Download Hodgkin 1980

MORE SEX-DETERMINATION MUTANTS OF
CAENORHABDITIS ELEGANS
JONATHAN HODGKIN
M . R. C. Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 ZQH, England
Manuscript received April 21, 1980
Revised copy received September 24, 1980
ABSTRACT
Sex determination in Cnenorhabditis elegans is controlled by the X chromosome : autosome ratio, i.e. 2A;XX animals are hermaphrodite, and 2A;XO
animals are male. A procedure for isolating 2A;XO animals that are transformed into hermaphrodites has been developed. Nine mutations causing this
transformation have been obtained: eight are recessive, and all of these fall into
a new autosomal complementation group, her4 V. The remaining mutation
(her-2) is dominant and has a genetic map location similar to that of tra-I Ill.
Recessive mutations of tra-I cause the reverse transformation, transforming
2A;XX animals into males. Therefore, the her-2 mutation may result in constitutive expression of tra-I. Mutations in her4 are without effect on XX
animals, but the her-2 mutation prevents sperm production in both XX and
XO animals, in addition to its effect on the sexual phenotype of XO animals.
The epistatic relationships between tra and her genes are used to deduce a
model for the action of these genes in controlling sex determination.
HERE is substantial sexual dimorphism in the nematode Caenorhabditis
elegans: The adult hermaphrodite has a pair of ovaries and about 810 nongonadal nuclei; whereas, the adult male has a single testis and about 970 nongonadal nuclei. The two sexes exhibit many differences in post-embryonic development (SULSTON
and HOHVITZ
1977; KIMBLEand HIRSH1979), although
embryonic development is probably very similar in male and hermaphrodite.
Mutations that switch development from one sexual path to the other are of interest because they must alter the developmental behavior of many different
cells in precisely defined ways.
The primary mechanism of sex determination is chromosomal: hermaphrodites have five pairs of autosomes and two X chromosomes (abbreviated 2 A ; X X ) ,
while males have five pairs of autosomes and one X chromosome ( 2 A ; X O ) .
MADL and HERMAN
(1979), extending earlier work by NIGON (1949), have
shown that sex determination is controlled by the ratio of X chromosomes to
autosomes, as in Drosophila, rather than by the number of X chromosomes. Also,
as in Drosophila, this ratio seems to be determined by the cumulative action of
multiple sites on the X chromosome. However, there are a few genes that can
exert an overriding influence on sexual phenotype. Previous work has identified
,four such autosomal genes, tra-1, tra-2, tra-3 and isz-2. The tra (transformer)
and BRENNER
1977) appear to
genes (KLASS,WOLFand HIRSH1976; HODGKIN
Genetics 96:649-664 November, 1980.
650
J. H O D G K I N
be necessary for normal hermaphrodite development since mutations in these
genes cause X X animals to develop into phenotypic males or intersexes; X O animals are not affected and develop into normal males. The gene isx-l (intersex:
NELSON,
LEWand WARD1978) appears to be necessary for normal gonad development because the temperature-sensitive mutation isz-l (hcl7) causes feminization of the gonad in both sexes at restrictive temperature. However, isx-1
has little effect on nongonadal differentiation in either sex, unlike the tra genes.
This paper describes the isolation of mutations that cause X O animals to develop into hermaphrodites rather than males. Mutants of this type have not
previously been detected in C . ekgans, probably because X O animals are rare
in stock cultures (predominantly X X hermaphrodites) and because an X O hermaphrodite might be phenotypically indistinguishable from an X X hermaphrodite. However, frequencies of X O animals in hermaphrodite cultures can be
greatly increased by him mutations, which cause meiotic nondisjunction of X
chromosomes (HODGKIN,
HORVITZ
and BRENNER1979). Also, X O animals can
be distinguished from X X animals by the mutation dpy-21, which has dumpy
expression in X X animals and no expression in X O animals, regardless of phenoand BRENNER1977). A strain carrying dpy-21 and a him
typic sex (HODGKIN
mutation segregates both dumpy hermaphrodites ( X X ) and wild-type males
( X O ), and mutations transforming X O animals into hermaphrodites can be
detected by the appearance of wild-type hermaphrodites. Nine independent mutations have been obtained by this technique, of which eight fall into one complementation group, her-1 V (her standing for “hermaphroditizatio”) . One of
these alleles is temperature sensitive. The ninth mutation, her-2 ZZZ, is dominant
and has a map location similar to that of the gene tra-1. The interactions of tra
and her genes are described and discussed.
MATERIALS A N D METHODS
Strains: The following genes and mutations were used in this study. Strain names are given
only when relevant or convenient. A simplified genetic map is shown in Figure 1. The gene
unc-67, previously reported to lie a t the right end of LGIII (BRENNER
1979), is probably not
linked to LGIII (R. ROSENBLUTH,
personal communication; J. HODGKIN,
unpublished data).
LGI : unr-151e73). LGII : dpy-lO(e128), tra-2(elO95), unc-4(el20), sqtd(sc1) (J. Cox, personal communication). LGIII : Zon-l(el85), sup-5(e1464) (WATERSTON
and BRENNER1978),
unc-32(e189), wnc-69(e587), uab-7(el562) (J. G. CULOTTI
and E. M. HEDGECOCK,
personal communication), her-2(e1575), tra-1(e1076, el099, e1516), dpy-l8(e364, e499), unc-64(e246).
LGIV : unc-17(e245), isx-l(hc17) (NELSON,LEWand WARD1978), him-S(e1489), tra-3(e1107),
dpy-4(1166). LGV : dpy-f1(e224),her-l(e1518, e1519, e1520, e1558, e1559, e1561, e1564, e1574),
unc-42(e270), sma-1 (e3O), him-5(el 490), unc-39(e257), unc-76(e9Il), dpy -21 (e428), eDfl
(e1405) (RIDDLEand BRENNER
1978). LGX : unc-l(e94),dpy-3(e27), urtc-2(e55), unc-78(e1217>
(R. H. WATERSTON,
personal communication), dpy-8(e130), lon-2(e678), mecc-2(e75),dpy7(e88), mec-7(e1506), mec-l0(e1515), unc-lO(el02), dpy-6(e14), mc-l(el392) (SULSTON
personal communication),
1976), him-4(e1207), unc-9(eZM), unc-84(e1410) (H. R. HORVITZ,
unc-7(e5), mec-5(el504), mec-41e1343, e1506).
