Functional analysis of Prdm14 during Xenopus embryogenesis 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 Rolf Patrick Berndt born in Tokio, Japan Göttingen, April 2015 Members of the Thesis Committee: Dr. Kristine A. Henningfeld (Supervisor, Reviewer) Institute of Developmental Biochemistry, University of Göttingen Prof. Dr. Gregor Bucher (Reviewer) Department of Evolutionary Developmental Genetics, University of Göttingen Prof. Dr. Andreas Wodarz Department of Microscopic Anatomy and Molecular Cell Biology, University of Cologne Affidavit Herewith, I declare that I prepared the PhD thesis "Functional analysis of Prdm14 during Xenopus embryogenesis" on my own and with no other sources and aids than quoted. Submission date Göttingen, 30.04.2015 _________________________ Rolf Patrick Berndt Table of Contents I Table of Contents Table of Contents ............................................................................................... I Acknowledgements ......................................................................................... V Abstract ........................................................................................................... VI List of Figures ............................................................................................... VIII List of Tables ................................................................................................... XI Abbreviations ................................................................................................. XII 1. Introduction ................................................................................................... 1 1.1 Xenopus neurogenesis ................................................................................ 1 1.2 Neural induction ........................................................................................... 2 1.3 Stabilization of the neural fate ..................................................................... 5 1.4 Neuronal differentiation ............................................................................... 6 1.5 Neurulation and neuronal subtype specification ...................................... 7 1.6 Lateral inhibition ........................................................................................... 9 1.7 Neural crest ................................................................................................. 11 1.8 The Prdm protein family ............................................................................. 13 1.8.1 Structural properties of Prdm proteins ...................................................... 13 1.8.2 The role of Prdms in early vertebrate neurogenesis and neural crest formation ............................................................................................................... 15 1.8.2.1 Prdms in neural crest formation ......................................................................... 15 1.8.2.2 Regulation of neuronal subtype specification by Prdms .................................... 16 1.8.2.3 Prdms in axon outgrowth ................................................................................... 16 1.8.3 Prdm14 is an epigenetic regulator and stemness factor ........................... 17 1.9 Aims ............................................................................................................. 18 2. Material and Methods .............................................................................. 19 2.1 Material ........................................................................................................ 19 2.1.1 Model organism ........................................................................................ 19 2.1.2 Bacteria ..................................................................................................... 19 2.1.3 Antibiotics and Media ................................................................................ 19 2.1.4 Oligonucleotides ....................................................................................... 19 2.1.4.1 RT-PCR Oligonucleotides (Primer) .................................................................... 20 2.1.4.2 Sequencing oligonucleotides ............................................................................. 20 2.1.4.3 Morpholino oligonucleotides .............................................................................. 20 2.1.5 Overexpression constructs .......................................................................... 21 Table of Contents II 2.1.6 antisense-RNA-constructs ........................................................................... 22 2.2 Methods ............................................................................................................. 23 2.2.1 DNA-standard methods ............................................................................... 23 2.2.1.1 Polymerase chain reaction (PCR) ...................................................................... 23 2.2.1.1.1 Reverse transcription-PCR (RT-PCR) ........................................................ 23 2.2.1.1.2 PCR Cloning ............................................................................................... 23 2.2.1.2 Agarose gel electrophoresis .............................................................................. 23 2.2.1.3 Gel purification of PCR and restriction fragments .............................................. 24 2.2.1.4 DNA restriction digestion ................................................................................... 24 2.2.1.5 Ligation ............................................................................................................... 24 2.2.1.6 Chemical transformation of bacterial cells ......................................................... 24 2.2.1.7 Plasmid preparation ........................................................................................... 25 2.2.1.8 DNA sequencing ................................................................................................ 25 2.2.2 RNA standard methods ............................................................................... 25 2.2.2.1 In vitro synthesis of capped sense mRNA ......................................................... 25 2.2.2.2 In vitro synthesis of antisense RNA ................................................................... 26 2.2.2.3 Total RNA isolation from ectodermal explants and whole embryos .................. 26 2.2.2.4 Reverse transcription ......................................................................................... 27 2.2.2.5 RNA-sequencing ................................................................................................ 27 2.2.2.5.1 Total RNA isolation from ectodermal explants ........................................... 27 2.2.2.5.2 Sample preparation and sequencing .......................................................... 28 2.2.2.5.3 Sequencing alignment (performed by TAL) ................................................ 28 2.2.2.5.4 Statistical analysis (performed by TAL) ...................................................... 28 2.2.3 X. laevis embryo culture and micromanipulations ....................................... 29 2.2.3.1 Stimulation of eggs ............................................................................................ 29 2.2.3.2 Preparation of X. laevis testis ............................................................................ 29 2.2.3.3 In vitro fertilization .............................................................................................. 29 2.2.3.4 Microinjections ................................................................................................... 30 2.2.3.5 X. laevis ectodermal explants (“animal caps”) ................................................... 30 2.2.3.6 Dexamethasone treatment ................................................................................. 30 2.2.3.7 β-Gal staining ..................................................................................................... 31 2.2.4 Whole mount in situ-hybridization (WMISH) ................................................ 31 2.2.5 Phosphorylated Histone 3 (pH3) staining .................................................... 33 2.2.6 Fluorescent immunostaining ........................................................................ 34 2.2.7 Luciferase reporter assay ............................................................................ 35 2.2.8 Vibratome sectioning ................................................................................... 35 2.2.9 Protein standard methods ........................................................................... 36 2.2.9.1 SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) ...................................... 36 2.2.9.2 Western blotting ................................................................................................. 36 3. Results ......................................................................................................... 38 Table of Contents III 3.1 Temporal and spatial expression analysis of X. laevis prdm14 ................... 38 3.2 Prdm14 gain of function .................................................................................. 43 3.3. Prdm14 promotes proliferation and the expansion of neural progenitors 44 3.4 Prdm14 promotes ectopic