IntroductionAscidian oocytes are highly polarized along the animal-vegetal(a-v) axis before fertilization (Sardet et al., 1989; Sardet et al.,1992). This polarity principally concerns two peripheraldomains: a mitochondria-rich and ER-poor subcortical layer(the myoplasm); and a cortical endoplasmic reticulum (cER)network (Roegiers et al., 1999). The peripheral region of theegg is also characterized by an (a-v) gradient of maternalmRNAs (called postplasmic mRNAs or, because they make aposterior end mark in zygotes and embryos, PEM mRNAs)including the muscle determinant macho1 (Nishida andSawada, 2001; Sardet et al., 2005; Sardet et al., 2003; Satou etal., 2002; Yoshida et al., 1996). We have recently shown that,in Halocynthia, the cER network is associated with somepostplasmic/PEM RNAs, constituting a cortical domain thatwe call the cER-mRNA domain throughout this article (Sardetet al., 2003; Sardet et al., 2005). We did not, however, provide

a detailed description of domain reorganizations in wholeHalocynthia zygotes. Neither did we analyse crucial stages ofcortical reorganization before cleavage or describe thesimultaneous changes in cytoskeleton organization at the levelof the cortex. This prompted us to extend our initialobservations made in Halocynthia in two other ascidian speciesused for research, Ciona intestinalis and Phallusiamammillata, and in particular to include a thorough study ofreorganizations of cortical domains and the cytoskeleton afterfertilization in these two species.

The cortical cER-mRNA domain and subcortical myoplasmdomain (Fig. 1, Fig. 2I) are relocalized in two major phases ofreorganizations between fertilization and first cleavage(Roegiers et al., 1999). First, a sperm-triggered actomyosin-driven contraction moves the sperm aster vegetally andconcentrates the cER-mRNA domain and the myoplasm in andaround the vegetal/contraction pole (Fig. 2IC, cp). The position

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The mature ascidian oocyte is a large cell containingcytoplasmic and cortical domains polarized along aprimary animal-vegetal (a-v) axis. The oocyte cortex ischaracterized by a gradient distribution of a submembranemonolayer of cortical rough endoplasmic reticulum (cER)and associated maternal postplasmic/PEM mRNAs (cER-mRNA domain). Between fertilization and first cleavage,this cER-mRNA domain is first concentrated vegetally andthen relocated towards the posterior pole viamicrofilament-driven cortical contractions and sperm-aster-microtubule-driven translocations. The cER-mRNAdomain further concentrates in a macroscopic corticalstructure called the centrosome attracting body (CAB),which mediates a series of asymmetric divisions starting atthe eight-cell stage. This results in the segregation ofdeterminant mRNAs and their products in posterior cellsof the embryo precursors of the muscle and germ line.

Using two species of ascidians (Ciona intestinalis andPhallusia mammillata), we have pursued and amplified thework initiated in Halocynthia roretzi. We have analysed thecortical reorganizations in whole cells and in corticalfragments isolated from oocytes and from synchronouslydeveloping zygotes and embryos. After fertilization, weobserve that a cortical patch rich in microfilaments

encircles the cER-mRNA domain, concentrated into acortical cap at the vegetal/contraction pole (indicating thefuture dorsal pole). Isolated cortices also retainmicrotubule asters rich in cER (indicating the futureposterior pole). Before mitosis, parts of the cER-mRNAdomain are detected, together with short microtubules, inisolated posterior (but not anterior) cortices. At the eight-cell stage, the posteriorly located cER-mRNA domainundergoes a cell-cycle-dependant compaction into theCAB. The CAB with embedded centrosomal microtubulescan be isolated with cortical fragments from eight-cell-stage embryos.

These and previous observations indicate thatcytoskeleton-driven repositioning and compaction of apolarized cortical domain made of rough ER is a conservedmechanism used for polarization and segregation ofcortical maternal mRNAs in embryos of evolutionarilydistant species of ascidians.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/118/11/2393/DC1

Key words: Oocytes, Embryos, Cortex, RNA localization,Endoplasmic reticulum, Unequal division, Polarity, Development

Summary

Polarity of the ascidian egg cortex and relocalizationof cER and mRNAs in the early embryoFrançois Prodon1,*, Philippe Dru1, Fabrice Roegiers2 and Christian Sardet1,‡

1BioMarCell, UMR7009 Biologie du Développement, CNRS/Université Pierre et Marie Curie, Station Zoologique, Observatoire, Villefranche sur Mer 06230, France2Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, R356, Philadelphia, PA 19111, USA*Present address: Department of Biology, Graduate School of Science Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan‡Author for correspondence (e-mail: sardet@obs-vlfr.fr)

Accepted 9 March 2005Journal of Cell Science 118, 2393-2404 Published by The Company of Biologists 2005doi:10.1242/jcs.02366

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of this vegetal protrusion defines the future site of gastrulationand dorsal pole (Fig. 2IC, d) of the embryo (Roegiers et al.,1995). Maternal determinants for muscle, endoderm, unequalcleavage and gastrulation are concentrated in this vegetalregion after fertilization (Nishida, 1997; Nishida, 2002b;Roegiers et al., 1999). The sperm centrosome is also movedvegetally in the cortex, predicting the position of the futureposterior pole (Fig. 2IC, P). After meiosis is complete (20-30minutes after fertilization), determinants of muscle (macho1)and unequal cleavages, as well as more than two dozenpostplasmic/PEM RNAs are displaced posteriorly towards thefuture posterior pole of the zygote (Chiba et al., 1999; Roegierset al., 1999; Sardet et al., 2005; Sardet et al., 2003). This phasestarts after the male and female pronuclei form and it extendsthroughout mitosis (25-50 minutes). This second major phaseof reorganization is caused mainly by the translocation of thesperm aster with respect to the posterior cortex (Sardet et al.,1989; Roegiers et al., 1999). During first cleavage,determinants and the cortical and subcortical domains arepartitioned equally in the posterior region of the two firstblastomeres. At the eight-cell stage, a macroscopic corticalstructure called the centrosome attracting body (CAB) formsin the most posterior region of vegetal blastomeres (Nishida,2002b; Nishikata et al., 1999). The CAB is characterized bythe accumulation of the cER-mRNA domain, includingmacho1 (Nishida and Sawada, 2001; Sardet et al., 2003). It isthought that the CAB is involved in asymmetric divisions andacts as a source of factors (probably translation products oflocalized mRNAs) directing development and differentiation in

the posterior region of the embryo (Kobayashi et al., 2003;Kondoh et al., 2003; Nishida, 2002a; Nishida, 2002b; Sardetet al., 2005; Sardet et al., 2003; Yoshida et al., 1998).