Strains were obtained from the Cambridge collection (BRENNER1974), except where noted
and BRENNER(1977) ; for him-4, him-5,
otherwise. For tra-1, tra-2, tra-3 and dpy-4, see HODCKIN
him4 and dpy-21, see HODGKIN,
HORVITZ
and BRENNER(1 979) ; for her-1 and her-2, see below.
65 1
SEX-DETERMINATION MUTANTS
The mec mutations were obtained from M. L. CHALFIE
and J. E, SUMTON.I am grateful to all
the workers cited for providing strains and communicating unpublished data.
Nomenclature conforms to the guidelines of HORVITZ
et al. (1979). Most genetic procedures
1974; HODGKIN,
HORVITZ
and BRENNER
1979). Photowere as described previously (BRENNER
graphs of relevant phenotypes may be found elsewhere (e.g., KLASS,WOLFand HIRSH1976;
HODGKIN
and BRENNER1977; NELSON,
LEWand W m 1978).
lsolation of her mutants: A strain (CB2300) was constructed, of genotype him-51ei490) dpy21(e428) V ; unc-7(e5) X . The unc-7 marker was included to prevent crossing between males and
hermaphrodites, which is otherwise likely to reduce homozygosis and prevent expression of recessive mutations. This strain was mutagenized with 0.05 M ethyl methanesulfonate (EMS) for 4
hr according to the standard protocol (BRENNER
1974), and L4 or young adult animals were
picked and allowed to produce self-progeny. Thirty mutagenesis experiments were carried out,
and a total of 3,600 Po hermaphrodites were picked. CB2300 animals normally produce an average
of 65 hermaphrodite and 40 male self-progeny per animal, but after EMS mutagenesis the average brood is reduced to I1 hermaphrodites and 5 males. Therefore, Po hermaphrodites were
placed on 9 cm culture plates in sets of 10 to 20 per plate. F,, F, and F, generations were examined for the appearance of non-Dpy hermaphrodites. This screening was not exhaustive, but many
candidates were picked, mainly from the F, generation. The F, worms were very numerous and
mostly starving, so that they were difficult to screen.
outcrossing, complemntation and mapping of her-I: The mutation her-l(e1518) was initially isolated as a strain her4 him-5 dpy-21 V ; unc-7 X . This was crossed w i k him-5 ma$es,
and a strain her-I him-5 was isolated. The mutation was assigned to LGV because the multiple
mutant dpy-11 her4 him5 was difficult to construct; whereas, multiple mutants with dpy
markers on other linkage groups were easily obtained. In order to isolate her-I alone, hermaphrounc-76 were constructed, and 6 recombinant
dites of constitution her-1 him-5 +/dpy-i1
Unc progeny picked. These were crossed with dpy-11 him-5 males: none of 6 wild-type progeny
her-1 unc-76/dpy-11+ him-5 or f f u m segregated males; thus, all must have been
76/dpy-11 him-5
Unc progeny were picked, crossed with wild-type males, and F, males
backcrossed with the Unc parents. Three of 6 gave few or no Unc male progeny; these must have
been her-1 unc-76. They were crossed with dpy-I1 males, and wild-type male progeny crossed
with dpy-11 unc-76 hermaphrodites. Wild-type hermaphrodite progeny were picked, which must
have been her-l/dpy-Il unc-76 or +/dpy-11 unc-76. A wild type hermaphrodite was picked from
the next generation of self-progeny, which segregated neither dpy-21 nor unc-76, and subsequent
tests confirmed that the progeny of this animal were all homozygous for her-I and carried no
other detectable markers. The other 7 isolates of her-1 mutations were outcrossed in an analogous
manner, but using unc-39 rather than unc-76.
The mutation h&-1(eI518) confers no visible phenotype on an X X animal, whether heterozygous or homozygous, so that the presence of her-I in a strain was usually assayed either by the
suppression of a Him phenotype (because him her-1 homozygotes segregate no phenotypic
males), or by crossing with wild-type males and then crossing the progeny males with hermaphrodites of a test strain her-I(e1518) unc-76; dpy-3 X . Segregation of non-Unc Dpy hermaphrod p y 3 / 0 ) indicated the presence of a her-1 mutation. Compledites (i.e., her-1 unc-76/her-l
mentation tests on the 7 other isolates of her-1 were carried out in this manner; all 7 failed to
complement e1518, except for e1561, which gave partial complementation at 20" and no complementation at 25".
A precise map location for her-1 was obtained by the following crosses: Hermaphrodites of
constitution her-l/dpy-Ii unc-42 were constructed: 29 of 30 Dpy recombinant progeny carried
her-1, whereas I of 30 Unc recombinants carried her-1. Tight linkage between unc-42 and her4
was confirmed by crossing dpy-I1 b r - 1 unc-42 hermaphrodites with dpy-11 her-1 unc4 2 / f $- males. Total male counts of 1,468 wild-type : 23 Dpy : I Unc : 0 Dpy Unc were
obtained. This gives p 5 1.5% for dpy-11-unc-42 linkage in males as compared to p = 2.9% for
the same linkage in hermaphrodites. Therefore, her-1 is located 0.1% or less to the left of unc-42.
The deficiency eDf1 (e1405), described by RIDDLEand BRENNER
1978) fails to complement
unc-42, unc-41 and sup-3, but it does complement her-I. This was shown. by crossing UGC-15
++
'+ +
+
+.
+;
+
+
652
J. HODGKIN
++
Ce73) I ; smu-i/eDfl V hermaphrodites with her-I unc-42 sma-i/+
males: many Unc-42
male progeny were obtained, which were presumably of constitution unc-l5/+; eDfi/her-1
unc-42 sma-i; X O .
A useful chromosomd aberration of LGIIZ: The analysis of the her-2 and tra-I mutations has
been facilitated by the fortuitous isolation, after acetaldehyde mutagenesis, of strain CB1517,
which has the constitution eDf2/eDf2 ZII; eDpb(lZZ;f), where the extents of the deficiency
(eDf2) and the duplication (eDp6) are as shown i n Figure 1. Animals of strain CBI517 are
severely uncoordinated, but animals of genotype eDf2/+ or eDf2/+; eDp6 or
eDp6
are viable and fertile in both sexes. The duplication is meiotically unstable and can act as a
balancer for markers in this region; for example, hermaphrodites of genotype uab-7 dpy-i8/
uab-7 dpy-i8; eDph are wild type and produce only 2 kinds of self-progeny: those of the parental
type and vab-7 dpy-18 homozygotes that have lost the duplication. These 2 types are produced in
roughly equal numbers; no Vab or Dpy recombinant self-progeny are observed. I am indebted to
R. P. ANDERSON
for assistance in the analysis of CB1517.
