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Characterization of transgenic zebrafish lines for studying role of platelet derived growth factor (PDGF) signaling on coronary vessel formation in regenerating zebrafish heart after cryoinjury

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Characterization of transgenic zebrafish lines for studying role of platelet derived growth factor (PDGF) signaling on coronary vessel formation in regenerating zebrafish heart after cryoinjury

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Characterization of transgenic zebrafish lines for studying role of Platelet Derived
Growth Factor (PDGF) signaling on coronary vessel formation in regenerating
zebrafish heart after cryoinjury
by
Gayatri Nair
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
Master of Science
(Biochemistry and Molecular Biology)
December 2013
1

TABLE OF CONTENTS

LIST OF FIGURES

3
LIST OF ABBREVIATIONS

4
ACKNOWLEDGEMENT

6
ABSTRACT

7
Chapter 1. Introduction
1.1 Coronary Heart Disease 8
1.2 Zebrafish as a model

9
Chapter 2. Heart Regeneration in zebrafish
2.1 Zebrafish Heart 10
2.2 Injury Models
2.2.1 Amputation
2.2.2 Cryoinjury
2.2.3 Genetic Ablation
10-11
2.3 Mechanism of Heart Regeneration in zebrafish after amputation

12

2

Chapter 3. Background
3.1 Blood Vessel formation during embryogenesis in zebrafish 14
3.2 Platelet Derived Growth Factor (PDGF) help in the stability of blood vessels
in mammals
16
3.3 Previous work on pdgfrβ in zebrafish from our lab

17
Chapter 4. Characterization of transgenic line hsp70:Gal4; UAS:dnpdgfrβ-
YFP;fli1a:GFP

4.1 Introduction 18
4.2 Working of UAS-Gal4 System with respect to hsp70:Gal4; UAS:dnpdgfrβ-
YFP
20
4.3 Results and Discussion 21
4.4 Materials and Methods

29
Chapter 5. Characterization of transgenic line Ubi:CreERT2;Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ

5.1 Introduction 32
5.2 Working of Cre-LoxP System with respect to Ubi:CreERT2;Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ
32
5.3 Results 34
5.4 Materials and Methods

38
Bibliography 40
3

LIST OF FIGURES

Figure 1. Zebrafish heart regeneration 12
Figure 2. Phases of vasculogenesis in zebrafish embryo 15
Figure 3. Dominant negative Pdgfrβ blocks Pdgf signaling 19
Figure 4. Working of hsp70:Gal4; UAS:dnpdgfrβ-yfp 20
Figure 5. Confocal image of adult heart section from UAS:dnpdgfrβ-yfp
transgenic line with successful integration of transgene in
zebrafish genome
22
Figure 6. Genotype result for gal4 gene in UAS:dnpdgfrβ-yfp; hsp70:Gal4 23
Figure 7. pdgfrβ in-situ hybridization on uninjured UAS:dnpdgfrβ-yfp;
hsp70:Gal4 heart
25
Figure 8. Whole mount confocal result of UAS:dnpdgfrβ-yfp; hsp70:Gal4;
fli1a:eGFP zebrafish heart
28
Figure 9. In-situ hybridization result showing expression of dnpdgfrβ after
cryoinjury in UAS:dnpdgfrβ-yfp; hsp70:Gal4; fli1a:eGFP
zebrafish heart
29
Figure 10. Working of Ubi:CreERT2; Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ 33
Figure 11. Ubi:CreERT2; Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ zebrafish
embryo showing ubiquitous expression of GFP
34
Figure 12. Genotype result for cre gene in Ubi:CreERT2; Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ
35
Figure 13. pdgfrβ in-situ hybridization on zebrafish embryo to test for
induction in Ubi:CreERT2; Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ
36
Figure 14. Induction in Ubi:CreERT2;Ubi:loxP-GFP-loxP-mCherry 37

4

LIST OF ABBREVIATIONS
4OHT 4-Hydroxytamoxifen
cmlc2 cardiac myosin light chain 2
Cre Cyclization recombination
DA Dorsal Aorta
DAPI 4',6-diamidino-2-phenylindole
DNA Deoxyribonucleic acid
dpa days post amputation
dpc days post cryoinjury
eGFP Enhanced Green Fluorescent Protein
EtOH Ethanol
Fli1a Friend leukemia integration 1a
GFP Green Fluorescent Protein
hpa hours post amputation
hpf hours post fertilization
Hsp70 Heat shock promoter 70
ISV Inter-segmental vessels
loxP Locus of crossover [x] in P1 bacteriophage
MAPK Mitogen-activated Protein Kinase
MI Myocardial Infarction
NaOH Sodium Hydroxide
PC Pericytes
PCR Polymerase Chain Reaction
5

PCV Posterior Cardinal Vein
PDGF Platelet Derived Growth Factor
Pdgfr Platelet Derived Growth Factor Receptor
PFA Paraformaldehyde
PI 3-kinase Phosphatidylinositide 3-kinases
RA Retinoic Acid
SH2 Src Homology 2
SIV Sub-intestinal Vessel
Tm Annealing temperature
UAS Upstream Activating Sequence
vSMC Vascular Smooth Muscle Cell
Yfp/YFP Yellow Fluorescent Protein

6

ACKNOWLEDGEMENTS
I would like to thank my brother for encouraging me to pursue my Masters in United
States and for supporting me with his honest opinions and practical advice every step of
the way. It has been a learning experience inside and outside the academic realm. Also,
my heartfelt thanks to my parents for the constant support and faith they have shown in
me. I sincerely hope that I do not disappoint myself and my family.
I would like to thank all my humble lab members for their patience and immense help in
this one year. Especially, to Dr. Jieun Kim for taking me under her supervision and
helping me understand my project, plan my experiment and execute them properly, to
Ying Huang for patiently answering all my questions and clearing my doubts and
supporting me whenever I felt discouraged, to Dr. Michael Harrison, for his immense and
thorough knowledge in the field has helped me understand concepts clearly and to
Arthela Osorio. I would also like to thank Dr. Esteban Fernandez for his help on confocal
microscope. Lastly, I am grateful to Dr. Ellen Lien for giving me the opportunity to work
in her lab. Also, I want to thank her for being patient with me in spite of my lack of
enthusiasm towards research.

