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Developmental Biology 290 (2006) 297 – 311 www.elsevier.com/locate/ydbio

Establishment of –vegetal polarity during maturation in ascidian oocytes

Franc¸ois Prodon *, Janet Chenevert, Christian Sardet

BioMarCell, UMR7009, CNRS/UPMC, Station Zoologique, Observatoire Oce´anologique, Villefranche sur Mer 06230, France

Received for publication 27 June 2005, revised 21 October 2005, accepted 9 November 2005 Available online 5 January 2006

Abstract

Mature ascidian oocytes are arrested in metaphase of meiosis I (Met I) and display a pronounced animal–vegetal polarity: a small meiotic spindle lies beneath the animal pole, and two adjacent cortical and subcortical domains respectively rich in cortical endoplasmic reticulum and postplasmic/PEM RNAs (cER/mRNA domain) and mitochondria (myoplasm domain) line the equatorial and vegetal regions. Symmetry-breaking events triggered by the fertilizing sperm remodel this primary animal–vegetal (a–v) axis to establish the embryonic (D–V, A–P) axes. To understand how this radial a–v polarity of eggs is established, we have analyzed the distribution of mitochondria, mRNAs, microtubules and chromosomes in pre-vitellogenic, vitellogenic and post-vitellogenic Germinal Vesicle (GV) stage oocytes and in spontaneously maturing oocytes of the ascidian Ciona intestinalis. We show that myoplasm and postplasmic/PEM RNAs move into the oocyte periphery at the end of oogenesis and that polarization along the a–v axis occurs after maturation in several steps which take 3–4 h to be completed. First, the Germinal Vesicle breaks down, and a meiotic spindle forms in the center of the oocyte. Second, the meiotic spindle moves in an apparently random direction towards the cortex. Third, when the microtubular spindle and chromosomes arrive and rotate in the cortex (defining the animal pole), the subcortical myoplasm domain and cortical postplasmic/PEM RNAs are excluded from the animal pole region, thus concentrating in the vegetal hemisphere. The actin cytoskeleton is required for migration of the spindle and subsequent polarization, whereas these events occur normally in the absence of microtubules. Our observations set the stage for understanding the mechanisms governing primary axis establishment and meiotic maturation in ascidians. D 2005 Elsevier Inc. All rights reserved.

Keywords: Oogenesis; GVBD; Meiosis; Polarization; Cortical RNAs; Cortex; Ciona

Introduction axis) is established during oogenesis. This axis is often apparent in the mature oocyte as a polarized distribution of The polarization of oocytes, eggs and embryos arises from a organelles and cytoskeletal elements. The a–v axis together rupture of symmetry caused by localized signaling resulting in with another cue such as factors locally introduced by the redistributions of organelles and/or macromolecular complexes sperm plays an important role in the establishment of the along axes (Sardet et al., 2004; Prodon et al., 2004; Wodarz, embryonic antero-posterior (A–P), dorso-ventral (D–V) and 2002). In the fly Drosophila melanogaster, embryonic axes are consequent left–right (L–R) axes. Such is the case in ascidians established early during oogenesis (Huynh and St Johnston, in which the mature oocyte displays a visible primary a–v axis 2004), while in the worm C. elegans polarization is only which predicts the general orientation of the embryonic D–V established after fertilization (Nance, 2005). In most other axis and graded potentialities for the formation of ectoderm, organisms such as amphibians, mammals, echinoderms or mesoderm and endoderm (Conklin, 1905; Roegiers et al., 1995, molluscs, a primary axis (so called animal–vegetal or a–v 1999; Nishida, 2005). The primary a–v axis of mature oocytes of the principle solitary ascidian species used for research (Halocynthia roretzi, * Corresponding author. Present address: Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Ciona intestinalis and savigny, Phallusia mammillata)is Osaka 560-0043, Japan. characterized by the presence of a meiotic spindle situated at E-mail address: [email protected] (F. Prodon). the animal pole and the polarized distribution of two concentric