Mapping of her-2: The location of her-2 on the genetic map was difficult to determine because
of the dominant self-sterility caused by this mutation. Linkage to unc-32 and dpy-I8 was deterhim-5 dpy-21 hermaphromined by means of the following crosses: (1) unc-32 her-2/+
dites x unc-32/+; him-5 dpy-Zi males gave 16 Unc male progeny and 313 non-Unc male
progeny. The frequency of Unc males relative to all males in such a cross is given by p/2, SO
that p = 10% for linkage between unc-32 and h0-2. ( 2 ) her-2 dpy-i8b+
him-5 dpy-21
hermaphrodites x dpy-i8/+ males gave 3 Dpy-18 male progeny and 347 non-Dpy male
progeny; hence, p = 1.7% for linkage between her-2 and dpy-18.
These linkage values refer to meiosis in the oocytes of him-5 animals, and are therefore not
strictly comparable to linkage values as conventionally measured (BRENNER1974). The tight
linkage to dpy-i8 suggested that her-2 might have a location near tra-1, and therefore a 3-factor
her-2 +/vab-7 $- dpy-18; him-5 with
cross was performed by crossing animals of constitution
males of constitution vab-7 dpy-i8; eDp6; him-5. These males will mate because eDp6 complements uab-7 and dpy-i8, but there is no recombination between these two markers and the duplication in the male. Therefore, all Vab and Dpy recombinants arise from recombination in the
hermaphrodite, Of 12 Vab recombinants, 7 carried her-2 and 5 did not; of 15 Dpy recombinants,
8 carried her-2 and 7 did not. Therefore, her-2 is located between these 2 markers, which are
about 2.5 map units apart. This is similar to the location of the gene tra-2. Another 3-factor cross
was carried out by constructing hermaphrodites of constitution uab-7 k+ dpy-ill/+ tra-1
Five out of 12 Vab recombinant self-progeny carried tra-1, and 7 out of 12 Dpy
(e1516)
recombinants carried tra-i.
Epistatic interactions: The phenotypes of various tra; her double mutants are summarized
in Table 1. They were determined by means of the following crosses: For tral(ei076), tra-I;
eDp6 hermaphrodites were crossed with him-5 males, and progeny X O males were crossed with
her-i him-5 dpy-2i hermaphrodites. Three of 6 wild-type progeny segregated both wild-type
and Tra-I males; they must have been tra-Zj+; her-2 him-5 dpy-21/+ him-5
One of 6
wild-type self-progeny from those animals segregated one-fourth Tra-I males (both Dpy and
non-Dpy), and no wild-type males; it must have been tra-I/+; her-I him-5 dpy-21; X O . The
cross was repeated without dpy-2i to yield hermaphrodites of constitution tra-i/+; her-1 him-5.
All male progeny had a n incomplete male phenotype like that of ei076 X X animals, although
one-third must have been X O rather than X X .
For tra-Z(eI095), tra-Z/dpy-iO unc-4 hermaphrodites were crossed with him-5 dpy-21 males,
and progeny males were crossed with her-i him-5 dpy-21 hermaphrodites. One of 6 Dpy-21
hermaphrodite progeny yielded wild-type males and Tra-2 Dpy-21 incomplete males; it must
have been tra-2/+; her-I him4 dpy-21/+ him-5 dpy-2i. Non-Dpy hermaphrodite (i.e.,her-2
X O ) animals were picked, and 2 of 4 gave some Dpy male and non-Dpy male self- progeny; they
must have been tra-2/;+; her-i him-5 dpy-2i; X O . The Dpy ( X X ) male progeny had a Tra-2
(incomplete male) phenotype, but the non-Dpy ( X O ) male progeny resembled wild-type XO
males; a minority (about one-fourth) were fertile and sired progeny.
+/+;
+;
+;
+
+.
+.
653
SEX-DETERMINATION MUTANTS
0
w
H
FIGUREl.-A simplified genetic map of C. elegans.
X
654
J. HODGKIN
TABL,E 1
The phenotypes of some sex determination mutants
X X phenotype
Genotype
XO phenotype
hermaphrodite
male
incomplete male
incomplete male
incomplete male
hermaphrodite
hermaphrodite/female
female
male
male
incomplete male
male
incomplete male
incomplete male
incomplete male
hermaphrodite/female
hemaphrodite/female
wild type
tra-I (s)
tra-I ( w )
tra-2
tra-3
her-I
her-2/+
her-2
tra-l(s);tra-2
ira-l(s);tra-3
tra-2;tm-3
tra-1(s); her-I
tra-l(w);her-I
tra-2;her-I
tra-3;her-I
tra-2;her-2/+
tra-3;her-2/+
male
male
male
male
male
hermaphrodite
hermaphrodite/female
female
male
male
male
male
incomplete male
male
male
hermaphrodite/female
hermaphrodite/female
tra-l(s) is one of the strong alleles osf tra-I, such as e1099 or e1516; t r - l ( w ) , is the weak
allele of tra-I, e1076. Most of these single and double mutants are described in this paper or in
a previous paper (HODGKIN
and BRENNER1977); a few have been analyzed in unpublished work.
For tra-3(e1107), allowance must be made for the fact that expression of the Tra phenotype
is delayed one generation : tra-3/tra-3 X X self-progeny of tra-3/+ hermaphrodites are also
hermaphrodites, but all of their X X self-progeny are Tra-3 incomplete males. Thus, tra-3; him-5
hermaphrodites were obtained from tra-3
dpy-4; him-5 parents, and these segregated only
Tra-3 X X males and wild-type X O males. These tra-3; him-5 X O males were crossed with her-I
him-5 dpy-21 hermaphrodites. Four of 4 non-Dpy progeny segregated wild-type males; thus, all
must have been tra-3/+; her-I him-5 dpy-21/+ h i m 5
From those animals, three Dpy-21
hermaphrodites were obtained that segregated no males in the Fl self-progeny, but many males
in the F,; these animals must have been tra-3/+; her-I him-5 dpy-21. From their F, self-progeny,
3 hermaphrodites were picked that segregated only male self-progeny; these must have been
tra-3; her-I him-5 dpy-21 hermaphrodites. The Dpy ( X X ) males had a Tra-3 (incomplete male)
phenotype, but the non-Dpy ( X O ) males resembled wild-type X O males, and a minority (about
1 in 10) were fertile males that sired progeny.
