Structure-function analysis and aspects of evolution of the transcription factor Sox10 [Elektronische Ressource] = Struktur-Funktionsanalyse des Transkriptionsfaktors Sox10 und Aspekte der Evolution / vorgelegt von François Cossais

Structure-function analysis and aspects of evolution of the transcription factor Sox10Struktur-Funktionsanalyse des Transkriptionsfaktors Sox10 und Aspekte der Evolution Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-NürnbergzurErlangung des Doktorgrades Dr. rer. nat.vorgelegt vonFrançois Cossaisaus La Roche-sur-Yon, FrankreichAls Dissertation genehmigt von der Naturwissen-schaftlichen Fakultät der Friedrich-Alexander-UniversitätErlangen-NürnbergTag der mündlichen Prüfung: 19.05.2010Vorsitzender der Promotionskommission: Prof. Dr. Eberhard BänschErstberichterstatter: Prof. Dr. Michael WegnerZweitberichterstatter: Prof. Dr. Andreas Feigenspanto my family and friends,Table of contents IVTable of contents : Zusammenfassung ..........................................IX..................................................................Summary .................................................X.............................................................................1. Introduction ............................................................................. 1 ............................1.1 Sox protein.s...............................................1.........................................................................1.2 The SoxE gro.u.p...........................................4......................................................................1.2.1 Evolution of SoxE genes..................................
Publié le : vendredi 1 janvier 2010
Lecture(s) : 31
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Source : D-NB.INFO/1003762344/34
Nombre de pages : 155
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Structure-function analysis
and aspects of evolution
of the transcription factor Sox10
Struktur-Funktionsanalyse
des Transkriptionsfaktors Sox10 und
Aspekte der Evolution
Der Naturwissenschaftlichen
Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
François Cossais
aus La Roche-sur-Yon, FrankreichAls Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Tag der mündlichen Prüfung: 19.05.2010
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Michael Wegner
Zweitberichterstatter: Prof. Dr. Andreas Feigenspanto my family and friends,Table of contents IV
Table of contents :
Zusammenfassung ..........................................IX..................................................................
Summary .................................................X.............................................................................
1. Introduction ............................................................................. 1 ............................
1.1 Sox protein.s...............................................1.........................................................................
1.2 The SoxE gro.u.p...........................................4......................................................................
1.2.1 Evolution of SoxE genes.........................................4.......................................................
1.2.2 SoxE propertie.s..............................................5...............................................................
1.2.3 So.x8.....................................................7.........................................................................
1.2.4 So.x9.....................................................8.........................................................................
1.2.5 Sox1.0....................................................9........................................................................
1.2.5.1 Sox10, general informati.o.ns..................................9...............................................
1.2.5.2 Sox10 and disea.se.s......................................1.0.....................................................
1.3 Neural developme.nt.........................................1.3................................................................
1.3.1 NC development..............................................13.............................................................
1.3.1.1 Induc.t.io.n............................................1.3...............................................................
1.3.1.2 Specifica.ti.o.n..........................................1.4...........................................................
1.3.1.3 Migrati.o.n.............................................1.6...............................................................
1.3.1.4 Fate specification/determination and differentia.t.io.n. ................1.8 ........................
1.3.1.5 NC evolut.io.n...........................................2.1...........................................................
1.3.2 Oligodendrocyte development ....................................2.2...............................................
2. Aim of the study ........................................................................ 26 ........................
3. Results ................................................................................... 28 ............................
3.1 Functional analysis of SOX10 during early neural crest develo.p.m.e.nt.......2.8..............
3.1.1 Altered gene expression after ectopic SOX10 expression in the chick spina.l. .co.28.rd..
3.1.2 Structure-function studies in the electroporated chicken n.eu.r.a.l .t.u.b.e ....3.0...............
3.1.3 Consequences of human mutations on SOX10 functions on the NC....... ................34
3.1.4 SOX10 mutations, apoptosis and dominant negative funct.io.n.s.............3.7...................
3.1.5 The unusual features of the Sox10 Dom muta.t.io.n.....................4.1...............................Table of contents V
