dc.contributor.author
Vallecillo García, Pedro
dc.date.accessioned
2018-06-07T17:33:47Z
dc.date.available
2016-04-08T08:50:10.558Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/3993
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-8193
dc.description
Contents Acknowledgements 1 Abstract 2 Zusammenfassung 3 Résumé 4 Introduction
4.1 Embryological origins of skeletal muscles 4.1.1 Genetic networks
controlling myogenesis 4.1.2 Generic program 4.2 Genetic cascades underlying
skeletal muscle specification differ depending on muscle position in the embryo
4.2.1 Head Muscles 4.2.2 Axial Muscles 4.2.3 Limb muscles 4.3 Extrinsic
signals regulating axial muscle formation 4.4 Limb muscle patterning 4.4.1
Migration 4.4.2 Establishment of the ventral and dorsal muscle masses 4.4.3
Limb muscle splitting 4.5 Lateral plate mesoderm derived tissues and their
roles in muscle patterning 4.5.1 Cartilage and muscle interactions 4.5.2
Tendon and muscle interactions 4.5.3 Muscle connective tissue and muscle
interactions 4.6 Odd-skipped related gene 1 (Osr1) 4.7 Chemokines signaling
molecules orchestrate tissue formation 4.8 Extracellular matrix involvement in
myogenesis 4.9 Aims of the study 5 Material 5.1 Instruments 5.2 Chemicals 5.3
Buffers 5.4 Kits 5.5 Plasmids 5.6 Antibodies 5.7 Bacteria 5.8 Primer 5.9
Imaging software 5.10 Other software 5.11 Internet resources 5.12 Mouse lines
6 Methods 6.1 Molecular Biological Methods 6.1.1 Isolation of genomic DNA
6.1.2 Total RNA isolation 6.1.3 cDNA synthesis 6.1.4 Synthesis of digoxigenin
labeled RNA-transcript 6.1.5 Polymerase chain reaction (PCR) 6.1.6 Sanger
sequencing 6.2 Preparation of animal tissue 6.2.1 Fixation of prepared
embryonic tissue 6.3 Histological methods 6.3.1 Paraffin embedding and
sectioning 6.3.2 Cryo-embedding and sectioning 6.3.3 In situ hybridization
(cryosections) 6.3.4 Immunohistochemistry 6.3.5 Whole-mount
immunohistochemistry 6.3.6 Oil Red O staning 6.4 Cell culture methods 6.4.1
Extraction and culturing of primary embryonic cells 6.4.2 Immunocytochemistry
(ICC) 6.5 Biochemical Methods 6.5.1 Total Protein isolation and protein
concentration 6.5.2 SDS PAGE 6.5.3 Western blot (WB) 6.6 Statistical analyses
7 Results 7 7.1 Characterization of Osr1 expression and cell lineage fate
during mouse embryonic development 7.1.1 Expression pattern of Osr1 during
embryonic limb development . 7.1.2 Contribution of Osr1 cells to limb tissues
7.2 Lack of Osr1 in Osr1GCE/GCE mutants leads to muscle defects 7.2.1 Muscle
patterning defects in Osr1GCE/GCE embryos 7.2.2 Myofiber disorganization in
Osr1GCE/GCE embryos 7.2.3 Formation of ectopic muscles and ectopically located
muscle progenitors in Osr1GCE/GCE embryos 7.2.4 Correlation of Osr1 expression
with phenotypic changes in muscle patterning 7.3 Transcriptome analysis of
embryonic Osr1+ cells 7.3.1 RNA-sequencing of Osr1+ sorted cells 7.3.2 Gene
ontology analysis of deregulated genes 7.4 Osr1 is required for connective
tissue identity in the embryo 7.5 Extracellular matrix impairment in
Osr1GCE/GCE embryos 7.5.1 Osr1 is required for the correct production and
organization of structural components of the extracellular matrix 7.5.2 Basal
lamina disruption in muscles fibers of Osr1GCE/GCE embryos 7.6 Osr1GCE/GCE
embryos display defects in tendon formation 7.7 Impaired Cxcl12/Cxcr4 axis in
muscle progenitors of Osr1GCE/GCE embryos 7.8
ReducedproliferativecapacitiesofmuscleprogenitorscellsinOsr1GCE/GCE mutants
7.9 Increased apoptosis of myogenic cells in the limb of Osr1GCE/GCE embryos
7.10 Reduced number of myogenic cells in the limb of Osr1GCE/GCE embryos 7.11
MyoblastsexhibitimpairedterminaldifferentiationinOsr1GCE/GCE mutants 7.12
DifferentialfusionimpairmentsofmyogeniccellsinOsr1GCE/GCE embryos in vitro and
in vivo 7.13 Patterning defects are preceded by muscle progenitor mislocation
8 Discussion 8.1 Osr1 expression and potential during mouse development 8.1.1
Osr1 labels connective tissue cells associated with skeletal muscle during
mouse limb development 8.1.2 Contribution of Osr1+ connective tissue cells to
fetal tissues 8.2 Osr1 involvement in skeletal muscle formation 8.2.1 Muscle
patterning impairments in Osr1 knockout mice 8.2.2 Muscle patterning defects
are preceded by limited and mislocated myogenic cells 8.3 Defects in tendon
formation 8.4 Transcriptome analyses of Osr1GCE/+ and Osr1GCE/GCE sorted cells
8.4.1 Maintenance of connective tissue identity 8.4.2
ImportanceofOsr1+connectivetissuecellsinextracellularmatrix formation 8.4.3
Osr1+ connective tissue cells and secreted molecules 9 Future work 10
References Supplementary Material List of figures List of tables List of
publications
dc.description.abstract
The musculoskeletal system allows body motion. Despite the distinct mesodermal
origins of its components, the development of muscle, connective tissue (CT)
and bone is highly coordinated. Osr1 encodes a zinc-finger transcription factor
expressed in muscle CT in limbs. The aim of the PhD thesis was to elucidate
Osr1 function in the non-cell autonomous regulation of mouse limb muscle
formation. Genetic lineage tracing revealed that Osr1+ cells are progenitors
for several CTs, including muscle, dermal and lung CTs, but also for smooth
muscle and brown adipocytes. Comprehensive phenotypic analysis of skeletal
muscles in E13.5Osr1GCE/GCE mouseembryos revealed impaired muscle formation.
