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Genetic Analysis of the Sall Transcription Factor Family in Murine Development

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Genetic Analysis of the Sall Transcription Factor Family in Murine Development Genetic Analysis of the Sall Transcription Factor Family in Murine Development DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN FAKULTÄT III – BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Ulrich Elling aus Freising 2005 Promotionsgesuch eingereicht am: 21. November 2005 Die Arbeit wurde angeleitet von: Dr. M. Treier Doktorvater: Prof. Dr. Schneuwly Thesis advisory committee, EMBL: Dr. S. Cohen Dr. C. Nerlov Prüfungsausschuß: Vorsitzender: Prof. Dr. Kunzelmann 1. Gutachter Prof. Dr. Schneuwly 2. Gutachter Prof. Dr. Witzgall 3. Prüfer: Prof. Dr. Seufert Ersatzperson: Prof. Dr. Warth Zusammenfassung Zusammenfassung Spalt Proteine bilden eine Familie von Transkriptionsfaktoren, die von Caenorhabdits elegans über Drosophila melanogaster bis hin zu Vertebraten konserviert sind. In Säugetiergenomen findet man jeweils vier Gene, die als Spalt-like (SALL1 bis SALL4) bezeichnet werden. Mutationen in SALL1 und SALL4 verursachen im Menschen Townes- Brocks-Syndrom (TBS) beziehungsweise Okihiro-/Duane Radial Ray-Syndrom (DRRS). Beide Syndrome variieren stark in ihrem klinischen Befund, beschrieben sind unter anderem Verdopplung oder Fehlen von Fingern und Zehen, Schäden im zentralen Nervensystem sowie Missbildungen im Urogenitalsystem. Wie es zu diesen multiplen Fehlbildungen kommt ist nicht verstanden. Aufbauend auf eine systematische, vergleichende Untersuchung der Expressionsmuster wurden in der vorliegenden Arbeit die Auswirkungen von Mutationen in Sall1 bis Sall4 auf die Embryonalentwicklung von Mäusen einzeln oder in Kombination untersucht. Bisher wurde nur gezeigt, dass Verlust von Sall1 zur Nierenagenese führt. Das Ziel war deshalb, durch Untersuchung der verschiedenen Sall Mutationen in unterschiedlichen Organsystemen zugrunde liegende Gemeinsamkeiten herauszuarbeiten, die eine generelle Rolle von Sall Genen während der Embryonalentwicklung aufzeigen. Sall4 ist der erste Sall Transkriptionsfaktor, der während der Embryonalentwicklung exprimiert wird. Die innere Zellmasse (IZM) der Blastula, aus der die embryonalen Stammzellen und das Extraembryonale Endoderm hervorgehen, exprimiert Sall4 besonders stark. Im Trophoblast hingegen wird die Transkription von Sall4 ab dem Tag e5.5 vollständig reprimiert. Die Deletion von Sall4 aus dem Mausgenom führt bei homozygoten Tieren zum Fehlen der beiden IZM Zellinien und damit zum Absterben des Embryos direkt nach der Einnistung in die Gebärmutter. Das Studium dieses Prozesses in einem Kultursystem bestätigte den sukzessiven Verlust der IZM und führte zu der Aussage, dass Sall4 für den Erhalt der embryonalen Stammzellen und des primitiven Endoderms notwendig ist. In Abwesenheit von Sall4 kommt es jedoch nicht zum Zelltod sondern zu reduzierter Zellteilung dieser Zellinien und zu deren Differenzierung. Auch embryonale Stammzellen verlieren in Kultur die Fähigkeit zur Zellteilung, wenn Sall4 deletiert wird. Um zu testen, ob Sall4 die Identität von Zellen zellautonom steuert, wurden chimäre Mäuse aus Zellen mit und ohne Sall4 hergestellt. Dabei wurde festgestellt, dass Zellen, deren Sall4 Gen entfernt ist, auch im Verband mit Wildtyp-Zellen nicht zum Embryo beitragen können. Dies lässt die Schlussfolgerung zu, dass die Funktion von Sall4 zellautonom ist. I Zusammenfassung Im Gegensatz zum frühen Embryo werden in Vorläuferzellen des zentralen Nervensystems alle vier Sall Gene abgelesen. Es konnte gezeigt werden, dass die Mutation einzelner Gene nur milde Effekte auf die Entwicklung hat wohingegen das Entfernen mehrerer Sall Faktoren zu Verlust, Verkleinerung oder mangelnder Organisation verschiedener Bereiche des Nervensystems führt. Dies wurde auf die Beobachtung zurückgeführt, dass die Zellteilungsrate verringert ist. Beispiele hierfür sind die Grosshirnrinde und der Bulbus olfactorius. In Sall1/Sall2 Doppelmutanten kommt es so zum kompletten Verlust des Geruchssinns. Auch die wachsende Extremitätenknospe aktiviert mehrere Sall Gene, in späteren Stadien vor allem Sall1 und Sall3. Hier konnte gezeigt werden, dass die vollständige Ausbildung aller Elemente des Skeletts von der Dosis der Sall Aktivität abhängt und totale Abwesenheit von Sall1 und Sall3 Oligodaktylie zur Folge hat. Mutation mehrerer Gene führt in Mäusen demnach zu Entwicklungsschäden vergleichbar denen, wie sie in Menschen, für TBS und DRRS beschrieben sind. Funktionen von Sall Faktoren überlappen stark. In allen untersuchten Entwicklungsprozessen kommt es in Abwesenheit aller hier exprimierten Sall Transkriptionsfaktoren zum Fehlen von differenzierten Zellen in den betroffenen Geweben, das sich nicht durch Zelltod sondern durch den Verlust der Vorläuferpopulation erklärt. Da Sall