1 Department of Genetics, Cell Biology and Development, University of Minnesota,
321 Church Street SE, Minneapolis, MN 55455, USA
2 Developmental Biology Center, University of Minnesota, 321 Church Street SE,
Minneapolis, MN 55455, USA
3 Department of Biochemistry, University of Wisconsin, 433 Babcock Drive,
Madison, WI 53706-1544, USA
4 Howard Hughes Medical Institute, University of Wisconsin, 433 Babcock Drive,
Madison, WI 53706-1544, USA
Author for correspondence (e-mail:
zarkower{at}gene.med.umn.edu)
Accepted 25 November 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sex determination, Gonad, Gonadogenesis, C. elegans, Forkhead
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The global regulators of sexual development presumably act via more
tissue-restricted downstream genes. Indeed, several tissue-specific sexual
regulators have been identified in worms and flies; for example, in the
nematode C. elegans the DM domain protein MAB-3 controls male
development of the intestine and parts of the nervous system
(Shen and Hodgkin, 1988;
Yi et al., 2000
), the related
protein MAB-23 regulates male development of mating muscles, nervous system
and posterior hypodermis (Lints and
Emmons, 2002
), and FOG-1 and FOG-3 act in the germline to promote
spermatogenesis (Barton and Kimble,
1990
; Ellis and Kimble,
1995
). Similarly, in Drosophila, Fruitless controls male
development of the CNS and musculature
(Ito et al., 1996
;
Ryner et al., 1996
), Takeout
acts in fat cells in the head to promote male courtship behavior
(Dauwalder et al., 2002
) and
Dissatisfaction regulates courtship behavior in the nervous system of both
sexes (Finley et al., 1997
).
Although the action of such proteins begins to reveal how global regulators
impose sexual dimorphism on individual tissues, the downstream regulators
remain unknown in many tissues. In particular, no gonad-specific
sex-determining gene has been identified in any invertebrate, despite the
central role in sexual reproduction of the gonad and its highly dimorphic
anatomy in most species.
This paper focuses on control of sexual dimorphism in the gonad of C.
elegans. The two sexes of C. elegans are the self-fertilizing
hermaphrodite (chromosomally XX) and the male (XO). Hermaphrodites can be
considered females with sperm, a view underscored by the existence of closely
related nematodes that reproduce as typical females and males. Therefore,
C. elegans sex determination is likely to rely on a primal
male/female control mechanism, with more recent regulatory modifications that
permit hermaphroditism (Fitch and Thomas,
1997). Arguably the most crucial global regulator of sex
determination in C. elegans is tra-1, which promotes female
development in hermaphrodites and encodes TRA-1A, a zinc-finger protein of the
Ci/GLI class of transcription factors
(Hodgkin, 1987
;
Zarkower and Hodgkin, 1992
).
tra-1 activity is essential for all aspects of female sex
determination in somatic cells and also plays a crucial role in the germline
(Hodgkin, 1987
;
Schedl et al., 1989
). TRA-1A
controls some downstream targets by direct transcriptional repression
(Chen and Ellis, 2000
;
Conradt and Horvitz, 1999
;
Yi et al., 2000
). The picture
emerges, therefore, that TRA-1A promotes female cell fates by repressing the
expression or activity of genes that otherwise would direct male development
in specific tissues (reviewed by Zarkower,
2001
).
Despite major differences in mature gonad morphology between the two sexes,
most regulators of gonadogenesis identified so far play similar roles in both
sexes (Hubbard and Greenstein,
2000; Friedman et al.,
2000
; Mathies et al.,
2003
; Miskowski et al.,
2001
; Siegfried and Kimble,
2002
). Indeed, even tra-1, in addition to its essential
role in directing hermaphrodite gonadal development, also has a minor role in
male gonadogenesis (Hodgkin,
1987
; Schedl et al.,
1989
) (L.M. et al., unpublished). Therefore, sexual dimorphism in
the gonad may not arise by execution of unrelated parallel developmental
programs in each sex, but rather by sex-specific modulation of an underlying
common gonadogenesis program. In this respect, control of C. elegans
gonadogenesis may resemble genital disc development in Drosophila, in
which the Doublesex branch of the sex-determination pathway
sex-specifically modulates the response to cell signaling pathways found in
both sexes (reviewed by Christiansen et
al., 2002
).
In the work described here, we identify the forkhead transcription factor FKH-6 as a regulator of sexual dimorphism in the C. elegans gonad. The gonad of fkh-6 mutant males is feminized: hermaphrodite-specific gonadal reporters are expressed; a vulva is frequently present; and early gonadogenesis resembles that of hermaphrodites. By contrast, the overall morphology of mutant hermaphrodite gonads is normal in most animals, although mutants are infertile, because of defects in the differentiation of the hermaphrodite somatic gonad. Extra-gonadal tissues in both sexes appear to be normal in fkh-6 mutants, and fkh-6 reporters are expressed only in the gonad. Genetic and molecular analyses suggest that fkh-6 acts downstream of tra-1 in the first division of the somatic gonadal precursor cells, which establishes gonadal sexual dimorphism. Collectively, these results indicate that FKH-6 is an organ-specific regulator of sexual dimorphism.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrated transgene arrays were as follows: qIs56[lag-2::gfp, unc-119+]V; ayIs2[egl-15::gfp, dpy-20+]IV; ezIs1[K09C8.2::gfp, pRF4]X; bxIs13[egl-5::gfp, lin-15+]X;
syIS50[cdh-3::gfp, dpy-20+]; tnIs5[lim-7::gfp, pRF4]; leIs8[pes-8::gfp; pRF4];
ezIs2[fkh-6(pro)::gfp, unc-119+]III;
and qIs76[tra-1::gfp, pRF4].
