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Keywords:

  • maternal mRNA;
  • polarity;
  • cortex;
  • translation;
  • posterior pole;
  • CAB;
  • unequal cleavage;
  • embryogenesis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

Ascidian is a good model to understand the cellular and molecular mechanisms responsible for mRNA localization with the discovery of a large family of localized maternal mRNAs, called postplasmic/PEM RNAs, which includes more than 40 members in three different ascidian species (Halocynthia roretzi, Ciona intestinalis, and C. savignyi). Among these mRNAs, two types (Type I and Type II) have been identified and show two different localization patterns from fertilization to the eight-cell stage. At the eight-cell stage, both types concentrate to a macromolecular cortical structure called CAB (for Centrosome Attracting Body) in the posterior-vegetal B4.1 blastomeres. The CAB is responsible for unequal cleavages and the partitioning of postplasmic/PEM RNAs at the posterior pole of embryos during cleavage stages. It has also been suggested that the CAB region could contain putative germ granules. In this review, we discuss recent data obtained on the distribution of Type I postplasmic/PEM RNAs from oogenesis to late development, in relation to their localization and translational control. We have first regrouped localization patterns for Type I and Type II into a comparative diagram and included all important definitions in the field. We also have made an exhaustive classification of their embryonic expression profiles (Type I or Type II), and analyzed their functions after knockdown and/or overexpression experiments and the role of the 3′-untranslated region (3′UTR) controlling both their localization and translation. Finally, we propose a speculative model integrating recent data, and we also discuss the relationship between postplasmic/PEM RNAs, posterior specification, and germ cell formation in ascidians. Developmental Dynamics 236:1698–1715, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

One hundred years after the three monumental papers on ascidian development published by Edwin Conklin (1905a–c; see also Gehring,2004; Sardet et al.,2005), ascidians have re-emerged as a promising model to understand in detail how important mRNAs responsible for axis establishment, cleavage pattern, tissue differentiation, and germ line formation are localized (Nishida,1997,2002,2005; Sardet et al.,2003,2005; Davidson and Christiaen,2006). The positioning of mRNAs to a particular region of eggs and embryos constitutes an important strategy for regulating protein expression at the right time in the right place and contributes, therefore, to the establishment of cell polarity (King et al.,2005). Ascidian embryos develop in a typical mosaic manner (Conklin,1905b), and they undergo rapid cell-fate determinations, which are regulated by determinants localized to specific regions of unfertilized eggs and early embryos (reviewed by Nishida,2005; see also Nishida and Kumano in this issue).

THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

Ascidians are simplified chordates and constitute a third embryological system model, with the fruit fly Drosophila melanogaster and the frog Xenopus laevis, in which mRNAs localized in the cortex during oogenesis play an important role in development, exemplified by the discovery of a class of maternal mRNAs, called postplasmic/PEM mRNAs (Yoshida et al.,1996; Satou,1999; Sasakura et al.,2000; Nishida and Sawada,2001; Nakamura et al.,2003,2005,2006; Sardet et al.,2005). The family of postplasmic/PEM RNAs is composed of two types (Type I and Type II), and both accumulate at the posterior pole of developing embryos (Sasakura et al.,2000) (Fig. 1). Recently, oligonucleotide-based microarray analyses using differential screening demonstrated that this presence of postplasmic/PEM RNAs at the posterior pole appears to be the predominant localization pattern of maternal transcripts in early ascidian embryos, emphasizing the importance of this class of mRNAs (Yamada et al.,2005). A few other distribution patterns for localized maternal mRNAs have been observed in ascidian eggs and embryos, for example, a mitochondria-like distribution, a uniform cortical localization, or an accumulation in cell junctions at the eight-cell stage (Jeffery et al.,1983; Swalla and Jeffery,1995; Nishikata et al.,2001; Makabe et al.,2001).

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Figure 1. Schematic representation of postplasmic/PEM RNAs distribution (Type I and Type II) from oocytes to tail bud stage. Periods of development are indicated in boxes at the top of the figure: oogenesis (gray), phases of reorganizations (purple) triggered by fertilization (pink, arrowhead), cleavage stages (orange), and gastrulation/neurulation (green). Distribution patterns for Type I (yellow stars) and Type II (light pink and pink spot) postplasmic/PEM RNAs are shown on the upper (A1–K1/K2) and lower (A2–K1/K2) panels, respectively. A1–A2: Previtellogenic stage I oocytes. B1–B2: Postvitellogenic stage III oocytes. C1–C2: Stage IV oocytes or unfertilized eggs. D1–D2, E1–E2: Zygotes after the first (D1–D2) and second (E1–E2) major phases of cortical and cytoplasmic phase of reorganization. F1–F2: Two-cell embryos. G1–G2: Four-cell embryos. H1–H2: Eight-cell embryos. I1–I2: The 16-cell embryos. J1–J2: The 110-cell initial gastrula stage embryos. K1–K2: Tail bud stage embryos. B8.11 and B8.12 cells are indicated, respectively, by arrowheads and arrows. Important steps for Type I and Type II are indicated in gray boxes. The presence of postplasmic/PEM RNAs in blastomeres different from those containing the CAB is indicated by small light yellow stars (Type I) and light pink zones (Type II). The drawings in the blue square highlight the difference of distribution between Type I and Type II postplasmic/PEM RNAs before fertilization. After the eight-cell stage (red square) Type I and Type II RNAs show the same posterior accumulation in the CAB region. Movements that occur during their respective relocalization are represented by black arrows. a, animal; v, vegetal; CP/future D, contraction pole/future dorsal pole; A, anterior; P, posterior; D, dorsal; V, ventral. Important terms are defined and illustrated in the Definitions panel.

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In this review, we focus on the distribution of Type I postplasmic/PEM RNAs from oogenesis to late development and discuss recent data obtained concerning the relation to cellular and molecular mechanisms underlying their localization and translational control at the posterior pole of embryos. We have first integrated the redistribution pattern of Type I and Type II postplasmic/PEM RNAs from oocytes to tail bud stages into a comparative schematic representation (Fig. 1) and also compiled an exhaustive list of postplasmic/PEM RNAs including their Type I or Type II classification and zygotic expression profiles (Table 1). Second, we summarize what is known about their functions in Halocynthia roretzi, Ciona intestinalis, and C. savignyi based on phenotypes obtained using injections of antisense Morpholino Oligonucleotides (MO) and/or synthetic mRNAs (knockdown/overexpression). Then, we describe (1) how the polarized distribution of Type I postplasmic/PEM RNAs along the animal–vegetal (a–v) axis occurs concomitantly with the establishment of (a–v) polarity during meiotic maturation, and (2) how they accumulate to the posterior pole of embryos at the eight-cell stage. We also analyze the mechanisms involved in the localization of postplasmic/PEM RNAs and their translational control after fertilization. We propose a speculative model integrating recent data and aspects that remain to be elucidated to understand mechanisms controlling both their localization and translation. And finally, we discuss the relationship between postplasmic/PEM RNAs, posterior specification, and germ cell formation after cleavage stages in ascidians.

Table 1. Catalogue of Postplasmic/PEM Genes of Three Species of Ascidian, Ciona intestinalis, Ciona savignyi, and Halocynthia roretzi
GeneOriginal gene nameGene nameIdentificationaTypebExpression patterns of genescTissues expressed zygoticallydFunction/characterizationReference
  • a

    Identification by NCBI accession number (http://www.ncbi.nlm.nih.gov/entrez) or C. intestinalis cDNA clone ID (ciXXXXXXXX) in the database (http://ghost.zool.kyoto-u.ac.jp/indexr1.html) or C. savignyi cDNA clone ID (csXXXXXXXX) in the database (http://hoya.zool.kyoto-u.ac.jp/cgi-bin/gbrowse/cs).

  • b

    Classification of genes depending on criteria described in Sasakura et al. (2000). 'n.d.” means “ not determined”.

  • c

    The gene expression at eight-cell stage embryo were described in the left column. “PL” indicates that transcripts of gene showed the posterior-most localization pattern, whereas “unloc” indicates that they unlocalized in the embryo. Whether the redistribution to B8.12-line cells during gastrulation exists or not is discribed in the middle culumn. “B8.12” indicates that transcripts of the gene were redistributed to B8.12 cells. “n.d.” means “not determined”. Whether the transcripts of the gene were expressed only maternally (“M”) or expressed also zygotically in addition to matternally expression (“MZ”) were described in the right culumn. “n.e.” means “not examined”.

  • d

    NS, nervous system; Me, mesencyme; Ep, epidermis; Mu, Muscle, En, endoderm; ES, endodermal strand; No, notochord.

