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Drosophila myogenesis as a model for studying cis-regulatory networks [Elektronische Ressource] : identifying novel players and dissecting the role of transcriptional repression / Lucia Ciglar

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Drosophila myogenesis as a model for studying cis-regulatory networks: identifying novel players and dissecting the role of transcriptional repression Lucia Ciglar (Kayserová) 2010 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 Presented by Bachelor of Science Lucia Ciglar born in Šala, Slovakia Oral examination: th6 July 2010 Drosophila myogenesis as a model for studying cis-regulatory networks: identifying novel players and dissecting the role of transcriptional repression Referees: Dr. Jürg Müller Prof. Dr. Herbert Steinbeisser Inaugural-Dissertation zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg vorgelegt von Bachelor of Science Lucia Ciglar aus Šala, Slovakia Tag der mündlichen Prüfung: 6. Juli 2010 ACKNOWLEDGMENTS My foremost gratitude belongs to Eileen Furlong, in whose lab my PhD. research was performed. She has been a truly exceptional, supportive, and caring supervisor and I feel deeply indebted for all her tremendous knowledge and precious time that she shared with me.
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Drosophila myogenesis as a model
for studying cis-regulatory networks:
identifying novel players and dissecting the role of
transcriptional repression






Lucia Ciglar
(Kayserová)
2010


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












Presented by

Bachelor of Science Lucia Ciglar
born in Šala, Slovakia



Oral examination:
th6 July 2010


Drosophila myogenesis as a model for
studying cis-regulatory networks:
identifying novel players and dissecting the role of
transcriptional repression













Referees: Dr. Jürg Müller
Prof. Dr. Herbert Steinbeisser







Inaugural-Dissertation

zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen Gesamtfakultät
der
Ruprecht-Karls-Universität
Heidelberg








vorgelegt von

Bachelor of Science Lucia Ciglar
aus Šala, Slovakia




Tag der mündlichen Prüfung:
6. Juli 2010
ACKNOWLEDGMENTS
My foremost gratitude belongs to Eileen Furlong, in whose lab my PhD.
research was performed. She has been a truly exceptional, supportive, and caring
supervisor and I feel deeply indebted for all her tremendous knowledge and precious
time that she shared with me.
I am also very grateful to my academic advisors Jürg Müller, Herbert
Steinbeisser, François Spitz, Walter Wahli, and Carl Neumann, for their guidance and
advice during our yearly meetings.
A big THANK YOU goes to the entire Furlong lab, I am very happy to have
had a chance to work with you all!
Martina Braun and Hilary Gustafson gave me a lot of assistance throughout
my PhD. and were very kind and patient all the time. I really enjoyed all the lunches,
coffees, and little chats that we had together!
Guillaume Junion brought a lot of joy to the everyday life and thanks to his
unique French charm I handled with smile even the most difficult situations.
I cannot thank enough my desk and benchmate, Robert Zinzen, from whom I
have been learning a lot every day and who also critically read parts of my thesis and
translated the summary. His extraordinary expertise saved me a lot of time and
inspired me throughout my PhD.
To Stefan Bonn I am very grateful for his exciting, phenomenal ideas about
my projects as well as science in general. I also deeply appreciate all his advice on
scientific writing and presentations and help with translation of the summary.
I am very happy that I had a chance to work with Ya-Hsin Liu, who has
always been kind and encouraging, and very helpful, especially with the histological
techniques and embryo imaging.
Without Junaid Akhtar my life in the lab would have been a lot less
entertaining. He also helped me a lot with the subcloning strategies and cell culture
based assays.
I thank Jelena Erceg for spreading her enthusiasm for science and being
encouraging all the time, keep smiling!
Without the expertise of our informaticians, the work presented here would not
have been possible. Bartek Wilczynski performed all the statistical analysis and
processed the results of the ChIP-on-chip experiments. Even more, he did an excellent job explaining to me patiently the analysis that he has done. Charles
Girardot similarly helped with the analysis of ChIP-on-chip data, provided the input
for the molecular screen, and was always extremely helpful. I also learnt a lot from
Nicolas Delhomme, Julien Gagneur, Mikhail Spivakov, and Zhen Xuan Yeo.
I would like to thank our new lab members Yad Ghavi Helm, Pierre Khoueiry,
and Enrico Cannavo for contributing to the great atmosphere and making the lab an
even more pleasant place.
Equally, I am indebted to the past members of the Furlong lab, Thomas
Sandmann, Janus Jakobsen, and Michal Karzynski, for a warm welcome and help at
the beginning of my PhD. I am especially thankful to Paulo Cunha for our nice
collaboration on the Lame duck project.
I am also very grateful to the core facilities at EMBL, in particular the
Advanced Light Microscopy Facility, GeneCore, and Vladimír Beneš for (not only)
technical support. My gratitude belongs to Sandra Müller who has performed all
embryonic injections.
Last, but not least, I would like to acknowledge the Louis-Jeantet Foundation
for making my PhD. at EMBL possible.

