Characterization of the zebrafish Orb/CPEB-related RNA-binding ...

Characterization of the zebrafish Orb/CPEB-related RNA-binding proteinand localization of maternal components in the zebrafish oocyte

Laure Bally-Cuif*, William J. Schatz, Robert K. Ho

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

Received 9 June 1998; accepted 30 June 1998

Abstract

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification areunknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributedmaternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocytematuration at which they concentrate to the future animal pole. We have further characterized one of these mRNAs,zorba, which encodes ahomologue of theDrosophilaOrb andXenopusCPEB RNA-binding proteins.Zorbabelongs to the group of earliest mRNAs to localize atthe animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization isindependent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA. 1998 ElsevierScience Ireland Ltd. All rights reserved

Keywords:RNA localization; Determinant; RNA-binding protein; Orb; CPEB; Zebrafish oogenesis

1. Introduction

Understanding the molecular bases of axes establishmentis an important challenge of developmental biology. Inextensively studied model systems such asDrosophila mel-anogasterandXenopus laevis, maternal factors non-ubiqui-tously localized within the egg have been demonstrated toplay a major role in imparting asymmetries. These ‘deter-minants’ generally consist of mRNAs deposited or anchoredin a localized fashion within the oocyte, following localproduction by accessory cells and/or a passive or activetransport to a specific binding site (see for reviews: Mick-lem, 1995; Gru¨nert and St Johnston, 1996; King, 1996).These factors are responsible for setting up both the ante-roposterior (AP) and dorsoventral (DV) axes inDrosophila,and the animal-vegetal (An/Vg) axis in theXenopusoocyte.An additional step of local factor ‘activation’ is required inXenopusembryos to specify the DV axis following cortical

rotation (see for a review Gerhart et al., 1989). Differentstrategies have been illustrated in other systems. Forinstance in theCaenorhabditis elegansegg, no localizedmRNAs have so far been isolated. Rather, polarities seemestablished by a later process, relying on the localizeddegradation or translational control of some ubiquitousmRNAs and on subsequent inductive cell–cell interactionsafter the first embryonic cleavage divisions (Goldstein et al.,1993; Goldstein and Hird, 1996; Seydoux et al., 1996). Itremains unclear at present how widely these different stra-tegies are used within the animal kingdom.

As an entry point towards a comparative analysis of axesspecification strategies in vertebrates, we have turned to theteleost zebrafish,Danio rerio. In zebrafish, the An/Vg axisis established during oogenesis as it can be observed atlaying by the asymmetrical (animal) location of the germ-inal vesicle (the oocyte nucleus) and of the micropyle(Wacker et al., 1994), a specialized structure of the vitellineenvelope allowing sperm entry (Hart et al., 1992; Hart andDonovan, 1983). After fertilization, the egg cytoplasm(endoplasm) will converge to the animal pole, forming ayolk-free blastodisc where meroblastic divisions occur andwhich will give rise to the embryo proper (see Driever,

Mechanisms of Development 77 (1998) 31–47

0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reservedPII S0925-4773(98)00109-9

* Corresponding author. Present address: GSF Forschungszentrum, Insti-tut fur Saugetiergenetik, Ingolsta¨dter Landstrasse 1, D-85758 Neuherberg,Germany. Tel.: +49 89 31874204; fax: +49 89 31873099;e-mail: [email protected]

1995; Kimmel et al., 1995 for reviews). The DV axisbecomes morphologically visible much later (Schmitz andCampos-Ortega, 1994). However, lithium, UV- and cold-sensitive periods (Stachel et al., 1993; Stra¨hle and Jesutha-san, 1993; Jesuthasan and Stra¨hle, 1997) in zebrafish andother teleosts (Oppenheimer, 1936; Tung et al., 1945; Kos-tomarova, 1969) suggest that the DV axis may also be deter-mined prior to the onset of zygotic transcription (or mid-blastula transition, 1024-cell stage). Together, these obser-vations suggest that both An/Vg and DV axes in zebrafishare established under the control of maternal factors.

As a first step towards the identification of such factors,we initiated a search for zebrafish homologues of moleculesinvolved in the asymmetrical localization, activation orrepression, of maternal mRNAs inDrosophilaandXenopus.In fact, these species seem to use conserved determinantsonly in rare instances (Mosquera et al., 1993), but they sharecommon strategies for the localization or translational reg-ulation of these determinants. In particular, specific RNA-and/or cytoskeleton-binding proteins are involved in bothspecies (see for reviews Micklem, 1995; Hesketh, 1996;King, 1996; Macdonald and Smibert, 1996). Here we reportthe cloning and expression analysis of the zebrafish homo-logue ofDrosophila orbandXenopus CPEB. orbencodes amaternal RNA-binding protein of the RRM (RNA recogni-tion motif) family which is required at multiple steps ofDrosophilaoogenesis (Lantz et al., 1992). The Orb proteinis present from the earliest stages of germ line development,and first accumulates in the developing oocyte shortly afterthe formation of the 16-cell cyst. It is later redistributed tothe posterior, and subsequently to the anterior, pole of theoocyte (Christerson and McKearin, 1994; Lantz et al.,1994). The most severeorb mutations, orbdec andorbF343, likely protein-nulls, block ovarian developmentat the 8-cell cyst stage (Lantz et al., 1994). Mutants retain-ing weak residualorb function,orbF303, are blocked later asthey fail to properly differentiate an oocyte within the 16-cell egg chamber. Finally, the least affectedorb mutantsorbmel differentiate an oocyte; however, it is abnormallypolarized along both the AP and DV axes (Christersonand McKearin, 1994). Maternal mRNAs such asBicau-dal-D, fs(1)K10, oskarandgurken,involved in establishingthese polarities, are mislocalized in theorb mutants (Chris-terson and McKearin, 1994; Lantz et al., 1994). Togetherwith the distribution of Orb protein in the egg chamberduring the course of oogenesis, these phenotypes are con-sistent with the idea that Orb may be involved in the trans-port and/or anchoring of specific maternal mRNAs in theoocyte. In parallel to these studies, a maternalXenopusprotein possessing a very similar RNA-binding domain,CPEB, was isolated for its capacity to bind the matura-tion-type cytoplasmic polyadenylation element (CPE)(Hake and Richter, 1994). The maturation-type CPE is ashort U-rich nucleotide stretch (of consensus sequenceUUUUAAU) present in the 3′UTR of several mRNAsinvolved inXenopusoocyte maturation. The CPE promotes

the cytoplasmic polyadenylation and resultant translationalactivation of these mRNAs (see Macdonald and Smibert,1996; Richter, 1996; Hake and Richter, 1997; Stebbins-Boaz and Richter, 1997 for reviews). CPEB was in particu-lar proven to be necessary for the polyadenylation of thec-mosmRNA and, consequently, forXenopusoocyte matura-tion (Stebbins-Boaz et al., 1996).

