Baicalin, which is a flavonoid, was previously shown to exert neuroprotective effects against ischemic injury and oxidative insults. In this study, baicalin was found to induce neuronal differentiation on both C17.2 NSC and primary mouse NSC originated from hippocampuses of E14.5 mouse embryos. The baicalin-mediated differentiation of C17.2 NSC was noted in dose- and time-dependent manners. Baicalin-treated NSC displayed long processes of neurites. The gene expression of neuronal markers, NF-L, TUBB3 and MAP2 was also significantly increased after treated with 20 to 50 muM baicalin on C17.2 NSC. Treating C17.2 NSC with baicalin significantly increased the number of TUBB3 positive cells by 300%. A significant increase in the gene expression of TUBB3 was also observed on primary NSC upon baicalin treatment at 5 to 10 muM. The number of TUBB3 positive cells was increased by 100% after treating with 10 muM baicalin. C17.2 NSC treated with baicalin also increased the gene expression of GABAergic and serotonergic neuronal subtype specific enzymes GAD1 and TPH1. / Nature provides a vast pool of natural compounds with neuroprotection and neurotrophism. A few of these compounds can induce the differentiation of neural stem cells (NSC). There are ample opportunities to discover more natural compounds with differentiation inducing effect on NSC. One of the objectives of this project is to look for novel natural compounds showing neurogenic effect on NSC. This project has established a platform for screening medicinal materials and natural compounds with neural differentiation promoting effect on C17.2 mouse neural stem cell line. Screening results identified total Sanqi saponins, total Renshen saponins, Huangqin extracts and baicalin as potent candidates for inducing this differentiation of NSC. / This project also aims at characterizing the mechanisms involved in the neuronal differentiation effect of baicalin on NSC. Annotation from microarray analysis indicated that baicalin treatment on C17.2 NSC is related to development of tissue and nervous system. qPCR study attested the increased gene expression of nerve growth factor-beta, neurotrophin-3, pro-neural transcriptional factors Ngn1, Ngn2 and NeuroD2. Western blotting showed that baicalin activated ERK1/2 MAP kinase but not JNK and p38 MAP kinases. / This project demonstrated the neurogenic potential of natural resources on NSC. A novel neuronal induction effect of baicalin on NSC was also demonstrated with its mechanisms characterized. This project also revealed that baicalin can be used for promoting functional recovery of post-ischemia animals. / This study showed for the first time that baicalin exerts neuronal differentiation inducing effect on NSC. Another objective of this project is to study whether baicalin can promote functional recovery of animals with ischemia brain injury. Mice having undergone transient occlusion of the bilateral common carotid arteries with blood-reperfusion to induce global cerebral ischemia were treated with baicalin and/or EGFP-NSC. Ischemia animals received implantation of EGFP-NSC into the caudate putamen and/or intravenous injection of baicalin on alternate days for two-week on day seven post-ischemia displayed significant improvement of the cognitive function in terms of the incident of error and escape time in the water T-maze task compared to the control arm of ischemia mice. Data of the study suggested that the therapeutic effect of baicalin would be comparable to that of neural stem cell transplant in improving the cognitive function in a mouse ischemic stroke model. / Li, Ming. / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 199-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke. / Li, Jin. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts in English. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [in Chinese] --- p.v / TABLE OF CONTENT --- p.vi / LISTS OF FLOWCHARTS --- p.xii / LISTS OF FIGURES --- p.xiii / LIST OF TABLES --- p.xvi / LIST OF EQUIPMENTS --- p.xvii / LIST OF ABBREVATIONS --- p.xvii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- Central nervous system disorder --- p.1 / Chapter 1.1.1 --- Stroke --- p.1 / Chapter 1.1.2 --- Spinal cord injuries --- p.4 / Chapter 1.1.3 --- Parkinson's disease --- p.6 / Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8 / Chapter 1.2 --- Stem cell therapy --- p.10 / Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11 / Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11 / Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15 / Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16 / Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18 / Chapter 1.3 --- Stem cells --- p.20 / Chapter 1.3.1 --- Embryonic stem cells --- p.21 / Chapter 1.3.1.1 --- Derivation and characterization --- p.21 / Chapter 1.3.1.2 --- Biology of ES cells --- p.21 / Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21 / Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24 / Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25 / Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27 / Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28 / Chapter 1.3.2 --- Neural stem cells --- p.30 / Chapter 1.3.2.1 --- Derivation and characterization --- p.30 / Chapter 1.3.2.2 --- Biology of NS cells --- p.32 / Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34 / Chapter 1.3.4 --- Mesenchymal stem cells --- p.35 / Chapter 1.4 --- Serotonin (5-HT) --- p.36 / Chapter 1.4.1 --- Distribution --- p.37 / Chapter 1.4.2 --- Metabolism --- p.37 / Chapter 1.4.3 --- Biological effects of 5-HT --- p.38 / Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40 / Chapter Chapter2 --- Aim --- p.43 / Chapter 2.1 --- Hypothesis and study objectives --- p.43 / Chapter Chapter3 --- Materials and Methods --- p.49 / Chapter 3.1 --- Chemicals and Reagents --- p.49 / Chapter 3.1.1 --- Cell culture --- p.49 / Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51 / Chapter 3.1.3 --- Cell proliferation assay --- p.52 / Chapter 3.1.4 --- Cell apoptosis assay --- p.52 / Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52 / Chapter 3.1.6 --- Western blotting --- p.55 / Chapter 3.1.7 --- Molecular biology --- p.56 / Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58 / Chapter 3.1.9 --- MAO activity assay --- p.58 / Chapter 3.1.10 --- Endogenous ROS production assay --- p.58 / Chapter 3.2 --- Consumable --- p.58 / Chapter 3.3 --- Cells --- p.60 / Chapter 3.3.1 --- Feeder cell --- p.60 / Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60 / Chapter 3.3.2 --- ES cells --- p.61 / Chapter 3.3.2.1 --- ES cell D3 --- p.61 / Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61 / Chapter 3.3.3 --- NS cells --- p.61 / Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61 / Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61 / Chapter 3.4 --- In-house prepared solutions --- p.62 / Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63 / Chapter 3.4.2 --- Gelatin solution 01% --- p.62 / Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62 / Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63 / Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63 / Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63 / Chapter 3.4.7 --- Laminin solution --- p.64 / Chapter 3.4.7 --- MEF Maintenance medium --- p.64 / Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64 / Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65 / Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65 / Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65 / Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66 / Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66 / Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67 / Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67 / Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67 / Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68 / Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68 / Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68 / Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69 / Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69 / Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69 / Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70 / Chapter 3.4.22 --- RIPA --- p.70 / Chapter 3.4.23 --- Resolving buffer (8X) --- p.70 / Chapter 3.4.24 --- Stacking buffer (4X) --- p.71 / Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71 / Chapter 3.4.26 --- Transfer buffer (10X) --- p.72 / Chapter 3.4.27 --- Transfer buffer (IX) --- p.72 / Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72 / Chapter 3.4.29 --- TBS (10X) --- p.73 / Chapter 3.4.30 --- TBS-T (IX) --- p.73 / Chapter 3.4.31 --- Stacking gel --- p.73 / Chapter 3.4.32 --- Resolving gel --- p.74 / Chapter 3.5 --- Methods --- p.75 / Chapter 3.5.1 --- Cell culture --- p.75 / Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75 / Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75 / Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76 / Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76 / Chapter 3.5.1.5 --- Passage of culture --- p.77 / Chapter 3.5.1.5 --- 6 Cell count --- p.78 / Chapter 3.5.1.7 --- Cytospin --- p.78 / Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78 / Chapter 3.5.1.9 --- Cryopreservation --- p.79 / Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79 / Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81 / Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83 / Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84 / Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85 / Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85 / Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86 / Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87 / Chapter 3.5.2 --- Cell proliferation assay --- p.87 / Chapter 3.5.2.1 --- Cell morphology --- p.87 / Chapter 3.5.2.2 --- WST-1 assay --- p.88 / Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88 / Chapter 3.5.2.4 --- NCFC assay --- p.89 / Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89 / Chapter 