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First published online June 22, 2006
doi: 10.1242/10.1242/dev.02446
1 Laboratory for Germline Development, RIKEN Center for Developmental Biology,
Kobe, Hyogo 650-0047, Japan.
2 Department of Biology, Konan University, Kobe, Hyogo 658-8501, Japan.
3 Department of Marine Biotechnology, Fukuyama University, Fukuyama, Hiroshima
729-0292, Japan.
4 Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama 351-0198,
Japan.
* Authors for correspondence (e-mail: shirae{at}cdb.riken.jp; akiran{at}cdb.riken.jp)
Accepted 15 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ciona intestinalis, vasa, Germ cell, Germ plasm, Nuage, Asymmetric cell division
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
germ plasm contains many maternally inherited RNAs and proteins, which play
crucial roles in germ cell formation. These germ plasm components often form
electron-dense granulofibrillar structures, called germinal granules. In later
embryogenesis, germinal granules are reorganized to form perinuclear granules,
or nuage, a hallmark of germ cells in a wide range of animal species. Thus,
the identification and characterization of the germ plasm and the germinal
granules are crucial for understanding the mechanism of germline
formation.
Ascidian embryos are well known to develop in a typical mosaic fashion
(Conklin, 1905), and they
undergo rapid cell-fate determinations, which are regulated by maternal RNAs
and proteins localized to specific regions of unfertilized eggs and early
embryos (reviewed by Nishida,
2005). As several lines of evidence have indicated that the
somatic cell-fate determination is completed by the 110-cell stage
(Conklin, 1905;
Nishida, 1987), it is believed
that germline cells are also specified by this stage.
Within ascidian embryos, a specific cytoplasm, called the postplasm, is
sequestered to a region at the posterior pole
(Sasakura et al., 1998;
Sasakura et al., 2000), where
many maternal RNAs and proteins are highly concentrated
(Yoshida et al., 1996;
Satou, 1999;
Imai et al., 2004;
Yamada et al., 2005) (reviewed
by Sardet et al., 2005). The
postplasm contains a specialized cytoplasmic structure, called the
centrosome-attracting body (CAB). The CAB is composed of electron-dense masses
(EDM), which are morphologically very similar to germinal granules, and the
cortical endoplasmic reticulum (cER) domains
(Hibino et al., 1998;
Iseto and Nishida, 1999).
Recent observations have shown that in Ciona intestinalis, the RNA
and protein of CiVH, a homolog of the Drosophila
germline-specific gene vasa, are highly concentrated in the
postplasm, presumably in the CAB (Fujimura
and Takamura, 2000; Takamura
et al., 2002). The CiVH-positive cells are later incorporated into
the primitive gonads of juveniles to form germ cells. Vasa is an integral
component of Drosophila germinal granules (polar granules and nuage),
and its homologs have also been identified as germline-specific genes in a
wide range of animals (reviewed by Raz,
2000). The postplasm-containing blastomeres are also likely to be
transcriptionally inactive, as the zygotic transcriptions of several genes,
including the housekeeping gene elongation factor-1
, are
detected at a high level in all blastomeres, except for the
postplasm-containing cells (Tomioka et
al., 2002) (M.S.-K. and A.N., unpublished). Furthermore, it has
been proposed that, following the cleavage stage, a pair of the
postplasm-containing blastomeres at the posterior pole of the embryo, the B7.6
cells, are mitotically inactive during the subsequent embryogenesis. This
transcriptional quiescence and cell division arrest are also seen in the
primordial germ cells (PGCs) in Drosophila and C. elegans
during early embryogenesis (reviewed by
Leatherman and Jongens, 2003).
Based on these observations, it has been proposed that in ascidians, the
postplasm acts as the germ plasm, and that the B7.6 cells develop into
PGCs.
Although the postplasm is thought to act as the germ plasm, the CAB and
several postplasmic components are also known to play crucial roles in unequal
cleavages and somatic cell differentiation. First, the CAB functions in
promoting unequal cleavages during early embryogenesis
(Hibino et al., 1998;
Iseto and Nishida, 1999;
Nishikata et al., 1999).
Second, posterior end mark (PEM) RNA, which was the first
molecule identified as a postplasmic component
(Yoshida et al., 1996),
appears to control the positioning of the cleavage planes, promoting unequal
cleavages (Nishida, 2005).
