University of Southern California Dissertations and Theses

Tools to study the epicardium's response during cardiac regeneration

(USC Thesis Other)

Tools to study the epicardium's response during cardiac regeneration

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

TOOLS TO STUDY THE EPICARDIUM’S RESPONSE IN CARDIAC
REGENERATION
by
Nicole Rubin

A Thesis presented to the
FACULTY of the USC GRADUATE SCHOOL
University of Southern California
In Partial Fulfillment of the
Requirements for the Degree
Doctor of Philosophy
(Pathology)

May 2013

Copyright 2013 Nicole Rubin
ii
ACKNOWLEDGEMENTS
This thesis and my thesis work are dedicated to my mother. She has been my
biggest cheerleader and the main reason for pursuing an education. She is a role model as
a strong woman and a survivor. Thank you for all your encouragement and giving me
enthusiasm when things got tough. I hope that I make you proud, as you make me proud.
I would also like to thank all the people who kept me going during this process:
new and old lab-mates and friends. Especially to Ali Darezerehshki, who helped me at
the bench but also maintained a cool, clear head when things were frustrating. Also thank
you to Gianluca Turcatel, who is not only my friend, but also my go-to when I had so
many technical questions. Your passion for science is infectious and at times
intimidating, but it will take you far. All the lab members who became more than
colleagues but lifetime friends (Hyuna! Katie!).
Last but not least, I would also like to acknowledge my mentors. Gary Martin,
who was my original role model scientist, who gave me the passion to make science my
career. Ellen Lien, I thank you for giving me the opportunity to be part of something
great by welcoming me to her lab. Vesa Kaartinen, although geographically separated, he
made time to give me guidance and mentorship.

iii
TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
ABSTRACT vii…viii
BACKGROUND p. 1…13

Chapter I: Transgenic mouse model for studying a gene in cardiac repair after
ischemic injury in adult mouse p. 14…48
Introduction p. 14…17
Results p. 18…36
Discussion p. 37…39
Materials and Methods p. 40…47

Chapter II: Cryoinjury in neonatal mouse heart to study mammalian cardiac
regeneration p. 48…81
Introduction p. 48…49
Results p. 50…72
Discussion p. 73…75
Materials and Methods p. 76…81

Chapter III: Primary culture of zebra fish (Danio rerio) epicardium: an in vitro tool
to study the role of epicardium in cardiac regeneration p. 82…97
Introduction p. 82…83
Results p. 84
Discussion p. 85
iv
Materials and Methods p. 86…97

CONCLUSION p.98…100

TABLES p. 101
BIBLIOGRAPHY p.102…112
FIGURES
Introduction
1. Zebrafish heart regeneration model. p. 3
2. Fgf signaling is required for zebrafish regeneration: neovascularization p. 8
3. Differential gene expression during the zebrafish heart regeneration. p. 10
Transgenic mouse model for studying a gene in cardiac repair after ischemic injury in
adult mouse
4. Coronary artery ligation surgery is a model for cardiac ischemia. p. 17
5. Triple- transgenic mouse to target inducible expression of Fgf10 in the
epicardium does not have significant transgene induction. p. 20
6. Tbx18-cre lineage labeling of epicardium changes after birth. p. 22
7. Adult Tbx18-cre mice have no epicardial Cre recombinase activity without
injury. p. 24
8. Chicken beta Actin promoter Cre recombinase for ubiquitous Fgf10
overexpression has dramatic transgene overexpression. p. 27
9. Fgf10 overexpression does not change scar size after ischemic injury in adult
mouse. p. 30
v
10. Fgf10 overexpression does not alter vascularization of border zone of scar in the
left ventricle after injury. p. 32
11. Overexpression of Fgf10 did not improve survival after cardiac injury. p. 34
12. Fgf10 does not increase the expansion of Wt1-positive epicardial cells due to
ischemic injury in adult mouse. p. 36

Fgf10 in neonatal mouse cardiac cryoinjury
13. Cryoinjury is a modifiable model of cardiac injury p. 51
14. Differential phosphorylation of p42/ p44 (Erk) after cardiac injury in the neonatal
mouse. p. 53
15. Erk phosphorylation is found in the epicardium and at the injury site of cryo-
injured mice. p. 54
16. Induction of Fgf10 can be controlled with a double transgenic mouse model to
study the effect of Fgf10 overexpression in the neonatal mice after injury. p. 56
17. Induction of Fgf10 transgene in double transgenic mice increases Fgf10 and
downstream genes in neonatal mice. p. 58
18. Tet(o)H2b-GFP is a reporter for assaying inducibility and tissue specificity in the
double-transgenic mouse model. p. 60
19. Fgf10 overexpression in the absence of injury had no effect on the morphology of
the epicardium (Wt1-positive cells). p. 62
20. Fgf10 overexpression does not increase vimentin expression Wilm’s Tumor 1-
positive cells in the injury area. p. 64
vi
21. Continuous overexpression of Fgf10 does not alter scar size after cryoinjury. p. 67
22. Temporary overexpression of Fgf10 from 3 dpc to 10 dpc does not alter scar size
after cryoinjury. p. 68
23. Cryoinjury in neonatal mouse heart leads to increased Wt1-positve cells in the
injury, Fgf10 enhances it further by day 21 post surgery. p. 69
24. Fgf10 overexpression increased the proliferation of Wilm’s Tumor 1- positive
cells at 14 dpc. p. 72

In vitro model to study epicardium in regeneration
25. Representation of the epicardial culture on fibrin. p. 90
26. Outgrowth of heart explants are epicardial cells, but amputated hearts have
increased EMT markers in the outgrowth. p. 95
27. Zebrafish heart explants select for non-cardiomyocyte cells but can allow for
endothelial cell contamination in the primary culture of epicardial cells. p. 97
TABLES
1. Genotyping Primers p. 101
2. qPCR Primers p. 101
vii
ABSTRACT
Cardiovascular disease is the number one cause of mortality worldwide and when
not fatal contributes to chronic morbidity and decreased lifespan. Treating cardiovascular
disease is a medical priority and suitable treatments continue to elude the medical field.
Increasing interest in adult tissue regeneration as a solution to the medical burden of
adulthood disease, including cardiac pathologies, has occurred with the identification of
adult stem cells and organ regeneration in vertebrate animals. I used vertebrate animals as
tools for studying cardiac regeneration. We utilized the adult mouse with a coronary
artery ligation to evaluate the effects of Fgf10 on cardiac repair after ischemic injury. We
also developed a neonatal mouse injury model to evaluate Fgf10 on the endogenous
regeneration process, specifically the effect on epicardial cells. Additionally we
developed a primary zebrafish epicardial cell culture for in vitro assays on the epicardial
cells during regeneration. These tools allowed us to study the effect of a gene’s
expression, in this study Fgf10, on the heart after injury and in endogenous repair/
regeneration processes. It was determined that Fgf10 does not have significant
improvement on the cardiac regeneration process since it did not reduce the fibrosis in the
heart in adult or neonatal mice, but does have an effect on epicardial cell proliferation
during the injury response. Overexpression of Fgf10 increased proliferation of the
Wilm’s tumor -1 expressing cells after injury, resulting in increased epicardial cells in the
injury site. The significance of influencing epicardial cells during injury corroborates
prior publications indicating epicardium response to injury is beneficial to repair and
makes it an ideal target for therapies. This study identified Fgf10 as candidate growth
viii
factor in improving the response of the epicardium to cardiac injury and suggests further
evaluation is warranted.

1
BACKGROUND
Adult regeneration: Increasing interest in regenerative ability of adult tissues has
occurred especially with the characterization of stem cells and identification of adult
stem/ progenitor cells. Resident stem or progenitor cells in adult tissues have been
implicated as responsible for, or at least a contributor to the repair and regeneration of
tissue in the adult organism. The number of tissues with identifiable stem cells is few,
including liver and skin, and the capacity for regeneration after injury in those tissues
with stem cells is limited. Additionally, the rethinking of the potential for proliferation of
(what was once considered perpetually quiescent) adult cells, such as cardiomyocytes,
and its contribution in regeneration (Bergmann et al., 2009) have fueled new hypotheses
for unlocking regenerative potential.
Mammals appear to be the most limited vertebrate in their capacity for tissue
regeneration. However, reptilian, amphibian and teleost organisms demonstrate increased
capacity for regeneration as adults, including tissues that are particularly static in
mammals, such as nerve and cardiac tissue (Gardiner, 2005; Stoick-Cooper et al., 2007).
This has made these organisms, such as the zebrafish (Danio rerio) useful models in
understanding the mechanisms of adult regeneration in organs such as the heart. The
standard murine model for mammals, the well-defined Mus musculus, remains useful as a
stand-in for mammalian regenerative potential while providing a greatly controllable
genome for dissecting out the roles of specific genes in this process.

2
The zebrafish model for regeneration has elucidated mechanisms and important
factors in the process. These mechanisms have many similarities with the developmental
processes for organogenesis in mammals (reviewed in Steinhauser and Lee 2011).
Extrapolating from zebrafish regeneration and developmental processes in mouse,
vasculogenesis and cardiac cell differentiation and proliferation for mammalian
regeneration can be activated or enhanced. Ischemia in the human (mammalian) heart
causes significant tissue damage with loss of many cardiac cells through apoptosis and
necrosis, only to be replaced with non-functional fibrotic tissue and hypertrophy but not
hyperplasia of remaining cardiomyocytes. After an ischemic episode, the heart is
permanently scarred and functionally impaired leading to lifelong morbidity or ultimately
death. Cardiovascular disease affects 27.1 million people in the USA, 599,413 deaths in
2009, and is the leading cause of all mortality (Center for Disease Control and
Prevention, 2012). Determining the precise mechanisms and initiators for regeneration
may lead to possible therapeutic agents for human patients, decreasing mortality and
morbidity from ischemic heart diseases.

3

Stem/progenitor cells in the adult: In contrast with animal models with well-characterized
regenerative potential, the process of regeneration in mammalian heart is not as well
Figure 1. Zebrafish heart regeneration model. Artistic depiction of the timeline of
cardiac regeneration process in the zebrafish. Ventricular resection injures the heart,
and subsequent cellular changes, such as epicardial activation, cardiomyocyte
proliferation and neovascularization, occur that contribute to the regeneration of the
lost tissue. (Lien et al., 2012)

4
understood. Adult stem cells in the mammalian heart may be capable of differentiating to
replace multiple cardiac cell types damaged or lost from injury. During embryogenesis, a
stem cell population does exist. Nkx 2.5/c-Kit
+
cells from embryonic heart comprise a
multipotent group of cells capable of differentiating into smooth muscle cells and
cardiomyocytes (Wu et al., 2006). Early cardiac progenitors in the early anterior heart
field (Isl1
+
) can be regulated through Wnt signaling, which increases Fgf10 ligands and
Fgf signaling through mitogen-activated protein kinase pathway (Cohen et al., 2007). In
the adult human heart, evidence supports the identification of resident cardiac cells which
are positive for c-Kit expression and fulfill the criteria for stem cells: self-renewing,
clonogenic and multipotent (Beltrami et al., 2003). These cardiac stem cells continue to
differentiate to functional cells and can be captured in a transient stage progressing from
stem cell to differentiated cardiomyocytes. Most importantly, these cardiac stem cells
were able to replace myocardium as well as blood vessels after myocardial infarction by
incorporating into the damaged heart tissue (Bearzi et al., 2007 and Hsieh et al., 2007).
The embryonic progenitor cells may persist in the adult organ, comprising the population
of cardiac progenitors cells capable of replenishing blood vessels and myocardium after
injury. The regenerative capacity of resident progenitor/ stem cells in the heart may only
require the proper stimulus to activate.

Biological activators of adult regeneration: The factors that activate and orchestrate
progenitor/ stem cell qualities in cardiac cells are not clear: either the actions of ever-
present resident adult stem cells or a process of de-differentiation of other mature cardiac

5
cell types. Fin regeneration in zebrafish can be studied as a generic example for
regenerative processes, such as in blastema formation. The adult zebrafish fin model of
regeneration has been used to identify the important factors necessary for regeneration,
such as growth factors (including fibroblast growth factors, or Fgf’s ), metalloproteinases,
inflammatory factors (Whitehead et al., 2005 and Lien et al., 2006: Figure 3) and heat
shock proteins (Makino et al., 2005). The zebrafish fin model is a useful generic model to
understand how adult tissue in fully formed structures respond to injury and can replace
lost cells of various types, such as vascular cells.
The development of a heart regeneration model in the zebrafish has clarified more
specifically what mechanisms occur in the heart after injury in order to regenerate (figure
1). Upregulation of expression of growth factors and metalloproteinases during the injury
and regeneration process suggest stimulation of cells for processes including proliferation
of cells and restructuring of the tissue (Lien et al., 2006) (figure 3), similar to the process
of embryonic development. Zebrafish model systems also revealed a progenitor cell
subpopulation within the epicardium that can give rise to new blood vessels within the
myocardium after amputation (Lepilina et al., 2006). Similarly in mice, a secreted protein
from injured myocardium, thymosin β4, is necessary for activation of epicardium-
derived cells to give rise to new vessels by undergoing EMT (Smart et al., 2007). This
demonstrates the importance of epicardium and myocardium interaction during
neovascularization in mammalian heart as well as zebrafish. Upregulation of growth
factors and epithelial and mesenchymal interactions (the epicardium to myocardium

6
cross-talk) suggests that the zebrafish regeneration is a process that recapitulates
developmental organogenesis.

Cardiomyocyte proliferation The zebrafish heart regeneration model has illustrated the
source of new cardiomyocytes after injury. The loss of functional cardiac tissue,
specifically beating cardiomyocytes, is the main obstacle after heart damage and the
source of morbidity and mortality after the injury. The key to replenishing these cells
remains unclear; however, it is evident in the zebrafish heart regeneration model, the new
cardiomyocytes after injury are derived mainly from the proliferation of existing
cardiomyocytes (Jopling et al., 2011). Additionally these studies identify a subpopulation
of cardiomyocytes that are proliferative in the response to injury (Kikuchi et al., 2011).
The cues or factors that are required to trigger this proliferative response aren’t yet
determined, but growth factors would be obvious candidates. Potentially an interaction,
similar to embryonic development, between epicardium and myocardium via growth
factor intermediaries would be necessary for cardiomyocyte proliferation
Since cardiomyocyte proliferation would be an essential component to the
regenerative process, initiating and sustaining it in the mammalian system is a priority in
mammalian cardiac repair research. The zebrafish model illustrates how the proliferation
of cardiomyocytes was the major contributor to replacing lost cardiomyocytes (Kikuchi
et. al, 2010 and Jopling et al., 2010), and also the abrogation of proliferation via the mps1
mutation (Poss et al., 2005), can result in fibrosis and inability to regenerate. The
hypotheses for the mouse model would be of which factors contribute to cardiomyocyte

7
proliferation. In the embryonic cardiac development of the mouse many signals from the
epicardium, via growth factors, stimulate the cardiomyocyte proliferation (Stuckmann et
al. 2003). Since the regenerative process recapitulates the developmental processes, a
similar mechanism of epicardium-to- myocardium signaling may be required for
proliferation in adult cardiomyocytes after injury. The potential for this paracrine
signaling might already be in place, as the epicardium has been identified as a modulator
of the injury response in the adult mouse’s myocardium (Zhou et al., 2011). Although
signaling between the epicardium and myocardium is endogenous to the injured adult
heart, the mammalian heart lacks sufficient response to fully regenerate the damaged
tissue. Improving the regenerative ability, especially cardiomyocyte proliferation, has
been of particular interest. Growth factors have become common targets for improving
the regeneration in mammals by triggering cardiomyocyte proliferation.

Growth factors in adult repair: Of all the gene candidates, growth factors have been the
most appealing as they can stimulate proliferation or have other improvements to the
overall regeneration outcome. Activating Fgf signaling with a constitutively active
receptor had improvement in overall repair (Matsunaga et al., 2009). Other growth
factors such as periostin (Kuhn et al., 2007), neureglin (Bersell et al., 2009) and Fgf1
(Engel et al., 2005) have all shown promise in increasing cardiomyocyte proliferation for
improvement in repair in the mouse heart after injury.
Fgf signaling is of particular interest and has shown promise in improving repair
in the mammalian system in various processes that are required for regeneration. Fgf

8
ligands such as Fgf1 (Engel et al., 2005) and Fgf2 (Shiekh et al. 1996) contribute
positively to repair after myocardial infarction in animal models. Additionally, targeting
the Fgf receptor using transgenic animals has also demonstrated how the Fgf signaling
axis functions in cardiac repair. The zebrafish model of heart regeneration was inhibited
at the neovascularization level (figure 2) when a dominant- negative Fgf receptor (dn-
fgrfr) was expressed after injury (Lepilina et al., 2006). Conversely overexpression of the
constitutively activated Fgf receptor in a mouse model after cardiac injury improved the
blood vessel formation after injury for an overall protective effect (Matsunaga et al.,
2009). During development, Fgf signaling acts upstream of the hedgehog pathway for
vascularization of the heart (Lavine et al., 2006), and likewise the hedgehog pathway is
required for the maintenance of vasculature in the adult (Lavine et al., 2008) of which
Fgf signaling might likely continue to be acting upstream. Overall, Fgf signaling has
demonstrated promise in the vascularization process, which is necessary for cardiac
repair.

