Characterization of the Macrostomum lignano ageing phenotype
Jeroen CRUCKE
Thesis submitted to obtain the degree of Master in Biology
Academic year 2009/2010
Supervisor: Dr. Maxime Willems Co-Supervisor: Prof. Dr. Wouter Houthoofd Tutor: Drs. Stijn Mouton Nematology Section
Characterization of the Macrostomum lignano ageing phenotype
Jeroen CRUCKE
Thesis submitted to obtain the degree of Master in Biology
Academic year 2009/2010
Supervisor: Dr. Maxime Willems Co-Supervisor: Prof. Dr. Wouter Houthoofd Tutor: Drs. Stijn Mouton Nematology Section
© May 10 Faculty of Sciences – Nematology Section
All rights reserved. This thesis contains confidential information and confidential research results that are property to the UGent. The contents of this master thesis may under no circumstances be made public, nor complete or partial, without the explicit and preceding permission of the UGent representative, i.e. the supervisor. The thesis may under no circumstances be copied or duplicated in any form, unless permission granted in written form. Any violation of the confidential nature of this thesis may impose irreparable damage to the UGent. In case of a dispute that may arise within the context of this declaration, the Judicial Court of Gent only is competent to be notified.
Deze masterproef bevat vertrouwelijk informatie en vertrouwelijke onderzoeksresultaten die toebehoren aan de UGent. De inhoud van de masterproef mag onder geen enkele manier publiek gemaakt worden, noch geheel noch gedeeltelijk zonder de uitdrukkelijke schriftelijke voorafgaandelijke toestemming van de UGent-vertegenwoordiger, in casu de promotor. Zo is het nemen van kopieën of het op eender welke wijze dupliceren van het eindwerk verboden, tenzij met schriftelijke toestemming. Het niet respecteren van de confidentiële aard van het eindwerk veroorzaakt onherstelbare schade aan de UGent. Ingeval een geschil zou ontstaan in het kader van deze verklaring, zijn de rechtbanken van het arrondissement Gent uitsluitend bevoegd daarvan kennis te nemen. Acknowledgements
ACKNOWLEDGEMENTS
Toen ik halverwege het eerste semester plots vernam dat ik van thesisonderwerp moest veranderen, was ik toch wel wat in paniek. Al het werk van voordien leek plots voor niets geweest. Achteraf bekeken was dit gevoel onterecht en kijk ik heel tevreden terug naar het afgelopen thesisjaar. Zonder de hulp van tal van mensen zou dit alles nooit mogelijk geweest zijn. Zij verdienen hier daarom een speciaal woordje van dank. Allereerst mijn promotor, Maxime. Bedankt voor al je advies en tips. Je opmerkingen waren steeds terecht en hebben me gestuurd tot een eindresultaat waar ik best wel trots op ben. Wat zou een thesis verder zijn zonder een fantastische begeleider? Bedankt Stijn voor alle uitleg bij de experimenten, het nalezen van mijn teksten en alles wat je het voorbije jaar gedaan hebt om deze thesis te doen slagen! Natuurlijk ook ontzettend bedankt voor de taart en de pittaschotel. Je hebt er wel een mooie databank van je artikels aan overgehouden, niet? Bij deze wil ik je alvast ook veel succes wensen met het schrijven van je doctoraat. Ook een woordje van dank aan Wouter en Freija voor alle hulp wanneer er problemen waren en aan Marjolein voor het gieten van de plaatjes voor mijn culturen. Myriam, bedankt om me kennis te laten maken met de wereld van de elektronenmicroscopie. Ik ben bijzonder blij dat ik deze techniek van jou heb mogen leren. Ook bedankt voor het vele snijwerk en de fixaties die je uitvoerde voor mijn thesis. Geen coupe was je te veel en je stond altijd voor me klaar. De leuke sfeer op de bureau zal ik ook niet snel vergeten. Pam, Hanne en Barbara met jullie was het altijd ontzettend fijn! Bovendien ook bedankt Pam voor de positieve feedback na het lezen van mijn inleiding. Dank gaat ook uit naar Ineke. Het was aangenaam om jou als medestudent in het labo te hebben. Bedankt voor al het overleg, voor het nalezen van mijn Nederlandstalige samenvatting en ook om er voor te zorgen dat er altijd verse BSA-T klaarstond als ik nog maar eens een kleuring moest uitvoeren. Verder wil ik ook nog het labo bedanken om vorig jaar me de unieke kans te geven om mee te gaan naar het platwormencongres te Hasselt. Deze ervaring was de ideale manier om gemotiveerd met het praktische werk van mijn thesis te starten. Een speciaal woordje van dank gaat ook uit naar mijn ouders. Bedankt om mijn studies te financieren en mij de kans te geven om de laatste twee jaar op kot te gaan. Tenslotte wil ik ook nog de persoon bedanken die zoveel voor me betekent, mijn vriendin, Carolien. Na vijf
Characterization of the Macrostomum lignano ageing phenotype i
Acknowledgements jaar samen aan de universiteit is het einde eindelijk in zicht! Het waren soms hectische dagen en ook al had het je zelf heel erg druk met je eigen thesis, toch kon ik altijd bij je terecht. Je kritische opmerkingen en de vele tips qua lay-out hebben er zeker voor gezorgd dat deze thesis is wat hij geworden is. Zonder jou was het me nooit gelukt!
Characterization of the Macrostomum lignano ageing phenotype ii
Table of Contents
TABLE OF CONTENTS
Acknowledgements ...... i Table of Contents ...... iii List of Tables ...... v List of Figures ...... vi Part 1: Introduction ...... 1 1.1 Ageing ...... 1 1.1.1 Definitions of ageing ...... 1 1.1.2 Causes of ageing ...... 1 1.1.3 Stem cells and ageing ...... 2 1.2 Ageing model systems ...... 3 1.2.1 Comparative biology ...... 3 1.2.2 Advantages and disadvantages of current model organisms ...... 4 1.2.3 Is there a need for new ageing model systems? ...... 4 1.3 A flatworm ageing model system ...... 5 1.3.1 Flatworms and stem cells ...... 5 1.3.2 The free-living flatworm, Macrostomum lignano ...... 6 1.3.3 Macrostomum lignano as an ageing model system ...... 7 1.3.4 Ageing in Macrostomum lignano ...... 8 1.4 Hypothesis ...... 9 Part 2: Objectives ...... 10 Part 3: Material and Methods ...... 11 3.1 Overview ...... 11 3.2 Ageing cultures ...... 12 3.3 Light microscopical analysis ...... 12 3.3.1 Principle ...... 12 3.3.2 Procedure ...... 12 3.4 Transmission electron microscopy ...... 13 3.4.1 Principle ...... 13 3.4.2 Procedure ...... 13 3.4.3 Software ...... 14 3.5 3D-Reconstruction ...... 14 3.6 Neoblast labelling ...... 15 3.6.1 Principle ...... 15
Characterization of the Macrostomum lignano ageing phenotype iii
Table of Contents
3.6.2 Protocol: BrdU immunostaining ...... 15 3.6.3 Protocol: EdU immunostaining ...... 16 3.6.4 Confocal microscopy ...... 16 3.7 Experimental design: Tissue maintenance during ageing ...... 16 3.8 Experimental design: Experimental induction of cysts ...... 17 3.9 Statistical Analysis ...... 18 Part 4: Results ...... 19 4.1 Cyst diversity ...... 19 4.2 Cyst structure ...... 22 4.2.1 Cyst morphology ...... 22 4.2.2 Tissue morphology ...... 22 4.2.3 Cyst-opening ...... 28 4.2.4 Branching ...... 28 4.2.5 3D-Reconstruction ...... 29 4.3 Tissue maintenance during ageing ...... 31 4.3.1 EdU immunostaining ...... 31 4.3.2 BrdU immunostaining ...... 33 4.3.3 EdU versus BrdU ...... 35 4.4 Experimental induction of cysts ...... 36 Part 5: Discussion ...... 39 5.1 Characterization of cysts ...... 39 5.1.1 Cyst diversity ...... 39 5.1.2 Cyst and tissue morphology ...... 41 5.1.3 Cysts in Macrostomum lignano versus cysts in the animal kingdom ...... 45 5.2 Causes of cysts ...... 46 5.2.1 Tissue maintenance during ageing ...... 46 5.2.2 Experimental induction of cysts ...... 48 5.3 Hypothetical model for the development of cysts ...... 50 Part 6: Conclusions ...... 53 Part 7: Summary ...... 55 Part 8: Samenvatting ...... 57 References ...... 60
Characterization of the Macrostomum lignano ageing phenotype iv
List of Tables
LIST OF TABLES
Table 1: Listing of all grids photographed with TEM
Table 2: Number of proliferating cells in the head region for EdU immunostaining
Table 3: Summary of p-values (EdU)
Table 4: Number of proliferating cells in the head region for BrdU immunostaining
Table 5: Summary of p-values (EdU versus BrdU)
Table 6: Number of proliferating cells in the rostrum compared between control- and punctured group
Table 7: Development of cysts during the observation period of eight days
Characterization of the Macrostomum lignano ageing phenotype v
List of Figures
LIST OF FIGURES
Figure 1: Ageing of stem cell functionality (Rando, 2006)
Figure 2: Macrostomum lignano
Figure 3: Body deformities in 238 days old individuals (Mouton et al., 2009b)
Figure 4: Overview material and methods
Figure 5: Puncturing experiment
Figure 6: Puncturing
Figure 7: Overview cyst diversity
Figure 8: Range in size of cysts at tail plate
Figure 9: Extreme sizes of cysts
Figure 10: Opening of cysts
Figure 11: Small versus large cysts
Figure 12: Detail of tissues surrounding small cysts
Figure 13: Influence of large cysts on the surrounding tissues
Figure 14: Effect of cyst on the position and organization of the testis
Figure 15: Cyst-opening
Figure 16: Branching
Figure 17: 3D-reconstruction of a region containing a cyst in the rostrum
Figure 18: Number of proliferating cells in function of age (EdU immunostaining)
Figure 19: Number of proliferating cells in function of age (BrdU immunostaining)
Figure 20: Wound healing after puncturing
Figure 21: Number of proliferating cells in the rostrum compared between control- and punctured group
Figure 22: Hypothetical model for the development of cysts
Characterization of the Macrostomum lignano ageing phenotype vi
Introduction
PART 1: INTRODUCTION
1.1 AGEING
1.1.1 DEFINITIONS OF AGEING
One of the biological processes most known to man is ageing. We can see the effects of it with others, or experience them ourselves (Kirkwood, 2005). Despite the fact that everyone is familiar with this process, defining ageing is not so straightforward, because it is a complex process which has an effect on every aspect of life. A multitude of definitions exist to support the large number of different theories that have been put forward in the past. All of these definitions have two aspects in common, namely, a decline in both performance and fitness with advancing age, resulting in an increased risk of death (Comfort, 1956; Finch, 1990; Harman, 1981; Hughes & Reynolds, 2005).
1.1.2 CAUSES OF AGEING
Many theories have been developed throughout history to account for the ageing process. However, most of these theories focus on but one single part of this complex mechanism. In order to fully understand what makes us grow old, all of these theories should be put together and looked at as a whole (Harman, 1998). When trying to explain the ageing process, it is important to explain both the underlying mechanical background, as well as the occurrence of ageing in the light of evolution (Hughes & Reynolds, 2005). Evolutionary theories of ageing try to explain how ageing has originated, and why it has not been more effectively opposed by natural selection during the course of evolution (Kirkwood, 1977). Molecular theories on the other hand, explain how different cellular processes can cause senescence. For example, the production of reactive oxygen species (ROS) is thought to play an important role in the ageing of cells by causing oxidative damage to macromolecules (Beckman & Ames, 1998).
Characterization of the Macrostomum lignano ageing phenotype 1
Introduction
1.1.3 STEM CELLS AND AGEING
Ageing in organisms is characterized by a functional decline, due to changes in the organ and tissue system. Alongside this declining functionality, is a diminished capacity to respond to injury or stress (Rando, 2006). This response usually results in the proliferation of stem cells. Stem cells are characterized by their capacity of self-renewal, and by their ability to differentiate into new tissues. Their capacity for self-renewal allows them to replace damaged cells while sustaining their own population (Morrison et al., 1997). As a consequence, ageing can be the result of the reduced functionality of stem cells to maintain and repair tissues (Rando, 2006).
Figure 1: Ageing of stem cell functionality
The reduction of stem cell functionality with advancing age can be the result of interactions on several levels. This figure illustrates several possibilities using a skeletal muscle fiber and a stem cell. Upon injury, stem cells are induced locally to proliferate and generate new progeny in order to repair damaged tissues (a). Changes in the stem cells themselves (b), in the niche (c) or in the systemic environment (d) could reduce the proliferative capacities of stem cells (reprinted from Rando, 2006).