3.5.3.1 --- RNA extraction --- p.89 / Chapter 3.5.3.2 --- RNA quantitation --- p.90 / Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90 / Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91 / Chapter 3.5.3.5 --- RNA Integrity Check --- p.91 / Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91 / Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92 / Chapter 3.5.4 --- Microarray --- p.94 / Chapter 3.5.5 --- Immunofluoresent staining --- p.94 / Chapter 3.5.6 --- Western blot --- p.95 / Chapter 3.5.6.1 --- Harvesting samples --- p.95 / Chapter 3.5.6.2 --- Protein extraction --- p.96 / Chapter 3.5.6.3 --- Protein quantification --- p.96 / Chapter 3.5.6.4 --- SDS-PAGE --- p.97 / Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97 / Chapter 3.5.6.6 --- Blocking the membrane --- p.97 / Chapter 3.5.6.7 --- Immunoblotting --- p.97 / Chapter 3.5.6.8 --- Signal detection --- p.98 / Chapter 3.5.7 --- Cell apoptosis assay --- p.98 / Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98 / Chapter 3.5.7.2 --- TUNEL --- p.99 / Chapter 3.5.8 --- Endogenous ROS assay --- p.100 / Chapter 3.5.9 --- In vivo studies --- p.101 / Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101 / Chapter 3.5.9.2 --- Transplantation --- p.101 / Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102 / Chapter 3.5.10 --- Histological analysis --- p.103 / Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103 / Chapter 3.5.10.2 --- Cryosectioning --- p.103 / Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104 / Chapter 3.6 --- Data analysis --- p.104 / Chapter Chapter4 --- Results --- p.113 / Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113 / Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113 / Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115 / Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115 / Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115 / Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117 / Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119 / Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119 / Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119 / Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120 / Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121 / Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122 / Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122 / Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123 / Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123 / Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127 / Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130 / Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130 / Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130 / Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131 / Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132 / Chapter 4.7.2 --- Cell labelling --- p.132 / Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133 / Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135 / Chapter Chapter5 --- Discussion --- p.180 / Chapter Chapter6 --- Conclusions --- p.192 / References --- p.195

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Sexual dimorphism has been long noted in human neurobiology, apparent most notably in sex-biased distribution of multiple neurological disorders or diseases, from autism spectrum disorder to Parkinson's disease. With the advances in molecular biology, genetics and epigenetics have come into focus as key players in sexually dimorphic neural development; and yet, many studies in the field of neuroscience overlook the importance of sex for the human brain. For this project, human embryonic and neural stem cells were chosen for three main reasons. Firstly, they provide an easily obtainable, scalable and physiologically native model for the early stages of development. Secondly, neural stem cells populations are retained within the adult human brain, and are implicated to play a role in cognition and mental illness, and as such are of interest in themselves. Thirdly, stem cell lines are widely used in research, including clinical trials of transplantation treatments, and for this reason should be meticulously examined and characterized. Here, the morphology, behaviour, and expression of selected genes in four stem cell lines, two of female and two of male origin, was examined in side-by-side comparisons prior to and during neuronal differentiation using a variety of methods including light microscopy, time-lapse two-photon microscopy, quantitative real-time PCR and immunocytochemistry. The obtained results have shown previously uncharacterised differences between those cell lines, such as a higher rate of proliferation but a slower rate of neuronal differentiation in male cell cultures compared to female cells cultivated in the same conditions, and a sex-biased expression of several markers of neuronal maturation at late stages of differentiation, as well as diverse patterns of expression of X- and Y-linked genes involved in stem cell proliferation and neural development.