9

Another route of Fgf signaling effects is through epicardial cell epithelial-to
mesenchymal transition (EMT). As mentioned earlier, EMT of the epicardium is of
interest as a mechanism necessary for cardiac regeneration. We have shown in our lab
that growth factor signaling pathways such as platelet-derived growth factor, or PDGF,
are necessary for the activation of the epicardium as well as the neovascularization for
cardiac regeneration in zebrafish (Kim et al., 2010). . Utilizing similar Fgf stimulus may
trigger vasculogenesis even in the adult mammalian heart. The roles of growth factors in
development make them candidates for initiating cell proliferation and vasculogenesis
after injury, yet they already make some contribution to the repair process in regenerative
and non-regenerative tissue. Fgf signaling is a strong candidate as it has already been
studied in its role for neovascularization in the adult heart, but it also has the capacity for
activating the EMT of epicardial cells in cardiac development (Vega-Hernandez et al.,
2011). In this case the ligand-receptor axis of Fgf10-Fgfr2b was necessary for the EMT
of epicardial cells that developed into fibroblasts.

Figure 2. Inhibition of Fgf signaling abrogates new vessel formation. Fluorescent
image of transgenic fli1: EGFP zebrafish demonstrate a lack of new vessels in the
injury area at 30 days post ampuatation in the hsp70:dn-fgfr1 transgenic zebrafish.
(Lepilina et al., 2006)

10

Figure 3. Differential gene expression during the zebrafish heart regeneration.
Zebrafish regeneration initiates the expression or upregulation of inflammatory genes,
growth factor genes, and matrix metalloproteinases. Increased expression of growth
factors in the zebrafish indicates a potential role for growth factors in the regeneration
process, as it may be missing or deficient in the mammalian injury response. (Lien et
al., 2006)

11

Developmental Organogenesis: Utilizing animal model systems to study the process of
regeneration in vivo, the processes required for regeneration has become elucidated. The
characterization of gene regulation during regeneration and the identification of genes
essential to regeneration show remarkable similarity to the process of embryonic
organogenesis. The interactions between the epicardium and myocardium orchestrate the
development of the organ, and individually the epicardium and myocardium contribute
uniquely. The cross-talk is essential for activating important processes in embryonic
development, such as proliferation, which have recently been identified as critical for the
regeneration process as well (Kikuchi et al., 2010 and Jopling et al. 2010).

EMT in the heart: Epithelial-to – mesenchymal transition is a process necessary for the
formation of various structures of the heart. The endocardial cells that migrate into the
cardiac jelly of the heart by EMT contribute to the cardiac cushions and eventually the
formation of the septa and valves of the heart (Srirondigrit et al. 2008). Epicardium also
predominantly functions in the formation of various structures of the heart by a process of
EMT. Epicardial cells undergo EMT to form epicardium- derived cells (EPDC’s) that
reside within the mesenchyme to give rise to smooth muscle cells of blood vessels,
fibroblasts that generate the heart’s connective tissue and arguably the endothelial cells
(reviewed in Winter and Gittenberger-de Groot, 2007).

12
Epicardium and myocardium interaction: The growth factor activation of epicardium is
essential in the development of the functional heart including the cardiomyocytes and
coronary vessels. Epicardium acts as a modulator in myocardium development via
growth factor signaling. Without epicardium and its signals cardiomyocytes don’t
proliferate (Stuckmann et al., 2003). Retinoic acid is an essential participant in this
sequence of epicardium induction of myocardium proliferation (Stuckmann et al., 2003
and Chen et al., 2002). A retinoic acid- erythropoietin- Igf2 (insulin-like growth factor 2)
axis between the liver, epicardium and myocardium (Brade et al., 2011) utilizes paracrine
growth factor signaling to contribute to the proliferation of cardiomyocytes (Li et al.,
2011). Additionally, knockout of another growth factor signaling pathway of Fgf
receptors (one and two) in myocardium, not endothelial cells (Lavine et al., 2006),
confirms that the myocardium proliferation and vessel development depends on Fgf
signaling. Although the epicardium develops normally without the activity of Fgf’s,
cardiomyocyte proliferation decreases and leads to a hypoplasia and lower vascularity of
the heart (Lavine et al., 2005 and 2006). Fgf ligands act on the myocardium cells to
trigger the hedgehog signaling pathway and further downstream angiogenic genes such as
VEGF, which in turn controls coronary vasculature development (Lavine et al., 2006.
Additional evidence that signaling between the epicardium and myocardium is
responsible for coronary vessels is the origination of blood vessels in the subepicardial
space (Pennisi and Mikawa, 2009). The activation of the epicardial genes and
differentiation- inducing retinoic acid signaling in regeneration of the heart in adult

13
zebrafish is remarkably similar to the role of epicardium in development with the
requirement of functional Fgf signaling (Lepilina et al., 2006).

14
Chapter I: Transgenic mouse model for studying a gene in cardiac
repair after ischemic injury in adult mouse

INTRODUCTION
In order to study repair in adult mammalian heart, necrotic injury with tissue deep-
freezing or ischemic injury have been implemented. Development of a model
representative of the injury to the heart that occurs with myocardial infarction in humans
is important in dissecting the injury process and experimentally testing potential
therapies. The simplified injury model consisting of freezing the cardiac tissue, usually
using a super-cooled metal probe, or cryo-probe, has been effective in taking a broad
approach to understanding the process of cardiac damage and subsequent necrosis,
apoptosis and inflammation in adult mouse experimental models (Van Amerongen et al.,
2008). But in order to refine the molecular aspects of these processes and determine
targets for potential therapeutic intervention, the ischemic injury with a coronary artery
ligation surgery was necessary. The coronary artery ligation surgery requires greater
technical skill than the cryo- probe application but has replaced other cardiac tissue injury
models as the main model for studying cardiac ischemic injury similar to myocardial
infraction (MI) (figure 4). We employed this model to evaluate our candidate growth
factor, Fgf10, to determine if this sole growth factor can modulate the injury, scar or
repair process due to coronary artery ligation- induced ischemia in the heart. In order to
study this growth factor it was necessary to have an animal model for inducible Fgf10
gene expression control in addition to the injury model.

15
In order to test our hypothesis regarding the function of the Fgf10 gene in our
simulated MI model, the choice was to use the common laboratory mammalian animal
model the house mouse, Mus musculus. The larger mammalian in vivo models, such as
the laboratory rat, has the advantages over the mouse mostly due to size and the ease of
which the larger size simplifies the surgical procedure and some assays which require
more accessible equipment to measure cardiac function. However, a library of mutants,
knockins and transgenic mice make the Mus musculus a better choice to create an in vivo
tool to study gene function in conjunction with the injury model.
Approaches to administer bio-molecules, genes, or pharmacological agents for
evaluation have been widely varied and are of interest in modeling therapeutic
techniques. These have included transgenic animals, virus, biological scaffolds and more.
We intended to design a model that can be manipulated for temporal control as well as
tissue specificity utilizing readily available and established transgenic and knockin mice.
Considering the various transgenic systems, the well-defined available Cre recombinase
transgenes provide an assortment of promoter or knockin for cellular/ tissue specificity
and thus was chosen as a means to target cell or tissue gene expression (reviewed in
Nagy, 2000). Cre recombinase targets specific cells and their lineage irreversibly, thus
permanently ensuring the inducibility of gene expression in that cell population
regardless of fluctuating gene and/ or promoter expressions during the duration of the
experiment. Subsequently, the Cre recombinase transgene increases the complexity of the
transgenic model; however, provides expression consistency.

16
The temporal control of gene expression is equally important in the in vivo model.
A controlled form of gene activation required an established system that has been well
characterized as reversible and promptly activated. Various promoters can be activated
with pharmacological or other forms of manipulation (i.e.: heat). The tetracycline-
inducible system has been also well characterized for temporal control through
doxycycline inducibility via food or injection. A knockin of the reverse trans-activator
protein, rtTA, expresses after Cre- mediated recombination for tet system activation
(Belteki et al., 2005). Additionally it has been used successfully to activate our candidate
gene, Fgf10, within hours of doxycycline exposure using tet (o)Fgf10 transgene (Gupte et
al., 2009), thus made it an excellent candidate for control of gene expression timing. We
designed our model system for tissue specificity and temporal control by incorporating
the Cre recombinase and tet transgenic systems.

17

Figure 4. Coronary artery ligation surgery is a model for cardiac ischemia. Gross
views of the dissected heart showing the left ascending artery is permanently sutured
below the auricle to prevent blood flow to the tissue of the left ventricle, resulting in
tissue damage. Three days after the surgery, tissue damage can be seen grossly in the
left ventricle below the suture (arrow) in which the ischemic area appears red (A).
After seven days, the tissue of the ischemic area appears translucent (asterisk) as it
becomes thin and scarred (B). Scale bar represents 5mm.

18
RESULTS
Multi-transgenic mice evaluated as models to study the temporal role of Fgf10
overexpression in epicardium: The development of a refined in vivo mammalian model
that has control over tissue specificity of expression as well as when the gene would be
expressed was the aim of the generation of our mouse model in this study. We exploited
the vast number of already existing mutant, transgenic, and knockin mice to accomplish
the mouse model without having to generate a new mouse line. The tet-on system was
utilized to control temporal windows of expression of the transgene for overexpression of
Fgf10 growth factor. The tet system consists of the tet (o)fgf10, which has the promoter
for tet binding for inducing expression, and the transactivator protein, rtTA, which works
with doxycycline to bind to the tet promoter for gene expression. In order to achieve
tissue specificity the tet system was combined with a Cre recombinase transgenic mouse
line to remove the floxed neomycin- resistance gene sequence (which prevents further
transcription downstream) upstream of the tet -activator protein, rtTA (Belteki et al.,
2005). Tbx18-cre (Cai et al. 2008) was used to achieve epicardial cell specificity for
autocrine and paracrine signaling with Fgf10. Together this combination of three
transgenes/ knockin genes defined our mouse model for tissue specificity and temporal
control.
A schematic representation of the DNA constructs and recombination in the
genetic model illustrates how the Cre recombinase and tet-on system worked together.
Cre- mediated recombination removes the floxed neomycin resistance gene cassette,
allowing reverse tetracycline trans- activator protein, rtTA, gene expression. The rtTA
protein works in conjunction with doxycycline (when present) on the tet promoter of the

19
transgene, tet(o)Fgf10, for induction of Fgf10 in the Tbx18- derived cells (epicardium)
(figure 5). However quantification by qPCR analysis of cDNA for total Fgf10 showed a
non-significant (p=0.29) 1.35- fold expression in the triple-transgenic mice
(Tbx18:Fgf10) over the control littermates. This mouse model using Tbx18- cre had
insufficient gene overexpression in total RNA from the heart ventricle, as see by the
qPCR assay. However, the use of heterozygous mice (for all transgenes) may also
contribute to decreased gene expression in this system. But another factor that contributes
to the fold- change of gene expression is the contamination of the sample tissue’s RNA
by non- Tbx18 lineage cells to dilute the exogenous Fgf10 RNA. It subsequently resulted
in the validation of the recombination efficiency of Tbx18-cre.

20

Figure 5. Triple- transgenic mouse to target inducible expression of Fgf10 in the
epicardium does not have significant transgene induction. A schematic
representation of the DNA constructs and recombination in the genetic model with
Tbx18- inserted gene for Cre recombinase. Cre – mediated (red arch) recombination
removes the neomycin resistance (Neo) gene cassette in order for rtTA (orange half
donut) gene to express. In the presence of doxycycline (purple donut) rtTA protein
binds the tet promoter of the transgene, tet(o)Fgf10, for expression of exogenous
Fgf10 in the Tbx18- derived cells. Quantification of Fgf10 RNA by qPCR analysis of
cDNA for total Fgf10 showed a non-significant (p=0.29) 1.35- fold expression in the
triple-transgenic mice (Tbx18:Fgf10) over the control littermates as is represented
graphically (B).

21
Tbx18-cre transgenic line was assayed for specificity of cells with recombination using a
ubiquitously expressed knockin beta-galactosidase reporter line with the floxed neomycin
resistance gene (followed by a polyadenylation sequence), Rosa 26:neo
fl/fl
_beta-
galactosidase (lacZ) (Soriano, 1999), would express the enzyme to convert the substrate
X-gal ® into a visible blue chromagen in mice containing both knockin genes. When
assayed on embryonic day 11.5 mouse embryos containing both transgenes, the pattern of
blue staining in the epicardium resembled the published results of Cai et al., 2008. Thus
the Tbx18-cre line showed epicardial cell specificity for recombination and thus targeted
expression for our system (figure 6). When the assay was repeated on neonatal mice of
one day of age, labeled cells decreased to approximately 50% of epicardial cells showing
recombination for enzyme activity, and the beta-galactosidase expressing cells
aggregated around blood vessels. Epicardium’s expression of Tbx18 and cells derived
from Tbx18- expressing cells decrease over the course of the embryonic development.

22

Figure 6. Tbx18-cre lineage labeling of epicardium changes during development.
Bright field image of embryonic day 11.5 mouse embryo (A) and heart and lungs of
postnatal day 1 pup, derived from crossing Tbx18-cre mouse to Rosa: neo
fl/fl
_beta-
galactosidase, after beta- galactosidase activity assay. Blue- black staining (arrows) is
visible on the surface cell layer of the developing heart (A). After birth neonatal pups
have beta-galactosidase staining in cells clustered around vessels (B’) and non-
continuous expression in the epicardial cells with some epicardium without staining
(arrowheads) (B’). Scale bar represents 1 mm (A), 5 mm, and 50 microns (B’).

23
Tbx18-derived cells are evident with Cre- mediated recombination in the adult
epicardium after injury. I intended to use this transgenic mouse model for obtaining
epicardial cells’ expression after coronary artery ligation surgery. Literature on the
Tbx18-cre mouse indicates a loss of cells derived from Tbx18- expressing epicardium in
uninjured adults (Cai et al., 2008). Thus to clarify the potential for re- expression of
Tbx18 in the epicardium of adult mice and Cre- mediated recombination in epicardial
cells with Tbx18-cre, adult 10 week-old Tbx18-cre; Rosa26: neo
fl/fl
_ beta-galactosidase
mice underwent coronary artery ligation surgery and hearts were harvested one week
after the surgery for beta-galactosidase chromagenic staining with X-gal ® substrate.
Despite Tbx18-cre having a mainly epicardial cell- specific recombination activity in the
heart when assayed in embryos, in adult mice of 10 weeks of age, the epicardium no
longer contains any detectable Tbx18- derived cells, as demonstrated by the lack of
epicardial blue staining with chromagenic beta-galactosidase assay in transgenic mice,
Tbx18-cre; Rosa26: neo
fl/fl
_ beta-galactosidase. Light microscope images of hearts from
ten week old Tbx18-cre; Rosa26: neo
fl/fl
_ beta-galactosidase mice that underwent sham
surgery (n=3) or coronary artery ligation to create ischemic injury (n=3) and collected
seven days later were assayed to evaluate Cre recombinase activity in the heart of Tbx18-
cre mice. Sham operated mice showed patchy myocardial staining with a concentration of
staining in the septum and no evident epicardial cell staining. This confirmed the results
(at 6 weeks of age) seen in the paper reporting the transgenic line’s development (Cai et
al., 2008). The mice with ischemic injury have increased blue staining in the left ventricle
indicating greater number of cells with Cre- mediated recombination after the injury.

24
Seven days after the injury, Cre- mediated recombination was detected in epicardial cells
(figure 7). The assay revealed that when compared to the uninjured control littermates,
the mice with injured hearts had demonstrated staining in epicardial cells. Approximately
40% of the epicardial layer of injured hearts had blue staining whereas all uninjured
hearts still had no blue staining in the epicardium. Thus injury of the heart activates
expression of Tbx18 in the adult, since novel Tbx18- Cre- mediated recombination is
present in the adult epicardium after coronary artery ligation and not in the epicardium of
uninjured adult heart.