Third, macho-1 RNA, which encodes a transcription factor, is tightly
associated with the CAB and acts as a muscle determinant in
Halocynthia and Ciona
(Nishida and Sawada, 2001;
Satou et al., 2002a;
Kobayashi et al., 2003).
Maternal macho-1 activity in the postplasm-containing blastomeres
also promotes the posterior adjacent blastomeres to differentiate into
mesenchyme during early cleavage stages
(Kondoh et al., 2003;
Kobayashi et al., 2003).
Fourth, CiYB1 protein has been identified as a component of the postplasm in
C. intestinalis embryos (Tanaka
et al., 2004). CiYB1 is a member of the Y-box protein family,
which includes RNA-binding proteins that control the translation of specific
mRNAs (reviewed by Matsumoto and Wolffe,
1998; Sommerville,
1999). Consistent with this function, CiYB1 binds Ci-PEM
and Ci-macho-1 RNAs in vitro, and represses their translation in the
rabbit reticulocyte lysate system, suggesting that CiYB1 regulates the
translation of postplasmic/PEM RNAs
(Tanaka et al., 2004).
Previous studies have indicated that these postplasmic/PEM RNAs and proteins,
which are involved in somatic cell differentiation during the cleavage stages,
can be detected in the presumed B7.6 cells in the tailbud embryo. Therefore,
it is thought that postplasmic components with known functions in somatic cell
development may have additional roles in germ cell formation. However, how the
postplasm components regulate germ cell specification and somatic cell
differentiation remains elusive.
Here, we show that B7.6 cells undergo an asymmetric cell division during gastrulation to produce two distinct daughter cells: CAB-containing anterior cells, named B8.11, and CAB-negative posterior cells, the B8.12 cells. Most of the postplasmic components remain associated with the CAB and are partitioned only into the B8.11 cells, which later lose CiVH protein expression and associate with the gut wall in juveniles. By contrast, maternal CiVH RNA and protein are specifically released from the CAB and diffuse into the cytoplasm just before the B7.6 cell division, allowing the CiVH products to be inherited by the B8.12 cells. Furthermore, CiVH protein production in the B8.12 cells is upregulated through the translation of the maternal CiVH RNA, resulting in the formation of perinuclear CiVH granules, which may be the nuage structure. The B8.12 descendants are incorporated into the primitive gonad in juveniles. Our data indicate that the CAB and most of the postplasmic components involved in somatic cell differentiation are excluded from the germline progenitor cells through an asymmetric cell division, and suggest that the diffusion of specific postplasmic components into the cytoplasm prior to the B7.6 cell division is crucial for proper germline development during ascidian embryogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
After insemination, the eggs were reared at 18°C in Millipore-filtered
seawater (FSW) containing 50 µg/ml streptomycin sulfate. The larvae were
staged according to Chiba et al. (Chiba et
al., 2004).
In situ hybridization
Sense and antisense DIG-labeled RNA probes were synthesized from the cDNA
clones in the Ciona intestinalis Gene Collection Release I
(Satou et al., 2002b), using a
digoxigenin RNA-labeling kit (Roche). Whole-mount in situ hybridization of
staged C. intestinalis embryos was carried out as described
(Etani and Nishikata,
2002).
Antibody generation
The full-length CiVH-coding region was amplified by PCR and cloned
into pProExHTa (Gibco). Histidine-tagged CiVH protein was expressed in
Escherichia coli BL21 cells by IPTG induction and purified with
Ni-NTA agarose (Qiagen). The protein was further purified by preparative
SDS-PAGE, and dialyzed against PBS. Polyclonal antisera against the purified
protein were generated in rabbits by Kitayama Labes (Nagano, Japan). For
immunoblotting and immunohistochemistry, the rabbit antisera were affinity
purified with the antigen immobilized on a HiTrap NHS column (Amersham).
Immunoblotting
Immunoblotting was carried out using the E. coli cell lysate
expressing recombinant histidine-tagged CiVH, the adult gonad lysate, or total
lysates of 5 or 20 animals at different developmental stages. The
affinity-purified rabbit anti-CiVH antibody (1:1000 dilution) and mouse
anti-ß-tubulin antibody (3E7, DSHB; 1:1000 dilution) were used as primary
antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse
IgG (Jackson; 1:3000 dilution) was used as the secondary antibody, and signals
were detected with the ECL system (Amersham).