7

ABSTRACT
It is known that PDGF-B and PDGFRβ interaction is crucial in blood vessel formation in
mammals. PDGF-B is involved in proliferation and recruitment of vSMC (vascular
smooth muscle cells) and pericytes to blood vessels. Mutants lacking PDGF-B and
PDGFRβ show pericyte loss, capillary dilation and rupture. Not much has been known
about role of Pdgfb-Pdgfrβ mediated signaling in zebrafish until recently. Now it has
been shown that pdgf is upregulated in regenerating zebrafish heart and Pdgf signaling is
necessary for intersegmental vessel formation in zebrafish embryo. Also, with the help of
chemical inhibitors blocking Pdgfr signaling, it has been shown that Pdgf signaling plays
an important role in coronary vessel formation during regeneration in zebrafish. The aim
of this project is to help characterize an improved, cleaner and more specific genetic tool
which can overcome the shortcoming of chemical inhibitor to study the role of Pdgfrβ
signaling in coronary vessel formation during regeneration in zebrafish heart after
cryoinjury.

8

Chapter 1. Introduction

1.1 Coronary Heart Disease
Coronary Heart Disease is the leading cause of death in United States claiming around
600,000 lives every year according to 2012 statistics from the Center for Disease Control
and Prevention. Coronary Heart Disease is the narrowing or blockage of the coronary
arteries (the major blood vessels that delivers oxygen rich blood to myocardium) due to
deposition of cholesterol and fatty acids along the walls of the arteries; known as
atherosclerosis. The inadequate flow of blood to heart decreases the adequate supply of
oxygen and nutrients to the heart tissue needed for its proper functioning. Under
circumstances when the blood supply to a portion of the heart muscle is cut off, the
downstream myocardium undergo necrosis leading to myocardial infarction. The
impaired blood flow triggers the heart cells in the territory of the occluded coronary
artery to die through necrosis and apoptosis. The cells lost via necrosis are not replaced
by new cardiomyocyte in mammalian hearts, instead a non-contractile fibrotic collagen
scar is formed in its place and remaining cardiomyocytes in the post infarct heart undergo
hypertrophy or dilation to compensate for the loss. This in turn decreases the
functionality of the injured heart and makes it susceptible to future MI events and organ
failure. (American Heart Association, NIH: National Heart, Lung and Blood Institute,
Chilton RJ)

9

1.2 Zebrafish as a model
In contrast to humans, many other non-mammalian vertebrates show remarkable
regenerative ability. Urodele amphibians like newt can regenerate limb. Teleost fish such
as zebrafish can regenerate fin, retina, spinal cord and heart (Matthew Gemberling et al.
2013). Zebrafish (Danio rerio) is a powerful model organism for studying development
and regeneration. They are easy to maintain and reproduce quickly with large clutch size
of embryos. Zebrafish embryos develop externally following fertilization, making them
easily accessible for embryonic manipulation and imaging. In addition the optical
transparency of embryo allows researchers to study the formation of organs and
vasculature during development using transmitted light or fluorescent imaging
techniques. They are amenable to forward and reverse genetic approaches and with their
remarkable capacity to regenerate, they make a favorable model system for studying
tissue regeneration (Gore AV et al 2012, Poss 2006, Gemberling et al 2013, Major RJ et
al 2007).

10

Chapter 2. Heart Regeneration in Zebrafish
2.1 Zebrafish heart
Zebrafish heart is two chambered with one ventricle and one atrium. Like mammals, it
has three layers: epicardium, myocardium and endocardium. The epicardium is formed
by a single layer of mesothelial cells supported by basal lamina, and imbricated with
collagen, fibroblasts and vascular structure in the subepicardial space (Hu N et al, 2001).
The myocardium is composed of a thin, external layer of compact myocytes penetrated
by blood vessel and by internal myocytes organized into extensive and elongated
trabeculae (Poss et al, 2002). The endocardium is a single cell layer lining the inner
trabecular myocardium.

2.2 Injury Models
Zebrafish heart is microscopic, with ventricle being ~1mm
3
. Hence it is difficult to
generate a coronary artery occlusion to create an ischemic myocardial injury similar to
humans. However in order to study heart regeneration in zebrafish, various injury models
have been introduced.
2.2.1 Amputation: Kenneth D. Poss et al in 2002 introduced amputation as an injury
model to study zebrafish heart regeneration. In this model, ~20% of ventricular apex is
surgically removed with iridectomy scissors. New cardiomyocytes replace lost myocytes
and new blood vessels vascularizes cells in the injury area. Over a period of 1-2 months,
11

the zebrafish heart completely regenerates without scarring. Amputation had been the
most widely used injury model in zebrafish to study heart regeneration for a long time.
2.2.2 Cryoinjury: Another injury model was recently developed by Chablais F. et al,
González-Rosa et al and Schnabel K. et al to closely mimic myocardial infarction in
humans. This new model known as cryoinjury involves bringing the tip of a metallic
probe prechilled in liquid nitrogen in contact with the apex of an adult zebrafish heart for
approximately 20-30 sec. As a result, the cells which come in contact with the probe are
damaged by freezing and thawing. The dead cells and tissue remain in the injury area and
a collagen scar is formed closely resembling the pathogenesis observed during
myocardial infarction in humans. Interestingly, unlike human heart, in zebrafish the scar
is later absorbed or resolved and the zebrafish heart fully regenerates (González-Rosa et
al, 2012).
2.2.3 Genetic Ablation: Using an inducible double transgenic line cmlc2:CreER;
bactin2:loxP-mCherry-STOP-loxP-DTA, Jinhu Wang et al in 2011 facilitated ablation of
~60% of ventricular myocytes in adult zebrafish. Such an injury disrupts electric
conduction and elicits signs of cardiac failure such as lethargic appearance, gasping
phenotypes, reduced stress sensitivity. However, within 1 month, ventricle is fully
muscularized with regenerating cardiomyocytes and new blood vessels and no significant
scarring.
Gaining more insight into how zebrafish heart regenerates naturally can help design
therapeutic strategies to treat human heart after MI, unlock our own regenerative
potential such that heart can completely recover itself after an injury or cardiac failure
12

and unravel stem cells or progenitor cells which can be triggered to replace injury area in
heart with new functional cardiac muscle

