1 BioMarCell, UMR 7009, CNRS/UPMC, Station Zoologique, Observatoire
Océanologique, Villefranche sur Mer, 06230, France
2 Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta,
Midori-ku, Yokohama 226-8501, Japan
Author for correspondence (e-mail:
sardet{at}obs-vlfr.fr)
Accepted 6 August 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By using high-resolution, fluorescent, in situ hybridization in eggs, zygotes and embryos of Halocynthia roretzi, we showed that macho 1 and HrPEM are localized on a reticulated structure situated within 2 µm of the surface of the unfertilized egg, and within 8 µm of the surface the vegetal region and then posterior region of the zygote. By isolating cortices from eggs and zygotes we demonstrated that this reticulated structure is a network of cortical rough endoplasmic reticulum (cER) that is tethered to the plasma membrane. The postplasmic RNAs macho 1 and HrPEM were located on the cER network and could be detached from it. We also show that macho 1 and HrPEM accumulated in the CAB and the cER network. We propose that these postplasmic RNAs relocalized after fertilization by following the microfilament- and microtubule-driven translocations of the cER network to the poles of the zygote. We also suggest that the RNAs segregate and concentrate in posterior blastomeres through compaction of the cER to form the CAB. A multimedia BioClip `Polarity inside the egg cortex' tells the story and can be downloaded at www.bioclips.com/bioclip.html
Key words: Ascidian embryo, Maternal mRNA, Egg cortex, RNA localization, Endoplasmic reticulum
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ascidians are urochordates that develop into simple tadpoles of 3000
cells (Satoh, 1994
). They
display a profound degree of autonomous development
(Nishida, 1997
;
Satoh, 2001
). Removal and
transfer of small fragments of the periphery of the egg show that the
formation of three kinds of tissue (tail muscle, endoderm and epidermis)
involves peripherally localized maternal determinants that are repositioned in
two main phases of reorganisation that occur between fertilization and first
cleavage (Nishida, 1997
;
Roegiers et al., 1999
). An
essential element of the muscle determinant is a cortically localized maternal
mRNA that encodes a zinc-finger transcription factor called macho 1
(Nishida and Sawada, 2001
).
The mRNA for macho 1 is one of a large number of maternal mRNAs
(called postplasmic RNAs) that concentrate at the posterior pole of the
cleaving embryos. The original, most abundant posteriorly-localized mRNA,
called pem (for posterior end mark), was identified in Ciona
(Yoshida et al., 1996
). This
was followed by the discovery of several mRNAs (pem-2-pem-6)
with similar localization (Satou and
Satoh, 1997
). To date, 28 postplasmic mRNAs have been identified
in Halocynthia, in the MAGEST maternal mRNA database
(http://www.genome.ad.jp/magest/)
(Sasakura et al., 2000
;
Makabe et al., 2001
;
Nakamura et al., 2003
).
The Halocynthia homologue of Ciona pem, HrPEM, plus
macho 1 mRNA and some postplasmic mRNAs in Halocynthia are
already located before fertilization in an animal-vegetal gradient (classified
as type I postplasmic RNAs) (Nishida and
Makabe, 1999; Sasakura et al.,
2000
; Makabe et al.,
2001
; Nishida and Sawada,
2001
; Nakamura et al.,
2003
) (the present study). These type I postplasmic mRNAs then
become concentrated in the vegetal cortex after fertilization and relocalize
to the posterior cortex before first cleavage (see
Fig. 1 for HrPEM and
macho 1). Co-ordinated relocalization of cortical and cytoplasmic
domains has been well documented in ascidians since 1905, when Conklin
described the myoplasm in the egg. This is a pigmented peripheral cytoplasmic
domain that is rich in mitochondria and inherited by muscle cells
(Jeffery, 1995
;
Nishida, 1997
;
Satoh, 2001
). The
concentration and translocation of the myoplasm occurs between fertilization
and first cleavage in two main phases that consists of several subphases of
cytoplasmic and cortical reorganizations
(Sardet et al., 1989
;
Chiba et al., 1999
;
Roegiers et al., 1999
). These
reorganizations (historically called ooplasmic segregation) are driven by the
interplay of microfilaments and/or astral microtubules in the cortex and
subcortex. In this way, at least five cortical and cytoplasmic domains are
stratified in the vegetal hemisphere by the time meiosis ends
(Roegiers et al., 1999
). After
meiosis, two of the domains, the cortical ER-rich domain and the bulk of the
mitochondria-rich subcortical-myoplasm domain, move posteriorly and are
distributed equally between the first two blastomeres.
|
We have shown that it is possible to isolate the cortex of eggs of the
ascidian Phallusia mammillata by gently shearing eggs and embryos
fixed to a polycationic surface using a stream of isotonic solution that
mimicks the cytoplasm of the egg (Sardet
et al., 1992). Isolated cortices are constituted of characteristic
networks of cER, microfilaments and some microtubules, all of which remain
strongly attached to the plasma membranes during the isolation procedure
(Sardet et al., 2002
). In
unfertilized ascidian eggs the cER network is a monolayer distributed along
the animal-vegetal gradient (Sardet et
al., 1992
). It is characterized by the presence of numerous sheets
and tubes in the vegetal hemisphere and by a sparse distribution of tubes in
the animal hemisphere. After fertilization, this gradient distribution of the
cER network increases via microfilament-driven cortical contractions which
concentrate the cER into a 2- to 5-µm-thick layer around the contracted
vegetal pole (Gualtieri and Sardet,
1989
; Speksnijder et al.,
1993
). The vegetal cER network is then displaced towards the
posterior pole via microtubule-driven translocations mediated by the sperm
aster (Speksnijder et al.,
1993
). The bulk of the cER network and the myoplasm are
distributed equally in the posterior region of the first two blastomeres after
amphimixy, mitosis and cleavage (Roegiers
et al., 1999
). The cER is of particular interest because at low
resolution its location and displacement after fertilization seem to
correspond closely to the position and relocalizations of type I postplasmic
RNAs.