In order to analyse the structure of the cortex and itstransformation, it is possible to isolate cortical fragments rapidlyand reproducibly using simple techniques (Sardet et al., 2002;Sardet et al., 1992). Ascidian zygotes and embryos are ideal fora detailed study of the cortex because they developsynchronously in large numbers, are highly polarized and can beattached to polylysine-coated surfaces to prepare large fields ofcortical fragments coming from identifiable regions of zygotesand embryos at all stages. The isolated ascidian oocyte cortexconsists of the plasma membrane (PM), adhering cytoskeletalelements and a conspicuous polarized network of cortical roughendoplasmic reticulum (cER), which is covered with ribosomes.

Recently using the Japanese ascidian Halocynthia, our laband Nishida’s lab have shown that one of the most abundantcortical maternal mRNAs, Hr-PEM1, and the muscledeterminant Hr-macho1 are bound to the cER network in eggsand embryos. We have suggested that these maternal mRNAsare relocalized together with the cER network during the twomajor phases of reorganization and segregated into specificblastomeres to direct differentiation and asymmetric division(Sardet et al., 2003). However, in this study, we did notexamine other cortical components (MF, MT) or critical stagesof relocalization (second major phase). We also wonderedwhether what we described in Halocynthia roretzi held true forits distant relative C. intestinalis, the cosmopolitan speciesused by most laboratories.

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Fig. 1. Polarity along the animal-vegetal (a-v)axis of the ascidian oocyte (see Movies 1-3 insupplementary material). (A1,A2) Confocalequatorial section (30 µm from surface) showingthe distribution of the ER network (A1) and the7-8 µm thick mitochondria-rich subcorticalmyoplasm (m) (A2). (A3,A4) Enlarged views ofthe ER-poor myoplasm. (B) Electron-microscopy section of the vegetal-pole regionshowing the ER network (red), ERmicrodomains (ER), cER near the plasmamembrane (arrowheads), mitochondria (m,green) and yolk platelets (YP, blue). (C1,C2)Confocal subcortical section (3 µm fromsurface) passing through the myoplasm (m).(C3,C4) Enlarged views of the transition zone atthe edge of the myoplasm (m). (D1,D2) High-magnification confocal cortical sections (1 µmfrom surface) of the cER network in the vegetal(D1) and animal hemispheres (D2). (E) ERaround the meiotic spindle (ms, arrowhead) atthe animal pole. (F1,F2) Equatorial (F1, 30 µmfrom the surface) and cortical (F2, 1 µm fromsurface) confocal sections of fluorescent in situlocalization of Ci-PEM1 RNAs (arrowheads).(F3) Confocal cortical view (1 µm from surface)of the vegetal hemisphere at highermagnification. Ci-PEM1 RNA signal appears asa reticulated network. (a) Animal pole. (v)Vegetal pole. All confocal acquisitions are madeon Ciona intestinalis oocytes, the electronmicrograph is from a section of a Phallusiamammillata oocyte.

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We now report the evolution of cortical polarityincluding cytoskeletal elements in zygotes and embryosof C. intestinalis which belongs to theAplousobranchiata order and the European species P.mammillata belonging to the Phlebobranchiata order(Turon and Lopez-Legentil, 2004). Our resultscomplement our initial observations (Sardet et al., 2003)in the Japanese species H. roretzi which belongs to theStolidobranchiata, a third order. Our study shows thatthese three evolutionarily distant ascidian species usesimilar cytoskeleton-driven repositioning andcompaction of the cER network for polarization of corticalmaternal mRNAs (including the determinant macho1) and forsegregation of these mRNAs in small posterior blastomeres. Apart of this story is told in the form of a BioClip ‘Polarity insidethe egg cortex’, a multimedia document that can bedownloaded from the ‘Research’ section ofhttp://www.bioclips.com/. Further information on the cortex isavailable in supplementary materials (six films) and on ourlaboratory web site (http://biodev.obs-vlfr.fr/biomarcell/ascidies/eggcortex.html).

Materials and MethodsBiological materialAdults of the ascidians C. intestinalis and P. mammillata werecollected near Sète or Roscoff, France. Oocytes and zygotes weredechorionated using either 0.1% trypsin in seawater (pH 8.0) for 3

hours or with 1% thioglycolate, 0.05% pronase in filtered sea water,pH 10.0, for 2-5 minutes (Dumollard and Sardet, 2001; Sardet et al.,2003; Sardet et al., 1989). Oocytes were fertilized and embryoscultured as described (Roegiers et al., 1999).

Labelling of living eggs, zygotes and embryosMitochondria were labelled for 5-10 minutes with the carbocyaninedye DiOC2(3) (0.5 µg ml–1) or 1 µM TMRE mitotracker (MolecularProbes) and chromosomes were labelled for 15 minutes with 1 µgml–1 Hoechst 33342 (Molecular Probes) (Dumollard and Sardet, 2001;Sardet et al., 1989). For labelling endoplasmic reticulum, we injecteda small oil droplet saturated with DiIC16(3) (Speksnijder et al., 1993).

Confocal and electron microscopyWe used a Leica SP2 confocal microscope, and ZeissAxiophot/Axiovert and Metamorph/Metaview software (Universal

Fig. 2. (I) Isolation of cortices from oocytes, zygotes andembryos. (IA). Eggs or synchronous embryos deposited into adrop of cortex buffer and allowed to attach to a polylysine-coated coverslip are sheared with a stream of cortex bufferfrom a Pasteur pipette. (IB) Fields of isolated cortices arelabelled, mounted and observed using epifluorescence orconfocal microscopy. (IC) The position of grey coverslips (c)indicate the origin of the cortical fragments isolated fromoocytes, zygotes or embryos selected for observation. cER isred, the myoplasm is green, microtubules in light blue andDNA in dark blue. a, animal; v, vegetal; cp, contraction pole;d, dorsal; P, posterior; A, anterior. (ID) Field of corticesisolated from unfertilized eggs and labelled for cER. (II)Isolated cortices from oocytes retain a-v polarity. (IIA1)Cortex isolated from an oocyte labelled for cER (red) andmicrofilaments (yellow). Regions corresponding to the animal(a) and vegetal (v) hemispheres are shown. (IIA2) Detail ofthe region boxed in IIA1. (IIB1-B3) Cortex isolated from anoocyte. cER is double labelled with DiIC16(3) (B1, red) andby immunofluorescence for ribosomes (B2, green). Mergedimage of cER network and ribosomes (B3, colocalization inyellow). Dotted line indicates the edge of isolated cortex.Arrowhead indicates cytoplasmic ER that has fallen onto thecoverslip during the shearing process (B3, arrowhead). (IIC1-D2) Fragments of cortices isolated from an oocyte labelled forcER (C1, red) and Ci-PEM1 (C2, green) (IID1,D2). Cortexisolated and labelled after KCl-puromycin treatment; labelledfor cER (D1) and ribosomes (D2). (IIE). Replica of afragment of an isolated oocyte cortex (fast-freezing deep-etching electron microscopy). Cortical ER is red,microfilaments orange and particles on the cER surfaceyellow. Most particles are the size of ribosomes (arrowheads),coated pits are blue and the internal side of the plasmamembrane (PM) is grey. All cortices were obtained fromCiona oocytes except for E, which is from Phallusia.

