Localized molecules and the establishment of polarity in zebrafish

LOCALIZED MOLECULES AND THE ESTABLISHMENT OF POLARITY IN ZEBRAFISH ANIKET V. GORE (M. Sc. UNIVERSITY OF PUNE, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2006 i Dedicated to my parents, my brother and my wife Swarada, ii Acknowledgements: I am very grateful to Temasek Life Sciences Laboratory for providing excellent scientific infrastructure and financial support during the course of my Ph.D. I am thankful to my thesis committee members Drs. Suresh Jesuthasan, Naweed Naqvi and Yang Xiaohang for time and effort they have invested in this study. I am grateful to all the past and present members of Vertebrate Development group, for useful suggestions and insightful advice. I am especially thankful to Gayathri and Devpriya for their excellent support and encouragement during early days of my PhD. I am very grateful to my friends Mahendra, Richard and Volker for productive scientific discussions and support through out this study. I also want to thank Lim Shi Min, Patrick Gilligan, Mahendra D. Wagle and Sarada Bulchand for proof reading parts of my thesis. I am incredibly thankful to Dr. Karuna Sampath for accepting me as her first graduate student, for her valuable insight, constant encouragement, support and advise. Karuna’s excellent mentorship and scientific enthusiasm continue to be a great encouragement. Thank to Drs. Shingo Maegawa and Eric Weinberg at University of Pennsylvania for their incredible support and collaboration during the course of this study. iii I also want to thank to all the facility incharge for their support and help, especially to Chin Heng Goh for providing an excellent fish facility and to Connie for her help with the confocal microscopy. I also wish to thank everybody at TLL for helping me one or other way which helped me to finish this project. At last, I also want to thank all my family members, especially to my mother who encouraged me and helped build the confidence in me to finish my PhD. My father and brother for their constant support and encouragement through out this study. My lovely wife Swarada, who believed in me and always stood by me to give strength and support. iv Table of contents Acknowledgements: _____________________________________________________ iii Table of contents ________________________________________________________v List of Figures ________________________________________________________ viii List of abbreviations: ____________________________________________________ ix Abstract: ______________________________________________________________ x Summary: _____________________________________________________________ xi Publications:__________________________________________________________ xiii Chapter I: Introduction ______________________________________________ 1.1 1.1.1 1.1.2 1.1.3 1.1.4 1.1.5 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2.7 History of localized determinants and axis specification: _____________________ Polarity in S. cervisiae (Budding Yeast) ________________________________________ Polarity in C.elegans embryos________________________________________________ Polarity in Drosophila oocytes and embryos _____________________________________ Polarity in cultured fibroblast cells ____________________________________________ Polarity in Xenopus oocytes__________________________________________________ 2 4 Mechanisms of axes specification in different model systems: _________________ Mechanism of axes specification in Drosophila melanogaster _______________________ Mechanism of axis specification in insect species _________________________________ RNA localization and cell fate determination in ascidian eggs ______________________ 11 Mechanisms of axes specification in Xenopus ___________________________________ 14 RNA localization and oocyte/embryo polarity in zebrafish _________________________ 17 Early embryonic development of zebrafish _____________________________________ 21 The Nodal Signaling Pathway in zebrafish _____________________________________ 24 Chapter II: Materials and Methods____________________________________ 29 2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 Zebrafish Embryo and larval cultures: __________________________________ 29 Zebrafish Embryos _______________________________________________________ In vitro Fertilization ______________________________________________________ Oocyte activation_________________________________________________________ Drug treatment for cytoskeleton inhibitors _____________________________________ Microinjection into zebrafish embryos ________________________________________ Microinjection into zebrafish mature oocytes ___________________________________ Embryo dissections _______________________________________________________ Embryo Cleavage furrow labeling____________________________________________ 29 29 30 30 30 31 32 32 Molecular Biology and Recombinant DNA techniques: _____________________ 33 Molecular biology techniques _______________________________________________ DNA sequencing _________________________________________________________ Labelled capped mRNA synthesis____________________________________________ Antisense DIG/Fluorescein labeled probe synthesis ______________________________ Isolation of Mitochondrial Cloud structure _____________________________________ Total RNA isolation ______________________________________________________ Reverse Transcriptase (RT) reaction __________________________________________ Polymerase Chain Reaction (PCR) ___________________________________________ Real-Time RT PCR _______________________________________________________ 33 33 34 35 35 36 36 36 37 v 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 3.1.1 3.1.2 3.1.3 3.1.4 3.2 3.2.1 3.2.2 3.2.3 38 39 41 41 42 42 RNA localization during oogenesis: _____________________________________ 47 Stages of Zebrafish oocytes_________________________________________________ The follicle cells in zebrafish ovary___________________________________________ RNA localization pattern during zebrafish oogenesis _____________________________ RNA localization during zebrafish oogenesis and early embryogenesis _______________ 47 48 49 50 Localization of transcripts of Nodal signaling pathway molecules: ____________ 51 The mitochondrial cloud in young oocytes _____________________________________ 51 Localization of cyclops transcripts during oogenesis______________________________ 53 Isolation of mitochondrial cloud and generation of cDNA library____________________ 57 3.3 Localization of oep transcripts:_________________________________________ 61 3.4 Localization of squint transcripts: ______________________________________ 62 3.4.1 3.4.2 3.4.3 3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6 3.6 3.6.1 3.6.2 3.7 Localization of squint transcripts during oogenesis _______________________________ 62 Localization of squint transcripts in activated oocytes/embryos _____________________ 64 Role of cytoskeleton in squint RNA localization_________________________________ 64 Localization of squint transcripts during early embryogenesis:_______________ 69 Localization of squint transcripts during early cleavage stages ______________________ Squint RNA localization is an active process____________________________________ Localization of synthetic labeled squint RNA ___________________________________ Role of actin microfilaments in late squint