In the present work, we examined the precise localization of HrPEM and macho 1 in eggs, zygotes, embryos and isolated cortices using high-resolution in situ hybridization. Our results showed that HrPEM and macho 1 mRNAs could be isolated with the cortex and that both were associated with the network of rough cER. We propose that some postplasmic mRNAs translocate and relocalize with the cER network in the egg cortex following fertilization and accumulate with cER in the CAB during cleavages. A multimedia BioClip `Polarity inside the egg cortex' (www.bioclips.com/bioclip.html) describes this in more detail.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of cortices from eggs and embryos
Isolated cortices were prepared using cortex solutions (Buffer X, EMC and
CIM buffer) and observed fixed or live as described
(Sardet et al., 1992).
Devitellinated eggs (15-30), synchronously developing zygotes and embryos were
deposited in a Ca2+-free medium (EMC solution) onto
polylysine-coated coverslips. Unfertilized eggs that were attached, fertilized
eggs (20-30 minutes after fertilization) and 8-cell-stage embryos (4-5 hours)
were immediately sheared with a gentle stream of Millipore-filtered isotonic
cortex solution (Buffer X) to which was added 1 unit µl1
RNase inhibitor (Toyobo, Osaka, Japan). The cortices were fixed within 1-2
minutes after isolation in a freshly prepared mixture of paraformaldehyde (4%)
and glutaraldehyde (0.1%) in CIM buffer. After 30 minutes fixation in a humid
chamber at 11°C, cortices were washed in CIM buffer and stored for <6
hours in phosphate-buffered saline (PBS) to which was added 1 unit
µl1 RNase inhibitor. The ER network in isolated cortices
was labelled with carbocyanine dyes, DiIC16(3) (Molecular Probes, Eugene, OR,
USA) emulsion or a solution of DiOC6(3) (Molecular Probes) in either CIM
buffer or Buffer X as described (Sardet et
al., 1992
). Each cortex-loaded coverslip was inverted and mounted
on a microscope slide using two strips of double-sided sticky tape spacers,
then fixed firmly with epoxy glue on the left and right sides to form a
perfusion chamber. Several cortices were identified by their position on the
coverslip and photographed to record the cER distribution. These cortices were
identified again after in situ hybridization to record the distribution of
specific mRNAs. Cortices were observed and photographed by fluorescence
microscopy (Nikon) and confocal microscopy using a Nipkow disc-based apparatus
(Yokogawa, Tokyo, Japan) coupled to a fluorescence microscope (Zeiss).
In situ hybridization
Specimens were hybridized in situ with digoxigenin (DIG)-labelled
HrPEM and macho 1 antisense probes that cover the entire
cDNAs (GenBank accession numbers, AB045129 for HrPEM and AB045124 for
macho 1). Arginyl-tRNA synthetase was used as a probe for
ubiquitously distributed maternal mRNA (GenBank accession number, AV383566).
Detection of mRNA in whole-mount specimens was carried out essentially as
described previously (Miya et al.,
1994).
For cortex specimens, the in situ protocol was carried out without treating
fixed samples with detergent such as Tween 20, solvent such as EtOH, and
proteolytic enzymes such as proteinase K. Solutions between the coverslip and
the microscope slides of the perfusion chamber were changed by drawing
solutions from one side with a piece of filter paper whilst perfusing the next
solution from the opposite side. The slides were maintained in handmade humid
chambers throughout the in situ hybridization protocol. After washing with
PBS, samples were prehybridized for 1 hour at 42°C. Then the specimens
were allowed to hybridize with the DIG-labelled antisense probe (approximately
0.1 µg ml1) overnight at 42°C. The specimens were
treated with 20 µg ml1 RNase A (Sigma) for 30 minutes at
37°C and washed. The conventional coloring using alkaline-phosphatase
conjugated anti-DIG antibody was performed as described
(Miya et al., 1994).
The tyramide signal amplification method (TSA Biotin System, NEN Life
Science Products, Boston) was used to obtain high-resolution fluorescent
images of mRNA localization (Wilkie and
Davis, 1998). This technique depends on peroxidase-mediated
deposition of biotin-labelled tyramide at the location of the antisense RNA
probe labelled with DIG. After washing of hybridized embryos and cortices,
samples were treated with an HRP-conjugated anti-DIG antibody Fab fragment
(1:2000, Boehringer Mannheim, Mannheim, Germany) overnight at 4°C. The TSA
reaction was carried out for 10 minutes according to the supplier's
instructions. Samples were then washed with PBS for 30 minutes and reacted
with Alexa green 488-conjugated streptavidin (1:100, Molecular Probes, Eugene,
OR, USA) for 60 minutes. After washing with PBS for 60 minutes, samples were
perfused with 80% glycerol and observed under a fluorescent microscope or a
confocal microscope. This method gives high resolution because the peroxidase
reaction produces tyramide radicals that react covalently with proteins at the
site of HRP activity, which prevents appreciable diffusion of the signal and
fluorescently labelling the structures surrounding the target mRNA.
