The conserved nuclear receptor Ftz-F1 is required for
embryogenesis, moulting and reproduction in
Caenorhabditis elegans
Masako Asahina1,2, Takeshi Ishihara3, Marek Jindra1,4, Yuji Kohara5, Isao Katsura3
and Susumu Hirose1,*
1
Department of Developmental Genetics, 3Laboratory of Multicellular Organization, and 5Laboratory of Genome Biology, National
Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan
2
Institute of Parasitology, and 4Institute of Entomology, Czech Academy of Science, Ceske Budejovice, 37005 Czech Republic
Abstract
Background: Nuclear receptors are essential players in
the development of all metazoans. The nematode
Caenorhabditis elegans possesses more than 200
putative nuclear receptor genes, several times
more than the number known in any other organism. Very few of these transcription factors are
conserved with components of the steroid response
pathways in vertebrates and arthropods. Ftz-F1, one
of the evolutionarily oldest nuclear receptor types, is
required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila.
Results: We employed two complementary
approaches, direct mutagenesis and RNA interference, to explore the role of nhr-25, a C. elegans
Introduction
Nuclear receptors are vital for development, reproduction and many aspects of physiology from vitamin
and lipid metabolism to behaviour (Mangelsdorf et al.
1995). These specialized transcription factors are found
in organisms that range from the simplest Metazoa
(cnidarians) to mammals, but are absent from lower
eukaryotes (Escriva et al. 1997). Most of the structural
subclasses of nuclear receptors (Laudet 1997) existed
before deuterostomes had evolved, as exempli®ed by
their conserved Ftz-F1/SF-1 counterparts in insects and
vertebrates. Whether some of the roles of these
Communicated by: Hiroshi Handa
* Correspondence: E-mail: [email protected]
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ortholog of Ftz-F1. Deletion mutants show that
nhr-25 is essential for embryogenesis. RNA interference reveals additional requirements throughout
the postembryonic life, namely in moulting and
differentiation of the gonad and vulva. All these
defects are consistent with the nhr-25 expression
pattern, determined by in situ hybridization and
GFP reporter activity.
Conclusions: Our data link the C. elegans Ftz-F1
ortholog with a number of developmental processes. Signi®cantly, its role in the periodical
replacement of cuticle (moulting) appears to be
evolutionarily shared with insects and thus supports
the monophyletic origin of moulting.
molecules also remained conserved in the diverging
animal phyla is a challenging problem that requires
comparative studies on phylogenetically distant organisms (Ruvkun & Hobert 1998). To address this,
we chose the nematode Caenorhabditis elegans as the
simplest genetically tractable organism possessing
nuclear receptors.
Strikingly, mutants of only ®ve of the enormous
249 nuclear receptor genes predicted in C. elegans
(Miyabayashi et al. 1999) have been identi®ed. Of these,
odr-7 (Sengupta et al. 1994), unc-55 (Zhou & Walthall
1998) and fax-1 (Wightman et al. 1997) function in
speci®c neurones, daf-12 in the progression of larval
stages (Antebi et al. 1998) and sex-1 in sex determination (Carmi et al. 1998). RNA-mediated interference
(RNAi) revealed functions of two additional members,
of which nhr-2 is required during embryogenesis (Sluder
Genes to Cells (2000) 5, 711±723
711
M Asahina et al.
Drosophila
Ecdysteroids
(insect steroid hormones)
αFtz-F1
fushi tarazu
engrailed
segmentation
C.elegans
?
EcR-USP
(receptor complex)
?
DHR3
NHR-23
βFtz-F1
NHR-25
transcription factors
cuticle gene EDG84
metamorphosis
cuticle synthesis
molting
Genes to Cells (2000) 5, 711±723
SF-1
P450 steroidhydroxylases
StAR, MIS
molting
gonad and vulva formation
embryogenesis
et al. 1997) and nhr-23 for moulting (Kostrouchova et al.
1998). Interestingly, ®ve of these seven functionally
characterized nuclear receptors are conserved between
C. elegans and other animals (Sluder et al. 1999).
We have focused on another type of nuclear receptor
conserved in C. elegans, the ancient subfamily Ftz-F1
(Laudet 1997). The roles of Ftz-F1-like proteins in
vertebrates and insects appear quite different (Summary
®gure). Mammalian SF-1 directs sexual differentiation
and steroid hormone synthesis in primary steroidogenic tissues (reviewed by Parker & Schimmer 1997).
SF-1 de®cient mice fail to develop male gonads,
adrenal glands and speci®c brain regions, and die soon
after birth (Luo et al. 1994; Ikeda et al. 1995; Sadovsky
et al. 1995). In Drosophila, an aFtz-F1 isoform is
required maternally for proper segmentation. Alternate
segments are deleted in ftz-f1 mutant embryos in a
pair-rule fashion, as they are in mutants for the
homeodomain protein Fushi tarazu (Ftz) (Wakimoto
et al. 1984), with which Ftz-F1 interacts (Guichet et al.
1997; Yu et al. 1997). A postembryonic isoform bFtzF1 is expressed under ecdysteroid (insect moulting
hormone) control and plays a critical role in
metamorphosis (Woodard et al. 1994; Broadus et al.
1999).
Nematodes and insects develop differently in many
aspects, one being the apparent absence of segmentation, while sharing some intriguing similarities,
notably the periodic replacement of cuticle at larval
moults. Nematodes and arthropods have been recently
712
Mouse
steroidogenesis
gonad and adrenal
differentiation
Summary ®gure Comparison of Ftz-F1
roles in genetically studied representatives
of insects and vertebrates. In both groups,
steroid hormones are synthesized from
precursors such as cholesterol. Ftz-F1
type molecules are direct regulators of
the steroid conversion in mammals. In
insects, ecdysteroids act through speci®c
nuclear receptors (EcR-USP) to trigger a
complex cascade of transcription factors
including DHR3 and bFtz-F1. These in
turn regulate mostly unknown sets of
genes, executing the moulting and metamorphic events. In C. elegans, neither the
synthesis of ecdysteroids nor EcR and USP
orthologs have been detected. Shown in
blue are the conserved C. elegans orthologs
of two members of the insect ecdysteroid
pathway, whose common role is moulting.
StAR, steroidogenic acute regulatory protein; MIS, MuÈllerian-inhibiting substance.
placed under the newly de®ned clade `Ecdysozoa' and
moulting is thought to be of a monophyletic origin
(Aguinaldo et al. 1997). Several constituents of the
ecdysteroid response pathway in insects are members of
the nuclear receptor family, including the ecdysone
receptor complex EcR/Usp (Yao et al. 1992, 1993;
Thomas et al. 1993), DHR3, DHR38, bFtz-F1 and
others, and at least some are known to be involved in
moulting or cuticle synthesis in Drosophila (Bender et al.
1997; Murata et al. 1996; Hall & Thummel 1998;
Kozlova et al. 1998; Schubiger et al. 1998; Broadus et al.
1999). While orthologs of the EcR and usp genes are
missing in C. elegans, some of the downstream components of the ecdysteroid cascade are conserved
(Sluder et al. 1999), suggesting that they might regulate
cuticle synthesis and moulting in the worm. Thus far
only nhr-23, an ortholog of the insect ecdysteroidinduced DHR3 genes (Koelle et al. 1992; Palli et al.