Transcriptomic analysis highlighted two major molecular characteristics caused
by the lack of Osr1 activity. First,Osr1 activelyrepressed the expression of
genesassociated with cartilage and tendondevelopment, suggesting that Osr1
confers a muscle connective tissue identity. Second, Osr1 positively regulated
the expression of components of the extracellular matrix (ECM). In addition to
the decrease of ECM components, numerous signaling molecules were significantly
down-regulated in Osr1-deficient cells of mutant embryos. This highlights the
function of Osr1+ resident connective tissue cells in limb muscle formation.
It also establishes that Osr1 regulates the transcription of ECM components in
limb muscle CT. Lastly, it suggests that Osr1 exerts its function via
chemokines and secreted factors to ensure proper muscle development.
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dc.description.abstract
Das muskuloskeletale System ist essentiell für die Fähigkeit zur Fortbewegung.
Dieses aus mehreren Komponenten bestehende System erfordert eine koordinierte
Morphogenese während der Entwicklung. Das Osr1 (Odd skipped-related 1) Gen
kodiert für einen ZinkFinger Transkriptionsfaktor der im Muskel-Bindegewebe
der Extremität im Huhn, wie auch in der Maus exprimiert ist. Das Ziel dieser
Arbeit war, die Funktion von Osr1 in der nicht-Zell-autonomen Regulation der
Muskelentwicklung in der Extremität der Maus zu analysieren. Durchgeführte
Analysen der Osr1 Zelllinie zeigten, dass Osr1 Zellen Vorläufer für
verschiedene Arten von Bindegewebe sind, darunter das Muskelbindegewebe, das
Bindegewebe der Dermis oder retikuläre Fibroblasten der Lunge. Außerdem wurde
gefunden, dass Osr1 Zellen Vorläufer für glatte Muskelzellen darstellen und
für braune Fettzellen. Eine komprehensive Analyse des Muskelphänotyps in
Osr1-defizienten Mäusen(Osr1GCE/GCE) zeigte klare Defekte in der lokalen
Musterbildung. Durch eine Transkriptomanalyse konnte gezeigt werden, dass zwei
Hauptaspekte durch das Fehlen von Osr1 betroffen waren. Zum einen wird Osr1
benötigt, um die Aktivität von Genen, die mit der Entwicklung von Knorpel- und
Sehnenzellen assoziiert sind, zu reprimieren. Dies suggeriert, dass Osr1 an
der Festlegung einer zellulären "Bindegewebsidentitätïn den mesenchymalen
Vorläufern der Extremität beteiligt ist. Zum anderen wird Osr1 benötigt, um
Gene der muskulären extrazellulären Matrix zu aktivieren. Zusätzlich zu einer
Reduktion der Matrixkomponenten, zeigten zahlreiche Gene für Signalmoleküle
eine Herunterregulation in Osr1+ Zellen aus Osr1GCE/GCE Embryos.
Zusammengefasst zeigen diese Daten eine funktionelle Rolle der Osr1
BindegewebsZellpopulation im Prozess der Muskelentwicklung der Säugetier-
Extremität. Osr1 scheint in diesem Zusammenhang die Transkription von
extrazellulären Matrixkomponenten positiv zu regulieren. Schließlich
suggerieren die Daten, dass Osr1 einen Teil seiner Funktion auch darüber
bewerkstelligt, Chemokine und andere sekretierte Faktoren zu induzieren,
welche die Musterbildung der Muskeln bestimmen.
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dc.format.extent
140 Seiten
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
skeletal muscle
dc.subject
connective tissue
dc.subject
extracellular matrix
dc.subject
reduction in proliferation
dc.subject
tendon development
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie
dc.title
Function of the transcription factor Osr1 in the connective tissue-mediated
control of muscle formation
dc.contributor.inspector
Dr. Delphine Duprez
dc.contributor.inspector
Dr. Fabien Le Grand
dc.contributor.inspector
Dr. Doreen Janke
dc.contributor.firstReferee
Prof. Dr. Sigmar Stricker
dc.contributor.furtherReferee
Prof. MD. Simone Spuler
dc.contributor.furtherReferee
Prof. Dr. Ketan Patel
dc.contributor.furtherReferee
Prof. Dr. Manuel Koch
dc.date.accepted
2015-09-30
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000101698-9
dc.title.translated
Die Rolle des Bindegewebs-spezifischen Transkriptionsfaktors Odd skipped-
related (Osr1) in der Musterbildung der Muskulatur
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refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000101698
refubium.mycore.derivateId
FUDISS_derivate_000000018932
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access