Immunohistochemistry
Embryos and larvae were fixed with 4% paraformaldehyde in 0.5 M NaCl, 0.1 M
MOPS pH 7.5 at room temperature for 30-60 minutes, washed with PBS containing
0.1% Tween 20 (PBSTw) and kept at 4°C until use. The samples were
pretreated with blocking solution (1% bovine serum albumin in PBSTw) for 30
minutes and incubated with the primary antibody in blocking solution overnight
at 4°C. The primary antibodies used were: affinity-purified rabbit
anti-CiVH (1:1000 dilution), rabbit anti-CiYB1
(Tanaka et al., 2004) (1:1000
dilution) and mouse monoclonal anti-phosphohistone H3 (Cell signaling; 1:2000
dilution). The secondary antibodies used were Alexa488- or Alexa568-conjugated
anti-rabbit IgG, and Alexa568-conjugated anti-mouse IgG (Molecular Probes). To
visualize cell boundaries and nuclei, the samples were incubated with
Alexa660- or Alexa568-conjugated phalloidin and DAPI (Molecular Probes) for 2
hours at room temperature. The embryos and larvae were mounted in Vectashield
(Vector Laboratories) and observed under a laser confocal microscope (Leica
TCS SP2 AOBS).
Double staining for proteins and RNAs were carried out as follows. To detect hybridized signals, embryos were incubated with an HRP-conjugated anti-DIG antibody for 30 minutes (1:500; Roche), followed by amplification with biotinyl tyramide (1:50) for 20 minutes (Perkin Elmer). The samples were then incubated with the rabbit anti-CiVH antibody (1:1000 dilution) or rabbit anti-CiYB1 antibody (1:1000 dilution) overnight at 4°C. To visualize the signals, embryos were incubated with Alexa594-conjugated anti-rabbit IgG (1:1000 dilution) and FITC-conjugated streptavidin (Molecular Probes, 1:500 dilution) overnight at 4°C.
Lineage tracing of B7.6 blastomeres
CellTracer CM-DiI (Molecular Probes) was dissolved in soybean oil (Sigma)
to a concentration of 1 mg/ml. DiI solution was injected into the B7.6
blastomeres of the dechorionated 64-cell embryos and cultured individually in
24-well plates at 18°C. After metamorphosis, the juveniles were fed with
the diatom Chaetoceros gracilis until the primitive gonads were
formed. We observed both live and fixed animals to detect DiI signals alone
and both CiVH and DiI signals, respectively.
Inhibitor treatments
Embryos at the 110-cell stage were reared in FSW containing 100 or 200
µg/ml puromycin (Sigma) to inhibit translation, or 20 or 40 µg/ml
actinomycin D (Sigma) to inhibit transcription, as described
(Whittaker, 1977;
Nishida and Kumano, 1997). The
treated embryos were allowed to develop until the presumptive tailbud stage at
18°C (12-14 hours after fertilization). The embryos were stained for CiVH,
and classified into three types, according to the degree of CiVH staining in
the presumptive B8.12 cells: high (as high as the level in control embryos),
weak (detectable but obviously reduced compared with the control level) and
undetectable. A total of 100 embryos was counted under each condition. The
global effects of the reagents on zygotic transcription and translation were
analyzed by the expression of alkaline phosphatase (AP) mRNA
and AP activity, respectively. It is reported that, following the cleavage
stage, AP is produced through the translation of zygotically expressed mRNA in
Ciona (Imai et al.,
2000). Histochemical staining for AP was performed as described
(Imai et al., 2000). RT-PCR was
carried out using total RNA prepared from 50 embryos. The primer sets for
Ci-AP and Ci-ß-tubulin were designed from the
cDNA information (CLSTR13498 for Ci-AP and CLSTR00086 for
Ci-ß-tubulin) in the Ghost database
(http://ghost.zool.kyoto-u.ac.jp/indexr1.html).
To confirm the effects of inhibitors on the expression of Ci-AP, two
different regions of the Ci-AP mRNA coding region were analyzed by
RT-PCR. The following primer sets were used: AP-1,
5'-TTGTGCAAGTGTTGCGGTAATCGG-3' and
5'-TGTTTTCTTGCGCTCTGTCGTGCC-3'; AP-2,
5'-ATTGACCACGGTCACCATGCAGG-3' and
5'-TGTTTTCTTGCGCTCTGTCGTGCC-3'; CiVH,
5'-ATGTTTGACGACGATTGGGAACC-3' and
5'-CCATCATCTTTCTTGGAATACGGG-3'; ß-tubulin,
5'-ACCAAATTGGTGCTAAGTTCTGGG-3' and
5'-TGGTGTGGCACAGGTCGTATGG-3'.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, we detected a novel CiVH localization
pattern through an improved in situ hybridization procedure in the present
study. The previous report showed that CiVH RNA is distributed
broadly in unfertilized eggs (Fig.