1992; Carney et al. 1997), has been directly linked with
C. elegans moulting (Kostrouchova et al. 1998).
In this paper, we have examined the expression and
developmental roles of the closest ftz-f1 relative in
C. elegans, systematically designated nuclear hormone
receptor 25 (nhr-25). Disruption of the gene with a
short deletion caused an embryonic arrest. nhr-25 RNA
interference, in addition to con®rming the mutant
phenotype, disrupted moulting and the formation of
gonad and vulva. These results present the ®rst direct
evidence for a role of a Ftz-F1-like molecule in
C. elegans development.
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Ftz-F1 role in C. elegans development
Results
nhr-25 gene structure and sequence
comparison
To ®nd out whether C. elegans contains a Ftz-F1 relative,
we searched the DDBJ/GENBANK/EMBL database with
the DNA-binding domain (DBD) of Drosophila melanogaster Ftz-F1 (Lavorgna et al. 1991). The DBD consists
of zinc ®ngers and a Ftz-F1 box (Ueda et al. 1992),
characteristic of this nuclear receptor class. The search
yielded a cosmid F11C1, overlapping with terminally
sequenced cDNAs of the Y. Kohara EST project (NIG,
Japan), clones yk342d8 and yk175f2. These sequences
correspond to an X-linked gene, systematically designated nhr-25. We completed the cDNA sequencing,
con®rming the splicing pattern (Fig. 1A) and the
572-amino acid coding sequence predicted by the
program. The ®rst predicted methionine is
immediately preceded by a stop triplet and is within a
context close to the C. elegans initiation consensus
(Blumenthal & Steward 1997).
Figure 1B shows comparison of the deduced NHR25 sequence with selected representatives from insects
and vertebrates. NHR-25 is most similar to insect
Ftz-F1 from Drosophila and the silkworm Bombyx mori
in both the zinc ®ngers and the Ftz-F1 box. The
vertebrate sequences are products of three distinct
types of genes: the steroidogenic factors (SF-1) and
two groups of liver-speci®c transcription factors, one
activating the a-fetoprotein gene (FTF; Galarneau et
al. 1996), and the other regulating a cytochrome
hydroxylase gene (CPF; Nitta et al. 1999) and hepatitis
B virus (B1F; Li et al. 1998) expression. Interestingly,
GENEFINDER
Figure 1 Organization of the nhr-25 gene
and design of nhr-25::GFP fusions (A)
and sequence comparisons with related
proteins (B). (A) A 9315-bp XbaI-SpeI
genomic fragment encompassing the predicted nhr-25 open reading frame (ATG to
TAA) encoded by exons 1±10 (black
boxes) and a polyadenylation site (polyA)
was used for transformation rescue. The 50
end of exon 1 is left open because the
transcriptional start site is uncertain. Lines
between the exons represent introns. Zinc
®nger (Zn) and Ftz-F1 box (F1) motifs
constituting the DNA-binding domain
(DBD) and the conserved regions II, III
and AF-2 of the ligand binding domain
(LBD) are shown. Arrowheads indicate
nested primer sets used to isolate the
nhr-25(D2389) deletion (hatched frame).
Reporter constructs A, ANLS, BNLS and C
contain > 3 kb of upstream sequence and
the indicated exons fused with GFP. (B)
Percentage residue identities between
NHR-25 and selected insect (Dm, Drosophila melanogaster; Bm, silk moth) and
vertebrate (Dr, zebra®sh; Xl, Xenopus; Ts,
turtle; Gg, chicken; Mm, mouse; Hs,
human) Ftz-F1-related proteins. Numbers
above the NHR-25 protein represent
amino acid positions. Zn, F1, II and III,
as in Fig. 1A; hatched boxes indicate the
AF-2 domain.
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Genes to Cells (2000) 5, 711±723
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M Asahina et al.
FTF and CPF/B1F show a greater similarity to NHR25 in the conserved regions II and III of the ligandbinding domain (LBD) than does SF-1 (Fig. 1B).
Signi®cant conservation (41±47% amino acid similarity) is also found in a 15-amino acid region near
the carboxyl end, corresponding to the activation
domain AF-2 of some steroid receptors (Danielian et al.
1992).
nhr-25 expression patterns
In situ hybridization in wild-type animals detected
maternal mRNA that was evenly distributed in the early
embryo (Fig. 2A±C). By about 120 min after ®rst
cleavage, zygotic mRNA expression began in the
progeny of the E cell, the founder of intestine, then
progressed into the posterior hypodermal precursors
(Fig. 2D±F). Starting from the comma stage, the
mRNA expression localized to the hypodermis and
intestine (Fig. 2G±I), where it continued during early
larval stages (Fig. 2J). The most striking result of the
in situ hybridization was a strong expression in the
developing and adult germ-line (Fig. 3). The mRNA
appeared in the primordial gonads of L2-L3 larvae
(Fig. 3A) and continued to accumulate in the gonadal
loops as they expanded in L4 (Fig. 3B). The adult gonad
was loaded with the transcript (Fig. 3C). In contrast to
the gonad, the mRNA expression declined in the rest of
the body past L2 stage.
nhr-25 expression was further examined via transformation with nhr-25::GFP constructs A, ANLS, and BNLS
(Fig. 1A). Constructs A and ANLS comprised a 3.6-kb
nhr-25 genomic fragment including the complete exons
1 and 2, the ®rst two introns and the ®rst 39 bp of exon
3, fused to GFP. The introns and exons were included to
report any regulatory effect these sequences might have
on nhr-25 expression. Regardless of the presence or
absence of the SV40 nuclear localization sequence
(NLS), ¯uorescence was localized to the nuclei. Construct BNLS contained only the ®rst six amino acids of
NHR-25 (Fig. 1A).
The expression of GFP fusions A, ANLS and BNLS
practically mirrored the pattern that was seen from
in situ hybridization during early embryonic development (Fig. 4). It ®rst appeared in the four descendants
of the E cell and later in derivatives of the ABp and
C blastomeres, i.e. cells assuming hypodermal fate
(Fig. 4A±C). The expression continued in the
hypodermis (but not in the gut) throughout the
remainder of the embryogenesis (Fig. 4D±F). In L1
larvae, the strongest GFP signals were observed in the
seam cells and the pharyngeal and tail hypodermis
(Fig. 4G,H). We also noted expression in the excretory
duct cell.
Constructs A and ANLS were only active in early
larvae (no GFP was detected beyond L2), consistent
with the decline of mRNA in L3 and older worms
(Fig. 3). In contrast, the expression of BNLS persisted in
Figure 2 nhr-25 mRNA expression
detected with in situ hybridization in
wild-type (N2) embryonic and early
larval stages. (A±C) Maternal RNA is
evenly distributed in early dividing
embryos. (D±J) Zygotic transcription
starts in E cell progeny (D), then continues
in the gut and mainly in the posterior
hypodermis of late embryos (G, H),
hatchlings (I) and until L2 larvae (J). In
all images, anterior is to the left. Scale bars
are 10 mm (A±I) and 40 mm (J).
