Publications of the National Public Health Institute A 28/2005 Department of Molecular Medicine, National Public Health Institute Helsinki, Finland and Department of Medical Genetics, University of Helsinki Investigations on molecular aspects of Lethal Congenital Contracture Syndrome Niklas Pakkasjärvi Investigations on molecular aspects of Lethal Congenital Contracture Syndrome Niklas Pakkasjärvi Department of Molecular Medicine, National Public Health Institute, and Department of Medical Genetics, University of Helsinki Academic Dissertation To be publicly discussed with the permission of the Medical Faculty of the University of Helsinki, in the large lecture hall at the Haartman Institute, Haartmaninkatu 3, Helsinki, January 20 th , at 12 noon Helsinki 2006 Publications of the National Public Health Institute KTL A28 / 2005 Copyright National Public Health Institute Julkaisija-Utgivare-Publisher Kansanterveyslaitos (KTL) Mannerheimintie 166 00300 Helsinki Puh. vaihde (09) 474 41, telefax (09) 4744 8408 Folkhälsoinstitutet Mannerheimvägen 166 00300 Helsingfors Tel. växel (09) 474 41, telefax (09) 4744 8408 National Public Health Institute Mannerheimintie 166 FIN-00300 Helsinki, Finland Telephone +358 9 474 41, telefax +358 9 4744 8408 ISBN 951-740-598-7 ISSN 0359-3584 ISBN 951-740-599-5 (pdf) ISSN 1458-6290 (pdf) Kannen kuva - cover graphic: Painopaikka Helsinki 2005 Supervised by Academy Professor Leena Peltonen-Palotie Department of Medical Genetics University of Helsinki and Department of Molecular Medicine National Public Health Institute Helsinki, Finland and Docent Marjo Kestilä Department of Molecular Medicine National Public Health Institute Helsinki, Finland Reviewed by Professor Marja-Liisa Savontaus Department of Medical Genetics University of Turku Turku, Finland and Docent Anders Paetau Department of Pathology University of Helsinki Helsinki, Finland Opponent Professor Judith Hall UBC & Children's & Women's Health Centre of BC, Department of Pediatrics, BC's Children's Hospital, Vancouver, BC “Se kasvattaa luonnetta” -Antero Järvinen, s/y Antares, Baltic Sea, 1980s 5 Niklas Pakkasjärvi, Investigations on molecular aspects of Lethal Congenital Contracture Syndrome Publications of the National Public Health Insitute, A28/2005, 90 Pages ISBN 951-740-598-7; 951-740-599-5 (pdf-version) ISSN 0359-3584; 1458-6290 (pdf-version) http://www.ktl.fi/portal/4043 ABSTRACT Arthrogryposis is a clinical description of a phenotype caused by fetal immobility. More than 150 conditions presenting with congenital arthrogryposis are known. Prenatally lethal cases of arthrogryposis present a diagnostic challenge although they are more frequently encountered due to improved ultrasound methods. Post-mortem diagnostics is often hampered by fetal maseration. Therefore, accurate diagnosis is at times only descriptive and prognosis is not always possible to predict. The incidence of anterior horn cell disorders associated with arthrogryposis and early fetal demise is poorly known. Thus, more knowledge about the genetics and epidemiology of these disorders is needed. Importantly, identification of the defective pathways in these disorders would most probably expose critical elements in the normal development of human motoneurons. The Finnish Disease Heritage includes two lethal arthrogryposes. The pathogenetic mechanism of these diseases is still unknown. This thesis sheds light on the molecular mechanisms active during the pathogenesis of Lethal Congenital Contracture Syndrome. LCCS leads to death of the affected fetuses before the 32 nd gestational week. The hallmark of the syndrome is degeneration of the anterior horn of the spinal cord. We localized the defective gene to chromosome 9q34.1 and provided a scaffold to sequence the genomic region. We then continued by the analysis of the positional candidate genes in the critical DNA-region, but until now the LCCS gene and the corresponding mutation remains unknown. To obtain further clues of the character of the LCCS gene and involved pathways, we performed DNA microarray experiments on LCCS spinal cords to unravel the molecular pathways that are deranged during the disease process. We observed changes in central developmental themes, including Sonic Hedgehog in addition to an indication of oligodendrocyte dysfunction. To address the implicated oligodendrocyte dysfunction further, neural precursor cells were harvested from post-mortem LCCS CNS and cultured in vitro. Neural precursor cells from both LCCS patients and age-matched controls were succesfully enriched in culture. The LCCS neural precursor cells appeared denser and assessment of proliferation indicated increased mitotic 6 activity. Transcript analysis provided a molecular explanation to the observed phenomenon. The neural precursor cells of LCCS patients seemed to respond normally to differentiating stimuli of morphogens suggesting their normal initial development and implying problems at later stages of differentiation of the cells of spinal cord. However, distinct differences were observed in the transcript profiles of LCCS patients when compared to controls. Aberrant apoptosis during initial differentiation was not found in LCCS neural precursor cells. LCCS accounts for the majority of prenatally lethal arthrogryposes in Finland. To determine the true number of LCCS cases, we estimated the birth prevalence of LCCS to 1: 25300 births through a register based study during 1987-2002. The intricate developmental network of the spinal cord is subjective to disturbances at multiple occasions. This thesis shows that neural precursor cells can propagate and differentiate in culture, and thus, most developmental checkpoints are passed normally at least in culture in LCCS patients. Most probably, the disease mechanism is active at post-mitotic stages of the spinal cord cells. Keywords: LCCS, Arthrogryposis, Development, Motoneurons, Oligodendrocytes, Stem cells, Epidemiology 7 CONTENTS ABSTRACT................................................................................................................5 ORIGINAL PUBLICATIONS LISTED.................................................................9 ABBREVIATIONS .................................................................................................10 1. INTRODUCTION..............................................................................................12 2. SURVEY OF THE LITERATURE.................................................13 DEVELOPMENT OF THE HUMAN EMBRYO ..................13 The early stages .....................................................................13 Development of the central nervous system..........................14 The identity of motoneurons.................................................17 Glial cell origin......................................................................17 Programmed Cell Death during development........................18 Stem Cells .............................................................................19 Neural stem cells...................................................................21 Regulation of gene expression...............................................24 HUMAN GENETICS..............................................................26 The Human Genome Project ................................................27 Positional cloning to find disease genes.................................28 The Finnish Disease Heritage................................................30 LETHAL ARTHROGRYPOSIS..............................................32 Epidemiology........................................................................33 Pathogenesis .........................................................................34 Lethal Congenital Contracture Syndrome..............................35 Lethal Arthrogryposis with Anterior Horn Cell Disease ........37 Spinal Muscular Atrophy type I.............................................38 MOTONEURON DISEASES .................................................38 3. AIMS OF THE STUDY......................................................................................40 4. MATERIALS & METHODS .............................................................................41 5. RESULTS AND DISCUSSION .........................................................................46 Linkage studies – Search for shared chromosomal regions and assignment of the LCCS locus to chromosome 9q34.1 (Publication I + unpublished)....................................................46 Haplotype mapping to fine map the critical DNA region (Publication I, unpublished) ......................................................47 Physical mapping (Publication I + unpublished)........................48 Candidate genes (Publication I + unpublished) .........................48 8 Revised diagnostic criteria and incidence figures for LCCS (unpublished) ............................................................................51 Transcript analysis (Publication II) ............................................52 Neural precursor cells (Publication III) .....................................57 Synopsis....................................................................................64 6. CONCLUDING REMARKS..............................................................................66 7. ACKNOWLEDGEMENTS................................................................................68 8. REFERENCES ...................................................................................................71 9. ORIGINAL PUBLICATIONS...........................................................................90 9 ORIGINAL PUBLICATIONS LISTED The original publications listed below form the content of this thesis and are referred to in the text by their corresponding Roman numeral. Additional unpublished results are referred to as unpublished. I. Päivi Mäkelä-Bengs, Niklas Järvinen, Katri Vuopala, Anu Suomalainen, Jaakko Ignatius, Mari Sipilä, Riitta Herva, Aarno Palotie, and Leena Peltonen Assignment of the disease locus for lethal congenital contracture syndrome to a restricted region of chromosome 9q34, by genome scan using five affected individuals. Am J Hum Genet. 1998; 63(2):506-16 II. Niklas Pakkasjärvi, Massimiliano Gentile, Juha Saharinen, Jarno Honkanen, Riitta Herva, Leena Peltonen and Marjo Kestilä Indicative oligodendrocyte dysfunction in spinal cords of human fetuses suffering from a lethal motoneuron disease J Neurobiol. 2005; 65(3):269-81 III. Niklas Pakkasjärvi, Laura Kerosuo, Heidi Nousiainen, Massimiliano Gentile, Juha Saharinen, Satu Suhonen, Hannu Sariola, Leena Peltonen, Marjo Kestilä and Kirmo Wartiovaara Neural Precursor Cells from a fatal human motoneuron disease differentiate despite aberrant gene expression Submitted 10 ABBREVIATIONS ALS Amyotrophic Lateral Sclerosis APECED Autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy ARF Cyclin-Dependent Kinase Inhibitor ARL7 ADP-Ribosylation Factor-Like 7 BAC Bacterial Artificial Chromosome BHLH Basic Helix Loop Helix BMP Bone Morphogenetic Protein bp Base Pair BRDU Bromodeoxyuridine C.elegans Caenorhabditis elegans cM Centi Morgan CNS Central Nervous System DCTN1 Dynactin DNA Deoxyribonucleic Acid ds Double Stranded Dynamin-1 Dynamin-1 Gene EDG Endothelial Differantiation Gene EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor ERBB Epidermal Growth Factor Receptor FADS Fetal Akinesia Deformation Sequence FCS Fetal Calf Serum FDH Finnish Disease Heritage FGF Fibroblast Growth Factor FISH Fluorescence In-Situ Hybridisation FMRP Fragile-X Mental Retardation Protein GFAP Glial Fibrillary Acidic Protein GLI Glioma Associated Oncogene Homolog GTPase Guanosine 5´phosphate binding protein HB9 Homeobox Gene HB9 HGP Human Genome Project LIF Leukemia Inhibitory Factor IBD Identical By Descent IGF-1 Insulin-Like Growth Factor I Islet-1 LIM/Homeodomain Transcription factor Isl1 LAAHD Lethal Arthrogryposis with Anterior Horn Cell Disease LCCS Lethal Congenital Contracture Syndrome LD Linkage Disequilibrium Lhx 3 LIM Homeobox Gene 3 Lhx 4 LIM Homeobox Gene 4 LIM Lin11 and Mec3 domain LOD Logarithm of Odds MAB21L MAB21 Cell Fate Specification Gene MHC Myosin Heavy Chain 11 miRNA Micro RNA MMC Motor Column Motoneuron MNR2 MNR2 Homeobox Gene mRNA Messenger RNA NAIP Neuronal Apoptosis Inhibitory Protein NEFH Heavy Neurofilament subunit Nkx2.2 Nk2 Drosophila Homolog of B Transcription Factor NPC Neural Precursor Cell Olig2 Oligodendrocyte Lineage Transcription Factor 2 PAC P1-Artificial Chromosome PAK3 p21-Activated Kinase 3 PAK7 p21-Activated Kinase 7 PAX6 Paired Box Gene 6 Transcription Factor PCD Programmed Cell Death PCR Polymerase Chain Reaction PHACTR2 Phosphatase and Actin Regulator 2 PIPOX L-pipecolate oxidase pMN Motoneuron and Oligodendrocyte Precursor Domain PNS Peripheral Nervous System PRPH Peripherin RBPMS RNA-Binding Protein Gene with Multiple Splicing REST Repressor Element-1 Silencing Transcription Factor RNA Ribonucleic Acid RPCI Roswell-Park Cancer Institute RT-PCR Reverse Transcriptase-Polymerase Chain Reaction SD Standard Deviation SHH Sonic Hedgehog SH3GLB2 Endophilin B2 SMA Spinal Muscular Atrophy SMAD TGFbeta signaling protein 1 SMN Survival Motor Neuron Gene SNP Single Nucleotide Polymorphism SOD1 Cu/Zn Superoxide Dismutase SSCP Single Strand Conformation Polymorphism STS Sequence Tagged Site TGFbeta Transforming Growth Factor beta Family URB1 Steroid Sensitive Gene 1 WIF-1 WNT Inhibitory Factor-1 WNT Wingless-Type MMTV Integration Site Family YAC Yeast Articifial Chromosome ZFHX1B Zinc Finger Homeobox 1B 12 1. INTRODUCTION Development is an intricate process during which cells pass different stages and interact to establish an organism. Disturbances in development lead to congenital diseases. Lethal arthrogryposis results from fetal immobility and is manifested by distinct features consisting mainly of joint contractures and facial anomalies. It can be caused by intrinsic or extrinsic reasons. Lethal Congenital Contracture Syndrome (LCCS) is a lethal arthrogryposis part of the Finnish Disease Heritage. LCCS leads invariably to fetal death before the 32 nd gestational week. The fetuses show the Fetal Akinesia Deformation Sequence (FADS) with severly atrophic muscles. The hallmark lies in the developmental defect of the spinal cord, where degeneration of the anterior horn and descending tracts are observed. The molecular background of LCCS has not been characterized. Here we present investigations on the genetic background of Lethal Congenital Contracture Syndrome, a human disorder with an unknown defect. The characterization of the involved molecular pathways should result in the identification of a critical steps in the development of the spinal cord. 13 2. SURVEY OF THE LITERATURE DEVELOPMENT OF THE HUMAN EMBRYO The early stages Development relies on the transient stages of cells. An embryo is being built from a single cell, that through continuous symmetric and asymmetric cell division slowly gives rise to a remarkable organism (Gilbert, 1997). The embryo absorbs information from both its genes and the surrounding environment during development, establishing symbiosis of genotype and phenotype. Fertilization is initiated by the combination of the genetic material of the sperm and egg giving rise to a new organism, the zygote. In preparation for fertilization, the germ cells undergo meiosis to reduce the number of chromosomes to haploid. The germ cells also differentiate for proper function. Fertilization occurs in the uterine tube, from where the zygote undergoes a series of rapid mitotic divisions generating a blastula while journing to the site of implantation in the uterus (Adams et al., 1956; Egarter, 1990). Cleavage is asynchronous after the two-cell stage with one of the two blastomeres dividing to form a three cell embryo. Subsequently, embryogenesis is established, during which the developing embryo is growing and preparing for the life after birth. As the blastula reaches the 16-cell stage it is called a morula deriving from the latin word for mulberry. This period is followed by a decline of the mitotic rate and a displacement of the cells termed gastrulation starting during the second week. Gastrulation gives rise to the three germ layers, ectoderm, mesoderm and endoderm, situated from the outside progressing inside, respectively (Tam and Beddington, 1987). The embryo develops cephalocaudally meaning that gastrulation continues in caudal segments while cranial structures start to differentiate. 14 Formation of the germ layers is followed by a period relying on active cellular interactions, where tissues and organs are formed. The initiation of gastrulation is a highly sensitive period for extrinsic insult, but the sensitive period does continue during the whole period of organogenesis. After formation of tissues and organs, the embryo is mostly occupied with growth and maturation. These two features will continue well beyond birth and are subjective to surrounding signals. Development of the central nervous system The nervous system is one of the earliest organ systems to differentiate from the blastula in the embryo. The central nervous system (CNS) develops from the ectoderm, giving rise to the various cell types of the mature nervous system. The CNS appears during the 3 rd week as the neural plate, a thickened part of ectoderm located in front of the primitive pit. The lateral edges of the primitive pit elevate, approach and eventually fuse between 18 and 26 days of gestation to form the neural tube (Muller and O'Rahilly, 1987). By the 7 th week, the cephalic portion of the neural tube has processed dilations from which the brain is developed. The caudal neural tube increases in size parallel to the growth of the embryo and differentiates to form the spinal cord (Cowan, 1979; Herschkowitz, 1988). The peripheral nervous system (PNS) develops from the neural crest cells, which have differentiated from the developing neural tube and migrated to specific regions. Migration is paramount as well in establishing the CNS and during the first 20 weeks, when neurons and glial cells migrate from the germinal sites in the subventricular zones and central canal of the spinal cord to their destinations. The ventral midline portion of the neural tube is called the floor plate and it is central in orchestrating the development of the spinal cord. The floor plate cells of the notochord secretes paracrine factors that pattern the spinal cord to distinct domains. 