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.11.025 298 F. Prodon et al. / Developmental Biology 290 (2006) 297–311 peripheral domains: a subcortical domain rich in mitochondria teans and sea urchins, when fully grown GV-containing (called myoplasm) and a cortical domain rich in endoplasmic oocytes are taken out of the gonad and exposed to sea water, reticulum associated with some maternal postplasmic/PEM maturation and GVBD occur spontaneously to yield oocytes RNAs (cER/mRNA domain). Both peripheral domains line the which can be fertilized and develop normally (Stricker and equatorial and vegetal regions and are excluded from the Smythe, 2001; Voronina and Wessel, 2003). Similarly, oocytes animal pole (Sardet et al., 1992, 2003; Roegiers et al., 1995, of the ascidian H. roretzi and those of Molgula occidentalis 1999; Prodon et al., 2005). Additional differences along the a– mature spontaneously in sea water (Sakairi and Shirai, 1991; v axis include differences in the distribution of microfilaments Sawada and Schatten, 1988). Artificially matured oocytes of and microtubules which are respectively enriched and con- Halocynthia can be fertilized and develop (Sakairi and Shirai, spicuously absent from the subcortical myoplasm domain 1991; Bates and Nishida, 1998), and it has been mentioned that (Sardet et al., 1992). How and when this extensive polarization the same is true for Ciona oocytes (Satoh, 1994). of the mature ascidian oocyte is established are not known. Our objective was to describe precisely the timing of events Oogenesis has been studied in several ascidian species with leading to the distribution of organelles, cytoskeletal elements regard to the elaboration of accessory cellular structures and and mRNAs along the primary a–v axis in the cosmopolitan chorion which surround the oocyte (Sugino et al., 1987; Eisenhut ascidian species of reference: C. intestinalis. The older literature and Honegger, 1997), the location, size and appearance of (reviewed by Kessel, 1983) and more recent work on oocytes oocytes in the gonad (Kessel, 1966; Okada and Yamamoto, (Jeffery and Capco, 1978; Swalla et al., 1991) have distin- 1993) and the distribution of organelles, mRNAs, cytoskeletal guished 3 main stages of oogenesis based on the size, yolk proteins and structures in oocytes before and after maturation content and pigmentation of oocytes: smaller pre-vitellogenic (Conklin, 1905; Kessel, 1983; Jeffery and Capco, 1978; Swalla oocytes (stage I), vitellogenic oocytes (stage II) and larger post- and Jeffery, 1995; Yoshida et al., 1997; Tanaka et al., 2004; vitellogenic oocytes (stage III) which are those able to undergo Sawada and Schatten, 1988). Immature oocytes of various sizes maturation when released in sea water. The mature egg that is and stages of oogenesis are found in the ovary, and the fully ready for fertilization could logically be called a mature stage IV grown oocytes mature to meiotic metaphase I (Met I) upon oocyte. We have carefully examined C. intestinalis oocytes of all ovulation. In some ascidian species such as the Enterogona sizes dissected from the gonads and large oocytes spontaneously Ciona and Phallusia, large numbers of mature oocytes (Met I) maturing in sea water with respect to the changing appearance of are stored in a long oviduct before being released in batches and the surrounding structures (acellular and cellular layers) and to fertilized. In ascidian species such as the Halo- the distribution and relocalization of organelles (mitochondria/ cynthia, mature oocytes are not stored, but stocks of full grown nucleus), chromosomal and microtubular structures (meiotic immature (Prophase I) oocytes accumulate in the ovary to mature spindle) and maternal mRNAs which are localized in the cortex upon spawning. In all species, fertilization causes the oocyte to at the time of fertilization (the postplasmic/PEM RNAs Ci- reinitiate and complete the meiotic and mitotic cell cycles and to PEM3 and Ci-PEM1). undergo the phases of cytoplasmic and cortical reorganizations We come to the conclusion that stage I, II and III oocytes are (generally called ooplasmic segregation) which define A–P and fully symmetrical with no axis of polarity and that the process of D–Vaxes by the time of first cleavage (Satoh, 1994; Roegiers et oogenesis is accompanied by major changes in the location and al., 1995, 1999; Prodon et al., 2005; Nishida, 2005). state of organization of organelles, microtubules and postplas- Fully grown GV-containing oocytes from many organisms mic/PEM RNAs. We show that the establishment of the primary including molluscs, sea urchins, starfish, nemerteans worms, animal–vegetal axis occurs during maturation when the amphibians and mammals can mature in vitro. Activation of centrally located meiotic spindle migrates towards the maturing the kinase Cdk1/cyclin B (Maturation Promoting Factor or oocyte cortex, and that the polarization of the myoplasm and MPF) triggers the initiation of the meiotic cell cycle starting mRNA-containing peripheral domains occurs after the animal with Germinal Vesicle BreakDown (GVBD) and subsequent pole is defined. We further demonstrate that these events are changes such that the oocytes acquire the competence to be dependent on the actin cytoskeleton but not on microtubules, and fertilized and develop normally (Voronina and Wessel, 2003; we discuss the implications of these findings for the mechanism Smythe and Stricker, 2005; Jessus and Ozon, 2004). In of the establishment of a–v polarity in ascidians. molluscs, starfish, amphibians and mammals, the hormonal/ physiological signal and receptors for maturation produced by Materials and methods follicle cells have been identified as small organic molecules which can be used to induce in vitro maturation, whereas in sea Biological material and obtaining oocytes urchins, nemerteans and ascidians, natural signals responsible Adults of the ascidian C. intestinalis were collected at the Marine Biological for triggering maturation are not known. Nevertheless, in Station of Roscoff (Britany, France) and in Se`tes (Etang de Tau, Mediterranean Halocynthia, there is evidence that the in vivo trigger for coast, France). Collection of oocytes at different stages in C. intestinalis is maturation is a trypsin-like macromolecule produced by follicle straightforward as ovaries can be easily removed by dissection. Each Ciona adult cells (Sakairi and Shirai, 1991). It has also been observed that has one ovary located at the base, i.e. the settlement side opposite oral and atrial siphons (Figs. 1A, B). The ovary is enclosed by a simple germinal epithelium as removal of calcium from sea water and lowering pH can described by Okada and Yamamoto (1993, 1999), forming an individualized partially block GVBD in Halocynthia and other ascidian gland near the intestine separated from the spermiduct and embedded in the species (Sakairi and Shirai, 1991; Lambert, 2005). In nemer- mantle as visceral organs (Satoh, 1994)(Figs. 1B, C). We also observed that F. Prodon et al. / Developmental Biology 290 (2006) 297–311 299

Fig. 1. The ovary of the ascidian C. intestinalis. (A) Ovary (ova, dotted circle) and spermiduct (s, arrowheads) are visible through the tunic in intact animal. (B) Detunicated animal. Ovary is located at the base (b) of the animal, on the side opposite of atrial and oral siphons (as, os). Oviduct (ovd) is indicated by arrowhead. (C) Two ovaries isolated from 2 Ciona adults. The upper one is darker because pigmented maturing oocytes are abundant; the lower one is white and contains mostly young non-pigmented oocytes. Insert: detail of an isolated ovary indicating the presence of maturing oocytes (dark spots). (D, E) Fragments of ovaries (DIC optics) showing the distribution of GV-containing oocytes of different sizes and pigmentations. Arrowheads indicate the position of the Germinal Vesicle (GV). ovaries change in size and color depending on the abundance of maturing oocytes dehydrated in 25%, 50% and 75% ethanol (10 min each) and stored in 100% they contain (Figs. 1C, D, E). This convenient property allows selection of ethanol at À20-C. After rehydration by successive incubation in 75%, 50% and gonads and oocytes of desired stages. After dissection of ovaries, large ovarian 25% ethanol and in PBT (PBS containing 0.1% Tween 20) three times (10 min fragments were removed, and oocytes transferred to filtered sea water and each), the specimens were treated with 2 Ag/ml proteinase K in PBT (25 min at selected according to size, appearance of the vitelline coat and pigmentation. room temperature) and digestion was stopped by washing with PBT (three times, Once the largest GV-stage III oocytes are collected and deposited in sea water 5 min each). The specimens were post-fixed with 4% paraformaldehyde in PBS (corresponding to ? t0 X)at17-C, the process of maturation is initiated. The for 30 min followed by washing with PBT three times (5 min each). They were oocytes which matured in vitro (overnight) could be fertilized with sperm incubated in prehybridization buffer (50% formamide, 6Â SSC, 5Â Denhardt, 1 activated by dilution in sea water pH 9.0 (adjusted with 1N NaOH) for 10 min. To mg/ml yeast total RNA) for 1 h at 55-C. The prehybridization buffer was replaced label cellular components in vivo or after fixation, the vitelline coat was removed with hybridization buffer containing 50 ng/ml digoxygenin (DIG)-labeled chemically using a solution of 1% Thioglycolate–0.05% Pronase in filtered sea antisense or sense transcript. Hybridization reaction was carried out at 55-C water as described (Prodon et al., 2005). overnight. At the end of the hybridization, the specimens were washed as described by Wada et al. (1995). After blocking in 0.5% blocking reagent (Roche Labeling of fixed and live oocytes blocking reagent), the specimens were incubated with anti-DIG alkaline phosphatase 1/2500 at 4-C overnight. After four washes in PBT (30 min for After removing the chorion, full grown stage III oocytes were fixed at each), hybridized RNAs were revealed using the HNPP fluorescent detection kit different periods during maturation (Germinal Vesicle (GV) stage, Germinal (Roche) according to the manufacturer’s instructions. Oocytes were mounted in Vesicle BreakDown (GVBD) stage, 1 h/2 h/4 h after GVBD, mature oocytes) in citifluor and observed using confocal microscopy. methanol at À20-C blocked in PBS containing BSA (1%) and incubated with the mouse NN18 monoclonal antibody (1/200, ICN), which labels mitochon- Ovary sections dria (Prodon et al., 2005), and the YL1/2 rat anti-h-tubulin antibody (1/500, Expression of Ci-PEM3 mRNAs in ovaries was observed by in situ Amersham). Immunolabeled samples were then washed in PBS Tween (0.1%), hybridization using digoxigenin (DIG)-labeled Ci-PEM3 antisense probes incubated with fluorescent secondary antibodies (Jackson Labs), treated with (AK114726). Antisense and sense probes were prepared using in vitro Hoechst 33342 (1 Ag/ml in PBS) for 20 min to label DNA and mounted in transcription. Small pieces of the ovary were dissected, fixed, washed, Citifluor (Chemlab). For labeling with TRITC phalloidin (Molecular Probes), dehydrated as described by Koyanagi and Honegger (2003) and embedded in oocytes were fixed in 4% paraformaldehyde, 0.5 M NaCl in PBS and washed in JB4 wax, a clear polymer embedding matrix (Canemco and Marivac, Canada). PBS containing 0.1% Triton X 100. To trace the distribution of mitochondria in Ten-micrometer-thick sections were cut, dewaxed and rehydrated in a series of living oocytes, we labeled them with TMRE (Molecular Probes) as described SP15-100 bath (Permount, Fisher Scientific) and water for 10 min each and (Prodon et al., 2005). Oocytes were mounted and observed using epifluores- hybridized as described by Koyanagi and Honegger (2003). cence, Differential Interference Contrast (DIC), Bright Field optic or confocal microscopy (Roegiers et al., 1999; Prodon et al., 2005). Imaging oocytes using video, digital epifluorescence and confocal microscopy Treatment with cytoskeletal inhibitors For video-enhanced microscopy observations, we used a Zeiss Axiophot Oocytes were selected and dechorionated as for observation during microscope equipped with Newicon and SIT cameras (Lhesa and Hamamatsu). maturation, and nocodazole, cytochalasin D or DMSO as a control was added The images were analyzed with Universal Imaging Software (Metamorph v at the time when the first oocytes in the batch began GVBD. Concentrations 2.76). Confocal laser scanning microscopy was performed on an inverted Leica used were 5–10 AM for nocodazole and 4–16 Ag/ml for cytochalasin D SP2 confocal microscope equipped with argon neon, helium neon and green (sigma) with similar effects on polarization. After 4–5 h of incubation, oocytes neon lasers. For observations of living or fixed oocytes labeled with TMRE or werefixedincold(À20-C) methanol for labeling microtubules and Hoechst, YL1/2 and NN18 antibodies, images were acquired using the mitochondria or in 4% paraformaldehyde for labeling actin as described above. simultaneous confocal scan mode.