2.3 Mechanism of heart regeneration in zebrafish after amputation

As reviewed by Lien et al in 2012 (Figure 1), the early stage of heart regeneration is
similar to wound healing. Immediately after amputation a blood clot is formed at the
injury site to stop bleeding and seal the ventricle. An infiltration of inflammatory cells
Figure 1. Zebrafish heart regeneration
A diagram depicting the steps involved in the regeneration of zebrafish heart
after amputation. The zebrafish heart regenerates completely within 30 days after
20% ventricular resection. (Lien et al. 2012)
13

such as macrophages and neutrophils into the injury area has been reported. Within 1-3
hours post amputation (hpa), the endocardium undergoes organ-wide morphological
changes. Also the endothelial cells start expressing retinaldehyde dehydrogenase 2
(raldh2) (it encodes for the rate limiting enzyme for RA synthesis) at the same time.
Strong raldh2 expression persists in both atrium and ventricle until 6hpa and becomes
localized to the injury site by 1day after amputation (dpa). The blood clot is replaced by a
fibrin clot by 2-3dpa. By 1-2dpa, the epicardium of regenerating heart gets activated
throughout the ventricle and gradually becomes localized to apex. The activated
epicardium proliferates from 3-7dpa. It is suggested that the epicardial cells undergo
epithelial-to-mesenchymal transition (EMT) (Lepilina A et al 2006) giving rise to
fibroblasts and pericyte-like perivascular mural cells of coronary vessels. At 7dpa,
cardiomyocytes in the sub-epicardium initiates DNA synthesis and cell proliferation and
by 14dpc gata4+ cardiomyocytes localize to the apex and newly formed blood vessels
vascularize the newly formed myocardium. By 30dpa, the heart is almost fully
regenerated (Figure 1).

14

Chapter 3. Background
3.1 Blood vessel formation during embryogenesis in Zebrafish
Blood vessels are an integral component of all organs and are vital not only for their
function but also, as their development is important in physiological and pathological
processes. The formation of blood vessel involves two morphogenetic processes:
vasculogenesis and angiogenesis. Vasculogenesis involves the determination and
differentiation of endothelial progenitor cells from the mesoderm and their de novo
organization to a primitive vascular blood plexus. The further expansion and networking
of primary vessels from existing blood vessels by remodeling is known as angiogenesis.
Due to the small size, optical clarity and rapid development of zebrafish embryo, they are
good model for studying vascular morphogenesis in vivo. In zebrafish embryo,
angioblasts, the precursors of endothelial cells originate from ventrolateral mesoderm and
from 14hpf start migrating towards the embryonic midline where they aggregate to form
a vascular cord (Figure 2A). Cells located in the dorsal portion of the vascular cord give
rise to Dorsal aorta (DA) at 17hpf and the cells located ventrally give rise to Posterior
cardinal vein (PCV) at 21hpf (Figure 2B). By 30hpf, both vessels are fully formed and
result in the first circulation loop (Figure 2D). Once the primary axial vessels are formed,
the elaboration and networking of vasculature occurs via angiogenesis leading to the
development of inter-segmental vessels (ISV) and the sub-intestinal veins (SIV) (Herbert
et al, 2009; Jin et al., 2005 and Ellertsdóttir et al., 2009).

15

The regulation of vasculogenesis and angiogenesis involves multiple cell types and
communication between these cells is necessary to coordinate the formation of the
vascular system. A blood vessel in general consists of an inner endothelium lining the
lumen. Depending upon the type of vessel, the endothelium is surrounded by a basal
lamina and by mural cells which support and stabilize vessels. Depending on their
density, morphology, location and expression of specific markers mural cells are
Figure 2. Phases of vasculogenesis in zebrafish embryo (Elín Ellertsdóttir et al. 2009)
(A)Medial migration. From 14 hpf onward, angioblasts (purple) that originate in the
lateral plate mesoderm migrate over the endoderm towards the midline just below the
hypochord, where they aggregate to form a vascular cord (B). (B) Arterio-venous
segregation and ventral sprouting. At around 17hpf, angioblasts start to express markers
of arterio-venous differentiation in arterial cells (marked red). These cells are located in
the dorsal portion of the vascular cord and will give rise to the DA, whereas ephb4a
expressing cells are located more ventrally and will contribute to the PCV. At 21hpf,
angioblasts located in the ventral part of the vascular cord start migrating ventrally and
accumulate below the forming DA (B,C). (C) Lumen formation. The DA forms and
lumenizes prior to the PCV in the absence of blood cells (brown) by cord hollowing.
Venous angioblasts aggregate and coalesce around the blood cells to ultimately form a
tube. (D) Functional Vasculature. At 30hpf, both vessels are fully formed and carry blood
flow. Endothelial cell junctions are indicated in green
16

commonly subdivided into pericytes (PC) and vascular smooth muscle cells (vSMC)
(Konstantin Gaengel et al, 2009). Pericytes are associated with the smallest diameter
blood vessels and share their basal membrane with the endothelium. Vascular smooth
muscle cells form concentric layers around larger blood vessels.

