embryonic stem cells to detect developmental toxicants
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Bastian Zimmer
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
Konstanz 2011
Tag der mündlichen Prüfung: 02.12.2012 1. Referent: Prof. Dr. Marcel Leist 2. Referent: PD Dr. Gerrit Begemann 3. Referent: Prof. Dr. Marcus Groettrup 4. Referent: Prof. Dr. Gabsang Lee
Konstanzer Online-Publikations-System (KOPS)
Publications, integrated in this thesis Chapter C
Zimmer B, Kuegler PB, Baudis B, Genewsky A, Tanavde V, Koh W, Tan B, Waldmann T, Kadereit S, Leist M. Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing.
Cell Death Differ. 2011 Mar;18(3):383-95. Epub 2010 Sep 24.
Chapter D
Zimmer B, Schildknecht S, Kuegler PB, Tanavde V, Kadereit S, Leist M. Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure. Toxicol Sci. 2011 Jun;121(2):357-67. Epub 2011 Mar 7.
Chapter E
Zimmer B, Lee G, Meganathan K, Sacchinidis A, Studer L, Leist M. Genuine human neural crest cells as test system for developmental toxicity and potential rescue strategies. under review
Some text passages in the general introduction were taken from:
Kadereit S*, Zimmer B*, van Thriel C, Hengstler J.G., Leist M. Compound selection for modeling developmental neurotoxicity (DNT) and prenatal neurotoxicity with embryonic stem cells. accepted for publication in Frontiers in Bioscience
*: both authors contributed equally
Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17- 42.
Kuegler PB, Baumann BA, Zimmer B, Kadereit S, Leist M, GFAP-independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells, GLIA, in press
Waldmann, T., Weng, M., Zimmer, B., Pöltl, D., Scholz, D., Broeg, M., Kadereit, S.,Wuellner, U., Leist, M. Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment. submitted
A. Summary... 6
Zusammenfassung... 7
B. General introduction... 9
1. (Neuro)development in general... 9
1.1 Proliferation... 11
1.2 Differentiation/Patterning... 12
1.3 Migration... 14
1.4 Neurite outgrowth ... 15
1.5 Synaptogenesis / Neurotransmitter household ... 16
2. Environmental chemicals and (neuro)development... 17
2.1 Barker Hypothesis ... 18
2.2 Time of insult vs. time of phenotype onset ... 18
2.3 Susceptibility of the developing brain to chemicals... 19
2.4 Environmental chemicals and developmental disabilities ... 20
2.5 Phenotype vs. biological process ... 22
3 Development of in vitro test systems in the 21st century ... 23
4. Embryonic stem cells (ESC) as source for in-vitro testing ... 24
Aims of the thesis... 27
Chapter C... Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing... 29
Abstract ... 31
Introduction ... 32
Results ... 34
Discussion ... 45
Materials and Methods ... 49
Supplements ... 56
Chapter D... Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure... 73
Abstract ... 75
Introduction ... 76
Materials and Methods ... 78
Results ... 82
Discussion ... 91
Supplements ... 95
Chapter E... Genuine human neural crest cells as test system for developmental toxicity and potential rescue strategies... 101
Abstract ... 102
Introduction ... 103
Materials and Methods ... 105
Results ... 107
Discussion ... 114
Supplements ... 118
F. Concluding discussion... 131
G. Bibliography... 142
Record of contribution ... 169
A. Summary
Developmental disabilities and congenital malformations associated with neural development are increasing problems in western countries. More and more evidence emerges from human epidemiological studies that environmental chemicals as well as drug and food constituents contribute to such an increase. Unfortunately, developmental neurotoxicity is currently the least examined form of developmental toxicity. Less then 200 compounds worldwide, mostly pesticides, have been tested in vivo according to the OECD test guideline TG 426. This guideline requires expensive and labor intensive animal experiments which often lack human relevance. As embryonic stem cells are able to differentiate into every cell type of an organism and have been shown to recapitulate in vivo development in the culture dish, they are considered a powerful alternative to whole animal experiments. Since also human embryonic stem cells have been generated, it is possible to mimic effects of chemicals on human neural development in vitro today. In the framework of this doctoral thesis, we used murine and human embryonic stem cells to establish basic concepts of in vitro developmental toxicity testing and to develop new test systems based on these cells.
In a first step, we characterized and modified a published 2-step neural differentiation protocol based on mouse ESC to fulfill the requirements of an in vitro toxicity test system. By using whole genome transciptome analysis we were able to identify different waves of gene expression. In a second step, we correlated these waves of gene expression with important steps of neural development. Proof-of-principle experiments showed that the waves identified could be the basis for endpoint selection and exposure windows. In the next step, we analyzed the effects of low-dose chronic methylmercury exposure on late neuronal differentiation. We identified dopaminergic neurons as relevant targets of mercury toxicity. We thereby were the first to correlate gene expression findings with functional readouts in an embryonic stem cell based in vitro developmental neurotoxicity test system. Additionally, we were able to correlate in-cell toxicant concentrations with relevant in vivo concentrations in such a setting.
After having established the principles important for embryonic stem cell based developmental neurotoxicity testing, we developed a neural crest migration assay based on cells differentiated from human embryonic stem cells. We found that these cells detected adverse effects on cell migration, an important process during neural development, in a more sensitive way than non- neural cell lines. These findings contribute to the development of embryonic stem cell based in vitro assays and have set general principles on what needs to be assessed in such assays.
Zusammenfassung
Entwicklungsstörungen und angeborene Fehlbildungen, die durch Fehler in der Nervensystementwicklung verursacht werden, sind ein steigendes Problem in westlichen Ländern. Immer mehr epidemiologischen Studien deuten darauf hin, dass Umweltchemikalien sowie Medikamente und Nahrungsmittelbestandteile zu diesen Problemen beitragen. Ent- wicklungsneurotoxiziät ist zurzeit die am wenigsten untersuchte Form von Entwicklungs- toxizität. Weltweit wurden bisher weniger als 200 chemische Verbindungen, meist Pestizide, gemäß der OECD Richtlinie TG 426 getestet. Diese Richtlinie erfordert teure und arbeitsin- tensive Tierexperimente, die nur selten die Situation im Menschen widerspiegeln. Da mit embryonalen Stammzellen die Embryonalentwicklung in der Kulturschale nachvollzogen werden kann, gelten sie als eine vielversprechende Alternative zu Tierexperimenten. Dadurch ist es heute möglich, die Auswirkungen von Chemikalien auf die Entwicklung des menschlichen Nervensystems in vitro zu untersuchen. In dieser Doktorarbeit verwendeten wir embryonale Stammzellen von Maus und Mensch um grundlegende Konzepte für die Untersuchung von entwicklungsneurotoxischen Substanzen in der Kulturschale sowie neue Testsysteme zu entwickeln.