pemCi-pemCi-pem-1AK113383Type IPL M It plays roles in anterodorsal patterning and assymmetric division.Yoshida et al.,1997
Cs-pemCs-pem-1D83482Type IPL M Yoshida et al.,1996
HrPEMHr-pem-1AB045129Type IPL M Nishida and Sawada,2001
macho1Ci-macho1Ci-macho-1AB077368Type IPL MZNSA zinc finger transcriptional factor. It acts as a muscle determinant and a posterior patterning determinant responsible for mesenchyme formation.Satou et al.,2002; Imai et al.,2004; Yagi et al.,2004
Cs-macho1Cs-macho-1AB057740Type IPL MZNSSatou et al.,2002
HrMacho-1Hr-macho-1AB045124Type IPL MZNSNishida and Sawada,2001; Kobayashi et al.,2003; Kondoh et al., 2003; Sawada et al.,2005
POPK1Ci-POPK1Ci-POPK-1citb032l21Type IPLB8.12MZEpA Ser/Thr kinase protein similar to C. elegans Sad-1 gene. It is upstream of macho-1 and plays a role in concentration of the cortical ER, subsequent localization of Type I postplasmic/PEM mRNAs, and concentration of putative germinal granules.Yamada et al.,2005; Yamada,2006
Cs-POPK1Cs-POPK-1csga085p15n.e.PLB8.12MZEpYamada,2006
HrPOPK-1Hr-POPK-1AB014885Type IPLB8.12MZEP, NSSasakura et al.,1998b; Nakamura et al.,2005          
Wnt5Ci-Wnt5Ci-wnt-5AK222285Type IPL MZEp, MuSecreted intercellular sinaling protein. It plays roles in the primary notochord, mesencyme and muscle cell formation. Downstream of macho-1.Imai et al.,2004, Yamada,2006
Cs-Wnt5Cs-wnt-5csef023g20n.e.PL MZEp, MuYamada,2006
HrWnt-5Hr-wnt-5AB006608Type IPL MZEp, Mu, No, En, Me, NSSasakura et al.,1998a; Sasakura and Makabe,2001; Sasakura and Makabe,2002; Nakamura et al.,2006
pem-3Ci-pem-3Ci-pem-3AK114726Type IPLB8.12MZNS, Me, ESA RNA binding protein with KH and RING finger domain, similar to C. elegans MEX-3. It plays a role in the differentiation of the brain of the larvae.Fujiwara et al.,2002; Yamada,2006
pem-3Cs-pem-3AB001769Type IPLB8.12MZNS, Me, ESSatou,1999
HrPEM-3Hr-pem-3AB091123Type IPLB8.12MZNS, MeNakamura et al.,2003; Nakamura et al.,2006
pem-2Ci-pem-2Ci-pem-2AK114933Type IPL MZNS, MuContaining a SH3 and a GEF domain.Yamada et al.,2005; Yamada,2006
pem-2Cs-pem-2AB001770n.e.PLB8.12M Satou and Satoh,1997
ZF1Ci-ZF1Ci-ZF-1AK113919Type IPLB8.12M A C3H-type zinc finger protein similar to C.elegens PIE-1.Yamada et al.,2005; Yamada,2006
Cs-ZF1Cs-ZF-1csef025b24n.e.PLB8.12M Yamada,2006
HrZF-1Hr-ZF-1AB029332Type IPLB8.12M Sasakura et al.,2000
VHCi-VHCi-VHAB016603Type IIPLB8.12MZEnA Vasa orthologue.Fujimura and Takamura.,2000; Takamura et al.,2002; Shirae-Kurabayashi et al.,2006
Cs-VHCs-VHcsef009i10n.e.PLB8.12MZEnYamada,2006
Dll-BCi-Dll-BCi-Dll-BAK174652Type IPL MZNS, EpA homeobox transcriptional factor.Caracciolo et al.,2000; Imai et al.,2004
Cs-Dll-BCs-Dll-Bcsga004f13n.e.PL MZNS, EpYamada,2006
Prd-BCi-Prd-BCi-prd-BAK222276n.d.PL MZMe, MuA homeobox transcriptional factor.Imai et al.,2004
Cs-Prd-BCs-prd-Bcsef026o14-unloc-n.e. Yamada,2006
prepCi-prepCi-prepAB210651n.d.PLn.d.MZMeA homeobox transcriptional factor.Imai et al.,2004
Cs-prepCs-prepcscl041p13n.e.PL n.e. Yamada,2006
LAG1-like3Ci-LAG1-like3Ci-LAG1-like3AB210527n.d.PLn.d.MZEpA homeobox transcriptional factor.Imai et al.,2004
ets/pointed2Ci-ets/pointed2Ci-ets/pointed2AK222254-unloc-MZNS, EpA ETS-domain transcription factor, target of FGF/MAPK signaling. It is required for induction of notochord, mesenchyme, and brain.Imai et al.,2004; Bertrand et al., 2003
Cs-ets/pointed2Cs-ets/pointed2cstb008p02-unloc--n.e.Yamada,2006
HrEtsHr-etsAB092968Type IIPL MZNS, Ep, NoMiya and Nishida.,2003
Fli/ERG4Ci-Fli/ERG4Ci-FLI/ERG4citb004m11n.d.PLn.d.MZEn, NoA ETS-domain transcription factor.Imai et al.,2004
FoxJ2Ci-FoxJ2Ci-foxJ2AB210439n.d.PLn.d.M A Fox transcription factor.Imai et al.,2004
 Cs-FoxJ2Cs-foxJ2csef036e04n.e.PL n.e.  Yamada,2006
scalloped/TEF1Ci-scalloped/TEF1Ci-scalloped/tef1AB210674n.d.PLn.d.MZNSA transcription factor similar to scalloped and TEF.Imai et al.,2004
Cs-scalloped/TEF1Cs-scalloped/tef1csef028l12-unloc-n.e. Yamada,2006
Eph1Ci-Eph1Ci-eph-1AB210395Type IPL M A tyrosine kinase receptor for Ephyrins, beta-catenin downstream gene.Imai et al.,2004
Cs-EphCs-eph-1AB057735n.e.PL M  Imai,2003
Eph2Ci-Eph2Ci-eph-2cilv003g03Type IPL MZNS, EpA tyrosine kinase receptor for Ephyrins.Imai et al.,2004
GCNFCi-GCNFCi-GCNFAB210469Type IPLB8.12MZNS, Me, EpA nuclear receptor.Imai et al.,2004
 Cs-GCNFCs-GCNFcstb015h12n.e.PLB8.12MZNS, Me, Ep Yamada,2006
TolloidCi-TolloidCi-tolloidcibd030o19Type IPL MZNS, Me, EnA TGF-β signal transduction molecule.Imai et al.,2004
Raf1Ci-Raf1Ci-raf-1AB210659n.d.PLn.d.MZNS, EnA signal transduction molecule in MAPK signalling cascade.Imai et al.,2004
Cs-Raf1Cs-raf-1csef046k16-unloc-n.e. Yamada,2006
Nemo-LikeCi-Nemo-LikeCi-nemo-LikeAB210564n.d.PLn.d.M A signal transduction molecule in MAPK signalling cascade.Imai et al.,2004
Cs-NEMO-like kinaseCs-NEMO-like kinaseAB057742n.e.PL n.e. Yamada,2006
RhoGAP-aCi-RhoGAP-aCi-rhoGAP-aciad019a17Type IPL MZNoA Rho GTPase activating protein.Yamada et al.,2005; Yamada,2006
 Cs-RhoGAP-aCs-rhoGAP-acscl039a24n.e.PL n.e.  Yamada,2006
midnolinCi-midnolinCi-midnolincieg023k14Type IPLB8.12MZNS, Me, EpSimilar to midnolin containing ubiquitin-like domain.Fujiwara et al.,2002; Yamada et al.,2005; Yamada,2006
BL4/5-αCi-BL4/5-αCi-BL4/5-αAK173738Type IPL MZNS, EpA orthologue of Band 4.1-like protein.Yamada et al.,2005; Yamada,2006
 Cs-BL4/5-αCs-BL4/5-αcsef029j23n.e.PL n.e.  Yamada,2006
PTP-likeCi-PTP-likeCi-PTP-likecign044b03n.d.PLB8.12M A tyrosine phosphatase-like protein containin a PDZ domain.Yamada et al.,2005; Yamada,2006
Cs-PTP-likeCs-PTP-likecsga002k04-unloc-n.e. Yamada,2006
GLUTHrGLUTHr-GLUTAB091122Type IPL MZNS, Me, EpA glucose transporter. It is required for endoderm, mesencyme, and notochord formation.Nakamura et al.,2003; Nakamura et al.,2006
DPOZCi-DPOZCi-DPOZciad008a12n.d.PL M DNA polymerase zeta subunit.Yamada et al.,2005; Yamada,2006
 Cs-DPOZCs-DPOZcscl005g13n.e.PL n.e.  Yamada,2006
pen1Ci-pen1Ci-pen-1ciad056p19Type IPL MZNS, Ep, NoSimilar to mammalian g1-related zinc finger protein.Yamada et al.,2005; Yamada,2006
 Cs-pen1Cs-pen-1csef012n03n.e.PL n.e.  Yamada,2006
 HrPEN-1Hr-PEN-1AB091124Type IPL MZEP Nakamura et al.,2003; Nakamura et al.,2006
pen2HrPEN-2Hr-PEN-2AB091125Type IPL M It is required for mesenchyme, posterior endoderm, and muscle formation.Nakamura et al.,2003; Nakamura et al.,2006
pem-7Ci-pem7Ci-pem-7AK114446Type IPLB8.12M  Nishikata et al., 2002; Yamada,2006
 Cs-pem7Cs-pem-7cslv007b06n.e.unloc-n.e.  Yamada,2006
pem-8Ci-pem8Ci-pem-8AK114238n.d.PLB8.12M  Nishikata et al.,2001; Yamada,2006
 Cs-pem8Cs-pem-8csma090i19-unloc-n.e.  Yamada,2006
pem-9Ci-pem9Ci-pem-9AK116615-unloc-M  Nishikata et al.,2001; Yamada,2006
 Cs-pem9Cs-pem-9csga039h23-unloc-n.e.  Yamada,2006
pem-10Ci-pem-10Ci-pem-10AK114949Type IPL MZEn Fujiwara et al.,2002; Yamada,2006
pem-11Ci-pem-11Ci-pem-11AK222379Type IPLB8.12MZMeContaining TUDOR domains.Fujiwara et al.,2002; Yamada,2006
pem-12Ci-pem-12Ci-pem-12cilv037c08Type IPL MZunidentifiable Yamada et al.,2005; Yamada,2006
 Cs-pem-12Cs-pem-12csga101e23n.e.PL n.e.  Yamada,2006
pem-13Ci-pem-13Ci-pem-13cign072a14Type IPL M  Yamada et al.,2005; Yamada,2006
 Cs-pem-13Cs-pem-13cscl037b09n.e.PL n.e.  Yamada,2006
pem-14Ci-pem14Ci-pem-14cign047g05Type IPL MZNS Yamada,2006; Yamada,2006
 Cs-pem14Cs-pem-14cslv005l14n.e.PL n.e.  Yamada,2006
ZF097Ci-ZF097Ci-ZF097AK222421n.d.PL M Containing zinc finger domain.Miwata et al.,2006
Cs-ZF097Cs-ZF097csga050p08-unloc-n.e. Yamada,2006
ZF266Ci-ZF266Ci-ZF266AB210796n.d.PLn.d.MZNS, Me, NoContaining C2H2-type zinc finger domains.Imai et al.,2004; Miwata et al.,2006
ZF364Ci-ZF364Ci-ZF364cieg037b03Type IPL M Similar to Rabphilin 3AMiwata et al.,2006
pem-4Ci-pem-4Ci-pem-4AK222372-unloc-M Containing C2H2-type zinc finger domain and a signal for nuclear localization.Yamada,2006
pem-4Cs-pem-4AB001771Type IIPL M Satou and Satoh,1997
pem-5Ci-pem-5Ci-pem-5AK115396-unloc-M  Nishikata et al., 2002; Yamada,2006
 pem-5Cs-pem-5AB001772Type IIPL M  Satou and Satoh,1997
pem-6Ci-pem-6Ci-pem-6AK113695-unloc-MZNS, MeContaining A20-like and AN1-like zinc finger domains.Yamada,2006
pem-6Cs-pem-6AB001773Type IIPL M Satou and Satoh,1997
Pet-1Ci-Pet-1Ci-pet-1cieg030f20-unloc-M  Yamada,2006
 Cs-Pet-1Cs-pet-1csga037e12-unloc-n.e.  Yamada,2006
 HrPet-1Hr-pet-1AB029333Type IIPLn.e.M  Sasakura et al.,2000
Pet-2HrPet-2Hr-pet-2AB029334Type IIPLn.e.M  Sasakura et al.,2000
Pet-3Ci-Pet-3Ci-pet-3AK115296-unloc-M  Yamada,2006
 Cs-Pet-3Cs-pet-3csef043e15-unloc-n.e.  Yamada,2006
 HrPet-3Hr-pet-3AB029335Type IIPLn.e.M  Sasakura et al.,2000; Sasakura and Makabe,2002

