Developing a Zebrafish model for muscle regeneration [Elektronische Ressource] / presented by Sandeep Paul

De
DISSERTATION SUBMITTED TO THE COMBINED FACULTIES FOR THE NATURAL SCIENCES AND FOR MATHEMATICS OF THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY FOR THE DEGREE OF DOCTOR OF NATURAL SCIENCES   SANDEEP PAUL INSTITUTE FOR TOXICOLOGY AND GENETICS FORSCHUNGSZENTRUM KARLSRUHE Examination Committee: Prof. Dr. Uwe Strähle Prof. Dr. Stephan Frings Prof. Dr. Nicholas Foulkes Prof. Dr. Gabriele Petersen Date of the oral exam:……………………..   DEVELOPING A ZEBRAFISH MODEL FOR MUSCLE REGENERATION Presented by SANDEEP PAUL Institute for Toxicology and Genetics Forschungzentrum Karlsruhe DISSERTATION SUBMITTED TO THE COMBINED FACULTIES FOR THE NATURAL SCIENCES AND FOR MATHEMATICS OF THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY FOR THE DEGREE OF DOCTOR OF NATURAL SCIENCES  For my parents…    Zusammenfassung Ein besseres Verständnis der Muskelregeneration würde es uns ermöglichen, effektivere Therapien für Patienten zu entwickeln, die unter degenerativen Muskelerkrankungen leiden, wie zum Beispiel muskuläre Dystrophien. Modellorganismen erleichtern das Verständnis von Muskelregeneration, jedoch wurden bis jetzt nur Nagetiere und Hühner entsprechend untersucht. In der vorliegenden Arbeit wurden drei verschiedene Ansätze angewendet, um ein Modell der Muskelregeneration im Zebrafisch zu erstellen.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 43
Tags :
Source : ARCHIV.UB.UNI-HEIDELBERG.DE/VOLLTEXTSERVER/VOLLTEXTE/2009/9697/PDF/SANDEEP_THESIS.PDF
Nombre de pages : 233
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DISSERTATION

SUBMITTED TO
THE COMBINED FACULTIES FOR
THE NATURAL SCIENCES AND
FOR MATHEMATICS OF

THE RUPERTO-CAROLA
UNIVERSITY OF HEIDELBERG,
GERMANY
FOR THE DEGREE OF
DOCTOR OF NATURAL
SCIENCES

 
SANDEEP PAUL
INSTITUTE FOR TOXICOLOGY AND GENETICS
FORSCHUNGSZENTRUM KARLSRUHE



Examination Committee:
Prof. Dr. Uwe Strähle
Prof. Dr. Stephan Frings
Prof. Dr. Nicholas Foulkes
Prof. Dr. Gabriele Petersen

Date of the oral exam:……………………..


 
DEVELOPING A ZEBRAFISH
MODEL FOR MUSCLE
REGENERATION
Presented by
SANDEEP PAUL
Institute for Toxicology and Genetics
Forschungzentrum Karlsruhe

DISSERTATION SUBMITTED TO
THE COMBINED FACULTIES FOR THE NATURAL
SCIENCES AND FOR MATHEMATICS OF
THE RUPERTO-CAROLA UNIVERSITY OF
HEIDELBERG, GERMANY
FOR THE DEGREE OF
DOCTOR OF NATURAL SCIENCES
 
For my parents…






 
 
