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First published online 11 June 2008
doi: 10.1242/dev.017046
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1 Center for Neural Development and Disease, University of Rochester, Rochester,
NY 14642, USA.
2 Department of Biology, University of Rochester, Rochester, NY 14642,
USA.
3 Department of Biomedical Genetics, University of Rochester, Rochester, NY
14642, USA.
* Author for correspondence (e-mail: douglas.portman{at}rochester.edu)
Accepted 22 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Sexual dimorphism, Sex determination, Sexual differentiation, Sex differences, DM domain, DMRT, Cell fusion, Developmental timing, Selector gene
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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In the nematode C. elegans, one of the most prominent sexual
dimorphisms is in tail tip morphology. Although the hermaphrodite tail tip is
whip-like, the male tail is blunt-ended and harbors several copulatory
structures (Sulston et al.,
1980
; Emmons,
2005). In larvae of both sexes, the tail tip comprises four nested
larval cells, hyp8 through hyp11 (referred to here as hyp8-11). In
hermaphrodites, this architecture remains static throughout development. By
contrast, the male tail tip undergoes dramatic remodeling in the final (L4)
larval stage (Sulston et al.,
1980; Nguyen et al.,
1999). During this morphogenesis, hyp8-11 fuse together and
retract anteriorly to form a rounded tail tip. Subsequently, the distinct
process of anterior tail retraction occurs, in which the entire tail is pulled
anteriorly, leaving the elongated rays in its wake
(Sulston et al., 1980;
Nguyen et al., 1999).
Defects in male tail tip retraction result in a pointed or `leptoderan'
(Lep) tail tip phenotype (Nguyen et al.,
1999). Previous work has shown that mutations in both Wnt
signaling and the heterochronic (developmental timing) pathways result in Lep
phenotypes. Mutations in the Wnt gene lin-44 cause weak tail tip
retraction defects (Zhao et al.,
2002); additionally, loss of TLP-1, an Sp1-family Zn-finger factor
that may act downstream of Wnt signaling, causes pronounced failure of tail
tip morphogenesis (Zhao et al.,
2002). In males carrying a gain-of-function allele of the
heterochronic gene lin-41 (Slack
et al., 2000), the retraction program is delayed, resulting in a
partially unretracted tail tip. By contrast, lin-41(lf) males have an
over-retracted tail, with premature retraction initiating in L3
(Del Rio-Albrechtsen et al.,
2006). However, the means by which Wnt signaling and developmental
timing converge on tail morphogenesis are not clear. Moreover, the mechanism
that brings sex-specificity to tail morphogenesis is unknown.
All sex differences in C. elegans ultimately arise from sex
chromosome content: XX in hermaphrodites, X0 in males
(Brenner, 1974;
Madl and Herman, 1979).
Downstream of this primary cue, a regulatory hierarchy controls the activity
of the master sexual regulator TRA-1A, a Gli-family transcriptional repressor
(Hodgkin, 1987;
Zarkower and Hodgkin, 1992).
tra-1 activity is necessary and sufficient to generate essentially
all somatic sexual dimorphism. Though TRA-1A is expressed in both sexes, it is
fully active only in hermaphrodites, where it represses male-specific genes
(Zarkower, 2006). Only three
direct targets of TRA-1A in the soma are known: mab-3 in the
intestine (Yi et al., 2000),
egl-1 in the HSN neurons (Conradt
and Horvitz, 1999) and ceh-30 in the CEM neurons
(Peden et al., 2007;
Schwartz and Horvitz, 2007).
However, these targets account for only a small subset of sex-specific
development and control single-cell-level processes (yolk production and cell
death). By contrast, it is not known how tra-1 specifies sex-specific
organogenesis, where sexual information must regulate cell fate,
differentiation and morphogenesis.
Despite the great variety in sex-determination pathways of animal species,
the conservation of DM family genes indicates that these mechanisms may derive
from a common ancestor. The DM domain is an unusual DNA-binding Zn finger
initially identified in the Drosophila sex-determination gene
doublesex and the C. elegans sexual differentiation gene
mab-3 (Erdman and Burtis,
1993; Raymond et al.,
1998). Genes of this family have since been implicated in
sex-specific development across the animal kingdom. Interestingly, DM genes
act at a variety of points in these pathways, from very early steps [e.g.
DMY is the primary sex determining cue in Medaka
(Matsuda et al., 2002;
Matsuda et al., 2007)] to
later sex-specific differentiation [e.g. DMRT1 is necessary for
differentiation of testes and the germline in mice
(Kim et al., 2007a;
Kim et al., 2007b)]. As a
result, the nature of the ancestral conserved function of DM genes in sex
determination and differentiation remains unclear.