Electron microscopy of isolated cortices
Isolated cortices fixed as described above were treated briefly with
OsO4, fast-frozen and processed for deep-etching and replication as
described (Sardet et al.,
1992). Replicas were examined in a Hitachi electron
microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The changing localization of HrPEM and macho 1 maternal mRNAs is shown in Fig. 1. In unfertilized eggs, HrPEM mRNA, the most abundant postplasmic RNA and macho 1 (not shown) formed a thin cortical layer along the animal-vegetal gradient, with a maximum concentration in the vegetal hemisphere (Fig. 1A). High-resolution examination of the surface by confocal microscopy showed that HrPEM and macho 1 are located in a reticulated structure <2 µm thick situated beneath the plasma membrane (Fig. 1A1). The mRNAs became further concentrated in the vegetal pole region as a consequence of the first major phase of ooplasmic segregation, which occurs 20-30 minutes after fertilization (Fig. 1B,B1). After completion of meiosis (40-50 minutes), postplasmic mRNAs moved to the future posterior pole during a second major phase of relocalization (80-100 minutes, Fig. 1C,C1). As a result of these cortical relocalizations, the cortical structure decorated by HrPEM and macho 1 thickened to 5-8 µm (Fig. 1A1-C1). This mRNA-rich cortical structure generally appears reticulate (see particularly the tangential section shown in Fig. 1A2,B2) and sometimes punctate (possibly because the formaldehyde fixation does not preserve the continuity of the structure). Some labelled structure apparently detached from the surface after the second phase of relocalization (Fig. 1C1).
Zygotes of Halocynthia start to cleave at about 140 minutes. The
first cleavage partitioned the posteriorly and cortically localized mRNAs,
such as HrPEM and macho 1, equally between the first two
blastomeres (data not shown). At the 8-cell stage, only posterior-vegetal
(B4.1) blastomeres inherited postplasmic mRNAs. These RNAs are concentrated in
a tiny area of the posterior region of the B4.1 blastomeres
(Fig. 1D-G). This region
corresponded to the CAB, the cortical structure that attracts the centrosome
and nucleus towards the posterior pole of 8- to 64-cell-stage embryos
(Nishikata et al., 1999).
Posterior-animal views of the 8-cell embryos displayed the moustache-like
shape of the mRNA localization, which corresponds to the characteristic shape
of the CAB just before unequal cleavage starts
(Fig. 1F,G).
HrPEM and macho 1 mRNAs are retained in cortices
isolated from eggs and embryos
Because the appearance and movements of the reticulated structure decorated
by HrPEM and macho 1 reminded us of the cER situated beneath
the egg surface (Roegiers et al.,
1999; Speksnijder et al.,
1993
), we isolated cortices from eggs, zygotes and embryos of
Halocynthia using the methods previously described for unfertilized
eggs of Phallusia (Sardet et al.,
1992
). We isolated cortices after the first phase of ooplasmic
segregation, when postplasmic RNAs are concentrated in the vegetal-pole region
(Fig. 2A-D), and from 8-cell
embryos where they make a small posterior mark
(Fig. 2E-I). Characteristic
fields of pancake-like imprints that represent fragments of cortices could be
observed on the coverslip (Fig.
2A,C,E). To investigate the localization of mRNAs at the
subcellular level, it was essential to preserve the structures of these
isolated cortices and, in particular, their membranes (plasma membrane, cER
network and occasional membranous organelles). To this end, we omitted the
permeabilization, digestion and extractions steps (using Triton X100,
proteinase and ethanol) that are generally included after fixation in
conventional in situ hybridization procedures.
|
Cortices could also be isolated from the 8-cell embryos, which are cube-like. Groups of four, cortical, pancake-like fragments were detected (Fig. 2E,F). In roughly 1 in 6 cases, which represented embryos that fell on the posterior side, isolated cortices retained the characteristic mRNA-rich moustaches (Fig. 2E-I). These moustaches correspond to the postplasmic RNA-rich CAB in Fig. 1D-G. By contrast, pieces of cytoplasm that occasionally adhered to isolated cortices did not hybridize appreciably to HrPEM and macho 1 maternal mRNAs (data not shown).
To verify the specificity of the localization of HrPEM and
macho 1 mRNAs in the cortex, we examined whether a ubiquitously
distributed maternal mRNA in egg cytoplasm (arginyl-tRNA synthetase;
T. Okada and H. Nishida, unpublished) was retained in isolated cortices. The
prevalence of this mRNA in eggs is thought to be comparable to that of
macho 1, based on the EST data of MAGEST, an EST database of maternal
mRNA in Halocynthia (Makabe et
al., 2001). When cortices isolated from eggs and 8-cell embryos
were examined, the signal from arginyl-tRNA synthetase mRNA is very
weak compared with HrPEM and macho 1 mRNAs (see
Fig. 2F, in which overexposure
was used to increase the background signal), which indicates that noncortical
mRNAs are not retained appreciably in isolated cortices.
Isolated cortices are characterized by a polarized cER network that
adheres to the plasma membrane
The cER network can be made visible in live cortices with the lipophilic
dyes DiIC16(3). As in Phallusia, the unfertilized egg cortex of
Halocynthia was characterized by the presence of a continuous
monolayer of cER tubes and sheets, which form a carpet that adheres firmly to
the underside of the plasma membrane (Fig.
