Markus Holotta
The influence of hyproxia an GATA-1 and Epo expression levels in developing zebrafish
ISBN: 978-3-8366-0759-9
Druck Diplomica® Verlag GmbH, Hamburg, 2008
Zugl. Leopold-Franzens-Universität Innsbruck, Innsbruck, Österreich, MA-Thesis /
Master, 2007
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Acknowledgement
I would like to thank Prof. Bernd Pelster for enabling this masters the-
sis. My sincere thanks applies to Prof. Thorsten Schwerte for the excellent
supervision of this work.
Of course I would like to thank Dr. Renate Kopp, Dr. Nikolaus Medgyesy
and Dr. Margit Egg for technical support and the pleasant atmosphere in
the laboratory.
Finally my thanks goes to all the members of the Institute of Zoology
which helped with words and deeds.
Contents
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
1
Introduction
6
1.1
Zebrafish hematopoiesis
. . . . . . . . . . . . . . . . . . . . .
7
1.2
GATA-1 and Co-factors
. . . . . . . . . . . . . . . . . . . . .
8
1.2.1
The role of GATA-1
. . . . . . . . . . . . . . . . . . .
8
1.2.2
GATA-1 related factors . . . . . . . . . . . . . . . . . .
10
1.3
Epo and EpoR
. . . . . . . . . . . . . . . . . . . . . . . . . .
11
1.4
Oxygen dependent development . . . . . . . . . . . . . . . . .
13
1.5
Intention of this study . . . . . . . . . . . . . . . . . . . . . .
14
2
Material and Methods
16
2.1
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
2.2
Imaging system . . . . . . . . . . . . . . . . . . . . . . . . . .
17
2.3
Epo mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
2.4
Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
3
Results
21
3.1
General aspects . . . . . . . . . . . . . . . . . . . . . . . . . .
21
3.2
Total cell number . . . . . . . . . . . . . . . . . . . . . . . . .
21
3.3
Manually counted fluorescent cells . . . . . . . . . . . . . . . .
22
3.4
GATA-1 expression . . . . . . . . . . . . . . . . . . . . . . . .
22
3.5
Epo expression
. . . . . . . . . . . . . . . . . . . . . . . . . .
25
2
CONTENTS
CONTENTS
4
Discussion
27
4.1
General aspects and observations . . . . . . . . . . . . . . . .
27
4.2
Pros and cons of DsRed
. . . . . . . . . . . . . . . . . . . . .
29
4.3
Epo and GATA-1 . . . . . . . . . . . . . . . . . . . . . . . . .
30
4.4
In vivo imaging . . . . . . . . . . . . . . . . . . . . . . . . . .
33
4.5
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
34
3
Abbreviations
bpm . . . . . . . . . . . . . . . . . . . . . . beats per minute
CBP . . . . . . . . . . . . . . . . . . . . . cAMP response element-binding protein bind-
ing protein
dpf . . . . . . . . . . . . . . . . . . . . . . . days post fertilisation
Epo . . . . . . . . . . . . . . . . . . . . . . erythropoietin
EpoR . . . . . . . . . . . . . . . . . . . . . erythropoietin receptor
ERBP . . . . . . . . . . . . . . . . . . . . Epo mRNA-binding domain
FCM . . . . . . . . . . . . . . . . . . . . . flow cytometry
FOG . . . . . . . . . . . . . . . . . . . . . Friend of GATA
GC . . . . . . . . . . . . . . . . . . . . . . . glucocorticoid
GR . . . . . . . . . . . . . . . . . . . . . . . glucocorticoid receptor
Hb . . . . . . . . . . . . . . . . . . . . . . . haemoglobin
HIF . . . . . . . . . . . . . . . . . . . . . . hypoxia inducible factor
hpf . . . . . . . . . . . . . . . . . . . . . . . hours post fertilisation
ICM . . . . . . . . . . . . . . . . . . . . . . intermediate cell mass
IGFBP . . . . . . . . . . . . . . . . . . . insulin-like growth factor binding protein
IS . . . . . . . . . . . . . . . . . . . . . . . . intensity stage
LDH . . . . . . . . . . . . . . . . . . . . . lactate dehydrogenase
pf . . . . . . . . . . . . . . . . . . . . . . . . post fertilisation
RT-PCR . . . . . . . . . . . . . . . . . . reverse transcription polymerase chain reaction
RTK . . . . . . . . . . . . . . . . . . . . . receptor tyrosine kinase
SCF . . . . . . . . . . . . . . . . . . . . . . stem cell factor
VVR . . . . . . . . . . . . . . . . . . . . . ventral vein region
4
Abstract
The transcription factor GATA-1 is essential for the development of the
erythroid cell lineage in vertebrates. In this article we introduce a method
to easily determine the approximately development status of red blood cells
and the progression of blood formation by intensity of fluorescence in GATA-
1/Ds-Red marked zebrafish. We classified the blood cells on the basis of
their fluorescence intensity in 5 intensity stages (IS) with the brightest in IS
1. The comparison with our erythropoietin (Epo) data showed a noticable
correlation between GATA-1, Epo mRNA and EPO protein level. Between
2 and 3 dpf we observed a major increase in blood cell concentration to
circa 1200 cells*nl
-1
, until 15 dpf this value decreased to about the half.
