1 Department of Biochemistry and Howard Hughes Medical Institute, University of
Wisconsin-Madison, Madison, WI 53706-1544, USA
2 Department of Genetics, North Carolina State University, Raleigh, NC
27695-7614, USA
3 Department of Molecular and Medical Genetics, University of Toronto, Toronto
M5S 1A8, Canada
4 Whitehead Institute, Cambridge, MA 02142-1479, USA
* Author for correspondence (e-mail: jekimble{at}facstaff.wisc.edu)
Accepted 5 June 2004
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SUMMARY |
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Key words: TRA-1, GLI, EHN-3, Cell polarity, Cell proliferation, Gonadogenesis, C. elegans
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Introduction |
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In C. elegans, XX embryos develop as hermaphrodites (essentially
females that transiently make sperm), and XO embryos develop as males
(Hubbard and Greenstein,
2000). Most adult tissues are sexually dimorphic. Of particular
importance to this paper are the gonads: hermaphrodite adults have two
U-shaped gonadal arms that are related to each other by two-fold rotational
symmetry, but males have only a single J-shaped gonadal arm that is
asymmetric. Each gonadal arm, whether hermaphrodite or male, possesses a
proximodistal axis that is established in early gonadogenesis. At the distal
end of each arm reside either one (hermaphrodites) or two (males) distal tip
cells, which govern germline proliferation; more proximal are the germline and
sex-specific somatic gonadal structures (e.g. uterus in hermaphrodites,
seminal vesicle in males). The proximodistal axis of the gonad departs from
the anteroposterior body axis and instead reflects coordinates internal to the
organ.
When a wild-type first stage larva (L1) hatches from its eggshell, it
possesses a four-celled gonadal primordium with two somatic gonadal precursors
(SGPs) and two primordial germ cells (PGCs)
(Hubbard and Greenstein,
2000). The anterior and posterior SGPs (Z1 and Z4, respectively)
reside at the poles of the primordium and the PGCs are cradled between them
(Fig. 1A). This primordium
assembles during embryogenesis and appears identical in the two sexes. The
genetic control of SGP development and assembly of the gonadal primordium is
not well understood. We previously described three genes that affect SGP
development. The hnd-1 gene encodes a Hand bHLH transcription factor
and influences SGP survival and SGP position within the primordium; the
ehn genes are genetic enhancers of hnd-1
(Mathies et al., 2003
). The
vertebrate Hand transcription factor has been implicated in controlling
development of several organs, including the gonad in zebrafish
(Weidinger et al., 2002
).
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The tra-1 gene is best known for its role in specifying female
fates: XX tra-1 null mutants are sexually transformed from
hermaphrodite to male (Meyer,
1997). In addition, tra-1 affects gonadogenesis: 20-50%
of both XX and XO null mutants possess small and variably mis-shapen gonads
(Hodgkin, 1987
;
Schedl et al., 1989
). However,
the role of tra-1 in gonadogenesis had not been explored. In this
paper, we demonstrate that tra-1 controls both SGP position and
polarity in XX and XO animals, and that TRA-1 protein is expressed in SGPs in
male and hermaphrodite gonadal primordia. We also find that tra-1
acts redundantly with ehn-3 to control SGP maturation and division.
Finally, we report that ehn-3 encodes a nuclear zinc-finger protein
that is expressed specifically in the SGPs. We conclude that TRA-1/GLI and
EHN-3 are key regulators of SGP development. This role for TRA-1 in the
control of SGP development is a major departure from its better-known role in
sex determination, and may be an ancient role of TRA-1/GLI in nematode
development.
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Materials and methods |
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LGII: ehn-1(q690)
(Mathies et al., 2003),
tra-2(ar221ts)
LGIII: dpy-18(e364), tra-1(e1099), tra-1(e1834), unc-119(ed3), eDp6
LGIV: ced-2(e1752), ced-3(n717), dpy-13(e184sd),
ehn-3(q689) (Mathies et al.,
2003), unc-5(e53), nDf41, mDf4, fem-3(e1996)
LGV: fog-2(q71), him-5(e1490)
LGX: hnd-1(q740)
(Mathies et al., 2003),
xol-1(y9)
Dominant GFP balancers: hT2[qIs48] for LGI and LGIII, and nT1[qIs50] for LGIV and LGV.