25

Multi-transgenic mice for analyzing Fgf10 during repair after cardiac ischemic injury in
adult mice: To overcome the insufficient Fgf10 transgene expression of the Tbx18-cre
model, another Cre recombinase was used to increase the target cells to achieve
ubiquitous expression. Transgenic mice with chicken beta actin promoter-driven cre
(CAG-cre, Sakai et al. 1997) was crossed with the Rosa26: neo
fl/fl
_ rtTA; tet(o)Fgf10
mice, referred to as Tg(Fgf10). Mice with Cre were used for subsequent breeding, and
due to germline recombination of the Rosa26: neo
fl/fl
_rtTA with this Cre transgenic
mouse, offspring didn’t require transmission of the Cre as the recombined Rosa26: neo
fl/fl
_ rtTA was transmitted for rtTA expression and could be used for Fgf10 induction
Figure 7. Adult Tbx18-cre mice have no epicardial Cre recombinase activity
without injury. Light microscope images show hearts from ten week old Tbx18-cre;
Rosa: neo
fl/fl
_ beta-galactosidase mice that underwent sham surgery (n=3) or
coronary artery ligation to create ischemic injury (n=3) and collected seven days later.
Ten micron sections were stained with X-gal ® for beta- galactosidase activity (blue)
and counterstained with nuclear fast red (pink) to determine Cre- mediated
recombination. Sham operated mice (A and A’) showed patchy myocardial staining
with a concentration of Cre- mediated recombination in the septum (A) and no evident
epicardial cell expression (A’). The mice with ischemic injury (B and B’) have
increased blue staining in left ventricle (B) indicating greater number of cells with
Cre- mediated recombination after injury. Seven days after injury, Cre- mediated
recombination was detected in epicardial cells (B’). Rectangles indicate magnified
area. Scale bar represents 2mm (A and B) or 100 microns (A’ and B’).

26
similar to littermates with all three transgenes (Sakai et al. 1997). The germline
recombination of this Cre transgenic mouse was confirmed by crossing with the Rosa26:
neo
fl/fl
_ lacZ reporter mice assay for ubiquitous beta-galactosidase expression even in the
absence of Cre recombinase gene transmission (not shown) as reported in Sakai et al.,
1997. The new mouse model for Fgf10 overexpression throughout all tissues, Tg(Fgf10),
was analyzed for Fgf10 induction by qPCR of cDNA from heart ventricle RNA after one
week of induction. Tg(Fgf10) mice have an average of 167 -fold expression of Fgf10 in
the heart (p<0.01) compared to control littermates (without tet(o)Fgf10)). This mouse
was subsequently used to study the effect of Fgf10 overexpression in the heart after
injury for post-inflammation induction, days seven to seventeen after injury (figure 8).
Using a combination of Cre recombinase technology and the tet-on system, rtTA with
tet(o)Fgf10, gene overexpression and temporal control of Fgf10 gene expression in the
heart is attainable.

27

Figure 8. Mouse model with Chicken beta actin promoter Cre recombinase for
Fgf10 overexpression has significant, ubiquitous transgene overexpression. A
schematic representation of the DNA constructs and recombination in the genetic
model with the gene for Cre recombinase under the chicken beta actin promoter. Cre –
mediated (red arch) recombination removes the neomycin resistance (Neo) gene
cassette in order for rtTA (orange half donut) gene to express. In the presence of
doxycycline (purple donut) rtTA protein binds the tet promoter of the transgene,
tet(o)Fgf10, for expression of exogenous Fgf10 (A). Quantification by qPCR analysis
of cDNA for total Fgf10 in the heart showed a significant 167- fold expression in the
induced transgenic mice Tg(Fgf10) over control littermates (B). Experimental
timeline of gene induction and BrdU labeling after injury for analysis of cardiac repair
in adult transgenic mouse (C).

28
Infarct size is not reduced by Fgf10 overexpression: Exogenous Fgf10 was
overexpressed, from days seven to seventeen post cardiac ischemia injury. AFOG
trichrome staining was done on several tissue section slides to delineate the scar/ injury
area from the uninjured myocardium on multiple planes of the transverse sections.
Microscope images of consecutive 10 micron sections of tissue stained for collagen with
the AFOG trichrome stain, which stains collagen blue and myocardium brown, of adult
mice that underwent coronary artery ligation and were collected 21 days after the injury
are arranged consecutively from base to apex (left to right) demonstrating the relative
ratio of scar area changes according to location of the section in both the control and
Tg(Fgf10). Analysis of the seven transgenic mice (Tg(Fgf10)) and eight control mice for
the tissue thickness of the left ventricular wall at the injury and the length of the infarcted
area of the left ventricle were done. In order to quantify scar length, three sections were
evaluated for the length of the blue scar area and the circumference of the left ventricle.
The ratio, scar length: left ventricle circumference, of Tg(Fgf10) specimens (n=7) and the
control specimens (n=8), were averaged The averages for each group are represented on
the graph, they showed no significant difference (figure 9). Three sections were evaluated
for scar thickness and septum thickness, expressed as a ratio, scar thickness: septum
thickness, of which Fgf10 and the control group showed no significant difference
(p=0.17). After measuring the scar thickness (normalized to total left ventricle
circumference), Tg(fgf10) =29.30%, control= 27.00%, and scar length, Tg(Fgf10)= 0.23
and control = 0.26, the normalized ratios were compared for both groups with statistical
analysis to reveal no significant difference in either parameter (p=0.17 ,0.19,

29
respectively). Fgf10 overexpression did not alter the scar size, length or thickness, in
mice after ischemic injury.

30

Figure 9. Fgf10 overexpression does not change scar size after ischemic injury in
adult mouse. Microscope images of consecutive 10 micron heart tissue sections
stained for collagen with AFOG trichrome stain, which stains collagen blue and
myocardium brown, of adult mice that underwent coronary artery ligation and were
collected 21 days after injury. They are arranged consecutively from base to apex (left
to right) demonstrating the relative ratio of scar area changes according to location of
the section in both the control and Tg(Fgf10) (A). Quantification of the average ratio,
scar length: left ventricle circumference, of Tg(Fgf10) (n=7) and the control (n=8),
29.3% and 27.0% respectively, showed no significant difference (p=0.19) (B).
Quantification of the average ratio, scar thickness: septum thickness, quantified and
expressed as a ratio, scar thickness: septum thickness, of which Tg(Fgf10) was 25.0%
and the control group was 23.0%, thus showed no significant difference (p=0.17) (C).

31

Fgf10 does not increase blood vessel density in the infarct border: In order to assess the
effect of Fgf10 on the injured myocardium’s vasculature, I quantified the amount of
blood vessels (PECAM-positive lumens) in the border region of the infarct adjacent to
the unscarred myocardium. Immunofluorescent staining for blood vessels using PECAM
antibody on 10- micron tissue sections from the control and Tg(Fgf10) adult mice 21
days after the ischemic injury, the number of lumens in the end scar regions adjacent to
the histologically normal myocardium were counted and summed (figure 10). Three
sections per sample were averaged for statistical analysis; the Tg(Fgf10), samples (n=7)
had no significant difference in the number of lumens in the border zone compared to
control group (n=8). Tg(Fgf10) had an average of 110.30 PECAM-positive lumens in the
border regions and control had an average of 94.25 PECAM-positive lumens in the
border regions, but were not statistically (p=0.38) more prevalent with expression of
exogenous Fgf10.

32

33

Fgf10 does not increase survival after ischemic injury: Mortality occurred throughout the
experiment as a consequence of the ischemic injury. The experimental groups are
Tg(Fgf10), which had Fgf10 overexpression and the control group, which had no Fgf10
overexpression, with doxycycline administration to both groups (those with tet(o)Fgf10
transgene and those without). The control group also contained three uninduced mice, or
no doxycycline exposure, with tet(o)Fgf10 transgene. Mortality from cardiac ischemia
was not significantly (p=0.15) decreased by the overexpression of Fgf10 (46.7 % survival
at day 21 post surgery) compared to the control (68.8% survival at day 21 post surgery)
(figure 11). The overexpression of Fgf10 showed no histological benefits hence the lack
of significant improvement in survival.

Figure 10. Fgf10 overexpression does not alter vascularization of border zone of
scar in the left ventricle after injury. Immunofluorescent staining for blood vessels
using anti- PECAM antibody (red) and DAPI (blue) on 10 micron tissue sections from
the control and Tg(Fgf10) adult mice 21 days after the ischemic injury (B), the
number of lumens (asterisks) in the end scar regions adjacent to the histologically
normal myocardium (green autofluorescence) were counted and summed. Three
sections per sample were averaged for statistical analysis (p= 0.38); the Tg(Fgf10),
samples (n=7) had no significant difference in the number of lumens in the border
zone compared to control group (n=8) (A).

34

Fgf10 does not increase the expansion of Wt1-positive epicardial cells due to ischemic
injury in adult mouse: Cardiac ischemic injury in adult mice leads to a marked thickness
in the epicardium, which is usually a single layer thick, to multiple layers of Wt1-positive
epicardial cells (Zhou et al., 2011). This study revealed a marked expansion of these cells
in the injury site/ scar area as well as adjacent myocardium so that the epicardium no
longer had a single layer of squamous epithelium of Wt1-positive cells, but the Fgf10
Figure 11. Overexpression of Fgf10 did not improve survival after cardiac
injury. Kaplan-Meier survival graph shows that the Fgf10 overexpression mice (red
line) did not have a significant improvement in survival (46.7%) over the pooled
control littermates (black line) (68.8%).

35
overexpression did not further increase Wt1-positive cell expansion. After the injury, the
epicardium had a consistent multiple-layered epicardium of at least two Wt1-positive cell
layers of thickness up to five layers. Fluorescent microscopy images of injured hearts
stained for Wt1 with immunohistochemistry demonstrate the epicardial cells are
expanded to multicellular thickness after injury. Quantification of the ratio of Wt1-
positive cells to total cells in the expanded epicardium of the injured hearts averaged 0.45
in Tg(Fgf10) (n=7) compared to 0.33 in control hearts (n=8) but shows no significant (p=
0.42) difference (figure 12). Fgf10 does not enhance the epicardium’s response to injury
when expressed in the post-inflammatory phase (days 10 to 17 post surgery).

36

Figure 12. Fgf10 does not increase the expansion of Wt1-positive epicardial cells
due to ischemic injury in adult mouse. Fluorescent microscopy images of injured
hearts, of Fgf10 overexpressing mice (B) and control littermates (A), stained for Wt1
(magenta) with immunohistochemistry (A and B) and nuclei were stained with DAPI
(blue). The epicardial cell layers (myocardium has red auto-fluorescence) are
expanded to multicellular thickness (white arrows) after injury. Quantification of the
ratio of Wt1-positive cells in the hearts after injury (C) averaged 0.45 in Tg(Fgf10)
(n=7) compared to 0.33 in control hearts (n=8) but shows no significant (p= 0.42)
difference. Scale bar represents 20 microns.

37
DISCUSSION
In this study we created an in vivo mammalian model to study the function of a gene,
Fgf10, and the effect of its overexpression. Combining the genetic technology of Cre
recombinase cell/ tissue specificity with the inducibility of tet genetic models, temporal
control and tissue targeting of our candidate gene was achieved. The experimental
window of expression was possible by providing doxycycline to the experimental mice in
the desired time frame. The inducible tet/ rtTA system allowed us to bypass the
deleterious effects of excess growth factor (in this case Fgf10 has many potent effects on
multiple tissues after long-term exposure) prior to the injury and after the repair/
regeneration. This means it can be used as a tool to evaluate administration windows that
would be beneficial for human patient therapies. Additionally since studies have
delineated phases of the injury/ repair process, these can be addressed so gene or growth
factor function can be clearly defined. In this study, the expression of the transgene was
targeted to the period after inflammation in order to avoid contributing to the
inflammatory response. The Tbx18-cre mouse chosen for this study did not provide
sufficient gene expression and complete epicardial cell expression, although partially re-
initiated by injury, was not expected based on the Cre- mediated recombination assay;
however, switching to another Cre recombinase transgenic mouse, in this case a Cre
recombinase gene expressed under the chicken beta actin promoter, overcame this lack of
gene induction and provided ubiquitous expression. Therefore, this mouse model can
easily be manipulated with choice of Cre recombinase transgenic mouse for different cell
specificity or amount of gene expression. This also highlights the importance of
validating the cell/ tissue target of the mouse model. Additionally, the choice of gene for

38
overexpression can be switched to any of the available tet- responsive genes. Together
the combination allows for a great amount of variability to achieve the desired system for
studying a specific gene in a specific tissue type for a specified amount of time. The tet
mouse model has potential to be a valuable tool for evaluating various candidate genes in
their role for cardiac injury and the repair process and the potential for regeneration.
Evaluation of Fgf10 overexpression in the injured heart in the post-inflammatory
phase was done using the inducible multi-transgenic model. Standard analysis of tissue
and cellular changes revealed insignificant changes with Fgf10 overexpression. The
model provided significant Fgf10 induction, yet Fgf10 overexpression affected none of
the parameters assayed in this study, including scar size or neovascularization of the
injury. An interesting epicardial phenotype in response to injury was noted and was
confirmed in a later publication (Zhou et al., 2011). The epicardial cell response to the
injury was a dramatic histological change that thickened the epicardium through
hyperplasia. Our candidate growth factor has been identified as activating Wt1-positive
epicardial cells to migrate into the subepicardium and express vimentin in the embryo
(Vega-Hernandez et al., 2011), indicating the receptor is expressed in epicardial cells.
Potentially, the adult mouse epicardial cells may have turned off expression of the
primary receptor for Fgf10, Fgfr2b, by adulthood and did not regain expression with the
injury. However, the lack of significant effects on any of the parameters assayed despite
dramatic gene induction, suggests that the induction period in this study was not ideal for
affecting them. Unfortunately, this study only included the single induction period, but
the flexibility of this inducible mouse model makes it relatively simple to modify the

39
experiment for another gene expression period to re-evaluate the effects of Fgf10
overexpression.

40
MATERIALS AND METHODS

Generation of multi-transgenic mice: In order to create a tissue-specific and temporally
controlled gene expression model, we crossed a tissue-specific Cre recombinase to the
Rosa26: neo
fl/fl
_rtTA knockin mouse. The tet-on (doxycycline inducible) transgene
expression is limited to the cells derived from Cre- mediated recombination for rtTA
expression.
Mice were genotyped at fourteen days of age using a tail tissue which was lysed in
proteinase K (100ug/ml) and used for PCR amplification. PCR primers for transgenes,
alpha-MHC: rtTA and tet(o)Fgf10 are listed in the table below. Thirty-five cycles of
94°C for one minute, 58°C for thirty seconds to anneal, 72°C for one minute were
performed in a PCR machine to generate the amplicons. PCR products were separated on
a 1.5% agarose / TAE gel.

Flowchart for experimental analysis of Fgf10 in adult mouse heart injury.

41
Induction of gene expression by doxycycline: Mouse had a diet of normal feed and water
as indicated by CHLA animal facilities, until adulthood, about 8 weeks of age.
Approximately 2 weeks prior to ischemic-injury surgery, the normal feed was replaced
with a doxycycline- supplemented rodent diet (Harlan Teklad). They remained on the
doxycycline-supplemented diet until tissue was harvested. For embryonic induction of
growth factors by transgenes, pregnant females will be supplied the doxycycline-
supplemented feed at day 11 of gestation, and the embryos will be harvested at E14 for
expression analysis.

Ischemic injury/ sham surgery: Adult mice over 8 weeks of age underwent surgery, under
the guidance of the CHLA facility veterinarian, Dr. Baer, after being anaesthetized with a
isofluorane 4% in O
2
by intubation. Throughout the surgery the mice were maintained on
a respirator: the mice were intubated with a polyethylene catheter (22 gauge) inserted
through the mouth into the trachea in order to provide continuous anesthesia and
ventilation at 120 cycles per minute of 200-300ul/ cycle utilizing the Harvard Apparatus
® small animal ventilator. Fur on the chest and abdomen was shaved and the surgical
area was disinfected with ethanol and then chlorhexadine prior to the first incision. An
incision to the chest exposed the heart at which point a 6-0 silk suture will be tied around
the LAD, and was determined as successful when a white pallor on the left ventricle
down to the apex was seen. Control/sham operated mice were sutured on the left
ventricle. Air in the mouse’s chest was removed by syringe as the chest wall was sutured
closed, first the muscle layer, using 6-0 prolene sutures, and secondly the skin, using

42
surgical grade tissue glue. They recovered from anesthesia on a heating pad and were
extubated from the respirator once they were breathing independently. They were
monitored for morbidity throughout the first 24 hours and ketoprofen (2mg/kg) will be
administered to reduce pain, both prior to the end of the surgical procedure and 24 hours
later. Ibuprofen was diluted in drinking water and consumed ad libitum for the first three
days.