Proteine immer in Vorläufer- und Stammzellen aktiv sind lässt sich also ein allgemeines Modell postulieren, nachdem Sall Proteine für den Erhalt dieser nicht differenzierten Zellen nötig sind. Dieser Blickwinkel eröffnet neue Interpretationsmöglichkeiten für das Zustandekommen der Vielzahl an Missbildungen in TBS und DRRS. II Table of Contents Table of Contents ZUSAMMENFASSUNG........................................................................................................................I TABLE OF CONTENTS....................................................................................................................III INTRODUCTION................................................................................................................................. 1 1. THE SPALT-LIKE TRANSCRIPTION FACTOR FAMILY............................................................................. 1 1.1. ORTHOLOGS AND SIMILARITIES ........................................................................................................ 1 1.2. PROTEIN STRUCTURE AND SUBCELLULAR LOCALIZATION................................................................ 3 2. MUTATIONS IN SPALT-LIKE GENES CAUSE INHERITED DISEASES IN HUMANS..................................... 5 2.1. TOWNES-BROCKS SYNDROME (TBS) ................................................................................................ 6 2.2. OKIHIRO SYNDROME/RADIAL RAY SYNDROME................................................................................ 6 3. MUTATIONS IN SPALT AND SEM-4 ....................................................................................................... 7 4. SPALT-LIKE TRANSCRIPTION FACTORS AND CELL SIGNALLING ........................................................... 8 5. AIM OF THIS STUDY ............................................................................................................................. 9 RESULTS............................................................................................................................................. 11 1. COMPARISON OF SALL PROTEINS....................................................................................................... 11 1.1. COMPARATIVE EXPRESSION ANALYSIS OF SALL MRNA TRANSCRIPTS IN MOUSE.......................... 11 1.2. EXPRESSION DOMAINS OF SPALT LIKE PROTEINS OVERLAP. .......................................................... 16 2. GENERATION OF MOUSE LINES.......................................................................................................... 17 2.1. KNOCKOUT VECTOR DESIGN FOR SALL2 ........................................................................................ 17 2.2. ESTABLISHMENT OF A MOUSE LINE CARRYING A SALL2 NULL ALLELE .......................................... 19 2.3. MICE MUTANT FOR SALL2 ARE VIABLE AND FERTILE .................................................................... 20 2.4. EGFP KNOCKIN OF SALL4............................................................................................................... 20 2.5. CONDITIONAL KNOCKOUT OF SALL4 .............................................................................................. 22 2.6. THE FLOXED SALL4 ALLELE IS FUNCTIONAL................................................................................... 24 2.7. BREEDING AND STRAIN MAINTENANCE.......................................................................................... 25 3. SALL4 IS REQUIRED FOR PERIIMPLANTATION DEVELOPMENT .......................................................... 26 3.1. NO HOMOZYGOUS EMBRYOS WERE FOUND AT E10.5 .................................................................... 26 III Table of Contents 3.2. GENERATION AND TESTING OF AN ANTIBODY RAISED AGAINST MURINE SALL4........................... 26 3.3. PRECISE EXPRESSION ANALYSIS FOR SALL4 .................................................................................... 28 3.4. IN VIVO ANALYSIS OF EMBRYOS DELETED OF SALL4 ..................................................................... 31 Sorting Embryos at Blastocyst Stage.................................................................................................... 31 Immunohistochemical Analysis of Expanded Blastocysts. ................................................................ 32 Histological Analysis at e6.5 and e7.5 ................................................................................................. 35 Lineage Marker Analysis on Implantation Sites................................................................................. 35 3.5. EX VIVO ANALYSIS.......................................................................................................................... 37 Outgrowth of Blastocysts in Culture..................................................................................................
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