Zuerst passten wir ein bereits veröffentlichtes Protokoll, mit dem man embryonale Stamm- zellen der Maus in Nervenzellen differenzieren kann, an die Anforderungen eines in vitro Toxizitätstestsystems an. Dabei konnten wir unterschiedliche Wellen von Genexpression während der Entwicklung von Nervenzellen aus embryonalen Stammzellen identifizieren. In ersten Experimenten konnten wir zeigen, dass diese Wellen als eine Grundlage für spätere toxikologische Analysen dienen können. Anschließend analysierten wir die Auswirkungen von geringen, über einen längeren Zeitraum dosierten Quecksilberkonzentrationen auf die späte Nervensystementwicklung. Dabei fanden wir heraus, dass speziell dopaminerge Neuronen durch Quecksilber geschädigt werden. Außerdem konnten wir in einem auf embryonalen Stammzellen basierenden Entwicklungsneurotoxizitäts-Testsystem erstmals Genexpressionsdaten mit funktionellen Analysen kombinieren. Nachdem wir wichtige Grundlagen zur Untersuchung von Entwicklungsneurotoxizität gelegt hatten, entwickelten wir einen Zellwanderungstest basierend auf Neuralleistenzellen, die aus menschlichen embryo- nalen Stammzellen differenziert worden waren. Diese Zellen konnten nachteilige Effekte von Chemikalien auf die Zellwanderung besser als nicht neurale Zellen anzeigen. Mit dieser Arbeit konnten wir zur Entwicklung von Testsystemen, die auf embryonalen Stammzellen basieren, beitragen und grundlegende Prinzipien für diese Art von Tests etablieren.
Abbreviations
ADHD: Attention deficit hyperactivity disorder ASD: autism spectrum disorder
AVE: anterior visceral endoderm BBB: blood brain barrier
BMP: bone morphogenic protein CNS: central nervous system DNT: developmental neurotoxicity ECM: extracellular matrix
EMT: epithelial-to-mesenchymal transition ESC: embryonic stem cell
ESNATS: Embryonic Stem cell-based Novel Alternative Testing Strategies FGF: fibroblast growth factor
GABA: γ-Aminobutyric acid GD: gestational day
GSK3: Glycogen synthase kinase 3 HTS: high-throughput screening IPS: induced pluripotent stem cells IQ: intelligence quotient
LIF: Leukemia inhibitory factor
LOAEL: lowest-observed-adverse-effect-level MeHg: methylmercury
MLK: mixed lineage kinase NCC: Neural crest cell NSC: neural stem cell
PCBs: Polychlorinated biphenyls PNS: peripheral nervous system PoT: pathways of toxicity RA: retinoic acid
SHH: sonic hedgehog SVZ: sub ventricular zone US: United States
B. General introduction
In order to establish in vitro based test systems to detect developmental toxicants, which are normally tested in whole animals, it is crucial to understand the key biological processes taking place during normal development. The following general introduction therefore describes key events during neurodevelopment and tries to bridge current knowledge about those processes with toxicological concepts.
1. (Neuro)development in general
The development of a complex vertebrate organism requires several tightly controlled and exactly timed mechanisms (Aylward 1997). The development of the sophisticated mammalian, and particularly the human, nervous
system is one of the most complex processes in nature. During the development of the nervous system, several different cellular processes such as apoptosis, cell differentiation, patterning, neurite outgrowth and migration (which will be discussed in detail later) have to take place in a tightly regulated manner (Rao and Jacbson 2005).
As illustrated in Figure 1, the development of the nervous system starts with a process called neurulation (Greene and Copp 2009). The notochord thereby induces the formation of the neural plate within the ectoderm (around gestational day (GD) 8 in rats and GD 14-19 in humans) (Rice and Barone 2000). Already at that stage, two different cellular identities can be identified based on the expression of different markers and specifier genes (Betancur et al.
2010; Betters et al. 2010; Sauka-Spengler and Bronner-Fraser 2008). On the one hand, the neural plate border, giving rise to the neural
Figure 1: Schematic illustration of neurulation
Upon cell proliferation, the neural plate starts to develop into a neural fold. Already at that stage NCC progenitors (grey) can be identified. The neural groove deepens and ultimately fuses to become the neural tube.
NCCs (grey) now start migrating to their target sites. For more details see text
crest cells (NCC) (indicated as grey cells in Figure 1), which among many other structures develop into the peripheral nervous system (PNS) (Huang and Saint-Jeannet 2004), on the other hand the designated neural tube, which later gives rise to the central nervous system (CNS) (Gilbert and Singer 2006) (indicated as blue cells in the lower part of Figure 1).
In humans, the neural plate develops into the neural groove due to massive cell proliferation at the boarders of the neural plate around GD 20. The two neural folds then fuse in the area later developing into the hindbrain to form the beginning neural tube in humans around GD 21 (Hood 2005). From now on, the neural groove closure proceeds in a zipper-like manner in both directions (anteriorly and posteriorly) (Wilson and Maden 2005). In humans, neural tube closure is completed between GD 26 and 28 (in rats GD 10.5 -11) with the anterior neuropore closing first (GD 24 – 26) and the posterior end closing later (between GD 25 – 28) (Hood 2005). Incomplete closure of the neural tube results in developmental congenital disorders such as spina bifida (Harris and Juriloff 2010; Mitchell et al. 2004) which is characterized among other symptoms by an open back. Approximately 1 in 1000 children born is affected by this birth defect. Chemicals like valproic acid have been described to increase the risk for spina bifida and craniofacial malformations. Especially the use of valproic acid during the first trimester of pregnancy increases the risk (Jentink et al. 2010; Ornoy 2009). The timeline of rat neural development is illustrated in Figure 2.
Figure 2: Timing of important processes of neural development in the rat. Modified from (Rice and Barone 2000)
After neurulation, early brain development is initiated within the different areas of the neural tube. The anterior part of the neural tube will develop into the brain (including fore-, mid- and hindbrain), while the posterior part will develop into the spinal cord (Altmann and Brivanlou 2001; Nishi et al. 2009). These dramatic morphological and cellular changes are
accomplished by different cellular processes such as proliferation, differentiation, migration and synaptogenesis (some of them are summarized in Figure 3). As these cellular processes need to be understood to be able to model them in vitro, some of them will be described in more detail.
1.1 Proliferation
After closure of the neural tube, the early brain consists of a single layer of cells. In order to develop into the sophisticated structure of an adult brain, massive cellular proliferation has to take place. Within the developing brain, 4 different main proliferative regions can be found, some of which persist into adulthood: The ventricular zone, the external granule layer of the cerebellum, the subventricular zone (SVZ) and the subhilar proliferative zone within the dentate gyrus, the two latter one persisting into adulthood (Rao and Jacbson 2005).
Figure 3: Key biological processes involved in differentiation of the nervous system.
Modified from (Kadereit et al. 2011) and en.wikipedia.org/wiki/Central_nervous_system The cerebral cortex may be the most remarkable structure of the mammalian, and especially the human, brain. It is made up by 10 – 20 billion interconnected neurons and 5 – 10 times more glial cells (Nowakowski and Hayes 1999). This enormous number of cells is created mainly by two different types of cell division. In the beginning, a limited number of
progenitor cells, is expanded by vertical cell division. Around 6 weeks after conception (in humans), the progenitor cells divide symmetrically (vertically), giving rise to two identical daughter cells, both of which re-enter the cell cycle (Choi 1988), expanding the progenitor pool.
Next, the SVZ forms around 7 weeks after conception (Zecevic 1993). From week 9 to 10, the cortical plate compacts and the intermediate zone is expanded. During this process, cells start to exit the cell cycle due to asymmetric or horizontal cell division. During this process the two resulting daughter cells can be separated into an apical and a basal daughter cell. The cell attached to the ventricular surface remains within the progenitor pool, while the daughter cell which has lost contact to the lumen of the ventricle exits cell cycle and starts to differentiate.