Based on structural homology, we have clonedzorba, thepotential zebrafish homologue oforb/CPEB. We demon-strate here thatzorbamRNA and protein are maternal andbecome restricted to a subcortical crescent at the futureanimal pole during early stages of oocyte maturation. Ourresults suggest a localized function for the maternal RNA-binding protein Zorba in zebrafish, possibly related to thespecification of the animal pole. In addition, we character-ized the distribution of other maternal mRNAs in the zebra-fish oocyte. Surprisingly, our comparison highlights theexistence of several classes of maternal mRNAs redistribut-ing to the animal region at different stages of oocyte matura-tion. These findings demonstrate that the distribution of thedifferent maternal mRNAs in the zebrafish oocyte is speci-fically regulated, and also that their accumulation at theanimal pole occurs prior to the redistribution of the endo-plasm, which follows fertilization.

2. Results

2.1. Cloning of zorba, a zebrafish homologue of Drosophilaorb

As a first step towards the identification of putative mater-nal determinants in zebrafish, we looked for potentialupstream factors involved in the localization or in the trans-lational regulation of maternal mRNAs. We used degener-ate oligonucleotides directed against the RNA-bindingdomain (RNA-BD)-encoding region ofDrosophila orband Xenopus CPEBto RT–PCR amplify a single 450 bpfragment under low stringency annealing conditions (seeSection 4) from pre-midblastula transition zebrafishcDNA. This fragment encoded an RNA-BD comprised oftwo RRM motifs, overall 62% and 90% similar to those ofthe Orb andXenopusCPEB proteins, respectively. The frag-ment was extended by 3′RACE PCR and the resulting 1.6 kbcDNA fragment was used to screen at high stringency azebrafish 4–8 cell-stage cDNA library. Ten positive cloneswere isolated, all encoding parts of the same protein. Fourclones, 2.5–3 kb in length, contained a full length 1800 bpORF, which was preceded by 15 bp of 5′UTR containing anin-frame stop codon, and followed by 1–1.5 kb 3′UTRsdiffering in their polyadenylation sites (Fig. 1A). Thededuced protein sequence, of calculated molecular weight60 kDa, is aligned in Fig. 1B with Orb andXenopusCPEB.The RNA-BD of this zebrafish protein contains the samearrangement of two closely linked RRM motifs as found inOrb and CPEB, with the same atypical RNP2 peptide in the

32 L. Bally-Cuif et al. / Mechanisms of Development 77 (1998) 31–47

second RRM, in which Phe residues are replaced by Arg andTyr (Lantz et al., 1992) (Fig. 1B). The RNA-BD is followedby a short zinc-finger domain 58% and 67% identical tothose of Orb and CPEB, respectively (Fig. 1B). These fea-

tures are characteristic of Orb and CPEB among the RRM-containing RNA-binding proteins. While homologybetween the three proteins is much lower beyond thesedomains, significant stretches of similarity are observed in

Fig. 1. (A) Nucleotide and amino acid sequences of the zorba mRNA and protein. The START and STOP codons are in capital letters, an in-frame STOPcodon upstream of the ATG is double-underlined, the polyadenylation sites of the differentzorbaclones sequenced are indicated by the asterisks, and theRNA-binding domain-encoding region is underlined. The 3′UTR domain aligning with that of X.CPEB (C) is in italics. (B) Sequence alignment of the Zorba,XenopusCPEB (Hake and Richter, 1994) andDrosophilaOrb (Lantz et al., 1992) proteins. The RRM motifs of the RNA-binding domains are in italics, withthe characteristic RNP-1 and -2 peptides underlined. The atypical S and R residues within the second RNP2 are indicated (asterisks). The C and H residues ofthe single zinc finger domain located C-terminal to the RNA-BD are underlined. Short stretches of homology outside the RNA-BDs are underlined with adotted line. (C) Sequence alignment of the 3′UTRs ofzorba(position + 2518) and CPEB (position+ 2638; Hake and Richter, 1994). Note, underlined, thetwo conserved CPEs. (D) Temporal expression ofzorba, revealed by Northern blot. 10 pg of total RNA from oocytes (Oo), 64-cell (64), sphere (Sph), 50%epiboly (50%), tail bud (tb), 28-h- and 36-h-stage embryos were loaded in each lane and probed withzorbafull length clone. Ribosomal RNA, visualized byethidium bromide staining, was used as an internal standard for quantification of deposited RNA. A single 3 kb mRNA species is detected during oogenesisand early cleavage stages, but not after mid-blastula transition (MBT).

33L. Bally-Cuif et al. / Mechanisms of Development 77 (1998) 31–47

Fig. 1b–d.


the N-terminal halves of the three proteins, and C-terminalto the zinc finger domain (Fig. 1B). We thus believe that wehave isolated the zebrafish orthologue oforb, hence named‘zebrafish orb-type a’ (Zorba). The extensive similarity ofZorba and CPEB RNA-BDs further suggests that both areRNA-binding proteins of similar specificity. It is thereforeinteresting to note that the 3′UTRs of zorba and XenopusCPEBalign at position+2518 over a restricted 53 nt stretchcomprising two closely linked potential CPE elements (seeFig. 1C), suggesting possible binding of the Zorba protein toits own mRNA (see Section 3).

2.2. zorba mRNA is strictly maternal and is asymmetricallylocalized in maturing zebrafish oocytes

Northern blot experiments were performed to determinethe time-course ofzorba transcription (Fig. 1D). A single2.5–3 kb mRNA species was detected, abundant in oocytesand decreasing in pre-mid-blastula transition embryos. Noexpression was detected at post-mid-blastula transitionembryonic stages, at larval stages, and in various adult tis-sues other than ovaries (including skeletal muscle, brain andtestes, not shown). Therefore,zorba is unique in that itsexpression is restricted to pre-mid-blastula transition stages,since all the other maternally expressed genes described todate in zebrafish also display a zygotic component duringembryonic development. This suggests thatzorba has aspecific maternal function.

The spatial distribution ofzorba transcripts was deter-mined by whole-mount in situ hybridization. At post-ferti-lization stages, thezorbamessage was uniformly distributed

within the blastomeres (not shown). The intensity of thesignal then progressively decreased to become undetectableby mid-blastula transition. By contrast, localization of thezorbamessage followed a dynamic and non-ubiquitous pro-file during oogenesis (Fig. 3). Oogenesis in zebrafish pro-ceeds through five stages of maturation, identifiable on thebasis of oocyte size and of the presence or location of spe-cific subcellular components, such as the germinal vesicle(GV) and yolk granules (Fig. 2, and Selman et al., 1993). Atstage III, a micropylar follicle cell (the future site of spermentry) becomes visible at the future animal pole (see Selmanet al., 1993). The GV is located at the center of the oocyteuntil stage IV, when it migrates to the future animal pole ofthe oocyte and breaks down. These features are the firstmorphological asymmetries described during oocytematuration. Strikingly, we observed that thezorbamessagewas ubiquitous at stages I and II, but gradually accumulatedin one restricted spot at the periphery of the oocyte duringstage III (Fig. 3A, arrowheads). Expression was maintainedin that region at stage IV (Fig. 3B), and then became delo-calized at stage V to cover a much broader area presumablyencompassing the future blastodisc (Fig. 3C, and seebelow).