localization ____________________________ Localization of squint RNA is independent of cleavage furrow______________________ Localization of squint RNA is independent of the centrosome ______________________ 69 72 74 78 80 82 Localization of squint RNA is dependent on its 3’ UTR:_____________________ 84 Minimal localization element in squint 3’ UTR__________________________________ 84 Squint 3’ UTR can localize a lacZ reporter gene to future dorsal side _________________ 87 Discussion: _________________________________________________________ 93 Chapter IV: Cellular functions of localized Squint _______________________ 97 4.1 4.1.1 4.1.2 4.1.3 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 In situ hybridization ______________________________________________________ In situ hybridization on sections _____________________________________________ Antibody staining ________________________________________________________ Actin microfilaments staining _______________________________________________ Nuclei/DNA staining______________________________________________________ Imaging Techniques ______________________________________________________ Chapter III: RNA localization during zebrafish oogenesis and embryogenesis _ 47 3.1 Stainings and imaging techniques: ______________________________________ 38 DV axis specification in zebrafish: ______________________________________ 97 Role of Nodal signaling in DV axis specification _______________________________ 100 Squint marks dorsal by 4-8 cell stage ________________________________________ 102 Analysis of shh and hgg1 expression in operated embryos ________________________ 107 Squint is required for dorsal specification: ______________________________ 112 Injection of sqtMO into fertilized embryos ____________________________________ Injection of sqtMO into mature oocytes ______________________________________ b-catenin independent localization of squint RNA ______________________________ Squint donor site morpholino selectively block RNA processing ___________________ Unprocessed squint transcripts in the mature oocytes ____________________________ 112 114 118 119 120 Discussion: ________________________________________________________ 123 Chapter V: Discussion _____________________________________________ 128 vi 5.1 5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.3 Localization of squint mRNA:_________________________________________ 128 Dynamic process of squint mRNA localization _________________________________ Translational control of squint RNA _________________________________________ Events prior to localization ________________________________________________ Conserved steps in RNA localization in zebrafish and other model organisms _________ Role of other localized RNAs in anchoring squint RNA __________________________ 128 131 133 134 135 Function of Squint in dorsal specification:_______________________________ 137 Role of Squint in early embryonic development ________________________________ Squint signaling independent of Oep_________________________________________ Maternal unprocessed Squint_______________________________________________ Role of BMPs in DV patterning ____________________________________________ Canonical Wnt signaling pathway and axis specification _________________________ Conserved mechanism of DV axis specification amongst vertebrates ________________ 137 141 144 146 147 149 Proposed model for dorsal axis specification in zebrafish: __________________ 151 References: ______________________________________________________ 154 vii List of Figures Figure 1. Schematic showing A-P axis specification in Drosophila _____________________________ 10 Figure 1. Schematic showing RNA localization pathways during Xenopus oogenesis _______________ 13 Figure 1. Schematic of Nodal signaling pathway ___________________________________________ 26 Figure 3.1 Detection of mitochondrial cloud in young oocytes __________________________________ 52 Figure 3.2 Different RNA localization patterns during oogenesis ________________________________ 55 Figure 3.3 Isolation of mitochondrial cloud material from young oocytes _________________________ 58 Figure 3.4 Localization of squint RNA during oogenesis ______________________________________ 63 Figure 3.5 Localization of squint RNA during egg activation ___________________________________ 65 Figure 3. squint RNA transport is microtubule dependent ____________________________________ 67 Figure 3. squint RNA localization in cleavage stage embryos _________________________________ 70 Figure 3.8 squint RNA localization during blastula and gastrula stages___________________________ 71 Figure 3.9 Real time RT-PCR analysis of squint transcripts ____________________________________ 73 Figure 3. 10 Dynamic localization of squint RNA in real time __________________________________ 75 Figure 3. 11 Dynamic localization of squint RNA in real time __________________________________ 76 Figure 3. 12 squint RNA transport is microtubule dependent ___________________________________ 77 Figure 3. 13 Role of actin in squint RNA anchoring at cleavage stages ___________________________ 79 Figure 3. 14 squint RNA localization is independent of cleavage plane ___________________________ 81 Figure 3. 15 squint 3’ UTR is required for its localization _____________________________________ 85 Figure 3. 16 Dynamic localization of squint RNA in real-time with its minimum localization element ____ 86 Figure 3. 17 Dynamic localization of lacZ:sqt 3’ UTR in real-time ______________________________ 88 Figure 3. 18 Co-localization of lacZ:sqt 3’ UTR with endogenous squint RNA______________________ 89 Figure 3. 19 lacZ:sqt 3’ UTR RNA localizes to one side of the embryo____________________________ 91 Figure 3. 20 lacZ:sqt 3’ UTR localizes to the dorsal side of the embryo ___________________________ 92 Figure 4. Schematic representation of blastomere ablation experiments ________________________ 106 Figure 4. Live embryo phenotypes of blastomeres ablated ___________________________________ 108 Figure 4. shh and hgg1 expression analysis in blastomeres ablated embryos ____________________ 110 Figure 4. sqtMO injections into oocytes develop ventreralised embryos ________________________ 116 Figure 4. RT-PCR results showing presence of maternal unspliced squint transcripts______________ 121 viii List of abbreviations: AP AV BMP bp DV dpf FGF hpf LR MBT MO PAR PCR RT TGF-b UTR UV Wnt WT YSL Anterior-Posterior Animal-Vegetal Bone morphogenic protein Base pairs Dorso-Ventral days-post fertilization Fibroblast growth factor hours-post fertilization Left-Right Mid-blastula transition Morpholino Partitioning defective Polymerase chain reaction Reverse transcriptase Transforming growth factor – beta Un-translated region Ultra-violet Wingless related MMTV integration site Wild-Type Yolk syncytial layer ix Abstract: The generation of polarity and patterning during both vertebrate and invertebrate embryogenesis depends in part on the asymmetric localization of molecules. In the zebrafish, Danio rerio maternally deposited molecules of Wnt and TGF-b signaling pathways contribute to DV axis formation. Specifically, the Nodal related molecules Cyclops, Squint and Southpaw (which belong to TGF-b super family) are essential for dorsal and mesendoderm specification as well as specification of left-right asymmetry. In this study, I have found that transcripts of the nodal related gene squint are