3A-C). Outside the attached cortices, the inner cytoplasmic tubes
of ER that connected with the cER were usually projected onto the glass
surface by the stream of cortex solution, forming a characteristic comet tail
in the direction of shear (Fig.
4A,C,D,F,G). High-resolution electron microscopy of replicas of
cortices generated by the fast-freeze deep-etch technique showed that the
tubes and sheets of the cER were studded with particles
(Fig. 3C,D). Most particles on
the cER had the same size and shape, and were most likely ribosomes
(Fig. 3D), as shown for
Phallusia cortices (Sardet et
al., 1992). Therefore, the cER of Halocynthia cortices is
rough ER. However, a few particles were smaller or had elongated shapes,
different from those of ribosomes.
|
|
HrPEM and macho 1 bind to the cER network in
cortices isolated from eggs and zygotes
The cER network in the unfertilized egg is a monolayer of interconnected
tubes and sheets attached to the plasma membrane, so this is the easiest stage
at which to analyse colocalization of fluorescently labelled mRNAs and cER. At
the highest resolution possible with the fluorescent microscope, mRNAs for
HrPEM and macho 1 were distributed in a network that
coincided precisely with the cER network
(Fig. 4A,E-G). It was also
clear that there was no appreciable hybridization of the probes for
HrPEM-1 and macho 1 on ER tubes that originated in deeper
cytoplasmic regions. Such inner cytoplasmic strands of ER often lie outside
the cortex in the comet tails produced during shearing of the eggs, and are in
continuity with the cER network (Fig.
4A,D,F). The lack of signal on noncortical ER indicates that the
mRNA signal detected on cER is not caused by nonspecific binding of the
postplasmic mRNAs to the ER of the whole egg. Further evidence for the
specificity of localization of HrPEM and macho 1 on the cER
network was provided by the low amount of binding of the sense probes for
HrPEM and macho 1, and the antisense probes for the
ubiquitously distributed maternal mRNA of arginyl-tRNA synthetase on the cER
network (Fig. 2F). To further
test the binding of mRNAs for HrPEM and macho 1 to the cER
network, we exposed live cortices to hypotonic treatment, which vesiculates
the cER network. Previously, we showed that many of the ER vesicles remain
attached to the plasma membrane even after hypotonic treatment
(Sardet et al., 1992). The
HrPEM mRNA hybridization pattern in this case appeared as lines of
fluorescently double-labelled spots that coincided with vesiculated ER
(Fig. 4H). These observations,
therefore, provide strong evidence that cortical postplasmic mRNAs, such as
HrPEM and macho 1, are localized on the monolayer of cER
that is attached to the cytoplasmic face of the plasma membrane in the
unfertilized egg.
We have previously shown that we could strip the cER of particles
(ribosomes and others) by treating isolated Phallusia cortices with a
solution of puromycin and KCl (Sardet et
al., 1992). The puromycin-KCl treatment dramatically decreased the
signal for HrPEM RNA on the cER network of unfertilized
Halocynthia eggs (Fig.
4A,C).
The colocalization of HrPEM and macho 1 mRNA with the cER is also evident in egg cortices after the cortical contraction triggered by fertilization, at the end of the first phase of ooplasmic segregation, 20 minutes after fertilization (Fig. 4B,D). However because the cER network in the vegetal region of isolated cortices is much thicker and compacted by the cortical contraction (Fig. 1B), precise colocalization is seen only using confocal microscopy (Fig. 4B,D). In these isolated cortices, microvilli at the vegetal pole, which resulted from fertilization-induced cortical contraction, were labelled with the lipophilic dye DiIC16(3) but did not contain significant HrPEM mRNA. This indicates that RNA localization is restricted to the cER network (Fig. 4B).
HrPEM and macho 1 mRNA is concentrated in the CAB
together with the cER
As described previously (Fig.
2E-I) and shown at higher resolution in
Fig. 5A, cortices isolated from
mid-8-cell-stage embryos typically contained striking patches of cER
accumulation in the shape of moustaches. The two sides of the moustaches
correspond to the two CABs present in the cortex of the two B4.1
posterior-vegetal blastomeres (Fig.
5A). Our observation that tight packing of cER filled most of the
volume of the CAB that were isolated with Halocynthia cortices fits
with the previous detection of many vesicles in the CAB by electron microscopy
(Iseto and Nishida, 1999). The
CAB of 8-cell-stage Halocynthia embryos is a macroscopic cortical
structure
4-7 µm thick, 5-10 µm wide and 30-50 µm long. Both
DiIC16(3) and DiOC6(3) gave identical labelling patterns that revealed the
topography of the ER network in isolated cortices
(Fig. 3E,F,
Fig. 5F). The ER in the CAB
seems to be a continuous network of tightly packed tubes
(Fig. 3E,F), which are unfolded
occasionally by shearing force (Fig.
5D). The ER network compacted in the CAB was connected to tubes of
the cER network that extended as a monolayer to line the plasma membrane
outside the CAB (Fig. 3E,
Fig. 5C). In some cases, large
vesicles were embedded in the ER-rich CAB; we presume that this represents an
early stage of compaction of the CAB (Fig.
3E). As expected, the tubular ER network inside and outside the
CAB vesiculated when live, 8-cell-stage cortices were perfused with
hypo-osmotic solutions (Fig.
