PDGF in Cerebellar Development and Tumorigenesis

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1018

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JOHANNA ANDRÆ

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Pathology presented at Uppsala University in 2001

Abstract Andræ, J. 2001. PDGF in Cerebellar Development and Tumorigenesis. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1018. 50 pp. Uppsala. ISBN 91-554-4987-5

Medulloblastoma is a highly malignant cerebellar childhood tumor. As in many other brain tumors, expression of platelet-derived growth factor (PDGF) and its receptors has been shown in medulloblastoma. To reveal the importance of this growth factor in cerebellar development and tumorigenesis, analyses were performed on human medulloblastoma cell lines and on tissue from normal mouse brain at different stages of development. The in vivo effect of a forced expression of PDGF-B in the cerebellar primordium was examined in transgenic mice. In the normal mouse embryo, we found PDGF receptor-α-positive cells in the early neuroepithelium and on neuronal precursors. In the postnatal cerebellum, cells in the external germinal layer and Purkinje cells expressed the receptor. In the medulloblastoma cells, expression of all the three PDGF isoforms and PDGF receptors was seen and correlated to neuronal differentiation. Endogenously activated, i.e. tyrosine phosphorylated, PDGF receptors were identified. To reveal the role of PDGF in normal cerebellar development, we established transgenic mice where a PDGF-B cDNA was introduced via homologous recombination into the engrailed-1 gene. Engrailed-1 is specifically expressed at the mid-/hindbrain boundary of the early neural tube, i.e. in an area from which the cerebellar primordium develops. The ectopic expression of PDGF-B caused a disturbance of cerebellar development. Midline fusion of the cerebellar primordium did not occur properly, which resulted in cerebellar dysplasia in the adult mouse. In a parallel study, the expression pattern of a glial fibrillary acidic protein (GFAP)-lacZ transgene was followed in the embryonic mouse central nervous system. It was shown that the human GFAP promoter was already active by embryonic day 9.5 and as development proceeded, expression occured in different, independent cell populations. Among these cell populations were the radial glial cells in the neocortex.

Key words: Cerebellum, medulloblastoma, PDGF, GFAP, transgenic mice.

Johanna Andræ, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden

ISSN 0282-7476 ISBN 91-554-4987-5

Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2001

2 Before you go on a hunt It is wise to ask someone What you are looking for Before you start to look for it

Winnie the Pooh

To my parents

3 This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I Andræ, J., Molander, C., Smits, A., Funa, K. and Nistér M. Human medulloblastoma cells express different platelet-derived growth factor isoforms and carry endogenously activated receptors. Submitted

II Andræ, J., Hansson, I., Afink, G.B. and Nistér, M. (2001) Platelet- derived growth factor receptor-α in ventricular zone cells and in developing neurons. Molecular and Cellular Neuroscience, in press

III Andræ, J., Afink, G.B., Wurst, W. and Nistér, M. Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium causes defective midline fusion and cerebellar dysplasia. Manuscript

IV Andræ, J., Bongcam-Rudloff, E., Hansson, I., Lendahl, U., Westermark, B. and Nistér, M. A 1.8 kb GFAP-promoter fragment is active in specific regions of the embryonic CNS. Submitted

4 TABLE OF CONTENTS ______Abbreviations ...... 6

BACKGROUND ______Introduction ...... 7 Central Nervous System (CNS)...... 7 The neural stem cell Neuronal cells Astrocytes Oligodendrocytes Radial glial cells Intermediate filaments Cerebellum and its role ...... 10 Cerebellar cell types and their organization Development of cerebellum Granular cells and the external germinal layer Sonic hedgehog in cerebellar development Mouse mutants affecting the cerebellum Medulloblastomas ...... 14 Brain tumors in children Genetic alterations in medulloblastomas Patched and Sonic hedgehog in medulloblastomas Other mouse models for PNET/medulloblastomas Platelet-derived growth factor (PDGF)...... 17 Biological functions of PDGF Expression of PDGF and PDGFR in the CNS PDGF mutant mice Transforming activity of PDGF PDGF and PDGFR in human brain tumors

METHODS ______Transgenic mice and the “knock-in” technique...... 21 Embryonic stem (ES) cells Replacement vector for gene targeting Electroporation of ES cells and screening of clones for homologous recombination Blastocyst injection and generation of chimeric mice Whole mount β-galactosidase staining Fixation of mouse tissue for histological analysis Immunohistochemistry

5 PRESENT INVESTIGATION ______Hypothesis and Aims ...... 26 Human medulloblastoma cells express different platelet-derived growth factor isoforms and carry endogenously activated receptors (Paper I)...... 27 Platelet-derived growth factor receptor-α in ventricular zone cells and in developing neurons (Paper II)...... 29 Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium causes defective midline fusion and cerebellar dysplasia (Paper III)...... 30 A 1.8 kb GFAP-promoter fragment is active in specific regions of the embryonic CNS (Paper IV)...... 33

GENERAL DISCUSSION ______35

ACKNOWLEDGEMENTS ______36

REFERENCES ______38

Abbreviations bFGF basic fibroblast growth factor CNS central nervous system CNTF ciliary neurotrophic factor DAB 3,3’-diaminobenzidine tetrahydrochloride E embryonic day EGF epidermal growth factor EGL external germinal layer ES cells embryonic stem cells FCS fetal calf serum GFAP glial fibrillary acidic protein HRP horseradish peroxidase IF intermediary filament IGF insulin-like growth factor IGL internal granular layer NSC neural stem cell O-2A oligodendrocyte type-2 astrocyte OP oligodendrocyte precursor PDGF platelet-derived growth factor PDGFR platelet-derived growth factor receptor PNET primitive neuroectodermal tumor Ptch patched Shh sonic hedgehog TGFβ transforming growth factor beta VZ ventricular zone

6 Introduction Most brain tumors in children are derived from astrocytes or belong to a group of tumors called primitive neuroectodermal tumors (PNETs). PNETs are highly malignant, invasive tumors that are difficult to treat. Often the treatment is a combination of surgery, cytostatic drugs and radiation. PNETs that are localized to the cerebellum are called medulloblastomas. The cell of origin for medulloblastomas is not known, but it is commonly believed that these tumors arise from undifferentiated cells in the external germinal layer (EGL) in the cerebellum. Many brain tumors express the platelet-derived growth factor (PDGF) and its receptors, and a few years ago expression was also reported in medulloblastomas (Black et al., 1996; Smits et al., 1996). The aim of this thesis was firstly to investigate the role of PDGF in normal cerebellar development in mice and in human medulloblastomas. The embryonic expression pattern of the human GFAP promoter in transgenic mice was also characterized.

CENTRAL NERVOUS SYSTEM (CNS)

The nervous system can be divided into the central nervous sytem (CNS), consisting of the brain and spinal cord, and the peripheral nervous sytem (PNS) that includes nerves, ganglions and receptors. Closure of the neural tube and formation of the brain vesicles are early steps in CNS development. The brain vesicles form specialized regions, e.g. the cerebrum, cerebellum and oblongate medulla. Initially, the neural tube consists of a single cell-layered neuroepithelium, from which all CNS cell types later arise. The three main groups of cells in the CNS are neurons, oligodendrocytes, and astrocytes.

The neural stem cell Neural stem cells (NSC) are cells in the CNS that have the capacity to differentiate into neurons, astrocytes and oligodendrocytes and that are able to self-renew. If grown in vitro in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) or transforming growth factor-β (TGFβ) the cells proliferate, but upon removal of the factor the cells start to differentiate (reviewed by Kilpatrick et al., 1995 and McKay, 1997). The cells can be induced to differentiate into specific cell types by the addition of extra-cellular factors. Stimulation with PDGF turns the cells into neurons, ciliary neurotrofic factor (CNTF) promotes the development of astrocytes and thyroid hormone (T3) forces the cells to become both astrocytes and oligodendrocytes (Johe et al., 1996). It has also been shown that the choice of cell fate depends on the density of the NSC culture, in which densely grown cells differentiated into smooth muscle cells in the presence of serum (Tsai and McKay, 2000). It is now known that neurogenesis also takes place in the adult human brain (Eriksson et al., 1998; Johansson et al., 1999). From studies on adult mouse brain, it has been suggested that NSCs are subventricular zone astrocytes (Doetsch et al., 1999; Laywell et al., 2000) or ependymal cells (Johansson et al., 1999). There are also reports of oligodendrocyte precursor cells and radial glia cells that show NSC-like properties (Kondo and Raff, 2000; Malatesta et al., 2000). The main question is probably whether

7 there are any true tissue-specific stem cells. It has been shown that adult NSCs can differentiate into cells representing all three germ layers, for example into blood, heart and liver cells (Bjornson et al., 1999; Clarke et al., 2000).

Neuronal cells Neurons are the messenger cells of the nervous system. In the adult brain, neurons are organized in specific cell layers. Initially, undifferentiated neuroepithelial cells stretch from the pial lining to the ventricular surface. DNA synthesis takes place at the pial side of the neuroepithelium. The cell bodies then move to the ventricular surface where they undergo mitosis and subsequently, move back to the pial side to differentiate into neurons and glia (Frisen et al., 1998; Johansson et al., 1999; Sauer, 1935). The post mitotic (newborn) neurons migrate back towards the pial side along radial glial processes to their final position. Late born neurons end up closest to the pial surface and an “inside-out” organisation is formed. The settling of neurons in the cerebellum follows a different pattern (see below). As differentiation into specific neuronal cell types progesses, new proteins are sequentially expressed. Some, like microtubule associated protein (Map2), β-III-tubulin (Tuj1) and neurofilaments (NF), are expressed in many neurons, while others are specific for certain subpopulations of neurons during a limited time period. Specific neurons can be identified by the level of expression of certain marker proteins; for example the neuronal nuclei protein (NeuN) is not expressed in Purkinje cells in the cerebellum (Mullen et al., 1992) whereas Calbindin-D28K is specifically expressed in these cells (Nordquist et al., 1988). Neuronal cells can also be identified by their expression of different neurotransmitters and associated proteins, e.g. the acetylcholine receptor, the dopamine transporter and γ-aminobutyric acid (GABA).

