1 Department of Biological Sciences, Graduate School of Science, Osaka
University, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
2 Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuda,
Midori-ku, Yokohama 226-8501, Japan
3 Faculty of Integrated Arts and Sciences, Tokushima University, 1-1
Minami-Josanjima, Tokushima 770-8502, Japan
* Author for correspondence (e-mail: yorinaka{at}bio.sci.osaka-u.ac.jp)
Accepted 19 August 2005
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SUMMARY |
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Key words: Ascidian embryo, Halocynthia roretzi, RNA localization, postplasmic/PEM RNA, Cortical endoplasmic reticulum, Germ plasm, POPK-1 kinase, Sad-1 kinase
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Introduction |
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In ascidians, several maternal mRNAs are located in the posterior-vegetal
cortex (PVC) of fertilized eggs just before cleavage. Micromanipulation
experiments in which PVC is deleted and transplanted have shown that it is
possible to remove and transplant the potential for formation of the posterior
tissues, including muscle, and for unequal cleavages that are characteristic
of the posterior pole. By contrast, the removal and transplantation of the egg
cytoplasm of other regions have no effect
(Nishida, 1994). When PVC is
removed from eggs, mirror-image duplication of the anterior half occurs in the
cell fates and cleavage pattern. Muscle and mesenchyme precursor blastomeres
are converted to nerve cord and notochord, respectively, so that central
endoderm blastomeres are encircled by these blastomeres
(Nishida, 1994
;
Kobayashi et al., 2003
). The
transplantation of the PVC to the anterior region of the PVC-deficient eggs
reversed the anteroposterior axis. Therefore, localized factors in the PVC
play critical roles in the determination of the anteroposterior axis, which is
involved in autonomous specification of muscle fate, generation of differences
in responsiveness to inductive signals in mesenchyme and notochord precursor
blastomeres, and control of cleavage pattern (reviewed by
Nishida, 1997
;
Nishida, 2002
;
Nishida, 2005
).
In eggs of Halocynthia roretzi, nine maternal mRNAs localized to
the PVC have been identified so far, including macho-1, the muscle
and posterior determinant. They are called Type I postplasmic/PEM
mRNAs (Sasakura et al., 1998a;
Sasakura et al., 1998b
;
Nishida and Sawada, 2001
;
Makabe et al., 2001
;
Nakamura et al., 2003
)
(reviewed by Sardet et al.,
2005
). They show identical localization during cytoplasmic and
cortical reorganization, so-called ooplasmic segregation in ascidians, and are
concentrated in the centrosome-attracting body (CAB) during early cleavages.
The CAB was first found as a small subcellular structure that operates
cleavage planes during successive unequal cleavages at the posterior pole
(Hibino et al., 1998
;
Nishikata et al., 1999
)
(reviewed by Nishida et al.,
1999
). Every Type I postplasmic/PEM mRNA is localized to
the CAB at the 8-cell stage (Sasakura et
al., 2000
; Nakamura et al.,
2003
; Sardet et al.,
2003
). Thus, the CAB serves as the core structure of a
multifunctional complex that operates cleavage planes and anchors Type I
postplasmic/PEM RNAs. Having both functions together, the CAB ensures
that Type I postplasmic/PEM RNAs are infallibly partitioned into one
of the daughter cells after cell divisions.
Another conspicuous characteristic of the CAB is that it is enriched in
putative germ plasm. An electron microscopic study revealed that the CAB
contains an electron-dense matrix (EDM) that resembles germ plasm in other
animals (Iseto and Nishida,
1999). The CAB is eventually segregated into the putative germline
cells, the posteriormost and smallest blastomeres (B7.6 cells) of the 64-cell
embryos (Fujimura and Takamura,
2000
; Takamura et al.,
2002
) (reviewed by Nishida,
2005
). Various observations support the view that B7.6 cells are
primordial germ cells in ascidians, and that the CAB, enriched in
postplasmic/PEM RNAs, also contains putative germ plasm. As
Halocynthia eggs are translucent, the CAB can be seen in extracted
and cleared embryos. In such extracted embryos, only the EDM seems to persist
in the CAB as a highly refractive structure under an optical microscope
(Iseto and Nishida, 1999
).
Maternal mRNAs accumulated in the CAB are categorized into two groups.