Mouse care post surgery/ pain management Mice were kept according to NIH regulations
and monitored throughout the surgery and up until tissue harvest. Mice were observed at
least every 24 hours if they appeared to show signs of distress or discomfort administered
ketoprofen and provided ibuprofen diluted in drinking water until they appeared normal,
ambulatory and groomed. In the case that mice were highly moribund such as excessive
water retention, festering wounds or greater than 20% weight loss, they were euthanized
and tissue harvested for examination.

Tissue Procurement: Mice will undergo CO2 euthanasia and hearts will be surgically
removed in PBS and then 2M KCl and processed for histology or RNA, DNA or protein
recovery. Gravity-assisted pressure fixation with 4% paraformaldehyde standardizes the
tissue morphology for histological processes.

Whole mount beta- galactosidase enzyme assay (lacZ stainining): Embryo tissue was
procured from pregnant dams eleven days after a seminal plug was detected. Adult mice
were euthanized by CO2 asphyxiation and neonatal mice were euthanized by

43
supercooling on ice followed by decapitation. Heart tissue (or embryos) was removed and
briefly washed in PBS (0.1 M phosphate buffered solution). The tissue was then briefly
fixed in 4% paraformaldehyde/ 2% glutaraldehyde for 15 minutes. The tissue was
bleached in 3%H2O2 in methanol for 10 minutes. A detergent wash of (0.02% Igepal,
0.01% Sodium Deoxycholate, and 2mM MgCl2in 0.1M phosphate buffer [pH 7.3] ) was
used to permeabilize the tissue for three times 10 minutes. The detergent was replaced
with X gal ® (Promega) staining solution (0.02% Igepal, 0.01% Sodium Deoxycholate,
5mM Potassium Ferricyanide, 5mM Potassium Ferrocyanide, and 2mM MgCl2 diluted in
0.1M phosphate buffer [pH 7.3]) and the tissue was submerged in the solution and
allowed to develop at 37 C, while protected from light until desired color was seen.
Whole mount images of the stained tissue or embryos were taken with dissecting
microscope. If tissue was to be sectioned: the tissue was then embedded in cryo-
embedding media, and cryosectioned. Sections were collected on glass slides and rinsed
of cryo- embedding media and briefly stained with nuclear fast red. The sections were
imaged with bright field microscopy.

In vivo cellular proliferation: In order to assess proliferation of the Wt1-positive cells,
nucleotide labeling with bromo-deoxyuridine (BrdU) for the final ten days prior to
collection. Surgically injured mice were administered an intra-peritoneal shot of BrdU
diluted in sterile saline for a final concentration of 100 mg/kg (BrdU weight: body
weight) starting on day ten after surgery and were left in the cage to recover as normal.
They received another injection every second day until the end of the experiment. Tissue

44
was recovered as described and processed for histology as described. Tissues sections
were used for immunohistochemistry against BrdU with rat anti-BrdU (Abcam) as
described for PECAM or Wt1 or in conjuction with Wt1. Tissue sections were stained
with Wilm’s Tumor 1 antibody (Santa Cruz) followed with secondary antibody goat anti-
rabbit conjugated to Alexa 594 (Molecular Probes) and co-stained with Vectashield©
mounting media with Propridium iodide. Confocal microscope images were taken on
Zeiss LSM 710® single photon confocal microscope. The average ratio of the number of
BrdU positive nuclei (double stained with antibody and DAPI) to WT1-positive nuclei
are quantified by at least three sections of each sample for Wt1-positive cells for injury
area (scar region determined by consecutive tissue slides stained by AFOG and lack of
green autofluorescence from myocardium for reference) for each heart. The average of
seven hearts was taken for the experimental group and eight hearts was taken for the
control group. The measurements are analyzed for statistical significance with Wilcoxon
rank sum test.

RNA isolation and Quantitative/ RT PCR: Ventricle tissue were bead-disruption
homogenized in Trizol ® and RNA isolated as indicated with Trizol ® extraction. RNA
was quantified using Nanodrop® spectrophotometer. Generation of cDNA was done in
vitro using First Strand cDNA synthesis kit ® (Invitrogen™). The cDNA was measured
using Nanodrop® spectrophotometer. Total RNA total quantity was assessed by qPCR
amplification of the cDNA using primers and probe combinations designed by Roche
Assay Design Center Forty cycles at 94°C for ten seconds, 60°C for thirty seconds to

45
anneal. The software calculated the C.P. and the target gene was compared to the
housekeeping genes to determine the relative fold change in expression.

Histological analysis: Tissue harvested is fixed in 4% paraformaldehyde overnight at
4°C. Fixed tissue was than dehydrated, cleared in toluene and then paraffinized. Paraffin-
embedded tissue was sectioned at 10µm. Sections were stained with hematoxylin and
eosin and examined by light microscopy for histological changes.

Blood vessel density analysis: Tissue sections of 10 microns on glass slides are processed
by immunohistochemistry: paraffin is removed with toluene washes, then rehydrated with
graded ethanol until phosphate buffered solution. Heat and acid antigen retrieval was
done with Vector ©Antigen Retrieval solution and a microwave. Tissue sections were
stained with PECAM antibody (Thermo Fisher Scientific) by immunohistochemistry
followed by secondary antibody goat anti-rabbit conjugated to Alexa 594 and co-stained
with Vectashield© mounting media with Propridium iodide. Confocal images were taken
on Zeiss LSM 710® single photon confocal microscope. The average number of vessels
is quantified by at least three sections of each sample for PECAM-positive lumens for
both border areas (scar region adjacent to non-scarred myocardium) for each heart. The
average of seven hearts was taken for the experimental group and eight hearts was taken
for the control group. The measurements are analyzed for statistical significance with
Wilcoxon rank sum test.

46
Wilm’s tumor 1 protein immunostaining and quantification: Tissue sections of 10
microns on glass slides are processed by immunohistochemistry: paraffin is removed
with toluene washes, then rehydrated with graded ethanol until phosphate buffered
solution. Heat and acid antigen retrieval was done with Vector ©Antigen Retrieval
solution and a microwave. Tissue sections were stained with Wilm’s Tumor 1 antibody
(Santa Cruz) followed with secondary antibody goat anti-rabbit conjugated to Alexa 594
(Molecular Probes) and co-stained with Vectashield© mounting media with Propridium
iodide. Confocal images were taken on Zeiss LSM 710® single photon confocal
microscope. The average ratio of the number of Wt-1 positive nuclei (double stained with
antibody and DAPI) to total nuclei (DAPI- stained) was quantified by at least three
sections of each sample for Wt1-positive cells for injury area (scar region determined by
consecutive tissue slides stained by AFOG and lack of green autofluorescence from
myocardium for reference) for each heart. The average of seven hearts was taken for the
experimental group and eight hearts was taken for the control group. The measurements
were analyzed for statistical significance with Wilcoxon rank sum test.

Scar measurement analysis: Ten-micron tissue sections on slides were deparaffinized and
rehydrated to phosphate buffered solution. The tissue slides were placed in Bouin’s
fixative for 2.5 hours at 56 degrees Celsius then one hour at room temperature. The slides
were washed of Bouin’s fixative for 20 minutes in running water. The slides were treated
with 1% phosphomolybdic acid for five minutes then washed briefly with deionized
water. Slides were immersed in and stained with Acid fuschin, Orange G, Aniline blue

47
(AFOG) trichrome staining solution for 5 minutes and then briefly rinsed with deionized
water. Slides were then quickly processed with alcohol to dehydrate and then cleared in
toluene and mounted with Cytoseal ®. Tissue sections were imaged with bright-field
microscopy. Images of sections were calibrated for measurement on Image J software.
Three to five measurements spanning the width of the tissue were taken and averaged for
scar length and left ventricle circumference and the ratio of the average scar length to
average left ventricle circumference was calculated. Three to five measurements
spanning the length of the tissue were taken and averaged for scar thickness and septum
thickness and the ratio of the average scar thickness to average septum thickness was
calculated. The measurements were analyzed for statistical significance with Wilcoxon
rank sum test.

Imaging/ Microscopy Whole mount imaging was performed with Leica dissecting scope
and using Spot Advanced ® imaging program. Fluorescent imaging of sections was
performed using the Zeiss LSM 710 © single photon confocal microscope with Zen ®
imaging software.

48
Chapter II: Cryoinjury in neonatal mouse heart to study mammalian
cardiac regeneration

INTRODUCTION
Although the adult mouse model has been a staple for studying mammalian
response to injury, the neonatal mouse model has recently been introduced as an
alternative. The neonatal mouse hearts were amputated on the first day after birth at the
apex in order to study regeneration mechanisms (Porrello et al., 2011). The regenerative
capacity rapidly decreases by postnatal day seven. The surgical process is substantially
simpler more analogous to the zebrafish surgical process and can accomplish many
studies that are only possible with the extensive mouse transgenic and mutant lines
available, not yet available to zebrafish. However, ventricular resection removes healthy
heart tissues and is very different from the pathogenesis of myocardial infarction.
Recently, it has been shown that adult zebrafish hearts can also fully regenerate after
cryoinjury (Chablais et al., 2011; Gonzalez-Rosa et al., 2011; Schnabel et al., 2011). In
order to determine if neonatal mouse hearts can regenerate after cryoinjury, we have
established a cryoinjury model for neonatal mouse hearts to replicate the adult model of
pathogenesis.
The novel neonatal mouse model for cardiac regeneration studies has provided a
unique in vivo mammalian model of injury and regeneration, but similar to the zebrafish
it utilizes ventricular resection as a mode of injury. We enhanced this model to better test
hypotheses regarding the processes of regeneration especially, the mechanisms associated

49
with necrosis, tissue resorption and inflammation. In order to do this we created an injury
to the neonatal mouse heart using extreme cold, cryoinjury. This was a logical choice
since it has been previously used successfully to achieve tissue damage to adult mouse
hearts (Van Amerongen et al., 2008) and zebrafish heart regeneration model has also
progressed to the cryoinjury (Chablais et al., 2011; Gonzalez-Rosa et al., 2011; Schnabel
et al., 2011). The cryoinjury is capable of damaging the myocardium of the ventricle
while leaving the apoptotic and necrotic tissue to be resorbed and remodeled prior to
regeneration of the lost cardiac tissue. We established this as a novel and useful model of
injury for neonatal mice to be used for our studies on mammalian cardiac regeneration.

50
RESULTS
Cryoinjury on the neonatal heart is a model to study cardiac injury response and repair: In
order to accelerate the study of cardiac regeneration in the mammalian heart, a novel
cryoinjury model in neonatal mice of 1 day of age was established. The model combines
the injury, occasionally used in adult mice, which utilizes extremely cold temperature to
damage tissue with the recent mouse model of surgical manipulation of neonatal mouse
hearts. The use of a metal probe of 1mm super-cooled in liquid nitrogen creates a
reproducible damage to the heart when applied to the surface of the left ventricle, which
is modifiable to be either mild or severe. Cardiac tissue becomes necrotic and eventually
scarred to the full thickness of the ventricular wall after severe injury (figure 13). This
model sufficiently mimics the adult injury while using mice at a young age for quick
experimental study and utilizing their increased capacity for regeneration.

51

Phospho-Erk expression is present at injury and epicardium after cryoinjury: Protein
homogenate from mouse ventricles after cryoinjury or sham operation at multiple time
points to evaluated by Western blot for phosphorylation of the p42 and p44 proteins (Erk)
by probing for phospho- Erk and total Erk. The representative samples are shown for
example (figure 14). Quantification of the average of at least 3 samples for each
Figure 13. Cryoinjury is a modifiable model of cardiac injury. Light microscopy
images of the neonatal heart after mild (A, C, E) or severe injury (B, D, F) show
damage to the myocardium (arrowheads) which can be up to full thickness (D) that
will result in fibrosis of the tissue, as seen by AFOG staining (F).

52
condition at each time point was made of the ratio for phospho-Erk: total Erk and
analyzed for statistical difference to demonstrate changes in Erk phosphorylation after
injury at various time points. Three phases of Erk activity changes were detected. ERK
activity increased immediately after cryoinjury (5 hr) and in the middle of regenerative
process (10 dpc). However, Erk activity decreased at the completion of the experiment
(21 dpc).
Since I cannot determine the tissue types that have Erk activity using Western
blotting of whole ventricle tissue, I performed immunohistochemistry for phosphorylated
Erk (p42/ p44) of heart tissue at the various time points after injury. The staining as seen
with immunohistochemistry indicates a common pattern prominent in the heart after
injury, not seen in sham operated age matched controls (figure 15). Specifically the
appearance of phospho-Erk is seen concentrated to the injury site and epicardium with
the cryoinjury compared to the sporadic staining throughout the heart in the sham
samples. Immunohistochemistry for phosphorylated Erk (p42/ p44) of heart tissue at the
various time points after injury indicates the presence of phospho-Erk is more prominent
in the heart after injury, in comparison to sham operated age matched controls at multiple
time points. Injury activates sustained Erk phosphorylation specifically in injured cells
and epicardium which corroborates the Western blot data indicating differential
phosphorylation overall in heart tissue.

53

Figure 14. Differential phosphorylation of p42/ p44 (Erk) after cardiac injury in
the neonatal mouse. Representative Western blot of protein homogenate from mouse
ventricles after cryoinjury “C” or sham “S” operation at various time points were
evaluated for phosphorylation of the p42 and p44 proteins (Erk) by immunoblotting
for phospho- Erk and total Erk and detected b fluorescence(A). The average of at
least 3 samples for each condition at each time point was made of the ratio for
phosphor-Erk: total Erk and analysed for statistical difference and represented
graphically (B) to demonstrate differential phosphorylation of Erk after injury at 5 hp,
10 dp and 21 dp.

54

55

Fgf10 and downstream genes increase in transgenic neonatal mice after induction without
persistent increased Erk phosphorylation: A schematic of the DNA construct
demonstrates the alpha-Myosin Heavy Chain (MHC) promoter for rtTA is used to obtain
cardiomyocyte expression which is combined with the tet(o)Fgf10 transgene for
inducibility in the presence of doxycycline. Additionally an experimental timeline
demonstrates the induction periods used in this study, the inducibility allows for temporal
control which was used to test either a short -term induction (3 days post surgery to 10
days post surgery) or continuous induction (the day of the surgery until tissue collection
21 days post surgery) throughout the experiment. The transgenic mouse model was
determined to be sufficient to achieve significant Fgf10 overexpression in neonatal
stages.
Figure 15. Erk phosphorylation is found in the epicardium and at the injury site
of cryoinjured mice. Light microscopy of heart tissue sections stained by
immunohistochemistry for phospho-Erk at various time points after P1 cryoinjury to
the heart. Positive staining for phospho-Erk protein (bluish black color) can be seen
localized in the myocardium of the injury (C, F, I, L, O, R) and epicardium (B, E, H,
K, N, Q) (arrowheads) in injured hearts. Similar time points in sham hearts (A, D, G,
J, M, P) show no staining in the epicardium and no increased localized staining in the
myocardium. Scale bar represents 25 microns except in A and C it represents 100
microns.

56

Figure 16. Induction of Fgf10 can be controlled with a double transgenic mouse
model to study the effect of Fgf10 overexpression in the neonatal mice after
injury. DNA construct of the alpha-Myosin Heavy Chain (aMHC) promoter for rtTA
(orange half donut) is used to obtain cardiomyocyte expression which is combined
with the tet(o)Fgf10 transgene for inducibility in the presence of doxycycline (purple
donut) (A). Experimental timeline used in this study, the inducibility allows for
temporal control which was used to test either a short -term induction (3 days post
injury to 10 days post injury) or continuous induction (the day of the surgery until
tissue collection 21 days post surgery) throughout the experiment (B). Arrows indicate
collection timepoints: 10 dp, 14 dp, 21 dp (after cryoinjury or sham surgery).

57
The double transgenic mouse utilizing alpha MHC-rtTA and tet(o)Fgf10 can be induced
to express exogenous Fgf10 which can be detected by RT PCR with primers that detect
the transgenic Fgf10 mRNA in the heart only in double transgenic mice, Tg(Fgf10), not
control littermates after doxycycline-induction which contributed to a quantified 98.99
fold increase of total Fgf10 over control littermates (p<0.01) with qPCR analysis .
However, significant increased downstream signaling, Erk phosphorylation, was not
detected. Western blot analysis of heart ventricle protein from induced neonatal mice
shows that Tg(Fgf10) mice and control littermates have a similar amount of Erk
phosphorylation, 0.44 to 0.43 phospho-Erk: Erk ratio, respectively, at the time of
collection, Real time PCR assay showed that Erm and Pea3, genes found downstream of
Fgf signaling, were significantly (p<0.05) increased to 2.53- fold and 2.01-fold,
respectively, of control littermates. Additionally, with qPCR detected Fgfr2 in the
transgenic mice after induction but was not detectable in control mice.