3.2 Physiological relevance of dimerization of Sox10 in. .v.iv.o...............4.5 ..........................
3.2.1 General overview of the Sox10aa1 mice..............................4.6.......................................
3.2.2 Analysis of the ENS in Sox10aa.1. m.ice............................4.7.........................................
3.2.3 Analysis of melanocyte development in Sox10aa1 mice....................4.8.........................
3.2.4 Analysis of the peripheral nervous system in Sox10aa.1. .mi.ce.............4.9....................
3.2.5 Analysis of oligodendrocyte development in Sox10aa1 .mi.ce...............5.2.....................
3.3 Aspects of Evolution of the Sox10 pr.ot.e.in.........................54..........................................
3.3.1 Comparison of sequences and biochemical properties between mouse Sox10 and
Sox100B from Drosophi.l.a.........................................5..4........................................................
3.3.2 Sox100B electroporation into the early neural tube of. .ch..icke..n..........5..7...................
3.3.3 Generation of Sox100B expressing mi.ce.............................5.9......................................
3.3.4 Ability of Sox100B to rescue melanocyte development in the absence of S.o.x1.0.6..4...
3.3.5 Ability of Sox100B to rescue ENS development in the absence of .S.o.x1.0.....6.5..........
3.3.6 Ability of Sox100B to rescue sympathetic nervous system and adrena l gland
development in the absence of Sox10 ...................................6..6.............................................
3.3.7 Ability of Sox100B to rescue DRG development in the absence of So.x1.0.......6.8..........
3.3.8 Ability of Sox100B to rescue Schwann cell development in the absence of. .So.x7.10.0..
3.3.9 Ability of Sox100B to rescue oligodendrocyte development in the absence of .S7.o2x10
4. Discussion .....................................................................................75 ....................
4.1 Structure function analysis of So.x.10.............................7.5...............................................
4.1.1 Sox10 and early NC induct..io.n..................................7.5................................................
4.1.2 Lessons of early NC development for human disease.s....................7.7..........................
4.1.3 Lessons from Sox10 knock-in mice for development of NC derivatives and
oligodendrocytes.................................................7.9.................................................................
4.2 Aspects of evolution of the Sox10 p.ro.te.i.n........................8.4..........................................
4.3 Concluding remarks and future directio.n.s..........................8.8.........................................
5. Material .........................................................................................................92 ......
5.1 Organisms................................................92..........................................................................
5.1.1 Mouse lin.es................................................9.2................................................................
5.1.2 Bacterial cul.t.u.re.s.........................................9.2............................................................
5.1.3 Cell .l.in.e................................................9.2....................................................................
5.1.4 Chicken l.i.n.e ..............................................92.................................................................
5.2 Reagents and chemica.l.s.....................................9.2...........................................................
5.3 Buffer and solution.s.........................................93................................................................Table of contents VI
5.4 Oligonucleotide.s...........................................94....................................................................
5.4.1 Oligonucleotides used for genoty.pi.n.g .............................9.4.........................................
5.4.2 Oligonucleotides used for cloning and sub.-.cl..o.n.in.g.................9.5..............................
5.4.3 Oligonucleotides used for RT-.P.C.R ...............................9.5............................................
5.4.4 Oligonucleotides used for sequencing of the Sox100B se.q.u.e.nce..........9.6.................
5.5 Probes used for in-situ hybridizati.on. .............................96...............................................
5.6 Probes used for Southern-b.l.ot.................................9.6.....................................................
5.7 Antibodie.s...............................................9.6.........................................................................
5.7.1 Primary antibo.d.ie.s.........................................9.6...........................................................
5.7.2 Secondary antibo.d.ie.s.......................................9.8........................................................
6. Methods ........................................................................ 100 ...................................
6.1 Standard metho.ds.........................................1.0.0...............................................................
6.2 Gene targeting strategy and generation of mouse m.u.t.an.t.............1.0.0........................
6.2.1 Southern-.b.l.o.t...........................................1.0.1.............................................................
6.2.2 Radioactive labeling of DNA using „Random Prim.er.“...................1.0.1..........................
6.3 Genotypin.g..............................................1.0.2......................................................................
