Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEMRNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEMmRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.

mRNA localization occurs in a diversity of organisms, ranging from yeasts to plants and animals. It plays important roles in embryonic axis formation,cell polarity, asymmetric cell divisions and cell differentiation in eggs,embryos and somatic cells such as fibroblasts and neurons(Kloc et al., 2002). In many cases, mRNA localization is coupled with translational control. In flies and vertebrates, the restriction of some localized mRNAs to a particular region is important for their translation. This enables proteins to be synthesized at the right time in the right place(Lipshitz and Smibert, 2000; Johnstone and Lasko, 2001; Palacios and Johnston, 2001; Yoshida et al., 2004). In Xenopus and Drosophila, maternal factors segregate in distinct regions of the egg and blastomeres, and in many cases these factors are maternal mRNAs produced during oogenesis. For example, bicoid and oskar mRNAs are localized to the anterior and posterior poles in Drosophila eggs and determine the anterior-posterior axis and germ cell formation (Riechmann and Ephrussi,2001). In Xenopus eggs, several maternal transcripts are specifically localized in the animal or vegetal cortex of eggs. In particular, VegT mRNA localization in the vegetal cortex is crucial for endomesoderm formation, and Xcat2 for germ cell differentiation(King et al., 1999).

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/PEMmRNAs (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/PEMmRNAs. 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/PEMmRNA 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.

Animals and embryos

Adults of the ascidian Halocynthia roretzi were collected near the Asamushi Research Center for Marine Biology, Aomori, Japan, and the Otsuchi International Coastal Research Center, Iwate, Japan. Naturally spawned eggs were fertilized with a suspension of non-self sperm and raised in Millipore-filtered seawater containing 50 μg/ml streptomycin sulfate and 50μg/ml kanamycin sulfate at 9-13°C.

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-1primers: 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).

POPK-1 is required for muscle formation

To investigate the function of POPK-1 in ascidian development, we used specific MOs to inhibit its translation. MOs are effective in specifically preventing the functions of various genes in ascidian embryos(e.g. Satou et al., 2001). We designed two MOs against different non-overlapping sequences of Hr-POPK-1 (MO1 and MO2) to confirm their specificity. As both of them gave very similar results, the specificity of these MOs were supported, and we present the results without discrimination between the two (most results were obtained with MO2). Most embryos injected with POPK-1 MO developed normally up to the 110-cell stage, although a few showed a radialized cleavage pattern, as described later. Then gastrulation became aberrant. Eventually,the morphology of larvae was severely affected. The tail was not obvious, and no sensory pigment cells were present (Fig. 2A′). The degree of malformation was dose dependent. The morphology of larvae injected with standard control MO and 5-mismatch control MO was normal. However, the aberrant development of POPK-1MO-injected embryos was not rescued by co-injection of POPK-1 mRNA lacking morpholino target sequences.

We examined muscle formation in embryos injected with MO by monitoring the expression of acetylcholinesterase (AChE), a muscle-specific enzyme. Uninjected and control MO-injected larvae looked normal, and AChE was expressed in tail muscle cells (Fig. 2B). In embryos injected with POPK-1 MO, however, AChE expression was severely reduced or abolished(Fig. 2B′). Suppression of muscle formation was confirmed by immunostaining with the myosin antibody Mu-2 (Fig. 2C,C′). Absence of muscle cells was further confirmed in embryos in which cleavage was arrested at the 110-cell stage. Even when cleavage is permanently arrested at the 110-cell stage by treatment with cytochalasin B, ascidian embryos continue some differentiation and eventually express tissue differentiation features(Whittaker, 1973). In cleavage-arrested embryos uninjected or injected with control MO, ten muscle precursor blastomeres expressed Mu-2 and AChE, as expected from the fate map(Fig. 2D,E). By contrast, in embryos injected with POPK-1 MO, the number of positive blastomeres was greatly reduced (Fig. 2E′, Table 1).

The phenotype of POPK-1-deficient embryos resembles that of macho-1-deficient embryos

In macho-1-deficient embryos, primary muscle cells are lost, and the formation of other tissues is also affected(Nishida and Sawada, 2001; Kobayashi et al., 2003). To investigate the possibility that POPK-1 might have a similar role to macho-1, we analyzed the formation of mesenchyme, notochord and endoderm in embryos with MO. The presence of mesenchyme cells was examined with the monoclonal antibody Mch-3. When cleavage was arrested at the 110-cell stage, four presumptive mesenchyme blastomeres expressed the Mch-3 antigen in control embryos (Fig. 2F,G). By contrast, the mesenchyme marker was rarely expressed in MO-injected embryos(Fig. 2G′, Table 1). Notochord formation was evaluated with Not-1 antibody. Inhibition of POPK-1 function did not affect the expression of the Not-1 antigen at the tailbud stage(Fig. 2H,H′). In cleavage-arrested embryos, the arrangement and maximum number of Not-1-positive cells coincided well with notochord-lineage cells at the 110-cell stage in controls (Fig. 2I,J). There was no notable difference between control embryos and POPK-1 MO-injected embryos (Fig. 2J′, Table 1).