This process is considered a very early step characteristic for differentiation of precursor cells into neurons (Chan et al. 2002).
During week 12 of development, the proliferative activity in the human cortex peaks but the ventricular zone is no longer increasing in width, due to a balance of cell division and young neurons leaving this zone (Simonati et al. 1999). In week 18 of gestation, neurogenesis in the human cortex ends and the generation of new neurons after week 18 becomes negligible for the development of the human brain (Chan et al. 2002). Although neurogenesis and cell proliferation mostly ends within this period, cell division and neurogenesis persist into adulthood within the SVZ (also a well known source of adult neural stem cells (NSC)) and the dentate gyrus (Lennington et al. 2003), as discovered by (Eriksson et al. 1998).
1.2 Differentiation/Patterning
Another very important process during development, and especially during development of the nervous system, is cell differentiation and the correct patterning of these cells.
Differentiation of progenitor cells into more mature cell types begins as soon as the precursor cells have completed their last cell division (asymmetric) and are ready for cell migration.
Cell differentiation and patterning are initiated by a complex interplay between intracellular and extracellular signals such as signaling through growth factors which lead to the expression of genes influencing either neuronal or glial cell differentiation (Kuegler et al.
2010).
Such growth factors are present within the developing brain at different concentrations in different areas (Sansom and Livesey 2009). Those gradients are generated due to specific cell populations, such as the floor plate or the isthmic organizer secreting different types of
growth factors (Fasano et al. 2010). The different growth factors, secreted by different areas, and the resulting complex mixture of signals, which also differ in their intensity in different brain areas, modulate and refine the regional differentiation and patterning of differentiating cells (Takahashi and Liu 2006).
For a long time, the early patterning of the nervous system was believed to be a default mechanism. As inhibitors of the bone morphogenic protein (BMP) signaling pathway such as noggin play an important role in promoting early neurodevelopment (Gaulden and Reiter 2008), it was thought that ectodermal cells differentiate into neural tissue as long as they are not exposed to BMPs (Munoz-Sanjuan and Brivanlou 2002). More recently, an additional important concept has emerged. Besides the important aspect of the absence of BMP signaling, the presence of fibroblast growth factor (FGF) signaling appears to play an important role (Levine and Brivanlou 2007; Stern 2006). FGFs seem to promote a “pre”- neural state (Stern 2005) priming the cells for neural differentiation. Besides BMP inhibition and FGF signaling, integrating patterning signals such as Wnt/GSK3 signaling which for instance regulates the duration of BMP/Smad1 signaling are important for correct neural development (Pera et al. 2003).
After the initial specification of primitive neuroectodermal tissue, other factors in addition to BMPs and FGFs become more and more important. As it is believed that the early neural tissue has an innate anterior identity (Wilson and Houart 2004), structures prone to become anterior, such as the early telencephalon, need to be protected from caudalizing factors. This task is achieved again by specialized structures within the developing organism, such as the anterior visceral endoderm (AVE) in the mouse (Stern 2001). Not only structures like the AVE guarantee the identity of more anterior brain parts. Also the fact that signals, such as Wnts, FGFs, nodals and retinoic acid (just to name a few), required for directing neural tissue more caudally, are located in more caudal parts of the developing brain, plays an important role (Agathon et al. 2003).
Since the mature mammalian brain has not only an anterior – posterior identity, but also a dorsal – ventral pattern, again different gradients and morphogens such as SHH (directing neural tissue to more ventral brain areas) are needed to guarantee a proper brain development (Gaspard et al. 2008; Gaspard and Vanderhaeghen 2010; Götz and Huttner 2005).
During this complicated process of patterning, the signals not only have to be separated in space, they also have to be separate in the timing. For instance it is a well established concept in vivo and in vitro, that neurogenesis takes place before gliogenesis (Sugimori et al. 2007;
Temple 2001). To make the whole process of differentiation and cell patterning even more
complicated, the timing of differentiation is not only important for different cell types such as astrocytes and neurons, also the differentiation of different sub-types of neurons depends on the correct timing of signals, which result in characteristic neurogenic waves (Gaspard et al.
2008).
This concept of a wave-like process of neural development, in which different processes ranging from proliferation over differentiation to network formation overlap in a wave-like pattern, is well established in the literature. Meanwhile, these biological processes have been broken down to gene expression and specific gene signatures associated with different cell types and biological processes describing the initial morphological observations (Abranches et al. 2009; Aiba et al. 2006; Wei et al. 2002).
1.3 Migration
Besides cell proliferation and differentiation, correct cell migration is essential for the development of the nervous system (Ruhrberg and Schwarz 2010; Valiente and Marin 2010).
Due to the complexity of cell migration during neural development and the scope of this PhD thesis, migration in the CNS will only be discussed to a minor extend, whereas neural crest cell (NCC) migration which plays an important role in the development of the PNS will be discussed in more detail. Nevertheless, general aspects such as migration along defined paths in a strictly timed manner are applicable and important for neural cell migration in general.
CNS migration can be divided into two different modes of migration. The principle mode of migration within the cortex is the so-called radial migration. During radial migration, the neurons move along radially oriented glial fibers orthogonal to the surface of the brain.
During tangential migration, the neurons (in rodents interneurons of the cerebral cortex) move parallel to the brain surface along axons or other neurons (Metin et al. 2008; Nadarajah and Parnavelas 2002).
The migration of NCCs, in contrast to the migration of CNS progenitors, starts shortly after neurulation. At the end of neurulation, NCCs receive signals including BMPs, FGFs and Wnts secreted from the surrounding tissue, which lead to a process called epithelial-to- mesenchymal-transition (EMT) (Sauka-Spengler and Bronner-Fraser 2008). The cells then delaminate from the neural tube, sort into segregated migratory streams and migrate along distinct paths to their target sites in the periphery giving rise to a large variety of cells including bone and cartilage of the head, melanocytes, Schwann cells and sensory neurons (Harris and Erickson 2007; Krull 2001; Le Douarin and Kalcheim 2009).
The migration of the 4 known different populations of NCCs, namely cranial, cardiac, vagal and trunk, is thereby tightly controlled as to the timing, the region along the neural tube the cells emerge from, and their route of migration (Ruhrberg and Schwarz 2010). These tightly regulated processes include dynamic events such as reorganization of the cytoskeleton and membrane compartments, rearrangements of the extracellular matrix (ECM) and cell junctions as well as detachment and reattachment via adhesion molecules (Kadereit et al.
2011). Thereby the whole process of migration is again guided by gradients of proteins which serve as chemoattractans such as CXCR4 (Kasemeier-Kulesa et al. 2010) or repellents such as Sema3A (Anderson et al. 2007; Schwarz et al. 2009a; Schwarz et al. 2009b). Ultimately, much of the cellular response to such chemokines is mediated via members of the Rho family of small GTPases, such as Rac, Rho or Cdc42. Signaling through those enzymes results in a reorganization of the actin cytoskeleton including actin polymerization, contraction via interaction with myosin and adhesion to the substrate, amongst others, via integrins (Becchetti and Arcangeli 2010; Kadereit et al. 2011; Kurosaka and Kashina 2008; Nobes and Hall 1995).