Characterization of the Macrostomum lignano ageing phenotype 2
Introduction
A diminished functionality of stem cells can be the result of changes on different levels of complexity (Figure 1) (Rando, 2006). Mutations that arise in the DNA of stem cells are readily passed on to their daughter cells and accumulate during ageing. If these genetic lesions provide a growth advantage they will in return produce a positive selection for the stem cell clone, resulting in a malignant transformation or cancer. To reduce the chance of developing cancer, there are mechanisms which detect defects in the genome of stem cells, and which reduce the proliferative capacity of these cells. However, these mechanisms which reduce the replicative capacity of stem cells, such as apoptosis and cellular senescence, may cause a declining cell replacement and contribute to the ageing process. This is called the „cancer- ageing hypothesis‟ and it implies intrinsic stem-cell ageing (Campisi, 2001; Sharpless & Depinho, 2007). A reduction in the function of stem cells, however, does not necessarily involve intrinsic stem cell ageing. Age-related changes in the local niche, the systemic environment or any combination of these factors can contribute significantly to a decline in stem cell functionality (Rando, 2006). Age-associated changes in the stem cell niche have already been shown to play a role in initiating age-related dysfunction of tissue-specific stem cells in mice (Mayack et al., 2010). Changes in the systemic environment can involve immunological and neuroendocrine changes, or signals from damaged cells (Rando, 2006). The role of stem cells in ageing still needs a lot of research. There is an obvious need for new validated systems in which the interaction between stem cells and the surrounding tissues can be easily studied (Sharpless & Schatten, 2009). Such a model system, however, is currently unavailable.
1.2 AGEING MODEL SYSTEMS
1.2.1 COMPARATIVE BIOLOGY
Scientists face several problems when studying ageing in humans. Besides the obvious ethical issues, there is also the main problem of duration. The life of humans is too long to serve as a proper study object. Making use of adequate model systems can therefore aid in deducing universal aspects of ageing, that can afterwards be generalized to a larger number of species (Buffenstein et al., 2008). A good model organism has to meet certain criteria: it needs to have a short generation time, produce a large number of offspring, it should be relatively small, easy to care for, breed well in laboratory conditions, and it should be easy to
Characterization of the Macrostomum lignano ageing phenotype 3
Introduction manipulate (Bolker, 1995). The most commonly used model organisms in ageing research are the single-celled yeast, Saccharomyces cerevisiae, the free-living nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the laboratory mouse Mus musculus (Buffenstein et al., 2008).
1.2.2 ADVANTAGES AND DISADVANTAGES OF CURRENT MODEL ORGANISMS
In ageing research, these four model organisms have been around for quite some time. They have provided scientists with a good basis for studying certain aspects of the ageing process. However, they are not perfect and all have advantages and disadvantages (Austad, 2009). The vertebrate model systems, such as the laboratory mouse Mus musculus, have the advantage of being phylogenetically closely related to humans. Conclusions based upon mouse models are therefore more likely to be valid in humans as well. On the other hand, vertebrate models are complex systems. The stem cell system, for example, is not readily accessible, and ageing research is difficult because of the long life span (Jenner & Wills, 2007). The invertebrate models are usually small, short-living, inexpensive to grow or culture, and thus amenable as laboratory animals (Austad, 2009). Mutants can be made fairly easy, and this has contributed substantially to the study of certain genes or biochemical pathways important in senescence. Weak points include the fact that results cannot always be generalized to other species due to a large phylogenetic distance. Conclusions based upon results from research in invertebrates, should be analyzed carefully before extrapolating to humans (Jenner & Wills, 2007). Another disadvantage is that the cells of these organisms are completely (C. elegans) or for the largest part (D. melanogaster) post-mitotic during adulthood. This makes the study of the relation of stem cells and ageing a very difficult task (Buffenstein et al., 2008; Gershon & Gershon, 2000; Johnson, 2003; Yu, 1998).
1.2.3 IS THERE A NEED FOR NEW AGEING MODEL SYSTEMS?
Ageing is a very complex process, and finding one organism in which all aspects can be studied is not possible. Hence, it is of paramount importance that we look for other model systems that are particularly useful for studying certain aspects of ageing. The involvement of stem cells in the ageing process is one of those aspects for which new validated systems are needed.
Characterization of the Macrostomum lignano ageing phenotype 4
Introduction
The role of stem cells in ageing research has become more important over the last few years. Stem cells are responsible for the renewing of tissues and maintaining homeostasis. Ageing, however, is characterized by an impaired tissue homeostasis. Stem cells could therefore be an important factor in what causes senescence (Rando, 2006; Sharpless & Depinho, 2007). Studying the influence of stem cells on the ageing process, however, is difficult in the current model systems for the reasons mentioned in 1.2.2. One type of organisms who do not posses these limitations, are flatworms (Austad, 2009). These animals are known for possessing an experimentally accessible and totipotent population of stem cells, called neoblasts (Baguñà et al., 1994).
1.3 A FLATWORM AGEING MODEL SYSTEM
1.3.1 FLATWORMS AND STEM CELLS
Flatworms and their powers of regeneration have intrigued scientists throughout history. Their remarkable capability of forming new individuals out of small body fragments remains unique within the bilaterian clade. It has even led Dalyell in 1814 to the conclusion that flatworms could “almost be called immortal under the edge of the knife”. Cutting an animal in half will result in two genetically identical individuals. Body pieces even as small as 10000 cells are still able of forming an entirely new individual (Montgomery & Coward, 1974). This unique feature can be ascribed to the presence of a permanent population of totipotent stem cells, called neoblasts (Rossi et al., 2008; Saló, 2006). Besides their dominant role in regeneration, they are also constantly being recruited to maintain/renew differentiated and aged tissues. Their role in tissue maintenance is thus of vital importance to these animals (Rossi et al., 2008). In adult flatworms, the neoblasts are the only remaining dividing and differentiating cells. Studying them can be done by using several methods. The techniques of importance for this master thesis are the use of thymidine analogues and transmission electron microscopy (TEM). The thymidine analogues, such as 5‟-bromo-2‟-deoxyuridine (BrdU), incorporate into the DNA of the cells in S-phase of the cell cycle. Afterwards, they can be visualized by using specific primary and secondary antibodies. This technique is mainly used to examine the location and the distribution of the stem cell population. Transmission electron microscopy is used to study the ultrastructure of the cells (Bode et al., 2006).
Characterization of the Macrostomum lignano ageing phenotype 5
Introduction
Most of the research in the past has focused on triclad species (called planarians), but here a different member of the Platyhelminthes will be used, namely Macrostomum lignano (Macrostomorpha).
1.3.2 THE FREE-LIVING FLATWORM, MACROSTOMUM LIGNANO
Macrostomum lignano is a free-living, marine flatworm that was originally found on the beaches of the Northern Adriatic sea in Lignano, Italy. It is a member of the Macrostomorpha, the most basal taxon within the rhabditophorans (Ladurner et al., 2005). They are very active, negative phototactic worms. Dorsal of the mouth are two eyes used for photoreception. M. lignano possesses a transparent body ranging in size from 1-2 mm long and 0.3 mm wide (Figure 2A). The epidermal cells are covered with cilia and contain on the dorsolateral side numerous glands, called rhabdites. Food uptake is performed by a ventral opening of the digestive system. Food can enter the sack-like gut via a pharynx bearing a cluster of pharyngeal glands. The nervous system encompasses several main structures such as a brain and two longitudinal nerve cords (Figure 2A, D) (Ladurner et al., 2005). These flatworms are cross-fertilizing hermaphrodites, so they possess both testis and ovaria (Figure 2A). The stylet, the male reproductive organ, can be found at the tail plate (Figure 2A, C) (Ladurner et al., 2005). The neoblasts can be subdivided into three distinct sets. The first set is located in the mesoderm, between the epidermis and the gastrodermis (Figure 2E). They are spread across the body along two lateral bands that are most likely to be associated with the lateral nerve cords (Figure 2B). A second population of neoblasts are spread out in between the lateral bands of mesodermal stem cells and are called, the gastrodermal neoblasts. The third and last population of neoblasts resides in the gonads, called germline stem cells. In front of the eyes there is a complete lack of neoblasts (Figure 2B) (Bode et al., 2006).
Characterization of the Macrostomum lignano ageing phenotype 6
Introduction
Figure 2: Macrostomum lignano
Several photographs illustrating important characteristics of M. lignano. (A) 0verview of an adult animal, showing the brain (b), eyes (e), pharynx (p), gut (g), testis (t), ovarium (o), and stylet (s). (B) Confocal image showing the distribution of neoblasts in an adult individual. Green dots are S-phase labelled neoblasts, red dots are mitosis labelled neoblasts. (C) Detail of stylet (s) with seminal vesicle (sv). (D) Detail of rhamnites (r) going through the brain (b). (E) Detail of epidermis and mesoderm with arrowheads indicating position of neoblasts.
1.3.3 MACROSTOMUM LIGNANO AS AN AGEING MODEL SYSTEM
Macrostomum lignano has a lot of potential as a model organism for ageing research. It is an ideal system for studying the reciprocal interaction between stem cells and the ageing process, which could provide new insights in the causing agents of senescence. There are a lot of tools available for this organism, and the first demographic analysis has been published (Mouton et
Characterization of the Macrostomum lignano ageing phenotype 7
Introduction al., 2009a). First of all, its transparent body and the fact that the animals can be relaxed easily using MgCl2, make it very amenable for light microscopical analysis. For example, cysts or other abnormalities can be easily spotted and examined under the microscope in vivo. The stem cell system is well described and can be visualized using thymidine analogues such as 5‟-bromo-2'-deoxyuridine. Contrary to other model organisms for ageing research (e.g. Drosophila melanogaster, Mus musculus), there is a large set of easily accessible stem cells, and studying the interaction with the surrounding tissues does not pose a problem. For example, by simply cutting these animals, the stem cell dynamics during regeneration can be examined. These analyses can be performed at any given time and make it therefore possible to assess the relationship between age and neoblasts (Mouton et al., 2009b). Its small size also allows a quantification of the stem cells of the entire organism. Furthermore, its short generation time and low cultivation needs make it a perfect animal for housing under laboratory conditions.
1.3.4 AGEING IN MACROSTOMUM LIGNANO
The unique stem cell system of flatworms and their exceptional capacity for regeneration could led one to think that these animals do not age at all. This is, however, not true. Macrostomum lignano has a median life span of about 205 days (29.5 weeks) (Mouton et al., 2009a). The 90th percentile is about 373 days and the maximum lifespan 861 days (Mouton S., pers. comm.). So what causes ageing in flatworms? It was hypothesized that with advancing age, the renewing of tissues can no longer be maintained, and the balance between young and old cells is disturbed (Abeloos, 1930). Morphological data seems to support this hypothesis. As these animals grow older, their transparent body changes into an opaque grey, and several types of distinct body deformities start to appear (Mouton et al., 2009b). These deformities encompass small bulges, grooves in the epidermis, and liquid-filled cysts (Figure 3). In addition to ageing, these same deformities can be seen in animals that have been cut repeatedly (Mouton et al., 2009b). However, these deformities have not been properly characterized.
Characterization of the Macrostomum lignano ageing phenotype 8
Introduction
Figure 3: Body deformities in 238 days old individuals
(A) The presence of a groove in the head region. (B) Another body deformity commonly seen in older individuals is the presence of a liquid-filled cyst. Scalebars are 100 µm (reprinted from Mouton et al., 2009b).
1.4 HYPOTHESIS
We know that cysts develop in older animals and in animals after repetitive cutting. Why these cysts develop, which types of cells are involved in this sort of structure, or how they originate, is still unknown. Therefore, the characterization of these deformities by describing their diversity, along with a study of the morphology of the cysts and its surrounding tissues, will aid in providing an answer to what type of structure this is. Furthermore, it is also important to assess what causes these deformities to develop. This could be because of a reduced control of tissue maintenance, an incomplete regeneration of local wounds, or a combination of both. Finding answers to these questions will provide better insights into the ageing process of M. lignano, and will allow a more in depth analysis of this flatworm as a model organism for studying the reciprocal interaction between stem cells and the ageing process.
Characterization of the Macrostomum lignano ageing phenotype 9
Objectives
PART 2: OBJECTIVES
The main objective of this master thesis is to perform a more detailed study of the cysts, which appear in M. lignano during ageing and repetitive cutting. Here, the focus will be on ageing animals. Light microscopy will be used for documenting the morphology of animals containing cysts. Using this technique, we will try to give an overview of the several distinct locations at which these deformities can appear, how they can vary in size, and the amount of cysts that can be found in a single individual. Transmission electron microscopy will provide us with a better understanding of which cells are involved in this type of structure. It will also allow us to see if these cysts have an influence on and cause any significant alterations in the surrounding tissues. Next, we will use neoblast labelling methods for evaluating the stem cell functionality in function of age. We will study tissue homeostasis in both young and aged animals and test whether the control of tissue maintenance diminishes as these flatworms grow older, and if this results in deformities. Finally, we will use a more experimental approach to see how irregularities are repaired by making a local wound. Combined with the labelling of neoblasts, this experiment will show whether or not stem cells are involved in an initial reaction for healing the wound, or if we get a remodeling of the existing tissues. These results can provide us with clues of the involvement of stem cells and tissue remodeling in cyst formation.
Characterization of the Macrostomum lignano ageing phenotype 10
Material and Methods
PART 3: MATERIAL AND METHODS
3.1 OVERVIEW
An overview of the three main techniques used during this thesis is given in Figure 4.
Figure 4: Overview material and methods
Schematic representation illustrating the main techniques used during the course of this master thesis. Different approaches were used resulting in a more in depth analysis of the cysts in M. lignano.