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Genes to Cells (2000) 5, 711±723
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Ftz-F1 role in C. elegans development
Figure 3 In situ hybridization shows nhr25 mRNA in the developing gonads of L3
(A) and L4 (B) larvae. Adult hermaphrodites continue to accumulate the transcript
in the germ-line (C). Anterior is to the left
and ventral side down. Bar ˆ 100 mm.
the hypodermis of later stages (Fig. 4I). None of the
constructs showed expression in the gonad, revealed by
in situ hybridization (Fig. 3), as GFP reporters are
normally silenced in the germ-line.
We ®nally tested construct C, encompassing the
entire nhr-25 coding sequence and including all but the
last intron (Fig. 1A). Unfortunately, transformation
with this plasmid caused an early developmental arrest,
Figure 4 Developmental expression of nhr-25::GFP fusions in N2 background. (A±F) Top panels show Nomarski images
corresponding to the ¯uorescent images below them. Activity of the A (A±D), A NLS (F) and B NLS (E) constructs mirrors zygotic
expression of nhr-25 mRNA (Fig. 2) throughout embryogenesis. D, comma stage; E, 1.5-fold stage; F, 2-fold stage. (G,H) ANLS
expression in hypodermal cells of the pharynx and tail, seam cells and excretory duct (arrows) of L1 larvae. (I) Expression of BNLS in the
hypodermis of a late L3 larva. In all images, anterior is to the left. Scale bars are 10 mm (A±G) and 50 mm (H, I).
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Genes to Cells (2000) 5, 711±723
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M Asahina et al.
such that none of the embryos showing GFP ¯uorescence hatched. This could be due to over-expression of
the functional NHR-25, as suggested by negative effects
of a similar full-length fusion with another nuclear
receptor NHR-82 (Miyabayashi et al. 1999).
Isolation and phenotype of an nhr-25 deletion
mutant
We screened for nhr-25 mutants based on the combined
effects of trimethylpsoralen (TMP) and UV radiation,
followed by the PCR detection of deletions within the
nhr-25 sequence. A single deletion allele of 2389 bp
(D2389) was found after screening 24 000 genomes.
Sequencing of the PCR product showed that the DNA
between nucleotide 213 of intron 1 and nucleotide 187
of intron 7 had been deleted (Fig. 1A). This removed
the coding region from Gly40 in the P-box of the ®rst
zinc ®nger motif to Ser377 in the middle of the conserved region II within the LBD. Exons 1 and 8 are in
the same reading frame, so this deletion might generate
a shortened protein with preserved NHR-25 terminal
structures. This potential mutant protein should lack its
DNA-binding capacity (Ueda et al. 1992), suggesting
that this allele is null.
The effect of the mutation was ®rst seen in
nhr-25(D2389)/‡worms, identi®ed using PCR with
primers Fw3 and Rev4. On average, 24.2 6 3.1%
of eggs laid by 13 heterozygotes in 4 days failed to
hatch (n ˆ 2424), indicating that the nhr-25 mutation
is embryonic lethal. To establish a genetic stock
without introducing an embryonic lethal balancer,
nhr-25(D2389)/‡worms were crossed with males
hemizygous for a ¯r-1 allele (ut11) (Katsura et al.
1994), which is closely linked with nhr-25. Consistent
with the previous result, 28.7 6 5.5% of embryos from
nhr-25(D2389)/¯r-1 hermaphrodites did not hatch,
showing that ¯r-1 did not worsen the nhr-25(D2389)
effect. The remaining progeny consisted of normal
heterozygous and slow growing ¯r-1 progeny. The
presumed nhr-25(D2389) homozygous embryos developed without visible abnormalities until at least 200 min
after ®rst cleavage. They arrested at the 1.5 to 2-fold
stage and their posterior ends were disorganized and
failed to elongate (Fig. 5). Formation of the pharynx
structure was seen and gut granules localized in the
posterior half.
The embryonic lethality was partially rescued with
a plasmid carrying an XbaI-SpeI nhr-25 genomic
fragment (Fig. 1A). This DNA was co-injected into
nhr-25(D2389)/¯r-1 worms with a plasmid carrying
rol-6(su1006) but since no Rol line could be selected,
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Genes to Cells (2000) 5, 711±723
Figure 5 Terminal phenotype of nhr-25(D2389) deletion
mutants. The bottom left panel shows a typical appearance of
approximately 25% embryos produced by nhr-25(D2389)/
‡worms, arresting at 1.5-fold stage, in comparison with wildtype embryos (wt, top left). Right panels show auto¯ourescent
gut granules in the posterior body part. Bar ˆ 30 mm.
the rescue was scored directly in F1. Compared to
untreated mutants with 25% embryonic lethality, only
12.2 6 4.3% embryos from 10 injected parents failed to
hatch (n ˆ 384). The rescued nhr-25(D2389) larvae died
early and so their numbers were dif®cult to score. The
rescue was insuf®cient to produce homozygous nhr25(D2389) adults, as we failed to detect those using
PCR. To con®rm the loss-of-function phenotype of
the mutation in an independent way, we employed
RNA interference (see below).
Effects of RNA interference
The broad expression of nhr-25 detected by in situ
hybridization in post-embryonic stages suggests a role
for the gene in larvae and adults. Since our mutant
screening produced a single embryonic lethal allele, we
performed RNAi to reveal the later effects of the loss of
nhr-25 function. We injected 12 wild-type adults with
double stranded (ds) nhr-25 cRNA and scored the
survival of their offspring over two time intervals
(Table 1). In eggs laid between 14 and 19 h after
injection, an average of 44% embryos failed to hatch.
Their appearance was essentially identical to the terminal phenotype of the nhr-25(D2389) homozygotes
shown in Fig. 5.
Around 25% of the progeny died as L1-L2 larvae.
This lethal phase correlates with the expression of nhr25 mRNA and constructs A and ANLS (Figs 2±4). The
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Ftz-F1 role in C. elegans development
Table 1 Effects of nhr-25 RNA-mediated interference on survival and adult appearance
Terminal phase
dsRNA
Antisense
Frequency of defects seen in adults
Time after
injection
n
Embryo
L1-L2
Adult
Gonadal
defect
Vulvaless
Protruding
vulva
Tail
defect
14±19 h
19±43 h
14±19 h
19±43 h
807
1129
684
1207
44.3 6 17.9
28.6 6 12.5
3.3 6 5.7
0.4 6 0.8
24.5 6 14.8
24.1 6 13.2
4.7 6 5.6
0
31.2 6 15.1
47.3 6 15.4
92.0 6 9.2
99.6 6 0.8
99.2 6 2.6
ND
0
ND
70.7 6 17.4
ND
0
ND
28.5 6 17.6
ND
26.8 6 21.0
ND
72.5 6 20.7
ND
0
ND
Data are mean percent values 6 SD. The `n' values represent total numbers of eggs produced by 12 worms injected with dsRNA
and 12 worms injected with anti-sense RNA in each time interval of egg collection. The same microgram concentrations of ds and
ssRNA were used. Defects in progeny surviving into adulthood are scored in the right section of the table. ND, not determined.
larvae had severe defects in moulting and formation
of the integument (Fig. 6A±C). The integrity of
their surface was disturbed, probably due to incomplete
cuticle synthesis. Old cuticle remained attached to
various body parts. The tail also failed to develop
properly in many larvae (Fig. 6C).