1A) and gradually accumulates at the posterior pole of the embryos
during the 4- to 16-cell stage (Fujimura
and Takamura, 2000). However, using our improved procedure, we
found that a small amount of CiVH RNA had already started to
accumulate at the posterior cortex of the fertilized one-cell stage embryo
(Fig. 1B). During the cleavage
stages, CiVH RNA was incorporated into a pair of blastomeres located
at the posterior pole (Fig.
1C). At the gastrula stage, CiVH RNA showed a single
dot-like distribution in the embryos (Fig.
1D, arrowheads). However, in the neurula, the dot-like
CiVH RNA distribution was found in two points of the posterior part
of the embryo (Fig. 1E,
arrowhead and arrow). In tailbud embryos, the CiVH RNA signals were
clearly segregated into the middle and distal regions of the endodermal strand
of the tail (Fig. 1F,G,
arrowheads and arrows, respectively). Under our staining conditions, 100% of
the tailbud embryos showed this CiVH RNA distribution pattern.
By contrast, Ci-PEM RNA, which accumulated in the postplasm after
fertilization and partitioned into the posterior-most blastomeres during the
cleavage stage (Fig. 1H-J,
arrowheads), was, unlike the CiVH RNA, detected only in the middle of
the tail region in the tailbud embryo (Fig.
1M,N, arrowheads). We found that the maternal Ci-macho-1
RNA had the same distribution pattern as the Ci-PEM RNA during
embryogenesis (data not shown) (Satou et
al., 2002a). Thus, although the distribution patterns of
CiVH RNA overlapped with those of the Ci-PEM and
Ci-macho-1 RNAs during the cleavage stages, in subsequent stages, the
CiVH RNA, but not the Ci-PEM or Ci-macho-1 RNAs,
was distributed into two discrete cell masses in the middle and distal regions
of the tail.
|
In 64-cell stage embryos, the CiVH protein was concentrated at the
posterior pole of the posterior-most cells, the B7.6 cells
(Fig. 2A,A'), where
postplasmic/PEM RNAs and the CAB also accumulate
(Yoshida et al., 1996;
Iseto and Nishida, 1999).
Notably, this region was also highly enriched with F-actin, as revealed by
phalloidin staining (Fig.
2A',A'', arrowheads), suggesting that the CAB
associates tightly with an F-actin mass. During the initial stage of
gastrulation, the position of the CiVH protein within the B7.6 cells changed
from posterior to anterior, owing to the movement of the cells towards the
interior of the embryo (Fig.
2B,B'). At the beginning of the invagination, the CiVH
protein in the B7.6 cells was associated with the F-actin layer at the cortex
(Fig. 2B',B''
arrowheads), suggesting that CiVH protein remained in the CAB. However, during
gastrulation, although some CiVH protein remained anchored to the CAB at the
anterior cortex (Fig.
2C', arrowheads), some was released from the anterior cortex
and diffused into the bulk cytoplasm to form cytoplasmic granules
(Fig. 2C', arrows). At
this stage, we found that the chromosomes of the B7.6 cells were positive for
phosphohistone H3, a mitosis marker (Fig.
2D',D'', arrows), and the B7.6 cells subsequently
underwent asymmetric cell division to form small anterior and large posterior
daughter cells (Fig. 2E;
arrowhead and arrow, respectively). Thus, the B7.6 cells became mitotically
active during early gastrulation to produce two morphologically distinct cells
that both contained CiVH protein. Following the previous lineage nomenclature
for ascidians (Conklin, 1905),
the small anterior daughter cells were named B8.11, and the large posterior
daughter cells B8.12.
CiVH protein in the B8.12 cells forms perinuclear granules
After the asymmetric cell division, the level of CiVH staining in the B8.12
cells increased (Fig.
2E',F', arrows), and the CiVH protein formed clear
granules around the nucleus at the tailbud stage
(Fig. 2F', arrows). Each
B8.12 cell then divided once to form four CiVH-positive cells by the late
tailbud stage (Fig. 2G''',
arrows). The CiVH staining in the B8.12 daughter cells remained high during
the larval stage (Fig. 3A-D).