1.4 Neurite outgrowth
Once neuronal progenitor cells have reached their target site, they have to differentiate to fully mature neurons and generate the complex neurite network that is characteristic for the highly developed mammalian nervous system. Neurite outgrowth relies on intrinsic (e.g. expression of receptors) and extrinsic factors. An important extrinsic aspect is the interaction of the differentiating cells with components of the extracellular matrix (ECM) as well as with other cells, e.g. glial cells, via cell adhesion molecules (CAMs) and integrins (Kiryushko et al.
2004; Powell et al. 1997; Tarone et al. 2000). Similar to the processes already described for cell migration, gradients of attractants and repellents are sensed by the growth cone of growing neurites, leading to actin reorganization via GTPases and a directed growth of the neurite. It has been proposed that the signals from the ECM or extracellular guidance cues resulting in neuron polarization converge at the level of GSK3 (Kadereit et al. 2011;
Yoshimura et al. 2006).
1.5 Synaptogenesis / Neurotransmitter household
After the axonal growth cones have reached their targets, the functional units of the brain, the synapses, have to be established. The mammalian brain comprises 1011 neurons, which are connected to each other by up to 1015 synapses (Drachman 2005). The detailed wiring of the complex neuronal circuits in mammalian brains, which is to a large degree self-generated, depends on neurotransmitters and neuromodulators (Ruhrberg and Schwarz 2010). For example disturbances of the fetal dopamine transporter by cocaine resulted in a delayed postnatal synaptic maturation (Bellone et al. 2011).
It has been shown that neurotransmitters, such as catecholamines can be detected in vertebrate as well as in invertebrate embryos before neurons are differentiated. As there is no synapse- based target yet, those early appearing neurotransmitters are thought to play an important role during the development of the nervous system. Therefore, a switch in the function of these neuroreactive molecules from the developmental to the maturation phase has been proposed (Pendleton et al. 1998). This theory is supported by the expression of different neurotransmitter receptors, such as the dopamine receptor D1A which regulates neuronal growth very early during development (Todd 1992). Furthermore, it has been shown that specific neurotransmitter receptors are expressed on progenitor cells of the CNS before
Table 1: Summary of neurotransmitter effects during neural development neurotransmitter known function during neural development reference GABA Exerts a variety of trophic influences through the
stimulation of the GABAAR; controls cell cycle kinetics in neuronal progenitors
(Barker et al. 1998;
Haydar et al. 2000;
Li and Xu 2008;
Nakamichi et al.
2009) Glycine Increases the number of primary neurites and total
neurite length
(Tapia et al. 2000) Glutamate Promotes neuronal growth and differentiation;
activation of NMDA-R promotes neurite outgrowth
(Aruffo et al. 1987;
Pearce et al. 1987) Acetylcholine Exerts chemoattractance and guidance for nerve
growth cones
(Zheng et al. 1994)
synapses are established, where they function as growth regulators during specific developmental periods (Nguyen et al. 2001). Table 1 gives a small summary of different types of neurotransmitters and their known functions during development.
Besides, their function during early development described above, neurotransmitters play an essential role during the establishment of the neuronal connections via synapses within the brain.
The process of synapse formation, called synaptogenesis can be broken down into two major stages. First, initial contact is made between the two cells and an immature synapse is formed.
In the next step, synaptic connectivity is fine-tuned by the elimination (pruning) and strengthening of synapses. In order to achieve the goal of forming a functional synapse, the synapse-forming and the synapse-receiving cell exchange signals and initiate the second step of synaptogenesis. Dendrite as well as axon-specific protein complexes (e.g. active zone proteins, synaptic vesicle proteins) are recruited to the initial contact site and a functional synapse is formed (Bury and Sabo 2010; Colon-Ramos 2009; Garner et al. 2002; McAllister 2007; Munno and Syed 2003).
2. Environmental chemicals and (neuro)development
Due to this complexity and interplay of processes it is not surprising that already small mistakes in one of these processes have great impact on the whole development of the nervous system. It is estimated, that around 22% of the adults in the United States suffer from at least one mental illness and according to the World Health Organization, this percentage is going to increase in the near future (Andersen 2003; Holden 2000). Additionally, 3 – 12%
(depending on the source) of the children under the age of 18 in the US bear at least one neurodevelopmental disorder (Boyle et al. 1994; Hass 2006; Schettler 2001). Many of these mental illnesses can be associated to genetic mutations (Rosenberg et al. 2007), but data from twin studies indicate that the influence of the environment on such disorders should not be regarded as minor (Fishbein 2000; McGuffin et al. 2001).
Environmental chemicals such as mercury or lead are known to disturb neurodevelopment in humans and are therefore - among many other chemicals, which do not necessarily have the strong epidemiological supportive data of these two metals - suspected risk factors for neurodevelopmental disorders (Grandjean and Landrigan 2006).
The following section describes why the developing brain is particularly vulnerable to toxic insults and why neurodevelopmental disorders might be associated to exposure to environmental chemicals.
2.1 Barker Hypothesis
Barker developed the concept that parameters of growth, such as birth weight or head circumference, can be used to predict the risk of adult diseases, such as coronary heart disease, stroke, insulin resistance and diabetes (Osmond and Barker 2000). Initially, the Barker Hypothesis was developed by David Barker in the 1990s (Barker 1997; Zadik 2003).
By using large epidemiological studies as well as reconstruction of birth cohorts in the UK, Barker was able to correlate such adult disease with reduced fetal growth and impaired development during infancy (Barker et al. 1992; Barker et al. 1993). The influence of e.g.
poor nutrition resulting in low birth weight as risk factor for disease later in life is nowadays firmly established in human epidemiology (Calkins and Devaskar 2011). Whether environmental chemicals could also lead to such effects still remains an unproven theory (Silbergeld and Patrick 2005). However, this theory is supported by studies correlating smoking during pregnancy with reduced birth weight and ultimately with cardiovascular disease (Bakker and Jaddoe 2011; Geelhoed et al. 2011). Additionally, it has been shown that effects of chemicals during early development can be transmitted through the germline up to 3 generations (Nilsson et al. 2008; Skinner et al. 2010). All these results suggest that negative events during early development lead to negative outcomes in the adult organism.
As a result of the suspected relation between exposure to environmental chemicals during embryonic or fetal development and adult mental disease, the Barker Hypothesis was expanded to neurodevelopmental disorders during the Mount Sinai Conference on Early Environmental Origins of Neurological Degeneration in 2003 (Landrigan et al. 2005).
2.2 Time of insult vs. time of phenotype onset
Another concept in (neuro)developmental toxicology is in line with the Barker Hypothesis.
This is the long latency period of many (developmental)neurotoxicants. It has been shown, that e.g. signs of toxicity of MeHg emerged several years after the cessation of a 7 year exposure period in nonhuman primates (Rice 1996). Furthermore, it has been shown that
effects of perinatal exposure to MeHg may emerge as late as 9 years after birth (Davidson et al. 2006).
The long latency period of many suspected neurodevelopmental toxicants is often explained by using the two hit theory of neurodevelopmental toxicology (Wang and Slikker 2011). The theory explains the long latency period by the need for a second, not necessarily toxic, event for the symptoms to manifest. The theory also includes the possibility that a toxic event (second hit) in the adult or aging brain is more severe in a brain which was exposed to a toxic agent during development (first hit). This is supported by studies showing that. effects of developmental exposure to e.g. triethyltin only manifest in aged organisms (Barone et al.
