Development 121, 601-612 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
601
The biogenesis of the coiled body during early mouse development
João Ferreira and Maria Carmo-Fonseca*
Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal
*Author for correspondence
SUMMARY
The coiled body is an ubiquitous nuclear organelle that
contains essential components of the pre-mRNA splicing
machinery as well as the nucleolar protein fibrillarin. Here
we have studied the biogenesis of the coiled body in early
mouse embryos. The results show that coiled bodies form
and concentrate splicing snRNPs as early as in the
maternal and paternal pronuclei of 1-cell embryos. This
argues that the coiled body is likely to play a basic role in
the nucleus of mammalian cells. In order to correlate the
appearance of coiled bodies with the onset of transcriptional activity, embryos were incubated with brominated
UTP and the incorporated nucleotide was visualized by fluorescence microscopy. In agreement with previous studies,
transcriptional activity was first observed during the 2-cell
stage. Thus, coiled bodies form before activation of
embryonic gene expression.
The appearance of coiled bodies in 1-cell embryos was
preceded by the formation of morphologically distinct
structures that also contain coilin and which we therefore
refer to as pre-coiled bodies. At the electron microscopic
level pre-coiled bodies have a compact fibrillar structure,
whereas coiled bodies resemble a tangle of coiled threads.
Although both pre-coiled bodies and coiled bodies contain
the nucleolar protein fibrillarin, the assembly of coiled
bodies is separated both in time and in space from
ribosome synthesis. Our results suggest that the embryonic
‘nucleolus-like body’ is a structural scaffold that nucleates
independently the formation of the coiled body and the
assembly of the machinery responsible for ribosome
biosynthesis.
INTRODUCTION
and coiled bodies have been highly conserved in evolution and
therefore are expected to have an important function in the
nucleus.
Immunofluorescence and immunoblot analysis using antibodies directed against coilin show that coiled bodies are
dynamic structures that are induced to assemble or disassemble under different metabolic conditions (see Lamond and
Carmo-Fonseca, 1993a). In particular, the number of coiled
bodies per nucleus increases when the cells are stimulated to
grow rapidly or when high levels of gene expression are
induced. In situ localization data show that coiled bodies concentrate both splicing snRNPs (Carmo-Fonseca et al., 1992
Matera and Ward, 1993) and the protein splicing factor U2AF
(Zhang et al., 1992), thus suggesting an involvement in premRNA processing. However, as splicing appears to be cotranscriptional (see Rosbash and Singer, 1993) and the sites of transcription by RNA polymerase II are widely spread throughout
the nucleoplasm (Jackson et al., 1993; Wansink et al., 1993),
it is unlikely that coiled bodies correspond to major sites of
splicing. It is also unlikely that coiled bodies represent a
storage structure for excess or inactive snRNPs, since snRNPs
no longer concentrate in coiled bodies under conditions that
inhibit or decrease gene expression, when the level of inactive
snRNP increases. One possibility is that coiled bodies are
involved in the expression of a specific subset of genes and this
would be consistent with the observation that most cells
The coiled body is an intranuclear domain that has recently
attracted attention following the discovery that it contains high
concentrations of small nuclear ribonucleoprotein particles
(snRNPs) involved in splicing of pre-messenger RNAs
(reviewed by Lamond and Carmo-Fonseca, 1993a). The name
‘coiled body’ was proposed by Monneron and Bernhard in
1969 to describe a distinct nucleoplasmic structure that
resembles a tangle of coiled threads when visualized with the
electron microscope (Monneron and Bernhard, 1969). At the
ultrastructural level coiled bodies have been observed in a
variety of tissue and cultured cells, suggesting that they are
ubiquitous structures within the nucleus of higher eukaryotes.
A major advance in the study of this nuclear organelle was the
identification of autoimmune patient sera that specifically
labeled the coiled body and recognized a novel protein of
approximately 80×103 Mr called p80 coilin (Andrade et al.,
1991). The complete coding sequence of coilin has been
obtained (Carmo-Fonseca et al., 1994) but no consensus motif
was identified that gives a clue to the function of the protein.
Another coilin-related protein named SPH-1 has been recently
identified in Xenopus laevis (Tuma et al., 1993). Overall the
SPH-1 protein is 38.3% identical to coilin and it is present in
a specific nuclear structure of amphibian oocytes that may be
related to mammalian coiled bodies. This indicates that coilin
Key words: mouse development, coiled body, biogenesis, nuclear
structure, nucleolus, snRNPs, hnRNPs
602
J. Ferreira and M. Carmo-Fonseca
contain very few coiled bodies per nucleus (frequently 2 to 4).
Alternatively, coiled bodies may have a more general role in
some form of either pre- or post-splicing events, such as prespliceosomal snRNP assembly, post-spliceosomal snRNP
recycling or intron degradation (for discussion see Lamond and
Carmo-Fonseca, 1993a,b).
A striking characteristic of the coiled body is that it is
sometimes seen in close association with the periphery of the
nucleolus. In fact, coiled bodies were first identified by Ramony-Cajal in 1903, as ‘nucleolar accessory bodies’ in neuronal
cells stained with silver. Based on this association, it was
widely predicted that coiled bodies may have a role in
ribosome biogenesis (see Brasch and Ochs, 1992). This
hypothesis was strengthened by the observation that both
nucleoli and coiled bodies contain fibrillarin (Raska et al.,
1990), a protein that is known to bind several small nucleolar
RNAs required for the processing of pre-rRNA. However,
further studies have failed to detect either rRNA or ribosomal
proteins in the coiled body (Raska et al., 1990; Carmo-Fonseca
et al., 1993), arguing against a direct involvement of this
structure in rRNA synthesis or maturation. Alternatively, it has
been proposed that coiled bodies may function in the processing of small nucleolar RNAs that are transcribed from introns
of genes that code for ribosome proteins and other cellular
proteins (Tycowski et al., 1993; Lamond and Carmo-Fonseca,
1993a). At present several small nucleolar RNAs
(UsnoRNAs), including the U14, U15, U16 and U17snoRNAs
are known to be encoded within introns of pre-mRNAs
(reviewed by Filipowicz and Kiss, 1993). Thus, it is attractive
to postulate that snRNPs in coiled bodies could be involved in
the specific processing of these pre-mRNAs and targeting of
the excised introns to the nucleolus.
Apart from the spatial proximity and the presence of fibrillarin, very little is known about possible interactions between
the nucleolus and the coiled body. Do coiled bodies arise from
nucleoli by a budding mechanism, or do they form in the nucleoplasm and then fuse with the nucleolus? What drives the
assembly of a coiled body? Here we have sought to address
these questions by studying the stepwise assembly of the coiled
body during early mouse development.
