1 Howard Hughes Medical Institute and Department of Biochemistry, University of
Wisconsin-Madison, Madison, WI 53706-1544, USA
2 Institute for Behavioral Genetics, University of Colorado-Boulder, Boulder, CO
80309-0447, USA
* Author for correspondence (e-mail: jekimble{at}facstaff.wisc.edu)
Accepted 11 March 2003
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SUMMARY |
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Key words: C. elegans, Gonadogenesis, HAND, Organ primordium, hnd-1, ehn
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INTRODUCTION |
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We have focused on developmental controls of the gonadal primordium in the
nematode Caenorhabditis elegans. This organ primordium is unusually
simple: it is composed of two somatic gonadal precursor cells (SGPs) and two
primordial germ cells (PGCs). The SGPs generate all somatic tissues of the
gonad proper (i.e. ovary or testis), as well as genital ducts (e.g. uterus,
vas deferens), whereas the PGCs give rise to all germ cells, including
gametes. The SGPs and PGCs arise from distinct embryonic blastomeres and
assemble into the gonadal primordium midway through embryogenesis
(Fig. 1A,B) (Sulston et al., 1983). Within
the mature primordium, the SGPs (Z1 and Z4) reside at the distal poles and the
PGCs (Z2 and Z3) are situated proximally
(Fig. 1C)
(Kimble and Hirsh, 1979
). In
addition to this proximal-distal polarity, the primordium displays left-right
and dorsal-ventral polarity (Fig.
1C). Therefore, the four-celled gonadal primordium is patterned in
three axes.
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Several genes have been identified that control early gonadogenesis in
C. elegans (Fig. 1C)
(Hubbard and Greenstein,
2000). For example, gon-2 and gon-4 control the
onset and timing of gonadal cell divisions
(Friedman et al., 2000
;
Sun and Lambie, 1997
), and
lin-17, sys-1, wrm-1, lit-1 and pop-1 govern the asymmetric
division of the SGPs (Siegfried and
Kimble, 2002
; Sternberg and
Horvitz, 1988
). For germline development, pie-1 and
nos-2 control PGC fate and influence their incorporation into the
gonadal primordium (Seydoux et al.,
1996
; Subramaniam and Seydoux,
1999
; Tenenhaus et al.,
2001
). In this paper, we report that the C. elegans Hand
bHLH transcription factor hnd-1 is important for early gonadogenesis
as well as for embryogenesis. Specifically, hnd-1 influences the
number and position of SGPs in the gonadal primordium, and affects body shape
in the embryo. The hnd-1 gene is expressed broadly in the embryonic
mesoderm and then more specifically in the SGPs. Our results suggest that
hnd-1 governs maintenance of SGP fate and SGP survival. We also
report the discovery of two genetic enhancers of hnd-1, named
ehn-1 and ehn-3 (for enhancer of Hand), that have
overlapping functions with hnd-1 in embryogenesis and
gonadogenesis.
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MATERIALS AND METHODS |
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Plasmids and transgenes
All cloning was performed by standard methods
(Sambrook et al., 1989). PCR
products were sequenced. Primer sequences are available upon request.
Transgenes were generated as simple arrays unless otherwise noted.
hnd-1 cDNA (pJK849 and pJK901)
Using a probe from the coding region of C44C10.8, we isolated a
hnd-1 cDNA from an embryonic C. elegans cDNA library (a gift
from P. Okkema) and subcloned it to make pJK849. The hnd-1 5'
end was cloned by RT-PCR using embryonic total RNA, a primer to the SL1
trans-spliced leader and internal hnd-1-specific primers. A
full-length hnd-1 cDNA (pJK901) was assembled from the SL1 RT-PCR
product and pJK849.
hnd-1(FL)::GFP (pJK850)
GFP coding sequences were amplified by PCR from pPD95.81 (a gift from A.
Fire) and subcloned into a hnd-1 genomic fragment (pJK906). pJK850
includes 1568 bp of the hnd-1 sequence upstream of the 5'UTR
and 182 bp downstream of the 3'UTR. pJK850 was injected with pRF4[Rol]
(Mello et al., 1991) into
hnd-1 to generate qEx486; this array rescued hnd-1
gonadal defects completely (n=136) and reduced lethality from 28% to
7% (n=190).
hnd-1(N)::GFP (pJK848)
The first two exons and 1540 bp upstream of the hnd-1 5'UTR
were PCR amplified and cloned into pPD95.81 (a gift from A. Fire). pJK848 was
injected into unc-4(e120) with the co-injection marker
pNC4-21[unc-4+] (Miller and
Niemeyer, 1995) and N2 DNA to create qEx447 and,
subsequently, qIs55. With the exception of SGPs,
hnd-1(N)::GFP was detected in cells that also express hlh-1,
a marker for body muscle (Krause et al.,
1990
).
hnd-1::GFPlacZ (pJK900)
The hnd-1 promoter (plus 11 N-terminal codons) was PCR amplified
and cloned into pPD96.04 (a gift from A. Fire). pJK900 was injected with
pRF4[Rol+] to create qEx492 and, subsequently, qIs69. pJK850
and pJK900, but not pJK848, express GFP in several head cells that we have not
identified.