To determine the subcellular localization ofzorbamRNAaccumulation during oocyte maturation, we performed insitu hybridization on ovary sections (Fig. 3D–G). No tran-scripts were ever detectable in the follicle cells. Transcriptswere present in oocytes of all stages, distributed ubiqui-tously at stages I and II (Fig. 3D), and progressively loca-lized thereafter to a narrow subcortical domain by stage III(Fig. 3D,E). The position of this domain relative to the

Fig. 2. Stages of oogenesis in zebrafish (after Selman et al., 1993), showing the appearance of the first morphological animal/vegetal asymmetries: theformation of the micropylar cell at stage III, and the migration of the germinal vesicle towards the anima pole at stage IV. Diameter sizes of the oocytes, andmeiotic stages, are indicated. GVBD, germinal vesicle breakdown.


Fig. 3. Expression ofzorbaduring zebrafish oogenesis, as revealed by in situ hybridization on whole-mount (A–C) and sectioned (D–G) oocytes.zorbamRNA is ubiquitous at stages I and II (A, D), and starts to accumulate at the future animal pole (see text) at early stage III (A, D, arrowheads). In D, notethecentrally located germina vesicle, and the onset of accumulation of yolk granules, identifying stage II. The germinal vesicle is also visible as a transparentcentral region in the stage III oocyte shown in A.zorbamRNA is tightly tethered at the future animal pole at late stage III (E; note the central location of thegerminal vesicle and the abundant yolk granules) and IV (B, F). It redistributes over the entire future blastoderm at stage V (C). The future animal pole can bemorphologically identified at stage IV by the localization of the germinal vesicle (F) and of the micropyle (G). gv, germinal vesicle; m, micropyle. Scalebar = 0.4 mm.


future egg axes could not be defined at this stage. However,at stage IV, this subcortical crescent overlayed the site ofmigration of the GV (Fig. 3F), and lay under the micropyle(Fig. 3G), showing that the position of zorba mRNA loca-lization corresponds to the future animal pole of the egg.Low levels of expression could still be detected at stage IVin the yolk-free oocyte cytoplasm, as well as around theGV(see Fig. 3F).

Our data establish thatzorba transcripts are localized tothe presumptive animal pole of the oocyte from maturationstage III onwards, before morphological A/P determination.This observation demonstrates the existence of an earlymolecular asymmetry within the zebrafish oocyte whichidentifies the future animal pole. The localization of thezorbamessage at stage III cannot be dependent on redistri-bution of the oocyte endoplasm, which occurs later, afterfertilization (or activation) to form a protuberant blasto-derm.zorbamessage localization must therefore rely on adifferent, earlier mechanism.

2.3. mRNA localization in the zebrafish oocyte at stage IIIis a selective phenomenon

We wished to determine whether the localization ofzorbatranscripts reflected a general phenomenon (i.e. occurredwith all maternal mRNAs at stage III of oocyte maturation)or was a selective process (i.e. affected only a subset ofthese maternal mRNAs). We therefore compared the loca-lization of zorbamRNA with that of seven other maternalmRNAs, using ISH on adjacent ovary sections (Fig. 4). Thematernally expressed zebrafish genes tested were the fol-lowing: Vg1(Helde and Grunwald, 1993),Notch(Bierkampand Campos-Ortega, 1993),Taram-A(Renucci et al., 1996),Sox19(Vriz and Lovell-Badge, 1995),gsc (Stachel et al.,1993), PABP (polyA-binding protein gene, L.B-C andR.K.H., unpublished data), and a maternalActRIIB (L.BCand R.K.H., unpublished data). Overall, we observed fromthis sampling that maternal mRNAs could be subdividedinto three categories: (A) mRNAs which show the sameearly localization aszorba at stage III (namely:Notch,PABP, andTaram-A), (B) mRNAs which become concen-trated in the same restricted animal region slightly later, atearly stage IV (namely:Vg1), and, finally, (C) mRNAswhich remain ubiquitous at these stages and do not appearto follow any specific localization during oocyte maturation(namely:ActRIIB, gsc, andSox19). All mRNAs will, how-ever, localize to a very broad region in the animal hemi-sphere at stage V, corresponding to all or most of the futureblastoderms (seezorbaon Fig. 3C); mRNAs of categories Aand B are thus delocalized between stages IV and V. As anexample, Fig. 4 shows the localization ofzorbaandNotch,and the non-localization ofVg1, gscandActRIIBmRNAs atstage III; and Fig. 5 shows the localization ofzorbaandVg1mRNAs to the future animal pole at stage IV (see also Fig.6).

Therefore, it appears that only a subset of maternal

mRNAs becomes accumulated to the future animal pole atearly oogenesis stages, suggesting that this localization is ahighly regulated process.

Fig. 4. Comparison of the distribution ofzorba (A, C, E, G),Notch (B),Vg1 (D), gsc (F) and ActRIIB (H) mRNAs during zebrafish oogenesis.Adjacent sections of the same stage III oocyte are shown on each line(note, when visible, the central location of the germinal vesicle). At stageIII, zorbamRNA accumulates at the future animal pole (arrowhead),NotchmRNA localizes like zorba, most of Vg1 mRNA is still ubiquitous(arrows),gscmRNA is mostly found subcortically but around the wholeoocyte (arrows), andActRIIB mRNA is mostly central and surrounds thegerminal vesicle (not visible on H).Vg1 mRNA will redistribute to theanimal pole between stages III and IV (see Fig. 5), andgscandActRIIBmRNAs only at stage V and in a loose fashion. All mRNAs show the sameprofile at this stage (see for instancezorba in Fig. 3C). See text for moreexamples. gv, germinal vesicle. Scale bar= 0.4 mm.


2.4. Localization of zorba and Vg1 mRNAs to the animalpole is independent of microtubules and microfilaments

The non-ubiquitous localization of maternal mRNAs inXenopusandDrosophilaoocytes has been shown to rely oncytoskeletal elements, such as microtubules and microfila-ments. For instance, it was shown thatXenopus Vg1mRNAwas transported to the vegetal pole by a microtubule-depen-dent process, and was then anchored to actin microfilamentspresent at the vegetal cortex (Yisraeli et al., 1990). Usingimmunocytochemistry, we could reveal microtubules in thesubcortical domain of zebrafish oocytes from stage IIIonwards (see Fig. 7F), thus extending previous reports(see Hart and Fluck, 1995 for a review). In addition, weused phalloidin-FITC labelling and detected microfilamentsimmediately underlying the oocyte vitelline membranefrom stage II onwards (see Fig. 7H). To determine whethersuch cytoskeletal elements were involved in localizingzorbaand/orVg1mRNAs to the animal pole during zebra-fish oocyte maturation, we applied microtubule- or micro-filament-depolymerizing drugs to oocytes cultured in vitrofrom early stage III onwards, and assessed the distributionof zorbaandVg1mRNAs in these oocytes by whole-mountISH. Maturation stages were estimated at the time of cultureby oocyte size, the presence of vitellus and the location ofthe germinal vesicle, as reported in Selman et al. (1993).Oocytes were placed in culture at early (diameter 0.35–0.5mm) or late (0.5–0.7 mm) stage III.

