Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1018 _____________________________ _____________________________ PDGF in Cerebellar Development and Tumorigenesis BY 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 © Johanna Andræ 2001 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