One important characteristic of mouse embryogenesis that
favors this kind of study is that early development is quite
slow. By 10-12 hours after fertilization nuclear membranes
form around the maternal and paternal chromosomes giving
rise to the female and male haploid pronuclei and by 24-48
hours post-fertilization the embryo is still at the 2-cell stage.
This is much slower than the development of embryos from
other species such as sea urchin, Drosophila or Xenopus, that
by 24 hours after fertilization already contain hundreds or even
thousands of cells. Another relevant feature of mouse embryogenesis is that most embryonic genes, including the rRNA
genes, are only switched on during the 2-cell stage (for reviews
see Schultz, 1986; Santiago and Marzluff, 1989; Telford et al.,
1990; Majumder and DePamphilis, 1994). This contrasts
markedly with the situation observed in post-mitotic somatic
cells where both nucleolar and nucleoplasmic transcription
starts within minutes after the assembly of daughter nuclei
(Scheer and Benavente, 1990; Ferreira et al., 1994). Thus, early
mouse embryogenesis provides a unique model system to
study the assembly of nuclei that remain largely devoid of productive gene expression.
MATERIALS AND METHODS
Animals and embryo recovery
Random-bred Swiss albino mice (Charles River Breeding Laboratories) were used. Superovulation was induced by injection of human
chorionic gonadotrophin (hCG) as described by Hogan et al. (1986).
Unfertilized oocytes were recovered 14-16 hours post-hCG from the
oviducts of unmated females, according to conventional procedures
(Hogan et al., 1986). Mated females were used to obtain early, mid
and late 1-cell embryos (at 16-20 hours, 20-24 hours and 24-29 hours
post-hCG, respectively), as well as early and late 2-cell embryos (at
30-34 hours and 44-48 hours post-hCG, respectively), 4-cell embryos
(at 50-54 hours post-hCG) and 8-cell embryos (at 69-72 hours posthCG).
Immunofluorescence
Groups of 30-60 oocytes or embryos were rinsed twice in phosphatebuffered saline (PBS), 2× in HPEM buffer (60 mM Pipes, 25 mM
Hepes, 10 mM EGTA, 2 mM MgCl2, pH 6.9) and fixed/permeabilized in 3.7% paraformaldehyde in HPEM buffer containing 0.5%
Triton X-100, for 40 minutes at room temperature with gentle
agitation. Alternatively, oocytes and embryos were rinsed in PBS,
fixed in 3.7% paraformaldehyde in PBS and then permeabilized in
0.5% Triton X-100 in PBS for 10-15 minutes at room temperature.
After fixation and permeabilization the cells were washed in PBS containing 0.05% Tween 20 and 0.05% NaN3 (PBS-Tw), incubated with
5% normal goat serum in PBS-Tw for 15 minutes, washed in PBSTw and incubated with primary antibodies diluted in PBS-Tw for 1
hour at room temperature. For double-labeling experiments both
primary antibodies were incubated simultaneously for 2 hours at room
temperature. Antibody-binding sites were detected using secondary
antibodies conjugated to either fluorescein (FITC), rhodamine
(TRITC) or Texas Red (TxRed) (Dianova, Germany). The cells were
mounted in 50% glycerol in PBS containing 100 mg/ml DABCO (as
an anti-fading agent) and 1 µg/ml DAPI (as a DNA stain).
Antibodies
Coilin was detected using rabbit polyclonal antibodies raised against
a β-galactosidase fusion protein containing the carboxy-terminal
region of p80 coilin (Andrade et al., 1991). Fibrillarin was labeled
using either an autoimmune patient serum or the monoclonal antibody
72B9 (Reimer et al., 1987). HnRNP C, A and L proteins were revealed
by monoclonals 4F4, 4B10 and 4DII, respectively (Choi and
Dreyfuss, 1984). Splicing snRNPs were labeled with the anti-Sm
monoclonal antibody Y-12 (Lerner et al., 1981), anti-2,2,7-trimethylguanosine cap (m3G cap) monoclonal antibody (Bochnig et al., 1987),
anti-U1snRNP protein 70K monoclonal antibody (Billings et al.,
1982), and anti-U2 snRNP protein B″ monoclonal antibody 4G3
(Habets et al., 1989). Non-snRNP splicing factors were detected using
SC-35 (Fu and Maniatis, 1990) and 3C5 (Turner and Franchi, 1987)
monoclonal antibodies. Controls were performed using non-immune
sera and a variety of rabbit and human sera with distinct specificities.
In situ hybridization
Biotinylated 2′-O-alkyl oligoribonucleotide probes were used to label
the U1 and U2 snRNAs, as previously described (Carmo-Fonseca et
al., 1991). Unfertilized oocytes and embryos were washed in PBS,
rinsed in HPEM buffer, extracted with 0.5% Triton X-100 in HPEM
buffer containing 1mM PMSF for 5 minutes at 4°C and fixed in 3.7%
paraformaldehyde in HPEM buffer containing 0.5% Triton X-100, for
40 minutes at room temperature with gentle agitation. In situ hybridization was performed as described by Carmo-Fonseca et al. (1992).
Visualization of transcription sites
Visualization of transcription sites was performed according to the
method of Jackson et al. (1993). Embryos were washed in PB buffer
Biogenesis of the coiled body
603
Fig. 1. Immunofluorescent localization of coilin in early mouse embryos. In recently ovulated unfertilized oocytes (A,B), the anti-coilin
antibodies produce a widespread staining of the cytoplasm (A). At 20 hours post-hCG (early 1-cell stage; C,D) both female and male pronuclei
contain discrete patches of coilin at the periphery of the ‘nucleoli’ (C, arrows). At 27 hours post-hCG (late 1-cell stage; E,F) the anti-coilin
antibodies label discrete foci in the nucleoplasm (E, arrows). B and D depict DNA staining and F is a phase contrast micrograph. Bars, 25 µm.
(Jackson et al., 1993) at 4°C and the plasma membrane was permeabilized with 0.05% Triton X-100 in PB buffer at 4°C, for 1-2
minutes, until a slight swelling of the cells was observed. Then, the
embryos were washed in PB buffer (3× 2 minutes, at 4°C), incubated
with transcription mix containing 0.1 mM bromo-UTP (Sigma) at
33°C for 20-25 minutes. After washing in PB at 4°C for 2 minutes,
604
J. Ferreira and M. Carmo-Fonseca
the nuclear membranes were permeabilized with 0.2% Triton X-100
in PB at 4°C for 3-4 minutes with gentle agitation. Then the cells were
washed in PB at 4°C for 3 minutes, fixed in 3.7% paraformaldehyde
in PB for 15 minutes at room temperature with gentle agitation,
washed in PB and then rinsed in PBS. The incorporated bromo-UTP
was detected using a primary antibody anti-bromodeoxyuridine
(Boehringer) and a secondary antibody coupled to FITC (Dianova).