58

Figure 17. Induction of Fgf10 transgene in double transgenic mice increases
Fgf10 and downstream genes in neonatal mice. Image of UV detected amplicon
from RT PCR with primers that detect the transgenic Fgf10 mRNA in the heart only
in double transgenic mice, Tg(Fgf10), not control littermates after doxycycline-
induction (A) which contributed to a quantified 98.99 fold increase of total Fgf10 with
qPCR analysis (D). Western blot analysis (B) of heart ventricle protein from induced
neonatal mice shows that Tg(Fgf10) mice and control littermates have a similar
amount of Erk phosphorylation (0.44 to 0.43, respectively) at the time of collection
(C). However, Fgf signaling downstream genes, Erm and Pea3, still have a 2.53- and
2.01- fold expression, respectively, of control littermates as seen in this graph of
quantification of relative gene expression (E).

59
Double-transgenic mouse with GFP-tagged histones (H2b) validates in vivo models for
temporal control and heart specificity: The alpha- MHC: rtTA transgenic mouse was
crossed with another tet responsive transgenic mouse, tet(o)H2b-GFP in order to
visualize inducibility and specificity of the tissues of expression. Pups were induced from
birth until neonatal day ten, at which point they were collected and sectioned for imaging
and co-stained with DAPI (4',6-diamidino-2-phenylindole). Fluorescent microscopy
images of heart lung, and liver tissue of the alpha- MHC: rtTA crossed with the
tet(o)H2b-GFP line illustrate green fluorescent nuclei were visible throughout the
myocardium of the heart with no expression in the epicardium (figure 18). Other tissues
were examined for the expression of H2b-GFP, of which the liver was completely
negative, while a thin layer in the lung was positive. GFP-labeled histones (H2b) were
identified in cells of the myocardium of the heart and few cells of the lung but not in the
liver. Overall, the expression was inducible and mostly exclusive to the heart in neonatal
mice.

60

Figure 18. Tet(o)H2b-GFP is a reporter for assaying inducibility and tissue
specificity in the double-transgenic mouse model. Schematic of DNA constructs of
the two transgenes that compose an inducible, heart- specific expression model of
GFP- tagged histone (H2b) for assaying inducibility and tissue specificity using α-
MHC: rtTA in neonatal mice (A). Induction in neonatal mice up to P10 was used to
label cells with both transgenes expressed. Fluorescent microscopy images of heart (B
and B’) the rectangle indicates magnified area, lung (C), and liver (D) tissue of α-
MHC: rtTA; tet(o)H2b-GFP transgenic mouse (A). GFP-labeled histones were
identified in cells of myocardium of heart (B and B’) and few cells of lung (C)
bronchi (Br) but not in liver (D).

61
Exogenous Fgf10 overexpression did not affect the epicardium in the absence of injury:
We employed this transgenic mouse model to determine the effect of Fgf10
overexpression on the Wt1-positive epicardial cells without injury. Fgf10 was
overexpressed from birth until collection at fourteen days of age. First staining for Wt1
by immunohistochemistry and imaging with fluorescent microscopy for Wt1-positive
staining and tissue morphology was performed to evaluate the hearts’ epicardial cells.
Fluorescent microscopy images of hearts from the double transgenic mice for Fgf10
overexpression (Tg(Fgf10)) and control littermate without cryoinjury were stained with
antibody to Wt1 to detect changes to the thickness of the epicardium (layers of Wt1-
positive cells). Control littermates and Tg(Fgf10) both demonstrated a single layer of
Wt1-positive cells in the epicardium. Control littermates and Tg(Fgf10) mice both
exhibited apparently normal histology of the epicardium with a single squamous
epithelial morphology and positive, nuclear staining for Wt1 (figure 20). In the absence
of injury overexpression of Fgf10 had no effect on the epicardial cells’ morphology in
neonatal hearts.

62

Figure 19. Fgf10 overexpression in the absence of injury had no effect on the
morphology of the epicardium (Wt1-positive cells). Fluorescent microscopy images
of hearts from the double transgenic mice for Fgf10 overexpression (Tg(Fgf10)) and
control littermate without cryoinjury were stained with antibody to Wt1 to identify the
epicardium (layers of Wt1-positive cells). Control littermates (A and A’) and
Tg(Fgf10) (B and B’) both demonstrated a single layer of Wt-1-positive cells in the
epicardium with no remarkable histological changes. Scale bar represents 20 microns.

63
Fgf10 did not increase vimentin expression in epicardial cells (Wt1-positive cells) in
cardiac injury: Although Fgf10 increased the final number of Wt1-positive cells in the
injury by proliferation; it did not contribute to the expression of the fibroblast marker,
vimentin. Similar to the increase of epicardial cell thickness, cardiac injury also led to
Wt1- positive cells also expressing vimentin. Fluorescent microscopy images of the
injury area of the heart at 10 dpc, 14 dpc and 21 dpc cryo-injured hearts stained with
immunohistochemistry for Wt1 and vimentin marked cells with expression of both
proteins in the injury area (figure 21). Quantification of cells with vimentin in the
cytoplasm and Wt1 in the nuclei as ratio of total cells with Wt1-positive nuclei was done
for the Fgf10 overexpression specimens (n=3 for all time points) and compared to control
littermates (n=3 for all time points except 21 dpc Tg(Fgf10) n=4). Ratio of vimentin-
positive, Wt1-positive cells were not significantly different in the Tg(Fgf10) mice than
controls at any of the time points.

64

65

Infarct size is not reduced by Fgf10 overexpression: Two induction periods of Fgf10
were tested in the cryoinjured mice. A continuous induction period beginning from the
day of surgery and lasting until tissue procurement on day 21 post surgery was evaluated
for changes to scar size: scar length or scar thickness. Artistic schematic of how the
transverse sections of the heart (at 21 days post-surgery) stained for AFOG were
analyzed: the length of the scar (depicted as blue) was measured and normalized to the
circumference of the left ventricle (LV). The thickness (yellow line) of the scar area was
also measured and normalized to the septum between the left and right ventricle.
Continuous Fgf10 overexpression resulted in the average scar thickness ratio for
Tg(Fgf10) hearts was 0.24 and not significantly different than control hearts (0.27). The
Figure 20. Fgf10 overexpression does not increase vimentin expression Wilm’s
Tumor 1-positive cells in the injury area. Fluorescent microscopy images of the
injury area of the heart at 10 dpc, 14 dpc and 21 dpc cryo-injured hearts stained with
immunohistochemistry for Wt1 (magenta) and vimentin (green) (A). Graph
representing quantification of cells with vimentin (green) in the cytoplasm and Wt1
(magenta) in the nuclei as ratio of total cells with Wt1-positive nuclei, in the injury
compares the Fgf10 overexpression (Tg(Fgf10)) (n=3 for all time points) to control
littermates (n=3 for all time points). Ratio of vimentin-positive, Wt1-positive cells
were not significantly different in the Tg(Fgf10) mice than controls at any of the time
points.

66
average scar length to left ventricle circumference ratio in Tg(Fgf10) was 0.15 and the
average for the control was 0.19, which were not significantly different (figure 22). In the
seven day overexpression experiment (overexpression of Fgf10 from 3 dpc to 10 dpc)
The average scar thickness ratio for Tg(Fgf10) hearts was 0.27 and not significantly
different than control hearts (0.26). The average scar length to left ventricle
circumference ratio in Tg(Fgf10) was 0.35 and the average for the control was 0.33,
which were not significantly different (figure 23). Overexpression of exogenous Fgf10
for seven days or continuously throughout the experiment didn’t lead to a reduction in
fibrosis.

67

Figure 21. Continuous overexpression of Fgf10 does not alter scar size after
cryoinjury. Transverse sections of the heart at 21 days post surgery were stained for
AFOG and the length of the scar (depicted as blue) was measured and normalized to
the circumference of the left ventricle (LV). The thickness (yellow line) of the scar
area was also measured and normalized to the septum between the left and right
ventricle (A). The average scar thickness ratio for Tg(Fgf10) hearts was 0.24 and not
significantly different than control hearts (0.27) (B). The average scar length to left
ventricle circumference ratio in Tg(Fgf10) was 0.15 and the average for the control
was 0.19, which were not significantly different.

68

Figure 22. Temporary overexpression of Fgf10 from 3 dpc to 10 dpc does not
alter scar size after cryoinjury. Transverse sections of the heart at 21 dpc were
stained for AFOG and length of the scar (depicted as blue) was measured and
normalized to the circumference of the left ventricle (LV). The thickness (yellow
line) of the scar area was also measured and normalized to the septum between the left
and right ventricle (A). The average scar thickness ratio for Tg(Fgf10) hearts (n=4)
was 0.27 and not significantly different than control hearts (n=4) (0.26) (B). The
average scar length to left ventricle circumference ratio in Tg(Fgf10) was 0.35 and the
average for control was 0.33, which were not significantly different.

69

70

Epicardial cells respond to injury with an expansion Wt1-positive epicardial cells that
were proliferative in response to Fgf10: Cardiac injury in neonatal mice by cryoinjury
leads to an increase of epicardial cells, which is usually a single layer thick to multiple
layers of Wt1-positive epicardial cells. Confocal microscopy images of
immunofluorescent staining for Wt1 and proliferating cell nuclear antigen (PCNA) to
analyze Wt1-positive cells in the injury area for proliferation with double staining for
PCNA and Wt1 at 10, 14 and 21 days post surgery. The ratio of PCNA-positive
epicardial cells were low at all time points for both control (0.08 at 10 dpc, 0.11 at 14
dpc, 0.20 at 21 dpc) and Tg(Fgf10) (0.16 at 10 dpc, 0.25 at 14 dpc, and 0.24 at 21 dpc),
but only at 14 dpc Tg(Fgf10) had a statistically (p<0.05) greater number of double-
stained nuclei compared to controls at the same time point.
Figure 23. Cryoinjury in neonatal mouse heart leads to increased Wt1-positive
cells in the injury, Fgf10 enhances it further by day 21 post surgery. Fluorescent
microscopy images of the neonatal heart at 10 dpc (A and B), 14 dpc (C and D) and
21 dpc (E and F) after immunohistochemistry staining for Wt1. The graph (G)
represents the average number of Wt1- positive nuclei in in the injury for each
timepoint. Tg(Fgf10) mice had no changes in the number of Wt1-positive cells in the
injury until day 21 post surgery, Fgf10 contributed to a greater number (p<0.05) of
Wt1-positive cells compared to the control littermates. Scale bar represents 20
microns.

71
Analysis of the epicardial expansion in the injury area by immunostaining for Wt1
and confocal imaging indicates Wt1- positive cells are increased to more than a single
layer. Quantification of the ratio of Wt1- positive nuclei: total nuclei in the injury area
indicates that a significant increase (p<0.05) of Wt1- positive cells with Fgf10
overexpression (Tg(Fgf10)) by 21 dpc, but not at earlier timepoints 10 pc and 14 dpc.
The increased proliferation of Wt1-positive cells detected at 14 dpc contributes to an
overall increase in this population, as seen at 21 dpc.

72

Figure 24. Fgf10 overxpression increased the proliferation of Wilm’s Tumor 1-
positive cells at 14 dpc. Confocal microscopy images of immunofluorescent staining
for Wt1 and proliferating cell nuclear antigen (PCNA) to analyze Wt1-positive cells in
the injury area for proliferation with double staining for PCNA (green nuclei) and
Wt1 (red nuclei) at 10, 14 and 21 days post surgery. PCNA-positive epicardial cells
were infrequent at all time points for both control and Tg(Fgf10), but only at 14 dpc
Tg(Fgf10) had a statistically greater number of double-stained nuclei compared to
controls at the same time point (B). Scale bar represents 20 microns.

73
DISCUSSION
The use of the neonatal mouse in a cardiac injury model to study cardiac repair and
regeneration is fairly new and was initiated by the Porrello et al. study in 2011 depicting
the enhanced ability for cardiac regeneration in neonatal mice as opposed to adult mice.
In this original study, the injury replicated the adult zebrafish amputation model by using
ventricular resection as a mode of injury. However, in order to create a model more
similar to the human myocardial infarction model, our lab modeled our cardiac injury
after the adult mouse cryoinjury model so that resolution of the necrotic tissue could also
be examined. By using the mice at the neonatal stage, the increased regenerative capacity
is utilized while adapting the cryoinjury model to improve the similarity of our injury to
the human MI. The mechanisms involved in regeneration, such as cardiomyocyte
proliferation and epicardial cell expansion, were roughly addressed with the Porello et al.
study, the change to the cryoinjury model indicated some of the mechanisms during the
repair and regeneration were confirmed. The identification of increased Erk
phosphorylation, particularly at the site of injury was expected and could be linked to the
response to the injury, such as necrosis, apoptosis, inflammation, fibrosis, cellular
proliferation, cellular migration as the MAPK pathway has been identified as upstream of
these processes in various tissues and various conditions. As this study did not conclude
which processes were affected by the MAPK signaling and it was seen at all assayed time
points, it is only in speculation that it might be stated that the role for signaling was
cellular dependent. This is further supported by the detection of Erk phosphorylation in
epicardium and endocardium after the injury, whereas none was detected in the sham

74
controls. The role of epicardium or endocardium is not clearly defined in the neonatal
mouse cryoinjury model at this point, but extrapolating from the zebrafish model, the
epicardium likely is activated in the response to injury and can recapitulate
developmental processes such as EMT as well as proliferation and paracrine signaling to
the myocardium. Endocardium may also be increasing the phoshporylation of Erk in
order to activate these processes as well. EMT and proliferation of the epicardium is of
particular interest since it was the hypothesis for the role of Fgf10 in the repair and
regeneration of the heart after injury. Detection of increased proliferation of Wt1-positive
cells at 14 dpc that leads to a total increase of Wt1-positive cells in the epicardium of the
injury in the Fgf10 overexpressing hearts compared to controls supported this hypothesis.
The mouse model for overexpression of Fgf10 is instrumental in testing the hypothesis. It
not only had dramatically increased the expression of Fgf10, it was sufficient enough to
increase genes downstream of Fgf10. However, the lack of a significant increase in Erk
phosphorylation with the overexpression of Fgf10 indicated that potentially signaling
downstream of Fgf10 was not through Erk or that it was too brief to have been detected
in the period it was assayed. Either of these conclusions suggest that Erk phosphorylation
seen naturally in the regeneration process at many time points could be due to funneling
of various upstream signals and could also diverge into various downstream
consequences than as seen with Fgf10. The hypothesis for the epicardium’s response to
Fgf10, the increase of proliferation of epicardial cells and detection of cells’ co-
expression of Wt1 and vimentin, further suggests that signaling such as MAPK leads to
the epicardial cells’ proliferation (PCNA expression) and EMT (vimentin expression), of

75
which we only saw a significant effect on proliferation. Expansion of epicardial cells that
can undergo EMT suggested that this might lead to increased fibrosis. Upon assaying for
scar formation, no difference in scar size in the presence of increased Fgf10 compared to
controls determined that this is not the case for the neonatal cardiac repair. Our model
with inducibility allowed for temporal control of the gene expression in the case that
there might be a critical window for Fgf10 to affect fibrosis. Limiting expression to only
a week did not demonstrate any benefit or detriment to scar size, as the Fgf10 group has
no difference in scar size compared to controls. Overall, there appears to be a role for the
epicardium in the response to cardiac injury, as seen with increased Erk phosphorylation,
the proliferation of epicardial cells and expression of vimentin in epicardial and sub-
epicardial cells. Additionally the epicardium after injury has the ability to respond to a
growth factor, in this case Fgf10, which was not detected in the absence of injury. It is
not concluded whether the role of epicardial cells in this injury response is beneficial, but
if previous models of cardiac injury in either the adult mouse or the zebrafish are
indicators, most likely there is potentially a positive role. If expanding the epicardium did
not enhance fibrosis or improve scar resolution, it might have potential to contribute to
the neovascularization in the injury through similar mechanisms to the embryonic
development, in which Wt1-positive epicardial cells undergo EMT and contribute to the
supportive cells of the new vessels. This could subsequently contribute to the overall
outcome of the regenerative process and could be investigated further.