Zusammenfassung
Ein besseres Verständnis der Muskelregeneration würde es uns ermöglichen,
effektivere Therapien für Patienten zu entwickeln, die unter degenerativen
Muskelerkrankungen leiden, wie zum Beispiel muskuläre Dystrophien.
Modellorganismen erleichtern das Verständnis von Muskelregeneration, jedoch
wurden bis jetzt nur Nagetiere und Hühner entsprechend untersucht.
In der vorliegenden Arbeit wurden drei verschiedene Ansätze angewendet, um
ein Modell der Muskelregeneration im Zebrafisch zu erstellen.
Erstens, wurde ein ENU-Mutagenese-Screen durchgeführt, um Mutanten mit
defekter Muskelerhaltung zu identifizieren, die möglicherweise auf fehlerhafter
Regeneration basiert. Es wurde eine Mutante identifiziert und charakterisiert (gum),
die einen fortschreitenden Verlust von Beweglichkeit und myofibrillärer Organisation
aufweist. Charakterisierung der gum Mutanten ließ multiple Defekte in der
Muskulatur und in neuronalem und Neuralleisten-Gewebe erkennen.
Zweitens, wurde durch Verwendung eines Acetylcholinesterase-Inhibitors
(GAL) ein chemisch induzierbares Modell für Myopathie im Zebrafisch entwickelt.
Entfernung von GAL erlaubte es den Muskeln sich zu regenerieren und ihre normale
Funktion wiederzuerlangen. Basierend auf Elektronenmikroskopie und
Antikörperfärbung wurden vermeintliche Muskelstammzellen (Satellitenzellen) des
Zebrafischs identifiziert. Pax7, ein Hauptmarker für Satellitenzellen in allen
Wirbeltieren, markiert im Zebrafisch eine Zellschicht, das Dermomyotom, auf der
Oberfläche der Somiten ab dem 24 Stunden Stadium (24 hpf). Diese Zellen bilden
zunächst FT Muskelfasern (weiße Muskelfasern) und später Satellitenzellen. In dieser
Arbeit wurde beobachtet, dass Pax7-positive Dermomyotomzellen erhöhte
  iProliferation aufweisen und eine gesteigerte Bewegung in tiefere Schichten des
Myotoms nach Beschädigung der Muskulatur.
Drittens, zeigte die Erstellung eines genomweiten Transkriptionsprofils von
mit GAL behandelten Zebrafisch-Larven eine Heraufregulierung von zahlreichen
Genen als Reaktion auf die Myopathie. Ein Vergleich dieser Gene mit
Muskelregenerationsmodellen in der Maus zeigte eine signifikante Übereinstimmung
(ca. 25%). Eine Expressionsanalyse einiger dieser Gene (cmya1, zgc:100919) deutet
darauf hin, dass sie möglicherweise eine Rolle in der Biologie von Satellitenzellen
spielen.
















ii
Abstract
A better understanding of muscle regeneration would allow us to devise
therapies that are more effective for patients suffering from myodegenerative diseases
such as muscular dystrophies. Animal models facilitate the understanding of muscle
regeneration, but so far, only rodents and chicken have been suitably exploited in this
regard.
In the present study, I adopted three different approaches to establish a model of
muscle regeneration in zebrafish. Firstly, an ENU mutagenesis screen was performed for
mutants with defective muscle maintenance that might result from faulty regeneration. I
identified and characterized one mutant (gum) showing progressive loss of motility and
myofibrillar organization. Characterization of gum mutants revealed multiple defects in
muscle, neuronal and neural crest derived tissues.
Secondly, using an inhibitor of acetycholinesterase (GAL), I established a chemically
inducible model of myopathy in zebrafish. Removal of GAL allowed the muscles to
regenerate and restored their normal function. Based on electron microscopy and
immunohistochemistry, the zebrafish putative muscle stem cells (satellite cells) were
identified. Pax7, a key marker for satellite cells in all vertebrates, labels a layer of cells, the
dermomyotome, on the surface of zebrafish somites from 24 hpf onwards. These cells give
rise initially, to the fast muscle fibers and later to the satellite cells. In this study, it was
+veobserved that the Pax7 dermomyotome cells show increased proliferation and migration
into deeper myotome upon muscle damage.
Thirdly, unbiased genome wide transcriptional profiling of GAL treated zebrafish
larvae showed numerous genes upregulated in response to the myopathy. Comparison of these
genes to mouse models of muscle regeneration showed a significant (about 25%) overlap.
Expression analysis of some of the genes (cmya1, zgc:100919) indicates that they might have
a role in satellite cells biology.