HS-hnd-1 (pJK902)
The hnd-1 cDNA from pJK901 was cloned into pPD49.78 (a gift from
A. Fire) to generate pJK902, which was injected into qIs61 with the
co-injection marker pRF4[Rol+] to make qEx493. Embryos were subjected
to two 30-minute heat pulses at 33°C, with a one hour recovery interval.
Resulting L1 larvae were scored for extra SGPs using pes-1::GFP.
hlh-1::hnd-1GFP (pJK904)
A hnd-1::GFP fusion was generated by inserting GFP into the
RsrII site of the full-length hnd-1 cDNA (pJK901).
hnd-1::GFP was then cloned into pPD51.45
(Krause et al., 1990) to
generate pJK904, which was injected into hnd-1 with the co-injection
marker pRF4[Rol+] to make qEx496; this array rescued hnd-1
gonadal defects and marginally rescued lethality (20%, n=372).
lag-2::hnd-1GFP (pJK905)
A hnd-1::GFP fusion (see above) was cloned into pJK590
(Blelloch et al., 1999) to
generate pJK905, which was injected into hnd-1 with the co-injection
marker pRF4[Rol+] to make qEx497; this array partially rescued
hnd-1 gonadogenesis defects and did not rescue lethality (24%,
n=192).
hnd-1 genomic DNA (pJK906)
A plasmid carrying hnd-1 genomic DNA was amplified by PCR; it
contained the same upstream and downstream sequences as in
hnd-1(FL)::GFP. pJK906 was injected with pPD136.64
[myo-3::YFP] (a gift from A. Fire) and pJK907 [pes-1::CFP]
into hnd-1 to make qEx495; this array was used for mosaic
analysis.
pes-1::CFP (pJK907)
The pes-1 promoter from pUL#MJA1
(Molin et al., 2000) was
cloned into pPD136.64 (a gift from A. Fire).
hnd-1 RNA interference and deletion
Double-stranded hnd-1 RNA was generated, using pJK849 as template,
and injected at 1 mg/ml. The hnd-1(q740) deletion was isolated
essentially as described by Kraemer et al.
(Kraemer et al., 1999), and
backcrossed eight times. To test for maternal effects, hnd-1 females,
generated by fog-1 RNAi (Jin et
al., 2001
), were crossed with N2 males [17% of the cross-progeny
died as embryos or young larvae (n=313), and all adult progeny had
normal gonads (n=260)]. To test for zygotic lethality, we scored
progeny of unc-9 hnd-1/++ mothers [6% died as embryos or larvae
(n=235)]. To investigate whether hnd-1(q740) was a null
allele, RT-PCR was performed on mutant and wild-type worms, using primers to a
region retained in the hnd-1 deletion. Template RNA was prepared from
20 gravid adults using TRI reagent (Molecular Research Center). A PCR product
was obtained only from wild-type worms.
Tests for hnd-1 genetic interactions
hlh-1
Progeny of hlh-1/+; hnd-1 mothers had 55% embryonic and larval
lethality, compared with 25% defects for hlh-1/+
(Chen et al., 1994) and 28%
defects for hnd-1 (this work).
The following were evaluated using number of gonadal arms as a measure:
sys-1
100% of sys-1/+; hnd-1/+ worms had two arms (n=89).
sys-1/+; hnd-1 had 68% gonadal arms, compared with 70% for
hnd-1 alone and <1% for sys-1/+. sys-1 dominantly
enhances other Sys mutants (K. Siegfried, unpublished).
gon-4
100% of gon-4/+; hnd-1/+ worms had two arms (n=112).
gon-4; hnd-1 double mutants had 30% gonadal arms (n=46),
compared with 43% for gon-4
(Friedman et al., 2000) and
70% for hnd-1.
gon-2
At 20°C, the progeny of gon-2; hnd-1 worms had 78% gonadal
arms (n=89), compared with 70% for hnd-1 and 100% for
gon-2 (Sun and Lambie,
1997). The progeny of gon-2; hnd-1 worms shifted to
25°C as L4s resembled gon-2 alone (most had no visible
gonad).