As a test for our culture conditions, the maturation of latestage III oocytes to stage IV was monitored in the absence ofdrug treatment. After 5 h of culture, 23% of late stage IIIoocytes (n = 148) had matured to stage IV (as assessed bytheir translucent appearance, and by the disappearance ofthe germinal vesicle), a proportion similar to that reportedby Selman et al. (1994). This proportion was brought to 97%(n = 54) after addition of 5 pg/ml DHP hormone for 5 h,

again in agreement with the report by Selman et al. (1994).These results show that our culture conditions are compa-tible with at least some steps of oocyte maturation fromstage III onwards. We then tested the effect of cytoskeletaldrugs treatment. In all cases, a low proportion of culturedoocytes (16%,n = 562) showed no detectable expression ofeither zorba or Vg1 after 5 h. When identically stagedoocytes were cultured in parallel after injection with cappedlacZ mRNA, a similar proportion (20%,n = 30) showed nobeta-galactosidase activity (not shown). We assume thatthese oocytes were dead at the time of culture, or did notsurvive the culture conditions, and they were not taken intoaccount in the following analysis of our results. We alsoverified that the proportion of oocytes maturing to stageIV was unaffected by drug treatment.

To test for a role of microtubules in mRNAs localization,isolated oocytes were cultured in the presence of 2 or 20mg/ml nocodazole for 5 h. Whole-mount immunocytochemistryusing the anti-beta tubulin antibody KMX1 assessed that nopolymerized microtubules were detectable after a 20mg/mltreatment (not shown). However, this treatment did not leadto any change inzorba(n = 44) andVg1 (n = 46) mRNAslocalization at any stage compared to untreated oocytes(n = 26 and 32, respectively) (Fig. 6A–D). Therefore,microtubules do not appear to be a primary component ofthe mRNAs localization process.

To test for a role of actin microfilaments, oocytes weretreated with 2.5 or 25mg/ml cytochalasine B for 5 h. Phal-loidin–FITC staining demonstrates a gradual disruption ofthe subcortical actin network at these doses, with totalbreakdown obtained at the highest dose (not shown). Thedistribution ofzorbaor Vg1mRNAs in oocytes cultured atearly stage III was not modified by these treatments. How-ever,zorbaor Vg1mRNAs distribution was affected whenoocytes were treated with cytochalasine B at late stage IV.We observed an enlargement and an increase in the staining

Fig. 5. Localization ofVg1mRNA at the future animal pole of a stage IV oocyte. Ovary sections were hybridized with thezorba(A) or Vg1(B) probes, andstage IV oocytes were identified by the animal location of the germinal vesicle (gv). Staining of adjacent sections for either mRNA demonstrates thatVg1mRNA accumulates in the same restricted animal domain aszorbamRNA from stage IV onwards (arrowheads). Scale bar= 0.4 mm.


Fig. 6. Distribution ofzorba(A, C, E) andVg1(B,D,F) mRNAs in oocytes cultured for 5 h from late stage III onwards and treated with: 20mM nocodazole todisrupt microtubules (C, D), or 25mM cytochalasine B to disrupt microfilaments (E, F). (A, B) mock treatment.zorbamRNA is localized in all oocytesstudied (A, arrowheads); in comparison,Vg1 localization was consistently noted in only those oocytes that underwent maturation under culture conditions (B,arrowheads). Nocodazole treatments (C, D) had no effect upon the localization of eitherzorbaor Vg1. However, cytochalasine B treatments enhanced thelocalization of bothzorbaandVg1mRNAs. Note in E and F that the expression domains are wider and more intensely labeled at the animal pole (bars). Inaddition, note that unlike the oocytes shown in B and D, all oocytes treated with cytochalasine B exhibited strong localization ofVg1; however, cytochalasineB treatments did not significantly affect maturation rates (see text for details). Scale bar= 0.4 mm.


intensity of thezorbamRNA localization spot at the animalpole in all cytochalasine B-treated oocytes cultured at thisstage (n = 20, Fig. 6E). In rare instances (10%, Fig. 6E,arrows), the staining spot appeared split.Vg1 mRNA nor-mally localized later thanzorba, between stages III and IV.We observed thatVg1 mRNA, like zorba, concentrated tothe animal pole in late stage III oocytes treated with cyto-chalasine B (localization in 28% cases,n = 18, at 2.5mg/mlcytoB, and in 100% cases,n = 26, at 25mg/ml cytoB; Fig.6F). Again, the cytochalasine B-induced localization waswider than normal. After a control culture of late stage IIIoocytes,Vg1 mRNA was localized in only 22% of cases(n = 46, Fig. 6B), i.e in the oocytes having matured tostage IV. However, the cytochalasine B-induced localiza-tion of Vg1 was not related to oocyte maturation, as weverified that the proportion of oocytes maturing to stageIV was not affected by cytochalasine treatment.

These results suggest that microfilaments are notinvolved in the localization of eitherzorba or Vg1mRNAs at the animal pole. Rather, a likely interpretationis that the depolymerization of the actin network most prob-ably released an internal non-localized pool ofzorba andVg1 mRNAs (visible in Figs. 3F and 4D), which subse-quently accumulated at the animal pole following anactin-independent process. Alternatively, microfilamentsmight be necessary for the tight tethering ofzorbamRNAto a restricted spot at the animal pole.

2.5. zorba mRNA localization is reflected at the proteinlevel

To test whetherzorbamight perform a localized functionin the zebrafish oocyte, we wished to determine whether,like its mRNA, the Zorba protein was localized early duringoogenesis. Therefore, we raised a polyclonal antibodyagainst the N-terminal (non-RNA-BD) portion of theZorba protein (see Section 4). Western blot analyses usingthis antibody revealed a major band of approx. 62 kDa inoocyte extracts (Fig. 7A), a size in agreement with theexpected molecular weight of the Zorba protein (60 kDa)and with the size of the in vitro translated product ofzorbamRNA in a reticulocyte lysate. No protein is detected byWestern blot (Fig. 7A) or immunocytochemistry in post-fertilization embryos and in adult tissues except ovaries

(not shown). However, the 62 kDa band was specificallydetected in extracts of embryos which had been previouslyinjected with cappedzorba mRNA (not shown). Furtherconfirmation that the detected protein came from theinjected mRNA was obtained by whole-mount immunos-taining of embryos injected with a mixture of 2× 106 kDafixable rhodamine dextran and cappedzorba mRNA: theimmunocytochemistry and rhodamine stainings werealways largely overlapping (data not shown). Takentogether, these results suggest that our antibody specificallyrecognizes the Zorba protein. The undetectable level of pro-tein between fertilization and mid-blastula transition sug-gests that at this stage thezorba mRNA (see Fig. 1D) isnot translationally active, and/or that the Zorba protein israpidly degraded.