Characterization of the Macrostomum lignano ageing phenotype 11
Material and Methods
3.2 AGEING CULTURES
Macrostomum lignano can be cultured in the laboratory under standardized conditions.
Worms are kept in Petri dishes containing F2, a nutrient-enriched artificial seawater medium at a salinity of 32‰ (Guillard & Ryther, 1962). The animals are fed ad libitum using the diatom Nitzschia curvilineata, and are incubated at a constant temperature of 20°C and a
13h:11h light: dark cycle (Rieger et al., 1988). For this thesis, however, animals are housed individually in multiwell plates, containing 12 wells each. This way there are no other variables (e.g. group size, reproduction, mutual interactions, etc.) influencing ageing besides the medium, and there is no mixing of animals of different ages. When starting new cultures, adults are placed together overnight in a Petri dish and removed the next morning. Eggs laid that night will already hatch five days later. One-day-old juveniles are then transferred one by one into multiwell plates. Each three to four weeks all the animals are transferred into new plates containing F2 medium and diatoms. This is to prevent a depletion of food and to keep the worms well fed. A total of five cultures were started during this thesis. The first four cultures contained ten plates and thus 120 worms, and were started on 19th October, 2009; 28th November, 2009; 1st March, 2010 and 8th March, 2010. A fifth and final culture contained five plates and thus 60 worms, and was set up on 24th March, 2010.
3.3 LIGHT MICROSCOPICAL ANALYSIS
3.3.1 PRINCIPLE
Light microscopy is used to study the morphology of animals with cysts in a non destructive manner. This allows us to monitor the same animal in vivo through time. The transparent body of M. lignano is a major advantage for this technique as internal structures can be easily studied. The light microscope used was an Olympus BX 51, and photographs were taken with the digital camera Olympus C5060.
3.3.2 PROCEDURE
When examining morphology using a light microscope, squeeze preparations need to be made. This is an essential process for immobilizing the worm and studying the internal
Characterization of the Macrostomum lignano ageing phenotype 12
Material and Methods structures. Animals displaying cysts are isolated from the cultures. In a first step, the animals are relaxed using MgCl2 (7.14%) isotonic to artificial seawater (ASW), and put in a drop (50 µl) on a microscope slide. Minute amounts of plasticine are then attached to the corners of a cover slip (20x20 mm) in order to make little feet. The cover slip is then placed on top of the drop containing the specimen, and is gently pressed down on all sides. Excess water can afterwards be removed using a filter paper. Finally, the animal can be studied using the microscope, and pictures can be taken on different magnifications (10x, 20x, 40x, 60x, 100x) (adapted from Schärer et al., 2005) .
3.4 TRANSMISSION ELECTRON MICROSCOPY
3.4.1 PRINCIPLE
Studying details of tissue/cell morphology is done by means of transmission electron microscopy (TEM). This type of microscope can achieve far greater magnifications (up to 80000x) then a traditional light microscope and permits us to better distinguish differentiated cells.
3.4.2 PROCEDURE
Animals displaying cysts, and control animals without cysts, were selected from the ageing cultures. They were relaxed using a 1:1 ASW-MgCl2 solution and fixed in 2.5% glutaraldehyde buffered with 0.1 M NaCacodylate containing 10% sucrose. Following post- fixation in 1% osmium tetroxide in the same buffer and standard dehydration in acetone (Eisenman & Alfert, 1982), the specimens were embedded in Spurr's low viscosity resin (Spurr, 1969) for the preparation of ultrathin sections to be viewed with the TEM, and semithin sections to be viewed with the light microscope. Semithin sections (1 µm) were made using a Reichert-Jung ultra-cut ultramicrotome (Leica, Vienna, Austria). These sections were stained using a 1% methylene blue and 1% azur II in 1% borax solution (Richardson et al., 1960), and mounted in DePeX (Gurr, BDH laboratory, UK). Serial ultrathin sections (70 nm) were made with a Leica-Ultracut-S-ultramicrotome (Leica, Vienna, Austria) and placed on formvar-coated single-slot copper grids (Agar Scientific, Stansted, UK). The contrast of the ultrathin sections was improved with lead citrate and uranyl acetate (EMstain, Leica). Observations of the ultrathin sections were made using a
Characterization of the Macrostomum lignano ageing phenotype 13
Material and Methods
Jeol JEM-1010 transmission electron microscope (Jeol, Tokyo, Japan) operating at 60kV. The photographs were digitalized using the DITABIS system (Pforzheim, Germany). A total of 26 animals were fixed during this thesis. Out of 26, 10 were used for ultrathin sectioning, and studied with TEM. First, semithin sections were made until at the position of the cyst. Next, there was an alternation of 30 ultrathin sections followed by 10 semithin sections, until the cyst was fully sectioned. Another six animals were used exclusively for semithin sectioning. For a complete listing of all grids photographed with the electron microscope, see Table 1.
Table 1: Listing of all grids photographed with TEM Gridbox Grids photographed Jeroen1 A7, A9, A13, A16, A18, Jeroen2 A1, A2 Jeroen3 A9, C9, E11, E13, D12, A18, D16, E18, B20 Jeroen4 E6, C1, B2, C5, B8, B7, D10, B11, A16 Jeroen5 D4, A5, C5, B6, E6, B7, E8, B9, C9, A10, E10, E11, D13, A15, E16, A18, D19 Jeroen6 A1, E1, D2, C3, E3, B4, E4, A6, C6, C8, D9, D10, E14, E16 Jeroen7 A2, C5, C6 Jeroen8 A8, A10 Jeroen9 A1, C3, D4, A5, C6, A8, C13, A4, C1, B2, E3 Jeroen10 S1 Jeroen11 A4, B4, C2, D4, E3, F3, G2, G4, H2, I1, I3, N4,N5, O4, P4, R3 Jeroen12 A5, B5, C5, H2 Jeroen 13 B2
3.4.3 SOFTWARE
Sections viewed at magnifications of 3000x and higher (up to 12000x), had to be subdivided into 10-15 photographs, and afterwards reconstructed using Adobe® Photoshop® CS4 Extended (version 11.0). This was done through the „Automate-Photomerge‟ function.
3.5 3D-RECONSTRUCTION
A 3D-reconstruction of a region containing a cyst in the rostrum was made using a series of 35 semithin sections covering a distance of 47 µm. These sections were made as described in 3.4.2, and mounted in DePeX. Photographs were taken with a digital camera attached to the light microscope at 60x magnification. The reconstruction was made using the software
Characterization of the Macrostomum lignano ageing phenotype 14
Material and Methods program Amira® (v3.1.1.), and photographs from different perspectives were made using Rhinoceros® (v4.0).
3.6 NEOBLAST LABELLING
3.6.1 PRINCIPLE
The neoblasts are the only dividing cells in adults of M. lignano (Ladurner et al., 2000). This allows us to use thymidine analogues, which will incorporate in the nucleus of cells in S- phase, to identify and visualize the neoblasts (Newmark & Alvarado, 2000). Two types of thymidine analogues were used: 5-bromo-2‟-deoxyuridine (BrdU) and 5-ethynyl-2‟- deoxyuridine (EdU). Contrary to BrdU, EdU labelling lacks the DNA denaturing steps involving strong acids, heating, and proteases needed for exposing the epitope in BrdU immunostainings (Chehrehasa et al., 2009). The use of proteases in BrdU labellings results in the loss of a certain amount of epidermal cells depending on the length of the treatment. Thus, EdU immunostainings result in a better preservation of the morphology of the organisms.
3.6.2 PROTOCOL: BRDU IMMUNOSTAINING
Animals were pulsed for 30 minutes (5 mM BrdU). After washing three times for five minutes with ASW, the animals were relaxed using a 1:1 ASW-MgCl2 solution, and fixed with 4% paraformaldehyde. Organisms were washed five times for five minutes with phosphate buffered saline (PBS) and permeabilized for 60 minutes in PBS-T (0.1% Triton X- 100 in PBS). Next, specimens were treated with 0.2 mg/ml protease XIV at 37°C under visual control, for degradation of the epidermis, allowing easier access of the primary and secondary antibodies. Worms were then incubated in 2N HCl at 37°C for one hour to denature the DNA, and washed five times for five minutes with PBS. Animals were blocked for 30 minutes with bovine serum albumin-triton (BSA-T, 1% BSA in PBS, 0.1% Triton X-100) and incubated overnight at 4°C with rat anti-BrdU (AbD Serotec) primary antibody (1:800 in blocking solution). After washing with PBS, animals were incubated for one hour with secondary antibody FITC-conjugated donkey anti-rat (Rockland) (1:600 in blocking solution). Finally, animals were mounted using Vectashield (Vector Laboratories Inc, Burlingame, CA).
Characterization of the Macrostomum lignano ageing phenotype 15
Material and Methods
3.6.3 PROTOCOL: EDU IMMUNOSTAINING
EdU immunostaining was performed using the Click-iT™ EdU imaging kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. This protocol is normally intended for use in cell cultures, but was adapted for whole mount stainings of M. lignano as follows. Animals were pulsed for two hours (200 µM EdU). After washing three times for five minutes with ASW, the animals were relaxed using a 1:1 ASW-MgCl2 solution, and fixed with 4% paraformaldehyde. Organisms were then washed five times for five minutes with PBS and blocked for two hours in BSA-T (1% BSA in PBS, 1% Triton X-100). Next, the animals were incubated with a Click-iT™ reaction cocktail containing Click-iT™ reaction buffer, CuSO4, Alexa Fluor® 488 Azide, and reaction buffer additive for two hours at 4°C while protected from light. After washing five times for five minutes with PBS, animals were incubated in PBS-T (1% Triton X-100 in PBS) for one hour, and afterwards washed again five times with PBS. Finally, animals were mounted using Vectashield (Vector Laboratories Inc, Burlingame, CA).
3.6.4 CONFOCAL MICROSCOPY
Visualization of the neoblasts after immunostaining is realized with a confocal laser scanning microscope (cLSM) of the type, Nikon Eclipse TE2000-S. This type of microscope uses an argonlaser as excitation source, which allows us to take optical sections through the specimen over a preset distance (z-stacks). This results in high resolution, horizontal projections in which we can easily quantify the number of labelled stem cells using the appropriate software (cell counter, ImageJ 1.43i). Both fluorochromes used, FITC (BrdU immunostaining) and Alexa fluor (Edu immunostaining), are excited at a wavelength of 488 nm, which causes them to emit green light at a wavelength of 540 nm.
3.7 EXPERIMENTAL DESIGN: TISSUE MAINTENANCE DURING AGEING
The goal of this experiment is to test whether the formation of cysts is caused by a declining tissue homeostasis in older animals. To test this, the rate of tissue maintenance is compared between both young and old worms. Cultures were set up, and animals were allowed to age until the desired age of one, four and six months old. These ages were chosen based upon the demographic analysis of the species. Since worms are adult at about three weeks, and the
Characterization of the Macrostomum lignano ageing phenotype 16
Material and Methods median life span is around seven months (Mouton et al., 2009a), the age of one month was used for young animals, six months for aged animals and four months as an intermediate age. As a marker for tissue maintenance, the number of neoblasts in the head region anterior to the eyes is used. EdU immunostainings were performed instead of BrdU labellings, because there is no loss of epidermal cells and a better preservation of morphology. Worms were pulsed and chased for four days (“pulse-chase experiment”), before the labelling was performed. During the chase period, cells can divide, migrate and differentiate. Since there is a complete lack of neoblasts in front of the eyes, all stem cells present in that region can be considered as either migrating cells that are going to differentiate, or as cells that have migrated and are already differentiated. All the neoblasts in the head region, anterior to the eyes, are then counted using the cell counter of ImageJ 1.43i.
3.8 EXPERIMENTAL DESIGN: EXPERIMENTAL INDUCTION OF CYSTS
The aim of this experiment is to make a local wound, and see how this is repaired. This will show if stem cells are involved in an initial reaction for healing the wound, or if we get a remodeling of the existing tissues. These results can provide us with clues of the involvement of stem cells and tissue remodeling in cyst formation. The experiment can be divided in several steps (Figure 5):
Figure 5: Puncturing experiment
A visual representation of the different steps performed during this experiment. Numbers colored in green are animals that were punctured. Numbers depicted in blue are worms that were not punctured (control).
Step 1: 90 animals were pulsed (BrdU) Step 2: Immediately after the pulse, 60 animals were punctured as illustrated in Figure 6. The remaining 30 served as control animals.
Characterization of the Macrostomum lignano ageing phenotype 17
Material and Methods
Step 3: 24 hours later the first staining was performed to test whether there is an initial stem cell reaction for healing the wound. This staining included 15 animals that were punctured and 15 from the control group. Step 4: The remaining 45 animals that were punctured were observed on a daily basis for the following eight days, to quantify the amount of cysts that developed and at which location. Step 5: A final immunostaining was performed after eight days to see if cells involved in cysts/wound healing are formed by differentiating labelled neoblasts. This labelling was, however, not successful and is therefore not depicted in Figure 5, nor is it included in the results section.
Figure 6: Puncturing
A small hole was made in the head region anterior to the brain (outline above the eyes) by means of a fine needle. Before puncturing, animals were immobilized
using MgCl2 (7.14%), and put in a small drop on the lid of a Petri dish.