In C. elegans, only two of the 11 DM genes predicted from genome
sequence, mab-3 and mab-23, have been characterized. As a
direct target of tra-1, mab-3 represses yolk production in the male
intestine (Shen and Hodgkin,
1988; Yi et al.,
2000). mab-3 is also necessary for the male-specific
expression of lin-32, a gene that triggers development of the
male-specific sensory rays (Zhao and
Emmons, 1995; Portman and
Emmons, 2000), though this function seems to be indirectly
regulated by tra-1 (Ross et al.,
2005). mab-23 is also necessary for a variety of
male-specific events, including ray sensory neuron patterning and
male-specific muscle differentiation
(Lints and Emmons, 2002).
These sex-specific functions of mab-23 also seem to be indirectly
regulated by tra-1. Whether additional DM genes control other
sex-specific characteristics in C. elegans is unknown, as is the
extent to which DM genes act as the primary effectors of tra-1
function.
Here, we find that a previously uncharacterized DM gene, dmd-3, is necessary for male-specific morphogenesis of the tail tip. Moreover, supplying dmd-3 to the hermaphrodite tail is sufficient to bring about male-like morphogenesis. By coordinating sexual, temporal and spatial information, dmd-3 occupies a crucial node in the regulatory network that coordinates tail remodeling. In addition, mab-3 plays a secondary, partially redundant, role in tail tip morphogenesis. dmd-3 and mab-3 trigger at least two independent processes necessary for morphogenesis, including the male-specific expression of the cell fusogen EFF-1. Together, our studies identify a crucial role for two DM genes in a genetic mechanism that couples sex determination to the sex-specific modification of a set of shared cells.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The following
mutations were used: lin-44(n1792), lin-41(bx42), lin-41(bx37),
lin-41(ma104), mab-3(e1240), eff-1(ok1021), tra-1(e1099), pha-1(e2123),
tlp-1(bx85), dmd-3(ok1327), dmd-3(tm2863), him-5(e1490), mab-23(bx118)
and ozDf2. lin-41(bx37) and lin-41(bx42) were provided by D.
Fitch (New York University) and dmd-3(tm2863) was provided by the
National Bioresource Project (S. Mitani, Tokyo Women's Medical University).
All other mutants were obtained from the Caenorhabditis Genetics Center. All
strains except those carrying tra-1(e1099) contained
him-5(e1490) to increase the frequency of spontaneous males.
Transgenes
The following integrated transgenic strains were used in this study:
syIs78 [AJM-1::GFP, unc-119(+)], fsIs2
[dmd-3::YFP, cc::GFP], fsIs3
[dmd-3::YFP, cc::GFP], fsIs7 [pUR13
(E(ht)
TRA-1::DMD-3::GFP),
cc::GFP], fsIs9 [pUR12
(E(ht)::DMD-3::GFP), cc::GFP],
fsIs10 [pUR15 (MAB-3::GFP), cc::GFP] and
fsIs12 [pUR14
(E(ht)TRA-1-G
A::DMD-3::GFP),
cc::GFP]. The following extrachromosomal transgenic strains were used in the
study: fsEx110 [pUR18(E(ht)::nlsGFP, pBX1(pha-1+)],
fsEx114 [pUR4 (E(ht)::GFP), pBX1
(pha-1(+))], fsEx118 [pUR5
(E(ht)TRA-1::GFP),
pBX1(pha-1(+))], fsEx135
[pJDC41(EFF-1::GFP [translational]),
pBX1(pha-1(+))], fsEx136 [pJE3(eff-1::GFP
[transcriptional]), fsEx154 [DMD-3::YFP,
cc::GFP], fsEx182
[pUR25(E(ht)dmd-3mab-3::nlsGFP,
pBX1(pha-1+)], fsEx183 [pUR17
(EFF-1::GFP::mCherry), pBX1(pha-1+)],
fsEx184 [pUR27
(EFF-1dmd-3mab-3amab-3b::GFP::mCherry),
pBX1(pha-1+)]. pJDC41 and pJE3 were generously provided by W. Mohler
(Mohler et al., 2002;
del Campo et al., 2005). All
conclusions drawn in the text were supported by additional independently
derived transgenes.
dmd-3 alleles
The dmd-3(ok1327) deletion removes 926 bp, including the 3';
end of the final dmd-3 exon, leaving a predicted mutant protein in
which the 50 C-terminal residues are replaced with five novel amino acids.
This deletion also removes much of the 3'; UTR of dmd-3. In
dmd-3(tm2863), a 407 bp region that comprises much of exons 2 and 3
is replaced with a 10 bp insertion (Fig.
1A). This results in a predicted open reading frame encoding 55
N-terminal amino acids (including all but six amino acids of the first DM
domain) followed by 12 novel amino acids and a stop codon. Thus,
dmd-3(tm2863) is likely to be a molecular null allele. Both
dmd-3(ok1327) and dmd-3(tm2863) are recessive, and have
essentially identical male tail defects.
dmd-3 RNAi
Double-stranded dmd-3 RNA was prepared as previously described
(Fire et al., 1998) and
injected into young adult hermaphrodites. F1 adult male and hermaphrodite
offspring were examined for phenotypes.
