feminizing Mutations 537 strain of genotype dpy-26; eDp26 xol-I. This strain shows A significantly stronger feminization than either mutation L R I alone (Table 1) I . Chromatids of normal unpaired at meiosis I The preceding data suggest that the properties of x eDp26 arise from duplication ofkey numerator ele- ments, which act as primary sex determinants for female " development. If this is so, then they should act zygoti- Chromatids of unpaired X with eDp26 at meiosis I cally. In the experiments above, the duplication was always present in the maternal germ line, so it is con- ceivable that bothlethality and feminization could have L X been exacerbated by maternal effects. Experiments in- troducing eDp26zygotically, viasperm, were carried out Alternative pairing and recombination between sister chromatids I using tra-l(e1099); e26XX males. Such males were I crossed with him-8; unc-l(dm) females, which gen- A fern-I /\ erate nullo-Xoocytes and can therefore beused to gen- /\ erate patroclinous X0individuals. One series of crosses / \ eDp27 \ \ yielded 1092 Unc hermaphrodites, five non-Unc her- \ maphrodites, two Unc males, eight non-Unc males, and R numerous unhatched eggs and arrested Xol larvae. A XAX tL R control cross with tra-1 XXmales yielded 216 Unc her- Products of sister chromatid exchange maphrodites and 134 non-Unc males. Consequently, the expected number of patroclinous X0 progeny in B the cross with tra-1;eDp26 XX males is 677 (1092 X 134/216). Only eight of these survived, as normal or eDp2 7 slightly abnormal males. The five non-Unc hermaphro- X dites were unexpected and might have been trans- x2 formed X0 progeny, but they produced self-progeny Meiotic pairing of eDp27 and a normal X chromosome inconsistent with such a genotype. Instead, they were I probably XXanimals carrying attached Xchromosomes, I which can be generated from eDp26 (see below). The I I eight males were testcrossed to establish whether they eDp26 1 still carried eDp26 or not. Three were not fertile, one was a revertant to a wild-type Xchromosome, and four transmitted apparently unaltered eDp26 chromosomes. Re-creation of eDp26 by recombination Therefore, eDp26 X0 males can occasionally survive,at FIGURE 6.-Diagrams illustrating the structures of eDp26 a frequency of -0.6%, if the duplication is supplied and eDp27, and proposed recombination eventsinvolving paternally. This survival rate is higher than seen if the these duplications. The left telomere of the X chromosome duplication is present maternally, implying that there is marked by an arrowhead, and the duplicated material in is a slight maternal enhancement of the X0 lethality. eDp26 isshown as a thicker line. (A) In an X0 individual However, the zygotic X0 lethality is still in excess of carrying eDp26, alternative sister chromatid pairings are possi- ble. Recombination between the sister chromatids can then 99%, demonstrating that zygotic presence of eDp26 is give rise to a double duplication, eDp27, and to an attached sufficient to kill almost all X0 progeny. Xchromosome as the reciprocal product. The same products A duplicated free derivativeof eDp26: The reversion could also be generatedby meiosis in an XXindividual homo- to wild type of eDp26 chromosomes, apparently by loss zygous for eDp26, as a result of end-toend pairing (not illus- of the duplicated part of the chromosome, raised the trated). (B) Recombination between eDp27 and a normal X possibility that this duplication could be recovered a chromosome can regenerate an attached duplication, pre- as sumably identical in structure to the original eDp26. free fragment,which wouldbe useful for furthermanip ulations and dosage studies. Crosses to recover such a free derivative were carried out as described in MATERI- duplication of the left end of the X chromosome was ALS AND METHODS.Several candidates were recovered confirmed. However, the FISH data demonstrated that with apparently similar properties, and one of these, eDp27 is not a simple duplication; instead, it carries two eDp27, was examined in more detail. Genetically it be- copies of the left end of the Xchromosome,in inverted haves as a free duplication covering all tested markers orientation (Figure 4D). A free duplication with this on the left end of the Xchromosome, up to and includ- structure could be generated from an eDp26 chromo- ing unc-2. It retains the Xol property of eDp26, consis- some by aberrant meiotic pairing, as shown in Figure tent with expectation. Animals carrying eDp27were ex- 6A, so in retrospect it is not surprising that a duplication amined by FISH analysis, and the presence of a free such as eDp27was recovered. 538 andJ. Hodgkin D. G. Albertson