The appearance of IS 1 cells correspond approximately to the peaks in Epo
cRNA copies and the highest values in EPO protein emerged about 1 day
later. Our data show that the blood cell concentration, Epo and Gata-1
expression in zebrafish larvae is subjected to large fluctuations in the first
few days of development.
Chapter 1
Introduction
The zebrafish Danio rerio, also known as Brachidanio rerio, Cyprinus re-
rio and others, is a omnivorous, tropical fish of the family Cyprinidae and
was at first described by Hamilton in 1822. Its natural range within Asia
are slow moving or stagnant water bodies in India, Bangladesh, Nepal and
Pakistan [1, as cited in [2]]. In the past few decades the zebrafish became
an important model organism for genetical, developmental and physiological
studies. Fish are vertebrates and thus the genetic program is more similar
to that of mammals than invertebrate models like the fruitfly (Drosophila
melanogaster ). Because of this relationship most of the zebrafish genes have
human orthologs [3]. Due to its short generation time of approximately 3
month, the rapid development and the transparency up to adulthood this
tropical teleost is predestined for in vivo hematologic studies and digital
imaging techniques. Especially digital imaging is a gentle, non-invasive and
thereby seminal method for researcher who are working with transparent
animals. The combination with fluorescent reporter genes allows real time
imaging of gene expression and cell migration studies over their whole lifespan
[4].
6
1.1. Zebrafish hematopoiesis
1. Chapter: Introduction
1.1
Zebrafish hematopoiesis
Zebrafish hematopoiesis can be classified into a primitive (embryonic) and
definitive (fetal and adult) part. The primitive hematopoiesis produces pri-
marily erythrocytes and some macrophages, whereas the definitive part is
responsible for cells of the erythroid, lymphoid and myeloid lineage [5]. The
embryonic hematopoiesis occurs in a region called "intermediate cell mass"
(ICM), which is located dorsally to the yolk tube. The ICM arises from
posterior-lateral mesoderm at the 5-somite stage (11,5 hpf) and contains,
amongst others, primitive hematopoietic precursors and gives rise to the en-
dothelia of the major trunk vessels [6, 7].
The first markers of definitive hematopoiesis are detected in the ICM af-
ter about 30 h. It has been shown that a few hematopoietic cells exist at
other locations like the ventral wall of the dorsal aorta, which may be the
first site of definitive hematopoiesis, and the "ventral vein region", a region in
the tail ventral to the axial vein. This ventral vain region (VVR) comprises
of the posterior part of the ICM and is proposed to be an larval site for en-
dothelial tissue development [5, 8]. The major site for hematopoiesis through
adulthood is the kidney. Additionally, Willett et al. found circulating ery-
throblasts and immature erythrocytes associated to the heart endocardium
from 24 hpf onward, which could be confirmed by other authors. There is
evidence to suggest that the heart is a blood forming organ between the
disappearance of the ICM and the onset of definitive hematopoiesis in the
pronephros at about hour 96 pf [9, 10].
Zebrafish erythropoiesis consists of two waves of red blood cell produc-
tion, a primitive and a definitive one. The primitive erythropoiesis generates
erythrocytes (EryP) which express embryonic hemoglobin, erythrocytes of
the definitive wave (EryD) synthesise the adult type of globin [11]. After 10
somites (10 hpf) blood precursors express mature erythroid markers such
as embryonic globin [6]. Later, at about the 18-somite stage (15,5 hpf),
primordial blood cells are differentiated out of it [1214]. The 20 h ICM
already contains proerythroblasts and about 24 hpf the first blood cells en-
ter the developing circulatory system. Till about 30 hpf more and more cells
7
1.2. GATA-1 and Co-factors
1. Chapter: Introduction
will be received and the ICM disappears [9]. The circulating proerythroblasts
mature to flattened elliptical erythrocytes during the next 4 days [15].