DNA constructs
For the dmd-3::YFP transcriptional reporter, a genomic
fragment from -4264 to +549 bp with respect to the dmd-3 start codon
was amplified. For the DMD-3::YFP translational reporter, a
genomic fragment from -4264 to +4674 bp was amplified. These fragments were
fused to YFP-coding sequence by overlap extension PCR
(Boulin et al., 2006). A
transcriptional mab-3::GFP reporter (pUR11) was generated by
cloning a mab-3 genomic fragment (-8786 to +12 bp) into pPD107.94. A
translational MAB-3::GFP reporter (pUR15) was generated by
inserting the mab-3-coding region (+13 to +3868 bp) into pUR11.
To generate E(ht)::GFP, the -2740 to -1595 region of the dmd-3 promoter was first cloned into pPD107.94 to generate pUR18. The NLS was then removed by digesting with KpnI and religating to generate E(ht)::GFP (pUR4).
To mutate the putative TRA-1A binding site within E(ht), the
3'; end of the TRA-1A site was replaced with a SalI site by
cloning two PCR products into the SphI and XbaI sites in
pPD107.94 (WT TRA-1A site: TTTCTGTGTGGGTGTTC, mutant site:
TTTCTGTGTGTCGACTC). The NLS was removed to generate
E(ht)TRA-1::GFP (pUR5). A point mutation
in the TRA-1A site was generated with the QuickChangeII-XL Site-Directed
Mutagenesis Kit (Stratagene) using complementary primers that changed the pUR5
TRA-1A site to TTTCTGTGTGAGTGTTC to generate
E(ht)TRA-1-GA::GFP (pUR10).
To express dmd-3(+) from E(ht)::GFP, the
dmd-3-coding sequence (with GAAAAA added upstream of the start codon
to aid translation) was cloned into pUR4 to generate
E(ht)::DMD-3::GFP (pUR12). To put
DMD-3::GFP downstream of the mutant TRA-1 sites, the
wild-type E(ht) fragment was removed from pUR12 and replaced with an
SphI-XbaI fragment from pUR5 or pUR10 to generate
E(ht)TRA-1::DMD-3::GFP (pUR13)
and E(ht)TRA-1-GA::DMD-3::GFP
(pUR14), respectively.
The putative DMD-3 and MAB-3 binding sites in E(ht), at -2516 and -2465, respectively, were mutated using pUR18 as the starting template. The putative DMD-3 site was changed from TGTAACA to TGGATCC and the putative MAB-3 site was changed from CCCAACA to CTCGAGA to generate pUR25.
To generate an operon containing the EFF-1::GFP translational reporter followed by an mCherry transcriptional reporter, an outron and mCherry sequence were inserted into the EFF-1::GFP translational reporter 24 bp downstream of the GFP stop codon and 105 bp upstream of the unc-54 3'; UTR. To generate this construct, a NotI site was inserted at this position by mutating pJDC41 from CCGGTCGC to GCGGCCGC to generate pUR16. The outron and mCherry-coding sequence were amplified from pENTRY-SrfI-mCherry (a gift from J. White and E. Jorgensen) and cloned into the NotI site to generate pUR17.
The putative DMD-3 binding site (at position -4121 in the eff-1 promoter) and the two putative MAB-3 binding sites (-5501 and -3201) were mutated using pUR17 as the starting template. The putative DMD-3 site was changed from TGCAACA to TGCATGC and the putative MAB-3 sites were changed from CGCAACA to CGGATCC to generate pUR27. Unexpectedly, pUR27 also contained a 11 bp deletion (-3892 to -3882) that does not appear to affect EFF-1::GFP expression.
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Microscopy
Images were obtained using a Zeiss Axioplan 2 with epifluorescence
illumination and ApoTome structured illumination (Carl Zeiss Microimaging) or
by confocal microscopy using a Leica TCS NT. Digital images were processed
using Adobe Photoshop. L4 larvae were staged according to linker cell
migration, tail tip retraction and anterior tail retraction. In early L4, the
linker cell has just completed migrating to the ventral side and the tail tip
cells hyp8-11 are unfused and unretracted. In early mid-L4, the linker cell
has progressed roughly halfway from its ventral turn to the hindgut, and the
tail tip is unfused and unretracted. By mid-L4, the linker cell has migrated
completely to the hindgut and the tail tip is undergoing cell fusion but not
retraction. In late mid-L4, the tail tip is fully fused and retraction is
under way. In early late L4, the tail tip is fully retracted but anterior
retraction has not yet begun. In late L4, anterior retraction is under way,
generating elongated rays and the fan.
MH27 antibody staining
Mid-L4 fsIs7; him-5 and fsIs9; him-5 larvae were
permeabilized by freeze-cracking (Hurd and
Kemphues, 2003) and fixed with methanol
(Miller and Shakes, 1995).
Larvae were stained with the anti-AJM-1 antibody MH27 (Developmental Studies
Hybridoma Bank, University of Iowa)
(Francis and Waterston, 1991;
Koppen et al., 2001) followed
by Texas Red-labeled goat anti-mouse IgG (Jackson ImmunoResearch).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Y43F8C.10 is predicted to encode a 251-amino acid protein
with two DM domains (Fig. 1A).