Type I postplasmic/PEM mRNAs are already localized to the PVC before
cleavage starts. Some of them, such as macho-1 and Hr-PEM1,
have been shown to associate with the cortical endoplasmic reticulum (cER)
tethered to the plasma membrane of the egg, and they are concentrated into the
CAB together with the cER by the 8-cell stage
(Sardet et al., 2003;
Sardet et al., 2005
).
Consequently, the CAB is enriched in cER and Type I postplasmic/PEM
mRNAs. By contrast, Type II postplasmic/PEM mRNAs are distributed
evenly throughout the egg cytoplasm, and then gradually concentrate into the
CAB during cleavages. Cs-PEM is the Type I postplasmic/PEM
mRNA first found in ascidians (Yoshida et
al., 1996
). A complete list of Type I and Type II
postplasmic/PEM mRNAs in three ascidian species are available in Makabe
et al. (Makabe et al., 2001
)
and Sardet et al. (Sardet et al.,
2005
). Experiments with cytoskeletal inhibitors showed that
distinct mechanisms are involved in the localization of Type I and
Type II mRNAs to the CAB
(Sasakura et al., 2000
),
although the localization mechanism still remains largely unknown. The results
of the removal and transplantation of the PVC of eggs indicate that Type I
postplasmic/PEM mRNAs are more important than Type II. The
crucial functions in early development of macho-1 and three other
Type I postplasmic/PEM mRNAs (Hr-Wnt-5, Hr-GLUT and
Hr-PEN2) in Halocynthia have been investigated
(Nishida and Sawada, 2001
;
Kobayashi et al., 2003
;
Nakamura et al., 2005
).
Halocynthia roretzi-posterior protein kinase-1
(Hr-POPK-1) is a Type I postplasmic/PEM mRNA and encodes a
serine/threonine kinase (Sasakura et al.,
1998b). The expression is strictly maternal during embryogenesis.
Hr-POPK-1 protein shares high similarity to Sad-1 of Caenorhabditis
elegans and SAD-A of mouse throughout its entire length
(Crump et al., 2001
;
Kishi et al., 2005
)
(Fig. 1). Humans and
Drosophila also have several proteins very similar to
POPK-1/Sad-1/SAD-A, although their functions in these animals are not known.
Therefore, POPK-1 is a member of a group of proteins widely conserved among
metazoans. Three domains are conserved between these proteins: a kinase
domain, a domain next to the kinase domain and a domain in the C-terminal
half. The latter two domains show no similarity to domains with known
functions. Crump et al. (Crump et al.,
2001
) reported that the kinase domain of Sad-1 has extensive
similarity to that of Par-1, which plays a central role in specification of
the anterior-posterior polarity in C. elegans eggs. However,
similarity is lower in the Par-1 kinase domain, and Par-1 has no conserved
domains other than the kinase domain (Fig.
1). To investigate the functions of Hr-POPK-1, we
injected eggs with specific antisense morpholino oligonucleotides (MOs). The
results indicate that Hr-POPK-1 is required for proper transport of the
Type I postplasmic/PEM mRNAs during cleavages via regulation of
concentration and positioning of the cER, as well as for proper CAB
formation.
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Materials and methods |
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Microinjection of MOs and synthetic mRNA
To suppress the function of Hr-POPK-1, we used two MOs (Gene
Tools). The sequences of the MOs against Hr-POPK-1 (Accession No.
AB014885) were as follows: Hr-POPK-1 MO1
(5'-CGGCGCATTTGACATTTTAAAGAAA-3'), which covers the starting
methionine, and Hr-POPK-1 MO2
(5'-TGTTCAGTTCAAATGACACAATAAA-3'), which covers the 5' UTR.
As a control MO, we used standard control oligo
(5'-CCTCTTACCTCAGTTACAATTTATA-3'), 5-mismatch control MO
(5'-TCTTGAGTTGAAATCACAGAATAAA-3';
mismatches underlined), and PEN1 MO
(5'-CGTAAACAGTAGGAACAATTTCATA-3'). macho-1 MO was the
same as used previously (Kobayashi et al.,
2003). We injected 500 pg of Hr-POPK-1 MO1 and 750 pg of
Hr-POPK-1 MO2 and control MOs into the fertilized eggs.