The remaining 31% of the progeny formed mostly
sterile hermaphrodites with combinations of defects,
summarized in Table 1. Virtually all the animals showed
malformations of the gonad, detectable as early as stage
L3 when the gonad begins to grow. Instead of extending
along the body axis, the gonad grew like a tumour,
containing an abnormally large number of small nuclei
(Fig. 7). When present, the gonad arms were often thin,
and in some animals only one arm formed or two arms
extended posteriorly (Fig. 7C). The second most frequent defect was the absence of vulva or formation of
an abnormal protruding vulva (Fig. 7B,E). Finally, 73%
of adults had deformed tails (Fig. 7E).
In summary, the observed defects were reproducible
and in a good agreement with nhr-25 expression and its
timing in the hypodermis and gonad. All of the above
described defects were also observed in the later egg
collection (19±43 h after injection), only with reduced
penetrance (Table 1; not quanti®ed for adult phenotypes). In contrast, injections of equal concentrations of
anti-sense nhr-25 cRNA caused little damage, except
for the formation of protruding vulvae (Table 1).
Dominant-negative effects of nhr-25::GFP
constructs
Figure 6 nhr-25 dsRNA causes moulting defects. Attached
pieces of old cuticle (arrows) and irregularities or holes in the
integument probably cause lethality during L1 and L2 larval
stages. Some animals have deformed posterior ends (A,C).
Anterior is to the left. Bar ˆ 30 mm.
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Dominant-negative effects have been reported for GFP
fusions with several nuclear receptors or their DBDs in
C. elegans (Kostrouchova et al. 1998; Miyabayashi et al.
1999). By truncating the NHR-25 DBD within the
second zinc ®nger in constructs A and ANLS (Fig. 1A),
we aimed to avoid DNA-binding and thus any such
negative action. Nevertheless, both A and ANLS exerted
adverse effects on the development of two transgenic
lines (data not shown). The most frequent defect was
the absence of vulva, a phenotype similar to the effect of
RNAi. However, the vulvaless animals were fertile,
such that progeny hatched inside of the parent (bag-ofworms). We scored this phenotype in GFP-positive
worms that had been selected as L1 larvae (because
Genes to Cells (2000) 5, 711±723
717
M Asahina et al.
Figure 7 RNAi affects gonadal development and adult morphology. Gonads of
RNAi-treated animals undergo tumorous
growth since L3 and L4 (A) and fail to
elongate normally. Panel (C) shows an
adult with abnormally thin gonadal arms
extending in the same orientation. DAPI
staining (D) reveals hyperplasia in the adult
germ-line. Surviving adults often suffer
from no vulva (E) or protruding vulva
(B, arrow). Panel B shows that occasional
oocytes are formed in these animals
(arrowhead). Cuticle secretion is clearly
affected and some animals also display
deformed tails (E). See Table 1 for a
summary of adult phenotypes. Except in
D, anterior is to the left and ventral side
down. Bars ˆ 50 mm in (E) and (C) (also
applies to A, B), and 10 mm (D).
the expression of A constructs is undetectable at later
stages) and grown to adulthood. On average, 65% adults
carrying either transgene were of the bag-of-worms
phenotype (Table 2).
To support the idea that the egg-laying defect was due
to a compromised nhr-25 function, we injected construct A into nhr-25(D2389)/¯r-1 worms. This
enhanced the bag-of-worms phenotype in their F1
heterozygous progeny from 64 to 81% (Table 2). To
determine if this effect was caused by the NHR-25
coding sequence, N2 worms were transformed with the
BNLS construct (Fig. 1A). The egg-laying defects were
again observed, albeit at a lower frequency (Table 2).
Table 2 Effect of nhr-25::GFP constructs on egg laying is
enhanced by nhr-25 de®ciency
Construct
Host genotype
n
Egl adults
A
A
ANLS
BNLS
N2
nhr-25(D2389)/¯r-1
N2
N2
39
21*
41
33
64%
81%
66%
30%
Wild-type (N2) or nhr-25(D2389)/¯r-1 hermaphrodites were
injected with the indicated plasmids shown in Fig. 1(A). Egg
laying defects (Egl), were scored as `bag-of-worms' phenotype
in adults arizing from their GFP-positive progeny. *, only
heterozygous progeny was scored and slow growing ¯r-1
homozygotes were excluded.
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Genes to Cells (2000) 5, 711±723
These effects may be speci®c, as egg laying was not
affected in another transgene mbf1::GFP, strongly
expressed in the hypodermis (unpublished results).
Whether the extrachromosomal nhr-25 constructs act
by silencing the endogenous NHR-25 expression will
be tested when an NHR-25 antibody becomes
available.
Discussion
nhr-25 is essential for embryogenesis
Inactivation of nhr-25 function either by a mutation
deleting the essential regions of nhr-25 or by RNAi
caused embryonic lethality. The introduction of an
nhr-25 plasmid into the (D2389) mutants partially
restored hatching, indicating that the embryonic lethal
phenotype is due to a loss-of-function of nhr-25.
Incomplete rescue may be due to the detrimental
effects of excessive copies of a functional gene or to
the mosaic partition of the extrachromosomal array
during development. nhr-25 is expressed in embryonic
epithelial cells. Although early descendants of the E
blastomere are the ®rst cells to express nhr-25, this
expression seems to be transient and is soon followed
by lasting expression, mainly in the progeny of ABp
and C cells, forming the hyp7 syncytium, seam and
other hypodermal cells (Sulston et al. 1983). Consistent with the nhr-25 expression in hypodermal cells,
both the nhr-25(D2389) mutant and RNAi affected
embryos failed to elongate, arresting at the 1.5-fold
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Ftz-F1 role in C. elegans development
stage. These embryos were not properly enclosed,
showing extruding tissues, particularly at their posterior. This suggests that nhr-25 is required for the
secretion of cuticle by the hypodermis.
In situ hybridization shows strong expression in the
germ-line and maternal deposition of the mRNA.
Although dsRNA is known to interfere ef®ciently with
maternal genes (Fire et al. 1998; Montgomery et al.
1998), we have not observed defects earlier than those
seen in nhr-25(D2389) mutant embryos using RNAi.
We assume that either suf®cient NHR-25 protein is also
deposited into the eggs or that the maternal RNA is not
essential.
Contrasting with nhr-25 is Drosophila Ftz-F1, expressed
in perhaps all organs except for the gonads (H. Ueda,
personal communication). Consistently, ftz-f1 mutant
phenotypes do not suggest defects in reproduction
(Broadus et al. 1999).
In the case of vulval development, nhr-25 expression
in hypodermal vulva precursor cells may be required for
their competence to form vulva. In this sense, bFtz-F1
has been shown to render Drosophila tissues competent
to undergo changes in response to the ecdysteroid signal
(Broadus et al. 1999). The posterior end defects possibly
result from faulty expression of cuticle constituents,
such as the sqt-1 and rol-6 collagen genes (Kramer et al.
1988, 1990; Park & Kramer 1994).