Concept of Maternal Determinant and the Discovery of postplasmic/PEM RNAs

Micromanipulation experiments performed by Nishida in the 1990s and involving removal of peripheral regions of the zygote and ablation and transfer of the Posterior-Vegetal Cytoplasm & cortex (PVC) in H. roretzi first revealed the existence of three determinants in the periphery of the zygote involved in tissue formation (epidermis, endoderm, and muscle), and of two determinants in morphogenetic processes (unequal cleavages and gastrulation; Nishida,1994,1997,2002,2005; Fig. 1E1–E2, and see the Definitions section). Maternal determinants are factors (mRNAs/proteins, macromolecular complexes, or structures) inherited from the unfertilized egg that direct axis formation and/or cell fate specification in the embryo. When maternal determinants are inhibited or removed, axis formation or development are abnormal. In contrast, when determinants are injected or induced in ectopic sites or blastomeres, they can redirect developmental fate (see review by Sardet et al.,2005).

During the same period, RNA differential screening experiments using oocyte fragments prepared by centrifugation of the ascidian C. savignyi led to the discovery of the first posterior localized mRNA, called pem because its detection signal by in situ hybridization formed a posterior end mark in the posterior-most vegetal blastomeres (called B4.1) at the eight-cell stage (Marikawa et al.,1994,1995; Yoshida et al.,1996). In addition, maternal transcripts of pem were cortically localized to the future posterior pole of C. savignyi zygotes, a region equivalent to the PVC containing maternal determinants in H. roretzi. It was also shown that overexpression of pem mRNA (now designated pem-1 or PEM-1; see Fig. 1, Nomenclature for postplasmic/PEM RNAs) perturbed anterior and dorsal structures in larvae (Yoshida et al.,1996). This remarkable discovery made by Satoh and his colleagues was followed by the identification of other pem-family genes in C. intestinalis, C. savignyi, and H. roretzi with the same localization pattern as pem (Satou and Satoh,1997; Yoshida et al.,1997; Sasakura et al.,1998a,b,2000; Satou,1999; Caracciolo et al.,2000; Fujimura and Takamura,2000; Nishida and Sawada,2001; Sasakura and Makabe,2002; Satou et al.,2002; Miya and Nishida,2003; Yamada,2006). Actually, we know now clearly that at least one of the maternal determinants defined by micromanipulation experiments can be attributed to a postplasmic/PEM RNA with macho-1 mRNA, the muscle determinant, which codes for a transcription factor responsible for the differentiation of primary muscle cells located in the tail of the larvae (Nishida and Sawada,2001).

Large databases of mRNA localization patterns obtained from whole-mount in situ hybridization projects have been established using two ascidian species: H. roretzi (MAGEST: http://www.genome.ad.jp/magest/) and C. intestinalis (cDNA project: http://ghost.zool.kyoto-u.ac.jp/indexr1.html). These in situ screens resulted actually in the identification of many maternal transcripts showing a restricted localization at the posterior region of eight-cell stage embryos (Kawashima et al.,2000; Makabe et al.,2001; Nishikata et al.,2001; Fujiwara et al.,2002; Nakamura et al.,2003). Furthermore, a genome-wide analysis of ascidian postplasmic/PEM mRNAs was achieved with the release of draft genome sequences of C. intestinalis, and thus more than 40 such localized mRNAs were detected in C. intestinalis, C. savignyi, and H. roretzi (Dehal et al.,2002; Imai et al.,2004; Yamada et al.,2005; Miwata et al.,2006; Yamada,2006; see also Table 1). Originally, the terms “postplasmic” and “pem” were used to designate these localized maternal mRNAs in H. roretzi, C. savignyi, and C. intestinalis, respectively. Recently, it has been suggested that the term postplasmic/PEM should be used to name these mRNAs accumulated at the posterior region of embryos regardless of the ascidian species (reviewed by Sardet et al.,2005; Fig. 1, Definitions).

Definition of postplasmic/PEM RNAs

Postplasmic/PEM mRNAs form a large family of localized maternal mRNAs, whose definition was originally based on their stereotyped and invariant relocalization pattern from fertilization to cleavage stages (Fig. 1, see Definitions). In 2000, Sasakura and colleagues identified two pathways for the localization of postplasmic/PEM RNAs at the posterior-vegetal cytoplasm and cortex in the eight-cell stage embryo of the ascidian H. roretzi and, thus, defined two groups of postplasmic/PEM RNAs (Type I and Type II). Furthermore, this specific region located in the posterior-vegetal B4.1 blastomeres from the eight-cell stage was originally named “postplasm” (Sasakura et al.,1998a,b,2000). The postplasm also contains a macromolecular cortical structure called CAB (for Centrosome Attracting Body) forming at the eight-cell stage (Hibino et al.,1998; Nishikata et al.,1999; Iseto and Nishida,1999; Sasakura et al.,2000; Patalano et al.,2006; review by Sardet et al., this issue; Fig. 1). The CAB is responsible for three successive asymmetric divisions at the posterior pole of embryos from the 8-cell stage to the 64-cell stage, and also ensures the infallible partitioning of Type I postplasmic/PEM RNAs into one of the smaller daughter cells after each division (Nakamura et al.,2005; Fig. 1H–J). This is the reason why we will use indiscriminately the terms “unequal cleavage” or “asymmetric cell division” to describe these posterior cleavages. Because the ascidian terminology is a bit confusing, we also intend to clarify this aspect by regrouping all important definitions in the same panel (see Fig. 1, Definitions). In this review, we will use only the term “CAB region” instead of the original definition “postplasm” to designate this posterior region of embryos containing postplasmic/PEM RNAs from the eight-cell stage.

The main differences between Type I and Type II postplasmic/PEM RNAs concern their localization and redistribution between fertilization and the eight-cell stage, and also their abundance (Yoshida et al.,1996,1997; Satou,1999; Sasakura et al.,2000; Yamada,2006; Fig. 1). Abundant Type I postplasmic/PEM RNAs display a polarized cortical distribution along the a–v axis in unfertilized eggs (Fig. 1C1). Between fertilization and first cleavage, they are transiently concentrated in the vegetal/contraction pole after a first phase of cytoplasmic and cortical reorganization (microfilament-dependent; Fig. 1D1) and are subsequently translocated to the PVC after a second phase of cytoplasmic and cortical reorganization (microtubule-dependent; Fig. 1E1). After the first cleavage, Type I postplasmic/PEM RNAs are equally partitioned into blastomeres (Fig. 1F1). Then, after the second cleavage, Type I postplasmic/PEM RNAs are segregated into the posterior pair of cells (Fig. 1G1), and finally concentrate in the CAB region at the eight-cell stage (Sasakura et al.,2000; Prodon et al.,2005; see also Sardet et al., this issue; Fig. 1H). Interestingly, some recent reverse transcriptase-polymerase chain reaction (RT-PCR) and microarray experiments performed using blastomeres isolated from eight-cell embryos indicate both that some Type I mRNAs are also present in blastomeres other than those containing the CAB (Yamada et al.,2005; Fig. 1H, see small light yellow stars and also Distribution of Type I postplasmic/PEM RNAs During Early Oogenesis and Meiotic Maturation Section). Furthermore, some Type I postplasmic/PEM RNAs, such as pem-1 and macho-1 for example, have been shown to be bound to a network of rough cortical Endoplasmic Reticulum (cER) tethered to the plasma membrane of the oocyte, thus forming a cER-mRNA domain (Sardet et al.,2003,2005; Prodon et al.,2005; Patalano et al.,2006). This cER-mRNA domain is relocated after fertilization and concentrates into the CAB at the eight-cell stage (Sardet et al.,2003,2005; see Sardet et al. in this issue, and also Fig. 1, Definitions). Finally, Type I postplasmic/PEM RNAs are generally conserved in four ascidian species (C. intestinalis, C. savigny, H. roretzi, and Phallusia mammillata) and code for proteins with diverse functions (Sasakura et al.,2000; Sardet et al.,2005; Yamada et al.,2005; Yamada,2006; see also Fig. 1, section 2, and Table 1).

In contrast, the localizations and functions of Type II postplasmic/PEM RNAs are less well documented (reviewed in Sardet et al.,2005). They are uniformly distributed throughout the egg cytoplasm (Fig. 1C2), and then progressively accumulate in the CAB region during early cleavage stages (Fig. 1E2–H; Sasakura et al.,2000). More specifically, the posterior accumulation of most Type II postplasmic/PEM RNAs starts to be detectable from the four-cell stage (Fig. 1G2). Nevertheless, their relocalization from fertilization to the eight-cell stage remains controversial, since some variable patterns have been observed (Sasakura et al.,2000; Shirae-Kurabayashi et al.,2006; Yamada,2006). Even if experiments with cytoskeletal inhibitors showed that distinct mechanisms are involved in the posterior localization of Type I and II to the CAB region (Sasakura et al.,2000; Yamada,2006; Fig. 1D1–E1, 1C2–D2), the localization mechanism still remains largely unclear (Nakamura et al.,2005; see Fig. 1, Speculative Scenario About the Relocalization and the Translational Control of postplasmic/PEM RNAs).

Type I and Type II postplasmic/PEM RNAs: A Notion to Revisit?