Hr-POPK-1 mRNA was transcribed from pBluescriptHTB containing the
Hr-POPK-1 open reading frame with a mMessage mMachine T3 kit (Ambion)
and a Poly (A) Tailing kit (Ambion). macho-1 mRNA was synthesized as
described previously (Kobayashi et al.,
2003
). MO and synthetic mRNA were dissolved in sterile distilled
water and injected into ascidian eggs as described by Miya et al.
(Miya et al., 1997
).
Immunostaining, histochemical staining and in-situ hybridization
The monoclonal antibody Mu-2 was used for monitoring muscle formation
(Nishikata et al., 1987). This
antibody recognizes the myosin heavy chain in tail muscle cells of
Halocynthia larvae (Makabe and
Sato, 1989
). The monoclonal antibody Mch-3 was used to detect
mesenchyme formation (Kim and Nishida,
1998
). The specimens were fixed after the hatching stage for 10
minutes in methanol at 20°C. Formation of notochord cells was
monitored by staining with the Not-1 monoclonal antibody
(Nishikata and Satoh, 1990
;
Nakatani and Nishida, 1994
).
Specimens were fixed at the tailbud stage. Indirect immunofluorescence was
carried out by standard methods using a TSA fluorescein system (Perkin-Elmer
Life Sciences). Then specimens were mounted in 80% glycerol and examined under
a fluorescence microscope. In some cases embryos were allowed to develop up to
the 110-cell stage and transferred to seawater containing 2.5 µg/ml
cytochalasin B (Sigma) to permanently arrest further cleavage.
To detect muscle formation, we also used histochemical detection of
acetylcholinesterase (AChE) as described by Karnovsky and Roots
(Karnovsky and Roots, 1964).
Specimens at the tailbud stage were fixed in 5% formalin in seawater for 10
minutes at room temperature. The reaction was performed at 4°C for 16
hours to reveal the presence of the AChE (brown products). Formation of
endoderm was monitored by histochemical detection of alkaline phosphatase
(ALP) activity by using the methods described by Meedel and Whittaker
(Meedel and Whittaker, 1989
).
Embryos were fixed for 1 minute in 70% ethanol at 20°C. Specimens
were treated with ALP detection buffer for monitoring purple products.
Whole-mount in-situ hybridization was performed as described by Miya et al.
(Miya et al., 1994;
Miya et al., 1997
). Specimens
were hybridized with digoxigenin (DIG)-labeled macho-1, Hr-PEM1
(Nishida and Sawada, 2001
),
Hr-POPK-1 (Sasakura et al.,
1998b
), Hr-ZF1
(Sasakura et al., 2000
),
Hr-Wnt-5 (Sasakura et al.,
1998a
) and Hr-PEN1
(Nakamura et al., 2003
)
antisense RNA probes.
Reverse transcription-polymerase chain reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was carried out
with a Cells-to-cDNA II kit (Ambion) according to the manufacturer's protocol.
Ten embryos at the 8-cell stage, which were devitellinated with a fine
tungsten needle, were lysed in 100 µl Cell Lysis II Buffer and used for
cDNA synthesis. PCR was carried out using the following macho-1
primers: 5'-GAATAATCCACACGCTT-3' and
5'-GCTTGGTTTCGCCTAA-3', Hr-POPK-1 primers;
5'-GTATCGCATACACTGTTG-3' and 5'-AAATGGAGCAGTTCCTTG-3',
Hr-ZF1 primers; 5'-AATTCCTCCCCTGGTTGA-3' and
5'-TGATTTGGTGGAACACAAC-3', and Hr-Notch primers as a
loading control; 5'-TCTACCCTTTTGCTATTCC-3' and
5'-ATTTGTCACTTAGAATTAAGA-3'. PCR was performed for 34 cycles for
Hr-POPK-1and HrZF-1, and 35 cycles for macho-1 and
Hr-Notch, at 94°C for 1 minute, 50°C (53°C for
Hr-POPK-1) for 1 minute and 72°C for 1 minute. The PCR products
were resolved by 1-2% agarose gel electrophoresis.
Extraction of embryos
To visualize the CAB, embryos were extracted and cleared. Dechorionated
8-cell stage embryos were rinsed twice with Ca2+-,
Mg2+-free artificial seawater containing 1 mmol/l EGTA, and
transferred to an extraction buffer composed of 50 mmol/l MgCl2, 10
mmol/l KCl, 10 mmol/l EGTA, 2% Triton X-100, 20% glycerol, and 25 mmol/l
imidazole (pH 6.9) for 1-2 hours
(Nishikata et al., 1999).