1995).
These effects are explained by a decrease in reserve/repair capacity of the brain caused by the first hit, due to which aging or a second hit have more severe outcomes later in life. Thereby the timing of disturbance of neurodevelopment by the first hit might also determine the type of defect or mental illness later in life (Watson et al. 1999).
2.3 Susceptibility of the developing brain to chemicals
Another well established concept is, that a developing organism/organ is much more susceptible to toxic insults than an adult organism/organ. It has been shown by many studies that low doses of chemicals not toxic for the mature CNS can cause defects in the developing nervous system (Claudio et al. 2000; Tilson 2000). Many studies support the fact, that the timing of a toxic insult is much more important than the type (e.g. type of chemical) (Fan and Chang 1996; Rice and Barone 2000). A particular sensitive time window seems to be the period of organogenesis (GD 20 – 40). It has been shown that radiation during this period often leads to malformations (De Santis et al. 2007). It is estimated, that 90% of all human embryos that experience a disturbance during early organogenesis are spontaneously aborted (Opitz et al. 1987). Additionally, protection and detoxification mechanisms are not fully developed in the early stages of development. An example would be PON1, the enzyme metabolizing chlorpyrifos and other organophosphate pesticides, which is not fully active in humans until the age of 9 (Huen et al. 2009). Other protection mechanisms, such the blood brain barrier (BBB) or DNA repair systems are either not present or not fully functional during development (Adinolfi 1985; Saunders 1986). Another important aspect is the lack of a liver detoxification system in the early fetus. During development, the fetus relies on the detoxification processes of the mother. Current existing exposure limits, aiming to protect the
nervous system, often do not take these aspects into consideration. They are designed to protect workers, not a developing fetus. Concentrations considered to be safe for adults might not necessarily be safe for a developing organism (Ginsberg et al. 2004; Tilson 2000).
Additionally, many chemicals known to have an effect on the developing nervous system, such as e.g. MeHg, tend to accumulate in fetal blood or lipid rich compartments, such as the mother milk, resulting in much higher exposure concentrations for the fetus/child compared to the mother (Jensen 1983; Landrigan et al. 2002; Sonawane 1995).
2.4 Environmental chemicals and developmental disabilities
As already described earlier, neurodevelopmental disorders and mental illnesses are a real and increasing problem in western countries. It is well established that mental disorders like autism or schizophrenia are the result of the complex interplay between genetic and environmental factors. It is believed that the individual genetic background influences the response to environmental factors. This concept is often referred to as G x E interaction (Gene-Environment interaction) (Tsuang et al. 2004). A famous example would be the alcohol flush syndrome. Due to genetic mutations, the activity of the aldehyde dehydrogenase (ALDH) is decreased, resulting in flushing of the face after alcohol consumption. Such genetic mutations are mainly observed in the Asian population (Takeshita et al. 1996;
Wermter et al. 2010). Whether such G x E interactions also play a role in environmental chemicals leading to mental disorders is still under debate and needs further investigation.
The following paragraphs try to summarize the evidence for the correlation of environmental chemicals with some of the most well-known developmental disabilities.
• Mental retardation
Mental retardation is a disorder appearing before adulthood, characterized by a low IQ (below 70), deficits in adaptive behaviors and impaired cognitive function (Fredericks and Williams 1998). Roughly 3% of the children in the US are affected by some form of mental retardation. Genetic disorders account for only 25-30% of the causes of mental retardation (Daily et al. 2000). Due to the fact that nervous system malformations ultimately lead to mental retardation, the disease should be regarded as a result of impairment to the CNS development in general, and not as an individual well defined disease.
Although the genetic background accounts for almost a third of diagnosed mental retardation, it is nowadays well established that environmental factors significantly contribute to this disorder (Matilainen et al. 1995; Simonoff et al. 1996). Several chemicals, including ethanol, lead, MeHg and cadmium, have been associated with mental retardation and more subtle forms of reduced IQ (Beattie et al. 1975;
Grandjean and Landrigan 2006; Marlowe et al. 1983; Mendola et al. 2002; Sokol et al. 2003).
• Schizophrenia
Schizophrenia is characterized by delusions, sensory hallucinations and impairment of speech organization (Goldner et al. 2002). Schizophrenia and autism may be the only mental disorder for which a possible cause in neurodevelopment, namely a delay in neurodevelopment, is widely accepted and therefore resulted in the neurodevelopmental hypothesis of schizophrenia (Powell 2010).
Substances such as lead, amphetamine, ketamine, phencyclidine or cigarette smoke have also been associated with schizophrenia (Keilhoff et al. 2004; Mouri et al. 2007; Opler et al. 2008; Zammit et al. 2003).
• Attention deficit hyperactivity disorder (ADHD)
About 3-5% of the children worldwide suffer from the psychiatric disorder ADHD (Nair et al. 2006; Polanczyk et al. 2007). It usually starts before the age of 7 and often continues into adulthood (Azmitia and Whitaker-Azmitia 1991; Elia et al.
1999) and is more commonly diagnosed in boys than in girls (Dreyer 2006; Malhi and Singhi 2001). Environmental chemicals are suspected to account for the increase in children diagnosed with ADHD over the last years. Although many scientists believe that this increase is rather due to better diagnostic tools, awareness, or even faking of the disease (Cormier 2008; Sansone and Sansone 2011; Simpson et al. 2011), exposure to environmental factors and chemicals such as smoking, manganese, lead, ethanol and PCBs during pregnancy has been shown to be associated with ADHD (Aguiar et al. 2010; Bouchard et al. 2007; Braun et al. 2006; Eubig et al. 2010; Ha et al. 2009; Kukla et al. 2008).
• Autism spectrum disorders (ASD)
Autism spectrum disorders, including autism itself, asperger syndrome or pervasive developmental disorder, are neurodevelopmental disorders characterized by impairment of social interactions and communication, restricted and repetitive pattern of behavior and/or interest (Levy et al. 2009). These symptoms all reliably manifest before the age of 3 (Filipek et al. 1999; Nash and Coury 2003). As already discussed for ADHD boys bare a higher risk of developing ASD (Brugha et al. 2011; Newschaffer et al. 2007). It is estimated that 60 – 70/10 000 births are affected by this lifelong disorder (Fombonne 2009).
What autism has in common with most neurodevelopmental disorders is that the causes are not really known or understood. Many possible causes have been proposed including genetics such as mutations in Mecp2 or Fmr1 (de Leon- Guerrero et al. 2011; Moy and Nadler 2008) and teratogenic agents (Arndt et al.
2005; Trottier et al. 1999). Teratogenic compounds suspected to be a possible cause for autism include, amongst others, thalidomide, heavy metals such as mercury, PCBs or pesticides (Bernard et al. 2001; Jolous-Jamshidi et al. 2010;
McGovern 2007; Stromland et al. 1994).
2.5 Phenotype vs. biological process
All these mental disorders and their characteristic phenotypes are caused by a complex interplay of different biological processes. For psychiatric disorders, endophenotypes have been established to divide behavioral symptoms into stable phenotypes with a clear genetic background. Although the existing definition of endophenotypes is very strict and based on genetic criteria, requiring heritability, illness state independence and illness co-segregation within families (Berti et al. 2011; Gottesman and Gould 2003; Gould and Gottesman 2006;
Hasler and Northoff 2011), the principle behind the concept could be very useful for developmental neurotoxicity testing in vitro (Kadereit et al. 2011).