This duplication, eDp27, carries two copies of the left animals from this strain were scored, giving average end of X, so it might be expected to have stronger progeny of 90 Lon hermaphrodites, nine Lon males, effects than eDp26. This was examined by constructing six 3-X hermaphrodites, and 84 inviable zygotes. This animals of genotype dpy-26; unc-2; eDp27 XO. These are brood composition is very different from that of normal viable hermaphrodites, segregating self progeny con- lon-2 hermaphrodites from strain CB678 (lon-2(e678)) sisting of many dead eggs, some viable hermaphrodites (average single brood for five animals: 277 Lon her- and intersexes, aswell as many Unc-2 males (which maphrodites, 25 inviable zygotes). The production of have presumably lost the duplication). Feminization is Lon males, and also of 3-X hermaphrodites, suggests comparable or stronger than with eDp26, because most thatthe putative attachedX chromosome structure dpy-26; unc-2; eDp27 X0 animals appear to be hermaph- breaks down at a significant frequency, generating nor- rodite in phenotype (8/11 in one brood). However, mal X chromosomes, and consequently to Lon male they are rather small and sickly. It is not possible to self-progeny. Two such maleswere crossed with unr- maintain a stable strain ofthis genotype, because l(e719) hermaphrodites, siring wild-type hermaphro- healthier and more fertile hermaphrodites arise sponta- dite progeny. Some of these were selfed and produced neously, and outgrow the parentalhermaphrodite a normal hermaphrodite broodconsisting of wild-type, strain. These hermaphrodites do not segregate Unc-2 Unc and Lon hermaphrodites, no males, and very few progeny and therefore probably result from recombina- inviable zygotes. The Lon segregants were examined, tion between eDp27 and the X chromosome,which will and none behaved like the parental CB5042 strain; in- generate an attachedsingle duplication, similar or iden- stead, they appeared identical toCB678 hermaphro- tical in structure to eDp26. Such an event is diagrammed dites. Thus, it appears that the attached Xstructurecan in Figure 6B. break (presumably at the attachment site at the left Generation of apparent attached X chromosomes: end), regenerating a normal X chromosome. The event giving rise to eDp27 should also give rise, as Meiotic chromosomes of hermaphrodites expected a reciprocal meiotic product, to an attached Xchromo- to carry attached X chromosomes were examined cyto- some (XX ), that is, two X chromosomes joined attheir genetically and by FISH analysis. The meiotic figures left ends (Figure 6A). Rare patroclinous hermaphro- showed substantial levelsof abnormal chromosomes dites were indeed generated in the cross described in and chromosome breakage. Definitive evidence of an an earlier section (cross between tra-1; eDp26 XX males attached Xstructurewas not obtained, butthis structure andfem-1 him+ unc-l(dm)fema!es), and it seemed likely seems to be the simplest explanation of the genetic that these might have been XX individuals. Attached phenomenology described above. chromosomes have not been generatedin any previous work on C. elegans, so it was of interest to see if convinc- DISCUSSION ing evidence of such a genotype could be obtained and to examine the behavior-of an attached Xstructure. The strategy outlined in the Introduction, for selec- To generate marked XX chromosomes, males of ge- tion of dominant feminizing mutations of C. elegans, notype dpy-26; eDp26 lon-2 X0 were crossed with fern-l evidently workseffectively, yielding both known and him-8; unc-l(dm) females. Rare (fewer than 1%) Lon novel kinds of mutation. Four separate components of non-Unc hermaphrodites were seen among the prog- this investigation will be discussed. eny of thiscross. One of these was selfed and then First, methodological points: the general approach crossed with wild-type males.The self-progeny included used is likely to be relevant in other areas of C. elegans many dead eggs as well as Lon hermaphrodites, a few genetics. A major advantage of genetic analysis in this Lon males and some shorter hermaphrodites with phe- organism is the ability to apply powerful selections or notypes similar to lon-Z/lon-2/lon-2 3X hermaphrodites efficient screens to very large numbers of individuals, (HODGKINet al. 1979). Thecross yielded predominantly to isolate suppressors or enhancers of a great variety of wild-type males, Lon hermaphrodites and 3-Xhermaph- mutant phenotypes. C. elegans reproduces normally by rodites. Both self-progeny and cross-progeny are there- meiotic self-fertilization, permitting isolation of both fore consistent with an attached X genotype. The 3-X recessive anddominant modifiers. Sometimes selec- cross-progeny were selfed, yielding predominantly 3-X tions will be swamped by common recessive suppres- hermaphrodites and Lon hermaphrodites. Several of sors, impeding the isolation of rare dominant modifi- the latter were selfed and produced broods like the