Download Autonomy and nonautonomy of sex determination in

863
Development 112, 833-879 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Autonomy and nonautonomy of sex determination in tripioid intersex
mosaics of C. elegans
PEPPER SCHEDIN*, CRAIG P. HUNTER and WILLIAM B. WOODt
Department of Molecular, Cellular and Developmental Biology, Box 347, University of Colorado, Boulder, Colorado 80309, USA
•Present Address: AMC Cancer Research Nutrition Center, 1600 Pierce Street, Lakewood, Colorado 80214, USA
t Corresponding author
Summary
The primary sex-determining signal in Caenorhabditis
elegans is the ratio of X chromosomes to sets of
autosomes (X/A ratio), normally 1.0 in hermaphrodites
(XX) and 0.5 in males (XO). XX triploids (X/A=0.67)
are males, but if these animals carry a partial
duplication of the X chromosome such that X/A=0.7,
they develop as intersexes that are sexually mosaic. We
have analyzed these mosaics using Nomarski microscopy
and in situ hybridization to obtain information on
whether sex determination decisions can be made
independently in different cells and tissues, and when
these commitments are made. The observed patterns of
male and female cells in individual animals indicate that
sex determination decisions can be influenced by
anterior-posterior position and that sex determination
decisions can be made as late as the third larval stage of
postembryonic development. Although these decisions
clearly can be made independently in different lineages,
they show substantial biases toward one sex or the other
in individual animals. We interpret these results to
suggest that sex determination in C. elegans is not
entirely cell autonomous.
Introduction
The primary signal that determines sex in C. elegans
is the ratio of the number of X chromosomes to the
number of sets of autosomes (X/A ratio). Wild-type
diploid hermaphrodites have two X chromosomes and
two sets of autosomes (2X;2A), whereas diploid males
have only one X chromosome (1X;2A). 2X;4A
tetraploids and 2X;3A triploids develop as fertile
males. 3X;4A tetraploids develop as fertile hermaphrodites, as do 4X;4A tetraploids and 3X;3A triploids.
Thus X/A ratios of g0.75 lead to hermaphrodite
development, and ratios of g0.67 lead to male
development (Nigon, 1951; Madl and Herman, 1979).
The X/A ratio determines the activities of a set of
interacting regulatory genes (Fig. 1), which ultimately
dictate the activity of the tra-1 gene, the terminal
regulator of sexual differentiation (Hodgkin, 1988;
Villeneuve and Meyer, 1990). The activity of the tra-1
gene is required cell autonomously for hermaphrodite
development (Hunter and Wood, 1990). However, it is
not known whether the sex determination process that
sets the state of tra-1 is executed autonomously by each
cell in the embryo, or whether information regarding
X/A or the states of the upstream sex determining
genes is communicated between cells. We have
approached this question by artificially setting the X/A
In the soma of adult C. elegans, more than 30% of the
959 cells in hermaphrodites and 40 % of the 1031 cells in
males make up tissues that exhibit sexual dimorphism
or sex-specific biochemical differences. These tissues
include the hypodermis, musculature, intestine, somatic gonad, germ line and nervous system (Sulston
and Horvitz, 1977; Sulston et al. 1980; Kimble and
Sharrock, 1983). The hermaphrodite has a simple tail, a
vulva arising from the midventral hypodermis, and a
symmetrical gonad with an anterior and a posterior
reflexed arm, in which the germ line produces sperm
during the fourth larval (L4) stage and oocytes in the
adult. In addition, the hermaphrodite intestine late in
the L4 stage begins to produce vitellogenins, which are
secreted into the pseudocoelom and taken up by
developing oocytes during oogenesis (Sharrock, 1983;
Kimble and Sharrock, 1983; Blumenthal et al. 1984).
The male has a more complex tail and accompanying
specialization of the nervous system for copulation, in
which the male is the activ.e partner. The male gonad
consists of a single reflexed arm, and the germ line
produces sperm only. The male intestine does not
synthesize vitellogenins.
Key words: Caenorhabditis elegans, development,
differentiation, commitment, in situ hybridization,
mosaicism, gradient, sex determination, intersex,
vitellogenins.
864
P. Schedin, C. P. Hunter and W. B. Wood
—
X/A
\
fem-1
fem-2 — /
' fan-3
'
.?
Sexual
V
Dosage
Compensation
Fig. 1. Regulatory relationships of the genes controlling
sex determination in C. elegans. Based on the results of
genetic analysis, each gene or set of genes negatively
regulates the succeeding gene or set in the pathway at an
unspecified level. Modified from Villeneuve and Meyer,
1990.
this result to indicate that assessment of the X/A ratio
could be made independently in different tissues during
development. In earlier experiments we confirmed the
production of several classes of mosaic intersexes in
2X;3A strains carrying duplications of about 20 % of
the X chromosome (Wood etal. 1985). We present here
a more extensive analysis of such intersexual animals,
whose patterns of mosaic development provide information regarding both the cell autonomy and timing of
sex determination decisions.
Materials and methods
ratio to approximately 0.7, between the values that
unambiguously dictate male or hermaphrodite development, so that the animals develop as mosaics of male
and hermaphrodite tissues (Madl and Herman, 1979).
In these animals, we have examined the resulting
patterns of sexual differentiation at the cellular level. If
sexual identity decisions (tra-1 ON or OFF) are made
autonomously then clones of lineally related cells will
express the same sexual phenotype. Alternatively, if
sexual identity decisions involve non-autonomous interactions then non-clonally related cells will consistently
express identical sexual phenotypes. If decisions must
be made early, then clones expressing the same sexual
phenotype will be large; if the decision can be reversed
or postponed until later in development, then clones
may be small.
This approach was taken earlier with Drosophlla
(Bridges, 1925), in which sex is also determined by the
X/A ratio, normally 2X:2A in females and 1X:2A in
males. Triploid animals with two X chromosomes
(2X:3A) develop as intersexual' mosaics, with, large
patches of male and female tissue in the same
individual. This observation is consistent with an
ambiguous X/A signal that is assessed autonomously by
individual cells or groups of cells fairly early in
development, at about the cellular blastoderm stage,
such that sexually determined progenitors give rise to
patches of contiguous, similarly sexed progeny cells (for
review see Baker and Belote, 1983). More recent work
indicates that assessment of the X/A ratio involves
irreversibly setting the expression state of the cellautonomous Sex lethal {Sxl) gene. In addition to
regulating downstream sex determination genes, Sxl
also sets the level of X chromosome expression,
ensuring that similar levels of X-linked gene products
are made in both XY and XX cells (Cline, 1984).
In C. elegans, Madl and Herman (1979) showed that
if 2X;3A animals carry one copy of an X duplication
representing about 20 % of the chromosome, then they
exhibit intersexual phenotypes. If such animals carry a
smaller X duplication, then they develop as morphologically normal males, like 2X;3A animals without a
duplication; if they carry an even larger duplication,
they develop as complete hermaphrodites, like 3X;4A
animals. These authors observed wide variability of
sexual phenotypes in the intersexual animals, ranging
from nearly fully hermaphrodite to nearly fully male,
with examples of mosaic individuals. They interpreted
Nematode strains, nomenclature and culture
The wild type of Caenorhabditis elegans var. Bristol (designated N2) used in these studies was originally obtained from
the Cambridge England strain collection (Brenner 1974).
Robert Herman (University of Minnesota) provided the
tetraploid strain SP344, of genotype dpy-11 (e224)V;unc3(el51)X, and the two diploid strains SP75, of genotype
mnDP25(X:l);unc-3(el51)X, and SP116, of genotype
mnDp9(X:l);unc-3(el51)X. The duplications mnDp25 and
mnDp9 each represent approximately 20% (genetically) of
the X chromosome and are actually translocations stably
attached to linkage group (LG) I (Herman et al. 1979).
Genetic nomenclature in this paper conforms to established
conventions for C. elegans (Horvitz et al. 1979), with the
following exception: because the convential representation of
karyotypes (nA;mX) leads to confusion when discussing X/A
ratios, we have reversed this representation to nX;mA, as
used for Drosophila karyotypes; we urge that this latter
convention be adopted for C. elegans as well.
General culture methods have been compiled by Sulston
and Hodgkin (1988). To obtain synchronized animals,
embryos isolated by hypochlorite treatment of gravid hermaphrodites were plated onto NGM plates seeded with OP50
and allowed to hatch at 20 °C. Every hour the plates were
gently flooded with 5 ml of M9 salts to dislodge the newly
hatched first-stage larvae, which were removed by aspiration
and replated to provide synchronous populations.