3.9 STATISTICAL ANALYSIS
The quantified number of neoblasts, resulting from the different immunostainings, was compared between groups. When dealing with multiple groups, an analysis of variance (ANOVA) was performed. If only two groups were present, a Two Sample t-test was used. Normality of the dataset was tested using a Shapiro-Wilk normality test. Homogeneity of variances was tested using a Levene‟s test. Data was transformed if necessary. All statistical analyses were carried out using R 2.11.0.
Characterization of the Macrostomum lignano ageing phenotype 18
Results
PART 4: RESULTS
4.1 CYST DIVERSITY
Ageing cultures were screened at regular time intervals. Individuals displaying cysts were isolated, and squeeze preparations were made for light microscopical analysis as described in 3.3.2. The aim was to document the diversity in location, size and number of cysts that can appear in Macrostomum lignano. The youngest individual, in which cysts were studied, was 81 days of age. An overview of the range in different phenotypes that was studied is given in Figure 7.
Location and number of cysts Cysts can be found from anterior to posterior, throughout the entire adult body. The regions that are most prone to develop cysts are the head and tail region. When looking at the rostrum, usually one or several smaller cysts can be found (Figure 7A), while at the tail plate the cysts can vary from small (Figure 8A) to large (Figure 8B). Cysts can also develop at all other locations in between the rostrum and the tail plate. These locations include: the region between the pharynx and the testis (Figure 7B), the region of the gonads (Figure 7C), and the area between ovarium and tail plate (Figure 7D, E). The number of cysts found, varies amongst individuals. This can go from the presence of a single cyst (Figure 7B), to multiple cysts located at different areas in the body (Figure 7A). When multiple cysts are present, a combination of both large and small cysts can often be found (Figure 7D, F).
Size and consequence for the animal The size of these deformities is variable. They can range from small (Figure 7A), to big (Figure 7B, D, F). Some individuals were found to possess only small cysts (Figure 7A), while others had a single large one (Figure 7B). Furthermore, small and large cysts can also occur simultaneously in the same individual (Figure 7D, F). Some of these deformities were also observed to take on a more extreme size. Animals were observed in which a single cyst was present encompassing up to half the body size of the individual (Figure 9). A closer look at the wall of these cysts revealed that the surface is not smooth, but contains multiple grooves causing different currents of fluid inside this structure, due to the movement of cilia inside these grooves (Figure 9B). Nevertheless the size of these deformities, some animals are still
Characterization of the Macrostomum lignano ageing phenotype 19
Results capable of moving around unhindered through the medium (Figure 9A). However, as these cysts grow even larger the movement of the animals is no longer directed when compared to individuals free of cysts (Figure 9C).
Age of the animals There is a positive correlation between the age, and the number of individuals displaying cysts. The age of the animals, however, does not seem to correlate with size, number or location of these deformities. When comparing animals of different ages, sometimes the largest cysts can be found in the younger individual (Figure 8B), while the older animal has only a small cyst (Figure 8A).
Figure 7: Overview cyst diversity
Figure illustrating the range in size, the number
per animal and the several locations at which cysts can occur. Both small (arrowheads) and large (*) cysts can be seen throughout the entire adult body. Arrow indicates the position of remnants of tissue inside a liquid-filled cyst. Age
of the animals at time of photograph was 121 days (A, F); 134 days (E); 146 days (C) and unknown (B, D). Scalebars are 150 µm. Anterior is to the right.
Characterization of the Macrostomum lignano ageing phenotype 20
Results
Figure 8: Range in size of cysts at tail plate
(A) Tail of a 134-day old individual containing a single small cyst (arrowhead). (B) Tail of 81-day old individual with a large cyst (*), giving the tail a hollow appearance. Also note the fact that here, the younger animal has a much larger cyst than the older individual. Scalebars are 100 µm. Posterior is to the left.
Figure 9: Extreme sizes of cysts
(A) 122-day old individual possessing a large cyst about half its own body size. This animal was not hindered in its movement. (B) Enlargement of A, showing that the wall of the cyst is not smooth but contains several grooves, giving rise to multiple currents inside this structure due to the movement of cilia on the inside of these grooves. (C) 81-day old individual having a very large deformity, which severely impairs its mobility. Scalebars are 100 µm (A, C) and 50 µm (B). Anterior is to the left.
Characterization of the Macrostomum lignano ageing phenotype 21
Results
4.2 CYST STRUCTURE
4.2.1 CYST MORPHOLOGY
The deformities themselves appear to be empty, liquid-filled cavities. Some exceptions were found in which the cysts contained remnants of tissue (Figure 7E). Noteworthy, is the fact that the cysts appear to have an opening, which connects the internal cavity to the external environment (Figure 10).
Figure 10: Opening of cysts
Cysts were found to possess an opening (arrowhead), which connects the inside of these deformities to the outside environment. When multiple cysts were present in the head, most of them contained this opening. Age of the animals at the time of study was 94 days (A), 121 days (B), and 134 days (C). Scalebars are 100 µm. Anterior is to the left.
4.2.2 TISSUE MORPHOLOGY
The cysts were studied at the ultrastructural level by means of transmission electron microscopy (TEM). Animals displaying deformities, together with control individuals free of cysts, were selected from the ageing cultures and fixed as described in 3.4.2. From the beginning, a distinction could be made between small and large cysts (Figure 11). Both types appear as well-defined structures within the animal (Figure 11). Large cysts, however, cause a loss of organization in the surrounding tissues (Figure 11A), while this is not the case in small deformities (Figure 11A) Hence, both types of cysts will be dealt with separately below.
Characterization of the Macrostomum lignano ageing phenotype 22
Results
Figure 11: Small versus large cysts
(A) Electron micrograph of a 114-day old individual with a small cyst (c, orange) in the head region. Small cysts are well-defined structures encircled by a single cell layer (light green), which do not appear to have a great impact on the surrounding tissues. (B) Micrograph of an individual with multiple cysts (c, orange), both small and large, in the gut (g) region between the pharynx and the testis. The organization of the tissues seems to be lost. Scalebars are 8 µm (A) and 20 µm (B).
4.2.2.1 SMALL CYSTS
The first thing to notice when looking at these deformities by means of TEM is that the cysts appear as well-defined, hollow structures. They are ciliated on their inside, and are completely encircled by a single cell layer. The cells from this layer appear to be of the same type as the ones part of the epidermis covering the animal (Figure 12B). Both layers consist of large, tightly packed together cells with a lobed nucleus in which numerous cilia are anchored on the apical side. Therefore, the cell layer encircling the cysts can be considered as a second layer of epidermis within the mesoderm. Notable as well, is the significant loss of mesodermal structures (e.g. muscle cells, nervous tissue, glands, etc.) when compared to control animals, because of the presence of the cavity. This, however, did not prove to be lethal. Furthermore, when comparing the remaining structures within the mesoderm between control animals (Figure 12C), and worms containing cysts (Figure 12D), no structural differences can be seen.
Characterization of the Macrostomum lignano ageing phenotype 23
Results
Figure 12: Detail of tissues surrounding small cysts
(A) 22-day old control animal showing the position of the outer cell layer, the epidermis (ep, purple). (B) Micrograph of a 114-day old individual showing the position of both the outer epidermis (ep, purple) and the cell layer surrounding the cyst (purple). Note the loss in mesodermal structures because of the presence of the cavity. (C) Detail of area indicated by the black square in A. Typical mesodermal structures such as muscle cells (mu, green), nervous tissue (nv, yellow), glands (gl), etc., can be seen below the epidermis (ep, purple). (D) Detail of area indicated by the black Square in B. Both the outer epidermal cell layer (ep, purple, left), and the inner epidermal layer encircling the cyst (ep, purple, right) are identical to each other. In between the two layers all other typical mesodermal structures, such as muscle cells (mu, green), nervous tissue (nv, yellow), glands (gl)… are still present. Scalebars are 6 µm (A, C), 8 µm (B) and 2000 nm (D).
Characterization of the Macrostomum lignano ageing phenotype 24
Results
4.2.2.2 LARGE CYSTS
Large cysts have the same structural morphology as the small ones, namely, well-defined, hollow structures encircled by a single epidermal layer, which is ciliated on its apical side. The difference between the two, besides the size of the cyst itself, lies in the impact on the surrounding tissues and organs (Figure 13 and 14). The different cell types in the mesoderm close to a large cyst can still be distinguished, but there is a loss of tissue organization. Notable as well, is the occurrence of more apoptotic cells (Figure 13C) in comparison to control animals and animals with small cysts. These cells are easily recognized on the ultrastructural level because of their irregular shape and condensed chromatine (Schweichel & Merker, 1973). Furthermore, the presence of multiple smaller cysts surrounding a single large one was observed in this animal (Figure 13B). When looking at a series of ultrathin sections, the multiple smaller cysts are the result of branching. Noteworthy as well, was the presence of tissue inside a cyst, which is indicated by the black square D in Figure 13B. This has also been observed through in vivo light microscopical analysis (Figure 7E). Closer study of this tissue revealed the presence of membrane bodies (Figure 13D) (term adopted from De Mulder et al., 2009).
Characterization of the Macrostomum lignano ageing phenotype 25
Results
Figure 13: Influence of large cysts on the surrounding tissues
(A) Electron micrograph of a 22-day old control individual at the level of the gut (g) between the pharynx and testis. The mesoderm can be seen in between the gut (g) and the overlying epidermis (ep). (B) Section at the region of the gut (g). The presence of multiple cysts (c) is observed alongside disorganization in the mesodermal tissues. (C) Magnification of the respective area (black square marked C) indicated in B. There is a loss of structure in the mesoderm below the epidermis (ep, purple). Several scattered structures such as, muscle cells (mu, green) and nervous tissue (nv, yellow) can still be recognized. Note the presence of an apoptotic cell (ap, red). (D) Enlargement of the respective area (black square marked D) indicated in B. Several membrane bodies (mb, blue) are present in the tissue inside the cyst. Scalebars are 10 µm (A), 20 µm (B), 4 µm (C), and 1000 nm (D).
Besides the impact on the surrounding tissues, large cysts were also found to influence the location and structure of organ systems (Figure 14). For example, further study of the same animal as depicted in Figure 13B, revealed the displacement of the testis. In control animals the testis can be found in the mesoderm just below the epidermis on both sides of the animal (Figure 14A). However in this case, it was moved more towards the inside of the body because of the presence of a large cyst (Figure 14B). The usual organization within the testis of spermatids, spermatagonia, spermatozoids, spermatocytes and primordial germ cells could no longer be distinguished (Figure 14C).
Characterization of the Macrostomum lignano ageing phenotype 26
Results
Figure 14: Effect of cyst on the position and organization of the testis
(A) 22-day old control animal demonstrating the position of the testis (te, light green) in the mesoderm, above the gut (g) and just below the epidermis (ep). (B) Displaced position of the testis (te, light green) in between the gut (g) and a large cyst (c). Note the presence of the single large cyst which is the result of the fusion of the several small cysts in Figure 13B. (C) Enlargement of the area indicated in B (black square), showing the loss of organization within the testis (te, light green). Scalebars are 20 µm (A, B), and 6µm (C).
Characterization of the Macrostomum lignano ageing phenotype 27
Results
4.2.3 CYST-OPENING
When first looking at a series of ultrathin sections, the presence of an opening, which connects the inside of the cyst to the outside environment, became apparent. This finding has been corroborated by results from in vivo light microscopical analysis, and the study of semithin sections (Figure 15). Further quantification of the occurrence of exits to the external environment was made based upon semithin sections. A total of six animals were fully sectioned. These six animals contained 15 cysts in total, of which 12 (80%) displayed a connection from the inside of this structure to the outside.
Figure 15: Cyst-opening
Cysts displaying an opening (black square), which connects the inside of this structure to the outside environment, have been found through different analyses. (A) In vivo light microscopical analysis of a 134-day old individual. (B) Semithin section of an animal aged 155 days at time of fixation. (C) Electron micrograph of a 114 day-old animal. Scalebars are 50 µm (A), 30 µm (B), and 6 µm (C).
4.2.4 BRANCHING
Besides the typical, simple structure of the cyst, some deformities were also observed to display a more complicated pattern (Figure 16). The cavities of some of these cysts were found to form side branches. A single deformity then consists of several branches coming together at the epidermis, into a single opening. This was found to be present both by means of TEM (Figure 16A), and light microscopical analysis (Figure 16B).
Characterization of the Macrostomum lignano ageing phenotype 28
Results
Figure 16: Branching
Some animals were found to possess cysts that started to form side branches. This was observed to be present by means of TEM (A) and light microscopical analysis (B). Arrow indicates the point of branching. Note the presence of a large cyst (*) close to the branches in B. Scalebars are 10 µm (A) and 100 µm (B).
4.2.5 3D-RECONSTRUCTION
A 3D-reconstruction was made of a small cyst in the head region (Figure 17A). This image was based upon a series of 35 semithin sections, covering a distance of 47 µm. The use of 3D- images makes it possible to visualize both the exterior as well as the interior of a structure (Figure 17B-D). The outer surface clearly indicates the presence of an opening, which makes a connection between the cyst and the outside environment. This opening is positioned on the dorsal left side of the animal. The entire cyst itself appears as a well-defined, sac-like structure within the animal. The cyst starts on the anterior side, where the opening is located, and then extends longitudinally in the posterior direction. Further extension towards the dorsal and ventral side can be seen.