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.

Then we analyzed the epistatic relationship between POPK-1 and macho-1 by monitoring muscle formation. When macho-1 MO(60-120 pg, low dose) was injected into fertilized eggs, the number of muscle cells in larvae was significantly reduced(Nishida and Sawada, 2001)(compare Fig. 3C,D). Co-injection with POPK-1 mRNA (230 pg) had no rescuing activity, as tail formation was still severely affected, and the number of muscle cells was similar to that in embryos injected only with macho-1 MO (compare Fig. 3D,E). However, when POPK-1 MO2 (750 pg) and macho-1 mRNA (120 pg) were co-injected, excess muscle formed (Fig. 3F, Table 2). These results suggest that the suppression of Hr-POPK-1 function affects muscle cell formation through macho-1, and that POPK-1 acts upstream of macho-1.

POPK-1 is required for proper localization of macho-1 mRNA during early cleavage stage

As macho-1 mRNA is a localized muscle determinant and POPK-1 acts upstream of macho-1, we then examined whether the localization of macho-1 mRNA in the CAB at the 8-cell stage is altered in embryos injected with POPK-1 MO. In normal embryos, macho-1 mRNA accumulated in a moustache-shaped CAB in the posterior cortex of posterior-vegetal B4.1 blastomeres (79% of 29 cases, Fig. 4A, arrowheads). At the early 8-cell stage, embryos injected with POPK-1 MO, macho-1mRNA was more diffuse (all 18 cases, Fig. 4A, arrowheads). By contrast, at the late 8-cell stage, macho-1 mRNA became concentrated in smaller bilateral globular dots,instead of a normal elongated distribution (all 38 cases, Fig. 4A, arrowheads). In normal embryos the localization was connected on both sides of the midline, as the CAB is connected, but in injected embryos small dots were always visible apart from the midline. It is noteworthy that the small staining was always present but never lost in every case. Hr-PEN1 is a Type I postplasmic/PEM mRNA and has similarity to mammalian g1-related protein (Nakamura et al.,2003). To carry out more precise control experiments, we injected MO against PEN1, and macho-1 mRNA was normally localized like a moustache (data not shown). The result further supports that the aberrant localization of macho-1 mRNA is caused by specific inhibition of the POPK-1 function.

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/PEMmRNAs 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-1MO, 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.

Hr-POPK-1 regulates cER concentration into the CAB

macho-1 and Hr-PEM1 mRNAs has been shown to associate with a cER that is tethered to the egg plasma membrane, translocate to the posterior region during ooplasmic segregation, and then concentrate into the CAB during cleavages together with the cER(Sardet et al., 2003). In the present study, we prepared isolated cortices from the unfertilized eggs and the 8-cell embryos and labeled the cER in red with the lipophilic dye DiIC18 (3). The distribution of other Type I postplasmic/PEM mRNAs (Hr-POPK-1, Hr-ZF1 and Hr-Wnt-5)was visualized with green fluorescence at high resolution using fluorescence in-situ hybridization in the same cortex. These three Type I postplasmic/PEM mRNAs were also co-localized with the cER network in unfertilized eggs (Fig. 7A-D),and they were highly accumulated in the CAB at the 8-cell stage(Fig. 7E-G). However, unlike macho-1 (Fig. 7D) and Hr-PEM1, which are uniformly distributed on cER, the signals on entire cER were relatively weaker and, especially in Hr-POPK-1, some small granules with bright fluorescence were sparsely detected on tubes and sheets of the cER in unfertilized eggs(Fig. 7A-C, arrows).

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-PEM1mRNA-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.