Astrocytes In the mature brain, neurons are surrounded by glial cells. Several classes of glial cell precursors have been identified, for example the glial restricted precursor, the oligodendrocyte type-2 astrocyte (O-2A) progenitor cell and the astrocyte precursor cell (reviewed by Lee et al., 2000). The star-shaped astrocytes comprise the major part of all glial cells in the CNS. They extend processes with end-feet that adhere to blood vessels or neurons. They are also the predominant cell type in the blood-brain barrier. Mature astrocytes express glial fibrillary acidic protein (GFAP) (see below). A specific type of astrocyte (type-2 astrocyte) was found in cultures of rat optic nerve (Raff et al., 1983). Type-2 astrocytes are characterized by a neuron/oligodendrocyte-like morphology and immuno-reactivity with antibodies to the ganglioside A2B5, whereas type-1 astrocytes have a more fibroblast-like morphology and do not bind to the A2B5 antibody. The CNTF was shown to induce type-2 astrocytes in cultures (Hughes et al., 1988). However, the existance of the type-2-astrocyte in vivo remains unproven.

Oligodendrocytes Oligodendroglial cells myelinate and isolate the axons of developing neurons in the CNS. Early markers for oligodendrocytes are the transcription factors Olig1 and Olig2 (Lu et al., 2000; Zhou et al., 2000). Expression of the Olig genes is induced by sonic hedgehog (see below) and is the earliest known “marker” for these cells. Olig2 is also

8 expressed in neuronal progenitor cells (Takebayashi et al., 2000). PDGFRα positive oligodendrocyte precursors are found in the ventral spinal cord and diencephalon (Hardy and Friedrich, 1996; Pringle and Richardson, 1993). These precursors were previously referred to as O2-A progenitors, because they were believed to give rise to oligodendrocytes and type-2-astrocytes. Since there is a doubt whether the type-2- astrocyte exists in vivo, most people today depict the cell type as oligodendrocyte precursor (OP). Other protein “markers” used to identify OPs are the proteoglycan NG2 (Nishiyama et al., 1996) and A2B5 (Grinspan et al., 1990).

Radial glial cells A special type of glial cell is the radial glial cell. These cells are radially distributed with endfeet at the pial side and at the ventricular surface. Radial glial cells guide migratory neurons to their correct positions (Hatten, 1990). There are several markers for radial glia; RC2 (Misson et al., 1988), brain lipid-binding protein (BLBP) (Feng et al., 1994), and the glutamate transporter GLAST (Shibata et al., 1997). It has been suggested that radial glia can develop into astrocytes, oligodendrocytes and ependymal cells (Edwards et al., 1990). Recently, it was shown that a radial glial cell can differentiate into either neurons or astrocytes and could therefore be regarded as a NSC (Malatesta et al., 2000). This finding was confirmed by Noctor et al., who elegantly showed that radial glial cells generate neurons that migrate on the very same radial glial process (Noctor et al., 2001). The radial organization of the neocortex has been described earlier in “the radial unit hypothesis” (Rakic, 1988).

Intermediate filaments Cells at different stages of differentiation can be identified by their expression of cell type-specific proteins. Among these are the intermediate filaments (IFs), which are part of the cytoskeleton. These 10 nm thick filaments are divided into different classes with respect to homology and tissue distribution; for example, desmin is expressed in muscle cells, neurofilaments in neuronal cells and keratins in epithelial cells (Lazarides, 1980). All IF proteins have a common structure and assemble into filaments in a similar way (Stewart, 1993 and references therein). The class VI filament, nestin is a marker for NSCs and radial glial cells (Lendahl et al., 1990). Nestin expression is present in most proliferating NSCs and is down-regulated when the cells differentiate (Dahlstrand et al., 1995). It has also been shown that nestin requires vimentin for proper assembly (Marvin et al., 1998). Vimentin belongs to the class III filaments and is widely expressed in mesenchymal cells. In the CNS, vimentin is transiently expressed in neuronal and glial cell precursors and is progressively replaced by GFAP and neurofilaments, respectively (Lazarides, 1980). However, reactive astrocytes that appear after CNS injury, express nestin, vimentin and GFAP (Frisen et al., 1995). Vimentin “knock-out” mice develop and reproduce normally (Colucci-Guyon et al., 1994), but electron microscopy studies show that loss of vimentin affects the development of Bergman glia in the cerebellum (Colucci-Guyon et al., 1999). No nestin mutant mouse has yet been described. GFAP (class III IF) has been considered an astrocyte specific protein, however, several reports show GFAP expression in other cell types (Bertelli et al., 2000; Holash et al., 1993; Marino et al., 2000). The GFAP gene has been disrupted in several mouse

9 models (Gomi et al., 1995; Liedtke et al., 1996; McCall et al., 1996; Pekny et al., 1995), but the biological functions of GFAP are still basically unknown (Rutka et al., 1997). In the adult mouse CNS, GFAP-containing astrocytes are involved in tissue repair and the expression of GFAP increases during reactive astrocytosis (Norton et al., 1992 and references therein).

CEREBELLUM AND ITS ROLE

Cerebellum is a center for the coordination of movement. Motor cortex in the cerebrum sends signals to the muscles, telling them how to contract. Cerebellum receives a copy of that signal, as well as a report from the body with information on how the movement was performed. Cerebellum compares the two signals and decides if the movement was satisfactory. If not, a signal is sent from the cerebellum to the motor cortex, saying how to correct the movement. Recent data suggest that the cerebellum also contributes to mental and social functions. Investigations on children, who had undergone surgery to remove cerebellar tumors, showed disturbances in, for example, speech and emotions (Riva and Giorgi, 2000). Herrup & Kuemerle (1997) wrote ”...it is safe to say, in 1996, that we are still not exactly sure what the cerebellum does”, which is most likely still true today.

Cerebellar cell types and their organization The cells in the adult cerebellum are organized in defined layers; the molecular layer, the Purkinje cell layer, the internal granular layer (IGL) and the central medullary layer. During early postnatal development, there is also a transient external germinal layer (EGL). Cells in the different layers form a complex network (Altman and Bayer, 1997 and references therein).

molecular layer stellate cell

basket cell

Purkinje cell layer Purkinje cell

granular layer Golgi granule cell cell

medullary layer

r e

ib f

in efferent signal b

lim c mossy fiber

Fig. 1 Interactions between different neuronal cell types in the cerebellum 10 Afferent signals to the cerebellum go through climbing fibers and mossy fibers. Each climbing fiber synapses with only one Purkinje cell, whereas mossy fibers divide to different lobes and form synapses with granule cells and Golgi cells. Granule cells and Golgi cells synapse with each other, and the granule cells also form synapses with stellate cells, basket cells and Purkinje cells. Stellate cells and basket cells also signal directly to the Purkinje cells. Out of this complex network, it is the Purkinje cells alone that generate efferent signals from the cerebellum. Cerebellum is believed to contain more neurons than the rest of the body. It has even been suggested that more than 80% of all human neurons are cerebellar granule cells (Lange, 1975).

Development of the cerebellum The cerebellar development is a complex story that can be told with the emphasis on the anlage, gene expression, anatomical changes, cell movements, etc. The first events are controlled by the isthmic organizer, the area at the mid-/hindbrain boundary (MHB) (Fig.2) that induces and polarizes the midbrain and the anterior hindbrain (reviewed by (Simeone, 2000). Expression of pattern forming genes in this area is tightly controlled. Control of the expression of En1, Gbx2, Wnt1, Fgf8 and Otx2 is mutually interdependent and their relative spatial distribution is important for the formation of the whole region. If, for example, the expression of Otx2 is dorsalized, the result is an enlargement of the colliculus and absence of vermis (Broccoli et al., 1999). Early in cerebellar development, the neural tube bends and a wide opening appears in the dorsal aspect. The edges of this rhomboid opening are called the rhombic lip. The opening over the 4th ventricle is covered by a thin membrane (the medullary velum), anterior to which the cerebellar anlage is located. The anlage expands and will subsequently fuse in the dorsal midline. The cerebellar fusion initiates around E16 in mice by an apposition of the cerebellar primordia (Altman and Bayer, 1997). Before fusion, the neuroepithelium that covers the primordia is in a proliferative phase. This ceases when the anlage fuses and the neuroepithelium covering the area involved in fusion is dissolved.

mid-/hindbrain boundary

midbrain cerebellar primordium

hindbrain

forebrain

spinal cord

Fig. 2 Sagittal section through the head region of an E12 mouse

11 Granular cells and the external germinal layer The granule cells originate from the rostral rhombic lip (Alder et al., 1996) and until recently, it was believed that exclusively granule cells were derived from this area. However, a recent report shows that cells of the rostral rhombic lip also differentiate into hindbrain neurons (Lin et al., 2001). Precursor cells begin to move from the rhombic lip over the cerebellar anlage at E13 in the mouse (Hatten and Heintz, 1995). These EGL cells gradually cover the newly formed cerebellum, but they do not cover the midline areas until the fusion is complete and a solid tissue base has been created (Altman and Bayer, 1997). The EGL is a transient cell layer. It is highly proliferative and reaches its maximum thickness at P5-P10. Two distinct zones can be distinguished in the EGL; the outer mitotic/proliferative zone and the inner differentiating zone. The postmitotic cells in the inner zone differentiate and start to migrate inwards to form the IGL. Initially, the granule cells migrate along Bergmann glia (Komuro and Rakic, 1998). Around P20, the entire EGL is gone. The site(s) of origin for many cerebellar interneurons is controversial. There are reports showing both that basket cells, stellate cells and Golgi cells originate from the EGL (Altman and Bayer, 1997; Hausmann et al., 1985) and other reports showing that interneurons are generated from progenitors in the cerebellar white matter (Zhang and Goldman, 1996). Purkinje cells originate in the neuroepithelium of the 4th ventricle and gather in cell clusters, which supply different cerebellar lobes with Purkinje cells (Altman and Bayer, 1997; Yuasa et al., 1991).

inferior colliculus * ~E16 cerebellar primordium

oblongate medulla

inferior colliculus

cerebellum ~ birth oblongate medulla

sagittal section coronal section

Fig. 3 Sections through the developing cerebellum. * marks the fusing cerebellar anlage.