3G).
|
At the highest possible magnification of the CAB with confocal microscopy, we observed partial colocalization of ER labelled with DiIC16(3) and of mRNA fluorescently labelled with Alexa green 488. Inside the CAB, the ER network was so tightly compacted that it was difficult to determine the exact degree of colocalization of the ER and mRNAs (Fig. 5F,G). Fortunately, the network of ER tubes and sheets compacted in the CAB occasionally stretched like spaghetti under the shear force applied to prepare cortices (Fig. 2E, Fig. 5D,E). In these `stretched' CABs, the large patches of colocalization of ER and HrPEM mRNA were evident. However, although some mRNA patches coincided with the ER network, others seemed to fill the space between ER sheets and tubes (see green regions indicated by arrowheads in Fig. 5E).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The association of HrPEM and macho 1 mRNAs with the cER network, which is present initially as a gradient in the unfertilized egg, provides a simple explanation for the polarization and relocalization of at least some type I postplasmic RNAs, and their ultimate accumulation in the CAB, the structure responsible for unequal cleavages at the 8-64-cell stage. Our observations also indicate that the primary muscle determinant the cortical maternal mRNA macho 1 probably reaches its site of localized accumulation in the CAB as a passenger of the cER network. Localization and translocation of cER therefore appear to provide a simple mechanism for the localization of specific cortical mRNAs to a subset of blastomeres.
Postplasmic mRNAs are associated with the cortical ER network of
eggs
Microtubules, microfilaments and ER have been implicated in the
translocation and anchoring of mRNAs to the cortex in yeast, somatic cells,
oocytes and embryos (reviewed in Bassell et
al., 1999; Lasko,
1999
; Jansen,
2001
; Kloc et al.,
2002
; Palacios,
2002
). However, it has been difficult to directly visualize the
association between mRNAs and these subcellular structures, because fixation,
digestion and permeabilization procedures used for high-resolution in situ
localization studies alter the integrity of cellular membranes and subcellular
organization. High-resolution localization studies of HrPEM and
macho 1 in isolated cortices (an open-cell preparation that needs no
permeabilization) provided direct evidence that the rough cER network that is
attached to the plasma membrane is a main site of localization for some
cortical mRNAs in unfertilized ascidian eggs. We cannot presume, however, that
all type I postplasmic RNAs associate with the cER. At present, we do not know
how type I postplasmic mRNAs reach their cortical location during oogenesis.
In Xenopus oocytes it has been suggested that that localizing mRNAs
(such as maternal Veg1 mRNA) might move with the ER to the vegetal
cortex during oogenesis (Deshler et al.,
1997
; Schnapp,
1999
). It will be interesting to see whether the cER network and
cortical mRNAs such as HrPEM and macho 1 reach their
cortical location separately or together during oogenesis and maturation, and
whether this happens at the same time as the polarized mitochondria-rich
myoplasm becomes established as a subcortical basket
(Swalla et al., 1991
). Another
important question is the nature of the localization signal in these
postplasmic RNAs. Recently, Sasakura and Makabe
(Sasakura and Makabe, 2002
)
showed that small elements in the 3'-UTR region are sufficient for
posterior localization of Halocynthia type I postplasmic mRNAs such
as HrWnt-5 and HrPOPK-1, as is the case for localized
Drosophila and Xenopus mRNAs
(Betley et al., 2002
).
For the moment we can only speculate about the nature of the binding of
HrPEM and macho 1 to the cER network. We have examined the
distribution of cER, microtubules and microfilaments in the isolated cortex
(Sardet et al., 1992). The
fine distribution of microtubules and microfilaments is very different from
that of cER. Therefore, the possibility that these cytoskeletal elements
mediate association of cER and mRNAs is unlikely. Sasakura at al.
(Sasakura at al., 2000
)
observed the localization of some type I postplasmic RNAs (HrZf-1,
HrPOPK-1 and HrWnt-5) in fertilized eggs treated with
cytoskeletal inhibitors. Treatment with cytochalasin and nocodazole (which
disassemble microfilaments and microtubules) disturbed the translocation of
those postplasmic RNAs as well as the first and second phases of ooplasmic
segregation respectively, as observed previously (Sardet, 1989;
Speksnijder et al., 1993
).
However, these inhibitors do not cause postplasmic RNAs to detach from the
cortex. This indicates that cytoskeletal elements are required for
translocation of cER but not for the association of mRNAs to cER. At present
there is no way to disrupt intermediate filaments in ascidian embryos.
One possibility is that these mRNAs bind to ribosomes or other particles
present at the surface of the rough cER network. This is indicated by the fact
that puromycin-KCl treatment detaches the bulk of HrPEM and macho
1 from the cER network in isolated cortices. This treatment was used
originally to detach ribosomes from the rough ER-microsome fraction
(Sabatini et al., 1966) and
also detaches ribosomes and other particles from the cER in the isolated
cortex of Phallusia eggs (Sardet
et al., 1992
). In Phallusia, the cER network can be
labelled with the RNA dye thiazole orange and the staining is abolished by the
puromycin-KCl treatment (Sardet et al.,
1992
). Because rough ER is generally thought to be a site of
localization for mRNAs that encode membrane or secreted proteins, it might
seem surprising to find that mRNAs such as HrPEM and macho
1, which do not code for such proteins, are also associated with the cER.