6.3.1 Genotyping of the Sox10Sox100B a.l.le.l.e .........................1.0.2.....................................
6.3.2 Genotyping of the Sox10aa1 a.l.le.l.e .............................1.0.2..........................................
6.3.3 Genotyping of the Sox10LacZ a.l.l.el.e............................1.0.3.........................................
6.3.4 Genotyping of the Sox10Dom al.le.le..............................1.0.3.........................................
6.4 Chicken in ovo electroporat.io.n................................1.0.4..................................................
6.5 Quantitative RT-PC.R........................................1.0.4.............................................................
6.5.1 Total RNA iso.l.a.ti.o.n......................................1.0.4........................................................
6.5.2 Complementary DNA-synthesis (Reverse Transcriptio.n.). ................10.5.......................
6.5.3 Lightcycler .P.CR ...........................................1.05...........................................................
6.6 Histological staining.s.......................................1.0.5............................................................
6.6.1 Immunohistochemist..ry.......................................1.0.6.....................................................
6.6.2 Tunel a.ssa..y.............................................1.0.6..............................................................
6.6.3 in situ hydri.d.iza.t.io.n......................................1.0.7........................................................
6.6.3.1 DIG labeling of cRNA probes by in vitro tr.a.n.scr..i.p.ti.o.n.........1.0.7......................
6.6.3.2 In situ hydridization on cr.y.o.-.se..ct.i.o.ns....................10.7....................................
6.6.3.3 Whole mount in situ hybr.i.d.iza.t.io.n.........................1.08.......................................
6.6.4 NADPH-Diaphorase sta.in.i.n.g.................................1.0.9...............................................
6.7 Microscopy, image editing and statistical ana.l.ys.i.s..................1.0.9 ...............................
6.8 Cell culture methods and biochemical anal.y.si.s.....................1.10...................................Table of contents VII
6.8.1 Cultivation and tranfection of HEK293 .ce.l.l.s........................11.0...................................
6.8.2 Preparation of protein extract from. .ce.l.l.s ........................1.1.0.....................................
6.8.3 Electrophoresis of pro.t.ei.n.s ..................................1.10.................................................
6.8.4 Western bl.o.t..............................................11.1...............................................................
6.8.5 Electromobility shift .a.ssa...y..................................1.11...................................................
7. Abbreviations................................................................................................. 114 ..
8. References ....................................................................................118 ...................
Publications .............................................1.4.1......................................................................
Curriculum vitae...........................................1.4.2..................................................................
Acknowledgements .........................................14.4..............................................................Zusammenfassung IX
Zusammenfassung :
Die Entstehung von Neuralleistenzellen und Oligodendrozyten war ein wichtiger Schr itt in
der Evolution der Wirbeltiere. Mutationen des Transkriptionfaktors SOX10 beeinträch tigen
die Entwicklung dieser Zelltypen im Menschen und verursachenN eurocristopathien u nd
demyelinisierende Schädigungen. Es wird angenommen, dass die Art und die Position de r
Mutationen einen direkten Einfluß auf den Schweregrad der Krankheit haben. Die genau e
Wirkungsweise der verschiedenen Mutationen, sowie die Bedeutung von verschiedenen
Domänen des Sox10 Proteins für Entwicklungprozesse wurden bisher allerdings n icht
genau analysiert.
Im Rahmen dieser Arbeit wurden die funktionelle Bedeutung von verschiedenen SOX10
Domänen und der Einfluß von SOX10 Mutationen auf die frühe Entwicklung der
Neuralleistenzellen untersucht. Die Überexpression von SOX10 im Neuralrohr von
Hühner-Embryonen zeigte, dass die Funktion in der frühen Entwicklung d er
Neuralleistenzellen auf der D NA-Bindefähigkeit und Transaktivierungsfähigkeit d es
Proteins beruht. Außerdem zeigen meine Ergebnisse, dass verkürzte SOX10 Proteine, die
aufgrund von Nonsense-Mutationen innerhalb des vierten Exons desSO X10 Gens
produziert wurden, sich dominant negativ verhalten, dass aber die Herstellung so lcher
Proteine im Menschen durch „nonsense mediated deca“y vermieden wird. Verschiedene
Mutationen des SOX10 Proteins hatten weiterhin unterschiedlichen Einfluß auf
Oligodendrozyten und verschiedene Neuralleistenderivate. Diese Aussage wurde mittels
einer knock-in Mausvariante bestätigt, in der das Wildtyp Sox10 Protein durch eine
Mutante ohne kooperative DNA-Bindefähigkeit ersetzt wurde. Die Entwicklung vo n
Neuralleistenderivaten war in diesen Mäusen stark beeinträchtigt, wohingegen die
Oligodendrozytenentwicklung normal war.
