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Modeling of Neural Differentiation by Using Embryonic Stem Cells to Detect Developmental Toxicants

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Modeling of Neural Differentiation by Using Embryonic Stem Cells to Detect Developmental Toxicants Modeling of neural differentiation by using embryonic stem cells to detect developmental toxicants Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Bastian Zimmer an der Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie Konstanz 2011 Tag der mündlichen Prüfung: 02.12.2012 1. Referent: Prof. Dr. Marcel Leist 2. Referent: PD Dr. Gerrit Begemann 3. Referent: Prof. Dr. Marcus Groettrup 4. Referent: Prof. Dr. Gabsang Lee List of publications Publications, integrated in this thesis Chapter C Zimmer B, Kuegler PB, Baudis B, Genewsky A, Tanavde V, Koh W, Tan B, Waldmann T, Kadereit S, Leist M. Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing. Cell Death Differ. 2011 Mar;18(3):383-95. Epub 2010 Sep 24. Chapter D Zimmer B, Schildknecht S, Kuegler PB, Tanavde V, Kadereit S, Leist M. Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure. Toxicol Sci. 2011 Jun;121(2):357-67. Epub 2011 Mar 7. Chapter E Zimmer B, Lee G, Meganathan K, Sacchinidis A, Studer L, Leist M. Genuine human neural crest cells as test system for developmental toxicity and potential rescue strategies. under review Some text passages in the general introduction were taken from: Kadereit S*, Zimmer B*, van Thriel C, Hengstler J.G., Leist M. Compound selection for modeling developmental neurotoxicity (DNT) and prenatal neurotoxicity with embryonic stem cells. accepted for publication in Frontiers in Bioscience *: both authors contributed equally Publications, not integrated in this thesis Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17- 42. Kuegler PB, Baumann BA, Zimmer B, Kadereit S, Leist M, GFAP-independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells, GLIA, in press Waldmann, T., Weng, M., Zimmer, B., Pöltl, D., Scholz, D., Broeg, M., Kadereit, S.,Wuellner, U., Leist, M. Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment. submitted Table of contents A. Summary .............................................................................................................................. 6 Zusammenfassung.................................................................................................................... 7 B. General introduction ........................................................................................................... 9 1. (Neuro)development in general...................................................................................... 9 1.1 Proliferation.......................................................................................................... 11 1.2 Differentiation/Patterning..................................................................................... 12 1.3 Migration.............................................................................................................. 14 1.4 Neurite outgrowth................................................................................................ 15 1.5 Synaptogenesis / Neurotransmitter household ..................................................... 16 2. Environmental chemicals and (neuro)development..................................................... 17 2.1 Barker Hypothesis................................................................................................ 18 2.2 Time of insult vs. time of phenotype onset .......................................................... 18 2.3 Susceptibility of the developing brain to chemicals............................................. 19 2.4 Environmental chemicals and developmental disabilities ................................... 20 2.5 Phenotype vs. biological process ......................................................................... 22 3 Development of in vitro test systems in the 21st century ............................................. 23 4. Embryonic stem cells (ESC) as source for in-vitro testing .......................................... 24 Aims of the thesis.................................................................................................................... 27 Chapter C.................................................................................................................................... Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing............. 29 Abstract ................................................................................................................................ 31 Introduction .......................................................................................................................... 32 Results .................................................................................................................................. 34 Discussion ............................................................................................................................ 45 Materials and Methods......................................................................................................... 49 Supplements ......................................................................................................................... 56 Chapter D.................................................................................................................................... Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure ....................................................................................................... 73 Abstract ................................................................................................................................ 75 Introduction .......................................................................................................................... 76 Materials and Methods......................................................................................................... 78 Results .................................................................................................................................. 82 Discussion ............................................................................................................................ 91 Supplements ......................................................................................................................... 95 Chapter E.................................................................................................................................... Genuine human neural crest cells as test system for developmental toxicity and potential rescue strategies.................................................................................................................... 101 Abstract .............................................................................................................................. 102 Introduction ........................................................................................................................ 103 Materials and Methods....................................................................................................... 105 Results ................................................................................................................................ 107 Discussion .......................................................................................................................... 114 Supplements ....................................................................................................................... 118 F. Concluding discussion ..................................................................................................... 131 G. Bibliography .................................................................................................................... 142 Record of contribution ....................................................................................................... 169 Summary A. Summary Developmental disabilities and congenital malformations associated with neural development are increasing problems in western countries. More and more evidence emerges from human epidemiological studies that environmental chemicals as well as drug and food constituents contribute to such an increase. Unfortunately, developmental neurotoxicity is currently the least examined form of developmental toxicity. Less then 200 compounds worldwide, mostly pesticides, have been tested in vivo according to the OECD test guideline TG 426. This guideline requires expensive and labor intensive animal experiments which often lack human relevance. As embryonic stem cells are able to differentiate into every cell type of an organism and have been shown to recapitulate in vivo development in the culture dish, they are considered a powerful alternative to whole animal experiments. Since also human embryonic stem cells have been generated, it is possible to mimic effects of chemicals on human neural development in vitro today. In the framework of
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