Roles of nhr-25 in post-embryonic
development and reproduction
NHR-25 as a core regulator of moulting
Post-embryonic expression of nhr-25 suggests that this
gene plays a role in larvae and adults. RNAi caused
lethality in L1-L2 but not in older larvae, corresponding
with nhr-25 expression and its decline in the hypodermis past L2 stage. Defects in cuticle replacement
(moulting) and morphogenesis, consistent with the
spatial expression, again point to an nhr-25 requirement
in the hypodermis. Lesions and irregularities of the
integument showed that not only the shedding of the
old cuticle, but also synthesis of a new one were
compromised.
Those larvae that reached L3-L4 instars usually
formed sterile adults, invariably with tumorous gonad
and often with a missing or abnormal vulva and
deformed tail. Since we lack any genetic data about
involvement of nhr-25 in pathways controlling gonad
and vulva differentiation, it is dif®cult at this point to
discuss how nhr-25 participates in these events. The
hyperplasia of the RNAi-affected gonad shows that
excessive mitoses occur in the germ-line. Similar gonad
appearance results from loss of cul-1, a cullin gene
necessary for cell cycle exit in C. elegans (Kipreos et al.
1996). Unlike in cul-1, however, we have not found
extra neurones in RNAi treated worms carrying the
mec-7::GFP marker (Hamelin et al. 1992), suggesting
that nhr-25 is not a general regulator of the cell cycle.
Similar defects of gonadal differentiation also result from
mutations in the gon-1 metalloprotease gene, implicated
in the shaping of extracellular matrix (Blelloch &
Kimble 1999). Gonad hyperplasia is also caused by a
constitutive activation of the Notch relative GLP-1
(Berry et al. 1997), required for signalling between the
distal tip cell and the germ-line. We speculate that
nhr-25, which is expressed strongly in the germ-line,
might be a downstream component of such a pathway.
Both arthropods and nematodes need steroids for
moulting. In insects, precursors such as cholesterol are
converted into ecdysteroids and used as extracellular
signals to synchronize the moulting of distant body
parts. In nematodes, the absence of dietary sterols compromises moulting (Coggins et al. 1985). More directly,
both sterol starvation and mutations in a megalinrelated protein LRP-1, which presumably mediates
sterol endocytosis by hyp7 hypodermis, have been
shown to prevent the shedding and degradation of the
old cuticle at all C. elegans moults (Yochem et al. 1999).
Whether ecdysteroids are required for nematode
moulting is not clear, as their synthesis from cholesterol
has not been demonstrated (Chitwood 1992).
In the epidermis of insects, moulting is regulated by
ecdysteroids acting through an ecdysone receptor complex EcR/USP and a cascade of transcription factors,
including bFtz-F1, on expression of stage-speci®c genes
(Riddiford et al. 1999). bFtz-F1 is induced by an
increase and a consequent decline of ecdysteroid titre
(Sun et al. 1994) and is likely to control genes acting in
cuticle formation (Hiruma et al. 1995). In Drosophila,
bFtz-F1 is necessary for metamorphosis (Woodard et al.
1994; Broadus et al. 1999) and for activation of at least
one pupal cuticle gene EDG84A (Murata et al. 1996).
The importance of bFtz-F1 for the larval moult is
suggested by a rescue of ftz-f1 mutants with heat shock
induction of bFtz-F1 around the moulting period
(H. Ueda, personal communication). Expression of
Drosophila bFtz-F1 is activated by another nuclear
receptor, DHR3 (Kageyama et al. 1997; Lam et al.
1997).
How can NHR-25 act in the moulting of C. elegans?
Since NHR-25 and NHR-23 are conserved with
Ftz-F1 and DHR3, it is possible that they are part of a
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Genes to Cells (2000) 5, 711±723
719
M Asahina et al.
pathway analogous to that which exists in insects.
Signi®cantly, RNAi targeting of nhr-23 caused moulting
defects (Kostrouchova et al. 1998) that were similar to
those shown here for nhr-25. We are now preparing an
antibody to test whether the genetic epistasis between
nhr-23 and nhr-25 is the same as in Drosophila. However,
the upstream part of this pathway is unclear, because C.
elegans lacks orthologs of EcR and USP (Sluder et al.
1999), which are thus far the only known receptors
capable of conveying the ecdysteroid signal to gene
expression. There is an alternative possibility that
instead of being downstream of a steroid receptor,
NHR-25 could mediate the conversion of dietary
sterols to compounds active in moulting. Encouraging
this idea is that Ftz-F1 type proteins direct steroid
conversion in mammals (reviewed by Parker &
Schimmer 1997).
Our analysis of NHR-25 function suggests that the
roles of ftz-f1 orthologs in Drosophila and C. elegans
moulting are parallel (Summary ®gure) and supports the
monophyletic origin of moulting (Aguinaldo et al.
1997). We propose that Ftz-F1/NHR-25 and DHR3/
NHR-23 are the central, evolutionarily oldest regulators of genes executing cuticle replacement in all
Ecdysozoa. While these orphan receptors are suf®cient
to control the moulting program in small, simple forms
such as C. elegans, the synchronized moulting of distant
and diversi®ed body parts in arthropods demands
coordination by an extracellular signal (a moulting
hormone) and its receptor.
Experimental procedures
Nematode strains and culture
C. elegans var. Bristol N2 (Brenner 1974) was the wild-type strain
used in this study. Strain MT1401: ‡/szT1[lon-2(e678)] I;
nDf19/szT1 X (Ambros & Horvitz 1984), obtained from the
Caenorhabditis Genetics Center (Minnesota, USA) and strain ¯r1(ut11) (Katsura et al. 1994) were used for balancing of our nhr-25
mutant. Except during the mutant screening, worms were grown
at 20 8C on the rich nematode growth medium (RNGM) agarose
plates as described (Brenner 1974).
DNA clones and sequence analysis
cDNA clones yk342d8, yk175f2 and yk663g4 encoding NHR25 originated from the Y. Kohara EST project (National Institute
of Genetics, Japan). The genomic cosmid clone F11C1 was
obtained from Alan Coulson at the Sanger Center (Cambridge,
UK).
The cDNA clones yk342d8 and yk175f2 in pBluescript IISK were sequenced from the vector T3 and T7 primers and
720
Genes to Cells (2000) 5, 711±723
with several internal primers to verify their entire sequence.
Sequencing reactions with the Perkin Elmer dye terminator
system were resolved on an ABI 373 automatic sequencer.
Deduced protein sequences were compared with the DDBJ/
GENBANK/EMBL databases using BLAST algorithms.
PCR and primers
All PCR reactions were performed on a Takara temperature
cycler with the ExTaq (Takara) thermostable DNA polymerase,
possessing the proofreading ability. The nhr-25 speci®c primers
(forward and reverse) used in this study are listed below.