Molecular markers: qIs47 [X-linked GFP], qIs55
[hnd-1(N)::GFP] (Mathies et al.,
2003), qIs56 [lag-2::GFP]
(Blelloch et al., 1999
),
qIs61 [pes-1::GFP]
(Molin et al., 2000
),
qIs74 [GFP::POP-1]
(Siegfried et al., 2004
),
qIs76 [tra-1::GFP], a transcriptional reporter of tra-1
expression (Chang et al.,
2004
), qIs77 [unc-122::GFP]
(Miyabayashi et al., 1999
),
and rdEx1 [GFP::TRA-1], a translational TRA-1 reporter with
partial rescuing activity.
Phenotypic analysis and laser ablation
XO tra-1 males were identified as non-GFP animals from a cross of
tra-1(e1099)/hT2[qIs48] females to tra-1(e1099)/hT2[qIs48];
qIs47 XO males. qIs47 is an X-linked GFP marker and
hT2[qIs48] is a GFP balancer chromosome. tra-1/hT2[qIs48]
females were generated using fog-1(RNAi)
(Jin et al., 2001). Cell
lineages were followed as described
(Sulston and Horvitz, 1977
).
Laser ablations were performed using a Micropoint Ablation Laser System
(Photonic Instruments) as described
(Bargmann and Avery, 1995
). For
SGP daughter isolation, Z1 or Z4 was ablated, the animal rescued, then one SGP
daughter ablated. All ablations were validated. The fate of the remaining SGP
daughter was assayed in L3-L4 using lag-2::GFP.
tra-1 and ehn-3 RNAi
Double-stranded RNA was synthesized using Megascript T7 kit (Ambion) and
injected at 1 mg/ml. Template for RNA synthesis contained over 500 nucleotides
and targeted all ehn-3 and tra-1 transcripts.
ehn-3 molecular biology
We mapped ehn-3 between dpy-13 and unc-5 on
chromosome IV: 1/34 Unc non-Dpy animals from ehn-3(q689)/unc-5
dpy-13 were Ehn; mDf4 complemented and nDf41 failed to
complement ehn-3(q689). We cloned ehn-3 by transformation
rescue. Two independent transgenic lines carrying a PCR-generated fragment
from 1817 bp upstream to 1024 bp downstream of the predicted ZK616.10-coding
region rescued ehn-3(q689) completely (n=66). Using RT-PCR
with the SL1 trans-spliced leader, we confirmed the predicted gene structure;
this transcript is ehn-3B1. We also identified a minor splice variant
(ehn-3B2) that skips the second exon. The entire rescuing region was
sequenced from ehn-3(q689) genomic DNA, and found to carry only one
change 788 bp upstream of the ehn-3B1 ATG. We isolated an
ehn-3 deletion by a PCR-based screen
(Kraemer et al., 1999):
ehn-3(q766) removes 1074 bp upstream of the ehn-3B ATG plus
DNA encoding the N-terminal 14 amino acids of EHN-3B. Using semi-quantitative
RT-PCR to a region contained in all three transcripts, we examined ZK616.10
expression in ehn-3 mutants: hlh-2 was the template control
and amplifications were performed within the linear range (32 cycles for
ehn-3, 27 cycles for hlh-2). Both ehn-3(q689) and
ehn-3(q766) mutants expressed about tenfold less ZK616.10 RNA than
did wild-type. Since ehn-3(q689) appeared to be the stronger allele,
we searched for additional transcripts by RT-PCR and found one that included
two unpredicted upstream exons trans-spliced to SL1; ehn-3(q689)
causes a frame-shift mutation in the second exon of this larger transcript
(ehn-3A); ehn-3(q766) removes the second exon and part of
the first exon common to ehn-3A and ehn-3B.