ers, which are often uniquely informative. Carrying out original Lon hermaphrodite.These were crossed selections enmasse in an obligate male/female strain against wild-type males for four more generations, to will focus attention on dominant mutations. In this in- ensure removal of the original marked chromosomes vestigation, the mutation tra-2(e2046)was used to en- carryingfem-1 him-8, or dpy-26. sure propagation as a male/female strain, but other One of the Lon hermaphrodites resulting from this mutations could be used instead, such as fog-2 (which, series of crosses was used to found a strain CB5042, of like tra-2(e2046), results in a purely female germ-line in presumed genotype Zon-21on-2. Complete broods for five XX individuals) (SCHEDLet al. 1988) or either spe-8 or Nematode Feminizing Mutations 539 spe-12, mutations that lead to nonfunctional sperm in caused by loss-of-function mutations in the gene xol- hermaphroditesalthough male spermare normal 1. Consistent with this, the X0 lethality is rescued by (L’HERNAULTet ul. 1988). mutations of dosage compensation genes such as dpy- In addition, we used an approach that started with 26, dpy-27 and dpy-28. When eDp26 X0 animals are re- an already hypermorphic allele of tru-2 and selected stored to viability by this means, they are transformed for enhancers, in the hope of identifylng additional either partly or wholly into viable fertile hermaphro- regulatory sites. Important developmental control dites, although aminority develop as fertile males. Simi- genes may often besubject to multiple modes of regula- lar behavior is observed with xol-1 mutations in combi- tion, as in the case of both tra-2 and tru-1 (DONIACH nation with these dosage compensation genes (MILLER 1986; ZARKOWER et ul. 1994), so selections of this type et al. 1988), although the feminizing effects of xol-1 are may be appropriate and effective elsewhere. significantly stronger than those of eDp26 (Table 1). The second feature of this work is the isolation of The doubly mutantX chromosome, eDp26 xol-1, is tru-2(eg) mutations. Our results demonstrate that a tru- stronger still. 2(eg, gf) allele is able, in asingle dose supplied by either The similarity between eDp26 and xol-1 mutations is female or male parent, to completely transform an X0 confirmed by the interaction between eDp26 and sdc-2. individual from the normal male fate to a functional The eDp26 duplication has been combined with a loss- female fate, in all somatic and germ-line tissues. Obser- of-function mutation of sdc-2, which has opposite prop- vations on these alleles confirm and extend previous erties by itself: X0 sdc-2 animals are viable males, but work defining tru-2 as a major switch gene acting atthis XX sdc-2 animals are inviable and masculinized (NUS level in the sex determination pathway (DONIACH1986; BAUM and MEYER1989). X0 animals of genotype eDp26 HODGKIN1986; KUWABARAet al. 1992). Ten indepen- sdc-2are normal fertile males, and XXanimals are dead, dent tru-2(eg, gf) mutations were obtained with appar- demonstrating that the sdc-2 mutation is fully epistatic ently identical properties. The existence of these new to eDp26. Interactions with the other sdc genes, sdc-1 alleles raises new questions and possibilities for the fur- and sdc-3, have not yet been examined, but mutationsof ther investigation of tru-2. The data reported here are these genes appear to have weaker and more complex consistent with the possibility that the tru-2(eg) muta- propetiesthan sdc-2 (VILLENEUVEand MEYER 1987; tions render TRA-2A insensitive to inhibition by HER- DELONCet ul. 1993). The fact that an sdc-2 mutation is 1. However, proper assessment of the properties of the epistatic to eDp26indicates that theduplication is acting tru-2(eg) mutations entails their separation from the an- at a very early step in the sex determination pathway. tecedent tru-2(gf)allele. This separation and the further The duplications eDp26 and eDp27 cover a region, analysis of these mutations at both molecular and ge- between lin-32 and unc-2, that has not been previously netic levels has been carried out by P. E. KUWABARA duplicated. There appear to be only a few such regions (unpublished results). on the X chromosome (MENEELY1994), though it is The third significant feature of these experiments is not possible to exclude the possibility that some of the the isolation of theunusual Xol duplication, eDp26. existing duplications are discontinuous and therefore As documented in RESULTS, this duplication has the fail to cover some small interstitial regions. The contrast following properties: it is almost 100% lethal to X0 between eDp26 and mnDp73, a duplication only slightly individuals, but entirely viable in homozygous XX ani- smaller than eDp26, is dramatic: X0 males carrying mals. Genetic analysis indicates that it is a