Fw1:
50 -ATTGCCATACTCACACGTTTG
Fw2:
50 -TTAGTTGACCCACAAGACAG
Fw3:
50 -ATCGACGTTCACTATTTCAGG
Fw4:
50 -CAGCTTATCAGTTGAATGAAG
Rev1:
50 -ACAAGTGAGGTGTGTTGATTG
Rev2:
50 -ATTGCCAAGAAGAGCTACAAG
Rev3:
50 -TCCGTTTTGCAGAAGAGAACAG
Rev4:
50 -GCTCGTTTTCACAACAATTACG
Rev5:
50 -GTAGGATCCACGTGGCAGTTGG
Rev6:
50 -CAGAAGGATCCTCTCGACGTCAG
Plasmid vectors and transformation of
C. elegans
A 9315-bp XbaI-SpeI fragment of the F11C1 cosmid encompasses the nhr-25 sequence from nucleotide 3043 upstream of
the ®rst methionine to nucleotide 1198 downstream of the
polyadenylation site (Fig. 1A). This fragment was subcloned into
pBluescript II-KS and used for the construction of nhr-25::GFP
reporter fusions as follows. Constructs A and ANLS consisted of a
PCR product of the T7 promoter primer in pBluescript and an
anti-sense primer Rev5 within nhr-25 exon 3 (Fig. 1A), subcloned into the XbaI and BamHI sites of vectors pPD.95.75 and
pPD95.67, respectively. Construct BNLS was analogically prepared in pPD.95.70 using primer Rev6, placed at the start of the
nhr-25 open reading frame (ORF). Construct C included the
entire nhr-25 ORF and consisted of the genomic sequence
between the XbaI and XhoI sites, spliced with the cDNA of exons
9 and 10, and fused to GFP in vector pPD.95.79 (Fig. 1A). All of
the reporter fusions were veri®ed by restriction mapping and by
the sequencing of regions containing PCR products.
For transformation (Mello & Fire 1995), adult hermaphrodites
were microinjected in both gonad arms with 100 ng/mL of
plasmid DNA. Transformants were selected under a Leica
¯uorescent stereomicroscope for many generations, but the
constructs were not chromosomally integrated. The GFP
expression patterns were observed using a Zeiss ¯uorescent
microscope.
In situ hybridization
In situ hybridization was performed on wild-type (N2) animals
with a digoxigenin labelled yk663g4 cDNA probe as described
(http://watson.genes.nig.ac.jp/db/method/index.html).
q Blackwell Science Limited
Ftz-F1 role in C. elegans development
Mutagenesis and mutant screening
An nhr-25 deletion mutant was isolated in a PCR-based
screening after mutagenesis with trimethylpsoralen (TMP) and
UV radiation (Jansen et al. 1997). Animals were grown at 15 8C
throughout the screening. Wild-type L4 larvae were immersed
for 30 min in 15 mg/mL TMP (Sigma), washed and then
irradiated in a UV crosslinker (Stratagene) under 361.5 nm for
1 h. Treated worms were fed on agarose plates seeded with E. coli
OP50 for 24 h, then their eggs were recovered by basic hypochlorite treatment and distributed on fresh plates. Grown F1
larvae were divided into about 200 per 6-cm plate. On a total of
120 plates, worms were allowed to reproduce for 8 days, then
samples of each plate's population were harvested for DNA
preparation and the rest were kept at 15 8C. DNA was extracted
by dissolving the worms overnight at 65 8C in 10 mM Tris-HCl
(pH 8.0), 50 mM NaCl, 2.5 mM MgCl2, 0.45% Tween-20, 0.05%
gelatin and 200 mg/mL Proteinase K. After inactivation of
Proteinase K (95 8C, 15 min), aliquots of the extracts were pooled
in tens, and then 2 mL of each pool were used in 25-mL PCR
reactions with sets of nhr-25 speci®c primers. These reactions
were then subjected to second-round PCR with appropriate
nested primers (Fig. 1A).
Primers Fw3 and Rev4 ampli®ed a short-size band in a single
pool and in one of the unpooled DNA preparations, identifying
the plate with nhr-25 mutant worms. The population on this
plate was expanded and subjected to two additional rounds of sib
selection until the nhr-25 deletion was narrowed down to the
progeny of a single worm. Individuals in this progeny were then
backcrossed and mated with males of two strains, MT1401
(balancer szT1(X)) and ¯r-1(ut11), and stocks were established.
The mutant allele obtained is a 2389-bp deletion within nhr-25
and is therefore provisionally referred to as D2389.
Transformation rescue
The 9315 bp XbaI-SpeI segment of the F11C1 cosmid (Fig. 1A)
in pBluescript served to supply nhr-25‡ function. nhr25(D2389)/¯r-1(ut11) or control adults were injected in both
gonad arms with the rescue construct DNA at concentrations of
25, 50 or 100 ng/mL, mixed with 75, 50 and 10 ng/mL,
respectively, of plasmid pRF4, carrying the dominant marker
rol-6(su1006) (Kramer et al. 1990). After 12 h, individual worms
were allowed to lay eggs on fresh plates and dead embryos and
hatched larvae were counted. The rescue was scored directly in
the progeny of the injected worms, because the Rol phenotype
was not visible and no transformed line could be isolated.
RNA interference
RNA-mediated interference with anti-sense and doublestranded (ds) RNA was employed to disrupt nhr-25 function
in vivo (Fire et al. 1998). The complementary RNA strands
were synthesized from the yk175f2 cDNA in separate reactions
with T3 and T7 RNA polymerases, respectively. After DNase
treatment, the RNA was phenol/chloroform extracted and
q Blackwell Science Limited
precipitated with ethanol. For dsRNAi, 5 mg of each RNA
strand were combined in 20 mL of injection buffer (Mello &
Fire 1995) and annealed by heating at 68 8C for 10 min and at
37 8C for 30 min. After checking its double-strandedness on a
nondenaturing agarose gel, the dsRNA was injected into the
gonads of adult hermaphrodites. Antisense RNA was used at the
same concentration (10 mg in 20 mL injection buffer).
Acknowledgements
We thank Alan Coulson for providing the cosmid F11C1, J. M.
Kramer for the pRF4 plasmid and Andrew Fire lab for GFP
vectors. Sharing of unpublished data by Hitoshi Ueda, critical
reading of the manuscript by David Champlin, and technical
assistance of T. Motohashi and Y. Takada are appreciated. We
thank the Caenorhabditis Genetics Center (funded by the NIH
National Center for Research Resources) for C. elegans strains.
This work was supported by Grants-in-Aid for Scienti®c
Research from the Ministry of Education, Science, Sports and
Culture of Japan (to S.H., I.K. and Y.K.) and a grant 204/00/
0811 from the Grant Agency of the Czech Republic (to M.J.).
M.J. was supported by a Center of Excellence Program of Japan.
Note added in proof
After submission of the manuscript, the following article on nhr25 has been reported: Gissendanner, C.R. & Sluder A.E. (2000)
nhr-25, the Caenorhabditis elegans ortholog of ftz-f1, is required for
epidermal and somatic gonad development. Dev. Biol., 221,
259±272.
References
Aguinaldo, A.M., Turbeville, J.M., Linford, L.S., et al. (1997)
Evidence for a clade of nematodes, arthropods and other
moulting animals. Nature 387, 489±493.
Ambros, V. & Horvitz, H.R. (1984) Heterochronic mutants of
the nematode Caenorhabditis elegans. Science 226, 409±416.