Type I postplasmic/PEM mRNA associates with the cER but not with CAB precursors at 2- and 4-cell stages

As mentioned before, in extracted embryos formation of the CAB is initiated as dozens of small dots at the 2- and 4-cell stages. However, the distribution of the Type I postplasmic/PEM mRNAs at the 2- and 4-cell stages did not show such a punctate and dotted appearance in whole-mount in-situ hybridization (Fig. 8C,D)(Sasakura et al., 1998a;Sasakura 1998b; Nakamura et al.,2003). In our previous study, the co-localization of Type I postplasmic/PEM mRNAs and the cER was observed only in eggs and 8-cell embryos (Sardet et al., 2003). Therefore, we examined the cER/mRNA distribution at the 2- and 4-cell stages in isolated cortices to investigate it in more detail. Hr-PEM1 mRNA and cER were also clearly co-localized at these stages(Fig. 8F). However, as observed in whole mounts, a dotted distribution corresponding to the CAB precursors at these stages was never observed. Therefore, cER/mRNA distribution is not identical to that of CAB precursors in extracted embryos (probably corresponding to EDM/putative germ plasm) at these stages, although the distribution of both becomes overlapped in the CAB in the 8-cell embryos. POPK-1 is probably involved in proper concentration and positioning of both of cER/mRNA domain (Figs 4 and 8) and CAB precursors(Fig. 5) by the 8-cell stage.

The role of Hr-POPK-1 in localization of cER/mRNA domain

Organisms use mRNA localization to locally produce cytoplasmic factors in particular regions within cells and embryos. In ascidians, maternal Type I postplasmic/PEM mRNAs are localized to the PVC in fertilized eggs, and some of them have been shown to play important roles in establishment of the anteroposterior axis (Nishida and Sawada,2001; Nakamura et al.,2005). In this study, we have presented evidence that the postplasmic/PEM mRNA Hr-POPK-1 is required for the proper spatial distribution of postplasmic/PEM mRNAs and CAB materials at the posterior pole of cleavage-stage embryos.

When POPK-1 MO was injected into eggs, macho-1 mRNA became diffuse at the posterior pole at the 2-, 4 and early 8-cell stages,then detected in tiny spots after the late 8-cell stage. The spots were much smaller, rounded and always laid apart from the midline, unlike the normal moustache shape. This was also observed for all six postplasmic/PEMmRNAs, including POPK-1 itself. It is not clear whether this remarkable transition from diffused to compact distribution observed in POPK-1-deficient embryos reflects the events that also take place in normal embryos. The results of Sardet et al.(Sardet et al., 2003) and the present data indicate that several postplasmic mRNAs are co-localized with cER and move with it. In isolated cortices of the POPK-1 MO-injected 8-cell stage embryos, cER was also present in bilateral compact regions apart from the midline. The distribution matches that of the postplasmic/PEM RNA. Therefore, POPK-1 is involved in the proper distribution of mRNAs by controlling cER movements such as compaction and positioning.

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.

The role of Hr-POPK-1 in localization of EDM/putative germ plasm

The morphology of the CAB in extracted embryos was also altered in POPK-1 knockdown embryos. It became smaller, rounded and separate from the midline. The morphology nicely corresponded to that of the cER/mRNA domain at the 8-cell stage. The CAB precursors were more dispersed at the earlier stage compared with normal distribution(Fig. 5). One of the conspicuous characteristics of the CAB is that it is enriched in EDM, which resembles germ plasm of various kinds of animals(Iseto and Nishida, 1999). The blastomeres that inherit the CAB are thought to become primordial germ cells in ascidians (Fujimura and Takamura,2000; Takamura et al.,2002) (reviewed by Nishida,2005), supporting the idea that the EDM corresponds to germ plasm. In extracted embryos, only the EDM seems to persist in the CAB in electron microscopic observation (Iseto and Nishida,1999), and it is likely that EDM is visible as a highly refractive structure under the optical microscope. Therefore, POPK-1 is also likely to be required for proper concentration and positioning of the EDM/putative germ plasm, although further analysis will be required for elucidation of the hypothesis that EDM in the CAB is equivalent to germ plasm in ascidians by detecting germ-plasm-specific molecular markers such as Xcat2 in Xenopus (King et al.,2005).

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.

We thank members of the Asamushi Research Center for Marine Biology and the Otsuchi International Coastal Research Center for help in collecting live ascidian adults, and members of the Misaki Marine Biological Station and Seto Marine Biological Laboratory for help in maintaining them. We also thank Dr T. Nishikata (Konan University) for providing the Not-1 and Mu-2 monoclonal antibodies, Drs T. Kawashima (Kyoto University) and Y. Kohara (National Institute of Genetics) for the MAGEST plasmid, and Dr C. Sardet (Villefranche marine station) for critical reading of the manuscript and useful discussion. This work was supported by Grants-in-Aid for Scientific Research from JSPS and MEXT, Japan (16107005 and 13044003), and by Toray Science and Technology Grant to H.N.

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