duplication mnDp73 can develop as fertile males, whereas those car- of the left end of the X chromosome, attached to the rying eDp26 are inviable. left tip of the X. Cytological observations confirm this These results are most easily explained by proposing and demonstrate that it is attached in inverted orienta- that eDp26 duplicates one or more key sites for the tion, a property that would have been difficult to de- assessment of XA ratio, and that all its effects on viabil- duce from genetics alone. ity and sexual phenotype arise from this. Furthermore, Under most circumstances the attachment of eDp26 the extreme difference between mnDp73 and eDp26 sug- to the Xchromosome is very stable, but the duplication- gests that a major numerator site is located between lin- bearing chromosome does revert to a wild type state on 32 and unc-2. rare occasions, and more frequently if the X chromo- As a result of these observations, a specific search was some is unpaired. The reversion to wild type and the carried out for a dose-sensitive element in the interval homozygous viability of eDp26/eDp26 individuals dem- between theright end-points of mnDp73 and eDp26 onstrates that no sequences have been lost from the X (HODGKINet ul. 1994). Cosmid clones from this region chromosome, and hence that the XO-lethal effects are were identified and tested for the ability to confer an likely to be dominant, This was confirmed by the isola- XO-lethal effect, by constructing transgenic lines car- tion of eDp27, afree derivative of eDp26, which has rying additional copies of each tested cosmid. One set comparable dominant XO-lethal effects. of overlapping cosmids did have such an effect, confer- The XO-lethalityof these duplications (eDp26 and ring both X0 lethality and feminization at much the eDp27) appeared to be phenotypically similar to that same level as does eDp26. This is the first time such an 540 andJ. Hodgkin D. G. Albertson effect has been seen, although many transgenic lines effect of Xchromosome duplications. Increased dosage carrying extra copies of other X chromosome clones of mnDp66, which covers the left end of the X, leads to have beenonstructed by other laboratories, in the reduced expression from this transgene in X0 animals. course of cloning specific genes (for example, ZHAO In contrast, yDpl4, a duplication that includes the lin- and EMMONS1995) or in searches for numerator ele- 32-unc-2 interval, had no apparenteffect on expression, ments (MCCOUBREYet al. 1988). The molecular data although its X0 lethal effects are stronger. It may be define a region of <25 kb with lethal and feminizing that the reporter transgene does not accurately reflect properties, which we have named fox-1 (standing for regulation of the endogenous xol-1, or possibly there &minizing locus on X). are additional genes as well as xol-1 that act at this level The observations on fox-1 suggest, but do notprove, in the hierarchy, as discussed below. that most or all of the effects of em26 are due to the Two additional properties of eDp26 are noteworthy extra dosage of this locus and that the rest of the dupli- in this context. First, it appears that X0 animals carrying cation does notcarry major numerator elements. Some eDp26 can sometimes survive to develop as normal fer- contrasting data suggesting the existence of additional tile males, especially if the duplication is supplied via elements have been obtained by &RIB and MEYER sperm. It is conceivable that these animals are in fact (1994). As a result of our observations on eDp26, these mosaic,having lost the duplication in the soma but authors searchedfor additional duplications of parts of retained it in the germ line, but this explanation seems the left end of the X chromosome and tested existing less likely. One interpretation of the survival of these duplications more stringently for numerator activity. animals is that EA ratio is assessed at an early point in They obtained evidence for a region (region 1) at the development, but thereafter sexual identity is main- left end of the X, duplicated by mnDp66, which has tained by some other means. In the survivors, e26 some deleterious effect on X0 animals when present may have failed to switch the program into the female, in three doses. Duplications covering the lin-32-unc-2 XO-lethal mode of development at this early point, so interval (region 3) exert a significant X0 lethal effect they develop normally as healthy males. (25% lethality) in a single extra dose, consistent with Second, the fact that eDp26 has an enhancing effect the predictionof a major site in this interval. This lethal- on the feminization caused byxol-1 is significant, be- ity is increased by the presence of an extra copy of an cause it suggests that eDp26 is exerting a feminizing interval to the left of lin-32 (region 2) and by mnDp66. effect