Ďakujem aj naším českým priateľom za všetky skvelé akcie a výlety, ktoré
sme spolu podnikli. Vďaka vám sme sa tu cítili ako doma, budete nám veľmi chýbať!

Túto prácu s láskou venujem svojím najbližším, ktorým patrí moja najväčšia vďaka.
V prvom rade ďakujem môjmu milovanému manželovi za všetku jeho lásku,
pochopenie, starostlivosť, a trpezlivosť. Mojím úžasným, milujúcim rodičom,
bratom, a starým rodičom, za ich nikdy nekončiacu lásku a podporu, aj keď to pre
nich znamenalo mnoho neľahkých chvíľ. Ďakujem aj mojím druhým rodičom a
švagrovcom za láskyplné prijatie do rodiny a manžela, o akom sa mi ani nesnívalo.
Veľká vďaka patrí aj všetkým ďalším členom bližšej i širšej rodiny a rodinným
priateľom za vytvorenie úžasného zázemia, ktorému som nesmierne šťastná.

TABLE OF CONTENTS

ACKNOWLEDGMENTS _______________________________________ 5

LIST OF FIGURES AND TABLES ______________________________ 10

SUMMARY__________________ 12

ZUSAMMENFASSUNG_______ 14

ABBREVIATIONS____________________________________________ 17

GENE SYMBOLS_____________ 18

1
 INTRODUCTION__________ 20

1.1
DIFFERENTIAL
GENE
EXP RESSION:
THE
BASIS
FOR
DEVELOPMENT...............................20 

1.1.1
 The
initiation
of
transcription
is
key
to
differential
gene
expression............... 20

1.1.2
 Transcriptional
repression
in
developmental
decision
making.......................... 22

1.1.3
 The
Mediator
complex
and
its
role
in
the
regulation
of
gene
expression ........ 23

1.1.4
 General
principles
of
developmental
cis­regulatory
networks............................ 28

1.1.5
 Drosophila
muscle
development
as
a
model
for
dissecting
the
logic
of

regulatory
networks................................ ................................ ................................ ............ 29

1.2
EARLY
STAGES
OF
MESODERM
DEVELOPMENT
IN
DROSOPHILA 
EMBRYOS...................31

1.2.1
 A
nuclear
gradient
of
the
transcription
factor
Dorsal
subdivides
the
dorsal­
ventral
axis
of
the
blastoderm
embryo ....... 31

1.2.2
 Twist
and
Snail:
two
major
transcriptional
regulators
in
the
presumptive

mesoderm ................................ ................................ ................................ ................................ .33

1.3 
MUSCLE
DEVELOPMENT
IN
DROSOPHILA 
EMBRYOS.........................37

1.3.1
 Inductive
signals
from
the
ectoderm
subdivide
the
mesoderm
into
four

do mains ..... 37

1.3.2
 Specification
and
morphogenesis
of
the
visceral
musculature............................ 38

1.3.3
 Cardioblasts
specification
and
the
formation
of
a
functional
heart................. 39

1.3.4
 The
sp ecification
of
somatic
muscle
progenitors
is
orchestrated
by
crosstalk

between
multiple
pathways................................ ................................ ............................. 40