12 Sonic hedgehog in cerebellar development The murine Sonic hedgehog (Shh) was cloned as a homologue of the Drosophila gene hedgehog (Echelard et al., 1993). The Drosophila hedgehog belongs to the family of segment polarity genes, i.e. genes that maintain a repeated pattern in the segments during development. Other genes in this family include engrailed, wingless, cubitus interruptus, and patched. The secreted protein, Shh, binds to the 12-transmembrane protein, patched (Ptch) (Marigo et al., 1996; Stone et al., 1996), a homologue of the segment polarity gene, patched, in Drosophila (Nusslein-Volhard and Wieschaus, 1980). Normally, ptch inhibits the 7-transmembrane protein, smoothened (Smo), but on binding with Shh, this inhibition is abrogated and an intracellular signal cascade starts. The signaling cascade involves activation of Gli and the transcription of target genes as Ptch and Gli. Vertebrate Shh genes are expressed e.g. in notocord and floorplate cells (Echelard et al., 1993; Krauss et al., 1993; Riddle et al., 1993). The expression of Shh in the notocord induces differentiation of floor plate cells and motor neurons. The fate of the cells depends on the concentration of Shh (Marti et al., 1995; Roelink et al., 1995). In the cerebellum, Purkinje cells express Shh, where it functions as a mitogen for granule cells in the EGL (Dahmane and Ruiz-i-Altaba, 1999; Wallace, 1999; Wechsler- Reya and Scott, 1999). EGL cells cultured in vitro respond to Shh by proliferating and do not differentiate, as unstimulated cells do. In a thymidine incorporation assay, Shh increased cell proliferation more than 100-fold, while other known mitogenic substances for granule cell precursors (IGF-1, EGF, bFGF) only gave a 2- to 6-fold increase. In vivo experiments show that Shh is required to properly expand the EGL during postnatal development. Blocking of the Shh signal alters the differentiation of granule cells and Bergmann glia and disturbs the development of Purkinje cells.

Mouse mutants affecting the cerebellum Many spontaneous murine mutants, as well as transgenic or knockout mice with cerebellar defects, have been described. Mutations affecting the cerebellum often lead to ataxia, and the phenotypes are easily scored. The list of cerebellar mutants is long and in most cases, the phenotype was characterized long before the causative mechanism was known. Drosophila engrailed is a protein that maintains the anterior/posterior boundary in each segment, and mutations in the engrailed gene result in fusion of adjacent segments (Gilbert, 1994). The mouse engrailed genes (En1 and En2) were identified due to their homology to Drosophila engrailed (Joyner et al., 1985). The genes code for homeodomain-containing transcription factors which during embryonic development are specifically expressed at the mid-/hindbrain boundary (Davidson et al., 1988; Davis and Joyner, 1988). Mice, homozygous for an En1 null mutation, die at birth with a mid- /hindbrain deletion (Hanks et al., 1995), whereas heterozygous mice are viable and have no known defects. The En2 mutation is less severe. The cerebellum is reduced in size and the foliation pattern is altered (Joyner et al., 1991). Fusion of the cerebellar primordium is delayed at E15.5, but no differences are seen after two further days (Millen et al., 1994). Some mutations target a specific cell population. In mice lacking Math1 the granule cell population is affected. Math1 codes for a transcription factor that is expressed in

13 EGL cell precursors and if Math1 is mutated, EGL cell precursors fail to form or to proliferate (Ben-Arie et al., 1997). The number of Purkinje cells is not affected, but they are disorganized and the cerebellum is smaller. In the weaver mutant, EGL cells proliferate normally but they are blocked in early differentiation and fail to migrate along radial glia. A mutation in the G-protein gated K+channel GIRK2 causes influx of Na+ (Kofuji et al., 1996). Weaver granule cells can differentiate normally if the Na+ influx is blocked pharmacologically. There are also examples of mutations that hit different levels of the same pathway. The Reeler mutant has cell migration problems, leading to a disorganized layering in different brain regions. The size of the cerebellum is reduced and the foliation is absent (reviewed by (Curran and D'Arcangelo, 1998). Purkinje cells fail to migrate properly and the number of granule cells are greatly reduced. The reeler gene was cloned and shown to code for an extra-cellular protein, called reelin (D'Arcangelo et al., 1995). Almost identical phenotypes are seen in the scrambler and yotari mutants; i.e. small unfoliated cerebelli, reduction in granule cell numbers and ectopic location of the Purkinje cells (Goldowitz et al., 1997; Yoneshima et al., 1997). The scrambler and yotari mutants are caused by mutations in the same gene, i.e. the mouse disabled-1 (mDab1) (Sheldon et al., 1997). These mice still express reelin, the basis for the hypothesis that mDab1 acts downstream of reelin. Later it was shown that reelin binds to two lipoprotein receptors (VLDLR and ApoER2) which induces tyrosine phosphorylation of Dab1 (Hiesberger et al., 1999; Howell et al., 1999). Double mutant mice that lack both VLDLR and ApoER2 display a phenotype that is identical to the Reeler mutant. The list of cerebellar mutants could be made much longer, and it should be noted that many of these mutants also show defects outside the cerebellum.

MEDULLOBLASTOMAS

Brain tumors in children Around 100 new cases of brain tumors in children (0-19 years of age) are diagnosed per year in Sweden. In 1998, 96 new cases were reported (Cancer Incidence in Sweden 1998, Socialstyrelsen). Brain tumors in children are mainly of two kinds; astrocyte- derived tumors and primitive neuroectodermal tumors (PNET). PNETs are malignant, embryonal CNS tumors consisting of mostly small, round, undifferentiated cells (Giangaspero et al., 2000). PNETs that occur in the cerebellum are called medulloblastomas and correspond to WHO grade IV (World Health Organization). These tumors metastasize via the cerebro-spinal fluid. Medulloblastoma is a childhood tumor and most cases occur by the age of 0-20 years, with a peak around 7 years. However, medulloblastomas are also found in adults, even late in life. The name Medulloblastoma was given by Bailey and Cushing (Bailey and Cushing, 1925). Since neurons, glial cells and muscle cells were found, they named the tumors after a hypothetical cerebellar stem cell – the medulloblast. So far, no human medulloblast has been identified, but the rat cerebellar cell line ST15A was suggested to fulfil the criteria (Valtz et al., 1991). When cultured under defined conditions these

14 nestin-positive, neuroectodermal cells differentiate into neuronal-, glial- and muscle cells. The diagnosis of medulloblastoma is mainly based on morphological criteria, but immuno-reactivity to several cellular proteins can also be found in the tumors. Among these are synaptophysin, intermediate filament proteins, neural cell adhesion molecules and nerve growth factor and its receptors (Giangaspero et al., 2000). Antibodies used routinely in the clinic to make a diagnosis in a suspected medulloblastoma case are directed against synaptophysin, GFAP, vimentin and also an antibody that marks proliferative cells (Pathologist Leonie Saft, University Hospital, Uppsala, personal communication).

Genetic alterations in medulloblastomas The most common genetic alteration in medulloblastomas is isochromosome 17q. The tumor suppressor gene p53 is situated on chromosome 17p, but very few p53 mutations have been found in medulloblastomas (Adesina et al., 1994; Ohgaki et al., 1991). A bad prognostic factor for patients with medulloblastoma is expression of the oncogene c- myc (Herms et al., 2000). Amplification and rearrangements of the c-myc locus are found in many medulloblastoma cell lines (Bruggers et al., 1998; Friedman et al., 1988) but are rare in primary tumor biopsies (Badiali et al., 1991; Nozaki et al., 1998; Reardon et al., 2000). It has been suggested that it is easier to establish cell lines from medulloblastomas with over expression of c-myc. Generally, proliferating cells express c-myc and when cells are differentiating, c-myc expression is down regulated (Henriksson and Lüscher, 1996). C-myc is normally expressed in developing cerebellar neurons (Ruppert et al., 1986), and high c-myc mRNA levels are found before birth and during proliferation of cells in the EGL. A better prognosis than average is linked to expression of TrkC (Segal et al., 1994), a tyrosine kinase receptor for neurotrophins. The most common effects of neurotrophins on neurons are to promote differentiation and survival. Neurotrophin 3 (NT-3), for example, acts as a mitogen for cultured neural crest cells (Kalcheim et al., 1992). Medulloblastoma cell lines transfected with TrkA (high affinity receptor for nerve growth factor) respond with massive apoptosis when stimulated with nerve growth factor (Muragaki et al., 1997). The paired box-containing genes (Pax) encode transcription factors, that are expressed in specific brain regions and contribute to pattern formation. Some of these genes are only expressed during development but some are also present in the adult tissue. Pax5 is normally found at the mid-/hindbrain boundary in the embryo. Many desmoplastic medulloblastomas express Pax5 in proliferating cells (Kozmik et al., 1995). From studies in genetically modified mice, it has been suggested that Pax5 is involved in the regulation of neuronal precursor cells (Urbanek et al., 1994). Mice lacking Pax5 have a reduced inferior colliculus and the foliation of the rostral cerebellum is affected. The zinc finger protein, Zic, is expressed in cerebellar granule cells throughout development (Aruga et al., 1994). Mice deficient in Zic1 have a hypoplastic cerebellum with altered foliation (Aruga et al., 1998). In an investigation of human medulloblastomas, 26/29 cases expressed Zic (Yokota et al., 1996) whereas 0/22 other human cancers expressed the protein.