Antebi, A., Culotti, J.G. & Hedgecock, E.M. (1998) daf-12
regulates developmental age and the dauer alternative in
Caenorhabditis elegans. Development 125, 1191±1205.
Bender, M., Imam, F.B., Talbot, W.S., Ganetzky, B. & Hogness,
D.S. (1997) Drosophila ecdysone receptor mutations reveal
functional differences among receptor isoforms. Cell 91,
777±788.
Berry, L.W., Westlund, B. & Schedl, T. (1997) Germ-line tumor
formation caused by activation of glp-1, a Caenorhabditis elegans
member of the Notch family of receptors. Development 124,
925±936.
Blelloch, R. & Kimble, J. (1999) Control of organ shape by a
secreted metalloprotease in the nematode Caenorhabditis
elegans. Nature 399, 586±590.
Blumenthal, T. & Steward, K. (1997) RNA processing and gene
structure. In: C. Elegans II (eds D.L. Riddle, T. Blumenthal,
B.J. Meyer & J.R. Priess), pp. 117±145. Plainview, NY: Cold
Spring Harbor Laboratory Press.
Brenner, S. (1974) The genetics of C. elegans. Genetics 77,
71±94.
Genes to Cells (2000) 5, 711±723
721
M Asahina et al.
Broadus, J., McCabe, J.R., Endrizzi, B., Thummel, C.S. &
Woodard, C.T. (1999) The Drosophila bFTZ-F1 orphan
nuclear receptor provides competence for stage-speci®c
responses to the steroid hormone ecdysone. Mol. Cell 3,
143±149.
Carmi, I., Kopczynski, J.B. & Meyer, B.J. (1998) The nuclear
hormone receptor SEX-1 is an X-chromosome signal that
determines nematode sex. Nature 396, 168±173.
Carney, G.E., Wade, A.A., Sapra, R., Goldstein, E.S. & Bender,
M. (1997) DHR3, an ecdysone-inducible early-late gene
encoding a Drosophila nuclear receptor, is required for
embryogenesis. Proc. Natl. Acad. Sci. USA 94, 12024±12029.
Chitwood, D.J. (1992) Nematode sterol biochemistry. In:
Physiology and Biochemistry of Sterols (eds G.W. Patterson &
W.D. Nes), pp. 257±293. Champaign, IL: American Oil
Chemists' Society.
Coggins, J.R., Schaefer,F.W. III & Weinstein, P.P. (1985)
Ultrastructural analysis of pathologic lesions in sterol-de®cient
Nippostrongylus brasiliensis larvae. J. Invertebr. Pathol. 45,
288±297.
Danielian, P.S., White, R., Lees, J.A. & Parker, M.G. (1992)
Identi®cation of a conserved region required for hormone
dependent transcriptional activation by steroid hormone
receptors. EMBO J. 11, 1025±1033.
Escriva, H., Sa®, R., Hanni, C., et al. (1997) Ligand binding was
acquired during evolution of nuclear receptors. Proc. Natl.
Acad. Sci. USA 94, 6803±6808.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. &
Mello, C.C. (1998) Potent and speci®c genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391,
806±811.
Galarneau, L., Pare, J.F., Allard, D., et al. (1996) The alpha1fetoprotein locus is activated by a nuclear receptor of the
Drosophila FTZ-F1 family. Mol. Cell. Biol. 16, 3853±3865.
Guichet, A., Copeland, J.W.R., Erdely, M., et al. (1997) The
nuclear receptor homologue Ftz-F1 and the homeodomain
protein Ftz are mutually dependent cofactors. Nature 385,
548±552.
Hall, B.L. & Thummel, C.S. (1998) The RXR homolog
ultraspiracle is an essential component of the Drosophila
ecdysone receptor. Development 125, 4709±4717.
Hamelin, M., Scott, I.M., Way, J.C. & Culotti, J.G. (1992) The
mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed
primarily in the touch receptor neurons. EMBO J. 11,
2885±2893.
Hiruma, K., Carter, M.S. & Riddiford, L.M. (1995) Characterization of the dopa decarboxylase gene of Manduca sexta and
its suppression by 20-hydroxyecdysone. Dev. Biol. 169,
195±209.
Ikeda, Y., Luo, X., Abbud, R., Nilson, J.H. & Parker, K.L.
(1995) The nuclear receptor steroidogenic factor 1 is essential
for the formation of the ventromedial hypothalamic nucleus.
Mol. Endocrinol. 9, 478±486.
Jansen, G., Hazendonk, E., Thijssen, K.L. & Plasterk, R.H.
(1997) Reverse genetics by chemical mutagenesis in Caenorhabditis elegans. Nature Genet. 17, 119±121.
Kageyama, Y., Masuda, S., Hirose, S. & Ueda, H. (1997)
Temporal regulation of the mid-prepupal gene FTZ-F1:
DHR3 early late gene product is one of the plural positive
regulators. Genes Cells 2, 559±569.
Katsura, I., Kondo, K., Amano, T., Ishihara, T. &
Kawakami, M. (1994) Isolation, characterization and epistasis
722
Genes to Cells (2000) 5, 711±723
of ¯uoride-resistant mutants of Caenorhabditis elegans. Genetics
136, 145±154.
Kipreos, E.T., Lander, L.E., Wing, J.P., He, W.W. & Hedgecock,
E.M. (1996) cul-1 is required for cell cycle exit in C. elegans and
identi®es a novel gene family. Cell 85, 829±839.
Koelle, M.R., Segraves, W.A. & Hogness, D.S. (1992) DHR3: a
Drosophila steroid receptor homolog. Proc. Natl. Acad. Sci.
USA 89, 6167±6171.
Kostrouchova, M., Krause, M., Kostrouch, Z. & Rall, J.E. (1998)
CHR3: a Caenorhabditis elegans orphan nuclear hormone
receptor required for proper epidermal development and
molting. Development 125, 1617±1626.
Kozlova, T., Pokholkova, G.V., Tzertzinis, G., Sutherland, J.D.,
Zhimulev, I.F. & Kafatos, F.C. (1998) Drosophila hormone
receptor 38 functions in metamorphosis: a role in adult cuticle
formation. Genetics 149, 1465±1475.
Kramer, J.M., French, R.P., Park, E.C. & Johnson, J.J. (1990)
The Caenorhabditis elegans rol-6 gene, which interacts
with the sqt-1 collagen gene to determine organismal
morphology, encodes a collagen. Mol. Cell. Biol. 10,
2081±2089.
Kramer, J.M., Johnson, J.J., Edgar, R.S., Basch, C. & Roberts, S.
(1988) The sqt-1 gene of C. elegans encodes a collagen critical
for organismal morphogenesis. Cell 55, 555±565.
Lam, G.T., Jiang, C. & Thummel, C.S. (1997) Coordination of
larval and prepupal gene expression by the DHR3 orphan
receptor during Drosophila metamorphosis. Development 124,
1757±1769.
Laudet, V. (1997) Evolution of the nuclear receptor superfamily:
early diversi®cation from an ancestral orphan receptor. J. Mol.
Endocrinol. 19, 207±226.
Lavorgna, G., Ueda, H., Clos, J. & Wu, C. (1991) FTZ-F1, a
steroid hormone receptor-like protein implicated in the
activation of fushi tarazu. Science 252, 848±851.
