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GREEN-DISSERTATION-2019.Pdf (9.107Mb) Characterization of the Temporal Dynamics of Wnt-mediated Posteriorization of the Vertebrate Neural plate A Dissertation Presented to the Faculty of the Department of Biology University of Houston In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy By David Green August, 2019 Characterization of the Temporal Dynamics of Wnt-mediated Posteriorization of the Vertebrate Neural plate ......................................................................................... David Green APPROVED: ......................................................................................... Dr. Arne C. Lekven, Chair ......................................................................................... Dr. Amy K. Sater ......................................................................................... Dr. Brigitte Dauwalder ......................................................................................... Dr. Daniel S. Wagner Rice University ......................................................................................... Dr. Dan E. Wells, Dean, College of Natural Sciences and Mathematics ii ACKNOWLEDGEMENTS I would first like to thank my committee Dr. Daniel Wagner, Dr. Brigitte Dauwalder and Dr. Amy Sater for their helpful comments and suggestions in writing this dissertation. I would also like to thank my Master's thesis committee Dr. Bruce Riley, Dr. Alvin Yeh and Dr. Rene Garcia who helped in many of the early stages of my doctoral research. A special thanks to Dr. Arne Lekven for all his help in making me the scientist I am today. Thank you to my parents for always supporting me throughout these years. This dissertation is dedicated to Katie, without you none of this would have been possible. iii Characterization of the Temporal Dynamics of Wnt-mediated Posteriorization of the Vertebrate Neural plate An Abstract of a Dissertation Presented to the Faculty of the Department of Biology University of Houston In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy By David Green August, 2019 iv ABSTRACT The vertebrate central nervous system is divided into a series of functional domains along the AP axis; forebrain, midbrain, hindbrain, and spinal cord. Failure to properly pattern the AP axis has been associated with a range of neuropathologies including schizophrenia and autism. In recent years, there have been numerous findings that have challenged dogmatic beliefs in how the AP axis is established during embryogenesis. After induction, the AP axis of the neural plate is established via several posteriorizing morphogens, including wnt8a. Wnt8a is secreted from the margin and forms a signaling gradient. The current dogma suggests that this signaling gradient is achieved by diffusion. Recent evidence supports an alternative model where Wnt8a is transported along cellular protrusions called filopodia. The filopodial model introduces several inconsistencies that must be resolved. Here we provide evidence that primary Wnt-mediated posteriorization of the neural plate occurs in a narrow window of time between 4.6 hpf and 7 hpf. The interpretation of this signal by the neural plate is complex and outcomes of these signals are sometimes not detected until several hours later. A secondary phase of Wnt-mediated posteriorization is regulated by Wnts expressed in the neural plate. These findings provide rationale for how filopodial-mediated Wnt-signaling occurs early in epiboly while the neural plate is still in range. Additionally our findings establish new temporal elements to the Wnt-mediated posteriorization paradigm, showing that the neural plate responds dynamically to Wnt signaling both in regards to competency to Wnt as well as the time it takes for Wnt signaling to affect transcription. Together these findings provide important steps to redefining the Wnt-signaling paradigm in lieu of recent challenges to the previous model. v Contents 1 INTRODUCTION 1 1.1 Patterning of the Anterior Posterior Axis of the Central Nervous System in Development and Disease . .1 1.2 Neural Induction and Patterning . .3 1.3 Mechanisms of Wnt Signaling . .4 1.4 Role of Morphogens in Patterning in the Neural Plate . .6 1.5 Summary of Zebrafish Development and Formation of the Nervous System . .7 1.6 Establishment and Early Patterning of the Midbrain Hindbrain Boundary . .8 1.7 Establishing the Wnt Signaling Gradient . .9 1.8 Defining the Temporal Elements of Wnt-Mediated Neural Posteri- orization . 10 1.9 Project Outline . 12 2 DISTINCT PHASES OF WNT REPSONSE IN THE NEURAL vi PLATE DURING EPIBOLY 14 2.1 Introduction . 14 2.2 Materials and Methods . 17 2.2.1 Zebrafish care . 17 2.2.2 Heat shocks and in situ hybridizations . 17 2.2.3 Drug treatments . 18 2.2.4 Screening transgenic lines . 