15 Patched and sonic hedgehog in medulloblastomas Most medulloblastomas occur spontaneously, but in rare cases they are seen as part of an inherited syndrome; Nevoid basal cell carcinoma syndrome (NBCCS), also called Basal cell nevus syndrome or Gorlins syndrome (Gorlin, 1987). This syndrome is characterized by developmental defects and a predisposition to a variety of tumors, most frequently basal cell carcinomas. The gene responsible for NBCCS is the human homologue of patched (Hahn et al., 1996; Johnson et al., 1996). Tumor formation seems to take place when there is a constitutive activation of the Shh signalling pathway, the result of overexpression of Shh or Smo or if Ptch is inactivated. The Ptch gene has been disrupted in two independently generated mouse models (Goodrich et al., 1997; Hahn et al., 1998). Ptch -/- mice die at E9 with neural tube closure defects, whereas Ptch +/- mice survive but develop tumors, for example medulloblastomas. No transgenic mice that overexpress Shh or Smo in the cerebellum have been reported. In human medulloblastomas, Ptch mutations have been found (Pietsch et al., 1997; Vorechovsky et al., 1997), whereas mutations in Shh and Smo are rare. Oro and colleagues found a Shh mutation in 1/14 samples (Oro et al., 1997), whereas another investigation failed to detect either Shh or Smo mutations in 37 samples (Zurawel et al., 2000). However, Smo mutations have been found in basal cell carcinomas (Xie et al., 1998). Medulloblastomas also occur as part of Turcot´s syndrome (Hamilton et al., 1995), which is associated with mutations in the adenomatous polyposis coli (APC) gene. APC is part of the Wnt-signaling pathway. Downstream of APC is β-catenin, and mutations in both of these genes are found in a few percent of sporadic medulloblastomas (Huang et al., 2000; Zurawel et al., 1998). It can be concluded that not all medulloblastomas can be explained by mutations in the same genes or even in the same signalling pathway. There are most likely several pathogenetic mechanisms involved in medulloblastoma development. It is open to question whether or not all tumors start from the same cell type.

Other mouse models for PNET/medulloblastomas Mouse models for PNET/medulloblastomas have been generated in multiple ways; xenografts of established human medulloblastoma cell lines, in vivo viral infection of CNS cells, transplantation of tissues transfected with oncogenes, and introduction of a SV40 Tag transgene in mice (Fung and Trojanowski, 1995 and references therein). The oncoprotein encoded by SV40 T inactivates both pRb and p53, two tumor supressors that control cell growth and proliferation. Medulloblastoma-like tumors are also found in transgenic mice that express the early region of the papovavirus JCV (CY) (Krynska et al., 1999). The histopathological differences between different brain tumors are not always clear. In the same way, it might be hard to predict the kind of tumor that will appear in any particular mouse model. Using a Cre-LoxP system, Marino et al wished to establish an astrocytic glioma model by inactivating pRb in GFAP promoter active cells in a p53 null background (Marino et al., 2000). Unexpectedly, the mice displayed medulloblastomas that arose from GFAP promoter active cells in the EGL.

16 PLATELET-DERIVED GROWTH FACTOR (PDGF)

The platelet-derived growth factor (PDGF) was first identified as a mitogen in human serum (Kohler and Lipton, 1974; Ross et al., 1974; Westermark and Wasteson, 1976). It has several biological functions such as cell proliferation, differentiation and chemotaxis, and it is involved in embryonic development (reviewed by Heldin and Westermark, 1999). The PDGF molecule consists of polypeptide chains that form hetero- and homodimers. For a long time the PDGF-A and PDGF-B chains were thought to be the only PDGF chains (Heldin and Westermark, 1999), but recently a novel member (PDGF-C) was characterised (Li et al., 2000). PDGF-C is the only isoform that is secreted in an inactive form. It carries a CUB domain that needs to be released before binding to the PDGFR-α can occur. CUB domains bind to proteins and carbohydrates but its specific role in PDGF-C is not yet known. All PDGFs are coded from different genes and the active form of the proteins is around 30 kDa in size.

PDGFs bind with different affinities to specific tyrosine kinase receptors; PDGFR-α and PDGFR-β (Li et al., 2000; Seifert et al., 1989). The PDGFR-β can only bind PDGF-B whereas the PDGFR-α can bind all three subunits. Binding studies to test whether PDGF-CC binds to PDGFR-αβ have not been performed. Upon ligand binding the receptors dimerize and become autophosphorylated (Heldin and Ostman, 1996; Heldin et al., 1998). Molecules with Src homology 2 (SH2) domains bind to the phosphorylated tyrosines and an intracellular signaling cascade starts. Among the SH2- domain-containing molecules are phosphatidylinositol 3’kinase, phospholipase C-γ and the tyrosine phosphatase SHP-2. Several down-stream, interacting intracellular signal transduction pathways are known, for example the Ras/MAPK pathway.

PDGF-AA PDGF-AB PDGF-CC PDGF-BB

PDGFR−αβ −ββ PDGFR−αα PDGFR

Fig. 4 Schematic drawing of high-affinity interactions between PDGFs and PDGF receptors 17 Biological functions of PDGF Many studies on cellular functions are performed in vitro, and it is not always certain that the effect will also apply in vivo. It is also important to bear in mind that certain PDGF isoforms will affect different cell types in different ways. Culture conditions are also a variable factor. PDGF induces proliferation in many cell types, and the mitogenic response depends on the PDGF isoforms. PDGF-AA is a potent mitogen for O2-A progenitors from rat optic nerve, whereas PDGF-BB is more mitogenic for rat fibroblasts (Pringle et al., 1989). PDGF can also act as a survival factor. After addition of PDGF to O2-A cells the number of surviving cells increased (Barres et al., 1992) and rat pheochromocytoma cells (PC12) are prevented from going into apoptosis when incubated in serum-free medium containing PDGF (Yao and Cooper, 1995). This effect is mediated via phosphatidylinositol-3 kinase. In addition, PDGF added to PC12 cells induces neuronal differentiation and neurite outgrowth, an effect that is mediated through the PDGFR-β (Heasley and Johnson, 1992).

Expression of PDGF and PDGFR in the central nervous system All PDGF receptors and chains are expressed in the CNS. The first detailed information on PDGF-A, -B and PDGFR-β expression in the CNS was reported the same year (Sasahara et al., 1991; Smits et al., 1991; Yeh et al., 1991). Yeh et al. (1991) perfomed in situ hybridisation and immunohistochemistry for PDGF-A on embryonic and adult mice. They found expression in spinal cord neurons as early as E12. At E15, the signal increased and was detected also in dorsal root ganglia and in the brain. In the adult CNS, most neurons were PDGF-A positive, e.g. cerebellar Purkinje cells and pyramidal cells in the hippocampus. Glial cells were also found to express PDGF-A but at lower levels. Hutchins & Jefferson (1992) detected PDGF-A in cells of the ventricular zone in E11.5 mice. The expression was in structures which were similar to neuronal growth cones. Neurons also express PDGF-B. Using immunohistochemistry, Sasahara et al. (1991) detected protein expression in a wide range of neurons in the non-human primate Macaca menestrina. The intensity of the stainings varied between different areas of the brain. High expression was detected e.g. in the hippocampus and in brain stem nuclei. In a following paper, the authors describe expression of PDGF-B in embryonic and adult rat brain (Sasahara et al., 1992). At E14, they found PDGF-B in the subventricular zone of the 3rd ventricle, in the ependyme and in olfactory nerve fibers. Expression in the olfactory bulb was very high at all stages examined. Cerebellar cells were not investigated until E19, by which time both Purkinje cells and granule cells were positive. The expression in all these areas persisted throughout adulthood. PDGF-C expression in brain has only been investigated by in situ hybridization in E9.5-E15.5 mice (Ding et al., 2000). At this stage of development, PDGF-C was detected in early EGL cells in the cerebellar primordium. No other cells in the CNS were reported as positive. Expression of PDGFR-α is generally attributed to glial cells in the CNS. A subpopulation of PDGFR-α positive cells were found in rat ventral spinal cord (E12), cells that were thought to be early oligodendrocyte precursors (Pringle et al., 1992). A similar finding was reported in mice (Yeh et al., 1993). However, PDGFR-α has also been found in neuronal cells. Oumesmar et al. detected the receptor in a variety of

18 neurons, e.g. in the Purkinje cell and granule cell layer of the cerebellum (Oumesmar et al., 1997). The choroid plexus and the meninges also express the α-receptor (Pringle et al., 1992). Immunohistochemical analysis of the PDGFR-β showed expression in many neuronal cell types in the rat CNS (Smits et al., 1991). High expression was seen in pyramidal cells of the hippocampus and on cerebellar granule cells.