In situ hybridization Results

Whole mounted oocytes Classification of oocytes Oocytes were hybridized in situ using DIG-labeled Ci-PEM1 (AK113383) antisense probes. Detection of Ci-PEM1 was carried out as described previously (Prodon et al., 2005). Dechorionated oocytes were fixed with 4% paraformal- In order to understand the progression of oocytes through dehyde in 0.5 M sodium chloride, 0.1 M MOPS (pH 7.5) at 4-C overnight, oogenesis and maturation and to determine at which period the 300 F. Prodon et al. / Developmental Biology 290 (2006) 297–311 a–v polarity of the Ciona oocyte is established, we examined Previous reports on oogenesis in several ascidian species the distribution of the mitochondria-rich myoplasm, a good (reviewed by Kessel, 1983; and see Swalla et al., 1991) consider indicator for a–v polarity. We simultaneously examined that there are basically 3 stages of GV-containing oocytes: small microtubules, chromosomes and postplasmic/PEM maternal transparent pre-vitellogenic (stage I), growing vitellogenic mRNAs in live and fixed oocytes. Based on the organization of oocytes (stage II) and post-vitellogenic, pigmented, full grown these intracellular indicators as well as the size, pigmentation GV-stage oocytes (stage III). We could clearly distinguish of the oocytes and changes in the cellular layer surrounding the subcategories using criteria of chorion structure and oocyte size, oocyte, we were able to define subcategories in GV-containing pigmentation, organization and shape of microtubules and oocytes. mitochondria (Fig. 2). In this study, we use the term chorion to

Fig. 2. Stages of oocyte maturation with accompanying microtubule and mitochondrial reorganizations. Periods of oogenesis (pre-vitellogenesis, vitellogenesis) and maturation (Germinal Vesicle BreakDown abbreviated GVBD, mature oocytes) are represented above each oocyte stage. For each stage, the average oocyte diameter is indicated in micrometers. Note that oocytes about double in size from stage I to IV. Oocytes in stage I are represented in column (A), in early and late stage II in (B and C), in stage III in (D) and in stage IV in (E). (A1–E1) Aspect of chorion and oocyte pigmentation (DIC optics). The oocyte in (E1) appears smaller because the image was reduced to include the surrounding test cells (tc) and follicle cells (fc) and the vitelline space (vs). (A2–E2) Detail of chorion at higher magnification (DIC optics) showing the presence of accessory cells (ac, arrowhead in A2, B2), follicle cells (fc), test cells (tc) and vitelline space (vs, arrowhead in D2). Dotted lines indicate the periphery of oocytes. (A3–E3, A4–E4) Microtubule distribution from stage I to stage IV after immunostaining (equatorial confocal sections). In (E3), the meiotic spindle (ms, arrowhead) is present at the animal pole in mature stage IV oocyte. Inserts at lower right in (A3) and (E3) show chromosomes. (A4–E4) Detail of microtubule distribution (equatorial confocal sections). In (A4–D4), the GV is located on the right side and the cell cortex on the left side. (E4) Detail of the animal pole region showing the meiotic spindle in stage IV oocytes (mature oocyte). (A5–E5, A6–E6) Mitochondria localization and polarization in live oocytes. Equatorial (A5–E5) and tangential (A6–E6) confocal sections. Dotted circle in (D5) indicates the periphery of the GV. (a) Animal pole, (v) vegetal pole. (A7–E7) High magnification tangential confocal sections showing change in size, shape and aggregation state of mitochondria. F. Prodon et al. / Developmental Biology 290 (2006) 297–311 301 describe the cellular (follicle cells and test cells) and acellular definition is the animal pole (Figs. 2E3, E4), and chromosomes structures (vitelline coat and space) surrounding the oocytes are aligned onto a metaphase plate (Fig. 2E3, insert). (Eisenhut and Honegger, 1997). In the smallest GV-containing The vast majority of mitochondria are concentrated in a ring oocytes, which correspond to pre-vitellogenic stage I oocytes around the GV prior to vitellogenesis (stage I oocyte) (Fig. (less than 50 Am in diameter), the chorion is not fully formed, 2A5), and only a few mitochondria are present in the cortex. At and only a few undifferentiated accessory cells (ac) are present at this stage, mitochondria are long and stringy, about 5–6 Amin the periphery of these young non-pigmented oocytes (Figs. 2A1, length (Figs. 2A6, A7). Early stage II vitellogenic stages A2). The morphology of the chorion changes during vitello- oocytes contain an abundance of shorter mitochondria often genesis. In early stage II oocytes (50–70 Am in diameter), an present as aggregates in the cytoplasm and cortex (Figs. 2B5– increasing number of accessory cells forms a monolayer B7). In later vitellogenic stages II and III, mitochondria acquire surrounding completely these transparent oocytes (Figs. 2B1, the short and thick rod shapes (Figs. 2C5–C7, D5–D7) like B2). In late stage II vitellogenic oocytes (70–80 Am in diameter those in mature oocytes (Figs. 2E5–E7). We could clearly and pigmented), fully individualized cube-shaped follicle cells distinguish early and late stage II oocytes due to the increased (fc) surround the oocytes. Test cells are not apparent at this stage density of mitochondria and microtubules near the surface of (Figs. 2C1, C2). In full grown pigmented stage III oocytes (about larger stage II oocytes. In post-vitellogenic stage III oocytes, 100 Am in diameter), the vitelline space (vs) begins to appear mitochondria are found exclusively at the cell periphery, (Figs. 2D1, D2). The follicle cells located on the outside of the forming a 7- to 10-Am-thick subcortical myoplasm domain vitelline coat enlarge, taking the appearance of petals around a (Fig. 2D5). Along equatorial confocal sections, this peripheral flower. At this stage, fully grown and pigmented stage III ring of mitochondria is symmetrically distributed around the oocytes acquire the ability to mature spontaneously when stage III oocyte, with no indication of the presence of animal or exposed to sea water as noted previously (Satoh, 1994). After vegetal poles. After meiotic maturation, however, the subcor- disruption of the Germinal Vesicle (GVBD), the cellular chorion tical mitochondria-rich myoplasm domain is excluded from the expands and mature Ciona oocytes (stage IV) arrest in meiotic animal pole region (Figs. 2E5, E6). In these mature stage IV metaphase I. At this stage, the chorion is stratified, and spherical oocytes, the myoplasm forms a typical basket-shaped domain test cells become visible on the inside of the vitelline coat close characterizing unfertilized eggs of numerous ascidian species to the plasma membrane of these strongly pigmented oocytes as recognized by Conklin in his classical description of this (Figs. 2E1, E2). Some of these mature stage IV oocytes can be yellow pigmented region in Styela partita (1905). fertilized (data not shown). All of these stage IV oocytes are These observations made on individual oocytes of various surrounded by a vitelline coat and fully differentiated follicle and stages suggested that the polarization of mitochondria distri- test cells which resemble the chorion of naturally matured and bution in oocytes occurs between stage III and stage IV during spawned oocytes (Fig. 2E1 and see Eisenhut and Honegger and/or after GVBD. We therefore analyzed these changes in (1997) for details). more detail in maturing oocytes.

