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From the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm Sweden
Heart Regeneration: Lessons from the Red Spotted Newt
Nevin Witman
Stockholm 2013
All previously published papers herein were reproduced with permission from the publisher. Printed in Sweden by Universitetsservice US-AB Stockholm 2013. Distributor: Stockholm University library © Nevin Witman, 2013 ISBN 978-91-7447-726-9 Front Cover: Shows a photomicrograph of a regenerated newt ventricle immunolabled with MHC (red) and BrdU (green) at 65dpi.
ABSTRACT Unlike mammals, adult salamanders possess an intrinsic ability to regenerate
complex organs and tissue types, making them an exciting and useful model to study
tissue regeneration. The aims of this thesis are two fold, (1) to develop and
characterize a reproducible cardiac regeneration model system in the newt, and (2) to
decipher the cellular and molecular underpinnings involved in regeneration.
In Paper I of this thesis we developed a novel and reproducible heart
regeneration model system in the red-spotted newt and demonstrated for the first time
the newt’s ability to regenerate functional myocardial muscle, following resection
injury, without scarring. The observed findings coincide with an increase in several
developmental cardiac transcription factors, wide-spread cellular proliferation of
cardiomyocytes and non-cardiomyocyte populations in the ventricle and reverse-
remodeling at later time points during regeneration. Of further interest was the
identification of functionally active Islet1+ve and GATA4+ve cardiac precursor cells in
and around regenerating areas. The observation of such cell types further compels the
similarity between mammalian cardiac development and newt cardiac regeneration
and justifies these animals as suitable model organisms for studying heart
regeneration.
In Paper II we wanted to decipher the molecular cues possibly driving
cardiac regeneration in newts. Here we used qualitative and quantitative methods to
delineate the function microRNAs (miRNAs) have in this process. One interesting
candidate, namely miR-128, a known tumor suppressor miRNA and regulator of
myogenesis, was found to have a regulatory role in controlling hyperplasia during
newt cardiac regeneration. We show that treating regenerating newt hearts with
miR128 antagomirs evoked an increase in cellular proliferation within non-
cardiomyocyte populations. Of further interest is the discovery of a novel binding site
of miR-128 in the 3’UTR of Islet1. We speculate that the natural increase in miR-128
expression levels during newt cardiac regeneration functions as a fine-tuning
mechanism to control cellular proliferation of precursor cells.
The question of how newts regenerate has long been postulated. It has been
thought that although gene products found in the newt are highly homologous to those
of mammals, there may be functional differences. It has also been debated whether
newts are able to more efficiently and effectively utilize transcriptional or post-
transcriptional modifiers to diversify their gene pool. In Paper III of my thesis we
sought to examine these questions by exploring if a link exists between RNA editing,
a wide-spread post-transcriptional process and regeneration. We observed that A-to-I
editing enzymes (ADARs) are present and active in regenerating newt tissues. In
regenerating tissues, at times concomitant with increased cellular proliferation, we
discovered a localized nuclear to cytoplasmic shift of ADAR1 expression. This
activity of ADAR1 during regeneration may be partly responsible for driving the
cellular plasticity that is needed during multiple phases of tissue regeneration in the
red-spotted newt.
The major findings of this thesis can be summarized as follows: that the red
spotted newt is able to regenerate cardiac tissue, in the absence of scarring, and
restore function to the heart following resection injury. The response of the newt heart
to injury is governed by an increase in known developmental cardiac transcription
factors, which peak at a time when cellular proliferation is also heightened. We have
also shown that miRNAs are differentially expressed during various phases of heart
regeneration. One novel candidate, miR-128, appears to have some control over
proliferating cell types in the heart. We also show that Islet1 is a novel target of miR-
128, indicating that miR-128 may also contribute to the re-specification of
differentiating cardiac precursors. Finally, we observe that ADAR enzymes are
functionally active in regenerating tissue. This implies a notion that RNA editing may
act as a global regulator involved in several aspects of tissue regeneration. These
discoveries provide additional information as to understanding the molecular
pathways that are activated during regeneration and may one day help to amend the
regeneration deficiencies that are found in humans.
TABLE OF CONTENTS
LIST OF PUBLICATIONS ..................................................................................................... 7 LIST OF ABBREVIATIONS .................................................................................................. 8 PROLOGUE ............................................................................................................................. 9 1. REGENERATION ............................................................................................................. 11
1.1 ORGAN REGENERATION IN A HISTORICAL PERSPECTIVE.................................................. 11 1.2 TYPES OF REGENERATION ............................................................................................... 13
1.2.1 Reparative regeneration ...................................................................................... 14 1.2.1.1 Cellular regeneration ................................................................................................... 15 1.2.1.2 Tissue regeneration ...................................................................................................... 15
1.2.2 Physiological regeneration .................................................................................. 15 1.2.3 Hypertrophic regeneration ................................................................................. 16
2. NEWT AS AN IDEAL REGENERATION MODEL ..................................................... 17 2.1 ECOLOGY AND HABITAT ................................................................................................. 17 2.2 WHY THE NEWT IN RESEARCH ON REGENERATION? ........................................................ 17 2.3 EXAMPLES OF NEWT REGENERATION .............................................................................. 18
2.3.1 Limb regeneration ............................................................................................... 19 2.3.2 Lens regeneration ................................................................................................ 19 2.3.3 Spinal chord and neuronal regeneration ........................................................... 20
3. CARDIOGENESIS ............................................................................................................ 22 3.1 VERTEBRATE CARDIAC DEVELOPMENT .......................................................................... 22
3.1.1 Cardiac progenitors and organogenesis ............................................................. 22 3.1.2 Transcriptional regulation .................................................................................. 24
3.2 CARDIOVASCULAR DISEASE ........................................................................................... 25 3.3 CARDIAC THERAPIES ...................................................................................................... 26
3.3.1 Stem cells .............................................................................................................. 26 3.3.2 Reprogramming ................................................................................................... 28
4. MODEL ORGANISMS OF HEART REGENERATION .............................................. 29 4.1 MAMMALS...................................................................................................................... 29 4.2 FISH ................................................................................................................................ 30 4.3 AMPHIBIANS ................................................................................................................... 31
5. MICRORNAS ..................................................................................................................... 35 5.1 MICRORNA BIOGENESIS ................................................................................................ 35 5.2 MICRORNAS IN REGENERATION .................................................................................... 37 5.3 MICRORNAS AND CARDIAC REGENERATION .................................................................. 38
6. RNA EDITING ................................................................................................................... 42 6.1 A-TO-I RNA EDITING ..................................................................................................... 42
6.1.1 ADAR enzymes .................................................................................................... 42 6.2 EDITING SUBSTRATES ..................................................................................................... 44
6.2.1 Editing in protein-coding regions ....................................................................... 44 6.2.2 Editing of non-coding RNA ................................................................................. 45
6.3 FUNCTIONAL ASPECTS OF RNA EDITING ........................................................................ 46 7. PRESENT INVESTIGATIONS ........................................................................................ 48
7.1 AIMS .............................................................................................................................. 48 7.2 PAPER I ........................................................................................................................... 49
7.2.1 Results and discussion ......................................................................................... 49 7.3 PAPER II ......................................................................................................................... 51
7.3.1 Results and discussion ......................................................................................... 51 7.4 PAPER III ........................................................................................................................ 53
7.4.1 Results and discussion ......................................................................................... 53 7.5 FUTURE PERSPECTIVES ................................................................................................... 55
8. CONCLUSIONS ................................................................................................................ 58
9. ACKNOWLEDGEMENTS ............................................................................................... 59 10. REFERENCES ................................................................................................................. 63
7
LIST OF PUBLICATIONS
I. Nevin Witman, Barri Murtuza, Ben Davis, Anders Arner, and Jamie I.
Morrison (2011). Recapitulation of developmental cardiogenesis governs
the morphological and functional regeneration of newt hearts following
injury. Developmental Biology, 354:1. pp.67-76.
II. Nevin Witman, Jana Heigwer, Barbara Thaler, Weng-Onn Lui, and Jamie I.
Morrison (2013). miR-128 regulates non-myocyte hyperplasia, deposition
of
extracellular matrix and Islet1 expression during newt cardiac
regeneration. Manuscript under consideration by Developmental Biology. Original
submission date: June 7, 2013
III. Nevin M Witman, Mikaela Behm, Marie Öhman, and Jamie I. Morrison
(2013). ADAR-Related activation of adenosine-to-inosine RNA editing
during regeneration. Stem Cells and Development, 22:16. pp.2254-67
8
LIST OF ABBREVIATIONS
ADAR Adenosine Deaminases Acting on RNA
BrdU 5-bromo-2’-deoxyuridine
CVD Cardiovascular Disease
CHF Congestive Heart Failure
CDC Cardiac derived cell
CMC Cardiomyocyte
CPC Cardiac progenitor cell
DNA Deoxyribonucleic acid
dpi days post-injury
dsRBD double-stranded RNA binding domain
dsRNA double-stranded RNA
EPDC Epicardial derived progenitor cells
ESC Embryonic Stem Cell
FGF Fibroblast Growth Factor
FHF First Heart Field
GFP Green Fluorescent Protein
LAD Left anterior descending (artery)
SCI Spinal Chord Injury
SHF Secondary Heart Field
MI Myocardial Infarction
miRNA microRNA
mRNA messenger RNA
ncRNA non-coding RNA
NES nuclear export signal
NLS nuclear localization signal
lncRNA long non-coding RNA
RISC RNA induced silexing complex
RNA Ribonucleic acid
RNAi RNA interference
UTR Untranslated Region
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PROLOGUE The capacity of the human body to repair injured tissues and organs following
damage, disease or degeneration via the natural ageing process is limited and
decreases with age. The field of regenerative biology aims to repair, replace or in
someway augment damaged tissues and organs. At the time of this thesis, a great
majority of treatments for chronic and life threatening diseases are merely palliative.
That is, they are aimed at alleviating the symptoms associated with disease, not
repairing the injury.
An organ with a very limited ability to self-repair following injury is the heart,
with many cardiovascular diseases (CVD) becoming more prevalent with age. The
onset of vascular disease can be gradual, usually with weak symptoms until the onset
of an acute heart attack, which leaves the heart muscle damaged. Cardiac failure can
also develop as a result of several other conditions, e.g. genetic changes in the
musculature or hypertension. The damaged sustained from heart disease is often
irreversible and patients who are living with CVD may suffer a diminished quality of
life. Novel therapies need to be developed.
Unlike humans, some non-mammalian vertebrates such as fish and
amphibians have emerged as excellent models to study regeneration, as they are
natural regenerators. One such example of these amazing natural regenerators is the
red-spotted newt, Notophthalmus viridescens. In what seems like a boundless list,
newts can regenerate many complex organs and cell types including the limb, lens of
the eye, retina, jaw and neurons of the central nervous system. The regeneration of all
these organs and tissues are done intrinsically, without the delivery of stem cells or
transplantations. Furthermore, these aquatic salamanders do not seem to have a
decline in their regenerative capabilities throughout their adult life. Taken together
these characteristics make newts an ideal model to study the processes underlying
tissue regeneration.
The work carried out during my PhD tenure focused on mechanisms of
regeneration of the newt heart after a resection injury. The heart of the newt is able to
regenerate the myocardium following a resection injury during a 60-day period. The
magnitude of this injury casued by the resection corresponds to the functional loss of
heart cells in patients suffering from severe cardiac injury. Additionally I examined
gene-expression profiles of known cardiac genes during heart regeneration and show
that the newt heart regenerates following a proliferative response. I also examined
regulation of the newt heart response to injury at a molecular level, by studying the
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involvement microRNAs (miRNAs) had during the regeneration process. Finally I
studied a post-transcriptional regulatory mechanism, RNA editing, which may act to
some degree as a global regulator of tissue regeneration.
In the background of this thesis I will provide a detailed description of
regenerative research, including types of regeneration and mechanisms involved in
regenerating organisms. I will then highlight why the red-spotted newt is an excellent
model organism in which to study regeneration. Next, I will introduce the reader to
cardiac development and disease. The importance of cardiac therapies as well as
benefits from using model organisms to study heart regeneration will be discussed.
Subsequently, I will introduce non-coding RNAs, highlighting miRNAs as a
significant avenue for further exploring molecular mechanisms involved in
regenerative research. Lastly, I will briefly discuss the importance of RNA editing as
a post-transcriptional modifier that may be involved as a global regulator in tissue
regeneration.
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1. REGENERATION
1.1 Organ regeneration in a historical perspective
The regeneration of body parts has captured the attention of many different
cultures and mythologies for thousands of years. One of the earliest dated tales of
regeneration comes from Greek mythology, when Homer and Hesiod recount stories
of the half human, half god Prometheus. The story goes that after Prometheus stole
fire from Olympus to aide mankind, Zeus punished him by chaining him to a rock
where every night he would have his liver pecked out by an eagle. Doomed by
immortality, the following day his liver would regenerate and so he would endure the
same punishment night after night for thousands of years. Whether the Ancient
Greeks were aware of the livers natural ability to regenerate at that time is uncertain,
but ironically we know today the liver is an organ with a remarkable ability to
regenerate throughout adult life (Pack et al., 1962).