If added, α-amanitin (100 µg/ml) or actinomycin D (10 µg/ml) were
present for 10 minutes at 4°C prior to, and during, transcription.
RNasin (50 U/ml) was added to all solutions.
Fluorescence microscopy
Samples were examined with both a Leitz Orthoplan and a Zeiss
Axiophot fluorescence microscope. Images were either photographed
directly or recorded using a Hamamatsu SIT-camera and a ARGUS
10 image processor (Hamamatsu Photonics, Japan). Confocal
microscopy was performed using the Zeiss LSM 310 equipped with
argon ion (488 nm) and HeNe (543 nm) lasers.
Electron microscopy
Immunoelectron microscopy was performed using anti-coilin antibodies and a pre-embedding technique. The oviducts were flushed
with M2 medium (Hogan et al., 1986) and the embryos were
incubated with 0.5% pronase (Sigma) in M2 medium for 6-9 minutes
at 37°C until complete dissolution of the zona pellucida. When
present, the cumulus cells were removed by incubation with 0.03%
hyaluronidase (Sigma) in M2 medium at 37°C for approximately 5
minutes, before digestion with pronase. After washing in M2 medium,
the embryos were rinsed in HPEM buffer, attached to poly-L-lysine
coated plastic Petri dishes, extracted with 0.5% Triton X-100 in
HPEM buffer containing 1 mM PMSF for 3 minutes at 4°C and fixed
in 3.7% paraformaldehyde in HPEM buffer containing 0.5% Triton
X-100, for 40 minutes at room temperature with gentle agitation.
After fixation, the cells were washed in PBS-Tw, blocked with 5%
normal goat serum for 15 minutes at room temperature and then
incubated with anti-coilin antibody for 1 hour at room temperature.
After washing (3× 20 minutes) in PBS-Tw, the embryos were rinsed
in TT buffer (20 mM Tris, pH 8.2, 20 mM NaN3, 0.05% Tween 20,
0.1% BSA, 0.5 M NaCl), blocked with 5% NGS in TT buffer for 15
minutes and incubated with goat-anti rabbit IgG conjugated to 5 nm
gold particles (Amersham) diluted 1:20 in TT buffer containing 5%
normal goat serum, for 2 hours at room temperature. After washing
(3× 20 minutes) in TT buffer, the cells were fixed in 2% glutaraldehyde in TT buffer, post-fixed in 0.5% OsO4, dehydrated in ethanol
and embedded in Epon according to the method of Langanger et al.
(1984). Sections were examined with a JEOL 100CXII electron
microscope operated at 80 kV.
RESULTS
The assembly of coiled bodies is preceded by the
formation of pre-coiled bodies
In order to analyze the formation of coiled bodies during early
mouse development, we have used coilin-specific antibodies to
perform both immunofluorescence and immunoelectron
microscopy. By immunofluorescence, the anti-coilin antibodies produced a widespread staining in the cytoplasm of
recently ovulated unfertilized oocytes (Fig. 1A). This labeling
pattern was considered specific when compared to the background staining produced by non-immune sera (data not
shown). Following fertilization, in both the maternal and
paternal pronuclei of early 1-cell embryos (at 16-20 hours posthCG), the anti-coilin antibodies labeled discrete patches asso-
Fig. 2. Immunoelectron microscopic
localization of coilin in early mouse
embryos. In fertilized eggs obtained at
16 hours post-hCG the anti-coilin
antibodies label compact
homogeneous fibrillar aggregates that
appear as spheroids in the
nucleoplasm (A). In early 1-cell
embryos (obtained at 16-20 hours
post-hCG) the anti-coilin antibodies
label similar compact fibrillar
structures that associate with the
periphery of the ‘nucleolus’ (B; nu,
‘nucleolus’). In late 1-cell embryos (at
27 hours post-hCG) the anti-coilin
antibodies label nucleoplasmic
structures, which consist of coiled
fibrillar threads and are therefore
likely to correspond to coiled bodies
(C,D). Bars, 0.25 µm.
Biogenesis of the coiled body
ciated with the periphery of large compact structures that have
been called ‘nucleolus-like-bodies’ or ‘nucleolar precursors’
(Fig. 1C, arrows). Later after fertilization, during the mid to
late 1-cell stage (at 20-29 hours post-hCG) the anti-coilin antibodies labeled both peri-‘nucleolar’ structures and a few
discrete foci in the nucleoplasm of both maternal and paternal
pronuclei (Fig. 1E, arrows). These nucleoplasmic foci were
never observed in embryos recovered at 16-20 hours posthCG, but were detected in 6% of embryos (from a total of 67)
recovered at 20-24 hours post-hCG, and in 57% of embryos
(from a total of 99) recovered at 27-29 hours post-hCG. This
suggests that in 1-cell embryos coilin associates first with the
periphery of the ‘nucleolus’ and later appears in nucleoplasmic foci.
Immunoelectron microscopy of fertilized eggs obtained at
14-16 hours post-hCG showed that at this very early stage,
anti-coilin antibodies label compact homogeneous fibrillar
aggregates that appear either free in the nucleoplasm (Fig. 2A)
or in association with the periphery of the ‘nucleolus’
(Fig. 2B). In early 1-cell embryos (at 16-20 hours posthCG) the labeling was exclusively detected in fibrillar
structures associated with the ‘nucleolus’ (data not
shown), and in late 1-cell embryos (at 27-29 hours posthCG) the anti-coilin antibodies labeled two distinct type
of structures, i.e., peri-‘nucleolar’ fibrillar aggregates
and nucleoplasmic bodies, which consist of coiled
fibrillar threads and are therefore likely to correspond to
coiled bodies (Fig. 2C,D).
In summary, we observe that coilin is detected in both
the male and female pronuclei shortly after fertilization.
Within each pronucleus, coilin assembles first into
compact fibrillar structures that associate with the
periphery of the ‘nucleolus’, and later coiled bodies are
formed (Table 1). Since the compact fibrillar structures
that associate with the periphery of the ‘nucleolus’
contain coilin and precede the appearance of coiled
bodies, we refer to them here as pre-coiled bodies. Both
pre-coiled bodies and coiled bodies are observed in 2-,
4- and 8-cell embryos (Fig. 3A and data not shown) and
occasionally coiled bodies are seen in close proximity
to pre-coiled bodies (Fig. 3B), suggesting that one
structure may arise from the other.
Pre-coiled bodies and coiled bodies contain
fibrillarin
As fibrillarin is known to be present in the coiled body
of somatic cells, double-immunofluorescence experiments were performed in early mouse embryos using
antibodies specific to coilin and fibrillarin (Fig. 4).
