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Goosecoid in Der Mesodermalen Differenzierung Von Embryonalen Stammzellen Und Der Teratokarzinomazellinie P19 Der Maus

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Goosecoid in Der Mesodermalen Differenzierung Von Embryonalen Stammzellen Und Der Teratokarzinomazellinie P19 Der Maus Forschungszentrum Karlsruhe Technik und Umwelt Wissenschaftliche Berichte FZKA 6382 Die Rolle des Homöoboxgens goosecoid in der mesodermalen Differenzierung von embryonalen Stammzellen und der Teratokarzinomazellinie P19 der Maus Michael W. Schäffer Institut für Toxikologie und Genetik vom Fachbereich Chemie der Universität Kaiserslautern genehmigte Dissertation Forschungszentrum Karlsruhe GmbH, Karlsruhe 1999 Zusammenfassung Zusammenfassung Das Homöoboxgen goosecoid, das zuerst in Xenopus identifiziert wurde, ist im Organisator bzw. seinem Äquivalent während der Gastrulation in Frosch, Hühnchen, Zebrafisch und Maus exprimiert. Um die Rolle von goosecoid in der frühen Mausentwicklung zu untersuchen, wurden embryonale Stammzellklone isoliert, die das Maushomolog stabil unter der Kontrolle des Ubiquitin-Promotors überexprimierten. Diese Klone zeigten bereits ohne weitere Induzierung von Differenzierungsvorgängen eine von Wildtyp-ES-Zellen unterschiedliche Morphologie, die von fibroblastenähnlichem, mesenchy-malem Aussehen geprägt war. goosecoid-ES-Zellklone exprimieren im Gegensatz zu den Wiltdtypzellen Vimentin, ein Markergen für mesenchymale Zelltypen. Dieses intermediäre Filamentprotein ist wie goosecoid in den ersten mesodermalen Zellen während der Embryonalentwicklung exprimiert. Die Analyse der Vimentin-Promotorsequenz zeigte, daß die Aktivierung der Vimentin- Expression direkt durch die Bindung von Goosecoid an spezielle, möglicherweise relevante Bindesequenzen erfolgen könnte. Die goosecoid-ES-Klone exprimieren einige wichtige Markergene des Organisators, wie die neuralen Induktoren Chordin und Follistatin, sowie Transkriptionsfaktoren, wie das Homöoboxgen Otx2 und das "Forkhead"-Gen HNF3β. Interessanterweise waren mRNA- Transkripte für das organisatorspezifische lim-1 in den Proben der Klone nicht nachweisbar. Ebenfalls negativ verlief der Nachweis des Markers für Primitivstreifenmesoderm, Brachyury. In Zusammenarbeit mit Diane Boucher (UCSF, USA) konnte mit Hilfe semiquantitativer RT- PCR goosecoid-ES-Zellklone (Expression unter der Kontrolle des CMV-Promotors) untersucht werden, die unterschiedliche Expressionsniveaus von goosecoid besaßen. Nur in Klon g202, der das höchste Niveau an goosecoid-Transkripten aufwies, konnte keine Brachyury-mRNA nachgewiesen werden. Diese Experimente deuten auf eine inverse Korrelation von goosecoid- und Brachyury-Expression hin. Die Brachyury-Repression erfolgt in Xenopus über zwei definierte Homöobox-Bindestellen, von denen nur eine auch im 650 bp-Promotor der Maus zu finden ist. Die gezielte Mutagenese dieser potentiellen Bindestelle führte jedoch nicht zu einem Verlust der Repression durch goosecoid in transienten Promotorreporter-Experimenten in P19 Zellen. Die schrittweise Verkürzung um jeweils ca. 100 bp des Brachyury-Promotors führte zu einem interessanten "Promotorprofil". Der 535 bp-, sowie der 334 bp- Promotor konnten in seiner Aktivität durch goosecoid am stärksten reprimiert werden. Der 441 bp- Promotor konnte nicht durch goosecoid reprimiert werden. Die homologen Bereiche enthalten potentielle Bindestellen für entwicklungsbiologisch relevante Transkriptionsfaktoren, die als Reaktionspartner für goosecoid denkbar wären. III Abstract The role of the homeobox gene goosecoid in mesodermal differentiation of embryonic stem cells and the teratocarcinoma cell line P19 of the mouse Abstract The homeobox gene goosecoid, originally identified in Xenopus, is expressed in the organizer or its equivalent during gastrulation in the frog, chick, zebrafish and mouse. To investigate the role of goosecoid in mouse development, embryonic stem cells were generated that stably overexpress the murine homologue of goosecoid under the control of the ubiquitin-promotor. These clones revealed a mesenchymal-like morphology distinct from wildtype ES-cells without any further differentiation steps. Vimentin, a marker gene for mesenchymal cells, is expressed in these cell clones. The morphological changes of these cells did not correlate with a reduction of the expression level of the cell adhesion molecule E-Cadherin. The goosecoid ES-clones express important organizer-specific genes like the neural inducers chordin and follistatin, the homeobox gene Otx2 and the forkhead gene HNF3β. Surprisingly, lim-1-mRNA as well as the expression of gastrulation-associated gene Brachyury could not be detected in these cells. In collaboration with Dr. Diane Boucher (UCSF, USA) embryonic stem cells were generated that stably overexpress the murine homologue of goosecoid under the control of the CMV- promotor. Brachyury expression