over and above that caused by downregulation of Surprisingly, none of the duplications or combinations xol-1. It might be argued that the xol-1 mutation retains examined by these authors has X0 lethality quite as some residual activity, which can be further reduced by high as seen with eDp26. At present, themost straighfor- the action of eDp26, but it is known that xol-l(y9) is a ward interpretation of the data obtained in our work deletion of 225 kb, which entirely removes the xol-1 (this paper and HODGKINet al. 1994) and by &RIB gene (WINDet al. 1995), so this does not seem a possi- and MEYER(1994) is that thereexists a major numerator bility. The normal process of sex determination in the site, probably fox-1, between lin-32 and unc-2, plus a wild type may therefore entail some gene or genes in limited number of additional sites elsewhere on the X addition to xol-1, though it seems probable that xol-1 chromosome. Further genetic and molecular work will activity is an important factor. It was postulated long undoubtedly clarify the nature and importanceof these ago that there may be minor effects of X chromosome various contributions to measuring X chromosome dosage that bypass the main control genes (HODGKIN dose. However, it is reasonable to conclude now that a 1980). rather small number of sex-linked factors is involved, A final feature of these experiments is the generation much as in Drosophila (CLINE1993), and that most of of apparent attached X chromosomes, by recombina- the X chromosome material does not contribute sig- tion events involving eDp26 (diagrammed in Figure 6). nificantly to assessment of the XA ratio. Cytological obsyvations on hermaphrodites predicted Molecular investigations of the effects of these dupli- to carry such XX chromosomes provided evidence of cations should also be rewarding. Both genetic and mo- abnormality, but no unequivocal proof of such a struc- lecular experiments on xol-l (MILLERet al. 1988; RHIND ture. However, it is hard to explain the transmission et al. 1995) indicate that this gene plays a major role in properties of these chromosomes in anyother way, and the initial response to the XA ratio. Loss-of-function the structureof eDp26 makes it likely that they wouldbe mutations of xobl lead to X0lethality and feminization, generated. In organisms such as Drosophila, attached while overexpression of xol-1leads to XXlethality. More- chromosomes are very stable, making them useful as over, a xoL1::ZacZreporter transgene responds embryon- balancers and for chromosomal manipulations. In the ically to the EA ratio: X0 embryos exhibit substantially siplation we have examined, however, theapparent higher lacZ staining than do XX embryos, suggesting XX chromosomes appear to give rise to normal Xchro- xol-1is under sex-specific transcriptional regulation mosomes ata significant frequency, presumably by (WINDet aZ. 1995). This reporter construct has also breakage at the attachment site during meiosis. They been used by AKERIB and MEXER (1994) to examine the are thereforeunlikely to be very useful as tools. Possibly Fem inizing MutationsNematode Feminizing 541 the attachment site in eDp26and the resulting chro- HODGKIN,J., 1986 Sex determination in the nematode Cmorhab- 2% ditis elegans: analysis of tra-3 suppressors and characterization of mpsomes is unusually fragile, in whichcase stable fern genes. Genetics 114: 14-52. XX chromosomes (or attached autosomes) might be HODGKIN,J., 1987a A genetic analysis of the sex determining gene, generated by other means. More probably, the mecha- tra-1, inthe nematode Caenorhabditis elegans. Genes Dev. 1: 731 -745. nism of meiotic segregation in C. elegans precludes the HODGKIN,J., 1987b Primary sex determination in the nematode C. formation of any stable attached chromosomes. After elegans. Development 101 (Suppl.): 5-16. exchange at meiosis I, chiasmata terminalize to the ends HODGKIN,J., 1988 Sexual dimorphism and sex determination, pp. 491-584 in The Nematode Cmmhabditis elegans, edited by W. B. of paired chromosomes, and either end of each chro- WOOD.Cold SpringHarbor Laboratory, Cold Spring Harbor, NY. mosome has the potential to act as a kinetochore (AL HODGKIN,J., 1990 Sex determination compared in Drosophila and BERTSON and THOMSON1993). Inan attached X Caenorhabditis. Nature 344: 721-728. HODGKIN,J. A,, and S. BRENNER,1977 Mutations causing transfor- chromosome, if terminalization occurs toward the at- mation of sexual phenotype in the nematode Cmorhabditis ele- tachment site, then two interlocked chromosomes may guns. Genetics 86: 275-287. result, with chromosomal breakage at disjunction as a HODGKIN,J., H. R. HORVITZand S. BRENNER,1979 Nondisjunction mutants of the nematode Caenmhubditis elegans. Genetics 91: likely consequence. 67-94. 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