We further used this antibody on ovary sections to deter-mine the distribution of the Zorba protein during oocytematuration. At early stage I (stage Ia, Selman et al.,1993), no or little protein is detected (Fig. 7D). It becomesdetectable at stage Ib (Fig. 7C), and remains ubiquitouslydistributed within the oocyte cytoplasm until stage II (Fig.7C). It then follows a gradual accumulation to the futureanimal pole during stage III (Fig. 7D,E), and remains loca-lized to this pole at stage IV (Fig. 7G). No staining wasobserved with the pre-immune serum (not shown). There-fore, the distribution of the Zorba protein corresponds to thatof its mRNA, suggesting that Zorba may perform a localizedfunction.

To determine whether Zorba might be linked to cytoske-letal elements when it becomes localized to the animal pole,the proteins from detergent-soluble and insoluble fractionsof stages I–II and III–IV oocytes were isolated under highsalt conditions solubilizing most of the yolk proteins. Sur-prisingly, Western blot analyses of these extracts demon-strate that the Zorba protein is always associated with theinsoluble fraction (Fig. 7B) (which contains most of thecytoskeletal elements of the cell, as revealed by microtubuledetection, not shown), suggesting that Zorba could be boundat all stages to cytoskeletal components. Double-labellingsof oocyte sections with anti-Zorba and anti-beta tubulinantibodies reveal a region of overlap between the two stain-ings at the site of Zorba protein accumulation from stage IIIonwards (Fig. 7E,F). However, we were unable to copurifymicrotubules and Zorba by ultracentrifugation and immu-

Fig. 7. Temporal and spatial distribution of the Zorba protein. (A) Protein extract from oocytes (Oo), 1–4 cell (1–4), sphere (Sigh), 10 somite (10s) and 24-h-stage embryos was resolved by electrophoresis, blotted to nitrocellulose and probed with rabbit anti-Zorba antiserum (or pre-immune serum at the oocytestage (PI)). Zorba protein is detected in oocytes, but not at any stage after fertilization. (B) Detergent-soluble (S) and -insoluble (I) fractions of oocyte extractsat stages I–II (lanes 1–2) and III–IV (lanes 3–4) analysed by the same Western blot procedure. Zorba protein is always associated with the detergent-insolublefraction. The detection of a minor faster migrating species of approx. 60 kDa in some instances suggests that the 62 kDa band represents a post-translationallymodified form of Zorba. (C, D) Immunolocalization of the Zorba protein during oogenesis. Ovary sections were incubated with rabbit anti-Zorba antibody,later revealed with HRP. The protein is absent or in low amounts at stage Ia (D), ubiquitous at stages Ib and II (C), and tethered at the future animal polefromstage III (D, note the central location of the germinal vesicle). (E, F) Double-labelling of an ovary section with anti-Zorba (E) and anti-beta tubulin KMX-1(F). In stage III oocytes, tubulins and Zorba distributions overlap at the site of Zorba accumulation (arrowhead). (G, H) Double-labeling of an ovarysectionwith anti-Zorba (G) and phalloidin–FITC (H). The limits of the different oocytes are indicated by dotted white lines in G. Note that at stage IV, the Zorbaprotein (arrowhead) is not in immediate contact with the subcortical actin network.


noprecipitation (data not shown); thus, whatever the stageconsidered, the majority of Zorba protein is unlikely to bebound to microtubules. In addition, double labellings atstage III with phalloidin–FITC and anti-Zorba show thatthe Zorba accumulation spot is located relatively far fromthe subcortical actin network (Fig. 7G,H). The subcorticalaccumulation of Zorba at this stage thus may not be due tobinding to this actin network. The Zorba protein may accu-mulate at the animal pole as a result of the previous locali-zation mRNA, ensuring a localized protein production, and/or as a result of binding to intracellular components otherthan microfilaments and microtubules.

In conclusion, we observed that the Zorba protein speci-fically accumulates at the animal pole from stage III ofoocyte maturation, demonstrating that some maternal pro-teins can be non-ubiquitously distributed in the zebrafishoocyte. The Zorba protein may thus exert a localized rolein the zebrafish oocyte.

3. Discussion

3.1. The zorba gene of zebrafish encodes an RNA-bindingprotein

The zorba gene encodes a protein of 600 amino acidswhich shows significant homology toDrosophila Orb(40% overall identity) (Lantz et al., 1992) andXenopusCPEB, and the recently identified mouse CPEB (67% over-all identity) (Hake and Richter, 1994; Gebauer and Richter,1996). In particular, two atypical RRM motifs, closelylinked to a single zinc finger, are present in the C-terminalhalf of all four proteins. The N-terminal halves are muchmore divergent; however, they show two short stretches ofsignificant homology. This structural conservation stronglysuggests that theorb, CPEBsand zorba genes belong tothe same family. Suggestive evidence for the existence ofa single gene inDrosophila, zebrafish (this report) andmouse (Gebauer and Richter, 1996) suggests thatorb,CPEBandzorbaare orthologous genes. In support of thisconclusion, these genes share expression characteristics asall four are transcribed during oocyte development.

The RNA-binding domain has been demonstrated to beone of the diagnostic determinants of the binding specificityof RNA-binding proteins (see Biamonti and Riva, 1994 for areview). TheXenopusand mouse CPEB proteins can recog-nize the same RNA target in vitro, an A/U rich nucleotidesequence designated as CPE element (Gebauer and Richter,1996). The similarity between the RNA-binding domains ofZorba and the CPEBs thus strongly suggests that Zorba alsorecognizes the CPE. Accessory domains of RNA-bindingproteins have been proposed to help form supramolecularstructures via protein–protein or protein–RNA interactions,thereby modulating the functional specificity of the RNA-binding proteins (see Biamonti and Riva, 1994). In spite ofthe fact that Zorba, Orb and the CPEBs probably bind simi-

lar target sequences, the divergence of their N-terminaldomains may therefore introduce important functional dif-ferences between these proteins.XenopusCPEB has beenisolated for its role in the regulation of polyadenylation(Hake and Richter, 1994), and Orb has been hypothesizedto play a role in RNA localization (Christerson andMcKearin, 1994; Lantz et al., 1994). Whether both proteinsshare both functions is under investigation. The N-terminaldomain of Zorba does not possess any typical motif of clas-sical RNA-binding protein-accessory domains, rendering itdifficult to predict its biochemical function. It is, however,more closely related to the N-terminus of the CPEBs than tothat of Orb, suggesting that Zorba may also be involved inthe control of translation.

3.2. zorba mRNA and protein become localized at earlyoogenesis stages

We found that the distribution ofzorbamRNA and pro-tein was biphasic during oocyte maturation. An initial phaseof ubiquitous distribution until stage II is followed by aphase of sharp localization from stage III onwards. This isreminiscent of the redistribution oforb mRNA and proteinduringDrosophilaoogenesis, but differs from the non-loca-lization of mCPEBmRNA in the mouse oocyte (Gebauerand Richter, 1996). The distinct phenotypes of the differentorb mutant alleles show that Orb has several functions,probably associated with the different phases of Orb loca-lization (Christerson and McKearin, 1994; Lantz et al.,1994). By analogy, it is tempting to speculate thatzorbamight successively exert different roles in the zebrafishoocyte.