Characterization of the Macrostomum lignano ageing phenotype 29
Results
Figure 17: 3D-reconstruction of a region containing a cyst in the rostrum
(A) Habitus of Macrostomum lignano. A reconstruction of the area indicated (outlined in green) in this control animal, was made of an individual displaying a cyst at that location. (B-D) Reconstruction of both cyst (cy) and epidermis (ep), photographed from different angles. Images on the left visualize the external surface, while images on the right were made transparent for visualization of the internal structures. The opening (op) of the cyst is visible in all views on the dorsal left side of the animal. (B) Top view with the upper side of the picture representing posterior, and the lower side anterior. (C) Front view, looking at the structure from an anterior perspective. (D) Side view with the left side of the picture being anterior, and the right side posterior. Scalebars are 150 µm (A) and 20 µm (B-D).
Characterization of the Macrostomum lignano ageing phenotype 30
Results
4.3 TISSUE MAINTENANCE DURING AGEING
The goal of this experiment was to test if the formation of cysts can be caused due to a declining control of tissue homeostasis in older animals. To test this, the rate of tissue maintenance is compared between both young and old worms. As a measure for tissue maintenance, the number of neoblasts in the head region was counted after a chase period of four days. Immunostainings were performed as described in 3.5. Each labelling included a control group of young animals, and a group of the age to be tested. The mentioned age of the animals coincided with the time of pulse. Both EdU and BrdU were used to make a comparison between both techniques. Results of both methods are described below.
4.3.1 EDU IMMUNOSTAINING
The first labelling included a control group with animals aged 25 days (± one month), and a group of animals aged 118 days (± four months). The second labelling included a control group of animals aged 28 days (± one month), and a group of individuals aged 182 days (± six month). Results are summarized in Table 2. A graphical representation of these results can be found in Figure 18.
Table 2: Number of proliferating cells in the head region for EdU immunostaining
Control 118 days Control 182 days (C4M_EdU) (4M_EdU) (C6M_EdU) (6M_EdU) Worm 1 63 46 105 46 Worm 2 53 34 91 53 Worm 3 48 67 100 50 Worm 4 74 32 132 56 Worm 5 64 71 126 38 Worm 6 56 65 102 64 Worm 7 57 54 89 52 Worm 8 50 71 88 61 Worm 9 122 35 103 45 Worm 10 49 110
Worm 11 67
Worm 12 81
Mean ± SD 65 ± 23 56 ± 17 105 ± 15 52 ± 8
C4M_EdU: Control group for the corresponding aged group of four months
4M_EdU: Group of animals aged four months C6M_EdU: Control group for the corresponding aged group of six months 6M_EdU: Group of animals aged six months
Characterization of the Macrostomum lignano ageing phenotype 31
Results
Tissue Maintenance (EdU) 140
120
100
80
60
40 Number proliferatingof cells 20
0 118 days 182 days
Control Aged
Figure 18: Number of proliferating cells in function of age (EdU immunostaining)
Above: Upper panel shows DIC image of an animal used for immunostaining. Lower panel („) shows an overlay of confocal stacks of the same animal. Green dots represent labelled neoblasts while the arrow indicates the position of the eyes. All the cells in front of the eyes after a chase of four days were counted. Animals are aged 28 days (A), 118 days (B), and 182 days (C). Scalebars are 100 µm. Below: Graphical representation of the number of proliferating cells in function of age.
Characterization of the Macrostomum lignano ageing phenotype 32
Results
Statistical analysis (ANOVA) of these results gave the following p-values which are summarized in Table 3.
Table 3: Summary of p-values (EdU)
groups p-value 4M_EdU - C4M_EdU 0.4785308 C6M_EdU - C4M_EdU 0.0004172 6M_EdU - C4M_EdU 0.3104880 C6M_EdU - 4M_EdU 0.0000017 6M_EdU - 4M_EdU 0.9730038 6M_EdU - C6M_EdU 0.0000018
Significant differences (p-value<0.05) between the groups of interest were found between the control groups of both four (C4M_EdU) and six months (C6M_EdU), and between the control group (C6M_EdU) and its respective aged group (6M_EdU) at six months. No significant difference (p-value>0.05) was found between the control group (C4M_EdU) and its respective aged group (4M_EdU) at four months.
4.3.2 BRDU IMMUNOSTAINING
For this experiment only one labelling was performed. This staining included a control group aged 38 days (± one month), and a group of worms aged 202 days (± six months). The results are summarized in Table 4. A graphical representation of the results can be found in Figure 19.
Table 4: Number of proliferating cells in the head region for BrdU immunostaining
38 days 202 days (C6M_BrdU) (6M_BrdU) Worm 1 56 14 Worm 2 51 28 Worm 3 63 39 Worm 4 31 25 Worm 5 46 34 Worm 6 44 24 Worm 7 48 31 Worm 8 42 37 Mean ± SD 48 ± 10 29 ± 8
C6M_BrdU: Control group for the corresponding aged group of six months 6M_BrdU: Group of animals aged six months
Characterization of the Macrostomum lignano ageing phenotype 33
Results
Tissue Maintenance (BrdU) 70 60 50 40
30 proliferating cells 20 10
Number of 0 202 days
control aged
Figure 19: Number of proliferating cells in function of age (BrdU immunostaining)
Above: Upper panel shows DIC image of an animal used for immunostaining. Lower panel („) shows an overlay of confocal stacks of the same animal. Green dots represent labelled neoblasts while the arrow indicates the position of the eyes. All the cells in front of the eyes after a chase of four days were counted. Animals are aged 38 days (A), and 202 days (B). Scalebars are 100 µm.
Below: Graphical representation of the number of proliferating cells in function of age.
Characterization of the Macrostomum lignano ageing phenotype 34
Results
Statistical analysis (Two-Sample t-test) showed a significant difference in number of neoblasts in the head region between the control group (C6M_BrdU) and its corresponding aged group (6M_BrdU) at six months (p-value=0.0008926).
4.3.3 EDU VERSUS BRDU
Both techniques were compared in order to establish which of the two methods was most optimal for obtaining the results. A statistical analysis (ANOVA) was performed on the above data of the animals aged ± six months, along with their respective control groups, of both the EdU and BrdU labelling. The resulting p-values are summarized in Table 5.
Table 5: Summary of p-values (EdU versus BrdU)
groups p-value 6M_EdU - C6M_EdU 0.0000000 C6M_BrdU - C6M_EdU 0.0000000 6M_BrdU - C6M_EdU 0.0000000 C6M_BrdU - 6M_EdU 0.8665327 6M_BrdU - 6M_EdU 0.0007997 C6M_BrdU - 6M_BrdU 0.0083066 C4M_EdU: Control group for the corresponding aged group of four months 4M_EdU: Group of animals aged four months C6M_EdU: Control group for the corresponding aged group of six months 6M_EdU: Group of animals aged six months C 6M_BrdU: Control group for the corresponding aged group of six months 6M_BrdU: Group of animals aged six months
There is a significant difference (p-value<0.05) at the age of six months, between both control groups (C6M_BrdU and C6M_EdU), and a significant difference between the corresponding aged groups (6M_BrdU and 6M_EdU) of the two labelling methods. Despite these differences, both methods still succeed in yielding the same information involving the proliferative capacity of stem cells, namely, a significant difference between the control group and their respective aged groups at six months (6M_EdU and C6M_EdU ; C6M_BrdU and 6M_BrdU ).
Characterization of the Macrostomum lignano ageing phenotype 35
Results
4.4 EXPERIMENTAL INDUCTION OF CYSTS
The goal of this experiment was to gain more insights in the development of cysts, and see how irregularities, such as a small wound, are repaired. Hypothetically, this can be either through an initial reaction of the stem cells, by means of remodeling of existing tissue, or a combination of both. The experiment was performed as described in 3.8. All the animals included at the beginning of this experiment, in this experiment were young adults, aged 30 days. Initially, a small hole was made in the head region anterior to the brain by means of puncturing (Figure 6) in 60 animals. When observed 24 hours later, 100% of the animals that were punctured, were able to regenerate the wounded tissue (Figure 20). At this time a first BrdU immunostaining was performed to test for an initial stem cell reaction. This staining included a group of non-punctured individuals, and a group of worms that was punctured. The number of stem cells in the rostrum, anterior to the eyes, was counted. The results are summarized in Table 6. A graphical representation of the results can be found in Figure 21.
Figure 20: Wound healing after puncturing
(A) Head region of an animal before puncturing. (B) Animal that was punctured in the rostrum, anterior to the brain. Arrow indicates position of the wound. (C) Same animal as in B, 24 hours later. The wound is no longer visible and is regenerated. Scalebars are 50 µm. Anterior is to the right.
Table 6: Number of proliferating cells in the rostrum compared between control- and punctured group Control Punctured
Worm 1 23 52 Worm 2 10 89 Worm 3 12 62 Worm 4 4 85 Worm 5 13 87 Worm 6 20 62 Worm 7 18 70 Worm 8 15 63 Mean ± SD 14 ± 6 71 ± 14
Characterization of the Macrostomum lignano ageing phenotype 36
Results
BrdU Immunostaining 90
80
70
60 50 40
30
20 Number proliferatingof cells 10 0 Control Punctured
24h chase
Figure 21: Number of proliferating cells in the rostrum compared between control- and punctured group
Above: Upper panel shows DIC image of an animal used for immunostaining. Lower panel („) shows an overlay of confocal stacks of the same animal. Green dots represent labelled neoblasts while the arrow indicates the position of the eyes. All the cells in front of the eyes after a chase of 24 hours were counted.
(A) Control group. (B) Group of animals that were punctured. White circle indicates an accumulation of cells at the wound region. Scalebars are 100 µm. Below: Graphical representation of the number of neoblasts compared between control and punctured animals.
Characterization of the Macrostomum lignano ageing phenotype 37
Results
Statistical analysis (Two-Sample t-test) showed a significant difference in number of stem cells in the head region, between the control group and the group that was punctured (p- value=4.610e-08). The remaining 45 animals that were punctured were then observed on a daily basis for the following eight days. Out of 45 animals, 12 (27%) developed cysts by the end of this period. This number consisted of five animals that developed cysts in the head region, six individuals developed cysts at the tail, and one worm developed a cyst at the region of the gonads. Results are summarized in Table 7.
Table 7: Development of cysts during the observation period of eight days Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
H 0 1 3 3 3 5 5 5 T 0 0 1 4 6 6 6 6 B 0 1 1 1 1 1 1 1 Total 0 2 5 8 10 12 12 12 H: cyst in the head region; T: cyst in the tail region; B: cyst in the region between head and tail
Characterization of the Macrostomum lignano ageing phenotype 38
Discussion
PART 5: DISCUSSION
In this master thesis, the cysts that develop in Macrostomum lignano during ageing were studied. The presence of these deformities was already described by Mouton et al. (2009b), but a more detailed analysis has never been performed. The goal of this research was twofold. On the one hand, this study aimed at characterizing these cysts by giving an overview of the diversity, and describing what sort of structure it is. On the other hand, several experiments were performed to assess what causes these cysts to form. Below the results are discussed.
5.1 CHARACTERIZATION OF CYSTS
5.1.1 CYST DIVERSITY
Initially the diversity of cysts was documented using light microscopy. Parameters taken into account were the location, the size and the number of cysts, along with the age of the animals.
Location The regions that are most susceptible to develop cysts are the head and tail region. This could be because these areas are most intensively used by the animal. While eating, the rostrum is constantly coming into contact with the sharp surface of the diatoms. This could result in small wounds, which at a later age can no longer regenerate properly, and can potentially develop into cysts. This illustrates that the process of regeneration can sometimes go wrong, resulting in morphological abnormalities, which was also demonstrated by our puncturing experiment. This kind of sequence resembles the development of grooves in the rostrum after experimental amputation of the head anterior to the brain. After amputation of this region, 20% of the rostrum regenerates developed distinct grooves (Egger et al., 2006). The tail plate contains the duo gland system, which is used for adhesion to the substrate. The constant detaching and reattaching to the surface puts a lot of pressure on this area, which makes it more susceptible than other regions to different influences (e.g. mechanical stress, diatoms, salinity, etc.). An example that illustrates the vulnerability of the tail plate are the pathologies caused by a parasite of a species of thraustochytrid, namely, Thraustochytrium caudivorum sp. nov. (Schärer et al., 2007). This parasite was isolated from laboratory cultures
Characterization of the Macrostomum lignano ageing phenotype 39
Discussion of M. lignano, and causes lesions which start at the tip of the tail plate, and can result in the complete dissolution of the posterior part of the animal (Schärer et al., 2007). Here, however, the presence of a parasite can be ruled out based on the study of numerous sections. The way in which the neoblasts are distributed throughout the body could also explain the susceptibility of the head and tail region to develop cysts. In both the rostrum and the tip of the tail plate there is a complete lack of stem cells. Hence, recruitment of neoblasts to these regions involves the migration of cells. This migration delays the response at these areas and therefore could be one of the reasons why sometimes problems, such as cysts, occur. Apart from the anterior and posterior region, cysts could also be found at all other locations in between. These could also be the result of an erroneous regeneration. The occurrence of heteromorphoses, malformations manifested in regeneration experiments, has already been widely described (Brønsted, 1969; Egger, 2008; Egger et al., 2007). These deformities include, for example, the presence of multiple heads and tails. While these phenotypes are the result of amputating certain regions in the body, local wounds might develop into less pronounced phenotypes such as the occurrence of cysts.