By contrast, the CiVH staining in the B8.11 cells remained low in the F-actin
aggregates (Fig.
2G',G'' arrowheads), and the anterior CiVH foci in the
tail became undetectable by the beginning of metamorphosis
(Fig. 3A-D).
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B8.12 descendents develop into germ cells
The B8.12 daughter cells divided further in the tail to form 8-16
CiVH-positive cells during the larval stage
(Fig. 3A-D, arrowheads).
Subsequently, these cells were carried into the trunk region along with other
resorbed tail cells, 
24 hours after hatching (stage 3a;
Fig. 3E, arrowhead). At stage
3b, the CiVH-positive cells (Fig.
3F,F', arrowheads) remained in the tail debris. After the
tail debris was mostly absorbed at stage 4
(Fig. 3G,G'), the
CiVH-positive cells aligned within the sparse remaining tail debris
(Fig. 3G', arrowheads).
In stage 5 juveniles, the CiVH-positive cells aligned within a tube-like
structure that extended from the tail debris toward the stomach
(Fig. 3H,H', arrowheads).
By stage 6, this tube-like structure had changed into a drop-like shape
containing the CiVH-positive cells (Fig.
3I,I'). Subsequently, the CiVH-positive cells were
surrounded by somatic cells to form the primitive gonad at stage 7
(Fig. 3J,J' arrowheads)
(Okada and Yamamoto, 1999;
Chiba et al., 2004). Within the
primitive gonad, the CiVH-positive cells proliferated, and their number
increased with subsequent development (Fig.
3J', arrowheads; data not shown).
|
). Even 14 days after the DiI labeling, we successfully detected the DiI signals in the CiVH-positive cells, which assembled in the primitive gonads in stage 6-7 juveniles (Fig. 4C). Notably, juveniles that showed relatively strong DiI labeling in the B8.11 cells at the tailbud stages (arrowheads in Fig. 4B) had DiI-positive but CiVH-negative cells attached to the hind-gut wall near the anus (data not shown), suggesting that the B8.11 cells never coalesce into the primitive gonad. To confirm the fate of the B8.11 cells in the juveniles, we removed the B8.12 descendants from DiI-labeled larvae by cutting off the distal region of the tail where the B8.12 descendants clustered (Fig. 4D). When such larvae were allowed to develop, the DiI-positive cells were still detected at the gut wall in stage 5 juveniles [Fig. 4E, arrowhead; in the images we obtained, the DiI signal was very faint, and the auto-fluorescence of the tail debris (arrow) was thus relatively greater under microscopic observation]. These results demonstrate that the B8.12 descendants coalesce into the primitive gonads. Although it is still possible that the B8.11 cells migrate to the gonad in later stages, these results strongly suggest that, under normal developmental conditions, the B8.12 descendants, but not the B8.11 cells, develop into germ cells.
|
We next examined the localization of CiVH protein and Ci-PEM RNA. Although the Ci-PEM RNA was strictly colocalized with the CiVH protein in the CAB during the cleavage stage (Fig. 6C-E), it remained concentrated in the CAB at the gastrula stage (Fig. 6H-J, arrowheads). After the B7.6 cell division, the Ci-PEM RNA segregated only into the B8.11 cells (Fig. 6M-O, arrowheads), while the CiVH protein was distributed to both the B8.11 and 8.12 cells (Fig. 6M, arrows). We also observed that maternal Ci-macho-1 RNA never diffused from the CAB and segregated only into the B8.11 cells (data not shown). These observations indicate that CiVH RNA and protein are specifically released from the CAB prior to the B7.6 cell division and that their specific diffusion into the bulk cytoplasm is likely to be crucial for their distribution into the B8.12 cells.
It has remained elusive whether the release of CiVH protein from the CAB is
a specific mechanism for certain proteins or a general one for all the protein
components in the postplasm. To discriminate between these possibilities, we
examined the distribution of another protein component in the postplasm, CiYB1
(Tanaka et al., 2004). We
found that CiYB1 protein tightly colocalized with Ci-PEM and
Ci-macho-1 RNA and was partitioned only into the B8.11 cells
(Fig. 7; data not shown). These
results indicate that the release of CiVH RNA and protein from the
CAB prior to the B7.6 cell division is a specific mechanism that enables their
distribution into the B8.12 cells.