76
MATERIALS AND METHODS
Generation of multi-transgenic mice: In order to create a tissue-specific and temporally
controlled gene expression model, we crossed a tissue-specific alpha-MHC driven rtTA
to the tet(o)Fgf10 mouse. The tet-on (doxycycline inducible) transgene expression is
limited to the cells expressing alpha-MHC and thus rtTA.
Mice were genotyped at fourteen days of age using a tail tissue which was lysed in
proteinase K (100ug/ml) and used for PCR amplification. PCR primers for transgenes,
alpha-MHC: rtTA and tet(o)Fgf10 are listed in the table below. Thirty-five cycles of
94°C for one minute, 58°C for thirty seconds to anneal, 72°C for one minute were
performed in a PCR machine to generate the amplicons. PCR products were separated on
a 1.5% agarose / TAE gel.

Induction of gene expression by doxycycline: Mouse had a diet of normal feed and water
as indicated by CHLA animal facilities, until adulthood, about 8 weeks of age.
Approximately 2 weeks prior to ischemic-injury surgery, the normal feed was replaced
with a doxycycline- supplemented (0.0625%) rodent diet (Harlan Teklad). They remained
on the doxycycline-supplemented diet until tissue is harvested. For embryonic induction
of growth factors by transgenes, pregnant females were supplied the doxycycline-
supplemented feed at day 11 of gestation, and the embryos will be harvested at E14 for
expression analysis.

77
Cryo-probe injury/ sham surgery: Neonatal pups at 1 day of age (P1) will undergo
surgery, under the guidance of the CHLA facility veterinarian, Dr. Baer, after being
anaesthetized with cold submergence. Throughout the surgery the mice were maintained
anaesthetized by remaining on a cold pack until completion of the surgery. The surgical
area was disinfected with ethanol and then chlorhexadine prior to the first incision on the
skin. An incision to the chest and ribs exposed the heart at which point the heart was
coaxed out of the chest through the opening by gentle pressure on the right side, then the
liquid nitrogen-cooled probe was applied until the tissue is cold-damaged, as seen by a
white pallor on the left ventricle down to the apex. Control/sham operated mice were not
exposed to the cold probe. Air in the mouse’s chest was removed by pressure as the chest
wall was sutured closed, first the muscle layer and secondly the skin, using 6-0 prolene
suture. They recovered from anesthesia on a heating pad and. They were continually
monitored for morbidity throughout the first 24 hours and ketoprofen (2mg/kg) was
administered to reduce pain, prior to the end of the surgical procedure.

Mouse care post surgery/ pain management Mice were kept according to NIH regulations
and monitored throughout the surgery and up until tissue harvest. Mice were observed at
least every 24 hours if they appeared to show signs of distress or discomfort administered
ketoprofen and provided ibuprofen diluted in drinking water until they appeared normal,
ambulatory and groomed. In the case that mice were highly moribund such as excessive
water retention, festering wounds or greater than 20% weight loss, they were euthanized
and tissue harvested for examination.

78
RNA isolation and Quantitative/ RT PCR: Ventricle tissue was bead-disruption
homogenized in Trizol ® and RNA isolated as indicated with Trizol ® extraction. RNA
was quantified using Nanodrop ® spectrophotometer. Generation of cDNA was done in
vitro using First Strand cDNA synthesis kit ® (Invitrogen©). The cDNA was measured
using Nanodrop ® spectrophotometer. Total RNA total quantity was assessed by qPCR
amplification of the cDNA using primers and probe combinations designed by Roche
Assay Design Center. The program was forty cycles at 94°C for ten seconds, 60°C for
thirty seconds to anneal. The software calculated the C.P. and the target gene was
compared to the housekeeping genes to determine the relative fold change in expression.

Protein collection/ isolation and Western blot: Mouse heart tissue was collected as stated
for tissue procurement in PBS, with as much blood removed from ventricles by cutting
open ventricles and allowing blood to be released before coagulation. Tissue pieces were
immediately placed in RIPA buffer (25mM Tris-HCl (pH 7.6), 150mM NaCl, 1% NP-40,
1% sodium deoxycholate, 0.1% SDS) with protease and phophatase inhibitors (in final
concentration of 1 µg/ ml each) in a microcentrifuge tube and placed on ice. They were
immediately homogenized by bead disruption homogenization in NextAdvance®
homogenizer. When no more tissue pieces were visible, the microcentrifuge tube was put
on ice for ten minutes. The remaining solids were separated from the protein in solution
by spinning for ten minutes at 4 degrees Celsius cooled microcentrifuge) at 4000 G.
Protein supernatant was recovered from the pellet, mixed with Laemmle buffer and
frozen immediately or used for Western blot immediately. Undiluted protein supernatant

79
was used for protein concentration with BioRad ® DC Protein Assay kit. For Western
blot use, the protein solution with Laemmle buffer, equivalent to 40 µg total protein, was
loaded into 10% polyacrylamide separation gels. Gels were placed in gel running
apparatus and run at 300 V for approximately 45 minutes or until loading buffer was
within 1 centimeter of the bottom of the gel. Protein was transferred onto PVDF
membranes (75 V for 75 minutes in 4 degrees Celsius cold room). PVDF membranes
were then blocked with SEA Block buffer (Thermo Fisher) and then immuno-labeled
with antibody against Erk and phospho-Erk (Cell Signaling) diluted in blocking buffer
overnight. Membranes were washed with TBS buffer and then detected with secondary
antibody (goat anti-rabbit IgG 680 and goat anti-mouse IgG 800).

Histological analysis: Tissue harvested was fixed in 4% paraformaldehyde overnight at
4°C. Fixed tissue was than dehydrated, cleared in toluene and then paraffinized. Paraffin-
embedded tissue was sectioned at 10µm. Sections were stained with hematoxylin and
eosin and examined by light microscopy for histological changes.

BrdU labeling: In order to assess proliferation of the Wt1-positive cells, nucleotide
labeling with bromo-deoxyuridine (BrdU) was done for the final 24 hours prior to
collection. Nursing mothers were administered an intra-peritoneal shot of BrdU diluted in
sterile saline for a final concentration of 100 mg/kg (BrdU weight: body weight) and pups
were allowed to nurse as normal.

80
Blood vessel density analysis: Tissue sections were stained with PECAM antibody by
immunohistochemistry. Images were taken of serial sections and number of vessels
quantified per area. The average of three hearts was taken for each experimental group.

Wilm’s tumor 1 protein immunostaining and quantification: Tissue sections of 10-
microns on glass slides are processed by immunohistochemistry: paraffin was removed
with toluene washes, then rehydrated with graded ethanol until phosphate buffered
solution. Heat and acid antigen retrieval was done with Vector ©Antigen Retrieval
solution and a microwave. Tissue sections were stained with Wilm’s Tumor 1 antibody
(Santa Cruz) followed with secondary antibody goat anti-rabbit conjugated to Alexa 594
(Molecular Probes) and co-stained with Vectashield© mounting media with Propridium
iodide. Confocal images were taken on Zeiss LSM 710® single photon confocal
microscope. The average ratio of the number of Wt-1 positive nuclei (double stained with
antibody and DAPI) to total nuclei (DAPI- stained) were quantified by at least three
sections of each sample for Wt1-positive cells for injury area (scar region determined by
consecutive tissue slides stained by AFOG and lack of green autofluorescence from
myocardium for reference) for each heart. The average of seven hearts was taken for the
experimental group and eight hearts was taken for the control group. The measurements
are analyzed for statistical significance with Wilcoxon rank sum test.

Scar measurement analysis: Ten-micron tissue sections on slides were deparaffinized and
rehydrated to phosphate buffered solution. The tissue slides were placed in Bouin’s

81
fixative for 2.5 hours at 56 degrees Celsius then one hour at room temperature. The slides
were washed of Bouin’s fixative for 20 minutes in running water. The slides were treated
with 1% phosphomolybdic acid for five minutes then washed briefly with deionized
water. Slides were immersed in and stained with Acid fuschin, Orange G, Aniline blue
(AFOG) trichrome staining solution for 5 minutes and then briefly rinsed with deionized
water. Slides were then quickly processed with alcohol to dehydrate and then cleared in
toluene and mounted with Cytoseal ®. Tissue sections were imaged with bright-field
microscopy. Images of sections were calibrated for measurement on Image J software.
Three to five measurements spanning the width of the tissue were taken and averaged for
scar length and left ventricle circumference and the ratio of the average scar length to
average left ventricle circumference is calculated. Three to five measurements spanning
the length of the tissue were taken and averaged for scar thickness and septum thickness
and the ratio of the average scar thickness to average septum thickness was calculated.
The measurements were analyzed for statistical significance with Wilcoxon rank sum
test.

Imaging/ Microscopy Whole mount imaging was performed with Leica dissecting scope
and using Spot Advanced ® imaging program. Fluorescent imaging of sections was
performed using the Zeiss LSM 710 © single photon confocal microscope with Zen ®
imaging software.

82
Chapter III: Primary culture of zebra fish (Danio rerio) epicardium: an
in vitro tool to study the role of epicardium in cardiac regeneration

INTRODUCTION
Heart pathologies in youth and adults contribute to morbidity and mortality
worldwide. Defects of the heart are the predominant lethal birth deformity worldwide
(AHA, 2009), while heart disease is the number one contributor to morbidity and
mortality in aging adults (AHA, 2009). Studies of the developing heart have unraveled
some of the etiologies of congenital heart defects. On the other hand, much has yet to be
understood about repair of the injured heart. In contrast to mammals, lower vertebrates
such as newts and the zebrafish (Danio rerio), have demonstrated the capacity for full
organ regeneration (Poss et al., 2002; Gardiner, 2005) and have thus been a useful
experimental model to explore the potential for adult tissue regeneration.
Regeneration of the heart requires reconstitution of all cardiac cell types as well
as blood vessels and connective tissue. Zebrafish heart regeneration occurs via
proliferation of remaining cells (Poss et al., 2002) and trans/ de-differentiation of cells
(Lepilina et al., 2006; Kikuchi et al., 2010; Jopling et al., 2010). Although evidence of
mammalian heart stem (Bearzi et al., 2005) and progenitor cells (Cai et al., 2002,
Laugwitz, et al., 2005) exists, their contribution to repair or cell replenishment in adults is
unclear. Epicardial cells are another cardiac cell that has demonstrated plasticity and
multi-potentiality. Epicardial- derived progenitor cells (EPDC’s) originate from the
epicardium and are characterized in embryonic heart as capable of entering the

83
myocardium to contribute to fibroblasts, smooth muscle cells (reviewed in Winter and
Gittenberger-de Groot, 2007) and cardiomyocytes (Zhou et al., 2008; Cai et al., 2008).
Epicardium in adult zebrafish hearts undergoes a dedifferentiation and induction of early
developmental genes during regeneration after ventricular resection (Lepilina et al., 2006;
Lien et al. 2006). Primary cultures of cardiac cell types, including cardiomyocytes
and epicardial cells, are important tools that have been established. However, the primary
cultures require the plasticity of the embryonic cardiac cells in order to obtain and
maintain the cells. Epicardial cell cultures from embryonic mouse hearts (Chen et al.
2002) or proepicardium explants (Kruithof et al., 2006 and Torlop et al., 2010) have
become a staple in many embryonic studies. Adult heart tissue explants for deriving the
epicardial cells has limitations and requires the proper stimulus, such as growth factors
(Smart et al., 2006) to induce. Few studies have determined which growth factors or
other migration- inducers can stimulate the adult epicardium. Since adult epicardium has
a terminally differentiated phenotype that has lost much of the plasticity and potential of
embryonic tissue, a system unique from the current proepicardium and the embryonic
heart culture techniques is required.
In order to understand the specific role of epicardium in the regeneration process
of adult zebrafish heart, we have established a protocol for isolating and culturing
epicardium using a 3D matrix of fibrin, a precursor matrix for the regenerating area.

84
RESULTS
Amputated hearts cultured on fibrin will yield epicardial monolayer within about 5 days
with 10% FBS in DMEM. After the first day some initial epicardial cells can be seen,
with a migratory morphology, extending from the explant. The culture should be
followed daily for the next week until a sufficient outgrowth is obtained, then the explant
should be removed immediately. This protocol should yield pure epicardial cells that can
be immediately used for experimental manipulation if not allowed to overgrow and allow
for contaminating cells such as endothelial cells (figure 27). Unamputated hearts may
also be exchanged for regenerating hearts with some reduction in the amount of
epicardial cells obtained (figure 26).

Gelatin can be substituted for fibrin coating of the culture wells, which will also reduce
the rate and number of epicardial cells obtained. The coverslip base is transferable and
can be moved as done with the epicardin immunofluorescence analysis. The fibrin
matrix’s three dimensions allow for assessment of EMT of epicardium in some
experimental scenarios. The cells derived from this protocol can be used for RNA
extraction and gene expression analysis, which we have done successfully. RT PCR
analysis for epithelial markers (figure 26) and staining for ZO-1 and actin confirms the
epithelial phenotype with ZO-1 associated at the cellular membrane coinciding with
cortical actin filaments. Staining for epicardial cells by epicardin (Tcf21)
immunodetection will demonstrate the presence of this transcription factor in the nuclei
of cells obtained.

85
DISCUSSION
Thus far, very few zebrafish cell lines or epicardial cell lines are established for
experimental use. The technique described in this new protocol allows for a relatively
simple means of isolating epicardial cells from zebra fish in order to use them for
molecular and cellular experimental analyses. Initiating a primary culture fresh from the
source bypasses the necessity of transforming and immortalizing the cells for in vitro
studies, thus making them more suitable for comparison to cells in vivo. This protocol
also describes how they can be further analyzed for gene expression with RNA
quantification techniques such as qPCR, or they can be stained with
immunohistochemistry techniques to identify protein expression and /or localization.
Pharmacological agents or small molecules can be tested for their effects on the
epicardial cells with this in vitro protocol, and increasing the sample sizes is achievable
with zebra fish in order to create high-throughput assays.

86
MATERIALS AND METHODS
1. Amputation: Regenerating zebrafish hearts have dynamic epicardia that
demonstrate embryonic characteristics including enhanced migratory potential.
The regeneration model for zebrafish hearts indicate that the epicardium expresses
early epicardial markers, such as tbx18, as early as 3 days into the regeneration
(Poss et al., 2002, Lien et al., 2006). Although the unregenerating, adult heart has
some potential for migration in our culture system, regenerating hearts remain the
most consistent for obtaining a sheet of epithelial cells without the addition of
exogenous stimulants (figure 26) Zebrafish can undergo ventricular resection of
the heart as described (Poss et al., 2002). The hearts can be recovered after 3 days
of unhindered repair and after up to 14 days post ventricular amputation to
acquire pure monolayers of epicardium on the fibrin matrix. In summary, fish can
be anaesthetized and the heart cavity opened to expose the heart. Customarily,
twenty percent of the ventricle is amputated. The zebrafish can be allowed to
recover and maintained similarly to uninjured adult fish. The regenerating heart
can be collected for culture by euthanizing the fish and opening the chest cavity
similar to the amputation resection, removing the entire heart at the atrium. The
regenerating portion of the heart may still be visible as a pale region at the apex
composed mostly of fibrin. The activated epicardium of the regenerating heart
will adhere and migrate over the culture fibrin as it does with the native fibrin
clot.

87
2. Explant culture/ initiation of epicardium culture: In order to establish the
epicardial culture, the heart is optimally placed on the proper matrix for adherence
and migration of the epicardium. Activated epicardium in the amputated heart
migrates over and into the fibrin clot at the regeneration site. Fibrin provides an
ideal in vitro matrix for the epicardium culture as well. Although similar
epicardial culture systems for embryonic hearts (Chen et al., 2002) can be done
on conventional culture plates, or on gelatin, adult zebrafish heart shows a less
dynamic capacity for migration on these scaffolds. (figure 27) On the fibrin,
epicardial cells from unamputated (un-regenerating) hearts can be seen after
several days as well. Prolonged culturing of the explant will allow for
mesenchymal cell types and endothelial cells to grow over the initial layer of
epicardial cells as well as allow cardiomyocytes to adhere under the explant
(when the explant is removed). Once the heart explant is surrounded by a
monolayer of epithelial cells, the remaining whole organ explant should be
removed to prevent the contamination of other cell types.
Epicardial cell markers are limited, especially for the differentiated adult tissue. Cells
can be analyzed for phenotype and cell marker, epicardin, to verify the epicardial
origin. The monolayer of epicardial cells demonstrates positivity for the nuclear
expression of epicardin and the epithelial characteristics of membrane- associated
tight junctions (zona occludins 1) and actin. Few cells on the periphery demonstrate
some mesenchymal phenotype, as they are more migratory.