It will be very hard, most of the time impossible, to model a complex disorder such as e.g.
schizophrenia with all its behavioral aspects 1:1 in vitro. Therefore, the concept of endophenotypes, adapted to DNT testing, could facilitate modeling such disease in vitro.
Instead of correlating a phenotype to a genetic connection, phenotypes such as neuroanatomical or neurobehavioral changes could be associated to basic biological processes which can be modeled in an in vitro system. It is important to bear in mind that the biological
process might be disturbed long before the phenotype manifests (Collman 2011). The effects of many known DNT chemicals have already been linked to such basic biological processes.
A prominent example would be schizophrenia, which has been linked to impaired neurogenesis and neuronal migration. Lead, an environmental chemical suspected to be a possible cause for schizophrenia (Opler et al. 2008) has been shown to impair neurogenesis and alter migration of neuronal progenitors (Dou and Zhang 2011; Jakob and Beckmann 1986). Therefore, modeling such processes in vitro, based on data how these processes are involved in impaired neural development or mental disorders, would facilitate the identification of chemicals causing such impairments.
3 Development of in vitro test systems in the 21st century
As already mentioned in the previous paragraph, modeling complex disease or adverse effects of chemicals in vitro is extremely challenging. Additionally, assessing outcomes of exposure to DNT chemicals, such as a reduced IQ level, is extremely complicated and labor intensive to achieve in animal models. Moreover, human beings are not 70 kg rats (Hartung 2009).
Besides different metabolic features, the normal ontogeny of neural development in rodents is also different from humans. A striking difference is the considerably longer prenatal maturation of the nervous system in humans compared to rats. Such differences result in e.g.
different exposure routes during critical periods of neural development. The exposure route in rats might therefore be via lactational transfer during the first postnatal week, whereas the same biological process would be targeted by a chemical via transplacental transfer during the 3rd trimester of pregnancy in humans (Clancy et al. 2007; Rice and Barone 2000). Although the general sequence of brain development is the same in humans and rodents, the total length of neural development is dramatically different, from days in rodents to weeks or months in humans. Besides those differences in timing of development, clear structural differences exist.
The human neocortex and the visual system are larger compared to those structures in the rats, whereas in rodents the olfactory system, in relation to other brain regions, is much bigger than in humans.
In addition to tragic events, such as the thalidomide catastrophe in the early 1960s or the recent failure of TGN1412 (Ances 2002; Annas and Elias 1999; Attarwala 2010; Stebbings et al. 2007; Stirling et al. 1997; Woollam 1978), data from pharmaceutical companies indicate that animal models are not always able to predict effects of chemicals or drugs on human development or disease situations. During the 1990s, only 11% of new possible drugs
entering clinical trials were registered. Out of these 11%, 23% even failed after registration.
Both, late manifestation of low efficacy and safety issues (account for 30%) are major reasons for late failures of novel drugs (Kola and Landis 2004). Consequently, new and improved test systems, able to predict efficacy and especially toxicity in a human-specific manner are urgently needed (Wobus and Loser 2011).
In order to solve some of these problems, the US National Research Council has developed a strategy and vision for toxicity testing in the 21st century (Gibb 2008; Krewski et al. 2010).
This strategy includes the use of human-based cellular assays which are applicable to high- throughput screening (HTS). Such new test systems, in combination with e.g. systems biology and bioinformatics, would help to understand how chemicals affect normal cellular function, and how this altered function results in a disease/disorder phenotype (NRC 2007).
Furthermore, pathways of toxicity (PoT) could be identified by using such an approach.
Those PoTs, when they are established, well validated and - most important complete - would then allow risk assessment of chemicals based on disturbance of these PoT (Leist et al.
2008b).
4. Embryonic stem cells (ESC) as source for in-vitro testing
In order to achieve the goal of “Toxicology in the 21st century”, new assays, and as the vision proposes the use of cellular systems applicable to HTS, also new sources of unmodified, non cancerous, reliable cells are needed.
Since the establishment of the first mouse (Evans and Kaufman 1981; Martin 1981) and human (Thomson et al. 1998) ESC lines and the generation of induced pluripotent stem cells (IPS) (Takahashi et al. 2007; Takahashi and Yamanaka 2006), a lot of effort has been undertaken to use these cells for regenerative medicine (Menendez et al. 2006; Nsair and MacLellan 2011), modeling of disease (Lee and Studer 2011) and development (Dvash and Benvenisty 2004) as well as for toxicity testing of chemicals (Anson et al. 2011; Wobus and Loser 2011) and drug screening (Laustriat et al. 2010; Pouton and Haynes 2007). Although mouse and human ESC, besides common characteristics, show several differences in e.g.
culture requirements or marker expression (summarized in Table 2), ESC from both species have been shown to be useful tools to test the toxicity of different chemicals (Seiler and Spielmann 2011; Stummann and Bremer 2008; Stummann et al. 2008, 2009). An important feature of ESC for the use in screening of developmental toxicants is their ability to recapitulate in vivo development in vitro. It has been shown, that the expression of different markers in mouse ESC differentiating into the neural lineage closely resembles the onset of
expression of those markers during in vivo development (Barberi et al. 2003). Furthermore, it has been shown that in vitro differentiation of ESC responds to morphogens and growth factors such as sonic hedgehog (SHH) or retinoic acid (RA) in similar ways, as in vivo (Cazillis et al. 2006; Murry and Keller 2008; Okada et al. 2008).
The potential of this new technology in toxicology has therefore also been taken up by large pharmaceutical companies like ROCHE, which use stem cells to screen drugs for cardiotoxicity and effects on neurogenesis (Baker 2010) as well as funding agencies like the European Union, which fund large consortia such as ESNATS to develop robust ESC-based assays to screen for toxic compounds (Wobus and Loser 2011).
As part of this large European consortium we, and especially myself during my PhD thesis, developed ESC-based assays which are able to detect developmental, particularly neurodevelopmental, toxicants. The results of my thesis are included in the following 3 chapters each representing an individual publication.
Table 2: Comparison of mouse and human ESC modified from (Wobus and Boheler 2005)
marker expression mouse ES cells human ES cells reference
Oct3/4 + + (Pesce et al. 1999;
Thomson et al. 1998)
Nanog + + (Chambers et al.
2003; Mitsui et al.
2003)
Sox2 + + (Avilion et al. 2003;
Ginis et al. 2004)
SSEA1 + - (Ginis et al. 2004;
Solter and Knowles 1978)
SSEA3/4 - + (Ginis et al. 2004)
TRA-1-60/80 - + (Ginis et al. 2004)
morphology high nucleo-cytoplasmatic ratio (Wobus and Boheler 2005)
in vitro growth characteristics
tight round clumps flat loose colonies (Wobus 2001) Teratoma
formation in vivo
+ + (Thomson et al.
1998; Wobus et al.
1984) regulation of self
renewal LIF, BMPs FGF2, feeder cells or
matrigel (Wobus and Boheler 2005)
differentiation potential
pluripotent Pluripotent, able to differentiate into
trophoblast-like cells
(Draper and Fox 2003; Odorico et al.