Some of the first documented scientific discoveries of regeneration were
recorded by the well know philosopher and scientist, Aristotle (384-322BC). In his
book “Historia Animalium” Aristotle notes that the tails of lizards and snakes are able
to regenerate (as cited in (Dinsmore, 1996)). Although we now know the regenerated
lizard tail is not a perfect replica, with many anatomical and functional differences to
uninjured tails (Ritzman et al., 2012).
Though there were observational scientific discoveries of regeneration in the
ancient times, experimental documentation of regeneration came much later. In 1712
René-Antoine Ferchault de Réaumur (1683-1757), a French scientist, reported a
descriptive study of limb and claw regeneration in crayfish (as cited in (Dinsmore,
1991)). He concluded that one possible explanation as to why crustaceans can
regenerate appendages and humans cannot regenerate theirs is because crustacean
appendages break easily at the joints. This fascinated other scientists of the 18th
Century such as Abraham Trembley, Charles Bonnet and Lazzaro Spallanzani.
Abraham Trembley (1710-1784) was a Swiss naturalist who would later
become known for his scientific discoveries of regeneration in freshwater polyps. He
discovered when this animal is bisected, the half containing the head will regenerate a
new tail and the half containing the tail a new head. These animals would later be
named Hydra, due to the regenerative characteristics they shared with the Greek
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mythological creature Hercules battled. Interestingly Hercules was also the hero who
freed Prometheus from his daily hepatectomies.
During the same time regeneration was being discovered in Hydra,
Trembley’s cousin Charles Bonnet (1720-1793) documented the annelid worm’s
capability to regenerate new segments multiple times following resection (as cited in
(Birnbaum and Alvarado, 2008)). He noted the rate in which a part regenerated
correlated to the temperature in which the organism was kept, i.e. the cooler the
temperature the slower the regenerative response. Although Bonnet dabbled in
regeneration biology for a few years, he is perhaps more well known for his discovery
of parthenogenesis – a form of asexual reproduction, which he discovered whilst
studying plant lice (aphids). This concept would later fuel scientists to learn more
about embryo development (Davis, 2012).
In 1769 an Italian scientist named Lazzaro Spallanzani (1729-1799) would be
the first to describe detailed studies on vertebrate regeneration. He showed that
amphibians, such as prematamorphic frogs and toads could regenerate their tails. He
would also be the first to show that salamanders possessed a remarkable ability to
regenerate their limbs, tails and jaws. Spallanzani sketched and documented the
formation of a thin round stump near the injury site that formed shortly after
amputation. Today we know this as the blastema, a small bud-like structure
containing a cell mass which gives rise to the new structure (Tsonis and Fox, 2009).
Regeneration would even attract the attention of the well-known British
scientist, Charles Darwin (1809-1882). On Darwin’s famous voyage aboard the
Beagle he observed the regenerative properties of several species of planarian.
However, even Darwin’s work in this area remained primarily descriptive. In the
years to come questions around regeneration would be depicted in higher resolution
and more detailed experimental studies would surface.
Thomas Hunt Morgan (1866-1945) was an American evolutionary biologist
and is well known for receiving the Nobel Prize in medicine in 1933 for discoveries
linking the chromosome and heredity. However, Morgan also explored regenerative
research and believed that by studying regeneration phenomena one could learn more
about the development of organisms. He hypothesized this because he felt that
regeneration, like development, required differentiation of cell types and thus was part
of a fundamental growth process, (as cited in (Esposito, 2013)). This view was at
arms with other theoretical positions of the time, such as those belonging to August
Weismann (1834-1914), which pinned regeneration as a special adaptation to some
13
organisms, (as cited in (Esposito, 2013)). The latter theory coincides with the notion
that lower organisms adapted regenerative traits particularly in organs that were most
frequently subject to injury. In one investigation Morgan provided evidence against
Weismann’s theory by showing hermit crabs (Eupagurus longicarpus) were able to
regenerate their distal limbs following amputation, even though distal limbs were not
prone to injury (as cited in (Esposito, 2013)). Whether or not regeneration is an
adaptive trait or an evolutionary trait lost in more complex organisms remains widely
debated amongst developmental biologists even today.
So why is the evolution of regeneration important? If regeneration is
evolutionarily ancestral across metazoan species, there should exist conserved traits
making it possible to coax repair mechanisms in mammals to better regenerate.
However, if regeneration has been adapted independently of evolution across different
species, then understanding the species-specific selectivity of regeneration may also
be helpful in identifying new molecular activities that could be adapted in human
molecular pathways.
1.2 Types of regeneration
It was Thomas Hunt Morgan who first began to standardize terminology in
regenerative biology (as cited in (Sanchez Alvarado, 2000)). He classified the
regeneration of Metazoans into two basic groups 1) regeneration that occurs via
rearrangement in the absence of cell proliferation and 2) regeneration that requires
cellular proliferation. The first is referred to as Morphallaxis, a term coined by
Morgan in 1901, which describes the replacement of a missing part via the
reorganization of pre-existing parts without the need for cellular proliferation.
Morphallaxis is most commonly seen in several species of invertebrates such as
Hydra. Although it had previously been shown that Hydra can regenerate parts of the
body when DNA synthesis is inhibited, more recent work supports the notion of
proliferating cells with a blastema-like feature that contribute to the regenerative
process (Chera et al., 2009; Cummings and Bode; 1984, Hicklin and Wolpert, 1973).
Another model organism considered to undergo morphallactic regeneration is
the planaria. Planaria are flatworms with bilateral symmetry and a remarkable ability
to regenerate. Morgan himself demonstrated the ability of these flatworms to
regenerate a whole animal from as little as 1/279th the body (as cited in (Sanchez
Alvarado, 2000)). Recent research has shown a small population of pluripotent stem
cells named neoblasts undergo proliferation and differentiation to replace lost tissue
14
following resection in the planaria (Wagner et al., 2011). Indeed the reorganization of
tissue does occur in regenerating organisms making these two terms not mutually
exclusive. However it would more recently appear that cellular proliferation is a
requirement for most regenerating organisms, making the strict term of morphallaxis
slightly outdated (Agata et al., 2007).
According to Morgan’s specific definition, the second group of regeneration,
epimorphosis, refers to the case of regeneration where the proliferation of material
precedes and interacts with the development of the new part, (as cited in (Reddien and
Alvarado, 2004)). There is a broad range of divergent species capable of epimorphic
regeneration including worms (Wagner et al., 2011), insects (Anderson and French,
1985) and starfish (Thorndyke et al., 2001). Here the activation of proliferating cells
is the underpinning regulatory effect in restoring resected or damaged tissue. The
most prominent examples of epimorphic regeneration are seen in urodele amphibians
(see section on newt limb regeneration).
To date the classification of regeneration has been slightly modified.
Epimorphic regeneration is generally discussed as reparative regeneration that is
mediated through a blastema, utilizing a mechanism involving dedifferentiation,
proliferation and transdifferentiation (Sanchez Alvarado, 2000). Transdifferentiation
describes the process of a committed cell type in one tissue lineage that is able to
convert into a cell of an entirely distinct lineage. These cells lose their tissue-specific
markers along with their tissue specific function of their previous lineage type and
acquire the onset of new markers and functions in the transdifferentiated cell type.
One mammalian example of transdifferentiation occurs during development of the
oesophagus. It has been shown that smooth muscle cells in developing rodents can
switch phenotype favoring that of skeletal muscle immediately after birth
(Patapoutian et al., 1995). In lower vertebrates perhaps the most prominent and well
known example of transdifferentiation occurs during lens regeneration in the newt
(see section on newt lens regeneration and reviewed extensively (Barbosa-Sabanero et
al., 2012)).
1.2.1 Reparative regeneration
The common element in reparative regeneration is generally the exact
functional replacement of a lost or resected part of the body, with no scarring. Aquatic
salamanders, such as the red-spotted newt (Notophthalmus viridescens) possess much
more extensive regenerative capabilities than mammals. Amongst them is cellular
15
dedifferentiation – a process that involves the regression of a differentiated cell to a
simpler, more embryonic-like form (Jopling et al., 2011). Following injury,
dedifferentiation can create a milieu of stem cell-like cells that can proliferate and
undergo re-differentiation to form mature tissue specific cell types. Reparative
regeneration can occur at the single cell level or occur in multicellular body parts.
1.2.1.1 Cellular regeneration
Apart from neurons, few mononuclear cell types are known to regenerate or
repair in mammals. Axonal transections lead to a short period of degeneration,
followed by an extension phase providing new cytoplasmic material and interaction
with its immediate environment (Gillen et al., 1997). Following pathological or
alterations in physiological conditions, oligodendrocyte progenitors have been shown
to proliferate and provide mature oligodendrocytes (Barnabe-Heider et al., 2010).
1.2.1.2 Tissue regeneration
The replacement of damaged or worn out tissue such as bone and skeletal
muscle often rely on cells that are not the mature, dominant cell type in order to
regenerate. For example, satellite cells are adult, tissue-specific stem cells and are the
major contributors to the regeneration of mammalian skeletal muscle (Collins et al.,
2005; Mauro, 1961). Although these cells have been shown to give rise to the
myofiber component of skeletal muscle, muscle regeneration often involves a number
of other cell types, such as fibroblasts, endothelial cells, smooth muscle cells and
Schwann cells (Mauro, 1961).
1.2.2 Physiological regeneration
Physiological regeneration within adult vertebrates generally occurs in organs
or tissue sources that require the reconstitution of differentiated cell types that are
generally short-lived. In mammals, the most prominent examples include the
replacement of blood lineage cell types, skin cells and the gut epithelium, which rely
on regular homeostatic replacement. Other mammalian and non-mammalian examples
include the shedding of exoskeletons in crustaceans and snake skin, the annual cycle
of feather replacement in birds and the regrowth of deer antlers (Stoick-Cooper et al.,
2007). With the large variation of examples of physiological regeneration, what
categorizes the phenomena is reconstitution in order to meet physiological needs to
ultimately maintain the equilibrium of tissues.
16
1.2.3 Hypertrophic regeneration
Most adult organisms have some means of dealing with injury, damage or
disease even if complete tissue regeneration is no alternative. A regulatory process
developed in most mammals (including humans) to deal with such loss or damage
includes the process of hypertrophic responses. For example, humans can survive up
to two-thirds hepatectomy, which results in severe remodeling and an increase in
overall mass in the remaining lobe(s) of the liver in order to compensate for the
increase in functional demand (Michalopoulos, 2007). In mammals, and apart from
the liver, some smooth muscle organs also elicit hypertrophic responses to damage or
disease. Following urinary infection or urinary outlet obstruction in the rat, the
smooth muscle cells that make up muscle bundles of the bladder become larger and
longer. Although no mitoses are generally found, cells with two nuclei are often
present (Gabella and Uvelius, 1990). In mammals, nephrectomy results in a
compensatory renal enlargement of the other kidney (Nagaike et al., 1991). Toxic
damage to the kidney is often counteracted by the growth of the remaining nephrons,
however some evidence supports the presence of kidney stem cells (Reule and Gupta,
2011). Hypertrophic regeneration generally encompasses a restorative response
throughout the entirety of the organ, as opposed to a regenerative response that only
replaces tissue at the precise site of damage.
An adult organism with a remarkable ability to regenerate many complex
tissue types is the red-spotted newt and will be discussed in greater detail in the next
section.
17
2. NEWT AS AN IDEAL REGENERATION MODEL
2.1 Ecology and habitat
Newts are aquatic vertebrates that belong to the Caudata group of urodele
amphibians. Urodele amphibians make up the group of metazoans containing newts
and salamanders, which sets them apart from frogs as they retain their tails as adults.
In particular, the red-spotted newts (Notophthalmus viridescens) (Rafinesque, 1820)
are studied extenisvely in experimental biology due to their natural abilities to
regenerate. These newts are inhabitants of North-Eastern America. They are known
for having a complex life cycle that encompasses three distinct life stages: aquatic
larva or tadpole, red eft or terrestrial juvenile and aquatic adult. Larval newts hatch
with external gills, short front legs but the hind legs are absent. Within a week of
hatching the larvae become active, feeding on small aquatic life and grow quickly
(Hamilton, 1940). During larval metamorphosis the animals reabsorb their gills,
develop lungs and emerge from the water as terrestrial juveniles (Shi and Boucaut,
1995). The juvenile newt usually appear orange in color with bright red spots and this
stage of the life cycle can last anywhere from two to seven years, although two to
three years on land is most common (Hurlburt, 1969). The newt may migrate
relatively far distances until finding a suitable aquatic habitat (Gill, 1978). Once the
newts have discovered a new suitable environment they undergo a second form of
metamorphosis, after which they are sexually mature adults and appear olive-green in
color but keep their juvenile red spots (Figure 1) (Brockes and Kumar, 2005). Newts
will spend the majority of their adult life primarily in water. In the wild newts are
estimated to live up to 15 years, but in captivity newts have been reported to live
longer- an impressive life-span for an animal of its size (Hillman et al,. 2009).
2.2 Why the newt in research on regeneration?
By utilizing organisms that retain the ability to regenerate complex body parts
and tissue-types throughout adult life, scientists can construct model systems in the
hope of understanding natural regeneration and thus understand why it may be
blocked in humans. Contrary to mammalian organisms, which have very little
postnatal regenerative ability, urodele amphibians such as the newt are an ideal model
system for studying regeneration. Urodele amphibians share similar biological
pathways with mammals, thus allowing comparative analysis to be performed with
18
humans. Additionally, by comparing how mammals and salamanders respond to
injuries we can identify pertinent genes or proteins that can either be introduced or
inhibited in the mammalian system in the hope that regeneration efficiency can be
increased. The majority of somatic cell types found in the newt, including those that
make up the heart, express similar markers as those found in humans. Furthermore, as
mentioned previously the newt undergoes several stages of metamorphosis, ultimately
providing us with a comparative adult-vertebrate model system.
Figure 1. Photograph of an adult red spotted newt. An adult newt submerged in an aquatic environment. Note the olive-green color and characteristic red spots. The adult newt grows to approximately 8cm in length with an average weight of approximately 2g. Photograph by Nevin Witman.
2.3 Examples of newt regeneration
Salamanders have been of great interest to experimental biologists, beginning
with the work of previously mentioned Lazzaro Spallanzani. In this section I will
concentrate on discussing some of the most well characterized models of regeneration
in red-spotted newts.
19
2.3.1 Limb regeneration
Newt limbs, whether distally (mid-ulna and radius bone) or proximally
amputated (mid-humerus bone), proceed through three timed stages of regeneration
(Iten and Bryant, 1973). Following amputation, migrating epidermal cells quickly
cover the amputation surface forming the wound epithelium, which protects the
internal structures from the external environment and delivers the signals necessary to
initiate and maintain regeneration. Dedifferentiated cells become concentrated just
below the newly formed wound epidermis that produces a bud-like growth at the tip
of the limb stump called the blastema, which becomes vascular and neurogenic as it
matures (Rageh et al., 2002). The rapid morphologic outgrowth of the blastema leaves
the amputated limb structurally identical to that of the uninjured limb, although
slightly smaller than the original. The regenerate will continue to grow in size until
reaching the same dimensions of an uninjured limb. The exact origin and identity of
the dedifferentiated cells in the blastema remain uncertain, but it was long believed
these cells are multipotent with no lineage restrictions. Interestingly, it was shown
that amputating the forelimbs stimulates a population of satellite cells, which
contribute to new limb tissues in a similar manner to mammalian repair (Morrison et
al., 2006). Moreover through the use of GFP transgenic tracking experiments in
another salamander, the axolotl (Ambystoma mexicanum), a study showed that the
mass of undifferentiated progenitors from each tissue source produces progenitors
that are lineage restricted (Kragl et al., 2009). Further work is necessary to fully
understand the origin of cells needed to regenerate the full complement of cells
present within the newt limbs. Worth mentionining is additional work that has shown
limb regeneration is nerve dependent. A newt-specific secreted nerve factor called
anterior gradient protein (nAG) rescues regeneration in de-nervated newt limbs
(Kumar et al., 2007). This nerve factor is expressed in Schwann cells of the mylinated
nerve sheaths and has a role in improving blastemal cell proliferation in vitro.
2.3.2 Lens regeneration
Adult newts possess the ability to regenerate a new lens following lentectomy
(Tsonis and Del Rio-Tsonis, 2004). Following removal of the lens, pigmented
epithelial cells of the iris transdifferentiate. The epithelial cells of the dorsal iris first
dedifferentiate and subsequently change phenotype by differentiating into lens cells.
However, the same epithelial cells from the ventral iris do not appear to undergo these
events.
20
In an effort to address the question of whether ageing or repetitive injury
stunts tissue regeneration, this same group conducted a 16 year-long experiment on
lens regeneration (Eguchi et al., 2011). No significant changes in growth rates or sizes
of regenerated lenses were reported upon repeated removal of the lens after they had
re-grown. Additionally, no significant differences were observed in morphological
and gene expression characteristics compared between control animals that had never
undergone regeneration and animals undergoing repetitive regeneration. The findings
signify regenerative capabilities are not diminished upon repetition in aged animals.
Fibroblast growth factors (FGFs) have additionally been shown to elicit an effect on
lens regeneration. For example, inhibition of FGF-1 blocks lens regeneration from the
dorsal iris, yet the up-regulation of FGF-1 stimulates excess dedifferentiation of the
iris and retina cells (Del Rio-Tsonis et al., 1998).
2.3.3 Spinal chord and neuronal regeneration
Adult newts possess the ability to recover function following damage to the
spinal cord. Following a complete transection injury, the newt spine and tail
regenerates, with the animals regaining use of their hind limbs in as little as 4 weeks
(Davis et al., 1990). The exact mechanisms of spine regeneration are unclear, but
some elegant work suggests that subsequent to tail amputations a blastema forms, the
spinal cord elongates and axons restore connections as they grow into the newly
developing tissues (Singer et al., 1979). The process appears to be governed in a
manner that reiterates developmental programming and processes. A more recent
model has been developed to follow spinal cord injury (SCI) in newts without the
need to perform tail amputations, with the idea being that this type of SCI mimics
more closely the spinal injuries most commonly seen in humans (Zukor et al., 2011).
Following SCI in the newt, an environment consisting of a loose extracellular matrix
permits axon regeneration across the lesion. However, following SCI in humans, the
environment of the injury site becomes inhibitory to regeneration due to the formation
of dense scarring (Norenberg et al., 2004). Of further interest is the recent discovery
that transplanted cultured neurospheres can contribute to the regeneration of the
damaged spinal chord (McHedlishvili et al., 2011).
To determine tissue regenerative abilities in the newt brain, previous studies
attempted to surgically resect small portions of the frontal lobe. After 8 months it was
reported that most the resected area had been restored, but the origin of new cells was
not thoroughly investigated (Okamoto et al., 2007). Additionally it has been shown
21
that newts can regenerate neurons of the central nervous system following toxin
induced cell death (Parish et al., 2007). The authors here reported a 30-day post-lesion
regeneration time and the presence of BrdU+ve dopaminergic neurons, indicating
regeneration fueled by neurogenesis. Interestingly the same group has recently shown
the importance of dopamine as a senescence activator of stem cells and also a
controller of neuronal homeostasis (Berg et al., 2011).
Over the last 10 years the heart has become another organ of interest in
regenerative biology. I will next discuss why heart regeneration has gained significant
attention in the field of regeneration as well as present current research and
considerable discoveries within the field.
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3. CARDIOGENESIS
“How can you describe this heart in words, without filling a whole book?” -Leonardo da Vinci, 1513
(Note left by Leonardo da Vinci next to an anatomical sketch of a human heart.)
As this quote illustrates, Leonardo da Vinci was highly fascinated by the heart.
He would become one of the first to recognize the heart as a muscle and begin to
provide detailed medical illustrations of the heart. Apart from discovering anatomical
and physiological characteristics of the heart, da Vinci observed post-mortem
degeneration to the heart and blood vessels in the elderly thus linking the ageing
process to degeneration. A better understanding of developmental cardiac biology
offers the potential to broaden current knowledge of mechanisms involved in both
acquired and congenital heart diseases.
3.1 Vertebrate cardiac development
To discuss vertebrate heart development in great detail across vertebrates
would be too extensive for this thesis. More in-depth information regarding distinct
comparisons between cell types and transcription factor networks across vertebrate
heart development have been made previously (Pandur et al., 2013). However, I will
concentrate on the more general findings, features and mechanisms between
vertebrates, with the main focus relating to mammalian heart development.
3.1.1 Cardiac progenitors and organogenesis
During vertebrate embryonic development the heart is the first organ to form
and function, and is paramount for survival during embryonic life. The precise
development of the vertebrate heart requires highly controlled cellular organization.
The migration and differentiation of cardiac progenitor cells contribute to the
morphology of the heart and it’s distinct compartments. The events leading to heart
formation in vertebrates is homologous. The sequence of events include the formation
and looping of the heart tube, elongation and growth via the extensive addition of
progenitor cells and morphogenesis of the cardiac chambers, cushions and valves
(extensively reviewed (Vincent and Buckingham, 2010)).
In zebrafish, it has been shown that myocardial lineages separate during
midblastula stage and will provide cells needed to create atrial and ventricular
23
myocardium (Stainier et al., 1993). Notable discoveries using the zebrafish have
helped to further describe the cellular and molecular mechanisms activated during
vertebrate heart development (Bakkers, 2011).
In amphibians and mammals two myocardial lineages appear to diverge from
a common progenitor, which contribute to two distinct phases of heart development,
the first heat field (FHF) and the second heart field (SHF) (Figure 2A) (Brade et al.,
2007; Meilhac et al., 2004). The FHF makes up the left ventricle and partial atria
formation, whereas the adjacent SHF makes up the right and left ventricle as well as
the outflow tract (Figure 2) (Buckingham et al., 2005; Kelly et al., 2001). However, it
is speculated that both lineages probably share in some common contributions to
other regions of the heart. Interestingly the FHF is a focused assortment of
differentiating cell types and morphological growth, whereas the SHF actively
contributes proliferating cardiac precursors in the early stages of heart development.
Figure 2. Diagram depicting the major steps during the development of the vertebrate heart. First a cardiac crescent is formed which comprises of two heart fields that contain different populations of cardiac precursors. The first heart field (FHF, red) contributes to the left ventricle (lv) and the secondary heart field (SHF, blue) contributes to the right ventricle (rv) left and right atria (la, ra), and outflow tracts (ot). From (Bruneau, 2008), modified and reprinted with permission.
The cells that make up the early heart originate from mesoderm lineage. These
cells form the primitive streak at the midline of the embryos, creating a contracting
linear heart tube (Figure 2B). The linear tube will proceed to expand and undergo
major twisting and looping events, while septa and valves begin to form between the
chambered compartments of atria and ventricles (Figure 2C). The heart will continue
to undergo morphological changes and maturation events during later stages of fetal
life (Figure 2D).
A B C D
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T-box transcription factors are expressed in the cells that make up the
primitive streak and first reveal a cardiac niche (Costello et al., 2011). Mesp1 has also
been shown to be a primitive marker of cardiac progenitor cells and may be a master
regulator for early cardiac cell fate (Bondue et al., 2008). Several other early stage
markers of cardiac progenitors include members of the GATA family of zinc finger
transcription factors (i.e. GATA 4/5/6) and the NK-2 class of homeodomain-
containing transcription factors (Nkx2-5). These are amongst the earliest known genes
to be expressed in cells facing cardiac lineage fate and their expression plays
paramount roles in heart formation (Peterkin et al., 2005).
The LIM homeodomain family Islet1 is a critical transcription factor with
essential roles in cardiac development, specifically the SHF. Although recently Islet1
protein has been found in the cardiac crescent, which suggests an earlier role during
the FHF (Prall et al., 2007). Evidence supporting the importance of this transcription
factor was previously reported, showing that murine Islet1 knockouts have severely
deformed hearts (Cai et al., 2003). Despite the function of Islet1 previously being
defined as a progenitor marker recent data suggests Islet1 may regulate multi-potency
of epicardial cells into cell types harboring mesenchymal features (Brønnum et al.,
2013; Bu et al., 2009; Zhou et al., 2008).
It was generally believed that the generation of endothelial, cardiac and
smooth muscle cells arise from distinct embryonic precursors during cardiogenesis.
However, it has recently been demonstrated that Islet1 progenitor cells can generate
all three cardiovascular lineages (Moretti et al., 2006). A subset of Islet1 positive
undifferentiated cells are detected shortly after birth as well as in the postnatal heart in
rodents and humans (Laugwitz et al., 2008; Laugwitz et al., 2005). Islet1 has not been
detected in terminally differentiated cardiomyocytes in the adult mammalian heart.
However, these cells can develop into functional cardiomyocytes in vitro based on
their marker gene expression, contractile properties and intracellular calcium release
(Laugwitz et al., 2005).
3.1.2 Transcriptional regulation
The activation of genes encoding cardiogenic transcription factors
meticulously manages the development of the heart along with its endless function.
Transcription factors are required for regulating cardiogenesis and morphogenesis, as
well as components regulating contractility (Olson, 2006). For example, the
expression of Nkx2-5 is a paramount transcription factor critical for terminal
25
differentiation of myocardial cells (Lyons et al., 1995). It was more recently shown
that Nkx2-5 expression functions to maintain a balance between progenitor
proliferation and differentiation (Prall et al., 2007).
Serum response factor (SRF) and the GATA factors have been shown to
regulate the expression of multiple cardiac genes and initiate cardiac differentiation
(Niu et al., 2008; Zhao et al., 2008). The heart and neural crest derivatives (HAND)
basic helix-loop-helix family of transcription factors control aspects of chamber
differentiation (Srivastava et al., 1997). The HAND proteins are selectively expressed
in adult ventricular CMCs (HAND1 and HAND2) and atrial CMCs (HAND2), which
are severely down-regulated in patients with cardiomyopathies (Natarajan et al.,
2001).
Often cardiac transcription factors work synergistically, by regulating gene
expression to co-regulate heart development (He et al., 2011). Networks of these
interacting transcription factors are slowly being defined, however their full
complement of target genes has yet to be determined. One example is the ability for
Nkx2-5 to interact with the GATA factors, which can regulate cardiac genes such as
Mef-2c and connexin40 (Dodou et al., 2004; Linhares et al., 2004). Gene and protein
expression involved in signal transduction pathways including transcriptional
programs, can be severely affected following biomechanical stress to the heart. As
such, developmental signaling pathways (which involve cardiogenic transcription
factors) and stem cells are emerging as novel promising candidates to study in relation
to improving the treatment of cardiovascular disease.
3.2 Cardiovascular disease
Cardiovascular diseases include a broad range of diseases that affect the
cardiovascular system, which comprises the heart and blood vessels. Cardiovascular
disease continues to account for more deaths then any other disease worldwide.
Ischemic heart disease is the most common debilitating cardiac injury in humans,
which often results in heart failure. Ischemia is usually caused by coronary artery
obstruction and leads to a myocardial infarction (MI). An MI occurs when there is an
interruption of blood supply to the heart, causing heart cells to die due to lack of
oxygen. The subsequent loss of cardiac cell number along with ischemic induction of
necrosis, results in the replacement of damaged heart muscle with fibroblasts, which
help to form a fibrotic scar. Even though the scar tissue elicits some mechanical
support shortly after the infarct, it predisposes for arrhythmias. Additionally, the
26
inability for the newly formed scar formation to contract consequently diminishes the
overall function of the heart and if left untreated can lead to heart failure and death
(Anversa et al., 2006). It is interesting to note that heart attacks were very uncommon before the 20th
Century. It was in 1912 that James Herrick (1861-1954), an American physician
would become one of the first physicians to describe heart disease as a result of
hardening of the arteries (James, 2000). Since then, the global scourge of heart
disease has been described as a pandemic. With this said, rather then relying on heart
transplants it has become essential that we produce novel therapies to combat heart
disease.
3.3 Cardiac therapies
The field of regenerative medicine is rapidly evolving, with much hope
recently being placed into stem cell therapies and molecular interventions. Stem cells
are cell types which exist in many organs of the human body, they are self-renewable
and act to replace worn out or damaged cells. Stem cell transplantations are being
widely used across the globe where injection into the diseased part of a body can
stimulate an endogenous healing response or be used to replace damaged or diseased
cells and tissues. Ultimately, scientists are turning to stem cells to replace damaged
and dead cardiomyocytes (CMCs), the major “powerhouse” cell type of the heart.
3.3.1 Stem cells
Embryonic stem cells (ESCs) are perhaps the most suitable cell type for
cardiovascular regeneration, as they are self-renewing and pluripotent. ESCs are
capable of giving rise to all three embryonic germ layers (the mesoderm, the
endoderm and the ectoderm) and therefore retain the potential to differentiate into any
of the functionally mature cell types of the human body (Thomson et al., 1998).
Along with the legal and ethical hurdles that are currently limiting the use of ESCs,
there are many technical issues to overcome. These challenges include the viability of
the transplanted cells that may lead to aggressive tumor formation, coupling of ESCs
to host cardiomyocytes and overcoming susceptibility to immunological rejection
(Yoon et al., 2005). Although there have been some reports of injected ESCs
improving rodent heart contractility and diminished scar sizes following ischemic
injuries, together the ethical and technical obstacles have hindered the use of ESCs for
in vivo myocardial regeneration in humans (Min et al., 2003; van Laake et al., 2007).
27
The presence of adult cardiac stem cell / progenitor cell (CPCs) populations in
the heart have been reported by several groups. Such cell types have been identified
based on different cell surface markers and in vitro assays (Beltrami et al., 2003; Cai
et al., 2003; Matsuura et al., 2004; Messina et al., 2004; Smart et al., 2011). The use
of such cell types for the potential generation of de novo cardiomyocytes are looking
promising, however the mechanisms by which CPCs facilitate improvement in the
heart may be paracrine affiliated (Gnecchi et al., 2008; Huang et al., 2011). For a
detailed overview of cardiac progenitor cell biology and other adult stem cell
therapies, the reader may refer to the following review (Leri et al., 2011).
Of even greater interest, and avoiding some of the ethical implications that are
embryonic stem cell accountable, is the ability of cardiac derived cells (CDCs) to
form 3-dimensional cardiospheres. Explant cultures of surgical or endomyocardial
biopsies can be cultured in order to isolate cardiospheres (Messina et al., 2004; Smith
et al., 2007). Cardiospheres form spherical clusters of cardiac progenitor cells that are
clonogenic and undifferentiated cells (Figure 3) (Davis et al., 2009). Of critical
importance is the fact that CDCs can be expanded many times over on fibronectin
coated plates, providing suitable cell numbers for cell therapies. Very recently the
cardiosphere approach was used in a phase I clinical trial that yielded positive results
(Makkar et al., 2012). Following delivery of CDCs in a randomized trial, patients with
MI showed improved ejection fractions, reduction in scar formation and increases in
heart mass after 6 months.
Figure 3. Examples of cardiospheres immunostained with different cardiac antigens. Cultured cardiac explants can form 3-dimensional spherical clusters of cells that can either express myogenic lineages such as (A) myosin heavy chain (MHC) and (B) cardiac troponin I (cTnI), or express progenitor cell markers such as Nkx2.5 (C). From (Davis et al., 2009). Reprinted with permission from PLOS.
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3.3.2 Reprogramming
The technique of reprogramming adult cells into pluripotent cells or into an
alternative cell type could hold great potential for regenerative therapies. The
technology to convert one cell type into another by forced expression of foreign DNA
or transcription factors has been known for sometime (Davis et al., 1987; Takahashi
and Yamanaka, 2006).
For example, it has been shown that by overexpressing fibroblasts with the
skeletal muscle transcription factor MyoD, these cells could be converted to skeletal
muscle cells (Davis et al., 1987). Moreover it has been shown that the forced
expression of myocardin can convert fibroblasts into smooth muscle cells (Wang et
al., 2003). However, to date, no single factor has been shown to force fibroblasts into
a cardiomyocyte phenotype. Previously it was reported that fibroblasts could be
converted into CMC-like cells in vitro by expressing a cocktail of transcription factors
including GATA-4, Mef2c, and Tbx5, albeit only a small percentage of these
fibroblasts appeared to be fully reprogrammed into beating CMC-like cells (Ieda et
al., 2010). Even more recently it was shown that resident cardiac non-myocytes such
as fibroblasts could be reprogrammed with this same cocktail of transcription factors
in vivo (Qian et al., 2012). Intriguingly these in vivo reprogrammed CMC-like cells
showed similar contractile properties to that of normal CMCs and were able to
electrically couple with endogenous CMCs. Despite some functional improvement in
the mice treated with these reprogrammed CMC-like cells following ligation injury,
the efficiency of such cells to be reprogrammed remained very low. In short, the combinations of cell and gene therapies are slowly showing
promise, but many problems are limiting their use in a clinical setting. Over-coming
such shortfalls of the activation of immune responses, improving delivery of
reprogrammed factors and advancing the survival rates of transplanted cells will
accelerate the potential for these therapeutic options.
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4. MODEL ORGANISMS OF HEART REGENERATION
Due to the limited self-renewal capacity of the mammalian heart following
injury, original research approaches need to be explored in order to find ways to
ameliorate cardiac regeneration in humans. Another such avenue to pursue is to study
vertebrate animal models that can regenerate the heart. Lower vertebrate orders such
as teleost fish, and urodele amphibians contain model organisms that have previously
been shown to possess intrinsic abilities to regenerate a plethora of body parts and
tissues as adult organisms. Here I will discuss some of the vertebrate models of heart
regeneration and key findings in the field.
4.1 Mammals
In spite of the limited regenerative capacity of the four-chambered adult
mammalian heart to regenerate, coronary artery ligation models in rodents have been
employed as a tool to study damage associated with post myocardial infarction. One
of the first reports to describe coronary ligation to induce MI in rodents was produced
over 30 years ago (Zolotareva and Kogan, 1978). However, only more recently have
the morphological and functional effects been documented in more detail (Patten et
al., 1998; Salto-Tellez et al., 2004). The implementation of such a model system has
made it feasible to examine the effects of stem cell transplantations in repairing
damaged myocardium (see section on cardiac therapies). Although initial studies
report encouraging evidence for stem cells to transdifferentiate and functionally repair
ischemic damage, more recent reports have begun questioning these findings and
report new short-comings (Balsam et al., 2004; Murry et al., 2004; Orlic et al., 2001).
Major issues currently being noted in these model systems include the formation of
tumors following stem cell transplantations and the need for immunosuppressive
therapy to prevent host rejection (Swijnenburg et al., 2005). These investigations may
provide an advanced understanding of how to overcome several hurdles employing
stem cell technology in humans.
Given the capacity of neonatal cardiomyocytes to divide, a recent report
investigated whether the heart of the 1-day-old neonatal mouse could overcome
partial resection injury. It was shown that in as little as 3 weeks following resection
injury the morphological architecture of the mouse heart was restored and after 2
months the newly formed apex had normal contractile function (Porrello et al., 2011).
30
Interestingly, this ability to regenerate myocardium is lost after 7 days of age, a time
when cardiomyocytes become binucleate and withdraw from the cell cycle (Li et al.,
1996). Of further interest was the recent release of another report also revealing the
ability of the 1 day old neonatal mouse heart to respond to ischemic LAD – ligation
injury, an ability which is also lost after 7.5 days of age (Haubner et al., 2012). Taken
together these seminal investigations indicate a time when regenerative capacity of
the neonatal mouse heart is lost. By understanding the mechanisms that alter this
regenerative behavior, we may one day be able to restore the regenerative capabilities
that have been lost in adult mammalian hearts.
4.2 Fish
Interestingly, teleosts such as zebrafish (Danio rerio) and the giant danio
(Devario aequipinnatus) also possess a high degree of natural regenerative potential.
For over a decade the descriptive regenerative vigor of the two-chambered zebrafish
heart to overcome approximately 20% apical ventricular resection has been
acknowledged (Poss et al., 2002). The complete regenerative process is largely
unknown, although more molecular and genetic tools are being applied providing
critical clues. Previous evidence supported the notion that the regenerated
myocardium of the zebrafish heart arises from a subclass of epicardial progenitor cells
(Lepilina et al., 2006). It was proposed that the cardiac injury stimulates expansion of
the epicardium, where FGFs crosstalk with migrating epicardial cells to the injury
site, which slowly revascularize the heart. At a similar time myocardial progenitors
originate in the damaged areas where they begin to express pre-cardiac markers and
eventually associate with previously existing muscle.
The notion of resident progenitor cells influencing natural heart regeneration
has more recently been opposed using inducible transgenic zebrafish models to
specify cell types within the heart (Jopling et al., 2010; Kikuchi et al., 2010). These
more recent studies describe a mechanism of cardiomyocyte dedifferentiation and
proliferation to restore lost or damaged cardiac tissue in zebrafish. Interestingly, one
of the studies supported the notion of a heterogeneous population of adult
cardiomyocytes with proliferative potential (Kikuchi et al., 2010). This group found
that a subpopulation of GATA4 inducing cardiomyocytes is a major contributor to the
regeneration of resected zebrafish hearts. Interestingly GATA4 remained expressed
throughout the regeneration process. As GATA4 is a gene required for normal cardiac
development, this may imply that these cells activated a more embryonic program.
31
Cryocauterization injuries have recently been applied to zebrafish and are
believed to be an alternative model to coronary artery ligations based on the damage
sustained by the technique (van den Bos et al., 2005). This injury model in zebrafish
causes massive tissue necrosis, followed by large amounts of collagen deposits, which
are subsequently replaced with new cardiac muscle (Chablais et al., 2011; Gonzalez-
Rosa et al., 2011; Schnabel et al., 2011). The dynamics of regeneration appears to
vary between injury models, as recovery from cryocauterization appears to take
longer and perfect ventricular shape is not restored, as it appears to be following
resection (Gonzalez-Rosa et al., 2011; Poss et al., 2002). Another cauterization
damage model has recently emerged in the giant danio (Lafontant et al., 2011). Some
of the data obtained in this model system reveals the importance of neovascularization
to the damaged areas of the heart. It is proposed that the neovascularization may
mitigate the relationship between diminishing fibrosis and proliferating
cardiomyocytes to regenerate the damaged myocardium. Additional evidence
gathered in this report reveals the presence of proliferating cardiomyocytes, although
the authors do not address whether progenitor cells may also support the regeneration.
In an effort to understand if zebrafish can overcome larger injuries to the
cardiac system that relay signs of acute cardiac failure, a recent study employed an
inducible transgenic ablation system in CMCs (Wang et al., 2011). Following cell
specific depletion of 60% of the ventricular myocardium, activated cellular and
molecular responses rapidly regenerate the myocardium. It is interesting to note that
myocyte death alone is substantial enough to prompt a regenerative response in this
model, as opposed to signals emanating from resected tissue or blood clot formation.
A more recent report employing a similar cardiac specific transgenic ablation model
system showed that zebrafish utilize differentiated atrial cardiomyocytes to
transdifferentiate into ventricular cardiomyocytes ultimately aiding in ventricular
regeneration following ablation damage (Zhang et al., 2013). Although it is unknown
if mammals contain a similar transdifferentiation capacity in atrial myocytes, this
pivotal finding warrants a new possible source for endogenous cellular regenerative
therapy in patients with heart failure.
4.3 Amphibians
Previous studies sought to address whether the three-chambered newt heart in
contrast to mammalian hearts, can efficiently regenerate lost cardiac muscle. Initial
studies established that following apical ventricle resections, the adult newt
32
cardiomyocytes in adjacent areas could re-enter the cell cycle and were capable of
forming new cardiac fibers following injury (Oberpriller and Oberpriller, 1974).
However, this response was minimal with dense connective tissue replacing the
absent cardiac tissue, resulting in scarring. Intriguingly, subsequent work observed
that replacing the area of resected cardiac tissue with minced newt cardiac muscle
gave rise to a continuous ventricular wall and lumina, which was formed in the
absence of scarring (Bader and Oberpriller, 1978). However, this grafted area
appeared to be morphologically and functionally separate from the remaining
ventricle.
Unlike their mammalian counterparts, cultured cardiomyocytes from adult
newt hearts continually proliferate (Bettencourt-Dias et al., 2003; Matz et al., 1998).
Also cultured cardiomyocytes from newt hearts provided evidence for disparate sub
populations of cardiomyocytes (Bettencourt-Dias et al., 2003). The authors reported a
sub-population of cardiomyocytes, which stably arrest following mitosis or
cytokinesis, but uncovered another small percentage of cardiomyocytes that retain
proliferative potential. Understanding why such a block arises in some
cardiomyocytes but not others holds potential for augmenting cardiac regeneration in
mammals.
More current studies implemented a mechanical crush-injury model to the
newt heart, which damages the cardiac tissue without the need for resection. Using
forceps to squeeze newt heart ventricles until hemorrhage, a dramatic decrease of
sarcomeric protein expression levels was observed, coinciding with an increase in
mitotically-active cardiomyocyte-like cells in the damaged areas (Laube et al., 2006).
This group revealed for the first time that the regenerative response of the newt heart
to injury was not limited to a wound-healing and scarring scenario. A current in-depth
investigation lead by the same group analyzed morphological and ultrastructural
changes in crush-injury induced newt hearts. This study has provided insight that
extracellular matrix components may be more important then previously believed, as
this not only provided some mechanical stability, but considerably influenced the
reconstitution of damaged myocardium by providing guidance cues for new CMCs
(Piatkowski et al., 2012). With the previous evidence that newt CMCs have high
plasticity, it was believed that dedifferentiating CMCs provide the regenerate (Laube
et al., 2006). This more recent report supports a notion of immature CMCs as a source
for regeneration, as damaged CMCs immediately appear to undergo necrosis and are
removed by macrophages. This group could not exclude newly emerging CMCs may
33
come from a pool of progenitor cells and as such the source of cells responsible for
regenerating newt hearts remains undetermined.
A previous report revealed that the axolotl, Ambystoma mexicanum, could
positively respond to a partial ventricular resection injury (Cano-Martinez et al.,
2010). The axolotl heart showed recovery of contractile activity in as little as 30dpi,
with full morphological recovery between 30-90dpi. Interestingly, evidence for CMC
and non-CMC proliferation was evident based on proliferative responses seen using
BrdU pulse chase experiments.
Newts can impressively regenerate following substantial ventricular injury,
but how they manage to survive in the first place is just as astonishing. In an effort to
understand newt survival following needle puncture to the ventricle, a recent article
provided evidence that the heart of the Cynops pyrrhogaster, another urodele
amphibian, can redirect blood flow away from the injury site (Miyachi, 2011).
According to this report, the onset of valve hyperplasia immediately following
damage consequently changes the anatomical flow of the ventricular inflow and
outflow tracts, preventing additional hemorrhage, allowing the rapid induction of
repair and regeneration mechanisms to proceed.
In summary, different regeneration models provide different theories, which
endeavor to explain the regeneration phenomena in vertebrate species. Although
growth factors and other major signaling networks are most likely involved, the
bigger question remains – where are the cells coming from that contribute to natural
cardiac regeneration? There are two major speculations; 1) Differentiated cells re-
enter the cell cycle. There has been some evidence for the activation and proliferation
of cardiomyocyte populations contributing to the regenerating zebrafish hearts
following ventricular resection (Jopling et al., 2010; Kikuchi et al., 2010). Additional
information obtained in neo-natal mouse heart resection injuries also propose new
cardiomyocytes coming from pre-existing cardiomyocytes (Porrello et al., 2011). The
mechanism of action would require the mature cells of the heart to undergo
dedifferentiation, where they down-regulate their contractile genes allowing them to
re-enter the cell cycle and proliferate. The cells can then redifferentiate back to adult
cardiomyocytes where they can replenish the missing tissue. 2) Recruitment of
progenitor cells. There is also some support for the recruitment of epicardial
progenitor cells (EPDCs) that become active following cryocauterization induced
damage in zebrafish or induced myocardial infarction in mouse (Gonzalez-Rosa et al.,
2011; Smart et al., 2011). These cells may act to migrate into the myocardium and
34
repopulate dead cells in the damaged heart. Of further interest is the recent discovery
of in vivo reprogramming where atrial cardiomyocytes transdifferentiate and migrate
into ventricular cardiomyocytes providing an alternative method for repopulating
damaged myocardial tissue (Zhang et al., 2013).
Investigating how newts and teleosts remove fibrotic lesions, regulate
molecular pathways and control cell proliferation following severe cardiac damage,
provides an incentive to overcome the limitation of cardiac regeneration in mammals.
There are many aspects driving the control, maintenance and regulation of heart
regeneration in these lower vertebrate species. One such regulator is the involvement
of non-coding RNA’s such as microRNAs and they will be discussed in detail in the
following section.
35
5. MicroRNAs
In humans over 20,000 proteins are produced from just 1.2% of our genome
(Clamp et al., 2007; Mattick, 2011). It is also known that many more genes are
transcribed that give rise to non-protein-coding transcripts. Recently non-coding
RNAs (ncRNAs) have begun to emerge with diverse and significant roles in cellular
pathways that can affect physiological processes, including those found in cardiac
development and regeneration (Hudson and Porrello, 2013).
Two classes of ncRNAs include microRNAs (miRNAs) and long non-coding
RNAs (lncRNAs). The human genome encodes thousands of lncRNAs, many of
which have demonstrated affects on biological processes such as regulating gene
expression (Guttman and Rinn, 2012). Presently miRNAs are more widely
characterized so I will focus on the biogenesis and modulation of miRNAs
particularly in the context of regeneration below.
5.1 MicroRNA biogenesis
Since the initial discovery of miRNAs in the early 1990’s, it has been shown
that these small, evolutionary conserved, non-coding RNAs act as a novel mechanism
of post-transcriptional regulation (Fire et al., 1998; Lee et al., 1993). Recent
speculations have estimated that over one-third of mRNAs in the mammalian genome
are regulated by miRNAs (Lewis et al., 2005).
The processing of miRNAs occurs both in the nucleus and the cytoplasm of
the cell and involves a number of complex regulatory enzymes and binding proteins
(for a detailed review see (Winter et al., 2009)) (Figure 4). miRNAs are first
transcribed in the nucleus by RNA polymerase II and are referred to as primary
miRNAs (pri-miRNAs). These pri-miRNAs are 5’ capped, polyadenylated and range
in size, but on average are approximately 2kb in length (Cai et al., 2004). The pri-
miRNA is further microprocessed in the nucleus into hairpin RNAs by Drosha (an
RNase type-III enzyme) with the help of a double stranded RNA binding protein
DiGeorge syndrome critical region 8 (DGCR8) to form precursor miRNAs (pre-
miRNAs) approximately 70 nucleotide bases in length (Landthaler et al., 2004). The
pre-miRNAs are exported out of the nucleus into the cytoplasm via exportin-5, where
they are further processed by the ribonuclease Dicer, leaving a miRNA duplex. One
strand of the miRNA duplex (approximately 18-25 nucleotides long) guides the RNA-
36
induced silencing complex (RISC) to partial complimentary sequences of target
mRNAs where they then interfere with the regulation of mRNA stability or
translation (Mourelatos et al., 2002; Schwarz et al., 2003). The other strand is
typically but not always degraded (Yang et al., 2011). It is commonly supported that
the 5’ end of the miRNA, specifically from nucleotides 2 to 8 (known as the ‘seed
region’), plays a critical role in the pairing with the target-associated mRNAs in
metazoans (Bartel, 2009). The miRNA interaction with the mRNA transcript is
mediated via Watson-Crick base pairing with most miRNAs binding to the 3’-UTR of
their target mRNAs. Recent evidence suggests miRNAs can also interact with the 5’-
UTR and protein-coding regions of targeted mRNAs (Chi et al., 2009). The current
notion for the primary mechanism of action of miRNA – mRNA interactions are to
induce post-transcriptional gene silencing via decay (Guo et al., 2010) or repression
of translational initiation (Gregory et al., 2005). This is the mechanism of RNA
degradation seen by the RNAi machinery, where the Argonaute protein Ago2 is the
endonuclease responsible for cleaving the RNA target (Fire et al., 1998; Liu et al.,
2004; Meister et al., 2004; Rand et al., 2004; Rivas et al., 2005). Although the classic
dogma is that miRNAs most commonly act to repress their mRNA targets, some
evidence suggests that miRNAs could be involved in stimulating mRNA translation
(Vasudevan et al., 2007).
37
Figure 4. Schematic overview of miRNA biogenesis. The process of miRNA transcription is controlled by RNA-polymerase II, which produces long primary miRNAs (pri-miRNAs) typically with hairpin morphology. These pri-miRNA products are cleaved by a nuclear endonuclease, Drosha, which crops the stem of the pri-miRNA leaving a pre-miRNA. Pre-miRNAs are exported out of the nucleus by Exportin-5 and are further cleaved by Dicer, an RNAse III enzyme leaving a mature double-stranded miRNA. Mature miRNAs interact with Argonaute endonuclease proteins (Ago2) to become incorporated into the RNA-induced silencing complex (RISC), which together are directed towards mRNA targets and bind to their 3’ untranslated region (UTR). Image adapted from (Goettsch & Aikawa, 2013).
5.2 MicroRNAs in regeneration
Regeneration encompasses a plethora of dynamic and rapid alterations in gene
expression. Several modes of regulation must exist in order to meet the demands of
quick-acting and fine-tuned changes in altered gene expression ongoing in
regenerating tissue. Compelling evidence would then nominate miRNAs as one such
candidate for this mediation during complex tissue regeneration.
It has previously been shown through both gain-of-function and loss-of-
function experiments that FGF signalling depeletes miR-133 expression, which is a
critical component for optimal caudal fin regeneration in the zebrafish (Yin et al.,
2008). A more recent study comparing data sets from different species revealed many
38
similarities between axolotl spine regeneration and zebrafish fin regeneration,
including the down-regulation of miR-133 during regeneration (Sehm et al., 2009).
However, this study found miR-196 to be the most up-regulated and strongest
candidate in axolotl tail regeneration. miR-196 down-regulates Pax7 protein levels,
which in turn drove cells in the regeneration zone to proliferate and migrate out of the
blastema. This process helps mediate the development of the new ependymal tube,
illuminating the functional importance of miR-196 in tissue regeneration.
Additionally the inhibition of miR-196 resulted in excessively high levels of Pax7
proteins, which can inversely inhibit proliferation, leaving a defective regenerative
response.
As previously mentioned newt lens regeneration occurs via the
transdifferentiation of the epithelial cells of the dorsal iris, but not the ventral iris. To
try to elucidate the mechanisms underlying such specificity, the function of specific
miRNAs were explored to delineate their function during lens regeneration. Here, it
was shown that the up-regulation of miR-148 and let-7b not only affected cellular
proliferation, but interestingly regulated the levels of other miRNAs, indicating a
possible involvement of a miRNA network (Nakamura et al., 2010).
In order to reveal the importance of miRNAs during axolotl limb regeneration,
a current study employed and paired microarrays to identify key players of regulation.
Reportedly the relationship between miR-21 and its target Jagged1 (member of WNT
signaling pathway), are speculated to crucially regulate the commitment of
proliferating blastemal cells during limb regeneration (Holman et al., 2012). This
group only considered one time point during regeneration (mid-blastemal
development) and as such it would be interesting to identify other important highly
regulated miRNAs at different time points during the regenerative timeline.
5.3 MicroRNAs and cardiac regeneration
The importance of miRNAs in the heart was initially reported via the cardiac-
specific deletion of Dicer, the response of which depleted mature miRNAs
specifically in the heart and resulted in lethality shortly after birth (Chen et al., 2008).
Moreover miRNA profiling studies have provided a range of feedback indicating
miRNAs have regulatory functions in specific cardiac cellular processes such as
cardiac dedifferentiation, cell-cycle control, hypertrophy and remodeling in the adult
heart (Chen et al., 2008; Sayed et al., 2007; Thum et al., 2007). The importance of
several miRNAs during heart regeneration has come to light in mouse and zebrafish
39
models. A recent study identified a novel role for the miR-15 family following
ischemic injuries. Specifically, by over-expressing miR-195, a miR-15 family
member, regenerating neo-natal mouse hearts revealed fewer proliferating
cardiomyocytes (Porrello et al., 2013). The authors speculate these proliferating
cardiomyocytes contribute to the regenerate. Additionally these mice showed severe
fibrosis and had irregular cardiac function, indicating a similar response to adult-like
remodeling in non-regenerating scenarios. Intriguingly, the authors also showed in
adult mouse models undergoing the same procedure, inhibition of miR-15 family
members of miRNAs increased cardiomyocyte proliferation and improved cardiac
function. These results speculate that members of the miR-15 family may be one
potential post-natal regulator responsible for the natural arrest of cardiomyocyte
proliferation shortly after birth.
Rodent hearts that were diminished of the cardiomyocyte-specific miR-133
using antagomirs, resulted in a hypertrophic phenotype (Care et al., 2007). The role of
miR-133 was also recently addressed in zebrafish hearts where it’s expression is
naturally decreased upon injury (Yin et al., 2012). Moreover this group showed that
depleting miR-133 in the damaged heart enhanced the regenerative response, while
hearts that were over-expressed with miR-133 alternatively impeded muscle
regeneration, resulting in increased scar tissue formation. This group concluded miR-
133 acts in part to restrict cardiomyocyte proliferation.
The expression of muscle contractile proteins is tightly regulated and
interesting hallmark discoveries include the finding of a family of miRNAs that
regulate muscle-specific myosin genes. Such microRNAs have been coined
“MyomiRs” and consists of miR-1, miR-133, miR-208a, miR-208b and miR-499. It
has been shown that these miRNAs maintain the balance between βMHC and αMHC
expression, which alter contractility and function of cardiomyocytes in the heart
during injury and repair (Gupta, 2007). Of further interest are miRNAs that have been
shown to target several key cardiac transcription factors. One such example is the
effect miR-1 has on downregulating cardiac growth by targeting HAND2 during
development (Zhao et al., 2005). In addition miR-1 is the most abundant miRNA in
the mammalian heart and although all of the cardiac related functions of this miRNA
are not known, the upregulation of miR-1 has been shown to lead to improved cardiac
growth (Chen et al., 2008; Ivey et al., 2008).
Additionally it is interesting to note the heightened expression of certain
miRNAs in patients with cardiovascular disease. For example, miR-21 has been
40
shown to be the most up-regulated miRNA in cardiac disease, although its role is
largely unknown (Small et al., 2010). Some reports delineate a hypertrophic
phenotype with the induction of miR-21 (Thum et al., 2008). Other studies contradict
these results by finding an antihypertrophic effect of miR-21 on isolated
cardiomyocytes and cardiomyocyte growth (Sayed et al., 2008; Tatsuguchi et al.,
2007). The discrepancies highlight a note of caution when considering using these
small non-coding RNAs as therapeutic tools to overcome cardiovascular illnesses.
Other studies have also indicated the use of miRNAs to reprogram cardiac
fibroblasts into cardiomyocyte like cells. Using a small set of only four miRNAs, one
study showed the efficient reprogramming potential of mouse cardiac fibroblasts into
CMC-like cells (Jayawardena et al., 2012). Another study revealed the importance of
several miRNAs to initiate cardiac gene expression programs via the reprogramming
of human fibroblasts (Nam et al., 2013). Unexpectedly, this report also highlighted
muscle-specific miRNAs that further improved myocardial conversion without the
need for key cardiac transcription factors. Of further importance were novel findings
in patients and experimental MI models in mice, revealing muscle-specific miRNAs
such as miR-1, miR-133, miR-208, and miR-499 elevated in the plasma (Wang et al.,
2010). The exact mechanisms mediating the release of miRNAs into the blood and the
biological benefits or functions of circulating miRNAs has not yet been clearly
defined. A recent study reported findings of a mircrine response, in which miR-499
controls gene expression and is responsible for CSC growth and differentiation by
traversing gap-junctions (Hosoda et al., 2011). The discovery of circulating miRNAs
in the blood not only increases the complexity of miRNA involvement in an inter-
cellular manner, but also opens possibilities for the use of miRNAs as biomarkers
against cardiovascular diseases.
Taken together the reports and findings discussed here cover a multitude of
different species and tissue sources all relying on miRNAs for development,
functional repair and regeneration. The avenue for implementing miRNAs as a novel
therapeutic approach to one-day combat congenital heart disease is looking optimistic.
Meanwhile, using lower vertebrate species such as the newt will allow us to continue
to investigate the functional roles of miRNAs in cardiac biology.
In order to consider mechanisms of global regeneration, we considered how
mRNA transcript expression is controlled and regulated. One of many mechanisms
that regulate mRNA is via the post-transcriptional editing of mRNA. Below I will
41
introduce the enzymes that are responsible for A-to-I RNA editing and explain the
significance of RNA editing in relation to regeneration.
42
6. RNA EDITING
6.1 A-to-I RNA editing
RNA editing is a widespread co- and post-transcriptional molecular
phenomenon where changes in RNA sequences are introduced so that it differs from
the DNA template. This is a strategy for metazoans to increase the protein repertoire
without extending the number of genes. The most prevalent type of RNA editing is A-
to-I RNA editing. Here, adenosine deaminases acting on RNA (ADAR) are the
enzymes that mediate hydrolytic deamination, which are responsible for converting
adenosines to inosines in double stranded RNA substrates (Bass, 2002, Wagner et al.,
1989).
6.1.1 ADAR enzymes
The ADAR family is well conserved in vertebrates and there are three family
members (ADAR1-3) (Chen et al., 2000; Kim et al., 1994; O'Connell et al., 1995).
ADAR1 and ADAR2 have a wide tissue distribution, (Bass, 2002; Valente and
Nishikura, 2007), whereas expression of the ADAR3 enzyme is refined to the brain.
To date no enzymatic activity has been attributed to the ADAR3 enzyme (Chen et al.,
2000).
ADARs appear in great diversity across the metazoan spectrum (Figure 5). A
single ADAR2 homologue (dADAR) exists in Drosophila melanogaster, two ADAR
genes exist in Caenorhabditis elegans, which have shown to be critically important
for normal development and only one ADAR1 homologue expressed in Xenopus
laevis (Hough and Bass, 1994; Palladino et al., 2000; Tonkin et al., 2002). Moreover
ADAR homologues have been found in some invertebrates including squids and sea
urchins but interestingly, ADARs are absent in protozoa, yeast and plants (Jin et al.,
2009; Palavicini et al., 2009).
Members of the ADAR family share a common catalytic deaminase domain in
their C-terminus and two or three double-stranded RNA binding domains (dsRBD) at
the N-terminus (Saunders and Barber, 2003). The dsRBDs are required for the
successful binding to the double stranded RNA targets (Valente and Nishikura, 2007).
43
Figure 5. The ADAR family members compared between mammals and amphibians. Three mammalian ADAR proteins are presented with two isoforms of ADAR1, (p150 and p110), ADAR2 and ADAR3. Xenopus laevis express a common ADAR1 homologue (Xl ADAR1). The red spotted newt has an ADAR1 homologue (Nv ADAR1) as well as an ADAR2 homologue (Nv ADAR2). Common features to all ADAR enzymes are a catalytic deaminase domain (in blue), two or three dsRBDs (in green) and nuclear localization signals (in red). Image adapted from (Wahlstedt and Ohman, 2011). See paper III and section 7.4.
Two protein isoforms of the ADAR1 enzyme have been discovered in
mammals and are referred to as p150 and p110 according to their molecular weight.
The ADAR1(p150) has a promoter that is interferon inducible. The shorter isoform,
ADAR1(p110) is constitutively expressed from two different promoters (George and
Samuel, 1999; Kawakubo and Samuel, 2000). One current axiom is that the longer
isoform is involved in an immune host defense mechanism against RNA viruses, as it
is upregulated upon viral infection (Toth et al., 2009).
All the members of the ADAR enzymes contain a nuclear localization signal
(NLS) and are catalytically active in the nucleus. However, ADAR1(p150) also
contains a nuclear export signal (NES) in the N-terminus and as such has been shown
to shuttle between the nucleus and cytoplasm (Poulsen et al., 2001). Interestingly, it
was recently reported that the short ADAR1(p110) lacking this signal also shuttles
between the nucleus and cytoplasm with the help of exportin (Fritz et al., 2009).
Intriguingly this is the same chaperone protein used to export pre-miRNAs from the
nucleus to the cytoplasm (see section 5.1 microRNA biogenesis).
44
Several splice variants have been reported for both ADAR1 and ADAR2
although it has not yet been fully elucidated if these splice variants are translated into
enzymatically active proteins (Agranat et al., 2010; Lykke-Andersen et al., 2007). The
pre-mRNA of ADAR2 has been shown to be a target of the ADAR2 enzyme and
therefore ADAR2 can regulate its own protein expression by an auto-regulatory
feedback loop mechanism (Rueter et al., 1999). Here, editing creates an alternative
splice site, generating a frame shift, resulting in a non-functional truncated protein.
6.2 Editing substrates
6.2.1 Editing in protein-coding regions
The deamination of adenosines into inosines results in inosines being
recognized as guanosines by the splicing and translational machinery. The
replacement of an adenosine with a guanosine can create codon changes. A-to-I RNA
editing has two different modes of action, site-selective editing and hyper editing,
which occur in different regions of RNA. Site selective editing occurs more
frequently in coding regions, where one or a few adenosines are deaminated into
inosines. Hyper-editing occurs in a more promiscuous manner often in non-coding
sequences where many adenosines are targeted for deamination (Wahlstedt and
Ohman, 2011). The majority of site-selective A-to-I RNA editing events is limited in
number and most have been found in neurotransmitter receptors and ion channels. A
few examples include the glutamate receptor (Higuchi et al., 1993), the alpha subunit
of the GABA receptor (Ohlson et al., 2007), the serotonin receptor (Burns et al.,
1997) and the voltage-gated potassium channel Kv1.1 (Hoopengardner et al., 2003).
The effects of editing on these transcripts change amino acid residues, which results
in altered ion channel permeability as well as having an affect on ligand-binding
affinity (Burns et al., 1997; Higuchi et al., 1993; Seeburg et al., 1998; Wang et al.,
2000).
It is important to mention that mechanisms underlying ADAR substrate
determination are not fully known. Although some reports have indicated certain
neighboring nucleotides are favored over others, there is no strict consensus sequence
in which all ADARs act on, nor are there required co-factors for the deaminase
activity (Lehmann and Bass, 2000). It would also appear ADAR activity is influenced
by certain characteristics of dsRNAs including their size and mismatched base pairs
that create bulges (Enstero et al., 2009; Ohman et al., 2000).
45
6.2.2 Editing of non-coding RNA
As mentioned in the previous section hyper-editing occurs in non-coding
RNAs, often within Alu repeats found in 5’ and 3’ UTRs, but also in other regions of
UTRs and introns. Of further interest is accumulating evidence for A-to-I RNA
editing involved in the RNAi pathway. An interesting notion has recently come to
light that A-to-I RNA editing mechanisms interact with the RNAi pathway via the
competition of dsRNA substrates. As discussed previously, miRNA-mediated gene
silencing can control many different processes in development and regeneration
including differentiation and proliferation. To intensify the complexity of miRNAs
and editing further, pri-miRNAs are generally quite long double stranded RNA
structures with miss-matches and bulges (see section 5.1 miRNA biogenesis and
Figure 4), making them ideal targets of ADARs and editing.
The editing of certain pri-miRNA structures can inhibit or enhance Drosha
activity. For example, the editing of pri-miR-142 effects Drosha processing. This
inability for Drosha to process accordingly interrupts the overall mature micro-
processing of miR-142, which instead becomes rapidly degraded by a specific
ribonuclease (Scadden, 2005). Interestingly, reports have begun to show that pri-
miRNAs that escape editing in the nucleus may meet ADARs in the cytoplasm.
Although ADARs are enzymatically active in the nucleus, they can still bind to pre-
miRNAs in the cytoplasm thus preventing continuation of the miRNA maturation
process. Additionally, editing at the pri-miRNA level may later prevent Dicer
processing, as is the case with pre-miR-151 (Kawahara et al., 2007a). It is
hypothesized that ADAR activity may reduce the production of small RNAs if Dicer
prefers only Watson-Crick base pairs, not I:U wobbles created from editing. The
significance of A-to-I RNA editing in miRNAs is also emphasized by the fact that
editing within the “seed region” may lead to the mature miRNAs silencing different
targets as is the case for miR-376 (Kawahara et al., 2007b). In turn, A-to-I RNA
editing is yet another mechanism the cellular machinery utilizes to control abundance
of miRNA and thus their functions. However, the relationship is reciprocal, as recent
studies have shown mature miRNAs can target ADARs. For example, the expression
of ADAR1 has been shown to be down-regulated by miR-1 (Lim et al., 2005). It is of
further interest to note that miR-1 plays a critical role in the development of the heart,
and that during mammalian development the highest expression of ADAR1 is seen in
46
the heart (Srivastava, 2006; Wang et al., 2004). This implies a symbiotic relationship
between RNA editing, miRNAs and the regulation of heart development.
6.3 Functional aspects of RNA editing
The inactivation of ADAR1 leads to an embryonic lethal phenotype due to
widespread apoptosis and therefore is required for life in mammals (Wang et al.,
2004; Wang et al., 2000). Mice with ADAR2 mutations die shortly after birth and
experience repetitive episodes of epileptic seizures due to under-editing of the
glutamate receptor, a major target of ADAR2 (Higuchi et al., 2000).
Human pathophysiology affiliated with RNA-editing deficiencies is most
closely related to disorders of the central nervous system. Editing deficiency in the
Q/R site of the glutamate receptor can disrupt blood flow to the brain and editing
disorders of the serotonin receptor have been linked to neuropsychiatric disorders
such as depression and schizophrenia (Peng et al., 2006; Schmauss, 2005). In
addition, the effect of reduced RNA editing has been linked to human gliomas and
cancer (Maas et al., 2001; Paz et al., 2007). How this reduced activity of editing
manifests malignant cell growth is not yet known.
Of further interest was a recent report that linked RNA editing to congenital
heart disease (Borik et al., 2011). The gene encoding thyroid hormone related protein
1 is implicated in cardiac hypertrophy, angiogenesis and is targeted by the MyomiR
miR-208. The A-to-I editing of this substrate was significantly higher in children with
cyanotic congenital heart disease. These authors concluded that post-transcriptional
regulations such as RNA editing may play a role in regulating cellular and metabolic
pathways that influence hypoxic conditions in the heart.
The physiological importance of editing transcripts has been documented in
mutant flies with deletions of the dADAR gene, which are known to edit glutamate
channels. Here, brain-related physiological complications arise such as uncoordinated
locomotion and signs of neurodegeneration (Palladino et al., 2000).
It is interesting to note that several seminal studies revealed a critical role for
ADAR1 in maintenance of hematopoietic stem cells. In one such study, the loss of
ADAR1 in mouse hematopoietic cells led to a global upregulation of type-I and type-
II interferon-inducible transcripts resulting in cellular apoptosis (Hartner et al., 2009).
Moreover, it has also been shown that ADAR1 deficient hematopoietic progenitor
cells suffer an inability to form differentiated colonies (XuFeng et al., 2009). The
47
inability to form differentiated colonies in vitro is most likely a result of increased
apoptosis in these cells.
48
7. PRESENT INVESTIGATIONS
7.1 Aims
The aims of this thesis were to elucidate if newt hearts could regenerate
myocardial tissue following a novel resection injury and to decipher the molecular
and cellular involvement in response to tissue/organ injury and regeneration.
The specific aims of the different studies are:
Paper I: Create a standardized cardiac resection injury model. Use this new model
system in order to determine the structural and functional responses the newt heart has
to resection injury. To investigate what cell types and transcriptional regulators may
be involved in regenerating the heart.
Paper II: Delineate a possible role for miRNAs in regenerating adult cardiac tissue by
applying a miRNA micro-array screen. Then create a small miRNA cDNA library to
obtain newt specific sequences. Perform quantitative and qualitative methods to
characterize a role for miRNAs in newt cardiac regeneration.
Paper III: Examine if A-to-I RNA editing is active in a variety of different regeneration
tissues in the red-spotted new. Investigate how this post-transcriptional modification
could be responsible for mediating signaling components necessary to drive the
creation of cellular plasticity and regeneration.
49
7.2 Paper I
7.2.1 Results and discussion
It had initially been reported that the heart of the adult red-spotted newt had a
limited regenerative response to cardiac injury. This was based on apical resections of
the ventricle, which left a partial repair response that included some cardiomyocyte
turnover while the presence of fibrotic lesions and scarring triumphed over tissue
regeneration (Oberpriller and Oberpriller, 1974). More recent reports provided
evidence that newt hearts could restore damage done to the myocardium following
crush injury (Laube et al., 2006). Although this group showed the remarkable
plasticity potential of newt cardiomyocytes to control their identity, the further
investigation of cellular and molecular mechanisms involved in newt cardiac
regeneration could go someway in amending the regeneration deficit found in
mammals. In Paper I, we aimed to produce a standardized and reproducible resection
injury model. As opposed to apical resections that incur variations in damage size,
this new procedure would allow us to obtain precise tissue removal. In addition, by
resecting a lateral portion of the ventricle we minimize the chances of breaching the
ventricular lumen and thus reduce morbidity rates. We could then assess on a more
standardized level, the extent of which the newt heart responds to injury over longer
regeneration times.
First, we employed histological staining methods that were able to show the
newt myocardium undergoes full morphological restoration in as little as 2 months
following injury. The morphological recovery could be characterized as follows; first
around 1 week following resection we observed a thick thrombin clot covering the
amputation plane. Over the course of the next few weeks, fibrin and collagen deposits
receded, as the epicardium appears to restore structure to the myocardium. Infiltrating
cardiomyocytes begin to invaginate the damaged areas and by 45dpi minimal signs of
resection injury are noticed.
In an effort to trace cardiac functional output, we next conducted
echocardiogram studies. Interestingly, the inner cardiac wall measurements provided
fractional shortening values similar to humans, which revealed full functional
recovery in newt hearts in as little as 3 weeks following resection injury. At 60dpi, a
time when we observe morphological recovery, fractional shortening values are
similar to that seen in uninjured hearts.
50
We conducted BrdU pulse chase experiments to assess the cell cycle re-entry
of cell populations in the cardiac milieu. Together with immunohistochemical
approaches, we found that the newt hearts response to injury was a result of increased
and widespread cellular proliferation. We observed a gradual increase in proliferation
in both cardiomyocytes and non-cardiomyocytes throughout the heart. A peak of
cellular proliferation was noted between 21-45dpi, thereafter the numbers of BrdU+ve
cells began to subside.
Next we decided to conduct a molecular signature of several well-known
cardiac transcription factors known to play an important role in cardiogenesis, as
molecular events during regeneration often recapitulate those during development.
These results showed similar mRNA expression patterns for all cardiac transcription
factors studied. In general, immediately following resection, expression levels
decreased but were later heightened at times synonymous with increased cellular
proliferation. Worthy of note was the fact that a significant drop in expression was
also observed at 45dpi, possibly indicating that the cells needed for cardiac
replacement have been reprogrammed and increased cardiac transcriptional activity is
no longer required.
Lastly, we were able to examine separate populations of proliferating
GATA4+ve and Islet1+ve expressing cells localized in and around the regeneration
zones. We identified mature cardiomyocytes that expressed these markers. We also
found Islet1 and GATA4 expression in proliferating non-myocytes. That these cells
were also functionally active provides more evidence that there could be a population
of resident cardiac progenitors becoming activated in response to the resection injury.
51
7.3 Paper II
7.3.1 Results and discussion
As described in section 5 of this thesis, miRNAs have previously been shown
to be involved in many aspects of cardiac biology and regeneration. In Paper II we
wanted to assess what kind of miRNAs may be involved in the regulation of the
different phases of newt cardiac regeneration.
Information regarding miRNAs in the newt was quite limited at the time of
this study. As such we decided to conduct a miRNA microarray screen to gauge
differentially expressed miRNAs involved in cardiac regeneration. As newt miRNA
sequences were not available, detection probes that were complementary to Xenopus,
zebrafish and human miRNAs were employed in order to identify conserved
miRNAs. We looked at miRNA expression levels at two different time points, 7dpi
which corresponds to the early wounding response and 21dpi, which is a time-point
when hyperplasia is at its peak.
The microarray revealed 37 miRNA candidates that were significantly up- or-
down-regulated between 7dpi and 21dpi. We next constructed and sequenced small
RNA libraries in order to secure newt specific sequencing information for
downstream experiments. Sequenced data that was aligned to miRBase revealed 22
sequences corresponding to the previously identified miRNAs from the microarray
screen. From here four highly conserved candidates were selected for a thorough
investigation regarding their expression patterns over the duration of the regeneration
timeline.
One candidate, miR-128, showed the most heightened expression pattern at a
time when cellular proliferation is known to be at its peak. Using in-situ hybridization
techniques we were able to localize the expression pattern of miR-128, which was
primarily restricted to the cardiomyocytes near the epicardial border in uninjured
animals. Of great interest was the specific localization of miR-128 directly in and
adjacent to the regeneration zone several weeks following injury and during
regeneration. Furthermore during regeneration miR-128 expression became localized
in non-myocytes in the regenerating areas.
To examine the effect miR-128 may have in vivo, we ablated miR-128
expression with specific antagomirs and conducted BrdU pulse chase experiments to
observe the effect on proliferating CMCs and non-CMCs during regeneration. Using
immunohistochemistry we were able to observe a significant positive hyperplastic
52
response in non-CMCs in animals that received miR-128 antagomirs. These results
suggest that the natural increase in miR-128 levels during regeneration could serve to
halt over-excessive cellular proliferation.
Interestingly, through the use of several bioinformatics programs we observed
that the 3’UTR of Islet1 contains a novel binding site for miR-128. An earlier study
by our group has shown the existence of Islet1+ve proliferating cells during newt heart
regeneration (PAPER I). To examine if Islet1 expression is altered by miR-128, we
cloned the newt Islet1 3’ UTR into a luciferase vector and conducted 3’UTR miRNA
luciferase assays. We confirmed that miR-128 suppresses Islet1 expression and that
Islet1 transcript and protein levels are decreased at a time when miR-128 is normally
elevated. Furthermore, Islet1 transcript and protein levels were dramatically increased
in animals treated with miR-128 antagomirs. Morphological assessment of the
animals treated with miR-128 antagomirs showed a delay in extracellular matrix
removal and myocardial regeneration following resection injury to the heart.
The role of miR-128 during newt cardiac regeneration could therefore be two-
fold. First miR-128 may act as a global regulator to avoid neoplasia at times of
extensive cellular proliferation in regenerating tissues. Alternatively this may be a
mechanism that allows dedifferentiated cells or proliferating cells to be fine-tuned
into their committed lineages. These results show that differentially expressed
miRNAs may coordinate several factors governing the response of the newt heart to
regenerate.
53
7.4 Paper III
7.4.1 Results and discussion
It has been speculated that the ability for the newt to regenerate many complex
tissue-types may be linked to cellular plasticity in differentiated cells or progenitor
cell types. In Paper III we demonstrated that an important post-transcriptional gene
regulator known as A-to-I RNA editing shows interesting connections with a variety
of regeneration scenarios in the newt.
In order to determine if A-to-I RNA editing is an active post-transcriptional
phenomenon in adult newts, we cloned GABRA3 mRNA, a known target for A-to-I
RNA editing. We found editing of the GABRA3 subunit transcript increased during
neuronal regeneration, as it does during neuronal development in mammals. We next
identified two functionally active RNA editing enzymes in the newt, ADAR1 and
ADAR2. Of further interest was the newt ADAR2, which also edits its own mRNA
transcript as it does in mammals. By targeting its own transcript, ADAR2 regulates its
own level of expression providing a mechanism to regulate editing of ADAR2
substrates, which could alter signaling components necessary to drive cellular
plasticity in regenerating tissue.
Immunohistochemistry analysis revealed an interesting nuclear to cytoplasmic
shift of ADAR1 expression during regeneration in all tissues studied. Of further
interest was the co-localization of ADAR1 within a stem cell population of Pax7+ve
satellite cells in the limb. These results indicate a possible involvement between RNA
editing and the behavior of proliferating cell types needed to regenerate the limb. The
editing efficiency between such satellite cells could be one mechanism used by the
newt to regulate plasticity of proliferating cells or to determine their lineage fate.
To investigate the importance of ADARs in newt tissue regeneration, we
cloned newt ADAR1 and ADAR2 and examined the transcriptional and translational
expression of these enzymes during regeneration. The gene expression profiles of
ADAR1 showed an increase in all tissues investigated, with heightened expression
appearing at times concomitant with increased cellular proliferation in the models
studied. This indicates that RNA editing of particular mRNA transcripts are most
likely targeted at intricate times during regeneration. The gene expression profile of
the functionally active unedited ADAR2 showed variation between all tissues studied.
One explanation for the variation of expression between tissues could be due to the
editing efficiency of tissue-specific substrates of ADAR2. Intriguingly, the protein
54
levels of each enzyme in all tissues studied remained relatively unaltered during
regeneration. This would indicate that protein levels may not have a direct correlation
with the functional quantity of RNA editing, but rather the sub-cellular re-localization
of the enzymes may be a better way to control editing efficiency.
55
7.5 Future Perspectives
The culmination of the work driven during this thesis can be summarized in
two different aspects. First we created a novel heart resection model in which we
could decipher how heart regeneration is regulated in the red spotted newt. Secondly
we sought to explore global regulators of regeneration in these animals. Although we
developed a functional model to study heart regeneration, we have not ascertained
what controls this response or which cell types are needed to harness regeneration.
However, one key finding is the identification of two disparate populations of
GATA4+ve and Islet1+ve proliferating cells in regenerating newt hearts. It would be
interesting to generate transgenic cell lines in order to delineate where the cells are
coming from and how they contribute to the regenerate. To date, there are no
transgenic red-spotted newts. Considering the animals take at least 2 years to reach
sexually maturity (adulthood), the process of creating such animals is not optimal and
other methods need to be adopted. Possible methods could include the induction of a
reporter line with a cardiac enhancer. One could try to transiently or stably integrate
marker genes via viral vectors to label and determine which cells are pertinent for
regeneration. Currently, such techniques have not been optimized for use in the red
spotted newt. Apart from addressing cell types in regeneration newt hearts, we also
found the up-regulation of key cardiac transcription factors during different intervals
along the regeneration time-line. As such, it would be interesting to conduct
functional assessment assays to uncover the importance of these transcripts at
different times during regeneration. Possible methods include silencing the expression
of such factors through the use of small interfering RNAs (siRNA) or morpholinos.
The novel model we constructed removes standardized portions of cardiac
tissue from the lateral portion of newt hearts. This model may one day answer pivotal
questions regarding how new muscle, through the formation of new cardiomyocytes,
can be restored in patients with heart disease. As discussed earlier in this thesis,
cryoinjury models are also becoming more popular in zebrafish heart regeneration
studies. To date there are no reports of cryoinjury models on newt hearts. As different
injury models address different questions, it would be of great interest to see how
newt hearts respond to cryoinjury. These model systems could unlock critical
information in how to reduce scarring, a major concern in post MI patients, as scar
tissue formation diminishes heart function. Together, it is the information gained and
compared across these model systems that would go some way to understanding why
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mammals have a functional deficit when it comes to regenerating damaged
myocardial tissue.
One major caveat regarding newt regeneration model systems is the lack of
genomic information. The large genome size and the lack of a closely related
reference genome have halted any attempts to assemble the newt genome. Several
groups have now invested energy in de novo sequencing of salamander
transcriptomes, and uncovered novel newt protein families (Abdullayev et al., 2013;
Looso et al., 2013). Recently EST database libraries derived from Notophthalmus
viridescens and Ambystoma mexicanum are beginning to provide large amounts of
sequence information, made publicly available to researchers (Bruckskotten et al.,
2012; Smith et al., 2005). Establishing information such as the newt transcriptome or
proteome may provide molecular insights into whether these animals utilize an
explicit set of genes for tissue regeneration.
In order to gauge a further molecular perspective in heart regeneration we
employed microarray and deep-sequencing strategies, which uncovered a large group
of known and conserved mature miRNAs within the newt heart. Several of these
miRNAs showed different expression patterns along the extended course of the
regeneration time-line. This could highlight the different functional roles miRNAs
play at specific time-points during newt cardiac regeneration. Of further interest was
the discovery of small RNA sequences that did not match those found in miRBase. By
using a reference genome such as the Xenopus, it could be possible to map these small
RNAs to larger pre-mRNA sequences, which could provide an indication of novel
miRNAs in the newt. Furthermore we only selected one conserved miRNA based on
the particular expression at a certain time point of interest. Additionally, the
microarray we conducted represents only two time points during regeneration, one
responding to the early wounding response (7dpi) and the other during the
hyperplastic peak (21dpi). Therefore this array may have only revealed a small subset
of differentially expressed miRNAs during a small window of newt cardiac
regeneration. A further and more detailed analysis of miRNAs at all intricate points
during heart regeneration would undoubtedly uncover novel candidate miRNAs
related to every aspect of heart repair. To delineate functional roles for such
candidates, other methods could include using antagomirs to ablate the expression of
several significantly regulated miRNAs during regeneration and study the impact they
have on functional regeneration. In coordination with these miRNA knockdowns, one
57
could then apply deep-sequencing methods to identify potential and putative targets in
regenerating tissue.
Of greater interest would be to further explore the relationship of Islet1 and
miR-128. Although in vitro we conclusively observe that miR-128 can target the 3’
UTR of Islet1, we can not confirm that the result of the diminished transcriptional
expression of Islet1 during regeneration in vivo is due solely to miR-128 or if there
exists alternative contributing factors. Finally, it would be of interest to examine the
relationship between Islet1 and miR-128 in mammalian organisms. The extensive role
of miR-128 during newt heart regeneration is not completely known. We speculate
that miR-128 may regulate Islet1 expression in order to control proliferation in
potential precursor cells. An interesting notion would be that miR-128 is partly
responsible for down-regulating Islet1 expression in mammalian cardiomyocytes,
limiting the plasticity of CMCs in mammalian hearts. Therefore, it is speculative that
through the use of antagomirs one could stimulate an endogenous proliferative
potential in patients suffering from congestive heart failure by unlocking the
dormancy of Islet1 expression.
To gauge if regenerating tissues are regulated in a globally related manner, we
investigated the involvement of one post-transcriptional mechanism, A-to-I RNA
editing. Although ADAR enzymes are active in a variety of regenerating tissues, the
functional relevance of RNA editing in regeneration remains unclear. It would be of
interest to impair the expression of ADAR enzymes in different regeneration
scenarios and address if this elicits a functional defect in regeneration. One possible
method would include the administration of morpholinos to knock down ADAR
expression during regeneration. This would indicate which tissue types absolutely
require RNA editing activity in order to functionally regenerate. To identify and study
the role of other substrates involved in RNA editing during regeneration in the newt,
one could ablate ADAR enzyme activity as previously mentioned and perform large-
scale mRNA sequencing to detect altered levels of mRNA editing. The expression of
ADAR1 enzymes within stem cell populations in the regenerating limb is an
intriguing find. It would be of further interest to explore which cell types are
harboring ADAR expression in regenerating hearts and brains. Further
immunohistochemistry approaches could be used in order to determine if ADARs
favour cell specific niches or if they are promiscuously expressed in a broad range of
cell types throughout regenerating tissue.
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8. CONCLUSIONS In this thesis I have shown that the adult red-spotted newt can fully respond to
partial ventricular resection in a process that is fueled by proliferation. A molecular
signature during regeneration revealed developmental cardiogenic transcription
factors most likely play a key role in regulating similar genes involved in cardiac
development. Additionally, I show that different miRNAs are involved in various
phases of newt cardiac regeneration. The inhibition of one miRNA has an affect on
altering the levels of proliferating cells within the heart, ultimately delaying the
regenerative response. Finally, I show that the red spotted newt may harness a global
post-transcriptional process, namely A-to-I RNA editing, which could work to micro-
manage the re-specification and phenotypic plasticity needed for cells to regenerate
injured tissue.
I hope that anyone who has read this far along into my thesis can appreciate
the red spotted newt as an incredible model organism to study heart regeneration.
Currently the biggest drawbacks found in these organisms are 1) the lack of genomic
information and 2) the fact that creating transgenic lines of newts is not optimal due to
its prolonged transition into a sexually mature adult. Due to advances in high-
throughput whole genome sequencing, I can foresee the newt genome being
sequenced and annotated in the near future.
The advantages of using these animals as model organisms for heart
regeneration are many. Apart from being ideal adult vertebrate models, they also
have a very complex cardiopulmonary circuitry, similar to that seen in mammals. In
addition they are also easy to maintain. The morphology and size of the heart is ideal
to study whole mount imaging. Strategies for future work in newt cardiac
regeneration should include efforts addressing which cells are responsible for
repairing the damaged newt heart and from where do they arise. Understanding the
molecular and cellular aspects driving heart repair in lower vertebrate species such as
the red spotted newt may one day help us abridge the regeneration deficiencies found
in humans suffering from cardiac diseases.
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9. ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to the following individuals who have
been a big part of my life over the last 4.5 years during my PhD.
First, it is difficult to overstate my gratitutde to my supervisor Jamie Morrison.
Thank you for accepting me as your PhD student. You introduced me to the exciting
field of regenerative biology and drove me to become the best scientist that I could
be. Thanks for all the great ideas and helping me to drive projects to completion. I
have thoroughly enjoyed working alongside you over the years and discussing great
science. I am sorry we did not manage to publish in Poultry Science, but you never
know what the future may hold.
To my Co-supervisor Anders Arner, thank you for all the professional and personal
advice over the years. I will never forget some late night evenings in the lab where we
discussed our shared musical interests and maybe did some scientific research too? I
only regret we did not have the opportunity to work on more projects together.
Current and past members of the Morrison group:
Claudia García González, you came at the right time to help look after me in the
final year of my PhD. I’ll never forget to put sliced potatoes on any injury from a
sunburn to an amputated limb. :P It has really been a pleasure working with you.
Christofer Ott, thanks for being a good friend and always being optimistic. I look
forward to more visits in Austria. Jana Heigwer, thanks for keeping me laughing
even during the hard days. Jessika Tibblin, thanks for always being encouraging and
helping me to keep my head up and moving forward. Siavish Javaheri, I am glad we
have stayed in touch and hope to see you in Göteborg. Barbara Thaler thanks for
your help with the microRNA projects.
To present and previous members of the old “Molbio” gang:
In grad school it’s important to have a good support system to help you survive and
stay sane. They may not have kept me sane, but the first few mentions tried, very
hard. Thank you Johan Waldholm, too many good memories to include here. Thanks
for being a great friend, giving me years of laughter and so much helpful advice in
60
and outside of the lab. I don’t know what will happen to Club Kungshamra when we
leave. But I do know although it may change, we certainly won’t! ; ) Ylva Ekdahl,
you’re my half-American sister I’m pretty sure. The offer is still on the table to swap
passports if you’re up for it. I’m pretty sure we’ll go down in history as the best
dynamic teaching duo ever. You’ve become a great friend. Thanks for looking after
me for all of these years. I’m sure we’ll meet up in NYC ; ) Mikaela Behm “doesn’t
care”. I’m not sure if we ever managed even one conversation together? So as normal
here come some familiar noises: “giggity” “bang” (the guns, naturally) and “cheap
sheeps are not sheap cheeps” – I bet you’re trying really hard to say that one right
now. ; ) Fredrik Lackmann, I wish you spent less time in Scotland and more time in
Stockholm. My advice to you if you ever get stuck with one of your projects is to just
ask WWYD “what would yeast do?” ; )
To Anne Beskow, you have no idea how quiet the hallways and labs have become
since you left. But don’t worry, “it’s exactly what it looks like.” Helene Wahlstedt,
thanks for always breaking my phone, stealing my milk and being one of the funniest
people I have ever met. Chammiran Daniel, thank you for volunteering so many
times to cut my hair. It was more a privilege for you then me, but I did appreciate it. :
P Vikoria Hessle, thanks for warmly welcoming me into the department as a
newcomer. I have thoroughly enjoyed and will never forget my relaxing summer stays
in Älvsjö. Widad Dantoft, thanks for the fun tea and coffee fikas på svenska. Om jag
har lärt mig någon svenska, så har jag nog dig att tacka. : )
To Micke Crona thanks for showing me around in the beginning of my PhD and also
teaching me how to climb. David Nord thanks for making me feel welcomed in that
big lab when I first started. To Sara Jupiter, thanks for welcoming me in the
department and always greeting me with a friendly smile.
I also want to thank some other current or previous members from old Molbio for
technical support and advice or just smiling faces that brought me encouragement
every day, including Ylva Engström (thanks for being an extra mentor and always
having time for me), Marie Öhman (sorry if I spent more time in your lab then my
own – but can you blame me? Also sorry for causing all those distractions during your
office meetings), Lars Wieslander (did I ever apologize for flooding “your
department” in my first week here?), Anne von Euler (thanks for introducing me to
Lidingö ice hockey), Petra Björk, Patrick Young, Andrea Eberle, Simei Yu,
Ulrich Theopold, Nadja Neumann, Solveig Hahne, Zhi Wang, Robert Marcus,
Monica Davis, Shiva Esfahani, Badrul Arefin, Gunnel Björklund, Robert
61
Krautz, Thomas Hauling, Josefin Plathan, Margareta Sahlin, Britt-Marie
Sjöberg, Fredrik Tholander, Neus Visa, and Gilad Silberberg.
I wish to thank some others in the department who helped me with all the practical
stuff. Britta Tumlin, ‘ett stort tack’ for making buffers and speaking Swedish with
me. Christina Jansson, thanks for your help with all the paperwork over the years.
Marina Meyer, thank you for helping me with all the paperwork as well as the
additional “American forms” I always threw your way. I appreciated your patience
and help so much.
Thanks to all the others who have passed through Molbio over the years and
influenced the department.
To more distant-colleagues and friends at the Karolinska Institute:
Matt Kirkham, thanks for making collaborations between our groups so easy and for
many good scientific discussions about regeneration. Valentina Paloschi thanks for
all the fun in and out of the labs, as well as the good scientific discussions in the
CMM lunchroom. Your Italian-English has really improved, even if I still don’t
understand you! Lasse Folkersen, thanks for your friendship, fun hat parties and
great discussions about space and science. I will definitely be visiting more in
Copenhagen.
To some new friends in MBW including Mattias Engelbrecht, Olle Dahlberg, Ali
Mirzaei, Alice Sollazo and Daniel Vare, thanks for helping to keep me fit in weekly
innebandy sessions.
To my “Stockholm family” who gave me a great life outside of work:
The other horsemen of the apocalypse: Oliver Adfeldt-Still, I’m so grateful we met
on a course at the KI the first week I was in Stockholm. You have been like a brother
to me, introducing me to Stockholm and many great friends, I am forever grateful.
Gregor McKechnie, thanks for possibly being the only friend I have with a voice of
reason and for taking good care of me. Remember, one can always train “IT”. Jack
Corkette, thanks for being my own personal court jester over the years. Your laugh is
contagious. Other members of my Stockholm family, you know who you are - thank
you for taking care of me, my time in Stockholm would not have been nearly as
memorable or complete without all of you.
62
To my best friends from home, Chris Gant and Andrew Maynard, thank you for
unrestricted support and a friendship that has spanned three decades.
And finally, to my family. To my grandparents, aunts, uncles, and other relatives,
your interest in my life has kept me going.
My heartfelt gratitude goes out to my hard-working parents, my dad Ray Witman
and my mum Kathy Witman, for giving me emotional support and guidance from a
far-distance. They have sacraficed their lives to giving me and my brothers
unconditional love and care. I can not express in words how grateful nor how lucky I
am to be your son, even if I’m just “the middle one”. :P To my two wonderful
brothers, Matt Witman and Colin Witman, you have no idea what I’ve been doing
over the years but thankyou for your understanding and support anyway. You have
always been there for me.
Det har varit en häpnadsväckande möjlighet att utvecklas som forskare i
Sverige. Det har varit ännu mer av ett privilegium att växa som person i
Stockholm. Jag kommer aldrig att glömma min tid här, både i och utanför
labbet. Jag har erhållit professionella allianser, vetenskapliga
erfarenheter och ännu flera goda vänner och minnen under 4 år än de
flesta kan hoppas på under en halv livstid. Dessa år kommer alltid att
saknas men kommer aldrig att glömmas. - Nevin Witman, 2013
63
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