We renamed this gene dmd-3, as the third characterized C.
elegans DM-domain gene (Raymond et
al., 1998; Lints and Emmons,
2002). Because DM domains have been implicated in sex-specific
development, we examined hermaphrodites and males carrying the dmd-3
deletion alleles dmd-3(ok1327) and dmd-3(tm2863)
(Fig. 1A). dmd-3
mutant hermaphrodites appeared morphologically wild type. By contrast,
dmd-3(ok1327), dmd-3(tm2863) and dmd-3(RNAi) males displayed
marked abnormalities in tail morphology. Most notably, they possessed
partially unretracted Lep tail tips reminiscent of those of adult
hermaphrodites (Fig. 1B,
Table 1; data not shown). This
phenotype resulted from a developmental defect in tail tip retraction: the
hypodermal cells hyp8-11 failed to pull back completely from the larval
cuticle (Fig. 1C). In addition,
the anterior region of the tail failed to retract completely in dmd-3
mutants (Fig. 1B,C). We
observed similar partial retraction defects in dmd-3(ok1327),
dmd-3(tm2863), dmd-3(RNAi) and hemizygous
dmd-3(ok1327)/ozDf2 males
(Table 1; data not shown),
indicating that this is probably the null phenotype.
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), we
constructed mab-3; dmd-3 double mutants
(Fig. 1B,C;
Table 1). Strikingly, the
mab-3; dmd-3 adult male tail was almost identical to that of a
hermaphrodite, with a long, smoothly tapered tail tip and no fan or rays. No
tail tip or anterior retraction movements were seen in mab-3; dmd-3
L4 males. Thus, dmd-3 and mab-3 act in a partially redundant
fashion to bring about male tail tip morphogenesis, with dmd-3
playing the primary role. We also examined mab-23; dmd-3 double
mutants, but found no indication that mab-23 also acts redundantly in
tail tip morphogenesis (data not shown). Interestingly, the consensus DNA
binding site for DMD-3, as determined by in vitro site-selection experiments
(M. Murphy and D. Zarkower, personal communication) closely matches that of
MAB-3 (Yi and Zarkower, 1999),
suggesting that the partial functional redundancy between dmd-3 and
mab-3 arises from an ability to regulate common downstream
targets.
Tail tip retraction is preceded by the male-specific fusion of hyp8-11
(Nguyen et al., 1999). In wild
type, 100% of mid-L4 males (n=28) showed hyp8-11 fusion, compared
with 0% of hermaphrodites (n=41). However, in dmd-3 single
mutant males, we observed cell fusion in only 44% of mid-L4 males
(n=50), and these fusions usually occurred only between hyp9 and
hyp10 (Fig. 1D). Again, the
loss of mab-3 enhanced this phenotype, such that no hyp8-11 fusions
were detectable in mid-L4 mab-3; dmd-3 males (0% of animals showed
cell fusion; n=35) (Fig.
1D). By contrast, cell fusion occurred normally in mab-3
single mutants (100%; n=40). In both dmd-3 and
mab-3; dmd-3 males, cell boundaries often persisted into
adulthood (Fig. 1E). Thus,
dmd-3 acts with mab-3 to coordinate both tail tip cell
fusion and retraction.
dmd-3 and mab-3 are expressed male specifically in the tail tip coincident with retraction
To better understand how dmd-3 mediates tail tip morphogenesis, we
constructed transcriptional (dmd-3::YFP) and translational
(DMD-3::YFP) reporter genes
(Fig. 1A). These reporters
exhibited essentially identical cellular expression patterns, and
DMD-3::YFP was able to rescue the dmd-3 tail
phenotype (data not shown). In males, we found that
dmd-3::YFP was expressed in a number of sexually dimorphic
or sex-specific cells, including the tail tip, hindgut, B lineage, ray RnA
neurons and somatic gonad (Fig.
2A,C). By contrast, hermaphrodites exhibited strong
dmd-3::YFP expression only in the anchor cell (not shown), a
hermaphrodite-specific somatic gonad cell that induces development of the
vulva (Kimble, 1981).
Non-sex-specific expression of these reporters was weak, occurring primarily
in the body hypodermis. In addition, expression in phasmid neurons of both
sexes was sometimes seen during L3 and L4 (not shown).
In hyp8-11, dmd-3::YFP expression was male specific and
coincided with morphogenesis (Fig.
2B,C; see Materials and methods for a description of L4
sub-stages). Tail tip expression initiated in early-mid L4 males, first in
hyp8, hyp9 and hyp11, and shortly thereafter in hyp10
(Fig. 2C). We occasionally
observed weak expression in hyp9 in late L3 males (see
Fig. 4B). Expression levels
peaked during tail tip retraction and decreased rapidly upon its completion.
dmd-3 was also expressed in hyp13, a male-specific, bi-nucleated
hypodermal cell thought not to have a role in tail tip morphogenesis (D. H. A.