1.2
GATA-1 and Co-factors
1.2.1
The role of GATA-1
GATA-1/2/3 are involved in hematopoiesis, while GATA-4/5/6 regulate heart,
gut and lung development in vertebrates [11]. Transcripts encoding GATA-1
are first detected at the two to three somite stage (10,3 hpf) in two stripes
of cells flanking paraxial mesoderm which later will fuse to the ICM [16].
No GATA-1 expression could be detected in the posterior ICM at 23 hpf
and the cells seems to be less differentiated than those in the anterior ICM
[8]. Long et al. even reported GATA-1 expression approximately after 8 hpf
(mid-gastrula stage) in the ventral region of the embryo [10].
Due to the fact that zebrafish are vertebrates, the DNA sequence mo-
tive GATA is well conserved from fish to humans [16]. GATA factors typ-
ically share the consensus binding sequence WGATAR (W = A or T; R
= A or G) and the GATA motive is present in cis-regulatory elements of
many erythroid-expressed genes [17, 18]. The expression of this zinc finger
protein is limited to erythroid, eosinophil, megakaryocyte, mast cell lineages
and multipotential myeloid progenitors. GATA-1, formerly known as GF-
1, NF-E1 and Eryf-1, was the first characterised factor of the six members
including GATA family and is normally the most abundant GATA factor
in late erythroid differentiation [16, 19, 20]. The protein contains two zinc
fingers comprising a zinc atom linked to four cysteines per finger. The car-
boxyl terminal is responsible for binding the WGATAR recognition sequence,
whereas the amino terminal zinc finger stabilises this interaction and binds
several cofactors. The C-terminal tail is responsible for specific DNA bind-
ing and wraps around into the minor groove of the DNA [21, 22]. GATA-1
mutants lacking the N-terminal domain do not show transcriptional activity,
but are able to restore differentiation of GATA-1 deficient embryonic stem
cells in vitro [23].
8
1.2. GATA-1 and Co-factors
1. Chapter: Introduction
Studies on GATA-1 deficient mice showed, that the mutation don't re-
duce the number of erythroid progenitors, or affect colony-forming potential.
Hematopoietic cells lacking GATA-1 are able to enter erythroid lineage, but
cease differentiation mostly at the proerythroblast stage. At the same time
GATA-2 expression is increased in erythroid progenitors [17, 19]. This may
be due to the fact, that GATA-2 partly overlaps in function with GATA-1
[19]. In contrast, mast cell and megacaryocyte lines are able to complete
differentiation in absence of GATA-1. A lack of GATA-2 in mice leads to a
insufficient number of erythroid progenitors, megacaryocytes and mast cells
[17, 24]. In GATA-1 deficient zebrafish embryos hematopoietic cells in the
ICM were found to differentiate into myelomonocytes [6]. Thus GATA-1 ini-
tiates terminal erythroid differentiation and besides suppresses cell growth by
suppressing transcription of responsible genes or interference with proteins
like the myeloid transcription factor PU.1 [25, 26]. GATA-1 is even able to
reprogram common lymphoid progenitors (CLPs) and granulocyte-monocyte
progenitors (GMPs) to form erythroid colonies through its antagonistic ef-
fect on PU.1. It has been shown that in hematopoiesis GATA expression is
switched from GATA-2 in early hematopoietic progenitors to GATA-1 during
terminal differentiation by suppressing GATA-2 gene expression by GATA-1
[17, 26]. A restoration of GATA-1 in GATA-1 deficient cell lines effect a
synchronous cell cycle arrest in the G
1
phase and a common differentiation
about 12 h after induction [27].
GATA-1 may serve as a direct activator of transcription or as a mediator
of promoter-enhancer activity and is moreover a regulator of its own pro-
motor in a positive feedback loop [28, 29]. Globin and heme enzyme genes
for example are target genes of the GATA-1 protein, their activation results
in hemoglobin production [20]. Additionally Briegel et al reported a hyper-
phosphorylated GATA-1 species preferentially located in the nucleus of avian
erythroid progenitor cells after differentiation induction. Before differentia-
tion induction the vast majority of GATA-1 is restricted to the cytoplasm.
This indicates that phosphorylation is an important process for translocation
of GATA-1 through the nuclear membrane and moreover enhances the DNA
binding affinity [22, 25].
9
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