Shortly after fertilization (at 14-16 hours post-hCG),
both anti-coilin and anti-fibrillarin antibodies labeled
fine punctate structures in the nucleoplasm, some of
which are associated with the periphery of the ‘nucleoli’
(Fig. 4A,B, arrows and arrowheads). These fine structures are likely to correspond to the compact fibrillar
aggregates observed by electron microscopy (see Fig.
2A,B). In the pronuclei of early 1-cell embryos both
antibodies predominantly stained discrete structures at
the periphery of the ‘nucleoli’ and in late 1-cell embryos
the two antibodies labeled both peri-‘nucleolar’ structures and nucleoplasmic foci (Fig. 4C,D, and data not
605
Table 1. Timetable of coiled body (CB) development in
fertilized mouse eggs
Time postTime post- fertilization
hCG (hours) (hours)
14-16
16-20
20-29
~2-4
~4-8
~8-17
Stage in coiled body development
pre-CBs (nucleoplasmic and peri-‘nucleolar’)
pre-CBs (predominantly peri-‘nucleolar’)
nucleoplasmic CBs and peri-‘nucleolar’ pre-CBs
shown). This indicates that in 1-cell embryos fibrillarin colocalizes with coilin in both pre-coiled bodies and coiled bodies.
In the nuclei of early 2-cell embryos (30-34 hours post-hCG)
coilin and fibrillarin also colocalized in both pre-coiled bodies
at the periphery of the ‘nucleoli’ and in nucleoplasmic coiled
bodies (Fig. 4E,F). However, during the late 2-cell stage (4448 hours post-hCG) the anti-fibrillarin antibodies labeled additional patchy structures localized at the periphery of the
Fig. 3. Coiled bodies may arise from pre-coiled bodies. A coiled body from
an 8-cell embryo reveals the typical ultrastructure reminiscent of a tangle of
coiled threads (A). Occasionally, a coiled body (cb) is seen in close
association with a pre-coiled body (pre-cb) at the periphery of the
‘nucleolus’ (nu) (B). Bars, 0.25 µm.
606
J. Ferreira and M. Carmo-Fonseca
Fig. 4. Embryonic coiled
bodies contain fibrillarin.
Double-immunofluorescence
experiments were performed
in early mouse embryos
using antibodies specific to
coilin and fibrillarin. At 16
hours post-hCG, both anticoilin and anti-fibrillarin
antibodies labeled fine
punctate structures in the
nucleoplasm of the
decondensing sperm head
(A,B, arrows), some of
which are associated with the
periphery of the ‘nucleoli’
(A,B, arrowheads). In 1-cell
embryos (at 25 hours posthCG) both antibodies stain
predominantly cap-shaped
structures at the periphery of
the ‘nucleoli’ (C,D,
arrowheads). In early 2-cell
embryos (at 30 hours posthCG) coilin and fibrillarin
colocalize in nucleoplasmic
coiled bodies (E,F,
arrowheads). Later in the 2cell stage (at 45 hours posthCG) the anti-fibrillarin
antibodies label both
nucleoplasmic coiled bodies
(G,H, arrowheads) and
additional structures
localized at the periphery of
the ‘nucleoli’, which are not
labeled by anti-coilin
antibodies (G,H, arrows).
Bars, 10 µm (A,B); 25 µm
(C-H).
‘nucleoli’, which are not labeled by anti-coilin antibodies (Fig.
4G,H, arrows and arrowheads). Although these new patches
containing fibrillarin are also associated with the periphery of
‘nucleoli’, they are clearly distinct from pre-coiled bodies and
coiled bodies (Fig. 5A,B, arrows and arrowheads). Previous
immunoelectron microscopic and autoradiographic studies
have demonstrated that fibrillarin localizes predominantly in
the dense fibrillar component of active nucleoli (reviewed by
Scheer and Benavente, 1990), and that in mouse embryos the
fibrillar component appears during the late 2-cell stage at the
Biogenesis of the coiled body
607
Fig. 5. Only a subfraction of
fibrillarin colocalizes with
coilin. High magnification
micrographs from an early 4cell embryo (52 hours posthCG) double-labeled using
anti-coilin (A) and antifibrillarin (B) antibodies. Coilin
is detected in foci that also
contain fibrillarin (arrows).
However, fibrillarin is
additionally present in patches
that do not contain coilin
(arrowheads). Bar, 10 µm.
periphery of the ‘nucleoli’, coincident with the activation of
rRNA synthesis (Tesarik et al., 1986, 1987). Therefore, it is
likely that the peripheral patches labeled by anti-fibrillarin antibodies in late 2-cell embryos correspond to sites of rRNA
synthesis and processing.
In conclusion, the results provide evidence for the presence
of two functionally distinct pools of fibrillarin in the nucleus:
one colocalizes with coilin in both pre-coiled bodies and coiled
bodies and is independent of rRNA synthesis, the other is
involved in rRNA processing and does not colocalize with
coilin.
Embryonic coiled bodies contain splicing snRNPs
In order to investigate whether the embryonic coiled bodies
also contain snRNPs, unfertilized oocytes and embryos were
double-labeled with anti-coilin polyclonal antibodies and a
monoclonal antibody specific for Sm proteins, a set of peptides
common to all snRNPs involved in splicing (reviewed by Will
et al., 1993). In unfertilized oocytes and recently fertilized eggs
(at 14-16 hours post-hCG) the anti-Sm monoclonal antibody
produced a widespread homogeneous staining, without any
clear concentration in discrete structures (Fig. 6A-D). In 1-cell
embryos the staining was predominantly detected in both the
male and female pronuclei, as previously reported (Lobo et al.,
1988; Dean et al., 1989). Within the pronuclei the labeling was
particularly concentrated both in discrete structures at the
periphery of the ‘nucleoli’ and in nucleoplasmic foci (Fig.
6E,G, arrows). As these structures were also labeled by anticoilin antibodies (Fig. 6F,H, arrows), they are likely to correspond to pre-coiled bodies and coiled bodies. It is important to
note that this result was only obtained using anti-Sm monoclonal antibodies, as polyclonal antibodies produced a very
intense overall staining of the nucleoplasm (data not shown).
Pre-coiled bodies and coiled bodies were also labeled by
several other probes specific for splicing snRNPs, namely
monoclonal antibodies that react either with the m3G cap modification of snRNAs or proteins specific for the U1 and U2
snRNPs, and oligonucleotide probes directed to the U1 and
U2snRNAs (data not shown). This indicates that snRNPs
interact with both pre-coiled bodies and coiled bodies in 1-cell
mouse embryos.
Having established that splicing snRNPs are concentrated in
coiled bodies as early as the 1-cell stage, we sought to examine
the embryonic distribution of other nuclear proteins known to
be involved in the metabolism of mRNA. The data show that
hnRNP proteins A, C and L, as well as protein splicing factors
recognized by the monoclonal antibodies SC-35 (Fu and
Maniatis, 1990) and 3C5 (Turner and Franchi, 1987; Bridge et
al., 1995) are all detected within the pronuclei of 1-cell
embryos (Fig. 7A-D and data not shown).