  iii“Sandeep, you should be as creative about your sequences as you are about coffee.”Uwe
Acknowledgements
This is the most difficult part: to sum up the experience of a few years in a few words,
that convention demands but comprehension defies. I would start by thanking Uwe for
accepting me as his graduate student, and then forgetting me for large tracts of time. That
helped me become independent (I believe, Uwe hopes) to a level where I am now. I would
like to thank Lixin for all the initial help in the lab especially for microarray experiments.
Yavor made it a lot easier for me to settle down in a new place, both within and outside the
lab. Christelle deserves a special mention for being hypercritical of each experiment I did
(and still do). Anticipating her questions helps me to improve the quality of my work. I would
like to thank Maryam and Sepand, for maintaining the nice social interaction within the lab,
and also Maryam for help with microscopy and Sepand for help with especially difficult
clonings. I would like to thank Masanari for showing me how to make your data visually
appealing and many, many, many discussions/suggestions about labeling and live imaging.
Urmas deserves special thanks for maintaining my (in)sanity in trying times with his humor,
which I appreciate even more than his skills in photography. I would like to thank Martin for
the excellent (I have been told so by native speakers) German translation of the abstract of my
thesis. I would also like to thank Olivier for not only all the help with molecular biology, gene
ontology, microarrays etc, but also for entertaining us with his adorable French accent. I
should also thank the fish facility staff; without their indefatigable care I would have never
learned how to take care of my fish.
That this report has so few mistakes is due to the diligent, untiring and (mostly) brutal
editing of Thomas. Not that he did it willingly; I had to cajole, beg, grovel, and finally
threaten him in equal measure. If it has many mistakes it is due to my confusion between
picking corrected copies vs. uncorrected ones.
And finally, I would like to thank Simone, for everything else and more.
iv
Table of Contents
Zusammenfassung..........................................................................................i
Abstract….…………………………………………….……….....…….….iii
Acknowledgements………………………………………….………..........iv
List of Abbreviations……………………………………………………….x
List of Tables……………………………………………………………....xi
List of Figures……………………………………………………...………xi
1 Introduction ............................................................................................ 1
1.1 Regeneration in metazoans 1
1.2 Mechanism of Regenerations: Stem Cells vs. De-differentiation.................................. 2
1.2.1 Stem cells and stem cell niche................................................................................... 2
1.2.2 Strategies of stem cell propagation: Asymmetric vs. Symmetric division ................ 5
1.2.3 Cellular dedifferentiation........................................................................................... 7
1.3 Muscular Dystrophies: The need for understanding Muscle Regeneration ................... 9
1.4 Embryonic myogenesis and the origin of muscle stem cells ....................................... 12
1.4.1 Early mesoderm and its derivatives......................................................................... 13
1.4.2 The formation of somites......................................................................................... 14
1.4.3 Muscle development from the somites: A morphological overview....................... 18
1.4.4 The spatial patterning of somite occurs in response to signals emanating from
adjacent tissues ...................................................................................................................... 23
1.5 Skeletal muscle regeneration: The role of satellite cells.............................................. 31
1.5.1 The role of pax3/7 genes in satellite cell biogenesis and function .......................... 34
1.5.2 Satellite cell activation............................................................................................. 35
1.5.3 Myogenic progression of satellite cells ................................................................... 37
1.5.4 Satellite cell self renewal and the stem cell potential: the role of the niche and
the heterogeneity within ........................................................................................................ 42
1.5.5 Therapeutic Potential of Satellite cells .................................................................... 49
  v1.5.6 Non-satellite cell mediated muscle regeneration ..................................................... 50
1.6 Myogenesis in zebrafish............................................................................................... 51
1.6.1 Somite patterning in zebrafish ................................................................................. 52
1.6.2 Patterning of the zebrafish myotome...................................................................... 55
1.6.3 Gene networks in skeletal muscle development in zebrafish .................................. 59
1.7 Zebrfaish as a model for developmental genetics ........................................................ 62
1.7.1 Genetic methods to study zebrafish muscle development and regeneration ........... 62
1.7.1.1 Forward genetics approaches to study zebrafish .............................................62
1.7.1.2 Reverse genetics appr zebrafish ..............................................64
1.7.2 Zebrafish genomics.................................................................................................. 65
1.7.3 Transgenic reporters: live imaging possibilities ...................................................... 66
1.7.4 High throughput analysis......................................................................................... 67
1.8 The need for a new model: using zebrafish to study muscle regeneration................... 67
2 Materials and Methods ........................................................................ 69
2.1 General Procedures ...................................................................................................... 69
2.1.1 Fish breeding and maintenance ............................................................................... 69
2.1.2 Using the escape response as an assay for motility ................................................. 69
2.1.3 Birefringence assay to assess the myofibrillar structure.......................................... 70
2.1.4 Galanthamine treatment........................................................................................... 70
2.2 Molecular Biology Methods ........................................................................................ 70
2.2.1 PCR, semi-quantitative RT-PCR, and cDNA Cloning ............................................ 71
2.2.2 Restriction Digestion and Ligation of DNA............................................................ 71
2.2.3 Extraction of DNA from agarose gel....................................................................... 71
2.2.4 TOPO-cloning of genes perturbed in microarray studies ........................................ 72
2.2.5 Transformation of competent E. coli cells and electroporation of targeting vector
fragment in electrocompetent BAC containing EL250 cells................................................. 73
2.3 Histological Methods................................................................................................... 74
2.3.1 Whole mount in-situ hybridization (WISH) ............................................................ 74
2.3.2 Plastic sections of epon mounted WISH embryos................................................... 75
vi

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