During extraction, the embryos become transparent. The CABs of the extracted
embryos were observed using Nomarski optics.
Isolation of cortices
Isolation of cortices of eggs and of 2-, 4-, and 8-cell-stage embryos and
fluorescence (TSA) in-situ hybridization with isolated cortices were carried
out as described previously (Sardet et
al., 2003). The cER network of isolated cortices was labeled in
red with a lipophilic dye, DiIC18 (3) (Molecular Probes).
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Results |
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Endoderm differentiation was monitored by detecting the expression of alkaline phosphatase (ALP). The suppression of POPK-1 function did not seem to affect ALP expression at the larval stage (Fig. 2K,K'). This was essentially confirmed in cleavage-arrested embryos. In both control embryos and MO-injected embryos, ALP activity was observed in ten endoderm precursors and two presumptive trunk lateral cells (TLCs) (Fig. 2L,M). However, in 15% of MO-injected embryos, ectopic ALP activity was observed in the posteriormost (B7.5) cells, which are presumptive muscle blastomeres (Fig. 2M', arrowheads; Table 1, parentheses).
Loss of muscle and mesenchyme, normal formation of notochord, and
transformation of B7.5 blastomere into endoderm were common to embryos
injected with a low dose of macho-1 MO
(Kobayashi et al., 2003). A
high dose of macho-1 MO injection resulted not only in the loss of
muscle and mesenchyme, but also in ectopic notochord formation in the
posterior region in place of original mesenchyme blastomeres, so that central
endoderm blastomeres were encircled by notochord blastomeres. This
anteriorization was also observed in embryos from which the posterior-vegetal
cortex (PVC) was removed (Kobayashi et
al., 2003
). However, ectopic notochord formation was never
observed in POPK-1 MO-injected embryos. These observations suggest
that the phenotype of POPK-1 MO-injected embryos resembles that of
embryos in which the function of macho-1 is partially inhibited. This
is supported by the results of the following experiments.
Hr-POPK-1 acts upstream of macho-1
Fertilized eggs injected with Hr-POPK-1 mRNA (100-300 pg) cleaved
normally, and the larvae had almost normal morphology, with sensory pigment
cells, palps and elongated tails, which were sometimes kinked
(Fig. 3A,A'). There was
no excess muscle formation in cleavage-arrested embryos, by contrast to those
injected with macho-1 mRNA (normal in 97% of 44 cases,
Fig. 3B,B'). The results
indicate that POPK-1 is required but not sufficient for muscle formation.
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|
We then examined macho-1 localization at the 2-, 4-, 16- and 32-cell stages. In the 2- and 4-cell embryos, localization in the mRNA-rich posterior cortical region seemed to be broader and more diffuse than that in control embryos (Fig. 4B, arrowheads). The diffuse distribution coincides well with that observed at the early 8-cell stage. Because the POPK-1 MO was injected into fertilized eggs after the completion of ooplasmic segregation, MO did not affect ooplasmic segregation. The effect observed at the 2-cell stage suggests that POPK-1 translation starts as early as the 2-cell stage, and MO interfered with its functions. At the 16- and 32-cell stages, macho-1 mRNA was localized in smaller dots at the posterior pole (Fig. 4B, arrowheads), as observed in the late 8-cell embryos. Therefore, transition from diffuse to compact distribution occurs at the middle 8-cell stage.
To evaluate the quantity of macho-1 mRNA, we carried out semi-quantitative RT-PCR using 8-cell embryos. macho-1 mRNA was amplified from ten embryos with and without MO. As shown in Fig. 4C, the amount of macho-1 mRNA seems to be slightly reduced in MO-injected embryos relative to uninjected embryos at the late 8-cell stage, but not completely abolished. This coincides with the result of in-situ hybridization. The result was confirmed in three independent experiments using different batches of eggs. We tentatively quantified the intensity of the bands with the software NIH Image. The intensity was reduced to 60% on average.
Hr-POPK-1 is involved in every Type I postplasmic/PEM mRNA localization and in proper CAB formation
To test whether the distributions of other Type I postplasmic/PEM
mRNAs are also affected, embryos were probed for five other Type I
postplasmic/PEM mRNAs: Hr-PEM1, Hr-POPK-1, Hr-ZF1, Hr-Wnt-5 and
Hr-PEN1 mRNAs. In embryos injected with control MO, the distributions
of all these mRNAs were normal (Fig.
