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Bptf Is Essential for Murine Neocortical Development

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Bptf Is Essential for Murine Neocortical Development Bptf is essential for murine neocortical development Gerardo Zapata Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Master’s degree in Biochemistry with specialization in Bioinformatics Department of Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa © Gerardo Zapata, Ottawa, Canada, 2020 Abstract Chromatin remodeling complexes modulate DNA accessibility permitting neuronal progenitor cells to proliferate and differentiate to form the mammalian neocortex. In the case of BPTF (Bromodomain PHD transcription Factor), the major subunit of a chromatin remodelling complex called NURF (Nucleosome Remodelling Factor), mutations leading to its haploinsufficiency have been linked to cause a recently annotated human neurodevelopmental disorder called NEDDFL (Neurodevelopmental disorder with dysmorphic facies and distal limb anomalies). Patients with this syndrome are mainly characterized with microcephaly and intellectual disability. We conditionally knockout (cKO) the Bptf gene during neocortical neurogenesis to analyze its role during embryonic and postnatal brain development. The Bptf cKO animals reveal significant forebrain hypoplasia. During cortical neurogenesis, the Bptf cKO mice show a reduction in intermediate neuronal progenitor (INP) cells, an increase in apoptosis as well as a prolonged cell cycle within proliferating progenitors. Similarly, the postmitotic pyramidal neurons of the Bptf cKO mice contained lower levels of Ctip2 and Foxp1. Lastly, our RNA-seq analysis delineated gene pathways deregulated by Bptf removal, which are involved in neurogenesis and neuronal differentiation. Our results indicate that Bptf is critical for murine telencephalon neurogenesis. The hypoplasia demonstrated in the mouse model can resemble the microcephaly displayed by the human NEDDFL patients, highlighting the relevance of chromatin remodelling complexes during intricate neural developmental processes. II Acknowledgements Tonia, your undying support, love and encouragement strengthened my desire to strive for greater things in life and pushed me further to seek a higher level of education. The completion of this thesis is a result of our accumulated efforts. Mamá, Papá y Ana María, todo lo que soy y todo lo que eh logrado es gracias a ustedes, esta tesis es tanto suya como mía. Sin su guía, apoyo y consejos, yo no hubiera sido capaz de haber empezado esta segunda etapa de mi vida. Juan, siempre has sido un gran modelo a seguir. Asimilar tu buen humor me ha ayudado a disfrutar lo que eh logrado y, aunque de lejos, tu ejemplo me dio las fuerzas necesarias para seguir luchando en tiempos difíciles y poder finalizar mi maestría. Dave, thank you for providing me with the opportunity to become part of the lab. I have learned a great deal, and in your lab, I have begun the next and exciting chapter of my life. Raies and Keqin, I learned almost all the techniques and experiments used in this thesis from both of you. Thank you so much for your patience, understanding and great company. III Table of Contents Abstract ......................................................................................................................................................... II Acknowledgements ..................................................................................................................................... III List of Abbreviations ................................................................................................................................... VI List of Figures ...........................................................................................................................................VIII List of Tables ............................................................................................................................................... IX 1. Introduction ........................................................................................................................................... 1 1.1. Cortical Neurogenesis ................................................................................................................... 1 1.1.1. Progenitor pool of the neocortex ........................................................................................... 1 1.1.2. Neurons of the cortical plate and their transcription factors ................................................. 3 1.1.3. Gliogenesis and Microglia Origins ........................................................................................ 5 1.2. Neurodevelopmental disorders and chromatin remodelers ........................................................... 6 1.3. Chromatin and nucleosome organization ...................................................................................... 7 1.4. Chromatin remodelers, their mode of function and role in neurodevelopmental disorders ........ 11 1.4.1. SWI/SNF ............................................................................................................................. 12 1.4.2. CHD .................................................................................................................................... 14 1.4.3. INO80 .................................................................................................................................. 15 1.4.4. ATRX .................................................................................................................................. 16 1.4.5. ISWI .................................................................................................................................... 16 1.4.5.1. ISWI mouse models ........................................................................................................ 18 1.5. Bromodomain PHD transcription factor (BPTF) ........................................................................ 19 1.6. Neurodevelopmental disorder with dysmorphic facies and distal limb anomalies ..................... 22 1.7. Hypothesis & thesis aims ............................................................................................................ 24 2. Materials & Methods ........................................................................................................................... 25 2.1. Transgenic mice .......................................................................................................................... 25 2.1.1. Animal Husbandry .............................................................................................................. 25 2.1.2. Mouse lines.......................................................................................................................... 25 2.1.2.1. Bptf loxp lines .................................................................................................................. 25 2.1.2.2. Cre driver lines ................................................................................................................ 25 2.1.3. Genotyping .......................................................................................................................... 26 2.1.4. Timed Breeding ................................................................................................................... 27 2.2. Tissue dissection for nucleic acid or protein extraction .............................................................. 28 2.3. Analysis of cortical tissue ............................................................................................................ 29 2.3.1. Cryo-sectioning of fixed tissue ............................................................................................ 29 IV 2.3.2. Nissl staining ....................................................................................................................... 29 2.3.3. Immunofluorescent staining ................................................................................................ 30 2.3.4. EdU pulse labelling ............................................................................................................. 30 2.3.5. In-situ Hybridization ........................................................................................................... 31 2.3.6. Quantification of stained tissue ........................................................................................... 33 2.4. Nucleic acid isolation from frozen tissue .................................................................................... 33 2.4.1. RNA isolation ...................................................................................................................... 33 2.4.2. cDNA preparation ............................................................................................................... 34 2.4.2.1. RT-PCR ........................................................................................................................... 35 2.4.2.2. RT-qPCR ......................................................................................................................... 35 2.4.3. RNA-sequencing data processing and analysis ................................................................... 36 3. Results ................................................................................................................................................. 38 3.1 Bptf conditional Knockouts – Nestin Cre .................................................................................... 38
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