The epistasis of tra-l(e1099) over tra-3 was demonstrated by obtaining tra-3 hermaphrodites
from a heterozygote tra-3
dpy-4, and crossing these hermaphrodites with tra-I X X males.
Wild-type hermaphrodite progeny were obtained, which must have been +/tra-I ; tra-3/+. From
their self-progeny, 4 hermaphrodites were obtained that segregated three Tra-3 to one Tra-I
males; these hermaphrodites must have been tra-I/+; tra-3. The tra-I; tra-3 X X self-progeny
resFmbled tra-i X X males and were at least as fertile.
The epistasis of her-2 over tra-2 and tra-3 was demonstrated by constructing stable strains
containing both tra and tra; her-2/+ animals, as explained below. The tra-2; her-2/+ strain
tra-2 +/dpy-10 I+sqt-I; him-5
was obta'ned by generating tra-2 X O males from a strain
(tra-2 homozygotes are recognizable because the trans-linked marker, sqt-I, is dominant, causing
rolling in heterozygotes), and crossing these males with unc-32 her-2 hermaphrodites (obtained
hermaphrodite parents). This cross yielded only non-Unc hermaphrofrom unc-32 her-2/+
and these were crossed again with
dites, which must have been tra-2/+; unc-32 her-2/+
tra-2 X O males to yield tra-2/tra-2; unc-32 her-2/+
These animals were recognized by their
+/+
+.
+/+
+
+
+.
+,
655
SEX-DETERMINATION MUTANTS
failure to yield anything but “female” ( t m - 2 ; her-2/f+) and tra-2 progeny when crossed with
tra-2 XO males. The unc-32 marker was subsequently lost by segregation.
The her-2/+; tra-3 strain was constructed in an analogous manner.
RESULTS
A strain carrying the markers him-5 and dpy-21 was constructed: this strain
segregates approximately 65 % Dpy (dumpy) hermaphrodites ( X X ) and 35 %
non-Dpy males ( X O ). After mutagenesis, rare non-Dpy hermaphrodites were
observed in the progeny. These were picked and tested to see if any were XO
hermaphrodites. Altogether, 261 candidates were picked, which fell into four
classes on the basis of self-progeny in the next generation: (a) Animals segregating the normal complement of Dpy hermaphrodites and non-Dpy males.
These were animals in which the expression of the dpy-21 mutation (which is
variable) was weak. (b) Animals segregating only non-Dpy hermaphrodites
and non-Dpy males. These were animals carrying homozygous suppressors of
dpy-21, most probably mutations such as Zon-1 or Zon-2, which partially or completely suppress dpy-21 expression. Most of the candidates picked (150 of 261)
fell into one or the other of these first two classes. (c) Animals segregating both
Dpy and non-Dpy hermaphrodites, and no males. Eight such animals were independently isolated, and all carried mutations in the gene her-1, described below. (d) Sterile animals: 103 animals produced no self-progeny. Fifty-one of
these were crossed with him-5 dpy-21 males, in the hope of obtaining crossprogeny, but almost all (48 of 51) produced no viable cross-progeny, nor did
they lay any fertilized or unfertilized eggs. Of the three that did produce crossprogeny, two failed to segregate non-Dpy hermaphrodites in any subsequent
generation, and one carried a dominant mutation her-2, described below.
The her-1 mutations: The first isolate of a fertile X O hermaphrodite was
found to be homozygous for a recessive mutation, e1518, which was mapped as
described in MATERIALS AND METHODS. Seven additional independent isolates of
such mutations all had the same map location, and all failed to complement
e1518. These mutations define the autosomal gene, her-1 V , so called because
her-1 mutants are hermaphrodite in phenotype regardless of the number of X
chromosomes.
The following crosses demonstrate that her-1(e1518) is a recessive mutation
linked to unc-42 V that transforms XO animals into hermaphrodites, and that
the mutation has no obvious lethal or maternal effects. (1) her-1 unc-42 X X
hermaphrodites x wild-type X O males gave non-Unc progeny of 169 hermaphrodites : 166 male : 0 inviable zygotes, i.e., 1 : 1 : 0. (2) her-1 unc-42 sma-1 X X
hermaphrodites x her-1 unc-42/+ 4- X O males gave non-Sma progeny of 209
wild hermaphrodite : 211 wild male : 396 Unc hermaphrodite : 0 Unc male : 7
inviable zygotes, i.e., 1 : 1 : 2 : 0 : 0. (3) Izer-1 unc-42/+
X X hermaphrodites x her-1 unc-42/+ X O males gave total progeny (under conditions favoring complete outcrossing) of 463 wild hermaphrodite : 456 wild male : 281 Unc
hermaphrodite : 0 Unc male : 6 inviable zygotes, i.e., 3 : 3 : 2 : 0 : 0.
+
+
656
J. HODGHIN
The first cross shows that the mutation is recessive, because half of the crossprogeny are males, which must be her-1 unc-42/+ X O animals. The second
cross gives twice as many Unc hermaphrodites as wild hermaphrodites, and no
Unc males, indicating that all of the her-2 unc-42 X O animals have been transformed into hermaphrodites. The expected numbers of wild-type male progeny
are obtained in both of these crosses (and in analogous crosses using XO hermaphrodite parents) showing that the mutant her-1 gene has no maternal effect
on the sexual phenotype of the progeny. The third cross shows that the wild-type
her-1 gene has no maternal or paternal rescue effect, because no Unc male progeny are obtained although both parents carry one copy of the wild type gene.
None of the crosses gives excess numbers of inviable zygotes, showing that there
i s no lethality associated with the mutation.
It might have been conceivable that the her-1 mutations cause duplication of
the X chromosome early in development, so that a 2A;XO genotype is converted
into a 2 A ; X X genotype. However, this is demonstrably not the case for the germ
line, as shown below. It is also unlikely to happen in somatic tissues, because
her-1 dpy-21 X O animals do not express the Dpy-21 phenotype characteristic of
X X animals, and it is reasonable to assume that this phenotype results from
somatic events.