Embryonic transcription is first detected during the
2-cell stage
In order to correlate the assembly of coiled bodies and their
interaction with splicing snRNPs with the onset of pre-mRNA
synthesis and processing, we decided to visualize the transcriptional activity of mouse embryos at the single cell level.
This was achieved by incubation of mildly permeabilized
embryos with bromo-UTP and subsequent visualization of the
incorporated nucleotide using the fluorescence microscope
(Jackson et al., 1993, Wansink et al., 1993; Fig. 8). The nuclei
of 4-cell (Fig. 8A,B, arrows) and late 2-cell (Fig. 8C) embryos
were intensely labeled, confirming that at these stages the
embryonic genes are actively transcribed. However, no significant labeling was ever observed in 1-cell or early 2-cell
embryos (Fig. 8A,B, arrowheads). Control experiments
performed in the presence of actinomycin D or α-amanitin
show that under these conditions the nuclei of late 2-cell
embryos were not labeled (Fig. 8D).
DISCUSSION
In this study we have analyzed the biogenesis of the coiled
body during early mouse development. We found that coiled
bodies are first detected in both maternal and paternal pronuclei
of 1-cell embryos, at approximately 16 hours after fertilization.
Thus, the assembly of the coiled body precedes the onset of
embryonic gene expression, which occurs during the 2-cell
stage (see Schultz, 1986; Santiago and Marzluff, 1989; Telford
et al., 1990; Majumder and DePamphilis, 1994).
Although expression of embryonic genes starts in 2-cell
embryos, splicing snRNPs are already detected within the
pronuclei of 1-cell embryos, as previously described (Lobo
et al., 1988; Dean et al., 1989). In addition, we have also
detected hnRNP proteins and non-snRNP protein splicing
factors in the pronuclei of early 1-cell embryos (Fig. 7). This
suggests that the pronuclei contain all the molecular components required for mRNA synthesis and maturation. Accordingly, microinjection of cloned genes into the pronuclei of 1cell mouse embryos revealed that there is transcription and
processing of mRNA from the injected DNA (Chen et al.,
608
J. Ferreira and M. Carmo-Fonseca
Fig. 6. Embryonic coiled
bodies contain splicing
snRNPs. In unfertilized
oocytes (A) and recently
fertilized eggs (C) the antiSm monoclonal antibody
produces a widespread
homogeneous staining,
without any clear
concentration in discrete
structures (B and D depict
the corresponding DNA
staining). In 1-cell embryos
(E,F, 25 hours post-hCG;
G,H, 29 hours post-hCG),
Sm antigens are widespread
in the nucleoplasm and in
addition concentrate in
perinucleolar patches and
foci (E,G, arrows). Doublelabeling with anti-coilin
antibodies indicates that
these structures correspond
to pre-coiled bodies and
coiled bodies (F,H, arrows).
Bars, 25 µm (A-D) and 10
µm (E-H).
1986). Interestingly, embryonic genes can also be expressed
in 1-cell embryos when these cells are arrested and thus do
not develop to 2-cell embryos (Martínez-Salas et al., 1988,
1989; Wiekowski et al., 1991), suggesting that the onset of
gene expression appears to be primarily determined by the
time elapsed after fertilization, independently of the
formation of a 2-cell embryo. Consistent with this view, it
has been shown that expression of genes injected into arrested
1-cell embryos at 22-35 hours post-hCG was delayed until
the normal time that embryonic gene expression began, i.e.
Biogenesis of the coiled body
609
Fig. 7. hnRNP proteins and
SR protein splicing factors
are present in the pronuclei
of 1-cell embryos. 1-cell
embryos (25-27 hours posthCG) were labeled with
antibodies directed to hnRNP
proteins A (A), C (B) and L
(C), and the monoclonal
antibody 3C5 which
recognizes protein splicing
factors (D). Bar, 25 µm.
until 35-40 hours post-hCG (Chen et al., 1986; MartínezSalas et al., 1989; Wiekowski et al., 1991). Based on these
and other data, Wiekowski et al. (1991) proposed the
existence of a biological clock that applies to all RNA polymerase II promoters and regulates the onset of embryonic
transcription. According to this model, it is conceivable that
the machinery required for mRNA synthesis and processing
is present in the pronuclei of 1-cell embryos, but it remains
silent until the ‘zygotic clock’ activates gene expression.
Thus, if coiled bodies are actively involved in the metabolism of pre-mRNA, it is not surprising that they form during
the 1-cell stage. It is also conceivable that early embryos may
inherit unspliced pre-mRNAs of maternal origin, and
therefore require coiled bodies before activation of transcription.
The presence of hnRNP A proteins within the pronuclei of
1-cell embryos is particularly intriguing, considering that at the
end of mitosis in somatic cells the import of hnRNP A proteins
into the nucleus is transcription-dependent (Piñol-Roma and
Dreyfuss, 1991). Despite all the evidence arguing against any
embryonic gene expression at this stage (see reviews by
Schultz, 1986; Santiago and Marzluff, 1989; Telford et al.,
1990), one may speculate that the pronuclei of 1-cell embryos
may synthesize some form of RNA and this would trigger the
import of hnRNP A proteins. In fact, Clegg and Pikó (1982;
1983a) have reported that 1-cell embryos synthesize a low
level of large heterogeneous poly(A)− RNA, which turns over
rapidly. However, studies from these and other authors agree
that significant synthesis of all the major classes of RNA is
only evident in the 2-cell embryo (Knowland and Graham,
1972; Levey et al., 1978; Clegg and Pikó, 1983a,b; Taylor and
Pikó, 1987).
Based on our previous visualization of the onset of transcriptional activity at the end of mitosis using incorporation of
bromo-UTP into nascent transcripts (Ferreira et al., 1994), we
decided to apply this technique to early mouse embryos.
Although transcription was clearly observed in late 2-cell (at
44-48 hours post-hCG) and 4-cell embryos (at 50 hours posthCG), we did not detect any significant transcriptional activity
in either late 1-cell embryos (at 27-29 hours post-hCG) or early
2-cell embryos (at 30-34 hours post-hCG). However, as there
is no information available on the sensitivity of this method,
we cannot exclude that transcription starts before the 2-cell
stage. In conclusion, if there is some form of transcription in
1-cell embryos it is clearly very low level and it does not
appear to be required for normal embryo development, since
treatment of 1-cell embryos with inhibitors of RNA polymerases does not prevent progression to the 2-cell stage
(Braude et al., 1979). Alternatively, if there is no transcriptional activity in 1-cell embryos, one has to consider that the
mechanisms controlling the nuclear transport of hnRNP A
proteins in early embryos may differ from those operating in
somatic cells.