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Imaging) for digital imaging (Roegiers et al., 1999). Thin-sectionelectron microscopy of properly orientated oocytes and embryos, andfast-freezing deep-etching and replication of cortices were performedas described (Sardet et al., 2003; Sardet et al., 1992).

Isolation and characterization of corticesOocytes or synchronous populations of zygotes and embryos atdifferent stages were deposited on a polylysine-coated coverslip intoa drop of EMC or one of three types of cortex buffer that all gavesimilar results (Buffer X, CIM buffer and Vacquier buffer) (Sardet etal., 1992). Oocytes, zygotes or embryos attached to a polylysine-coated glass surface were opened and emptied of their cytoplasm witha stream of cortex buffer squirted from a Pasteur pipette, leavingcortical fragments attached to the glass (Fig. 2I). Immunolabellingswere done essentially as described (Roegiers et al., 1999). Primaryantibodies were rat YL1/2 anti-β-tubulin or mouse anti-β-tubulinantibody (Amersham), and rabbit antibody against the S6 ribosomalsubunit (Cell Signaling Technology). Live cortices were alsoimmunolabelled using short incubations (15 minutes) with antibodiesand secondary antibodies in Buffer X. The cER network was labelledusing DiOC6(3) or DiIC16(3) and microfilaments were labelled usingfluorescent phalloidins (rhodamine- or Alexa-633-conjugated) asdescribed (Sardet et al., 1992). Surface sugars of live eggs and zygoteswere labelled with rhodamine/concanavalin-A (conA) (50 µg ml–1,Molecular Probes) for 1 minute before cortex isolation. To detachribosomes from the surface of the ER, isolated cortices were incubatedin KCl-puromycin (Sigma) buffer for 20 minutes as reported (Sardetet al., 1992; Sardet et al., 2003)

Polarity of the egg cortexIn order to determine the origin of the piece of cortex attached to thecoverslip with respect to the polarity of oocytes, zygotes or embryos[animal-vegetal (a-v), dorsoventral or anteroposterior], we applied thestrategy described previously for oocytes (Sardet et al., 1992). Cellswere first labelled with Hoescht 33342, TMRE mitotracker(Molecular Probes) and/or rhodamine/conA. We recorded the positionof individual zygotes attached to the coverslip and the orientation ofaxes, using scratch marks on the coverslip as reference points. Wethen sheared zygotes, fixed the resulting cortices and labelled themwith DiOC6(3) to observe the cER network. Using the scratch markson the coverslip, we matched the images of the zygote or embryo andthe corresponding piece of cortex to determine its origin along thepolarized axes.

In situ hybridizationThe fixations and fluorescent (tyramide signal amplification, TSA) insitu hybridization procedures (without extractions to preserve the cERnetwork in cortices) on whole cells and cortices isolated from themwere as described (Sardet et al., 2003). Hybridized antisense maternalmRNAs [Ci-PEM1 corresponding to CLSTR1544 and Ci-PEM3corresponding to CLSTR865 on the Ghost Database(http://ghost.zool.kyoto-u.ac.jp/indexr1.html)] labelled withdigoxigenin were revealed using the phosphatase precipitationmethod, the HNPP fluorescent detection kit (Roche) or the TSAmethod (Sardet et al., 2003). We have used a nomenclature forcortically localized mRNAs we call postplasmic/PEM RNAs thatharmonizes the current terminology (for review, see Sardet et al.,2005).

ResultsExperiments were performed on oocytes, zygotes and embryosof Phallusia and Ciona, and they gave similar results. To

facilitate understanding, we have selected the best illustrationsin either species (specified in figure legends). Phallusiazygotes were easier to handle for cortical orientationexperiments and most mRNA localization experiments weredone with Ciona.

Polarity along the a-v axis of the ascidian oocyteThe myoplasm is a mitochondria-rich, ER-poor domain andforms a 5-7 µm thick subcortical basket lining the vegetalhemisphere and opened in the animal pole region of the oocyte(Fig. 1A1-4,C1-4; see also Movies 1-3 in supplementarymaterial). The animal pole region itself is characterized by alow density of mitochondria and an accumulation of ERsurrounding the meiotic spindle (Fig. 1E). A transition zone inwhich the distribution and density of subcortical mitochondriaand the ER network change is clearly visible above the equator(Fig. 1C1-3). The distribution of the cER network in Ciona isseen in confocal tangential sections made 0.5-2 µm under thesurface (Fig. 1D1,D2). In the vegetal hemisphere, corticalsections reveal a very dense network of tightly knit tubules andER cisternae (Fig. 1D1), contrasting with the sparser corticalcER network observed in the animal hemisphere (Fig. 1D2).This cER network can be detected lining the PM in thin-sectionelectron micrographs, which also reveal strands of ERtraversing the mitochondria-rich myoplasm and the presenceof ER microdomains in the deeper cytoplasm (Fig. 1B).Maternal mRNAs Ci-PEM1 (Fig. 1F1) and Ci-PEM3 (data notshown) are located within 0.5-2 µm of the surface along an a-v gradient best seen in equatorial confocal sections. At thehighest possible resolution, the fluorescent in situ hybridizationsignal appears as a reticulated network (Fig. 1F2-3), suggestingthat Ci-PEM1 might be associated with the cER network.