4C, left, arrowheads). In embryos injected with POPK-1
MO, the distribution of these mRNAs was reduced in size
(Fig. 4C, right, arrowheads:
Hr-PEM1, 83% of 30 cases; Hr-POPK-1, 75% of 8 cases;
Hr-ZF1, 86% of 22 cases; Hr-Wnt-5, 100% of 10 cases; and
Hr-PEN1, 100% of 21 cases). The phenotype was identical to that
observed for macho-1 distribution. These results demonstrate that
POPK-1 MO affects the distribution of every Type I
postplasmic/PEM mRNA, including POPK-1 itself. We also carried
out semi-quantitative RT-PCR at the late 8-cell stage to evaluate the quantity
of Hr-POPK-1 and Hr-ZF1 mRNA, and Hr-Notch mRNA as
loading control. By contrast to macho-1, there was no remarkable
difference in the amount of POPK-1 and Hr-ZF1 mRNA between
uninjected embryos and MO-injected embryos
(Fig. 4D). The result was
confirmed in three independent experiments using different batches of eggs.
The intensity of the band was 94%, 110% and 91% on average for Hr-POPK-1,
Hr-ZF1 and Hr-Notch, respectively, compared to uninjected
embryos.
These postplasmic/PEM mRNAs are present in the CAB at the 8-cell stage. Therefore, we observed the shape of the CAB in extracted and cleared embryos. In uninjected embryos, control MO- and PEN-1 MO-injected embryos, the CAB appeared as two bars connected at the midline in the posterior cortex of the posterior blastomeres after extraction at the late 8-cell stage (Fig. 5A, arrowhead). By contrast, in embryos injected with POPK-1 MO, the CAB appeared as two small dots apart from the midline in the posterior cortex (Fig. 5B, arrowhead; 90% of 31 cases). In spite of this shrinkage, the small CAB was always present and never lost. This observation indicates that not only mRNA distribution, but also the CAB itself, shrank.
The CAB in extracted embryos is likely to correspond to the electron-dense
matrix (EDM) (Iseto and Nishida,
1999). It is first recognizable as precursors, which appear as
dozens of small dots in the posterior cortex of the 2-cell embryos. These
particles gradually assemble and form a slender cluster by the 4-cell stage.
During the 8-cell stage, the particles fuse together to form the CAB, which
has a uniform appearance (Hibino et al.,
1998
; Iseto and Nishida,
1999
). Injection of POPK-1 MO also affected the
distribution of CAB precursors in the 4-cell embryos. In normal embryos, the
particles had already gathered to a single line
(Fig. 5C,E). However, in
embryos injected with POPK-1 MO, the particles were still apart from
each other and were distributed in a broader region of the posterior cortex
(Fig. 5D,F). It was hard to
tell whether or not the total amount of granules was less.
We noticed that POPK-1 MO-injected embryos occasionally showed a radialized cleavage pattern (Fig. 6D; 34% of 112 cases), whereas all control MO-injected embryos showed unequal cleavages at the posterior pole after the 8-cell stage (Fig. 6A,B). Therefore, the correlation of the failure of unequal cleavage and shrinkage of the CAB was examined in more detail. Embryos were extracted at the 16-cell stage, and unequal cleavage and the shape of the CAB were monitored in the same embryos. Essentially, every POPK-1 MO-injected embryo had a small CAB, but unequal cleavage still took place in most of them (Fig. 6C,E; 92% of 12 cases). However, equally cleaved embryos also had a small CAB (Fig. 6D,F). There was no difference in CAB size between the two populations.
|
Then, we examined cER distribution in POPK-1 MO-injected embryos. As Hr-PEM1 is the most abundant postplasmic RNA, we observed the distribution of Hr-PEM1 in isolated cortices of the 8-cell embryos. In uninjected embryos, both cER and Hr-PEM1 mRNA were concentrated in the moustache-shaped CAB, confirming the previous observation (Fig. 8A). In embryos injected with POPK-1 MO, the cER- and Hr-PEM1 mRNA-rich regions were present in smaller rounded shape apart from the midline. The Hr-PEM1 mRNA-rich region always coincided with the small cER-rich region (Fig. 8B, arrowheads). These observations indicate that POPK-1 is required for proper concentration and positioning of cER, and that it affects the mRNA distribution via cER movements.