During telophase of somatic cells, snRNPs, hnRNP proteins,
non-snRNP protein splicing factors and coilin are rapidly transported into daughter nuclei, but coiled bodies assemble only
later during G1 (see Ferreira et al., 1994 and references
therein). Similarly, coilin is readily detected in both male and
female pronuclei but coiled bodies form only after a lag period,
during the late 1-cell stage. The finding that coiled bodies form
610
J. Ferreira and M. Carmo-Fonseca
and concentrate snRNPs as early as in the 1-cell embryo is conwhich consist of large compact spherical aggregates approxisistent with the view that the coiled body is playing a basic role
mately 2 µm in diameter. Although these pre-nucleoli are
in the nucleus of mammalian cells.
usually surrounded by a continuous rim of condensed
According to the idea that snRNPs cycle between distinct
chromatin, they do not contain DNA and are devoid of RNA
nuclear compartments and that the coiled body may be
synthesis. Activation of rDNA transcription is paralleled by an
involved in post-splicing events, e.g., recycling of snRNPs
infiltration of chromatin into the precursor structures, which
(Lamond and Carmo-Fonseca, 1993a,b) we could speculate
progressively acquire the morphology typical of functionally
that upon fertilization the maternal snRNPs previously stored
active nucleoli (Tesarik et al., 1987). In the mouse embryo this
in the oocyte must interact with the coiled body in order to be
activation process occurs at the late 2-cell stage and is charac‘reactivated’ for splicing of embryonic pre-mRNAs. This
terized by the appearance of fibrillar centers and dense fibrillar
would explain why coiled bodies assemble before the onset of
component at the periphery of the pre-nucleoli (Geuskens and
embryonic gene expression.
Alexandre, 1984). As fibrillarin binds nascent pre-rRNAs and
Preceding the appearance of coiled bodies in late 1-cell
localizes primarily in the dense fibrillar component of funcembryos (24-29 hours post-hCG), the pronuclei of early 1-cell
tional nucleoli (Scheer and Benavente, 1990), it is conceivable
embryos (16-24 hours post-hCG) contain discrete structures
that the discrete patches stained by anti-fibrillarin antibodies at
that are labeled by anti-coilin antibodies and are frequently
the periphery of the precursor nucleoli in late 2-cell embryos
associated with the periphery of precursor nucleoli. Viewed
represent the sites of rRNA synthesis and processing. These
with the electron microscope these structures have a compact
patches of fibrillarin do not contain coilin and may coexist with
fibrillar morphology and are therefore distinct from
coiled bodies, which resemble a tangle of coiled
threads. As they precede the formation of coiled
bodies, the compact fibrillar structures are referred
to here as pre-coiled bodies. Although the data do
not allow us to conclude that pre-coiled bodies are
the precursors of coiled bodies, it shows that coilin
interacts with precursor nucleoli before the
assembly of mature coiled bodies.
Immediately after fertilization (14-16 hours
post-hCG), pre-coiled bodies are detected either as
spheres in the nucleoplasm or as peri-‘nucleolar’
patches (Fig. 4A, arrows and arrowheads). Later, at
16-20 hours post-hCG, the pre-coiled bodies are
predominantly observed at the periphery of the
precursor nucleoli (Table 1). Since the pre-coiled
bodies also contain fibrillarin, they are very similar
to the so called prenucleolar bodies, which initiate
the formation of nucleoli in post-mitotic somatic
cells (reviewed by Scheer and Benavente, 1990).
However, in cycling somatic cells pre-nucleolar
bodies are transient structures that form at
telophase and rapidly coalesce around the chromosomal nucleolus organizer regions (NORs) upon
activation of rRNA synthesis. In nuclei that lack
NORs or where RNA polymerase I was inhibited,
the prenucleolar bodies fail to organize into
nucleoli (Benavente, 1987, 1988). In mouse
embryos, the fibrillar structures containing coilin
and fibrillarin associate with the periphery of
precursor nucleoli long before the activation of
rDNA transcription, which occurs during the late
2-cell stage (Pikó and Clegg, 1982; Clegg and Pikó,
Fig. 8. Visualization of transcription in early mouse embryos. Late 1-cell (at
1982, 1983a,b). Therefore, despite their morpho27hours post-hCG) and 4-cell (at 52 hours post-hCG) embryos were pooled
logical similarities, it is likely that these fibrillar
together and incubated with bromo-UTP as described in Materials and Methods
aggregates are functionally distinct from pre(A). Alternatively, early 2-cell (at 34 hours post-hCG) and 4-cell (at 58 hours
nucleolar bodies.
post-hCG) embryos were pooled and incubated with bromo-UTP (B).
It is well established that early mammalian
Transcriptional activity was clearly detected in the nuclei of 4-cell embryos
embryos do not synthesize rRNA and do not have
(A,B, arrows) but not in 1-cell or early 2-cell embryos (A,B, arrowheads).
typical nucleoli (see Tesarik et al., 1986, 1987).
However, transcription was already detected in late 2-cell embryos (at 48 hours
However, both pronuclei and early embryonic
post-hCG) (C). As a control, late 2-cell embryos (at 48 hours post-hCG) were
nuclei contain large precursor structures called
incubated with bromo-UTP in the presence of 10 µg/ml actinomycin D (D). Bar,
25 µm.
‘nucleolus-like bodies’ or ‘precursor nucleoli’,
Biogenesis of the coiled body
pre-coiled bodies at the periphery of the same ‘nucleolus’, but
the two structures are always clearly separated from each other
(see Fig. 5). This shows that the assembly of the coiled body
is disassociated from ribosome biogenesis both in time and in
space.
A major conclusion from the present study is that the
assembly of coiled bodies is preceded by the formation of precoiled bodies, which arise at the periphery of a precursor
nucleolus. The data provides evidence that embryonic
precursor nucleoli represent a specialized scaffold with the
potential to nucleate independently the assembly of coilin into
pre-coiled bodies and the formation of a machinery active in
ribosome biogenesis. A similar scaffold is probably present in
all somatic cells but it is difficult to demonstrate due to the
rapid formation of active nucleoli at the end of mitosis. It will
be important in the future to identify and characterize the
molecular components of this intranuclear organizing center.
The authors are grateful to Professor David-Ferreira for support and
for providing access to laboratory facilities in the Gulbenkian Institute
of Science, and to Dr A. Lamond for critical reading of the manuscript, as well as for providing snRNA oligonucleotide probes. We
are also grateful to Mr Romão for animal care facilities and to
Conceição Alpiarça, Fernanda Barreto and Dora Brito for help with
electron microscopy. We wish to thank the following laboratories for
generously providing antibodies used in this study: Dr Eng Tan for
anti-p80 coilin and anti-fibrillarin antibodies; Dr Ian Mattaj for antiSm Y12 antibody; Dr Gideon Dreyfuss for anti-hnRNP antibodies; Dr
Bryan Turner for the 3C5 monoclonal antibody. Dr Walther van
Venrooij for anti-B’ and anti-70K antibodies; Dr Reinhard Lührmann
for anti-m3G-cap antibody; Dr Tom Maniatis for anti-SC-35
antibody.