PDGF mutant mice All PDGF and PDGF receptor genes (except PDGF-C) have been experimentally disrupted in mice (Bostrom et al., 1996; Leveen et al., 1994; Soriano, 1994; Soriano, 1997). All phenotypes are lethal, which implies an important role for PDGF during development. The PDGF-A null mice go through three critical restriction points. Most mice are growth retarded and die at E10 or at birth. The ones that survive birth lack lung alveolar smooth muscle cells and die of lung emphysema after a few weeks (Bostrom et al., 1996). This is due to a failure in migration and proliferation of PDGFR-α positive smooth muscle cells (Lindahl et al., 1997). Both the PDGF-B (Leveen et al., 1994) and the PDGFR-β mutant mice (Soriano, 1994) die perinatally. They both lack mesangial cells and the kidney glomeruli are not properly formed. The mice also have dilated blood vessels leading to oedemas. As with PDGF-A null mice, cells fail to migrate and proliferate. In the PDGF-B mutant mice, PDGFR-β positive pericytes fail to migrate and follow the sprouting vessels (Lindahl et al., 1997). The PDGFR-α null mice die in utero between E14-E16 with neural tube defects and death of neural crest cells (Soriano, 1997). The Patch mice with a spontaneous pdgfr- α deletion also show collapsed ventricles, reduced metencephalon and abnormal choroid plexus (Stephenson et al., 1991). Only the PDGFR-α null mice display severe CNS defects. However, in PDGF-A deficient mice oligodendrocyte precursors fail to develop (Fruttiger et al., 1999). The lack of more severe phenotypes in the CNS might be due to the redundancy of PDGF receptors and ligands, i.e. each receptor can bind several ligands and each ligand can bind to more than one receptor. Finally, the PDGF-C null phenotype has not yet been described and the possibility of the existance of more than two receptors must still be considered.

Transforming activity of PDGF In 1980, Deinhardt and colleagues showed that the simian sarcoma virus (SSV) could induce glioblastomas when injected in the brain of newborn marmosets (Deinhardt 1980). The amino acid sequence of the transforming gene, p28sis/v-sis, in SSV was soon thereafter shown to be a homologue of the PDGF-B gene (Devare et al., 1983; Doolittle et al., 1983; Waterfield et al., 1983). The transforming activity of v-sis was shown to be mediated via binding to and phosphorylation of the PDGF receptor (Johnsson et al., 1985; Leal et al., 1985). Both PDGF-A and -B have transforming properties and induce foci when transfected into immortalized mouse fibroblasts (NIH 3T3 cells), but the transforming capacity is much higher for PDGF-B (Beckmann et al., 1988). Also, the level of PDGF-B expression determines the capacity of malignant

19 transformation, as shown by growth in soft agar and in nude mice (MacArthur et al., 1992). Cells that express PDGF-B at a high level are more prone to transform. Further evidence for the involvement of PDGF-B in in vivo transformation came from Uhrbom et al., who injected a PDGF-B bearing retrovirus into the brains of newborn mice. These mice developed glioblastomas/PNETs (Uhrbom et al., 1998). The finding of both PDGFs and PDGF receptors in tumor cells led to the hypothesis that v-sis/PDGF driven transformation is mediated via an autocrine stimulatory loop (Betsholtz et al., 1984; Hermansson et al., 1988). Some authors claim that the autocrine activation of the receptors takes place intracellularly (Bejcek et al., 1989; Keating and Williams, 1988), while other evidence suggests that the transforming activity mediated through the receptors can be inhibited at the cell surface (Fleming et al., 1989; Johnsson et al., 1985).

PDGF and PDGFR in human brain tumors Several types of human brain tumors express PDGF and PDGFR, for example astrocytomas (Guha et al., 1995), glioblastomas (Hermanson et al., 1996; Hermansson et al., 1988) and medulloblastomas (Black et al., 1996; Mapstone et al., 1991; Smits et al., 1996). In most of these cases, ligands and receptors are co-expressed in the same tumor, which potentially enables autocrine stimulation. Amplification and/or overexpression of the PDGFRA have been detected in a few malignant gliomas (Fleming et al., 1992). Both overexpression and amplification of the PDGFRA were, however, only seen in one of fifty analyzed tumor samples. In another glioma study, the authors found rearrangement and amplification of the PDGFRA in one single case (Kumabe et al., 1992). Constitutively active receptors were identified in human meningiomas by immunoblotting and immunostaining with an antibody against phosphorylated tyrosine 751 in the PDGFR-β (Shamah et al., 1997). The intriguing question is whether PDGF autocrine stimulatory loops are responsible for the transformed phenotype in these tumors. One piece of evidence that favors this hypothesis is the fact that transfection of dominant negative PDGF into astrocytoma cell lines, reverses the transformed characteristics (Shamah et al., 1993), as does extracellular addition of anti-PDGF antibodies (Vassbotn et al., 1994).

20 METHODS

Transgenic mice and the “knock-in” technique Transgenic mice are genetically modified mice that have received an extra gene or in which a gene has been inactivated. An extra gene can be introduced by pronuclear injection into fertilized eggs or by homologous recombination in embryonic stem cells (ES-cells), the so-called “knock-in” technique. Basically, the result of the two methods is the same, but there are some advantages with the “knock-in” technique. For pronuclear injection, a DNA construct is made in which the cDNA of interest is put under the control of a specific gene promoter. By using tissue specific promotors, one can choose in which tissues the transgene will be expressed. When injected into a fertilized egg, the construct will integrate randomly in the host genome. If the construct is integrated close to an enhancer sequence or nearby a silencer site the expression of the transgene might not reflect the endogenous activity of the chosen promotor. The “knock-in” technique also starts with a DNA construct. The 5' and 3' ends of the construct should contain sequences that are homologous to the endogenous gene. By homologous recombination in embryonic stem cells, the construct will integrate into the host genome and replace the coding part of the endogenous gene. The transgene is then under the control of the endogenous promotor, including all natural silencers and enhancer elements, causing transgene expression in exactly the “right” cells and be regulated like the endogenous gene.

Embryonic stem cells Embryonic stem cells (ES cells) are undifferentiated cells that have the ability to differentiate into any cell type in the body. ES cells are derived from multipotent cells in the inner cell mass of blastocysts i.e. the cells that will develop into the embryo.

embryonic stem cells

culture of blastocyst ES cells Fig. 5 Establishment of embryonic stem cells (ES cells) At this early stage, the embryo is tolerant to manipulation and will develop normally even if a few cells are taken away or if extra cells are added. The culture conditions for ES cells are very strict. It is important to keep the cells undifferentiated, otherwise they can not be used to generate chimeric mice. Rule number one is to prevent the cultured cells from becomming dense. To maintain the ES cells in an undifferentiated stage in culture for a longer time, the cells are grown on top of a monolayer of embryonic fibroblasts (feeder cells) or on gelatinized plates together with leukaemia inhibitory factor (LIF) (Smith et al., 1988; Williams et al., 1988). The exact function of LIF is not

21 known. The first established ES cell line was derived from 129 mice (Evans and Kaufman, 1981). Today, ES cells from 129 mice are frequently used even though there are ES cell lines with other genetic backgrounds.

Replacement vector for gene targeting DNA constructs for “knock-in” purposes must contain sequences that are homologous to the target gene, markers for selection and a unique restriction site for linearization of the construct (Hasty and Bradley, 1993). The length of the homologous sequences are important for proper recombination to occur (Hasty et al., 1991). The gene targeting frequency increases with the length of homology from 1.3 to 6.8 kb. To identify cells where homologous recombination has taken place, a positive selection marker is included. A commonly used marker for positive selection is the bacterial gene for neomycin resistance (neo), which confers resistance to G418. A negative marker is sometimes included to exclude clones where random integration has taken place. The negative marker can be the viral thymidine kinase gene (tk), which confers suspectibility to gancyclovir.

DNA construct transgene

electroporation ES cell

endogenous gene

homologous recombination

transgene

Fig. 6 The transgene is integrated into the host genome through homologous recombination ("knock-in" technique).

Electroporation of ES cells and screening of clones for homologous recombination Before electroporation, the ES cells are grown on gelatinized plates, in order to obtain an ES cell suspension free from fibroblasts and to therefore enhance the uptake of the construct into ES cells. Electroporation with the linearized construct is performed and

22 the cells are seeded in 10-cm diameter plates. After 8-9 days of incubation in selective medium, only resistant colonies remain. These colonies are then picked one by one in 96-well plates, a time during which they are sensitive and need daily care. As soon as possible the cells are split and transfered to several identical plates, which are then either frozen or used in the preparation of DNA. The DNA from all cell clones are subjected to Southern blot analysis to identify clones in which homologous recombination has occured. Positive clones are then thawed and expanded for blastocyst injection.

Blastocyst injection and generation of chimeric mice ES cells, in which homologous recombination has occured, are injected into blastocysts to generate chimeric mice. A chimeric mouse is formed when cells of different genetic constitutions become established in the same mouse. Successfully injected blastocysts are implanted into the uterus of a pseudopregnant fostermother, i.e. a female mouse that was mated with a sterile male. By using ES cells from agouti mice and blastocysts from black mice, the chimeric mice can directly be identified by their fur color. The crucial step when generating a stable transgenic line is whether the ES cells have contributed to development of the germ cells or not. This can be determined by mating the chimera with a wild type mouse and analyzing the genomic DNA from the offspring. If the recombined locus is found in the offspring, then this is confirmation that germline transmission has occured and the chimera can then be used for further matings.

inject ES cells inject blastocysts into into blastocyst pseudopregnant female

Fig. 7 Generation of chimeric mice through injection of recombinant embryonic stem cells into blastocysts and implantation in uterus of chimeric pseudopregnant foster mother. offspring

23 Whole mount β-galactosidase staining The bacterial gene for β-galactosidase (lacZ) is a marker gene that is used to label plasmids or cells into which a gene construct has been integrated. The enzyme, β- galactosidase, has the ability to cleave galactose from X-gal. X-gal (5-bromo-4-chloro- 3-indolyl-β-D-galactoside) is a colorless compound that turns blue after cleavage. To analyze whole areas of lacZ expressing cells in vivo, X-gal staining can be performed on whole mount embryos and tissue that are then cleared in benzyl alcohol/benzyl benzoate (1:2). The blue staining is stable and can be analyzed under a light field microscope. The clearing step is excluded for tissues that are embedded in paraffin.