Cortices isolated from oocytes retain a-v polarityCortices isolated from oocytes, zygotes and embryos constitutean ideal open cell preparation in which to analyse the spatialdistribution of cortical organelles, cytoskeletal elements,macromolecular complexes and mRNAs associated with thecytoplasmic side of the PM (Fig. 2I). Live and fixed corticesisolated from oocytes of Ciona and Phallusia (~0.5-1.0 µmthick) show an obvious polarity in the organization of the cERnetwork and retain some microfilaments (MFs) andmicrotubules (MTs) that adhere to the PM or macromolecularcomplexes attached to it (Fig. 2IIE). Cortical fragments derivedfrom the vegetal hemisphere of oocytes are characterized by acER network made of tightly knit tubules or sheets (Fig.2IIA1,A2). The fluorescent signal for Ci-PEM1 RNAscolocalizes with this vegetal cER network (Fig. 2IIC1,C2) asdescribed for Halocynthia (Sardet et al., 2003). Isolatedcortical fragments issued from the animal hemisphere arecharacterized by a sparse tubular cER network (Fig. 2IIA1,A2).Observations of the cER in isolated cortices from Cionaoocytes are in agreement with confocal observations of wholeeggs (Fig. 1D1,D2) and previous observations (Sardet et al.,1992). MFs are also distributed along an a-v gradient, beingmore abundant where the cER network is the most tightly knit(Fig. 2IIA1,A2). Double labelling of membranes withDiIC16(3) and ribosomes (with antibody against S6 ribosomalprotein) demonstrates that the cER network retained on

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isolated cortices is rough ER (Fig. 2IIB1-B3). Ribosomes canbe detached from the cER network using KCl-puromycintreatment (Fig. 2IID1,D2) (Sardet et al., 2003; Sardet et al.,1992). That the cER is rough ER is confirmed by the fact that,in electron micrographs (fast-frozen, deeply etched replica inFig. 2IIE), many particles the size of ribosomes (Fig. 2IIE,yellow) can be detected on the cER (Fig. 2IIE, red).

Amplification of cortical polarity after fertilization andduring the first major phase of reorganizationFertilization triggers a first major phase of reorganization ofthe oocyte’s cortex which concentrates ConA-labelled surfaceglycoproteins and glycolipids, the cER-mRNA domain, and themitochondria-rich myoplasm in the vegetal hemisphere (Fig.3A,B,D-G). A vegetal/contraction pole (labelled cp/d in thefigures) forms in fertilized Ciona eggs (Fig. 3F1,F2,G) as inPhallusia (Roegiers et al., 1995). In both Phallusia and Ciona,the vegetal/contraction pole is not always exactly opposite thepolar bodies (marking the animal pole) but can be off by 45-

60° (Roegiers et al., 1995) (data not shown). Many corticalfragments isolated during and after the contraction 2-5 minutesafter fertilization contain tightly packed sheets of cER networkforming a patch (20 µm in diameter, 2-5 µm thick) positionedin the centre of the vegetal/contraction pole (Fig. 3C).Ribosomes associated with cER are abundant in this cER-richpatch (data not shown). Interestingly, actin microfilamentsform a ring around the cER-rich patch, which is particularlynoticeable at the end of the contraction (Fig. 3C1-C3,H). Wealso find that surface glycolipids and glycoproteins recognizedby ConA in the contraction pole (Fig. 3F1,F2) define amicrovillus-rich surface region corresponding to the corticalER-rich plaque situated beneath the PM (Fig. 3J1,J2). Amicrovillus-poor zone adjacent to the microvillus-rich zoneprecisely corresponds to the location of the MF ring (Fig.3H,J1,J2). Another feature of these postfertilization cortices isthe presence of conspicuous MTs emanating from spermasters, which are probably retained in the isolated corticalfragments through interaction of MTs with the PM or MFs

Fig. 3. Amplification of cortical polarity after fertilization (first major phase). (A1-C3) Zygotes and cortices during the contraction wave (seeMovie 4 in supplementary material). (A1-A4) Confocal subcortical sections (3 µm from surface) of a zygote during the fertilization contractionwave, showing the distribution of cER network and mitochondria-rich subcortical domain (myoplasm). (B) Confocal equatorial section (35 µmfrom surface) of Ci-PEM1 RNA fluorescent in situ localization (arrowheads). Dotted line marks the edge of zygote. (C1) Cortex isolated from thevegetal-pole region during the contraction. Merged images of cER (red) and microfilaments (green) show colocalization in yellow. The externaldotted line shows the edge of cortex and the internal dotted line shows the area of the highest cER accumulation. (C2,C3) Enlarged views of theregion boxed in C1 showing cER (C2, red) accumulation (arrowhead) and corresponding microfilaments (C3, green). (D1-J1) Zygotes and corticesat the end of the contraction wave. (D1,D2) Confocal cortical to subcortical section (1 µm to 3-4 µm from the surface). (Insert) Merged image ofcER (red) and mitochondria (green) in the vegetal/contraction pole region. (E) Cortical section of the vegetal/contraction pole and future dorsalpole of the embryo (cp/d) after a double labelling for cER (red) and mitochondria (green). (F1,F2) Equatorial (F1, 40 µm from surface) and surface(F2) sections of the vegetal/contraction pole region after labelling surface sugars with fluorescent ConA. (G) Equatorial section (35 µm fromsurface) of Ci-PEM1 RNA fluorescent in situ localization signal (arrowheads). The dotted line marks the edge of zygote. (H) Merged image of acortex triple labelled for cER (red), microfilaments (green) and microtubules (blue). The yellow area indicates the vegetal/contraction pole (cp/d)area, which is rich in both cER and microfilaments. sa, sperm aster. (I1,I2) Detail of microtubules (MT) of a sperm aster (I2). This area correspondsto an accumulation of ER in the cortex (I1). (J1,J2) Detail of a cortex isolated from the vegetal/contraction pole double labelled for cER (J1) andsurface sugars (J2). Whole-zygote confocal sections are from Ciona and cortices are from Phallusia zygotes. Dotted line indicates the border of thevegetal/contraction pole.

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and/or local accumulation of the cER network in the aster’scentre (Fig. 3I1,I2).