15 These domains further continue to specialize into distinct cell types. Sonic Hedgehog (Shh) is central in the patterning of the early spinal cord (Stamataki et al., 2005). The Shh pathway is relayed by the GLI family of transcription factors (Jessell, 2000). Mutations of Shh in human have been noted as causative of holoprosencephaly (Roessler et al., 1996) and also to impair neural patterning activity (Schell-Apacik et al., 2003). The actions of Shh regulates the expression of a distinct set of homeodomain and basic- helix-loop-helix transcription factors which fall into two categories: class I proteins, that are repressed by Shh and class II proteins that are activated by Shh (Briscoe et al., 2000). These transcription factors promote the development of the pMN domain in the anterior horn of the spinal cord (see figure 1 for specification of the pMN domain by the transcription factors Pax6, Nkx2.2, Olig2 and Irx3), from which both motoneurons and oligodendrocytes develop (Jessell, 2000; Briscoe and Ericson, 2001). Some controversy has been raised on the common origin of motoneurons and oligodendrocytes and it remains unclear whether or not these two cell types share a common progenitor cell (Rowitch et al., 2002; Noble et al., 2004). The formation of both cell types is dependent on the action of Olig2, a bHLH transcription factor first identified in oligodendrocyte development (Zhou et al., 2000; Takebayashi et al., 2002). Olig2 functions in concert with other transcription factors. When Olig2 is coexpressed with Neurogenin2, motoneurons are developed from the pMN domain (Takebayashi et al., 2002). As development proceeds a little further, the expression of Neurogenin2 is downregulated and Olig2 is coexpressed with Nkx2.2, oligodendrocytes are generated from the same pMN domain (Zhou et al., 2001). 16 In addition to hedgehog-related signaling, other components play specific roles during the development of the spinal cord. Wnt signaling is essential for the maintenance of neural progenitor cell proliferation, for controlling the size of the progenitor population, and to influence the decision of progenitor cells to differentiate or proliferate (Zechner et al., 2003). Further, Wnt signaling has been identified as dorsal factors that directly inhibit oligodendrocyte development. The addition of a Wnt antagonist rmFz-8/Fc, increases the number of immature oligodendrocytes in vitro spinal cord explants, demonstrating that endogenous Wnt signaling controls oligodendrocyte development (Shimizu et al., 2005). FGF signaling is involved in motoneuron development by controlling Hox expression (Dasen et al., 2003). Figure 1. The formation of the motoneuron precursor domain and the postmitotic cells determined by the expression of specific transcription factors. P represents for precursor domains, V for postmitotic cells. 17 The identity of motoneurons As motoneurons exit the cell cycle, they acquire columnar subtypes revealed by the position of the cell soma in the spinal cord and by the pattern of peripheral projections (Landmesser, 1978; Tosney et al., 1995). There are five columnar groups of motoneurons; two are found in the median motor column supplying to axial muscles, one set is the pre- ganglionic autonomic motoneurons and the remaining two groups are found within the lateral motor column at limb levels of the spinal cord (Landmesser, 1978; Prasad and Hollyday, 1991). The medial lateral motor column motoneurons supply ventrally derived limb muscles whereas lateral motor column motoneurons project axons to dorsally derived limb muscles (Landmesser, 1978; Tosney et al., 1995). The columnar identity of motoneurons can be distinguished by the expression profile of LIM homeodomain transcription factors. Islet-1 expression is required for the generation of all spinal motoneurons (Pfaff et al., 1996), Lhx 3 and Lhx4 denote medial MMC identity (Sharma et al., 1998). The expression of the homeodomain protein MNR2 persists in motoneurons of the medial MMC, whereas HB9 is expressed more widely in somatic motoneurons. The downregulation of MNR2 and HB9 expression is needed for the generation of preganglionic autonomic motoneurons (William et al., 2003). Glial cell origin Glial cell maturation partly parallels and overlaps neuronal differentiation. The precursor cells maturate in the specific domains and aggregate with neurons to form the specific brain regions. The number of glial cells is regulated such, that the proportion of neurons to glia is maintained at 1:10 (Sommer and Rao, 2002). The two main classes of glial cells, the oligodendrocytes and astrocytes, serve different functions in the nervous 18 system. Astrocytes are more versatile and they can be distinguished by the expression of Glial Fibrillary Acidic Protein (GFAP) (Eng et al., 1971; Bignami et al., 1972). Other glial cell types also exist including radial glia, pituary glia and olfactory ensheathing cells. As the precursor cells in the specific domains differentiate, they generate more restricted precursors that undergo progressive maturation. According to this model at least three types of restricted precursors exist: the neuronal precursors, the glial precursors and the neural crest precursors (Rao, 1999). In reality, additional precursors are likely to exist as several other precursors have also been identified (Rowitch et al., 2002). These include oligodendrocyte and type-2 astrocyte precursors (Raff et al., 1983), glial restricted precursors (Rao et al., 1998), polydendrocytes (Nishiyama et al., 1997), motoneuron-oligodendrocyte precursors (Lu et al., 2002; Sun et al., 2003), astrocyte precursor cells (Liu and Rao, 2004), and white matter precursors (Roy et al., 1999). How these precursors relate to others remains unclear. Multiple developmental pathways may exist, however, revision to current models is needed. Programmed Cell Death during development As motoneurons mature, they start sending axons out to the periphery during the 5 th week (Sariola et al., 2003). When they reach their target muscles, a period of programmed cell death (PCD) ensues, during which superfluous or nonfunctional motoneurons are eliminated. Approximately 50% of the developed motoneurons undergo PCD. Most cell types of the CNS encounter a similar destiny and it has been shown that PCD is an essential component of development (Oppenheim, 1991; Sendtner et al., 2000; Sommer and Rao, 2002). Much of developmental PCD is executed via apoptosis (Vaux and Korsmeyer, 1999). Central components in the 19 apoptosis cascade are the caspase family of proteases (Troy and Salvesen, 2002). The regulation of caspases is complex and is achieved by interactions of pro- and antiapoptotic regulators (Danial and Korsmeyer, 2004). Shh has been implicated as a regulator of apoptosis, where too much or too little Shh expression results in an increase of apoptosis (Oppenheim et al., 1999; Charrier et al., 2001; Paganelli et al., 2001). Survival of motoneurons through the period of PCD requires activity in the form of both afferent input and efferent response, trophic factors and cell interaction between the neurons and adjacent Schwann cells (Hamburger, 1934; Levi-Montalcini and Levi, 1942; Riethmacher et al., 1997). In addition, motoneuron PCD can be experimentally reduced by various factors including steroids (Nakamizo et al., 2000), growth factors (Ang et al., 1992), neurotrophic factors (Zhao et al., 2004) and neuromuscular blocking agents (Oppenheim, 1991). Stem Cells As cells differentiate, they reach a state of full function that is considered irreversible. The next step of differentiation is apoptosis, which eliminates the cell. These cells, if needed, are compensated for by less differentiated cells that are capable of self-renewal and retain the potential to differentiate depending on their specific environments. These cells are called stem cells. In the early embryo, the stem cells are uncommitted and before the blastocyst stage they are totipotent. The potency of cells describes the amount of options the cell has to differentiate. As development proceeds, the fate of the cells becomes somewhat more restricted (see figure 2 for schematics of cell potency as a function of time). According to current knowledge, adult stem cells have a pluripotent capacity and their fate is dependent on regional and developmental 20 contexts. The definition of stem cells is based on their capacity to self renew and differentiate under the appropriate stimuli. It has however been suggested that stemness is a state in the life cycle of the cell (Zipori, 2004). What actually determines the outcome is the environment in which the cell resides. Stem cells are dependent on their niche, which keeps them undifferentiated (Doetsch, 2003). Creating of these niches for stem cells in vitro is a challenge to be solved. Each tissue is constructed from tissue specific stem cells which respond to activating signals when needed. As differentiation proceeds, the number of stem cells is reduced. In regions of ongoing development, small numbers of stem cells are still encountered in the adult. Adult tissues also contain cells that display pluripotent capacity in vitro cultures. Multipotential adult progenitor cells capable of differentiating into mesodermal derivates, ectodermal cells and to endodermal products have been found from bone marrow (Jiang et al., 2002). Further, skin progenitor cells have been shown to differentiate into mesodermal derivatives and neuroectoderm (Toma et al., 2001). Thus, pluripotent stem cells prevail in the adult organism. Embryonic stem cells can in contrast to adult stem cells be amplified for long periods in vitro. An embryonic stem cell stems from the blastocyst and hence can generate all functional adult cell types. Much anticipation Figure 2. Potency of progenitor cells as a function of developmental stage. Straigth line indicates ES cells, curved line adult stem cells. 21 has therefore been laid on tissue replacement therapies for various conditions, including myocardial disorders, diabetes and neurological disorders (Mayhall et al., 2004; Davani et al., 2005). The dark side of stem cells is their close resemblance to cancer cells. Cancer cells are defined by their properties to reproduce beyond normal restraints and to invade regions of other cells. In these terms, stem cells bear close resemblance to cancer cells, and indeed several solid cancer forms have been found to originate from stem cells (Reya et al., 2001; Pardal et al., 2003; Singh et al., 2004). Thus, for therapeutics to be safe, we need to understand how stem cells are controlled before interventions become daily practice. To turn the coin around, by understanding stem cell characteristics, we gain much in the knowledge of cancer cells. Neural stem cells Neural stem cells or neural precursor cells (NPC) contain the blueprint for constructing the individual cells of the CNS (Gage, 2000). The hardwiring of the connections between the network of neural cells is influenced by activity on the other hand. Early studies led to the isolation of progenitor cells with stem cell like capacities from the embryonic CNS and PNS (Reynolds et al., 1992). Thereafter, NPCs have been identified from various regions of the nervous system (Mignone et al., 2004). The isolation of NPCs from adult tissues opened up new avenues for the utilization of these pluripotent cells. Adult NPCs are found to reside in at least the hippocampus and the subventricular zone and in the spinal cord (Emsley et al., 2005). Much hope has been set on NPCs in the therapeutic field, and time will tell how successful they will be (Lindvall et al., 2004). Neural precursor cells have already shown some of their potential in modeling neurological disease (Jakel et al., 2004). Early studies reported the capacity of transdifferentiation of adult stem cells, including neural stem cells, but 22 recent studies have not succeeded in proving these claims (Galli et al., 2000). However, stemness has also been described as a certain state of cells and the state would thus govern what the outcome of the cells is at later stages (Zipori, 2004). The creation of an appropriate niche remains precarious despite recent progress in the field (Shen et al., 2004). Thus, the true nature of stem cells is still warrants further delineation. Neural precursor cells can be isolated directly from embryonic or adult mammalian CNS by selecting cells responsive to mitogens. NPCs enrich in the presence of Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF) (Vescovi et al., 1999). These mitogens keep most of the NPCs undifferentiated. To increase the amount of undifferentiated human NPCs, Leukemia Inhibitory Factor (LIF) can be added to the culture (Wright et al., 2003), but is not obligatory for the enrichment of NPCs. Once the mitogens are withdrawed, the NPCs initiate differentiation. Differentiation can be expedited and instructed by various signals (see figure 3 for instructed differentiation of NPCs). Fetal Calf Serum (FCS) can be used for the generation of neurons and astrocytes from NPCs and IGF-1 instructs them to generate oligodendrocytes (Hsieh et al., 2004). Figure 3. Differentiation of neurosphere cells by instruction of external factors. 23 Differentiation of NPCs isolated from the subventricular zone of postnatal day 7 mice has been studied in detail by transcript analysis, where distinct genetic programs of NPC differentiation were found (Gurok et al., 2004). Interestingly, that study found almost no overlap between genes identified from previous studies performed by different methods using rat derived NPCs (Zhou et al., 2001; Wen et al., 2002). Similar studies using fetal human samples have not been performed. Neural precursor cells have been utilized in the study of Down syndrome, where the gene expression of neuronal target genes was analyzed with neurosphere derived material (Bahn et al., 2002). It was hypothesized that the disease mechanism of Down syndrome is present in the neural stem cells, where the authors describe a set of genes whose mRNA content was extremely low in the case neurospheres. This set of genes included transcription factors, adhesion molecules and synaptic components, all of which are concerned with neuronal maturation in some stage. All of the genes are also under the control of neuron-restrictive silencing factor REST (repressor element-1 silencing transcription factor). To test the validity of these results, the authors differentiated the neural stem cells, where only a diminutive proportion of the Down syndrome derived cells differentiated into neurons as compared to the controls. This study demonstrated the effect of neural stem cells in the investigation of developmental neurologic diseases. Fragile X syndrome has also been studied by the use of neural progenitor cells from an adult male with fragile X syndrome (Schwartz et al., 2005). The fragile X derived progenitor cells were cultured and differentiated into neuronal and astrocytic lineages. The expression of FMRP was reduced in neural cell culture in the patient as compared to the unaffected control, demonstrating the use of neural stem cells in the study of neurologic 24 diseases. Further studies using human neural stem cells are still under way and the true potential of neural stem cells in these are awaiting discovery. Regulation of gene expression Cell and tissue interaction during development relies on signaling. Hormones act over long distances, whereas paracrine factors influence the fate of their vicinity. The coordination of a group of cells changing the behavior of adjacent cells making them change in some way (eg differentiate) is termed induction. The proximate interactors are comprised of a limited set of proteins that the body uses for numerous processes. These proteins can be grouped into four major families based on their structures: (1) the fibroblast growth factor family (FGF)(Wilkie et al., 1995), (2) the Hedgehog family (Ingham and McMahon, 2001), (3) the Wingless (Wnt) family (McMahon et al., 1992), and (4) the transforming growth factor beta superfamily (TGFbeta) (Kingsley, 1994). Tissue interactions have been intensely studied especially during tooth and kidney formation, where epithelial-mesenchymal interaction is central (Thesleff et al., 1989; Sariola et al., 1991). The response of a group of cells depends on its genome. The regulation of gene expression is accomplished on several levels. Genes can be transcribed differentially, nuclear RNAs can be regulated by selection of which get into the cytoplasm and become mRNA, mRNAs can be selected for translation into proteins and proteins can be modificated differentially (Derman et al., 1981; Darnell, 1982). Differential gene transcription can be regulated by transcription factors and regulative elements. Transcription of genes is a highly regulated process controlling the interaction of transcription factors with cis-regulatory elements of DNA 25 and additional co-factors (Kadonaga, 2004). Trans-acting proteins bind cis- regulatory elements and control the rate of transcription of individual genes. Transcription factors have proven to be central during development and it has been estimated that one transcription factor exists for ten genes in human (Levine and Tjian, 2003). Non-coding small endogenous RNAs have been discovered as important regulators of gene expression. The first small non-coding RNAs found were the small temporal RNAs lin-4 and let-7, identified in Caenorhabditis elegans as key regulators of developmental timing (Lee et al., 1993; Reinhart et al., 2000). According to current knowledge microRNA (miRNA) genes constitute about 1-2% of the known genes in human and miRNA genes are known to be important regulators of translation and stability of target mRNAs and it has been estimated that they regulate protein production for 10% of all human genes (John et al., 2004). Neuronal differentiation has been reported to be influenced by small, noncoding double-stranded RNAs where the mechanism appeared to be mediated by a dsRNA/protein interaction (Kuwabara et al., 2004). The involvement of miRNAs have also been implicated in diseases such as Spinal Muscular Atrophy and Fragile X syndrome (Caudy et al., 2002; Ishizuka et al., 2002; Dostie et al., 2003). Regulation of gene expression is a context dependent dynamic process that requires the interaction of several proteins. Transcription factors can both activate and repress the transcription of any given gene determined by the surroundings and its own concentration and physical form (Ma, 2005). The expression of a large number of genes can be studied by microarray methods. RNA microarray studies have proven their validity in global transcript analysis in many different fields from study of 26 pathogenesis to classification of malignancies and diseases (DeRisi et al., 1996; Pomeroy et al., 2002). HUMAN GENETICS The human haploid genome contains 3 billion base pairs of DNA harbored in 22 autosomal and one sex chromosome. DNA is also present in mitochondria in addition to the chromosomes. Variations in DNA may contribute to disease and based on the role of genetic factors the diseases can be roughly divided into categories: (i) monogenic disorders, (ii) multifactorial disorders, (iii) mitochondrial disorders, (iv) chromosomal aberrations and (v) aquired somatic genetic disorders. Our present understanding of genetics lies on the foundations of work by Gregor Mendel, who studied the inheritance of genes in the garden pea, establishing the basic laws of inheritance (Mendel, 1866). Experiments by Oscar Avery showed that a nucleic acid was the chemical basis for specific heritable transformations in bacteria (Avery et al., 1943). Avery’s discovery sparked research into the nature of DNA and almost a decade passed before Francis Crick and James Watson discovered the chemical structure of DNA which by the order of its bases, encodes the genes (Watson and Crick, 1953). Watson and Crick based their proposal of the DNA structure on the work of Rosalind Franklin who had used X-rays to discover that DNA had phosphate groups on the outside and that DNA existed in two forms (Maddox, 2003). 