3.2 PDGF helps in the stability of blood vessels in mammals
Platelet-derived growth factor (PDGF) is a family of cationic homo- or heterodimers of
disulfide-bonded A- and B- polypeptide chains. They activate two structurally related
protein tyrosine kinase receptors: α and β. These receptors contain an extracellular ligand
binding domain, a transmembrane domain and an intracellular tyrosine kinase domain.
Binding of pdgf ligands to the receptor results in receptor dimerization followed by
transphosphorylation of tyrosine residue on the intracellular domain. This creates docking
sites for signal transduction molecules containing SH2 domain in turn activating
numerous signaling transduction pathways such as PI 3-Kinase pathway.
It has been reported that PDGF ligand and receptor are involved in the development of
blood vessels in mammals. Platelet-derived growth factor receptor-β is expressed on the
surface of developing vSMC/PC and in mammals, lack of its signaling leads to pericyte
loss, endothelial changes followed by capillary dilation and rupture (Lindahl et al 1997a).
PDGF-B ligand on the other hand is secreted from the endothelium of angiogenic sprouts
where it serves as an attractant for comigrating pericytes/vSMC expressing PDGFRβ
(Lindahl et al 1997, David M. Brown et al 1995). It stimulates proliferation of vSMC
probably via MAPK (mitogen-activated protein kinases) pathway and induces mural cell
17

fate in undifferentiated mesenchymal cells (Hellstrom et al 1999; Abramsson et al 2003;
Hirschi et al 1999).

3.3 Previous work on pdgfrβ in zebrafish from our lab
Unlike mammals, not much has been known about the role of pdgf in zebrafish until
recently. Dr. Ellen Lien in 2006 in a microarray analysis observed that pdgf-a and pdgf-b
is upregulated in regenerating zebrafish heart. With the help of an in-vitro zebrafish
cardiomyocyte culture, Dr. Lien reported that Pdgf-bb increases DNA synthesis in
zebrafish cardiomyocyte and inhibiting pdgf signaling using Pdgfr inhibitor decreases
DNA synthesis in zebrafish cardiomyocytes in-vitro as well as in-vivo. This laid the
initial step toward studying the molecular signaling behind regeneration in zebrafish.
Katie Wiens (2010) reported that Pdgfrβ is critical for intersegmental vessel formation
and extension during development in zebrafish. A deficit in ISV formation was observed
after Pdgfr chemical inhibition, pfgdrβ2 morphilino knockdown and dominant negative
pdgfrβ transgene expression in zebrafish embryo.
By inhibiting Pdgf signaling using Pdgfr inhibitor, Jieun Kim in 2010 showed that Pdgf
signaling is important for epicardial cell proliferation in-vitro and in-vivo, expression of
mesenchymal and mural cell markers and coronary blood vessel formation during heart
regeneration in zebrafish after amputation.

18

Chapter 4. Characterization of transgenic line hsp70:Gal4; UAS:dnpdgfrβ-YFP;
fli1a:eGFP

4.1 Introduction
Although it is convenient to treat the fish or embryos with chemical inhibitors, the
specificity and off target effects of chemical inhibitors is always a concern. Furthermore,
chemical inhibitors are generally dissolved in fish water and the fish is exposed to the
chemical by bath incubation and takes in the inhibitors via systemic circulation thus
failing in achieving spatial control.
In order to address these concerns one of the post-doc in our lab (Katie Wiens, 2010)
created a heat shock inducible dominant negative Pdgfrβ transgenic line based upon
UAS-Gal4 system. The dnPdgfrβ differs from the endogenous Pdgfrβ as the intracellular
tyrosine kinase domain in dominant negative PDGFRβ is replaced by YFP (Yellow
Fluorescent Protein) which creates an inactive protein (Figure 3). The receptor though
dimerizes with endogenous Pdgfrβ when the Pdgf ligand binds but prevents auto-
phosporylation of tyrosine residues thus inhibiting the downstream signaling
(Figure 3).
19

Figure 3. Dominant negative Pdgfrβ blocks Pdgf signaling
An endogenous Pdgfrβ (top) consists of an extracellular ligand binding domain, a
transmembrane segment and an intracellular tyrosine kinase domain. The binding
of Pdgf ligand leads to dimerization of the receptor followed by
autophosphorylation of tyrosine residue on the kinase domain. This creates
docking sites for signal transduction molecules containing SH2 domain and in
turn leads to downstream signaling.
A dnPdgfrβ (below) however is generated such that it either lacks the intracellular
tyrosine kinase domain or the intracellular tyrosine kinase domain is replaced with
YFP. Under both circumstances, an inactive protein is formed. The receptor
though dimerizes after ligand binding, no autophosphorylation occurs and hence
the downstream signaling is blocked.
20

4.2 Working of UAS-Gal4 system with respect to hsp70:Gal4; UAS:dnpdgfrβ-yfp

In the UAS-Gal4 system, the yeast transcription activator Gal4 binds its target sequence
UAS (Upstream Activating Sequence) and activates transcription of UAS- linked genes
(Zhan, 2010). The Gal4 protein activates transcription of only those genes bearing Gal4
binding sites (UAS) (Phelps et al, 1998).
In the transgenic line hsp70:Gal4; UAS:dnpdgfrβ-YFP (Figure 4), the Gal4 gene is
downstream of an hsp70 (heat shock 70) promoter. Hsp70 promoter activates Gal4 gene
expression ubiquitously, and Gal4 in turn directs transcription of UAS-target gene, here
dnpdgfrβ in an identical ubiquitous pattern.
Hsp70 expresses at low, often undetectable level at normal temperature (28.5°C) but is
robustly induced in all tissues at 37°C (Zhan, 2009) and hsp70 mRNA is selectively
Figure 4. Working of hsp70:Gal4; UAS:dnpdgfrβ-yfp
A schematic diagram depicting how in the double
transgenic line hsp70:Gal4; UAS:dnpdgfrβ-yfp
dnpdgfrβ is expressed after heat shock induction. The
fish are heat shocked at 38°C for 1 hour every day for
the duration of the experiment using water bath. Heat
shock results in expression of gal4 which in turn binds
to UAS promoter thus activating the expression of
dnpdgfrβ.