Fitch, personal communication) (Nguyen et
al., 1999). Importantly, dmd-3::YFP was not
expressed in hermaphrodite hyp8-11 at any stage
(Fig. 2C, part vii). Thus
dmd-3 expression parallels tail tip remodeling, consistent with a
cell-autonomous, instructive role for dmd-3 in the control of
morphogenesis.
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) was
not reported to be expressed in the tail tip, nor could it rescue the tail tip
retraction defects of mab-3; dmd-3 males (data not shown). We
therefore generated a MAB-3::GFP translational reporter
carrying 7.3 kb of additional upstream regulatory sequence. This transgene was
able to rescue the tail tip retraction defects of dmd-3; mab-3 males
to a dmd-3-like phenotype (Table
1). MAB-3::GFP was expressed in numerous cells
of the male tail, including weak expression in hyp8-11
(Fig. 2D). In early L4 males,
MAB-3::GFP was predominantly found only in hyp10, although
hyp9 expression was occasionally observed. By mid-L4, we observed expression
in all tail tip hypodermal cells. As with dmd-3, no
MAB-3::GFP expression was detected in hyp8-11 in
hermaphrodites at any stage (Fig.
2D, part vii). Together, these results suggest that dmd-3
and mab-3 act in hyp8-11 to bring about male-specific
morphogenesis.
dmd-3 is likely to be a direct tra-1 target
The male-specific expression of dmd-3 in two sets of cells present
in both sexes - the tail tip hypodermis and the hindgut - indicated that
dmd-3 could be a direct target of repression by TRA-1A in
hermaphrodites. Consistent with this, we found that dmd-3 is both
expressed during and is necessary for the tail tip retraction that occurs in
tra-1(e1099) XX pseudomales (Fig.
3A; Table 1)
(Hodgkin, 1987).
Interestingly, the severity of the dmd-3 phenotype is slightly
exaggerated in tra-1 pseudomales, though the reasons for this are
unclear. Nevertheless, these data show that dmd-3 lies genetically
and molecularly downstream of tra-1.
To ask how tra-1 regulates dmd-3, we first identified a
1.1 kb region 1.6 kb upstream of the dmd-3 start codon,
E(ht), that was both necessary and sufficient to direct male-specific
expression in the hindgut and tail tip
(Fig. 1A,
Fig. 3C; data not shown) when
placed upstream of the basal promoter pes-10
(Seydoux and Fire, 1994).
Expression of DMD-3 under the control of
E(ht)::pes-10 was sufficient to rescue the
dmd-3 tail morphology defect
(Table 1). Thus E(ht)
contains cis-acting elements that direct the sexual and temporal
regulation of dmd-3 in the tail tip hypodermis and hindgut.
E(ht) contains a nearly exact match to the TRA-1A consensus
binding site (Zarkower and Hodgkin,
1993; Yi et al.,
2000), varying at only one nucleotide
(Fig. 3B). To disrupt this
site, we replaced the central GGTGT with TCGAC to create
E(ht)TRA-1::DMD-3::GFP.
Strikingly, this change led to clear DMD-3::GFP expression
in the L4 hermaphrodite tail tip (93%; n=67 compared with 0%;
n=51 for E(ht)::DMD-3::GFP) and hindgut
(Fig. 3C, part xii). In
addition, a single point mutation (TGGGTGAG) in the putative
TRA-1A site led to similar expression in the L4 hermaphrodite tail tip (100%;
n=40) and hindgut (data not shown). This specific GA change has
also been demonstrated to disrupt TRA-1A binding in the context of the
egl-1 and ceh-30 promoters
(Conradt and Horvitz, 1999;
Schwartz and Horvitz, 2007).
We cannot rule out the possibility that this site indirectly mediates sexual
regulation of dmd-3 by TRA-1A. However, together with the finding
that TRA-1A can bind to nearly identical sites in vitro
(Zarkower and Hodgkin, 1993;
Conradt and Horvitz, 1999;
Yi et al., 2000), our results
indicate that dmd-3 is very likely to be a direct target of
repression by TRA-1A in the hermaphrodite tail tip and hindgut.
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TRA-1 in both sexes to ask whether providing
DMD-3 to the hermaphrodite would be sufficient to masculinize the
tail tip. Although all adult hermaphrodites carrying the wild-type
E(ht)::DMD-3::GFP transgene fsIs9
displayed normal whip-like tail tips (n=88), the mutant
E(ht)TRA-1::DMD-3::GFP transgene
fsIs7 produced a male-like rounded tail tip in 94% of adult
hermaphrodites (n=101) (Fig.
3C, part xiv). Consistent with this, the tail tip hypodermal cells
of L4 hermaphrodites carrying fsIs7 exhibited clear retraction-like
movements and some cell fusion (Fig.
3C, parts xii, xiii). Thus, sexually dimorphic dmd-3
expression determines the sexual specificity of tail tip morphogenesis.