Antibody staining
Embryos were fixed essentially as described by Miller and Shakes
(Miller and Shakes, 1995), and
then stained with
-PGL-1, a component of P granules
(Kawasaki et al., 1998
),
-HLH-1 (Krause et al.,
1990
) or
-UNC-54
(Miller et al., 1983
) for body
muscle. Secondary antibodies were used at 1:400 (Jackson Labs, West Grove,
PA). DAPI staining was performed as described by Kadyk and Kimble
(Kadyk and Kimble, 1998
).
ehn-1 and ehn-3 genetics
ehn-1(q638) was identified in a EMS mutagenesis screen for
gonadogenesis mutants (L.D.M., K. Siegfried, F.-H. Markussen, unpublished).
ehn-1(q690) and ehn-3(q689) were obtained in an
ehn-1 non-complementation screen of 2251 haploid genomes: EMS
mutagenized males were crossed to ehn-1(q638) rol-6 hermaphrodites,
and F2 progeny screened for gonadal defects. By three-factor mapping, we
positioned ehn-1 between unc-104 and rol-6 on
linkage group II, and ehn-3 between dpy-13 and
unc-5 on linkage group IV. ehn-1 is almost maternally and
zygotically sufficient for gonadogenesis. From an ehn-1(q638) rol-6
mother, 1% of heterozygous cross progeny had defects (n=221); from an
ehn-1(q638) rol-6/++ mother, none of the ehn-1 rol-6
homozygous progeny had defects (n=199). ehn-3 has minor
dominance: <1% of ehn-3 unc-5/++ had gonadogenesis defects
(n=222).
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RESULTS |
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hnd-1 affects embryo morphogenesis
hnd-1 mutants can die as embryos or young larvae with variable
body shape defects, typically in the posterior
(Fig. 3A,B). Most
hnd-1 embryos contained pharynx and gut
(Fig. 3C,D), as well as muscle,
as evidenced by twitching. Because hnd-1::GFP is expressed in
mesodermal precursors (see below), we compared body wall muscles in wild-type
and hnd-1 embryos using an -myosin antibody
(Miller et al., 1983
). Both
wild-type and hnd-1 late-stage embryos have four quadrants of body
muscle (Fig. 3E,F)
(Miller et al., 1983
),
although muscle fibers were sometimes disorganized in mutants
(Fig. 3F).
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hnd-1 governs SGP number and position
Wild-type hermaphrodites possess two gonadal arms. By contrast, adult
hermaphrodites depleted for hnd-1 displayed a range of gonadal
shapes: two gonadal arms (Fig.
4A); a single gonadal arm (Fig.
4B); no apparent gonad (Fig.
4C); or abnormal gonads (Fig.
4D). `Abnormal gonads' include a variety of shapes, most typically
an amorphous mass (Fig. 4D).
One-armed and two-armed gonads were frequent and were usually fertile, whereas
absent and abnormal gonads were less common and were always sterile
(Table 1). A similar, but less
penetrant effect was seen in males (not shown). We conclude that
hnd-1 is important, but not essential, for gonadogenesis.
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SGP number and position are crucial for gonadogenesis
We used hnd-1 mutants born with aberrant gonadal primordia to
investigate how organization of that primordium affected gonadogenesis.
Specifically, we used pes-1::GFP to score SGPs in hnd-1 L1
larvae, permitted the animals to develop and then examined them again as L4s.
Our results (Table 3) led to
three conclusions. First, most hnd-1 primordia with a wild-type
appearance (two SGPs placed at the poles) generated wild-type appearing adults
with two gonadal arms (93%, n=70). Therefore, hnd-1 appears
to play little or no role in gonadogenesis after formation of the gonadal
primordium. Second, most primordia containing one SGP generated adult gonads
with only a single arm (98%, n=53); none made two arms. Finally,
primordia with wild-type SGP number but aberrant SGP position often generated
defective gonads (26%, n=23). Therefore, SGP position within the
primordium may be important for gonadogenesis.
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hnd-1 expression during wild-type development
To investigate hnd-1 expression, we constructed three GFP
reporters (Fig. 2C).
hnd-1(FL)::GFP inserts GFP coding sequences into the third exon of
the full-length HND-1 protein; this reporter rescued hnd-1 mutants
(Materials and Methods). hnd-1(N)::GFP inserts GFP more N-terminally
and replaces the hnd-1 3'UTR with the unc-54
3'UTR. hnd-1::GFPlacZ replaces most of the hnd-1
coding region with GFP and ß-galactosidase coding sequences, and the
unc-54 3'UTR. All three hnd-1 reporters expressed GFP
in largely the same cells, but hnd-1(N)::GFP and
hnd-1::GFPlacZ expressed GFP at a higher level and expression
persisted longer. The rescue by hnd-1(FL)::GFP suggests that its
expression is relevant to hnd-1 function.