2001)
As already mentioned at the beginning this general introduction aimed to bridge current knowledge about key events in neurodevelopment with toxicological concepts. Other important aspects for the work presented here such as neural crest markers and function as well as toxicity of MeHg or compounds like CEP-1347 are well introduced and discussed in the respective sections of the following chapters (including 2 accepted publications and 1 submitted manuscript).
Aims of the thesis
Only very few chemical substances in our environment and in consumer products are fully characterized for their toxicity. Developmental neurotoxicity (DNT) is currently the least examined form of developmental toxicity. If at all, chemicals are tested for DNT in vivo according to the OECD guideline TG 426 (Makris et al. 2009). Subtle chemically-induced changes in e.g. cell positioning (migration) or cell patterning may result in a complex phenotype like reduced IQ. Such phenotypes are extremely difficult to assess in vivo and even harder in vitro.
A recently published review reported testing of about 100 substances, mainly pesticides, and another study reported neurobehavioral risk assessment for 174 compounds (Makris et al.
2009; Middaugh et al. 2003). Apart from this small group, the chemicals in our environment have not been tested for DNT (Grandjean and Landrigan 2006). In addition, a clear association with human DNT has been shown in epidemiological studies only for a handful of chemicals such as some heavy metals (arsenic, lead, mercury), polychlorinated biphenyls (PCBs), solvents (alcohol, toluene), and pesticides (Grandjean and Landrigan 2006;
Walkowiak et al. 2001). For about 100 additional chemicals, developmental toxicity can be inferred from animal studies (Crofton et al. 2011). To put it in a nutshell, our knowledge about the DNT potential of the chemical universe is extremely limited.
To address this issue, the work described in this thesis was undertaken to develop new toxicological test systems based on the differentiation of embryonic stem cells into the neural lineage. The aims of this thesis were:
1. to characterize in vitro neural differentiation of embryonic stem cells according to the requirements of a toxicological test system
2. to develop differentiation protocols and test systems to model the different steps of neural development
3. to validate these test systems by using pharmacological tool compounds known to affect the processes modeled in the test systems and by providing a mechanistic rational for their action
4. to optimize test systems to detect functional effects of known developmental neurotoxicants
Chapter C
Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel
approaches to developmental neurotoxicity testing
Bastian Zimmer1, Philipp B. Kuegler1, Birte Baudis1, Andreas Genewsky1, Vivek Tanavde2, Winston Koh2, Betty Tan2, Tanja Waldmann1, Suzanne Kadereit1, and Marcel Leist1
1Doerenkamp-Zbinden Chair for In Vitro Toxicology and Biomedicine, University of Konstanz, D-78457 Konstanz, Germany
2Bioinformatics Institute, 30 Biopolis Street, #07-01, 138671 Singapore, Singapore
Cell Death Differ. 2011 Mar;18(3):383-95. Epub 2010 Sep 24
Abbreviations
CNS: central nervous system DNT: developmental neurotoxicity DoD: day of differentiation
EB: embryoid body
ESC: embryonic stem cells GO: gene onthology
mESC: murine embryonic stem cells N: gene onthology neuronal differentiation NPC: neural precursor cell
RA: all-trans retinoic acid Shh: sonic hedgehog
Abstract
As neuronal differentiation of embryonic stem cells recapitulates embryonic neurogenesis, disturbances of this process may model developmental neurotoxicity (DNT). To identify the relevant steps of in vitro neurodevelopment, we implemented a differentiation protocol yielding neurons with desired electrophysiological properties. Results from focused transcriptional profiling suggested that detection of non-cytotoxic developmental disturbances triggered by toxicants such as retinoic acid or cyclopamine was possible. Therefore, a broad transcriptional profile of the 20-day differentiation process was obtained. Cluster analysis of expression kinetics, and bioinformatic identification of overrepresented gene ontologies revealed waves of regulation relevant for DNT testing. We further explored the concept of superimposed waves as descriptor of ordered, but overlapping biological processes. The initial wave of transcripts indicated reorganization of chromatin and epigenetic changes.
Then, a transient upregulation of genes involved in the formation and patterning of neuronal precursors followed. Simultaneously, a long wave of ongoing neuronal differentiation started.
This was again superseded towards the end of the process by shorter waves of neuronal maturation that yielded information on specification, extracellular matrix formation, disease- associated genes, and the generation of glia. Short exposure to lead during the final differentiation phase, disturbed neuronal maturation. Thus, the wave kinetics and the patterns of neuronal specification define the time windows and endpoints for examination of DNT.
Introduction
Ultimately, the entire complexity of the mammalian central nervous system (CNS) is generated during ontogenesis from a few single cells. Neuronal generation and differentiation, can be recapitulated by embryonic stem cells (ESC) under appropriate culture conditions (Abranches et al. 2009; Barberi et al. 2003; Conti and Cattaneo 2010; Gaspard et al. 2008;
Götz and Huttner 2005; Kuegler et al. 2010)}. ESC-based studies of neurodevelopment allow investigations not easily possible in vivo (Leist et al. 2008a) .However, known differentiation protocols differ in their suitability for toxicological studies. For instance, older protocols involve a step of embryoid body (EB) formation (Strübing et al. 1995). Frequently, only a small number of the initially-present ESC form neurons and the observation of individual cells is hardly possible. Other protocols use co-cultures with stromal cell lines to differentiate ESC towards neurons, and would therefore introduce additional complexity into models for developmental neurotoxicity (DNT). A recently developed monolayer differentiation protocol allows monitoring of the differentiation procedure and of possible effects of different chemicals during the whole period of differentiation on a single cell level (Ying and Smith 2003).
DNT is the form of toxicity least examined and hardest to trace, as it is not necessarily related to cell loss. Less than 0.1% of frequently used industrial chemicals have been examined, and for the few known toxicants the mechanism of action is still elusive (reviewed in (Bal-Price et al. 2009; Grandjean and Landrigan 2006; Makris et al. 2009)). Behavioral pathology in the absence of cell loss is also known from disease models, e.g. for Huntington’s disease (Hansson et al. 1999) or schizophrenia (Penschuck et al. 2006). Toxicants, such as mercury or lead may trigger behavioral or cognitive deficits without histophathological hallmarks (Grandjean and Landrigan 2006). Cellular physiology may be affected during the period of exposure (Rossi et al. 1993). This may eventually lead to changes in differentiation and patterning in the CNS, which is the basis for long term effects that are observed after the exposure to toxicants has ceased.
CNS development is assumed to be orchestrated by waves of gene expression (Aiba et al.
2006; Wei et al. 2002) that determine different intermediate cell phenotypes. Some periods may be more sensitive to certain toxicants than others. Epidemiological proof for such
“windows of sensitivity” in organ development with long term consequences for the organism
comes from thalidomide exposure in man (Kuegler et al. 2010) and various animal models (Jongen-Relo et al. 2004).
Current test systems based on the differentiation of stem cells to either cardiomyocytes (Marx-Stoelting et al. 2009) or neural cells (Bal-Price et al. 2009) neither yield mechanistic info, nor do they account for the complexity of CNS development, i.e. the establishment of a balance between multiple neuronal cell types (Kuegler et al. 2010; Rao and Jacbson 2005).