expressed maternally and distributed uniformly through all stages of oocytes. Upon fertilization, squint transcripts are initially localized to the blastoderm and subsequently to two blastomeres at the 4-cell stage, which depends on its 3’ untranslated region. Disruption of the microtubule cytoskeleton by nocodazole treatment affects squint RNA transport to the blastoderm. Removal of cells containing squint transcripts from 4-8 cell embryos or injection into oocytes of antisense morpholino oligonucleotides targeting squint can cause loss of dorsal structures. Thus, the DV axis is apparent by the 4-8 cell stage during embryogenesis, and requires the maternally encoded morphogen Squint and its associated factors. x which maternal Sqt along with maternal b-catenin initiates the signaling cascade. Maternal squint RNA, presumably along with its binding proteins localizes in the 4- cell embryo asymmetrically. This localization is dependent on the microtubule cytoskeleton (Gore et al., 2005). Once localized, the cells inheriting the squint RNA are specified as dorsal cells, which accumulate nuclear b-catenin later during development. Localized squint along with b-catenin starts zygotic squint expression and maintains the dorsal fates (Dougan et al., 2003). Apart from zygotic squint, b-catenin also activates zygotic expression of boz/dharma, and this, in parallel with squint, activates downstream target genes (Fekany et al., 1999; Shimizu et al., 2000; Sirotkin et al., 2000a). Zebrafish embryos that lack maternal ichabod, which encodes for b-catenin2, fail to maintain zygotic squint expression (Bellipanni et al., 2006; Kelly et al., 2000). However, in these embryos, injected squint RNA localization at the 4- cell stage is unaffected (Gore et al., 2005). These embryos fail to localize nuclear b-catenin and the phenotypes can be rescued by over-expression of members of the canonical Wnt signaling pathway. Overexpression of squint RNA also rescues the dorsal defects without nuclear accumulation of b-catenin in the injected embryos (Gore et al., 2005; Kelly et al., 2000). This indicates that Sqt may act downstream of b-catenin. However, in WT embryos, squint expression is activated by b-catenin via a feedback loop (Dougan et al., 2003; Feldman et al., 2002). This makes a strong case for exogenously injected and localized squint RNA to activate its own expression in ichabod embryos, independent of b-catenin2. This zygotic squint expression alone is enough to rescue the dorsal defects in the ichabod embryos (Gore et al., 2005; Kelly et al., 2000). Thus, maternal squint and b-catenin co-operate to specify dorsal axis in zebrafish embryos. 152 The maternal zygotic squint mutants show reduction of dorsal structures like reduced or absent prechordal plate mesoderm and breaks in the notochord (Aoki et al., 2002; Dougan et al., 2003; Feldman et al., 1998; Sirotkin et al., 2000a). However, loss of maternal and zygotic Squint activity by injection of squint morpholinos into the oocytes shows severely ventralized embryos (Gore et al., 2005). Undoubtedly, the localization of squint RNA during zebrafish oogenesis and embryogenesis and its role in dorsal axis specification has led to more questions than answers. The present study has provided insights into zebrafish dorso-ventral axis formation. Future studies will need to focus on the interaction between squint RNA and specific RNA-binding proteins, the mechanism of microtubule dependent squint RNA transport and dorso-ventral axis specification in zebrafish. 153 References: Abdelilah, S., Solnica-Krezel, L., Stainier, D. Y. and Driever, W. (1994). Implications for dorsoventral axis determination from the zebrafish mutation janus. Nature 370, 46871. Agius, E., Oelgeschlager, M., Wessely, O., Kemp, C. and De Robertis, E. M. (2000). Endodermal Nodal-related signals and mesoderm induction in Xenopus. Development 127, 1173-83. Alarcon, V. B. and Elinson, R. P. (2001). RNA anchoring in the vegetal cortex of the Xenopus oocyte. J Cell Sci 114, 1731-41. Altmann, C. R., Chang, C., Munoz-Sanjuan, I., Bell, E., Heke, M., Rifkin, D. B. and Brivanlou, A. H. (2002). The latent-TGFbeta-binding-protein-1 (LTBP-1) is expressed in the organizer and regulates nodal and activin signaling. Dev Biol 248, 118-27. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-10. Aoki, T. O., Mathieu, J., Saint-Etienne, L., Rebagliati, M. R., Peyrieras, N. and Rosa, F. M. (2002). Regulation of nodal signalling and mesendoderm formation by TARAM-A, a TGFbeta-related type I receptor. Dev Biol 241, 273-88. Arn, E. A., Cha, B. J., Theurkauf, W. E. and Macdonald, P. M. (2003). Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins. Dev Cell 4, 41-51. Arnold, C. and Clewley, J. P. (1997). From ABI sequence data to LASERGENE's EDITSEQ. Methods Mol Biol 70, 65-74. Bally-Cuif, L., Schatz, W. J. and Ho, R. K. (1998). Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte. Mech Dev 77, 31-47. Beck, S., Le Good, J. A., Guzman, M., Ben Haim, N., Roy, K., Beermann, F. and Constam, D. B. (2002). Extraembryonic proteases regulate Nodal signalling during gastrulation. Nat Cell Biol 4, 981-5. Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96, 195-209. Bellipanni, G., Varga, M., Maegawa, S., Imai, Y., Kelly, C., Myers, A. P., Chu, F., Talbot, W. S. and Weinberg, E. S. (2006). Essential and opposing roles of zebrafish {beta}-catenins in the formation of dorsal axial structures and neurectoderm. Development 133, 1299-309. Betley, J. N., Heinrich, B., Vernos, I., Sardet, C., Prodon, F. and Deshler, J. O. (2004). Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes. Curr Biol 14, 21924. Birsoy, B., Berg, L., Williams, P. H., Smith, J. C., Wylie, C. C., Christian, J. L. and Heasman, J. (2005). XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development. Development 132, 591-602. 154 Birsoy, B., Kofron, M., Schaible, K., Wylie, C. and Heasman, J. (2006). Vg1 is an essential signaling molecule in Xenopus development. Development 133, 15-20. Blagden, C. S., Currie, P. D., Ingham, P. W. and Hughes, S. M. (1997). Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev 11, 216375. Boyd, L., Guo, S., Levitan, D., Stinchcomb, D. T. and Kemphues, K. J. (1996). PAR2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C. elegans embryos. Development 122, 3075-84. Braat, A. K., Yan, N., Arn, E., Harrison, D. and Macdonald, P. M. (2004). Localization-dependent oskar protein accumulation; control after the initiation of translation. Dev Cell 7, 125-31. Branford, W. W. and Yost, H. J. (2002). Lefty-dependent inhibition of Nodal- and Wnt-responsive organizer gene expression is essential for normal gastrulation. Curr Biol 12, 2136-41. Cha, B. J., Koppetsch, B. S. and Theurkauf, W. E. (2001). In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway. Cell 106, 35-46. Chang, P., Torres, J., Lewis, R. A., Mowry, K. L., Houliston, E. and King, M. L. (2004). Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum. Mol Biol Cell 15, 4669-81. Charby, L. (1887). Contribution a l'embrologie normale teratologique des ascidies simples. J. Anat. Physio. Norm. Pathol. 