The hnd-1 reporters expressed GFP in the MS, C and D embryonic
lineages (Fig. 6A). Expression
was first observed in four MS great-granddaughters, four C
great-granddaughters and two D daughters
(Fig. 6B). These MS descendants
give rise to the SGPs and other mesodermal cells
(Fig. 6A) (Sulston et al., 1983); the C-
and D-expressing cells all generate body wall muscle
(Sulston et al., 1983
).
Expression continued through one cell division
(Fig. 6C) and then became
difficult to detect using hnd-1(FL)::GFP. hnd-1(N)::GFP remained
detectable in some cells within these MS and C lineages
(Fig. 6D), but disappeared from
most body muscle cells by the comma stage of embryogenesis
(Fig. 6E). Then, the
hnd-1 reporters were expressed in the SGPs (Z1 and Z4) as they
approached the PGCs to form the gonadal primordium
(Fig. 6E,F). Shortly after the
primordium was assembled, hnd-1(N)::GFP expression was reduced or
disappeared (Fig. 6G). GFP was
not detected in the SGPs at hatching or post-embryonically (not shown).
Therefore, hnd-1 appears to be expressed during embryogenesis in
mesodermal precursor cells that generate predominantly body wall muscle, and
then in SGPs.
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After formation of the gonadal primordium, SGPs remained associated with PGCs in hnd-1 embryos; none were seen detaching. Instead, SGP nuclei sometimes became smaller and hnd-1 reporter expression faded prematurely (38%, n=13). The simplest hypothesis is that hnd-1 is required for maintenance of SGP fate and possibly SGP survival.
We next investigated whether ectopic hnd-1 expression could transform other cells to the SGP fate. To this end, we used a heat-inducible promoter to express hnd-1 during embryogenesis but found no ectopic SGPs, as assayed by pes-1::GFP (Materials and Methods). We also expressed a full-length HND-1::GFP fusion protein under control of either of two mesodermal promoters (see below), but again did not observe ectopic SGPs. These results are consistent with the proposed role for hnd-1 in controlling SGP maintenance or survival.
What becomes of SGPs in hnd-1 mutants?
The missing SGPs in hnd-1 mutants might be explained by
transformation to a different cell type, or by cell death. To explore the
first idea, we reasoned that the most likely transformation would be to a
different mesodermal cell type. We tested this using hlh-8::GFP to
mark the M mesoblast (Harfe et al.,
1998) and unc-122::GFP to mark coelomocytes
(Miyabayashi et al., 1999
).
All wild-type L1s had a single hlh-8::GFP-expressing M mesoblast, as
expected (n=61). Similarly, most hnd-1 mutants had a single
M cell, but a few had two M cells (5%, n=63) or no M cell (2%,
n=63). Those with an additional M cell had two SGPs, suggesting that
extra M cells were not transformed SGPs. Likewise, occasional extra
coelomocytes were seen, but overall hnd-1 mutants had marginally
fewer coelomocytes than wild type (5.5 versus 5.9 on average per animal,
n>30). Importantly, the extra coelomocytes could be in worms with
two gonadal arms. Therefore, hnd-1 appears to have a low-penetrance
effect on M cells and coelomocytes, but this is unlikely to account for the
missing SGPs.
To determine whether SGPs are lost as a result of programmed cell death in
hnd-1 mutants, we examined hnd-1; ced-3 double mutants using
the pes-1::GFP marker. The ced-3 gene is required for all
programmed cell deaths (Ellis and Horvitz,
1986). In hnd-1 single mutants, 42% were missing at least
one SGP (Table 2A), and, in
ced-3; hnd-1 double mutants, 45% lacked at least one SGP
(n=69). Therefore, SGP loss does not appear to rely on
ced-3-dependent programmed cell death.
Next, we investigated whether SGPs died in hnd-1 mutants. In
C. elegans, cell corpses resulting from either programmed or necrotic
cell death are engulfed by their neighbors
(Chung et al., 2000;
Ellis et al., 1991
). The
engulfment of cell corpses relies on several genes, including ced-2
(Ellis et al., 1991
). In
ced-2 single mutants, no cell corpses were evident near the gonad
(Fig. 7A,B; n=54);
however, in ced-2; hnd-1 double mutants, cell corpses were found near
the gonad (Fig. 7C,D; 28%,
n=50). Importantly, the presence and site of corpses correlated with
SGP absence. We observed no cell corpses near gonads with two SGPs
(n=53), mostly anterior or right cell corpses near gonads missing Z1
(4/5; Fig. 7C), and only
posterior or left cell corpses in those missing Z4 (8/8). Indeed, in one cell
corpse, hnd-1::GFP was faintly expressed, indicating that it had been
specified originally as an SGP (data not shown). Therefore, SGPs appear to die
in hnd-1 mutants.