The distribution of postplasmic/PEM RNAs is easily recognizable because of their characteristic accumulation in the CAB region from the eight-cell stage (this could be a reason for simplifying the nomenclature and calling them “posterior mRNAs,” see Fig. 1, Definitions). Nevertheless, at the moment, the distinction between Type I and Type II postplasmic/PEM mRNAs is blurred in many cases. A recent RT-PCR study indicates that all Type I postplasmic/PEM RNAs are not always localized exclusively in the CAB region, as some Types I (Hr-wnt-5, Hr-POPK-1) could be detected in blastomeres other than the B4.1. Moreover, individual Type II postplasmic/PEM RNAs can show heterogeneous distribution patterns in fertilized eggs, ranging from unlocalized to partially posterior with cytoplasmic background, approaching the Type I profile. These discrepancies may in part be attributed to the fact that the in situ hybridization technique is hardly quantitative, and the extent of “localization” versus “background” can vary as a function of the abundance of the mRNA examined and the sensitivity of the detection method used.

Little is known about the localization/relocalization of postplasmic/PEM RNAs during oogenesis and after cleavage stages (i.e., after the 110-cell stage), these aspects remain to be analyzed in detail. However, the localization pattern of two Type I postplasmic/PEM RNAs (Ci-pem-1 and Ci-pem-3) has been established in pre- and postvitellogenic oocytes of C. intestinalis and indicates that the most abundant Type I postplasmic/PEM RNAs, Ci-pem-1, is uniformly distributed at the cell periphery of oocytes just before meiotic maturation (Prodon et al.,2006; see also Fig. 1, section 4.2). In H. roretzi, Hr-pem-1 mRNA shows a similar cortical distribution, whereas Hr-vasa and 006C16 mRNAs (006C16 corresponds to a clone in MAGEST database, and encodes a protein without obvious homologies), two mRNAs classified as Type II, are respectively localized around the Germinal Vesicle (GV) and homogeneously distributed throughout the cytoplasm (Prodon and Nishida, unpublished observations), suggesting that some new subtypes among Type II—or at least a specific “Vasa-type” distribution—could be defined in oocytes.

Recently, Shirae-Kurabayashi and colleagues also showed in gastrula and tail bud stages of C. intestinalis that certain Type I postplasmic/PEM RNAs and Ci-VH mRNA (for VasaHomolog, a Type II) are distributed into two populations of daughter cells called B8.12 and B8.11, resulting from an additional asymmetric division in the posterior-most vegetal B7.6 blastomeres (Shirae-Kurabayashi et al.,2006; see Fig. 1K1–K2, and also Distribution of postplasmic/PEM RNAs After Cleavage Stages and Germline Formation Section). In contrast, some Type I postplasmic/PEM RNAs are distributed exclusively in B8.11, suggesting that two distinct mechanisms are involved in their redistribution during this additional posterior unequal cleavage. These new observations taken together reinforce the idea that several subcategories may be distinguished among Type I and Type II postplasmic/PEM RNAs.

EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

We have listed genes encoding postplasmic/PEM RNAs previously reported in C. intestinalis/savignyi and H. roretzi along with their relevant information in Table 1. We have also indicated their distribution into the both B8.11 and B8.12 cells/ or only into B8.11 cells at tail bud stages. In addition to these listed genes, some other postplasmic/PEM RNAs were identified in the Mediterranean species P. mammillata (Pm-tolloid, Pm-vasa, and Pm-macho-1) and in the anural ascidian Molgula tectiformis (Mt-macho-1) (Sardet et al.,2005; Gyoja,2006; Patalano et al.,2006). In C. intestinalis, Yamada recently classified 23 postplasmic/PEM genes into Type I among the 37 genes identified as posteriorly localized at the eight-cell stage (Yamada,2006; Table 1). In C. savignyi, 24 Type I postplasmic/PEM mRNAs have also been recently identified (Yoshida et al.,1996; Satou,1999; Satou et al.,2002; Imai,2003; Imai et al.,2004; Yamada,2006). Of the 23 Type I postplasmic/PEM genes found in C. intestinalis, 17 and 7 orthologues were reported in C. savignyi and H. roretzi, respectively. The mRNAs of almost all genes display a posterior-most localization pattern (PVC and CAB region), except for Cs-pem-7 (see Table 1). This fact indicates that the localization pattern of most Type I postplasmic/PEM RNAs is well conserved between the different ascidian species than those of Type II. Finally, some postplasmic/PEM RNAs are also expressed zygotically during embryogenesis, suggesting that they play additional roles during late embryogenesis (Table 1).

ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

Antisense Morpholino Oligonucleotide (MO) injection is a powerful approach to knockdown genes of interest in ascidian embryos (Satou et al.,2001; Yamada et al.,2003). Indeed, the advent of MO has considerably accelerated the functional analysis of postplasmic/PEM RNAs, and consequently our understanding of their roles during ascidian development. In H. roretzi, knockdown and overexpression experiments were performed for nine Type I postplasmic/PEM RNAs. Knockdown experiments using MO reveal a function for six of the nine Type I postplasmic/PEM: pem-1 (T. Negishi, K. Sawada, and H. Nishida, personal communication), macho-1 (Nishida and Sawada,2001; Sawada et al.,2005), POPK-1 (Nakamura et al.,2005), wnt-5 (Sasakura and Makabe,2001; Nakamura et al.,2006), GLUT (Nakamura et al.,2006), and PEN-2 (Nakamura et al.,2006). On the other hand, injection of Hr-pem-3, Hr-PEN-1, and Hr-ZF-1 MO (and their overexpression by mRNA injection) does not affect embryogenesis. Therefore, the functions of these three genes remain elusive for the moment (Nakamura et al.,2006; Akanuma and Nishida, unpublished data).

Because the functions of Type II postplasmic/PEM RNAs have been less well investigated (reviewed in Sardet et al.,2005), in this section, we will review maternal and zygotic functions of the well-studied Type I postplasmic/PEM RNAs in the ascidian species H. roretzi, and C. intestinalis, and C. savignyi.

pem-1

In C. savignyi, pem-1 plays a role in anterodorsal patterning. It was shown that overexpression of Cs-pem-1 by injection of synthetic mRNA into fertilized eggs results in deficiencies of the anterior and dorsal structures (the anterior-most adhesive organ, dorsal brain, and sensory pigment cells) in the tadpole larvae, causing the shifting of the larval anterior neural and epidermal tissues toward the posterior (Yoshida et al.,1996). No drastic effects were observed in the formation of endoderm, mesenchyme, muscle, or notochord (Yoshida et al.,1996). In H. roretzi, the function of Hr-pem-1 is not yet well understood, but Hr-pem-1 appears to be involved in the positioning of cleavage planes. Indeed, Hr-pem-1 MO injection alters unequal cleavages that occur at the posterior pole of embryos (Negishi, Sawada, and Nishida, personal communication).

macho-1

Macho-1, which encodes a zinc-finger transcription factor (Zic family), was identified as the first localized maternal determinant responsible for muscle formation in H. roretzi (Nishida and Sawada,2001; Sawada et al.,2005). Indeed, depletion of macho-1 mRNA results in the loss of primary muscle cells in the tail of the tadpole larvae. In contrast, overexpression of synthetic macho-1 mRNA in an animal anterior blastomere, which normally never gives muscle cells, causes the ectopic formation of muscle cells. These results taken together indicate that macho-1 is both necessary and sufficient for specification of muscle fate. In addition, macho-1 plays a central role in generating the difference in responsiveness to inductive signals between notochord and mesenchyme precursor blastomeres (Kobayashi et al.,2003), indicating that macho-1 also acts as a posterior patterning determinant responsible for mesenchyme formation (reviewed by Nishida,2005).

Orthologs of macho-1 have been isolated in C. intestinalis and C. savignyi (Satou et al.,2002). Interestingly, in both Ciona species, the macho-1 gene is expressed both maternally and zygotically, the zygotic transcript being produced in cells of the central nervous system. Furthermore, it has been also shown in Ciona that macho-1 plays similar but not identical roles in embryonic muscle cell differentiation (Satou et al.,2002; Yagi et al.,2004). Satou and colleagues have reported that Cs-macho-1 is essential for initiating the zygotic expression of muscle-specific structural genes, such as the muscle actin gene Cs-MA-1. In contrast to H. roretzi, Cs-macho-1–depleted embryos activated Cs-MA-1 during gastrulation (Satou et al.,2002). Yagi and colleagues (2004) identified genes downstream of Ci-macho-1 and demonstrated that Ci-Tbx-6b and Ci-Tbx-6c are key mediators of Ci-macho-1 function in muscle cell differentiation. The maternal expression of Cs-macho-1 is also required for heart specification in metamorphosed juveniles (Satou et al.,2004).

POPK-1

In H. roretzi, Nakamura et al. (2005) recently demonstrated that Hr-POPK-1, which encodes a Ser/Thr kinase protein, plays multiple roles and orthologs were identified in C. intestinalis and C. savignyi. Both Ci-POPK-1 and Cs-POPK-1 mRNAs also show the characteristic localization pattern of Type I postplasmic/PEM RNAs (Yamada et al.,2005; Yamada,2006), but their functions remain to be analyzed in detail.

Suppression of Hr-POPK-1 function by MO leads to a significant phenotype, characterized by defects in the formation of muscle and mesenchyme tissues, in the translocation of the cER-mRNA domain (and, therefore, in proper CAB formation), and in posterior unequal cleavages. Epistatic analysis performed to understand the relationship between Hr-POPK-1 and macho-1 indicates that Hr-POPK-1 acts upstream of macho-1. The Hr-POPK-1 gene presents strong similarities with the Sad-1 gene in C. elegans, which plays a role in the presynaptic vesicle clustering in neurons. This finding suggests that POPK-1/Sad-1 kinase is involved in the traffic of membranous components.

wnt-5

wnt-5 is a secreted intercellular signalling molecule. In H. roretzi, overexpression of Hr-wnt-5 mRNA results in the disruption of morphogenetic movement of notochord cells, which occurs during and after gastrulation, whereas the differentiation of notochord is not affected (Sasakura and Makabe,2001). In contrast, functional suppression of Hr-wnt-5 with antisense MO results in failure of notochord and mesenchyme formation, and in a reduction of the number of muscle cells at the posterior pole (Nakamura et al.,2006). Because Hr-wnt-5 MO-injected embryos also fail to express an early notochord marker, Hr-bra, Hr-wnt-5 is also probably involved in early notochord specification. In addition, when Hr-wnt-5 MO is injected into anterior and vegetal A-line blastomeres (which normally do not inherit maternal Hr-wnt-5 mRNA) in embryos at the eight-cell stage, the primary notochord cells do not form, suggesting that the zygotic function of Hr-wnt-5 is responsible for the primary notochord formation. It is likely that maternal and/or zygotic Hr-wnt-5 have roles in the secondary notochord, mesenchyme, and muscle cell formation, as these tissues are derived from blastomeres that inherit maternal Hr-wnt-5 and also express Hr-wnt-5 zygotically. Wnt-5 was also identified in C. intestinalis and C. savignyi; both the localization of the maternal transcript and the zygotic expression pattern are similar for these three orthologous genes (Imai et al.,2004; Yamada,2006). In C. intestinalis, zygotic expression of Ci-wnt-5 is regulated by Ci-macho1 in early embryos (Yagi et al.,2004). In addition, it has also been hypothesized that maternally localized wnt-5 mRNA in the small B7.6 blastomeres may have a role in the formation of the cardiac mesoderm (Davidson and Levine,2003).