Extrachromosomal transgene arrays were leEx780[ZK813.3::gfp, pRF4]; and ezEx133[fkh-6(FL)::gfp, pRF4].
fkh-6 mutations were maintained in trans to the balancer
mIn1[dpy-10(e128) mIs14]II, where mIs14 carries the
integrated array ccEx9747. ccEx9747 is an extrachromosomal array
composed of three GFP constructs, one driven by a gut-specific enhancer and
the others by myo-2 and pes-10 promoters
(Edgley and Riddle, 2001).
Heterozygotes were identified as animals of normal size with GFP expression in
the pharynx and intestine, and mutants were identified as animals of normal
size lacking GFP expression in these tissues.
Isolation, mapping, and genetic manipulation of fkh-6 mutations
fkh-6(ez16) was isolated in an F2 EMS mutagenesis screen
(Sulston and Hodgkin, 1988) of
him-8(e1489) animals carrying the integrated array ezIs1,
which contains the male seminal vesicle and vas deferens marker
K09C8.2::gfp. K09C8.2 was identified in a cDNA microarray screen for
sex-enriched L4 mRNAs, comparing XX wild-type (N2) hermaphrodites with XX
pseudomales (tra-2; xol-1) (K.T., W. Yi, V. Reinke, and D.Z.,
unpublished). Mutant lines with abnormal or absent K09C8.2::gfp
expression were identified using a dissecting microscope with fluorescence
optics. The fkh-6(q641) allele was isolated in a F2 EMS mutagenesis
screen of him-5(e1490) animals for gonadogenesis defects.
The ez16 and q641 mutations were mapped between
lin-31 and let-172 on LGII by three-factor mapping, and
further localized by deficiency mapping. The deficiencies maDf4, nDf3
and nDf4 complemented ez16 and q641, whereas
ccDf4, ccDf7, ccDf5 and ccDf1 failed to complement. Single
nucleotide polymorphism (SNP) recombinant mapping
(Wicks et al., 2001) placed
ez16 to the left of a polymorphism contained in the cosmid M03A1. A
cosmid from this region (B0286) was injected with pRF4
[rol-6(su1006sd)] (Mello et al.,
1991
) into ez16/mIn1 hermaphrodites and found to rescue
the ez16 mutation. Fertile fkh-6 homozygous hermaphrodites
and fkh-6 males with normal gonadal morphology were scored as
positive for rescue. A PCR fragment containing 6.7 kb of fkh-6
upstream of the predicted start codon and 2 kb downstream of the predicted
stop codon also rescued ez16 mutants. In addition, RNAi directed
against fkh-6 gave the same phenotypes as ez16 and
q641. RNAi directed against fkh-6 was performed by feeding
bacteria expressing double-stranded RNA corresponding to fkh-6
(B0286.5; a gift from J. Ahringer) as described
(Ashrafi et al., 2003
;
Kamath et al., 2001
).
Molecular analysis of fkh-6 alleles
Template DNA for sequencing was made by amplifying exons from genomic DNA
prepared from N2 or fkh-6(ez16, q641) animals using the
ExpandLongTM or ExpandTM High Fidelity PCR System (Roche). Three
independent PCR reactions were sequenced for each exon from each strain using
Big-Dye Terminator Ready Reaction Mix (PE/Applied Biosystems). Sequencing of
ez16 DNA identified a mutation in the predicted first codon, changing
ATG to ATA (methionine to isoleucine). Sequencing q641 DNA identified
a G to A change in the first position of intron 2.
To confirm that the fkh-6(q641) mutation affects splicing, RT-PCR
was performed. RNA was isolated from N2 or fkh-6(q641) L1 and L4 XO
and XX animals using Tri ReagentTM (Molecular Research Center), and cDNAs
were prepared using Superscript II (Gibco). Sequencing of PCR products from
two overlapping primer sets identified identical transcripts in wild-type XO
and XX animals at L1 and L4. To predict the consequences of the q641
insertion for the FKH-6 structure, the primary amino acid sequence of FKH-6
was aligned with residues in the Foxd3 (2HDC) solution structure
(Jin et al., 1999) using
Clustal-W (Thompson et al.,
1994
). The insertion caused by the q641 mutation is in
helix 2, which is immediately prior to two hydrophobic amino acids that are
integral to the forkhead domain hydrophobic core (Phe-32 and Ile-33 in 2HDC;
Phe-50 and Ile-51 in FKH-6). By altering the register of helix 2, the
insertion is likely to disrupt formation of the hydrophobic core in FKH-6.
Lineage analysis and laser ablations
Cell divisions and migrations were followed by DIC microscopy using
standard methods (Sulston and Horvitz,
1977). To determine whether Z1.a and Z4.p can divide, we laser
ablated Z1.p and Z4.a (plus two of the four germ cells present at the time, to
simplify the analysis). In wild-type males, this ablation does not affect the
divisions of remaining cells (Kimble and
White, 1981
) (J. Kimble, unpublished). Ablations were performed as
previously described (Bargmann and Avery,
1995
) using a Micropoint Ablation Laser System (Photonics
Instruments, Arlington, IL). L1 XO fkh-6(q641) and
fkh-6(ez16) homozygotes were obtained as self-progeny from
fkh-6(q641)/mIn1; him-5(e1490) or fkh-6(ez16)/mIn1;
him-8(e1489) mothers. XO animals were identified based on the presence of
an enlarged B blast cell. L1 fkh-6(q641); tra-1(e1099) double
homozygotes were identified as self-progeny with a large B cell from
fkh-6(q641)/mIn1; tra-1(e1099)/+ mothers.
fkh-6 reporters
fkh-6(pro)::gfp (pWC1) was made by inserting a genomic DNA PCR
fragment, extending from 6708 bp upstream of the predicted start codon to 239
bp downstream, into the pPD95.69 GFP vector (gift of A. Fire). The
fkh-6(FL)::gfp (pWC2) rescuing GFP was made by inserting a genomic
DNA PCR fragment, from 6708 upstream of the predicted start codon to 3402
downstream, into pPD95.67. Transgenic strains were created by microinjection
as previously described (Mello and Fire,
1995). pWC1 was injected with pMM106b (unc-119+; a gift
from D. Pilgrim) into unc-119(ed3) worms to generate
ezEx147, and this strain was subsequently integrated to generate
ezIs2. pWC2 was injected with pRF4
(Mello et al., 1991
) into
fkh-6(ez16) to generate the extrachromosomal array
ezEx133.
pWC1 contains a single consensus TRA-1A binding site. pWC7, a derivative of this reporter with the consensus sequence changed from TTGGTGGTC to TTCTGCAGC was made by site-directed mutagenesis with the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene).
Genetic mosaic analysis
fkh-6(ez16) mutant hermaphrodites carrying an extrachromosomal
array of fkh-6(FL)::gfp and pRF4 were assayed for the ability to lay
eggs, indicating rescue of the fkh-6 somatic gonadal defects. Progeny
of rescued animals were examined for viability and inheritance of pRF4 (which
causes a dominant roller, or Rol, phenotype). Five hermaphrodites produced
large broods of fully viable mutant progeny, all non-Rol. In these
hermaphrodites, the extrachromosomal array must have been lost, prior to the
embryonic cell division forming P4, the blastomere from which the germline
precursors Z2 and Z3 derive. Male and hermaphrodite progeny of germline mosaic
hermaphrodites had the same gonadal phenotypes as homozygous fkh-6
progeny from fkh-6 heterozygotes. This indicates that any maternal
contribution of FKH-6 to the germline is not functionally significant.
tra-1 reporter
The tra-1::gfp reporter (pJK876) was generated by inserting a
PstI to BamHI fragment from cosmid F56C2 into pPD96.04 (a
gift from A. Fire). This construct contains 8138 bp upstream of the
tra-1 start codon and fuses the first six amino acids of TRA-1 to GFP
and ßGAL. pJK876 was injected with pRF4 to generate qEx480,
which was integrated to make qIs76.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We cloned the gene defective in ez16 and q641 by a
combination of fine genetic mapping, cosmid rescue and RNAi (Materials and
methods). The gene identified was fkh-6, one of a previously
described family of C. elegans forkhead-related genes
(Hope et al., 2003). The FKH-6
protein contains 323 amino acids, including a 96 amino acid region with high
similarity to the DNA-binding domain of the forkhead/winged-helix family of
transcription factors (Hope et al.,
2003
; Weigel and Jackle,
1990
) (Fig. 1A).
Sequencing genomic DNA from ez16 and q641 homozygotes
revealed their molecular lesions: ez16 is a G-to-A transition
predicted to alter the initiation codon from methionine to isoleucine, and
q641 is a G-to-A transition at the first nucleotide of intron 2,
predicted to affect splicing between exons 2 and 3
(Fig. 1A). To confirm the
effect of q641 on splicing, we sequenced fkh-6 cDNAs
prepared by RT-PCR from wild-type and fkh-6(q641) L1 XO animals, and
from mixed-stage fkh-6(q641) XX animals. fkh-6(q641) cDNA
has a six nucleotide insertion at the junction of exons 2 and 3, owing to use
of a downstream cryptic 5' splice site. This is predicted to insert two
amino acids into helix 2 of the forkhead domain. Alignment of FKH-6 to known
forkhead domain structures (Materials and methods) suggests that the
q641 insertion destabilizes the hydrophobic core of the forkhead
domain by shifting the orientation of two conserved hydrophobic residues in
helix 2 (Phe-50 and Ile-51). The two fkh-6 alleles have essentially
identical phenotypes, either as homozygotes or in trans to a deficiency, and
closely resemble fkh-6(RNAi); in addition, fkh-6(RNAi) of
fkh-6 homozygotes does not enhance the mutant phenotype
(Table 1, and data not shown).
Collectively, these results indicate that both fkh-6 alleles are
strong loss-of-function mutations and may be null.
|
|
The gonadal defects in fkh-6 mutant hermaphrodites, by contrast, do not include sexual transformation and usually do not affect overall gonadal morphology. Wild-type hermaphrodites have two symmetrical ovotestes with sheath, spermatheca and uterus (Fig. 1G,I; Table 1), and most fkh-6 hermaphrodites have this same morphology (Fig. 1H; Table 1). In addition, sheath/spermathecal precursors are present at the normal time and position in early L3 (Fig. 1K,L; n=30) and form a spermatheca that expresses the spermathecal marker leEx780 (n=50; data not shown). However, during L4, sheath/spermathecal daughters assume unusual and variable positions, and the adult spermatheca appears blocked, with embryos accumulating in the proximal sheath (Fig. 1H). A rarer hermaphrodite defect is the presence of only one of the two normal ovotestes (compare Fig. 1I with 1J; Table 1), which appears to result from the loss of a distal tip cell (see below). We conclude that fkh-6 affects gonadogenesis in both sexes, but its affect on overall gonadal morphogenesis is largely male specific.
Gametogenesis appears normal in fkh-6 hermaphrodites: spermatogenesis is followed by oogenesis and fertilization occurs (Fig. 1H). However, embryonic development arrests prior to hatching and all fkh-6 hermaphrodites are infertile (n>500). This maternal effect lethality could reflect a requirement in the somatic gonad, the germline, or the early embryo. To distinguish between these possibilities, we analyzed germ line mosaics (Materials and methods). In these animals, a rescuing fkh-6 array was present in the mother but was not transmitted to progeny, indicating loss of the array in the embryonic cell lineage leading to the germline (n=5). All germline mosaic hermaphrodites produced large broods of viable fkh-6 mutant progeny, demonstrating that neither germ line nor zygotic expression of fkh-6 is required for viability. Instead, we suggest that the embryonic lethality results from somatic gonadal defects, perhaps owing to insufficient support of developing oocytes by somatic cells.
The fkh-6 defects appear to be restricted to the gonad. Hermaphrodites and males are of normal size and have a growth rate similar to that of wild type. In addition, we observed no obvious defects in male tail differentiation (Fig. 1M,N) or sex muscle morphology and function (Fig. 1O,P). Males also display normal mating behavior, and do not accumulate yolk, indicating that the nervous system and intestine, respectively, are not feminized (data not shown).
fkh-6 males lack gonadal leader cells
Gonadal morphogenesis is controlled by `leader' cells, which guide
elongation of the growing gonadal arms
(Kimble and White, 1981). In
males, the `linker' cell has leader function, and two male distal tip cells
(DTCs) control germline proliferation. The severe defects in elongation of
fkh-6 male gonads suggested that the linker cell might be missing or
defective. To test this, we used lag-2::gfp, a marker intensely
expressed in the large round linker cell and more faintly in the two small
flat male distal tip cells (Siegfried and
Kimble, 2002
). Most fkh-6(q641) males lacked cells
intensely expressing lag-2::gfp with linker cell morphology (94%,
n=64), and many had abnormal numbers of DTCs
(Table 2). Because the linker
cell and male DTCs are generated during L1, these results suggest that
fkh-6 acts early in male gonadogenesis.
|
Feminization of cell fates in the fkh-6 male gonad
In wild-type hermaphrodites, vulval development is induced by the anchor
cell, a hermaphrodite-specific cell in the somatic gonad
(Kimble, 1981). The vulvae
present in fkh-6 males suggested that the gonad might be feminized,
possessing an anchor cell and possibly other hermaphrodite cell types. We
tested this possibility using reporter transgenes specific for hermaphrodite
gonadal cells. First, we asked whether fkh-6 male gonads make an
anchor cell. In wild-type animals, the cdh-3::gfp marker is expressed
specifically in hermaphrodite anchor cells and is not expressed in the male
gonad (Pettitt et al., 1996
)
(Fig. 2A,B). Remarkably, almost
all fkh-6(ez16) males (97%, n=64), expressed
cdh-3::gfp in at least one anchor cell
(Fig. 2C), and most had more
than one (83%), with an average of four. The anchor cells present in
fkh-6 XO gonads demonstrate that the fkh-6 male gonad is
feminized and are likely to be responsible for induction of male vulvae.
fkh-6 hermaphrodites do not have supernumerary anchor cells: all
animals examined had a single cdh-3::gfp-expressing cell in the gonad
(n=50). The frequency of vulvae in fkh-6 males is lower than
the frequency of cdh-3::gfp-expressing cells (25% versus 97%). This
suggests that some of the cdh-3::gfp-expressing cells may not have
full anchor cell function.
|
To test whether the feminization of fkh-6 male gonads is complete,
we examined expression of two male-specific gonadal markers. We used
ezIs1 to score seminal vesicle and vas deferens cells. In wild type,
ezIs1 is not expressed in hermaphrodites (n>>1000)
(Fig. 2M), but is expressed in
all male gonads from L4 through adulthood (n>>1000)
(Fig. 2N). In
fkh-6(ez16) males, a few gonadal cells expressed ezIs1 in
25% of animals (n=52), indicating the presence of seminal vesicle or
vas deferens cells (Fig. 2).
Similarly, egl-5::gfp, which is expressed in four seminal vesicle
valve cells in wild-type adult males
(Ferreira et al., 1999), was
expressed in one cell in 21% of fkh-6 mutant male gonads
(n=30). Neither male marker was expressed in fkh-6
hermaphrodites (not shown). We conclude that the feminization of
fkh-6 XO gonads, although extensive, is not complete, and that
fkh-6 XX gonads are not masculinized.
Feminization of early gonadogenesis in fkh-6 males
We next asked whether early gonadogenesis during the first larval stage
(L1) in fkh-6 mutants resembles that of males or hermaphrodites. At
hatching, the L1 gonadal primordium consists of four cells: two somatic
gonadal precursor cells (Z1 and Z4) occupy the poles and flank two central
germ line precursor cells (Z2 and Z3)
(Hubbard and Greenstein,
2000). This four-cell primordium is morphologically
indistinguishable in the two sexes; sexual dimorphism is established during
and after the first Z1/Z4 division and involves three sex-specific events
(Kimble and Hirsh, 1979
)
(Fig. 3A,C). First, Z1/Z4
daughter cells have a more pronounced size asymmetry in XO than XX gonads: in
males, Z1.a and Z4.p are visibly smaller than their sisters Z1.p and Z4.a,
while in hermaphrodites these sisters appear similar in size. Second, in males
Z1.p and Z4.a migrate anteriorly, while in hermaphrodites they retain their
central positions. Third, Z1.a and Z4.p do not divide in males, but do in
hermaphrodites.
|
We also examined L1 gonadogenesis in fkh-6 XX hermaphrodites. The four-cell gonadal primordium appeared normal (n=30), and the first divisions of Z1 and Z4 were typical of wild-type hermaphrodites (n=2). However, Z1/Z4 cell divisions were slower than wild-type in both XX and XO fkh-6 mutants (Fig. 3B,D). By contrast, no delay occurred in the first two rounds of the B lineage (n=8), suggesting that fkh-6 does not affect cell divisions generally.
fkh-6::gfp expression is gonad-specific
To study fkh-6 expression, we made two GFP reporters. One, a
transcriptional reporter, fkh-6(pro)::gfp, contains 6.7 kb upstream
of the predicted initiation codon and fuses the gfp-coding region in
frame at the beginning of exon 2. The other reporter, fkh-6(FL)::gfp,
contains the same 6.7 kb of upstream sequences, followed by genomic sequences
containing the complete fkh-6 coding region, and the
gfp-coding region fused in frame immediately 5' to the FKH-6
stop codon. This reporter fully rescued fkh-6 male gonadal defects
and restored hermaphrodite self-fertility, suggesting appropriate spatial and
temporal expression.
The two reporters have the same expression pattern. Both are expressed in Z1 and Z4 of XO (Fig. 4A,B) and XX (Fig. 4C,D) L1 larvae. However, later during L1, the timing of expression is sexually dimorphic: in XO larvae reporter expression persists until late L1 in Z1/Z4 descendants, whereas in XX larvae it decreases in mid L1 and is undetectable by late L1 (Fig. 4E,F, not shown). Later expression also is sexually dimorphic. In L3 hermaphrodites, the fkh-6 reporters resume expression in sheath-spermathecal precursor cells, continuing through adulthood in spermatheca and weakly in proximal sheath (Fig. 4G,H; data not shown). By contrast, no expression was observed in XO animals past the L1 stage. Reporter expression is consistent with fkh-6 mutant defects in L1 male gonadal morphogenesis and in hermaphrodite gonadal differentiation during L3 and L4. No expression was detectable outside the gonad in larvae or adults of either sex.
|
|
|
We next examined the relationship of tra-1 and fkh-6 in controlling Z1/Z4 divisions. We found that Z1/Z4 divisions in fkh-6; tra-1 mutants resemble those in fkh-6 single mutants rather than those in tra-1 single mutants (Fig. 5C-F). The detailed cellular analysis of tra-1 single mutants will be described elsewhere (L.M., unpublished). The Z1/Z4 cell division and movements of their daughters in tra-1 mutants have characteristics typical of wild-type XO males (e.g. asymmetric division, anterior migration) (Fig. 5D,F). By contrast, in both fkh-6 single mutants (Fig. 5C) and in fkh-6; tra-1 double mutants (Fig. 5E), the Z1 and Z4 divisions are not markedly asymmetric, and none of the daughters move to the anterior (summarized in Fig. 5F). Thus, for these early events of gonadogenesis, fkh-6 is epistatic to tra-1, suggesting that fkh-6 acts downstream of tra-1 to specify a male-specific division of Z1 and Z4 and the male-specific anterior migration of Z1/Z4 daughters.
To investigate the relationship of tra-1 and fkh-6 at the
molecular level, we employed GFP reporters for each gene
(Fig. 5G-J; summarized in
Fig. 5K). A tra-1::gfp
transgene is expressed in Z1 and Z4 and their descendents, both in wild-type
XX and XO L1 gonads (L.M. and J.K., unpublished) and in fkh-6 mutant
L1 gonads, with no obvious change in pattern or timing
(Fig. 5G,H). This lack of
effect is consistent with the genetic epistasis result placing tra-1
upstream of fkh-6 at this early stage of gonadogenesis. By contrast,
fkh-6::gfp expression in the L1 XX gonad was extended by removal of
tra-1 activity. Thus, whereas fkh-6::gfp in wild-type XX
animals was undetectable by late L1, its expression continued into late L1 in
tra-1(null) XX pseudomales (Fig.
5I,J); likewise, the L3 to adult expression of fkh-6::gfp
observed in wild-type XX hermaphrodites was absent in tra-1 XX
pseudomales (not shown). This dependence of fkh-6::gfp expression on
tra-1 but not vice versa (summarized in
Fig. 5K) is consistent with the
genetic epistasis observed in early L1 gonads of fkh-6; tra-1 double
mutants. To ask whether TRA-1A might directly repress fkh-6
transcription, we examined the 5' flanking region of fkh-6
present in the GFP reporters, and identified one close match to the TRA-1A
DNA-binding consensus (TTGGTGGTC from -6523 to -6531 relative to the
initiation codon). However, we did not find this site in the related nematode
C. briggsae, and mutating it to TTCTGCAGC, a change that should
eliminate regulation by TRA-1A in vivo (Yi
et al., 2000), did not affect the level or timing of
fkh-6::gfp expression in either sex (data not shown). We conclude
that tra-1 affects the timing of early fkh-6 expression, but
probably not by direct transcriptional repression (see Discussion).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
fkh-6 controls multiple aspects of male gonadogenesis
The L1 gonad in fkh-6 mutant males is sexually transformed in
several respects: the Z1/Z4 divisions lack male-specific size asymmetry, and
their proximal daughters do not undergo their male-specific anterior
migration. Furthermore, Z1.a and Z4.p, which do not divide in wild-type males,
do divide in fkh-6 XO gonads. The expression of hermaphrodite gonadal
markers from L3 through adult stages in fkh-6 mutant males confirms
the sexual transformation. In these fkh-6 males, the number and
position of cells expressing each gonadal marker gene varies. This is likely
to reflect variability in the extent of feminization of the mutant gonad,
resulting in a variably intersexual cell lineage. In wild-type males,
fkh-6 reporters are expressed only during L1, suggesting that it is
FKH-6 activity during this stage that determines the later male
differentiation of gonadal cells.
We have considered two models to account for the range of defects in
fkh-6 male gonadogenesis. First, FKH-6 might independently control
male-specific asymmetric division, cellular movement and cellular
differentiation, canalizing each into the male mode. Alternatively, the
initial division of Z1 and Z4 might determine whether subsequent events of
gonadogenesis occur in the male mode, and FKH-6 might control the entire
process by regulating only this first step. In this latter model, FKH-6 could,
for example, regulate a male-specific determinant that is partitioned during
the first Z1 and Z4 divisions, leading to male-specific cell migration and
differentiation. There are a number of precedents for FKH transcription
factors controlling asymmetry and cell polarity. For example, in C.
elegans, UNC-130 acts in neuronal precursors to regulate their asymmetric
division and confer distinct identities to daughter cells
(Nash et al., 2000;
Sarafi-Reinach and Sengupta,
2000
). Similarly, in Drosophila the Jumeaux protein is
required for proper localization and segregation of Numb in neuronal precursor
cells (Cheah et al., 2000
).
fkh-6 affects hermaphrodite and male gonadogenesis differently
The pattern of early gonadal cell divisions, migration and differentiation
appear largely normal in fkh-6 mutant hermaphrodites until early L3,
in sharp contrast with the situation in mutant males. Most importantly, there
is no evidence of sex reversal in fkh-6 hermaphrodites. Normal
hermaphrodite structures (sheath, spermatheca, uterus) are present, but
malformations become apparent from L3 onwards, and fkh-6
hermaphrodites are infertile. The onset of defective gonadogenesis correlates
with the hermaphrodite-specific expression of fkh-6::gfp reporters
from L3 onwards. Germline mosaic analysis demonstrates that embryonic
viability requires maternal fkh-6 activity in somatic cells, but not
in the maternal germ line or in zygotic cells.
Thus, while fkh-6 affects gonadogenesis in both sexes, its functions are mostly distinct in hermaphrodites and males. An exception is that early divisions of somatic gonadal cells are slower than normal in both sexes. One possibility is that fkh-6 controls an early cellular process that not only is intrinsic to the rate of cell division in gonads of both sexes, but also is essential for asymmetric division in the male gonad.
FKH-6 is required in the early XO gonad for male gonadogenesis and cell fate determination, and fkh-6 reporters are expressed longer in the L1 gonad in males than in hermaphrodites. Is persistent FKH-6 expression sufficient to direct male gonadal fates in hermaphrodites? To address this question, we overexpressed FKH-6 using either of two promoters (W.C. and D.Z., unpublished). Overexpression using a heatshock promoter was highly toxic even with very mild heatshock conditions. Overexpression using the lag-2 promoter to drive expression of FKH-6::GFP specifically in Z1/Z4 lineages showed no clear signs of masculinization.
In summary, fkh-6 has largely distinct functions in the two sexes. In XO animals it specifies male-specific cellular events in the early gonad and is required for commitment to male rather than female cellular differentiation, whereas in XX animals it controls later gonadal differentiation. In both sexes it affects the rate of gonadal cell division.
fkh-6 links global and organ-specific sex determination
The tra-1 gene encodes the terminal regulator in the global
somatic sex-determination pathway
(Hodgkin, 1987). We suggest
that fkh-6 acts downstream of tra-1 to establish sexual
dimorphism during the division of Z1/Z4 and in their daughters. This
suggestion is based on two lines of evidence. First, fkh-6 defects in
the early L1 gonad are epistatic to those of tra-1; and second,
fkh-6::gfp expression in XX animals is extended during L1 by a
tra-1(null) mutation. As tra-1 promotes female development
and fkh-6 promotes male development, tra-1 activity is
likely to inhibit fkh-6 in the early XX gonad, allowing Z1 and Z4 to
divide in the hermaphrodite mode. How does this occur? Mutating the only
consensus TRA-1A binding site in a fkh-6::gfp reporter had no
apparent effect on reporter expression
(Fig. 5; data not shown). The
simplest conclusion is that the inhibition of fkh-6 expression by
TRA-1A is indirect; however, it remains possible that TRA-1A regulates
fkh-6 transcription through a binding site we did not recognize or
redundantly with another factor.
The roles of fkh-6 and tra-1 are not limited to sex
determination. fkh-6 mutants exhibit delayed cell divisions in both
sexes, and fkh-6;tra-1 double mutant males have a synthetic
phenotype: shortly after the first division of Z1 and Z4, gonadogenesis
arrests. By contrast, fkh-6 tra-2 double mutant gonads do not arrest,
and they resemble the fkh-6 single mutant throughout gonadal
development. This difference suggests that tra-1 and fkh-6
share a function distinct from that of the global sex determination pathway. A
simple model is that both genes regulate proliferation of somatic gonadal
cells, and are partially redundant for this function. This idea helps
reconcile previous observations that tra-1 null mutant gonads, while
strongly masculinized, often are smaller than normal and disorganized
(Hodgkin, 1987;
Schedl et al., 1989
), whereas
null mutants in tra-2 and other upstream genes develop gonads of
normal size and morphology.
Is fkh-6 regulation of gonadal sexual development conserved?
fkh-6 is a gonad-specific regulator of sex determination. Is this
role unique to C. elegans, or might forkhead genes be conserved
regulators of gonadal sex determination? At least one forkhead gene,
Foxl2, is expressed in developing gonads of a variety of vertebrates
(Loffler et al., 2003), and is
required in the mammalian gonad (Crisponi
et al., 2001
; De Baere et al.,
2001
). Like fkh-6, Foxl2 is required in the female gonad
in somatic cells that support the germline. However, unlike fkh-6,
Foxl2 is not required in the male gonad and Foxl2 mutations have
not been shown to cause sexual transformation. It is difficult to assess
whether gonadal regulation by fkh-6 and Foxl2 in different
phyla reflects evolutionary conservation or is an example of convergent
evolution. C. briggsae has an unambiguous fkh-6 homolog
(Hope et al., 2003
), but
sequence conservation of the forkhead domain is insufficient for robust
phylogenetic analysis in more distant species, and FKH-6 lacks other conserved
sequence motifs that might aid in identifying orthologs.
fkh-6 provides an entry point for elucidating the process of gonadal sex determination in C. elegans. Once functionally equivalent genes are found in other phyla and more components of the fkh-6 regulatory pathway are discovered, the evolutionary history of gonadal sex determination should become much clearer.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashrafi, K., Chang, F. Y., Watts, J. L., Fraser, A. G., Kamath, R. S., Ahringer, J. and Ruvkun, G. (2003). Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421,268 -272.[CrossRef][Medline]
Bargmann, C. I. and Avery, L. (1995). Laser killing of cells in Caenorhabditis elegans. Methods Cell Biol. 48,225 -250.[Medline]
Barton, M. K. and Kimble, J. (1990).
fog-1, a regulatory gene required for specification of
spermatogenesis in the germ line of Caenorhabditis elegans.
Genetics 125,29
-39.
Cheah, P. Y., Chia, W. and Yang, X. (2000).
Jumeaux, a novel Drosophila winged-helix family protein, is required
for generating asymmetric sibling neuronal cell fates.
Development 127,3325
-3335.
Chen, P. and Ellis, R. E. (2000). TRA-1A
regulates transcription of fog-3, which controls germ cell fate in
C. elegans. Development
127,3119
-3129.
Christiansen, A. E., Keisman, E. L., Ahmad, S. M. and Baker, B. S. (2002). Sex comes in from the cold: the integration of sex and pattern. Trends Genet. 18,510 -516.[CrossRef][Medline]
Cline, T. W. and Meyer, B. J. (1996). Vive la difference: males vs females in flies vs worms. Annu. Rev. Genet. 30,637 -702.[CrossRef][Medline]
Conradt, B. and Horvitz, H. R. (1999). The TRA-1A sex determination protein of C. elegans regulates sexually dimorphic cell deaths by repressing the egl-1 cell death activator gene. Cell 98,317 -327.[Medline]
Crisponi, L., Deiana, M., Loi, A., Chiappe, F., Uda, M., Amati, P., Bisceglia, L., Zelante, L., Nagaraja, R., Porcu, S. et al. (2001). The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat. Genet. 27,159 -166.[CrossRef][Medline]
Dauwalder, B., Tsujimoto, S., Moss, J. and Mattox, W.
(2002). The Drosophila takeout gene is regulated by the
somatic sex-determination pathway and affects male courtship behavior.
Genes Dev. 16,2879
-2892.
De Baere, E., Dixon, M. J., Small, K. W., Jabs, E. W., Leroy, B.
P., Devriendt, K., Gillerot, Y., Mortier, G., Meire, F., van Maldergem, L. et
al. (2001). Spectrum of FOXL2 gene mutations in
blepharophimosis-ptosisepicanthus inversus (BPES) families demonstrates a
genotype-phenotype correlation. Hum. Mol. Genet.
10,1591
-1600.
Edgley, M. L. and Riddle, D. L. (2001). LG II balancer chromosomes in Caenorhabditis elegans: mT1(II;III) and the mIn1 set of dominantly and recessively marked inversions. Mol. Genet. Genomics 266,385 -395.[CrossRef][Medline]
Ellis, R. E. and Kimble, J. (1995). The
fog-3 gene and regulation of cell fate in the germ line of
Caenorhabditis elegans. Genetics
139,561
-577.
Erdman, S. E. and Burtis, K. C. (1993). The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain. EMBO J. 12,527 -535.[Abstract]
Ferreira, H. B., Zhang, Y., Zhao, C. and Emmons, S. W. (1999). Patterning of Caenorhabditis elegans posterior structures by the Abdominal-B homolog, egl-5. Dev. Biol. 207,215 -228.[CrossRef][Medline]
Finley, K. D., Taylor, B. J., Milstein, M. and McKeown, M.
(1997). dissatisfaction, a gene involved in sex-specific
behavior and neural development of Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA
94,913
-918.
Fitch, D. H. A. and Thomas, W. K. (1997). Evolution. In C. elegans II (ed. D. L. Riddle T. Blumenthal B. J. Meyer and J. R. Priess), pp.815 -849. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Friedman, L., Santa Anna-Arriola, S., Hodgkin, J. and Kimble, J. (2000). gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans. Dev. Biol. 228,350 -362.[CrossRef][Medline]
Hall, D. H., Winfrey, V. P., Blaeuer, G., Hoffman, L. H., Furuta, T., Rose, K. L., Hobert, O. and Greenstein, D. (1999). Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germ line and soma. Dev. Biol. 212,101 -123.[CrossRef][Medline]
Harfe, B. D., Branda, C. S., Krause, M., Stern, M. J. and Fire,
A. (1998). MyoD and the specification of muscle and
non-muscle fates during postembryonic development of the C. elegans
mesoderm. Development
125,2479
-2488.
Hodgkin, J. (1987). A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev. 1, 731-745.[Abstract]
Hodgkin, J. A. and Brenner, S. (1977).
Mutations causing transformation of sexual phenotype in the nematode
Caenorhabditis elegans. Genetics
86,275
-287.
Hodgkin, J., Doniach, T. and Shen, M. (1986). The sex determination pathway in the nematode Caenorhabditis elegans: variations on a theme. Cold Spring Harbor Symp. Quant. Biol. 50,585 -593.
Hope, I. A. (1991). `Promoter trapping' in Caenorhabditis elegans. Development 113,399 -408.[Abstract]
Hope, I. A., Mounsey, A., Bauer, P. and Aslam, S. (2003). The forkhead gene family of Caenorhabditis elegans. Gene 304,43 -55.[CrossRef][Medline]
Hubbard, E. J. and Greenstein, D. (2000). The Caenorhabditis elegans gonad: a test tube for cell and developmental biology. Dev. Dyn. 218,2 -22.[CrossRef][Medline]
Hunter, C. P. and Wood, W. B. (1992). Evidence from mosaic analysis of the masculinizing gene her-1 for cell interactions in C. elegans sex determination. Nature 355,551 -555.[CrossRef][Medline]
Ito, H., Fujitani, K., Usui, K., Shimizu-Nishikawa, K., Tanaka,
S. and Yamamoto, D. (1996). Sexual orientation in
Drosophila is altered by the satori mutation in the
sex-determination gene fruitless that encodes a zinc finger protein
with a BTB domain. Proc. Natl. Acad. Sci. USA
93,9687
-9692.
Jin, C., Marsden, I., Chen, X. and Liao, X. (1999). Dynamic DNA contacts observed in the NMR structure of winged helix protein-DNA complex. J. Mol. Biol. 289,683 -690.[CrossRef][Medline]
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G. and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2,RESEARCH0002 .[Medline]
Kimble, J. (1981). Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans. Dev. Biol. 87,286 -300.[Medline]
Kimble, J. and Hirsh, D. (1979). The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev. Biol. 70,396 -417.[Medline]
Kimble, J. E. and White, J. G. (1981). On the control of germ cell development in Caenorhabditis elegans. Dev. Biol. 81,208 -219.[Medline]
Lints, R. and Emmons, S. W. (2002). Regulation
of sex-specific differentiation and mating behavior in C. elegans by
a new member of the DM domain transcription factor family. Genes
Dev. 16,2390
-2402.
Loffler, K. A., Zarkower, D. and Koopman, P. (2003). Etiology of ovarian failure in blepharophimosis-ptosis-epicanthus-inversus syndrome (BPES): FOXL2 is a conserved, early-acting gene in vertebrate ovarian development. Endocrinology 144,245 -251.
Madl, J. E. and Herman, R. K. (1979).
Polyploids and sex determination in Caenorhabditis elegans.
Genetics 93,393
-402.
Mathies, L. D., Henderson, S. T. and Kimble, J.
(2003). The C. elegans Hand gene controls embryogenesis
and early gonadogenesis. Development
130,2881
-2892.
Matsuda, M., Nagahama, Y., Shinomiya, A., Sato, T., Matsuda, C., Kobayashi, T., Morrey, C. E., Shibata, N., Asakawa, S., Shimizu, N. et al. (2002). DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417,559 -563.[CrossRef][Medline]
Mello, C. and Fire, A. (1995). DNA transformation. Methods Cell Biol. 48,451 -482.[Medline]
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Abstract]
Miskowski, J., Li, Y. and Kimble, J. (2001). The sys-1 gene and sexual dimorphism during gonadogenesis in Caenorhabditis elegans. Dev. Biol. 230, 61-73.[CrossRef][Medline]
Nanda, I., Kondo, M., Hornung, U., Asakawa, S., Winkler, C.,
Shimizu, A., Shan, Z., Haaf, T., Shimizu, N., Shima, A. et al.
(2002). A duplicated copy of DMRT1 in the
sex-determining region of the Y chromosome of the medaka, Oryzias
latipes. Proc. Natl. Acad. Sci. USA
99,11778
-11783.
Nash, B., Colavita, A., Zheng, H., Roy, P. J. and Culotti, J.
G. (2000). The forkhead transcription factor UNC-130 is
required for the graded spatial expression of the UNC-129 TGF-beta guidance
factor in C. elegans. Genes Dev.
14,2486
-2500.
Nef, S. and Parada, L. F. (2000). Hormones in
male sexual development. Genes Dev.
14,3075
-3086.
Pettitt, J., Wood, W. B. and Plasterk, R. H.
(1996). cdh-3, a gene encoding a member of the cadherin
superfamily, functions in epithelial cell morphogenesis in Caenorhabditis
elegans. Development
122,4149
-4157.
Raymond, C. S., Murphy, M. W., O'Sullivan, M. G., Bardwell, V.
J. and Zarkower, D. (2000). Dmrt1, a gene related to
worm and fly sexual regulators, is required for mammalian testis
differentiation. Genes Dev.
14,2587
-2595.
Raymond, C. S., Shamu, C. E., Shen, M. M., Seifert, K. J., Hirsch, B., Hodgkin, J. and Zarkower, D. (1998). Evidence for evolutionary conservation of sex-determining genes. Nature 391,691 -695.[CrossRef][Medline]
Ryner, L. C., Goodwin, S. F., Castrillon, D. H., Anand, A., Villella, A., Baker, B. S., Hall, J. C., Taylor, B. J. and Wasserman, S. A. (1996). Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell 87,1079 -1089.[Medline]
Sarafi-Reinach, T. R. and Sengupta, P. (2000).
The forkhead domain gene unc-130 generates chemosensory neuron
diversity in C. elegans. Genes Dev.
14,2472
-2485.
Schedl, T., Graham, P. L., Barton, M. K. and Kimble, J.
(1989). Analysis of the role of tra-1 in germline sex
determination in the nematode Caenorhabditis elegans.
Genetics 123,755
-769.
Shen, M. M. and Hodgkin, J. (1988). mab-3, a gene required for sex-specific yolk protein expression and a male-specific lineage in C. elegans. Cell 54,1019 -1031.[Medline]
Siegfried, K. R. and Kimble, J. (2002). POP-1 controls axis formation during early gonadogenesis in C. elegans. Development 129,443 -453.[Medline]
Sulston, J. and Hodgkin, J. (1988). Methods. InThe Nematode Caenorhabditis elegans (ed. W. W. Wood), pp. 587-606. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Dev. Biol. 56,110 -156.[Medline]
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,4673 -4680.[Abstract]
Weigel, D. and Jackle, H. (1990). The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell 63,455 -456.[Medline]
Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H. and Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat. Genet. 28,160 -164.[CrossRef][Medline]
Yi, W., Ross, J. M. and Zarkower, D. (2000).
mab-3 is a direct tra-1 target gene regulating diverse
aspects of C. elegans male sexual development and behavior.
Development 127,4469
-4480.
Zarkower, D. (2001). Establishing sexual dimorphism: conservation amidst diversity? Nat. Rev. Genet. 2,175 -185.[CrossRef][Medline]
Zarkower, D. and Hodgkin, J. (1992). Molecular analysis of the C. elegans sex-determining gene tra-1: a gene encoding two zinc finger proteins. Cell 70,237 -249.[Medline]
Related articles in Development: