Institut für Entwicklungsgenetik GSF Forschungszentrum für Umwelt und Gesundheit TUM Zebrafisch Neurogenetik The induction and early development of the midbrain-hindbrain in the embryonic zebrafish Alexandra Tallafuß Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grads eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Erwin Grill Prüfer der Dissertation: 1. Univ.-Prof. Dr. Wolfgang Wurst 2. Univ.-Prof. Dr. Alfons Gierl 3. Univ.-Prof. Dr. Heinrich H.D. Meyer Die Dissertation wurde am 21.02.03 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 15.04.03 angenommen. TABLE OF CONTENTS i Table of Contents Abstract......................................................................................................................................iv Zusammenfassung......................................................................................................................v Abbreviations used in the text..................................................................................................vii Chapter I: Introduction...............................................................................................................1 1. Neural plate patterning...................................................................................................2 1.1. Neural plate induction..........................................................................................2 1.2. Common features of early neural plate patterning in vertebrates........................5 1.3. Patterning refinement along AP by local signaling centers.................................7 1.4. Early AP neural plate patterning in zebrafish......................................................8 2. The midbrain-hindbrain domain...................................................................................15 2.1. Position and functional derivatives....................................................................15 2.2. The MHD follows an atypical mode of development that responds to an organizer.............................................................................16 2.3. Molecular bases of MHD development and IsO activity...................................18 2.4. Major unanswered questions of MHD development..........................................21 2.5. Formation of the MHD in zebrafish...................................................................22 3. Neurogenesis and bHLH factors...................................................................................24 3.1. Delimiting neurogenesis sites within the vertebrate neural plate: proneural cluster..................................................................................................24 3.2. The neurogenesis process and lateral inhibition.................................................26 3.3. Main actors of the neurogenesis process: bHLH factors....................................27 Chapter II: Aims of the Thesis..................................................................................................32 Chapter III: Results and Discussion..........................................................................................34 1. Bts1 is the earliest selective regulator of pax2.1 in the MHD...................................35 2. Evolutionary consideration about the vertebrate MHD............................................35 3. Signals from the PCP control the size of the six3-positive neuronal cluster.............35 4. The transgenic line herPAC::egfp identifies her5 regulatory elements and allows to follow the fate of the entire prospective MHD.......................................36 5. Molecular and functional analyses of the bHLH factor her5........................................38 6. Identification of a new hairy/E(spl) gene, called him, and preliminary functional results.................................................................................39 7. To do ... .........................................................................................................................43 References.................................................................................................................................45 TABLE OF CONTENTS ii Appendix 1: The zebrafish buttonhead-like factor Bts1 is and early regulator of pax2.1 expression during mid-hindbrain development............................................56 Appendix 2: Formation of the head-trunk boundary in the animal body plan: an evolutionary perspective..........................................................................57 Appendix 3: Selective control of neuronal cluster size at the di-mesencephalic boundary by long-range signaling from the prechordal plate...............................58 Appendix 4: Tracing of her5 progeny in zebrafish transgenics reveals the dynamics of midbrain-hindbrain neurogenesis and maintenance.............................................59 Appendix 5: Regional inhibition of neurogenesis by the zebrafish bHLH transcription factor Her5 shapes the midbrain-hindbrain neuronal differentiation pattern............................................60 Chapter IV: Materials and Methods..........................................................................................61 1. Abbreviations and symbols used in the chapter Materials and Methods..................61 2. Recipes of often used Buffers and Solutions............................................................63 3. Animal model organism: Zebrafish...........................................................................66 3.1. Description and origin................................................................................66 3.2. Animal keeping and breeding.....................................................................67 3.3. Embryos......................................................................................................67 4. Molecular Techniques...............................................................................................67 4.1. Preparation of nucleic acids........................................................................67 4.1.1. Isolation of genomic DNA from embryos...................................67 4.1.2. Isolation of genomic DNA from tail-fins....................................68 4.1.3. Isolation of total RNA from embryos..........................................68 4.1.4. Preparation of cDNA...................................................................68 4.1.5. Plasmid DNA preparation...........................................................68 4.2. Preparation of digoxigenin- or flourescein- labeled anti-sense probes......68 4.3. Amplification of DNA by polymerase chain reaction (PCR)....................69 4.4. PCR purification.........................................................................................69 4.5. Isolation and purification of DNA fragments using Gel Extraction...........69 4.6. Ligation and Transformation......................................................................69 4.7. Transfer and detection of nucleic acids......................................................70 4.7.1. Southern Blot...............................................................................70 4.7.2. Detection of DNA probes............................................................70 4.7.3. Hybridization and detection.........................................................70 4.7.4. Autoradiography..........................................................................71 4.8. Analysis of sequences.................................................................................71 TABLE OF CONTENTS iii 5. Staining of embryos using in situ hybridization (ISH) and immunocytochemistry (IC).....................................................................................71 5.1. Preparation of embryos for ISH and IC......................................................71 5.2. Whole-mount ISH on zebrafish embryos...................................................71 5.2.1. Single color ISH..........................................................................71 5.2.2. Two-color ISH.............................................................................72 5.3. Immunocytochemistry (IC)........................................................................72 5.3.1. Whole-mount IC..........................................................................73 5.3.2. IC after ISH.................................................................................73 5.3.3. IC on cryostat sections.................................................................73 6. Preparation of flatmounts and sections.....................................................................74 6.1. Flatmounts..................................................................................................74 6.2. Cryostat sections.........................................................................................74 6.3. Plastic sections............................................................................................74 7. Scoring of the embryos.............................................................................................74 8. Additional and more detailed description of methods used in this Ph.D. work........74 Danksagung...............................................................................................................................75