GLUT

GLUT protein shows similarity to mammalian glucose transporter (Nakamura et al.,2003). Hr-GLUT is expressed zygotically in the mesenchyme and nervous system starting at the neurula stage. In Hr-GLUT MO-injected embryos, endoderm and mesenchyme do not differentiate, and the number of notochord cells appears to be increased (Nakamura et al.,2006). However, Hr-GLUT–overexpressing embryos do not display the ectopic formation of endoderm. This study indicates that Hr-GLUT mRNA is necessary, but not sufficient, for endoderm differentiation and its maternal and/or zygotic function are required for normal mesenchyme and notochord formation.

PEN-2

Hr-PEN-2, which is exclusively maternally expressed, is required for the formation of various tissues. In embryos injected with Hr-PEN-2 MO, mesenchyme formation is suppressed, and the amount of endoderm and muscle cells is reduced. Interestingly, endoderm cells derived from the anterior-vegetal blastomeres form, but not those derived from the posterior-vegetal blastomeres. These observations indicate that Hr-PEN-2 acts maternally in mesenchyme differentiation, formation of posterior endoderm, and partially for muscle differentiation (Nakamura et al.,2006).

pem-3

In C. savignyi, the zygotic function of Cs-pem-3, which encodes an RNA-binding protein, has been analyzed (Satou,1999). The zygotic transcript of Cs-pem-3 and its protein are detected in cells of neural plate, mesenchyme, and epidermis after the neural plate stage. Maternal Cs-pem-3 protein is also present at a low level in eggs and localized to the posterior region of the blastomeres where the Cs-pem-3 mRNA is detected in the 8-, 16-, and 32-cell embryos. The KH domains (RNA binding modules) of pem-3 show a high similarity to those of C. elegans MEX-3 (Muscle EXcess protein-3) and mammalian TINO (Satou,1999; Donnini et al.,2004). It has been demonstrated that MEX-3 has a role in anterior–posterior asymmetry of the C. elegans embryo and is a component of germ line–specific granules called P granules (Draper et al.,1996). In ascidians, inhibition of zygotic transcripts with an antisense oligonucleotide (S-oligo) results in the abnormal development of larvae without sensory pigment cells, indicating that the zygotic Cs-pem-3 in the neural plate cells might play a role in the differentiation of the brain of the ascidian larvae (Satou,1999). The role of maternal Cs-pem-3 has not been addressed. As described above, in H. roretzi, the role of the Hr-pem-3 gene remains enigmatic, as the larvae injected with Hr-pem-3 MO develop normally.

DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

Oogenesis and Meiotic Maturation

Oogenesis is a crucial period in the establishment of a–v polarity in most animals (Albertini and Barrett,2004; Sardet et al.,2005; Prodon et al.,2005). Mature ascidian oocytes are arrested in Metaphase I of meiosis and are highly polarized along the a–v axis: a small meiotic spindle (MS) is present at the animal pole, and two adjacent domains, respectively, enriched in cER and Type I postplasmic/PEM RNAs (cortical cER-mRNA domain) and mitochondria (subcortical myoplasm) line the vegetal hemisphere (Roegiers et al.,1999; Prodon et al.,2005; review by Sardet et al., this issue). Recent data obtained in C. intestinalis, indicate that the a–v polarization of the myoplasm, and two Type I postplasmic/PEM RNAs (Ci-pem-1 and Ci-pem-3) located at the cell periphery of fully grown oocytes, occurs during meiotic maturation, after the MS and chromosomes have moved to the cortex defining the animal pole (Prodon et al.,2006).

Ascidian meiotic maturation corresponds to the Prophase I–Metaphase I transition, during which the nuclear membrane of the GV disappears (also called GVBD for Germinal Vesicle BreakDown) and the spindle forms (Voronina and Wessel,2003). Even if external stimuli and signaling pathways responsible for the triggering of GVBD are not yet well understood in ascidians (Lambert,2005), the ability of postvitellogenic oocytes of C. intestinalis and H. roretzi, to mature spontaneously in sea water (Satoh,1994; Numakunai,2001) makes them a particularly attractive model for the study of the a–v polarization (Conklin,1905b; Prodon et al.,2006; see Sardet et al., this issue). In both C. intestinalis and H. roretzi, ovaries can be easily removed by dissection and maturing oocytes can be selected manually (Fig. 2A–C). In the enterogona Ciona, the ovary contains oocytes at different stages of oogenesis (previtellogenic, vitellogenic, and postvitellogenic oocytes) in contrast to the pleurogona Halocynthia in which ovary contains mainly fully grown oocytes (Prophase I) during the spawning season. Furthermore, the eccentric positioning of the GV in Prophase I–arrested oocytes of H. roretzi constitutes the first sign of asymmetry in contrary to C. intestinalis (Fig. 2A).

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Figure 2. Localization pattern of postplasmic/PEM RNAs in oocytes and after cleavage stages. The distribution of Ci-/Hr-pem-1 mRNAs during oogenesis is shown in the left panel [oocytes (pre-/postvitellogenic stages)], and the distribution of Ci-VH (mRNA and protein) and Ci-pem-1 mRNA in late stages in the right panel (gastrula/tail bud/juvenile stages). Left panel: Oocytes (pre-/postvitellogenic stages). A: Chorionated germinal vesicle (GV) -containing stage III oocytes of H. roretzi (upper insert) and C. intestinalis (lower insert) just before meiotic maturation. B: Ovarian fragment isolated from H. roretzi (differential interference contrast optics). C: Distribution of Ci-pem-3 mRNA at stage I (left) and stage II (right) on ovary sections of C. intestinalis. Arrowheads indicate the presence of pem-3–positive granules around the GV. D1–E1, D2–D3, E2–E3: Distribution of pem-1 mRNA in oocytes of C. intestinalis (D1–E1) and H. roretzi (D2–D3, E2–E3) just before meiotic maturation (D1–D2–D3) and 4 hr after the complete breakdown of the GV (GVBD) (E1–E2–E3). The two halves of panels (D1–D2, E1–E2) show confocal equatorial (left) and surface (right) views of the same oocyte. The dotted white circle in D1, D2 indicates the position of the GV. D3–E3: Confocal surface views at higher resolution before (D3) and after (E3) GVBD. Arrowheads indicate the presence of cortical patches in (D3) which are spread after maturation (E3). Right panel: Gastrula/tail bud/juvenile stages. A: A mid-gastrula embryo with pH3-positive chromosomes in one of the B7.6 cells (yellow, arrow). B–D: Late-tail bud stage embryo labeled for filamentous actin (magenta) and Ci-VH protein (green). Ci-VH signal is detected in the B8.11 pair (arrowhead in B, C), and in the B8.12 pair that have divided to form four cells (small arrows in B, D). These four cells contain perinuclear Ci-VH granules (arrows in D). In the B8.11 cells, the faint Ci-VH signals were detected in the F-actin aggregates (B,C, arrowheads). E, F: Expression patterns of Type I and Type II postplasmic/PEM RNA in tail bud stage. E: Distribution of Ci-pem-1 mRNA (Type I) in B8.11 cells (arrowhead). F: Distribution of Ci-VH mRNA (Type II) in B8.11 and B8.12 cells (see arrowhead and arrow). G: Tail bud-derived from an embryo in which B7.6 cells had been injected/marked with 1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate (DiI) for lineage tracing. B8.11 and B8.12 cells contain the DiI label (see arrowhead and arrow). H,I: A stage 6 juvenile stained for Ci-VH protein (green) that was derived from an embryo in which the B7.6 cells were labeled with DiI (magenta). The arrow points to the primitive gonad. Insert in H: View at higher magnification. I: Five cells that are DiI-labeled (arrowheads) and Ci-VH–positive (green) coalesce into the gonads. J: Effects of translational and transcriptional inhibitors on the expression of Ci-VH protein at the tail bud stages. Embryos at the 110-cell stage were incubated in presence of puromycin (translational inhibitor) at 100 or 200 μg/ml, or with actinomycin D (transcriptional inhibitor) at 20 or 40 μg/ml and cultured until the tail bud stage (12–14 hr after fertilization, at 18°C). Then, embryos were stained for Ci-VH protein, and classified in three types according to the level of Ci-VH protein expression in the presumptive B8.12 cells (high/control, weak, undetectable). A total of 100 embryos was counted under each condition.

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A classification of the oocytes of C. intestinalis was recently established, based on the size and pigmentation of oocytes, the aspect of the chorion, and the distribution of organelles. Four main stages of oogenesis could be distinguished: small transparent previtellogenic oocytes (stage I); growing vitellogenic oocytes (stage II); and larger postvitellogenic oocytes (stage III), which are those able to undergo maturation when they are exposed to sea water; and finally mature oocytes, which can be fertilized (stage IV oocyte or unfertilized eggs; Prodon et al.,2006).

Distribution of Type I postplasmic/PEM RNAs During Early Oogenesis and Meiotic Maturation

Recent results indicate that the cortical accumulation of Type I postplasmic/PEM mRNAs occurs during early oogenesis (Ci-pem-3 mRNA) and their polarized relocalization along the a–v axis during meiotic maturation (Ci-pem-3 and Ci-pem-1 mRNAs), suggesting that two different mechanisms may be required during the final polarization of oocytes (Fig. 1A1–B1) as described below. It is essential now to examine the localization pattern of more Type I postplasmic/PEM RNAs during early oogenesis to determine whether there are different localization pathways (see Fig. 1, section 1.4, and also 1A1–B1).