Li, M., Xie, Y.H., Kong, Y.Y., Wu, X., Zhu, L. & Wang, Y.
(1998) Cloning and characterization of a novel human
hepatocyte transcription factor, hB1F, which binds and
activates enhancer II of hepatitis B virus. J. Biol. Chem. 273,
29022±29031.
Luo, X., Ikeda, Y. & Parker, K.L. (1994) A cell-speci®c nuclear
receptor is essential for adrenal and gonadal development and
sexual differentiation. Cell 77, 481±490.
Mangelsdorf, D.J., Thummel, C.S., Beato, M., et al. (1995) The
nuclear receptor superfamily: The second decade. Cell 83,
835±839.
Mello, C. & Fire, A. (1995) DNA transformation. In:
Caenorhabditis Elegans: Modern Biological Analysis of an Organism
(eds D.F. Epstein & D.C. Shakes), pp. 451±482. San Diego:
Academic Press.
Miyabayashi, T., Palfreyman, M.T., Sluder, A.E., Slack, F. &
Sengupta, P. (1999) Expression and function of members of a
divergent nuclear receptor family in Caenorhabditis elegans. Dev.
Biol. 215, 314±331.
Montgomery, M.K., Xu, S. & Fire, A. (1998) RNA as a target of
dsRNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 95, 15502±15507.
Murata, T., Kageyama, Y., Hirose, S. & Ueda, H. (1996)
Regulation of the EDG84A gene by FTZ-F1 during
metamorphosis in Drosophila melanogaster. Mol. Cell. Biol. 16,
6509±6515.
Nitta, M., Ku, S., Brown, C., Okamoto, A.Y. & Shan, B. (1999)
CPF: an orphan nuclear receptor that regulates liver-speci®c
q Blackwell Science Limited
Ftz-F1 role in C. elegans development
expression of the human cholesterol 7alpha-hydroxylase gene.
Proc. Natl. Acad. Sci. USA 96, 6660±6665.
Palli, S.R., Hiruma, K. & Riddiford, L.M. (1992) An
ecdysteroid-inducible Manduca gene similar to the Drosophila
DHR3 gene, a member of the steroid hormone receptor
superfamily. Dev. Biol. 150, 306±318.
Park, Y.S. & Kramer, J.M. (1994) The C. elegans sqt-1 and rol-6
collagen genes are coordinately expressed during development, but not at all stages that display mutant phenotypes. Dev.
Biol. 163, 112±124.
Parker, K.L. & Schimmer, B.P. (1997) Steroidogenic factor 1: a
key determinant of endocrine development and function.
Endocr. Rev. 18, 361±377.
Riddiford, L.M., Hiruma, K., Lan, Q. & Zhou, B. (1999)
Regulation and role of nuclear receptors during larval molting
and metamorphosis of Lepidoptera. Amer. Zool. 39, 736±746.
Ruvkun, G. & Hobert, O. (1998) The taxonomy of developmental control in Caenorhabditis elegans. Science 282,
2033±2041.
Sadovsky, Y., Crawford, P.A., Woodson, K.G., et al. (1995) Mice
de®cient in the orphan receptor steroidogenic factor 1 lack
adrenal glands and gonads but express P450 side-chaincleavage enzyme in the placenta and have normal embryonic
serum levels of corticosteroids. Proc. Natl. Acad. Sci. USA 92,
10939±10943.
Schubiger, M., Wade, A.A., Carney, G.E., Truman, J.W. &
Bender, M. (1998) Drosophila EcR-B ecdysone receptor
isoforms are required for larval molting and for neuron
remodeling during metamorphosis. Development 125,
2053±2062.
Sengupta, P., Colbert, H.A. & Bargmann, C.I. (1994) The
C. elegans gene odr-7 encodes an olfactory-speci®c member of
the nuclear receptor superfamily. Cell 79, 971±980.
Sluder, A.E., Lindblom, T. & Ruvkun, G. (1997) The
Caenorhabditis elegans orphan nuclear hormone receptor gene
nhr-2 functions in early embryonic development. Dev. Biol.
184, 303±319.
Sluder, A.E., Mathews, S.W., Hough, D., Yin, V.P. & Maina,
C.V. (1999) The nuclear receptor superfamily has undergone
extensive proliferation and diversi®cation in nematodes.
Genome Res. 9, 103±120.
Sulston, J.E., Schierenberg, E., White, J.G. & Thomson, J.N.
(1983) The embryonic cell lineage of the nematode
Caenorhabditis elegans. Dev. Biol. 100, 64±119.
q Blackwell Science Limited
Sun, G.-C., Hirose, S. & Ueda, H. (1994) Intermittent expression of BmFTZ-F1, a member of the nuclear hormone
receptor superfamily, during development of the silkworm
Bombyx mori. Dev. Biol. 162, 426±437.
Thomas, H.E., Stunnenberg, H.G. & Stewart, A.F. (1993)
Heterodimerization of the Drosophila ecdysone receptor with
retinoid X receptor and ultraspiracle. Nature 362, 471±475.
Ueda, H., Sun, G.-C., Murata, T. & Hirose, S. (1992) A novel
DNA-binding motif abuts the zinc ®nger domain of insect
nuclear hormone receptor FTZ-F1 and mouse embryonal
long terminal repeat-binding protein. Mol. Cell. Biol. 12,
5667±5672.
Wakimoto, B.T., Turner, F.R. & Kaufman, T.C. (1984) Defects
in embryogenesis in mutants associated with the Antennapedia
gene complex of Drosophila melanogaster. Dev. Biol. 102,
147±172.
Wightman, B., Baran, R. & Garriga, G. (1997) Genes that guide
growth cones along the C. elegans ventral nerve cord.
Development 124, 2571±2580.
Woodard, C.T., Baehrecke, E.H. & Thummel, C.S. (1994) A
molecular mechanism for the stage speci®city of the Drosophila
prepupal genetic response to ecdysone. Cell 79, 607±615.
Yao, T.-P., Forman, B.M., Jiang, Z., et al. (1993) Functional
ecdysone receptor is the product of EcR and ultraspiracle genes.
Nature 366, 476±479.
Yao, T.-P., Segraves, W.A., Oro, A.E., McKeown, M. & Evans,
R.M. (1992) Drosophila ultraspiracle modulates ecdysone
receptor function via heterodimer formation. Cell 71, 63±72.
Yochem, J., Tuck, S., Greenwald, I. & Han, M. (1999) A gp330/
megalin-related protein is required in the major epidermis of
Caenorhabditis elegans for completion of molting. Development
126, 597±606.
Yu, Y., Li, W., Su, K., et al. (1997) The nuclear hormone receptor
Ftz-F1 is a cofactor for the Drosophila homeodomain protein
Ftz. Nature 385, 552±555.
Zhou, H.M. & Walthall, W.W. (1998) UNC-55, an orphan
nuclear hormone receptor, orchestrates synaptic speci®city
among two classes of motor neurons in Caenorhabditis elegans.
J. Neurosci. 18, 10438±10444.
Received: 1 May 2000
Accepted: 30 May 2000
Genes to Cells (2000) 5, 711±723
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