21

degraded at non-heat shock temperature, hence controlling the timing and level of
expression by altering the timing and temperature of the heat shock delivered. Because of
heat conductivity in aquatic animals such as zebrafish, the use of hsp70 promoter helps
retain advantage in transgene expression (Shoji et al, 2008). Using the hsp70:Gal4;
UAS:dnpdgfrβ-YFP double transgenic fish embryos. Katie showed that the transgenic
lines have the same intersegmental vessels phenotypes as the embryos treated with Pdgfr
inhibitor.
The aim here is to characterize the transgenic line UAS:dnpdgfrβ-yfp;hsp70:Gal4;fli1a-
eGFP as adult fish and to test if the transgenic line can be used to study the effect of
Pdgfr signaling on coronary vessels. In order to test the efficacy, I looked at the coronary
vessel phenotype in cryoinjured zebrafish heart during regeneration, after overexpressing
dnpdgfrβ. The results were compared with the phenotype (lack of coronary vessel
formation) observed on treatment with Pdgfr inhibitor after amputation (Jieun Kim 2010)
and cryoinjury (Figure 8 C and F) (unpublished data; Jieun Kim). I have chosen
cryoinjury as my injury model over other models as it mimics myocardial infarction in
mammals thus giving the opportunity to understand, analyze the pathological process so
that the findings can be related and applied in mammals.

4.3 Results and Discussion
The triple transgenic zebrafish line UAS:dnpdgfrβ-yfp;hsp70:gal4;fli1a-eGFP was
generated by crossing UAS:dnpdgfrβ-yfp;hsp70:gal4 double transgenic line with the
double transgenic fli1a:eGFP;hsp70:Gal4 line. Fli1a-GFP (friend leukemia integration
22

1a is an endothelial cell marker) exhibits fluorescence to blood vessels due to the Green
Fluorescent Protein marker (Lawson ND et al 2002). This enables us to study coronary
vessels.
pBH vector which was used to generate UAS:dnpdgfrβ-yfp construct carry mCherry
reporter gene downstream of cmlc2 (cardiac myosin light chain 2) promoter. As a result
the embryo with successful integration of UAS:dnpdgfrβ-yfp transgene into its genome
exhibit red heart (Figure 5). The embryos were thus screened using the fluorescent
markers (GFP, mCherry) to select embryos with double positive fluorescence (gfp+,
mCherry+) over single positive and no fluorescence.

Figure 5. Confocal image of adult heart section from UAS:dnpdgfrβ-yfp
transgenic line (left) with successful integration of the transgene in zebrafish
genome. Red fluorescence in the heart is due to pBH vector which was used
to make the UAS:dnpdgfrβ-yfp transgene construct. pBH vector carry
mCherry reporter gene downstream of cmlc2 promoter. DAPI stains nucleus
blue. Image of wild type heart section (right) is for comparison.

23

The double positive embryos were grown into adult zebrafish. Since hsp70:gal4
transgene construct do not have a fluorescent marker tag, in order to select fish in which
the transgene was successfully integrated into the genome, the fish are genotyped once
they grow into adults (Figure 6). gal4(-) are used as single transgenic fish as control for
the experiment to study coronary vessels. In the absence of Gal4 protein, there would be
no expression of the dnpdgfrβ transgene hence no inhibition of Pdgf signaling.

Figure 6. Genotype result for gal4 gene in UAS:dnpdgfrβ-
yfp;hsp70:gal4. DNA was isolated from tail fin of transgenic fish and
a PCR was run using forward and reverse primers of Gal4 gene and β-
actin gene. Presence of band shows that the gene is present in genome.
β-actin was used as control to check for PCR validation.

24

Expression Analysis
To test if we can observe any expression of the transgene (dnpdgfrβ), I heat shocked
UAS:dnpdgfrβ-yfp;hsp70:gal4(+) fish without any injury at 38°C for 1 hr every day for 7
days and the heart was collected at the end of 7
th
day and paraffin embedded for
sectioning. The expression was analyzed by in-situ hybridization. The in-situ
hybridization result showed very strong dnpdgfrβ expression (Figure 7).

25

Next, I checked how the coronary vessel phenotype is affected in regenerating zebrafish
heart after cryoinjury, if DnPdgfrβ is able to successfully inhibit Pdgf signaling similar to
Pdgfr chemical inhibitors. Based on previously published inhibitor data (Jieun Kim,
2010), I hypothesized that DnPdgfrβ inhibits Pdgf signaling effectively and results in
decreased in coronary blood vessel formation in regenerating zebrafish heart. To test the
hypothesis, I took equal number of triple transgenic fish from both set (gal4+, gal4-),
cryoinjured their hearts and then subjected the fish to heat shock by bath incubation at
38°C for 1 hour every day.
Keeping in mind that heat shock and cryoinjury delays the process of regeneration, I
chose to look at regenerating zebrafish heart of the transgenic line 21 days after
cryoinjury to match the 14dpa time point of Pdgfr inhibitor treatment (Jieun Kim 2010).
Figure 7. pdgfrβ in-situ Hybridization on uninjured UAS:dnpdgfrβ-
yfp; hsp70:Gal4 heart
Zebrafish heart of UAS:dnpdgfrβ-yfp; hsp70:Gal4 line(right) after
7 days of heat shock at 38°C for 1 hr every day. The result showed
strong dnpdgfrβ expression. Control (left) showed no dnpdgfrβ
expression.
26