Microtubules and mitochondria reorganize during oogenesis Oocyte maturation

To further characterize different stages of oogenesis, we In order to analyze the events which occur during maturation examined the distribution of mitochondria, microtubules and of living stage III oocytes, we observed their spontaneous chromosomes, characteristic markers of the a–v polarity of maturation in sea water using time-lapse recordings. At 17-C, fertilizable mature oocytes (Sardet et al., 1992; Sawada and GVBD starts about 2.5 h after stage III oocytes are removed from Schatten, 1988). isolated ovaries and deposited into sea water (Fig. 3D). This Microtubules are increasingly abundant in the cytoplasm of process occurs in a relatively synchronous manner in isolated growing oocytes from stage I to early stage II. They radiate populations of maturing oocytes whether chorionated (Fig. 3A) from the periphery of the GV towards the cell cortex (Figs. or dechorionated (Fig. 3C). Surprisingly, follicle cells are able to 2A3, A4, B3, B4). In late stage II and III oocytes, microtubules elongate outside the ovarian tissue in about 10 h (Fig. 3B). The appear shorter and are essentially concentrated in both the chorion (follicle cells, test cells and vitelline space) elaborated cortex and around the GV (Figs. 2C3, C4, D3, D4). At these by the spontaneously maturing oocytes resembles that of stages, the GV is located in a central position and contains a naturally matured oocytes (see Eisenhut and Honegger, 1997). single nucleolus. Chromosomes are condensed, suggesting that Following GVBD, a central light zone (lz) can be discerned oocytes are arrested in prophase I (diplotene) prior to in the middle of the oocyte cytoplasm (Figs. 3A, C, and see maturation (Fig. 2A3, insert). Throughout these oogenic stages, Supplementary Material Movies 1 and 2). This light zone microtubules were radially symmetric with no obvious corresponds to the position of the meiotic apparatus which polarized distribution or center of nucleation. These findings forms in the center of oocytes after GVBD and then migrates indicate that in ascidian oocytes centrosomes are absent or not towards the cell cortex in about 2 h at the approximate speed of functional as microtubule organizing centers. 0.4 Am/min (Fig. 3C, and see further Fig. 6). Time-lapse After GVBD, in mature stage IV oocytes, most cytoplasmic recordings show that cortical and cytoplasmic contraction/ and cortical microtubules disappear, a small meiotic spindle relaxation waves sweep through maturing oocytes such that (¨10 Am in length) is observed in a cortical location which by oocytes appear to dance. These characteristic movements occur 302 F. Prodon et al. / Developmental Biology 290 (2006) 297–311

Fig. 3. Germinal Vesicle BreakDown (GVBD) and maturation in living oocytes. (A) Images from a time-lapse sequence (from Supplementary Material Movie1) showing the in vitro maturation of a chorionated C. intestinalis oocyte (Bright Field optics). Follicle cells (fc), test cells (tc) and vitelline space (vs) are indicated by arrowheads. (GV) Germinal Vesicle, (lz) light zone. ‘‘t0’’ corresponds to the beginning of the BreakDown of the GV (GVBD) 2 h 30 min after dissection of the ovary. Time is indicated in hours (h) and minutes (V). (B) Close up view of chorion from sequence in (A) showing changes in the morphogenesis of follicle cells (light blue) and test cells (light yellow) during GVBD and maturation. The fc progressively elongate from the beginning to the end of this sequence. A slight rotation of the oocyte gives the impression that the fc moves. During this period, tc moves in the vitelline space. (C) Images from a time-lapse sequence (from Supplementary Material Movie 2) of a dechorionated Ciona oocyte during and after GVBD (DIC optics). Arrowhead indicates the location of the meiotic spindle after GVBD. (D) Quantification and timing of GVBD in chorionated oocytes spontaneously maturing upon exposure to sea water (t0). Four series of 50 oocytes each were averaged. at specific times which correspond to the onset of GVBD and 2 RNAs (such as macho1, PEM1, PEM3) situated just beneath the h after GVBD when the meiotic spindle has reached the cortex plasma membrane and a subcortical layer of mitochondria-rich (see Supplementary Material Movie 3). myoplasm distributed in a polarized manner along the a–v axis We found that some of the Ciona oocytes which matured (reviewed in Sardet et al., 2005; Nakamura et al., 2003). We spontaneously in sea water are competent for fertilization and therefore examined when mitochondria and when postplasmic/ can develop, as has been observed for Halocynthia (Sakairi and PEM RNAs acquired their peripheral and polarized distribu- Shirai, 1991), therefore it is likely that the in vitro maturation tions. In situ hybridization of ovary sections reveals that, in stage and polarization processes recapitulate what happens in vivo. I and II oocytes, Ci-PEM3 is concentrated in aggregates around the GV (Figs. 4A, B). In larger oocytes, whose stages were Polarization of the maturing oocyte difficult to determine from random ovary sections, the RNA signal was located at the periphery (Fig. 4C). Mature ascidian oocytes from several species contain a To determine when the major postplasmic/PEM RNAs cortical layer of maternal mRNAs called postplasmic/PEM become polarized, we fixed populations of spontaneously F. Prodon et al. / Developmental Biology 290 (2006) 297–311 303