CiVH protein is produced from inherited maternal RNA in the B8.12 cells
We next examined whether CiVH protein production in the B8.12 cell lineage
requires zygotic CiVH transcription, or whether the translation of
maternally inherited CiVH RNA is upregulated in the B8.12 cells. For
this purpose, we examined the effects of transcription and translation
inhibitors on CiVH protein production in the B8.12 cells. Embryos at the
110-cell stage were treated with a transcription inhibitor, actinomycin D
(AcD), or a translation inhibitor, puromycin, and they were then allowed to
develop until the presumptive tailbud stage. Although both AcD and puromycin
treatments severely affected embryogenesis, we could still detect the B7.6
cell lineage by observing the CiVH signals in the posterior of the
embryos.
|
). RT-PCR
analysis confirmed that there was a severe reduction in zygotic AP
expression in the AcD-treated embryos (Fig.
8D). These data indicate that CiVH protein production in the B8.12
cells was not suppressed by inhibiting the zygotic transcription during
embryogenesis. By contrast, the accumulation of CiVH protein in the presumptive B8.12 cells was severely inhibited by the puromycin treatment (Fig. 8C'). When 110-cell stage embryos were treated with 100 µg/ml puromycin, only 3% of the embryos had a normal level of CiVH protein in their B8.12 cells at the presumptive tailbud stage, but 43% showed weak production, and in 54% the CiVH granules were undetectable. Furthermore, when embryos at the 110-cell stage were treated with 200 µg/ml puromycin, 6% showed a normal level of anti-CiVH staining and 33% showed weak staining (Fig. 8C'), and CiVH signals were not detectable in 61% of the embryos. Thus, the reduction of anti-CiVH staining in the B8.12 cells by the puromycin treatment occurred in a dose-dependent manner. RT-PCR analysis indicated that the level of CiVH RNA was not affected by the inhibitor treatments (Fig. 8D). These results indicated that it is the translation of maternal CiVH RNA, and not zygotic CiVH transcription, which is responsible for the production of CiVH protein in the B8.12 cells. These results further suggest that the specific release of CiVH RNA and protein from the CAB prior to the B7.6 cell division is crucial for germ cell specification in ascidians.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Takamura et
al., 2002). However, it remained unclear whether B7.6 cells
directly coalesce into the gonads to develop into germ cells and whether other
postplasmic components with known functions in somatic cell development
contribute to germ cell formation. In the present study, by tracing the B7.6
cell fate in detail, we clarify the process by which germ cells in ascidians
are specified.
Dynamic redistribution of CiVH protein and germ cell specification in Ciona
We showed here that a dynamic redistribution of CiVH protein occurs
following the cleavage stage. First, each of the two posterior-most
blastomeres, the B7.6 cells, divided asymmetrically during gastrulation
(Fig. 2D',D'') to
produce two distinct daughter cells, B8.11 and B8.12. Second, although most of
the postplasmic components segregated only into the small CAB-inheriting B8.11
cells, the CiVH RNA and protein were inherited by both the
CAB-containing B8.11 cells and the CAB-lacking B8.12 cells (Figs
2,
5). Third, the segregation of
CiVH RNA and protein into both daughter cells was mediated through
their specific diffusion into the cytoplasm prior to the B7.6 cell division
(Fig. 2C',
Fig. 5H-J). Fourth, after the
B7.6 cell division, the CiVH protein in the B8.12 cells formed perinuclear
granules (Fig. 2G'''). As
Vasa is an integral component of the germinal granules, or nuage, in a wide
range of animals, we propose that the CiVH-containing perinuclear granules are
the nuage in C. intestinalis. Finally, by tracing the B8.12 cell fate
in detail with DiI labeling and CiVH immunostaining, we found that the B8.12
cells divided further to form 8-16 CiVH-positive cells, which were
incorporated into the primitive gonads and developed into germ cells (Figs
3,
4). From these observations, we
conclude that B8.12 cells are the PGCs in this organism. Previous electron
microscopic observations in C. intestinalis showed nuage-like
structures in the putative PGCs located in the tail debris during
metamorphosis (Yamamoto and Okada,
1999). Our present observations suggest that the formation of this
structure begins in the germline cells at a much earlier stage: immediately
after the B7.6 cell division in the gastrula. Electron microscopic analyses of
the B7.6 cells and their descendants in the gastrula and tailbud-stage embryos
will confirm this issue.
|
Most postplasmic components are excluded from the germline lineage
In contrast to the B8.12 cells, we found that the B8.11 cells never
coalesced into the primitive gonads, but rather associated with the gut wall
in juveniles, where they were without CiVH expression
(Fig. 4E; data not shown). The
B8.11 cells contained a mass of F-actin, which was probably the CAB remnant.