In comparison however, I did not find a consistent lack of coronary blood vessel in the
injured area in the experimental fish (Figure 8D). There was not a considerable difference
in coronary blood vessel phenotype in the injury area of the regenerating ischemic
zebrafish heart between the control and experiment (Figure 8 A and D).
We reasoned that the lack of phenotype may be explained if either DnPdgfrβ is not
strongly expressed or in spite of overexpression, it is not able to overcome the
endogenous expression to inhibit Pdgf signaling. I performed in-situ hybridization on
sections of hearts collected from my experiment to check for the expression of dnpdgfrβ.
Unfortunately I did not observe a strong expression of dnpdgfrβ in the regenerating hearts
of cryoinjured fish after 21 days of heat shock (Figure 9). The expression we did observe
appears to be due to endogenous pdgfrβ and not due to overexpression of dnpdgfrβ.
It is thought that over a period of time the heat shock promoter weakens and probably
gets silenced. Since Gal4 is continuously required for the target gene expression, if heat
shock promoter fails to activate gal4, there would be no expression of DnPdgfrβ to
efficiently inhibit Pdgf signaling. Assuming 21 days is a late time point to look at, I
decided to repeat the experiment with an earlier time point of 14 days heat shock after
injury. Unfortunately, even at 14 days the confocal images failed to show a strong
phenotype (Figure 8 E) and the in-situ data showed inconsistent expression (Figure 9). It
indicates that for some reason, the dnpdgfrβ is unable to express efficiently and hence
unable to inhibit Pdgf signaling.
When Dr. Katie Wiens used this system to study intersegmental vessels in zebrafish
embryo in 2010, it showed strong expression which unfortunately I failed to see in adults.
27

Also, I cannot explain as why the line showed strong expression in uninjured adult heart
(after 7 day heat shock) but failed to show expression in case of injury (after 14 days and
21 days heat shock). However, in light of my findings I propose that probably the system
has become weak over the generation or is not as effective in adults. The weakening of
the line may be due to silencing of heat shock promoter or due to methylation of UAS
repeats after many generation (Halpern et al, 2008 and Akitake et al. 2011). As the
UAS:dnpdgfrβ-yfp;hsp70:gal4 transgenic line failed to express DnPdgfrβ in adult injured
zebrafish heart and thus failed to inhibit Pdgf signaling, I cannot draw any conclusion
regarding role Pdgf signaling in coronary vessel formation during heart regeneration

28

Figure 8. Whole mount confocal result of UAS:dnpdgfrβ-yfp;hsp70:gal4;fli1a-eGFP
zebrafish heart.
The top row showcase (A) confocal images of UAS:dnpdgfrβ-yfp; fli1a-eGFP;
hsp70:gal4(-) zebrafish heart after cryoinjury and 21 days heat shock treatment (B)
confocal images of UAS:dnpdgfrβ-yfp; fli1a-eGFP; hsp70:gal4(-) zebrafish heart after
cryoinjury and 14 days heat shock treatment and (C) wild type fish treated with DMSO
for 14 days after cryoinjury. The result showed that there is no effect on coronary vessel
formation in regenerating heart as expected.
The bottom row shows (D) confocal images of UAS:dnpdgfrβ-yfp; fli1a-eGFP;
hsp70:gal4(+) heart after cryoinjury and 21 days heat shock treatment (E) confocal
images of UAS:dnpdgfrβ-yfp; fli1a-eGFP; hsp70:gal4(+) heart after cryoinjury and 14
days heat shock treatment and (F) wild type fish treated with InhV inhibitor for 14 days
after cryoinjury. The inhibitor treated heart clearly shows decrease in coronary vessels in
injury area of regenerating heart. However in the transgenic heart, we observed
inconsistency in coronary vessel formation.
29

4.4 Material and Methods:
Genotyping: As gal4 gene in hsp70:gal4 transgenic line is not tagged to a reporter gene,
it was required to genotype the fish to separate hsp70:gal4(+) fish and hsp70:gal4(-) fish.
Fish were anesthetized in tricane (1 tsp of 0.4% tricane in 180 ml of fish water) and 50%
of their tail fin was cut using a sterilized surgical blade and collected in pcr tube with
60ul of 50mM NaOH. Each fish was kept in a separate genotype tank to avoid mixing
Figure 9. In-situ hybridization results showing expression of dnpdgfrβ after cryoinjury in
hsp70:gal4;UAS:dnpdgfrβ-yfp;fli1a:eGFP hearts. The UAS:dnpdgfrβ-yfp; fli1a-eGFP;
hsp70:gal4(-) heart shows no expression of dnpdgfrβ. The UAS:dnpdgfrβ-yfp; fli1a-eGFP;
hsp70:gal4(+) hearts at 21dpc show weak expression which we think is not due to
dnpdgfrβ but is the expression of endogenous pdgfrβ. In 14dpc heart, we observed 20% of
heart showing expression of dnpdgfrβ all over the heart similar to what we observed in
uninjured 7 day heat shock treated heart, however 80% showed expression only in injury
area which make us believe that the expression we are seeing in the injury area is the
expression of endogenous pdgfrβ.
30

and confusion. DNA was extracted from the collected tail fin by heating them at 95°C for
20 min using thermal cycler (GeneAmp® PCR System 9700 by Applied Biosystem). The
DNA was then used for PCR using forward and reverse primers for gal4 and β-actin with
Tm=55°C in GeneAmp® PCR System 9700 from Applied Biosystem. β-actin gene was
used for control as it is a housekeeping gene which is constitutively expressed, to confirm
the presence of DNA in the solution. The PCR result was run on 1% Agarose gel and fish
were separated into hsp70:gal4(+) or hsp70:gal4(-).

Cryoinjury: Fish were cryoinjured following the protocol as previously described by
Fabian et al (2011). Fish were anesthetized in 0.4% tricane and placed on a damp sponge
with ventral side up under a dissection microscope. A small incision was made through
the chest using sterilized forceps to access the heart. The ventricular apex was frozen
using a stainless steel cryoprobe (0.8mm diameter tip) precooled in liquid nitrogen. The
probe was held to the apex for 25-30 seconds. To stop the freezing of the heart, fish water
at room temperature was dropped on the tip of the cryoprobe. The fish were returned to
fish tank with fish water and left on bench.