Fixation of mouse tissue for histological analysis An essential step for histological analysis is the fixation and preservation of the tissue. Fixation stabilizes the tissue in different ways, and the choice of fixative is based on the requirements of the subsequent analysis (Farmilo and Stead, 1989). Formalin and formaldehyde-based fixatives form cross-linking methylene bridges in the tissue. The morphology is well preserved, but many monoclonal antibodies do not work on formalin-fixed and paraffin-embedded tissues. Alcohols fix by coagulation. As no further compounds are added to the tissue, this is an ideal fixation method for many immunohistochemical applications. However, alcohols are poor penetrators and are only suitable for small pieces of tissue. Cryostat sectioning followed by acetone fixation is optimal to preserve immunoreactive epitopes, but the quality of the morphology in cryostat sections is usually compromised.

sagittal section

horizontal section

coronal section

Fig. 8 Orientation of tissue sections through a brain

Immunohistochemistry and immunofluorescence Immunohistochemistry and immunofluorescence are methods by which proteins are visualized in tissues with the use of antibodies. For the indirect method, a primary antibody that recognizes the tissue antigen is used followed by a secondary antibody with affinity to the first one. The secondary antibody is coupled to a detection molecule, e.g. biotin, an enzyme or a fluorescent molecule (immunofluorescence). Avidin has high affinity for biotin, which is utilized in the avidin-biotin complex (ABC) method. The ABC is coupled to an enzyme, for example horseradish peroxidase (HRP). Several ABCs can bind to one biotinylated antibody and thereby enhance the signal. The staining is visualized by adding chromogens, substrates that form colored 24 products after a specific reaction. A commonly used chromogen is 3,3’- diaminobenzidine tetrahydrochloride (DAB), which turns into a brown product when bound to and oxidized by HRP. To enhance the signal further, the DAB reaction can be coupled to an oxidation reaction by glucose oxidase and β-D-glucose in the presence of metal ions, for example nickel ammonium sulfate or cobolt chloride. Metal ions are bound to the staining complex and intensify the signal. Some antigens can be masked if the tissue was fixed in a cross-linking fixative (see above). The antigens can be unmasked, for example, by treatment of the sections in citrate buffer in a microwave oven (Cattoretti et al., 1993) or by limited digestion with trypsin.

25 PRESENT INVESTIGATION

Hypothesis and Aims A few reports had shown that human medulloblastomas express PDGFs and PDGF receptors and we wanted to find out whether this was associated with autocrine stimulation and if it was of importance for tumor formation. Our hypothesis was (1) that there would be PDGF receptor positive cells in the developing cerebellum, (2) that a genetic change would lead to an over-production of PDGF ligand and (3) that this would be a starting point in medulloblastoma formation.

To test the hypothesis we performed the following studies: * Analysis of human medulloblastoma/PNET cell lines with respect to expression of PDGFs, PDGF receptors and the presence of endogenously activated receptors (Paper I).

* Investigation of PDGFR-α expression during embryogenesis in the forming cerebellar anlage and in the developing cerebellum (Paper II).

* Analysis of the effect of forced PDGF-B expression in the developing cerebellum in vivo in mice (Paper III).

In a parallel project, we wanted to characterize the activity of the human GFAP promoter in the embryonic mouse CNS, to determine if the promoter was suitable to target early glial cells in transgenic mice. In order to do this, we:

* characterized the activity of the human GFAP promoter in vivo in GFAP promoter lacZ transgenic mice (Paper IV).

26 Human medulloblastoma cells express different platelet-derived growth factor isoforms and carry endogenously activated receptors (Paper I) The involvement of PDGF in many brain tumors is an established fact and PDGF- induced transformation has been suggested to occur via an autocrine loop (Bejcek et al., 1989; Keating and Williams, 1988). When reports showed that human medulloblastomas express PDGF receptors (Black et al., 1996; Smits et al., 1996) the question arose whether these tumor cells were stimulated by this mechanism. The aim of this work was to reveal whether the PDGF receptors in medulloblastoma cell lines were endogenously activated, and what cellular effects PDGF had on medulloblastoma cells. We also wanted to find out if the presence of PDGF receptors was in some way correlated to the stage of differentiation of the cells. The study was performed on five cell lines; four cell lines derived from medulloblastomas (D283 Med, D324 Med, D341 Med and D425 Med) and one derived from a primitive neuroectodermal tumor (PFSK-1). Three of the cell lines were grown in suspension and two were adherent. Our first goal was to analyze the expression of mRNA for PDGFs and PDGFRs in relation to different cell type specific “markers” using Northern blot analysis. We used probes for PDGFs, PDGF receptors, glial and neuronal “markers”, as well as for different myc genes. Taken together, the results showed that; (1) cell lines with mRNAs for PDGFs and PDGFRs also expressed “neuronal markers” and had normal c-myc levels, whereas (2) cell lines without “neuronal markers” did not express PDGF, but had elevated c-myc levels. We found no expression of N-myc in the medulloblastoma cells, which is consistant with their neuronal differentiation. Expression of N-myc and L-myc was previously shown to influence at commitment of neuroepithelial precursor cells to glial and neuronal differentiation, respectively (Bernard et al., 1992). A very interesting finding was the presence of PDGF-C mRNA in three of the cell lines. The newly identified PDGF-C is normally found in EGL cells in the developing cerebellum (Ding et al., 2000). The question of whether the PDGFRs were endogenously phosphorylated was addressed using an in vitro kinase assay. A constitutively active PDGFR-α was found in the three cell lines with neuronal characteristics. Also the fibroblast cell line (AG 1518) contained endogenously active α-receptors, which is consistant with previous results (Paulsson et al., 1987). No endogenous phosphorylation of the PDGFR-β was detected. However, with the exception of one cell line, all displayed β-receptors that could be phosporylated upon stimulation with PDGF-BB. We also tried to detect PDGFR-α and -β protein by Western blot analysis, but this method was not sensitive enough to detect the receptors in more than one cell line. This indicates that the receptor levels were very low. The amount of endogenously produced PDGF-BB protein was estimated in a DELFIA assay. With this method PDGF-BB levels in cell lysates, but not secreted from the cells, were measured. All medulloblastoma cell lines expressed PDGF-BB protein, but high levels of protein did not correspond to high levels of mRNA in the Northern blot analysis. The highest protein levels were found in D283 Med, a cell line in which we could not detect any PDGF-B mRNA. Similar results were obtained in a study on sarcoma cell lines (Heller et al., 2000). PDGF-AA and -CC protein levels were not measured. The results show that most cell lines express PDGFRs that can be phosphorylated upon

27 stimulation with ligand, and that PDGF-B protein is expressed. To find out what biological effects PDGF might have on medulloblastoma/PNET cells we analyzed known cellular responses to PDGF, such as proliferation, differentiation and migration (Heldin and Westermark, 1999). The mitogenic response was investigated in several growth assays. Cells were sparsely or densely seeded and grown in the absence or presence of PDGF-AA or -BB (0.5 ng/ml to 30 ng/ml). The mitogenic response was estimated in cell counting experiments over three weeks or in 3H-thymidine incorporation assays during incubation for two days. The only effect seen was that the growth of sparsely grown cells was stimulated by the addition of low levels of PDGF- AA and -BB. No effect was recorded on migration of medulloblastoma cell lines. A pilot study on cell motility was performed on gold-coated culture dishes (Albrecht-Buehler, 1977), but we could not detect any difference between PDGF stimulated and untreated cells. However, only randomly migrating cells are scored with this assay and it is possible that the cells could migrate against a PDGF gradient. Differentiation and morphological changes were first investigated by viewing the cells under a phase contrast microscope. No morphological changes were seen, but it is possible that the cells differentiated by changing their protein expression pattern, i.e. a differentiation that did not include any morphological changes. Three cell lines were investigated by immunohistochemistry with antibodies to “neuronal proteins” after PDGF stimulation. Cells were grown for two days in 0.5% FCS with or without addition of PDGF-BB (5 ng/ml). Control cells were also grown in 10% FCS. Adherent cells were seeded on cover slips and cells in suspension were transfered to slides in the cytospin centrifuge before fixation in methacarn (se paper II). We used antibodies against synaptofysin, β-III-tubulin (Tuj1) and the neuronal protein NeuN. When D283 Med was grown in 0.5% and 10% FCS, only a few cells were synaptofysin positive, whereas after addition of PDGF-BB the synaptofysin expression was upregulated. This change was only detected in one of the three cell lines tested and none of the other two proteins were affected. We believe that this change is PDGF-induced differentiation. The ability to differentiate in response to PDGF is probably influenced by the stage of differentiation of the cell line at the time of stimulation and by other characteristics of the tumor cells. Neuronal differentiation in response to PDGF has also been described in other types of neuronally derived tumor cell lines (Heasley and Johnson, 1992; Pahlman et al., 1992). In summary, we show that endogenously activated PDGFRs are present in some medulloblastoma cell lines and that expression of PDGFs, PDGF receptors and “markers” for neuronal differentiation are co-expressed in some of the cell lines while absent in others.