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Discussion |
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What causes the reduction in size of the cER/mRNA domain? There are two possibilities. (1) In normal embryogenesis, postplasmic/PEM RNAs are highly concentrated into the small CAB region by the 8-cell stage. In POPK-1 knockdown embryos, the mRNAs were spread much more broadly at the early stages. This causes difficulty in gathering all the mRNAs into the small posterior region. (2) In normal 8-cell embryos, bilateral cER/mRNA domains are connected with each other at the midline. The elongated moustache morphology implies anchoring one end of the cER/mRNA domains to the midline. But in POPK-1 knockdown embryos, they are rounded and lie apart from the midline. This might be caused by breakup of the anchoring to the midline.
As POPK-1 has overall similarity with the proteins of a wide variety of
animals, it is probably a member of a group of widely conserved proteins with
shared common cellular functions among metazoans. The Sad-1 kinase of C.
elegans is present in synapse-rich regions of axons, and mutation affects
the size, shape and position of vesicle clustering in neurons
(Crump et al., 2001).
Considering their common function in C. elegans and ascidians, these
proteins may be involved in regulation of transport of intracellular
organelles, especially membranous components, because POPK-1 mediates
concentration and positioning of cER. The unknown conserved domains in the
C-terminal half may be domains interacting with membrane-transport machinery
or with cytoskeletal elements. A search in the Drosophila two-hybrid
protein interaction database (Drosophila Interaction Database:
http://portal.curagen.com/cgibin/interaction/flyHome.pl?modeIn=List)
indicated with high confidence that the Drosophila homolog (CG6144)
of POPK-1/Sad-1 interacts with CG11250. The function of CG11250 is not known,
but it also encodes conserved proteins among a variety of metazoans. As
POPK-1/Sad-1 is a kinase, one of the key future issues is to determine the
target of phosphorylation by POPK-1/Sad-1.
|
At the 8-cell stage, cER/mRNA and EDM/putative germ plasm domains overlap
at the posterior pole of the B4.1 blastomeres. From the 2- to 8-cell stage,
the extraction-resistant CAB materials such as EDM/putative germ plasm starts
to be assembled into the CAB as a number of dispersed particles, then the
particles gather to form single entity
(Hibino et al., 1998). By
contrast, we noticed that the distribution of postplasmic/PEM mRNAs
was broader than that of the CAB precursor particles, and not granulated
(Sasakura et al., 1998a
;
Sasakura et al., 1998b
;
Nakamura et al., 2003
). In the
present study, we confirmed the difference in detail by using isolated
cortices. The distributions of cER/mRNA and the CAB precursor particles are
not precisely identical at these early stages. It appeared that concentration
of cER/mRNA into the CAB and assemblage of EDM/putative germ plasm into the
CAB are parallel processes, but these processes share common mechanisms that
involve POPK-1. The above idea reminds us of the similar processes of maternal
mRNA localization of the Xenopus oocyte, where two major pathways
operate to localize maternal mRNAs to different but overlapping domains within
the vegetal cortex during oogenesis
(Heasman et al., 1984
;
Chang et al., 2004
;
King et al., 2005
). One is the
early pathway RNAs such as Xcat2, destined to become germ plasm, and
another is the late pathway RNAs such as Vg1, which is important for
animal-vegetal axis specification.
Localization of postplasmic/PEM mRNA into the CAB may be required for its function
In late embryogenesis, the phenotypes of embryos injected with
POPK-1 MO resembled those of the embryos injected with a low dose of
macho-1 MO. In addition, we noted that some embryos failed to undergo
unequal cleavages of the posteriormost blastomeres, although no relationship
was observed between the morphology of the CAB and failure of unequal
division. Hr-PEM1 is the most abundant Type I
postplasmic/PEM mRNA in ascidian eggs
(Yoshida et al., 1996;
Nishida and Sawada, 2001
;
Makabe et al., 2001
). Our
recent results suggested that Hr-PEM1 function is essential for
unequal cleavage, although the morphology of the CAB after extraction was
intact in Hr-PEM1-deficient embryos (H.N. and K. Sawada,
unpublished). Therefore, the presence of CAB materials in extracted embryos is
not exactly correlated with centrosome-attracting activity. Taking into
account these observations, the late phenotypes of POPK-1 knockdown
embryos are probably indirect and due to partial inhibition of
macho-1 and Hr-PEM1 functions.