27 The Human Genome Project The Human Genome Project (HGP) was initially launched in 1990 with the goal to obtain the euchromatic sequence of the human genome. The initial approach was biphasic, with mapping of the human genome to provide a scaffold for genome assembly and subsequent sequencing (Bentley et al., 1998; Deloukas et al., 1998). To facilitate and confirm the mapping approach, both human and mouse genomes were mapped and to gain knowledge before the actual sequencing effort, smaller and less complicated genomes were sequenced (Goffeau et al., 1996; Adams et al., 2000; Waterston et al., 2002). While the approaches gained success, the ultimate goal seemed within reach and finally, in February 2001, the International Human Genome Sequencing Consortium and Celera Genomics both provided a draft sequence as a first overall view of the human genome (Lander et al., 2001; Venter et al., 2001). Both reported draft sequences were far from complete and the finished sequence was published three and a half years later (Consortium, 2004). Although this sequence was published as complete, covering 99% of the euchromatic genome, it still contained gaps and requires further complementation. Most of the gaps are associated with segmental duplications requiring novel methods for solving them. The HGP opened the door to a new era in biomedical research, where a collaborative effort generated and characterized systematically a vast domain of biological knowledge. However, as most knowledge paves way for further questions, does the sequence lay several challenges ahead. All genetic polymorphisms need to be systematically identified to enhance their role in the study of diseases. Every functional element, including genes, proteins, regulatory controls and structural element needs systematic characterization and annotation. And finally, the functional 28 modules for genes and proteins need systematic identification. These issues require the comprehensive studies of numerous human genomes, comparative analyses with additional genomes and improved comprehension of expression, localization and interaction of functional modules. Positional cloning to find disease genes Identification of disease genes based on their location in the genome is called positional cloning (Collins, 1992). The approach consists of several steps, starting off with definition of the disease phenotype and collection an appropriate family material, followed by a genome wide scan and the identification of the disease susceptibility locus by linkage methods, advancing to linkage disequilibrium and haplotype mapping to further define the disease locus. The first human gene behind a genetic disease identified by a positional cloning effort was chronic granulomatous disease in 1986 (Royer-Pokora et al., 1986; Royer-Pokora et al., 1986), but positional cloning became a standard procedure only several years later (Collins, 1995). The late 1980s witnessed the evolution of the needed technologies e.g. polymerase chain reaction and the HGP for high throughput positional cloning (Saiki et al., 1985; Mullis et al., 1986; Collins and Galas, 1993). In genetic mapping, loci in the genome are studied in relation to each other. Two genetic loci are linked if they are inherited together more often than expected by chance. Linkage analysis tests two assumptions; two loci are linked with a given recombination fraction or they are not linked. The result of linkage analysis is expressed as a lod score, meaning the logarithm to the 10 base of the ratio of the two assumptions tested in linkage analysis. Linkage disequilibrium denotes the non-random distibution of alleles at linked loci. In linkage disequilibrium, the region adjacent to a disease marker will be overrepresentated by a specific allele. In the disease 29 chromosomes, the length of the chromosomal region in linkage disequilibrium depends on both the age of the mutation and the recombination frequency of the region of interest (Peltonen et al., 1995). Linkage disequilibrium offers shortcuts in the localization of disease genes in consanguineous families and in the search for chromosomal segments in isolated founder populations (Nikali et al., 1995). When the disease locus has been mapped, positional candidate genes are identified from the genomic sequence and positional cloning ends in the identification of the disease associated mutation. Prior to the availability of genomic sequences, an intermediate step of physical mapping was needed to establish a frame for identification of positional candidate genes. The HGP initiated with the mapping of the human and mouse genomes to facilitate the study of inherited diseases in addition to providing the scaffold for sequencing. The building of physical maps in the late 1990s utilized the isolation of artificial chromosome clones in the form of Yeast Artificial Chromosomes (YAC) (Burke et al., 1987), P1-Artificial Chromosomes (PAC) (Ioannou et al., 1994) and Bacterial Artificial Chromosomes (BAC) (Shizuya et al., 1992). The process begun with physically mapping these clones to the desired chromosomal region and thereafter organizing the clones relative to each other. A well utilized technique was Fluorescence In-Situ Hybridization, where the discovery of the use of extended DNA fibers paved way for increased resolution of the physical maps (Heiskanen et al., 1995). The ends of the clones were sequenced, which aided in the positioning of first candidate genes to the critical DNA region. As the HGP accelerated and made progress, preliminary sequences became available for gene prediction. Ab initio gene prediction software were set to high sensitivity and thus initial estimates of gene number in the genome may have been overly optimistic. In addition to gene prediction, the obtained genomic sequences were used for 30 construction of novel polymorphic genetic markers, which then might help in the further restriction of the targeted genetic region. As the physical map of the critical DNA region was finished, positional candidate genes were analyzed for disease associated mutations by way of e.g. motility assays, sequencing and expressional analyses. Nowadays positional cloning could be denoted a positional candidate gene approach, as the regional candidate genes are well listed in the databases and can be directly analyzed based on their position under the linkage peak. At times, the disease gene identification by positional cloning is not as straightforward as expected. Consequences of the mutations in non-coding regions are more difficult to detect than those found in coding regions of the DNA. A good example of such a mutation was the Finnish founder mutation in diastrophic dysplasia, a GT-to-GC transition in the donor splice site of the 5-prime untranslated exon of the disease gene. The mutation acts by severely reducing mRNA levels (Hastbacka et al., 1994; Hastbacka et al., 1999). Duplications and larger inversions may lead to the disruption of a gene or its regulatory element (Chen et al., 2004). Large chromosomal rearrangements are well documented to be associated to monogenic chromosome abnormalities and malignant transformation (Kolomietz et al., 2002). Hence, caution should be exercised when excluding mutations by traditional methods as they may not expose these alternative mechanisms behind the disease. The Finnish Disease Heritage In the light of current knowledge, the ancestors of the modern Finns have arrived both from eastern Ural some 4000 years ago and from a more 31 stable immigration of groups arriving from the South and West during some thousand years (Kittles et al., 1998; Norio, 2003). During centuries, the forefathers inhabited the coast lines, and only during the 16 th century did the migration reach the northern parts of Finland as the Swedish king Gustavus Wasa ordered migration to populate especially the Russian border (Cornell et al., 1966). Internal immigration was also propulsed by the need for new land and the avoidance of taxation by the Crown. The slow migratory movements in a proportionally vast country lead to the emergence of small regional isolates and village societies. Thus, the relatively small gene pool of the ancestors was spread unevenly, and lead finally to the regional clustering of heterozygote disease gene carriers and the incidence of autosomal recessive diseases in their offspring. Random drift was powerful in small founder populations, while some mutations got enriched, the incidence of others declined markedly. In a genetic isolate, one mutation can be assumed behind each disease that has enriched in the population. In other words, the genetic region of a distinct disease is derived most often from one early Finn carrying the gene defect in his or her genome. This leads to a definitive technical advantage in the identification of mutations behind rare diseases using the Finnish genome. The peculiar enrichment of mostly autosomal recessive diseases (carrying one major mutation in the Finnish genome) became known as the Finnish Disease Heritage. Early reports deviated from today’s criteria of the FDH. Poverty and socioeconomic factors leading to inadequate hygienic conditions in prewar Finland in the 19 th and 20 th century were reasons for high prenatal mortality. Thus, diseases leading to early childhood death were not recognized as own entities. Still, diseases that belong to the modern FDH-definition, such as Diastrophic Dysplasia were described (Lahdensuu, 1939). However, some of the diseases thought to have enriched in Finland (Gripenberg, 1953), were in reality prevalent 32 due to non-genetic causes. The first publications containing the FDH steps, the so called Perheentupas steps, appeared in the 1970s (Norio et al., 1973). Examples of success stories where the Finnish genome has been utilized to study the pathomechanism of disease include Polycystic lipomembraneous osteodysplasia with sclerosing leucoencephalopathy (Paloneva et al., 2002; Klunemann et al., 2005) and variant late neuronal ceroid lipofuscinosis (Savukoski et al., 1998; Holmberg et al., 2004). PLO- SL is good model for frontal lobe dementia and is intriguing due to mutations in two genes part of the same signaling cascade. VLINCL on the other hand represents a disease with a fairly young mutation where most families originate from a small village society in southern ostrobotnia. Nowadays the Finnish Disease Heritage is composed of 36 disorders (Norio, 2003). The genetic loci has been established for 34 of these and the gene has been characterized for 33 (www.findis.org). The FDH contains two lethal arthrogryposes, Lethal Congenital Contracture Syndrome (LCCS) and Lethal Arthrogryposis with Anterior Horn Cell Disease (LAAHD), which have not been so far found elsewhere. LETHAL ARTHROGRYPOSIS Arthrogryposis is derived from the Greek words arthron for joint and gryposis for crooked and is used to describe the phenotype of joint contractures. Lethal arthrogryposis is a clinical description of a phenotype of heterogeneous etiology. It represents the end-point of akinesia during pregnancy and can be due to neurological, connective tissue or extrinsic reasons schematically presented in figure 4 (Moessinger, 1983). The 33 descriptive findings consist of distinct facial features, joint contractures and a short umbilical cord. The facial features include an expressionless face, a petite mouth, a small receding chin and lowset poorly lobulated ears. Autopsy reveals pulmonary hypoplasia. At times, the joint contractures are accompanied by pterygia (Hall, 1986). The severity of the features depends on the time of onset of the fetal akinesia during pregnancy (Moerman et al., 1990). Epidemiology Estimations of the frequency of arthrogryposis in different populations have been variable depending on the definition of the condition and the type of study. Multiple congenital contractures (arthrogryposis) has been estimated to occur with a frequency of about 1:3000 live births and about 1 in every 200 newborns is estimated to be born with some form of contractures (Hall, 1997; Hall, 2002). Silberstein and co-workers reported a birth prevalence of 1 in 12000 for arthrogryposis multiplex congenita in Western Australia from a cohort born between 1980-1993 (Silberstein and Kakulas, 1998). Darin and co-workers reported the birth prevalence to be 1 in 5100 in a cohort from western Sweden born between 1979 and 1994 (Darin et al., 2002). However, none of these studies encountered very early onset forms associated with lethality in utero. Figure 4. Development of the Fetal Akinesia Deformation Sequence according to Spranger et al 1982 34 The incidence of anterior horn cell disorders associated with arthrogryposis and early fetal demise is poorly known. In one study from Finland, the incidence of LCCS only was estimated to be 1:12 700-1:19 000 births (Vuopala and Herva, 1994). In another study on LAAHD, a rough estimate of the incidence to be at least 1:50 000 was given (Vuopala et al., 1994). SMN-gene analyses were not done. The figures for all types of anterior horn cell diseases with arthrogryposis are lacking. Pathogenesis The reasons behind intrauterine fetal akinesia can be both intrinsic and extrinsic. The clinical picture remains fairly uniform despite the great number of conditions causing the disorder. Multiple explanations for this exists. Normal joint and adjacent tissue development is dependent on active fetal mobility (Drachman and Coulombre, 1962). As the joints become immobile, extra connective tissue develops around the joint, further limiting joint movement (Hall, 1997). The length of the umbilical cord is dependent on the proportion to the tractile tension which varies according to fetal size and mobility (Katsumata et al., 1991). Both fetal breathing and the volume of amniotic fluid are critical to the development and growth of the lung (Wigglesworth, 1997). The facial features are thought to be caused by disturbed fetal swallowing that accounts for the hypoplastic jaw (Moessinger, 1983). The ear abnormality stems from defective muscle function in the small ear muscle (Zerin et al., 1982). Fetal causes to akinesia can be grouped into anatomical locations: (I) muscular defects, (II) neuronal disorders and (III) connective tissue defects (Spranger et al., 1982; Moerman et al., 1990). The most common 35 cause to fetal akinesia and lethal arthrogryposis is neuronal dysfunction (Banker, 1986; Hageman et al., 1987; Hall, 1997). External causes of arthrogryposis can be divided into: space limitations, intrauterine circulation and maternal disorders (Hall, 1997). Lethal Congenital Contracture Syndrome Lethal Congenital Contracture Syndrome is a fetal motoneuron disease characterized by the Fetal Akinesia Deformation Sequence (Spranger et al., 1982). The fetuses are small for gestational age (<2SD) and present with a typical arthrogryposis phenotype (fig 5 displays a 23 week old LCCS fetus with all phenotypic features) consisting of multiple joint contractures, skeletal muscle atrophy with stick-like extremities, distinct facial features and hydrops (Herva et al., 1985). The facial features consist of ocular hypertelorism, lowset and posteriorly angulated ears and a hypoplastic jaw. Tissue pathology is characterized by severe muscle atrophy, lung hypoplasia and degeneration of the anterior horn of the spinal cord as the hallmark of the syndrome (see fig 6). The CNS is normal above the decussatio pyramidalis as compared to age-matched controls (Herva et al., 1988). The skeletal muscle atrophy is considered neurogenic as embryonic and neonatal myosin heavy chain (MHC) isoforms are expressed, but the intrafusal muscle spindles are lacking. Motor innervation is needed for differentiation of intrafusal fibers (Soukup et al., 1990; Soukup et al., 1993) and the lack of intrafusal fibers support the idea of defective motoneuron innervation in LCCS (Vuopala et al., 1996). Fetal akinesia and hydrops can be observed in early sonography during pregnancy, and LCCS invariably leads to death of the affected fetuses before the 32 nd gestational week. The incidence of LCCS has priorly been estimated to 1:19000 in Finland. LCCS is inherited in the autosomes, recessively. 36 The changes in LCCS spinal cord have been interpreted as degenerative, with the demise of motoneurons partly overlapping the period of natural spinal PCD. Indeed, LCCS is most often initially recognized due to fetal immobility during ultrasound examinations. The reason for prenatal death of LCCS fetuses remains unknown, but may depend on evolutionary factors. Figure 5. LCCS fetus displaying all the characteristic features including distinct facial features, multiple contractures and atrophic muscles. 37 Lethal Arthrogryposis with Anterior Horn Cell Disease Lethal Arthrogryposis with Anterior Horn Cell Disease (LAAHD) shares many similarities to LCCS. The fetuses may survive delivery, but die within one month. The neuropathological findings closely resemble those of LCCS. The locus for the LAAHD gene is currently unknown, but it is also inherited in the autosomes recessively (Vuopala et al., 1996). The incidence of LAAHD is at least be 1:50 000 in Finland (Vuopala et al., 1995). Figure 6. Histological sections of LCCS and age-matched controls of quadriceps femoris muscle and lumbar spinal cord. In the upper row are sections from a 23+4 week LCCS fetus shown, the lower row displays identical sections from the age-matched control. LCCS muscle shows scattered myotubes without clear organization. The spinal cord shows marked reduction at the anterior part with degeneration of the ventral and lateral columns (Pakkasjärvi et al, 2005). 