defined by the position of the meiotic chromosomes (Figs. 4E1–E3) and progressively merge into a homogeneous layer over the rest of the surface of the oocyte (Fig. 4F). The fluorescent signal for Ci-PEM1 RNAs does not appear as reticulated in maturing oocytes as it does in fully mature stage IV oocytes (Fig. 4F3, and see Sardet et al., 2003). From these observations, we conclude that the polarized distribution of Ci- PEM1 RNAs is achieved within4hofdisruption of the GV (Figs. 4F1, F2). We were able to follow the polarization of the mitochondria- rich subcortical myoplasm domain using time-lapse confocal microscopy in live maturing stage III oocytes labeled with the mitochondria dye TMRE (Fig. 5 and Supplementary Material Movie 4). Within 2 h of GVBD, a gradual polarization of the myoplasm was observed. Four hours after GVBD, the mitochondria-rich myoplasm clearly formed a polarized subcortical basket open at the animal pole (Figs. 5A–C). These observations show that the polarization of the myoplasm and of the layer of postplasmic/PEM RNA during maturation follow approximately the same time course.

Meiotic spindle displacement is the first sign of oocyte polarization

Having demonstrated that cortical postplasmic/PEM RNAs and subcortical myoplasm acquire their asymmetric distribu- tion within 4 h of GVBD, we examined the precise timing of this polarization with respect to the formation of the meiotic spindle and its localization at the cortex. We fixed populations of stage III oocytes 1, 2 and 4 h after Fig. 4. Cortical postplasmic/PEM RNAs become polarized after GVBD. (A, B, GVBD and labeled them for tubulin and DNA to follow the C) In situ hybridization of Ci-PEM3 RNA (arrowheads) on ovary sections formation and location of the meiotic apparatus. Mitochondria respectively at stages I, II and III. (GV) Germinal Vesicle. (D–F) Detection of were labeled as well to monitor the polarization of the fluorescent signal for Ci-PEM1 RNA using whole mount in situ hybridization in maturing oocytes. (D1–D3) Ci-PEM1 RNA distribution in GV-containing mitochondria-rich myoplasm domain (Fig. 6). One hour after oocytes (stage III). (D1) Confocal equatorial section. (D2) Detail at higher GVBD, the meiotic apparatus was already located excentrically, magnification of the fluorescent detection signal for Ci-PEM1 RNA (arrow- while the mitochondria-rich myoplasm still showed a concentric heads) shown in the boxed area in (D1); (D3) tangential confocal section (1 Am homogeneous subcortical distribution (Figs. 6B1, B2) as in GV- under the cell surface) showing Ci-PEM1 RNA patches (arrowheads) at higher containing oocytes with no indication of surface polarity (Figs. magnification. Insert: tangential confocal view of a whole GV-stage oocyte. (E1–E3) Ci-PEM1 RNA distribution in an oocyte 3 h after the complete 6A1, A2). The meiotic spindle is small and barrel-shaped and breakdown of the GV (GVBD stage) and chromosomes location. (E1) Confocal lacks conspicuous astral microtubules (Fig. 6 and see also Fig. equatorial section. Patches of Ci-PEM1 RNA (arrowheads) are excluded from 8). It first migrates from the oocyte center towards the cortex the animal pole region. (E2) Arrowhead indicates chromosomes location in the with its long axis perpendicular to the oocyte’s surface (Figs. oocyte shown in (E1). (E3) Tangential confocal section (1 Am under the cell 6B2, C2) and then rotates to become parallel to the oocyte surface). Arrowheads indicate patches of Ci-PEM1 RNA in the vegetal hemisphere (v). (F1–F3) Ci-PEM1 RNA distribution in a mature oocyte surface (Figs. 6D2, E2). Positioning of the meiotic spindle with spontaneously matured in sea water (4 h after the complete disruption of the aligned chromosomes beneath the cortex was completed 2–3 GV). (F1) Confocal equatorial section showing the polarized distribution of Ci- h after GVBD and at this time subcortical myoplasm began to PEM1 RNAs along the a–v axis. (F2) Confocal tangential section (1 Am under clear from the animal pole region (Figs. 6C1, C2, D1, D2). The the cell surface) of the oocyte shown in (F1). (F3) Higher magnification of the myoplasm acquired its final polarized distribution along the a–v reticulated fluorescent in situ hybridization signal at the vegetal pole for Ci- PEM1 RNAs corresponding to the boxed area in (F2). axis about 4 h after GVBD (Figs. 6E1, E2) as did Ci-PEM1 mRNA (Fig. 4). These observations show that the polarization of maturing oocytes at different times following GVBD and the myoplasm and of the maternal postplasmic/PEM RNAs localized the major postplasmic/PEM RNA Ci-PEM1 by in along the a–v axis do not occur during GVBD, but only after the situ hybridization (Figs. 4D1–D3, E1–E3, F1–F3). In stage III meiotic spindle has formed in a central position and migrated maturing oocytes, Ci-PEM1 RNAs were found concentrated in toward a cortical area of the oocyte which becomes the animal patches approximately 5 Am in thickness and 10 Amin pole. diameter uniformly distributed in the cell cortex (Figs. 4D1– To determine whether actin filaments also polarized during D3). These patches are first excluded from the animal cortex maturation, the distribution of actin was examined by 304 F. Prodon et al. / Developmental Biology 290 (2006) 297–311

Fig. 5. The subcortical mitochondria-rich myoplasm polarizes after GVBD. (A) Images from time-lapse sequence (from Supplementary Material Movie 4) along a tangential confocal section showing the polarization of subcortical mitochondria distribution. This sequence begins 10 min after the beginning of the BreakDown of the GV. (B, C) Confocal tangential sections of mitochondria in the GV-stage oocyte at the beginning (B) and at the end (C) of the sequence shown in (A). Time is indicated in hours (h) and minutes (V). (a) Animal pole, (v) vegetal pole. phalloidin labeling (Fig. 7). In GV-containing oocytes, actin is spindle and becomes reduced in intensity as the spindle excluded from the GV and enriched uniformly around the approaches the cortex (Figs. 7A3–A5, B3–B5). Throughout cortex (Figs. 7A1, B1). At the beginning of GVBD, actin maturation, cortical actin labeled brightly and uniformly; no accumulates around and within the GV as it breaks down (Figs. cortical inhomogeneity is detectable until the oocytes are fully 7A2, B2) condensing into a cloud-like clump by the end of mature, when cortical actin appeared rather less abundant at the GVBD. The actin cloud remains associated with the migrating animal pole. The cap of filamentous actin overlaying the