Unexpectedly, these mutations in E(ht) also disrupted the timing
of dmd-3 expression. In both sexes, the expression of
DMD-3::GFP from E(ht)TRA-1 and
E(ht)TRA-1-GA initiated prematurely in L2 and L3
larvae (Fig. 3C; data not
shown), suggesting that the regions mediating sexual and temporal input
overlap in the dmd-3 promoter. By contrast, only minor premature
expression was seen in L3 and early L4 males carrying the wild-type transgene;
this effect probably results from increased positive autoregulation caused by
DMD-3 overexpression (see below).
The premature expression of these transgenes also demonstrated that
dmd-3 activity was sufficient to trigger retraction at an
inappropriate time. fsIs7 induced precocious tail tip retraction in
both male and hermaphrodite L3 larvae, such that essentially all
fsIs7 L4 males and many L4 hermaphrodites had clearly pre-retracted
tail tips (Fig. 3C), a
phenotype not seen in fsIs9 L4 males. Furthermore, adult
fsIs7 males exhibited a clear `over-retraction' phenotype
(Del Rio-Albrechtsen et al.,
2006) (Fig. 3C,
part vi). Thus, dmd-3 expression in the tail tip can provide an
instructive cue for morphogenesis regardless of sex or developmental
stage.
Wnt signaling and heterochronic genes regulate tail tip morphogenesis through dmd-3
Both Wnt and heterochronic genes are necessary for normal male tail tip
morphogenesis (Zhao et al.,
2002; Del Rio-Albrechtsen et
al., 2006). However, the mechanisms underlying these functions are
unknown. We therefore tested the possibility that these phenotypes result from
misregulation of dmd-3. To determine whether the heterochronic gene
lin-41 regulates dmd-3, we examined
dmd-3::YFP in lin-41 mutants. Temporally delayed
lin-41(bx42gf) males have a Lep phenotype
(Del Rio-Albrechtsen et al.,
2006). We found that dmd-3::YFP expression in
these mutants initiated at the correct time in hyp8, hyp9 and hyp11, but was
frequently absent from hyp10 even into late L4
(Fig. 4A). We observed this
hyp10-specific expression defect in 89% of lin-41(bx42) mid-L4 males
(n=27) and 33% of lin-41(bx37) mid-L4 males (n=48),
but never in wild-type mid-L4 males (n=35). Interestingly, hyp10 is
generally the only cell that fails to fuse in lin-41(gf) mutants
(Nguyen et al., 1999;
Del Rio-Albrechtsen et al.,
2006). As the lack of dmd-3 expression specifically in
hyp10 is characteristic of early-mid L4 wild-type males
(Fig. 2C, part ii), we
interpret the lin-41(gf) phenotype to be a defect in the maturation
of dmd-3 expression. Conversely, we observed strong expression of
dmd-3::YFP in the pre-retracting tail tips of
lin-41(ma104lf) (Slack et al.,
2000) L3 males (87%; n=15, compared with 0%;
n=22 for wild type) (Fig.
4B). This indicates that the early retraction in these animals
(Del Rio-Albrechtsen et al.,
2006) probably results from premature dmd-3 expression.
Consistent with this, we found that the premature-retraction phenotype of
lin-41(lf) males (95% L4 pre-retracted tail; n=80) was
suppressed in lin-41; dmd-3 (13%; n=82) and lin-41;
mab-3; dmd-3 (2%; n=54) mutants. Thus, lin-41 controls
the stage specificity of tail tip morphogenesis by regulating dmd-3
expression.
|
) on dmd-3::YFP expression. In these
animals, tail tip dmd-3::YFP expression initiated at the
correct time, but was not sustained (Fig.
4C), such that we observed defects in dmd-3::YFP
expression in some or all tail tip cells in 98% of mid-L4 lin-44
males (n=40), compared with 0% in wild type (n=36). However,
the Lep defect of lin-44 mutants is subtle, and we found that its
severity was enhanced by a mutation in mab-3 (data not shown),
suggesting that mab-3 can compensate for the reduction of
dmd-3 expression in lin-44 males. Males carrying a mutation
in the putative Wnt effector tlp-1
(Zhao et al., 2002) displayed
a dmd-3::YFP expression defect similar to that of
lin-44 males: initial expression was normal but it was not properly
maintained in most mid-L4 males (75%, n=72)
(Fig. 4C). These results
indicate that tail tip dmd-3 expression is regulated in two distinct
phases: an initial, Wnt-independent induction of dmd-3 expression,
followed by Wnt-dependent maintenance and amplification.
An autoregulatory loop is important for tail tip morphogenesis
To determine whether dmd-3 autoregulation contributes to the
maintenance phase of dmd-3 expression, we examined
dmd-3::YFP in dmd-3, mab-3 and dmd-3;
mab-3 mutants (Fig.
4D). We observed a subtle decrease in dmd-3::YFP
expression in hyp8-11 in dmd-3 males. By contrast, mab-3
mutant males exhibited wild-type levels of dmd-3::YFP tail
tip expression. More clearly, dmd-3::YFP expression was
essentially abolished in hyp8-11 of mab-3; dmd-3 mid-L4 males.