However, using DNA microarrays for the large-scale identification of secreted
and membrane-associated gene products has revealed recently that many mRNAs
encoding cytosolic proteins are associated with microsomal membranes. This
indicates that the rough ER might be a major site of mRNA localization
(Diehn et al., 2000
).
Localization of specific mRNAs to a cER network has been shown most clearly in
rice seeds, in which a specialized subdomain of rough ER is the major
localization site for mRNAs that encode prolamin-storage proteins
(Choi et al., 2000
). It has
also been hypothesized that in Xenopus oocytes, developmentally
important mRNAs which code for secreted proteins, such as Vg1, are
associated with ER during their transport to the cortex, possibly via Vera,
the conserved RNA zipcode-binding protein
(Deshler et al., 1997
;
Mowry and Cote, 1999
).
Although anchoring of Vg1 to the cortex seems to depend principally
on the presence of keratin filaments in isolated Xenopus cortices, it
might also depend on the cER because Vg1 mRNA is lost from isolated
cortices after detergent treatment (Alarcon
and Elinson, 2001
).
In yeast, fibroblasts and nerve cells, translocation and anchoring of mRNAs
involves large irregular particles (0.2-0.7 µm) that, apparently, contain
many types of mRNAs, proteins, a large number of ribosomes and, in some cases,
ER (reviewed in Jansen, 2001;
Kloc et al., 2002
). This is
also apparently the case in Drosophila oocytes, in which
translocation and localization of bicoid mRNA in the cortex seems to
be mediated by large particles, in particular by ER-containing `sponge bodies'
(Wilsch-Brauninger et al.,
1997
). A role for intracellular membranes in the localization of
Oskar has also been recently proposed
(Jansen, 2001
;
Dollar et al., 2002
). In
addition, there are several indications that rough ER may play a role in mRNA
localization in neuronal dendrites because the translocating particles contain
the RNA-binding protein Staufen, which is clearly associated with rough ER in
these cells (Kohrmann et al.,
1999
). Because Staufen is also essential for the MT-dependent
localizations of the determinants oskar and bicoid to the
poles of the Drosophila oocyte, and for the microfilament-mediated
cortical localization of the homeobox-containing transcription factor
prospero in neuroblasts (Roegiers
and Nung Jang, 2000
), it is possible that cortical mRNA
localization in Drosophila also relies on binding to the ER and on
its translocation.
Postplasmic RNAs are relocated with the cER network after
fertilization
It is well established that in all ascidians examined, the cortically
localized postplasmic RNAs, and the determinants for muscle and endoderm
formation, concentrate in the vegetal cortex under the influence of the
cortical microfilament-driven contraction that is triggered by the fertilizing
sperm (Nishida, 1997;
Chiba et al., 1999
;
Roegiers et al., 1999
). We
have shown that, in Phallusia, this contraction is a consequence of
the Ca2+ wave that is triggered at the site of sperm entry, and
that it results in the accumulation of cER into a 2-5 µm-thick cortical
patch that is located in a microvillus-rich contraction pole in the vegetal
hemisphere (Roegiers et al.,
1995
; Roegiers et al.,
1999
). In the present work we showed that in the much larger egg
of Halocynthia, a cER accumulation also forms in the vegetal pole
region and that it is retained in the isolated cortex. This cER accumulation
concentrates the postplasmic RNAs in a 5-8 µm-thick layer in the vegetal
pole region. If contraction is inhibited by cytochalasin, type I postplasmic
RNAs do not concentrate vegetally, but form multiple cortical patches
(Sasakura et al., 2000
), a
behaviour we also noted in the cER network of Phallusia eggs
(Speksnijder et al.,
1993
).
In the Phallusia zygote, the vegetal accumulation of cER moves to
a posterior equatorial location after meiosis completion. This second major
phase of reorganization is initiated by a displacement of the sperm aster and
of its microtubules with respect to the cortex, and is completed by the
subsequent microfilament-mediated wave of cortical relaxation initiated in the
vegetal pole just before cleavage
(Roegiers et al., 1999). We
have not yet analysed this posterior translocation of cER in
Halocynthia. However, because cortical and cytoplasmic
relocalizations and cleavage patterns appear well conserved in ascidian
species, we assume that some postplasmic RNAs move posteriorly with the cER
accumulation that formed first in the vegetal pole region after
fertilization.
Postplasmic mRNAs and cER concentrate in the CAB
Our present work indicates that the extreme concentration of postplasmic
RNAs in a small posterior region of the B4.1 posterior-vegetal blastomeres is
related to the packing of most of the cER in the unfertilized egg into one
tight mass that fills a large part of the volume occupied by the CAB (Figs
3,
5). It has been observed by the
electron microscopy that a dense matrix containing vesicles is concentrated in
the CAB of Halocynthia (Iseto and
Nishida, 1999).
Confocal light microscopy sectioning of either live or fixed cortices
isolated at the 8-cell stage showed that the cER formed a tight network in the
CAB, and that this network is connected with a cER network that surrounds the
CAB and lines the plasma membrane. How compaction of the cER occurs during
early cleavages is unknown. Microtubules are, apparently, not involved in the
formation of the CAB, whose precursors first appear as a number of small
cortical particles at the 2-cell stage
(Hibino et al., 1998;
Nishikata et al., 1999
).
Cortical microfilaments or `adhesion complexes' on the surface of the ER might
participate in the progressive aggregation of cER into one compact CAB
domain.
