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Developmental Signaling by Noggin and Wnt in the Frog Xenopus By Developmental Signaling by Noggin and Wnt in the Frog Xenopus By John Joseph Young A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Richard M. Harland, Chair Professor Sharon Amacher Professor Michael S. Levine Professor Michael Freeling Spring 2013 Developmental Signaling by Noggin and Wnt in the Frog Xenopus © 2013 By John Joseph Young Abstract Developmental Signaling by Noggin and Wnt in the Frog Xenopus By John Joseph Young University of California, Berkeley Professor Richard M. Harland, Chair Xenopus has provided a powerful system to study cellular, developmental, and neuro- biology. The availability of their embryos and the advent of modern molecular techniques allowed investigators to revisit the observations of classical embryologists and begin to determine the molecular mechanisms underlying germ layer formation and axis induction. My thesis work took advantage of the frog Xenopus to first address the developmental role of Noggin, a Bone morphogenic protein (Bmp) antagonist, and then to determine the mechanism of Wnt-induced anterior-posterior patterning of the neural plate. The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. In the first part of this work, I address this problem by genome editing with zinc finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in X. tropicalis induced mutations that are consistent with results of non homologous end joining at the target site, resulting in mosaic loss of fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles. Founder animals yielded progeny that carry insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity among three germline mutant alleles. Breeding an identified null allele to homozygosity resulted in tadpoles with deformaties in the cranial skeleton. Anatomical analysis revealed severe reductions in Meckel’s cartilage with joint fusions. Gene expression analysis via in situ hybridization for chondrogenesis regulating factors in noggin mutants revealed a reduction in sox9 and col2a expression domains. Analysis of Bmp targets showed an expansion of hand2, edn1, and msx2 in the pharygeal arches (PAs) of mutants. This suggested a mechanism whereby incresed Bmp signaling inhibits chondrogenesis and ventralizes the PAs resulting in the jaw deformities observed in mutants. Neural development in amphibians occurs as a two-step process. First, ectodermal precursors adopt a neural fate in the absence of Bmp signaling. A second signal is then required to pattern the anterior posterior neuraxis. Signaling through Fibroblast growth factor (Fgf), retinoic acid (RA), and Wnt have each been demonstrated to be both necessary and sufficient for inducing 1 posterior fates in undifferentiated neural tissue. Wnt signaling in particular has been closely studied. However, the mechanism by which this pathway induces posterior fates remains unclear. To address this question, I used RNA-Seq to identify direct transcriptional targets in neural tissue by activating Wnt signaling in Xenopus neural explants pretreated with the translation inhibitor cycloheximide. Wnt-activated neural tissue resulted in over 200 genes with expression increased greater than 2-fold when compared to anterior neural tissue. in situ hybridization analysis of highly expressed transcription factors and RNA-binding proteins showed posterior expression. Of particular interest, the transcription factor sal-like 1 (Sall1) and sal-like 4 (Sall4) showed specific posterior neural expression suggesting a role in Wnt-induced neural patterning. The RNA-Seq screen found sall1 and sall4 expression to be induced by canonical Wnt signaling !'#%&!","+ ! &'&#%&!'!'.%&'!'%"!"sall4 were enriched in β-catenin chromatin imunoprecipitations. Knockdown of Sall4 resulted in the loss of spinal cord marker expression and an increase in the expression of pou25, pou60 and pou91 (pouV genes), the three Xenopus homologs of the stem cell factor pou5f1/Oct4. Overexpression of the pouV genes resulted in the loss of spinal cord identity, and knockdown of pouV function restored spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocked the posteriorizing effects of Fgf and retinoic acid signaling in the neurectoderm. These results suggest that Sall4, activated by Wnt signaling, represses the pouV genes to provide a permissive environment that allows for additional Wnt/Fgf/RA signals to posteriorize the neural plate. 2 To Nick Duesbery, Ph.D and Jeff McKelvey, Ph.D. I’m grateful to have been your student, proud to be your colleague, and most of all, honored to be your friend. i Table of Contents Table of Contents......................................................................................................................ii Table of Figures........................................................................................................................iv Acknowledgements..................................................................................................................vi Chapter 1: General Introduction.............................................................................................1 Classical Embryology.....................................................................................................1 The Molecular Era and Xenopus.....................................................................................2 Open questions................................................................................................................8 Goals of this thesis..........................................................................................................9 Chapter 2: Materials and Methods.........................................................................................11 Embryo and explant culture............................................................................................11 RNA and morpholino microinjections............................................................................11 Western Blotting.............................................................................................................12 Celery Extract Preparation..............................................................................................12 Mutation Detection by Celery Extract............................................................................12 Cartilage staining............................................................................................................13 Cycloheximide and dexamethasone treatments..............................................................13 Whole-mount in situ hybridization.................................................................................13 Embedding and Sectioning.............................................................................................14 RT-PCR and qPCR..........................................................................................................14 RNA-seq..........................................................................................................................14 ii Chromatin immunoprecipitation...................................................................................14 Chapter 3: Analysis of the developmental role of noggin in Xenopus tropicalis development via Zinc-Finger Nuclease mutagenesis...........................................................16 Introduction...................................................................................................................16 Results...........................................................................................................................17 Discussion.....................................................................................................................21 Chapter 4: Expression screen for direct targets of Wnt signaling in neural tissue.............................................................................................................................46 Introduction...................................................................................................................46 Results and Discussion..................................................................................................47 Chapter 5: Spalt-like 4 mediates Wnt-induced neural patterning via repression of pouV/Oct4 family members..................................................................................................................68 Introduction................................................................................................................................68 Results........................................................................................................................................69 Discussion...................................................................................................................................73 References...............................................................................................................................................97 Appendices.............................................................................................................................................118
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