The initial ubiquitous phase ofzorba expression mightcorrespond to a general role in oocyte development, via thetranslational regulation of mRNAs necessary for oocytesurvival or maturation. Specific oocyte mRNAs whose poly-adenylation is controlled by CPEB have been identified inXenopus(Hake and Richter, 1994; Stebbins-Boaz et al.,1996). CPEB was proven to be crucial for oocyte matura-tion: immunodepletion of CPEB fromXenopuseggs by theinjection of anti-CPEB antibody preventsc-mospolyadeny-lation and meiotic maturation (Stebbins-Boaz et al., 1996).The second, localized phase ofzorba expression mightreflect a role in oocyte polarization.zorba belongs to thegroup of earliest mRNAs to localize to the future animalpole. Localizing and/or locally regulating the translation oftarget mRNAs, the protein products of which are involvedin animal pole specification, might be a key step in thepathway setting up the An/Vg axis. In zebrafish, the animalpole is the region of the oocyte from which the embryoproper will develop. Specifying the animal pole thereforecontrols the choice between embryonic versus yolk fate.This may be reminiscent of the early role of Orb inDroso-phila, when it controls the choice of cell fate betweenoocyte and nurse cells within the egg chamber (Lantz etal., 1994).


Interestingly, the 3′UTRs of zorba and XenopusCPEBalign with 80% identity over a restricted 53 nt stretchincluding two potential CPEs. Such conservation suggestsfunctional significance, and this domain may mediate thebinding of zorba to its own mRNA, thus ensuring its loca-lization or local translational regulation. Production andlocalization of the Zorba protein would then be reinforcedvia an autoregulatory loop. Positive autoregulation mightalso occur inXenopus. Since no CPE is found in the3′UTR of mouseCPEB (see discussion in Gebauer andRichter, 1996) and in the fragment oforb 3′UTR necessaryto recapitulateorb mRNA distribution in transgenic flies(Lantz et al., 1994), the potential autoregulatory functionof members of the Orb/CPEB family might be species-spe-cific. A second potential candidate for Zorba binding isVg1mRNA, whose localization to the animal pole slightly fol-lows that of Zorba. The 3′UTR of Vg1possesses a potentialCPE (UUUUUAUU) 14 nt upstream of the polyA additionsite (see Fig. 1A in Helde and Grunwald, 1993). Severalproteins binding theVg1mRNA have been isolated inXeno-pus, although none of them appears to have the same mole-cular weight as full length CPEB (Elisha et al., 1995;Deshler et al., 1997; Mowry, 1997).

3.3. Maternal mRNAs distribution in the zebrafish oocyte isprecisely regulated

A major outcome of our study is the finding that mater-nal mRNAs are differentially distributed within the zebra-fish oocyte. Some become tightly tethered at the futureanimal pole as soon as stage III, others between stagesIII and IV, and a last pool redistributes more loosely tothe blastodermal region at stage V. With the unique excep-tion of Vg1, no localization studies of maternal mRNAshad previously been performed in zebrafish.Vg1 mRNAwas reported to be ubiquitous in early embryos (Helde andGrunwald, 1993); however, we have extended these studiesand we have observed a definite localization ofVg1 to thefuture animal pole of oocytes. Our finding has severalimportant implications. (i) It demonstrates that the zebra-fish oocyte is a polarized cell from much earlier than pre-viously thought. We observed a micropyle, the earliestasymmetry so far described (yet not in the oocyte properbut in the follicle cell layer) on oocytes larger than 0.5 mmin our preparations (see also Hart and Donovan, 1983; Hartet al., 1992). We have been able to now demonstrate theposition-specific expression of early localizing mRNAs inyounger, 0.3 mm oocytes. (ii) It shows that the redistribu-tion of most maternal mRNAs to the blastoderm probablyoccurs before, and therefore does not depend on, the redis-tribution of the endoplasm during and after fertilization.Indeed, all mRNAs tested were localized to the blastodermat stage V of oogenesis. This suggests that mRNA locali-zation to the embryonic region is not simply a by-productof cytoplasmic streaming. Experimental manipulations ofthe zebrafish egg have suggested that factors required for

DV axis formation are brought from the yolk cell into theblastoderm at the 32-cell stage by cytoplasmic streaming(Jesuthasan and Stra¨hle, 1997). If these factors are mRNAs,they would have to belong to a restricted population withvastly different localization properties to those we havesurveyed in this paper. (iii) The localization of somemRNAs and not others suggests that the localization pro-cesses are regulated and depend on specific RNAsequences. (iv) The mRNAs which localize early duringoogenesis suggest candidate factors and mechanisms forthe pathway(s) involved in animal pole specification.From our sampling, these appear rather numerous, butthe proportions of localized versus unlocalized mRNAsmay turn out to be different once the study is extendedto more mRNA species.

An important issue raised by our observations is the iden-tity of the factors regulating the differential mRNA locali-zation. All the mRNAs that we studied had an initialubiquitous distribution, and were never observed in the fol-licle cells. This suggests that they were transcribed by theoocyte itself, and later localized by an internal process, suchas regional stability, regional entrapment, or active trans-port. The two latter processes have been illustrated in bothXenopusand Drosophila. In Xenopusoocytes, early loca-lized mRNAs such asXcat2, are primarily trapped in themitochondrial cloud, and translocate to the vegetal regionvia the METRO pathway, independently of microtubulesand microfilaments (Kloc and Etkin, 1995; Forristall et al.,1995; Kloc et al., 1996). mRNAs localizing later, such asVg1, are actively transported along microtubules andbecome anchored to microfilaments at the vegetal cortex(Yisraeli et al., 1990; Forristall et al., 1995; Kloc andEtkin, 1995). In earlyDrosophila oocytes, a number ofmRNAs accumulate along the posterior or anterior cortexin an active movement involving microtubules and probablymicrotubule motors (see Gru¨nert and St Johnston, 1996).mRNAs localizing later (such asnanos, or oskar afterstage 10), i.e. after the dismantling of the microtubule net-work and the initiation of cytoplasmic streaming, are mostlikely trapped on a posterior pole anchor (see St Johnston,1995; Glotzer et al., 1997). Our results following treatmentswith cytoskeletal drugs in zebrafish suggest thatzorbaandVg1 mRNAs are localized independently of the microfila-ment and microtubule networks. Further, localization isenhanced when microfilaments are depolymerized, suggest-ing that an internal pool of these mRNAs has been releasedand accumulates at the animal pole. It is therefore mostprobable thatzorbaandVg1 localizations follow a trappingmechanism. Cytoskeletal elements other than microtubulesand microfilaments, or binding to other subcellular compo-nents or proteins, may explain entrapment within this spe-cific cytoplasmic domain. Which subcellular factors andmRNA sequences may be involved, and whether theseobservations can be generalized to other early localizingmRNAs, are important questions which may now beaddressed in the zebrafish.