in such clones displayed an inverse correlation with goosecoid transcript levels, suggesting that goosecoid was able to define discrete anterior mesendodermal cell fates. Interestingly, repression of Brachyury is conserved between Xenopus and mouse despite the lack of conservation of the Brachyury promoter. Mutation of the sole canonical homeobox binding site in the mouse Brachyury promotor did not abolish this repression. In order to narrow down sequences that mediate repression by goosecoid, Brachyury-promotor mutants with different length were cloned. Two additional sequences were identified that were able to repress Brachyury, following transient co-transfection of a goosecoid effector plasmid in P19 cells. These sequences contain conserved elements between the mouse and frog Brachyury promotor, representing potential binding sites for two other transcription factors that are important regulators of early vertebrate development: LEF-1 and caudal. These observations suggest that goosecoid may play a role in specification of anterior mesendodermal fates. IV Hinweis Hinweis Die vorliegende Arbeit wurde sorgfältig erarbeitet. Dennoch übernimmt der Autor für die Richtigkeit von Angaben und Hinweisen sowie für eventuelle Druckfehler keine Haftung. Die Wiedergabe von Gebrauchsnamen, Handelsnamen, Warenbezeichnungen usw. in dieser Arbeit berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, daß solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürften. V Abstract Die folgenden Abbildungen sind urheberrechtlich geschützt. Eine Reproduktion jedweder Art bedarf der ausdrücklichen Genehmigung der Autoren bzw. Verlage. Abbildung 1A und 1B: Kaufman TC, Seeger MA, Olsen G. (1990). Molecular and genetic organization of the antennapedia gene complex of Drosophila melanogaster. Adv Genet ; 27, Seite 313. Abbildung 1C: Edward B. Lewis aus: Gilbert, SF. (1994). Developmental Biology. 4th Edition, Seite 553, Abbildung 15.27. Sinauer Associates, Inc., Sunderland, MA. USA. Abbildung 2: Knippers, R. Molekulare Genetik., 6. Auflage, Seite 352, Abbildung 12.24 © 1995, Georg ThiemeVerlag, Stuttgart. Abbildung 3: Grafik von Patricia J. Wynne in: "Homeobox genes and the vertebrate body plan", Seite 47. © Juli 1990, Scientific American, New York, NY. USA. Abbildung 4: Linder Biologie, Abbildung. 406.1, 21. neu bearbeitete Auflage, © 1998 Schroedel Verlag GmbH, Hannover. Abbildung 5: Grafik von Thomas Schäffer, Höhr-Grenzhausen. Abbildung 8: Belo JA, Leyns L., Yamada G, De Robertis EM. (1998) The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants. Mech Dev, 72, Seite 15, Abbildung 3A. © 1998, Elsevier Science, Oxford, England. VI Inhaltsverzeichnis VII Inhaltsverzeichnis Seite Inhaltsverzeichnis Kapitel I Einleitung...................................................................... 1 1. Was ist Entwicklungsbiologie?.......................................................... 2 2. Homöoboxgene und Entwicklung...................................................... 3 3. Das Spemann'sche Organisatorphänomen........................................... 8 4. Die frühe Embryonalentwicklung der Maus......................................... 10 4.1 Vor der Implantation des Embryos................................................... 10 4.2 Nach der Implantation des Embryos................................................. 11 4.3 Die Gastrulation in der Maus......................................................... 11 5. Die Rolle Homöoboxgens goosecoid.................................................. 13 5.1 goosecoid -Expression in Xenopus.................................................. 14 5.2 goosecoid -Expressioni in der Maus................................................. 14 5.3 Gezielte Mutagenese des goosecoid-Gens.......................................... 15 5.4 Ist goosecoid das Organisatorgen?................................................... 16 6. Ein kurzer Schwanz, die Gastrulation und Brachyury.......................... 17 7. Modellsytem zur in vivo- und in vitro-Untersuchung der frühen Embryonalentwicklung der Maus Embryonale Stammzellen und Teratokarzinomazellinien.................... 19 7.1 Auf der Suche nach den Stammzellen: die Teratokarzinomazellinien........... 19 7.2 Embryonale Stammzellen............................................................. 19 VIII Inhaltsverzeichnis Seite Kapitel II Fragestellung der Arbeit........................................... 23 Kapitel III Materialien und Methoden........................................ 27 Materialien...................................................................................... 28 1. Feinchemikalien.............................................................................. 28 2. Radiochemikalien............................................................................ 29 3. Enzyme.........................................................................................
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