Furthermore, maternal Ci-PEM and Ci-macho-1 RNAs, which are
involved in unequal cleavage and somatic cell differentiation, and a putative
translational repressor, CiYB1 protein, were never detected in the B8.12
cells, but remained in the B8.11 cells with the putative CAB remnant
(Fig. 6; data not shown).
Although it is formally possible that protein products of these
postplasmic/PEM RNAs were partitioned into the B8.12 cells, we favor the idea
that these postplasmic components of the B8.11 cells have no role in germ cell
specification. Rather, it may be that these postplasmic components and/or the
CAB structure itself could interfere with proper germ cell specification, and
are therefore excluded from the B8.12 cells.
The mechanism by which the CiVH RNA and protein are specifically
released from the CAB prior to the B7.6 cell division remains uncertain. It is
reported that the postplasmic/PEM RNAs contain cis-acting signals in
their 3' untranslated region (UTR) that direct their localization to the
CAB (Sasakura and Makabe,
2002). Similarly, it is likely that the CiVH RNA contains
a specialized cis- acting sequence for its specific release from the
CAB prior to the B7.6 cell division.
|
|
;
Iseto and Nishida, 1999). It is
reported that in both Ciona and Halocynthia, macho1, PEM and
several other postplasmic/PEM mRNAs associate with the cER in the CAB to form
the cER/mRNA domain (Sardet et al.,
2005; Prodon et al.,
2005; Nakamura et al.,
2005). Although the precise distribution of CiVH RNA
within the CAB remains unknown, the specific release of CiVH RNA and
protein from the CAB may be due to a difference in the strength of their
association with the CAB; e.g. they might be associated with the EDM rather
than the cER.
Recently, the aPKC-PAR6-PAR3 complex (PAR complex) has been identified as a
component of the CAB (Patalano et al.,
2006). The PAR complex associates with the cortical F-actin under
the plasma membrane and is thought to regulate unequal cleavages in the
ascidian embryo. As the PAR complex is crucial for polarized RNA and protein
distribution within a cell and for asymmetric cell divisions in a wide range
of cell types (Macara, 2004),
the PAR proteins may be involved in the CiVH partitioning into the B8.12
cells.
Translational upregulation of maternal RNA in the B8.12 cells may promote germ cell formation
After the B7.6 cell division, CiVH protein production was dramatically
increased in the B8.12 cells, resulting in the formation of perinuclear
granules (Fig. 2G'''). We
found that the maternal CiVH RNA, released from the CAB prior to the
B7.6 cell division, was responsible for the CiVH protein production in the
B8.12 cells (Figs 5,
8). These data suggest that the
translation of CiVH RNA is partially repressed in the B7.6 cells but
is upregulated in the B8.12 cells, where the RNA is no longer associated with
the CAB. CiYB1 protein, which binds to and represses the translation of the
Ci-PEM and Ci-macho-1 RNAs in vitro
(Tanaka et al., 2004), may be
involved in the storage and/or translational control of CiVH RNA in
the postplasm. After the cleavage stage, when somatic cell-fate determination
is completed, the CiVH RNA was specifically released from the CAB,
distributed to the B8.12 cells, and translated to form perinuclear CiVH
granules. Thus, the CAB appears to be required for the storage of
CiVH RNA as well as for the repression of its translation during the
cleavage stage, when somatic cell-fate determination proceeds.
We have found that several other postplasmic/PEM RNAs are partitioned into
the B8.12 cells in a manner similar to that of CiVH RNA during
Ciona embryogenesis (M.S.-K. and A.N., unpublished). Furthermore, in
Halocynthia, it has also been observed that some postplasmic/PEM RNAs
are segregated into two regions within the embryos after gastrulation
(Nakamura et al., 2003) (H.
Nishida, personal communication). Thus, we propose that the dynamic
redistribution of specific postplasm components through the B7.6 cell division
is conserved mechanisms underlying germ cell specification in ascidians.
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ACKNOWLEDGMENTS |
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