Mechanisms Underlying the Temporal and Selective Induction of Ptf1a Target Genes
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Mechanisms underlying the temporal and selective induction of Ptf1a target genes Doctoral Thesis Dissertation for the award of the degree "Doctor rerum naturalium (Dr. rer. nat.)" in the GGNB program "Genes and Development“ at the Georg August University Göttingen Faculty of Biology submitted by Sven Richts born in Werl, Germany Göttingen, December 2017 Members of the Thesis Committee: Dr. Kristine A. Henningfeld (Supervisor, Reviewer) Department of Developmental Biochemistry, University of Göttingen Prof. Dr. Steven Johnsen (Reviewer) Clinic for General, Visceral and Pediatric Surgery, University of Göttingen Prof. Dr. Thomas Dresbach Department for Anatomy and Embryology University of Göttingen Affidavit Herewith, I declare that I prepared the PhD thesis "Mechanisms underlying the temporal and selective induction of Ptf1a target genes " on my own and with no other sources and aids than quoted. Submission date Göttingen, 31.12.2017 _________________________ Sven Richts Table of contents I Table of contents Table of contents .......................................................................................... I Acknowledgements .................................................................................... VI Abstract ...................................................................................................... VII List of figures ............................................................................................ VIII List of tables ................................................................................................. X Abbreviations ............................................................................................. XII 1. Introduction .............................................................................................. 1 1.1 Neurogenesis in Xenopus laevis ....................................................... 1 1.2 Neural induction ................................................................................. 2 1.3 bHLH transcription factors and their role during neuronal differentiation and determination ............................................................ 4 1.4 Lateral inhibition ................................................................................. 8 1.5 Neuronal subtype specification ....................................................... 10 1.6 Role of phosphorylation in regulating neurogenesis .................... 13 1.7 Epigenetic regulation of neurogenesis ........................................... 14 1.8 Changes in the epigenetic landscape during development .......... 19 1.9 Ptf1a ................................................................................................... 20 1.10 Ptf1a forms a trimeric transcription factor complex .................... 23 1.11 Gene induction and regulation by Ptf1a ....................................... 24 1.12 Aims ................................................................................................. 26 2. Materials and Methods .......................................................................... 27 2.1 Materials ............................................................................................ 27 2.1.1 X. laevis ........................................................................................ 27 2.1.2 Bacteria ........................................................................................ 27 2.1.3 Chemicals ..................................................................................... 27 2.1.4 Antibiotics and Media ................................................................... 27 Table of contents II 2.1.5 Oligonucleotides............................................................................ 28 2.1.5.1 RT-PCR oligonucleotides ........................................................ 28 2.1.5.2 Sequencing oligonucleotides .................................................. 29 2.1.6 Sense RNA constructs .................................................................. 30 2.1.7 Antisense RNA constructs ............................................................ 34 2.2 Methods ............................................................................................. 35 2.2.1 In vitro synthesis of capped sense RNA ........................................ 35 2.2.2 Xenopus methods ......................................................................... 35 2.2.2.1 Priming of X. laevis frogs ........................................................ 35 2.2.2.2 Preparation of the testis .......................................................... 36 2.2.2.3 In vitro fertilization ................................................................... 36 2.2.2.4 Microinjection .......................................................................... 36 2.2.2.5 Preparation of ectodermal explants (animal caps) .................. 36 2.2.2.6 Dexamethasone treatment ...................................................... 37 2.2.2.7 Bimolecular Fluorescent Complementation assay (BiFC) ....... 37 2.2.2.8 In vitro synthesis of antisense RNA ........................................ 37 2.2.2.9 Whole mount in-situ hybridization (WMISH) ........................... 38 2.2.2.9.1 Fixation and X-Gal staining............................................... 39 2.2.2.9.2 Rehydration ...................................................................... 39 2.2.2.9.3 Proteinase K treatment ..................................................... 40 2.2.2.9.4 Acetylation ........................................................................ 40 2.2.2.9.5 Hybridization reaction ....................................................... 40 2.2.2.9.6 Washings .......................................................................... 40 2.2.2.9.7 Antibody reaction .............................................................. 41 2.2.2.9.8 Staining reaction ............................................................... 41 2.2.2.9.9 Background removal and bleaching ................................. 41 2.2.3 Agarose gel electrophoresis .......................................................... 41 2.2.4.1 PCR cloning ............................................................................ 42 Table of contents III 2.2.4.2 Gel purification of PCR and restriction fragments .................. 42 2.2.4.3 DNA restriction digestion ........................................................ 42 2.2.4.4 Ligation................................................................................... 42 2.2.4.5 Chemical transformation of bacterial cells .............................. 43 2.2.4.6 Plasmid DNA preparation ....................................................... 43 2.2.4.7 DNA sequencing .................................................................... 43 2.2.5 ATAC sequencing ........................................................................ 44 2.2.5.1 Transposition reaction ............................................................ 44 2.2.5.2 Library preparation ................................................................. 44 2.2.5.3 Sequencing (by TAL) ............................................................. 45 2.2.5.4 Sequencing alignment and processing (performed by TAL)... 45 2.2.6 Gene expression analysis by semiquantitative RT-PCR or quantitative Nanostring analysis ............................................................ 46 2.2.6.1 Total RNA isolation from ectodermal explants and whole embryos ............................................................................................. 46 2.2.6.2.1 Reverse transcription ....................................................... 47 2.2.6.2.2 Reverse transcription-PCR (RT-PCR) ............................. 47 2.2.6.3 Quantitative Nanostring analysis ............................................ 47 2.2.7 RNA sequencing .......................................................................... 48 2.2.7.1 RNA isolation ......................................................................... 48 2.2.7.2 Sample preparation and sequencing ...................................... 49 2.2.7.3 Sequencing alignment (performed by TAL) ............................ 49 2.2.7.4 Statistical analysis (performed by TAL) .................................. 49 3. Results .................................................................................................... 50 3.1 Mutation of a single threonine residue in the Ptf1a C2 domain is sufficient to induce a mixed induced neuronal transmitter phenotype ................................................................................................................. 50 3.2 Introducing a negative charge at the Ptf1aT243 residue induces a more wildtype Ptf1a like phenotype ...................................................... 56 Table of contents IV 3.3 The mixed neuronal transmitter phenotype induced by Ptf1aT243A is not the result of an impaired interaction with Rbpj .......................... 57 3.4 Ptf1aT243 mutants strongly bind to Prdm13 .................................... 58 3.5 Ptf1a induces direct target genes at different time points............. 62 3.6 Temporal expression of Ptf1a target genes by RNA sequencing . 65 3.7 Ptf1a target genes are highly enriched for playing roles during transcription and neurogenesis ............................................................. 67 3.8 A knock-down of Brg1 affects the induction of indirect Ptf1a target genes ........................................................................................................ 69 3.9