Therefore, dmd-3 and mab-3 are necessary for strong
dmd-3 expression, and, at least in mab-3 mutants,
dmd-3 has a positive autoregulatory function. To determine whether
this autoregulation might occur through direct activation by DMD-3 itself, we
identified and disrupted two candidate DMD-3/MAB-3 binding sites in the
E(ht) region. However, mutating these sites did not result in a loss
of hyp8-11 expression (not shown). Thus, dmd-3-dependent expression
of dmd-3 may be mediated through intermediate regulators. As
maintenance-phase expression of dmd-3 requires lin-44 and
tlp-1, it is possible that dmd-3 activates a Wnt signal that
then directly promotes dmd-3 expression.
dmd-3 and mab-3 activate sex-specific expression of the cell fusogen EFF-1
Though tail tip morphogenesis involves both cell fusion and retraction, it
is unclear whether these two steps occur independently or, alternatively, if
retraction is simply a consequence of cell fusion. To investigate this, we
asked whether eff-1, the primary regulator of cell fusion in C.
elegans (Mohler et al.,
2002; Shemer et al.,
2004; Podbilewicz et al.,
2006), was necessary for hyp8-11 fusion and retraction. Consistent
with previous findings (Mohler et al.,
2002; Shemer and Podbilewicz,
2003), we detected no hyp8-11 fusion in males carrying the
putative null allele eff-1(ok1021)
(Fig. 5A). However, retraction
proceeded with only subtle abnormalities in these animals, resulting in a
non-Lep, blunt-ended tail (Fig.
5A). Thus, the fusion of hyp8-11 is not a prerequisite for
retraction, indicating that these two events are regulated in parallel.
Consistent with the requirement of eff-1 for hyp8-11 fusion, we
found that an EFF-1::GFP translational reporter
(del Campo et al., 2005) was
expressed in the male tail tip in a pattern that correlated closely with
dmd-3::YFP expression. EFF-1::GFP was
transiently expressed in hyp8-11 in mid-L4 males, peaking around the time of
cell fusion, but was expressed only very weakly in the tail tip of L4
hermaphrodites (Fig. 5B,D).
Interestingly, a transcriptional eff-1::GFP reporter lacking
eff-1 coding sequence (Mohler et
al., 2002) displayed only limited sex differences in expression
(not shown). To further explore the mechanisms of eff-1 regulation,
we generated a construct in which the eff-1 promoter drove expression
of an artificial operon (Blumenthal,
2005; White et al.,
2007) containing EFF-1::GFP-coding sequence
followed by an artificial `outron' and mCherry coding sequence
(EFF-1::GFP::outron::mCherry). In this
construct, mCherry fluorescence should reflect transcriptional regulation by
the eff-1 promoter, while GFP fluorescence reveals the net influence
of transcriptional and post-transcriptional controls on eff-1
expression. We observed mCherry expression in hyp8-11 of both sexes, but at
lower levels in hermaphrodites than in males. By contrast,
EFF-1::GFP expression was barely detectable in the
hermaphrodite (Fig. 5E).
Together, these results indicate that the male-specificity of tail tip
syncytium formation arises from the regulation of EFF-1, and that
this regulation probably occurs through both transcriptional and
post-transcriptional mechanisms.
|
To ask whether the transcriptional regulation of eff-1 by dmd-3 and mab-3 might be direct, we searched the eff-1 promoter for candidate DMD-3- and MAB-3-binding sites. We identified three putative DMD-3/MAB-3 elements and disrupted them in the context of the EFF-1::GFP::outron::mCherry reporter. However, this resulted in no detectable change in GFP or mCherry fluorescence in mid-L4 larvae of either sex (data not shown), indicating that the transcriptional activation of eff-1 by DMD-3 and MAB-3 may not be direct.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), indicating that let-7 also acts in this pathway,
though we have not tested this possibility directly.] Positional cues regulate
dmd-3 through a Wnt pathway that includes the ligand LIN-44 and its
downstream target tlp-1. Interestingly, this cue seems to be most
important for the maintenance and amplification of dmd-3 expression;
the initial positional or cell-type activator of dmd-3 remains
unknown. Finally, the male-specificity of dmd-3 expression arises
through regulation by the master sexual regulator TRA-1A.
The phenotype of mab-3; dmd-3 double mutants indicates that the
functions of these two genes partially overlap. Though only a small percentage
of mab-3 males have Lep defects, mab-3 enhances the
phenotype of every animal in a dmd-3 background
(Table 1). In addition,
overexpression of mab-3(+) in a mab-3; dmd-3 double mutant
can sometimes rescue animals to a nearly wild-type phenotype (not shown). As
the in vitro selected binding site for MAB-3 closely resembles that of MAB-3
(M. Murphy and D. Zarkower, personal communication)
(Yi and Zarkower, 1999), we
interpret this redundancy to reflect a partial overlap in the set of target
genes that DMD-3 and MAB-3 can regulate.
|
; Yi et al.,
2000). We note that this +15 position is likely to be important
for TRA-1A binding in vitro and in the TRA-1A target egl-1
(Zarkower and Hodgkin, 1993;
Conradt and Horvitz, 1999).