Size and consequence for the animal Cysts ranging in size from very small to extreme proportions, almost encompassing half the body of the animal, were observed. Finding an answer as to why some of these malformations remain small, while others tend to become enormous in size is not easy. One possibility could be that small cysts over time become larger due to a constant hydrostatic pressure on the inside of the cyst. Assuming that cysts could probably arise because of small wounds, larger cysts can then be considered as originating from older existing wounds which have had more time to develop. Since larger cysts were not seen in the rostrum, the body posterior from the head may be considered more susceptible to develop these larger deformities. This might be because of the presence of the gut, which is in essence a hollow sac. The gut is therefore not capable of counterbalancing the pressure caused by the liquid inside the cyst, resulting in a steady increase in volume. The rostrum on the other hand, consists of a solid mass of cells which limits the growth of the cyst. Alternatively, large cysts in the rostrum would probably be lethal if the brain were to be hit. Therefore, there is the possibility that animals with large cysts in the rostrum died before being noticed upon the next screening. The consequences these cysts have for the animals are minimal, when the cysts remain small. There is, however, a significant loss in mesodermal structures because of the presence of a cavity, but this does not appear to be lethal. Conversely, when cysts grow larger up to half the
Characterization of the Macrostomum lignano ageing phenotype 40
Discussion body size, an impaired movement can be observed. This is probably because of the large amount of liquid inside the cyst, causing these worms to float rather than swim through the medium. Also the loss of muscle and nervous tissue because of the large cavity could result in a lowered ability to move in a directed way. These worms often die within 4 weeks.
Number of cysts The amount of cysts per animal can go from a single cyst, to multiple cysts located at different areas in the body. In the case of multiple deformities, usually a combination of both small and large cysts was observed. When assuming that these malformations arise because of the failure of healing certain wounds, multiple cysts can be considered as several wounds at different locations which failed to regenerate properly. A combination of both small and large cysts is then the result of the different times at which wounds occurred.
Age of the animals A general tendency for the development of cysts with increasing age was observed. More individuals displaying deformities could be found in older animals when compared to control individuals aged one month. There was, however, no correlation between the age of the animals and the location, size, or number of cysts. Both small and large cysts seemed to occur randomly amongst the animals displaying these deformities. This is opposed as to what was assumed. Older worms were thought to develop larger deformities than younger individuals. These findings would indicate that the severity of these malformations is not age-dependent, while the development of them is, since no cysts can be found in young individuals. So, assuming that cysts start to develop from a certain age on, because of the failure of healing certain wounds, the severity of the cyst itself can be ascribed to the size of the initial wound, and the time it had to develop, not to the age at which the cyst develops.
5.1.2 CYST AND TISSUE MORPHOLOGY
Based on results from light microscopy, the cysts can be described as empty, liquid-filled cavities. The cysts were studied more in detail using transmission electron microscopy. A distinction could be made between both small and large cysts in their impact on the surrounding tissues.
Characterization of the Macrostomum lignano ageing phenotype 41
Discussion
Structure Results indicated that the cysts were hollow, isolated structures within the mesoderm, completely lined by a single layer of epidermal cells, which are ciliated on their apical side. Finding a layer of ectodermal tissue inside the mesoderm was an interesting observation. However, further analysis revealed the presence of an opening which connects the inside of the cyst to the exterior environment. This could indicate an invagination of the epidermis, thus explaining why there are epidermal cells present in the mesoderm. Additional implications of the cyst-opening are discussed further down below.
Influence on tissue morphology Besides the size of the cysts themselves, the difference between both small and large ones lies in the impact on the surrounding tissues. Small deformities appear to have no distinctive influence on the surrounding remaining cells in the mesoderm. Large deformities on the other hand have a much greater impact. The different cell types in the mesoderm close to a large cyst can still be distinguished, but there is a loss of tissue organization. Also in this case, the presence of multiple smaller cysts in proximity of a larger cyst was observed. Whether the loss of structural integrity in the mesodermal tissues is because of the size or the number of cysts is hard to tell. The loss of tissue architecture could also be an artifact resulting from the fixation procedure for TEM. However, when comparing a region close to the cyst, to a region much further away, the difference in tissue organization becomes clear. Tissues further away from the cysts appear in better shape than the ones close to these structures. Since differences can be observed, an artifact due to the fixation procedure can most likely be ruled out. If it would indeed be an artifact then the entire section would look the same. The influence of a cyst on the surrounding tissues has already been observed in the onespot snapper fish, Lutjanus monostigma (Al-Jahdali & Hassanine, 2010). In these fish, a myxosporean parasite is responsible for the development of deep protruding, digitiform, black cysts on the ovaries. These cysts, along with still developing ones, can occupy a considerable part of the ovary, while the other remaining regions appear vacuolated and degenerated. This suggests that cysts can induce histological lesions in the neighboring tissues (Al-Jahdali & Hassanine, 2010). Based on the latter example, the loss of architecture in the tissues of M. lignano is probably because of a combination of the size, the number, and the growth of the cysts in the animal. In this case, however, the presence of a parasite can be ruled out since this was never observed in the sections.
Characterization of the Macrostomum lignano ageing phenotype 42
Discussion
Apart from the loss in tissue structure, the amount of apoptotic cells seems to be elevated when compared to control animals, although this wasn‟t quantified. Apoptosis is an evolutionary highly conserved process which is essential during embryonic development in both invertebrates, such as Caenorhabditis elegans and Drosophila melanogaster, and vertebrates, such as Mus musculus (Campisi, 2003). In M. lignano, apoptosis is known to play an important role in tissue maintenance of the epidermis (Nimeth et al., 2002). The amount of apoptotic cells, however, should be tested in the future. Making a quantification purely based on the ultrastructure of the cells is a very difficult task. A possible solution for this problem could be the use of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). This is one of the most widely used histochemical methods for the detection of apoptotic cells (Nimeth et al., 2002). An increased amount of apoptotic cells might be one of the causes for the origin of these cysts. In humans, a defective apoptosis has already been shown to be the cause of numerous diseases. These disorders include autoimmune diseases, neurodegenerative disorders, cancer, and even age-related diseases such as osteoporosis and atherosclerosis (Fadeel et al., 1999). Apoptosis could thus also be one of the causes for abnormalities in M. lignano. In evolutionary terms, this can be explained by a phenomenon called antagonistic pleiotropy. Due to a declining force of natural selection with advancing age, certain genes or processes (e.g. apoptosis) which have been selected for their beneficial effects at an early age, have deleterious unselected effects in older animals resulting in aged phenotypes (Kirkwood & Austad, 2000). Alternatively, apoptosis could also be the result of infiltrating sea-water. Notable was also the fact that some of these cysts were found to contain remnants of tissue inside. This was observed both by TEM and light microscopy. When looking more into detail at this tissue, peculiar little lamellar bodies were observed. This sort of lamellar structure has already been described in earlier publications in association with apoptosis. A study which investigated the role of the piwi homologue macpiwi in M. lignano, found these bodies to be present in dying cells of RNAi treated animals, hinting their involvement in the process of cell death. Here, these structures were termed membrane bodies (De Mulder et al., 2009). In the literature, this same type of structure has already been described under a different name. A study performed by Castejón (2008), deals with the same type of structure under the name myelin bodies. They are described as concentric membranous lamellar formations encapsulating thin bands of cytoplasm, found in connection to several pathological processes, the process of apoptosis, and even in connection to the ageing process (Castejón, 2008). So,
Characterization of the Macrostomum lignano ageing phenotype 43
Discussion both the presence of apoptotic cells and membrane bodies would indicate a role for the process of cell death in the development of these cysts. Another observation was the influence of a large cyst on the position and structure of the testis within the animal. It shifted from its normal position in the mesoderm just below the epidermis, to an area more towards the inside of the animal. It is most likely that the testis was moved from its original position due to the developing cyst. This deformity probably kept extending inwards, and increasing in volume causing the testis to be moved away from its normal position in the body. This can probably be the case for other organs as well.
Cyst-opening Based upon results from the study of semithin sections, TEM, and light microscopy a large number of cysts were found to possess an opening which connects the inside of the cyst to the outside environment. This opening is probably the reason why these cysts are completely filled with liquid. The medium from outside the worm simply flows in through this connection and fills the inside of the cyst. Observations suggested that this opening is the result from an invagination of the epidermis which forms a hollow sac on the inside of the animal. This would explain the previous finding that these cysts are completely lined by epidermal cells. However, this does not rule out the possibility of de novo formation of epidermal cells inside the mesoderm, since the epidermis cannot keep expanding infinitely inwards without compromising its integrity. One possibility could be that mesodermal neoblasts are activated to differentiate into epidermal cells in order to keep up with the increasing surface of the growing epidermis. Why the neoblasts are activated to form epidermal cells instead of mesodermal structures is probably due to the contact of the interior milieu with the exterior environment inside this structure. A 3D-reconstruction was made based on semithin sections of an animal containing a cyst in the head region. This method provided a clear visual representation of how this structure looks like from a three dimensional point of view. The reconstruction confirms the observations already made with other methods and clearly shows the presence of an opening on the dorsal left side. A connection between a structure and the exterior environment is usually an indication of the way in which the structure develops. Due to the high amount of cysts that were found which had this opening, the cysts are probably formed from the outside going inwards. Contrary to what this opening might suggest, the fact that some cysts were found to contain remnants of tissue can indicate the opposite. It could be that the tissue on the inside for some reason starts
Characterization of the Macrostomum lignano ageing phenotype 44
Discussion to dissolve, creating a cavity which the neoblasts of the animal try to resolve by lining the borders with epidermal cells. Or, it could also be a byproduct of the way in which the cyst could develop. Some of the cysts were found to contain branches, and if these branches were to connect at the tip this would also cause the presence of tissue inside the cyst.
Branching Some cysts tend to become more complicated and start to form branches. Despite the fact that this stage seems more complicated than a simple hollow, liquid-filled sac, it might be an intermediate stage during the development of the cysts. Evidence to support this hypothesis comes from the observation that one section contains several smaller cysts surrounding a single large cyst, while sections more posteriorly located in the animal show only a single large cyst. The smaller cysts can then be considered as the branches from a single large cavity. Noteworthy as well, was an increased incidence of apoptotic cells near these smaller cysts. This could be an indication that the tissues separating the cysts are going to dissolve in order to connect the smaller branches and form a single large cavity.
5.1.3 CYSTS IN MACROSTOMUM LIGNANO VERSUS CYSTS IN THE ANIMAL KINGDOM
The occurrence of cysts is not unusual within the invertebrates. A group of animals where cysts widely occur are the parasitic flatworms (e.g. Digenea, Cestoda, etc.). These worms use cysts as an intermediate form in which further development occurs from the larval towards the adult stages (Reuter & Kreshchenko, 2004). These cysts, however, occur as a part of the natural life cycle and are therefore different from the cysts found in M. lignano. Searching for cyst-like structures within the invertebrates which also arise due to a defective process proves to be difficult. Within the vertebrates, however, several examples can be found of structures which have a good resemblance to the cysts in M. lignano. One of those examples are tissue cysts in the visceral organs (lungs, liver, and kidneys), or in muscular and neural tissues of warm-blooded animals, caused by an infection with the protozoan Toxoplasma gondii. These tissue cysts grow and remain intracellular, and consist of a cavity surrounded by a thin wall (Dubey, 2004). However, just like the cysts found in the life cycle of parasitic flatworms, these structures contain the infectious stages of a parasite, and are the consequence of an external influence coming into the body. Finally, one of the structures that show the most resemblances to the cysts studied in this thesis, are inclusions cysts found in humans, for example, the cysts found on the ovaries of woman. These cysts can vary greatly in size, just
Characterization of the Macrostomum lignano ageing phenotype 45
Discussion like the cysts in M. lignano, going from small to large, and consist of a liquid-filled structure lined by a ciliated epithelium. It is also thought that these are the precursors to ovarian epithelial cancer (Clow et al., 2002; Motta & Vanblerkom, 1975; Risch, 1999). Besides the ovaries, inclusion cysts can also be found at a wide range of different locations in the body. There exist epithelial inclusion cysts which can be found on the wrist, the nose, the sole of the foot, and even within the pancreas (Chang & Jin, 2008; Fisher & Macpherson, 1986). This illustrates why it is important to characterize aged phenotypes and age related diseases.