Developmental age of worms was determined by microscopic (Nomarski) examination of gonads to determine stage
of germ cell maturation, number of somatic gonad cells and
number of vulval precursor cells (Hirsh et al. 1976; Wolf et al.
1978).
Construction of triploid intersexes
Triploid intersex animals were produced as follows, where Dp
indicates either mnDp9 or mnDp25, both of which carry unc-3
(+)X (Madl and Herman, 1979). N2 males were mated to
Dp/Dp\unc-3/unc-3 hermaphrodites, and the male progeny,
of genotype Dp/ + ;unc-3/0, were mated to tetraploid dpyll;unc-3 hermaphrodites. The latter mating yields the
following classes of non-Dpy outcross progeny (in addition to
Dpy Unc tetraploid hermaphrodite and occasional male self
progeny):
(1) Unc triploid hermaphrodite (3X;3A, not carrying Dp),
of genotype dpy-11 / dpy-11 /+;unc-3 / unc-3 / unc-3,
(2) Unc triploid males (2X;3A, not carrying Dp), of
genotype dpy-11 /dpy-11 /+;unc-3/unc-3/0,
(3) non-Unc triploid hermaphrodites (3X;3A, carrying Dp)
of genotype Dpf+ /+;dpy-ll/dpy-11 /+;unc-3/unc-3/unc-3,
and
(4) non-Unc triploid males, intersexes, and possibly
Sex determination in triploid intersex mosaics of C. elegans
hermaphrodites (2X;3A, carrying Dp) of genotype Dp/+/
+ ;dpy-ll/dpy-ll/+;unc-3/unc-3/0.
Non-Unc progeny identified in the dissecting microscope as
males or intersexes (class 4) on the basis of morphology were
picked for analysis by in situ hybridization and Nomarski
microscopy. Note that non-Unc externally hermaphrodite
animals of class 4, if present, could not have been
distinguished from those of class 3 and therefore would not
have been picked. As a result the population of triploid
intersexes analyzed could have a bias against fully hermaphrodite pheno types.
Nomarski microscopy
Triploid intersex animals were examined by Nomarski
microscopy to determine the phenotypic sex of the somatic
gonad, germ line, tail hypodermis and ventral hypodermis
(presence or absence of vulva) based on gross morphology.
Gonads with two reflexed arms were scored as hermaphrodite; those with a single arm were scored as male. Germ cells
that were large, round, with prominent nuclei and usually
arranged in single file in the proximal gonad were scored as
oocytes. Small round germ cells with very compact nuclei
were scored as sperm. A protrusion of the ventral hypodermis
near the middle of the animal was scored as a vulva; no
distinction was attempted between functional vulvae and
nonfunctional protrusions of vulval cells. (Although the signal
for vulva formation originates from the somatic gonad, the
response of the ventral hypodermis appears to be sex-specific,
resulting in a functional vulva if the hypodermis is female and
a non-functional protrusion of hypodermal cells if it is male;
Hodgkin, 1987.) Tails with a hermaphrodite-specific tail whip,
either full length or stunted, were scored as hermaphrodite.
Tails with any male-specific rays or fan structure were scored
as male.
In situ hybridization
Sample preparation
Intestines and gonads were dissected from adult animals
according to the method of Kimble and Sharrock (1983),
mounted on a subbed (0.1% gelatin, 0.01% potassium
chromate) hybridization slide, - lightly squashed with a
coverslip, and frozen in liquid nitrogen as described by
Edwards and Wood (1983). The coverslip was removed and
the frozen tissues adhering to the slide were fixed in either
ethanol: acetic acid (3:1) at 4°C for 15 min or 4 % paraformaldehyde at 4°C for 15 min, and then dehydrated in ethanol.
Hybridization
Fixed tissues were hybridized to either a nick-translated
plasmid DNA or a primer-extended purified fragment of
either the vit-5 or vit-6 gene probes. The C. elegans
vitellogenin clones used in this study, pACYC184 and
pl3F9-2, were gifts from T. Blumenthal (Indiana University,
Bloomington). pACYC184 contains a 1.3 kb £coRI-//indIII
fragment of the vit-5 5'-upstream sequence and coding
sequence cloned into PUC-8. pl3F9-2 is a 1.1kb EcoRlHindlU. fragment of the vit-6 coding region cloned into PUC8. The hybridization was carried out as described by
Albertson (1984) with the following modifications. Probes
were labelled with 35S (107-108ctsmin~1/ig~1) rather than
biotin and were resuspended in 70% formamide, 0.4 M NaCl,
1.6mM EDTA, 0.04M Na2HPO4, 10mM DTT and 5xDenhardt's solution at 100-200//I final volume. Probe solutions
were heated at 65 °C for 5 min and quick-cooled on ice for
lmin. Approximately K^ctsmin" 1 in 10/il of hybridization
solution was applied to each sample, spread over the slide
865
with an 18-mm round coverslip, and put in an air-tight box
equilibrated with 70% formamide, at room temperature
overnight. Slides were given 5 washes of 15 min each in 70 %
formamide, lxSPE (0.165M NaCl, 20mM NaH2PO4, lmM
EDTA) one wash for 15min in lxSPE, and two more for
5min each in lxSPE, dried, and mounted for autoradiography.
Detection
Samples were mounted and autoradiographed according to
Edwards and Wood (1983). Exposure time was 1 day to 2
weeks depending on specific activity of probe. To make cell
nuclei visible by microscopy, developed slides were stained
with 1 /igml"1 aqueous solution of the DNA stain diamidinophenylindole dihydrochloride (DAPI, obtained from Boehringer-Mannheim).
Microscopy
A Zeiss photomicroscope was used to photograph the slides.
Autoradiographic grains were viewed either as bright spots on
a black background using dark-field optics or as dark spots on
a white background using bright-field optics. DAPI-stained
nuclei were observed under 365 nm epi-illumination. Cell
nuclei on DAPI-stained slides could be seen simultaneously
with autoradiographic grains by viewing with both visible
dark-field and 365 nm epi-illumination. In dissected, DAPIstained intestines, cell pairs int2 through int9 were identified
by counting nuclei posterior to intl. The intl quadruplet was
identified either by its association with the pharynx or by the
four characteristic intl nuclei. Only animals with 12 or more
dissected intestinal nuclei were scored to obtain the data
presented for in situ hydribidization experiments.
Results
Scoring of sex-specific differences in adult animals
To analyze intersexual animals, we scored sex-specific
differentiation in five tissues derived from four of the six
embryonic founder cells: AB, MS, E, and P 4 , which are
generated during the first four rounds of cleavage
division in embryogenesis (Fig. 2). The tail ectoderm,
us
_£*
TAL
VULVA
QERMLME
Fig. 2. Early embryonic cleavages that generate the six
founder cells: AB, MS, E, C, D and P4. Tissues showing
obvious sexual dimorphism or sex-specific differentiation
are derived from founder cells AB, MS, E, and P 4 .
Although tissues derived from C and D may also undergo
sex-specific differentiation, the differences are more subtle
and were not scored in these experiments. Adapted from
Sulston etal. (1983).
866
P. Schedin, C. P. Hunter and W. B. Wood
Table 1. Distinguishing characteristics and embryonic origins of sexually dimorphic tissues
Sexually
dimorphic
tissues
Somatic gonad
Germ line
Tail ectoderm
Ventral hypodermis
Intestine
Wild-type
hermaphrodite
phenotype
Wild-type
male
phenotype
Embryonic
origin
of tissue"
Two-armed
Oocytes+sperm
Simple: whip
Vulva present
vit mRNA present
Single-armed
Sperm only
Complex: rays+fan
Vulva absent
vit mRNA absent
MS
f«
AB
AB
B
" Founder cell. See Fig. 2.
midventral hypodermis, somatic gonad and germ line
we scored by Nomarski microscopy on the basis of
morphological differences described in the Introduction
and summarized in Table 1. In addition we could score
the intestine for synthesis of vitellogenins, using cloned
vit gene fragments (kindly furnished by T. Blumenthal,
Indiana University) as probes in an in situ hybridization
assay for presence of vitellogenin transcripts in individual cells. Of the six members in the vitellogenin gene
family vit-l-vit-6, the vit-5 probe hybridizes to transcripts of the highly conserved vit-l-vit-5 sub-family,
while the vit-6 probe is specific for the more divergent
vit-6 transcripts (Blumenthal et al. 1984; Spieth and
Blumenthal, 1985; Heine and Blumenthal, 1986).