All eight her-1 mutations were crossed into wild-type genetic backgrounds, as
described in MATERIALS AND METHODS. Most of the resulting X X strains were
indistinguishable from the wild-type strain N2 except in failing to segregate
males. The X X strain CB1518, carrying her-Z(e1528) and no other markers, was
examined in detail. Self-progeny broods f o r six animals gave an average brood
of 340 hermaphrodites : 0 males : 5 inviable zygotes, as compared to an average
of 330 hermaphrodite : 0.5 male : 3 inviable zygotes for the wild-type strain N2.
Therefore, her-1 has no apparent effect on X X animals.
When this strain was crossed with wild-type males, and progeny males
( h e r - l / + ) backcrossed with her-1 X X hermaphrodites, two kinds of hermaphrodite progeny were obtained. The majority class segregated many progeny and
few inviable zygotes and were presumably X X hermaphrodites (her-1 and
her-I/+) ;whereas, the minority class segregated fewer viable progeny and many
inviable zygotes. These were assumed to be her-1 X O hermaphrodites. Twentynine self-progeny broods were counted and gave an average brood of 36 hermaphrodite progeny : 38 inviable zygotes. Some animals were completely sterile
and exhibited gonadal abnormalities, but most (24 of 29) produced self-progeny:
between 17 and 160 zygotes. Thus, her-1 X O hermaphrodites are fertile, but produce only one-fifth as many zygotes as her-1 X X hermaphrodites.
The production of many inviable zygotes by these animals is expected if both
hermaphrodite gamete lines are X O rather than X X , because many zygotes will
consequently contain no X chromosome. Despite this massive genetic deficiency,
these zygotes go through early embryogenesis normally, and develop into abnormal embryos with up to 100 or more cells. Presumably this development is
supported by a maternal supply of essential sex-linked gene products.
+
SEX-DETERMINATION MUTANTS
65 7
The frequency of nullo-X zygotes (51%) is higher than the Mendelian expectation of 25%, suggesting that an excess of nullo-X gametes is produced by
one or both gamete lines in the XO hermaphrodite. This observation was confirmed by brood counts on a her-i' dpy-22 strain, in which XX and XO animals
can be distinguished by the dpy-21 marker. Four XX animals gave an average
self-progeny brood of 189 XX : 1 XO : 3 inviable zygotes, whereas six XO animals gave an average self-progenybrood of 9 XX : 41 XO : 62 inviable zygotes,
suggesting a nullo-X gamete frequency of 74% rather than 50%. The frequency
of nullo-X ova was measured directly by crossing X O hermaphrodites of constitution her-i' dpy-21; unc-7/0 with wild-type males. Wild-type progeny counts
of 73 hermaphrodites : 192 males were obtained, giving a nullo-X ovum frequency of 72%. Self-progeny counts for the same strain gave an average nullo-X
gamete frequency of 70%. Thus, it appears that both gamete lines are producing
an excess of nullo-X gametes, significantly different from the 50% expected if
the unpaired X chromosome always survives meiotic disjunction. Such a 50%
frequency is, however, reliably observed in XO male meiosis. These results confirm the conclusions reached previously (HODGKIN,
HORVITZ
and BRENNER
1979) that unpaired X chromosomes tend to be lost during hermaphrodite
meiosis, and that there is a special mechanism for handling the unpaired X
chromosome in male meiosis. This mechnasim must be inoperative or ineffective
in both gamete lines of the XO hermaphrodite. These results also demonstrate
that the germ-line of these animals is XO rather than XX, and that this abnormality does not prevent gametogenesis in the hermaphrodite.
Apart from the low fertility, most her-2 XO hermaphrodites are phenotypically indistinguishable from XX hermaphrodites with respect to all anatomical
personal communication). Seven of the eight
characters examined (J. SULSTON,
her-2 alleies are very similar in their effects. Two of them appear to reduce fertility in XX animals, as well as in XO animals, but it may be that this is due to
linked deleterious mutations rather than to the her-1 mutations themselves. One
of the her-i' isolates, ei'561,is temperature sensitive: at 25.5", 98% of the XO
animals are hermaphrodite in phenotype; whereas, at 15", only 10% are hermaphrodite. Most of the other 90% are fertile males. Temperature-shift experiin preparation) indicate that the her-I gene product is made
ments (J. HODGKIN,
early in embryogenesis, long before any overt sexual differentiation has occurred.
None of the eight her-? alleles showed any response to the pleiotropic supand BRENNER1978).
pressor sup-5 (WATERSTON
The her-2 mutation: A sterile (spermless) XO hermaphrodite was obtained as
described above, and found to carry a dominant mutation, her-2(e1575). It was
possible to maintain a stock of her-2/+ XO hermaphrodites by continued crossing of her-a/+; him-5 dpy-21 hermaphrodites with him-5 dpy-21 males, but no
stable or balanced strain could initially be obtained. The sterility of her-2/+
animals (both X X and XO) is high, but not complete: 18 of 24 XO animals
picked as L4's produced no fertilized eggs, but 6 of 24 produced a total of 28 XX
hermaphrodites: 71 XO hermaphrodites: 28 XO males: 134 inviable zygotes.
This sterility is largely due to a failure of spermatogenesis, because most XO
658
J. HODGKIN
animals will produce progeny when crossed with wild-type males: 12 of 12 such
animals produced cross-progeny, in all 435 hermaphrodites: 261 males: 385
inviable zygotes. Therefore, the average number of self-progeny zygotes is 11,
and the average number of cross-progeny zygotes is 90. The mutation also produces a high level of sterility in X X her-2/+ heterozygotes, again as a result of
the absence of sperm.
Animals homozygous for her-2 are difficult to obtain in view of the low selffertility of her-2/+ animals, and difficult to recognize in the absence of a recessive marker linked to her-2. However, they can be recognized by progeny testing: her-2 homozygotes produce only hermaphrodite progeny when crossed with
wild-type males, and all of these progeny hermaphrodites are her-2/+. The
homozygotes never produce any self-progeny, so that the phenotype of the homozygote is stronger than that of the heterozygote. Furthermore, her-2/+ X O animals are variable in tail morphology, indicating incomplete transformation, but
her-2 X O homozygotes are completely transformed with respect to tail structure.
