Molecular Regulation of Gliogenesis in Xenopus Laevis During Primary
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Molecular Regulation of Gliogenesis in Xenopus laevis during Primary Neurogenesis ------------------------------------------------------ A Dissertation Presented to the Faculty of the Department of Biology and Biochemistry University of Houston ------------------------------------------------------ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy ------------------------------------------------------ By Christina Helen Ulrich August 2019 Molecular Regulation of Gliogenesis in Xenopus laevis during Primary Neurogenesis ______________________________ Christina H. Ulrich APPROVED: ______________________________ Dr. Amy K. Sater, Chair ______________________________ Dr. Brigitte Dauwalder ______________________________ Dr. Arne Lekven ______________________________ Dr. Rachel K. Miller ______________________________ Dean, College of Natural Sciences and Mathematics ii In memory of my mom, and for my dad. iii Acknowledgements First, I would like to thank my mentor and friend, Dr. Amy K. Sater. She has provided countless hours of teaching and feedback and without her continual support and encouragement I could not have completed my studies. I would also like to thank my committee members, Dr. Brigitte Dauwalder, Dr. Rachel K. Miller, and Dr. Arne Lekven. They have provided valuable insight and asked questions that opened new avenues of thinking in my project. I would also like to recognize Dr. Robert Schwartz, he provided support and encouragement during difficult periods of my graduate studies. I am grateful to my family; my parents, Nancy and Sam Ulrich, and my siblings, Samantha, Marta, and Ben, who patiently listened to me practice my science communication on them. Without their love and support, I would never have started or finished graduate school. I want to thank my incredibly supportive partner Jon, his love of science and unflagging enthusiasm offsets my more pessimistic view of the world. I want to acknowledge Ella, who never passed up an opportunity to keep me company in lab and is an unparalleled frog detector. I would like to express my deepest thanks to my colleagues and the members of my lab, both past and present. Dr. Vrutant Shah, Dr. Ruth Ritter, Ray Torres, Melissa Zamora, Sydnee Eldridge, and Mahmoud Al Homouz have all provided wisdom, conversation, and assistance whenever I needed. Finally, I would like to recognize and thank the undergraduates I have been lucky enough to mentor, Anna Subonj, Devanshi Singh, Frida Mora, and Eric Murphy. iv Molecular Regulation of Gliogenesis in Xenopus laevis during Primary Neurogenesis ------------------------------------------------------ An Abstract of a Dissertation Presented to the Faculty of the Department of Biology and Biochemistry University of Houston ------------------------------------------------------ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy ------------------------------------------------------ By Christina Helen Ulrich August 2019 v Abstract Glia play essential roles in the vertebrate nervous system. In adults, glia can become mis-regulated, leading to the formation of tumors, including gliomas and glioblastoma (GBM). Understanding glial development is integral to elucidating the mechanisms responsible for the formation and progression of GBM. However, the signals required for initiation of glial development have yet to be completely characterized. Our preliminary studies in Xenopus laevis indicate that glial specification is initiated between Nieuwkoop and Faber (NF) stages 16-18 in the anterior spinal cord. Glial specification progresses posteriorly down the neural tube, occurring anterior of the midbrain-hindbrain boundary only at NF stage 24. Gene expression analysis on isolated explants consisting of either mid-gastrula neural ectoderm or animal cap ectoderm overexpressing the BMP inhibitor Noggin demonstrated that neural plates transcribed glial associated genes at levels akin to those of their whole embryo siblings. In contrast, the expression of glial genes in Noggin-injected animal caps (NogAC), such as olig3, sox10, glast, and glt-1, was significantly reduced. These findings suggest that (1) inhibition of BMP signaling alone is insufficient to induce gliogenesis; and (2) signals from the dorsal mesoderm during gastrulation are required for the initiation of gliogenesis at later stages. Pair-wise comparisons between the transcriptomes of mid-gastrula (NF Stage 11) and mid-neurulation (NF Stage 18) neural plates and NogACs elucidated underlying differences accounting for their distinct developmental potentials. Functional annotation of the differentially expressed genes revealed that glial transcription factors such as pou3f2, sox9, sox10, olig3, and rfx4 and members of Wnt, FGF, and RA signaling vi pathways were differentially expressed in neural plates. Pharmacological inhibition of these signaling pathways in neural plates suggested distinct functions in glial differentiation. Ectopic expression of TAZ, the master regulator of mesenchymal differentiation in GBM, in X. laevis embryos encouraged persistence of an undifferentiated neural progenitor population, and potentially inhibited glial specification. Our results indicate that signals important for gliogenesis are active in the Xenopus embryos as early as mid-gastrulation and have direct implications in further GBM research. vii Table of Contents Chapter I: Introduction………………………………………………………….