Fig. 6. Meiotic spindle positioning and the polarization of subcortical mitochondria-rich myoplasm. The drawings above schematically show the position of the meiotic spindle (in red), chromosomes (in blue) and myoplasm (in green) in spontaneously maturing oocytes at the end of GVBD (A) or at different times (B=1h;C= 2 h; D = 4 h) after the completion of GVBD. The position of the animal pole (a) becomes apparent 1 h after GVBD; the myoplasm begins to polarize vegetally (v) 2 h after GVBD. (A1–A2, B1–B2, C1–C2, D1–D2, E1–E2) Triple labeling of microtubules (in red), chromosomes (in blue) and mitochondria (in green). Confocal sections of oocytes along the animal (a)–vegetal (v) axis passing through the plane containing the meiotic spindle (indicated by an arrowhead). (A2–E2) Enlarged views of the meiotic spindle and chromosomes shown in (A1–E1). F. Prodon et al. / Developmental Biology 290 (2006) 297–311 305

Fig. 7. Actin filament rearrangement during oocyte maturation. (A1–A5) Equatorial confocal sections of oocytes labeled with phalloidin before GVBD (A1), during GVBD (A2), 1 h (A3), 2 h (A4) or 4 h (A5) after GVBD. Arrowheads indicate the accumulation of actin in the area of the meiotic spindle. (B1–B5) Equatorial confocal sections showing DNA labelling of the same planes shown in (A1–A5). Arrowheads indicate the position of chromosomes. (a) Animal pole; (v) vegetal pole. spindle evident in mature Phallusia oocytes (Sardet et al., stringy) to discrete condensed chromosomes aligned on a 1992) and in oocytes of many other species (Deng et al., 2005; metaphase plate, indicating that normal resumption of meiosis Weber et al., 2004) is not obvious in Ciona. has occurred in the oocytes matured in vitro. In all oocytes fixed after GVBD (Figs. 6–8 and see also Figs. 2A3, E3 inserts), the chromosomes are observed to Spindle migration and myoplasm polarization are transition from a prophase configuration (condensing and actin-dependent

In order to establish whether oocyte polarization involved the actin or microtubule cytoskeletons, oocytes were treated with cytochalasin or nocodazole just prior to maturation. After 4 to 5 h of treatment, when control oocytes were expected to have acquired a–v polarity, all samples were fixed and labeled for DNA, mitochondria and microtubules or actin (Fig. 8A). To quantitate the effect on the migration of the meiotic spindle, we drew three concentric spheres onto each oocyte and classified the position of the spindle or maternal chromatin as central, intermediate or peripheral (Fig. 8B). In over 97% of the control oocytes, the spindle was positioned at the cell periphery. In cytochalasin-treated oocytes, however, the spindle was nearly always found in a central (43%) or intermediate (52%) position and was peripheral in only 5% of cases. In nocodazole-treated oocytes, the chromatin was observed to be located in the peripheral zone 94% of the time, much like in the control sample. These results show that in the ascidian oocyte migration of the meiotic spindle to the surface requires actin microfilaments but not microtubules. Labeling of mitochondria revealed that, in the cytochalasin- treated oocytes, the myoplasm was never polarized (Fig. 8A, middle panel) even in the few cases when the spindle was Fig. 8. Effects of cytoskeleton inhibitors on meiotic spindle migration and found near the periphery. In nocodazole-treated oocytes, the myoplasm polarization. (A) Equatorial confocal sections of oocytes stained for myoplasm was clearly polarized (Fig. 8A, right panel), with mitochondria (green), microtubules (red) and chromosomes (blue) after mitochondria being sparse at the position where the chromatin treatment with DMSO (control, left panel), cytochalasin D (CytoD 8 Ag/ml, middle panel) and nocodazole (Noco 10 AM, right panel). Arrowheads indicate was located (the animal pole) and enriched in the opposite the position of the spindle and/or chromosomes. (B) Percentage of oocytes (vegetal) hemisphere, although the subcortical gradient of showing chromatin and/or a meiotic spindle located in the center (c, in blue), at mitochondria tended to be less steep than in control samples. the cell periphery (p, in orange) or in an intermediate position (i, in green) after These observations indicate that the polarization of surface treatments. The numbers above the bars are the totals from four experiments, domains from uniformly peripheral to vegetal enriched requires each of which gave similar results. (C) Confocal sections at high resolution of the meiotic spindle and chromosomes in oocytes incubated in DMSO (control, actin microfilaments but not microtubules, although micro- upper panel), cytochalasin D (16 Ag/ml, middle panel) and nocodazole (10 AM, tubules may be needed for refining the border between animal lower panel). Microtubules (MT) are in red and DNA in white. and vegetal regions. 306 F. Prodon et al. / Developmental Biology 290 (2006) 297–311

We also evaluated the effects of nocodazole and cytocha- – Within 30–60 min of GVBD, chromosomes condense onto lasin on meiotic spindle morphology (Fig. 8C). The a microtubular meiotic spindle (Fig. 9E). percentage of oocytes undergoing GVBD was normal in – The meiotic spindle arrested in meiotic metaphase (Met I) the presence of cytochalasin, and at low concentrations (4 or moves in an apparently random direction along its long axis 8 Ag/ml), the spindles which formed had an essentially towards the cortex thus defining the animal pole (Fig. 9F). normal appearance with the DNA aligned in a typical The meiotic spindle positions itself with its long axis metaphase plate (not shown)-like DMSO controls (Fig. 8C, parallel to the surface. Migration and cortical positioning of upper panel). At high concentrations of cytochalasin however the Met I spindle take about 1 h. (16 Ag/ml), the chromosomes were often observed to be – The subcortical mitochondria-rich myoplasm and cortical dispersed and the spindle disorganized (Fig. 8C, middle layer of postplasmic/PEM RNAs are excluded from the panel), indicating that actin may be involved in correct animal pole region and line the equator and vegetal regions chromosome congression during meiotic spindle formation as (Fig. 9H). Polarization along the primary a–v axis is has recently been shown for starfish oocytes (Lenart et al., completed about 4 h after GVBD (Fig. 9I). 2005). In the nocodazole-treated oocytes, microtubules were completely absent, and the DNA was not organized into a The initial asymmetry metaphase plate (Fig. 8C, lower panel), but generally chrosomomes were located near one another at the cell At this point, we can only speculate about what intrinsic surface. A slight delay or inhibition of GVBD was noted, or extrinsic cues might bias the direction of migration of the indicating that microtubules may be involved in breakdown meiotic spindle in Ciona oocytes. Prior to GVBD, no of the GV envelope. Interestingly, mitochondria labeling cytoplasmic or cortical asymmetry can be detected in the revealed a cleared zone always associated with the migrating oocyte: the GV lies in the center and the peripheral layers of DNA in nocodazole-treated samples, suggesting that some postplasmic/PEM mRNAs and myoplasm are radially sym- structure surrounds the chromatin even in the absence of the metric. The single nucleolus within the GV is a possible microtubular spindle. polarity cue, but time-lapse recordings show no correlation between the position of the nucleolus and the ultimate Discussion cortical location of the meiotic spindle. In some ascidian species including Halocynthia, Botryllus, Styela and Mol- We have defined and timed the events which lead to the gula, the GV adopts an eccentric location prior to maturation acquisition of the primary animal–vegetal axis in the oocyte of (Mukai and Watanabe, 1976; Sawada and Schatten, 1988; the ascidian reference species: C. intestinalis. This primary axis Sakairi and Shirai, 1991). It is likely that in these species the plays important roles at the time of fertilization and embryonic position of the GV prefigures the cortical location of the development since in some ascidians there is evidence that meiotic spindle (the animal pole), as is the case in many non- sperm enters preferentially in the animal hemisphere and the mammalian oocytes (Albertini and Barrett, 2004; Miyazaki et fertilization-triggered contraction always propagates in a al., 2005). In the ascidian Molgula manhattensis, an even general a–v direction (Speksnijder et al., 1989; Roegiers et earlier asymmetry has been described which consists of a al., 1995; Nishida, 2005). The a–v axis together with the site of large domain of basophillic cytoplasmic material (called the sperm entry defines polarized cues which direct a series of yolk nucleus or nuclear emission body) located on one side cortical and cytoplasmic reorganizations leading to the of the GV (Kessel, 1983; Crampton, 1899), but the nature of acquisition of D–V and A–P axes of the embryo (Roegiers this structure remains mysterious and has not been reported et al., 1995, 1999; Prodon et al., 2005). One major conse- in oocytes of other species. quence of these reorganizations is that peripheral determinants The direction of spindle migration in Ciona might be (such as the cortical postplasmic/PEM RNA macho1) distrib- influenced by some internal asymmetry such as polarized uted in a gradient of increasing density along the a–v axis in rupture of the GV membrane, as occurs in starfish oocytes the oocyte, concentrate and relocate in the posterior pole to (Terasaki et al., 2001), or by an asymmetry inherent in the influence differentiation of embryonic tissues, and, in partic- meiotic spindle itself, if the spindle poles are not equivalent ular, muscle along the A–P axis (Sardet et al., 2005; Nishida, (Vinot et al., 2004). Alternatively, it is possible that there exists 2005). a predetermined cortical destination marked by some as yet Here, we have shown that, in Ciona, the polarization of the unidentified molecular components. In species of many diverse myoplasm and important cortical mRNAs located at the phyla, spatial information is imparted to the oocyte by periphery of fully grown stage III oocytes occurs during surrounding cells or by a previous site of cell division (Sardet meiotic maturation, after the meiotic spindle and chromosomes et al., 2004). In oocytes of the colonial ascidian Botryllus, the have moved in the cortex defining the animal pole and that GV takes a polarized position farthest from the follicle stalk these events are actin-dependent. Chronologically, these which connects it to the gonad epithelium (Mukai and changes schematized in Fig. 9 are: Watanabe, 1976), and this appears to be the case in Halocynthia as well (F. Prodon and H. Nishida, personal – Meiotic maturation is triggered, and the Germinal Vesicle communication). Little is known about the origin of oocytes in breaks down in the center of the oocyte (Figs. 9D, E). most ascidian species, so the potential relationship between F. Prodon et al. / Developmental Biology 290 (2006) 297–311 307