J. F. was supported by a fellowship from Instituto Nacional de
Investigação Científica and M. C.-F. acknowledges support from
Junta Nacional de Investigação Científica e Tecnológica.
REFERENCES
Andrade, L. E. C., Chan, E. K. L., Raska, I., Peebles, C. L., Roos, G. and
Tan, E. M. (1991). Human autoantibody to a novel protein of the nuclear
coiled body: immunological characterization and cDNA cloning of p80coilin. J. Exp. Med. 173, 1407-1419.
Benavente, R., Rose, K. M., Reimer, G., Hügle-Dörr, B. and Scheer, U.
(1987). Inhibition of nucleolar reformation after microinjection of antibodies
to RNA polymerases I into mitotic cells. J. Cell Biol. 105, 1483-1491.
Benavente, R., Schmidt-Zachmann, M. S., Hügle-Dörr, B., Reimer, G.,
Rose, K. M. and Scheer, U. (1988). Identification and definition of
nucleolus-related fibrillar bodies in micronucleated cells. Exp. Cell Res. 178,
518-523.
Billings, P.B., R.W. Allen, F.C. Jenssen and S.O. Hoch. (1982). Anti-RNP
monoclonal antibodies derived from a mouse strain with lupus-like
autoimmunity. J. Immunol. 128, 1176-1180.
Bochnig, P., Reuter, R., Bringmann, P., and R. Lührmann. (1987). A
monoclonal antibody against 2,2,7-trimethylguanosine that reacts with
intact, class U, small nuclear ribonucleoproteins as well as with 7-methyl
guanosine-capped RNAs. Eur. J. Biochem. 168, 461-467.
Braude, P., Pelham, H., Flach, G. and Lobatto, R. (1979). Posttranscriptional control in the early mouse embryo. Nature 282, 102-105.
Brasch, K. and Ochs, R. L. (1992). Nuclear bodies: a newly rediscovered
organelle. Exp. Cell Res. 202, 211-223.
Bridge, E., Xia, D.-X., Carmo-Fonseca, M., Cardinali, B., Lamond, A. I.
and Pettersson, U. (1995). Dynamic organization of splicing factors in
adenovirus infected cells. J. Virol. (in press).
Carmo-Fonseca, M., Tollervey, D., Pepperkok, R., Barabino, S., Merdes,
A., Brunner, C., Zamore, P. D., Green, M. R., Hurt, E. and Lamond, A.
611
I. (1991). Mammalian nuclei contain foci which are highly enriched in
components of the pre-mRNA splicing machinery. EMBO J. 10, 195-206.
Carmo-Fonseca, M., Pepperkok, R., Carvalho, M. T. and Lamond, A. I.
(1992). Transcription-dependent colocalization of the U1, U2, U4/U6 and
U5 snRNPs in coiled bodies. J. Cell Biol. 117, 1-14.
Carmo-Fonseca, M., Ferreira, J. and Lamond, A. I. (1993). Assembly of
snRNP-containing coiled bodies is regulated in interphase and mitosis:
evidence that the coiled body is a kynetic nuclear structure. J. Cell Biol. 120,
841-852.
Carmo-Fonseca, M., Bohmann, K., Carvalho, M.T. and Lamond, A.I.
(1994). P80 coilin. In Manual of Biological Markers of Disease (ed. W. Van
Venrooij and R. N. Maini), section B. Dordrecht: Kluwer Academic
Publishers (in press).
Chen, H. Y., Trumbauer, M. E., Ebert, K. M., Palmiter, R. D. and Brinster,
R. L. (1986). Developmental changes in the response of mouse eggs to
injected genes. In Molecular Developmental Biology (ed. L. Bogorad), pp.
149-159. New York: Alan R. Liss.
Choi, Y.D. and Dreyfuss, G. (1984). Monoclonal antibody characterization of
the C proteins of heterogeneous nuclear ribonucleoprotein complexes in
vertebrate cells. J. Cell Biol. 99, 1997-2004.
Clegg, K. B. and Pikó, L. (1982). RNA synthesis and cytoplasmic
polyadenylation in the one-cell mouse embryo. Nature 295, 342-345.
Clegg, K. B. and Pikó, L. (1983a). Quantitative aspects of RNA synthesis and
polyadenylation in 1-cell and 2-cell mouse embryos. J. Embryol. Exp.
Morphol. 74, 169-182.
Clegg, K. B. and Pikó, L. (1983b). Poly(A) length, cytoplasmic
polyadenylation and synthesis of poly(A) RNA in mouse embryos. Dev. Biol.
95, 331-341.
Dean, W. L., Seufert, A. C., Schultz, G. A., Prather, R. S., Simerly, C.,
Schatten, G., Pilch, D. R. and Marzluff, W. F. (1989). The small nuclear
RNAs for pre-mRNA splicing are coordinately regulated during oocyte
maturation and early embryogenesis in the mouse. Development 106, 325334.
Ferreira, J. A., Carmo-Fonseca, M. and Lamond, A. I. (1994). Differential
interaction of splicing snRNPs with coiled bodies and interchromatin
granules during mitosis and assembly of daughter cell nuclei. J. Cell Biol.
126, 11-23.
Filipowicz, W. and Kiss, T. (1993). Structure and function of nucleolar
snRNPs. Mol. Biol. Rep. 18, 149-156.
Fu, X.-D. and Maniatis, T.(1990). Factor required for mammalian
spliceosome assembly is localized to discrete regions in the nucleus. Nature
343, 437-441.
Geuskens, M. and Alexandre, H. (1984). Ultrastructural and autoradiographic
studies of nucleolar development and rDNA transcription in preimplantation
mouse embryos. Cell Differ. 14, 125-134.
Habets, W. J., Hoet, M. H., DeJong, B. A. W., VanDer Kemp, A. and
VanVenrooij, W. J. (1989). Mapping of B cell epitopes on small nuclear
ribonucleoproteins that react with human autoantibodies as well as with
experimentally-induced mouse monoclonal antibodies. J. Immunol. 143,
2560-2566.
Hogan, B., Costantini, F. and Lacy, E. (1986). Manipulating the mouse
embryo. Cold Spring Harbor New York: Cold Spring Harbor Laboratory
Press.
Jackson, D. A., Hassan, A. B., Errington, R. J. and Cook, P. R. (1993).
Visualization of focal sites of transcription within human nuclei. EMBO J.
12, 1059-1065.