4. Experimental procedures

4.1. Cloning of zebrafish orb-type a (zorba)

4.1.1. Degenerate RT–PCR and 3′RACE PCR cloningA 450 bp cDNA fragment encompassing most of the

zorba RNA-binding domain-encoding region was PCR-amplified from pre-mid-blastula transition (,250-cell) zeb-rafish embryo mRNA using degenerate oligonucleotidesdirected against the RNA-binding domains ofD.orb(Lantz et al., 1992) andXenopusCPEB (Hake and Richter,1994). Sequences of the oligonucleotides were as follows:FP 5′ AA(A/G)GTITT(T/C)(T/C)TIGGIGGI(A/G)TIC-CITGGGA 3′ (D.orb amino acids 577–586), RP1 5′TGCCAITGCCA(G/A)CAI(C/G)(A/T)IC(TM)(G/A)CA-(G/A)AA(G/A)TA 3 ′ (D.orb amino acids 793–802), RP2 5′AIGG(G/A)TCIA(T/C)(T/C)TGIAC(T/C)TT(T/C)TTIGT(G/A)AA 3 ′ (D.orb amino acids 758–768). After reversetranscription from random primers (MMLV SuperscriptReverse Transcriptase, Amersham), two rounds of PCRwere performed with 100 pmol FP and RP1 primers, then100 pmol FP and RP2 primers, and the following cycles: 1min 94°C, 1 min 42°C, 1 min 72°C (2 cycles) and 1 min94°C, 1 min 48°C, 1 min 72°C (28 cycles). The resulting450 bp cDNA fragment was sequenced and an internal 5′non-degenerate oligonucleotide was designed to increasethe fragment length by 3′RACE PCR following the methodof Frohman (1993). The resulting 1.6 kb fragment, encom-passing the 3′ coding and non-codingzorba cDNA, wasligated to thezorba450 bp RNA-binding domain-encodingfragment using an internal restriction site, and the resultingpartial cDNA (1.6 kb) was used for library screening.

4.1.2. Construction and screening of a zebrafish pre-MBTcDNA library

PolyA+ RNA from 4–8-cell stage zebrafish embryoswas prepared using the Fast Track kit (Promega) accord-ing to the manufacturer’s instructions. cDNA was synthe-sized (M-MLV reverse transcriptase, Gibco) from 5mg ofpolyA+ RNA using a mixture of 1mg oligo dT primer and100 ng random hexamers in the first strand synthesis reac-tion. After ligation to EcoRI adaptors (Novagen), theresulting cDNA mixture was enriched in fragmentsabove 500 bp and ligated tolEX LoxTM arms (Novagen).150 000 phage plaques of the non-amplified library werescreened at high stringency using thezorba probe (seeabove) labelled by random priming (Boehringer Man-nheim kit). Hybridization was performed overnight at42°C in 50% formamide, 5× SSC, 5× Denhardt’s,0.5% SDS and 200mg/ml salmon sperm DNA, followedby rinses in 0.2× SSC, 0.1% SDS at 65°C. Ten positiveclones were isolated. The plasmids were excised into thepEX Lox vector according to Novagen’s instructions, andsequenced on both strands using the Sanger method(Amersham Sequenase version 2.0 kit). Sequences wereanalysed with the GCG software.

4.2. Northern blotting

Total RNA was extracted according to Chomczynskiand Sacchi (1987), electrophoresed (10mg per well) andtransferred onto nitrocellulose membranes (AmershamHybond N). Hybridization with 106 cpm/ml random pri-me-32P-labelled zorba full length probe was performedovernight at 42°C in 50% formamide, 50 mN sodiumpyrophosphate, 5× SSC, 5× Denhardt’s, 0.5% SDS and200 mg/ml salmon sperm DNA. The membrane wasrinsed at 65°C in 2× SSC, 0.1% SDS, and autoradio-graphed.

4.3. In situ hybridization (ISH)

4.3.1. ProbesRNA probes labelled by incorporation of digoxigenin-11-

UTP (Boehringer Mannheim) were synthesized from thefollowing templates:

zorba RNA-binding domain (RT–PCR fragment),zorba 3′ (1.6 kb RACE PCR fragment), andzorbafull length, all subcloned in pGEM-T (Promega) orpBS SK(−);full length zebrafishVg1cDNA (Helde and Grunwald,1993);full length zebrafishNotchcDNA (Bierkamp and Cam-pos-Ortega, 1993);zebrafishSox19cDNA (Vriz and Lovell-Badge, 1995);full length zebrafishgsccDNA (Stachel et al., 1993);zebrafishTaram-AcDNA (Renucci et al., 1996);zebrafish PolyA-binding protein (PABP) gene (L.B.-C.and R.K.H., unpublished data): 1.5 kb partial cDNAobtained by PCR amplification from pre-midblastulatransition zebrafish RNA;zebrafish maternalActRIIB gene (L.B.-C. and R.K.H.,unpublished data): zebrafish maternal activin receptortype II, 700 bp cDNA fragment obtained from ourzebrafish pre-midblastula transition library.

4.3.2. Whole-mount in situ hybridizationISH at post-fertilization stages were performed as

described in Prince et al. (1998). For ISH on oocytes, ovar-ies were dissected out from anaesthetized females, rinsed inPBS, and follicles were manually singled out, staged (seeSelman et al., 1993, and Section 2) and fixed in 4% paraf-ormaldehyde (PFA). Stage V oocytes were obtained bysqueezing females. After 1 h fixation at room temperature,the oocytes were manually dechorionated using forcepsunder the stereomicroscope. Oocytes were postfixed in 4%PFA overnight at 4°C, dehydrated in methanol and pro-cessed for ISH as above. Following ISH, oocytes orembryos were cleared and mounted in 80% glycerolbetween bridged coverslips. They were photographedunder bright field illumination using an Axioskop photomi-croscope (Zeiss).


4.3.3. In situ hybridization on paraffin sectionsOvaries were fixed in 4% PFA overnight at 4°C, then

dehydrated and embedded in Paraplast Plus. 7.5-mm micro-tome sections were processed for ISH as described in John-ston et al. (1997). The sections were mounted in Cytoseal.They were viewed and photographed in a Zeiss Axioskon.

4.4. Raising and purification of polyclonal anti-Zorbaantibodies

A His tag-zorbafusion construct was obtained by sub-cloning the N-terminal kbPstI fragment ofzorbafull lengthin frame into pQE31 (Qiagen). The fusion protein was pro-duced inEscherichia coliby IPTG induction according tostandard protocols (Qiagen), purified on nickel-agarose col-umns (Qiagen), dialysed to 150 mM NaCl/10 mM Tris–HCl, pH 7.5, and resuspended in PBS/0.1% SDS. Rabbitswere boosted with 250 mg of protein in Freund’s adjuvantevery month and bled at regular intervals. Antibody titrewas checked on Western blot against zebrafish oocyteextracts (see below). One batch showing high titre wasthen affinity-purified against the His tagged-Zorba proteinbound to nickel/agarose columns according to Gu et al.(1994) and used in Western and immunocytochemistry stu-dies.

4.5. Western blotting analyses

The following antibodies were used: affinity-purifiedpolyclonal anti-Zorba diluted to 1/200, and the mAb anti-beta tubulin KMX1 (Sigma) diluted to 1/50.