Amplification of posterior cortical polarity after meiosisduring the second major phase of reorganizationSperm aster localization in the cortex after the corticalcontraction triggered by the fertilizing sperm represents a firstindication of posterior polarization. This polarity is amplifiedduring a second major phase of reorganization (Chiba et al.,1999; Roegiers et al., 1999). The mitochondria-rich myoplasmdomain and the cER-mRNA domain, which first accumulatearound and in the vegetal/contraction pole, are thenprogressively translocated and relocalized in three subphasestowards the future posterior region and equator betweenmeiosis-II completion and cleavage (Fig. 2I, Fig. 4A-D; seeMovies 4 and 5 in supplementary material) (Roegiers et al.,1999; Sardet et al., 1989). We prepared cortical fragments from

synchronous zygotes during posterior translocation, identifiedtheir provenance (from vegetal, posterior, anterior regions) andlabelled them to observe cER, MT, MF and Ci-PEM1 RNAs.We observed that the posterior pole region of isolated corticeswas characterized by a more tightly knit network of cER (~1µm thick) composed of anastomosed tubules and sheets (Fig.4H1,H3,I1,I3,J1,J3,K1). This posterior cER network isdisplaced from the vegetal/dorsal region, where surface ConAbinding sites are the most abundant (Fig. 4F). The posteriorcortical fragments also retain many short MTs, which probablycorrespond to the tips of MTs emanating from the posteriorlylocalized sperm aster (Fig. 4E,K). In whole zygotes, part of thein situ signal for Ci-PEM1 RNAs is situated close to the cortex,but it is also present in deeper subcortical and cytoplasmiclocations (Fig. 4B,C) as previously observed in Halocynthia(Sardet et al., 2003). We observed that Ci-PEM1 RNAs areretained in posterior fragments of cortices isolated during thesecond major phase of reorganisation. Ci-PEM1 is clearly

associated with the tightly woven network of cERmarking the posterior pole, where the mRNAs formsmall patches (Fig. 4H,I). The Ci-PEM1 signal isalso detected on a subpopulation of ER tubulesprojected outside the isolated posterior cortex during

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Fig. 4. Cortical polarity along the anteroposterior axis(second major phase). (A1-A3) Successive confocalequatorial (40 µm from surface) sections of a zygote (31-41 minutes after fertilization) double labelled for ER(red) and mitochondria (green) (time-lapse confocalmicroscopy); see Movies 4 and 5 in supplementarymaterial). The direction of posterior translocation of themyoplasm is indicated by the white arrow and that of thecER by the white arrowhead. DiI indicates the injectedDiIC16(3) dye droplet. (B,C) Detection of the fluorescentin situ hybridization signal for CiPEM-1 RNA during (B)and at the end (C) of the second major phase ofreorganization (arrowheads). (D1-D4) Posterior view ofthe CiPEM-1 RNA signal at the end of the second phase.Views at higher magnification of the CiPEM-1 RNAsignal corresponding to the boxed area in (D1), 4 µm(D2; scale bar, 5 µm) and 1 µm (D3; scale bar, 5 µm)under the cell surface. (D4) A view at highermagnification of in situ localization of CiPEM-1 in theboxed area in D3 (scale bar, 2.5 µm). (E) Posterior viewof astral microtubules. (F) Equatorial view of a zygoteafter labelling surface sugars with fluorescent ConA.Labelled sugars remain centred around future dorsal pole(d) region (arrowheads). (G) Microfilament labelling ofsurface (arrowheads). (H1-H3,I1-I3) Cortex isolated froma zygote (40 minutes after fertilization) along theposterior-dorsal (P-d) axis. The cortical fragment isdouble labelled for cER (H1,I1, red) and Ci-PEM1(H2,I2, green). (I1-I3) Enlarged regions corresponding tothe boxed areas in (H). (H3,I3,J3) Merged images inwhich colocalized cER and Ci-PEM1 are shown inyellow (I3). (J1-J3) Cortical fragment from the posteriorcortex treated with KCl-puromycin and labelled for cER(J1, red) and Ci-PEM1 RNA (J2, green). (J3) Mergedimages shows that little Ci-PEM1 signal remains(arrowhead). (K1,K2) Fragment of isolated posteriorcortex labelled for cER (K1, red) and microtubules (K2,white). a, animal; d, dorsal; P, posterior. All observationsare made in Ciona except for F,G, which were made inPhallusia.

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the shearing process that breaks the egg open (data not shown).This probably corresponds to a part of the cER present in adeeper cytoplasmic location at this stage (Fig. 4A). As withoocyte cortices, we observe a loss of the signal for Ci-PEM1RNAs after KCl-puromycin treatment (Fig. 4I,J), suggestingthat Ci-PEM1 RNAs might be detached from the surface of thecER together with ribosomes.

cER-mRNA domain is inherited by the CAB in theposterior vegetal blastomeres at the eight-cell stageOwing to the orientations of the first three cleavage planes[along the a-v axis and along the anteroposterior (A-P) axis,respectively], the posterior myoplasm domain and the cER-mRNA domain are both equally partitioned into the twoposterior vegetal blastomeres (Fig. 5, B4.1) at the eight-cellstage (Fig. 2IC, Fig. 6D). From the eight-cell stage to the 64-cell stage, postplasmic/PEM RNAs are located in and aroundthe CAB (a cER-rich macroscopic structure sandwiched

between the myoplasm and the PM) (Fig. 5) (Iseto and Nishida,1999; Nishikata et al., 1999; Roegiers et al., 1999; Sardet etal., 2003). The formation and condensation cycle of the CABstructure can be observed using time-lapse confocal imagingof an embryo double labelled for ER and mitochondria (Fig.5A1-A3; see also Movie 6 in supplementary material), and infixed embryos and cortices with labelled chromatin and Ci-PEM1 RNAs. The cER-rich CAB domain is thinnest and mostextended along the PM at the eight-cell stage during interphase,compacting into a smaller and thicker cortical patch duringmitosis (Fig. 5A1-A3,B,C). This change in shape of the CABfrom spread out to concentrated repeats as a cycle during eachunequal cleavage (data not shown). Observation of the CABregion in Phallusia by electron microscopy (Fig. 5E1,E2)confirms the presence of a high density of ER tubes (rather thanvesicles) and of an electron-dense matrix as describedpreviously in Halocynthia and Ciona (Iseto and Nishida.,1999). We characterized the cER-rich zone of embryo cortices,taking advantage of the fact that the CAB is retained in cortical