38 Spinal Muscular Atrophy type I The neuropathological findings of LCCS resemble those of type I Spinal Muscular Atrophy (Hoffmann, 1893; Werdnig, 1894; Emery, 1971; Pearn, 1980). SMA type I is the most common autosomal recessive neurodegenerative disorder of childhood. It is at times encountered with artrogryposis. SMA is characterized by degeneration of motoneurons associated with muscle paralysis and atrophy. The most common form of SMA involves defects in the SMN-gene on chromosome 5q, where the severity of the disease depends on the defects in the neighboring NAIP- gene (Lefebvre et al., 1995; Mahadevan et al., 1995; Roy et al., 1995) and is at times associated with fetal arthrogryposis. Deletion of both copies of the SMN genes and the NAIP gene leads to most severe form of SMA designated SMA-0 (Burglen et al., 1996). The SMA-disease spectrum has proven to be heterogenic, and distinct entities can be found in SMA with pontocerebellar hypoplasia, and SMA caused by defects in the IGHMBP2- gene leading to SMA with early diaphragmatic affision called SMARD1 (MIM 604320), which usually is encountered with arthrogryposis (Rudnik- Schoneborn et al., 2003). One x-chromosomal form is also known that presents with arthrogryposis (Greenberg et al., 1988). The incidence of SMA in Finland has been estimated to 1:15 000 (Ignatius, 1992). MOTONEURON DISEASES Amyotrophic Lateral Sclerosis (ALS) is the most common motoneuron disease. The hallmark is the dysfunction and death of neurons in the motor pathways leading to spasticity, hyperreflexia, generalized weakness, muscle atrophy and paralysis (Mulder et al., 1986). ALS is clinically and genetically heterogeneous. Linkage has been found to several genetic loci 39 for familial ALS, which constitute about 5-10% of cases (Hand and Rouleau, 2002). Several predisposing genes have also been identified to the most common sporadic form of ALS. Among the familial cases, some 20% are associated with mutations in the Cu/Zn superoxide dismutase (SOD1) on chromosome 21q22.1 (Rosen et al., 1993). Sporadic cases are occasionally also encountered with novel mutations in SOD1 (Jones et al., 1993). Susceptibility is further associated with deletions or insertions in the gene encoding the heavy neurofilament subunit NEFH (Figlewicz et al., 1994), by deletions in the gene encoding peripherin PRPH (Gros-Louis et al., 2004) or by mutations in the dynactin DCTN1 (Munch et al., 2004). Several other ALS loci have also been identified. The gene mutated in ALS2, resides on chromosome 2q33 (Hadano et al., 2001; Yang et al., 2001), ALS3 on chromosome 18q21 (Hand et al., 2002), juvenile ALS or ALS4 on chromosome 9q34 in relative proximity to the LCCS region (Chen et al., 2004). ALS5 is linked to chromosome 15q15-q21 (Hentati et al., 1998), ALS6 to chromosome 16q12 (Abalkhail et al., 2003; Ruddy et al., 2003), ALS7 to chromosome 20q13.33 (Sapp et al., 2003) and ALS8 to chromosome 20q13.33 (Nishimura et al., 2004; Nishimura et al., 2004). Motoneuron diseases have sometimes been described as systemic diseases with primary manifestation in the spinal cord. What then makes motoneurons so vulnerable to extrinsic noci? Motoneurons are large post- mitotic cells that can extend the axons in length over 1m. They have a high metabolic rate and their cytoskeletons face large demands to sustains stresses encountered (Cookson and Shaw, 1999). Motoneurons possess large amounts of glutamate receptors and they lack calcium binding proteins and they express high levels of SOD1 rendering them vulnerable to oxidative stress (Shaw and Ince, 1997). So in addition to the complex and delicate development of motoneurons, they contain special features which makes them vulnerable to cell death. 40 3. AIMS OF THE STUDY I. Localize the LCCS gene in well characterized Finnish families to a chromosomal region II. Characterize the critical DNA-region and analyze the candidate genes for the disease mutation III. Decipher the molecular pathways disturbed in the pathogenesis of LCCS IV. Study the development of neurons and cells of glial lineage and the role of neural stem cells during pathogenesis of LCCS 41 4. MATERIALS & METHODS All methods and materials applied to this thesis are listed below and sited according to the original publications in which they appear. Ethical considerations All samples were collected after written informed consent from the parents. The project has been approved by the ethical committee of Helsinki University Central Hospital, Hospital District of Helsinki and Uusimaa, and all work has been done in accordance with the Helsinki declaration. Patients Initially, the family material consisted of 15 Finnish LCCS families, a total of 68 individuals, of whom 26 were affected. During the course of the study 13 new cases were included. The diagnosis of patients with LCCS has been confirmed in autopsy and histologic examination of the spinal cord. The material used in each publication is presented in the respective materials section of the corresponding work. The LAAHD family material consisted of 6 Finnish families, a total of 14 individuals, of whom 6 were affected. No new cases were included during the study. All of the LAAHD patient DNA was isolated from paraffin embedded sections. The diagnosis of LAAHD patients has been confirmed in autopsy and histologic examination of the spinal cord. 42 Method Original Publication DNA marker analysis I Linkage analysis I Linkage Disequilibrium Analyses I Fluorescence In Situ Hybridization I DNA Sequencing I RNA Isolation II, III Genechip Processing II, III Gene Expression Data Analysis II, III cDNA Synthesis II Reverse Transcriptase-PCR II, III Real Time Reverse Transcriptase- PCR II Transcription Factor Analysis II Neural Precursor Cell Culture III Cryopreservation III In Vitro Differentiation of Precursor Cells III Immunocytochemistry III Apoptosis assay III Proliferation assay III Methods not described in the individual publications are presented below: Bacterial clone contig The PAC (Pieter de Jong, Roswell Park Cancer Institute, Buffalo, NY) and BAC (Genome Systems Inc.) libraries were screened by PCR using primers for known polymorphisms and STSs in the region. New markers were generated by chromosomal walking. Positive clones were colony purified 43 and grown overnight in TB in the presence of the appropriate antibiotic. DNA was extracted by alkaline lysis. Radiation Hybrid mapping A whole genome radiation hybrid (rh) panel was analyzed (Research Genetics). Markers were amplified by PCR from each rh well. PCR was performed according to standard procedure and products were visualized on 1-2% agarose gels. The screening results for the panel were analyzed by the application of the rh server at stanford human genome center. Computational analyses The genomic sequence for the critical region was analyzed in sections by accessing individual programs Genscan (Burge and Karlin, 1997), Genie (Kulp et al., 1996), MZEF (Zhang, 1997) and Grail (Xu et al., 1994) in addition to the Genotator workbench (Harris, 1997), which represented the initial version of the current Genomebrowser (Karolchik et al., 2003) and Ensembl-platforms (Hubbard et al., 2002). MiRNA prediction was performed by accessing the mirSeeker server (Lai et al., 2003). In addition, sequences were compared using the basic local alignment search tool BLAST (Altschul et al., 1990). Sequences were also aligned locally using the Sequencher software (Gene codes, Inc.). Northern blot analysis PolyA+ RNA was isolated from skin fibroblasts, CNS biopsies, liver biopsies, lung biopsies, kidney biopsies or muscle biopsies of post- mortem LCCS fetuses and compared to RNA from similar tissues of age- matched control fetuses aborted for social reaseons. 5ug of polyA+ RNA was electrophoretically separated in a 0.8% agarose gel in the presence of formaldehyde and blotted onto a nylon membrane. Northern hybridizations were performed at 65C utilizing Express Hyb hybridization 44 solution (Clontech). RT-PCR products were processed to be used as probes according to standard protocols. Epidemiology To attempt a complete ascertainment, we collected information from multiple independent sources to identify all fetuses and infants affected with arthrogryposis in Finland between years 1987-2002. Information was collected from all live births, stillbirths and terminated pregnancies having the diagnosis or cause of death 3350A-3359X and 7558A (International Classificafion of Diseases-9), and G12.0-G12.9 and Q74.3 (ICD-10), respectively, corresponding to the diagnoses of spinal muscular atrophy or anterior horn cell disease or arthrogryposis. The following sources were used: 1) The death certificates of children who died before 12 months of age in Finland during 1987-2002 2) The National Registry for Congenital Malformations in Finland which also receives data on all terminated pregnancies (ref. www.stakes.fi) 3) All seven Departments of clinical genetics in Finland (five university hospitals (Helsinki, Turku, Tampere, Kuopio and Oulu), The Finnish Family Federation (Väestöliitto) and The Folkhälsan Department of Medical Genetics) 4) The five University hospital residential and outpatient registers 5) All those cases that had been analyzed for SMN-gene deletions at the department of medical genetics, University of Turku were listed. SMN- gene testing became available in Finland in 1996 and the laboratory of Turku has been the only diagnostic laboratory for SMA in Finland. In addition, further cases were looked for at the registers of the perinatalogy units as well as at the autopsy and muscle biopsy registers of the departments of paediatric pathology of the five university hospitals. 45 The National Research and Development Centre for Welfare and Health (STAKES) maintains a nationwide register with data on all malformations from live births and stillbirths in Finland that are diagnosed or suspected in children under 12months of age, or in terminated pregnancies. The information on malformations is continually updated nationwide. For all cases found, the clinical, laboratory, autopsy and ultrasonic data were re-evaluated. All cases with death in utero or before 12 months of age were included in the original cohort. Arthrogryposis was defined as joint contractures present in at least two regions of the body as seen at clinical examination or autopsy. For incidence calculations, numbers of livebirths and stillbirths for years 1987-2002 were obtained from the Central Statistical Office of Finland. Birth prevalence was defined as number of cases (live births or stillbirths) per total number of births (including live births and stillbirths). Incidence was defined as total number of cases (live births, stillbirths, intrauterine deaths and terminated pregnancies) per total number of births. Lethal arthrogryposis was defined as arthrogryposis with survival under 12 months of age. This study was approved by the ethical committee of Helsinki University Central Hospital, Hospital District of Helsinki and Uusimaa, and all work has been done in accordance with the Helsinki declaration. Approval for re-evaluation of patient records and analysis of the registrys from STAKES (the National Research and Development Centre for Welfare and Health), and the death certificate records from the Central Statistical Office of Finland was given by the Ministry of Social Affairs and Health. 46 5. RESULTS AND DISCUSSION Linkage studies – Search for shared chromosomal regions and assignment of the LCCS locus to chromosome 9q34.1 (Publication I + unpublished) LCCS shows enrichment in the genetically isolated subpopulation of the late settlement area of Finland (Vuopala and Herva, 1994). Therefore, the disease can be assumed to be caused by a single major mutation and linkage disequilibrium can be utilized in the final positioning of the gene (Hastbacka et al., 1992). Thus, the search for a shared chromosomal segment was used as the method to find the disease locus. We genotyped five affected individuals from two families, but found no evidence for a shared haplotype on any chromosomal region The haplotypes of the two affected siblings from family nr 2 (shown in publication I) used in this study were, however, identical by descent (IBD) for a total of 18 chromosomal regions and the siblings in family nr 3 were IBD for eight regions. (Two alleles are identical by descent when the identical allele is found in an earlier generation without any intervening mutations) Four of the identified IBD-regions were located in the same chromosomal regions. Linkage analysis using dense marker maps on those regions with the complete family material revealed evidence for linkage between LCCS and chromosome 9 markers. The maximum pairwise LOD score of 4.7 was obtained with marker D9S61. Obligatory recombinations restricted the LCCS locus to a 4cM interval between markers D9S1825 and D9S1830. The LAAHD family material proved to be insufficient for linkage studies or exclusion of this chromosomal locus. 47 Haplotype mapping to fine map the critical DNA region (Publication I, unpublished) Population isolates are beneficial for fine mapping of disease loci and the applicability of the haplotype mapping in the process aiming at the restriction of the critical DNA region. Limited numbers of recombinations have occurred in the original founder chromosome rendering the founder haplotype conserved in modern chromosomes. In the case of LCCS, haplotype mapping was utilised to significantly restrict the disease locus. Initial proof of linkage restricted the critical DNA-region to a vast stretch of DNA on chromosome 9q33-34. Extended haplotypes were built in the region to further restrict the critical region. The ancient recombinations observable on the core haplotype defined the critical region between markers D9S904 and D9S61, which was subsequently analysed in details for candidate genes. However, new markers derived from the accumulating sequences produced by the HGP and finer mapping and re- evaluation of the diagnoses eventually lead to the restriction of the LCCS locus to the adjacent region between markers D9S1827 and D9S752 (the positional relations of the critical region markers are depicted in figure 7). Sequencing of candidate genes was expected to yield at least novel polymorphisms to be utilized for haplotype mapping, but thus far, only a small amount of single nucleotide polymorphisms (SNP) were identified from the coding sequences of the analyzed candidate genes. Figure 7. Physical relationships of the critical region markers 48 Physical mapping (Publication I + unpublished) Linkage-based mapping of LCCS was concluded in 1998, during which the genome project was underway. The physical maps for the 9q34 region were non-existent and, thus initially a physical map for the region between markers D9S904 and D9S61 was constructed by PAC and BAC-maps. The clones were ordered mainly by FISH-methods as described previously. For certain markers, radiation hybrid-mapping was applied. The coverage for the PAC-library and a commercial BAC-library was not sufficient for the region and a collaboration with the Sanger Centre was established to obtain clones for the region from the RPCI-11 BAC library (Osoegawa et al., 2001). This collaboration yielded a complete physical map for the critical LCCS region and subsequently also the genomic sequence. The orientation of the individual genomic clones in relation to each other was still confirmed by STS-mapping (sequence tagged site) and a scaffold for candidate gene identification was established. At the time, an identical physical map was constructed by the HGP, but it did not increase the resolution or information content for the LCCS locus. Candidate genes (Publication I + unpublished) Initial positional candidate genes (NGAL, Notch1) were mapped by metaphase-FISH. By the construction of the physical map, further candidate genes were located in the critical region and analysed as candidate genes. As the genomic sequences were finished, a detailed transcript map for the region was obtained and candidate genes (see table 1) were sequenced through systematically to detect the causative mutation for LCCS. The structure of the candidate genes were based on published 49 cDNA or genomic sequences, which are continually updated online. The region contained several interesting positional and functional candidate genes including Dynamin-1. Dynamin-1 was considered the perfect candidate gene as it was functionally in line with the clinical picture of LCCS (van der Bliek et al., 1993). Namely, a temperature sensitive Drosophila mutant of Dynamin-1 designated Shibire showed paralysis at permissive temperatures (Clark et al., 1997). Homozygous mammalian mutations were suspected to be embryonically lethal (van der Bliek, personal communication). Dynamin-1 was involved in the recycling of endocytic vesicles in the synapse, where the role of the proteins is thought to be to cut off the throat of the budding vesicle (Sweitzer and Hinshaw, 1998). Another good regional and functional candidate gene was Syntaxin binding protein 1 (STXBP1), which was localized to the critical region by sequencing the ends of physical clones in the region (Swanson et al., 1998). STXBP1 resided reasonably close to Dynamin-1. It is involved in the localization of synaptic vesicles in the synapse (Verhage et al., 2000). It is similar to Endophilin B2 (SH3GLB2) located more telomeric in the critical region (Pierrat et al., 2001). The mouse model with a defective STXBP1 gene dies at the time of birth, it is paralyzed, but the brain is normally developed (Verhage et al., 2000). Hence, close resemblance to the LCCS phenotype can be observed. The other positional candidate genes did not evidently present as functional candidates, but were sequence analyzed in due order. However, none of the coding sequences of the positional candidates presented with mutations causative for LCCS. To provide further evidence for the exclusion of the positional candidate genes, we verified their expression levels and transcript sizes by reverse transcriptase PCR and/or Northern blotting. RNA from different tissues 50 and patients of different ages were selected to exclude possible developmental stage dependent variations. No differences between patients and controls could be detected in the steady state transcript level of the regional genes. As previously noted, non-coding small endogenous RNAs have