However, in contrast to egl-1, the TRA-1A site in dmd-3
matches the TRA-1A consensus at every other position, making it possible that
the +15 site is less important in the context of the dmd-3 promoter.
The C. briggsae dmd-3 ortholog, Cbr-dmd-3, also contains a
strong match to the TRA-1A consensus site within a 50 bp conserved region of
upstream sequence (not shown). Although we cannot rule out the possibility
that sexual regulation of dmd-3 occurs indirectly through a site
closely resembling that of TRA-1A, we consider this to be unlikely.
We believe that the sex-determination and heterochronic pathways probably
converge on a common cis-element in dmd-3, as disruption of
its TRA-1A site altered both sexual and temporal specificity of dmd-3
expression. These results support the short-range repression model proposed
for TRA-1A function (Conradt and Horvitz,
1999; Yi et al.,
2000; Zarkower,
2001), in which this factor acts locally to impart local sex
specificity to a single enhancer rather than to the entire locus. An
alternative possibility, that sexual and temporal regulation are both mediated
by TRA-1A, is unlikely, as precocious tail tip retraction is not observed in
tra-1 XX pseudomales (Fig.
3A). Interestingly, a similar phenomenon has been observed in the
Hox cluster gene egl-5: disruption of a putative upstream
TRA-1A-binding site was found to alter the sexual, temporal and spatial
specificity of egl-5 expression in seam cells
(Teng et al., 2004). Thus, the
overlap of TRA-1A sites with other regulatory elements may be a common
property of sexually regulated genes.
In contrast to sexual regulation, our results indicate that the regulation
of dmd-3 by lin-41 may be indirect. Previous work has
indicated that lin-41 controls its targets post-transcriptionally
(Slack et al., 2000). However,
as mutating the E(ht) promoter fragment altered the timing of its
expression, dmd-3 temporal control is likely to be mediated
transcriptionally. Although other known effects of lin-41 on
developmental timing are mediated through the transcription factor LIN-29
(Slack et al., 2000),
lin-29 mutant males do not exhibit an unretracted tail tip
(Euling et al., 1999). Thus, it
seems likely that an unidentified target of lin-41
(Del Rio-Albrechtsen et al.,
2006) regulates dmd-3.
The phenotypes of lin-44 and tlp-1 mutants indicate that Wnt signaling is important for dmd-3 maintenance and amplification, but not for its initial expression. Mutation of tlp-1 leads to a defect in maintenance of dmd-3 expression and a pronounced Lep phenotype. By contrast, though loss of lin-44 leads to a similar dmd-3 expression defect (Fig. 4C), the tail tip retraction phenotypes of these animals are relatively subtle. This could indicate that residual dmd-3 expression in lin-44 mutants is still able to exert a significant level of function. Our finding that mab-3 can enhance the lin-44 phenotype indicates that while dmd-3 is regulated primarily through lin-44, a different Wnt ligand might act preferentially on mab-3. Both of these Wnt signals would probably act primarily through tlp-1.
Because of its relatively simplicity, tail tip retraction serves as an
excellent model to explore the links between developmental signals and
morphogenesis. We have found that dmd-3 and mab-3 trigger
tail tip cell fusion by promoting expression of the fusogen EFF-1, probably
indirectly, through both transcriptional and post-transcriptional mechanisms.
As cell fusion and retraction can vary independently in related nematode
species (Fitch, 1997), and
eff-1 mutant males clearly undergo retraction, dmd-3 and
mab-3 must activate additional unknown effectors of morphogenesis.
Furthermore, yet other genes are likely to mediate the effects of
dmd-3 and mab-3 on the genetically separable process of
anterior tail retraction.
How do these findings inform our understanding of the role of DM genes in
sexual development? As discussed above, the surprising diversity in the nature
of the sex-specific functions of DM factors has made it difficult to
understand the basis for their conservation in these processes. Interestingly,
dmd-3, mab-3 and dsx all function at the interface between
the general sex determination hierarchy and the regulation of specific
developmental events. Thus, we suggest that the ancestral role of DM genes was
to act as cell-autonomous determinants of sexual information, directly linking
sex to the modulation of differentiation and morphogenesis. In a primitive
system, the differential expression of these genes could have allowed them to
act as `selector' genes of sexual information
(Mann and Carroll, 2002), much
as Hox cluster genes specify positional information. Once this crucial
function became fixed, upstream regulatory hierarchies could have evolved to
allow more complex mechanisms of interpreting the primary sex-determining cue
(Wilkins, 1995), giving rise
to the present day roles of dmd-3, mab-3 and dsx. The
selective expression of DM genes in one sex may also have allowed their
functions to be captured in further downstream steps. Further exploration of
this unique gene family will undoubtedly shed light onto the intersection of
sex determination and developmental patterning.
|
ACKNOWLEDGMENTS |
|---|
|
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TOP
SUMMARY
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RESULTS
DISCUSSION
REFERENCES |
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