In situ hybridization on ovary sections with Ci-pem-3 antisense probe indicates that aggregates enriched in Ci-pem-3 mRNAs are located uniformly around the GV at stage I (Fig. 2C) and fill the cytoplasm at stage II (Fig. 2C). After vitellogenesis, Ci-pem-3 and Ci-pem-1 mRNAs are both uniformly distributed at the periphery of full-grown stage III oocytes (Prophase I; Prodon et al.,2006), suggesting a migration of Type I postplasmic/PEM mRNAs from the periphery of the GV to the cell cortex as the oocyte grows. Because the in vitro culture of previtellogenic oocytes is not yet a reality, it is not possible to analyze the first steps of mRNA localization to the periphery of the oocyte and determine the possible roles of the cytoskeleton in this process. In full-grown oocytes just before maturation, at higher resolution, the detection signal for Ci/Hr-pem-1 mRNAs corresponds actually to cortical patches connected together by means of a reticulated network (Fig. 2D1–D3). Of interest, it has been reported that ascidian oocytes (stage I–II–III) do not possess a structure similar to the mitochondrial cloud, a large aggregate of mitochondria and ER, which lies between the future vegetal pole and the GV in stage I–II oocytes of X. laevis (Chang et al.,2004; Prodon et al.,2006).

After GVBD, when the MS reaches the cell cortex, Ci-/Hr-pem-1 mRNAs are excluded from the animal pole and display a reticulated and polarized distribution along the a–v axis (Fig. 2E1–E3). In C. intestinalis and H. roretzi, pem-1 mRNAs are anchored to a polarized network of rough cER distributed as a gradient of increasing density along the a–v axis, forming the cER-mRNA domain (Sardet et al.,2003; Prodon et al.,2005; see also Sardet et al. in this issue). It has been reported that at least five Type I mRNAs (pem-1, macho-1, POPK-1, ZF-1, wnt-5) are associated with this cER in eggs and early embryos (Sardet et al.,2003; Nakamura et al.,2005; Prodon et al.,2005). Recent experiments using cytoskeleton inhibitors indicate that the polarization along the a–v axis of Type I postplasmic/PEM RNAs is a microfilament-dependent mechanism like the redistribution of the mitochondria-rich myoplasm domain (Prodon et al.,2006; Prodon and Nishida, unpublished observations). Future experiments will have to determine whether (1) Type I postplasmic/PEM RNAs are already associated with the network of cER, and (2) their polarized redistribution along the a–v axis depends on the cER reorganizations during meiotic maturation.

Relocalization of postplasmic/PEM RNAs After Fertilization Toward the Posterior Pole

As mentioned in the introduction, between fertilization and first cleavage, the Type I postplasmic/PEM RNAs are relocated by two major phases (and several subphases) of cytoplasmic and cortical reorganizations (see review by Sardet et al. in this issue for details). Indeed, Type I postplasmic/PEM RNAs are first concentrated vegetally by means of microfilament-driven cortical contractions (Fig. 1D1), and then relocate toward the posterior region (PVC) by means of sperm-aster microtubule–driven translocations (Fig. 1E1). Between the two- and eight-cell stages, they are partitioned by a series of cleavages (Fig. 1F1–H1) and further concentrate in the CAB region. This finding results in the segregation of mRNAs and their products in the posterior pole of the embryo (Fig. 1I1–K1). In contrast, Type II postplasmic/PEM RNAs appear to be homogeneously distributed through the cytoplasm of mature oocytes and accumulate progressively in the CAB region after fertilization (Fig. 1C2–H2, see also CAB and Putative Germ Plasma Section).

LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

Messenger RNA localization is recognized as a key posttranscriptional mechanism to establish spatially restricted protein synthesis. In many organisms, the 3′-UnTranslated Region (3′-UTR) of mRNAs plays an essential role in the regulation of their stability, localization, and translation (reviewed in Bashirullah et al.,1998; Mowry et al.,1999; Lipshitz and Smibert,2000; Jansen,2001; Kloc et al.,2002; Lopez de Heredia and Jansen,2004; Shav-Tal and Singer,2005). Indeed, the 3′-UTR contains specific cis-acting elements called Localization Elements (LEs), short sequences recognized by trans-acting factors, which allow the proper localization of mRNAs to their final intracellular destination. In ascidians, several reports have shown that the localization of postplasmic/PEM RNAs to the posterior pole is also mediated by their 3′-UTRs (Sasakura et al.,1998a,b,2000, Sasakura and Makabe,2002; Yamada,2006).

Regulation of RNA Localization by 3′-UTR

In H. roretzi, experiments using green fluorescent protein (GFP) mRNA fused with or without the 3′-UTR of three Type I (Hr-wnt-5, Hr-POPK-1, Hr-ZF-1) and 3 Type II (Hr-pet-1, -2, and -3) postplasmic/PEM RNAs indicate that the 3′-UTR is necessary and sufficient to direct the trafficking of these GFP mRNAs to the posterior pole (Sasakura and Makabe,2002). Similarly, it has been shown that the 3′-UTRs of three Type I mRNAs in C. intestinalis (Ci-pem-1, Ci-wnt-5, Ci-ZF-1) and C. savignyi (Cs-pem-1, Cs-wnt-5, Cs-ZF-1) are also crucial for their proper localization, showing that 3′-UTRs play a conserved role in the posterior localization of postplasmic/PEM mRNAs (Yamada,2006).

LEs contained in the 3′-UTR of Hr-wnt-5 have been investigated in detail (Sasakura and Makabe,2002). To identify the minimal LEs, the 3′-UTR of Hr-wnt-5 mRNA was sequentially truncated. It was shown that a single sequence containing 279 nucleotides named WLE (for Wnt-5Localization Element) is required for the posterior localization of Hr-wnt-5 mRNA. More precisely, each of two small nucleotide sequences (20–25 nucleotides) called UGREs (for UG dinucleotide-Repetitive Elements) within WLE, is sufficient to drive this posterior localization (Fig. 3A3–A4). These UGREs correspond to the smallest LEs identified in ascidians. Furthermore, a 469 nucleotide-long LE identified in Hr-POPK-1, another Type I postplasmic/PEM mRNA, also contains some UGRE-like sequences, suggesting that UGREs could be conserved (Sasakura and Makabe,2002).

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Figure 3. Localization and translational control of Type I postplasmic/PEM RNAs. A1–A4, B1–B2: Several mRNA constructions labeled with digoxigenin (DIG) were introduced into C. savignyi unfertilized eggs. After overnight incubation, eggs were fertilized and embryos were fixed at 8- to 16-cell stages. Truncated or full-length mRNAs were detected using alkaline phosphatase–conjugated anti-DIG antibody and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP) substrate. Arrows indicate the CAB region. A1–A4: Detection of two exogenous Type I postplasmic/PEM RNAs (pem-1 and wnt-5) at the eight-cell stage. A1: Posterior accumulation of the 3′-untranslated region (UTR) of Cs-pem-1 mRNA. A2: Posterior localization of the full-length Ci-pem-1 mRNA. A3: Posterior accumulation of the truncated Hr-wnt-5 mRNA containing UGREs (from Sasakura et al.,2002). A4: No posterior accumulation of the full-length Hr-wnt-5 mRNA when two UGREs are deleted (described as 3′ET/3′d60/UaA in Sasakura et al.,2002). B1–B2: Identification of LEs in Ci-pem-1 mRNA. B1: Sequence alignment of LEs conserved between Cs-pem-1 and Ci-pem-1 3′-UTRs. Conserved regions (A and B) are underlined. The two CAC-motifs contained in Regions A and B are shown in capitals. B2: Localization activity in C. savignyi embryos of full-length and truncated forms of Cs-pem-1 mRNAs. CAC-motifs are shown by yellow ovals. The percentage of embryos in which the injected RNA localizes to the posterior is indicated, and the percentage showing strong signal in the CAB is in parentheses. Note that the localization efficiency of 3′ET/3′d162 is significantly lower than that of 3′ET. C: Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of two Type I postplasmic/PEM RNAs (Hr-wnt-5 and Hr-POPK-1) in isolated blastomeres. The posterior most vegetal B4.1 blastomeres were isolated at the eight-cell stage. RNAs from isolated B4.1 and remaining blastomeres (a4.2+b4.2+A4.1: abA) were subjected to RT-PCR using primers for Hr-wnt-5 and Hr-POPK-1. +RT and −RT, with or without reverse transcriptase. D1–D3: A translational repression activity is contained in the 3′-UTR of Type I postplasmic/PEM RNAs. The green fluorescent protein (GFP) open reading frame (orf) was fused with either the full-length 3′-UTR of Type I Hr-wnt-5 mRNA (D1), the truncated 3′-UTR of Type II Hr-pet-1 mRNA (D2), or the truncated 3′-UTR of Hr-wnt-5 mRNA (D3), and introduced into fertilized eggs of H. roretzi. E1–E2: Identification in the 3′-UTR of Hr-wnt-5 mRNA of elements responsible for its translational repression. E1: mRNA construct resulting from fusion of GFP orf to the 3′-UTR of Hr-wnt-5 mRNA. Position of the Localization Element “WLE” and restriction sites used for constructions (S, SacI; EV, EcoRV; ET, EcoT14I) are indicated, respectively, by a red arrow and black arrows. For an efficient translation and stability of mRNAs, UTRs of globin mRNA from X. laevis were added (Lemaire et al.,1995). E2: A series of deletion constructs from the 3′-UTR of Hr-wnt-5 mRNA and their effects on GFP expression. + and −, efficient or nonefficient translational repression activity. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Redundancy of LEs is probably the most evident in the case of Cs-pem-1 mRNA (Fig. 3B1–B2). Truncated fragments of the 3′-UTR of Cs-pem-1 do not localize with the same efficiency as the full-length 3′-UTR (Fig. 3B2), suggesting that several kinds of LEs are present on the full-length 3′-UTR and that they act synergistically to localize. Redundancy of LEs has also been reported in the 3′-UTR of one Type II postplasmic/PEM RNA, Hr-pet-3, as deletion of one element does not result in severe disruption of the posterior localization (Sasakura and Makabe,2002). The trans-acting factors that interact with these LEs have not yet been identified.