1.3.5
 Myoblast
fusion:
when
founder
cells
met
fusion
competent
myoblasts........... 42 

1.3.6
 From
myoblast
fusion
to
muscle
attachment .............................. 43

1.4
TRAMTRACK
IS
A
TRANSC RIPTIONAL
REPRESSOR
WITH
NUMEROUS
DEVELOPMENTAL

ROLES..................................................................46

1.4.1
 Molecular
characteristics
and
embryonic
expression
of
Ttk69
and
Ttk88 .... 46 

1.4.2
 Extensive
developmental
roles
of
Ttk69
and
Ttk88 .................. 49 

-7- TABLE OF CONTENTS

1.4.3
 Dynamic
expression
of
Ttk69
and
Ttk88
is
achieved
by
diverse
regulatory

mechanisms................................ ................................ ................................ ............................. 57

2
 AIMS OF THE THESIS_____________________________________ 62

3
 MATERIALS AND METHODS ______________________________ 64

3.1 
MATERIALS .......................................................................................................................64

3.1.1
 Instruments 64 

3.1.2
 Chemicals
and
reagents ........ 65

3.1.3
 Miscellaneous
materials ........ 67

3.1.4
 Oligonucleotides 67

3.1.5
 Antibodies ................................ .... 71

3.1.6
 Plasmids
and
vectors ................................ ................................ .............. 72

3.1.7
 Software ....... 72

3.1.8
 Media,
solutions,
and
buffers .............................. 72

3.1.9
 Fly
lines................................ ................................ ......... 76

3.1.10
Web
sites ................................ ...... 78

3.2
METHODS...........................................................79

3.2.1
 Molecular
biology
and
biochemistry ............... 79

3.2.2
 Histological
techniques ......... 81

3.2.3
 Generation
of
Drosophila
deletion
lines
by
FRT­mediated
recombination.... 81

4
 RESULTS ________________________________________________ 84

4.1
A
MOLECULAR
SCREEN
FOR
NOVEL
TRANSCRIPTION
FACTORS
ESSENTIAL
FOR

DROSOPHILA 
MESODERM
DEVELOPMENT.......................................................................84

4.1.1
 Experimental
design
of
the
screen .................... 84

4.1.2
 Classes
of
identified
muscle
phenotypes ......... 90

4.1.3
 Role
of
the
Mediator
complex
in
muscle
development ......... 103

4.2
THE
ROLE
OF
TRAMTRACK
IN
DROSOPHILA 
MUSCLE
DEVELOPMENT..........................108

4.2.1
 Analysis
of
Tramtrack
expression
and
its
loss­of ­function
and
gain­of ­
function
phenotypes................................ ................................ ................................ ......... 108

4.2.2
 Deciphering
the
molecular
function
of
Ttk69 120

4.2.3
 An
integrated
network
of
Lame
duck
and
Tramtrack69
activity
is
required

for
FCM
specification ........ 126

4.3
SNAIL
AS
A
TRANSCRIPT IONAL
ACTIVATOR
IN
THE
PRESUMPTIVE
MESODERM .........131

4.3.1
 Snail
binds
to
enhancer
regions
of
early
mesodermal
genes............................. 131

-8- TABLE OF CONTENTS

4.3.2
 Endogenous
expression
of
several
mesodermal
genes
is
dependent
on
Snail
................................ ................................ ................................ ................................ .................... 134

4.3.3
 Snail
enhances
the
activation
of
mesodermal
enhancers
in
vitro................... 136

4.3.4
 In
vivo
activity
of
mesodermal
enhancers
further
supports
the
ability
of
Snail

to
positively
regulate
transcription ........... 140

5
 DISCUSSION ____________________________________________ 148

5.1
DIFFERENT
APPROACHES
FOR
FORWARD
GENETIC
SCREENING
IN
 DROSOPHILA .......148

5.1.1
 The
advantages
and
limitations
of
deficiency
lines................................ ............... 148

5.1.2
 Alternative
approaches
to
large­scale
phenotypic
screening........................... 150

5.2
THE
 MEDIATOR
COMPLEX
IN
DROSOPHILA
 MYOGENESIS..............154

5.3
TRAMTRACK
IS
A
NOVEL
REGULATOR
OF
DROSOPHILA 
MYOGENESIS..........................156

5.3.1
 Functional
analysis
links
Ttk69
to
myoblast
fusion................................ ............... 156

5.3.2
 Regulation
of
Ttk69
expression
in
the
somatic
mesoderm .158

5.3.3
 In
vivo
activity
of
Ttk69
CRMs
suggests
that
Ttk69
might
contribute
to

transcriptional
activation................................ .............................. 162