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In C. elegans, mosaic animals can be made by loss of
extra-chromosomal arrays that carry transgenes and that are transmitted with
varying fidelity at each cell division
(Herman, 1984). For this
study, we created an extra-chromosomal array that carries a rescuing
hnd-1 genomic fragment and two fluorescent markers
(myo-3::YFP to mark body muscle and pes-1::CFP to mark
SGPs). We then identified `germline mosaics', animals that retained the array
in somatic tissues but failed to transmit it to their progeny; such animals
have lost the array in divisions generating the germline blastomere P4 (see
Fig. 6). All six germline
mosaics had a wild-type gonadal primordium, which suggests that hnd-1
activity acts in somatic tissues rather than in the germ line.
To further explore where hnd-1 acts, we used either of two
promoters: hlh-1, which is expressed in body muscle and not in SGPs
(Krause et al., 1990); or
lag-2, which is first expressed in the AB and MS lineages
(Moskowitz and Rothman, 1996
),
and then in SGPs (Miskowski et al.,
2001
). Each promoter was fused to a full-length, rescuing
hnd-1::GFP cDNA and expressed in hnd-1 mutants. Expression
of HND-1::GFP by the hlh-1 promoter rescued the hnd-1
gonadogenesis defects, from 52% to 5% defective (n=42). By contrast,
HND-1::GFP driven from the lag-2 promoter, which is expressed in the
two SGPs (Fig. 5G), did not
appreciably rescue hnd-1 gonadogenesis defects (36% defective,
n=108). The latter experiment has the caveat that this promoter is
switched on after SGPs assemble into the gonadal primordium and it may not be
expressed in dying SGPs. From the hlh-1::hnd-1GFP result, we suggest
that HND-1 acts in early mesodermal lineages.
hnd-1 acts independently of other early gonadogenesis
genes
The hnd-1 SGP defects are the earliest observed to date among any
genes controlling C. elegans gonadogenesis. To investigate whether
hnd-1 might function with other early gonadogenesis genes, we
explored genetic interactions between hnd-1 and two mutant classes.
The first type, represented by gon-2 and gon-4
(Friedman et al., 2000;
Sun and Lambie, 1997
),
controls the onset of cell divisions in the gonad but not in other tissues
(Fig. 1C). Gonadal divisions
are delayed in gon-2 or gon-4 single mutants
(Friedman et al., 2000
;
Sun and Lambie, 1997
), but not
in hnd-1 mutants (n=5). Moreover, hnd-1; gon-2 and
hnd-1; gon-4 double mutants have additive phenotypes (Materials and
Methods). Therefore, hnd-1 does not affect the onset or timing of
gonadal divisions and acts independently of gon-2 and
gon-4.
The second class of early gonadogenesis genes, represented by
sys-1 (Miskowski et al.,
2001), is required for SGPs to produce daughter cells with
different developmental potential (Fig.
1C). In wild type, each SGP generates one distal tip cell (DTC),
whereas in sys-1 mutants they make no DTCs
(Miskowski et al., 2001
). Most
hnd-1 SGPs that were properly positioned generated DTCs (96%,
n=193; Table 3), and
no genetic interactions were found with sys-1 (Materials and
Methods). Therefore, hnd-1 does not appear to affect SGP asymmetric
divisions, but instead ensures that two SGPs are present and properly
positioned in the gonadal primordium.
Identification of genetic enhancers of hnd-1
To identify additional genes controlling SGP development, we screened for
EMS-induced mutants with a hnd-1-like gonadogenesis phenotype and
discovered loss-of-function mutations of ehn-1 and ehn-3
[for enhancer of Hand (Materials and Methods)]. The ehn-1 and
ehn-3 mutants had low-penetrance gonadal defects
(Table 1). For ehn-1,
gonadal defects could be rescued either maternally or zygotically, but
ehn-3 exhibited no maternal effect (Materials and Methods).
Furthermore, ehn-1 had low-penetrance lethality, but lethality was
negligible in ehn-3 mutants (Table
4).