The her-2 mutation was mapped as described in MATERIALS AND METHODS; it
is located between the markers vab-7 and dpy-28 on LGIII. The gene tra-l has a
similar location between these two markers. Recessive mutations of tra-l cause
the opposite transformation to her-2: X X animals are transformed into males
(HODGKIN
and BRENNER1977). Further analysis of the her-2-tra-2 region is in
progress: the two loci appear to be tightly linked.
It is conceivablethat a translocation of X chromosome material to an autosome
could act as a dominant her mutation, but it is unlikely that her-2 is such a
translocation. The her-2 mutation causes no conspicuous interference with recombination on LGIII, such as might be expected for a translocation. Tests for
an X translocation were also carried out by studying the inheritance of many
sex-linked markers in crosses with her-2 X O hermaphrodites. Such hermaphrodites, when crossed with males carrying any sex-linked marker, e.g., Zon-2, will
segregate hermaphrodite progeny expressing the paternal marker (i.e.,her-2/+;
Zon-2/0 in this instance), but will not do so if the relevant region of the X chromosome has been translocated to LGIII. All the sex-linked markers indicated in
Figure 1 have been tested in this way, and all exhibited unambiguous patroclinous inheritance. Therefore, if her-2 is a translocation, it must involve only a
small and elusive part of the X chromosome.
Epistatic interactions between her and tra mutations: The phenotypes of X X
and X O animals of various her; tra genotypes are summarized in Table 1. The
crosses generating these double mutants are described in MATERIALS AND METHODS.
Preliminary experiments showed that all tra mutations are epistatic to the
her-2 mutations e2528 and e2520 in X X animals. The her-2 mutations appear
to act by making an X O genotype equivalent to an X X genotype; thus, further
crosses were carried out to see if this were also true in tra; her-2 animals, i.e.,
if tra; her-2 X O animals were identical in phenotype to tra; her-2 X X animals.
For tra-2, the mutation e2076 can be used to distinguish betwen X X and X O ;
X X animals are partially transformed males, but X O animals are normal
SEX-DETERMINATION MUTANTS
659
males. In the presence of homozygous her-l mutations, however, tra-I X O
animals are indistinguishable from tra-I X X animals: all are partially transformed. The double mutant tra-l(e1076); her-I is not quite identical to e1076
X X alone in that the gonads of these animals are more frequently intersexual
rather than male, but otherwise the epistasis is complete. Thus, with respect to
tra-I, her-I appears to act by making an X O genotype equivalent to an X X genotype.
Different behavior is observed for tra-2 and tra-3. Any mutation of these genes
can be used to distinguish X X and X O animals: X X animals are never more
than partially transformed; whereas, X O animals are normal males. In the
presence of homozygous her-1 mutations, tra-2 X X animals are partially transformed males, like tra-2 X X alone, and tra-2 X O animals are completely or
almost completely male, again like tra-2 alone. As with tra-1, epistasis is less
complete in the gonads, but at least some of the tra-2; her-1 X O animals are
capable of mating and siring progeny. The same result is obtained with tra-3.
Thus, her-I does not abolish the distinction between X X and X O , with respect to
tra-2 and tra-3, but it does abolish this distinction in a wild-type background and
in tra-I animals. This is a surprising result, in view of the fact that tra-l is
epistatic to tra-2 and tra-3 (HODGKIN
and BRENNER1977; this paper).
The epistatic suppression of her-1 by tra mutations can also be demonstrated
by inducing new tra mutations in a her-I stock. Strain CB2301 (her-l(el520)
him-5(e1#90)) was found to be most suitable for such experiments; this strain
never segregates phenotypic males, as expected, but rare males were observed in
the F, and subsequent generations after EMS mutagenesis. These males were
picked and crossed with unc-17 hermaphrodites, but in only 5 of 33 independent
crosses were non-Unc progeny obtained. Subsequent tests showed that in four
of these cases the fertile males were tra-2; her-I him-5; X O . In the fifth case,
only one non-Unc progeny hermaphrodite was obtained, which proved to carry
a new tra-I mutation. The original male could have been either X X or X O . Thus,
although this technique does yield new tra mutations, it is not very efficient.
Probably the inefficiency results from the variability of tra; her-I animals; as
noted above, most of them resemble wild-type males, but their gonads are frequently abnormal and fertile males are infrequent. This experiment also demonstrates that it is difficult or impossible to suppress her-l(e1520) except by
means of tra mutations.
These data show that tra mutations are epistatic to her-I mutations. By contrast, the opposite pattern is observed in the case of the her-2 mutation. Both
tra-2 and tra-3 mutations are efficiently suppressed by her-2, i.e.,the tra; her-2/+
animals (both X X and X O ) are hermaphrodite or female in phenotype, like
h.er-2/+ alone. This is demonstrated, in the case of tra-2, by constructing a stable
stock containing animals of genotype tra-2; her-2/+ ( X X and X O ) and tra-2
( X X and X O ) . All animals are homozygous for tra-2, but the stock survives because the her-2/+ heterozygotes are hermaphrodite or female; these animals are
fertilized by the tra-2 X O males, to yield more animals of the same four progeny
660
J. HODGKIN
types. An analogous stock consisting of her-2/+; tra-3 females and tra-3 males
has also been constructed.
The phenotype of a her-2 tra-1 double mutant has not yet been determined,
because the tight linkage between her-2 and tra-l mutations makes such a double
mutant difficult to construct.
DISCUSSION
The d p y S I ( e 4 2 8 ) mutation is expressed differentially in the two chromosomal sexes of C. elegans, and this differential expression depends on the X
chromosome to autosome ratio, not on sexual phenotype per se. 2 A ; X X animals
(normally hermaphrodite) are dumpy, while 2A;XO animals (normally male)
are wild type. This effect has been exploited as a means of identifying mutant
hermaphrodites carrying only one X chromosome. Two classes of mutants have
been isolated, those carrying recessive mutations in the gene her-l V (eight
independent isolates), and one mutant carrying a dominant mutation, her-2 111.