TRA-1 antibody staining
We generated two antisera against the TRA-1 N-terminus: -TRA-1(DZ)
against a peptide (Segal et al.,
2001
), and
-TRA-1(AS) against a GST fusion to an N-terminal
TRA-1 fragment. Both antibodies detected TRA-1 in SGPs of L1 larvae;
-TRA-1(AS) also detected TRA-1 in embryonic SGPs. Recognition was
specific, as antigen was not seen in tra-1(e1834) larvae (not shown)
(Segal et al., 2001
). Embryos
were fixed by freeze-cracking (Miller and
Shakes, 1995
), and larvae by a modified Finney-Ruvkun protocol
(Finney and Ruvkun, 1990
).
Primary antibodies were
-TRA-1 (pre-absorbed against tra-1(e1834);
eDp6 fixed worms),
-PGL-1
(Kawasaki et al., 1998
) and
-GFP (Clontech). Secondary antibodies were
-rabbit Cy3 and
-mouse FITC (Jackson ImmunoResearch). To distinguish between males and
hermaphrodites in L1s, we relied on sexually dimorphic coelomocyte positions:
they flank the gonad in males, but are anterior in hermaphrodites.
unc-122::GFP marked coelomocytes and him-5 increased male
frequency. We used tra-2(ts); xol-1 to generate XX L1 pseudomales
(Miller et al., 1988
), and a
fog-2 male/female strain to produce 50% XO progeny
(Schedl and Kimble, 1988
).
EHN-3::GFP (pJK939)
The ehn-3 genomic region, including 429 bp of upstream sequence
and all six exons of ehn-3, was PCR amplified, cloned into pT7blue
(Novagen), sequenced and cloned into pPD95.79 (a gift from A. Fire) to make
pJK939; this construct fuses GFP in frame to the last amino acid of EHN-3.
pJK939 was injected with pRF4 (Mello et
al., 1991) to make qEx488, and integrated to make
qIs68. EHN-3::GFP rescues ehn-3(q689) (from 18% to 1%
defective, n=604) and tra-1; ehn-3(q766) double mutants
(from 97% to 8% absent gonad, n=37).
GFP::TRA-1 (pJK946)
The tra-1 3' end (BamHI to ApaI) was
cloned from pDZ53 (Zarkower and Hodgkin,
1992) into pJK876 (Chang et
al., 2004
), replacing GFP and ß-gal-coding sequences.
GFP-coding sequences were cloned into the BamHI site. Finally, the
tra-1 cDNA 5' end was cloned downstream of GFP to make
GFP::TRA-1. pJK946 was injected with pRF4
(Mello et al., 1991
) to make
rdEx1. rdEx1 partially rescues tra-1(e1099): rescued animals
had a feminized tail and two-armed gonad, but were sterile. GFP::TRA-1
localization mirrors that of TRA-1 antibodies in L1 gonads, but is not
localized properly in all tissues. For example, GFP::TRA-1 accumulates in
intestinal nuclei of both males and hermaphrodites, while
-TRA-1
antibodies detect TRA-1 protein only in hermaphrodite intestinal nuclei.
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Results |
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tra-1 governs polarity of the Z1 division
In wild-type primordia, SGPs divide asymmetrically, producing daughters
with distinct sizes and fates (Fig.
1C,E) (Kimble and Hirsh,
1979). Moreover, the polarities of these asymmetric divisions are
opposite to each other: smaller SGP daughters are polar and larger ones are
central. In tra-1 mutants, SGPs divided asymmetrically, but the
polarity of the Z1 division was reversed compared with wild type
(Fig. 1D,F): the larger
daughter was generated at the pole and the smaller daughter was more central
(15/17). Thus, tra-1 regulates the polarity of the Z1 division.