Like the mRNAs themselves, the machinery for localization of postplasmic/PEM RNAs appears to be conserved among several ascidian species. When full-length Type I postplasmic/PEM RNAs from C. intestinalis (Ci-wnt-5, Ci-ZF-1, Ci-pem-1) are injected into unfertilized eggs of C. savignyi, these mRNAs are correctly localized to the posterior pole and vice versa (Yamada,2006; Fig. 3A2). The conservation of localization signals is also observed between H. roretzi and C. savignyi, because exogenous H. roretzi wnt-5 mRNAs accumulate in the CAB region in C. savignyi embryos (Fig. 3A3). In contrast, when UGREs are mutated, this localization activity is lost (Fig. 3A4), emphasizing the important role played by UGREs in the posterior localization of Type I postplasmic/PEM mRNAs. These cross-species localization experiments, thus, suggest a conservation of the LEs and trans-acting factors that localize postplasmic/PEM RNAs among different ascidian species.

Comparing the 3′-UTR sequences from C. savignyi pem-1 mRNA to that of C. intestinalis pem-1 mRNA reveals the existence of two conserved regions (Regions A and B, see Fig. 3B1–B2). Both regions A and B contain a CAC repeat in a neighborhood rich in uracils (5′-ucuCACcu-3′ motif, for example, in Region A). The deletion of these conserved motifs leads to a drastic reduction in localization efficiency (Fig. 3B2, 3′ET/3′d162). Interestingly, it has been shown in X. laevis oocytes that high density clusters of short CAC-containing motifs characterize the LEs of virtually all mRNAs localized to the vegetal cortex (Betley et al.,2002). Such abundant CAC-containing repeats were also found in the 3′-UTRs of many postplasmic/PEM RNAs (Sasakura and Makabe,2002; Sardet et al.,2005). This finding raises the possibility that conserved CAC motifs, in concert with uracil-rich element UGREs, could play crucial roles in the posterior localization.

Translational Repression and the 3′-UTR

Temporal control of translation of localized mRNAs is an important process to generate a gradient of morphogens and to direct differentiation by allowing distribution of these localized proteins to specific blastomeres or daughter cells. In ascidians, it is thought that the CAB could act as a source of factors (probably translation products of localized mRNAs) directing development and differentiation in the posterior region of the embryo (Nishida,2005; Sardet et al.,2005). Such a link between localization and translational regulation has already been reported in Drosophila, where translation of two posteriorly localized mRNAs, nanos and oskar, is repressed when they are not localized (reviewed in Lipshitz and Smibert,2000). Such translational repression may also be the case for ascidian postplasmic/PEM RNAs.

Although Type I postplasmic/PEM mRNAs highly accumulate in the CAB region, recent RT-PCR analyses reveal that some Type I (Hr-wnt-5 and Hr-POPK-1) postplasmic/PEM mRNAs are not limited to the posterior-most vegetal B4.1 blastomeres at the eight-cell stage (Fig. 3C). Microarray analysis also indicates the presence of postplasmic/PEM, such as Ci-pem-2 for example, in blastomeres other than B4.1 (Yamada et al.,2005). These observations taken together with MO experiments (see Fig. 1, section 3) suggest that proteins encoded by postplasmic/PEM mRNAs function at the posterior pole even if their mRNAs display a dispersed distribution outside the CAB. Indeed, in POPK-1 knocked down embryos, the posterior localization of postplasmic/PEM mRNAs, including macho-1, is disrupted (Nakamura et al.,2005). However, no ectopic muscle cells are formed. These observations are consistent with the possibility that translation of postplasmic/PEM mRNAs is repressed in nonposterior blastomeres to prevent abnormal cell fate specifications.

Of interest, recent studies (unpublished data) in which a GFP reporter is fused to the full-length or truncated 3′-UTRs of wnt-5 mRNA and injected into fertilized eggs of H. roretzi indicate that the 5′ promixal part of the 3′-UTR possesses a strong translational repression activity (Fig. 3D1–D3, E1–E2). The cis-elements critical for the translational repression of Hr-wnt-5 mRNA are not located in the WLE (Fig. 3E2), suggesting that trans-acting factors functioning in the translational repression are independent of those responsible for RNA localization.

In contrast to the Type I postplasmic/PEM RNAs, the analysis of the 3′-UTR of the Type II postplasmic/PEM RNA Hr-pet-1 mRNA suggests that there is no translational repression activity in the 3′-UTR (Fig. 3D2). Future studies may address whether the translational repression of Type I postplasmic/PEM RNAs also occurs in immature oocytes, and whether there is a conserved mechanism in different species of ascidians.

DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

CAB and Putative Germ Plasm

In many animals, the germ line is founded by a small group of Primordial Germ Cells (PGCs) set aside during early development. PGC fate is specified either by inductive signalling (e.g., in mammals and urodele amphibians) or by the inheritance of germ plasm, which is distinctive granular egg cytoplasm containing germ line determinants located at one pole of the zygote (Machado et al.,2005). At the eight-cell stage, Type I and Type II postplasmic/PEM RNAs are highly concentrated in the CAB region (see previous sections), which is presumed to be the location of putative ascidian germ plasm for four main reasons: first, electron microscopy of the CAB structure is characterized by an accumulation of cER trapping an electron-dense matrix resembling germinal granules identified in other animal species as germ plasm (Hibino et al.,1998; Iseto and Nishida,1999; Prodon et al.,2005). Second, the CAB-containing blastomeres are thought to be transcriptionally inactive during embryogenesis (Tomioka et al.,2002; Shirae-Kurabayashi, unpublished data). Third, B7.6 cells, the posterior-most blastomeres, produced after three successive unequal cleavages from the 8-cell stage to the 64-cell stage, stop dividing after the 64-cell stage. Such transcriptional quiescence and cell division arrest are characteristic features of the PGCs in D. melanogaster and C. elegans (reviewed by Leatherman and Jongens,2003). Finally, in C. intestinalis, RNA and protein of a Vasa Homolog (Ci-VH) are both highly concentrated in the posterior-vegetal blastomeres in the CAB region (Fujimura and Takamura,2000; Takamura et al.,2002). Because Vasa seems to be a germ line–specific gene in a wide range of animals (reviewed by Raz,2000; Extavour and Akam,2003), this finding reinforces the hypothesis that, in C. intestinalis, and probably H. roretzi and P. mammillata, small B7.6 blastomeres that contain the Vasa-positive CAB region develop into PGCs (Nakamura et al.,2005; Patalano et al.,2006). After metamorphosis, these Vasa-positive cells would be incorporated into the primitive gonads of juveniles and form germ cells.

Additional Posterior Asymmetric Cell Division Redistributes postplasmic/PEM RNAs into Two Populations of Cells

Recently, two different redistribution patterns of postplasmic/PEM RNAs after cleavage stages have been described, and a new hypothesis has been proposed about the contribution of these mRNAs to germ line formation (Shirae-Kurabayashi et al.,2006; Yamada,2006; Fig. 2, right panel). Using DiI-tracing and double labeling with antibodies to Ci-VH protein and phosphohistone H3 (a mitosis marker), it was found that the B7.6 cells, which have been regarded as mitotically inactive (see preceding section), actually undergo a supplementary asymmetric cell division during gastrulation to produce two distinct daughter cells (Fig. 2A, right panel; Shirae-Kurabayashi et al.,2006). Following the nomenclature for cell lineage established by Conklin (1905a), the two smaller anterior daughter cells containing the CAB should be called B8.11 (Fig. 2B, 2C, arrowheads), and the two larger posterior daughter cells B8.12 (Fig. 2B, 2D, arrows, right panel). Postplasmic/PEM mRNAs, including Ci-pem-1 and Ci-macho-1 mRNAs, which segregate into the B8.11 daughter cells (Fig. 2E, arrowhead, right panel), are associated with the gut wall of juveniles. These findings suggest that the postplasmic/PEM RNAs which are partitioned into B8.11 cells have no role in germ cell specification after gastrulation (Shirae-Kurabayashi et al.,2006).

In contrast to Ci-pem-1 mRNA, Ci-VH mRNA and its protein are distributed from the CAB-containing B7.6 cells to both B8.11 and B8.12 daughter cells (Fig. 2B–D, F, right panel). Later B8.12 cells divide further and are incorporated into the gonad of juveniles (Fig. 2G–H, right panel). Remarkably, the detection signal for Ci-VH protein increases in amount in B8.12 descendants, resulting in the formation of perinuclear Ci-VH granules reminiscent of electron dense “nuage” granules (Fig. 2D, right panel), a hallmark of germ cells in many animal species (Carre et al.,2002; Machado et al.,2005). Experiments using inhibitors of transcription and translation (Actinomycin D and Puromycin) were used to block the production of Ci-VH mRNA and its protein in the B8.12 cells. These treatments indicate that Ci-VH protein is translated from its maternal RNA source provided from the CAB region of the B7.6 cells (Fig. 2J, right panel), resulting in the formation of perinuclear germinal granules in B8.12 descendants as indicated by immunostaining (Fig. 2D, right panel; Shirae-Kurabayashi et al.,2006).

Recently, several other postplasmic/PEM RNAs (ZF-1, GCNF, pem-3, POPK-1, and so on) have been reported in both C. intestinalis and C. savignyi to be redistributed like Ci-VH mRNA into the B8.12 cells during embryogenesis (Yamada,2006; also see Fig. 1, section 2, and Table 1). This asymmetric division is apparently conserved in other ascidians because in H. roretzi, some postplasmic/PEM RNAs are also segregated into two distinct regions after gastrulation (Nakamura et al.,2003; Nishida, Nakamura, and Sasakura, unpublished observations). Thus, the redistribution of a specific set of the postplasmic/PEM RNAs through the B7.6 cell division may be a conserved mechanism underlying germ cell specification in ascidians.

CONCLUSION AND PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES

If we take into consideration all cellular and molecular data obtained in several ascidian species (C. intestinalis/C. savignyi, H. roretzi, and P. mammillata), a speculative scenario could be elaborated to explain the relocalization of postplasmic/PEM RNAs (Type I and Type II) and their translational control from fertilization to the eight-cell stage, even if detailed molecular and cellular mechanisms are not yet well understood. Roles of postplasmic/PEM RNAs in the posterior specification of embryos are also discussed as well as their posterior redistribution which occurs during the additional asymmetric division newly observed in B7.6 cells and their putative role in germ line formation.