5.2 CAUSES OF CYSTS
5.2.1 TISSUE MAINTENANCE DURING AGEING
It was hypothesized that with advancing age, the renewing of tissues can no longer be maintained, and the balance between young and old cells is disturbed (Abeloos, 1930). This loss of ability of tissues to maintain their structural integrity with advancing chronological age, has been associated with numerous morphological changes (Lange, 1968). One of those changes is the occurrence of ulceration, a discontinuity of the epidermis (Abeloos, 1930). This could indicate that the cysts observed in older animals of M. lignano could be because of a reduced control of cell renewal of aged tissues. Therefore, in order to confirm this hypothesis, the tissue maintenance in both young and old animals was tested, focusing on the role of stem cell functionality in this process. In this study two different methods were used. BrdU labellings rely on the incorporation of the thymidine analogue 5-bromo-2‟-deoxyuridine (BrdU) in the nucleus of cells in S-phase, which is generally used as a marker for the neoblasts in flatworms (Bode et al., 2006; Ladurner et al., 2008; Ladurner et al., 2000; Nimeth et al., 2002). Despite the fact that this method is highly reproducible, the problem when studying tissue maintenance is the use of proteases and other DNA denaturing steps. This often results in a loss of epidermal cells which is not beneficial if you want to make an accurate quantification of the number of neoblasts that have migrated to the rostrum and differentiated, especially since the head and tail region are the areas where most cells get lost. Therefore, the observed number of cells can be lower than the actual number of neoblasts. Nonetheless, the information should be the same since a similar amount of cells is lost in both the control and aged groups. Another advantage of BrdU labellings is that they are less
Characterization of the Macrostomum lignano ageing phenotype 46
Discussion subject to aspecific binding because of the presence of glands, improving the quantification of cells. The second labelling method did not require the use of proteases, strong acids and other DNA denaturing steps. This method was based on the incorporation of the thymidine analogue 5- ethynyl-2‟-deoxyuridine (EdU) (Chehrehasa et al., 2009). This method resulted in a much better preservation of morphology of the animal and no cell loss, allowing a more accurate quantification of the number of proliferating cells in the rostrum. The downside of this method, however, is the success rate. The labelling proved to be sensitive to numerous unknown variables which caused it to fail, resulting in a weak signal making quantification of the number of proliferating cells not possible. Another problem was the high amount of aspecific binding due to the presence of glands which complicated the counting of cells. Despite the fact that the protocol for EdU is much simpler to execute, and results in a much better preservation of morphology, the use of classical BrdU labellings would still be preferred because of the much higher success rate. The tissue maintenance of animals aged one, four and six months was determined using EdU. By means of BrdU labelling, the age of one and six months was tested. Each labelling included a control group of young individuals. The EdU labelling of the animals aged four months was, however, not completely successful. Nonetheless, no significant difference (p- value>0.05) could be observed when compared to its respective control group. The age of six months was tested using both EdU and BrdU. Significant differences (p-value<0.05) were found between both control groups and both aged groups of the two methods, indicating the loss of cells because of the use of proteases. Both methods were also capable of detecting a significant difference (p-value<0.05) in the number of proliferating cells in the head region at this age when compared to control animals. These results thus support the hypothesis that the malformations in M. lignano could be caused due to impaired tissue maintenance as a consequence of lower functionality of the stem cells in older animals. The decline of tissue maintenance capacity with increasing age is, however, not a new concept (Conboy & Rando, 2005). For example, in rodents, a decline in the number of new neurons produced by neural stem cells has been observed with increasing age. This decrease in neurogenesis has been associated with a progressive Parkinsonian disease (Wong et al., 2003). Another example is the ageing of haematopoietic stem cells in humans. This has been related to numerous diseases such as decreased immunity (Linton & Dorshkind, 2004), increased incidence of bone marrow failure and haematological neoplasia (Lichtman & Rowe, 2004), and anaemia (Beghé et al., 2004). These examples illustrate that
Characterization of the Macrostomum lignano ageing phenotype 47
Discussion the reduced stem cell functionality in older animals will have its consequences on the animal, and can cause a wide range of pathologies. In the case of M. lignano this could be the development of cysts. What exactly causes the stem cells to age? According to Sharpless & Depinho (2007), it is not so much the number of stem cells or their ability to self-renew that diminishes over time, but rather their function. Specifically, their capacity to generate progenitors and differentiated effectors cells declines with increasing age. An illustration of this is the accumulation of senescence markers in aged human skin. This is the result of a reduced number of stem cells able to respond to proliferative signals, rather than a reduction in the number of epidermal stem cells, or their ability to self-renew (Zouboulis et al., 2008). The reduced function of the stem cells can still be the result of intrinsic stem cell ageing, ageing of the stem cell niche, or signals from the systemic environment (Rando, 2006). Determining which factor could be the cause of the lowered functionality of the stem cells is very difficult. Especially, since evidence exists coming from various systems suggesting a role for all of these factors. A study performed by Carlson & Conboy (2007) established for the first time that both the local environment and the systemic milieu dramatically affect the regenerative potential of both human embryonic stem cells and mouse post-natal myogenic progenitor cells. Experiments exist in which the stem cells of aged mice are exposed to factors present in the serum of young mice through parabiotic pairings. This resulted in the restoration of the stem cell functionality, proving the role of the stem cell niche (Conboy et al., 2005). These results definitely indicate the influence of the stem cell niche, and the systemic environment on the function of stem cells. Conversely, morphological analysis of our own dataset suggested no significant differences in the niche/systemic milieu of six months old animals when compared to young individuals. Based on the previous, possible future experiments for determining the contribution of the niche, or the systemic environment in M. lignano, could involve the transplantation of neoblasts from aged animals into young individuals in which the resident stem cell population has been irradiated, and see if they are capable of properly regenerating after amputation.
5.2.2 EXPERIMENTAL INDUCTION OF CYSTS
This final experiment aimed at investigating how local irregularities, such as small wounds, are repaired. Hypothetically, this can be either through an initial reaction of the stem cells, by means of tissue remodeling, or a combination of both. These findings would provide more
Characterization of the Macrostomum lignano ageing phenotype 48
Discussion insights into the formation of cysts, and local wounding as a possible cause of cyst development. In the existing literature on Macrotstomum lignano, the role of neoblasts in the process of regeneration has always been studied by means of amputating certain parts of the body, such as the rostrum or tail (De Mulder et al., 2009; Egger et al., 2007; Egger et al., 2006; Nimeth et al., 2007). The stem cell response to local wounding, however, has never been tested and is performed for the first time in this experiment by puncturing the animals. Results from the BrdU labelling 24 hours after puncturing, revealed an initial response of the stem cells to local wounding, indicated by the significant increase (p-value<0.05) in number of proliferating cells which migrated to the location of the wound when compared to control animals. This was also shown by the fact that all the animals that were punctured were able to regenerate the lost tissue within 24 hours. Thus, our findings clearly indicate the role of stem cells in the healing of small wounds, which is similar to the involvement of neoblasts in the regeneration of whole body parts as already shown by numerous experiments performed in the past (De Mulder et al., 2009; Egger et al., 2007; Egger et al., 2006; Nimeth et al., 2007). To further study whether or not the epidermal cells lining the cysts are formed de novo by differentiating neoblasts, a second BrdU labelling was performed eight days after puncturing. This labelling was, however, not successful and did not yield any useful data. A possible alternative for this step would be the use of horseradish peroxidase. If the animals are then sectioned, the lining of the cysts would still be visible. This is, however, much more labor intensive then performing an immunostaining. After puncturing, the animals were observed on a daily basis over a period of eight days. By the end of this period, twelve of them (27%) developed deformities. The first cysts could already be seen starting from the second day after puncturing. From this period on there was a steady increase in individuals displaying deformities until day six. For the following two days, no new cysts developed. The deformities were located in the head region, at the tail plate, and one at the level of the gonads. These observations are interesting for two reasons. The first is that although all the animals that were punctured regenerated, still twelve of them developed malformations. This is very peculiar, since this implies that prior to developing cysts, a healing of the wound occurs. Nevertheless, the role of tissue remodeling cannot be neglected. The punctured animals were only controlled for regeneration of the wound using a stereo microscope. So what could seem like a perfectly healed wound, might have in fact displayed small irregularities if this would have been studied through light microscopy. These small irregularities which could have been
Characterization of the Macrostomum lignano ageing phenotype 49
Discussion missed upon the first observation could have further developed into cysts within the following days. On the other hand, checking each animal for regeneration of the wound using a light microscope would prove to be very labor intensive, and put additional stress on the animals due to the making of squeeze preparations which could also influence the results. The second reason which makes these observations so interesting is that instead of developing cysts in the puncture region as expected, some animals also developed deformities at their tail plate, and even one at the level of the gonads. Explaining why some of these animals developed cysts in other areas then the region where the wound was made, is not easy. One of the possibilities is that stem cells are required in the rostrum for regeneration of the wound, therefore rendering other regions more vulnerable. For example, if neoblasts are migrating to the rostrum, and the tail gets wounded during this process, the response of the neoblasts might be insufficient to properly heal this new wound at the tail plate, leading to the development of cysts at that location. A different explanation might be that in some animals the brain was damaged during puncturing. Egger et al. (2006) has already shown that damage to the brain by means of a longitudinal incision can result in abnormal structures in the body (e.g. bifurcated tail, extra tail plate, etc.) at other locations then the site of wounding. This indicates the key role of the brain in the process of regeneration (Egger et al., 2006). This experiment provided us with the vital information that neoblasts are involved in repairing local wounds, and more importantly, that it is possible to induce the formation of cysts by making a small wound. Further analysis of the involvement of tissue remodeling and the stem cells in the development of cysts could involve applying larger pulse and or chase periods. This would result in a larger number of cells, making it easier to distinguish the cysts in the animal on a confocal image. This larger pulse/chase period can, however, not be too long since after three weeks renewal of the epidermal cells during tissue homeostasis lining the cysts will occur.
5.3 HYPOTHETICAL MODEL FOR THE DEVELOPMENT OF CYSTS
All of our previous results can be put together in order to derive a process which accounts for the development of cysts. This process, which in the end leads to the establishment of a deformity, can be initiated by several triggers (Figure 22). A first trigger comes from the reduced functionality of stem cells in older animals (Figure 22B). In individuals of M. lignano there is a high turnover of cells in the epidermis. Old cells
Characterization of the Macrostomum lignano ageing phenotype 50
Discussion are removed by apoptosis and new ones are formed by differentiating neoblasts. In older animals, however, our tissue maintenance experiments (Figure 18 and 19) indicated that there is a loss of control of stem cell proliferation. As a consequence, cells that are removed in the epidermis through apoptosis can no longer be replaced at the same rate by newly formed cells. This results in weak spots in the epidermis. A second trigger comes from the loss of control of apoptosis, resulting in a much higher loss of epidermal cells (Figure 22C). The neoblasts can no longer keep up with the increased loss of cells, resulting in weak points in the epidermis. This was hypothesized based on our observations made by TEM (Figure 13), suggesting a role for the process of cell death. This hypothesis, however, needs to be further elucidated in the future. Finally, based on our puncturing experiment (see 4.5), the occurrence of wounds could also be one of the triggers which results in weaker spots at the epidermis, and eventually leads to the development of cysts (Figure 22D). The triggers described above all lead to the same result (Figure 22E). From then on, the further development is the same. Water will start to infiltrate at the weak epidermal locations, causing cell death in the underlying tissues. The cavity which is then created will be lined by epidermis formed by proliferating neoblasts. This is probably because of the contact between the external environment and the mesodermal neoblasts, giving them information regarding their position, causing them to form epidermis, and eventually closing the cavity resulting in a cyst (Figure 22F). Further extension of the cyst into the animal is then probably driven by a constant hydrostatic pressure of the medium on the inside of this structure.
Characterization of the Macrostomum lignano ageing phenotype 51
Discussion
Figure 22: Hypothetical model for the development of cysts
(A) In individuals of M. lignano, old epidermal cells are removed through apoptosis (orange stars), and replaced through the differentiation of neoblasts in the mesoderm. (B) Older animals have a decreased rate of stem cell proliferation (red crosses), and epidermal cells lost through apoptosis can no longer be replaced at the same rate, resulting in weak spots at the epidermis. (C) The control of the process of apoptosis is lost, resulting in a much higher loss of epidermal cells. (D) Wounding will also result in weak spots in the epidermis. (E) Water will start to infiltrate at the weak spots causing cell death in the underlying mesoderm. The cavity which then created will be lined by epidermal cells through the proliferation of neoblasts, resulting in the formation of a cyst (F).
Finding out which of the above scenarios is most likely to occur can be achieved by comparing this to the cause of structures similar to the cysts in this flatworm. Supporting evidence comes from the formation of inclusion cysts in humans. As discussed earlier, these are structures that show high similarity to the cysts in M. lignano. In most cases these structures arise as a consequence of lesions caused by surgical procedures, or by more natural causes such as wounding or mechanical pressure (Chang & Jin, 2008; Fajardo & Bessen, 1993; Song et al., 2006). This would thus provide support for the wounding scenario. Furthermore, epidermal inclusion cyst have been described to be caused by epidermal cells coming into contact with the underlying dermis (Farrer et al., 1992). In a similar way, contact between the exterior environment and the mesodermal tissue, could result in the differentiation of neoblasts into epidermal cells, resulting in the formation of a cyst. Based on the previous illustrations, our findings suggest that the cysts in M. lignano probably arise because of wounding to the epidermis, and the subsequent reduced capacity of older animals to adequately repair this.
Characterization of the Macrostomum lignano ageing phenotype 52
Conclusions
PART 6: CONCLUSIONS
The study of the cysts that appear in ageing animals of Macrostomum lignano has focused on two aspects. The first one was characterizing these deformities by describing both their diversity and morphology, and the influence they have on the surrounding tissues. The second aspect of the study was finding out why and how these structures are formed.