been proposed as key regulators of developmental timing. We sought to identify potential miRNA genes in the critical LCCS region by the application of the mirSeeker software (Lai et al., 2003). Multiple putative miRNA clusters were identified, which we subsequently analyzed for mutations. Unfortunately however, no disease associated mutations were revealed by this approach. This does not exclude the role of miRNA genes in the molecular pathogenesis of LCCS, as the identification of miRNA genes is still developing. Further, regulatory regions in the noncoding regions also demand additional studies. Thus, the next task is to analyze the whole critical region genomic DNA. The mutation can be in the regulatory regions of a gene, which can reside relatively far away from the coding region (Kondo and Duboule, 1999). Hence, the identification of the LCCS mutation will provide interesting times for future work. Gene Full name LCN2 Lipocalin2 STXBP1 Syntaxin binding protein 1 ZNF-X Zinc Finger X transcription factor DNM-1 Dynamin-1 KIAA1069 Novel gene CCBL Cysteine Conjugate Beta-Lyase SPTAN brain Spectrin alpha/Fodrin ZYG ZYG-11 Homolog B Endog Endonuclease G VSN Novel gene SH3GLB2 Endophilin B2 Table 1: Full genes excluded by sequencing and expression analysis as causative of LCCS 51 CEECAM cerebral endothelial cell adhesion molecule 1 DOLPP1 dolichyl pyrophosphate phosphatase 1 CRAT carnitine acetyltransferase LRRC8 LEUCINE-RICH REPEAT-CONTAINING PROTEIN 8 PHYHD1 phytanoyl-CoA dioxygenase domain containing 1 IER5L Immediate early response gene 5L PPP2R4 Phosphotyrosyl phosphatase activator 2A Revised diagnostic criteria and incidence figures for LCCS (unpublished) Since no mutations in the critical 9q34.1 region genes were verified, a re- evaluation (clinical features, gestational anamnesis, family history, genetic mapping) of all patients was performed. Combining clinical data with the extended haplotypes identified two fetuses that differed from the majority of patients. These two cases survived beyond the 32 nd gestational week and also restricted the critical DNA region differently from the other patients. When these two cases were omitted from the haplotype mapping, the region could be pinpointed more accurately between markers D9S1827 and D9S752. This process generated new diagnostic criteria for LCCS: (i) total immobility during pregnancy, (ii) death before 32 nd gestational week, (iii) typical verified spinal cord neuropathological findings and (iv) muscle atrophy in addition to the typical phenotype. Special emphasis should be laid on the fetal akinesia and prenatal death, as both excluded cases survived beyond this period and had recorded fetal movements in the 13week ultrasound examination. To clarify the true number of LCCS cases in Finland and to derive revised incidence figures, a register based study was initiated. Information on all fetuses and infants affected with arthrogryposis in Finland between 1987- 2002 were collected from multiple independent sources. During this time 52 period, a total of 214 cases, live births, stillbirths, intrauterine deaths or terminated pregnancies having the diagnosis of arthrogryposis were found. Of these, 74 were found due to arthrogryposis with prenatal death, either as intrauterine death or terminated pregnancy. Among this group, a distinct subset of 39 cases consisted of LCCS fetuses. During 1987-2002, the total number of recorded births was 984 743. Hence, the new incidence for LCCS was estimated at 1: 25 250 births (39 cases to 984743 births) as opposed to the prior estimate of 1:19 000 births. Previously, 8% of affected newborns have suffered death within the first year of life in a Canadian series of 350 cases (Hall, 1985). In our study, the observed frequency of lethal arthrogryposis was markedly higher, presumably due to the enrichment of LCCS and LAAHD in the finnish gene pool. Transcript analysis (Publication II) To determine which genes and underlying molecular mechanisms are disturbed in LCCS, and thus essential for normal development of anterior motoneurons, we compared gene expression profiles of LCCS spinal cords against their respective age-matched controls. By the application of contemporary bioinformatics during data analysis, changes in pathways central in the development of the CNS were observed. Particularly, Olig2 and Nkx2.2, two transcription factors crucial in the development of oligodendrocytes from the pMN domain, were observed to be downregulated in LCCS spinal cord. Further, several myelin related components were heavily downregulated fitting the hypothesis of an oligodendrocyte dysfunction shown in figure 8. 53 Data Analysis to identify deviating transcripts in LCCS spinal cord Global linear scaling was performed on the intensity values from the scanned images of the microarray chips. The normalizing of data for each individual gene to its median value across all probe arrays removed the occurrence of intensity-dependent bias in further analyses. After filtering out data of lesser quality and various data points pertaining to quality control probes, the initial 22 283 transcripts were reduced to 9 286. Within- and between group distribution of signal ratios, as well as their variance, were evaluated through analysis of log-log scatter plots of all possible pair-wise comparisons between probe arrays. This information, giving us an estimate of the variability caused by biological and technical noise, was used to determine cut-off levels to discriminate significant changes from non-significant ones in expression profiles of LCCS samples and controls. The cut-offs were applied individually to each sample- control comparison, thus resulting in three sets of upregulated and three sets of downregulated genes. To further increase stringency, the finalized result lists contained only genes found to share the direction of regulation in all three comparisons. This conservative approach identified a total of 34 genes as being significantly downregulated, the negative expression change between LCCS samples and controls ranging from two up to 15- Figure 8. Hypothesized pathogenesis of myelin downregulation in LCCS where the downregulation of Olig2 and Nkx2.2 leads to an oligodendrocyte dysfunction 54 fold. Only three genes were found to be significantly upregulated, expression levels changing from two up to over 5-fold. Genetic networks The upregulated genes in the spinal cord samples of the LCCS fetuses consisted of only three transcripts: WIF1, MAB21L1 and ARL7. Both WIF1 and MAB21L1 represent crucial genes for early fetal development and ARL7 is involved in cellular traffic. WIF1 is an inhibitor of Wnt signaling (Hsieh et al., 1999). Wnts are secreted molecules assigned to a vast amount of developmental processes, and they have been implicated in pre- and postsynaptic differentiation during Drosophila (Drosophila melanogaster) development (Packard et al., 2002). MAB21L is a homolog of the C. elegans (Caenorhabditis elegans) mab-21 cell fate specification gene (Mariani et al., 1999). Mab-21 has been implicated as a downstream target of TGFbeta (Morita et al., 1999). ARL7 represents a subgroup of the ARF family together with ARL4 and ARL6. The function of these genes is still unclear, and it has been speculated that they may be involved with protein transport between cell organelles (Jacobs et al., 1999). ZHFX1B, which shows a significant downregulation of expression in LCCS spinal cords, can be linked to the same TGFbeta pathway as MAB21L. ZHFX1B is a zinc finger homeobox 1b transcription factor that binds SMAD and represses SMAD-mediated transcriptional activation (Verschueren et al., 1999). SMAD transcription factors are mediators of the TGFbeta-family (Derynck and Zhang, 2003) and activation of SMAD is thought to lead to inhibition of MAB-21 (Morita et al., 1999). ZHFX1B is activated by Churchill, a zinc finger transcriptional activator that switches between different functions of Fibroblast Growth Factor (FGF) signaling (Sheng et al., 2003). ZHFX1B associates with SMAD1 only when the latter is phosphorylated, therefore acting as a putative sensor of Bone Morphogenetic Protein (BMP) activity in the cell (Postigo et al., 2003; 55 Sheng et al., 2003). FGF signaling on the other hand has been shown to be involved in motoneuron development by controlling Hox expression (Dasen et al., 2003). These data suggest links between different developmental pathways operating in the development of human spinal cord. Spinal cord development Motoneurons develop from the pMN domain of the anterior spinal cord under the influence of a set of transcription factors. Especially SHH has been implicated as central in the process of determining the identity of the progenitor cells of the spinal cord. Mutations of SHH in human have been noted as causative of holoprosencephaly (Roessler et al., 1996) and also to impair neural patterning activity (Schell-Apacik et al., 2003). The intensity levels of SHH were below the detection limit on the genechips, and thus we were not able to deduce any changes in expression between the LCCS- patients and controls. However, we could observe changes in the expression in factors linked to the SHH-signaling pathway, especially of a few involved in the specification of the pMN domain. In our experiments, the expression of PAX6 was marginally upregulated, and the expression of OLIG2 shows downregulation in the LCCS spinal cord. NKX2.2 also shows marginal downregulation of expression. PAX6 has been shown to repress the expression of NKX2.2 (Ericson et al., 1997; Jessell, 2000). From the phylogenetic footprinting and transcription factor analysis we identified PAX6 as a putative regulator of the downregulated genes. Furthermore, a member of the TGF� -signaling pathway, MAB21L1, and a factor capable of binding Smad, ZHFX1B, show altered expression in LCCS spinal cord. There is some evidence pointing towards a genetic interaction between mab-21 and mab-18, which is a PAX6 homolog, adding to the complexity of networks (Ericson et al., 1992). We could not see ISL1 expression in the microarray experiments, but at least 56 conventional RT-PCR reveals expression of ISL1 from LCCS spinal cords, thus making it possible that motoneurons are formed in LCCS-patients. We also looked at GLI-signaling to see if there were any significant changes in that signaling pathway, but we could only detect marginal upregulation of GLI2. By summarizing the findings above, we found alterations in several components in the pathways leading to mature motoneurons. These changes seem to be of a relatively high magnitude, since the cellular populations expressing such components define only a subset of the spinal cord and hence the signal may get diluted as we did not specifically dissect subregions of the spinal cord for this analysis. Identifying PAX6 as a regulatory transcription factor To identify common transcriptional motifs and transcription factors resulting in the observed expression patterns, phylogenetic footprinting methods were utilized. Amongst the set of 34 transcripts being downregulated in all LCCS patients, Paired-box gene 6 (PAX6) was found as a transcription factor with statistical significance in both tests compared to the control dataset and being overrepresented in the set of genes downregulated in LCCS. PAX6 plays an important role in central nervous system development and PAX6-null mice exhibit anopthalmia with central nervous system defects and lethality (Kioussi et al., 1999). PAX6 is conserved from invertebrates to vertebrates and functions as a regulator in central developmental processes of several organs (Chi and Epstein, 2002). Transcript analysis was also performed with one LAAHD spinal cord sample. The result was in line with the LCCS samples, indicating close similarities in the disease molecular pathways. However, detailed conclusions are not possible due to the analysis of only one sample. In addition, we looked at the transcript profiles of fetal diaphragmatic muscle from one LCCS patient as compared to an age-matched control. Severe 57 downregulation of myosin heavy chain expression could be observed as well as other findings. These data remain unpublish until additional samples are obtained for analysis. Neural precursor cells (Publication III) Since the global transcript analysis with LCCS fetuses suggested defects in the development of motoneurons and oligodendrocytes (Pakkasjarvi et al., 2005), we addressed the question of differentiation experimentally. We isolated neural precursor cells from fetal central nervous system by growing homogenized biopsies in non-adherent conditions where they form neurospheres and can be cultured in the presence of mitogens (EGF, FGF and LIF) for extended passages. No macroscopic differences between LCCS- and control neurospheres could be observed when the cells were maintained undifferentiated and both cell lines were passaged for extended periods (>P20). However, the LCCS neurospheres appeared to grow slightly denser as compared to the controls. To study the proportion of dividing cells in culture, neural precursor cells were subjected to BrdU to stain for mitotic cells. On average, 7% of LCCS NPCs stained positive for BrdU-incorporation, whereas only 3% of control NPCs were positive, indicating an increase in proliferation in LCCS NPCs. A neurosphere stained with anti-BrdU to detect mitotic cells is shown in figure 9, where the amount of dividing cells can be visually observed. To determine the molecular background of the observed increase in mitotic activity, the expression profiles of LCCS undifferentiated neural Figure 9. BrdU positive cells (red) in neurosphere 58 precursor cells were compared to their respective controls. This conservative approach identified a total of 49 genes as being significantly upregulated, the expression change between LCCS samples and controls ranging from two up to 27-fold. 63 genes were found to be significantly downregulated, the negative expression levels changing from two up to over 15-fold. The lists of significantly up-/down-regulated genes were examined for biologically relevant associations using the Web-based Gene Set Analysis Toolkit available at http://genereg.ornl.gov/webgestalt/. Annotation information defining the biological processes to which each gene could be ascribed to was retrieved from the classifications provided by the gene ontology (GO) consortium (Ashburner et al., 2000), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta. Statistical evaluation of enrichment of categories represented in each gene list, compared to the proportion observed in the total population of genes on the probe array, was performed using the hypergeometric test and p-values less than 0.01 were considered significant. This analysis indicated the Epidermal Growth Factor Receptor processing as being enriched in the upregulated undifferentiated NPC samples. The EGFR is a tyrosine kinase and signaling involves small GTPases of the Rho family (Wong and Guillaud, 2004). The oncogene ERBB is implicated to derive from EGFR and mutations in EGFR have been associated with small-cell lung cancer (Downward et al., 1984; Kobayashi et al., 1995). EGF is essential to the maintenance of neural precursor cells and EGFR levels are known to determine progenitor cell proliferation and differentiation (Lillien, 1995). These findings support the observed proliferative activity of LCCS NPCs. However, further experiments are warranted before definitive conclusions, as the biology of NPCs in neurosphereculture is still not completely understood. The in vitro setting for the creation of the appropriate niche remain precarious despite recent progress in the field (Shen et al., 2004). It is known that more than one 59 kind of precursor cells are present in the nervous system and that the different precursor cells exist at different times during development (Rao, 2004), which may limit our study with the human samples. Further, not only true precursor cells display self-renewal capacity (Trentin et al., 2004). Still, the transcriptome analysis revealed the molecules associated with proliferative activity and supports the notion that EGF-responsive cells are present at later stages of development (Svendsen et al., 1996). Indeed, a derangement of developmental timing may be critical for the survival of motoneurons (Hausmanowa-Petrusewicz and Vrbova, 2005) and the observed proliferation change may cause developmental delays that render the LCCS motoneurons vulnerable at later stages. To determine the potential to differentiate, the neural precursor cells were analysed during withdrawal of mitogens. Both LCCS and control derived neural precursor cells differentiated into neurons and cells of glial lineage. We stained cells for neuronal (neuronal class III � -tubulin), astrocyte (GFAP) and motoneuron (Islet-1, Hb9) markers as the cells were challenged by fetal calf serum addition after mitogen withdrawal. No macroscopic differences were observed between LCCS patients and controls in the number or appearance of the differentiated cells. Wu et al. have previously shown that by priming NPCs, many will aquire a cholinergic phenotype upon grafting into spinal cord (Wu et al., 2002). Gao et al. also showed how this approach was succesfully used for innervation of peripheral muscle with primed NPCs in rats suffering from motoneuron degeneration (Gao et al., 2005). In our hands, priming of neural precursor cells in vitro did not increase the percentages of cells positive for motoneuron markers. To address the question of oligodendrocyte development, the precursor cells were supplied with IGF- 1 and stained after differentiation for the oligodendrocyte marker O4. Again, no differences between patients and controls could be observed. 