Discussion These results are in line with the hypothesis that medulloblastomas originate from EGL cells, and the presence of PDGF-C was an interesting finding in this respect. We have also shown for the first time that PDGF α-receptors in medulloblastomas are most likely endogenously activated via an autocrine loop. However, the precise role of PDGF in this system is not known. A likely possibility is that PDGF is a marker for the neuronal differentiation stage of the cells, from which the tumor once developed. The

28 fact that high expression of c-myc mRNA did not occur together with PDGF mRNA expression might be due to a block in an early differentiation step. High c-myc mRNA levels are found in proliferating granule cell precursors in the EGL (Ruppert et al., 1986). If the cells are blocked at this stage, they will maintain high c-myc expression levels and they will not start to express PDGF and differentiate into neurons. C-myc amplification is rare in human medulloblastomas but it has been suggested to be more common in medulloblastomas that can be established as cell lines (Wasson et al., 1990). It has also been shown that high c-myc mRNA levels are associated with a bad prognosis in medulloblastoma patients (Herms et al., 2000).

Platelet-derived growth factor receptor-α in ventricular zone cells and in developing neurons (Paper II) Is it possible that medulloblastomas arise from early PDGFR-α positive cells in the cerebellum? To answer that question we needed to know if the α-receptor was present on cells in the developing cerebellum. In this study, we performed immunohistochemistry with a polyclonal anti-human PDGFR-α antibody, R7, (Eriksson et al., 1992) on sections from normal embryonic mouse brain. The antibody was purified from antiserum on a column with activated CH-sepharose coupled to the peptide (TIE, amino acids 1066-1084, Claesson-Welsh et al., 1989), against which the antibody had been raised. We used two different batches of purified antibodies with comparable results. To optimize the immunostaining, several different conditions were tested. The probably most important parameter, was the fixation of tissue. We tested 2% paraformaldehyde, buffered formalin, an alcohol-based fixative from Mercks (Neofix), ethanol and methacarn. Methacarn, a mixture of 60% methanol, 30% chloroform and 10% acetic acid, was the fixative that gave the best results. For immunostaining of intermediate filaments (vimentin), methacarn has previously been shown to work well (Urban and Hewicker-Trautwein, 1994). For best results we microwave-treated the slides 2 times for 10 minutes in citrate buffer. The stain was developed with DAB and Ni-enhancement. The glucose oxidase-enhancement method with nickel is an excellent method by which to visualize nerve fibers and terminals (Shu et al., 1988). Normal mouse embryos from E7.5 to a few days postnatal were dissected and fixed in methacarn. From E16.5 onwards, the brain was dissected out before fixation. Immunohistochemistry with the R7 antibody was performed, usually on sagittal sections, and with special focus on the developing cerebellum. In the early neuroepithelium, PDGFR-α positive cells were already detected by E8.5. Radially distributed cells with the nucleus near the VZ were present in most areas of the CNS. In the area of the cerebellar primordium these PDGFR-α positive cells disappeared from the VZ around E14-15, i.e. the timepoint when the cerebellar primordium thickens and starts to form. We performed immunohistochemistry with nestin, vimentin and Map2 antibodies to characterize the radial cells in the VZ and found overlapping of R7 staining with all the other antibodies. Nestin and vimentin, which are expressed in NSC, overlapped in the radial processes. The neuronal marker, Map2, was mostly found in the peripheral parts of the neuroepithelium, in PDGFR-α positive cells near the pial surface and in dorsal root ganglia.

29 Much of the cerebellar development and settling of cells is a postnatal event in the mouse. Cells migrate to their final positions and new connections are established. From the rhombic lip, EGL cells move medio/rostrally to first cover the whole anlage and thereafter migrate to the final positions in the IGL. Immunohistochemistry was performed at P5, and PDGFR-α was found on the axons of inwardly migrating granule cells. The intensity of the staining differed between different lobes, which is consistant with the fact that not all lobes develop at the same time. To confirm that the PDGFR-α staining was in the granule cells and not in other cells, we used antibodies against nestin, vimentin, GFAP, Map2, synaptofysin, NeuN and calbindin. Vimentin and GFAP label Bergman glia in the cerebellum (Shaw et al., 1981) and calbindin is a specific marker for Purkinje cells. The neuronal marker NeuN is expressed in postmitotic granule cells, and was present in the nuclei of the PDGFR-α positive cells. In adult mice, we detected the PDGFR-α in Purkinje cells. The results from this work show that there are PDGFR-α positive cells in the developing cerebellum, firstly in early neuroepithelial cells of the VZ and later, specifically in migrating EGL cells and in Purkinje cells.

Discussion These results verify our first hypothesis, namely that there are cells in the developing cerebellum that express the PDGFR-α. We show that PDGFR-α is expressed in cells in the early neuroepithelium of the CNS and in dorsal root ganglia, which have the capacity to differentiate into neuronal cells. Several studies have been performed with in situ hybridization to detect PDGFR-α mRNA in the embryonic CNS (Pringle et al., 1992; Yeh et al., 1993). The results have been consistent and show mRNA expression mostly in oligodendrocyte precursors and glial cells, starting around E15, but expression in the neuroepithelium has not been shown. However, it has been shown that a short exposure of PDGF during a limited time period, primes rat neuroepithelial cells to develop into neurons (Williams et al., 1997). Further, nestin positive neuroepithelial cells in culture express functional PDGFR-α (Park et al., 1999) and differentiate into neurons when exposed to PDGF. One possible explanation of the fact that PDGFR-α is easily detected in these cells by immunohistochemistry whereas the mRNA is hard to detect using in situ hybridization, is that the PDGFR-α gene can be regulated differently in different cell types. Expression of PDGF-A has been described in the VZ of the E11.5 mouse, in structures resembling neuronal growth cones (Hutchins and Jefferson, 1992). There is thus a possibility that an autocrine stimulatory loop is acting in the PDGFR-α positive cells at this time point. A further interesting finding is that EGL cells actually express the receptor, since medulloblastomas are believed to arise from these cells.

Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium causes defective midline fusion and cerebellar dysplasia (Paper III) If one is to speculate on the involvement of PDGF in cerebellar tumorigenesis, it is essential to know the role of PDGF in the normal development of the cerebellum. To answer that question, a “knock-in” mouse was created, in which human PDGF-B was expressed under the endogenous En1 promoter. A 700 bp-fragment representing the

30 whole coding region for PDGF-B (Forsberg et al., 1993) was ligated to an internal ribosomal entry-site (IRES) followed by a lacZ reporter gene. IRES is a sequence, to which ribosomes can bind and start translation. This enables protein translation from more than one position in the mRNA molecule, and two different proteins can thus be expressed under the same promoter. In our model, PDGF-B and β-galactosidase proteins were expressed in the same cells. PDGF-B-IRES-lacZ was cloned into a shuttle vector for the En1 3'promoter area (Hanks et al., 1995). Two different elements for selection were present in the shuttle vector; a neo cassette (flanked by loxP sites) for positive selection and an inversely oriented tk for negative selection. The two arms of homology (En1promoter and exon I) are both 3.7 kb and a unique Sal I restriction site for linearization is situated downstream from the second arm. The DNA construct was linearized and electroporated into embryonic stem cells (TBV2, 129sv). Recombinant clones were selected by growth in G418 and Gancyclovir containing medium and identified by Southern blot analysis. The site specific recombination frequency was relatively low. Three positive clones were found out of 710 screened (0.4%). All three clones were injected into blastocysts (C57Bl/6) and nine chimeras were born. After mating with wildtype mice (C57Bl/6), germline transmission was verified in one of the chimeras. The En1 3'promoter shuttle vector has been used in several earlier studies, where it was functional and gave expression in the correct areas. We were confident, therefore, that we could rely on this single chimera.

A -B F Sal 1 G S Z 1 1 E c o n n D e E tk E P IR la n

Fig. 9 The En1-PDGF-B-IRES-lacZ DNA construct. (A) Construct in shuttle vector, Sal 1 is a unique linearization site. (B) Linearized construct (C) Construct integrated into the endogenous En1 gene.

Expression of the transgene was first analyzed with X-gal staining, and the expression pattern was similar to that of endogenous En1 (Davidson et al., 1988; Davis and Joyner, 1988). In E9.5 embryos, β-galactosidase positive cells were present in the neural tube surrounding the mid-/hindbrain boundary, and in the adult mouse blue staining was seen in the inferior colliculi, in midline areas of the cerebellum and in pontine nuclei. We concluded that the integrated transgene was functional.

31 By “knocking-in” the transgene we also “knocked-out” the endogenous En1 gene. Mice that lack both En1 alleles display a mid-/hindbrain deletion and die around birth, whereas loss of one En1 allele gives no known defects (Hanks et al., 1995). Hence, all mice that survive are either heterozygous for the transgene or of wild type. Also, a potential phenotype observed postnatally would be due to a gain of function of the transgene and not due to loss of function of En1. The transgenic mice that were born behaved normally. Brains from different ages were serially sectioned in order to detect a potential phenotype. We first analyzed sagittal sections from adult brains, where the normal cell layering pattern seemed to be replaced by a dysplastic pattern. In coronal sections, the malformed area was centrally located. Immunohistochemistry identified these cells as normal cerebellar cells, i.e. cells that normally constitute the outer layers of the cerebellum. When the brains were sectioned horizontally, it was obvious that the fusion of the cerebellar anlage had not occured properly. The first signs of a malformation were found in sections from E16- 17 brains. At this time point before birth, the normal midline fusion of the cerebellar primordium takes place. In the En1-PDGFB-IRES-lacZ mice, fusion was defective and the EGL cells invaginated in the dorsal midline and settled centrally instead of bridging over the midline. To understand what is happening in the transgene expressing areas, we performed in situ hybridizations with mouse PDGFR-α and PDGFR-β probes. We also used a human PDGF-B probe to trace transgene expression. Transgene expressing cells were present in the cerebellar primordium at the time of fusion. Both Pdgfrα- and Pdgfrβ-expressing cell populations were detected in the primordium at the same time. The results to date indicate that PDGF is important for the normal formation of the cerebellum. We have not found any brain tumors in the transgenic mice, but a larger number of mice need to be followed for a long time before we can exclude that they are prone to tumors.