Asymmetry of the SGP division is controlled by Wnt/MAPK signaling
(Siegfried and Kimble, 2002),
including POP-1, the C. elegans TCF/LEF transcription factor
(Lin et al., 1995
). In
wild-type hermaphrodites, the central SGP daughters display more nuclear
GFP::POP-1 than their polar sisters
(Siegfried et al., 2004
). We
find that SGP daughters in wild-type males similarly exhibit GFP::POP-1
asymmetry: the larger central daughters have more nuclear GFP::POP-1 than do
smaller polar daughters (Fig.
1C, right). In tra-1 mutants, the polarity of GFP::POP-1
asymmetry was reversed in Z1, such that the larger polar daughter had more
nuclear GFP::POP-1 than its smaller central sister
(Fig. 1D, right). Therefore,
the tra-1 Z1 polarity reversal affects both cell size and abundance
of nuclear GFP::POP-1.
We next asked if the Z1 polarity reversal affected daughter cell fates. To
this end, we isolated SGP daughters from other somatic gonadal cells using
laser ablation and then assayed fates using functional and morphological
criteria. In males, the larger daughter normally generates a linker cell (LC),
which is a large, round cell that expresses lag-2::GFP brightly and
guides gonadal elongation; by contrast, the smaller daughter becomes a male
DTC, which is a small elongate cell that stimulates germline proliferation and
expresses lag-2::GFP dimly (Fig.
1G) (Blelloch et al.,
1999). In tra-1 mutants, the smaller central daughter of
Z1 expressed lag-2::GFP dimly and stimulated germline proliferation,
and the larger polar daughter generated a cell with typical LC morphology that
expressed lag-2::GFP brightly and led gonadal elongation
(Fig. 1H). Therefore, the fate
of each Z1 daughter correlated with its size and GFP::POP-1 level. We conclude
that the polarity of the Z1 division is fully reversed in tra-1
mutants. The polarity of Z4, by contrast, was normal in tra-1
mutants.
TRA-1 localization in SGPs
To assess tra-1 expression in SGPs, we examined TRA-1 protein
using -TRA-1 antibodies (Segal et
al., 2001
) (this work) and a GFP::TRA-1 translational reporter
(see Materials and methods). In wild-type XX embryos, TRA-1 was predominantly
nuclear in SGPs from formation of the primordium
(Fig. 2A,B), through
embryogenesis (Fig. 2C), and
into the first larval stage (Fig.
2D). The nuclear location suggests that TRA-1 is active in SGPs,
because the active state of TRA-1 has been correlated with nuclear
localization (Segal et al.,
2001
).
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After hatching, TRA-1 was still detected in SGPs, but its level and subcellular distribution were distinct in the two sexes. In hermaphrodite L1 SGPs, TRA-1 was largely nuclear (Fig. 2E), but in male L1 SGPs, TRA-1 was present at a lower level and was uniformly distributed between nucleus and cytoplasm (Fig. 2F). We observed a similar sexually dimorphic difference in the distribution of GFP::TRA-1 in L1 SGPs (Fig. 2G-J). The GFP::TRA-1 reporter partially rescued tra-1(0) mutants, indicating that it produces a functional protein (see Materials and methods). After the first SGP division, TRA-1 continues to be expressed in hermaphrodite gonads, but is no longer detectable in males (not shown). Therefore, after hatching, TRA-1 is predicted to be more active in hermaphrodite gonads than in male gonads, consistent with its role in promoting female fates.
tra-1 and ehn-3 redundantly control gonadal development
The hnd-1, ehn-1 and ehn-3 genes affect SGP position in a
manner reminiscent of tra-1: in hnd/ehn mutants, SGPs are
assembled correctly into the primordium, but they can lose their polar
position (Mathies et al.,
2003). We examined double mutant combinations of tra-1
with hnd-1, ehn-1 or ehn-3 to explore their relationships.
To our surprise, tra-1; ehn-3 double mutants had a striking
synergistic effect on gonadogenesis: whereas most tra-1 and
ehn-3 single mutants possessed easily detectable gonadal tissue by
Nomarski microscopy and DAPI staining (Fig.