Fig. 5. Cortical polarity of 8-16cell stage embryos (firstunequal cleavage). (A1-A3)Living embryo double labelledfor ER (red) and mitochondria(green); time-lapse confocalmicroscopy of eight-cell (A1),16-cell (A2) and 32-cell (A3)stages (see Movie 6 insupplementary material).Arrowheads indicate thelocation of the cER/mRNA-rich CAB. Inserts show fullviews of embryos at the eight-cell stage (lateral view) and atthe 16- and 32-cell stages(vegetal views). (B,C) Fixedembryos double labelled forCi-PEM1 RNA by fluorescentin situ hybridization (white)and DNA (blue). Arrowheadsshow the mRNA-rich CAB atthe beginning (interphase) ofthe eight-cell stage (B) and atthe end (mitosis) of the eight-cell stage (C). (D) High-magnification confocal corticalsection (6 µm from surface)showing Ci-PEM1 RNA signalat the end of the eight-cellstage. (E1,E2) Electron-microscopy section across the CAB of an eight-cell-stage embryo. The dotted area encircles the CAB. Enlarged view of the CAB is shown in(E1). ER tubules (cER, arrowhead) are red, mitochondria green and yolk platelets (YP) blue. Notice the presence of electron-dense matrix.Scale bars, 5 µm (E1) and 1 µm (E2). (F) Confocal observation of cortical fragments isolated from the posterior vegetal region of an eight-cell-stage embryo and labelled for cER (red). (Insert, top right) Lower-magnification view of four cortical imprints from two vegetal posterior B4.1and two animal posterior b4.2 blastomeres. Arrowheads indicate the cER accumulations that characterize the isolated moustache-shaped CAB.(Insert, bottom right) Higher-magnification view of cER accumulation observed in F. (G) Epifluorescence observation of the CAB isolated in amore condensed state and labelled for cER (arrowheads). Dotted lines show the edges of cortical fragments corresponding to blastomeres.(H1,H2) Confocal section of isolated cortex with condensed CAB double labelled for cER (H1) and microtubules (H2). Dotted lines delimitcortical fragments. (I) Confocal section of isolated cortex labelled for microfilaments in the area of a condensed CAB (arrowheads). (J) IsolatedCAB double labelled for cER (red) and ribosomes (green). Merged images show colocalization of cER and ribosomes in yellow andarrowheads indicate some rough cER tubes in continuity with the CAB. (Insert) The cER network (red) and attached ribosomes (green)normally compacted in the CAB have been stretched away from the attached cortex under the force of shear. All acquisitions were made inPhallusia except B-D, which were made in Ciona.

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fragments from the posterior face of the cube-like eight-cell-stage embryo. The CAB is easily recognized in isolatedcortices as cER-rich plaques (interphase, 20�10 µm, 4-5 µmthick; mitosis, 10�5 µm, 7-8 µm thick) (Fig. 5F,G).Ribosomes are particularly abundant and can stretch along ERstrands spread by the force of shear (Fig. 5J). Such isolatedCABs often have a hole in their centre possibly indicating theposition of the centrosome ripped away from the cortex duringthe shearing procedure (Fig. 5H1). A variable level ofcondensation of the cER network in the CAB is also evidentin isolated cortices. This probably reflects the cell-cycleposition of blastomeres at the time of cortex isolation. Doublelabelling of the isolated cortices demonstrates that the cER-richzone retains bundles of MTs enmeshed deep into the cERnetwork (Fig. 5H, one plane of a series of confocal z-axissections). These MTs probably originate from the centrosomeadjacent to the CAB. Actin MFs (Fig. 5I) and ribosomes (Fig.5J) are also abundant in the isolated CAB. Observations of Ci-PEM1 RNAs after fluorescent in situ localization indicate thatthey are present in a reticulated network compatible with theircER association as previously described for Halocynthia(Sardet et al., 2003).

DiscussionWe have analysed the reorganizations of the cell cortex inliving oocytes, zygotes and embryos, and in cortices isolatedfrom them in two species of ascidian: P. mammillata and C.intestinalis. The most conspicuous cortical changes concernthe distribution and relocalization of the submembranousnetwork of endoplasmic reticulum (cER) and of

postplasmic/PEM RNAs associated with it (Fig. 6). Thepolarization and compaction of the cER network followschanges in the activation status of the egg and its progressionthrough meiotic and mitotic cell cycles, and is correlated withcell-cycle-dependant modifications in the abundance andredistribution of cytoskeletal elements (MFs and MTs) andsurface topography (ConA-binding sites indicating abundantmicrovilli). Ciona, Phallusia and Halocynthia, the three mainspecies of solitary ascidians used for research, all seem to usecER-mRNA relocalization and compaction for polarization ofthe zygote and for the segregation of informationalmacromolecules (determinants/mRNAs) and the translationmachinery (ribosomes/cER) in small posterior blastomeres(Sardet et al., 2005; Sardet et al., 2003; Sardet et al., 1992).This suggests that ascidians species separated by millions ofyears of evolution share the same cellular and molecularmechanisms to establish developmental axes and translocateand segregate determinants acting on posterior blastomeres insimilar ways. Such conservation does not seem to havehappened in nematodes, which show great flexibility in cellularmechanisms of sperm-directed axis formation and domainlocalization (P granules) (Goldstein et al., 1998; Hasegawa etal., 2004). Our previous experiments with Halocynthia (Sardetet al., 2003) strongly suggested that there was a link betweenthe cER network established in the oocyte and the CAB, themacroscopic cortical structure mediating unequal posteriorcleavages, which acts as a signalling centre for muscle andposterior development (Nishida, 2002a; Nishida, 2002b).Wedid not, however, investigate whether the cER-mRNA domainthat moved posteriorly was retained as part of the isolatedposterior cortex. Our present observations show that the cER

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Fig. 6. Evolution of the cortex from fertilization to the eight-cell stage. Recapitulative diagram showing the rearrangements of the plasmamembrane, adhering cER network and associated postplasmic/PEM RNAs (cER/mRNA), and microtubules from fertilization to the eight-cellstage. An oocyte, zygotes during the first and second major phases of reorganization, and an embryo at the eight-cell stage are represented incross section and as isolated cortices in surface view and enlarged cross section.

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network established during oogenesis is undoubtedly astructural precursor of the CAB.

The concept of cERThe idea that a specific ER network exists in the cortex of eggsand embryos derives from the fact that, in Halocynthia (Sardetet al., 2003) and Ciona and Phallusia (this paper; F. Prodon etal., unpublished), the original and most abundantpostplasmic/PEM maternal RNA (Ci-PEM1) and others(including macho1) are associated with a layer of rough cERattached to the cytoplasmic face of the oocyte’s PM (Sardet etal., 1992; Sardet et al., 2005). In contrast to the cER,cytoplasmic ER (which is also mostly rough ER) does not bindCi-PEM1. After fertilization, another type of cER (formed ofsheets and tubes not coated with postplasmic/PEM RNAs) isalso present in isolated cortices of zygotes and blastomeres. Itis not clear whether this post-fertilization cER is a part of theoriginal oocyte cER from which postplasmic/PEM RNAs havebeen excluded (perhaps by lateral translation and/oraggregation) or whether it derives from deeper cytoplasmic ERthat has formed new connections to the PM after fertilization.Such rearrangements in the ER network are expected becausethe deeper cytoplasmic ER network is clearly in continuitywith the cER (Speksnijder, 1992). Our observations with anti-ribosome antibodies complement our previous conclusions(based on fast-freezing deep-etching electron microscopicobservations) that cER is rough ER (Sardet et al., 1992).

The fact that cER remains attached to the PM in corticalfragments isolated using large shearing forces implies that cERhas strong attachment sites to the PM. We have visualizeddiscrete attachment feet on the cER forming junctions with thePM in Phallusia (Sardet et al., 1992). Specialized junctionsbetween the PM and sarcoplasmic reticulum (SR) in musclecells are well documented and PM-ER junctions have beenrecently isolated and characterized from neurons andastrocytes, where they are suspected to play a role in calciumsignalling and the refilling of intracellular ER stores(Lencesova et al., 2004). In these cells, PM microdomains formjunctional units with the underlying ER throughmacromolecular complexes composed of specific subunits ofpumps, channels and submembranous proteins. It will beimportant to see whether such macromolecular complexes arecomponents of the PM-cER junctions we have described inascidian eggs (Sardet et al., 1992). We have argued that suchPM-cER junctions are likely to exist in other eggs (for review,see Sardet et al., 2002). Several recent reports also indicate thatspecific cortical ER compartments close to the PM are presentin yeast and plant cells, where they are thought to playimportant roles in polarization, membrane-protein insertion (inyeast bud) and mRNA and protein localization (in riceendosperm) (Du et al., 2004; Hamada et al., 2003).

Evolution of cortical polarity: from oocyte to the 8-16 cellstage embryoFig. 6 describes the cellular transformation of the originalnetwork of cER-mRNA present in the mature oocyte from amonolayer network of increasing density in the vegetal cortexinto a compact posterior cortical mass forming the bulk of theCABs of eight-cell-stage blastomeres. To achieve the

relocation and compaction the cER network passes throughtwo major phases of reorganization between fertilization andcleavage. We also detail changes in surface topography(microvilli) and the cortical MF and MT networks.

OocytesIn mature oocytes arrested in meiotic metaphase I (Fig. 6A),the cER forms a thin (less than 0.5 µm) monolayer made oftubes and sheets whose density increases from the animal tothe vegetal pole (Fig. 2A1, Fig. 6) (Sardet et al., 1992). Post-plasmic/PEM RNAs such as PEM1 are evenly distributed overthe whole surface of cER network at that stage. MFs areenriched in regions of abundant cER in both Ciona andPhallusia but, as previously noted, we do not know whetherthis represents the situation in living oocytes (Sardet et al.,1992). A few stable MTs are also present in the isolated cortexand are most frequent in the cER poor zone (animal region).No major differences in surface microvilli (ConA-bindingsites) are detected at this stage.

Zygote (first major phase)In the zygote (Fig. 6B), the spectacular actomyosin contractionthat follows fertilization by sperm drags the cER network,including the postplasmic/PEM RNAs, in the direction of thevegetal hemisphere. This contraction is triggered by calcium(Roegiers et al., 1995). The sperm aster, subcortical myoplasmlayer and surface macromolecules are also moved vegetally.This massive ‘capping’ of surface macromolecules togetherwith cortical and subcortical components results in theformation of a contraction/vegetal pole (Roegiers et al., 1999).During and at the end of the contraction, a remarkable ring ofMFs forms around the thick (3-5 µm) patch in which cERsheets and tubes, and MFs are intertwined. This cER/MF-richpatch corresponds to the microvillus-rich surface of thecontraction pole. The cER/MF-rich patch is surrounded by aMF ring, which is morphologically reminiscent of a cleavagering and corresponds to a microvillus-free zone on the surface(possibly a zone of membrane insertion, as seen duringcytokinesis). Few individual MTs adhere to the isolated cortexbut those that do are stable in live cortices. Just afterfertilization, large fragments of sperm aster are tethered to theisolated cortex, possibly via the centrosomal accumulation ofcER identified as the moving calcium-wave pacemaker sitesituated in the cortex (Dumollard and Sardet, 2001).

Zygote (second major phase)In the zygote (Fig. 6C) after meiosis completion, the spermaster situated in the future posterior pole of the embryo hasextended long MTs, which interact with the posterior cortexand the female pronucleus near the animal pole. An extensivetranslocation of the aster towards the centre of the egg androtational movement against the posterior cortex cause a hugedisplacement of the cER and postplasmic/PEM RNAsassociated with it, as well as of the bulk of the myoplasm andentrapped cytoplasmic elements (Chiba et al., 1999; Roegierset al., 1999). At this stage, some of the cER has moved awayfrom the surface during the rotational and folding motions ofthe posterior domains [as seen in Halocynthia (Sardet et al.,

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2003)]. It appears that some of this cER is positioned incytoplasmic ER folds located within the myoplasm or draggedtowards the centrosomal area along astral rays (Roegiers et al.,1999). At this stage postplasmic/PEM RNAs are no longerdistributed all over the cER in the cortex but form patchesassociated with the cER in the posterior region of the zygote.In contrast to the conspicuous MF-rich structures we observedat the vegetal/contraction pole, the mitotic zygote peripheryand cortical fragments including the posterior region lackedremarkable MF structures. We have previously observed thatthis region vibrates at high frequency owing to the buckling ofposterior MTs from the sperm aster against the surface(Roegiers et al., 1999). Therefore, it was not surprising to findmany short MTs (stable in live cortices) in isolated corticalfragments originating from the posterior region of the zygote.These MTs might represent MT ends in interaction with thecytoplasmic face of the PM and it would be interesting to knowwhether they are polarized and what motors and microtubule-associated proteins (MAPs) are associated with them. Toproceed further, an analysis similar to that conducted tounderstand the mechanism of the cortical rotation in Xenopuszygotes will be needed (Marrari et al., 2003; Marrari et al.,2004).

Embryo (8-16 cell stage)In the embryo (Fig. 6C), because the cER and associatedmRNAs are located in the vegetal posterior region at the timeof first cleavage, they naturally segregate in the two posteriorvegetal B4.1 blastomeres and their micromere progeny througha series of stereotyped symmetrical and asymmetricalcleavages. Our observations in Ciona and Phallusia confirmour previous observations in Halocynthia, in which the cER-rich CAB is proportionally bigger (about three times bigger)(Sardet et al., 2003). In addition, we could clearly see thatcortical ER and mRNAs in Ciona form an extended corticalplaque at interphase that is subsequently compacted duringmitosis just before unequal cleavage takes place. Although, inisolated cortices, a higher density of crisscrossing MFs aresituated beneath the PM in the CAB area, we do not knowwhether this represents the situation in whole embryos becauseno such cortical differences can be seen in fixed embryoslabelled with fluorescent phalloidins (Nishikata et al., 1999) (J.Chenevert, personal communication). MTs from thecentrosomal aster are present in great numbers enmeshedwithin the CAB and it is possible to imagine that multiplemotors situated within or on the surface of the CAB (on cERor electron dense matrix material) can manoeuvre and attractthe centrosomal MTs to mediate unequal cleavage between theeight-cell and 64-cell stages (Iseto and Nishida, 1999;Nishikata et al., 1999).

Some functions for polarized cERBecause ER is a multifunctional organelle important forcalcium regulation, signalling, mRNA localization andtranslation, it is interesting to consider what cellular anddevelopmental consequences localization of a large amount ofcER-mRNA domain at the poles might have.

Calcium regulation and signallingcER is endowed with special properties because it initiatescalcium signals triggered by the sperm factor even when it isinjected in the egg centre (Carroll et al., 2003; Kyozuka et al.,1998). Accumulation of cER in the centre of the sperm asterand in the vegetal/contraction pole are, successively, the sitesof two calcium-wave pacemakers that emit 6-12 repetitivecalcium waves from fertilization to meiosis completion. Theserepetitive calcium waves initiated in the zone of cERaccumulation are necessary for meiosis completion and alsoregulate mitochondrial metabolism (Specksnijder et al., 1990;Carroll et al., 2003; Dumollard et al., 2003). Because calciumwaves are followed by waves of enzymatic activations [proteinkinase C, calmodulin kinase II (Larabell et al., 2004;Markoulaki et al., 2004)], the vegetal/contraction pole, cER-mRNA domain and associated translation machinery will beexposed to the highest levels of these enzymatic activities andcalcium signals for 15-20 minutes during completion ofmeiosis II (Speksnijder et al., 1990). Therefore, during thisperiod, the vegetal/contraction pole might acquire specialproperties that affect later developmental events taking placeat that site, such as gastrulation. A memory of ER-generatedcalcium wave has been shown to play a role during thepostimplantation development of mammals (Ozil and Huneau,2001).

mRNA localization and translationIn contrast to Drosophila and Xenopus oocytes, in which themechanisms of mRNA localization are being uncovered (Klocand Etkin, 2005; Kloc et al., 2002), we know very little aboutthe initial localization of postplasmic/PEM RNAs in the cortexduring oogenesis. However, our observations in whole zygotesand embryos, and cortices isolated from them in Ciona (thispaper) and Halocynthia (Sardet et al., 2003) provide a possibleexplanation for the relocalization of some mRNA afterfertilization, namely that these mRNAs move and relocatetogether with the cER after fertilization. How a subset ofpostplasmic/PEM RNAs are bound to cER remains a mysterybut our working hypothesis is that they are associated with ER-associated Staufen-containing ribonucleoprotein particles ofthe type described in mammalian neurons or Xenopus oocytes(Ohashi et al., 2002; Yoon and Mowry, 2004).

In ascidians we know almost nothing of the translationalcontrol of cortical mRNAs, except for Cs-PEM3, whoseprotein product assumes a broader posterior distribution thanits mRNA situated in the CAB (Satou, 1999). The distributionof other proteins encoded by postplasmic/PEM mRNAs has notyet been examined. It is very likely that proteins coded by thesematernal RNAs and, in particular, the muscle determinantmacho1 are synthesized before the 16-cell stage, becausemacho1 must exert its effect on muscle precursor blastomeresthat do not inherit the cER-rich CAB. It is also probable that,like PEM3, most proteins translated from cortically localizedpostplasmic/PEM RNAs occupy a much larger area in theposterior pole. Recently, a Y-box protein (CiYB1; thought tobe involved in storage and translational control of mRNAs inoocytes) and its mRNA (CiYB1) have been shown to bepartially colocalized with postplasmic/PEM RNAs in theposterior of the embryo (Tanaka et al., 2004).

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Unequal cleavageAs discussed above, the cER/RNA domain and/or othermacromolecular complexes contained within the CAB mightbe responsible for pulling the centrosome and positioning onepole of the spindle near the cortex for the asymmetrical andunequal cleavages that generate posterior micromeres. Thismacroscopic structure might be related to the structure thatrepositions the spindle in posterior blastomeres ofCaenorhabditis elegans embryos (Skop and White, 1998).Isolated cortical fragments with adhering CABs and associatedMTs constitute an ideal open cell preparation to investigate themechanisms of MT translocation in an ER/MT-motor-richcortical domain using the strategy developed to reactivate MTtranslocation in isolated cortices of Xenopus (Marrari et al.,2003; Marrari et al., 2004).

Cortical polarity: ascidians compared with C. elegans,Xenopus and DrosophilaAscidians are an emerging model for cell and developmentbiology that offers the possibility to routinely isolate corticalfragments from synchronous oocytes, zygotes and embryos(the presence of chorions around oocytes and zygotes rendersthis approach difficult for the moment in C. elegans andDrosophila). In terms of the nature of the developmentalinformation contained in the cortex, it is clear that, as inXenopus and Drosophila, maternal mRNAs in the ascidiancortex play an essential role. There are strong indications that,in Xenopus, some of the maternal mRNAs (e.g. XCat2 andVg1) are associated with ER and ER-bound Staufen (Chang etal., 2004; Dollar et al., 2002; Yoon and Mowry, 2004). In termsof cellular mechanisms of polarity amplification afterfertilization, in both ascidians and in C. elegans, establishmentof anteroposterior polarity is linked to an actomyosin-dependent surface capping of many peripheral macromolecules(including the PAR3-PAR6-aPKC complex) and organelles(Munro et al., 2004; Schneider and Bowerman, 2003).Nothing, however, is known about reorganizations of corticalER in the C. elegans zygote. Similarities between Xenopus andascidian zygotes concern the MT-dependent cortical rotationthat, just before mitosis, displaces the cER-mRNA andmyoplasm domains posteriorly in ascidians and dorsalizingfactors and organelles in Xenopus (Weaver et al., 2003).

Because genomic and transgenic tools are now at hand inascidians (Satoh et al., 2003), we should be able to elucidatethe molecular and cellular mechanisms underlying corticalreorganizations, localization of mRNAs and informationprocessing in the cell cortex.

We thank ARC and AFM foundations, and ACI from the FrenchResearch Ministry for their support. We also thank C. Djediat and P.Chang for help with electron microscopy, C. Rouviere for adviceabout imaging, and J. Chenevert for critical reading of the manuscript.

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