Future studies The phenotypic analysis of the mice is an ongoing project. More extensive and detailed morphological analyses are needed to fully understand the function of PDGF in cerebellar development. We hope to be able to reveal if the malformation is due to a migration problem, or solely to a fusion defect or, thirdly, if a specific population of cells is missing or over-produced. Cerebellar development is dependent on the early activity of pattern forming genes, which are expressed in a tightly controlled manner around E8-E9 (Simeone, 2000). We do not believe that this early expression of the pattern forming genes is affected, but to exclude the possibility we will perform whole mount in situ hybridizations with probes against the corresponding mRNAs. Extensive analyses on the expression of endogenous PDGF and PDGFR mRNAs will also be performed using in situ hybridization on sections. To draw any conclusion from the transgene expression pattern and the observed phenotype it is important to know exactly how PDGF is normally expressed in this area. It is also important to analyze the expression at the level of protein.

32 Discussion The observed phenotype in the En1-PDGFB-IRES-lacZ mice might be due to a migration problem. Stimulation with PDGF can induce chemotactic activity in different cell types (Siegbahn et al., 1990). Here, it is likely that transgene expressing cells attract PDGFR positive cells of the EGL, which become invaginated when the cerebellum is supposed to fuse. Alternatively, there may a proliferative effect in which the transgene product stimulates PDGFR positive cells to proliferate and thereby inhibit fusion, allowing other cells to migrate in. Midline cerebellar dysplasias are sometimes found in humans. Cell clusters that resemble primitive neuroepithelial cells from the EGL can be found, for example, in the vermis (Yachnis et al., 1994). One type of cell clusters (heterotopias) consists of well-organized cell rests, arranged in a similar pattern to that in the normal cerebellar cortex. Dysplasias like these were found in newborns, especially in children with a trisomy (chromosome 13-15 or 18), and in adults.

A 1.8-kb GFAP promoter fragment is active in specific regions of the embryonic CNS (Paper IV) GFAP has been described as an astrocyte specific protein. We wanted to characterize the in vivo activity of the human GFAP promoter and evaluate the possibility of using the promoter to target early glial cells in transgenic mice. A GFAP-lacZ mouse was created and the expression pattern in the CNS was followed during embryogenesis using whole mount X-gal staining followed by sectioning and complemented by immunohistochemistry on tissue sections. GFAP activity was recorded in cells of apparently unrelated areas of the CNS. The first transgene expression was detected at E9.5 in a limited area of the neuroepithelium and at E10.5 in a subpopulation of cells in the trigeminal ganglia. These areas were well demarcated. The neuroepithelial staining was only present during that single day, whereas the transgene expression in the trigeminal ganglia increased and also included branches of the trigeminal nerve. A typical striated pattern was seen in the neuroepithelium close to the median sulcus at the bottom of the 4th ventricle and in the choroid invagination between the two lateral ventricles at E12.5. The roof of the lateral ventricles also expressed the transgene, and this staining increased day by day until E16.5 when the whole neocortex was intensively stained. Blue staining in thin, radial processes that spanned from the pial surface to the ventriclular surface was observed in coronal sections throughout the cortex at this time point. These cells were radial glial cells. In the area of the 4th ventricle, along the oblongate medulla, two bilateral bands of transgene expressing cells appeared at E12.5. This population of cells increased to also cover the cerebellar anlage, which stained intense blue at E16.5. A different type of staining pattern was seen, for example, in the ventral parts of pons and oblongate medulla and around the chiasma opticum. Here, the staining was localized to scattered, astrocyte-like cells. As the latter type of staining increased, it also included the glia limitans of the CNS, a staining that persisted in the adult mouse. We used immunohistochemistry to identify the transgene expressing cell types. Antibodies against GFAP, nestin and vimentin were used on tissue sections, cell smears and on primary cell cultures from the brain. Immunoreactivity to nestin and

33 vimentin were detected in radial transgene positive cells in the early CNS. Expression of GFAP protein was not detected until E17.5. However, we believe that the transgene expression does reflect endogenous Gfap activity. We performed RT-PCR and showed that Gfap mRNA was present from E10.5 onwards and GFAP mRNA was detected in the transgene expressing neocortex by in situ hybridization. We conclude that the GFAP promoter is a strong promoter that is active in the developing CNS and is therefore well suited to direct transgene expression in mice.

Discussion Recently, two reports showed that the human GFAP promoter is active in radial glial cells in mice and that these cells can differentiate into neurons (Malatesta et al., 2000; Noctor et al., 2001). The GFAP promoter could thus also be used to target neuronal precursor cells. Marino et al. (2000) targeted EGL cells and induced medulloblastomas in GFAP promoter active cells. The only way to really resolve the question if the endogenous Gfap promoter is active at this early stage, is to “knock-in” a reporter gene into the endogenous Gfap gene and follow the expression.

34 GENERAL DISCUSSION

Where do medulloblastomas originate from? Most likely, some are derived from EGL cells. Our results on PDGF-C expression in medulloblastomas and results on PDGFR-α expression on medulloblastoma cells and differentiating EGL cells support this theory, as well as case reports from the literature (Kadin et al., 1970). It does not follow, however, that all medulloblastomas develop from the same cerebellar cell type. The fact that NSCs have the ability to differentiate into multiple cell types, makes it possible that tumor cells, for example, express proteins specific for other cell types than their cell of origin. That would also explain the presence of neurofilament in the medulloblastoma cell lines, when cerebellar granule cells express α-internexin instead of neurofilament (Chien et al., 1996). The findings in this thesis imply that PDGF is involved in cerebellar development. “Knock-out” studies on the different PDGF genes have not shown any specific alterations in the fusing cerebellar anlage. However, the PDGFR-α mutant mouse, a mutation that is fatal in utero at E13-14, displays neural tube defects and a cleft face. It is possible that more CNS defects would appear in those mice if they could live longer. Our results, showing that the PDGFR-α is expressed in the early neuroepithelium and in the postnatal cerebellum, speak in favor of that possibility. Some reports have shown that PDGF is involved in proliferation and differentiation of neuronal cell in the CNS. We show here that over-expression of PDGF-B disturbs the normal fusion of the cerebellar anlage. The biological function that this process represents is still unknown. Migration is one likely possibility, perhaps in combination with both proliferation and differentiation. It is also likely that the effect of PDGF differs with time, developmental process and between different cell types in the developing brain. Expression of the PDGFR-α, for example, was detected on newly differentiated EGL cells migrating inwardly to the IGL. In these cells, PDGFR expression could be a marker for the neuronal phenotype or it could be involved in the migration process. It is also possible that these cells can give rise to medulloblastomas after a series of genetic alterations, but this remains an open question. With respect to tumor formation, more genetic changes than local overexpression of a growth factor are probably needed to generate medulloblastomas.

The work on the GFAP promoter, in combination with other reports (Malatesta et al., 2000; Noctor et al., 2001), shows that this promoter could be used to direct gene expression to radial glia and to cells that can differentiate into neurons. That, in combination with its early and strong transcriptional activity, means that the promoter has the potential to be put to many varied and interesting applications.

35 ACKNOWLEDGEMENTS

This work was carried out at the Department of Genetics and Pathology, Uppsala University, and was supported by grants from the Swedish Cancer Foundation, the Children´s Cancer Foundation of Sweden, the Mary Bevé Foundation, the Erik, Karin and Gösta Selanders Foundation, the James S. McDonnell Foundation and the Gunnar Nilsson Cancer Foundation. I want to express my sincere gratitiude to everyone who has supported me during this time. In particular I want to thank:

My supervisor Monica Nistér, for your enthusiasm and encouraging support, and for never giving up trying to make a scientist out of me.

Bengt Westermark, for suggesting Monica as my supervisor

My co-supervisor Gijs Afink, for fantastic support with everything related to cloning and plasmids, and for always ”being glad he´s a woman....”

Inga Hansson, my mentor in transgenic work and partner in antibodies

Marianne Kastemar, for all medulloblastoma cell experiments, in situ hybridizations and for being the one in the lab who understands the pleasure with cold weather and snow.

Professor Wolfgang Wurst at GSF, Munice, Germany for providing me a place in his lab, and Veronique Blanquet, Susanne Bourrier, Daniela Vogt Weisenhorn and everyone who helped me to establish and evaluate the En1-PDGFB-IRES-lacZ mice.

Erik Forsberg for giving me the opportunity to perform blastocystinjections.

Present and past members of the MN-lab and everyone in the BW/MN/NE/KFN- group for creating such an open and friendly atmosphere to work in.

”Fritidgården Tisteln”, especially Karin Ryd and Anna Dimberg for all coffee breaks, talks about everything but science and for creating such an impossible atmosphere to write in.

All coauthors, for their contribution to this thesis

The computer support William Schannong och Viktor Persson for always having time with pictures that couldn´t be printed, computers that died and for being able to find me new jaz discs, even when there were no ones to find.

My travel companions Lotta and Josefin for persuading me to go shopping in San Diego.

36 The remnants from Biomedicinarlinjen; Helena, Johanna, Cilla, Elisabet and Ulf, for nice Thursday lunches at Farmen.

Uppsala Klätterklubb and all friends who understand the fascination in climbing.

The best of Wijkmanska blecket: Ylva, Synne, Malin, Erika and Klossen for everything.

“Saxbrudarna” Maria, Kicki, Synne and Daniel for the times we actually managed to meet and play.

My parents Eva and Sven, my brothers David and Mattias with family Johanna, Filippa and Pauline. Thanks for always believing in me and for all your support!

Jonas for your love, for being You and for moving to Uppsala - the most flat area of Sweden

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