88
3. Culture Maintenance: Once the isolated epicardial cells are obtained, the culture
can be maintained with regular media changes and be used for experimental
analysis. The epicardial cells demonstrate a low proliferative index when
maintained with ordinary serum-supplemented media (even at 10% serum per
volume in DMEM). The cells can be further manipulated with growth factors/
pharmacological agents by addition of the respective agent to the media. Utilizing
the glass coverslip under the fibrin allows for removal of the cells from the well
for staining or other manipulations.

Tools:
Adult zebrafish (6 months or older),
Tricaine (4 mg/ml),
100 cm bowl with aquarium water,
sponge with center hollowed,
dissecting scope,
fine tip tweezers,
straight 4 mm spring scissors,
70% ethanol in spray bottle,
hot bead sterilizer,
fibrinogen (20 mg/ml),
thrombin (50 IU/ml) [Sigma],
circular glass coverslips [Fisher Scientific],

89
24- well culture plate
portable tank with aquarium water to temporarily house and transport fish,
Sterile DPBS [Cellgro],
DMEM (no additives) [Cellgro],
ice bucket,
petri dishes,
Sterile culture media (DMEM with 10% FBS, penicillin, streptomycin, and L-
glutamine);
sterile 1ml pipette tips,
cell culture incubator set at 29°C with 5% CO
2
and 95% humidity
Tcf-21 antibody [Abcam]
Goat serum [Sigma]
Goat anti- rabbit antibody conjugated with Alexa-594
Phalloidin- FITC [Dako]
Vectashield [Vector Laboratories]
Glass slides

90

Figure 25. Representation of the epicardial culture on fibrin. Bright-field image of
the heart placed on fibrin at beginning of the culture protocol (A). Artistic
representation of the heart in culture as seen transverse plane, after cells have grown
out from the heart explant (C). Bright-field image of the heart in cuture after three
days, in which epicardial cells have grown out (B). Kim et al., 2010.

91
Stepwise procedure:
I. Explant to in vitro epicardial cells
Heart Amputation: 4-14 days prior to tissue culture (5-7 minutes per fish)
Adult male and female fish of wildtype AB strain or transgenic lines of CMLC-nRFP
or Fli1-EGFP were used to identify cell types. Amputation of adult zebrafish was
performed as described (Poss et al. 2002).
Organ procurement for culture: 5 minutes per heart
1. Sterilize tools with 70% ethanol and hot bead sterilizer
2. Euthanize fish in Tricaine (10mls) dissolved in aquarium water (200mls) in
100cm bowl.
* Higher concentration of Tricaine (than for anesthesia) will euthanize the fish;
however, anaesthetizing the fish prior to heart extraction is sufficient.
3. Place fish in moist sponge platform under dissecting microscope to expose the
ventral side. Either spray or wipe the surface of the fish with 70% ethanol to
prevent contamination.
4. Open the fish pericardium in similar manner as for amputation and remove heart.
5. Heart can be placed temporarily in non-sterile DMEM (without additives) as a
holding media.
6. Heart is transferred to DPBS in a petri plate and cut transversely through apex and
not completely through the anterior end, in order to open the heart but keep in one
piece. The heart should be butterflied in this manner to create one side with high
surface area of epicardium.

92
* This should be done also while viewing through the dissecting scope and will be
important to wash and remove excess blood in and around the heart, which can
impede cell outgrowth on the matrix.
7. Transfer back to non-sterile- DMEM to hold and transfer.
Establishing culture of explant: 10-20min. All steps performed in a sterile culture
hood.
8. Fibrin matrix is prepared just prior to use (no more than 24hours prior to use).
And can be over coverslips for removal after staining.
a. Warm DMEM and fibrinogen in 37° C water bath for 30 minutes.
*The solutions should be warm and it should be noted that the fibrinogen
solution has no signs of un-dissolved fibrinogen (seen as swirls in the clear
liquid).
b. Thrombin can be thawed on ice up to 30 min before.
* It is important to avoid allowing the thrombin to get to warm or remain at
room temperature too long, as this begins to decrease the enzymatic activity.
c. Combine 4.5 mls of warm DMEM with 0.5mls of thawed fibrinogen in 15
ml conical tube, and agitate to mix.
d. In a sterile microcentrifuge tube combine 45 µl of DMEM and 5ul of
thawed thrombin, flick tube to mix.
e. Add 12.5 µl of the diluted (in DMEM) thrombin into the diluted (in
DMEM) fibrinogen, and mix quickly.

93
f. Quickly transfer 350ul of the mixed reaction solution to the 24 well
culture plate (with or without coverslips).
* The media may not cover the well, you can increase the volume or gently
agitate the plate to spread the liquid.
g. Place in 37 C incubator to allow the reaction to complete for solidified
fibrin.
* The concentrations of thrombin and fibrinogen are adjusted to get
polymerization in less than 20 minutes, usually about 5 minutes.
9. Heart is removed with sterile tweezers from DMEM and placed on the fibrin.
10. Leave the heart uncovered in the hood to allow any excess liquid to evaporate.
*This step is critical to encourage the tissue contact with the matrix/ coverslip/ well.
The liquid around the tissue should slowly shrink and the surface of the tissue should
remain moist but not be overly wet.
11. Add 200µl of media to the well slowly, avoiding force on the heart.
*Avoid dislodging the heart by pipetting on the side of the well. Only 200µl of media
is used for the first 24 hours to also prevent the tissue from disassociating and floating
in the media.
12. Keep in 29-31° C incubator with 5%CO
2

Maintaining culture 3-14 days
13. Culture media 300µl/ well (10% FBS in DMEM with penicillin/ streptomycin)
should be changed at least once every 5 days.

94
*The proliferation rate of epicardial cells is low even at high serum levels. The cells
do not need to be split when grown in a 24- well plate.
II. Phenotyping epicardial cells: 5- 24h
*Stringent selection for pure epicardial cells requires careful timing of the removal of
the explant. Epicardial cells can be confirmed with antibody staining. The fibrin
should be polymerized over a glass coverslip in order to do the staining.
1. Cells grown on a coverslip (with or without fibrin) can be fixed with 4%
paraformaldehyde for about 15 minutes.
2. Multiple washes with PBS + 0.1% Tween 20 (PBST) will remove fixative and
permeabalize the membrane for antibody penetrance (at least 1 hour total).
*The proper nuclear –specific staining of epicardin and reduced background
staining requires sufficient exposure to the detergents. PBS with Triton-X (1%) can
also be used for 30 minutes, in addition to PBST.
3. Non-specific protein binding is blocked with 10% goat (secondary antibody
species source) serum in PBST for 1 hour at room temperature.
4. Epicardin (1:100) or ZO-1 (1:100) can be labeled with antibody diluted in 10%
serum in PBST for 1hr at room temperature or overnight at 4 C.
5. Wash off the primary antibody with PBS with 0.1% NP40 (IgePal) in 3-5 minute
increments.
6. Secondary antibody conjugated with fluorescent tag (Alexa- 594 Goat anti-rabbit)
is diluted (1:500) and phalloidin conjugated with FITC is also added (1:1000) in
PBST.

95
7. Wash off secondary antibody with 3 to 5 minute washes in PBST.
8. The coverslips can be removed from the wells and placed on a glass slide with
DAPI mounting media, cell- side facing the slide.
*Be careful to note the side of the coverslip with the cells (if it isn’t fibrin coated,
it is not easy to differentiate).

96

Primary culture of zebrafish heart on fibrin: Zebrafish are anaesthetized using Tricaine
diluted in system water for approximately 90 seconds. The fish is dissected under
microscope to remove aorta from heart and placed in PBS over ice to stop heartbeat. The
heart is further dissected under aseptic conditions to remove atrium, aorta, and blood, and
the ventricle is dissected sagitally. Fibrin matrix is made in well of 24-well plate by
adding fibrinogen diluted in serum-free media (3mg/ml) to thrombin diluted in sterile
water (0.01 U/ml) into the bottom of the well. The dissected heart is placed on the fibrin
matrix and covered in 15% FBS in DMEM and incubated at 29°C with 5%CO
2
. The cells
will be supplemented (500 ng/ml) with recombinant human Fgf10 in the media.

Figure 26. Outgrowth of heart explants are epicardial cells, but amputated
hearts have increased EMT markers in the outgrowth. Bright-field microscopy
images of sham operated heart and amputated heart (7 dpa) for explant show
monolayers of cells exhibiting epithelial, cobblestone morphology, but explants from
amputated hearts exhibit greater cell outgrowth (B) than sham operated heart explants
(A). Images of PCR products from RT PCR of outgrowth cells’ RNA show presence
of epicardial genes, tbx18 and raldh2, as well as EMT markers, snail 1a, snail 2 and
snail 1b (C). The amputated heart has greater snail 2 expressed than sham and
detectable snail 1a versus undetectable expression in sham (C).

97
Figure 27. Zebrafish heart explants select for non-cardiomyocyte cells but can
allow for endothelial cell contamination in the primary culture of epicardial cells.
Bright-field and fluorescent microscopy images of transgenic zebrafish heart explants
in culture after seven days show outgrowth from the explant (A and B). Using
CMLC2-nRFP transgenic zebrafish hearts, cell outgrowth was determined to not be
CMLC2- positive cells, only localized within the explant (A’). Likewise Fli1-EGFP
zebrafish hearts, after seven days in culture, Fli1- positive cells were present in the
outgrowth as well as the explant (B’).

98
CONCLUSIONS
Appropriate tools are essential for testing a hypothesis therefore should be
evaluated critically. The field of regenerative research is nascent but growing rapidly,
requiring novel approaches to studying regeneration and the mechanisms, genes, et cetera
it entails. I described multiple tools that can be employed to answer questions about
regeneration or specific aspects of the regenerative process. In conjunction with the long-
standing mouse model utilized for heart disease research and the rodent cardiac ischemic
injury model, hypotheses for improving the outcome of human heart disease can now be
proposed as how to facilitate regeneration. The murine models of inducible gene
expression we employed for our study demonstrates potential as valuable tools to
evaluate the effects of a single gene function on the outcome of cardiac repair and
regeneration. This can be combined with the neonatal injury model, as was done in our
study, to test a hypothesis for heart regeneration. The neonatal model of heart
regeneration that we developed and used in this study is a novel yet useful model to study
cardiac regeneration in mammals. Additionally, the use of neonates and the injury
technique described here, cardiac injury is easily achievable with common dissecting or
surgical tools, alleviating the necessity for specialized surgical equipment. This is a
reasonable marriage of the favored mammalian research animal, which also has been
extensively genetically manipulated for gene function studies, and the more recent
surgical approach for heart regeneration with the zebra fish animal model.
The transgenic models for gene overexpression were used in our study to evaluate
the Fgf10 gene function in heart injury and repair/ regeneration. This mouse model

99
allowed for temporal modulation of expression that allowed this study to answer sub-
hypotheses regarding the length of Fgf10 overexpression and the effect on scar size. In
this study only an intermittent week-long and a continuous induction of Fgf10 were
evaluated, and no effect on scar size was observed. However, the induction period is
controllable with this mouse model. It could be further examined in a pulse-induction
approach as to overcome signaling modulation by negative feedback loops. Our modest
increase in proliferation of epicardial cells (WT1-positive) in our study was promising
but might have been hampered by suppression of Fgf10 signaling cascade after prolonged
expression. The use of a pulse induction approach might clarify this potential side effect.
Previous studies in other organ systems have demonstrated that the tet(o)Fgf10 transgenic
mouse can produce dramatic results with induction of the transgene, and with the
presence of the receptor in the epicardium, as seen in published studies and was
confirmed in our hands, a response to the excess growth factor would be expected. This
was seen in the published embryonic study for Fgf10 in the heart; however, a modified
approach as mentioned with either a pulse induction or change to the induction period
could enhance the response seen in this study, unaffected fibrosis or scar resolution and
modest increase of epicardial cells.
In addition to the mouse models in this study, another valuable tool we
developed to study the epicardium is the primary zebra fish epicardial cell culture
protocol. The zebra fish model of heart regeneration still continues to provide essential
information regarding the regenerative process and has progressed to more detailed
examination of the mechanisms and cellular roles. Since our lab has particular interest in

100
the role and activity of epicardial cells, an in vitro assay is beneficial to testing our
hypotheses. The plasticity of the zebra fish cardiac cells has been amenable to isolation
and manipulation in vitro. This protocol is advantageous in its ability to quickly analyze
large numbers of molecules on the epicardial cells. Screening for potential activators of
the epicardial cells using an in vitro assay such as this can be useful in determining a
candidate for in vivo studies. This protocol can serve as a preliminary assay to evaluate
growth factors that can be further studied using the transgenic mouse model.
Additionally, we demonstrated that the epicardial cells in vitro can be collected for RNA
analysis, or stained with immunohistochemistry techniques for protein analysis.

101
TABLES
Table 1:
Genotyping
Primers

Gene Sense Antisense Reference
CAGG-cre ctg cta acc atg ttc atg cc tcg acc agt tta gtt acc c Sakai et al., 1997
Cre generic cgt ttt ctg agc ata cct gga att ctc cca ccg tca gta cg Tessari et al., 2008
αMHC-
rtTA
cac ctg ggg ttc cca ccc
tta tgt
agc agc tcc agt gcgctg
tta unpublished
tbx18-cre
tcc ctg aac atg tcc atc
agg ttc
gcc aga gaa aga gga aac
ggc aaa
Sylvia Evans,
unpublished
tet (o)fgf10
gac gcc atc cac gct gtt
ttg acc
att tgc ctg cca ttg tgc tgc
cag Clark et al., 2000
rosa:
rtTA
f/f
gag ttc tct gct gc tcc tg
aag acc gcg aag agt ttg
tc Belteki et al., 2005
rosa (wt) gag ttc tct gct gc tcc tg
cga ggc gga tac aag caa
ta Belteki et al., 2005
rtTA
recombined aaa atc ttg cca gct ttc ccc aaa gtc gct ctg agt tgt tat
Wei Shi,
unpublished

Table 2:
qPCR
Primers
Gene Sense Antisense Probe
Fgf10 cgg gac caa gaa tga aga ct gca aca act ccg att tcc ac 80
Rnps1 aat ccc gat gaa gca gag aa tga tct ctt ggc cat caa ttt 58
Srp14 tac caa cca gct gca caa gt cat aca gag gtg ctg aaa gca g 6
Tpt1 gga tgg ctt aga gat gga gaa a tcc cat ttg tcc taa agt cct g 66
Erm tcc tac atg aga ggc gggta gta cag cct ggg gtc ctt ct 66
Pea3 cag act tcg cct acg act ca gcc ata acc cat cac tcc at 80
18s aaa tca gtt atg gtt cct ttg gtc gct cta gaa tta cca cag tta tcc aa 55
Fgfr2 cct acc tca agg tcc tga agc cat cca tct ccg tca cat tg 21

102

BIBLIOGRAPHY
Bearzi, C., Rota, M., Hosoda, T., Tillmanns, J., Nascimbene, A., De Angelis, A.,
Yasuzawa-Amano, S., Trofimova, I., Siggins, R.W., Lecapitaine, N., et al. (2007).
Human cardiac stem cells. Proc Natl Acad Sci U S A 104, 14068-14073.

Belteki, G., Haigh, J., Kabacs, N., Haigh, K., Sison, K., Costantini, F., Whitsett, J.,
Quaggin, S.E., and Nagy, A. (2005). Conditional and inducible transgene expression in
mice through the combinatorial use of Cre-mediated recombination and tetracycline
induction. Nucleic acids research 33, e51.

Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabe-Heider, F., Walsh, S.,
Zupicich, J., Alkass, K., Buchholz, B.A., Druid, H., et al. (2009). Evidence for
cardiomyocyte renewal in humans. Science 324, 98-102.

Bersell, K., Arab, S., Haring, B., and Kuhn, B. (2009). Neuregulin1/ErbB4 signaling
induces cardiomyocyte proliferation and repair of heart injury. Cell 138, 257-270.

Brade, T., Kumar, S., Cunningham, T.J., Chatzi, C., Zhao, X., Cavallero, S., Li, P.,
Sucov, H.M., Ruiz-Lozano, P., and Duester, G. Retinoic acid stimulates myocardial
expansion by induction of hepatic erythropoietin which activates epicardial Igf2.
Development (Cambridge, England) 138, 139-148.

103
Cai, C.L., Martin, J.C., Sun, Y., Cui, L., Wang, L., Ouyang, K., Yang, L., Bu, L., Liang,
X., Zhang, X., et al. (2008). A myocardial lineage derives from Tbx18 epicardial cells.
Nature 454, 104-108.