To demonstrate the sex and tissue specificity of
vitellogenin mRNAs, we hybridized the vit-5 probe to
dissected intestines and gonads of both diploid and
triploid male and hermaphrodite animals as described
in Materials and methods. Among a total of 145 diploid
(2X;2A) and 40 triploid (3X;3A) adult hermaphrodites,
all but a single animal showed strong hybridization of
the labeled probe to all cells in dissected portions of the
intestine and no hybridization to the gonad (Fig. 3A
and Table 2). In contrast, a total of 55 diploid (1X;2A)
and 45 triploid (2X;3A) adult males showed no signal
over either tissue (Fig. 3B and Table 2). These results
confirm the sex specificity of vitellogenin gene expression observed by Blumenthal et al. (1984) using
other methods. They also show that the tissue
specificity of vitellogenin protein synthesis observed by
Kimble and Sharrock (1983) reflects a corresponding
specificity of vitellogenin gene transcription.
Construction of triploid intersexes
We obtained animals with an X/A ratio of about 0.7 as
described in Materials and methods, by crossing diploid
(1X;2A) males carrying an autosomally attached
duplication of the right arm of X (mnDp9 or mnDp25),
representing about 20 % (genetically) of the chromosome, to marked autosomally tetraploid hermaphrodites (4X;4A). We picked all unmarked progeny
showing any discernible masculine characteristics as
representing 2X;3A animals carrying the duplication
(2X;3A+Dp). The markers used allowed us to distinguish these from all other progeny genotypes, except
that externally fully hermaphrodite 2X;3A+Dp animals, if present, would have been indistinguishable
from 3X;3A hermaphrodites and would not have been
Fig. 3. Sex and tissue specificity of vitellogenin mRNA
expression demonstrated by in situ hybridization. A
vitellogenin gene (vit-5) probe was hybridized as described
in Materials and methods to dissected intestines and
gonads of N2 hermaphrodites and N2 males. (A) Darkfield-epifluorescence image of a dissected 2X;2A wild-type
hermaphrodite, showing intense labeling of the intestine
and no labeling of the gonad. (B) Dark-fieldepifluorescence image of a dissected 1X;2A male, showing
no labeling of either intestine or gonad. Photographed as
in A.
picked. Therefore, the populations of triploid intersexes analyzed may be biased against hermaphrodite
phenotypes. We have pooled results obtained with
mnDp9 and mnDp25 in the experiments described
below, since both duplications resulted in similar
degrees and variability of masculinization.
Sex determination in triploid intersex mosaics of C. elegans
867
Table 2. Intestinal phenotypes in control and intersex animalsa
Karyotype,
Sex6
2X;2A, h
3X;3A, h
1X;2A, m
2X;3A, m
2X;3A;mnDp25, isx
2X;3A;mnDp9, isx
Animals
scored
All cells
expressing
vitellogenins
No cells
expressing
vitellogenins
144
0
l
145
40
55
45
85
78
39
0
0
€1
»
ss
Mosaic
intestines
i
a
•i
4S
27
U
m
n
fi
' Only animals with 12 or more intestinal nuclei dissected were included in the analysis.
b
h=hermaphrodite; m=male; isx=intersex.
Triploid intersex animals show tissue mosaicism for
sexual phenotype
We scored tissue sexual phenotypes in a total of 386
animals identified according to the above criteria as
triploid intersexes. Of these, 186 were live animals,
scored by Nomarski microscopy for hypodermal structures, gonad and germ line only; the remaining 200
were dissected and fixed for hybridization. In most of
the fixed animals, we were able to score one or more of
the above tissues by morphological criteria, in addition
to scoring the intestine for vitellogenin transcripts.
The five tissues scored in these animals generally
showed clear predominance of either male or hermaphrodite differentiation. Many animals were clearly
mosaic, with both male and hermaphrodite tissues in
the same individual. For example, Fig. 4A shows an
animal whose intestine contains vitellogenin transcripts, yet whose single-armed gonad and germ line
appear fully male. Fig. 4B shows the converse: an
animal with a male intestine (no vitellogenin synthesis)
and a female germ line. The germ line is scored as
female because the germ cells are large, have large
nuclei, and are ordered in a single row, all traits specific
to oocytes. The oocytes are smaller than normal,
possibly due to lack of vitellogenins.
Sexual identity decisions can be tissue-autonomous,
with one exception
The population of intersexes displayed a variety of
sexual phenotypes, ranging from morphologically fully
male to almost fully hermaphrodite. Between these
extremes, we observed animals displaying all possible
pairwise combinations of differently sexed tissues but
one, as shown in Table 3. We conclude that in general
the sex determination decision of one tissue is not
dictated by the sex of another, that is, decisions appear
to be tissue-autonomous. The ventral hypodermis was
exceptional in that vulva formation correlated essentially completely with the sexual phenotype of the
somatic gonad. In 208 of 209 animals, the presence of an
hermaphrodite (two-armed) gonad was associated with
the presence of a vulva, and the presence of a male
gonad with no vulva, as expected from the known
induction of vulval development by the anchor cell of
the hermaphrodite gonad (Kimble, 1981; Steinberg and
Horvitz, 1986).
Fig. 4. Tissues of opposite sex in mosaic intersexes. A vit-5
probe was hybridized as in Fig. 3 to dissected 2X;3A
animals that carried the duplication mnDp25. (A) Darkfield epifluorescence image showing an intersexual animal
with an hermaphrodite intestine as scored by labeling, a
male single-armed gonad as scored by morphology, and a
male germ line as scored by presence of sperm only.
(B) Dark-field-epifluorescence image of the converse
intersex. Intestine is male as scored by lack of labeling,
and germ line is hermaphrodite as scored by presence of
oocytes.
Tissues in an individual appear biased toward one sex
or the other
Among the animals in the intersex population examined, different tissues showed different frequencies of
868
P. Schedin, C. P. Hunter and W. B. Wood
Table 3. Pairwise combinations of oppositely sexed tissues seen in triploid intersex animals3
Tail
Intestine
Germ line
Somatic gonad
Ventral hypodermis
Tail
38
16
7
10
_
Ventral
hypodermis
28
12
<0.5
—
Somatic
gonad
27
10
-
Germ line
Intestine
23
* Each entry indicates the percent of animals showing opposite sexual identities for two tissues among the total number of animals in
which these two tissues could be scored. The total scored for each combination ranged from 180 to 330, except for the combination
intestine-ventral hypodermis, for which only 32 animals could be scored (see Table 5). See text and Table 1 for scoring criteria.
Table 4. Distribution of tissue sexual phenotypes in
triploid intersex animals
Experimental
Tissue
Sex"
Number
observed
Intestine
h
m
sum
Germ line
h
m
sum
h
m
sum
142
87
229
141
225
366
102
255
357
h
m
sum
h
m
sum
57
154
211
82
304
386
Somatic gonad
Ventral hypodermis
Tail
section; for example, hermaphrodite intestine-male
gonad is much more frequent than male intestinehermaphrodite gonad.
Frequency ± Error11
62%
38%
100%
6%
6%
39%
61%
100%
5%
5%
29%
71%
100%
27%
73%
100%
21%
79%
100%
5%
5%
6%
6%
4%
4%
° h=hermaphrodite, m=male
b
normal deviate, calculated as two standard deviations around
the mean assuming a binomial distribution: 1.96 (pXq/N) 1 ' 2 .
male and hermaphrodite phenotypes (Table 4). For
example, the percentages of animals showing the
hermaphrodite phenotype for intestine, germ line, and
somatic gonad were 62 %, 39 % and 29 %, respectively.
Thus tissues appear to differ in their response to an
intermediate primary sex-determining signal (see Discussion).
Most animals in the intersex population appeared to
be predominantly male or predominantly hermaphrodite. To quantitate this apparent correlation between
tissue sexual phenotypes, we compared the observed
frequencies of various pairwise combinations with the
frequencies expected assuming complete independence
(Table 5), calculated from the data in Table 4. Correlations are apparent; for every pair of tissues, there is a
clear bias toward the same-sex and against the oppositesex combination, showing that although tissue sexual
identity decisions can be made independently (Table 3),
these decisions are often correlated in individual
animals. Differences in the degree of non-correlation
are as expected from the apparently different responsiveness of the tissues described in the preceding
Internal mosaicism for vitellogenin expression in the
intestine
We examined 163 fixed intersex animals that had been
dissected so that at least half of the intestinal cells were
exposed, a sufficient number of cells to detect progeny
from all terminal intestinal cell divisions (see Fig. 5).
Among these animals, 26 exhibited mosaicism within
the intestine; that is, some cells showed the high level of
labeling characteristic of hermaphrodites and other
cells showed no labeling (Table 2). Fig. 6 depicts such a
mosaic intestine. The anterior end, up to and including
the cell pair designated int4, shows hybridization, while
the adjacent pair (int5) and cells posterior to it show
none (see Fig. 5 for nomenclature of intestinal cells).
The patterns of intestinal mosaicism in these animals
showed a consistent anterior-posterior polarity. In all
26 mosaics the intestinal cells, beginning at the anterior
of the intestine, showed labeling to a certain point,
beyond which no cells were labeled. In addition,
labeled cells were always contiguous. The position of
the boundary (/) between labeled and unlabeled cells
varied among different individuals from int2/int3 to
int6/int7. Cells within an intestinal pair also occasionally differed; Fig. 6C shows an intestine in which only
one cell of the int4 pair contained vitellogenin
transcripts. In some mosaic intestines, the level of
labeling in individual hybridizing cells appeared graded,
again always with the heavier labeling toward the
anterior. This phenomenon was observed with both the
generic vit-5 and the gene-specific vit-6 probes. It may
represent the first case of truly intersexual cellular
differentiation described in C. elegans.
In an attempt to control for an alternative explanation for the observed patterns, such as a general lack
of metabolic activity in the posterior intestinal cells, we
assayed intestines of triploid intersex and control
animals for expression of two non-sex-limited markers,
gut-specific esterase (Edgar and McGhee, 1986) and an
intestinal antigen stained by the monoclonal antibody
SP37 (S. Strome, personal communication). We found
no mosaic intestines among 200 triploid intersexes
scored for gut-specific esterase and 100 scored for
Sex determination in triploid intersex mosaics of C. elegans
869
Table 5. Distribution of pair-wise combinations of sexually differentiated tissues in triploid intersex animals
Tissue
pair
Tail,
Germ line
Tail,
Gonad
Tail,
Intestine
Tail,
Ventral hypodermis
Germ line,
Gonad
Germ line,
Intestine
Germ line,
Ventral hypodermis
Gonad,
Intestine
Gonad,
Ventral hypodermis
Intestine,
Ventral hypodermis
Sexual
phenotype
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
h,h
h,m
m,h
m,m
sum
Number
observed
72
2
48
200
322
74
3
21
231
329
41
1
66
72
180
37
3
18
151
209
87
30
3
210
330
73
7
37
73
190
52
20
2
111
185
49
3
42
77
171
55
0
1
153
209
13
9
0
10
32
Expected
frequency
±errorb
22%
1%
15%
62%
100%
22%
1%
6%
70%
100%
23%
1%
37%
40%
100%
18%
1%
9%
72%
100%
26%
9%
1%
64%
100%
38%
4%
19%
38%
100%
28%
11%
1%
60%
100%
29%
2%
25%
45%
100%
26%
0%
0%
73%
100%
41%
28%
0%
31%
100%
5%
1%
4%
5%
5%
1%
3%
5%
6%
1%
7%
7%
5%
2%
4%
6%
5%
3%
1%
5%
7%
3%
6%
7%
6%
4%
1%
7%
7%
2%
6%
7%
6%
0%
1%
6%
17%
16%
0%
16%
Expected
frequency
±errorc
8%
13%
30%
48%
100%
6%
15%
23%
56%
100%
13%
8%
49%
30%
100%
6%
16%
21%
57%
100%
11%
28%
18%
44%
100%
24%
15%
38%
23%
100%
10%
28%
17%
45%
100%
18%
11%
44%
27%
100%
8%
21%
19%
52%
100%
17%
45%
10%
28%
100%
Correlation"1
6%
6%
6%
6%
+
6%
6%
6%
6%
+
7%
7%
7%
7%
+
7%
7%
7%
7%
+
7%
7%
7%
7%
+
8%
8%
8%
8%
+
+
+
+
+
+
+
8%
8%
8%
8%
+
8%
8%
8%
8%
. +
8%
8%
8%
8%
+
8%
8%
8%
8%
+
+
+
+
+
* h=hermaphrodite, m=male
b
normal deviate; see Table 4.
c
Frequencies expected for pairs of tissues, if tissue sexual choices were independent; calculated as the products of the individual
frequencies for those tissues shown in Table 4. Errors in expected frequencies were calculated by propagating the errors shown in Table 4.
d
Correlation is indicated as ' + ' if more animals had the designated phenotype, and ' — ' i f fewer animals had the designated phenorype
than expected. All differences from expected values are highly significant (/ ) <0.001) with the exception of those for Tail, Intestine m,h
and m,m (P<0.025) and those for Intestine, Ventral hypodermis h,m (P<0.2), m,h, (P<0.1), and m,m (not significant).
expression of the intestinal antigen; both markers were
expressed uniformly throughout the intestine in all
animals.
Vitellogenin gene expression is initiated from posterior
to anterior
To ask if the observed polarity of transcript presence in
870
P. Schedin, C. P. Hunter and W. B. Wood
2V ) C 3V ) C 4V ) C 5L ) C 6L ) C 7L
Adult
20 cells
30-34 nuclei
pharynx
Fig. 5. Diagrammatic representation of intestinal cell lineage and morphology. The upper diagram shows the lineages of
the intestinal cells on the left side of the animal, deriving from the cells Eal and Epl (Sulston et al. 1983). The lineages on
the right side, deriving from Ear and Epr, are identical and have been omitted for simplicity. The lower portion of the
figure shows schematically the structure of the adult intestine, which consists of 20 cells arranged in 9 structural units
(designated intl through int9 as shown): 1 quadruplet and 8 pairs of cells, 14 of which are binucleate. Cross-hatched cells
are those deriving from the left side of the lineage. Because cells on each side of the animal exchange places with their
neighbors as shown between the 16- and 20-E-cell stages of intestinal development, the descendants of a given intestinal
precursor cell generally do not occupy adjacent positions in the adult intestine. For example, descendants of the Ea cell
(stippled) give rise to intl, int2, int3, and int5; the intervening unit int4 is derived from descendants of Ep.
mosaic intestines might reflect a polarity in the
initiation of vitellogenin gene expression, we examined
the onset of vit transcript accumulation in normal
diploid hermaphrodites. Vitellogenins are first produced in the hermaphrodite intestine in late L4 larvae,
just before the onset of oogenesis (Sharrock, 1983;
Kimble and Sharrock, 1983; Blumenthal, 1984). To
determine whether vitellogenin transcripts are also first
produced at this time, we dissected synchronized N2
larvae at various stages and assayed them by in situ
hybridization with the vit-5 probe.
We could first detect vitellogenin transcripts in late
L4 larvae, shortly after condensation of the germ-line
nuclei undergoing spermatogenesis in the proximal arm
of the gonad (Table 6). However, the transcripts
appeared with a polarity opposite to that observed in
adult mosaic intersexes. In animals assayed at the time
of onset, only the posterior cells of the intestine were
generally labeled; the most anterior cell pairs, intl and
int2, were consistently unlabelled (Fig. 7). At slightly
later stages, all cells were labeled, but a gradient of
labeling intensity was still apparent until the L4/adult
molt, when the labeling became uniform. Vitellogenins,
assayed by gel electrophoresis of proteins in parallel
experiments with the same synchronized populations,
became detectable about two hours after the first
vitellogenin transcripts (data not shown).
Characterization of internal mosaicism in germ line,
mesodermal and ectodermal tissues
In the preceding analyses, we scored tissues other than
the intestine only as hermaphrodite versus partially or
completely male, with no attempt at detailed characterization. To detect possible internal mosaicism in these
tissues, we examined over 300 additional intersex
mosaics by Nomarski microscopy. We scored these
animals for all the sex-specific structures deriving from
the postembryonic blast cells, listed in Table 7 (Sulston
and Horvitz, 1977; Sulston etal. 1980; 1983). In addition
to the germ line, clonally derived from P 4 , and the
somatic gonad, derived from the MS lineage, these
structures include the sex muscles (scored using
polarizing optics), derived postembryonically from the
M blast cell of the MS lineage, and several ectodermal
structures derived postembryonically from blast cells of
the AB lineage: the vulva in hermaphrodites and
various components of the tail in males (Figs 3 and 7;
Sex determination in triploid intersex mosaics of C. elegans
871
Fig. 6. Mosaidsm for vitellogenin mRNA expression
within the intestines of triploid intersexes. A vit-5 probe
was hybridized to dissected animals as in Fig. 4.