4.5.1. Time course analysis of Zorba expressionExtracts of oocytes or embryo were obtained by lysis at

4°C in 250 mM sucrose, 100 mM NaCl, 2.5 mM MgCl2, 10mM EGTA, 20 mM HEPES, 1% Triton X-100, 10 mM NaF,1 mM Na3VO4, 10mg/ml leupeptin, 1mg/ml aprotinin, 1mg/ml pepstatin A, 1mg/ml antipain, 20mg/ml phenylmethyl-sulfonyl fluoride. The extracts were then centrifuged at12 000 rpm for 10 min at 4°C to eliminate debris and yolkgranules, and the supernatant was collected. Samples wereloaded on a 12% acrylamide–bisacrylamide urea gel at 0.5–1 oocyte/embryo per lane. Nitrocellulose membrane transferand antibody incubation were performed according to stan-dard protocols, and the primary antibodies were revealedusing HRP-coupled secondary antibodies (Jackson Labora-tories) diluted to 1/2000, and enhanced chemiluminescence(Amersham).

4.5.2. Oocyte detergent soluble and insoluble fractionsOocyte detergent soluble and insoluble fractions were

obtained as described in Yisraeli et al. (1990). Oocyteswere singled out in PBS from whole ovaries, staged, andlysed in cold extraction buffer (0.5% Triton X-100, 10 mMPIPES, pH 6.8, 0.3 M KCl, 10 mM MgAc, 0.5 mM EGTA).Extracts were spun at 4°C for 20 min in a microfuge. The

detergent soluble fraction (DSF, supernatant) was recoveredand diluted in 1/3 volume of 4× SDS sample buffer. Thedetergent insoluble fraction (DIS, pellet) was resuspendedin an equal final volume of 1× SDS sample buffer. Thesame amount of each fraction, i.e. 0.5–1 oocyte per lane,was loaded on a 12% acrylamide–bisacrylamide urea geland processed for Western blotting as described above. Aparallel gel was dried and Coomassie-stained to ensure thatthe lysis procedure had solubilized most yolk proteins (notshown).

4.6. Immunocytochemistry and phalloidin labelling

4.6.1. Immunocytochemistry on sectionsOocytes or ovaries were fresh frozen at−50°C in isopen-

tane in liquid nitrogen and cryostat sectioned at 12.5mm.The sections were rinsed in PT (PBS, 0.025% Triton X-100), fixed in 4% paraformaldehyde for 30 min at roomtemperature (RT) and bleached in 6% H2O2 for 1 h atRT. They were incubated in 10mg/ml proteinase K inPT for 2 min at RT, then in 2 mg/ml glycine for 5 minat RT, and finally blocked in PTBN (2 mg/ml bovineserum albumin (BSA), 2% normal goat serum (NGS) inPT) for 1 h at RT. The primary antibodies (anti-zorba 1/200, KMX-1 1/50) diluted in PTBN were applied over-night at 4°C. Secondary antibodies (goat anti-mouse oranti-rabbit IgGs, FITC- or Cy3-coupled, Jackson) werediluted to 1/200 in PTBN and applied for 2 h at RT. Thesections were rinsed in PT, dehydrated and mounted inVectashield (Vector).

4.6.2. Phalloidin labellingOocytes and ovary sections were prepared and fixed as

described above. The sections were then processed in thedark to preserve fluorescence They were rinsed in PT andincubated in 0.1mg/ml phalloidin–PBS (Sigma) in PBS, 1 g/l gelatin, 0.025% Triton X-100 for 1 h at RT,rinsed in PT,blocked in PTBN for 2 h at RT and incubated in 1/200 anti-zorba antibody in PTBN at 4°C overnight. They wererevealed using 1/200 goat anti-rabbit, Cy3-coupled second-ary antibody (Jackson) as described above.

4.7. Oocyte culture and cytoskeletal drug treatments

Oocytes were cultured as described in Selman et al.(1994) with minor modifications. Briefly, ovaries fromanaesthetized females were immersed in 60% L-15 medium(Gibco), pH 7.5, 100mg/ml gentamycin sulfate (Sigma) at26°C, and follicles were singled out using fine forceps.Oocytes undergoing vitellogenesis (diameter.0.35 mm)(Selman et al., 1993) were arbitrarily separated into twogroups: early stage III (0.35–0.5 mm) and late stage III(0.5–0.7 mm), and were cultured at 26°C in the above med-ium at a density of 15–20 oocytes per ml. After 1 h ofculture, damaged oocytes were discarded and treatmentswere applied. Maturation of late stage III oocytes was


induced by treatment with 5mg/ml 17a,20b-dihydroxy-4-pregnen-3-one (DHP) (Sigma) for 5 h; ethanol carrierapplied in the same proportions was used as a control.Microtubule depolymerization was induced in both oocytesgroups by incubation with 2 or 20mg/ml nocodazole(Sigma) (see Gard, 1991), and microfilament depolymeriza-tion was induced by incubation with 2.5 or 25mg/ml cyto-chalasine B (Sigma) (see Kloc and Etkin, 1995). Bothtreatments were applied for 5 h at 26°C DMSO carrierapplied in the same proportions was used as a control.The oocytes were subsequently fixed in 4% paraformalde-hyde overnight at 4°C, in FGT fix (80 mM KPIPES, pH 6.8,5 mM EGTA, 1 mM MgCl2, 3.7% formaldehyde, 0.25%glutaraldehyde, 0.5mM taxol, 0.2% Triton X-100) (Gard,1991) overnight at 4°C, or were fresh frozen and cryostatsectioned, to be processed for whole-mount ISH, whole-mount KMX-1 immunocytochemistry or phalloidin-FITClabelling, respectively.

5. Note added in proof

The ActRIIB clone reported in this paper was found to beidentical to a full-length zebrafish ActRIIB isolated by Garget al. (pers. commun.). The zorba sequence reported in thispaper has been attributed the following GenBank accessionnumber: AF076918.

Acknowledgements

We are indebted to Drs. E. Boncinelli, J.A. Campos-Ortega, D. Grunwald, M. Halpern, F. Rosa and S. Vriz forgifts of probes, and to F. Rosa and M. Wassef for providinglaboratory facilities in the last stages of this work. We wouldlike to thank F. Rosa, A. Vincent, T. Vogt and M. Wasseffor their interest and advice, L. Hake and J. Richter fordiscussions when we initiated this work, and K. Selmanfor assistance in oocyte culturing techniques. We are grate-ful to S. Easter, C. Howley, V. Prince, F. Rosa, A. Vincentand M. Wassef for their critical reading of the manuscript.This work was supported by a donation from the RathmannFamily Foundation to the Molecular Biology Department atPrinceton University, by a Basil O’Connor Starter ScholarResearch Award from the March of Dimes to R.K.H. who isa Rita Allen Foundation Scholar, and by an EMBO LongTerm Fellowship and CNRS to L.B-C.

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Characterization of the zebrafish Orb/CPEB-related RNA-binding ...

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