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Functional relationships between the hnd-1 and EHN
genes
To investigate the functional relationships between the hnd-1 and
EHN genes, we first investigated double and triple mutants
(Table 4). Although hnd-1,
ehn-1 and ehn-3 single mutants all had relatively low-penetrance
gonadal defects, the double and triple mutants showed increased penetrance
(Table 4). For example, 80-90%
of ehn-1 and ehn-3 single-mutant adults had two gonadal
arms, but almost none of the ehn-1; ehn-3 double mutants had two
gonadal arms (2-4%; Table 4).
Similarly, only 5% of the double mutants had two SGPs at hatching
(Table 2A). By contrast, larval
lethality did not increase in the ehn-1; ehn-3 double mutant.
Therefore, ehn-1 and ehn-3 may be partially redundant for
SGP development.
The ehn-1; hnd-1 and ehn-3; hnd-1 double mutants were also more defective than any of the single mutants, but each double mutant was unique. For the gonadogenesis defects, ehn-3 enhanced hnd-1 more strongly than did ehn-1. Thus, some ehn-1; hnd-1 double mutants made two gonadal arms and only about one-third had no apparent gonad. By contrast, no ehn-3; hnd-1 double mutants had two gonadal arms, and most had no visible gonad (Table 4). Intriguingly, this situation was reversed for lethality: ehn-1 enhanced hnd-1 more strongly than did ehn-3 for both embryonic and larval lethality. One simple explanation is that the three genes are all partially redundant, but that each has acquired an individual role in the repertoire of activities normally carried out by hnd-1/EHN genes (see Discussion).
The ehn-1; ehn-3; hnd-1 triple mutant appears additive for the ehn-1; hnd-1 and ehn-3; hnd-1 defects. Thus, the penetrance of the triple mutant with respect to lethality is similar to that of the ehn-1; hnd-1 double mutant, and the penetrance of the triple mutant with respect to gonadal defects is similar to that of the ehn-3; hnd-1 mutant (Table 4). The fact that the triple mutant is not fully penetrant may suggest the existence of one or more additional genes involved in the process, or it may indicate that the ehn-1 or ehn-3 mutant is not a null.
To begin addressing relationships between the ehn genes and hnd-1 at a molecular level, we examined expression of hnd-1(N)::GFP to mark hnd-1 transcription and SGP formation. The ehn-1 and ehn-3 single mutants both expressed hnd-1(N)::GFP in two SGPs (ehn-1, n=35; ehn-3, n=31). Therefore, ehn-1 and ehn-3 do not control hnd-1 transcription in SGPs, which is consistent with the idea that they function in parallel to hnd-1. Furthermore, ehn-3; hnd-1 double mutant embyros made two SGPs (n=8), but few possessed SGPs at hatching (4%; Table 2A). Therefore, like hnd-1, the ehn-1 and ehn-3 genes do not affect SGP specification but instead influence SGP fate or survival.
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DISCUSSION |
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hnd-1 and control of SGPs in the gonadal primordium
Wild-type C. elegans has four gonadal precursors, two SGPs and two
PGCs, in stereotyped positions within the primordium. hnd-1 mutants
affect the presence and position of these precursors in the primordium and
within the animal. Thus, hnd-1 mutants can possess fewer than normal,
as well as mispositioned, SGPs or PGCs. The hnd-1 gene probably acts
cell autonomously in the SGPs or their precursors to control early
gonadogenesis. However, hnd-1 does not affect SGP specification,
because the correct number of SGPs is generated in all hnd-1-mutant
embryos. Nor does it cause SGPs to be transformed into either of two
mesodermal types (coelomocytes and the M mesoblast), although it remains
possible that they are transformed into muscle cells. Instead, we suggest that
hnd-1 is required for SGP survival.
What happens to SGPs in hnd-1 mutants? We used two classes of cell
death mutants: ced-3, which eliminates all programmed cell death
(Ellis and Horvitz, 1986), and
ced-2, which is defective in cell corpse engulfment
(Ellis et al., 1991
). At first
glance, our results appear contradictory: we observed extra cell corpses in
hnd-1; ced-2 double mutants, but saw no increase in the number of
SGPs in hnd-1; ced-3 double mutants. One simple explanation is that
SGPs die by a ced-3-independent pathway. Alternatively, if the SGPs
no longer expressed markers of their fate (e.g. pes-1), they would
not have been identified in our analysis of hnd-1; ced-3 mutants.
Therefore, it remains possible that hnd-1 mutant SGPs fail to
maintain their fate and die via programmed cell death. In either case, the
correlation between missing SGPs and extra cell corpses strongly supports the
idea that hnd-1 is required for SGP survival.
Why might SGPs die in hnd-1 mutants? One simple explanation is
that inhibition of apoptosis is part of the normal developmental program, as
has been suggested for the winged-helix transcription factor Fork head in
Drosophila salivary gland development
(Myat and Andrew, 2000).