Knowland, J. and Graham, C. (1972). RNA synthesis at the two-cell stage of
mouse development. J. Embryol. Exp. Morphol. 27, 167-176.
Lamond, A. I. and Carmo-Fonseca, M. (1993a). The coiled body. Trends Cell
Biol. 3, 198-204.
Lamond, A. I. and Carmo-Fonseca, M. (1993b). Localisation of splicing
snRNPs in mammalian cells. Mol. Biol. Rep. 18, 127-133.
Langanger, G., deMey, J., Moeremens, M., Daneels, G., deBrabender, M.
and Small, J. V. (1984). Ultrastructural localization of a-actinin and filamin
in cultured cells with the immunogold staining (IGS) method. J. Cell Biol.
99, 1324-1334.
Lerner, E. A., Lerner, M. R., Janeway, C. A. and Steitz, J. A. (1981).
Monoclonal antibodies to nucleic acid containing cellular constituents:
probes for molecular biology and autoimmune disease. Proc. Natl. Acad. Sci.
USA 78, 2737-2741.
Levey, I. L., Stull, G. B. and Brinster, R. L. (1978). Poly(A) and synthesis of
polyadenylated RNA in the preimplantation mouse embryo. Dev. Biol. 64,
140-148.
Lobo, S. M., Marzluff, W. F., Seufert, A. C., Dean, W. L., Schultz, G. A.,
612
J. Ferreira and M. Carmo-Fonseca
Simerly, C. and Schatten, G. (1988). Localization and expression of U1
RNA during early mouse embryo development. Dev. Biol. 12, 649-661.
Majumber, S. and DePamphilis, M. L. (1994). Requirements for DNA
transcription and replication at the beginning of mouse development. J. Cell
Biochem. 55, 59-68.
Martínez-Salas, E., Cupo, D. Y. and DePamphilis, M. L. (1988). The need
for enhancers is acquired upon formation of a diploid nucleus during early
mouse development. Genes Dev. 2, 1115-1126.
Martínez-Salas, E., Linney, E., Hassell, J. and DePamphilis, M. L. (1989).
The need for enhancers in gene expression first appears during mouse
development with formation of a zygotic nucleus. Genes Dev. 3, 1493-1506.
Matera, A. G. and Ward, D. C. (1993). Nucleoplasmic organization of small
nuclear ribonucleoproteins in cultured human cells. J. Cell Biol. 121, 715727.
Monneron, A. and Bernhard, W. (1969). Fine structural organization of the
interphase nucleus in some mammalian cells. J. Ultrastruct. Res. 27, 266288.
Pikó, L. and Clegg, K. B. (1982). Quantitative changes in total RNA, total
poly(A) and ribosomes in early mouse embryos. Dev. Biol. 89, 362-378.
Piñol-Roma, S. and Dreyfuss, G. (1991). Transcription-dependent and
transcription-independent nuclear transport of hnRNP proteins. Science 253,
312-314.
Raska, I., Ochs, R. L., Andrade, L. E. C., Chan, E. K. L., Burlingame, R.,
Peebles, C., Gruol, D. and Tan, E. M. (1990). Association between the
nucleolus and the coiled body. J. Struct. Biol. 104, 120-127.
Reimer, G., Pollard, K. M., Penning, C. A., Ochs, R. L., Lischwe, N. A. and
Tan, E. M.(1987). Monoclonal antibody from (New Zealand Black × New
Zealand White) F1 mouse and some human scleroderma sera target a Mr
34000 nucleolar protein of the U3-ribonucleoprotein particle. Arthritis
Rheum. 30, 793-800.
Rosbash, M. and Singer, R. H. (1993). RNA travel: tracks from DNA to the
cytoplasm. Cell 75, 399-401.
Santiago, C. L. and Marzluff, W. F. (1989). Changes in gene activity early
after fertilization. In The Molecular Biology of Fertilization (ed. H. Schatten
and G. Schatten), pp. 303-322. New York: Academic Press.
Scheer, U. and Benavente, R. (1990). Functional and dynamic aspects of the
mammalian nucleolus. BioEssays 12, 14-22.
Schultz, G. A. (1986). Utilization of genetic information in the preimplantation
mouse embryo. In Experimental Approaches to Mammalian Embryonic
Development (ed. J. Rossant and R. Pedersen), pp. 239-265. Cambridge:
Cambridge University Press.
Taylor, K. D. and Pikó, L. (1987). Patterns of mRNA prevalence and
expression of B1 and B2 transcripts in early mouse embryos. Development
101, 877-892.
Telford, N. A., Watson, A. J. and Schultz, G. A. (1990). Transition from
maternal to embryonic control in early mammalian development. Mol.
Reprod. Dev. 26, 90-100.
Tesarik, J., Kopecny, V., Plachot, M., Mandelbaum, J., Da Lage, C and
Fléchon, J.-E. (1986). Nucleologenesis in the human embryo developing in
vitro: ultrastructural and autoradiographic analysis. Dev. Biol. 115, 193-203.
Tesarik, J., Kopecny, V., Plachot, M. and Mandelbaum, J. (1987). Highresolution autoradiographic localization of DNA-containing sites and RNA
synthesis in developing nucleoli of human preimplantation embryos: a new
concept of embryonic nucleologenesis. Development 101, 777-791.
Tuma, R. S., Stolk, J. A. and Roth, M. B. (1993). Identification and
characterization of a spere organelle protein. J. Cell Biol. 122, 767-773.
Turner, B. M. and Franchi, L. (1987). Identification of protein antigens
associated with the nuclear matrix and with clusters of interchromatin
granules in both interphase and mitotic cells. J. Cell Sci. 87, 269-282.
Tycowski, K. T., Shu, M. D. and Steitz, J. A. (1993). A small nucleolar RNA
is processed from an intron of the human gene encoding ribosomal protein
S3. Genes Dev. 7, 1176-1190.
Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel, R.
and de Jong, L. (1993). Fluorescent labeling of nascent RNA reveals
transcription by RNA polymerase II in domains scattered throughout the
nucleus. J. Cell Biol. 122, 283-293.
Wiekowski, M., Miranda, M. and DePamphilis, M. L. (1991). Regulation of
gene expression in preimplantation mouse embryos: effects of the zygotic
clock and the first mitosis on promoter and enhancer activities. Dev. Biol.
147, 403-414.
Will, C. L., Behrens, S.-E. and Luhrmann, R. (1993). Protein composition of
mammalian spliceosomal snRNPs. Mol. Biol. Reports 18, 121-126.
Zhang, M., Zamore, P. D., Carmo-Fonseca, M., Lamond, A. I. and Green,
M. R. (1992). Cloning and intracellular localization of the U2 small nuclear
ribonucleoprotein auxiliary factor small subunit. Proc. Natl. Acad. Sci. USA
89, 8769-8773.
(Accepted 18 October 1994)