19 2.3 Results . 19 2.3.1 Temporal dynamics of Wnt regulation by hs:dkk1GFP and hs:wnt8aGFP . 19 2.3.2 A heat shock time series defines three distinct functional periods for Wnt-mediated MHB patterning. 20 2.3.3 Early gastrula Wnt signaling regulates primary neural plate anteroposterior pattern. 22 2.3.4 MHB formation is transiently disrupted with early-stage in- hibition of Wnt signaling. 25 2.3.5 A Wnt overexpression assay identifies two windows of re- sponse in the neural plate to Wnt signaling . 25 2.3.6 otx2b positive cells become refractory to Wnt signaling by 75% epiboly . 26 2.3.7 Determining the mechanism of the otx2b refractory period . 28 vii 2.3.8 Fgf and Wnt signaling synergize to suppress otx2b ...... 30 2.4 Discussion . 32 2.4.1 Wnt-mediated neural patterning can be divided into tem- porally distinct phases. 32 2.4.2 The dynamics of Wnt signaling response in MHB positioning 34 3 TRANSCRIPTIONAL RESPONSE OF THE NEURAL PLATE TO WNT SIGNALING IS TEMPORALLY DYNAMIC 36 3.1 Introduction . 36 3.2 Materials and Methods . 38 3.2.1 RNA-Seq analysis . 38 3.2.2 Cloning and injections . 38 3.3 Results . 39 3.3.1 Wnt modulation at time 1 and 2 induce a transcriptionally dynamic response in the embryos . 39 3.3.2 No correlation between changes in expression levels and pat- tern in neural AP patterning genes . 41 3.3.3 shisa2b overexpression induces morphogenetic failures in the neural tube . 43 3.4 Discussion . 47 3.4.1 Response of the neural Plate to Wnt signaling can be divided into three temporal classifications . 47 viii 3.4.2 Response of the neural plate to Wnt modulation is tempo- rally dynamic . 49 3.4.3 The role of shisa2b in patterning of the brain . 49 3.4.4 Conclusion . 50 4 GENERATING A GBX1 FLUORESCENT REPORTER LINE 51 4.1 Introduction . 51 4.2 Materials and Methods . 52 4.2.1 Modification of (CH211-89L23) to Create TgBAC(gbx1:H2b- egfp)............................... 52 4.2.2 Generating Tg(TgBAC(gbx1:H2b-egfp) Transgenic Zebrafish 53 4.2.3 Antibody Staining . 53 4.2.4 Fluorescent Imaging . 54 4.3 Results . 54 4.3.1 In situ hybridization of TgBAC(gbx1:H2b-egfp)....... 54 4.3.2 TgBAC(gbx1:H2b-egfp) EGFP protein localization . 56 4.3.3 Estimating the turnover of H2B-EGFP . 58 4.4 Discussion . 59 4.4.1 The perdurance of TgBAC(gbx1:H2b-egfp)) reveals regula- tory mechanisms of gbx1 .................... 59 ix 4.4.2 Assessment of TgBAC(gbx1:H2b-egfp) as a tool to assess the resolution of the otx2b/gbx1 interface . 59 4.4.3 The neural plate regulatory paradox . 60 5 Summary 62 5.1 Wnt-Mediated Posteriorization is Established Early in Epiboly and Interpreted Over an Extended Period of Time . 62 5.2 Wnt Signaling: Cascades and Patterning . 64 5.3 The Positioning of the MHB and Disease in Humans . 66 6 Appendix 67 x List of Figures 1 Zebrafish brain at 24hpf. ......................2 2 The activation/transformation model of neural induction. .3 3 The modified activation/transformation model of neural induction. ...............................4 4 The canonical Wnt signaling pathway. .............6 5 Filopodial Wnt gradient model. ................. 11 6 Temporal analysis of Wnt modulation by the hs:dkk1bGFP and hs:wnt8aGFP transgenes ................... 21 7 Timed overexpression of hs:dkk1GFP reveal distinct win- dows of Wnt response. ....................... 22 8 Wnt suppression temporarily disrupts MHB establishment during epiboly ............................ 23 9 MHB markers require Wnt signaling at different phases of maintenance .............................. 24 10 Timed overexpression of hs:wnt8aGFP reveal two distinct windows of Wnt competency ................... 27 xi 11 otx2b positive cells become unresponsive to Wnt signaling by 75% epiboly ............................ 29 12 gbx1 expansion detectable 4 h post heat shock ........ 30 13 otx2b is directly suppressed by Wnt signaling ......... 31 14 Wnt and Fgf co-overexpression suppresses midbrain fate .. 32 15 The Phases of Wnt-mediated neural patterning ....... 33 16 Schematic of wnt modulation and RNA-Seq analysis .... 39 17 PCA of RNA-Seq Analysis .................... 40 18 Temporally dynamic response of neural plate to Wnt mod- ulation ................................. 42 19 Validation of hsdkk1 RNA-Seq analysis ............. 44 20 Validation of hs:wnt8aGFP RNA-Seq analysis ........ 45 21 shisa2b overexpression induces convergent extension defects 47 22 Expression pattern of TgBAC(gbx1:H2b-egfp) reporter .. 55 23 Additional views of 8 hpf TgBAC(gbx1:H2b-egfp) embryos. 56 24 EGFP localization in TgBAC(gbx1:H2b-egfp) embryos ... 57 25 gbx1BAC:h2b2gfp expression in cerebellum clears within 13 hours ................................ 58 26 mir seed sequences identified in gbx1 3' UTR ......... 60 xii 27 Comparison of Wnt diffusion and filopodial transport models 63 28 A
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