5.3.4
 The
many
aspects
of
Ttk69
regulatory
roles................................ ............................. 164 

5.4
SNAIL
IS
A
BIMODAL
FA CTOR
THAT
CAN
BOTH
ACTIVATE
AND
REPRESS
 TRANSCRIPTION
............................................................................165

6
 CONCLUSIONS AND FUTURE DIRECTIONS _______________ 168

7
 APPENDICES ____________________________________________ 171

7.1
ANALY SED
 RNA I
LINES
AND
THEIR
PH ENOTYPES.........................................................171

7.2
EMBRYONIC
EXPRESSION
OF
GENES
ENCODING
THE
MEDIATOR
COMPLEX
SUBUNITS
............................................173

7.3
BINDING
OF
TRAMTRACK69
IN
THE
VICINITY
OF
KNOWN
DOWNSTREAM
TARGETS 175

7.4
SEQUENCES
OF
CLONED
ENHANCERS...............177

7.4.1
 Sequences
of
Tramtrack69 ­bound
enhancers .......................... 177

7.4.2
 Sequences
of
Snail­bound
enhancers ................................ ................................ ........... 179

REFERENCES ______________________________________________ 182

-9- LIST OF FIGURES AND TABLES

LIST OF FIGURES AND TABLES
Figures
1.1-1: Tissue-specific gene expression is driven by the activity of cis-
regulatory modules......................................................................................... 21

1.1-2: Organization of the Drosophila Mediator complex....................................... 24

1.1-3: Conservation and divergence of regulatory networks across evolution ........ 29

1.1-4: Muscle development in Drosophila melanogaster........ 30
1.2-1: The establishment of the dorsal-ventral axis in the blastoderm embryo........ 33
1.3-1: Subdivision of mesoderm by synergism between extrinsic and intrinsic
signals............................................................................................................. 38

1.3-2: Notch-dependent processes in somatic muscle development ........................ 42

1.3-3: Larval somatic muscles have a stereotypic pattern........ 45
1.4-1: Gene model of the ttk locus and basic features of Ttk proteins ..................... 47

1.4-2: Cell fate changes in the PNS of ttk loss- and gain-of-function
backgrounds ................................................................................................... 55

1.4-3: Mechanisms of Ttk69 and Ttk88 mRNA and protein control ....................... 60
4.1-1: Experimental design of the molecular screen ................................................ 84

4.1-2: Identifying transcription factors expressed in mesoderm .............................. 88

4.1-3: Distribution of identified phenotypes ............................................................ 90

4.1-4: Somatic, cardiac, and visceral musculature in wild type embryos ................ 92

4.1-5: Phenotypic class II: Pleiotropic defects......................... 93

4.1-6: Phenotypic class III: Aberrant somatic muscle number and/or
organization.................................................................................................... 97

4.1-7: Phenotypic class IV: Abnormal somatic muscle guidance and
attachment.... 100

4.1-8: Phenotypic class V: Excessive mononucleated myoblasts in the somatic
musculature .................................................................................................. 101

4.1-9: Defects in the heart development. 102

4.1-10: Deficiencies removing MED24 and MED14 show a similar somatic
muscle phenotype......................................................................................... 104

4.1-11: Generation of MED24, MED14, and MED14 MED24 deletion lines ......... 105

4.1-12: Muscle phenotypes in small deletion lines removing MED14, MED24,
and both genes together................................................................................ 107
4.2-1: Expression of Ttk69 mRNA, protein and enhancer..... 109

4.2-2: Ttk is essential for normal somatic muscle development ............................ 112

-10-

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