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. 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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