The “toxicology for the 21st century” initiative (Collins et al. 2008; Leist et al. 2008b) suggests the identification of pathways as opposed to the current black-box test systems. In the case of ESC-based models of DNT, this requires a detailed understanding of the developmental process leading to multiple different cell types. Detailed knowledge on the waves of gene induction controlling different developmental steps would be an essential prerequisite. However, CNS development is proceeding at different paces. For instance, the anterior and posterior part of the neural tube follow different kinetics, and some regions of the CNS continue neurogenesis, while in other regions cells have already reached fully postmitotic stages (Rao and Jacbson 2005).
Our study was undertaken to analyze the wave-like expression pattern of mESC neurodevelopment as a basis for the definition of test windows and markers. This knowledge should help to identify non-cytotoxic, but neuroteratogenic compounds able to shift neuronal composition or phenotypes. Finally, the markers should distinguish multiple cell types and differentiation stages, and be able to indicate subpopulations of cells.
Figure 1. Protein and mRNA-based markers of robust neuronal differentiation of mESC.
A. Cultures of mESC were fixed and stained on day 20 of differentiation. DNA, (blue) was stained with H-33342. Proteins are indicated as text on the micrograph in the same color as used for the display of their staining pattern. Tuj1: neuronal form of beta-III tubulin; NeuN: nuclear neuron-specific nuclear antigen, encoded by fox3) (Kim et al. 2009);
GAD: glutamate decarboxylase; SV2: synaptic vesicle glycoprotein 2a; PSD95: post-synaptic density protein 95. Scale bars: 20 µm. B. mESC cultures (n = 5 biological experiments) were differentiated towards neurons, and RNA was prepared at the indicated days of differentiation.
Gene expression of the stemness factor Oct4, ne neural stem cell marker Nestin, the mature neuronal marker Synaptophysin and the glial marker Gfap was quantified by quantitative RT-PCR. The means
± SD of the relative expression compared to day 0 (set to 1 on each diagram) was calculated and displayed (dotted lines). Relative gene expression data were also obtained by chip analysis and the means (n = 2) are displayed (solid line). Note the different scaling of the axes for chip or RT-PCR analysis, respectively, which was chosen for reasons of better comparability of the overall curve shapes.
The figures in the diagram indicate the relative expression level on DoD20 (DoD7 for nestin) vs DoD0, and thus define the axis scaling.
Results
Monolayer differentiation of mESC to neurons
On day-of-differentiation 20 (DoD20), the majority of cells was positive for the pan-neuronal markers Tuj1 and NeuN. Many cells also expressed the synapse associated markers SV2 and PSD95 (Fig. 1A). As a more quantitative overall measure for the robustness of the differentiation protocol, we chose mRNA expression, which we followed over time. The
kinetics for different markers were highly reproducible across experiments (Fig. 1B).
Differentiation to mature, electrophysiologically-active neurons was shown by the presence of voltage-dependent Na+ and K+ and Ca2+ channels in individual patch-clamped neurons (Fig.
2A-C, Fig. S1). Further experiments also identified spontaneous neuronal electrical activity (Fig. 2D) and action potentials (Fig. S1). Currents were also evoked by exposure to N-methyl- D-aspartate or kainic acid and blocked by the respective selective antagonists (Fig. 2E). Thus, our differentiation protocol yielded bona fide neurons.
Figure 2. Electrophysiological evidence for successful neuronal development.
Cells were differentiated on glass cover slips towards the neuronal lineage for 20-24 days and then placed into a temperature controlled recording chamber for whole cell patch-clamp studies. A. Representative example for the currents observed during the 20 ms voltage steps of the whole cell voltage clamp recording protocol displayed in B. Note that Na+ currents (downwards deflection) are observed at voltages ≥ -40 mV (solid line). Strong depolarizing and repolarizing (K+ currents; upwards deflection) are observed at depolarization to 0 mV (dashed line). C.
For voltage clamp recording (voltage step from – 80 mV to 0 mV) of Ca2x channels Na+ and K+ channels were blocked by addition of tetrodotoxin, tetraethylammoniumchloride (5 mM), 4- aminopyridine (10 mM), and substitution of intracellular K+ ions by 120 mM Cs+. Moreover, the measurement of Ca-currents was favoured by a bath solution containing barium ions (10 mM) instead of calcium ions. Current traces were obtained without Ca2+-channel blocker, or with the blockers nimodipine (1 µM) or Cd2+ (1 mM) added. Current data at 15 ms after the voltage step were corrected for cell capacitance (indirect measure for cell size) and displayed. Data represent means ± SD. ** p < 0.01. D.
Spontaneous action potentials were recorded in current clamp mode (0 pA). At the time indicated by an arrow, tetrodotoxin was added. The dashed line indicates 0 mV membrane potential. The scale bars indicate the dimensions of the membrane potential and the time domain. E. Recordings at individual neurons excited with specific glutamate receptor agonists in the presence or absence of blockers. Current traces were recorded after application of N-methyl-D- aspartate (NMDA) or kainic acid. All agonists were also tested in the presence of their respective specific antagonist (traces with 5-aminophosphovalerate (AP- 5), 6,7-dinitroquinoxalin-2,3-dione (DNQX)). The scale bars represent the current and time dimensions of the experiment. Data are representative for n ≥10 neurons (for agonists) and n = 3 for antagonists (on neurons with positive agonist response).
Transcription-based endpoints to identify disturbed neuronal differentiation
We next investigated whether subtle perturbations of the differentiation process below the cytotoxicity threshold would be detectable by mRNA-based readouts. Parallel mESC cultures
were differentiated for 7, 15 and 20 days and mRNA was prepared for quantitative RT-PCR analysis. These cells were treated during two different time windows (DoD1-7, DoD8-15) with two neuro-teratogens (Fig. 3A). With the concentrations used here cell death was not detectable (data not shown) and cells looked viable and were morphologically
Figure 3. Detection of non-cytotoxic developmental disturbances by transcriptional analysis
Cultures of mESC were neuronally differentiated for 7, 15 or 20 days as indicated in a-d. They were exposed to retinoic acid (RA) or cyclopamine (Cyclo) for the time periods indicated by the hatched boxes.
A. RNA was isolated at the indicated days (diamond) and used for quantitative RT-PCR analysis of selected differentiation and patterning markers. Headings indicate the overall biological effect, such as accelerated neuronal differentiation (e.g. Neuronal diff. (+)) or altered patterning (e.g. Caudalization). Names are the official gene names, apart from the following: Vglut1 = Slc17a7, HB9 = Mnx1. The data indicate relative expression levels in % compared to untreated controls at the same time point, and are means ± SD from two to three independent experiments for each treatment and exposure schedule. Significance levels (by ANOVA within a given experimental condition) are indicated (*: p < 0.05, **: p < 0.01, ***: p > 0.001).
The complete data set with standard deviations is given in Figure S2 B. Representative images of cultures on DoD15 in condition a. RA and Cyclopamine-treated cultures were viable indistinguishable from controls (ctrl.).
indistinguishable from untreated cells (Fig. 3B). We used the morphogen retinoic acid (RA) as a known in vivo and in vitro reproductive toxicant and cyclopamine for its ability to alter
sonic hedgehog (Shh) signaling resulting in the disruption of patterning gradients responsible for floor plate and ventral neurons (Gaspard et al. 2008; Rao and Jacbson 2005). As expected from the literature (Irioka et al. 2005), RA induced accelerated neuronal differentiation (increased synaptophysin expression) whereas cyclopamine reduced the expression of