Alternatively, cells may be programmed to die when they receive ambiguous
developmental cues. This idea is supported by the extensive apoptosis seen in
many developmental mutants [e.g. Pax6/eyeless mutants
(Halder et al., 1998
)].
Because hnd-1 SGPs initially show evidence of their fate (they
express SGP markers and migrate to the PGCs), we favor the idea that
hnd-1 is required for maintenance of cell fate and that in its
absence the SGPs die. Similarly, Pax6/eyeless mutants generate eye
primordia that express early markers of their fate (e.g. ey-eye
enhancer lacZ) and later undergo programmed cell death
(Halder et al., 1998
).
hnd-1 and embryonic viability
In addition to gonadal defects, hnd-1 mutants can die as embryos
or young larvae with body morphogenesis defects. Elongation of the embryo is
driven largely by cell shape changes in the hypodermis
(Priess and Hirsh, 1986).
However, mutants affecting muscle development also disrupt the process (e.g.
Bejsovec and Anderson, 1988
;
Chen et al., 1994
;
Waterston, 1989
). Of
particular interest to this work is the hlh-1 gene, which encodes the
C. elegans myoD homolog (Krause
et al., 1990
); its loss disrupts development of body wall muscles
and causes a characteristic morphogenesis defect
(Chen et al., 1994
). We
explored the possibility that hnd-1 may similarly be involved in body
muscle development. However, hnd-1; hlh-1 double mutants still make
body muscle, suggesting that these bHLH proteins control different aspects of
muscle development. Although speculative at the current time, we suggest that
hnd-1 may play a role in muscle fate that parallels its role in
controlling SGP fate.
Three genes with overlapping functions in SGP development
The hnd-1 deletion has incompletely penetrant gonadal and
embryonic defects. Yet, the mouse and zebrafish Hand mutants are completely
penetrant (Firulli et al.,
1998; Riley et al.,
1998
; Srivastava et al.,
1997
; Yelon et al.,
2000
). Why might a hnd-1-null mutant exhibit partially
penetrant defects? One simple explanation is genetic redundancy. We have
identified two genes, ehn-1 and ehn-3, that enhance the
hnd-1 phenotype. All three single mutants have partially penetrant
gonadal defects, and mutations in two of the three, hnd-1 and
ehn-1, also affect viability. Each of the double mutants has a more
severe gonadogenesis defect, which suggests at least two pathways control SGP
survival. Redundancy frequently results from gene duplication
(Ohno, 1970
). However, only
one Hand homolog exists in the C. elegans genome, and neither
ehn-1 nor ehn-3 maps to a region containing any predicted
bHLH protein. Therefore, the hnd-1 and EHN genes redundantly control
SGP development, but they are unlikely to represent paralogous pathways.
Intriguingly, ehn-1 enhances hnd-1 lethality more strongly than it enhances the hnd-1 gonadal defect, whereas ehn-3 enhances the hnd-1 gonadal defect but not its lethality. The identity of hnd-1 as a putative bHLH transcription factor provides a molecular framework for considering the enhancement of hnd-1 by ehn-1 and ehn-3. One idea is that ehn-1 and ehn-3 might encode, or control the activity of, transcription factors that cooperate with hnd-1 in the regulation of partially overlapping sets of target genes. Regardless of the molecular mechanism, the hnd-1/ehn genes clearly have overlapping, but non-equivalent, functions in embryonic development and gonadogenesis.
Regulation of mesoderm development by Hand transcription factors
The hnd-1 gene encodes the single Hand transcription factor in the
C. elegans genome (Ledent and
Vervoort, 2001). Higher vertebrates contain two Hand genes
(eHand/Hand1 and dHand/Hand2), whereas a single family
member has been identified in zebrafish
(Yelon et al., 2000
),
ascidians (Dehal et al., 2002
)
and flies (Moore et al.,
2000
). Vertebrate dHand is expressed in lateral plate
mesoderm and is important for development of mesodermal organs, including
heart and limbs (Firulli et al.,
1998
; Riley et al.,
1998
; Srivastava et al.,
1997
; Yelon et al.,
2000
). The Drosophila Hand gene is expressed in the
dorsal vessel (heart) and visceral mesoderm, but its function is not known
(Moore et al., 2000
). The
C. elegans Hand gene, hnd-1, is first expressed broadly in
mesodermal precursors that generate striated muscles, and then is restricted
to the somatic gonadal precursors; its function appears to affect both muscle
and gonadal development. Therefore, all Hand genes explored to date are
expressed in mesodermal cells and, where studied, are important for mesoderm
development.