The her-l mutations have no apparent effect on X X animals, but cause X O
animals to develop into fertile hermaphrodites rather than into males. However,
these X O hermaphrodites are less fertile than X X hermaphrodites, and sometimes exhibit gonadal abnormalities. The her-2 mutation has two effects: it
transforms X O animals into hermaphrodites, and it greatly reduces sperm production in both X X and X O animals. Heterozygotes (her-2/+) are frequently
completely spermless and on average produce only about 10 self-progeny ; homozygotes (her-2/her-2) are always completely spermless, i.e., they are female
rather than hermaphrodite. The transformation of X O animals by her-2 is also
more complete in homozygotes than in heterozygotes. The location of the her-2
mutation is similar to that of another gene controlling sex determination, tra-l
Ill.
Mutations causing incomplete transformation of X O animals would not have
been recovered in this search, because hermaphrodites that are both self-sterile
and cross-sterile can yield no progeny. Many putative X O hermaphrodites of this
type were seen, and it is possible that these carried her mutations causing incomplete transformation. Alternatively, they may have been X X hermaphrodites that carried mutations causing both sterility and suppression of the dpy-21
phenotype. Another possibility is that they were aneuploids, such as 4A;3X
hermaphrodites : dpy-21 is expressed in 4A;4X tetraploid hermaphrodites, but
not in 4A;3X hermaphrodites (unpublished observations).
The properties of the her mutations, and their interactions with tra mutations,
allow some speculation as to the mechanism of sex determination in C. elegans.
The existence of a small number of autosomal genes that can exert an overriding control on sex determination suggests that the X to autosome ratio is used
only as a signal that determines the level of activity of these genes, and the
products of these genes then act to control all aspects of sexual differentiation.
We have now shown that fertile males with two X chromosomes can be generated by tra-2 mutations, and fertile hermaphrodites with one X chromosome
can be generated by her-l mutations. I n both cases, the fertility is reduced rela-
661
SEX-DETERMINATIUN MUTANTS
tive to the normal fertility of the X O male and the X X hermaphrodite, which
may reflect some minor effect of the X to autosome ratio, but the major control
is plainly exerted via the tra and her genes.
The phenotypes resulting from various her and tra mutations, and the epistatic relationships between them, are summarized in Table 1.
Recessive mutations in the tra-1, tra-2, tra-3 and her-1 genes affect only one
sexual phenotype: tra mutations have no effect on the development of X O males,
and her-1 mutations have 110 effect on X X hermaphrodites. It is therefore likely
that the two sets of genes have complementary roles in wild-type sex determination, and the proposed activities of these genes in wild-type sex determination are
summarized in Table 2.
The mutation her-2 is dominant, unlike all the other sex-determination mutants so far isolated. It behaves as a point mutant, and it is tightly linked to the
tra-l mutations, which are recessive and cause the opposite transformation, turning X X animals into males. In addition, her-2 is expressed in both X X and X O
animals: both are feminized. Finally, it cannot be a simple amorph or hypomorph, because it is located in the region covered by the deficiency eDf2, and
this deficiency has no dominant effect on sex determination. These facts suggest
that her-:!is a mutation causing constitutive expression of the gene tra-2. This is
consistent with the interpretation of tra-1 presented previously (HODGKIN
and
BRENNER1977) that the wild type tra-1 gene product acts as a repressor of male
development. Constitutive expression or overproduction of this gene product
might then cause animals to develop into hermaphrodites or females regardless
of X chromosome genotype. Tests and discussion of this hypothesis will be provided in a future publication.
Both tra-2 and tra-3 alleles are suppressed by her-2, and her-1 alleles are suppressed by tra-l mutations. A simple explanation of these results is that the three
genes, her-1, tra-2 and tra-3, exert their effects entirely by acting (directly or indirectly) on the gene tra-1 (or its gene product). If this is so, then the sexual
phenotype of C . elegans is proximately controlled by the state of the gene tra-1:
when this gene is on ( i . q producing functional product), male development is
repressed and hermaphrodite development is activated; when this gene is off,
male development is permitted. The male sex can therefore be regarded as the
and
“neutral” sex and the hermaphrodite as the “dominant” sex (MCCARREY
ABBOTT
1979).
The epistatic relationships summarized in Table 1 can be used to construct
a more elaborate model, shown in Figure 2. The phenotypes resulting from all
TABLE 2
Proposed gene activities in wild-type sex determination
x Eellotype
her-I
xx
xo
ira-3
tr0-2
tra-l
OFF*
ON
ON*
OFF
ON
OFF
ON
OFF
Phenotype
hermaphrodite
male
* In this table, ON signifies presence of active gene product; OFF signifies absence.
662
J. HODGKIN
her-1
iigh X:A
ratio
tra-2
tra-1
~
\
\
\
rra-3
hermaphrodite
development
FIGURE
%--A model for the control of sex determination in C . elegans. The dotted line indicates an interaction that is not significant in wild-type sex determination. For further explanation,
see text.
the various mutant combinations can be explained by this set of regulatory interactions, if it is assumed that the recessive mutations of the tra and her genes
are amorphic or hypomorphic. The molecular nature of these interactions is
wholly unknown at present, but it seems preferable to try to construct formal
models such as this, rather than to postulate a complex and indecipherable interaction of all possible factors at once. In this model, the activity of her-I is
controlled by the X to autosome ratio; her-I acts negatively on tra-2, and tra-Z
acts positively on tra-I. Thus, in the wild-type hermaphrodite, a high X to autosome ratio turns off her-I; thus, tra-2 is active and turns on tra-I. In the male, a
low X to autosome ratio turns on her-I; hence, tra-2 is inactive and consequently
tra-I is also inactive.
The gene tra-3 acts in concert with tra-2 in this model. Alleles of tra-3 are
similar to weak alleles of tra-2 in phenotypic consequences and epistatic interactions. However, the transformed phenotype is seen only if the wild-type gene
is absent from both mother and zygote. This means that the tra-3 gene product
alone cannot determine sexual phenotype; if it did, the maternal inheritance of
tra-3 gene product would always prevent the appearance of males in the progeny
of wild-type hermaphrodites. The data can be explained if it is assumed that
the tra-3 gene product is normally present at all times, and that it acts as a
necessary co-factor for the action of tra-2. In tra-3/tra-3 progeny of tra-3/-1hermaphrodites, enough of the wild-type gene product is inherited via the egg
to allow tra-2 action, and only in the next generation is tra-2 action prevented.
If the tra-3/tra-3 hermaphrodite is mated with a wild-type male, zygotic expression of the wild-type tra-3 gene again supplies enough product to allow tra-Z
action.
In order to explain the different phenotypes of tra-I and tra-2 alleles, it is
necessary to postulate an additional control circuit, which is not normally active
in either sex of the wild type. In the presence of active tra-2 gene product, tra-I
is fully on, but in the absence of tra-2 product, tra-I is weakly affected by the X
to autosome ratio: it is partly on in the presence of a high X to autosome ratio,
off in the presence of a low X to autosome ratio. Partial activity of tra-I would
lead to a slight repression of male development, hence to an incomplete male
SEX-DETERMINATION MUTANTS
663
phenotype such as is seen in X X animals homoz.ygous for tra-2 mutations or
and BRENNER1977).
the weak tra-l mutation, el076 (HODGKIN
The additional circuit is also necessary to explain the different interactions
of her-l with tra-l and tra-2. That is, her-l X O animals resemble her-l X X
animals, and tra-I; her-I X O animals resemble tra-I; her-I X X animals, but
tra-2; her-2 X O animals are invariably more male in phenotype than are tra-2;
her-l X X animals. This result means that there must be another pathway, different from that involving her-2 and tra-2, by which the X to autosome ratio
can influence sexual phenotype, but that this pathway is apparent only in tra-2
mutants. The simplest explanation seems to be that presented. It is difficult to
reconcile these data (and also the suppression of tra-2 mutations by her-2) with
models in which tra-2 acts after tra-I, such as those proposed by KLASS,WOLF
and HIRSH(1976) and NELSON,
LEWand WARD(1978).
It should be noted that Table 1 simplifies the data, in that there is some variability in the phenotypes of most of the mutants and mutant combinations, and
epistasis is often incomplete. For example, her-I; tra-2 X O animals are male, but
usually of lower fertility than tra-2 X O animals. Therefore, models such as that
in Figure 2 do not explain all the details of the data. Another defect of the model
is that it treats all aspects of the sexual phenotype together; whereas, it is likely
that some aspects are controlled independently. For example, the mutation
i n - I (hcI7) causes transformation of the gonad in X O animals, but has no direct
effect on the secondary sexual characteristics of X O animals (NELSON,
LEWand
WARD
1978). The gene isx-l has therefore not been included in this model, but
i t could possibly be included by drawing up parallel pathways for gonadal and
nongonadal tissue.
The only other animal for which a comparable array of sex determination
genes has been identified is Drosophila. There are some striking parallels between the two systems: in both, the primary control of sex determination is an
X to autosome ratio, but this ratio is used mainly as a signal that determines the
activity of a small number of switch genes. These in turn govern most aspects
of sexual differentiation. Epistatic interactions can be used to deduce a simple
control pathway in both organisms (BAKERand RIDGE1980). Certain types of
mutation have been identified in Drosophila that have not been observed in C.
elegans; in particular, no sex-lethal locus (CLINE 1979) has been identified in
C . elegans. The most striking difference between the two systems is that all or
most of the genes identified in Drosophila affect only the somatic sexual phenotype (e.g.,MARSH
and WIESCHAUS
1978); whereas, most of the genes identified
in C. &gam affect both soma and germ line.
I thank ROBERTHORVITZ
and JOHN WHITEfor valuable discussion, and GARYSTRUHL
for his
comments on the manuscript.
LITERATURE CITED
BAKER,B. S. and K. A. RIDGE,1980 Sex and the single cell. I. On the action of major loci
affecting sex determinationin Drosophila melanoguster. Genetics 94: 383423.
BRENNER,
S., 1974 The genetics of Cuenorhabditis eleguns. Genetics 77: 71-94.
664
J. HODGKIN
CLINE,T. W., 1979 A male-specific lethal mutation i n Drosoph:la melanogaster that transforms
sex. Develop. Biol. 72 :266-275.
HODGKIN,J. A. and S. BRENNER,1977 Mutations causing transformation o f sexual phenotype
in the nematode Caenorhabditis elegans. Genetics 86: 275-287.
HODGKIN,
J., H. R. HORVITZ
and S. BRENNER,1979 Nondisjunction mutants of the nematode
Caenorhabditis e l g a n s . Genetics 91 : 67-94.
HORVITZ,
H. R., S. BRENNER.
J. HODGKIN
and R. K. HERMAN,
1979 A uniform genetic nomenclature for the nematode Caenorhabditis elegans. Molec. Gen. Genet. 175 : 129-133.
KIMBLE,J. and D. HIRSH,1979 Post-embryonic cell lineages of the hermaphrodite and male
gonads in Caenorhabditis elegans. Develop. Biol. 70: 396-417.
KLASS,M., N. WOLFand D. HIRSH,1976 Development of the male reproductive system and
sexual transformation in the nematode Caenorhabditis eleguns. Develop. Biol. 52 : 1-18.
MCCARREY,
J. R. and U. K. ABBOTT,1979 Mechanisms of genetic sex determination, gonadaI
sex differentiation, and germ cell development in animals. Advan. Genet. 20: 217-290.
MADL,J. E. and R. K. HERMAN,
1979 Polyploids and sex determination in Caenorhabdltis
elegans. Genetics 93 :393-402.
MARSH,J. L. and E. WIESCHAUS,
1978 Is sex determination in germ line and soma controlled by
separate genetic mechanisms? Nature 272 : 244-251.
NELSON,G. A., K. K. LEWand S . WARD,1978 Intersex a temperature sensitive mutant of the
nematode Caenorhabditis elegans. Develop. Biol. 66: 386-409.
NIGON,V., 1949 Effets de la polyploidie chez un Nematode libre C. R. Acad. Sci., Paris. 228:
1161-1 162.
RIDDLE,D. L. and S. BRENNER,
1978 Indirect suppression in Camorhabditis elegans. Genetics
89: 299-314.
J. E., 1976 Post-embryonic development in the ventral cord of Caenorhabditis elegans.
SULSTON,
Phil. Trans. Roy. Soc. Lond., B. 275: 287-297.
SULSTON,
J. E. and H. R. HORVITZ,
1977 Post-embryonic cell lineages in the nematode Caenorhabditis elegans. Develop. Biol. 56: 110-156.
WATERSTON,
R. H. and S. BRENNER,
1978 A suppressor mutation in the nematode acting on
specific alleles of many genes. Nature 275: 715-719.
Corresponding editor. R. K. HERMAN