…………1 1.1 Overview of Glial Cells……………………………………………………………. 2 1.2 Glial Development and Timing of the Gliogenic Switch…………………………...6 1.3 Xenopus laevis as a Developmental Model for Gliogenesis……………………….11 1.4 Xenopus Neural Development and Specification………………………………….12 1.5 Extrinsic Signals in Gliogenesis…………… ……………………………………15 1.6 Glia in Disease……………………………………………………………………..17 1.7 Project Outline……………………………………………………………………..21 Chapter II: Materials and Methods………………………………………………………23 2.1 Xenopus laevis Care and Embryo Culture…………………………………………24 2.2 Microinjection of Xenopus laevis Embryos………………………………………..25 2.3 Explant Isolation and Culture……………………………………………………...27 2.4 Cloning…………………………………………………………………………….28 2.4a Fragment Cloning…………………………………………………………..28 2.4b Vector Construction………………………………………………………...32 2.4c Plasmid DNA Isolation……………………………………………………..33 2.4d Sequence Confirmation.…………………………………………………….34 2.4e TAZ Subcloning…………………………………………………………….35 2.5 RNA Isolation……………………………………………………………………...35 2.6 cDNA Synthesis……………………………………………………………………38 2.7 Quantitative RT-PCR………………………………………………………………40 2.8 RNA Synthesis……………………………………………………………………..44 2.8a Plasmid DNA Linearization………………………………………………...44 2.8b In situ Probe Synthesis……………………………………………………...45 2.8c Capped RNA Synthesis……………………………………………………..46 2.9 In situ Hybridizations……… …………………………………………………….47 2.10 Sectioning, Imaging, and Image analysis………………………………………...51 viii 2.10a Sectioning………………………………………………………..………..51 2.10b Imaging………………………………………………………………...….52 2.10c Image Analysis……………………………………………...……………..53 2.11 Poly(A) Selected Total RNA Library Preparation………………………………..53 2.12 Transcriptome Sequencing Analysis……………………………………………...55 2.13 Small Molecule Inhibition of Wnt, FGF, RA, and Notch Signaling in NP………61 Chapter III: Initiation of Gliogenesis in Xenopus laevis…………………………………62 3.1 Overview…………………………………………………………………………...63 3.2 Temporal Expression of Glial Associated Genes in X. laevis …………………….67 3.3 Spatial Expression of Glial Associated Genes in X. laevis……………….………..73 3.4 Summary…………………………………………………………………………...81 Chapter IV: Regulation of the Initiation of Gliogenesis by Extrinsic Signals…………...82 4.1 Overview…………………………………………………………………………...83 4.2 Competency of Xenopus Explants in Initiating Gliogenesis……………...………..88 4.3 Sufficiency of sox9 and nfix in Gliogenesis During Primary Neurogenesis...……100 4.4 Discovery Based Identification of Differences Between Noggin Animal Cap and Neural Plate Explants During Gastrulation and Neurulation………….……………...103 4.5 In vivo Verification of Identified Genes and Signaling Pathways in Gliogenesis..130 4.6 Summary………………………………………………………………………….141 Chapter V: Effects of Aberrant Expression of TAZ on neurogenesis and gliogenesis...145 5.1 Overview of Canonical and Aberrant Roles of TAZ in Development and Disease146 5.2 Summary of Preliminary Xenopus laevis Research …………………………..….149 5.3 Replication of 2-cell Stage Injections……...……...……………………………...150 5.4 Effects of TAZ Overexpression on Neurogenesis……..…………………………156 5.5 Impact of Overexpression of Human TAZ on Gliogenesis……….……………...162 5.6 Effects of Overexpression of Human TAZ on Neural Crest Differentiation and Mesenchymal Gene Expression……………..………………………………………..165 5.7 Summary………………………………………………………………………….167 Chapter VI: Discussion…………………………………………………………………169 ix 6.1 Timing of the Gliogenic Switch in X. laevis……………………………………...170 6.2 Competence of Ectodermal Explants……………………………………………..172 6.3 Roles of Signaling in the Gliogenic Switch………………………………………174 6.4 Effects of Aberrant Expression of Taz on Glial Development………...…………177 6.5 Future Directions and Research Implications……………………………..……...178 References………………………………………………………………………………180 x List of Figures Figure 1.1 Early glial specification …………………………………………………..…...5 Figure 1.2 Glial specification transcription factor network ………………………………9 Figure 2.1 Fragment amplification scheme……………………………………………...29 Figure 2.2 cDNA synthesis scheme……………………………………………………...39 Figure 2.3 Quantitative reverse transcriptase reaction scheme…………………………..41 Figure 2.4 Transcriptome