Accordingly, the localization of every postplasmic/PEM mRNA was
aberrant but not lost in POPK-1 knockdown embryos. There could be two
possibilities for how the functions of postplasmic/PEM mRNAs are
partially inhibited. One is that mRNAs detached from the CAB-forming region
might be destabilized. Loss of POPK-1 function could cause problems
in gathering all the mRNAs into the CAB at the 8-cell stage. This idea is
supported by semi-quantification with RT-PCR of macho-1 mRNA.
However, the amount of Hr-POPK-1 and Hr-ZF1 mRNA was not
altered, indicating that this hypothesis is not applicable for every
postplasmic/PEM mRNA. Another possibility is that
postplasmic/PEM mRNAs could not be efficiently translated outside the
CAB, although we have no direct evidence to support this. However, when we
injected synthetic POPK-1 mRNA, larval development was normal. And
effects of MO were not rescued by co-injection of POPK-1 mRNA. These
results may suggest that non-localized mRNA is not efficiently translated,
although there are many other possibilities. Recently, it was shown that a
Y-box protein (CiYB1) is involved in translational control of localized mRNAs
in ascidian eggs and embryos of Ciona
(Tanaka et al., 2004). In
flies and vertebrates, the restriction of some localized mRNAs to a particular
region is important for their translation in various cases
(Lipshitz and Smibert, 2000
;
Johnstone and Lasko, 2001
;
Palacios and Johnston, 2001
;
Yoshida et al., 2004
).
In POPK-1 MO-injected embryos, localization of postplasmic mRNAs and formation of the CAB were aberrant but never completely abolished. This raises the possibility that the MOs used in this study are not able to completely inhibit the function of POPK-1. But this is not likely to be the case because of the following reasons. The effects of the POPK-1 MOs were dose dependent, and the severity of the phenotype seemed saturated at the dose we used. Further, we prepared two MOs against POPK-1. Co-injection of the two kinds of MOs synergistically worked at low doses, and reproduced the same phenotypes. Even when we co-injected the two MOs at the original concentration, the severity of the phenotypes was not increased. Therefore, the concentrations of MOs were high enough to produce the most severe phenotype. But we could not exclude the possibility that translation of POPK-1 starts as early as the first cell cycle before we injected the MO, and already-translated POPK-1 protein exerts its residual activity.
Postplasmic/PEM mRNA localization mechanism in ascidian embryos
There are several steps by which Type I postplasmic/PEM mRNAs are
eventually localized to the CAB. The mRNAs are located in the cortex of eggs.
During ooplasmic segregation, these mRNAs are relocalized to the PVC in
several cytoskeleton-driven phases
(Roegiers et al., 1999;
Sasakura et al., 2000
). Then
they concentrate into the CAB during cleavages. POPK-1 is involved in this
last process.
In the present study, co-localization of the postplasmic mRNA and cER was
further supported for Hr-POPK-1, Hr-ZF1 and Hr-Wnt-5.
Localization of Type I postplasmic/PEM mRNAs requires the presence of
cis-elements within the 3'-UTR of mRNAs, as in other organisms
(Sasakura and Makabe, 2002)
(reviewed by Kloc et al.,
2002
). The zip code is likely to be recognized by
trans-acting proteins that mediate attachment of the mRNAs to cER. In
Drosophila, gurken is localized in oocytes and plays essential roles
in defining the anterior-posterior and dorsoventral axes of the future embryo.
In this system, ER is also closely associated with the mRNA
(Saunders and Cohen, 1999
). A
mammalian homolog of Staufen, which is necessary for bicoid and
oskar localization in flies, is ER-binding protein
(Marion et al., 1999
). In
Xenopus oocytes, some of the maternal mRNAs co-localize with ER, and
ER associates with Staufen (Allison et al.,
2004
; Chang et al.,
2004
). Therefore, co-localization of mRNA with ER would be a
common phenomenon in the early development of different species. In this
study, we found that POPK-1 regulates the size and shape of the
cER/mRNA domain. The results of this study will provide novel information for
elucidating the localization mechanisms of maternal mRNAs in animal embryos.
As POPK-1/SAD-1 is a widely conserved protein, it will be informative to
examine whether the protein also works in other embryonic systems.
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ACKNOWLEDGMENTS |
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