The defects in hnd-1 have intriguing similarity to the defects in
the zebrafish Hand gene called hands off (han). Thus,
han mutants generate the normal number of precardiac cells, but these
cells cannot differentiate and a midline heart tube fails to form
(Yelon et al., 2000).
Similarly, SGPs are specified correctly in hnd-1 mutants, but they
often fail to maintain their fate and can subsequently die. The fate of the
cardiac precursors in zebrafish han mutants is not known
(Yelon et al., 2000
). We
suggest that the zebrafish and nematode Hand genes may play parallel roles in
controlling cardiac and gonadal precursor cells, respectively. Interestingly,
zebrafish han may also be important for gonadogenesis: han
mutants have defects in migration of germ cells to the gonad as well as
abnormalities in pax2.1 expression in the putative gonadal mesoderm
(Weidinger et al., 2002
).
Therefore, zebrafish han mutants, like hnd-1 mutants, might
have defects in development of the gonadal mesoderm. Our identification of
hnd-1 as a regulator of somatic gonadal development in C.
elegans raises the possibility that Hand genes are ancient regulators of
gonadogenesis.
Genetic controls of early gonadogenesis
How does the control of SGP development by hnd-1/ehn
genes compare to the genetic regulation of early gonadogenesis in other
animals? Although genes have been identified that govern formation of the
early gonad in both Drosophila and vertebrates, the genetic control
of early gonadogenesis remains relatively uncharted territory. Perhaps most
analogous to C. elegans hnd-1/ehn genes is Drosophila
clift, which encodes a novel nuclear protein required for both SGP
development in the gonad and for photoreceptor survival in the eye
(Boyle et al., 1997). The
clift effect on SGPs is remarkably similar to that of the
hnd-1/ehn genes: SGPs are generated in clift
mutants, but they do not coalesce into a gonadal primordium and are lost over
time (Boyle et al., 1997
).
Furthermore, ectopic clift expression, like ectopic hnd-1
expression, did not increase SGP number
(Boyle et al., 1997
).
Therefore, like hnd-1, clift may not be sufficient to direct SGP
development on its own. In mice, several transcription factors have been
implicated in development of the genital ridge, a mesodermal swelling destined
to generate the somatic gonad (Birk et al.,
2000
; Capel, 2000
).
In Sf1 and Wt1 knockout mice, the genital ridge forms
initially but it does not develop further; instead, the ridge regresses
because of programmed cell death
(Kreidberg et al., 1993
;
Luo et al., 1994
). Therefore,
although these genes all encode different transcription factors, the
similarities in mutant phenotype suggest parallels in the genetic control of
somatic gonadal precursors in flies, mammals and worms.
Controls of early organogenesis
How does C. elegans gonadogenesis compare with the development of
other organs? Some organs rely on `selector' genes, which regulate (directly
or indirectly) all the genes needed to generate a particular organ. One simple
example of an organ selector gene is C. elegans pha-4, which encodes
a forkhead transcription factor that appears to regulate most, and perhaps
all, pharyngeal genes (Gaudet and Mango,
2002; Horner et al.,
1998
). Another example is that of the Drosophila
Pax6/eyeless gene, which encodes a paired homeodomain transcription
factor that is crucial for eye development
(Quiring et al., 1994
).
Dramatically, Pax6/eyeless induces extra eyes when expressed
ectopically (Halder et al.,
1995
). Our data suggest that hnd-1 does not fit into the
organ selector model. Although loss of hnd-1 function can cause the
complete loss of gonadal development, global expression of hnd-1 did
not induce ectopic gonadal development. Therefore, gonad `identity' in C.
elegans might rely on the coordinate regulation of several genes. Based
on our analysis of hnd-1/ehn double mutants, the
ehn genes represent likely candidates for additional regulators of
the gonadal fate. Similarly, during Drosophila salivary gland
development, several genes, including the winged-helix transcription factor
encoded by fork head (fkh), are independently regulated and
required for the development of specific salivary gland cell types (reviewed
by Bradley et al., 2001
).
Interestingly, one aspect of fkh function is to inhibit apoptosis in
the salivary gland primordia (Myat and
Andrew, 2000
). Therefore, like hnd-1, fkh acts after the
salivary gland primordia are specified and is required for the survival of
specific salivary gland cell types. We suggest that the role of hnd-1
is to maintain the somatic gonadal fate and thereby prevent the death of the
somatic gonadal precursors. It remains to be seen whether the maintenance of
cell fate and cell survival are intimately linked during the development of
other organs.
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ACKNOWLEDGMENTS |
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