Speculative Scenario About the Relocalization and the Translational Control of postplasmic/PEM RNAs

Concerning Type I postplasmic/PEM RNAs, it clearly appears that some of them, such as pem-1 and macho-1 mRNAs, are already anchored to the surface of the polarized domain of cER (forming the cER–mRNA domain) at the end of meiotic maturation (see Fig. 1, see Distribution of Type I postplasmic/PEM RNAs During Early Oogenesis and Meiotic Maturation Section), and move with it after fertilization (Sardet et al.,2003,2005; Prodon et al.,2005; Nakamura et al.,2005). Although we do not know if all Type I postplasmic/PEM RNAs are associated with this cER domain (see Sardet et al. in this issue), it is tempting to hypothesize that some trans-acting factors recruit Type I postplasmic/PEM RNAs into RiboNucleoProteins complexes (RNPs) and mediate attachment to the cER. One of the most intriguing future questions is to understand how the localization machinery of Type I postplasmic/PEM RNA interacts with the cER network and its roles to promote efficient protein translation. Therefore, important aspects to investigate concern: (1) the anchoring of the Type I postplasmic/PEM RNAs at the surface of the cER, then (2) the relationship between the relocalization of the cER-mRNA domain and cytoskeleton reorganizations, and finally (3) the translational control of these polarized mRNAs.

One good candidate for a trans-acting factor involved in translational repression could be Ci-YB-1, a Y-box containing RNA-binding protein that is a component of ribosome-bound RNPs present in oocytes and zygotes, and enriched at the posterior pole of early embryos (Wada et al.,1998; Tanaka et al.,2004). Ci-YB-1 binds to Ci-pem-1 and Ci-macho-1 mRNAs (Tanaka et al.,2004), and in vitro translation analysis reveals that Ci-YB-1 protein can repress the translation of Ci-pem-1 mRNA (Tanaka et al.,2004). In addition, in C. intestinalis, H. roretzi, and P. mammillata, Ci-pem-1 and Ci-macho-1 mRNAs are associated with the cER domain which characterizes the CAB, suggesting indirectly that Ci-YB-1 may be also associated with this cER-mRNA domain (Sardet et al.,2003; Prodon et al.,2005). Recent work has identified individual components of the translation machinery that are specifically associated with the cER domain of the CAB (Paix and Sardet, personal communication). Ci-YB-1 protein is present in a large amount at the posterior pole at the 16-cell stage, the period during which the translational activity of some postplasmic/PEM RNAs starts. One possible explanation could be that Type I postplasmic/PEM RNAs may be released from Ci-YB-1 protein during development, leading to a loss of translational repression. Another possibility would be that the role of Ci-YB-1 activity as translational repressor may be inactivated by an interaction with other proteins at the posterior pole. Furthermore, it has been previously reported using in vitro translation systems that a Y-box protein can activate or repress translation, depending on the ratio of RNA and proteins (Evdokimova et al.,1998,2006), suggesting that, when some Type I postplasmic/PEM mRNAs and Ci-YB-1 protein are both strongly concentrated in the CAB region, translation could be activated.

Of interest, new observations obtained in the yeast S. cerevisae, the fly D. melanogaster, and the worm C. elegans raise the possibility that localization and translational regulation of mRNAs at the ER play a role in controlling the organization of this organelle (reviewed by Poteryaev et al.,2005; Decker and Parker,2006). In addition, a recent study in D. melanogaster oocytes indicates that trailer hitch (a gene required for the secretion of the Dorsal–Ventral patterning factor Gurken) and the vitellogenin receptor Yolkless are part of a RNP complex that is localized to subdomains of the ER, suggesting a link between RNP complexes and secretory pathway (Wilhelm et al.,2005). It was also shown that some factors (Me31B and Cup) important for translation and localization are associated with this large RNP complex (Wilhelm et al.,2005). We hypothesize that, in ascidians, some (nonsecreted) proteins encoded by postplasmic/PEM mRNAs could influence directly the integrity of the cER on which they are anchored, as suggested in Hr-POPK-1 MO-injected embryos (Nakamura et al.,2005; see Fig. 1, see Analyses of Functions of Type I postplasmic/PEM RNAs Section). Finally, it is also known that reorganizations of the ER network are under the influence of the cell cycle and/or physiological signals (Gillot et al.,1990; Stricker et al.,1998; Terasaki et al.,2001).

The relocalization mechanisms for Type II postplasmic/PEM RNAs is not yet understood, and this type displays variable patterns from fertilization to the eight-cell stage. Their weak signal after in situ hybridization (compared with Type I) makes it hard to determine clearly their relocalization pattern in early stages (from fertilized egg to the four- to eight-cell stage) and, therefore, to understand the mechanisms responsible for their posterior accumulation in the CAB region. Nevertheless, we can envision at least two possible mechanisms responsible for their redistribution in zygotes and early embryos. First, their relocalization could be mediated by a mechanism completely different from Type I postplasmic/PEM RNAs. For example, their accumulation in the CAB region at the eight-cell stage could be due to a progressive diffusion/entrapment restricted to the posterior pole, as is the case for vegetally localized RNAs in oocytes of X. laevis (Chang et al.,2004). Alternatively, the relocalization of Type II postplasmic/PEM RNAs could be actually driven by the same mechanism as for Type I postplasmic/PEM RNAs, and they could be accumulated at the posterior pole with the CAB precursor. Unfortunately, the weakness of their signal after in situ hybridization does not allow us to detect their distribution in the cell cortex before the eight-cell stage.

Future investigations will have to consider the fact that the balance between the translational repression and derepression of postplasmic/PEM mRNAs is a local and dynamic process subtly orchestrated in space and time. The identification of trans-acting factors combined with cellular approaches should also provide new insights about translational control mechanisms at the posterior pole of embryos.

Postplasmic/PEM RNAs and Posterior Specification

Several Type I postplasmic/PEM RNAs accumulated into the PVC of zygotes (see Fig. 1) play some critical roles in the determination of the A–P axis by directing the “posterior fate” of ascidian embryos. This posterior fate includes the autonomous muscle specification, the generation of differences in responsiveness to inductive FGF signals between mesenchyme and notochord precursor blastomeres, and the control of cleavage patterns (Nishida,1994).

Actually, proteins encoded by certain maternal Type I postplasmic/PEM mRNAs act in the formation of multiple tissues by influencing embryonic patterning. For example, the muscle determinant macho-1 is not only required for muscle specification, but also for the responsiveness in mesenchyme induction (Nishida and Sawada,2001; Kobayashi et al.,2003); pem-1 plays a crucial role for the positioning of cleavage planes (Negishi and Nishida, personal communication); POPK-1 is necessary for concentrating the cER-mRNA domain and putative germinal granules into the CAB during early cleavages (Nakamura et al.,2005); and finally many others act in mesenchyme formation (Kobayashi et al.,2003; Nakamura et al.,2006). Interestingly, posterior muscle cells are also reduced in PEN-2– and wnt-5–deficient embryos, indicating that PEN-2 and wnt-5 may be also involved in muscle specification. This finding also suggests that proteins encoded by several Type I postplasmic/PEM could act in the same process (Nakamura et al.,2006). Furthermore, proteins that are maternally and zygotically expressed could act in different processes during embryogenesis.

These data taken together strongly support the view that the a–p polarity of ascidian embryos is under the influence of the asymmetric distribution of Type I postplasmic/PEM mRNAs and theirs products that play some important roles in the posterior specification. Finally, it has been also proposed that Type I postplasmic/PEM mRNAs and their proteins might be involved in the formation of primordial germ cells, the control of the cell cycle length and endoderm differentiation, and the specification of the trunk ventral cells in the posterior region of the embryo (Nishida,2005).

Putative role of postplasmic/PEM RNAs in Germline Formation

After cleavage stages, postplasmic/PEM RNAs can be classified into two groups based on their redistribution in the tail bud (independently of their classification as Type I or Type II defined in early stages; see also Fig. 1). Certain mRNAs are inherited by both B8.11 and B8.12 cells, whereas others are only segregated into B8.11 cells (Shirae-Kurabayashi et al.,2006; Yamada,2006; see also Fig. 1K1–K2). An attractive idea is that some mRNAs that are involved in posterior specification are sequestered into B8.11 after having played their roles during cleavage stages and are not required for germ line specification. In contrast, only mRNAs partitioned into B8.12 may be essential for germ line specification.

It has been shown that the maternal Type II postplasmic/PEM RNA, Ci-VH, which is released from the CAB region before B7.6 cell division and accumulates into B8.12 primordial germ cells, is also translated (Shirae-Kurabayashi et al.,2006). This redistribution and translational up-regulation of Ci-VH mRNA suggest a role for postplasmic/PEM RNAs in germ line formation. These observations also suggest that the CAB structure and some unidentified components in the CAB region (trans-acting factors, see Fig. 1, see Translational Repression and the 3′-UTR Section and CAB and Putative Germ Plasma Section) would be required for the storage of Ci-VH mRNA as well as for its translational repression during cleavage stages when somatic cell fate determination proceeds. Of interest, Ci-YB-1, one of the putative translational repressors associated with the postplasmic/PEM RNAs in the CAB region, is only redistributed into B8.11 cells after cleavage stages (Shirae-Kurabayashi et al.,2006; see Fig. 1). In addition, we hypothesize that some other postplasmic/PEM RNAs distributed in B8.12 cells could be also involved in germ cell specification in a similar manner. For example, Pem-3 may be one such RNA, as it is the homolog of mex-3, which is associated with P granules in C. elegans germ line (Draper et al.,1996; Satou,1999). Finally, further identification of such postplasmic/PEM RNAs and analysis of their redistribution after cleavage stages will provide new insights into germ cell specification during ascidian embryogenesis and metamorphosis.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ASCIDIAN MODEL AND LOCALIZED MATERNAL mRNAs
  5. EMBRYONIC EXPRESSION PROFILES AND CLASSIFICATION OF POSTPLASMIC/PEM GENES
  6. ANALYSES OF FUNCTIONS OF TYPE I POSTPLASMIC/PEM RNAs
  7. DISTRIBUTION OF POSTPLASMIC/PEM RNAs FROM OOGENESIS TO THE EIGHT-CELL STAGE
  8. LOCALIZATION AND TRANSLATIONAL CONTROL OF POSTPLASMIC/PEM RNAs AT THE POSTERIOR POLE OF EMBRYOS
  9. DISTRIBUTION OF POSTPLASMIC/PEM RNAs AFTER CLEAVAGE STAGES AND GERMLINE FORMATION
  10. CONCLUSION AND PERSPECTIVES
  11. Acknowledgements
  12. REFERENCES