Dans les tumeurs hiérarchiques, les cellules souches du cancer (CSC), au sommet de la hiérarchie tumorale, peuvent s'auto-renouveler et se différencier en progéniteurs amplificateurs transitoires (TAP) avec un potentiel d'auto-renouvellement limité. Au cours du développement, les cellules souches neurales de Drosophile, appelées neuroblastes (NB), expriment en séquence deux protéines antagonistes se liant à l'ARN, Imp et Syncrip (Syp), qui respectivement favorise et réprime l'auto-renouvellement des NB. La perturbation de mécanismes de division asymétrique des NB peut générer leur amplification illimitée induisant de véritables tumeurs. À l’aide d’une analyse clonale et de modélisations mathématiques, nous avons démontré que les progéniteurs Imp+ dans les tumeurs agissent comme des cellules semblables aux CSC, capables de se renouveler indéfiniment et de se différencier en progéniteurs Syp+, qui, à l’instar des TAP, ont un potentiel d’auto-renouvellement limité et une forte tendance à entrer en quiescence. De plus, nous avons démontré que les tumeurs du NB suivent une organisation hiérarchique rigide dans laquelle la transition Imp-Syp est irréversible. Fait intéressant, en utilisant l’analyse transcriptomique, nous avons constaté que la transition Imp à Syp dans les NB de tumeurs induit une régulation négative des gènes glycolytiques et respiratoires, épuisant vraisemblablement la capacité de croissance et d’auto-renouvellement des progéniteurs Syp+. La conservation frappante de ces protéines de liaison à l'ARN ouvre la possibilité passionnante que des hiérarchies analogues puissent exister dans les cancers humain. / In hierarchical tumors, cancer stem cells (CSCs), at the top of the tumor hierarchy, can self-renew and differentiate in transient-amplifying progenitors (TAPs), with a limited self-renewal potential. Understanding the molecular mechanisms that drive tumor hierarchy and heterogeneity is crucial to develop effective therapies to eliminate CSCs. During development, Drosophila asymmetrically-dividing neural stem cells, called neuroblasts (NBs), sequentially express two antagonistic RNA-binding proteins, Imp and Syncrip (Syp), that respectively promote and repress NB self-renewal. Genetic perturbation of NB asymmetric division cause NB amplification and malignant tumors. By using lineage tracing, clonal analysis and stochastic mathematical modeling of tumor growth, we demonstrated that Imp+ progenitors act as CSCs. They are able to self-renew endlessly and differentiate in Syp+ progenitors, that have a limited self-renewal potential and the high tendency to undergo quiescence. NB tumors follow a rigid hierarchical organization, where the Imp-to-Syp transition is irreversible. Hence, Syp+ progenitors cannot revert to an Imp+ malignant state. Transcriptomic analysis revealed that the Imp-to-Syp transition in tumors induces a downregulation of glycolytic and respiratory genes that exhausts the growth and self-renewing potential of Syp+ progenitors. The striking conservation of these RNA-binding proteins opens the exciting possibility that analogous Imp-Syp hierarchies may exist in human cancers.

Sox proteins all contain a single ~70 amino acid High Mobility Group (HMG) DNA-binding domain with strong homology to that of Sry, the mammalian testisdetermining factor. In Drosophila melanogaster, there are four closely related members of the B group, Dichaete (D), Sox Neuro (Sox N), Sox 21a, and Sox 21b that each exhibit ~90% sequence identity within the HMG domain.The previous study has shown that Dichaete plays a major role in embryonic nervous system development and is expressed in several clusters of neurons in the brain, including intermingled olfactory LNs and central-complex neurons strongly expressed in local interneuron of the olfactory system. However, little is known about the possible expression and functions of the related group B Sox genes in the larval and adult brain. In particular, it is unclear if Sox N may function along with Dichaete in controlling the development or physiology of the adult olfactory system. Our data suggests Sox N potential role in the elaboration of the olfactory circuit formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

The human brain is the most complex structure in the known universe and one of the ultimate goals of humanity is to understand its function. The "bottom-up" approach to developmental neuroscience seeks to assemble a "parts list" of the genes expressed in each neuron and a map of the gene regulatory networks that determine the identity of the diverse neuronal types. A key part of building such a gene regulatory map is to identify the transcription factors that are key nodes in these networks. The goal of my PhD was to study the particular gene regulatory networks that govern the decision of the V5 skin cell to divide, lose its skin fate and decide to make dopamine and glutamate sensory neurons. We chose an unbiased forward genetic screen approach coupled with whole genome sequencing of mutants derived from these screens. In the process, we found several mutants that govern this process and developed a software pipeline that simplifies the analysis of mutants for others who perform forward genetic screens.