Heat Shock: A Microprocessor Shaker Bath was filled with water and maintained at
38°C. The fish tanks with cryoinjured fish , both gal4(+) and gal4(-) were placed in the
water bath at 38°C for 1 hour every day in the morning starting from the day after
cryoinjury till the day the heart were collected for imaging. The bath incubation induces
the hsp70 promoter in hsp70:gal4(+) fish.
31

Collecting heart and Imaging: Fish were culled using 0.4% tricane (2tsp tricane in 180
ml of fish water). They were mounted on a damp sponge with ventral side up under a
dissection microscope. A long vertical incision was made, running along the middle of
the chest using 70% EtOH sterilised forceps to access the heart. The heart was excised
carefully keeping the atrium, ventricle and the bulbous arteriosus intact. The heart was
then placed in a small petri dish containing DEPC-PBS and cleaned off of blood clots,
lipids sticking to the walls of the heart. Once cleaned, the heart was then placed on a
glass bottom petridish with low melting agarose in the desired orientation such that the
area of interest (containing the injury site) would face the objective of LSM 700 confocal
laser scanning microscope for imaging.

ISH (In-situ Hybridization): The collected hearts were fixed in 4% PFA for an hour,
equilibrated for several hours in 30% Sucrose and frozen for cryosectioning in Tissue
Freezing Medium. The heart were cryosectioned and were used for In-situ hybridization
using pdgfrβ antisense probe following the protocol as previously described by Kenneth
D Poss (2002) with modification

32

Chapter 5. Characterization of transgenic line Ubi:CreERT2;Ubi:loxP-GFP-STOP-
loxP-dnpdgfrβ
5.1 Introduction
In order to address the uncertainties we faced with the hsp-70 promoter and Gal4-UAS
system, we moved to a more stable and better inducible system to create the transgenic
line with dnpdgfrβ. Using the ubiquitin promoter (ubi) and the Cre-loxP system, our lab
generated Ubi:CreERT2 and Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ transgenic lines.
A triple transgenic line Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ;fli1a:eGFP
has been generated by crossing a double transgenic Ubi:CreERT2;Ubi:loxP-GFP-STOP-
loxP-dnpdgfrβ line with fli1a:eGFP line to facilitate study of coronary vessel in
regenerating zebrafish heart.

5.2 Working of Cre-LoxP system with respect to Ubi:CreERT2;Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ
The Cre (cyclization recombination) recombinase is a 38-kDa protein that recognizes and
mediates site specific recombination between two 34-bp DNA recognition sites referred
to as loxP (locus of crossover [x] in P1 bacteriophage) (Feil et al 2009, Kühn 2002).
CreER is a chimeric Cre recombinase that consists of Cre fused to mutated ligand –
binding domains of estrogen receptor (ERT2) [CreERT2 recombinase contains human
estrogen receptor ligand-binding domain with triple mutation]. CreER recombinases are
inactive and in cytoplasm until activated by the binding of 4OHT (4-Hydroxytamoxifen),
33

a synthetic estrogen receptor ligand. 4OHT is a chemical added/injected from outside and
is not produced by the cell naturally thus allowing for external temporal control of Cre
activity. Once activated, Cre recombinase is translocated into the nucleus and excises the
loxP-flanked DNA sequence. Once the Cre-loxP system works for the
Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ double transgenic fish, the Ubi:loxP-
GFP-STOP-loxP-dnpdgfrβ line can then be crossed to other Cre lines driven by different
promoters to achieve tissue specific expressions.

In the double transgenic line Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ (Figure
10), during the absence of an active Cre recombinase cells would express GFP protein
but not dnpdgfrβ as the STOP codon would prevent the downstream translation of
dnpdgfrβ. However when Cre recombinase is activated with 4OHT by the bath induction,
the recombinase would excise the loxP flanked ‘GFP-STOP’ sequence. As a result, cells
Figure 10. Working of
Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-
dnpdgfrβ
Treatment with 4OHT (4-hydroxy tamoxifen)
activates Cre recombinase which excise
‘GFP-STOP’ segment from Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ. This leads to the
expression of dnpdgfrβ.
34

would stop expressing GFP protein and would start expression of dnpdgfrβ transgene
hence inhibiting the Pdgf signaling (Figure 10).

5.3 Results
Embryo Screening:
The double transgenic Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-dnpdgfrβ embryos were
screened with the help of fluorescent marker GFP to separate embryo with integration of
transgene ‘Ubi:loxP-GFP-STOP-dnpdgfrβ’. Due to the ubiquitous promoter, embryo
with successful integration of the transgene in its genome exhibited green fluorescence
everywhere on its body (Figure 11).

Figure 11. Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-
dnpdgfrβ zebrafish embryo showing ubiquitous
expression of GFP (Green Fluorescent Protein)
35

On crossing the double transgenic line Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-
dnpdgfrβ with fli1a:eGFP, the resulting embryos were screened to select the ones which
showed green fluorescence throughout the body along with green fluorescence in blood
vessels which was more prominent along the gills. These embryos were Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ (+) and fli1a:eGFP (+). To know if they are Ubi:CreERT2 (+) as
well, the selected embryos are genotyped when they grow up into adults (Figure 12).

Figure 12. Genotype result for cre gene in Ubi:CreERT2;Ubi:loxP-GFP-
STOP-loxP-dnpdgfrβ. DNA was isolated from tail fin of transgenic fish
and a PCR was run using forward and reverse primers of Cre gene and β-
actin gene. Presence of band shows that the gene is present in genome. β-
actin was used as control to check for PCR validation.
36

Induction of transgenic line with 4OHT
To quantify Cre-mediated induction of dnpdgfrβ expression in Ubi:CreERT2;Ubi:loxP-
GFP-STOP-loxP-dnpdgfrβ, Dr. Jieun Kim from our lab did in-situ hybridization (pdgfrβ
probe) on embryos from the same line (Figure 13). The embryos were treated with 4OHT
from 1day post fertilization (dpf) to 6 days post fertilization and then paraffin embedded
for sectioning . The result showed positive induction with strong expression of dnpdgfrβ
(Figure 13).