Center for Disease Control and Prevention (CDC). (2012) FASTSTATS: Heart Disease.
http://www.cdc.gov/nchs/fastats/heart.htm.
Chablais, F., Veit, J., Rainer, G., and Jazwinska, A. The zebrafish heart regenerates after
cryoinjury-induced myocardial infarction. BMC Dev Biol 11, 21.

Chen, T.H., Chang, T.C., Kang, J.O., Choudhary, B., Makita, T., Tran, C.M., Burch, J.B.,
Eid, H., and Sucov, H.M. (2002). Epicardial induction of fetal cardiomyocyte
proliferation via a retinoic acid-inducible trophic factor. Dev Biol 250, 198-207.

Cohen, E.D., Wang, Z., Lepore, J.J., Lu, M.M., Taketo, M.M., Epstein, D.J., and
Morrisey, E.E. (2007). Wnt/beta-catenin signaling promotes expansion of Isl-1-positive
cardiac progenitor cells through regulation of FGF signaling. J Clin Invest 117, 1794-
1804.

Compton, L.A., Potash, D.A., Mundell, N.A., and Barnett, J.V. (2006). Transforming
growth factor-beta induces loss of epithelial character and smooth muscle cell
differentiation in epicardial cells. Dev Dyn 235, 82-93.

104
Engel, F.B. (2005). Cardiomyocyte proliferation: a platform for mammalian cardiac
repair. Cell Cycle 4, 1360-1363.

Gardiner, D.M. (2005). Ontogenetic decline of regenerative ability and the stimulation of
human regeneration. Rejuvenation Res 8, 141-153.

Gonzalez-Rosa, J.M., Martin, V., Peralta, M., Torres, M., and Mercader, N. Extensive
scar formation and regression during heart regeneration after cryoinjury in zebrafish.
Development (Cambridge, England) 138, 1663-1674.

Gupte, V.V., Ramasamy, S.K., Reddy, R., Lee, J., Weinreb, P.H., Violette, S.M.,
Guenther, A., Warburton, D., Driscoll, B., Minoo, P., et al. (2009). Overexpression of
fibroblast growth factor-10 during both inflammatory and fibrotic phases attenuates
bleomycin-induced pulmonary fibrosis in mice. American journal of respiratory and
critical care medicine 180, 424-436.

Horiba, M., Kadomatsu, K., Yasui, K., Lee, J.K., Takenaka, H., Sumida, A., Kamiya, K.,
Chen, S., Sakuma, S., Muramatsu, T., et al. (2006). Midkine plays a protective role
against cardiac ischemia/reperfusion injury through a reduction of apoptotic reaction.
Circulation 114, 1713-1720.

105
Hsieh, P.C., Segers, V.F., Davis, M.E., MacGillivray, C., Gannon, J., Molkentin, J.D.,
Robbins, J., and Lee, R.T. (2007). Evidence from a genetic fate-mapping study that stem
cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13, 970-974.

Jopling, C., Sleep, E., Raya, M., Marti, M., Raya, A., and Izpisua Belmonte, J.C.
Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation.
Nature 464, 606-609.

Keegan, B.R., Feldman, J.L., Begemann, G., Ingham, P.W., and Yelon, D. (2005).
Retinoic acid signaling restricts the cardiac progenitor pool. Science 307, 247-249.

Kikuchi, K., Holdway, J.E., Werdich, A.A., Anderson, R.M., Fang, Y., Egnaczyk, G.F.,
Evans, T., Macrae, C.A., Stainier, D.Y., and Poss, K.D. Primary contribution to zebrafish
heart regeneration by gata4(+) cardiomyocytes. Nature 464, 601-605.

Kim, J., Wu, Q., Zhang, Y., Wiens, K.M., Huang, Y., Rubin, N., Shimada, H., Handin,
R.I., Chao, M.Y., Tuan, T.L., et al. PDGF signaling is required for epicardial function
and blood vessel formation in regenerating zebrafish hearts. Proc Natl Acad Sci U S A
107, 17206-17210.

Kruithof, B.P., van Wijk, B., Somi, S., Kruithof-de Julio, M., Perez Pomares, J.M.,
Weesie, F., Wessels, A., Moorman, A.F., and van den Hoff, M.J. (2006). BMP and FGF

106
regulate the differentiation of multipotential pericardial mesoderm into the myocardial or
epicardial lineage. Dev Biol 295, 507-522.

Kuhn, B., del Monte, F., Hajjar, R.J., Chang, Y.S., Lebeche, D., Arab, S., and Keating,
M.T. (2007). Periostin induces proliferation of differentiated cardiomyocytes and
promotes cardiac repair. Nat Med 13, 962-969.

Laugwitz, K.L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin, L.Z., Cai,
C.L., Lu, M.M., Reth, M., et al. (2005). Postnatal isl1+ cardioblasts enter fully
differentiated cardiomyocyte lineages. Nature 433, 647-653.

Lavine, K.J., Kovacs, A., and Ornitz, D.M. (2008). Hedgehog signaling is critical for
maintenance of the adult coronary vasculature in mice. J Clin Invest 118, 2404-2414.

Lavine, K.J., Long, F., Choi, K., Smith, C., and Ornitz, D.M. (2008). Hedgehog signaling
to distinct cell types differentially regulates coronary artery and vein development.
Development (Cambridge, England) 135, 3161-3171.

Lavine, K.J., and Ornitz, D.M. (2008). Fibroblast growth factors and Hedgehogs: at the
heart of the epicardial signaling center. Trends Genet 24, 33-40.

107
Lavine, K.J., and Ornitz, D.M. (2009). Shared circuitry: developmental signaling
cascades regulate both embryonic and adult coronary vasculature. Circ Res 104, 159-169.

Lavine, K.J., White, A.C., Park, C., Smith, C.S., Choi, K., Long, F., Hui, C.C., and
Ornitz, D.M. (2006). Fibroblast growth factor signals regulate a wave of Hedgehog
activation that is essential for coronary vascular development. Genes Dev 20, 1651-1666.

Lavine, K.J., Yu, K., White, A.C., Zhang, X., Smith, C., Partanen, J., and Ornitz, D.M.
(2005). Endocardial and epicardial derived FGF signals regulate myocardial proliferation
and differentiation in vivo. Dev Cell 8, 85-95.

Lepilina, A., Coon, A.N., Kikuchi, K., Holdway, J.E., Roberts, R.W., Burns, C.G., and
Poss, K.D. (2006). A dynamic epicardial injury response supports progenitor cell activity
during zebrafish heart regeneration. Cell 127, 607-619.

Li, P., Cavallero, S., Gu, Y., Chen, T.H., Hughes, J., Hassan, A.B., Bruning, J.C.,
Pashmforoush, M., and Sucov, H.M. IGF signaling directs ventricular cardiomyocyte
proliferation during embryonic heart development. Development (Cambridge, England)
138, 1795-1805.

Lien, C.L., Schebesta, M., Makino, S., Weber, G.J., and Keating, M.T. (2006). Gene
expression analysis of zebrafish heart regeneration. PLoS Biol 4, e260.

108

Makino, S., Whitehead, G.G., Lien, C.L., Kim, S., Jhawar, P., Kono, A., Kawata, Y., and
Keating, M.T. (2005). Heat-shock protein 60 is required for blastema formation and
maintenance during regeneration. Proc Natl Acad Sci U S A 102, 14599-14604.

Matsunaga, S., Okigaki, M., Takeda, M., Matsui, A., Honsho, S., Katsume, A., Kishita,
E., Che, J., Kurihara, T., Adachi, Y., et al. (2009). Endothelium-targeted overexpression
of constitutively active FGF receptor induces cardioprotection in mice myocardial
infarction. J Mol Cell Cardiol 46, 663-673.

Murakami, M., Elfenbein, A., and Simons, M. (2008). Non-canonical fibroblast growth
factor signalling in angiogenesis. Cardiovasc Res 78, 223-231.

Nagy, A. (2000). Cre recombinase: the universal reagent for genome tailoring. Genesis
26, 99-109.

Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S., and Itoh, N.
(2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ
development. Biochem Biophys Res Commun 277, 643-649.

109
Pennisi, D.J., and Mikawa, T. (2005). Normal patterning of the coronary capillary plexus
is dependent on the correct transmural gradient of FGF expression in the myocardium.
Dev Biol 279, 378-390.

Pennisi, D.J., Rentschler, S., Gourdie, R.G., Fishman, G.I., and Mikawa, T. (2002).
Induction and patterning of the cardiac conduction system. Int J Dev Biol 46, 765-775.

Poss, K.D. (2007). Getting to the heart of regeneration in zebrafish. Semin Cell Dev Biol
18, 36-45.

Poss, K.D., Wilson, L.G., and Keating, M.T. (2002). Heart regeneration in zebrafish.
Science 298, 2188-2190.

Sakai, K., and Miyazaki, J. (1997). A transgenic mouse line that retains Cre recombinase
activity in mature oocytes irrespective of the cre transgene transmission. Biochem
Biophys Res Commun 237, 318-324.

Schnabel, K., Wu, C.C., Kurth, T., and Weidinger, G. Regeneration of cryoinjury induced
necrotic heart lesions in zebrafish is associated with epicardial activation and
cardiomyocyte proliferation. PLoS One 6, e18503.

110
Sheikh, F., Sontag, D.P., Fandrich, R.R., Kardami, E., and Cattini, P.A. (2001).
Overexpression of FGF-2 increases cardiac myocyte viability after injury in isolated
mouse hearts. Am J Physiol Heart Circ Physiol 280, H1039-1050.

Smart, N., Risebro, C.A., Melville, A.A., Moses, K., Schwartz, R.J., Chien, K.R., and
Riley, P.R. (2007). Thymosin beta4 induces adult epicardial progenitor mobilization and
neovascularization. Nature 445, 177-182.

Smart, N., Risebro, C.A., Melville, A.A., Moses, K., Schwartz, R.J., Chien, K.R., and
Riley, P.R. (2007). Thymosin beta-4 is essential for coronary vessel development and
promotes neovascularization via adult epicardium. Ann N Y Acad Sci 1112, 171-188.

Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain.
Nature genetics 21, 70-71.

Sridurongrit, S., Larsson, J., Schwartz, R., Ruiz-Lozano, P., and Kaartinen, V. (2008).
Signaling via the Tgf-beta type I receptor Alk5 in heart development. Dev Biol 322, 208-
218.

Steinhauser, M.L., and Lee, R.T. Regeneration of the heart. EMBO Mol Med 3, 701-712.

111
Stoick-Cooper, C.L., Weidinger, G., Riehle, K.J., Hubbert, C., Major, M.B., Fausto, N.,
and Moon, R.T. (2007). Distinct Wnt signaling pathways have opposing roles in
appendage regeneration. Development (Cambridge, England) 134, 479-489.

Stuckmann, I., Evans, S., and Lassar, A.B. (2003). Erythropoietin and retinoic acid,
secreted from the epicardium, are required for cardiac myocyte proliferation. Dev Biol
255, 334-349.

Torlopp, A., Schlueter, J., and Brand, T. Role of fibroblast growth factor signaling during
proepicardium formation in the chick embryo. Dev Dyn 239, 2393-2403.

van Amerongen, M.J., Harmsen, M.C., Petersen, A.H., Popa, E.R., and van Luyn, M.J.
(2008). Cryoinjury: a model of myocardial regeneration. Cardiovasc Pathol 17, 23-31.

Vega-Hernandez, M., Kovacs, A., De Langhe, S., and Ornitz, D.M. FGF10/FGFR2b
signaling is essential for cardiac fibroblast development and growth of the myocardium.
Development (Cambridge, England) 138, 3331-3340.

Whitehead, G.G., Makino, S., Lien, C.L., and Keating, M.T. (2005). fgf20 is essential for
initiating zebrafish fin regeneration. Science 310, 1957-1960.

112
Winter, E.M., and Gittenberger-de Groot, A.C. (2007). Epicardium-derived cells in
cardiogenesis and cardiac regeneration. Cell Mol Life Sci 64, 692-703.

Wu, S.M., Fujiwara, Y., Cibulsky, S.M., Clapham, D.E., Lien, C.L., Schultheiss, T.M.,
and Orkin, S.H. (2006). Developmental origin of a bipotential myocardial and smooth
muscle cell precursor in the mammalian heart. Cell 127, 1137-1150.

Zhou, B., Honor, L.B., He, H., Ma, Q., Oh, J.H., Butterfield, C., Lin, R.Z., Melero-
Martin, J.M., Dolmatova, E., Duffy, H.S., et al. Adult mouse epicardium modulates
myocardial injury by secreting paracrine factors. J Clin Invest 121, 1894-1904.

Abstract (if available)

Abstract Cardiovascular disease is the number one cause of mortality worldwide and when not fatal contributes to chronic morbidity and decreased lifespan. Treating cardiovascular disease is a medical priority and suitable treatments continue to elude the medical field. Increasing interest in adult tissue regeneration as a solution to the medical burden of adulthood disease, including cardiac pathologies, has occurred with the identification of adult stem cells and organ regeneration in vertebrate animals. I used vertebrate animals as tools for studying cardiac regeneration. We utilized the adult mouse with a coronary artery ligation to evaluate the effects of Fgf10 on cardiac repair after ischemic injury. We also developed a neonatal mouse injury model to evaluate Fgf10 on the endogenous regeneration process, specifically the effect on epicardial cells. Additionally we developed a primary zebrafish epicardial cell culture for in vitro assays on the epicardial cells during regeneration. These tools allowed us to study the effect of a gene’s expression, in this study Fgf10, on the heart after injury and in endogenous repair/ regeneration processes. It was determined that Fgf10 does not have significant improvement on the cardiac regeneration process since it did not reduce the fibrosis in the heart in adult or neonatal mice, but does have an effect on epicardial cell proliferation during the injury response. Overexpression of Fgf10 increased proliferation of the Wilm’s tumor -1 expressing cells after injury, resulting in increased epicardial cells in the injury site. The significance of influencing epicardial cells during injury corroborates prior publications indicating epicardium response to injury is beneficial to repair and makes it an ideal target for therapies. This study identified Fgf10 as candidate growth factor in improving the response of the epicardium to cardiac injury and suggests further evaluation is warranted.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Zebrafish as a blueprint for cardiac regeneration

PDF

Role of FGFR2b signaling pathway in the development of ectodermal derivatives

PDF

Epigenetic and genetic reprogramming during embryonic chicken feather bud morphogenesis, hair morphogenesis, and de novo hair regeneration

PDF

Developmental biology of epicardial adipose tissue

PDF

Investigating the role of cardiac fibroblasts in modulating angiogenesis during heart injury

PDF

Searching for mitogenic factors from the epicardium: PDGFA, Igf2 and more

PDF

Exploring the molecular and cellular underpinnings of organ polarization using feather as the model system

PDF

The role of fibroblast growth factor signaling on postnatal hepatic progenitor cell expansion

PDF

The roles of Tnni3k in heart regeneration, cardiac conduction system defects and cardiomyopathy

PDF

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

PDF

Retinoic acid and TGFβ signaling regulate cardiovascular development

PDF

Extracellular matrix regulation of mitochondrial function in engineered cardiac myocytes

PDF

Proliferation and maturation events in second heart field cells during cardiovascular development activated by the Delta like ligand-4 mediated notch signaling

PDF

Decoding the embryo: on spatial and genomic tools to characterize gene regulatory networks in development

PDF

Elucidating the role of neural crest specific Stat3 signaling in maintaining coronal suture patency during embryonic development

PDF

Investigation of butyrate’s effects on colonic stem cell development

PDF

Effect of vicrostatin on integrin based signaling molecules in cancer

PDF

The effects of prolonged fasting/ fasting mimicking diet (FMD) on CNS protection, regeneration, and treatment

PDF

Immunomodulatory and regenerative potential of amniotic fluid stem cells as a treatment strategy for pulmonary fibrosis

PDF

Study of bone morphogenetic protein-2 and stromal cell derived factor-1 in prostate cancer

Asset Metadata

Creator Rubin, Nicole (author)

Core Title Tools to study the epicardium's response during cardiac regeneration

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Pathobiology

Publication Date 04/29/2013

Defense Date 12/20/2012

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

Tag cardiovascular,epicardium,fgf10,ischemic injury,OAI-PMH Harvest,Regeneration

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hofman, Florence M. (committee chair), Kaartinen, Vesa (committee member), Lien, Ching-Ling (Ellen) (committee member), Maxson, Robert E., Jr. (committee member), Widelitz, Randall B. (committee member)

Creator Email kitnnicole@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-247186

Unique identifier UC11287910

Identifier etd-RubinNicol-1589.pdf (filename),usctheses-c3-247186 (legacy record id)

Legacy Identifier etd-RubinNicol-1589.pdf

Dmrecord 247186

Document Type Dissertation

Rights Rubin, Nicole

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA