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From the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm Sweden

Heart : 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 ...... 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

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. , 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 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

7 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 FGF Growth Factor FHF First Heart Field GFP Green Fluorescent Protein LAD Left anterior descending (artery) SCI Spinal Chord Injury SHF Secondary Heart Field MI 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

8 PROLOGUE The capacity of the human body to repair injured tissues and organs following damage, disease or degeneration via the natural 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

9 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 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.

10 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 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 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

11 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

12 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

13 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

14 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 , 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 , 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.

15 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 (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.

16 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 the animals reabsorb their gills, develop 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

17 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.

18 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 (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.

19 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

20 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.

21 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 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 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

22 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.

A B C D

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).

23 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

24 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 . Even though the scar tissue elicits some mechanical support shortly after the infarct, it predisposes for . Additionally, the

25 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 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).

26 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.

27

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.

28 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).

29 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.

30 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 (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 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

31 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

32 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

33 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.

34 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-

35 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).

36

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

37 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

38 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

39 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

40 introduce the enzymes that are responsible for A-to-I RNA editing and explain the significance of RNA editing in relation to regeneration.

41 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).

42

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).

43 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).

44 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

45 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

46 inability to form differentiated colonies in vitro is most likely a result of increased apoptosis in these cells.

47 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.

48 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.

49 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.

50 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

51 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.

52 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

53 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.

54 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

55 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

56 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.

57 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.

58 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

59 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

60 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.

61 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

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