(A) Epifluorescence image (800 x) showing DAPI-stained
nuclei. (B) Dark-field image of the same preparation as in
A, showing intense labeling over the anterior intestine
and no labeling of the posterior intestine. (C) Dark-fieldepifluorescence image (2OO0x) of DAPI-stained intestine
from a mosaic animal showing hybridization to only one
cell of the int4 cell pair.
Table 6. Onset of vitellogenin gene expression in N2 hermaphrodite intestines
Animals expressing vit genes as
measured by:
Stage
Hours at
25°C ± l h
Mid L3
Late L3
Early L4
Mid L4
22
26
28-30
32
Late L4
L4 Lethargusyoung adult
Young adult
32-34
35
37
Anatomical marker events"
hybridizationb
presence of
vitellogeninsc
about 60 primordial germ cells
50% of gonads beginning 180° turn
turn completed in 25 % of gonads; early vulval divisions
distal arm core formation; chromosome condensation in
nuclei of proximal arm
spermatids present, uterus near completion
germ cell nuclei in single file at loop, no oocytes
0/19
0/10
0/21
H
—
1/54
41/76d
10/10°
r*
-*
#
oocytes in both arms
11/11
#•
* The developmental stage of the gonad and germ line was determined to monitor the synchrony of the population.
b
Number of animals with positively hybridizing intestines using vit-5 probe.
c
Determined by SDS-PAGE of proteins from synchronized populations (data not shown).
d
30 of 41 intestines scored positive were mosaic: posterior E cells hybridized, anterior cells did not.
* 3 of 10 intestines scored positive were mosaic as in ( d ).
see Hunter and Wood, 1990 for more detailed lineage
diagrams).
In general the results corroborated those observed
for the whole organ phenotypes described above;
however, many of the lineages scored in these
experiments diverge during late embryogenesis and
larval development, increasing the resolution of the
analysis. The structures deriving from a given blast cell
were usually clearly hermaphrodite or male (Table 7).
Although we again observed a strong preference for
same-sex decisions among different blast-cell-derived
structures in individual animals (Table 8), we also
found instances of all pairwise combinations of sexual
phenotypes, indicating that the postembryonic blast
872
P. Schedin, C. P. Hunter and W. B. Wood
Fig. 7. Vitellogenin mRNA synthesis is initiated from posterior to anterior during the fourth larval stage. A vit-5 probe
was hybridized to dissected intestines from L4-stage N2 animals (32-34h at 25 CC). (A) Epifluorescence image showing
DAPI-stained intestinal nuclei. (B) Dark-field image of the preparation in A showing labelling over int5, less labeling over
int4, over the more anterior intl, int2, or int3. (C) Epifluorescence image of a second preparation showing DAPI-stained
intestinal nuclei. (D) Dark-field image of the preparation in (C) showing a high level of labeling over int5, possibly less
labeling over int4, less labeling over int3, very little labeling over int2, and no labeling over intl.
cells can make independent sexual identity decisions.
For example, the lateral (V5, T) and ventral (P3-8,
P10) hypodermal cell lineages diverge at about the 300cell stage (Sulston et al. 1983). Since the structures
derived from these cells can express different sexual
phenotypes, these cells must be able to make sexual
identity decisions as late as the end of the cell
proliferation stage of embryogenesis.
Some of the phenotypes that we observed for the
tissues and structures in Table 7 were intersexual,
suggesting that sexual identity decisions can be made
during postembryonic divisions in at least some blast
cell lineages. In some cases the mosaic phenotypes
suggested that, as in the intestine, anterior-posterior
position can affect the sexual identity of cells that divide
postembryonically. For example, mosaicism within the
germ-line was observed in five animals, all of which had
the normal bilobed hermaphrodite somatic gonad
morphology. In normal hermaphrodite development
the germ-line precursor cells Z2 and Z3 undergo many
rounds of proliferative division, beginning in the LI
larval stage. At first the descendants of Z2 and Z3
intermix, so that each gonadal lobe contains germ cells
descended from both blast cells. In each lobe the first
150 germ cells to mature become sperm and those
remaining mature as oocytes. The switch from spermatogenesis to oogenesis occurs just after the L4 to adult
molt, slightly earlier in the anterior lobe than in the
posterior lobe. In four of the mosaic animals the
anterior lobe showed hermaphrodite germ-line differentiation into oocytes and sperm, while the posterior
lobe showed male differentiation into sperm only. In
the remaining mosaic, the anterior lobe contained only
oocytes, while the posterior lobe contained oocytes and
sperm. These animals were scored as mature adults,
well after the switch to oogenesis. Thus the germ-line
mosaics, like the intestinal mosaics, appear to show a
consistent polarity with an anterior-hermaphrodite,
posterior-male bias, as if the sex determination decision
were positionally influenced. Moreover, germ-line
Sex determination in triploid intersex mosaics of C. elegans
873
Table 7. Distribution of blast cell sexual phenotypes in triploidintersex animals
Sexually dimorphic
Blast cell(s)
P3-P8"
Zl, Z4C
Z2, Z3 d
M*
Bg
PIO11
V5L1
V5R1
V6LJ
V6RJ
TR'
V5L/R, V6L/R, TL/R"
Sexual phenotype
tissue
Hermaphrodite
Male"
Intersex
Ventral hypodermis
Gonad
Germline
Intestine'
Sex muscles
Spicules (tail)
Hook (tail)
Ray 1 (tail)
Ray 1 (tail)
Rays 2-6 (tail)
Rays 2-6 (tail)
Rays 7-9 (tail)
Rays 7-9 (tail)
Tail
57
87
196
302
45
43
25
70
80
40
46
48
55
32
222
220
120
21
199
253
259
191
202
207
226
208
223
168
46
18
4
34
19
29
30
21
18
14
80
° An additional 92 animals were male in all scored tissues.
b
The P3-P8 ventral hypodermal cells were scored as hermaphrodite if the vulva was normal and male if the vulva was completely
absent. These cells were scored as intersexual if the vulva was herniated or grossly abnormal.
c
The Zl and Z4 cells were scored as intersexual if the gonad consisted of a single hermaphrodite lobe (either antenor or posterior), a
sac-like structure for either hermaphrodite lobe or the single male lobe, or any other odd or abnormal structure.
d
The Z2 and Z3 cells were scored as hermaphrodite if sperm and oocytes were present in both hermaphrodite lobes or in the single
male lobe. If oocytes or sperm only were present in one lobe and oocytes and sperm in the other lobe then the germline was scored as
intersexual. If sperm only were present in either a hermaphrodite or male gonad then Z2 and Z3 were scored as male.
e
The intestine was scored as hermaphrodite if vitellogenins, scored by Nomarski microscopy ('oily' appearing material in
pseudocoelom), were present and male if not.
f
The M cell was scored as intersexual if (1) male diagonal sex muscles were present at the vulva, (2) both some hermaphrodite vulva
muscles and some male posterior diagonal muscle were present, (3) hermaphrodite sex muscles were present at the vulva and the spicules
were not crumpled (male sex muscles are required for morphogenesis of the spicules), and (4) male diagonal sex muscles were present but
the spicules were crumpled. In addition, sex muscles were not found in 44 animals (not included as intersexes).
8
The B cell was scored as hermaphrodite if no spicules or refractory spicule blobs were present. If incomplete spicule blobs only or only
one spicule was present it was scored as intersexual. Normal or crumpled spicules were scored as male.
h
The P10 cell was scored as hermaphrodite if the hook was completely absent and there were no obvious posterior ventral cell
divisions. The P10 cell was scored as intersexual if the posterior ventral cells had divided, a partial hook was present, or an ectopic hook
only was present. A complete normal hook was scored as male.
1
The V5L and V5R cells were scored as male if ray 1 was present or there was an appropriate gap in the alae. Otherwise, it was scored
as hermaphrodite.
1
The V6L, V6R, TL, and TR cells were scored as hermaphrodite if all of their respective rays were absent. If a subset of the rays was
present, then that blast cell was scored as intersexual; if all the rays were present then that cell was scored as male.
k
The tail was scored as hermaphrodite if all six ray blast cells produced alae only. If all nine rays were present the tail was scored as
male. If any one of the six ray blast cells produced only a subset of the normal number of rays, or if at least one blast cell followed a
male fate and at least one other blast cell followed a hermaphrodite fate, then the tail was scored as intersexual.
sexual identity can be determined postembryonically,
since Z2- and Z3-derived cells in the two gonadal lobes
can make opposite decisions.
Intersexual or abnormal somatic gonads were observed in 17 animals. The predominant phenotype was a
sexually indeterminate gonad consisting of a large sac,
as if both distal tip cells or the linker cell failed to
migrate (Fig. 8). Several intersexes showed morphologies that could be interpreted as gonad mosaicism, in which one lobe apparently initiated normal
hermaphrodite development while the other failed to
elongate, resulting in a single-lobed hermaphrodite
gonad. Such a structure could result if one of the two
somatic gonad precursor cells (Zl and Z4) and its
descendants adopted a hermaphrodite fate while the
other adopted a male fate. Infiveof seven animals with
mosaic gonads the anterior Zl descendants apparently
followed hermaphrodite fates and formed an anterior
lobe while the posterior Z4 descendants apparently
followed the male fate of no posterior migration,
consistent with the gonad also being sensitive to
positional influences.
Two exceptional animals scored as having a male
gonad and a vulval protrusion in the mid-ventral
hypodermis (one listed in Table 3 and another in
Table 7) are likely to be gonad mosaics that developed a
hermaphrodite anchor cell and male linker cell. The
hermaphrodite anchor cell induces vulval formation
(Kimble, 1981; Sternberg and Horvitz, 1986) while
migration of the male linker cell leads the elongation of
the developing gonad to generate the characteristic
male morphology (Kimble and White, 1981).
The AB-derived tail structures scored arise from
eight blast cells, which in the male produce the
structures listed in Table 7 (Sulston and Horvitz, 1977;
Sulston and White, 1980). In hermaphrodites these
eight cells either do not divide or produce fewer
progeny that generate less specialized structures. In the
triploid intersexes, most of these blast cells exhibited
either normal male or normal hermaphrodite fates. In
874
P. Schedin, C. P. Hunter and W. B. Wood
Table 8. Distribution of pair-wise combinations of blast cell sexual phenotypes in triploid intersex animals0
V5R
V5L
obs
V5R
ss
OS
V6L
ss
OS
V6R
ss
OS
TL
ss
OS
TR
ss
OS
B
ss
OS
P9/10
ss
OS
M
ss
OS
b
232
12
226
30
225
25
227
28
224
26
211
45
201
55
198
34
exp
c
154
90
174
82
169
81
169
86
166
84
175
81
183
73
164
68
V6L
TL
V6R
B
TR
P9/10
obs
exp
obs
exp
obs
exp
obs
exp
obs
exp
obs
exp
obs
exp
223
34
243
34
222
32
239
37
221
56
211
65
214
32
174
83
183
94
168
86
178
98
186
91
193
83
171
75
255
9
248
16
241
22
248
24
242
30
223
19
202
61
197
67
196
67
212
60
223
49
190
52
240
21
266
19
258
30
247
40
236
19
193
68
208
77
222
66
232
55
198
57
248
14
240
29
231
38
215
26
189
73
204
65
214
55
184
57
250
37
240
46
228
26
214
73
225
61
191
63
267
38
234
35
252
53
209
60
228
37
216
49
Same sex (ss)
Opposite sex (os)
" See Table 7 for description of scored sexually dimorphic structures derived from the indicated blast cells. To condense the data for
display blast cells scored as intersexual in Table 6 are classified here as male. The differences between observed and expected values are
highly significant by chi-square test,with P<0.001 for all cases except those involving P9/10, where differences are less significant (/J<0.05
for all pairs in second from bottom row; P<0.1 for last pair in bottom row).
b
Observed number of triploid intersex animals in which the structures derived from the two designated blast cells (intersection of each
column and row) expressed either the same or opposite sexual phenotypes.
c
Expected number of tnploid intersex animals in which the structures derived from the two designated blast cells expressed either the
same or opposite sexual phenotypes. Calculated by multiplying together the fraction of animals expressing either a male or hermaphrodite
fate for each blast cell (Table 7) to determine the expected fraction. This was then multipled by the total number of animals in which both
blast cells were scored. For example, expected same sex for V5L and V5R=[(fraction male V5L)x(fraction male V5R) +(fraction
hermaphrodite V5L)x(fraction hermaphrodite V5R)]xtotal number of animals in which both V5L and V5R were scored.
individual animals all pairwise combinations of blast
cell clones showed strong biases toward same-sex
decisions (Table 8). However, exceptions were observed for every pair, arguing for the possibility of
autonomous decisions and against obligate inductive
effects between any of the sexually dimorphic structures
scored.
The T and V6 blast cells produce tail sensory rays in
Dorsal
A) Gonad
Posterior
Antefkx
B) Sex Muscles
no sex myoblasts
2 sex myoblasts
4 sex myoblasts
2 sex myobtasis
migrate anteriorly
rni^ate posieriorty
mJgrale posteriorly
dtleremiale Into
vutval and uterine
sex musdes
Differentiate irao
dagonal and other
sex musdes
cf flefenfate Into
norwSagonal
Fig. 8. Postembryonic migrations in development of the
male and hermaphrodite gonads and sex muscles. (A) In
hermaphrodites, the two distal tip cells (dtc) lead the
growing anterior and posterior gonad lobes first away from
and then toward each other, while maintaining the adjacent
germ-line cells (glc) in mitotic growth by locally inhibiting
meiosis. In males, the linker cell (lc) leads the growing
single lobe first anteriorly and then posteriorly, while the
two non-migrating dtcs locally inhibit the glcs from
entering meiosis (Hirsh et al. 1976, Klass et al. 1976;
Kimble and Hirsh, 1979; Kimble and White, 1981).
(B) The M blast cell in both sexes undergoes a dorsalventral division soon after hatching. In hermaphrodites the
ventral daughter cell generates two sex myoblasts that
migrate anteriorly during the L2 stage and stop near the
midpoint of the developing gonad, where they divide and
differentiate into vulval and uterine muscles. In males the
dorsal daughter cell generates four and the ventral
daughter cell two sex myoblasts that migrate posteriorly
and begin dividing during the L3 stage. The male diagonal
muscles scored in our experiments all arise from the four
myoblasts generated by the dorsal M-cell daughter (Sulston
and Horvitz, 1977; Sulston et al. 1980).
Sex determination in triploid intersex mosaics of C. elegans
males and posterior extensions of lateral alae in
hermaphrodites. In our analysis we detected apparently
intersexual fates for these blast cells (Table 7), seen
generally as presence of some but not all the rays
derived from a particular blast cell. These cases might
simply represent instances of incomplete male differentiation. Alternatively, however, they could result from
sexual mosaicism within these lineages. Since some of
the progeny cells that apparently made opposite sexual
identity decisions are generated during the L3 stage of
postembryonic development, these decisions may be
made near the point at which sex-specific cell differentiation begins.
Sex muscle development was often incomplete or
sexually ambiguous (Table 7), and many intersexual
animals appeared to lack sex muscles completely. The
most common muscle phenotype observed among
intersexes was both partial male and partial hermaphrodite sex muscle development. The hermaphrodite sex
muscles scored form a crossed pattern around the vulva
and function in egg-laying; the male sex muscles form a
diagonal parallel array at the base of the tail that
functions in mating, as well as a second group required
for normal morphogenesis and movement of the
spicules (Sulston and Horvitz, 1977; Sulston etal. 1980).
The sex muscles arise postembryonically, beginning
with division of the M blast cell during the LI stage to
produce two sex myoblasts in hermaphrodites and six in
males (Fig. 8). The hermaphrodite myoblasts migrate
anteriorly during the L2 stage to a point near the
developing gonad, where they divide during the L3
stage and later differentiate into vulval and uterine
muscles. The male myoblasts begin to migrate posteriorly, divide during the L3 stage, and later differentiate to form the male diagonal sex muscles (Sulston and
Horvitz, 1977). In one mosaic animal, we observed
distinctly male diagonal sex muscles located midventrally at the hermaphrodite position. This phenotype suggests that sex myoblasts reversed their sexual
identity during development, first migrating in the
hermaphrodite mode and then differentiating in the
male mode.
Discussion
By constructing and analyzing triploids carrying a
partial duplication of the X chromosome, we have
extended the observations of Madl and Herman (1979)
that intersexual phenotypes result from an X/A ratio of
about 0.7, between the values known to signal normal
male and hermaphrodite development, respectively.
Our results can be summarized as follows. The animals
are generally healthy, indicating that
the
X-chromosome dosage compensation mechanism can
accommodate intermediate X/A ratios. The intersexual animals are mosaics of male and female tissues and
cells within tissues. For any pairwise combination of
tissues or cells there is a clear bias toward same-sex
choices in individual animals. As expected, this bias is
absolute for the pair of tissues somatic gonad-
875
midventral hypodermis, since the hermaphrodite gonadal anchor cell is required to induce vulval development
in the midventral hypodermis (Kimble, 1981; Sternberg
and Horvitz, 1986). However, for all other combinations of tissues and cells, the bias is not absolute, and
opposite-sex decisions were observed. The mosaic
patterns indicate how late in development these
decisions can be made for the various cells and tissues
scored. In at least two tissues, intestine and germline,
the decisions appear to show global positional influences, so that mosaicism within these tissues shows a
consistent anterior-feminine,
posterior-masculine
polarity.
Our finding of all but one of the possible pairwise
combinations of opposite-sex phenotypes among the
tissues examined argues that in each of the sexually
dimorphic embryonic lineages, with the expected
exception noted above, the sexual identity decision can
be made autonomously, after these lineages have
diverged. However, several of our observations are not
consistent with a simple model, such as that generally
accepted for Drosophila, involving autonomous irreversible assessments of the X/A ratio in the early
embryo that commit large clones of cells to one sexual
identity or the other in triploid mosaic animals. Rather,
our results suggest the possibility that sexual identity
decisions in C. elegans can be influenced by cell
interactions until quite late in development. We discuss
this possibility and other interpretations below, in
connection with our observations on the same-sex bias
among tissues, the local same-sex biases among cells
within, certain tissues, the apparent global positional
influences on sexual identity, and the timing of sexual
identity decisions.
Tissue same-sex biases
One plausible explanation for the same-sex preference
among tissues in an individual is that although the
actual chromosomal X/A ratio is identical in each
embryo, its initial assessment, giving rise to the primary
signal, is imprecise in triploid intersexes, such that
individual embryos are likely to have either a male or
hermaphrodite bias. This would affect the sex determination decisions in all tissues; those likely to go against
the bias would be the apparently most responsive to
X/A increase in male-biased embryos and least
responsive in. hermaphrodite-biased embryos (see below) as in fact we have observed. The initial biases
could reflect variations in maternal contributions to the
assessment mechanism, for which there is evidence
although its nature is not understood (Villeneuve and
Meyer, 1987; Plenefisch et al. 1989; Villeneuve and
Meyer, 1990; R. Herman, personal communication). A
similar same-sex bias of tissue phenotypes has been
observed by Villeneuve and Meyer (1990) in individual
intersexual animals resulting from mutations in the sdc1 gene.
An alternative or additional effect contributing to
same-sex biases could be some non-autonomy in the sex
determination process, such that for example a hermaphrodite decision in one tissue could influence other
876
P. Schedin, C. P. Hunter and W. B. Wood
tissues toward the same decision. The finding of samesex biases in the hermaphrodite direction despite a
probable overall bias against hermaphrodite phenotypes in the population of animals analyzed would be
consistent with both of these explanations.
Two other possible causes of same-sex bias seem less
likely. One would be the influence, at threshold X/A
levels, of 'modifier genes', which segregate in different
proportions to different 2X;3A+Dp offspring in the
crosses producing these animals. Although such effects
are known to contribute to individual biases in
Drosophila 2X:3A intersex mosaics (Baker and Belote,
1983), they should not be a factor for C. elegans, in
which all genetic stocks derive from the ancestral N2
strain and should, therefore, be generally isogenic as
well as homozygous at almost all loci. Another
possibility would be that among the progeny of
tetraploids, which are karyotypically unstable, there
may be differences in autosome composition between
animals, resulting in actual X/A differences that could
account for the observed biases. Since some degree of
autosomal aneuploidy is known to be tolerated in C.
elegans (Sigurdson et al. 1986; C. P. H. unpublished),
we cannot rule out this possibility.
An additional puzzling observation at the tissue level
in triploid intersex mosaics is the apparent difference in
responsiveness of different tissues to feminization in
response to the intermediate X/A ratio. These differences could be related to the nature of the various
responses: feminization of the intestine as scored in our
experiments could result from expression of a single vit
gene, whereas feminization of the tail or somatic gonad
would require alteration of a more complex morphogenetic process controlled by many genes. Alternately,
responsiveness differences could be only apparent,
resulting from biases in the scoring of different tissues
as male or hermaphrodite. In the intestine, the tissue
found most responsive, an intersexual phenotype, that
is an intermediate level of vit transcription might well
have been scored as hermaphrodite, whereas in tail and
somatic gonad, the tissues found least responsive, an
intersexual phenotype would probably be scored as
male. However, it is also possible that intermediate
X/A ratios cause intermediate levels of tra-1 activity
(Fig. 1), and that different tissues have different
thresholds for response to tra-1. If so, this response
must in general be all or none; we did not observe
clearly intersexual cellular phenotypes except in mosaic
intestines, where cells at the border between vit gene
expression and non-expression often showed intermediate hybridization levels.
Local same-sex biases
At the local level, biases toward same-sex choices of
cells within tissues is difficult to explain except by nonautonomy in the sex determination process. In the
intestine, non-autonomy of the decision to express vit
genes is indicated by our finding that all labelled cells
were contiguous with each other, as were ah1 unlabelled
cells. This contiguity of labeled cells is unlikely to be an
artifact of leakage or transfer of vit transcripts between
cells, because individual unlabeled cells among labeled
neighbors (and vice versa) can be clearly seen using
similar procedures in the intestines of animals mosaic
for tra-1 gene function (Hunter and Wood, 1990). The
absence of individual unlabeled cells among labeled
neighbors and vice versa rules out the possibility that
clonally inherited commitments are made during
generation of the intestine, because of a peculiarity of
the intestinal lineage as determined by Sulston et al.
(1983). The 20 cells of the intestine arise from the E
founder cell in the embryo by the lineage diagrammed
in Fig. 5. Because two cells on each side of the animal
exchange places with their neighbors between the 16and 20-E-cell stages of intestinal development, the
descendants of certain precursor cells do not occupy
adjacent positions in the adult tissue. For example,
descendants of the Ea cell give rise to the intestinal
units intl, int2, int3, and int5; the intervening unit int4
is derived from descendants of Ep. Therefore, if
different heritable sexual commitments were made
early in the lineage, for example in the E-cell daughters
Ea and Ep, the result would be mosaic patterns with
non-contiguous cells expressing vitellogenin genes
(Fig. 5). Likewise, loss at one of the following left-right
divisions would result in patterns with cells on one side
of the intestine expressing and contiguous cells on the
other not expressing vitellogenins. These predicted
patterns are in fact observed in tra-1 mosaics (Hunter
and Wood, 1990), but never in triploid intersexes.
Additional evidence for local non-autonomy comes
from the same-sex preferences observed among blast
cells that give rise to postembryonic lineages, for
example in the tail (Tables 7, 8). Among the animals
exhibiting a male fate for any one of the blast cells
V5L/R, V6L/R, and TL/R (ranging from 191 to 223
scored), 168 or about 80% showed male fates for all
these cells (Table 7). If sexual fates were decided
independently, the expected percentage of animals with
all male fates for these cells, calculated from the
probabilities of maleness for the individual cells
(Table 7), would be about 25%, far less than the
observed value. The patterns' observed in mosaic
intestines as well as the biases observed in postembryonic blast cell lineages strongly suggest that cells in these
tissues can influence the sexual identity decisions of
their neighbors.
Global positional effects on sexual identity decisions
The observed polarity of the intestinal mosaics, with
hermaphrodite fates always anterior to male fates,
suggests a more global external influence on sex
determination or sexual differentiation. Its significance
is underscored by our observation, on smaller numbers
of animals, of the same polarity in germ-line mosaics
and the majority of apparent somatic-gonad mosaics.
The exclusive polarity observed in the intestine cannot
simply be an artifact of the dissection and in situ
hybridization procedures, since the same techniques
showed labeling with the reverse polarity in intestines
from diploid L4 hermaphrodites. The different responses of anterior and posterior cells could result from
Sex determination in triploid intersex mosaics of C. elegans
effects on either the sex determination process or on
downstream genes that more directly control sexspecific differentiation. In the intestine, for example,
the result could be explained by position-dependent
differences in activity of the mab-3 gene, which
negatively regulates vitellogenin synthesis in response
to the state of the sex-determining tra-1 gene (Shen and
Hodgkin, 1988).
We found no strict correlation among triploid
intersexes in general between vitellogenin synthesis in
the intestine and the sexual identity of any other single
tissue. Therefore, the external influence responsible for
the polarity of intestinal mosaics is not likely to be the
signal controlling normal intestinal sexual differentiation, unless such a signal emanates from a tissue that
was not scored or from several different tissues. Some
possibilities, for example, a signal from the midventral
hypodermis (scored only indirectly in most of our
experiments; see Materials and methods) is ruled out by
other experiments: animals in which the entire AB
lineage and therefore the ventral hypodermis is male
nevertheless express vitellogenin genes normally in the
intestine (Hunter and Wood, 1990), showing that a
female ventral hypodermis is not required to induce
intestinal vitellogenin synthesis.
Thus we can conclude only that in certain tissues,
when the primary sex-determining signal is poised at a
threshold level, some difference in positional information along the anterior-posterior axis can tip the
balance toward female differentiation in anterior cells
and male differentiation in posterior cells. Precedent
for such a phenomenon in C. elegans comes from
analyses of the mab-5 gene, which functions cellautonomously to control cell fates in the posterior of the
animal as if responding to positional information
(Kenyon, 1986; Waring and Kenyon, 1990).
There is no contradiction in our finding that the
gradient pattern of intestinal vitellogenin gene expression during larval development of normal diploid
hermaphrodites is opposite to the pattern in adult
intersexes; the triploid intersex patterns show that
intestinal cells can have position-dependent differences
in adult levels of vitellogenin transcripts, while the
pattern in L4 intestines reflects the kinetics of initiating
vitellogenin transcription. These results suggest that
vitellogenin transcription may be under the control of
two temporally distinct signals in normal diploids. The
first, establishing the sexual identity of intestinal cells,
probably leads to expression in XX animals of the tra-1
gene (Hodgkin, 1987) by each intestinal cell (Hunter
and Wood, 1990); this in turn may permit subsequent vit
gene expression by preventing the action of the mab-3
gene product. The second, a temporal signal probably
produced during the L4 stage and not necessarily
hermaphrodite-specific, could then trigger the onset of
vitellogenin gene transcription in hermaphrodite-committed intestinal cells, beginning at the posterior end.
Two such signals are also implicated in Drosophila,
where the hormones 20-hydroxyecdysone and juvenile
hormone signal initiation of vitellogenin synthesis in the
fat body of females, but have little effect on the fat body
877
cells of males, even at abnormally high concentrations
(Postlethwaite and Jowett, 1980).
Timing of sexual identity decisions
If two cells in a mosaic animal adopt opposite sexual
fates, then the choices must have been made after their
lineages diverged. The mosaic patterns that we have
observed indicate that choices of sexual identity can be
made during late embryonic and also during postembryonic development. For example, different sexual
identities of tail ectoderm hook and ray precursors in a
mosaic animal must result from decisions made after
their lineal divergence about midway through embryogenesis. Differences seen within mosaic germ lines, sex
muscles and tail ectodermal lineages that generate
multiple rays must result from decisions made after
hatching, because these tissues are all generated
postembryonically. The patterns observed indicate that
some of these decisions can be made as late as the L3
stage of postembryonic development. In mosaic intestines, the finding of many examples in which the vit
probe hybridized to only one of two sister cells in the
adult (e.g. to int2V but not int5L; see Fig. 5) shows that
the decision to express vitellogenin genes can be made
after all 20 cells of the adult intestine are generated by
the final E-cell divisions, which occur at the end of the
cell proliferation phase about halfway through embryogenesis (Sulston et al. 1983). However, this decision
could be made as late as the L4 stage when these genes
are expressed. In other tissues, our ability to score cells
reliably as either male, hermaphrodite, or intersexual
may be the limiting factor in ascertaining how late sex
determination decisions can be made.
These observations suggest the possibility that in
animals with ambiguous X/A ratios, some cells may
remain sexually uncommitted and able to vacillate
between feminine and masculine states until expression
of their sexually differentiated fates. This possibility is
supported by our observation of an animal with male
diagonal sex muscles at the mid-ventral hermaphrodite
position, indicating that the sex myoblasts must have
first migrated anteriorly in the hermaphrodite mode
and subsequently undergone the male pattern of muscle
differentiation. Because both migratory behavior and
production of sexually dimorphic muscle patterns are
probably cell-autonomous characteristics (Hunter and
Wood, 1990), the sex myoblasts in this animal must
have reversed their sexual identity between the L2 and
L4 larval stages.
Temperature-shift experiments on animals carrying ts
mutations in one of the sex-determining genes fem-2
(Kimble et al. 1984; Doniach and Hodgkin, 1984), tra-2
(Klass et al. 1979) and her-1 (P. S., P. Jonas, and W. B.
W., in preparation) have also shown that sex determination decisions can be reversed during larval development and even in adults. However, in these experiments, done with normal diploid strains, X/A ratios
were unambiguously assessed, and reversal of the
normal sexual phenotype was caused by inactivation of
a sex-determining gene.
X/A assessment in normal diploids must occur early
878
P. Schedin, C. P. Hunter and W. B. Wood
in embryogenesis, so that dosage compensation can be
set to either the male or the hermaphrodite mode
(Villeneuve and Meyer, 1990). However, the above
shift experiments and many other observations (Hodgkin, 1988) make it clear that X/A assessment does not
represent an irreversible commitment to sexual identity, because it can be overridden by subsequent effects
on the sex-determining genes that control tra-1 (Fig. 1).
The commitment becomes irreversible only when tra-1
activity has been definitively set to ON or OFF, or if this
activity is at an intermediate level, when the tra-1 signal
has been perceived as present or absent by its target
sexual differentiation genes. Thus our experiments
measure not the timing of X/A assessment, but rather
the timing of setting or perceiving the activity state of
tra-1.
We have shown that tra-1 and its target genes act cellautonomously to control sexual differentiation (Hunter
and Wood, 1990). However, if one or more of the
upstream genes that regulate tra-1 can act nonautonomously, then cells in triploid intersexes might
change their sexual identity through cell interactions at
any time in development, overriding any earlier X/A
assessment. Our results would then indicate the timing
of the last non-autonomous step in tra-1 regulation.
We conclude that some non-autonomy in the sex
determination process could explain the same-sex
biases of tissues and cells within tissues, the lateness of
sexual commitments, and possibly though less straightforwardly the positional effects on sex determination
observed in triploid intersex mosaics. If indeed present,
such non-autonomy should be demonstrable by analysis
of specific gene mosaics, which we are currently
pursuing (Hunter and Wood, in preparation).
Triploid mosaic intersexes in Drosophila produce
only large clones of oppositely sexed cells, indicating
early (pre-gastrulation) sex determination decisions
that appear irreversible (Baker and Belote, 1983) and
probably involve setting the activity state of the Sxl
gene (Cline, 1984), which maintains this state by
autoregulation. We have shown that such intersexes in
C. elegans can produce small clones of oppositely sexed
cells, indicating later, apparently unstable sex determination decisions and lack of an early commitment
maintained by autoregulation. These contrasting results
support the recent realization (Hodgkin, 1990) that
although Drosophila and C. elegans share a common
primary signal, the X/A ratio, these organisms differ
fundamentally in their mechanisms of sex determination.
We are grateful to A. Carpenter and A. Chisolm for critical
reading of the manuscript, to J. Taylor for help with its
preparation, and in particular to the reviewers for insightful
suggestions regarding revisions. This research was supported
by the National Institutes of Health through grants HD-11762
and HD-14958 to W.B.W. and predoctoral traineeships to
P.S. and C.P.H.
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{Accepted 25 March 1991)