To check for induction in adult zebrafish, we treated adult zebrafish of Ubi:Cre;
Ubi:loxP-GFP-STOP-loxP-mCherry line with 1µM 4OHT (Jopling C., 2010) and then
fin was imaged under green and red channel of fluorescent dissecting microscope. The
control (untreated transgenic fish) as well as the treated transgenic fish exhibited green
fluorescence with equal strength (Figure 14 A and D). However the fin of treated fish
Figure 13. pdgfrβ in-situ hybridization on zebrafish embryo to
test for induction in Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-
dnpdgfrβ.
Zebrafish embryo (Cre+ and Cre-) were treated with 4OHT
from 1dpf to 6dpf. Wild type is a negative control.
37

showed fragment of red fluorescence (Figure 14 E and F) which was absent in the control
(Figure 14 B and C). Thus we could see induction in treated adult fish and the strength of
induction increased with increased time of treatment. Once we are able to optimize the
condition for treatment to obtain uniform induction, we can study the coronary vessel
phenotype by inhibiting Pdgf signaling in Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-
dnpdgfrβ line.

Figure 14. Induction in Ubi:CreERT2; Ubi:loxP-GFP-loxP-mCherry
Ubi:CreERT2; Ubi:loxP-GFP-loxP-mCherry fish were treated with 1µM 4OHT every 2
days. The fin of the treated fish were imaged for GFP and mCherry fluorescence and
compared with untreated fish fin. (A), (B) and (C) are untreated fish acting as control. (D)
Fish treated for 7 days and fin imaged for GFP fluorescence on 7
th
day. As GFP is quite
stable protein, we observe GFP fluorescence similar to control(A) even after treatment. (E)
Fish treated for 7 days and fin imaged for mCherry fluorescence . We can observe specs of
fluorescence in the treated fin which is absent in the control (B). (F) Fish treated for 11 days.
We can see increased mCherry fluorescence which indicates that cells are continuously
getting induced.
38

5.4 Material and Methods:
Genotyping: As CreERT2 gene in Ubi:CreERT2 transgenic line is not tagged to a
reporter gene, it was required to genotype the fish to separate Ubi:CreERT2(+) fish and
Ubi:CreERT2(-) fish. Fish were anesthetized in tricane (1 tsp of 0.4% tricane in 180 ml
of fish water) and 50% of their tail fin was cut using a sterilized surgical blade and
collected in PCR tube (Polymerase Chain Reaction) with 60ul of 50mM NaOH. Each fish
was kept in a separate genotype tank to avoid mixing and confusion. DNA was extracted
from the collected tail fin by heating them at 95°C for 20 min using thermal cycler
(GeneAmp® PCR System 9700 by Applied Biosystem). The DNA was then used for
PCR using forward and reverse primers for Cre and β-actin with Tm=60°C in
GeneAmp® PCR System 9700 from Applied Biosystem. β-actin gene was used for
control as it is a housekeeping gene which is constitutively expressed, to confirm the
presence of DNA in the solution. The PCR result was run on 1% Agarose gel and fish
were separated into Ubi:CreERT2(+) or Ubi:CreERT2(-).

4OHT bath induction: A 13mM stock of 4OHT was prepared by dissolving 5mg (Z)-4-
Hydroxytamoxifen from Sigma-Aldrich® in 1ml EtOH and vortexed until the powder is
completely dissolved in EtOH to form a homogenous solution. In order to induce CreER
recombinase, adult fish from the Ubi:CreERT2;Ubi:loxP-GFP-STOP-loxP-mCherry
transgenic line is treated with 1µM 4-OHT (15.38ul 4-OHT in 200 ml of fish water)
every 2 days for one week using bath incubation.

39

Collecting heart: Fish were culled using 0.4% tricane (2tsp tricane in 180 ml of fish
water). They were mounted on a damp sponge with ventral side up under a dissection
microscope. A long vertical incision was made, running along the middle of the chest
using 70% EtOH sterilised forceps to access the heart. The heart was excised carefully
keeping the atrium, ventricle and the bulbous arteriosus intact. The heart was then placed
in a small petri dish containing DEPC-PBS and cleaned off of blood clots, lipids sticking
to the walls of the heart.

ISH (In-situ Hybridization): The collected hearts were fixed in 4% PFA overnight at
4°C, and then embeddedin paraffin following the protocol as described... The heart were
sectioned and were used for In-situ hybridization using pdgfrβ antisense probe following
the protocol as previously described by Poss (2002) with modification.

40

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Abstract (if available)

Abstract It is known that PDGF-B and PDGFRβ interaction in crucial in blood vessel formation in mammals. PDGF-B is involved in proliferation and recruitment of vSMC (vascular smooth muscle cells) and pericytes to blood vessels. Mutants lacking PDGF-B and PDGFRβ show pericyte loss, capillary dilation and rupture. Not much has been known about role of Pdgfb-Pdgfrβ mediated signaling in zebrafish until recently. Now it has been shown that pdgf is upregulated in regenerating zebrafish heart and Pdgf signaling is necessary for intersegmental vessel formation in zebrafish embryo. Also, with the help of chemical inhibitors blocking Pdgfr signaling, it has been shown that Pdgf signaling plays an important role in coronary vessel formation during regeneration in zebrafish. The aim of this project is to help characterize an improved, cleaner and more specific genetic tool which can overcome the shortcoming of chemical inhibitor to study the role of Pdgfrβ signaling in coronary vessel formation during regeneration in zebrafish heart after cryoinjury.

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Asset Metadata

Creator Nair, Gayatri (author)

Core Title Characterization of transgenic zebrafish lines for studying role of platelet derived growth factor (PDGF) signaling on coronary vessel formation in regenerating zebrafish heart after cryoinjury

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 11/22/2013

Defense Date 10/22/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cryoinjury,heart,OAI-PMH Harvest,PDGF,zebrafish

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lien, Ching Ling (Ellen) (committee chair), Stallcup, Michael R. (committee member), Tokes, Zoltan A. (committee member)

Creator Email gayathri6@gmail.com,gayatrin@usc.edu

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