3A), very few tra-1; ehn-3 double mutants had a
detectable gonad (Fig. 3B;
Table 1). We examined the gonad
in more detail using PGL-1, a germ cell marker that detects many cells in
wild-type (Kawasaki et al.,
1998
), ehn-3 (Fig.
3C) and tra-1 late larval gonads. By contrast, tra-1;
ehn-3 double mutants consistently had only a few PGL-1-positive cells
(Fig. 3D). The interaction
appears specific to tra-1 and ehn-3, as we observed only a
weak synergistic interaction for tra-1; hnd-1 and tra-1;
ehn-1 double mutants (Table
1). We conclude that tra-1 and ehn-3 have an
overlapping role in generation of gonadal tissue.
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SGPs in tra-1; ehn-3 double mutants
Why do tra-1; ehn-3 double mutants have so little gonadal tissue?
An examination of L1 larvae by Nomarski microscopy revealed the probable cause
- cells with morphology typical of SGPs were not seen (n=16). Whereas
all tra-1 and ehn-3 single mutants had normal SGPs
(Fig. 3E)
(Mathies et al., 2003),
tra-1; ehn-3 double mutants had either one or two tiny cells
associated with PGCs (Fig. 3G).
To determine whether the tiny PGC-associated cells were indeed SGPs, we used
pes-1::GFP and lag-2::GFP, which are molecular SGP markers
(Blelloch et al., 1999
;
Molin et al., 2000
). The SGPs
in tra-1 single mutants expressed pes-1::GFP
(Fig. 3F, 19/20), as did SGPs
in ehn-3 single mutants (53/53). However, the tiny cells in
tra-1; ehn-3 double mutants did not detectably express
pes-1::GFP (Fig. 3H;
0/16) or lag-2::GFP (not shown). The possibility of faint
lag-2::GFP expression by the tiny PGC-associated cells could not be
ruled out, because of bright expression in adjacent ventral neurons. However,
we searched for lag-2::GFP cells with SGP morphology elsewhere in the
animal and did not find them. Similarly, pes-1::GFP-expressing cells
were not found ectopically. We conclude that typical SGPs are missing from
tra-1; ehn-3 L1 larvae.
To determine whether SGPs were generated in tra-1; ehn-3 embryos,
we used hnd-1::GFP, which is first expressed in SGPs as they assemble
into the gonadal primordium (Mathies et
al., 2003). In tra-1(RNAi); ehn-3(q689) embryos, two
hnd-1::GFP-expressing SGPs were generated as normal, and they
assembled into the gonadal primordium (n=11). Therefore, tra-1;
ehn-3 SGPs express SGP-specific markers in embryos, but not in L1
larvae.
One simple explanation for the missing SGPs might have been cell death. The
hnd-1 gene is crucial for SGP survival
(Mathies et al., 2003), and
ehn-3 mutants enhance the SGP survival defect of hnd-1(RNAi)
(L.M., unpublished). We used cell death mutants to visualize cell corpses or
prevent programmed cell death in tra-1; ehn-3 mutants. SGP corpses
are easily seen in ced-2 mutants, which are defective in corpse
engulfment (Ellis et al.,
1991
; Mathies et al.,
2003
), and cells that normally die by programmed cell death are
able to survive in ced-3 mutants, which are defective in programmed
cell death (Ellis and Horvitz,
1986
; Yuan et al.,
1993
). We examined newly hatched tra-1; ehn-3(RNAi);
ced-2 L1 animals, but found no gonad-associated corpses (n=35),
and we examined tra-1; ehn-3(RNAi); ced-2 ced-3, but found no
increase in SGP number (n=15). Therefore, SGPs do not appear to
undergo programmed cell death in tra-1; ehn-3 mutants.