23, 167-321. Chartrand, P., Meng, X. H., Huttelmaier, S., Donato, D. and Singer, R. H. (2002). Asymmetric sorting of ash1p in yeast results from inhibition of translation by localization elements in the mRNA. Mol Cell 10, 1319-30. Chen, Y. and Schier, A. F. (2001). The zebrafish Nodal signal Squint functions as a morphogen. Nature 411, 607-10. Chen, Y. and Schier, A. F. (2002). Lefty proteins are long-range inhibitors of squintmediated nodal signaling. Curr Biol 12, 2124-8. Ciruna, B., Jenny, A., Lee, D., Mlodzik, M. and Schier, A. F. (2006). Planar cell polarity signalling couples cell division and morphogenesis during neurulation. Nature 439, 220-4. Clement, J. H., Fettes, P., Knochel, S., Lef, J. and Knochel, W. (1995). Bone morphogenetic protein in the early development of Xenopus laevis. Mech Dev 52, 35770. Collignon, J., Varlet, I. and Robertson, E. J. (1996). Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381, 155-8. Condeelis, J. and Singer, R. H. (2005). How and why does beta-actin mRNA target? Biol Cell 97, 97-110. Conklin, E. (1905). Mosaic development in Ascidian eggs. Journal of Experimental Zoology 2, 145-223. Constam, D. B. and Robertson, E. J. (2000a). SPC4/PACE4 regulates a TGFbeta signaling network during axis formation. Genes Dev 14, 1146-55. Constam, D. B. and Robertson, E. J. (2000b). Tissue-specific requirements for the proprotein convertase furin/SPC1 during embryonic turning and heart looping. Development 127, 245-54. 155 Davidson, B. and Levine, M. (2003). Evolutionary origins of the vertebrate heart: Specification of the cardiac lineage in Ciona intestinalis. Proc Natl Acad Sci U S A 100, 11469-73. Ding, J., Yang, L., Yan, Y. T., Chen, A., Desai, N., Wynshaw-Boris, A. and Shen, M. M. (1998). Cripto is required for correct orientation of the anterior-posterior axis in the mouse embryo. Nature 395, 702-7. Dohrmann, C. E., Kessler, D. S. and Melton, D. A. (1996). Induction of axial mesoderm by zDVR-1, the zebrafish orthologue of Xenopus Vg1. Dev Biol 175, 108-17. Dosch, R., Wagner, D. S., Mintzer, K. A., Runke, G., Wiemelt, A. P. and Mullins, M. C. (2004). Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I. Dev Cell 6, 771-80. Dougan, S. T., Warga, R. M., Kane, D. A., Schier, A. F. and Talbot, W. S. (2003). The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm. Development 130, 1837-51. Draper, B. W., Morcos, P. A. and Kimmel, C. B. (2001). Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis 30, 154-6. Driever, W. (1995). Axis formation in zebrafish. Curr Opin Genet Dev 5, 610-8. Driever, W. and Nusslein-Volhard, C. (1988a). The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, 95-104. Driever, W. and Nusslein-Volhard, C. (1988b). A gradient of bicoid protein in Drosophila embryos. Cell 54, 83-93. Dunn, N. R., Koonce, C. H., Anderson, D. C., Islam, A., Bikoff, E. K. and Robertson, E. J. (2005). Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile. Genes Dev 19, 152-63. Dunn, N. R., Vincent, S. D., Oxburgh, L., Robertson, E. J. and Bikoff, E. K. (2004). Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo. Development 131, 1717-28. Eimon, P. M. and Harland, R. M. (2002). Effects of heterodimerization and proteolytic processing on Derriere and Nodal activity: implications for mesoderm induction in Xenopus. Development 129, 3089-103. Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter, J. A., Moon, R. T. and Beachy, P. A. (1995). Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 5, 944-55. Elisha, Z., Havin, L., Ringel, I. and Yisraeli, J. K. (1995). Vg1 RNA binding protein mediates the association of Vg1 RNA with microtubules in Xenopus oocytes. Embo J 14, 5109-14. Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37-50. Ephrussi, A. and St Johnston, D. (2004). Seeing is believing: the bicoid morphogen gradient matures. Cell 116, 143-52. Erter, C. E., Solnica-Krezel, L. and Wright, C. V. (1998). Zebrafish nodal-related encodes an early mesendodermal inducer signaling from the extraembryonic yolk syncytial layer. Dev Biol 204, 361-72. 156 Fagotto, F., Guger, K. and Gumbiner, B. M. (1997). Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/beta-catenin signaling pathway, but not by Vg1, Activin or Noggin. Development 124, 453-60. Fekany, K., Yamanaka, Y., Leung, T., Sirotkin, H. I., Topczewski, J., Gates, M. A., Hibi, M., Renucci, A., Stemple, D., Radbill, A. et al. (1999). The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures. Development 126, 1427-38. Feldman, B., Concha, M. L., Saude, L., Parsons, M. J., Adams, R. J., Wilson, S. W. and Stemple, D. L. (2002). Lefty antagonism of Squint is essential for normal gastrulation. Curr Biol 12, 2129-35. Feldman, B., Dougan, S. T., Schier, A. F. and Talbot, W. S. (2000). Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish. Curr Biol 10, 531-4. Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin, H. I., Schier, A. F. and Talbot, W. S. (1998). Zebrafish organizer development and germlayer formation require nodal-related signals. Nature 395, 181-5. Feldman, B. and Stemple, D. L. (2001). Morpholino phenocopies of sqt, oep, and ntl mutations. Genesis 30, 175-7. Feng, B., Schwarz, H. and Jesuthasan, S. (2002). Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres. Exp Cell Res 279, 14-20. Ferrandon, D., Elphick, L., Nusslein-Volhard, C. and St Johnston, D. (1994). Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 79, 1221-32. Fujii, R., Yamashita, S., Hibi, M. and Hirano, T. (2000). Asymmetric p38 activation in zebrafish: its possible role in symmetric and synchronous cleavage. J Cell Biol 150, 1335-48. Fujimura, M. and Takamura, K. (2000). Characterization of an ascidian DEAD-box gene, Ci-DEAD1: specific expression in the germ cells and its mRNA localization in the posterior-most blastomeres in early embryos. Dev Genes Evol 210, 64-72. Gavis, E. R., Curtis, D. and Lehmann, R. (1996). Identification of cis-acting sequences that control nanos RNA localization. Dev Biol 176, 36-50. Gerhart, J., Danilchik, M., Doniach, T., Roberts, S., Rowning, B. and Stewart, R. (1989). Cortical rotation of the Xenopus egg: consequences for the anteroposterior pattern of embryonic dorsal development. Development 107 Supplement, 37-51. Glotzer, J. B., Saffrich, R., Glotzer, M. and Ephrussi, A. (1997). Cytoplasmic flows localize injected oskar RNA in Drosophila oocytes. Curr Biol 7, 326-37. Golling, G., Amsterdam, A., Sun, Z., Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K., Farrington, S. et al. (2002). Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat Genet 31, 135-40. Goodrich, J. S., Clouse, K. N. and Schupbach, T. (2004). Hrb27C, Sqd and Otu cooperatively regulate gurken RNA localization and mediate nurse cell chromosome dispersion in Drosophila oogenesis. Development 131, 1949-58. Gore, A. V., Maegawa, S., Cheong, A., Gilligan, P. C., Weinberg, E. and Sampath, K. (2005). The zebrafish dorsal axis is apparent at the four-cell stage. Nature In Press. 157 Gore, A. V. and Sampath, K. (2002). Localization of transcripts of the zebrafish morphogen Squint is dependent on egg activation and the microtubule cytoskeleton. Mech Dev 112, 153-6. Grant, P. and Wacaster, J. (1972). The amphibian grey crescent-A site of developmental information? Dev Bio 28, 454-471. Gritsman, K., Talbot, W. S. and Schier, A. F. (2000). Nodal signaling patterns the organizer. Development 127, 921-32. Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier, A. F. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97, 121-32. Guraya, S. S. (1979). Reccent advances in the morphology, cytochemistry, and function of Balbiani's vitelline body in animal oocytes. Int. Rev. Cytol. 59, 249-321. Hachet, O. and Ephrussi, A. (2004). Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization. Nature 428, 959-63. Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., van Eeden, F. J., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P., Heisenberg, C. P. et al. (1996). dino and mercedes, two genes regulating dorsal development in the zebrafish embryo. Development 123, 95-102. Haramoto, Y., Tanegashima, K., Onuma, Y., Takahashi, S., Sekizaki, H. and Asashima, M. (2004). Xenopus tropicalis nodal-related gene regulates BMP signaling: an essential role for the pro-region. Dev Biol 265, 155-68. Hatta, K., Kimmel, C. B., Ho, R. K. and Walker, C. (1991). The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system. Nature 350, 339-41. Heasman, J. (1997). Patterning the Xenopus blastula. Development 124, 4179-91. Heasman, J. (2006). Patterning the early Xenopus embryo. Development 133, 1205-17. Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994). Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell 79, 791-803. Heasman, J., Quarmby, J. and Wylie, C. C. (1984). The mitochondrial cloud of Xenopus oocytes: the source of germinal granule material. Dev Biol 105, 458-69. Heasman, J., Wessely, O., Langland, R., Craig, E. J. and Kessler, D. S. (2001). Vegetal localization of maternal mRNAs is disrupted by VegT depletion. Dev Biol 240, 377-86. Heisenberg, C. P. and Nusslein-Volhard, C. (1997). The function of silberblick in the positioning of the eye anlage in the zebrafish embryo. Dev Biol 184, 85-94. Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C. and Wilson, S. W. (2000). Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76-81. Hemmati-Brivanlou, A., Kelly, O. G. and Melton, D. A. (1994). Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77, 283-95. Herkowitz, I. (1985). Yeast as the universal cell. Nature 316, 678-9. 158 Holwill, S., Heasman, J., Crawley, C. and Wylie, C. (1987). Axis and germ line deficiencies caused by u. v irradiation of Xenopus oocytes cultured in vitro. Development 121, 2789-2798. Houston, D. W., Zhang, J., Maines, J. Z., Wasserman, S. A. and King, M. L. (1998). A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule. Development 125, 171-80. Howley, C. and Ho, R. K. (2000). mRNA localization patterns in zebrafish oocytes. Mech Dev 92, 305-9. Hyde, C. E. and Old, R. W. (2000). Regulation of the early expression of the Xenopus nodal-related gene, Xnr1. Development 127, 1221-9. Illmensee, K. and Mahowald, A. P. (1974). Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc Natl Acad Sci U S A 71, 1016-20. Ingham, P. W. and McMahon, A. P. (2001). Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15, 3059-87. Jesuthasan, S. and Strahle, U. (1997). Dynamic microtubules and specification of the zebrafish embryonic axis. Curr Biol 7, 31-42. Johnson, S. L., Midson, C. N., Ballinger, E. W. and Postlethwait, J. H. (1994). Identification of RAPD primers that reveal extensive polymorphisms between laboratory strains of zebrafish. Genomics 19, 152-6. Jones, C. M., Armes, N. and Smith, J. C. (1996). Signalling by TGF-beta family members: short-range effects of Xnr-2 and BMP-4 contrast with the long-range effects of activin. Curr Biol 6, 1468-75. Jones, C. M., Kuehn, M. R., Hogan, B. L., Smith, J. C. and Wright, C. V. (1995). Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation. Development 121, 3651-62. Kane, D. A. and Kimmel, C. B. (1993). The zebrafish midblastula transition. Development 119, 447-56. Kelly, C., Chin, A. J., Leatherman, J. L., Kozlowski, D. J. and Weinberg, E. S. (2000). Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish. Development 127, 3899-911. Kikuchi, Y., Trinh, L. A., Reiter, J. F., Alexander, J., Yelon, D. and Stainier, D. Y. (2000). The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors. Genes Dev 14, 1279-89. Kim-Ha, J., Kerr, K. and Macdonald, P. M. (1995). Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell 81, 403-12. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310. King, M. L., Zhou, Y. and Bubunenko, M. (1999). Polarizing genetic information in the egg: RNA localization in the frog oocyte. Bioessays 21, 546-57. Kinoshita, N., Minshull, J. and Kirschner, M. W. (1995). The identification of two novel ligands of the FGF receptor by a yeast screening method and their activity in Xenopus development. Cell 83, 621-30. Kishimoto, Y., Lee, K. H., Zon, L., Hammerschmidt, M. and Schulte-Merker, S. (1997). The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning. Development 124, 4457-66. 159 Klein, S. L. (1987). The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos. Dev Biol 120, 299-304. Kloc, M. and Etkin, L. D. (1995). Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development 121, 287-97. Kloc, M. and Etkin, L. D. (1998). Apparent continuity between the messenger transport organizer and late RNA localization pathways during oogenesis in Xenopus. Mech Dev 73, 95-106. Kloc, M. and Etkin, L. D. (2005). RNA localization mechanisms in oocytes. J Cell Sci 118, 269-82. Kloc, M., Larabell, C. and Etkin, L. D. (1996). Elaboration of the messenger transport organizer pathway for localization of RNA to the vegetal cortex of Xenopus oocytes. Dev Biol 180, 119-30. Kloc, M., Wilk, K., Vargas, D., Shirato, Y., Bilinski, S. and Etkin, L. D. (2005). Potential structural role of non-coding and coding RNAs in the organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes. Development 132, 3445-57. Kloc, M., Zearfoss, N. R. and Etkin, L. D. (2002). Mechanisms of subcellular mRNA localization. Cell 108, 533-44. Knaut, H., Pelegri, F., Bohmann, K., Schwarz, H. and Nusslein-Volhard, C. (2000). Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification. J Cell Biol 149, 875-88. Knaut, H., Steinbeisser, H., Schwarz, H. and Nusslein-Volhard, C. (2002). An evolutionary conserved region in the vasa 3'UTR targets RNA translation to the germ cells in the zebrafish. Curr Biol 12, 454-66. Kofron, M., Demel, T., Xanthos, J., Lohr, J., Sun, B., Sive, H., Osada, S., Wright, C., Wylie, C. and Heasman, J. (1999). Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors. Development 126, 5759-70. Kolev, N. G. and Huber, P. W. (2003). VgRBP71 stimulates cleavage at a polyadenylation signal in Vg1 mRNA, resulting in the removal of a cis-acting element that represses translation. Mol Cell 11, 745-55. Kondo, T., Kotani, T. and Yamashita, M. (2001). Dispersion of cyclin B mRNA aggregation is coupled with translational activation of the mRNA during zebrafish oocyte maturation. Dev Biol 229, 421-31. Koos, D. S. and Ho, R. K. (1999). The nieuwkoid/dharma homeobox gene is essential for bmp2b repression in the zebrafish pregastrula. Dev Biol 215, 190-207. Koprunner, M., Thisse, C., Thisse, B. and Raz, E. (2001). A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev 15, 2877-85. Ku, M. and Melton, D. A. (1993). Xwnt-11: a maternally expressed Xenopus wnt gene. Development 119, 1161-73. Lambert, J. D. and Nagy, L. M. (2002). Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages. Nature 420, 682-6. Larabell, C. A., Torres, M., Rowning, B. A., Yost, C., Miller, J. R., Wu, M., Kimelman, D. and Moon, R. T. (1997). Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway. J Cell Biol 136, 1123-36. 160 Lentz, T. L. and Trinkaus, J. P. (1967). A fine structural study of cytodifferentiation during cleavage, blastula, and gastrula stages of Fundulus heteroclitus. J Cell Biol 32, 121-38. Leung, C. F., Webb, S. E. and Miller, A. L. (2000). On the mechanism of ooplasmic segregation in single-cell zebrafish embryos. Dev Growth Differ 42, 29-40. Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. and Tabin, C. (1995). A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82, 803-14. Levitan, D. J., Boyd, L., Mello, C. C., Kemphues, K. J. and Stinchcomb, D. T. (1994). par-2, a gene required for blastomere asymmetry in Caenorhabditis elegans, encodes zinc-finger and ATP-binding motifs. Proc Natl Acad Sci U S A 91, 6108-12. Lin, S., Long, W., Chen, J. and Hopkins, N. (1992). Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos. Proc Natl Acad Sci U S A 89, 4519-23. Long, R. M., Singer, R. H., Meng, X., Gonzalez, I., Nasmyth, K. and Jansen, R. P. (1997). Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277, 383-7. Long, S., Ahmad, N. and Rebagliati, M. (2003). The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry. Development 130, 2303-16. Lopez de Heredia, M. and Jansen, R. P. (2004). mRNA localization and the cytoskeleton. Curr Opin Cell Biol 16, 80-5. Lowe, L. A., Supp, D. M., Sampath, K., Yokoyama, T., Wright, C. V., Potter, S. S., Overbeek, P. and Kuehn, M. R. (1996). Conserved left-right asymmetry of nodal expression and alterations in murine situs inversus. Nature 381, 158-61. Maegawa, S., Yamashita, M., Yasuda, K. and Inoue, K. (2002). Zebrafish DAZ-like protein controls translation via the sequence 'GUUC'. Genes Cells 7, 971-84. Mathieu, J., Barth, A., Rosa, F. M., Wilson, S. W. and Peyrieras, N. (2002). Distinct and cooperative roles for Nodal and Hedgehog signals during hypothalamic development. Development 129, 3055-65. Mathieu, J., Griffin, K., Herbomel, P., Dickmeis, T., Strahle, U., Kimelman, D., Rosa, F. M. and Peyrieras, N. (2004). Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations. Development 131, 629-41. Meno, C., Gritsman, K., Ohishi, S., Ohfuji, Y., Heckscher, E., Mochida, K., Shimono, A., Kondoh, H., Talbot, W. S., Robertson, E. J. et al. (1999). Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation. Mol Cell 4, 287-98. Meno, C., Ito, Y., Saijoh, Y., Matsuda, Y., Tashiro, K., Kuhara, S. and Hamada, H. (1997). Two closely-related left-right asymmetrically expressed genes, lefty-1 and lefty2: their distinct expression domains, chromosomal linkage and direct neuralizing activity in Xenopus embryos. Genes Cells 2, 513-24. Micklem, D. R. (1995). mRNA localisation during development. Dev Biol 172, 377-95. Minakhina, S. and Steward, R. (2005). Axes formation and RNA localization. Curr Opin Genet Dev 15, 416-21. 161 Mizuno, T., Yamaha, E., Kuroiwa, A. and Takeda, H. (1999). Removal of vegetal yolk causes dorsal deficencies and impairs dorsal-inducing ability of the yolk cell in zebrafish. Mech Dev 81, 51-63. Mizuno, T., Yamaha, E. and Yamazaki, F. (1997). Localized axis determinant in the early cleavage embryo of goldfish, Carassius auratus. Dev Growth Differ 206, 389-96. Mowry, K. L. and Cote, C. A. (1999). RNA sorting in Xenopus oocytes and embryos. Faseb J 13, 435-45. Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Heisenberg, C. P. et al. (1996). Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes. Development 123, 81-93. Nance, J. (2005). PAR proteins and the establishment of cell polarity during C. elegans development. Bioessays 27, 126-35. Nasmyth, K. (1983). Molecular analysis of a cell lineage. Nature 302, 670-6. Neuman-Silberberg, F. S. and Schupbach, T. (1993). The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. Cell 75, 165-74. Nieuwkoop, P. D. (1969). The formation of mesoderm in Urodelean amphibians. I. Induction by endoderm. Wilhelm Roux’ Arch. EntwMech. Org. 162, 341-373. Nojima, H., Shimizu, T., Kim, C. H., Yabe, T., Bae, Y. K., Muraoka, O., Hirata, T., Chitnis, A., Hirano, T. and Hibi, M. (2004). Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination in zebrafish. Mech Dev 121, 371-86. Ober, E. A. and Schulte-Merker, S. (1999). Signals from the yolk cell induce mesoderm, neuroectoderm, the trunk organizer, and the notochord in zebrafish. Dev Biol 215, 167-81. Olsen, L. C., Aasland, R. and Fjose, A. (1997). A vasa-like gene in zebrafish identifies putative primordial germ cells. Mech Dev 66, 95-105. Onuma, Y., Takahashi, S., Haramoto, Y., Tanegashima, K., Yokota, C., Whitman, M. and Asashima, M. (2005). Xnr2 and Xnr5 unprocessed proteins inhibit Wnt signaling upstream of dishevelled. Dev Dyn 234, 900-10. Onuma, Y., Yeo, C. Y. and Whitman, M. (2006). XCR2, one of three Xenopus EGFCFC genes, has a distinct role in the regulation of left-right patterning. Development 133, 237-50. Oppenheimer, J. (1936). The development of isolated blastoderms of Fundulus heteroclitus. J.Exp.Zool 72, 247-269. Palacios, I. M. and St Johnston, D. (2002). Kinesin light chain-independent function of the Kinesin heavy chain in cytoplasmic streaming and posterior localisation in the Drosophila oocyte. Development 129, 5473-85. Patel, N. H. (2000). It's a bug's life. Proc Natl Acad Sci U S A 97, 4442-4. Pelegri, F., Dekens, M. P., Schulte-Merker, S., Maischein, H. M., Weiler, C. and Nusslein-Volhard, C. (2004). Identification of recessive maternal-effect mutations in the zebrafish using a gynogenesis-based method. Dev Dyn 231, 324-35. Pelegri, F. and Maischein, H. M. (1998). Function of zebrafish beta-catenin and TCF-3 in dorsoventral patterning. Mech Dev 77, 63-74. 162 Pelegri, F. and Schulte-Merker, S. (1999). A gynogenesis-based screen for maternaleffect genes in the zebrafish, Danio rerio. Methods Cell Biol 60, 1-20. Piccolo, S., Sasai, Y., Lu, B. and De Robertis, E. M. (1996). Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589-98. Pogoda, H. M., Solnica-Krezel, L., Driever, W. and Meyer, D. (2000). The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for organizer formation. Curr Biol 10, 1041-9. Poulain, M. and Lepage, T. (2002). Mezzo, a paired-like homeobox protein is an immediate target of Nodal signalling and regulates endoderm specification in zebrafish. Development 129, 4901-14. Pozzoli, O., Gilardelli, C. N., Sordino, P., Doniselli, S., Lamia, C. L. and Cotelli, F. (2004). Identification and expression pattern of mago nashi during zebrafish development. Gene Expr Patterns 5, 265-72. Prodon, F., Dru, P., Roegiers, F. and Sardet, C. (2005). Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo. J Cell Sci 118, 2393404. Ramasamy, S., Wang, H., Quach, H. N. and Sampath, K. (2006). Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells. Dev Biol. Raz, E. (2003). Primordial germ-cell development: the zebrafish perspective. Nat Rev Genet 4, 690-700. Rebagliati, M. R., Toyama, R., Fricke, C., Haffter, P. and Dawid, I. B. (1998a). Zebrafish nodal-related genes are implicated in axial patterning and establishing left-right asymmetry. Dev Biol 199, 261-72. Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B. (1998b). cyclops encodes a nodal-related factor involved in midline signaling. Proc Natl Acad Sci U S A 95, 99327. Reissmann, E., Jornvall, H., Blokzijl, A., Andersson, O., Chang, C., Minchiotti, G., Persico, M. G., Ibanez, C. F. and Brivanlou, A. H. (2001). The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev 15, 2010-22. Renucci, A., Lemarchandel, V. and Rosa, F. (1996). An activated form of type I serine/threonine kinase receptor TARAM-A reveals a specific signalling pathway involved in fish head organiser formation. Development 122, 3735-43. Riechmann, V. and Ephrussi, A. (2001). Axis formation during Drosophila oogenesis. Curr Opin Genet Dev 11, 374-83. Sambrook, J. and Russell, D. (2001). Molecular Cloning, A Laboratory Mannual: Cold Spring Harbour Laboratory Press. Sampath, K., Cheng, A. M., Frisch, A. and Wright, C. V. (1997). Functional differences among Xenopus nodal-related genes in left-right axis determination. Development 124, 3293-302. Sampath, K., Rubinstein, A. L., Cheng, A. M., Liang, J. O., Fekany, K., SolnicaKrezel, L., Korzh, V., Halpern, M. E. and Wright, C. V. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395, 1859. 163 Sardet, C., Dru, P. and Prodon, F. (2005). Maternal determinants and mRNAs in the cortex of ascidian oocytes, zygotes and embryos. Biol Cell 97, 35-49. Sardet, C., Nishida, H., Prodon, F. and Sawada, K. (2003). Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex. Development 130, 5839-49. Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K. and De Robertis, E. M. (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79, 779-90. Saude, L., Woolley, K., Martin, P., Driever, W. and Stemple, D. L. (2000). Axisinducing activities and cell fates of the zebrafish organizer. Development 127, 3407-17. Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J., Strahle, U. and Haffter, P. (1998). Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development 125, 2983-93. Schier, A. F. and Shen, M. M. (2000). Nodal signalling in vertebrate development. Nature 403, 385-9. Schier, A. F. and Talbot, W. S. (2001). Nodal signaling and the zebrafish organizer. Int J Dev Biol 45, 289-97. Schier, A. F. and Talbot, W. S. (2005). Molecular Genetics of Axis Formation in Zebrafish. Annu Rev Genet. Schneider, S., Steinbeisser, H., Warga, R. M. and Hausen, P. (1996). Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech Dev 57, 191-8. Schnorrer, F., Bohmann, K. and Nusslein-Volhard, C. (2000). The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes. Nat Cell Biol 2, 185-90. Schroeder, K. E., Condic, M. L., Eisenberg, L. M. and Yost, H. J. (1999). Spatially regulated translation in embryos: asymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus. Dev Biol 214, 288-97. Schulte-Merker, S., Lee, K. J., McMahon, A. P. and Hammerschmidt, M. (1997). The zebrafish organizer requires chordino. Nature 387, 862-3. Selman, K., Wallace, R. A., Sarka, A. and Qi, X. (1993). Stages of oocyte development in zebrafish Brachydanio rerio. J. Morphol 218, 203-224. Shen, M. M. and Schier, A. F. (2000). The EGF-CFC gene family in vertebrate development. Trends Genet 16, 303-9. Shimizu, T., Yamanaka, Y., Ryu, S. L., Hashimoto, H., Yabe, T., Hirata, T., Bae, Y. K., Hibi, M. and Hirano, T. (2000). Cooperative roles of Bozozok/Dharma and Nodalrelated proteins in the formation of the dorsal organizer in zebrafish. Mech Dev 91, 293303. Shuttleworth, J. and Colman, A. (1988). Antisense oligonucleotide-directed cleavage of mRNA in Xenopus oocytes and eggs. Embo J 7, 427-34. Sirotkin, H. I., Dougan, S. T., Schier, A. F. and Talbot, W. S. (2000a). bozozok and squint act in parallel to specify dorsal mesoderm and anterior neuroectoderm in zebrafish. Development 127, 2583-92. Sirotkin, H. I., Gates, M. A., Kelly, P. D., Schier, A. F. and Talbot, W. S. (2000b). Fast1 is required for the development of dorsal axial structures in zebrafish. Curr Biol 10, 1051-4. 164 Smith, J. C. (1995). Mesoderm-inducing factors and mesodermal patterning. Curr Opin Cell Biol 7, 856-61. Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70, 82940. Sokol, S. Y. (1999). Wnt signaling and dorso-ventral axis specification in vertebrates. Curr Opin Genet Dev 9, 405-10. Spector, I., Shochet, N. R., Kashman, Y. and Groweiss, A. (1983). Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219, 4935. Spemann, H. and Mangold, H. (1924). Uber Induktion von Embryonenanlagen durch Implantation artfremder Organisatoren. Wilhelm Roux’ Arch. EntwMech. Org. 100, 599638. Strahle, U., Blader, P., Henrique, D. and Ingham, P. W. (1993). Axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev 7, 1436-46. Strahle, U. and Jesuthasan, S. (1993). Ultraviolet irradiation impairs epiboly in zebrafish embryos: evidence for a microtubule-dependent mechanism of epiboly. Development 119, 909-19. Sun, Z., Jin, P., Tian, T., Gu, Y., Chen, Y. G. and Meng, A. (2006). Activation and roles of ALK4/ALK7-mediated maternal TGFbeta signals in zebrafish embryo. Biochem Biophys Res Commun 345, 694-703. Sundell, C. L. and Singer, R. H. (1991). Requirement of microfilaments in sorting of actin messenger RNA. Science 253, 1275-7. Suzuki, H., Maegawa, S., Nishibu, T., Sugiyama, T., Yasuda, K. and Inoue, K. (2000). Vegetal localization of the maternal mRNA encoding an EDEN-BP/Bruno-like protein in zebrafish. Mech Dev 93, 205-9. Tabuse, Y., Izumi, Y., Piano, F., Kemphues, K. J., Miwa, J. and Ohno, S. (1998). Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans. Development 125, 3607-14. Takizawa, P. A., Sil, A., Swedlow, J. R., Herskowitz, I. and Vale, R. D. (1997). Actindependent localization of an RNA encoding a cell-fate determinant in yeast. Nature 389, 90-3. Tao, Q., Yokota, C., Puck, H., Kofron, M., Birsoy, B., Yan, D., Asashima, M., Wylie, C. C., Lin, X. and Heasman, J. (2005). Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120, 857-71. Tekotte, H. and Davis, I. (2002). Intracellular mRNA localization: motors move messages. Trends Genet 18, 636-42. Thisse, C. and Thisse, B. (1999). Antivin, a novel and divergent member of the TGFbeta superfamily, negatively regulates mesoderm induction. Development 126, 229-40. Thisse, C., Thisse, B., Halpern, M. E. and Postlethwait, J. H. (1994). Goosecoid expression in neurectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas. Dev Biol 164, 420-9. Thomsen, G. H. and Melton, D. A. (1993). Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell 74, 433-41. 165 Tian, J., Yam, C., Balasundaram, G., Wang, H., Gore, A. and Sampath, K. (2003). A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish. Development 130, 3331-42. van Eeden, F. and St Johnston, D. (1999). The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis. Curr Opin Genet Dev 9, 396-404. Vincent, J. P. and Gerhart, J. C. (1987). Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification. Dev Biol 123, 526-39. Wagner, D. S., Dosch, R., Mintzer, K. A., Wiemelt, A. P. and Mullins, M. C. (2004). Maternal control of development at the midblastula transition and beyond: mutants from the zebrafish II. Dev Cell 6, 781-90. Wagner, E. and Lykke-Andersen, J. (2002). mRNA surveillance: the perfect persist. J Cell Sci 115, 3033-8. Weaver, C., Farr, G. H., 3rd, Pan, W., Rowning, B. A., Wang, J., Mao, J., Wu, D., Li, L., Larabell, C. A. and Kimelman, D. (2003). GBP binds kinesin light chain and translocates during cortical rotation in Xenopus eggs. Development 130, 5425-36. Weaver, C. and Kimelman, D. (2004). Move it or lose it: axis specification in Xenopus. Development 131, 3491-9. Weeks, D. L. and Melton, D. A. (1987). A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell 51, 8617. Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B. and Raz, E. (2003). dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 13, 1429-34. Westerfield, M. (1994). The Zebrafish Book. Eugene, OR: University of Oregon. Wickens, M., Anderson, P. and Jackson, R. J. (1997). Life and death in the cytoplasm: messages from the 3' end. Curr Opin Genet Dev 7, 220-32. Williams, P. H., Hagemann, A., Gonzalez-Gaitan, M. and Smith, J. C. (2004). Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo. Curr Biol 14, 1916-23. Wilson, E. B. (1904). Experimental studies ongermin al localization. I The germ regions in the egg of Dentalium. II Experiments of the cleavage mosaic in Patella and Dentalium. J.Exp.Zool 1, 1-72. Wittbrodt, J. and Rosa, F. M. (1994). Disruption of mesoderm and axis formation in fish by ectopic expression of activin variants: the role of maternal activin. Genes Dev 8, 1448-62. Wolenski, J. S. and Hart, N. H. (1987). Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg. J Exp Zool 243, 259-73. Wolke, U., Weidinger, G., Koprunner, M. and Raz, E. (2002). Multiple levels of posttranscriptional control lead to germ line-specific gene expression in the zebrafish. Curr Biol 12, 289-94. Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M., Joseph, E. and Heasman, J. (1996). Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos. Development 122, 2987-96. 166 Xanthos, J. B., Kofron, M., Wylie, C. and Heasman, J. (2001). Maternal VegT is the initiator of a molecular network specifying endoderm in Xenopus laevis. Development 128, 167-80. Yabe, S., Tanegashima, K., Haramoto, Y., Takahashi, S., Fujii, T., Kozuma, S., Taketani, Y. and Asashima, M. (2003). FRL-1, a member of the EGF-CFC family, is essential for neural differentiation in Xenopus early development. Development 130, 2071-81. Yamanaka, Y., Mizuno, T., Sasai, Y., Kishi, M., Takeda, H., Kim, C. H., Hibi, M. and Hirano, T. (1998). A novel homeobox gene, dharma, can induce the organizer in a non-cell-autonomous manner. Genes Dev 12, 2345-53. Yang, J., Tan, C., Darken, R. S., Wilson, P. A. and Klein, P. S. (2002). Betacatenin/Tcf-regulated transcription prior to the midblastula transition. Development 129, 5743-52. Yeo, C. and Whitman, M. (2001). Nodal signals to Smads through Cripto-dependent and Cripto-independent mechanisms. Mol Cell 7, 949-57. Yokota, C., Kofron, M., Zuck, M., Houston, D. W., Isaacs, H., Asashima, M., Wylie, C. C. and Heasman, J. (2003). A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor. Development 130, 2199-212. Yoon, C., Kawakami, K. and Hopkins, N. (1997). Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124, 3157-65. Yoon, Y. J. and Mowry, K. L. (2004). Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin. Development 131, 303545. Zernicka-Goetz, M. (2002). Patterning of the embryo: the first spatial decisions in the life of a mouse. Development 129, 815-29. Zhang, J., Talbot, W. S. and Schier, A. F. (1998). Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92, 241-51. Zhou, X., Sasaki, H., Lowe, L., Hogan, B. L. and Kuehn, M. R. (1993). Nodal is a novel TGF-beta-like gene expressed in the mouse node during gastrulation. Nature 361, 543-7. Zunkler, B. J., Wos-Maganga, M. and Panten, U. (2004). Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents. Biochem Pharmacol 67, 1437-44. 167 [...]...Summary: The generation of polarity and patterning during both vertebrate and invertebrate embryogenesis depends in part on the asymmetric localization of molecules The growing oocytes achieve polarity by localizing different cell fate determinants in the form of mRNA or protein to separate parts of the oocytes and influence axis specification In vertebrates, the mechanism of DV axis specification... rotation” The molecules are transported to the equatorial region using the physical process cortical rotation and microtubules (Vincent and Gerhart, 1987) These proteins are mainly the signaling 15 molecules of the classical Wnt signaling pathway They include b-catenin, glycogen synthase kinase 3 (GSK 3), GSK 3 binding protein (GBP) and dishivelled (Dsh) (Weaver and Kimelman, 2004) Stabilization of b-catenin... localization of a actin mRNA to the leading edge of the cell (Sundell and Singer, 1991) The leading edge of a cell is the one which shows maximum lamellopodia or filopodia It was thought that the transcripts of a actin gene is utilized after localization for the maintenance and extension of cellular filopodia Presumably the crawling cells acquire polarity by localizing actin mRNA to the leading edge and delocalization... dorsal side is essential in frog eggs for the formation of the dorsal organizer GSK3 binding protein GBP is required on the dorsal side to inhibit the activity of the GSK3 and thus stabilizing b-catenin GBP binds directly to Dsh, a GSK3 inhibitor Binding of GBP to Dsh activates GSK3 and leads to degradation of b-catenin These molecules are transported from the vegetal cortex to the future dorsal side... accumulation of oskar mRNA is microtubule dependent During its localization, oskar RNA travels to the plus end of the microtubules along with the kinesin heavy chain at the posterior pole (Palacios and St Johnston, 2002) The splicing of oskar transcript is essential for oskar localization (Hachet and Ephrussi, 2004) Binding of BRUNO, an RNA binding protein, inhibits translation of the oskar RNA The 3’UTR of. .. formation Apart from these localized RNAs, several other proteins are present at the vegetal cortex of an oocyte in the form of protein In Xenopus embryos, fertilization leads to movement of these proteins from the vegetal cortex to a new equatorial position, on the side opposite to the sperm entry The cortex of the fertilized embryo starts rotating in the direction opposite to the sperm entry, the event known... This is so far the first known event of asymmetry during zebrafish embryogenesis that marks the DV axis Patterns of mRNA localization in zebrafish can be divided into two groups Group one, contains mRNAs getting localized to the precise position of the oocytes These transcripts are maternally expressed and they get segregated to the specific site of oocyte and may be involved in specifying the animal-vegetal... properly localized and upon perception of the correct signals Some of these RNAs are not actively synthesized in the developing oocyte but rather pumped into by surrounding nurse cells Bicoid transcripts are an example of such RNAs Localization of bicoid transcripts is dependent on its 3’UTR and RNA binding proteins One of such RNA binding protein, Staufen forms particles with bicoid RNA These particles... last few decades have identified that such determinants are either in the form of mRNA or as protein and influence early axis specification (Minakhina and Steward, 2005) 1.1.1 Polarity in S cervisiae (Budding Yeast) In budding yeast, S cerevisiae the mother cell expresses a set of proteins different from the daughter cell Mother cell expresses HO protein asymmetrically, while daughter cell accumulates... identified for the localization of different mRNAs In general, RNAs such as Xcat2 and Xdazl, which are components of the germplasm, localize during the early pathway and associae with the mitochondrial cloud (Kloc and Etkin, 1995; Kloc and Etkin, 1998) The transcripts of Vg1 and VegT involved in somatic cell fate specification localize to the vegetal cortex of an oocyte, and utilize the late localization . i LOCALIZED MOLECULES AND THE ESTABLISHMENT OF POLARITY IN ZEBRAFISH ANIKET V. GORE (M. Sc. UNIVERSITY OF PUNE, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK. layer x Abstract: The generation of polarity and patterning during both vertebrate and invertebrate embryogenesis depends in part on the asymmetric localization of molecules. In the zebrafish, Danio. localized RNAs are crucial players involved in generation of cell/oocyte polarity and patterning of early embryos. The molecules involved in this process change according to the requirement and