Fig. 9. Establishment of a–v axis in oocytes of C. intestinalis. The ovary is delimited by a red circle at the base of the adult of C. intestinalis. Different stages of oogenesis, maturation and polarization are shown using different colors. (A, B, C) correspond to stage I and II oocytes (light yellow), (D, E, F) to stage III oocyte undergoing GVBD (yellow), (F, G, H) to the migration of the meiotic spindle and polarization (orange), (H, I) to the completion of the polarization of mature stage IVoocyte (dark orange). The chorion (follicle cells, test cells and vitelline coat) is not represented. Gray arrows indicate periods during which oocytes display cortical and cytoplasmic movements (see the Results section ‘‘oocyte maturation’’ and Supplementary Material Movie 3 for details). The meiotic spindle is represented in red, chromosomes in blue, the mitochondria-rich myoplasm in green and postplasmic/PEM RNAs by yellow stars. (A–C) During vitellogenesis (stages I, II and early stage III), oocytes enlarge, the cellular chorion differentiates and mitochondria and postplasmic/PEM RNAs move from a position around the GV to a peripheral location. (D) GVBD occurs 2.5–3 h after exposure of GV-stage III oocytes to sea water (‘‘t0’’). (E) BreakDown of the Germinal Vesicle is achieved 30–45 min after ‘‘t0’’ (GVBD stage). (F) The meiotic spindle then forms and migrates along its long axis toward the cortex in 1 h. The symbol (?) indicates that the direction of migration of the spindle is apparently random. (G) Polarization does not occur during the migration of the meiotic spindle. (H) As the meiotic spindle arrives at a cortical position, defining the animal pole, the mitochondria-rich myoplasm begins to be excluded from that site. (I) The polarized distribution of the subcortical mitochondria-rich myoplasm and cortical Ci-PEM1 RNA is completed about 2 h after the positioning of the meiotic spindle and chromosomes in the animal cortex. position of the oocyte within the gonad and the ultimate oocytes, and we do not know whether these translocations are orientation of the a–v axis remains an open question. coordinated. Similar types of relocalizations occur in the Xenopus pre- Growth and structural organization of the ascidian oocyte vitellogenic oocyte in which microtubular reorganizations and the translocation of mRNAs to the cortex have been well In Ciona, oocyte growth is associated with characteristic studied (Gard, 1993; Kloc and Etkin, 2005). Xenopus maternal changes in the distribution of mitochondria, microtubules and mRNAs such as Xcat2 and Xdazl localize in a domain rich in cortical mRNAs which all translocate from the region germ plasm, mitochondria and ER called the ‘‘mitochondrial surrounding the GV (in stage I and II oocytes) to the cloud’’ via a mechanism of diffusion entrapment (Chang et al., periphery of the oocyte (in stage III oocytes). It is possible 2004). The mitochondrial cloud and associated mRNAs are that the aggregates of mitochondria and Ci-PEM3 mRNA we positioned near the GV in very young oocytes but then detach have observed in young oocytes correspond to Kessel’s from the vicinity of the GV, merge with one side of the oocyte electron dense material which transferred from the GV to cortex and fragment into vegetally localized germ plasm the cytoplasm and was found in large packets surrounded by islands (Wilk et al., 2005). Another class of mRNAs which mitochondria (Kessel, 1966). Because these centripetal include VegT and Vg1 localize after the mitochondrial cloud relocalizations of microtubules, mitochondria and postplas- has reached the cortex via a late pathway requiring micro- mic/PEM RNAs in oocytes take place within the Ciona tubules and kinesins (Betley et al., 2004). Thus, while ascidian ovary, they unfortunately cannot be studied in detail in living and Xenopus oocytes appear similar in that they both have 308 F. Prodon et al. / Developmental Biology 290 (2006) 297–311 vegetal layers of ER, mitochondria and mRNAs, it appears that dependent and occurs at the speed of 1.25 Am/s, much faster the pathways leading to this polarity are quite different: in than the rate we observe for meiotic spindle displacement in Xenopus, the contents of the mitochondrial cloud become Ciona (¨0.4 Am/min). In contrast, in Xenopus oocytes, a polarized as they disperse cortically so that the vegetal pole is myosin (Myo10) plays a critical role in GV anchoring, meiotic defined before maturation or even vitellogenesis has occurred. spindle assembly and anchoring to the cortex by integrating the In Ciona GV-proximal masses of mitochondria and mRNA actin microfilament and microtubule cytoskeletons (Weber et first distribute homogeneously throughout the cortex and then al., 2004). In C. elegans, the translocation of the meiotic polarize during maturation after the animal pole is defined. spindle to the oocyte cortex appears to be mediated by a However, the RNA localization mechanisms may use similar microtubule-associated kinesin (Yang et al., 2005). In mouse machinery as it has been noted that many cortically localizing oocytes, a large meiotic spindle forms in the center of the mRNAs in Xenopus and most of the cortical postplasmic/PEM oocyte after GVBD and migrates in an actin-dependent manner RNAs identified in Ciona and Halocynthia share a high towards the region of the cortex closest to one pole of the frequency of CAC-rich motifs in their 3VUTR regions which spindle (Maro and Verlhac, 2002). are thought to be a possible signature for targeting mRNAs to Thus, spindle migration in Ciona resembles the situation in the cortex in somatic cells and in the oocytes of mouse, but different localizing forces might operate consider- (Betley et al., 2002; Sardet et al., 2005). ing the size difference between the small mouse oocytes (¨80 Finally, we have noted characteristic changes of the cellular Am in diameter) with their large spindles (¨30 Am in length) chorion surrounding the oocyte during oocyte growth. It is compared with the small spindle (<10 Am in length) in the likely that these changes in chorion structure can be correlated larger Ciona oocyte (¨120 Am diameter). Another apparent with those described in intracellular organization of the oocyte similarity between oocytes of mouse and ascidians is that their which define stages of oogenesis (Fig. 9), as has been meiotic spindles lack conspicuous astral microtubules (Verlhac suggested previously (Sugino et al., 1987). We were surprised et al., 2000; Maro and Verlhac, 2002; this study), but it will be to observe that during in vitro maturation the extracellular layer necessary to examine this point more closely in both species of stage III oocytes undergoes significant reorganization and (Moore and Zernicka-Goetz, 2005). In mouse oocytes, mole- growth to form the beautiful petal-shaped chorion of mature cules which are implicated in meiotic spindle positioning or are Ciona oocyte (Fig. 3). This autonomous morphogenetic distributed asymmetrically during polarization include the transformation is certainly worth studying in detail. microfilament-binding protein Formin 2; Mos and MAP kinase; and the conserved polarity complex PAR3/PAR6/aPKC Spindle migration and establishment of oocyte polarity (Leader et al., 2002; Voronina and Wessel, 2003; Vinot et al., 2004; Duncan et al., 2005; Michaut et al., 2005). Ascidian A wide variety of strategies are used by maturing oocytes to homologs for some of these polarity proteins are known to be localize a meiotic spindle under the surface in preparation for present maternally (Sasakura et al., 2003; S. Patalano and J. the meiotic reduction of chromosomes in polar bodies at the Chenevert, personal communication), but their potential roles animal pole. In Ciona, as in some molluscs and mammals, the in a–v polarity remain to be addressed. reinitiation of meiosis causes GVBD and formation of a central One interesting question raised by our findings concerns how meiotic spindle which migrates towards the oocyte surface and where an actomyosin network might generate the force (Pielak et al., 2004). In contrast, in starfish or amphibian responsible for spindle movement in Ciona. Using phalloidin oocytes, the GV is located eccentrically just beneath the cortex staining, we observed no conspicuous tracks or polarized and defines the future position of the meiotic spindle distribution of actin filaments which could account for spindle (Yamamoto, 1997; Miyazaki et al., 2000; Gard, 1991). Finally, migration, however, a transient burst of actin polymerization in some species of sea urchins, sea cucumbers or fishes might have escaped detection in our fixed samples. Possibly, the meiosis, reinitiation triggers GV migration towards the surface weak actin cloud which accumulates around the GV and and subsequent GVBD and meiotic spindle formation at that accompanies the spindle as it migrates (Fig. 7) plays a role in site (discussed in Miyazaki et al., 2005). force generation. Alternatively, a flow of cortical actin vegetally Mechanisms involved in the migration and positioning of could generate a cytoplasmic movement in the opposite the meiotic spindle have been examined in a few species, and direction, similar to the ‘‘fountain flow’’ described in C. elegans there are examples of both microtubule-dependent and actin- zygotes in which cortical actomyosin flow towards the anterior dependent movements. In oocytes of starfish or sea cucumbers, sustains a cytoplasmic flow which drives organelles toward the well developed Microtubule Organizing Centers (MTOCs) posterior (Cowan and Hyman, 2004; Munro et al., 2004; Nance, present before the start of maturation nucleate asters between 2005). This hypothesis could explain phenotypes observed after the plasma membrane and the GV which clearly play a role in nocodazole treatment: in Ciona oocytes unlike those of mouse attracting and anchoring the GV to the surface (Miyazaki et al., (Maro and Verlhac, 2002), chromosomes stay packed together 2000, 2005). In oocytes of the worm Chaetopterus, microma- in the same cortical area even if the meiotic spindle is absent nipulation of the spindle demonstrated the existence of a (Fig. 8A, right panel), consistent with the idea of a polarized unique cortical site which can capture and attract the displaced flow. Future experiments examining actin dynamics in vivo and meiotic spindle (Lutz et al., 1988). Movement of the spindle the localization of myosin motors should shed light on the toward the cortical site in Chaetopterus is microtubule- mechanism of meiotic spindle translocation in ascidians. F. Prodon et al. / Developmental Biology 290 (2006) 297–311 309

Polarization of postplasmic/PEM RNAs and mitochondria-rich for their help with ovary sections and also Christian Rouvie`re myoplasm for aid in image processing and Philippe Dru for technical assistance. This research was supported by the Association Migration and positioning of the meiotic spindle and Franc¸aise contre les Myopathies (AFM), the Association contre chromosomes under the animal cortex of maturing Ciona pour la Recherche contre le Cancer (ARC) and the French oocytes precede the exclusion of postplasmic/PEM RNAs and Research Ministry (ACI). subcortical mitochondria-rich myoplasm from the animal pole region. The cytochalasin experiments indicate that actin is Appendix A. Supplementary data responsible for the transition of the myoplasm from a uniform layer of mitochondria to a polarized basket lining the equatorial Supplementary data associated with this article can be found and vegetal regions. We do not know if vegetal polarization of in the online version at doi:10.1016/j.ydbio.2005.11.025. the peripheral domains rich in mitochondria and RNAs requires the arrival of the meiotic spindle at the cell surface. References Determining whether this correlation is causative will require displacement of the spindle or components thereof using Albertini, D.F., Barrett, S.L., 2004. The developmental origins of mammalian centrifugation or micromanipulations as described recently in oocyte polarity. Semin. Cell Dev. Biol. 15, 599–606. starfish eggs (Kitajima and Hamaguchi, 2005) and in mouse Bates, W.R., Nishida, H., 1998. Developmental roles of nuclear complex oocytes (Deng et al., 2005). factors released during oocyte maturation in the ascidians Halocynthia We have shown previously that, in mature oocytes of roretzi and . Zool. Sci. 15, 69–76. Betley, J.N., Frith, M.C., Graber, J.H., Choo, S., Deshler, J.O., 2002. 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