We observed that the cER near the CAB is covered with patches of HrPEM mRNA, and that the contiguous cER in the same blastomere and the cER network in the animal or anterior blastomeres is devoid of HrPEM and macho 1 mRNA. This argues for the presence of a specific mRNA-rich cER domain in the unfertilized egg that is inherited selectively by posterior-vegetal blastomeres. This cER could also be considered the precursor of the CAB.
The egg cortex as a repository of spatio-temporal information
Our observations have interesting implications for the localization of some
type I postplasmic RNAs in the cortex, and in particular of the muscle
determinant macho 1. It is clear that the compaction of the cER
network underlying CAB formation represents a way to segregate important
cortical mRNAs, such as macho 1, in a small localized area of the
cortex in specific blastomeres. By analogy with what is known of the large
particles that localize mRNA in many cells, including yeast, neurons and
oocytes (reviewed in Jansen,
2001; Kloc et al.,
2002
), we suspect that in the 8-cell-stage ascidian embryo, a
translational machinery is concentrated in the mRNA patches that we observed
on the cER in and near the CAB. Localization and concentration of the
translational machinery and of macho 1 mRNA could be essential for
the activation of the translation of the muscle determinant macho 1.
Indeed, in order for macho 1 mRNA to be the primary muscle
determinant, macho 1 protein must be produced before or at the time the
compact CAB is formed, i.e. before the unequal cleavage of the 8-16-cell stage
separates the ER-rich CAB from the mitochondria-rich myoplasm domain inherited
by primary muscle cell blastomeres.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alarcon, V. B. and Elinson, R. P. (2001). RNA
anchoring in the vegetal cortex of the Xenopus oocyte. J.
Cell Sci. 114,1731
-1741.
Bassell, G. J., Oleynikov, Y. and Singer, R. H.
(1999). The travels of mRNAs through all cells large and small.
Faseb J. 13,447
-454.
Betley, J. N., Frith, M. C., Graber, J. H., Choo, S. and Deshler, J. O. (2002). A ubiquitous and conserved signal for RNA localization in chordates. Curr. Biol. 12,1756 -1761.[CrossRef][Medline]
Chiba, S., Miki, Y., Ashida, K., Wada, M. R., Tanaka, K. J., Shibata, Y., Nakamori, R. and Nishikata, T. (1999). Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs. Dev. Growth Differ. 41,265 -272.[CrossRef][Medline]
Choi, S. B., Wang, C., Muench, D. G., Ozawa, K., Franceschi, V. R., Wu, Y. and Okita, T. W. (2000). Messenger RNA targeting of rice seed storage proteins to specific ER subdomains. Nature 407,765 -767.[CrossRef][Medline]
Deshler, J. O., Highett, M. I. and Schnapp, B. J.
(1997). Localization of Xenopus Vg1 mRNA by vera protein and the
endoplasmic reticulum. Science
276,1128
-1131.
Diehn, M., Eisen, M. B., Botstein, D. and Brown, P. O. (2000). Large-scale identification of secreted and membrane-associated gene products using DNA microarrays. Nat. Genet. 25,58 -62.[CrossRef][Medline]
Dollar, G., Struckhoff, E., Michaud, J. and Cohen, R. S. (2002). Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development 129,517 -526.[Medline]
Gualtieri, R. and Sardet, C. (1989). The endoplasmic reticulum network in the ascidian egg: localization and calcium content. Biology of the Cell 65,301 -304.[CrossRef]
Hibino, T., Nishikata, T. and Nishida, H. (1998). Centrosome-attracting body: A novel structure closely related to unequal cleavages in the ascidian embryos. Dev. Growth Differ. 40,85 -95.[Medline]
Iseto, T. and Nishida, H. (1999). Ultrastructural studies on the centrosome-attracting body: electron-dense matrix and its role in unequal cleavages in ascidian embryos. Dev. Growth Differ. 41,601 -609.[CrossRef][Medline]
Jansen, R. P. (2001). mRNA localization: message on the move. Nature Rev. Molec. Cell Biol. 2, 247-256.[CrossRef]
Jeffery, W. R. (1995). Development and evolution of an egg cytoskeletal domain in ascidians. Curr. Top. Dev. Biol. 31,243 -276.[Medline]
King, M. L., Zhou, Y. and Bubunenko, M. (1999). Polarizing genetic information in the egg: RNA localization in the frog oocyte. BioEssays 21,546 -557.[CrossRef][Medline]
Kloc, M., Zearfoss, N. R. and Etkin, L. D. (2002). Mechanisms of subcellular mRNA localization. Cell 108,533 -544.[Medline]
Kohrmann, M., Luo, M., Kaether, C., DesGroseillers, L., Dotti,
C. G. and Kiebler, M. A. (1999). Microtubule-dependent
recruitment of Staufen-green fluorescent protein into large RNA-containing
granules and subsequent dendritic transport in living hippocampal neurons.
Mol. Biol. Cell. 10,2945
-2953.
Lasko, P. (1999). RNA sorting in Drosophila
oocytes and embryos. Faseb J.
13,421
-433.
Makabe, K. W., Kawashima, T., Kawashima, S., Minokawa, T.,
Adachi, A., Kawamura, H., Ishikawa, H., Yasuda, R., Yamamoto, H.,
Kondoh, K. et al. (2001). Large-scale cDNA analysis of the
maternal genetic information in the egg of Halocynthia roretzi for a gene
expression catalog of ascidian development.
Development 128,2555
-2567.