We suggest that the tiny PGC-associated cells in tra-1; ehn-3 mutants are, in fact, the missing SGPs. This idea is based on three lines of evidence. First, SGPs are born and assembled into the primordium normally in the embryo. Second, SGPs did not appear to die or be ectopically placed. Third, the tiny PGC-associated cells are in the right position and number to be SGPs (most animals had one or two tiny cells, but the cells were so small that one may have been missed). Therefore, we consider these tiny PGC-associated cells to be aberrant SGPs. We followed the aberrant SGPs of tra-1; ehn-3 mutants during the first three larval stages (L1-L3). During this interval, wild-type SGPs generate 10-12 daughter cells, depending on sex. The aberrant SGPs, however, did not grow in size or divide, but they did remain associated with the germ cells (n=2). Therefore, the SGPs appear to be present in tra-1; ehn-3 mutants, but they fail to enlarge, express L1-specific SGP markers and divide.
A dose sensitive interaction between tra-1 and ehn-3
In constructing tra-1(e1099); ehn-3(q689), we found that
tra-1 is a dominant enhancer of the ehn-3 gonadogenesis
defects. Only 18% of ehn-3(q689) mutants had gonadal defects and none
were seen in tra-1(e1099)/+ heterozygotes; however, 65% of
tra-1(e1099)/+; ehn-3(q689) mutants had defects
(Table 2). A similar, but less
dramatic, effect was seen for ehn-3(q766)
(Table 2). To determine whether
other sex determination regulators behaved similarly, we examined
ehn-3(q689) for enhancement by tra-2/+ and suppression by
fem-3/+, but found no striking interaction
(Table 2). We conclude that
gonadogenesis is sensitive to tra-1 dosage in ehn-3
mutants.
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Interdependence of tra-1, hnd-1 and ehn-3 expression
The hnd-1, tra-1 and ehn-3 genes are expressed in SGPs at
overlapping, but different, times of embryogenesis
(Fig. 6; this work)
(Mathies et al., 2003).
Because all three encode putative transcription factors, we asked if any of
them might regulate expression of the others. As described above and in a
previous work (Mathies et al.,
2003
), the hnd-1::GFP transcriptional reporter was
expressed normally in tra-1 and ehn-3 mutants. Similarly,
the EHN-3::GFP translational reporter was expressed in all SGPs in wild-type
(Fig. 5D,E) and tra-1
embryos (n=20). In hnd-1 embryos, about half the normal
number of SGPs expressed EHN-3::GFP (n=47), consistent with the
reduced number of SGPs seen in hnd-1 L1s. We conclude that the
expression of hnd-1 and ehn-3 appear to be initiated
independently of each other and of tra-1.
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Discussion |
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TRA-1 controls symmetry in the gonadal primordium
The wild-type C. elegans gonadal primordium is symmetrical, and
that symmetry is maintained in both sexes through the first SGP division
(Kimble and Hirsh, 1979).
Thus, the two SGPs reside at the poles of the primordium, and they divide with
opposite polarity to establish the opposing gonadal axes. The more central SGP
daughters are larger and have proximal fates, while polar daughters are
smaller and have distal fates. In tra-1 mutants, the gonadal
primordium is not symmetrical: tra-1 SGPs often move from their
normal polar positions, and they divide with the same polarity with respect to
the anteroposterior axis. Thus, their anterior SGP daughters are larger and
have proximal fates, while their posterior daughters are smaller and have
distal fates. This same defect is seen in XX and XO tra-1 mutants.
Therefore, in wild-type animals, the activity of TRA-1 controls symmetry of
the gonadal primordium.
The molecular mechanism by which TRA-1 governs gonadal symmetry is not known. One possibility is that the TRA-1 transcription factor regulates expression of specific cellular constituents in the SGPs that generate internal signals to create a gonad-specific polarity. For example, TRA-1 might direct assembly of a junction between Z1 and Z4 that polarizes these two cells with respect to each other rather than the anteroposterior axis. Now that TRA-1 has been identified as a key regulator of SGP position and polarity, it will be possible to identify target genes governing gonadal polarity and to learn the molecular mechanism by which proximodistal axes are organized within the early gonad.
Is tra-1 control of gonadal symmetry related to its sex-determining function?