No description available.

Production of large numbers of desirable human cell types in the laboratory is one of the major goals of stem cell research. Current experimental approaches have focused on the strategy of recapitulating the events of normal embryogenesis in culture, by treating cells - either tissue stem cells or pluripotent stem cells (iPS/ES cells) - with cocktails of growth factors, matrix proteins or pharmacological agents. This is challenging and often requires weeks or months of elaborate cell culture regimes. An alternative approach is the forced expression of master regulatory transcription factors; this can bypass developmental programs and drive conversion to the target cell type. Each of these strategies is inefficient and unreliable. Recently a new opportunity has arisen to exploit synthetic transcription factors (sTFs) to program and reprogram cell fate. To create such sTFs the CRISPR/Cas9 system is repurposed through tethering of catalytically dead Cas9 to various transcriptional regulatory effector domains (e.g. VP16, KRAB). In this thesis, we have explored sTFs as tools to reset transcriptional regulatory networks in neural stem cells and mouse embryonic fibroblasts. We tested transcriptional activation of key neural lineage target genes (e.g Olig2, Sox10 and Nkx6.2). We designed and validated a series of sTFs that could effectively activity these. We have found that activation of Sox10 by dCas9-VP160 in mouse neural stem cells can increase the amount of arising oligodendrocyte and oligodendrocyte precursors cells during the differentiation. The activity of sTFs strongly depends on cellular context: i.e. a specific sTF might work well in one cell type but not another. Importantly, these biological barriers are not easily overcome by increasing the strength of the sTF - either through levels or types of effector domains used. Our data inspecting single cells suggests that multiplex delivery of sTFs can indeed cooperate by both increasing the number of cells that activated the gene of interest and increasing the level of transcriptional activation in a given cell. To fully exploit these new technologies, we therefore developed a new construction pipeline that allows easy and efficient assembly of multiple sTFs. Using this approach, we were able to successfully activate three different target genes from a single expression plasmid (Olig2, Sox10 and Nkx6.2) in fibroblasts. These sTFs we able to force fibroblast transdifferentiation towards oligodendrocyte lineage. Future studies will explore further how to exploit these sTFs to augment or replace current reprograming strategies.

Au cours des 10 dernières années, le laboratoire de JP Hugnot s’est concentré sur les différents pools de progéniteurs et de cellules souches trouvés dans la moelle épinière adulte, chez l’homme comme chez la souris. Ceci est important pour mener ce type de recherche car la moelle épinière est affectée par plusieurs maladies neurodégénératives telles que la sclérose latérale amyotrophique (SLA) et des lésions traumatiques pour lesquelles il n'existe pas de traitement curatif. Chez des animaux comme le poisson zèbre, la moelle épinière peut se régénérer après une lésion en raison de l'activation de progéniteurs / cellules souches endogènes. Ainsi, en recherchant la présence et les propriétés de telles cellules chez les mammifères, en particulier les humains, on pourrait exploiter ces cellules pour la régénération, y compris les neurones. Nous avons procédé au profilage de l'ARN pour comparer la niche de cellules souches humaine et de souris et la niche de cellules souches de souris de la moelle épinière lésée ou non lésée. Cette niche est particulièrement intéressante dans la mesure où, chez les anamniotes, les cellules de l'épendymoglie radiale situées dans cette région sont multipotentes et peuvent générer de nouveaux motoneurones après une lésion. et des cellules similaires, mais non identiques, sont présentes chez la souris. Chez les mammifères, après la lésion, ces cellules de niche prolifèrent et migrent activement pour générer principalement des cellules astrocytaires et peu d'oligodendrocytes qui participent à la cicatrice gliale et à la régénération en fournissant un facteur neurotrophique tel que le CNTF, le HGF et l'IGF-1. Cette niche contient au moins 5 types de cellules et un nouveau type de cellules dorsales exprimant les facteurs de transcription Msx1 et Id4 a été identifié. Ces résultats indiquent que la niche de la moelle épinière adulte chez la Souris et chez l'homme est une mosaïque de cellules ayant différentes origines développementales et conservant des niveaux élevés de gènes de développement neural. Les interactions gliales-neuronales qui soutiennent et maintiennent les neurones intacts peuvent influer sur les maladies neurodégénératives. L'une de ces cellules gliales est l'oligodendrocyte satellite ou cellules satellites périneuronales (PNC). Les PNC sont étroitement associés au soma de gros neurones et largement répandus dans la substance grise du cortex et de la moelle épinière. Cependant, les propriétés cellulaires et les rôles fonctionnels de ces oligodendrocytes non myélinisants n'ont pas encore été découverts. Dans cette étude, les cellules positives à la nestine-GFP sont associées à des neurones immunocolorés pour l'antigène nucléaire neuronal dans le cortex et la moelle épinière. Nous avons identifié les PNC comme étant des cellules positives pour la CNPase qui ne sont ni des cellules progénitrices d'oligodendrocytes (PDGFRa) ni des oligodendrocytes myélinisants (MBP). Ces données suggèrent que les PNC pourraient affecter la survie neuronale ainsi que le processus de myélinisation dans des conditions de démyélinisation. En outre, il pourrait être impliqué dans des maladies neurodégénératives telles que la sclérose en plaques et la sclérose latérale amyotrophique en raison de leur interaction avec les motoneurones. / Over the last 10 years, JP Hugnot’s lab has been focusing on the different pools of progenitors and stem cells found in the adult spinal cord both in human and mouse. This is important to conduct this kind of research as the spinal cord is affected by several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and traumatic lesions for which there is no cure. In anamniotes such as Zebrafish, the spinal cord can regenerate after lesion due to endogenous progenitors/stem cells activation. So by investigating the presence and properties of such cells in mammals especially human, one could possibly harness those cells toward regeneration including neurons. We conducted RNA profiling to compare human vs mouse stem cell niche and lesioned vs non lesioned spinal cord mouse stem cell niche. This niche is particularly interesting as in anamniotes, radial ependymoglia cells located in this region are multipotent and can generate new motoneurons after lesion. And similar, albeit non identical, cells are present in mouse. In mammals, after lesion, these niche cells actively proliferate and migrate to generate mainly astrocytic cells and few oligodendrocytes which participate to the glial scar and regeneration by providing neurotrophic factor such as CNTF, HGF, and IGF-1. This niche contains at least 5 cell types and here a new dorsal cell type expressing Msx1 and Id4 transcription factors was identified. These results indicated that the adult spinal cord niche in mouse and human is a mosaic of cells with different developmental origin and maintaining high levels of neural developmental genes. Glial-neuronal interactions supporting and keeping neurons intact can be influence neurodegenerative diseases. One of these glial cells is the satellite oligodendrocyte or so called perineuronal satellite cells (PNCs). PNCs are tightly associated to the soma of large neurons and widely spread in the grey matter of the CNS both cortex and spinal cord. However the cellular properties and functional roles of these unmyelinating oligodendrocytes are not yet discovered. In this study, nestin-GFP positive cells are associated to neurons immunostained for neuronal nuclear antigen in both cortex and spinal cord. We identified PNCs as CNPase positive cells that are neither oligodendrocyte progenitor cells (PDGFRa) nor myelinating oligodendrocytes (MBP). These data suggest that PNCs might affect neuronal survival as well as the myelination process in demyelinating conditions. Also it could be implicated in neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis due to their interaction with motor neurons.