Mita-Miyazawa, I., Ikegami, S. and Satoh, N. (1985). Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replication. J. Embryol. Exp. Morphol. 87,1 -12.[Medline]
Miya, T., Makabe, K. W. and Satoh, N. (1994). Expression of a gene for major mitochondrial protein, ADP/ATP translocase, during embryogenesis in the ascidian Halocynthia roretzi. Dev. Growth Differ. 36,39 -48.
Mowry, K. L., and Cote, C. A. (1999). RNA
sorting in Xenopus oocytes and embryos. Faseb J.
13,435
-445.
Nakamura, Y., Makabe, K. W. and Nishida, H. (2003). Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi. Gene Expression Pattern 3,71 -75.[CrossRef]
Nishida, H. (1997). Cell fate specification by localized cytoplasmic determinants and cell interactions in ascidian embryos. Int. Rev. Cytol. 176,245 -306.[Medline]
Nishida, H. (2002). Specification of developmental fates in ascidian embryos: molecular approach to maternal determinants and signaling molecules. Int. Rev. Cytol. 217,227 -276.[Medline]
Nishida, H. and Makabe, K. W. (1999). Maternal information and localized maternal mRNAs in eggs and early embryos of the ascidian Halocynthia roretzi. Invert. Reprod. Dev. 36, 41-49.
Nishida, H. and Sawada, K. (2001). macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis. Nature 409,724 -729.[CrossRef][Medline]
Nishikata, T., Hibino, T. and Nishida, H. (1999). The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo. Dev. Biol. 209, 72-85.[CrossRef][Medline]
Palacios, I. M. (2002). RNA processing: splicing and the cytoplasmic localisation of mRNA. Curr. Biol. 12,R50 -52.[CrossRef][Medline]
Riechmann, V. and Ephrussi, A. (2001). Axis formation during Drosophila oogenesis. Curr. Opin. Genet. Dev. 11,374 -383.[CrossRef][Medline]
Roegiers, F. and Nung Jang, Y. (2000). Staufen: a common component of mRNA transport in oocytes and neurons? Trends Cell Biol. 10,220 -224.[CrossRef][Medline]
Roegiers, F., McDougall, A. and Sardet, C.
(1995). The sperm entry point defines the orientation of the
calcium-induced contraction wave that directs the first phase of cytoplasmic
reorganization in the ascidian egg. Development
121,3457
-3466.
Roegiers, F., Djediat, C., Dumollard, R., Rouviere, C. and
Sardet, C. (1999). Phases of cytoplasmic and cortical
reorganizations of the ascidian zygote between fertilization and first
division. Development
126,3101
-3117.
Sabatini, D. D., Tashiro, Y. and Palade, G. E. (1966). On the attachment of ribosomes to microsomal membrane. J. Mol. Biol. 19,503 -524.[Medline]
Sardet, C., Speksnijder, J. E., Inoue, S. and Jaffe, L. F. (1989). Fertilization and ooplasmic movements in the ascidian egg. Development 105,237 -249.[Abstract]
Sardet, C., Speksnijder, J., Terasaki, M. and Chang, P. (1992). Polarity of the ascidian egg cortex before fertilization. Development 115,221 -237.[Abstract]
Sardet, C., Prodon, F., Dumollard, R., Chang, P. and Chenevert, J. (2002). Structure and Function of the Egg Cortex from Oogenesis through Fertilization. Dev. Biol. 241, 1-23.[CrossRef][Medline]
Sasakura, Y. and Makabe, K. (2002). Identification of cis elements which direct the localization of maternal mRNAs to the posterior pole of ascidian embryos. Dev. Biol. 250,128 -144.[CrossRef][Medline]
Sasakura, Y., Ogasawara, M. and Makabe, K. W. (2000). Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos. Dev. Biol. 220,365 -378.[CrossRef][Medline]
Satoh, N. (1994). Developmental Biology of Ascidians. New York: Cambridge University Press.
Satoh, N. (2001). Ascidian embryos as a model system to analyze expression and function of developmental genes. Differentiation 68,1 -12.[CrossRef][Medline]
Satou, Y. and Satoh, N. (1997). posterior end mark 2 (pem-2), pem-4, pem-5, and pem-6: Maternal genes with localized mRNA in the ascidian embryo. Dev. Biol. 192,467 -481.[CrossRef][Medline]
Schnapp, B. J. (1999). RNA localization: A glimpse of the machinery. Curr. Biol. 9,R725 -R727.[CrossRef][Medline]
Speksnijder, J. E., Terasaki, M., Hage, W. J., Jaffe, L. F. and Sardet, C. (1993). Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg. J. Cell Biol. 120,1337 -1346.[Abstract]
Swalla, B. J., Badgett, M. R. and Jeffery, W. R. (1991). Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development. Development 111,425 -436.[Abstract]
Wilkie, G. and Davis, I. (1998). Visualizing mRNA by in situ hybridization using `high resolution' and sensitive tyramide signal amplification. Technical Tips Online (http://research.bmn.com/tto).
Wilsch-Brauninger, M., Schwarz, H. and Nusslein-Volhard, C.
(1997). A sponge-like structure involved in the association and
transport of maternal products during Drosophila oogenesis. J. Cell
Biol. 139,817
-829.
Yoshida, S., Marikawa, Y. and Satoh, N. (1996).
Posterior end mark, a novel maternal gene encoding a localized factor
in the ascidian embryo. Development
122,2005
-2012.