60 Figure 10 shows oligodendrocyte marker positive cells from differentiated LCCS NPCs. To further study the differentiation of NPCs into motoneurons, the cells were stained with choline acetyltransferase (Chat), a marker found in mature cholinergic motoneurons. We could not identify truly Chat-positive cells in our cultures. It is possible that our culture conditions do not favor the final maturation of motoneurons into cholinergic cells in the absence of further environmental clues. However, we are confident that our experiments show clear induction of differentiation and immunocytochemical evidence of cell fates that are affected in LCCS. To investigate whether excessive apoptosis occurs during the differentiation of LCCS derived neurospheres, we differentiated both LCCS and control neurospheres and measured apoptosis by fluorescence- activated cell sorting. Apoptosis was studied by detecting the translocation of phosphatidylserine to the extracellular side of the cytoplasmic membrane, an early indicator of apoptosis (Vermes et al., 1995). Necrotic cells were detected by DNA staining with propidium iodide to distinguish apoptotic and necrotic cells. Of the LCCS patients, 25% (n=5, SD=6.3) of the differentiated cells were apoptotic, whereas the number for controls was 17% (n=3, SD=6.8). Although the mean number of apoptotic cells was higher in LCCS derived progenitors, statistically significant differences could not be detected (student t-test p-value 0,29). The proportion of Figure 10. Oligodendrocyte O4- marker positive cells 61 necrotic cells was also similar in LCCS and control cultures (19%, SD=5.2 and 18%, SD=4.6, student t-test p-value 0,53). An exaggeration of naturally occuring PCD of motoneurons has been proposed as a part of the pathogenesis of spinal muscular atrophy (Fidzianska and Rafalowska, 2002; Soler-Botija et al., 2002). The pathogenesis behind the lack of anterior horn tissue in LCCS has remained unknown and speculations on an inability to produce the appropriate cell types or whether excessive PCD eliminates cells after differentiation have occured. We compared PCD during initial differentiation of NPCs, but statistically significant differences in PCD could not be observed. To further address the question of motoneuronal cell death, we would have to enrich for motoneurons and then pursue the apoptosis assays. To address any molecular disturbances during differentiation of neural precursor cells, RNA was collected from neurospheres and differentiated precursor cells from both LCCS-patients and the controls. The RNA was utilized in transcriptome analyses with commercial microarray chips. Aberrant gene expression in LCCS patients could be detected. Combining the gene lists of abberantly expressed transcripts in both neurospheres and differentiated cells yielded a set of 8 genes putatively active during initiation of the disease process. The transcripts that were upregulated in both conditions consisted of: (1) RBPMS, coding for an RNA binding protein; (2) URB, a steroid sensitive gene (Aoki et al., 2002); (3) GLT25D2, a gene containing a glycosyltransferase domain; (4) XM_371647, a transcript with a putative glycosyltransferase domain; (5) PIPOX, a gene with oxidase activity (Dodt et al., 2000). The downregulated transcipts common to both conditions consisted of: (1) PHACTR2 and (2) PHACTR3, two genes with phosphatase and actin regulator activity (Allen et al., 2004); and (3) PAK7, a brain specific gene encoding for a protein with serine/threonine kinase activity (Waterston et 62 al., 2002). Another member of the PAK-family was also observed to be downregulated in the undifferentiated NPCs, namely PAK3. In addition, EDG4, a lysophosphatidic acid G-protein-coupled receptor was observed to be downregulated. Lysophosphatidic acid has been recognized as an activator of the Rho family of GTPases, which use PAK7 as a downstream effector. In this study, we isolated and cultured NPCs from fetal LCCS brains. We demonstrate that despite the genetic defect leading to the absence of motoneurons in the patients, the NPCs retain the potential to differentiate into distinct cell types of the CNS. We also provide evidence that after differentiation the cells do not undergo excessive apoptosis. The gene expression changes support and explain the observation of an increased growth rate of NPCs. Thus, the disease may be due to a non-cell autonomous mechanism at the stem cell level meaning defective niche circumstances. It may as well be due to defects in interaction of cells at later stages. We also isolated neural precursor cells from the spinal cord of one LCCS fetus successfully. The growth kinetics of spinal cord derived NPCs are however too slow for further analysis with current in vitro niche conditions and thus, any true conclusions cannot be drawn based on these experiments. It is possible that the pool of spinal cord precursor cells is small and the culture conditions are not optimal for the necessary propagation of these cells to enable similar studies as was done for the brain NPCs. Of interest to note is, that priorly it has been unclear whether cells of spinal cord origin can be generated from brain derived neural precursor cells and only a small number of publications providing definitive evidence 63 are currently found (Wu et al., 2002; Gao et al., 2005). We show that cells expressing motoneuron markers can be differentiated from the human neural precursor cells in vitro. Their functional capacity awaits analysis. These CNS NPCs do not represent a developmentally relevant pool of motoneuron precursors, however, the results obtained provide further evidence to previous studies with CNS NPCs and motoneurons. 64 Synopsis The incidence of anterior horn cell disorders associated with arthrogryposis and early fetal demise is poorly known. Although obviously rare, early onset lethal arthrogryposes constitute a significant problem to the affected families due to the risk of recurrence. They are a diagnostic challenge to obstetricians performing ultrasonographic investigations as well as to geneticists giving genetic counseling. Thus, more knowledge about the genetics and epidemiology of these disorders are needed. Importantly, identification of the defective pathways in these disorders would most probably expose critical elements in the normal development of human motoneurons. This thesis describes the localization of the LCCS disease gene to chromosome 9q34.1 by linkage mapping. Despite high hopes and intense investigations, the true molecular background of LCCS remains unknown. The physical mapping of the region advanced paralleled to the progress of the Human Genome Project and provided at later stages novel information to the sequencing effort. Candidate genes were localized to the region, several of which had not previously been assigned to a specific genomic region. Sequencing of these genes witnessed the evolution of Sangers dideoxy chain termination reactions by manual propagation to the era of automated sequencers. However, none of the obvious candidate genes contained disease causing mutations, neither did their expression levels indicate any association to LCCS. To try an alternative approach, microRNA genes were predicted from the critical 9q34.1 region, but none of the identified microRNAgenes were mutated in LCCS. It may well be that the mutation lies in a regulatory element in 9q34.1, but the gene regulated is further away from this region. Therefore, interesting times are ahead once the gene defect is verified. 65 As another alternative avenue to identify involved genes and pathways and to gain further insight into the molecular pathways that are disturbed during the pathogenesis of LCCS, a global trancript analysis was performed. RNA from fresh LCCS spinal cord and age matched controls was isolated and analyzed with commercial microarray methods. An indicative oligodendrocyte dysfunction was recognized with specific changes in the expression of distinct transcription factors central in the development of the spinal cord. In detail, Olig2 and Nkx2.2 showed downregulation in expression in LCCS; two transcription factors of importance during both motoneuron and oligodendrocyte development. Further, downregulation in components of the oligodendrocyte product myelin were observed. The microarray study provided a still shot of a distinct phase in the pathogenesis of LCCS and many questions evolved. To analyze the differentiation of neurons and cells of glial origin during the LCCS pathogenesis further, neural precursor cells were harvested from post-mortem LCCS CNS. In vitro studies showed an increase in the proliferative activity of LCCS NPCs and the neurospheres also grew denser as compared to the controls. When differentiated, no morphological differences between patients and controls could be observed, and both lines readily differentiated into neurons, astrocytes, motoneuron-like cells and oligodendrocytes upon instruction. However, molecular changes could already be observed in microarray analyses from differentiating NPC cells. The true impact of these changes claim extensive further studies, but provide a glimpse into the molecular details of progenitor cell differentiation. 66 6. CONCLUDING REMARKS More than 150 conditions presenting with arthrogryposis multiplex congenita are known (Hall, 1985; Hall, 2002). Most of these conditions, however, can accurately be diagnosed only after birth and in living patients. Prenatally lethal cases of arthrogryposis still remain a challenge although they are more and more frequently encountered due to improved ultrasound methods. Post-mortem diagnostics is often hampered by fetal maseration as a function of intrauterine retention time. Therefore, accurate diagnosis is at times only descriptive and prognosis is not always possible to predict. When lethal arthrogryposis is suspected in ultrasound due to immobility, a regular and intense follow-up is recommended. The estimated birth prevalence of arthrogryposis varies greatly depending on the definition of the condition and the type of study. Multiple congenital contractures (arthrogryposis) has been estimated to occur with a frequency of about 1:3000 live births and about 1 in every 200 newborns is estimated to be born with some form of contractures (Hall, 1997; Hall, 2002). In this study, we estimated the birth prevalence of LCCS to 1: 25300 births through a register based study. The epidemiology of early onset lethal arthrogryposis is poorly known, but unpublished results indicate a relatively high frequency of the perinatally lethal cases which usually have been omitted in previous studies. This puts emphasis on the burden caused by these entities for the parents. Clearly more studies of the very early onset and prenatally diagnosed cases are needed to enable proper genetic counseling and prenatal diagnosis. This thesis work has used of different approaches towards the understanding of disease mechanisms. The first publication shows how linkage studies have been carried out using only a few individuals. 67 Previously unpublished results present the inherent difficulties in identifiying disease affecting mutations. The second publication utilizes global transcript analysis and contemporary bioinformatics in deciphering clues to the molecular pathways deranged during the disease process. In the third publication human neural precursor cells have been utilized in disease mechanisms. It provides an innovative approach to study a developmental neurologic disease and shows that cells expressing motoneuron markers can be generated directly from neural precursor cells in vitro. The intricate developmental network is subjective to disturbances at multiple occasions. This thesis shows that most of the developmental checkpoints of the spinal cord are passed normally at least in culture in LCCS patients. Most probably, the disease mechanism is active at post- mitotic stages of the spinal cord cells. The revelation of the genetic defect ensures interesting times ahead. 68 7. ACKNOWLEDGEMENTS During the years taken to complete this work, many have contributed, both knowingly and unknowingly. Of all people deserving to be acknowledged, only a few are mentioned here. This research was carried out in the Department of Molecular Medicine at the National Public Health Institute in Helsinki, Department of Human Genetics, University of California, Los Angeles, CA and the Developmental Biology laboratory, Institute of Biomedicine, University of Helsinki. I thank the former head of the institute, Professor Jussi Huttunen and the current director, Pekka Puska and professor Hannu Sariola for providing the excellent research facilities. This work has been financially supported by grants from Finska Läkaresällskapet, Suomen Lääketietenseura Duodecim, Biomedicum säätiö, Oskar Öflund-säätiö, and Stiftelsen för Dorothea Olivia, Karl Walter och Jarl Walter Perkléns minne. I have been privileged to be dually supervised by two distinguished scientists. My deepest appreciation goes to my invaluable mentor and colleague, Leena Peltonen- Palotie for guiding me through the obstacles of genetics and life as a scientist. I am grateful to Marjo Kestilä, who with diligence and patience made this a reality. I emphasize patience, something Marjo embodies, especially during the many desperate moments during this project. I am genuinely grateful to Riitta Herva, whose efforts have been of monumental proportions, requiring countless hours of tedious work in characterizing LCCS. I especially want to acknowledge her for sharing the insights into a fascinating disease. I am deeply indebted to Jaakko Ignatius for his extensive contributions in bringing this research to completion and adding the presence of a true academic medical mind. My thesis committee members Johanna Hästbacka and Anders Paetau deserve due credit for all their time and effort during this work. Anders is also thanked for the dual role of both thesis committee member and reviewer of this thesis. 69 Marja-Liisa Savontaus is deeply thanked for all the critical and improving comments provided to complete this thesis during the review process. My appreciation is also expressed to Kirmo Wartiovaara for leading me into developmental biology and stem cells. Nothing works in the modern world without data analysis and thorough biocomputational skills, something I have not been blessed with in the same proportions as my co-authors and friends Massimiliano Gentile and Juha Saharinen. Massi is also thanked for showing me all the trails to be explored in my spare time. Hanna Mikkola was the one waking my desire for scientific pursuit in setting an example by herself of how things should be done in research. Aarno Palotie is acknowledged for the initial discussions that sparked life into my scientific career. I thank Päivi Mäkelä-Bengs for, among other things, showing that working hours mean anything between 0-24. No time is too early or too late for work. Heidi Nousiainen is thanked for continuing on with the important task of sequencing what has not already been done. At times it can be hard, but you will be rewarded. Ville Holmbergs and Juha Palonevas presence merit my grateful thanks in making this a reality. At times, when the cozy athmosphere in the lab doesn’t feel that warm, you need friends to hang in there. Both of you provided that friendship needed. Ville is also thanked for sharing the first steps into the real clinics, experiences I treasure forever. Juha I also thank for taking care of the Jeep after we parted LA. It has been a genuine pleasure to work with the entire department, both present and past members. Their dedication to excellence is gratefully acknowledged. To mention only a few, Jenni, Mira, Henna, Jyrki, Jesper, Tuomas, Johanna, Markus, 70 Tero H., Jenny Anna, Tintti, Kaisu L, Nora, Anniina, Jussi, Pekka, Annika, Heli, Jonna, Jani, Maria, Petra, Päivi, Heidi L., Ilona, Teppo, Will, Joni, Jouni, Aino, Janna, Nabil, Tero Y., Mikko, Anu W., Anu J., Iski, Tuula, Lennu, Elli, Jarno, Harry, Chris, Ludy and Mark are especially thanked for their presence. I especially want to acknowledge Ritva Timonen for the always helpful and interested attitude. My appreciation is also expressed to the good people in the Sariola/Wartiovaara lab including Laura Kerosuo-Pahlberg, Katja Piltti, Nina Perälä and Satu Kuure. The whole HBGS with Tomi Mäkelä, Aija Kaitera and co-workers are thanked for making HBGS a true scientific family. I also want to thank Tomi for all the relentless struggle in favor for all the Ph.D-students. Without your help, this thesis would not have happened now. I am genuinely grateful to all my friends for showing me the good times off duty. Sebastian “Basse” Godenhjelm, Anders Karlsson and Roger Sittnikow fulfill the core group of friends I’ve had for as long as I can remember. PKU is also acknowledged. Preparing a thesis is a complex task, as one attempts to balance studying, working hours, lab&desktime with spare time (which usually does not exist). Many family members have helped in taking care of my family, when I’ve been busy. For this I thank Leena “Mumu” and Vesa “Muha” Pakarinen, Nelli and Sami Jerkku and Päivi and Tuomo Pakarinen in addition to my own family. My relatives deserve their part in acknowledgements as you are nobody without your family. My father, Antero Järvinen deserves my deepest appreciation for teaching me what perseverance is all about and for leading me into intellectual pursuits. All those adventures at sea with you and Riitta mean the world to me. My grandfather Klaus Järvinen was always there when you needed him. The example he set leads me to strive beyond what I can achieve. Together with Salme, their home was the safe harbor with answers to any question thinkable. My mother and Ingemar are of course responsible for the millions of pancakes my stomach has desired during my life ad hoc. Lara, my sister shows what it means to truly care for your compatriots. Lastly, for making everything worthwhile I thank Leila my wife and Felix, my son. Words are not enough to express my love and gratitude to my own family, without whom nothing matters. 71 8. REFERENCES Abalkhail H, Mitchell J, Habgood J, Orrell R, de Belleroche J. 2003. A new familial amyotrophic lateral sclerosis locus on chromosome 16q12.1-16q12.2. Am J Hum Genet 73:383-389. Adams EC, Hertig AT, Rock J. 1956. A description of 34 human ova within the first 17 days of development. Am J Anat 98:435-493. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, George RA, Lewis SE, Richards S, Ashburner M, Henderson SN, Sutton GG, Wortman JR, Yandell MD, Zhang Q, Chen LX, Brandon RC, Rogers YH, Blazej RG, Champe M, Pfeiffer BD, Wan KH, Doyle C, Baxter EG, Helt G, Nelson CR, Gabor GL, Abril JF, Agbayani A, An HJ, Andrews-Pfannkoch C, Baldwin D, Ballew RM, Basu A, Baxendale J, Bayraktaroglu L, Beasley EM, Beeson KY, Benos PV, Berman BP, Bhandari D, Bolshakov S, Borkova D, Botchan MR, Bouck J, Brokstein P, Brottier P, Burtis KC, Busam DA, Butler H, Cadieu E, Center A, Chandra I, Cherry JM, Cawley S, Dahlke C, Davenport LB, Davies P, de Pablos B, Delcher A, Deng Z, Mays AD, Dew I, Dietz SM, Dodson K, Doup LE, Downes M, Dugan-Rocha S, Dunkov BC, Dunn P, Durbin KJ, Evangelista CC, Ferraz C, Ferriera S, Fleischmann W, Fosler C, Gabrielian AE, Garg NS, Gelbart WM, Glasser K, Glodek A, Gong F, Gorrell JH, Gu Z, Guan P, Harris M, Harris NL, Harvey D, Heiman TJ, Hernandez JR, Houck J, Hostin D, Houston KA, Howland TJ, Wei MH, Ibegwam C et al. 2000. The genome sequence of Drosophila melanogaster. Science 287:2185-2195. Allen PB, Greenfield AT, Svenningsson P, Haspeslagh DC, Greengard P. 2004. Phactrs 1-4: A family of protein phosphatase 1 and actin regulatory proteins. Proc Natl Acad Sci U S A 101:7187-7192. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403-410. Ang LC, Bhaumick B, Munoz DG, Sass J, Juurlink BH. 1992. Effects of astrocytes, insulin and insulin-like growth factor I on the survival of motoneurons in vitro. J Neurol Sci 109:168-172. Aoki K, Sun YJ, Aoki S, Wada K, Wada E. 2002. Cloning, expression, and mapping of a gene that is upregulated in adipose tissue of mice deficient in bombesin receptor subtype-3. Biochem Biophys Res Commun 290:1282-1288. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel- Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. 2000. Gene ontology: tool 72 for the unification of biology. The Gene Ontology Consortium. Nat Genet 25:25-29. Avery OT, MacLeod CM, McCarty M. 1943. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. The Journal of Experimental Medicine 79:137-159. Bahn S, Mimmack M, Ryan M, Caldwell MA, Jauniaux E, Starkey M, Svendsen CN, Emson P. 2002. Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study. Lancet 359:310-315. Banker BQ. 1986. Arthrogryposis multiplex congenita: spectrum of pathologic changes. Hum Pathol 17:656-672. Bentley DR, Pruitt KD, Deloukas P, Schuler GD, Ostell J. 1998. Coordination of human genome sequencing via a consensus framework map. Trends Genet 14:381-384. Bignami A, Eng LF, Dahl D, Uyeda CT. 1972. Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res 43:429-435. Briscoe J, Ericson J. 2001. Specification of neuronal fates in the ventral neural tube. Curr Opin Neurobiol 11:43-49. Briscoe J, Pierani A, Jessell TM, Ericson J. 2000. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101:435-445. Burge C, Karlin S. 1997. Prediction of complete gene structures in human genomic DNA. J Mol Biol 268:78-94. Burglen L, Amiel J, Viollet L, Lefebvre S, Burlet P, Clermont O, Raclin V, Landrieu P, Verloes A, Munnich A, Melki J. 1996. Survival motor neuron gene deletion in the arthrogryposis multiplex congenita- spinal muscular atrophy association. J Clin Invest 98:1130-1132. Burke DT, Carle GF, Olson MV. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806-812. Caudy AA, Myers M, Hannon GJ, Hammond SM. 2002. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev 16:2491-2496. Charrier JB, Lapointe F, Le Douarin NM, Teillet MA. 2001. Anti- apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis. Development 128:4011-4020. Chen DC, Saarela J, Clark RA, Miettinen T, Chi A, Eichler EE, Peltonen L, Palotie A. 2004. Segmental duplications flank the multiple sclerosis locus on chromosome 17q. Genome Res 14:1483-1492. Chen YZ, Bennett CL, Huynh HM, Blair IP, Puls I, Irobi J, Dierick I, Abel A, Kennerson ML, Rabin BA, Nicholson GA, Auer-Grumbach M, Wagner K, De Jonghe P, Griffin JW, Fischbeck KH, Timmerman V, Cornblath DR, Chance PF. 2004. DNA/RNA 73 helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am J Hum Genet 74:1128-1135. Chi N, Epstein JA. 2002. Getting your Pax straight: Pax proteins in development and disease. Trends Genet 18:41-47. Clark SG, Shurland DL, Meyerowitz EM, Bargmann CI, van der Bliek AM. 1997. A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans. Proc Natl Acad Sci U S A 94:10438-10443. Collins F, Galas D. 1993. A new five-year plan for the U.S. Human Genome Project. Science 262:43-46. Collins FS. 1992. Positional cloning: let's not call it reverse anymore. Nat Genet 1:3-6. Collins FS. 1995. Positional cloning moves from perditional to traditional. Nat Genet 9:347-350. Consortium IHGS. 2004. Finishing the euchromatic sequence of the human genome. Nature 431:931-945. Cookson MR, Shaw PJ. 1999. Oxidative stress and motor neurone disease. Brain Pathol 9:165-186. Cornell J, Carlsson S, Rosén J. 1966. Den Svenska Historien - Vasatiden 1520-1611. Albert Bonniers Förlag, Stockholm. Cowan WM. 1979. The development of the brain. Sci Am 241:113-133. Danial NN, Korsmeyer SJ. 2004. Cell death: critical control points. Cell 116:205-219. Darin N, Kimber E, Kroksmark AK, Tulinius M. 2002. Multiple congenital contractures: birth prevalence, etiology, and outcome. J Pediatr 140:61-67. Darnell JE, Jr. 1982. Variety in the level of gene control in eukaryotic cells. Nature 297:365-371. Dasen JS, Liu JP, Jessell TM. 2003. Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature 425:926-933. Davani S, Deschaseaux F, Chalmers D, Tiberghien P, Kantelip JP. 2005. Can stem cells mend a broken heart? Cardiovasc Res 65:305-316. Deloukas P, Schuler GD, Gyapay G, Beasley EM, Soderlund C, Rodriguez-Tome P, Hui L, Matise TC, McKusick KB, Beckmann JS, Bentolila S, Bihoreau M, Birren BB, Browne J, Butler A, Castle AB, Chiannilkulchai N, Clee C, Day PJ, Dehejia A, Dibling T, Drouot N, Duprat S, Fizames C, Fox S, Gelling S, Green L, Harrison P, Hocking R, Holloway E, Hunt S, Keil S, Lijnzaad P, Louis-Dit-Sully C, Ma J, Mendis A, Miller J, Morissette J, Muselet D, Nusbaum HC, Peck A, Rozen S, Simon D, Slonim DK, Staples R, Stein LD, Stewart EA, Suchard MA, Thangarajah T, Vega- Czarny N, Webber C, Wu X, Hudson J, Auffray C, Nomura N, Sikela JM, Polymeropoulos MH, James MR, Lander ES, Hudson TJ, Myers RM, Cox DR, Weissenbach J, Boguski MS, Bentley DR. 74 1998. A physical map of 30,000 human genes. Science 282:744- 746. DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM. 1996. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet 14:457-460. Derman E, Krauter K, Walling L, Weinberger C, Ray M, Darnell JE, Jr. 1981. Transcriptional control in the production of liver-specific mRNAs. Cell 23:731-739. Derynck R, Zhang YE. 2003. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425:577-584. Dodt G, Kim DG, Reimann SA, Reuber BE, McCabe K, Gould SJ, Mihalik SJ. 2000. L-Pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases. Biochem J 345 Pt 3:487-494. Doetsch F. 2003. A niche for adult neural stem cells. Curr Opin Genet Dev 13:543-550. Dostie J, Mourelatos Z, Yang M, Sharma A, Dreyfuss G. 2003. Numerous microRNPs in neuronal cells containing novel microRNAs. Rna 9:180-186. Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J, Waterfield MD. 1984. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 307:521-527. Drachman DB, Coulombre AJ. 1962. Experimental clubfoot and arthrogryposis multiplex congenita. Lancet 2:523-526. Egarter C. 1990. The complex nature of egg transport through the oviduct. Am J Obstet Gynecol 163:687-688. Emery AE. 1971. The nosology of the spinal muscular atrophies. J Med Genet 8:481-495. Emsley JG, Mitchell BD, Kempermann G, Macklis JD. 2005. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol 75:321-341. Eng LF, Vanderhaeghen JJ, Bignami A, Gerstl B. 1971. An acidic protein isolated from fibrous astrocytes. Brain Res 28:351-354. Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, van Heyningen V, Jessell TM, Briscoe J. 1997. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90:169-180. Ericson J, Thor S, Edlund T, Jessell TM, Yamada T. 1992. Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256:1555-1560. Fidzianska A, Rafalowska J. 2002. Motoneuron death in normal and spinal muscular atrophy-affected human fetuses. Acta Neuropathol (Berl) 104:363-368. 75 Figlewicz DA, Krizus A, Martinoli MG, Meininger V, Dib M, Rouleau GA, Julien JP. 1994. Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis. Hum Mol Genet 3:1757-1761. Gage FH. 2000. Mammalian neural stem cells. Science 287:1433-1438. Galli R, Borello U, Gritti A, Minasi MG, Bjornson C, Coletta M, Mora M, De Angelis MG, Fiocco R, Cossu G, Vescovi AL. 2000. Skeletal myogenic potential of human and mouse neural stem cells. Nat Neurosci 3:986-991. Gao J, Coggeshall RE, Tarasenko YI, Wu P. 2005. Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats. Neuroscience 131:257-262. Gilbert S. 1997. Developmental Biology. Sinauer Associates. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG. 1996. Life with 6000 genes. Science 274:546, 563-547. Greenberg F, Fenolio KR, Hejtmancik JF, Armstrong D, Willis JK, Shapira E, Huntington HW, Haun RL. 1988. X-linked infantile spinal muscular atrophy. Am J Dis Child 142:217-219. Gripenberg U. 1953. [Diseases of significance in the study of genetics.]. Suom Laakaril 8:705-713. Gros-Louis F, Lariviere R, Gowing G, Laurent S, Camu W, Bouchard JP, Meininger V, Rouleau GA, Julien JP. 2004. A frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis. J Biol Chem 279:45951-45956. Gurok U, Steinhoff C, Lipkowitz B, Ropers HH, Scharff C, Nuber UA. 2004. Gene expression changes in the course of neural progenitor cell differentiation. J Neurosci 24:5982-6002. Hadano S, Hand CK, Osuga H, Yanagisawa Y, Otomo A, Devon RS, Miyamoto N, Showguchi-Miyata J, Okada Y, Singaraja R, Figlewicz DA, Kwiatkowski T, Hosler BA, Sagie T, Skaug J, Nasir J, Brown RH, Jr., Scherer SW, Rouleau GA, Hayden MR, Ikeda JE. 2001. A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat Genet 29:166-173. Hageman G, Willemse J, van Ketel BA, Verdonck AF. 1987. The pathogenesis of fetal hypokinesia. A neurological study of 75 cases of congenital contractures with emphasis on cerebral lesions. Neuropediatrics 18:22-33. Hall JG. 1985. Genetic aspects of arthrogryposis. Clin Orthop Relat Res:44-53. Hall JG. 1986. Analysis of Pena Shokeir phenotype. Am J Med Genet 25:99-117. 76 Hall JG. 1997. Arthrogryposis multiplex congenita: etiology, genetics, classification, diagnostic approach, and general aspects. J Pediatr Orthop B 6:159-166. Hall JG. 2002. Emery and Rimoin's Principles and Practice of Medical Genetics - Arthrogryposes (multiple congenital contractures). London: Churchill Livingstone. 4182-4235. Hamburger V. 1934. The effects of wing bud extirpation on the development of the central nervous system in chick embryos. J. Exp. Zool. 68:449 - 494. Hand CK, Khoris J, Salachas F, Gros-Louis F, Lopes AA, Mayeux-Portas V, Brewer CG, Brown RH, Jr., Meininger V, Camu W, Rouleau GA. 2002. A novel locus for familial amyotrophic lateral sclerosis, on chromosome 18q. Am J Hum Genet 70:251-256. Hand CK, Rouleau GA. 2002. Familial amyotrophic lateral sclerosis. Muscle Nerve 25:135-159. Harris NL. 1997. Genotator: a workbench for sequence annotation. Genome Res 7:754-762. Hastbacka J, de la Chapelle A, Kaitila I, Sistonen P, Weaver A, Lander E. 1992. Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland. Nat Genet 2:204- 211. Hastbacka J, de la Chapelle A, Mahtani MM, Clines G, Reeve-Daly MP, Daly M, Hamilton BA, Kusumi K, Trivedi B, Weaver A, et al. 1994. The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping. Cell 78:1073-1087. Hastbacka J, Kerrebrock A, Mokkala K, Clines G, Lovett M, Kaitila I, de la Chapelle A, Lander ES. 1999. Identification of the Finnish founder mutation for diastrophic dysplasia (DTD). Eur J Hum Genet 7:664-670. Hausmanowa-Petrusewicz I, Vrbova G. 2005. Spinal muscular atrophy: a delayed development hypothesis. Neuroreport 16:657-661. Heiskanen M, Hellsten E, Kallioniemi OP, Makela TP, Alitalo K, Peltonen L, Palotie A. 1995. Visual mapping by fiber-FISH. Genomics 30:31-36. Hentati A, Ouahchi K, Pericak-Vance MA, Nijhawan D, Ahmad A, Yang Y, Rimmler J, Hung W, Schlotter B, Ahmed A, Ben Hamida M, Hentati F, Siddique T. 1998. Linkage of a commoner form of recessive amyotrophic lateral sclerosis to chromosome 15q15-q22 markers. Neurogenetics 2:55-60. Herschkowitz N. 1988. Brain development in the fetus, neonate and infant. Biol Neonate 54:1-19. Herva R, Conradi NG, Kalimo H, Leisti J, Sourander P. 1988. A syndrome of multiple congenital contractures: neuropathological analysis on five fetal cases. Am J Med Genet 29:67-76. 77 Herva R, Leisti J, Kirkinen P, Seppanen U. 1985. A lethal autosomal recessive syndrome of multiple congenital contractures. Am J Med Genet 20:431-439. Hoffmann J. 1893. Über chronische spinale Muskelatrophie im Kindesalter, auf familiärer Basis. Dtsch Z Nervenheilk:427-470. Holmberg V, Jalanko A, Isosomppi J, Fabritius AL, Peltonen L, Kopra O. 2004. The mouse ortholog of the neuronal ceroid lipofuscinosis CLN5 gene encodes a soluble lysosomal glycoprotein expressed in the developing brain. Neurobiol Dis 16:29-40. Hsieh J, Aimone JB, Kaspar BK, Kuwabara T, Nakashima K, Gage FH. 2004. IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes. J Cell Biol 164:111-122. Hsieh JC, Kodjabachian L, Rebbert ML, Rattner A, Smallwood PM, Samos CH, Nusse R, Dawid IB, Nathans J. 1999. A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature 398:431-436. Hubbard T, Barker D, Birney E, Cameron G, Chen Y, Clark L, Cox T, Cuff J, Curwen V, Down T, Durbin R, Eyras E, Gilbert J, Hammond M, Huminiecki L, Kasprzyk A, Lehvaslaiho H, Lijnzaad P, Melsopp C, Mongin E, Pettett R, Pocock M, Potter S, Rust A, Schmidt E, Searle S, Slater G, Smith J, Spooner W, Stabenau A, Stalker J, Stupka E, Ureta-Vidal A, Vastrik I, Clamp M. 2002. The Ensembl genome database project. Nucleic Acids Res 30:38-41. Ignatius J. 1992. Childhood Spinal Muscular Atrophy in Finland. Tampere: University of Tampere. Ingham PW, McMahon AP. 2001. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15:3059-3087. Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, Batzer MA, de Jong PJ. 1994. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat Genet 6:84-89. Ishizuka A, Siomi MC, Siomi H. 2002. A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev 16:2497-2508. Jacobs S, Schilf C, Fliegert F, Koling S, Weber Y, Schurmann A, Joost HG. 1999. ADP-ribosylation factor (ARF)-like 4, 6, and 7 represent a subgroup of the ARF family characterization by rapid nucleotide exchange and a nuclear localization signal. FEBS Lett 456:384-388. Jakel RJ, Schneider BL, Svendsen CN. 2004. Using human neural stem cells to model neurological disease. Nat Rev Genet 5:136-144. Jessell TM. 2000. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet 1:20-29. 78 Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie CM. 2002. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 30:896- 904. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. 2004. Human MicroRNA targets. PLoS Biol 2:e363. Jones CT, Brock DJ, Chancellor AM, Warlow CP, Swingler RJ. 1993. Cu/Zn superoxide dismutase (SOD1) mutations and sporadic amyotrophic lateral sclerosis. Lancet 342:1050-1051. Kadonaga JT. 2004. Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell 116:247-257. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT, Roskin KM, Schwartz M, Sugnet CW, Thomas DJ, Weber RJ, Haussler D, Kent WJ. 2003. The UCSC Genome Browser Database. Nucleic Acids Res 31:51-54. Katsumata T, Miyake A, Aki T, Hirooka K, Hayashida M, Toyoda M, Tanizawa O. 1991. Length of the human umbilical cord in multiple pregnancy. Eur J Obstet Gynecol Reprod Biol 40:25-27. Kingsley DM. 1994. The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8:133-146. Kioussi C, O'Connell S, St-Onge L, Treier M, Gleiberman AS, Gruss P, Rosenfeld MG. 1999. Pax6 is essential for establishing ventral- dorsal cell boundaries in pituitary gland development. Proc Natl Acad Sci U S A 96:14378-14382. Kittles RA, Perola M, Peltonen L, Bergen AW, Aragon RA, Virkkunen M, Linnoila M, Goldman D, Long JC. 1998. Dual origins of Finns revealed by Y chromosome haplotype variation. Am J Hum Genet 62:1171-1179. Klunemann HH, Ridha BH, Magy L, Wherrett JR, Hemelsoet DM, Keen RW, De Bleecker JL, Rossor MN, Marienhagen J, Klein HE, Peltonen L, Paloneva J. 2005. The genetic causes of basal ganglia calcification, dementia, and bone cysts: DAP12 and TREM2. Neurology 64:1502-1507. Kobayashi H, Baumbach L, Matise TC, Schiavi A, Greenberg F, Hoffman EP. 1995. A gene for a severe lethal form of X-linked arthrogryposis (X-linked infantile spinal muscular atrophy) maps to human chromosome Xp11.3-q11.2. Hum Mol Genet 4:1213- 1216. Kolomietz E, Meyn MS, Pandita A, Squire JA. 2002. The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors. Genes Chromosomes Cancer 35:97-112. Kondo T, Duboule D. 1999. Breaking colinearity in the mouse HoxD complex. Cell 97:407-417. 79 Kulp D, Haussler D, Reese MG, Eeckman FH. 1996. A generalized hidden Markov model for the recognition of human genes in DNA. Proc Int Conf Intell Syst Mol Biol 4:134-142. Kuwabara T, Hsieh J, Nakashima K, Taira K, Gage FH. 2004. A small modulatory dsRNA specifies the fate of adult neural stem cells. Cell 116:779-793. Lahdensuu S. 1939. Chondrodystrophiasta. Duodecim:503-522. Lai EC, Tomancak P, Williams RW, Rubin GM. 2003. Computational identification of Drosophila microRNA genes. Genome Biol 4:R42. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M et al. 2001. Initial sequencing and analysis of the human genome. Nature 409:860-921. Landmesser L. 1978. The distribution of motoneurones supplying chick hind limb muscles. J Physiol 284:371-389. Lee RC, Feinbaum RL, Ambros V. 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843-854. Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M, et al. 1995. Identification and characterization of a spinal muscular atrophy- determining gene. Cell 80:155-165. Levi-Montalcini R, Levi G. 1942. Les consequences de la destruction d'un territoire d'innervationperipheique sur le developments des centres nerveux correspondents dans l'embryon de poulet. Arch. Biol. (Liege) 53:537-545. Levine M, Tjian R. 2003. Transcription regulation and animal diversity. Nature 424:147-151. 80 Lillien L. 1995. Changes in retinal cell fate induced by overexpression of EGF receptor. Nature 377:158-162. Lindvall O, Kokaia Z, Martinez-Serrano A. 2004. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat Med 10 Suppl:S42-50. Liu Y, Rao MS. 2004. Glial progenitors in the CNS and possible lineage relationships among them. Biol Cell 96:279-290. Lu QR, Sun T, Zhu Z, Ma N, Garcia M, Stiles CD, Rowitch DH. 2002. Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection. Cell 109:75-86. Ma J. 2005. Crossing the line between activation and repression. Trends Genet 21:54-59. Maddox B. 2003. The double helix and the 'wronged heroine'. Nature 421:407-408. Mahadevan MS, Korneluk RG, Roy N, MacKenzie A, Ikeda J. 1995. SMA genes: deleted and duplicated. Nat Genet 9:112-113. Mariani M, Baldessari D, Francisconi S, Viggiano L, Rocchi M, Zappavigna V, Malgaretti N, Consalez GG. 1999. Two murine and human homologs of mab-21, a cell fate determination gene involved in Caenorhabditis elegans neural development. Hum Mol Genet 8:2397-2406. Mayhall EA, Paffett-Lugassy N, Zon LI. 2004. The clinical potential of stem cells. Curr Opin Cell Biol 16:713-720. McMahon AP, Gavin BJ, Parr B, Bradley A, McMahon JA. 1992. The Wnt family of cell signalling molecules in postimplantation development of the mouse. Ciba Found Symp 165:199-212; discussion 212-198. Mendel G. 1866. Versuche über Pflanzen-Hybriden. Verhandlungen des naturforschenden Vereines in Brünn. Mignone JL, Kukekov V, Chiang AS, Steindler D, Enikolopov G. 2004. Neural stem and progenitor cells in nestin-GFP transgenic mice. J Comp Neurol 469:311-324. Moerman P, Fryns JP, Cornelis A, Bergmans G, Vandenberghe K, Lauweryns JM. 1990. Pathogenesis of the lethal multiple pterygium syndrome. Am J Med Genet 35:415-421. Moessinger AC. 1983. Fetal akinesia deformation sequence: an animal model. Pediatrics 72:857-863. Morita K, Chow KL, Ueno N. 1999. Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family. Development 126:1337-1347. Mulder DW, Kurland LT, Offord KP, Beard CM. 1986. Familial adult motor neuron disease: amyotrophic lateral sclerosis. Neurology 36:511-517. 81 Muller F, O'Rahilly R. 1987. The development of the human brain, the closure of the caudal neuropore, and the beginning of secondary neurulation at stage 12. Anat Embryol (Berl) 176:413-430. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51 Pt 1:263-273. Munch C, Sedlmeier R, Meyer T, Homberg V, Sperfeld AD, Kurt A, Prudlo J, Peraus G, Hanemann CO, Stumm G, Ludolph AC. 2004. Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS. Neurology 63:724-726. Nakamizo T, Urushitani M, Inoue R, Shinohara A, Sawada H, Honda K, Kihara T, Akaike A, Shimohama S. 2000. Protection of cultured spinal motor neurons by estradiol. Neuroreport 11:3493-3497. Nikali K, Suomalainen A, Terwilliger J, Koskinen T, Weissenbach J, Peltonen L. 1995. Random search for shared chromosomal regions in four affected individuals: the assignment of a new hereditary ataxia locus. Am J Hum Genet 56:1088-1095. Nishimura AL, Mitne-Neto M, Silva HC, Oliveira JR, Vainzof M, Zatz M. 2004. A novel locus for late onset amyotrophic lateral sclerosis/motor neurone disease variant at 20q13. J Med Genet 41:315-320. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. 2004. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet 75:822-831. Nishiyama A, Yu M, Drazba JA, Tuohy VK. 1997. Normal and reactive NG2+ glial cells are distinct from resting and activated microglia. J Neurosci Res 48:299-312. Noble M, Proschel C, Mayer-Proschel M. 2004. Getting a GR(i)P on oligodendrocyte development. Dev Biol 265:33-52. Norio R. 2003. The Finnish Disease Heritage III: the individual diseases. Hum Genet 112:470-526. Norio R, Nevanlinna HR, Perheentupa J. 1973. HEREDITARY DISEASES IN FINLAND -RARE FLORA IN RARE SOIL. Annals of Clinical Research 5:109-141. Oppenheim RW. 1991. Cell death during development of the nervous system. Annu Rev Neurosci 14:453-501. Oppenheim RW, Homma S, Marti E, Prevette D, Wang S, Yaginuma H, McMahon AP. 1999. Modulation of early but not later stages of programmed cell death in embryonic avian spinal cord by sonic hedgehog. Mol Cell Neurosci 13:348-361. Osoegawa K, Mammoser AG, Wu C, Frengen E, Zeng C, Catanese JJ, de Jong PJ. 2001. A bacterial artificial chromosome library for 82 sequencing the complete human genome. Genome Res 11:483- 496. Packard M, Koo ES, Gorczyca M, Sharpe J, Cumberledge S, Budnik V. 2002. The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation. Cell 111:319-330. Paganelli AR, Ocana OH, Prat MI, Franco PG, Lopez SL, Morelli L, Adamo AM, Riccomagno MM, Matsubara E, Shoji M, Affranchino JL, Castano EM, Carrasco AE. 2001. The Alzheimer- related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis. Mech Dev 107:119-131. Pakkasjarvi N, Gentile M, Saharinen J, Honkanen J, Herva R, Peltonen L, Kestila M. 2005. Indicative oligodendrocyte dysfunction in spinal cords of human fetuses suffering from a lethal motoneuron disease. J Neurobiol 65:269-281. Paloneva J, Manninen T, Christman G, Hovanes K, Mandelin J, Adolfsson R, Bianchin M, Bird T, Miranda R, Salmaggi A, Tranebjaerg L, Konttinen Y, Peltonen L. 2002. Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am J Hum Genet 71:656-662. Pardal R, Clarke MF, Morrison SJ. 2003. Applying the principles of stem- cell biology to cancer. Nat Rev Cancer 3:895-902. Pearn J. 1980. Classification of spinal muscular atrophies. Lancet 1:919- 922. Peltonen L, Pekkarinen P, Aaltonen J. 1995. Messages from an isolate: lessons from the Finnish gene pool. Biol Chem Hoppe Seyler 376:697-704. Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM. 1996. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84:309-320. Pierrat B, Simonen M, Cueto M, Mestan J, Ferrigno P, Heim J. 2001. SH3GLB, a new endophilin-related protein family featuring an SH3 domain. Genomics 71:222-234. Pomeroy SL, Tamayo P, Gaasenbeek M, Sturla LM, Angelo M, McLaughlin ME, Kim JY, Goumnerova LC, Black PM, Lau C, Allen JC, Zagzag D, Olson JM, Curran T, Wetmore C, Biegel JA, Poggio T, Mukherjee S, Rifkin R, Califano A, Stolovitzky G, Louis DN, Mesirov JP, Lander ES, Golub TR. 2002. Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 415:436-442. Postigo AA, Depp JL, Taylor JJ, Kroll KL. 2003. Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. Embo J 22:2453-2462. Prasad A, Hollyday M. 1991. Development and migration of avian sympathetic preganglionic neurons. J Comp Neurol 307:237-258. 83 Raff MC, Miller RH, Noble M. 1983. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303:390-396. Rao M. 2004. Stem and precursor cells in the nervous system. J Neurotrauma 21:415-427. Rao MS. 1999. Multipotent and restricted precursors in the central nervous system. Anat Rec 257:137-148. Rao MS, Noble M, Mayer-Proschel M. 1998. A tripotential glial precursor cell is present in the developing spinal cord. Proc Natl Acad Sci U S A 95:3996-4001. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G. 2000. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901-906. Reya T, Morrison SJ, Clarke MF, Weissman IL. 2001. Stem cells, cancer, and cancer stem cells. Nature 414:105-111. Reynolds BA, Tetzlaff W, Weiss S. 1992. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12:4565-4574. Riethmacher D, Sonnenberg-Riethmacher E, Brinkmann V, Yamaai T, Lewin GR, Birchmeier C. 1997. Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature 389:725-730. Roessler E, Belloni E, Gaudenz K, Jay P, Berta P, Scherer SW, Tsui LC, Muenke M. 1996. Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat Genet 14:357-360. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX, et al. 1993. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59-62. Rowitch DH, Lu QR, Kessaris N, Richardson WD. 2002. An 'oligarchy' rules neural development. Trends Neurosci 25:417-422. Roy N, Mahadevan MS, McLean M, Shutler G, Yaraghi Z, Farahani R, Baird S, Besner-Johnston A, Lefebvre C, Kang X, et al. 1995. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80:167-178. Roy NS, Wang S, Harrison-Restelli C, Benraiss A, Fraser RA, Gravel M, Braun PE, Goldman SA. 1999. Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter. J Neurosci 19:9986-9995. Royer-Pokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH. 1986. Cloning the gene for an inherited human disorder--chronic granulomatous disease--on the basis of its chromosomal location. Nature 322:32- 38. 84 Royer-Pokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH. 1986. Cloning the gene for the inherited disorder chronic granulomatous disease on the basis of its chromosomal location. Cold Spring Harb Symp Quant Biol 51 Pt 1:177-183. Ruddy DM, Parton MJ, Al-Chalabi A, Lewis CM, Vance C, Smith BN, Leigh PN, Powell JF, Siddique T, Meyjes EP, Baas F, de Jong V, Shaw CE. 2003. Two families with familial amyotrophic lateral sclerosis are linked to a novel locus on chromosome 16q. Am J Hum Genet 73:390-396. Rudnik-Schoneborn S, Sztriha L, Aithala GR, Houge G, Laegreid LM, Seeger J, Huppke M, Wirth B, Zerres K. 2003. Extended phenotype of pontocerebellar hypoplasia with infantile spinal muscular atrophy. Am J Med Genet 117A:10-17. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354. Sapp PC, Hosler BA, McKenna-Yasek D, Chin W, Gann A, Genise H, Gorenstein J, Huang M, Sailer W, Scheffler M, Valesky M, Haines JL, Pericak-Vance M, Siddique T, Horvitz HR, Brown RH, Jr. 2003. Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis. Am J Hum Genet 73:397- 403. Sariola H, Frilander M, Heino T, Jernvall J, Partanen J, Sainio K, Salminen M, Thesleff I. 2003. Kehitysbiologia. Duodecim. 311. Sariola H, Saarma M, Sainio K, Arumae U, Palgi J, Vaahtokari A, Thesleff I, Karavanov A. 1991. Dependence of kidney morphogenesis on the expression of nerve growth factor receptor. Science 254:571- 573. Savukoski M, Klockars T, Holmberg V, Santavuori P, Lander ES, Peltonen L. 1998. CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis. Nat Genet 19:286-288. Schell-Apacik C, Rivero M, Knepper JL, Roessler E, Muenke M, Ming JE. 2003. SONIC HEDGEHOG mutations causing human holoprosencephaly impair neural patterning activity. Hum Genet 113:170-177. Schwartz PH, Tassone F, Greco CM, Nethercott HE, Ziaeian B, Hagerman RJ, Hagerman PJ. 2005. Neural progenitor cells from an adult patient with fragile X syndrome. BMC Med Genet 6:2. Sendtner M, Pei G, Beck M, Schweizer U, Wiese S. 2000. Developmental motoneuron cell death and neurotrophic factors. Cell Tissue Res 301:71-84. 85 Sharma K, Sheng HZ, Lettieri K, Li H, Karavanov A, Potter S, Westphal H, Pfaff SL. 1998. LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons. Cell 95:817-828. Shaw PJ, Ince PG. 1997. Glutamate, excitotoxicity and amyotrophic lateral sclerosis. J Neurol 244 Suppl 2:S3-14. Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N, Vincent P, Pumiglia K, Temple S. 2004. Endothelial cells stimulate self- renewal and expand neurogenesis of neural stem cells. Science 304:1338-1340. Sheng G, dos Reis M, Stern CD. 2003. Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation. Cell 115:603-613. Shimizu T, Kagawa T, Wada T, Muroyama Y, Takada S, Ikenaka K. 2005. Wnt signaling controls the timing of oligodendrocyte development in the spinal cord. Dev Biol 282:397-410. Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M. 1992. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor- based vector. Proc Natl Acad Sci U S A 89:8794-8797. Silberstein EP, Kakulas BA. 1998. Arthrogryposis multiplex congenita in Western Australia. J Paediatr Child Health 34:518-523. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. 2004. Identification of human brain tumour initiating cells. Nature 432:396-401. Soler-Botija C, Ferrer I, Gich I, Baiget M, Tizzano EF. 2002. Neuronal death is enhanced and begins during foetal development in type I spinal muscular atrophy spinal cord. Brain 125:1624-1634. Sommer L, Rao M. 2002. Neural stem cells and regulation of cell number. Prog Neurobiol 66:1-18. Soukup T, Pedrosa-Domellof F, Thornell LE. 1993. Differentiation of supernumerary fibres in neonatally deefferented rat muscle spindles. Differentiation 53:35-43. Soukup T, Pedrosa F, Thornell LE. 1990. Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles. Histochemistry 94:245-256. Spranger J, Benirschke K, Hall JG, Lenz W, Lowry RB, Opitz JM, Pinsky L, Schwarzacher HG, Smith DW. 1982. Errors of morphogenesis: concepts and terms. Recommendations of an international working group. J Pediatr 100:160-165. Stamataki D, Ulloa F, Tsoni SV, Mynett A, Briscoe J. 2005. A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube. Genes Dev 19:626-641. Sun T, Dong H, Wu L, Kane M, Rowitch DH, Stiles CD. 2003. Cross- repressive interaction of the Olig2 and Nkx2.2 transcription 86 factors in developing neural tube associated with formation of a specific physical complex. J Neurosci 23:9547-9556. Swanson DA, Steel JM, Valle D. 1998. Identification and characterization of the human ortholog of rat STXBP1, a protein implicated in vesicle trafficking and neurotransmitter release. Genomics 48:373- 376. Sweitzer SM, Hinshaw JE. 1998. Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 93:1021-1029. Svendsen CN, Clarke DJ, Rosser AE, Dunnett SB. 1996. Survival and differentiation of rat and human epidermal growth factor- responsive precursor cells following grafting into the lesioned adult central nervous system. Exp Neurol 137:376-388. Takebayashi H, Nabeshima Y, Yoshida S, Chisaka O, Ikenaka K. 2002. The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages. Curr Biol 12:1157-1163. Tam PP, Beddington RS. 1987. The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development 99:109-126. Thesleff I, Vainio S, Jalkanen M. 1989. Cell-matrix interactions in tooth development. Int J Dev Biol 33:91-97. Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD. 2001. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 3:778-784. Tosney KW, Hotary KB, Lance-Jones C. 1995. Specifying the target identity of motoneurons. Bioessays 17:379-382. Trentin A, Glavieux-Pardanaud C, Le Douarin NM, Dupin E. 2004. Self- renewal capacity is a widespread property of various types of neural crest precursor cells. Proc Natl Acad Sci U S A 101:4495- 4500. Troy CM, Salvesen GS. 2002. Caspases on the brain. J Neurosci Res 69:145-150. van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ, Meyerowitz EM, Schmid SL. 1993. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol 122:553- 563. Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, Agarwala R, Ainscough R, Alexandersson M, An P, Antonarakis SE, Attwood J, Baertsch R, Bailey J, Barlow K, Beck S, Berry E, Birren B, Bloom T, Bork P, Botcherby M, Bray N, Brent MR, Brown DG, Brown SD, Bult C, Burton J, Butler J, Campbell RD, Carninci P, Cawley S, Chiaromonte F, Chinwalla AT, Church DM, Clamp M, Clee C, Collins FS, Cook LL, Copley RR, Coulson A, Couronne O, Cuff J, Curwen V, Cutts T, Daly M, David R, Davies J, Delehaunty KD, Deri J, Dermitzakis ET, Dewey C, 87 Dickens NJ, Diekhans M, Dodge S, Dubchak I, Dunn DM, Eddy SR, Elnitski L, Emes RD, Eswara P, Eyras E, Felsenfeld A, Fewell GA, Flicek P, Foley K, Frankel WN, Fulton LA, Fulton RS, Furey TS, Gage D, Gibbs RA, Glusman G, Gnerre S, Goldman N, Goodstadt L, Grafham D, Graves TA, Green ED, Gregory S, Guigo R, Guyer M, Hardison RC, Haussler D, Hayashizaki Y, Hillier LW, Hinrichs A, Hlavina W, Holzer T, Hsu F, Hua A, Hubbard T, Hunt A, Jackson I, Jaffe DB, Johnson LS, Jones M, Jones TA, Joy A, Kamal M, Karlsson EK et al. 2002. Initial sequencing and comparative analysis of the mouse genome. Nature 420:520-562. Watson J, Crick F. 1953. Molecular Structure of Nucleic Acids. Nature 171:737-738. Vaux DL, Korsmeyer SJ. 1999. Cell death in development. Cell 96:245- 254. Wen T, Gu P, Minning TA, Wu Q, Liu M, Chen F, Liu H, Huang H. 2002. Microarray analysis of neural stem cell differentiation in the striatum of the fetal rat. Cell Mol Neurobiol 22:407-416. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu- Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C et al. 2001. The sequence of the human genome. Science 291:1304-1351. Werdnig G. 1894. Die frühinfantile progressive spinale Amyotrophie. Arch Psychiatr Nervenkr:706-744. Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ, Sudhof TC. 2000. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864-869. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. 1995. A novel assay for apoptosis. Flow cytometric detection of 88 phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184:39-51. Verschueren K, Remacle JE, Collart C, Kraft H, Baker BS, Tylzanowski P, Nelles L, Wuytens G, Su MT, Bodmer R, Smith JC, Huylebroeck D. 1999. SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes. J Biol Chem 274:20489-20498. Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, Frolichsthal-Schoeller P, Cova L, Arcellana-Panlilio M, Colombo A, Galli R. 1999. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 156:71-83. Wigglesworth JS. 1997. Developmental pathology of the fetal lung. Pediatr Pulmonol Suppl 16:250-251. Wilkie AO, Morriss-Kay GM, Jones EY, Heath JK. 1995. Functions of fibroblast growth factors and their receptors. Curr Biol 5:500-507. William CM, Tanabe Y, Jessell TM. 2003. Regulation of motor neuron subtype identity by repressor activity of Mnx class homeodomain proteins. Development 130:1523-1536. Wong RW, Guillaud L. 2004. The role of epidermal growth factor and its receptors in mammalian CNS. Cytokine Growth Factor Rev 15:147-156. Wright LS, Li J, Caldwell MA, Wallace K, Johnson JA, Svendsen CN. 2003. Gene expression in human neural stem cells: effects of leukemia inhibitory factor. J Neurochem 86:179-195. Wu P, Tarasenko YI, Gu Y, Huang LY, Coggeshall RE, Yu Y. 2002. Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci 5:1271- 1278. Vuopala K, Herva R. 1994. Lethal congenital contracture syndrome: further delineation and genetic aspects. J Med Genet 31:521-527. Vuopala K, Herva R, Pedrosa-Domellof F, Thornell LE. 1996. Myosin heavy chain expression in muscles of two cases of lethal congenital contracture syndrome. Birth Defects Orig Artic Ser 30:369-378. Vuopala K, Ignatius J, Herva R. 1995. Lethal arthrogryposis with anterior horn cell disease. Hum Pathol 26:12-19. Vuopala K, Leisti J, Herva R. 1994. Lethal arthrogryposis in Finland--a clinico-pathological study of 83 cases during thirteen years. Neuropediatrics 25:308-315. Xu Y, Einstein JR, Mural RJ, Shah M, Uberbacher EC. 1994. An improved system for exon recognition and gene modeling in human DNA sequences. Proc Int Conf Intell Syst Mol Biol 2:376-384. 89 Yang Y, Hentati A, Deng HX, Dabbagh O, Sasaki T, Hirano M, Hung WY, Ouahchi K, Yan J, Azim AC, Cole N, Gascon G, Yagmour A, Ben-Hamida M, Pericak-Vance M, Hentati F, Siddique T. 2001. The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat Genet 29:160-165. Zechner D, Fujita Y, Hulsken J, Muller T, Walther I, Taketo MM, Crenshaw EB, 3rd, Birchmeier W, Birchmeier C. 2003. beta- Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev Biol 258:406-418. Zerin M, Van Allen MI, Smith DW. 1982. Intrinsic auricular muscles and auricular form. Pediatrics 69:91-93. Zhang MQ. 1997. Identification of protein coding regions in the human genome by quadratic discriminant analysis. Proc Natl Acad Sci U S A 94:565-568. Zhao Z, Alam S, Oppenheim RW, Prevette DM, Evenson A, Parsadanian A. 2004. Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their long-term survival following axotomy. Exp Neurol 190:356-372. Zhou FC, Duguid JR, Edenberg HJ, McClintick J, Young P, Nelson P. 2001. DNA microarray analysis of differential gene expression of 6-year-old rat neural striatal progenitor cells during early differentiation. Restor Neurol Neurosci 18:95-104. Zhou Q, Choi G, Anderson DJ. 2001. The bHLH transcription factor Olig2 promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31:791-807. Zhou Q, Wang S, Anderson DJ. 2000. Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25:331-343. Zipori D. 2004. The nature of stem cells: state rather than entity. Nat Rev Genet 5:873-878.