Im zweiten Teil dieser Arbeit sollte analysiert werden, wie Sox10 während der Ev olution
seine essentiellen Funktionen in Oligodendrozyten und Neuralleistenzellen erwarb. Zu
diesem Zweck untersuchte ich, ob das Sox100B Protein Darousso phila melanogaster die
spezifischen Funktionen von Sox10in vivo ausführen kann. Es wurde Sox100B im
Neuralrohr von Hühner-Embryonen überexprimiert und eine Mauslinie generiert, in der das
Sox10 Allel durch den offenen Leserahmen vonSo x100B ersetzt wurde. Die Analyse
dieser Tiere zeigte, dass Sox100B die Funktionen von Sox10 in der frühen Neuralleist en-,
Schwannzellen- und Oligodendrozytenentwicklung übernehmen kann.Di ese Ergebnisse
beweisen, dass eine Funktionsübernahme der ursprünglichen Eigenschaften (Ko-Option)
eines anzestralen SoxE Gens in das genregulatorische Netzwerk stattgefunden hat, d as
die Entwicklung dieser Zelltypen kontrol liertSp.ätere Entwicklungsdefekte in
Sox100B/Sox100B
verschiedenen Neuralleistenderivaten in Sox10 Mäusen zeigten aber, dass
gewisse funktionelle Spezialisierungen und Adaptationen der SoxE Proteinwirkungen i m
Nachhinein erfolgt sind. Summary X
Summary :
The invention of neural crest (NC) cells and oligodendrocytes played an import ant role in
the emergence of the vertebrate phylum during evolution. In humans, mutations of the
transcription factor SOX10 affect the development of these cell types and result in a wide
range of neurocristopathies and demyelinating disease.s A direct link betweSOen X10
mutations and the severity of the disease has been proposed. However the exact mode of
action of different mutations and more generally the importance of the different domain s of
the Sox10 protein on developmental processes have not been studied in detail.
The first aim of this study was therefore to better characterize the functional impo rtance of
different Sox10 domains and the influence SOof X10 mutations on early NC development.
Using overexpression in the chicken neural tube, I showed that Sox10 function s during
early NC development rely on its DNA binding and transactivation abilities. My results
moreover argue that truncated SOX10 proteins which result from nonsense mutatio ns in
exon 4 of thSOe X10 gene behave as dominant negatives but that production of t hese
proteins is prevented in human patients by nonsense mediated decay. Different mutation s
within the SOX10 protein can furthermore influence oligodendrocytes and NC-d erived
lineages differentially. This last conclusion was also confirmed in a knock-in mouse mutant
where wild-typSoe x10 was replaced by a mutant version without cooperative binding
capabilities. In these mice, development of most NC-derived-cell types was se verely
impaired whereas oligodendrocyte development was not.
The second aim of this study was to understand how Sox10 acquired its essential
functions in oligodendrocytes and NC cells during evolution. For that purpose I analyzed
the capability oSo f x 100B from Drosophila melanogaster to perform Sox10 specif ic
functionsi n vivo. Sox100B was overexpressed in the chicken neural tube and a mutant
mouse line was established where thSoe x10 allele was replaced by Sothxe 100B open
reading frame. Anaysis of these animals showed that Sox100B can take over the fu nctions
of Sox10 during early NC, Schwann cell and oligodendrocyte development arguing th at an
ancestralSo xE gene could have easily integrated into the gene regulatory networks that
control the development of these cell types following its co-option. How ever, later
developmental defects in several NC derived lineages in mice homozygous for th e
Sox100B replacement allele indicate that some degree of functional specialization and
adaptation of SoxE protein properties have taken place later on.

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