Characterization of cysts Cysts can be described as well-defined, hollow structures lined by a single epidermal cell layer, which is ciliated on its apical side. They can be found anywhere in the adult body going from head to tail, and can vary significantly in both size and number. The rostrum and tail plate were found to be more susceptible to develop cysts then other regions. Furthermore, there is a positive correlation between the age of the animals and the number of individuals displaying cysts, but not between the age and the size and number of these deformities per animal. Thus, the development of the cysts is age-dependent while the size of them isn‟t. The size is probably dependent on the time the cyst has to develop and the location at which it occurs. Cysts possess an opening to the outside environment which could be the result of an invagination of the epidermis covering the animal. Further analysis of these malformations indicates a greater impact of the large cysts on the surrounding tissues and organ systems when compared to small cysts. This results in a loss of architecture of the neighboring cells and tissues, along with the displacement and loss of structure of certain organs. Therefore, large cysts proved to be lethal to the animals, while small cysts weren‟t.
Causes of cysts Results have suggested that the reduced functionality of the stem cells with increasing age, along with the process of apoptosis, could play an important role in the development of cysts. Moreover, our data shows that local wounding can be a cause of cyst formation. We therefore conclude that in aged animals, irregularities in the epidermis caused by wounding or failing homeostasis can no longer be repaired properly and could lead to these deformities. Furthermore, the finding that cysts can still develop after an initial reaction of the stem cells for wound healing implies that the development of these structures is also accompanied with
Characterization of the Macrostomum lignano ageing phenotype 53
Conclusions de novo formation of epidermal cells. Different scenarios could be proposed explaining the degree in which these processes are involved in the development of the cysts.
Similar structures within the animal kingdom Within the invertebrates, no structures were found to be largely similar to the cysts in M. lignano. Vertebrates, however, and specifically humans, can develop inclusion cysts which consist of a fluid-filled cavity encircled by an epithelium. They can occur at a wide range of locations in the body, differ in size from small to large, and are also often caused by lesions that arise in the tissue. This structure thus seems fairly similar to the cysts in M. lignano.
Characterization of the Macrostomum lignano ageing phenotype 54
Summary
PART 7: SUMMARY
Ageing is a complex process which has an effect on every aspect of life. It is characterized by a functional decline, due to changes in the organ and tissue system. Alongside this declining functionality, is a diminished capacity to respond to injury or stress. This response often results in the proliferation of stem cells. One of the possible causes of ageing can thus be the reduced functionality of stem cells to maintain and repair tissues (Rando, 2006). Studying the role of stem cells in the ageing process, however, is difficult in the current model systems. The stem cell system is either, not readily accessible (Mus musculus), or the cells of these organisms are completely (Caenorhabditis elegans) or for the largest part (Drosophila melanogaster) post-mitotic during adulthood (Buffenstein et al., 2008). One group of organisms which do not have these limitations are flatworms. Flatworms possess a permanent population of likely totipotent stem cells, called neoblasts (Saló, 2006). Besides their dominant role in regeneration, they are constantly being recruited to maintain/renew differentiated and aged tissues during tissue homeostasis. Furthermore, the stem cell population in these animals is experimentally easily accessible as opposed to the vertebrate models (Bode et al., 2006). Hence, the free-living flatworm Macrostomum lignano will be used as a model organism for studying the reciprocal interaction between stem cells and the ageing process. As in other model organisms, it is suggested that ageing in flatworms results from the failure to maintain and renew tissues with advancing age (Abeloos, 1930). In M. lignano, morphological data exists to support this hypothesis. As these animals grow older, their transparent body changes into an opaque grey, and several types of distinct body deformities start to appear. These deformities encompass small bulges, grooves in the epidermis, and liquid-filled cysts (Mouton et al., 2009b). However, these deformities have not been properly characterized. In this master thesis, the cysts that develop in Macrostomum lignano during ageing were studied. The objective of this research was twofold. On the one hand, this study aimed at characterizing these cysts by giving an overview of the diversity, and describing what sort of structure it is. On the other hand, several experiments were performed to assess what causes these cysts to form. Analysis of the diversity of the cysts indicated that they can be found anywhere in the adult body going from head to tail, and that they can vary significantly in both size and number. The rostrum and tail plate, however, were observed to be more
Characterization of the Macrostomum lignano ageing phenotype 55
Summary susceptible to develop cysts then other regions. The development of cysts was found to be age dependent, while the size and number of them wasn‟t. The structure itself consists of a liquid- filled cavity, lined by epidermal cells, which are ciliated on their apical side. Cysts also possess an opening to the outside environment which could be the result of an invagination of the epidermis covering the animal. Furthermore, large cysts were observed to have an impact on the surrounding tissues and organ systems, while this was not the case with small cysts. This resulted in a loss of architecture of the neighboring cells and tissues, along with the displacement and loss of structure of the surrounding organs. TEM data also suggested a role for apoptosis in the development of cysts. Furthermore, several experiments were performed to unravel the causes of cyst formation. To test the hypothesis that ageing in flatworms is caused by a decreased control of stem functionality, and that this can be a possible cause for cyst development, the rate of cell renewal was quantified in both young and aged animals. Results indicated that there was indeed a reduced control of stem cell functionality in older animals for tissue maintenance. Moreover, we tested wounding as a possible cause of cyst development through puncturing. We found an initial reaction of the stem, namely, a migration to the location of the wound to replace the lost tissue. Furthermore, the animals were able to regenerate the lost tissue within 24 hours. However, cysts still developed starting from the second day after puncturing until the sixth day. We therefore conclude that in aged animals, irregularities in the epidermis can no longer be repaired properly, and could lead to the formation of cysts. Based on all the previous results, a model was proposed starting from three different triggers, which act independently or combined, to account for the origin of these irregularities in the epidermis, and the further development into cysts. These triggers are the reduced functionality of stem cells with increasing age, the loss of control of apoptosis, and the occurrence of wounds. When looking within the animal kingdom, we find similar structures to the deformities in this flatworm within the vertebrates, more specifically humans, namely, inclusion cysts. Just like the deformities in M. lignano, these inclusion cysts consist of a fluid-filled cavity encircled by an epithelium. They can occur at a wide range of locations in the body, differ in size from small to large, and are probably also caused by lesions that arise in the tissue. The study of the cysts that appear in ageing animals of M. lignano has provided better insights into its ageing process. This will allow a more in depth analysis of this flatworm as a model organism for studying the reciprocal interaction between stem cells and the ageing process. Furthermore, it has set the stage for possible future experiments in which the role of the stem cell niche or the systemic milieu on the functionality of stem cells can be further investigated.
Characterization of the Macrostomum lignano ageing phenotype 56
Samenvatting
PART 8: SAMENVATTING
Veroudering is een complex proces dat een invloed heeft op elk aspect van het leven. Het wordt gekenmerkt door een verminderde functionaliteit van het organisme, omwille van veranderingen in het orgaan- en weefselsysteem. Naast deze dalende functionaliteit, is er ook een verminderde capaciteit om te reageren op verwonding of stress. Deze reactie resulteert vaak in de proliferatie van stamcellen die een belangrijke rol spelen in het onderhouden en herstellen van weefsels. Bijgevolg kan een verminderde functionaliteit van de stamcellen een van de mogelijke oorzaken zijn van veroudering (Rando, 2006). Het is echter niet eenvoudig om de rol van stamcellen in het verouderingsproces te bestuderen in de huidige modelorganismen. Het stamcelsysteem is vaak niet toegankelijk (Mus musculus) en de cellen van deze organismen zijn ofwel volledig (Caenorhabditis elegans) of voor het grootste deel (Drosophila melanogaster) post-mitotisch tijdens het adulte leven (Buffenstein et al., 2008). Een groep van organismen die deze beperkingen niet heeft, zijn platwormen. Platwormen beschikken over een permanente populatie van totipotente stamcellen, de neoblasten (Saló, 2006). Naast hun dominante rol in het proces van regeneratie, worden ze constant gerekruteerd voor het onderhouden en vernieuwen van gedifferentieerde en verouderde weefsels tijdens weefselhomeostase. In tegenstelling tot de vertebrate modelorganismen is het stamcelsysteem bij M. lignano zeer toegankelijk (Bode et al., 2006). Bijgevolg wordt de vrijlevende platworm Macrostomum lignano gebruikt als modelorganisme voor het bestuderen van de reciproke interactie tussen stamcellen en veroudering. Net zoals bij de andere modelorganismen, werd in de literatuur gesteld dat veroudering in platwormen het gevolg kan zijn van het falen van het onderhouden en vernieuwen van weefsels met toenemende leeftijd (Abeloos, 1930). In M. lignano bestaat er morfologische data om deze hypothese te ondersteunen. Als deze dieren ouder worden, krijgt hun transparante lichaam een eerder ondoorschijnend, grijzig uiterlijk en beginnen er verschillende types van lichaamsmisvormingen op te treden. Deze misvormingen omvatten golvingen en groeves in de epidermis, en het voorkomen van cysten gevuld met vloeistof (Mouton et al., 2009b). Deze misvormingen zijn echter nog niet verder gekarakteriseerd. In deze masterthesis werden de cysten die optreden bij verouderende dieren van M. lignano bestudeerd. Het doel was tweedelig. Enerzijds werd de diversiteit aan cysten in kaart gebracht, en werd de structuur beschreven. Anderzijds werden verschillende experimenten uitgevoerd om te achterhalen wat de oorzaak is voor het ontwikkelen van deze misvormingen.
Characterization of the Macrostomum lignano ageing phenotype 57
Samenvatting
Onderzoek naar de diversiteit heeft aangetoond dat ze overal in het lichaam kunnen teruggevonden worden, en dat ze aanzienlijk kunnen variëren in zowel grootte als aantal. Uit de resultaten is gebleken dat het rostrum en de staartplaat meer vatbaar waren voor het ontwikkelen van cysten dan andere regio‟s in het lichaam. Het ontwikkelen van cysten bleek leeftijdsafhankelijk te zijn, in tegenstelling tot de grootte en het aantal van deze structuren. De misvorming zelf bestaat uit een met vloeistof gevulde holte, omlijnd door epidermiscellen die gecilieerd zijn aan hun apicale zijde. Verder werd gevonden dat cysten over een opening beschikken die het intern milieu van de cyste verbindt met de uitwendige omgeving. Vervolgens werd er een invloed van grote cysten geobserveerd op de omliggende weefsels en organen, terwijl dit bij kleine cysten niet het geval was. Dit resulteerde in het verlies van samenhang van naburige weefsels, in combinatie met de verschuiving en verlies van structuur van de omliggende organen. TEM data suggereerde verder ook nog een rol voor apoptose in de ontwikkeling van cysten. Vervolgens werden er enkele experimenten uitgevoerd om de oorzaak van de vorming van cysten te achterhalen. De snelheid waarmee celvernieuwing optreedt, werd gekwantificeerd in zowel jonge als oude dieren. Dit had als doel de hypothese te testen dat veroudering in platwormen veroorzaakt wordt door een dalende controle van stamcelfunctionaliteit, en of dit een mogelijke oorzaak kan zijn van cystevorming. Resultaten hebben aangetoond dat er inderdaad een verminderde controle was van stamcelfunctionaliteit in oudere dieren. Vervolgens hebben we verwonding onderzocht als mogelijke oorzaak voor cyste ontwikkeling door middel van de dieren te prikken. We zagen een initiële reactie van de stamcellen, namelijk een migratie naar de plaats van verwonding om het verloren weefsel te herstellen. Dit werd ondersteund door het feit dat alle dieren in staat waren om het verloren weefsel te regenereren binnen 24 uur. Daarentegen ontwikkelden er nog steeds cysten vanaf de tweede dag na het prikken tot de zesde dag. We kunnen daardoor concluderen dat onregelmatigheden in de epidermis niet altijd op een juiste manier hersteld kunnen worden, wat leidt tot de ontwikkeling van cysten. Verschillende oorzaken, die onafhankelijk van elkaar of gecombineerd werken, konden voorgesteld worden. Deze kunnen immers leiden tot onregelmatigheden in de epidermis en zo de verdere ontwikkeling tot een cyste in gang zetten. Mogelijke oorzaken zijn een verminderde controle van de stamcelfunctionaliteit met toenemende leeftijd, het verlies van controle van apoptose en verwonding. Wanneer men gaat zoeken in het dierenrijk naar structuren gelijkaardig met de cysten in deze platworm, komt men terecht bij het voorkomen van inclusie cysten in vertebraten, meer bepaald in mensen. Net zoals de cysten in M. lignano, zijn deze structuren opgebouwd uit een met een vloeistof gevulde holte omlijnd door een epitheel. Ze kunnen ook variëren in grootte,
Characterization of the Macrostomum lignano ageing phenotype 58
Samenvatting voorkomen op een brede waaier van plaatsen in het lichaam, en worden hoogst waarschijnlijk ook veroorzaakt door laesies in het weefsel. De studie van de cysten, die voorkomen in verouderende individuen van M. lignano, heeft geleid tot betere inzichten in het verouderingsproces van dit organisme. Dit zal een diepere analyse toelaten van deze platworm als een model om de reciproke interactie tussen stamcellen en het verouderingsproces te onderzoeken. Verder geeft dit onderzoek de aanzet voor toekomstige experimenten, waarbij de rol van de stamcelniche en het systemisch milieu verder onderzocht kan worden.
Characterization of the Macrostomum lignano ageing phenotype 59
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