TRA-1 is well known for specifying female fates in C. elegans
(Meyer, 1997), and female
gonadogenesis is a symmetrical process (hermaphrodites are essentially females
that make sperm transiently). The gonadal primordium is also symmetrical in
males, even though it develops into a one-armed asymmetric gonad. We have
found that TRA-1 promotes symmetry in the gonadal primordium of both XX and XO
animals. One idea is that the symmetrical primordium represents a female
aspect of early gonadogenesis in both sexes. According to this view, TRA-1
control of primordium symmetry is simply another manifestation of its sex
determination function to promote female fates.
A symmetrical gonadal primordium is thought to be an ancient feature of
nematode gonadogenesis (Chitwood and
Chitwood, 1950; Félix
and Sternberg, 1996
). In virtually all nematodes examined, this
same morphology is observed, despite a range of adult gonadal morphologies.
For example, some female nematodes possess asymmetrical adult gonads, but
nonetheless gonadogenesis begins with a symmetrical primordium
(Félix and Sternberg,
1996
). Conversely, some male nematodes possess symmetrical adult
gonads (Chitwood and Chitwood,
1950
). Therefore, the symmetry or asymmetry of adult gonads is not
a conserved sexual trait.
Why might an asymmetric adult gonad, such as that in C. elegans
males, begin development from a symmetrical primordium? In tra-1
mutants, the SGPs divide asymmetrically, and produce proximal and distal
daughters. However, the positions of those proximal and distal daughters are
abnormal in tra-1 mutants, and the resultant gonads are usually
mis-shapen and sterile (Hodgkin,
1987). Perhaps SGP position at the pole of the primordium is
crucial for the correct placement of proximal and distal daughters within the
developing gonad. That placement appears to be important, but not essential,
for generation of mature gonads with appropriate axes. Based on this
reasoning, we suggest that TRA-1 control of SGP positions within the gonadal
primordium may reflect a primitive role in patterning (see below) rather than
in specification of the female fate.
TRA-1 and EHN-3: redundant regulators of SGP maturation
The tra-1 and ehn-3 genes act redundantly to promote SGP
maturation. In tra-1 and ehn-3 single mutants, the SGPs
express markers and divide, albeit abnormally. By contrast, in tra-1;
ehn-3 double mutants, the SGPs do not mature and appear arrested in
development. Thus, whereas SGPs appear normal in tra-1; ehn-3
embryos, they are abnormal by hatching: in L1s, they do not express
SGP-specific markers, they do not grow in size and they do not undergo further
cell divisions. A similar effect on SGP maturation is not observed in either
tra-1 or ehn-3 single mutants. Therefore, either TRA-1 or
EHN-3 is sufficient to promote SGP maturation.
How might TRA-1 and EHN-3 interact to control SGP maturation? The TRA-1/GLI
zinc-finger proteins bind DNA and regulate transcription
(Alexandre et al., 1996;
Chen and Ellis, 2000
;
Conradt and Horvitz, 1999
;
Kinzler et al., 1988
;
Yi et al., 2000
;
Zarkower and Hodgkin, 1992
);
EHN-3 similarly possesses zinc fingers and a rescuing EHN-3::GFP reporter
protein is predominantly nuclear, suggesting a role for EHN-3 in the nucleus.
One simple idea is that TRA-1 and EHN-3 are both transcription factors, and
that they are essentially interchangeable for control of SGP maturation and
proliferation. Alternatively, TRA-1 and EHN-3 might cooperate to regulate
transcription, as suggested for murine GLI3 and ZIC1
(Koyabu et al., 2001
). Tests
of these molecular mechanisms must await identification of TRA-1/EHN-3 target
genes.
In a parallel study, we showed that TRA-1 is redundant with a forkhead
transcription factor, FKH-6, for gonadal divisions in both XX and XO animals
(Chang et al., 2004). In
fkh-6; tra-1 mutants, the SGPs express L1-specific SGP markers and
divide, but their daughters are arrested in development and fail to divide
further. This fkh-6; tra-1 cell division arrest is strikingly similar
to the tra-1; ehn-3 SGP arrest, albeit one division later. These two
parallel studies support the idea that TRA-1 regulates proliferation of the
SGPs and their descendants. This tra-1 control is masked by
redundancy with ehn-3 for SGP divisions and with fkh-6 for
later divisions. We do not yet know if the TRA-1 control of proliferation
extends to other tissues, a possibility that may also be masked by
tissue-specific redundant factors.
TRA-1 regulation of precursor cells may be ancient
The tra-1 gene encodes the single Ci/GLI homolog in the C.
elegans genome (Ruvkun and Hobert,
1998; Zarkower and Hodgkin,
1992
). In flies and vertebrates, Ci/GLI transcription factors
control embryonic patterning and cell proliferation in response to
hedgehog signaling (Berman et al.,
2003
; Ingham and McMahon,
2001
; Ruiz i Altaba,
1999
; Taipale and Beachy,
2001
; Thayer et al.,
2003
). In nematodes, Ci/GLI functions in patterning and
proliferation were thought to be lost: instead, TRA-1 appears to have been
co-opted for sex determination (Meyer,
1997
; Pires-daSilva and
Sommer, 2004
; Ruvkun and
Hobert, 1998
). In this paper, we show that TRA-1 is also a key
regulator of precursor cells, including SGP position, polarity, maturation and
proliferation. These TRA-1 functions are strikingly reminiscent of Ci/GLI
functions in embryonic patterning and proliferation in other organisms.
The findings reported in this paper, together with classical studies of
C. elegans sex determination
(Hodgkin and Brenner, 1977),
demonstrate that TRA-1 has two major developmental functions. It promotes
female development in most tissues, and it controls development of somatic
precursor cells in the gonad. The TRA-1 control of SGP positions and polarity
can be interpreted as imposing a female symmetry on male gonads, but its
control of SGP maturation and division is more difficult to envision as a
female character. Furthermore, other sex-determining genes (e.g.
tra-2 and fem-3) do not control SGP development, suggesting
that the TRA-1 control of SGP development is distinct from its control of sex
determination. We suggest that the TRA-1 regulation of precursor cells may be
an ancient function. This speculation is based in part on the similarity
between TRA-1 and Ci/GLI controls of patterning and proliferation (see above),
and in part on the well-documented rapid evolution of sex determining
mechanisms (Zarkower, 2001
).
Indeed, a crucial role for TRA-1 in development of the gonadal precursor cells
may have positioned this well-conserved regulator for its evolution to become
a sex-determination regulator.
The recruitment of common regulators into sex determination is not
unprecedented. The primary Drosophila sex-determining regulator,
Sxl, is a splicing regulator used in both sexes of closely related
flies, and Sry, the primary sex-determining regulator in mice,
appears to have evolved from a transcription factor used in both sexes
(reviewed by Zarkower, 2001).
The notion that the TRA-1/GLI sex-determining regulator was recruited in
C. elegans from the hedgehog signaling pathway is not new
(Ruvkun and Hobert, 1998
).
However, the idea that TRA-1/GLI may have retained ancient functions in
patterning and proliferation departs from the classic view.
Our findings pose two major questions for future studies. First, how does
TRA-1 control gonadal symmetry and SGP maturation in C. elegans? What
are its target genes and how do their products regulate precursor maturation
and primordium structure? Second, is the TRA-1-mediated control of SGP
development an ancient function? Do nematode GLI homologs generally control
SGP development? In light of the redundancy between tra-1 and
ehn-3, the recently described P. pacificus tra-1 mutations
(Pires-daSilva and Sommer,
2004) do not rule out an additional role in SGP development. If
the role of TRA-1 in controlling precursor cells is ancient, then analyses of
how TRA-1/GLI regulates SGPs in C. elegans may provide insights into
how GLI homologs control embryonic pattern and proliferation more generally in
the animal kingdom.
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ACKNOWLEDGMENTS |
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