Molecular regulation of forebrain development and neural stem/progenitor cells

Janne Hakanen

Department of Veterinary Biosciences Faculty of Veterinary Medicine University of Helsinki and Developmental Biology Program Institute of Biotechnology and Integrative life science doctoral program (ILS) University of Helsinki

Academic dissertation

To be presented for public examination with the permission of the Faculty of Veterinary Medicine of the University of Helsinki in the Lecture Hall 7 (Latokartanonkaari 7- B-Building) on 24th of October, 2014, at 12 noon. Supervisor

Marjo Salminen, Docent Department of Veterinary Biosciences University of Helsinki

Pre-reviewers

Juha Partanen, Professor Department of Biosciences University of Helsinki

Claudio Rivera, PhD, Research director Neuroscience Center University of Helsinki

Opponent

Dan Lindholm, Professor Institute of Biomedicine and Minerva Foundation Institute for Medical Research University of Helsinki

Custodian

Jyrki Kukkonen, Professor Department of Veterinary Biosciences University of Helsinki

ISBN 978-951-51-0135-8 (paperback) ISBN 978-951-51-0136-5 (pdf) ISSN 2342-3161 (print) ISSN 2342-317X (online) Table of contents

List of original publications Abstract Abbreviations 1. Review of the literature...... 1 1. 1 Development of mouse forebrain...... 1 1.1.1 Forebrain germinal zones...... 1 1.1.2 Stem/progenitor cells of forebrain germinal zones ...... 2 1.1.2.1 Self-renewal capacity ...... 3 1.1.2.2 Interkinetic nuclear migration ...... 3 1.1.2.3 Symmetric and asymmetric cell divisions...... 4 1.1.3 Forebrain neurogenesis ...... 4 1.1.3.1.1 Radial migration...... 5 1.1.3.1.2 Tangential migration...... 6 1.1.3.1 Specification of pallium ...... 6 1.1.3.2 Specification of pallial-subpallial boundary (PSB)...... 6 1.1.3.3 Specification of subpallium...... 7 1.1.3.3.1 Lateral ganglionic eminence (LGE)...... 7 1.1.3.3.2 Medial ganglionic eminence (MGE)...... 8 1.1.3.3.3 Caudal ganglionic eminence (CGE) ...... 8 1.1.3.3.4 Telencephalic stalk...... 9 1.1.4 Forebrain gliogenesis ...... 9 1.1.4.1 Specification of astrocytes...... 9 1.1.4.2 Origin and expansion of forebrain oligodendrocytes...... 10 1.1.4.3 Specification of oligodendrocytes...... 10 1.1.5 Development of adult germinal zones in forebrain...... 11 1.1.5.1 Specification of adult germinal zones ...... 12 1.1.5.2 Cellular composition of adult SVZ ...... 12 1.1.5.3 Adult ependymal cells as neural stem/progenitor cells...... 12 1.2 Olfactory system ...... 13 1.2.1 Development of olfactory system ...... 14 1.2.1.1 Radial glia of olfactory bulb...... 16 1.2.1.2 Development and origin of olfactory bulb projection neurons and LOT...... 16 1.2.1.3 Development and origin of olfactory bulb ...... 17 1.2.1.3.1 Temporal origin of olfactory interneurons...... 17 1.2.1.3.2 Lateral ventricle derived interneurons ...... 18 1.2.1.3.3 RMS derived interneurons ...... 18 1.2.1.3.4 Origin of adult olfactory bulb interneurons ...... 18 1.2.1.4 Development and origin of olfactory bulb oligodendrocytes...... 19 1.2.1.4.1 Origin of adult olfactory bulb oligodendrocytes...... 19 1.2.1.5 Development and origin of olfactory bulb astrocytes and OECs...... 19 1.3 Development of forebrain commissures ...... 20 1.3.1 Fusion of forebrain hemispheres...... 21 1.3.2 Role of midline cellular populations in development of CC...... 21 1.3.2.1 Glial wedge (GW) ...... 22 1.3.2.2 Indusium Griseum (IG) ...... 22 1.3.2.3 Subcallosal sling (SCS)...... 22 1.3.3 Callosal axon guidance during formation of CC...... 23 1.4 The Netrin family of guidance factors ...... 24 1.4.1 History of Netrins...... 24 1.4.2 Netrins in different animal species...... 24 1.4.3 Molecular structure of Netrins ...... 24 1.4.4 Interactions of Netrins...... 25 1.4.5 Interactions and developmental functions of NTN1 in animals...... 26 1.4.5.1 NTN1 receptor families...... 26 1.4.5.1.1 DCC and Neogenin ...... 26 1.4.5.1.2 UNC5 family...... 27 1.4.5.1.3 Down’s syndrome cell adhesion molecule (DSCAM)...... 27 1.4.5.1.4 Integrins ...... 27 1.4.5.2 NTN1 and its receptors in axon guidance ...... 28 1.4.5.2.1 NTN1 mediated signaling in axon chemoattraction ...... 28 1.4.5.2.2 NTN1 mediated signalling in axon chemorepulsion...... 29 1.4.5.2.3 Switches between NTN1 mediated axon attraction and repulsion ...... 30 1.4.5.2.4 NTN1 assisted axon guidance in mouse CNS ...... 31 1.4.5.3 NTN1 mediated cell migration in CNS...... 32 1.4.5.3.1 NTN1 mediated migration of neurons in mouse CNS...... 32 1.4.5.3.2 NTN1 mediated migration of glial cells in CNS ...... 33 1.4.5.3.3 NTN1 mediated migration of oligodendrocytes in mouse CNS...... 33 1.4.5.4 NTN1 mediated apoptosis through its receptors ...... 34 1.4.5.5 NTN1 also have a role in organogenesis outside of the nervous system ...... 35 1.5 Actin-bundling protein with BAIAP2 homology (ABBA)...... 35 1.6 Oncogenes affect basic developmental processes of stem/progenitor cells...... 36 1.6.1 Human Papilloma Virus (HPV) 16 E6/E7 oncogenes ...... 36 1.6.1.1 Tumor suppressor protein p53...... 37 1.6.1.2 Tumor suppressor protein pRb...... 37 2. Aims of the study ...... 39 3. Material and methods...... 40 4. Results and discussion ...... 42 4.1 NTN1 is required for the development of olfactory bulb (I) ...... 42 4.1.1 Ntn1 is expressed in forebrain regions which contribute to the development and renewal of olfactory bulb ...... 42 4.1.2 Ntn1 expressing cells extracted from embryonic and adult possess many characteristics associated with neural stem/progenitor cells ...... 43 4.1.3 NTN1 is not critical factor for proliferation, self-renew or differentiation capacity of neural stem/progenitor cells...... 44 4.1.4 Characterization of Ntn1/lacZ positive cells in postnatal RMS...... 44 4.1.5 Migration defects in Ntn1 deficient mouse forebrain...... 45 4.1.5.1 NTN1 may guide migration of the progenitor/precursor cells in a cell-autonomous or paracrine manner...... 45 4.1.5.2 Ntn1/lacZ expressing cells accumulate to the proximal RMS in Ntn1 deficient mice....46 4.1.6 Ntn1 deficient mice have reduced number of granular- and periglomerular cells in the olfactory bulb...... 46 4.1.6.1 Ntn1 deficient mice have reduced number of interneurons in their olfactory bulb...... 47 4.1.6.2 Development of distinct subpopulations was affected differently in Ntn1 deficient olfactory bulbs...... 47 4.1.6.3 Development of astroglia was normal in Ntn1 deficient olfactory bulbs...... 48 4.1.6.4 NTN1 has an impact on olfactory bulb oligodendrogenesis ...... 48 4.1.7 A summary of NTN1’s role in the cellular development of the olfactory bulb...... 50 4.2 Role of NTN1 in the development of CC (II)...... 50 4.2.1 Overall analysis of Ntn1 deficient CSB region in mice ...... 51 4.2.2 Expression of Ntn1 and NTN1 receptors during formation of CC ...... 51 4.2.3 Development of transient midline glial structures in Ntn1 deficient mice...... 52 4.2.4 NTN1 deficiency affects the distribution of neuronal guidepost cells in CSB region...... 53 4.2.5 Early callosal axons are guided close to the pial basal lamina in Ntn1 deficient mice...... 54 4.2.6 NTN1 deficiency affects interhemispheric midline fusion ...... 55 4.2.7 NTN1 may influence the development of CC in several ways...... 56 4.3 Characterisation of the expression and function of ABBA (III)...... 57 4.3.1 Developmental and adult expression of ABBA in mice CNS...... 57 4.3.1.1 ABBA protein is specifically present in radial glia-like cells and RGCs of CNS ...... 57 4.3.1.2 ABBA localizes to the interface between the plasma membrane and the cortical actin cytoskeleton in cultured radial-glial-like cells...... 58 4.3.2 ABBA interacts with actin monomers, but lacks actin bundling activity ...... 58 4.3.3 ABBA binds and deforms plasma membranes in vitro and in vivo ...... 59 4.3.4 ABBA participates in regulation of lamellipodial dynamics and process extension ...... 59 4.3.5 ABBA may participate in maintenance of radial migration in ...... 60 4.4 Impact of HPV16 E6/E7 oncoproteins on neural stem/progenitor cells (IV) ...... 60 4.4.1 HPV16 E6/E7 oncogenes increase cell proliferation and self-renewal of neural stem/progenitor cells...... 60 4.4.2 Neural stem/progenitor cells expressing E6/E7 oncogenes maintain their capacity to proliferate after initiation of differentiation...... 61 4.4.3 E6/E7 oncoproteins do not affect cell differentiation ...... 61 4.4.4 Proliferation and self-renewal capacity of neural progenitor/stem cells may not be coupled...... 62 4.4.5 E6/E7 oncoprotein mediated degradation of p53 and pRb protein family may facilitate formation...... 63 5. Conclusions...... 65 6. Concluding remarks ...... 66 7. Acknowledgements...... 68 8. References...... 69 List of original publications

This thesis is based on the following articles, which are referred to in the text by their roman numerals.

I Netrin1 is required for neural and glial precursor migrations into the olfactory bulb. Hakanen, J., Duprat, S. and Salminen, M. (2011) Dev Biol. 355, 101-114.

II Netrin1 is required for the fusion of the interhemispheric midline in mouse. Hakanen, J. and Salminen, M. Submitted.

III ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension. Saarikangas, J., Hakanen, J., Mattila, P.K., Grumet, M., Salminen, M. and Lappalainen, P. (2008) J Cell Sci. 121, 1444-1454.

IV Increase in self-renewal of neural stem cells by oncogenes affecting p53 and pRb. Piltti, K., Kerosuo, L., Hakanen, J., Eriksson, M., Angers-Loustau, A., Leppä, S., Salminen, M., Sariola, H. and Wartiovaara, K. (2006) Oncogene 25, 4880-4889.

The original publications have been reproduced with the permission of the copyright holders. Abstract

The development of the forebrain is dependent on controlled regulation of neural stem/progenitor cells, since the vast majority of all cells in the forebrain are generated by them. The cellular processes influencing forebrain development include cell proliferation, migration, differentiation, and apoptosis, which are regulated in a spatiotemporal manner by genetic programs and interactive protein networks in a given environment. In this thesis, the influence of Netrin1 (NTN1), Human papilloma virus (HPV) E6/E7, and actin-bundling protein with BAIAP2 homology (ABBA) proteins on neural stem/progenitor cells cellular processes was studied and evaluated. In vitro methods were used to uncover the cellular processes regulated and affected by these proteins. In addition, in vivo methods were used to assess their possible impact on forebrain development in mice. NTN1 belongs to a conserved family of laminin-related molecules and it is present in the developing and adult mouse brain. NTN1 has been thought to act as a diffusible long or short-range guidance cue, which influences growing axons and migrating cells in either a chemoattractive or repulsive manner, and NTN1 deficiency in the brain causes defects in axon guidance and cell migration. The main issue of this thesis was to determine the developmental impact of NTN1 in the formation of two forebrain structures, the olfactory bulb and the corpus callosum (CC). Olfactory bulb processes arriving odour information from the nasal cavity and transmit this information forward to various brain regions. During development, the different cell types of olfactory bulb are generated in distinct germinal regions of the brain. Projection neurons are mainly generated by the olfactory bulb stem/progenitor cells whereas majority of the interneurons are produced by the progenitors, which migrate from the forebrain germinal zones into the olfactory bulb via rostral migratory stream (RMS). The origin of non-neural cells, astrocytes and oligodendrocytes, is not as well understood as neurons. We observed that NTN1 has a significant impact on the beginning of stem/progenitor cells migration from the forebrain germinal zones into the olfactory bulb. In more detail, the migration of Ntn1 expressing stem/progenitor cells was delayed, which led to an accumulation of these cells in RMS, and to a substantial reduction of GABAergic interneurons and oligodendrocytes in the olfactory bulb. Thus, the results suggest that NTN1 acts mainly as a detachment/release factor for the Ntn1 expressing stem/progenitor cells in the beginning of their migration and this function is mediated in either cell-autonomous or paracrine manner. CC is the largest axon tract in the forebrain and it integrates motor, sensory and cognitive performances between cerebral hemispheres. In mice, the cerebral hemispheres have to fuse in the midline before the commissural axons can cross the midline and form the CC. This interhemispheric fusion normally includes the removal of leptomeningeal cells found between the hemispheres and disruption of pial basal lamina, which is produced and maintained by the leptomeningeal cells. In Ntn1 deficient mice the leptomeningeal cells were not removed and the pial basal lamina remained intact in the hemispheric midline. Thus, the results suggest that NTN1 is required for the interhemispheric fusion, which precedes midline crossing of commissural axons and is a prerequisite for the formation of the CC. ABBA belongs to the Bin–amphiphysin–Rvs167 (BAR) protein superfamily, which regulates plasma membrane morphology by directly influencing plasma membrane assembly or through rearrangement of the actin cytoskeleton. The functional and expression studies revealed that ABBA participates in the regulation of cell protrusions by enhancing actin dynamics and connecting plasma membrane deformation to actin cytoskeleton. ABBA was present in radial glia-like cells and radial glial cell extensions near meningeal pial basal lamina during CNS development. During early development of brain, interaction between radial glial end-feet and pial basal lamina is required to sustain radial migration and integrity of pial basal lamina. Thus, ABBA may have an important role in formation of radial glial extensions and their connections in CNS. Exquisite balance between proliferation, self-renew and differentiation of stem/progenitor cells is necessary to maintain appropriate tissue homeostasis. HPV16 E6/E7 oncoprotein mediated degradation of p53 and pRb family of proteins seemed to facilitate a defective coupling of proliferation, self-renewal, and differentiation processes inside neural stem/progenitor cells. In addition, these processes also seemed to be susceptible to the environmental signals. Hence, a defective coupling of proliferation, self-renewal, and differentiation processes in neural stem/progenitor cells may lead to an imbalance in normal tissue homeostasis and abnormal growth of tissue in the brain. In summary, even though NTN1, HPV E6/E7, and ABBA proteins affected different cellular processes of neural stem/progenitor cells, all these cellular processes participate in the development of forebrain conformation and, therefore, the future functionality of it. In addition, defects in regulation of neural stem/progenitor cells cellular processes may lead to congenital neural disorders in mice and humans. Thus, these results also increase our overall comprehension of the developmental cellular processes behind the different neural congenital disorders and diseases. Abbreviations

ABBA Actin-bundling protein with GLAST glutamate-aspartate transporter BAIAP2 homology GSX1 genomic screened homeobox 1 AC anterior commissure GTP guanosine triphosphate ACC agenesis of the corpus callosum GW glial wedge AEP anterior entopeduncular region HC hippocampal commissure aIPC astrocyte intermediate progenitor HPV human papilloma virus cell ID inhibitor of differentiation A-P anterior-posterior IG induseum griseum ASCL1 achaete-scute complex homolog 1 ION inferior olivary nucleus BAR Bin–amphiphysin–Rvs167 IPC intermediate progenitor cell bHLH basic helix-loop-helix IPL internal plexiform layer BLBP brain lipid-binding protein IRTKS insulin receptor tyrosine kinase BMP bone morphogenetic protein substrate cAMP cyclic adenosine monophosphate IRSp53 insulin receptor tyrosine kinase CB calbindin substrate p53 CC corpus callosum ISL1 insulin gene enhancer protein 1 CDC42 cell division cycle42 IZ intermediate zone CDK cyclin-dependent kinase JAK-STATjanus kinase signal transducer and CGE caudal ganglionic eminence activator of transcription signalling CGN cerebellar granule neuron pathway cGMP cyclic guanosine monophosphate LGE lateral ganglionic eminence CNS central nervous system LHRH luteinizing hormone-releasing CoupTF1 COUP transcription factor 1 hormone CR calretinin LHX2 LIM homeobox protein 2 CSB cortico-septal boundary LOT lateral olfactory tract DCC deleted in colorectal cancer LRN lateral reticular nucleus Diap3 diaphanous homolog 3 MAP1B microtubule-associated protein 1 B DLX1 distal-less homeobox 1 MAPK mitogen-activated protein kinase D-V dorsal-ventral Mbp myelin basic protein ECM extracellular matrix MCL mitral cell layer ECN external cuneatus nucleus Mena mammalian enabled EGL external granule layer MGE medial ganglionic eminence EMX1 empty spiracles 1 MIM missing in metastasis ENA enabled MZ marginal zone EPS8 epidermal growth factor receptor MZG midline zipper glia pathway substrate 8 NCK1 non-catalytic region of tyrosine FGF fibroblast growth factor kinase adaptor protein 1 ER81 ETS related protein 81 NeuN neuronal nuclei ERK extracellular signal-regulated kinase NFAT nuclear factor of activated T cells FAK focal adhesion kinase NG2 chondroitin sulphate proteoglycan FNIII fibronectin type III NGN2 neurogenin 2 FOXG1 forkhead box G1 nIPC neural intermediate progenitor cell FYN Src family kinase Fyn NKX2.1 NK2 homeobox 1 GABA Ȗ-aminobutyric acid Npn Neuropilin GCL granular cell layer NPY neuropeptide Y GEF guanine nucleotide exchange factors NSC neural stem cell GFAP glial fibrillary acidic protein NTN netrin GL glomerular layer NTR netrin-like N-WASP neuronal Wiskott–Aldrich syndrome RAC1 Ras related C3 botulinum toxin protein substrate 1 OB olfactory bulb RC2 anti-radial glial cell marker-2 OEC olfactory ensheating cells RGC radial glial cell oIPC oligodendocyte intermediate RGM repulsive guidance molecule progenitor cell RHOA Ras homolog gene family, member OLIG2 oligodendrocyte lineage A transcription factor gene 2 RMS rostral migratory stream ONL olfactory nerve layer ROBO roundabout OPC oligodendrocyte S100b S100 calcium binding protein B progenitor/precursor cell SCS subcallosal sling OSN olfactory sensory neuron Sema Semaphorin PAK1 p21- activated kinase 1 SGL subgranular layer PAX6 Paired box 6 SMADs sma and mad related proteins PDGFR platelet-derived growth factor SHH sonic hedgehog PICK1 C kinase-1 Sp8 specificity protein 8 PIP2 phosphatidylinositol 4,5- SST somatostatin bisphosphate SVZ subventricular zone 3,73Į phosphatidylinositol transfer protein TH tyrosine hydroxylase Į UNC uncoordinated PKC protein kinase C VASP vasodilator-stimulated PLC phospholipase C phosphoprotein Plp proteolipid protein VIP vasoactive intestinal peptide Pn postnatal day VZ ventricular zone POA anterior preoptic area WASP Wiskott–Aldrich syndrome protein POH preoptic– hypothalamic border WAVE2 WASP-family verprolin- region homologous protein 2 pRb retinoblastoma protein WNT Wingless-related PSB pallial-subpallial boundary PV parvalbumin 1. Review of the literature

1. 1 Development of mouse forebrain

Forebrain development, as a part of the central nervous system (CNS), begins during gastrulation when a group of embryonic ectodermal cells transforms into neuroepithelial cells in the centre of the embryo. This transformation (neural induction) is under the regulation of inductive signaling, which arises from a primary organizer region (known as primitive node) and the surrounding mesenchyme (Hebert and Fishell, 2008; Vieira et al., 2010). Neural induction is followed by neurulation process, where the neuroepithelial cells first organize into a flat sheet named the “neural plate” (Colas and Schoenwolf, 2001; Vieira et al., 2010). As development proceeds, the neural plate goes through subsequent steps of morphological shape changes to form the neural tube during neurulation (Colas and Schoenwolf, 2001). These shape changes include elongation and bending of the neural plate, which leads to formation of neural folds at the lateral edges of the neural plate. The neural folds elevate and move towards the dorsal midline, where they eventually contact each other and fuse, forming the roof of the neural tube. The elevation of the neural folds also establishes a space called the neural groove, which forms the future lumen of the neural tube after the dorsal closure of neural folds (Colas and Schoenwolf, 2001; Vieira et al., 2010). During neural induction and neurulation, the dorso-ventral (D-V) and antero-posterior (A-P) axes of the neural tube become gradually completed (Lumsden and Krumlauf, 1996). The development of D-V and A-P axes is regulated by extensive signaling arising from secondary signalling centers, which patterns the neural tube into distinct neuroepithelial regions and compartments (Lumsden and Krumlauf, 1996; Martinez-Ferre and Martinez, 2012). Later, the expanded anterior end of the neural tube becomes patterned into three brain vesicles: forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon) (Lumsden and Krumlauf, 1996; Wurst and Bally-Cuif, 2001). Subsequently, the forebrain will be further subdivided into the telencephalon and diencephalon (Hebert and Fishell, 2008; Martinez-Ferre and Martinez, 2012). In this thesis, the main objectives were related to the development of forebrain olfactory bulb and corpus callosum (CC), which are reviewed after general introduction of the forebrain development in separate paragraphs.

1.1.1 Forebrain germinal zones

As mentioned above, both the neural plate and neural tube are composed of a single layer of neuroepithelial cells, which form the neuroepithelium (Götz and Huttner, 2005). These neuroepithelial cells give rise to radial glial cells (RGCs), which have an elongated shape with a apical cilium extending into the ventricular lumen and a long basal process extending radially into the pial surface of the forebrain (Figure 1C; Tramontin et al., 2003; Götz and Huttner, 2005; Malatesta et al., 2008). This transition from neuroepithelial cells to RGCs begins in most of the brain regions simultaneously with neuronal differentiation (neurogenesis), around embryonic day (E) 9-10 in mice (Noctor et al., 2002; Götz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009). Furthermore, the onset of forebrain neurogenesis initiates the tranformation of single layered neuroepithelium into multilayered brain tissue, where the neuroepithelial cells and RGCs reside within germinal zones, located next to the ventricle walls (Götz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009). The first germinal zone to arise is called the ventricular zone (VZ) and it contains direct descendants of the primitive neuroepithelium. The second germinal zone is called subventricular zone (SVZ), which later emerges from the VZ and is located adjacent to the VZ along the ventricular wall (Figure 1A; Wichterle et al., 1999). The SVZ contains rapidly proliferating cells and it expands greatly during the last third of prenatal development, in parallel,

1 with progressive reduction of the VZ. This trend continues postnatally (first postnatal week in rodents), leading to persistence of SVZ as the only germinal zone in a mouse forebrain, after the VZ gradually disappears (Tramontin et al., 2003; Levison and Goldman, 1997; Malatesta et al., 2008). Altogether, the majority of different neuronal cells in the mouse forebrain are produced from these two germinal zones during development (Götz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009). Interestingly, developing human and ferret neocortex have additional RGC-like progenitors in the outer SVZ, which are thought to arise from the neuroepithelial cells and RGCs (Fietz et al., 2010; Fietz and Huttner, 2011).

Figure 1. Schematic illustration of stem/progenitor cells in forebrain germinal zones. (A) Coronal overview of the germinal zones next to the lateral ventricles. (B) Neuroepithelial, radial glial, and intermediate progenitor cells divide symmetrically or asymmetrically during development to generate different progeny. (C) Interkinetic nuclear migration, nuclei of neuroepithelial and radial glial cells move along apical-basal (A-B) axis during the cell cycle. (D) Radial migration occurs in two different modes (somal translocation and glia guided locomotion) during cortical development. CX, cortex; DC, differentiated cell; GGL, glia guided locomotion; IPC, intermediate progenitor cell; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; NEC, neuroepithelial cell; PM, pial membrane; RGC, radial glial cell; ST, somal translocation.

1.1.2 Stem/progenitor cells of forebrain germinal zones

It has been suggested in many studies that RGCs function as primary progenitors or neural stem cells (NSC) during brain development and throughout the majority of neurogenesis. RGCs generate either daughter neurons directly or a second, more restricted, intermediate progenitor cell (IPC), 2 also referred to as a basal progenitor cell, non-surface dividing cell, or transit amplifying cell (Kriegstein and Alvarez-Buylla, 2009). The IPCs are more restricted cells than RGCs and they generate neurons (nIPC) or glial cells, including oligodendrocytes (oIPC) and astrocytes (aIPC) (Kriegstein and Alvarez-Buylla, 2009). It has been suggested that IPCs populate the emerging embryonic SVZ, which starts contributing to ongoing neurogenesis together with the VZ (Tarabykin et al. 2001; Haubensak et al. 2004; Zimmer et al. 2004; Noctor et al. 2004). Although, IPCs populate the early SVZ they are also present within the early VZ, before formation of a distinct SVZ (Noctor et al. 2007; Kriegstein and Alvarez-Buylla, 2009). Thus, it seems that the VZ contains predominantly RGCs and to some extent IPCs whereas the early SVZ is mostly composed of IPCs and is lacking RGCs. In the embryonic brain, VZ stem/progenitor cells can be distinguished from SVZ stem/progenitors cells by the interkinetic nuclear migration (See Figure 1C and chapter 1.1.2.2) and by the contacts that VZ neuroepithelial and RGC cells make with the ventricular surface (Hansen et al., 2010; Kriegstein and Alvarez-Buylla, 2009). IPCs also differ from RGCs by their gene expression profile, together with lack of both the apical endfeet and ascending processes (Götz and Huttner, 2005; Corbin et al., 2008; Chojnacki et al., 2009). However, both IPCs and RGCs can divide either symmetrically or asymmetrically during development, but it is thought, that RGCs predominantly divide asymmetrically and IPCs undergo symmetrical divisions during the period of high neurogenesis (see Figure 1B and chapter 1.1.2.3; Götz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009; Taverna and Huttner, 2010).

1.1.2.1 Self-renewal capacity

During brain development, self-renewal ensures the maintenance and expansion of neural stem/progenitor cell populations in the germinal zones through symmetric or asymmetric cell divisions (Figure 1B). At first, NSCs undergo symmetric divisions generating two daughter cells with the same fate, namely neuroepithelial cells or RGCs. These divisions are followed by many asymmetric divisions, which generate NSC and a more differentiated cell such as IPC or neuron. In turn, IPCs mainly undergo symmetric divisions generating two differentiating postmitotic cells (Götz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009; Rowitch and Kriegstein, 2010).

1.1.2.2 Interkinetic nuclear migration

One feature shared by NSCs, but not IPCs, is a mitotic process known as interkinetic nuclear migration (Figure 1C). Thus, NSCs can be distinguished from the IPCs by interkinetic nuclear migration (Kriegstein and Alvarez-Buylla, 2009). In interkinetic nuclear migration, NSCs undergo mitosis (M-phase) at the apical side of the VZ, whereas S phase takes place at a more basal location (Miyata 2008; Taverna and Huttner, 2010). Furthermore, in transition phase G1 between M and S phase, nuclear migration proceeds in an apical-to-basal direction and in G2 phase in a basal-to- apical direction (Miyata 2008; Kriegstein and Alvarez-Buylla, 2009; Taverna and Huttner, 2010). Although the mechanism of interkinetic nuclear migration has been extensively explored, little is known about its functional significance. NSCs are known to progress in the cell cycle, even if interkinetic nuclear migration is arrested (Murciano et al. 2002; Taverna and Huttner, 2010). Recently it has been proposed that interkinetic nuclear migration regulates neurogenesis by modulating the exposure of NSC to neurogenic or proliferative signaling, such as Notch signaling (Murciano et al. 2002; Del Bene et al, 2008; Kriegstein and Alvarez-Buylla, 2009). Notch signaling in the retina has been shown to maintain NSCs in a proliferative state and prevent them from premature cell cycle exit (Del Bene et al, 2008). Furthermore, Notch signaling in the brain promotes RGCs identity and self-renewal (Gaiano et al. 2000; Gaiano and Fishell, 2002; Kriegstein and Alvarez-Buylla, 2009; Taverna and Huttner, 2010).

3 1.1.2.3 Symmetric and asymmetric cell divisions

Interkinetic nuclear migration partially induces the apico–basal polarisation of neuroepithelial cells and RGCs, which is apparent from the unequal distribution of transmembrane proteins in their plasma membrane (Götz and Huttner, 2005). For example, transmembrane protein prominin-1 (CD133) is exclusively found in the apical plasma membrane (Weigmann et al., 1997), whereas integrin Į6 is mainly found from the basal plasma membrane (Wodarz and Huttner, 2003; Götz and Huttner, 2005). In developing brain, these polarised transmembrane proteins, together with other cell fate constituents, can be inherited equally or unequally by the descendants of the neuroepithelial cells and RGCs (Götz and Huttner, 2005; Fish et al., 2008). This inheritance of cell fate constituents between the daughter cells is eventually determined by the orientation of the cleavage furrow, which ingresses perpendicular to the axis of a mitotic spindle (Fish et al., 2008; Fietz and Huttner, 2011). An equal distribution of cell fate constituents between the neuroepithelial- and RGCs descendants refers to symmetric divisions (Götz and Huttner, 2005). The symmetric divisions require a cleavage furrow axis, which proceeds precisely in the apical-to-basal direction, bisecting the apical plasma membrane vertically (vertical cleavage plane) (Huttner and Kosodo, 2005; Fietz and Huttner, 2011). Furthermore, the axis of the mitotic spindle has to be positioned precisely perpendicular to the cleavage furrow axis (horizontal spindle) (Fietz and Huttner, 2011). On the other hand, a cleavage plane opposite to vertical cleavage plane (horizontal cleavage plane) leads to asymmetric division and unequal distribution of cell fate constituents between daughter cells (Götz and Huttner, 2005; Fietz and Huttner, 2011). Hence, in asymmetric division the apical constituents will be distributed to one daughter cell and the basal constituents to another (Götz and Huttner, 2005; Huttner and Kosodo, 2005). Consequently, molecules involved in directing mitotic spindle and cleavage furrow orientation ensure the correct fate of neuroepithelial cells and RGC descendants (Fietz and Huttner, 2011). As mentioned above, neuroepithelial cells and RGCs are highly polarised and have both apical and basal contacts. The IPCs, on the other hand, are non-polar and lack both apical and basal contacts. Rodent IPC divisions are known to occur with a near random to horizontal cleavage plane orientation, which suggests that IPC divisions are mainly symmetric producing two daughter neurons or alternatively two IPCs, although, the second event is less frequent (Attardo et al., 2008; Farkas et al., 2008; Kriegstein et al., 2006; Fietz and Huttner, 2011).

1.1.3 Forebrain neurogenesis

It has become evident that RGCs and IPCs in different regions of the VZ/SVZ contribute to different neuronal cell types in the developing brain. It has been suggested that neuroepithelial cells and RGCs interpret gradients of morphogens like fibroblast growth factors (FGFs), wingless-related (WNTs), bone morphogenetic factors (BMPs) and sonic hedgehog (SHH) to unfold unique regional programs of transcription factor gene expressions. For example, Paired box 6 (Pax6), Empty spiracles homologue 1 (Emx1), Genomic screened homeobox 1 (Gsx1earlier known as Gsh1), Gsx2, ETS related protein 81 (Er81), Specificity protein 8 (Sp8), NK2 homeobox 1 (Nkx2.1), Distal-less homeobox 1 (Dlx1), Dlx2, and Oligodendrocyte lineage transcription factor gene 2 (Olig2), the LIM homeobox protein 2 (Lhx2), forkhead box G1 (Foxg1), COUP transcription factor 1 (CoupTF1) and insulin gene enhancer protein 1 (Isl1) together with proneural genes such as Neurogenin 2 (Ngn2) and Achaete-scute complex homolog 1 (Ascl1 earlier known as Mash1), participate in the specification of the postmitotic neuronal cell type produced by RGCs and IPCs in different VZ/SVZ regions (Campbell 2003; Puelles and Rubinstein, 2003; Guillemot 2007; Corbin et al., 2008; Long et al. 2009; Kriegstein and Alvarez-Buylla, 2009). In addition, Notch signaling through its pathway components, ligands, receptors, modulators and targets, generate stem/progenitor cell heterogeneity and, therefore, influence neuronal specification (Corbin et al., 2008; Caviness Jr et al., 2009). 4 Interestingly, the different combinations of above mentioned transcription factors as well as morphology, size, cell composition, and embryonic emergence determines division of the developing forebrain into distinct compartments in the D-V axis. Classically, the developing forebrain has been divided into the D-V axis to dorsal pallium (developing cortex and hippocampus) and ventral subpallial region, which are separated from each other by a boundary region (Figure 2; Corbin et al., 2000;Stoykova et al., 2000; Toresson et al., 2000;Yun et al., 2001). The ventral subpallial region can be further divided into four distinct regions. The medial ganglionic eminence (MGE), the lateral ganglionic eminence (LGE), the caudal ganglionic eminence (CGE), and the telencephalic stalk (the nonevaginated telencephalon), which includes the anterior entopeduncular region (AEP), the anterior preoptic area (POA), and the preoptic– hypothalamic (POH) border region (Figure 2), which resides close to the chiasmatic region. In addition, gene expression analyses suggest that a large part of the septum also belongs to the subpallium (Puelles et al., 2000; Brazel et al., 2003; Flames et al., 2007). In conclusion, although the embryonic pallial and subpallial VZ/SVZ regions may appear to be relatively homogeneous proliferative zones surrounding the ventricle, they contain heterogeneous RGCs and IPCs in relation to the postmitotic neuronal cell types they will produce (Kriegstein and Alvarez-Buylla, 2009). Thus, the pallium generates mainly excitatory glutamatergic projection neurons, whereas the subpallium produces inhibitory Ȗ-aminobutyric acid (GABA) and cholinergic interneurons (Hebert and Fishell, 2008; Moreno et al., 2009). In addition, neurons moving from a specific germinal subregion via radial migration to their differentiating/mantle zone are mainly projection neurons, whereas a substantial number of cells migrating to their final position through tangential migration correspond to interneurons (Hebert and Fishell, 2008; Moreno et al., 2009; Corbin and Butt, 2011)

Figure 2. Schematic illustration of the forebrain germinal regions. Forebrain is divided in dorso- ventral axis to dorsal pallium (cortex and hippocampus) and ventral subpallium (ganglionic eminences and septum), which are separated by pallial-subpallial boundary region. Interneurons generated in subpallial regions migrate into their final locations by using tangential migration (arrows). CX, cortex; HC, hippocampus; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; POA, preoptic area; PSB, pallial-subpallial boundary.

1.1.3.1.1 Radial migration

Radial migration is best understood in the cortex, where RGCs extend long processes from the VZ to pial surface of the cortex, providing permissive paths for the immature neurons to migrate into their destinations (Cayre et al., 2009). Early studies have shown that the cortex consists of layers, which are formed in an inside-out manner, with deep layers forming first and more superficial layers later (Angevine and Sidman, 1961; Rakic, 1974; Huang, 2009). As such, postmitotic neurons 5 originating from the VZ/SVZ must bypass earlier generated cells to reach their final destinations in the appropriate cortical layer, where they differentiate mainly into projection neurons (Angevine and Sidman, 1961; Rakic, 2007). This is achieved in close alignment to the processes of RGCs, which are guiding the radial migration in two distinct modes: somal translocation and glia guided locomotion (Figure 1D; Nadarajah et al., 2001; Kriegstein and Noctor, 2004; Rakic, 2007; Huang, 2009). Translocating neurons most probably inherit their parent RGC’s radial process, which is attached to pial surface or marginal zone, and moves the cell body to the appropriate position in the cortex (Nadarajah et al., 2001; Kriegstein and Noctor, 2004). In contrast, locomoting neurons do not have a contact to pial surface or marginal zone and they migrate along adjacent RGC processes to reach their appropriate locations (Nadarajah et al., 2001; Kriegstein and Noctor, 2004; Huang, 2009).

1.1.3.1.2 Tangential migration

Most cortical interneurons arise from ventral forebrain ganglionic eminences and migrate tangentially, parallel to tracks, before they turn and migrate radially into the cortex (Figure 2; Marin and Rubinstein, 2003; Metin et al., 2006; Huang, 2009). However, local interneuron progenitors have been identified in human cortical SVZ (Letinic et al., 2002). The main sites that give rise to cortical interneurons have been identified as the MGE and LGE in the ventral forebrain (Corbin et al., 2001). To reach the cortex, interneuron precursors use two main tangential migration routes; they either migrate through the cortical marginal zone (MZ) or along the subventricular/ intermediate zone (SVZ/IZ). After reaching their destination, interneurons integrate into the cortical plate via radial migration (Corbin et al., 2001; Marin and Rubenstein, 2003; Huang, 2009). In their long journey, interneurons are guided by several specific molecular cues. For example, MGE born interneuron precursors avoid entering the striatum by semaphorin (Sema3A and 3F) mediated chemorepulsion (Marin et al., 2001). Despite the fact that interneurons have distinct origins and migratory paths they seem to integrate into cortical layers in a similar inside-out manner as the projection neurons (Pla et al., 2006; Huang, 2009).

1.1.3.1 Specification of pallium

At the early stages of pallial development, morphogens such as FGFs, WNTs, SHH, and BMPs, as well as retinoic acid have been shown to control the graded expression of the Lhx2, Foxg1, CoupTF1, Pax6, Sp8 and Emx2 transcription factors in the germinal zones of the pallium. Together, these transcription factors have a crucial role in specifying the RGCs and IPCs that give rise to the different subclasses of projection neurons into the cortex and establish the cortical stem/progenitor domains in the VZ/SVZ (Molyneaux et al., 2007; Borello and Pierani, 2010; Hansen et al., 2011). Although some progress has been made to understand the transcriptional regulation behind the production of various cortical projection neuron types, the molecular mechanisms that govern the subtype of cortical neurons produced by RGCs and IPCs at different times remain largely unknown (Molyneaux et al., 2007; Hansen et al., 2011). For example, it is not clear whether the same genetic markers can be used to identify RGCs and IPCs of each neuronal subtype. Nevertheless, some of the neuronal subtype specific genes are expressed, both in particular cortical layers and in subpopulations of neurons in the VZ/SVZ, where they might label RGCs, IPCs or early postmitotic neurons of that same neuronal subtype (Molyneaux et al., 2007; Molyneaux et al., 2009).

1.1.3.2 Specification of pallial-subpallial boundary (PSB)

Recent studies have demonstrated that the developmental regulators PAX6 and GSX2 are required for the maintenance of dorso-ventral identity and, in particular, for the correct formation of the 6 boundary between the pallium and the LGE, the PSB (Figure 2; Corbin et al., 2000; Toresson et al., 2000; Yun et al., 2001; Cocas et al., 2011). GSX2 is known to repress dorsal genes like Pax6 and enhance the expression of ventral regulators including Ascl1 and Dlx in VZ/SVZ progenitors of the LGE (Toresson et al., 2000; Wang et al., 2009). For example, in Gsx2 mutant mice, the expression of Ascl1 and Dlx genes is lost from most of the progenitors in the LGE and is replaced by ventrally expanded dorsal regulators such as Pax6, Ngn1, and Ngn2 (Toresson et al., 2000; Stenman et al., 2003). Nevertheless, Gsx2 and Pax6 expressing progenitor cells in the PSB generate multiple inhibitory interneuron subtypes to the olfactory bulb and amygdala, and a subpopulation of amygdala excitatory neurons (Stenman et al., 2003; Carney et al., 2006; Waclaw et al., 2009; Cocas et al., 2011). Therefore, the role of PAX6 and GSX2 in the PSB is essential for the proper generation of specific neuronal subtypes and not only for patterning of stem/progenitor domains during embryonic development (Cocas et al., 2011)

1.1.3.3 Specification of subpallium

1.1.3.3.1 Lateral ganglionic eminence (LGE)

The LGE appears as an observable bulge between the PSB and MGE during the development of the subpallium (Figure 2). From a molecular point of view, the LGE is defined by the expression of Dlx1/2/5 and, in some sub-areas of Isl1, Gsx2, ER81 and Pax6, and by the lack of Nkx2.1 expression (Stoykova et al., 1996; Szucsik et al., 1997; Sussel et al., 1999; Flames et al., 2007). The dorsal border of the LGE with PSB is characterised by the expression of Tbr2, Ngn2 and Dbx1, and absence of Dlx2 expression. Whereas, the ventral limit of LGE with the MGE is defined by Nkx2.1 expression (Puelles et al., 2000; Yun et al., 2001; Moreno et al, 2009). The earlier impression was that the VZ of LGE contains two different progenitor pools, the dorsal and ventral LGE (Puelles et al., 2000; Toresson et al., 2000; Yun et al., 2001; Stenman et al., 2003). A more recent study, based on transcription factor expression profiles, proposes that the LGE contains at least four distinct progenitor regions (Flames et al., 2007). Flames et al. (2007) suggested that the dorsal LGE contains two distinct progenitor domains, named pLGE1 and pLGE2 (p stands for progenitor domain), which are distinguishable by their differential expression of Pax6 and ER81 transcription factors. The remaining pLGE3 and pLGE4 in the ventral portion of the LGE, contains Dlx1/2- and Isl1- expressing progenitor cell populations and, in addition, pLGE4 contains Nkx6.2 expressing progenitor cells (Flames et al., 2007; Moreno et al., 2009). From these distinct progenitor domains, LGE significantly contributes to the neuronal development of striatum, septum, and olfactory bulb. At later stages, the LGE also produces subtypes of cortical and hippocampal interneurons (Pleasure et al., 2000; Anderson et al., 2001; Stenman 2003; Moreno et al., 2009). From a molecular perspective, some of the earliest and most important genes expressed in the LGE are the Gsx genes (Gsx1 and Gsx2), which regulate the early specification of LGE progenitors (Pei et al., 2011). Interestingly, Gsx genes are not only required for the patterning of LGE progenitors, but also to control their proliferative characteristics, similarly, to Pax6 and Emx2 in the pallium (Toresson et al., 2001; Yun et al., 2001; Pei et al., 2011). More specifically, GSX2 is suggested to promote the expression of Ascl1 and Dlx genes in the VZ/SVZ of the LGE, but not in the MGE. This hypothesis is further supported by observations, which show that Gsx2 mutant mice have similar defects in the striatum and olfactory bulb as Ascl1/Dlx mutant mice (Corbin et al., 2000; Toresson et al., 2000; Toresson and Campbell, 2001; Yun et al., 2001). In addition to Ascl1/Dlx genes, expression of transcription factor Sp8 is partially regulated by GSX2 in dorsal LGE (Waclaw et al., 2006; Wang et al., 2009). Sp8 is known to be required for the survival, migration and specification of olfactory bulb interneuron subpopulations (Waclaw et al., 2006). Besides Gsx genes, it has been proposed that the Dlx/Isl1 transcription factor domain that occupies most of the ventral LGE gives rise to

7 striatal projection neurons, whereas the Dlx/Er81 domain in the dorsal LGE generates interneurons to the olfactory bulb (Stenman et al., 2003).

1.1.3.3.2 Medial ganglionic eminence (MGE)

While the border regions between the LGE, MGE and septum are anatomically easy to distinguish from each other, the ventral and caudal edges of the MGE with CGE or telencephalic stalk are difficult to determine (Flames et al., 2007). Gene expression analyses, however, have uncovered that the MGE VZ is defined by the strong expression of Nkx2.1, weak expression of Nkx2.2, and absence of both Pax6 and Shh expression (Sussel et al., 1999; Flames et al., 2007). A more detailed expression pattern analysis completed by Flames et al. (2007) with Dlx2, Gsx2, Er81, Nkx2.1, Nkx2.2, Nkx6.2, Lhx6, Lhx7, Couptf1, and Lhx2 genes have suggested an existence of at least five differently contributing progenitor domains within the MGE in early development (Flames et al., 2007). Altogether, the MGE contributes the majority of its progeny to the cortex by tangential migration, producing mainly GABAergic parvalbumin (PV) and somatostatin (SST) expressing interneurons (Xu et al., 2004; Butt et al., 2005; Ghanem et al., 2007). The MGE also contributes to the neuronal development of the septum, striatum, olfactory bulb, and is a source of some hippocampal neurons (Letinic and Kostovic, 1997; Wichterle et al., 1999; Pleasure et al., 2000; Wichterle et al., 2001). The primary patterning of the MGE progenitors is accomplished by FGF and SHH signaling, which either induces or enables the expression of Nkx2.1 (Fucillo et al., 2004; Gutin et al., 2006; Storm et al., 2006; Xu et al., 2010). On the other hand, the maintenance of Nkx2.1 expression during neurogenesis requires continuous SHH signaling (Anderson et al., 2001; Xu et al., 2004; Xu et al., 2005). The Nkx2.1 is an essential homeobox gene for specifying MGE and repressing LGE (and dorsal CGE) identity (Anderson et al., 1999; Sussel et al., 1999; Corbin et al., 2003; Butt et al., 2008). NKX2.1 exerts this role by regulating the expression of Lhx6 and Lhx7 genes, as the lack of expression of these genes in Nkx2.1 mutant mice has been shown to respecify MGE progenitors to a more dorsal fate resembling that of LGE progenitors (Sussel et al., 1999; Butt et al., 2008). Furthermore, the respecification leads to a reduction of MGE derived cell types in the forebrain (Sussel et al., 1999; Pleasure et al., 2000; Butt et al., 2008). Interestingly, in addition to specifying the MGE, Nkx2.1 expression is also required for the proper migration of postmitotic interneurons from the MGE to the cortex and striatum (Nóbrega-Pereira et al., 2008).

1.1.3.3.3 Caudal ganglionic eminence (CGE)

The CGE is defined as a posterior area, where the caudal extensions of the LGE and MGE fuse together with pallial regions to create a uniform structure (Nery et al., 2002; Flames et al., 2007; Hernandez-Miranda et al., 2010). Flames et al. (2007) have proposed that the anterior CGE contains progenitor domains similar to the LGE and MGE, while posterior CGE domains resemble more the pallial regions. Thus, the CGE does not seem to possess progenitors that are substantially different from those found in the LGE, MGE, or pallium (Flames et al., 2007). The CGE progenitors produce several different neuronal cell types to the cortex, amygdala, and hippocampus (Nery et al., 2002; Lopez-Bendito et al., 2004; Medina et al., 2004; Xu et al., 2004; Tole et al., 2005; Yozu et al., 2005). For example, CGE derived interneurons include cortical interneurons that express neuropeptide vasoactive intestinal peptide (VIP) and SST (Fishell and Rudy, 2011). CGE is also a source of calretinin expressing interneurons similar to the MGE (Wichterle et al., 2001; Xu et al., 2004). The difference between CGE - and MGE derived calretinin expressing interneurons seems to be the differential expression of Lhx6 (Butt et al., 2007). Lhx6 is expressed in all of the MGE- derived NKX2.1-dependent interneurons, whereas the CGE derived interneurons usually do not express Lhx6 (Fogarty et al., 2007; Hernandez-Miranda et al., 2010).

8 1.1.3.3.4 Telencephalic stalk

The AEP/POA lies adjacent to the MGE (Figure 2), and it is also specified by the expression of Nkx2.1 (Sussel et al., 1999). Although AEP/POA shares the expression of Nkx2.1 with MGE, the progenitors in these structures appears to have otherwise distinct gene expression profiles (Flames et al., 2007). As such, they contribute to the generation of cortical GABAergic interneuron populations (Xu et al., 2004; Butt et al. 2005; Hernandez-Miranda et al., 2010). Furthermore, POA derived cortical interneurons born from Nkx5.1 expressing progenitors commonly express neuropeptide Y (NPY), but not SST or other markers of interneurons (Gelman et al., 2009; Hernandez-Miranda et al., 2010).

1.1.4 Forebrain gliogenesis

Gliogenesis initiates later than neurogenesis in the developing mammalian CNS, as the stem/progenitor zones converts their neuron production to the generation of oligodendrocytes or astrocytes (Kessaris et al. 2006; Hochstim et al. 2008; Rowitch and Kriegstein, 2010). The switch from neurons to glial cells is regulated by several subtle changes in stem/progenitor cell programming. For instance, the activity of SHH and OLIG2 together with a dynamic pro-gliogenic transcriptional program is required to promote gliogenesis (Rowitch and Kriegstein, 2010), while a downregulation of proneural basic helix-loop-helix (bHLH) genes is needed to suppress ongoing neurogenesis (Bertrand et al., 2002; Rowitch and Kriegstein, 2010). Interestingly, it seems that BMP signaling inhibits oligodendrocyte production and induces astrocyte production, while SHH signaling seems to have an opposite effect, inducing oligodendrocyte production and inhibiting astrocyte production (Kessaris et al., 2008; Rowitch and Kriegstein, 2010).

1.1.4.1 Specification of astrocytes

It has been proposed that the same RGCs, which first produce neurons can transform into astroglial cells (Noctor et al., 2008) and/or produce more restricted glial IPCs, which then give rise to astrocytes (Kriegstein and Alvarez-Buylla, 2009). Thus, the switch from neuron to astrocyte production in RGCs and IPCs is thought to be mediated by the alterations occurring in the regulation of activators and suppressors related to cell fate specification. In early forebrain development, proneural bHLH transcription factors such as NGN1/2 and ASCL1 are known to suppress gliogenesis (Sun et al., 2001; Parras et al., 2007) by inhibiting the activity of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signalling pathway (JAK– STAT pathway) (Kessaris et al., 2008). As embryonic development continues, the NGN1/2 mediated inhibition of STAT1/3 pathway is released, which activates STAT-mediated transcription of astrocyte specific genes (Kessaris et al., 2008). Another system influencing spatial and temporal regulation of glial cell specification is gene silencing by epigenetic events. For example, the promoter regions of glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100b) are methylated during neurogenesis, repressing the astrocyte development (Takizawa et al., 2001; Namihira et al., 2004). On the other hand, Polycomb group complex promotes gliogenesis by epigenetic mechanisms repressing genes encoding proneural bHLH factors (Hirabayashi et al., 2009). Thus, epigenetic methylation and deacetylation seem to have a dual role in glial specification. Similarly, Notch signaling is promoting gliogenesis and inhibiting neurogenesis through proneural bHLH factors, but also upregulating astrocyte specific genes through activation of the JAK–STAT pathway (Kamakura et al., 2004; Rowitch and Kriegstein, 2010). It has also been proposed that BMPs induce astrocyte specification through forming an active complex between STATs, Sma and Mad related proteins (SMADs) and p300/CBP coactivator family (Allen, 2008; Sanosaka et al., 2009). In conclusion, astrocyte specific genes seem to be repressed at early 9 development and activated later by suppression of proneural genes and/or activating the JAK-STAT signalling pathway. The specification is generally followed by tangential migration of astrocytes along the white matter tracts, before they detach and move into their final destinations in the CNS (Hatton et al., 1993; Jacobsen and Miller, 2003; Rowitch and Kriegstein, 2010)

1.1.4.2 Origin and expansion of forebrain oligodendrocytes

In the forebrain, oligodendrocytes appear in temporal waves (Kessaris et al., 2006). At first, OPCs arise from the ventral neuroepithelium (AEP and MGE), where they spread throughout the forebrain by migration and proliferation (Spassky et al., 1998; Olivier et al., 2001; Spassky et al., 2001; Tekki-Kessaris et al., 2001; Kessaris et al., 2006; Kessaris et al., 2008). These first OPCs are generated from VZ cells expressing Nkx2.1 (presumably from RGCs) and they arrive to the cortex beginning at ~E16 in mouse (Pringle and Richardson 1993, Spassky et al., 1998; Tekki-Kessaris et al., 2001). This first wave is followed by Gsx2 expressing OPCs emerging from the LGE/CGE regions and later by Emx1 expressing OPCs from the cortex itself (Kessaris et al., 2006). Thus, there seem to be a progressively proceeding temporal wave of OPC production from ventral to dorsal regions in the forebrain (Kessaris et al., 2008; Rowitch and Kriegstein, 2010). Interestingly, the very first cortical Nkx2.1 expressing OPCs, which are generated in the ventral forebrain MGE/AEP regions are later overtaken by the Gsx2 expressing OPCs originating from the LGE/CGE and the endogenous Emx1expressing cortical OPCs (Kessaris et al., 2006; Kessaris et al., 2008). Consequently, the Nkx2.1 derived OPCs and mature oligodendrocytes almost disappear from the cortex by postnatal day 10 (Pn10), although, they remain as a major population in the subpallial region (Kessaris et al., 2006). This phenomenon has led to a proposal by Kessaris et al. (2008) that the selective advantage of later generated OPCs and/or continual turnover of oligodendrocytes from adult SVZ throughout adulthood might lead to the gradual loss of earlier generated OPCs and oligodendrocytes.

1.1.4.3 Specification of oligodendrocytes

The potential of neuroepithelial cells and RGCs to produce oligodendrocytes becomes regionally restricted through the action of organizing signals (similarly as in neurogenesis) such as SHH, FGFs, WNTs, and BMPs, all of which provide positional information through morphogen gradients (Rowitch and Kriegstein, 2010). For example, SHH in the MGE/AEP region is required for the proper production of the first Nkx2.1 expressing OPCs (Spassky et al., 2001). SHH is known to induce expression of two bHLH oligodendrocyte transcription factors, Olig1 and Olig2 (Nery et al., 2001; Kessaris et al., 2008). These Olig-genes mark the early oligodendrocyte lineage and the deletion of these genes leads to a complete absence of oligodendrocytes in mouse embryonic CNS (Zhou and Anderson, 2002; Kessaris et al., 2008). Another inducer of early oligodendrocyte specification is Ascl1, which is expressed at the same time with Olig2 in the MGE/AEP (Parras et al., 2004). In newborn mice, expression of Ascl1 can be observed in a large fraction of OPCs and Ascl1 deficient mice demonstrate severely reduced numbers of oligodendrocytes in their forebrain (Parras et al. 2004; Parras et al., 2007). The later OPC waves, which populate the forebrain from more dorsal domains have been shown to be SHH and ASCL1 independent (Nery et al., 2001; Parras et al., 2007). As OPC specification is activated by specific factors, it is also repressed by specific factors. For example, the homeobox proteins DLX1 and DLX2, which are required for GABAergic neuron production in the forebrain, repress OLIG2- dependent oligodendrocyte formation in the MGE/AEP (Petryniak et al. 2007). Similarly, Notch signaling is generally thought to suppress oligodendrocyte differentiation (Li et al., 2009). Notch signaling directly suppresses myelin basic protein (Mbp) expression and indirectly suppresses oligodendrocyte activators such as Ascl1 and Sox10 (Liu et al., 2006). Interestingly, SOX10 also directly regulates the transcription of 10 myelin genes Mbp and proteolipid protein (Plp), and Sox10 deficient mice have impaired terminal differentiation of oligodendrocytes (Stolt et al., 2002; Li et al., 2009). Another suppressor of oligodendrocyte lineage specification are BMPs, which act through the inhibitor of differentiation (ID) gene products to form heterodimers with OLIG1 and OLIG2 in oligodendrocytes, thereby inhibiting their differentiation (Samanta and Kessler, 2004)

1.1.5 Development of adult germinal zones in forebrain

The transformation of germinal zones from embryonic to adult is a gradual process, as the embryonic VZ/SVZ contains more cells and is remarkably broader than the adult counterpart (Peretto et al., 1999). In the first postnatal week, the germinal zones decrease significantly in size and by end of the second postnatal week they appear roughly similar to those observed in the adult brain (Peretto et al., 1999; Tramontin et al., 2003). Also, RGCs that persist in the germinal zones continue to generate neurons, astrocytes and oligodendrocytes in the first postnatal week. In the second postnatal week, RGCs begin quickly to transform into SVZ radial-like astrocytes and ependymal cells, which are thought to be stem/progenitor cells of the adult mouse brain (Tramontin et al., 2003; Merkle et al., 2004). This transformation of RGCs takes place with the gradual disappearance of VZ, leaving the SVZ in the walls of the lateral ventricles as the sole source of new neurons (Luskin, 1993; Lois and Alvarez-Buylla, 1994). Most of these new neurons migrate via the rostral migratory stream (RMS) into the olfactory bulb, where they continually replace local interneurons (Figure 3A; Lois and Alvarez-Buylla, 1994). Neurogenesis also continues in the hippocampus, particularly in the subgranular layer of the dentate gyrus (Figure 3A; Gould and Cameron 1996, Kempermann et al. 1997). The postnatal/adult neurogenesis in the lateral ventricle walls and in hippocampus is regulated by cell endogenous and exogenous mechanisms, which are significantly similar to embryonic ones, although, the origin and nature of the exogenous signals can vary considerably over time (Ming and Song, 2011).

Figure 3. Schematic drawings of the adult neural stem cell regions in the forebrain. (A) Stem/progenitor cells of the lateral ventricle walls generate new interneurons in the olfactory bulb and stem/progenitor cells of the hippocampus dentate gyrus produce new dentate granule neurons. (B) Illustration of the potential cell lineage relationships in the stem cell niche of the lateral ventricle wall. BV, blood vein; CX, cortex; DG, dentate gyrus; GCL; granule cell layer; GL, glomerular layer; HP, hippocampus; OB, olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular zone

11 1.1.5.1 Specification of adult germinal zones

During brain development, NSCs correspond to RGCs, whereas in the adult brain, a subpopulation of astrocytes in the lateral ventricle wall SVZ and radial astrocytes in the subgranular zone of hippocampus dentate gyrus function as NSCs (Lois and Alvarez-Buylla, 1994; Kempermann et al., 1997). As discussed above, neuroepithelial cells and RGCs in the developing brain become regionally determined for generating specific subtypes of neurons (Guillemot, 2007; Flames et al., 2007; Long et al., 2009; Kriegstein and Alvarez-Buylla, 2009). The adult SVZ-RMS-OB neurogenesis studies have shown that postnatal RGCs and adult radial-like astrocyte cells are also regionally determined (Hack et al., 2005; Kohwi et al., 2005; Lledo et al., 2008; Kriegstein and Alvarez-Buylla, 2009). The adult SVZ has been shown to express transcription factors Dlx1/2, ER81 (Stenman et al., 2003), Ascl1 (Parras et al., 2004), Pax6 (Kohwi et al., 2005; Hack et al., 2005), and Sp8 (Waclaw et al., 2006), which are also expressed in embryonic LGE during development (Lledo et al., 2008). Furthermore, functional studies and gene expression analyses have revealed that these transcription factors are required for the proper generation of different interneuron subtypes into the olfactory bulb (Toresson et al., 2001; Yun et al., 2003; Lledo et al., 2008).

1.1.5.2 Cellular composition of adult SVZ

In the adult SVZ, radial like astrocytes function as NSCs, also known as type-B cells (Figure 3B; Doetsch et al., 1999; Garcia et al., 2004). These GFAP-expressing stem cells are termed ‘‘astrocyte- like’’ because they display many typical glial properties (Liu et al., 2006). These type-B cells divide slowly and give rise to actively proliferating transit-amplifying cells (type C cells), which then divide rapidly and give rise to neuroblasts, the type A-cells (Doetch et al., 1997; Doetch et al., 1999; Lois and Alvarez- Buylla, 1994). The type-A cells migrate to the olfactory bulb via tangentially oriented chains and differentiate mainly into different subtypes of interneurons (Lois and Alvarez-Buylla, 1994). The neuroblast chains are ensheathed by the processes of type-B cells in the anterior and dorsal SVZ, where these chains condense to form the RMS (Lois et al., 1996; Lois and Alvarez-Buylla, 1994; Peretto et al., 1997). The adult SVZ niche also contains an extensive network of blood vessels, which provide an abundant extracellular matrix (ECM) next to the type-B and -C cells (Figure 3B; Mercier et al. 2002; Shen et al. 2008; Tavazoie et al. 2008). Interestingly, blood vessels and surrounding ECM is often connected to proliferating SVZ cells (Kerever et al., 2007; Tavazoie et al. 2008), suggesting that the blood vessels might provide a regulatory environment for the type-B and -C cells in the SVZ (Kriegstein and Alvarez-Buylla, 2009).

1.1.5.3 Adult ependymal cells as neural stem/progenitor cells

In the adult mouse brain, ependymal cells compose one cell thick lining, which separates the SVZ from the ventricle (Figure 3B). Ependymal cells are characterized by their cuboidal cell shape, surface microvilli and cilia together with adherens junctions with neighboring cells (Rakic and Sidman, 1968; Spassky et al., 2005). During brain development, most ependymal cells are generated from RGCs between E14 and E16 (Spassky et al., 2005). It has been proposed that RGCs give rise to ependymal cells by asymmetric division and/or by direct transformation of RGCs into ependymal cells (Tramontin et al., 2003). Although ependymal cells are generated early in development, they start to differentiate later, which is implicated by the appearance of cilia during the first postnatal week (Spassky et al., 2005; Jacquet et al., 2009). Furthermore, the temporal regulation of ependymal cell differentiation is delayed in the ventricular regions, where the neurogenesis continues through adulthood (Jacquet et al., 2009). The question of adult ependymal cells as NSCs has attracted great interest in many studies. It seems that embryonic/neonatal 12 ependyma contains RG, ependymal, and type-B cells in different spatiotemporal manners (Shen et al., 2008; Jacquet et al., 2009). Thus, the ependymal lining of lateral ventricles appear to contain cells in different maturation state depending on a regional position of a given cell. These reasons may also explain the opposite findings, which suggest that ependymal cells may or may not act as neural stem/progenitor cells (Coskun et al., 2008; Gleason et al; 2008; Capela and Temple, 2002; Pfenninger et al., 2011). Furthermore, it has been proposed that ependymal cells may act as neural stem/progenitor cells only in a specific situation, for example, after ependymal injury (Luo et al., 2006; Luo et al., 2008; Carlen et al., 2009; Nomura et al., 2010).

1.2 Olfactory system

The mammalian olfactory perception initiates, when molecular odorants in inhaled air dissolve into nasal mucus and bind to specific odorant receptors expressed on the cilia of olfactory sensory neurons (OSNs) in the olfactory epithelium (Figure 4; Buck, 1996; Schwob, 2002) Every OSN expresses only one odorant receptor belonging to a large odorant receptor family (~1000-2000 in mouse) (Buck and Axel, 1991; Whitman and Greer, 2009). The OSNs transform the olfactory perception into electrical impulses, which travel in the olfactory nerve through the cribriform plate into the glomerular layer of the olfactory bulb (Doucette, 1984; de Castro, 2009). In the olfactory bulb, the OSNs axons form synapses with the dendrites of periglomerular interneurons and secondary projection neurons, mitral- and tufted cells, within a spherical structure of neuropil called glomerulus (Buck, 1996; Wilson and Mainen, 2006; Zou et al., 2009). So, the information of one inhaled odour travels from olfactory epithelium through the OSN axon to a single specific glomerulus, where the information is processed by the intrabulbar circuits of secondary projection and interneurons. Furthermore, each secondary projection neuron only receives afferent input from one glomerulus, passing the odour information via the lateral olfactory tract (LOT) to the olfactory cortex (Buck, 1996; Zou et al., 2009; de Castro, 2009). In rodents, there are about 2000 glomeruli in the olfactory bulb, each receiving input from 1000 - 3000 OSNs, and each is innervated by about 25 mitral cells and 50 tufted cells (Buck, 1996). Interestingly, the olfactory system is not static throughout life, as OSNs in the olfactory epithelium and interneurons in olfactory bulb are continuously replaced (Alvarez-Buylla and Garcia-Verdugo, 2002; Beites et al., 2005). The newly generated OSNs and interneurons must find their specific locations, recreate their synaptic targets and integrate into existing circuitry, before they can be a new functional part of ongoing processes (Beites et al., 2005; Lledo et al., 2008). Nevertheless, the olfactory bulb projection neurons are not continuously replaced (Mizrahi and Katz, 2003). Interestingly, it seems that the human olfactory bulb neurogenesis declines gradually with aging and is almost lost during adulthood (Sanai et al., 2011; Bergmann et al., 2012; Ernst et al., 2014). However, Bergmann et al. (2012) reported that the turnover of the non-neural cells continues in the adult human olfactory bulb. Thus, the major difference between mice and humans seem to be that the neural precursor/progenitor cells generated in the lateral ventricle wall do not migrate into the olfactory bulb in adult humans (Ernst et al., 2014).

13 Figure 4. Schematic illustration of olfactory wiring in mouse. The odor information travels from olfactory epithelium through olfactory nerve to the olfactory bulb, where it is transmitted to several brain regions via lateral olfactory tract. A, amygdala; AON, anterior olfactory nucleus; EC, entorhinal cortex; GL, glomerulus; LOT, lateral olfactory tract; MTC, mitral cell; NC, nasal cavity; OE, olfactory epithelium; OM, odor molecule; ON, olfactory nerve; OSN, olfactory sensory neuron; OT, olfactory tubercle; PC, piriform cortex

1.2.1 Development of olfactory system

The early development of the olfactory system starts from the nasal placode, which forms into the pre-placodal non-neural ectoderm in the anterior region of the embryo (Figure 5A; Whitlock and Westerfield, 2000; Brugmann and Moody, 2005; Whitlock, 2008). The nasal placode becomes evident as a cellular thickening in the rostrolateral surface of the mouse embryo at E9 (Key, 1998). A day later at E10.5, the nasal placode invaginates to form a simple nasal pit, which is also the beginning of the nasal cavity. As development continues, the nasal pit invaginates further and divides into more complex olfactory and vomeronasal cavities with distinguishable epithelia. At the same time, OSNs in the olfactory epithelium begin to differentiate and send their axons through the basement membrane of the olfactory epithelium towards the forebrain (Figure 5A; Cuschieri and Bannister, 1975; Hinds, 1972). These OSN axons extend toward the forebrain in association with a heterogenic population of migrating cells, which are also derived from the nasal placode. Thus, in one day, bundles of OSN axons, as well as the migratory cells, have grown from the nasal pit and formed an aggregate referred to as the “migratory mass”, which contacts the rostral tip of the forebrain at E11.5 in mice (Key, 1998; Bailey et al., 1999). It has been suggested that the outward expansion of the olfactory bulb (evagination) starts from this contact area, also referred to as presumptive olfactory bulb, and is initiated by the penetration of OSN axons through the forebrain basement membrane deep into the brain VZ region (Figure 5B; Gong and Shipley, 1995; Key, 1998; Bailey et al., 1999). It has been proposed that these deep penetrating OSN axons inhibit progenitor proliferation and increase the number of differentiating cells, which leads to an evagination of the olfactory bulb at the anterior tip of the forebrain (Gong and Shipley, 1995). However, this view has been challenged by studies conducted with Pax6 and Dlx5 mutant mice, which suggests that evagination of the olfactory bulb can initiate without any deep penetrating OSN axons (Jimenez et al., 2000; Long et al., 2003; Levi et al., 2003) Thus, the signaling which induces the formation of the olfactory bulb requires further investigation. It seems, however, that FGF signaling is one of the inhibitors of the presumptive olfactory bulb progenitor proliferation (Hebert

14 et al., 2003). The presumptive olfactory bulb is first composed of two layers of cells: 1.) a deep VZ, which consists of rapidly proliferating cells organized in a radial orientation, and 2.) a superficial mantle layer, which contains mostly postmitotic cells that are more randomly orientated (Hinds, 1972, Gong et al., 1994). Later at E12-13, the olfactory bulb acquires its distinct bulbar shape and two new cell layers, a SVZ and IZ emerge between already existing VZ and mantle layer (Bailey et al., 1999; Blanchart et al., 2006). At the same time, OSN axon numbers start to increase, eventually forming the olfactory nerve layer surrounding most of the olfactory bulb surface and IZ starts to differentiate into a granular cell layer. At E14-16, glomerular initiation starts when the OSN axons begin to coalesce into axonal glomeruli and the radial glial processes start to form small tufts, called glial glomerulus (Figure 5C). Together these two processes form a protoglomeruli, which contains both axonal and glial components. The maturation of glomeruli will continue until postnatal ages by refinement of the axonal-, dendritical- and glial components of glomeruli. After gromerular initiation, the VZ disappears, leaving the SVZ as the deepest layer in the olfactory bulb. Also at this stage the granular cell layer and mitral cell layer begin to be separated by the developing external plexiform layer (Bailey et al., 1999; Blanchart et al., 2006). Finally, the mature olfactory bulb consists of six well defined layers; olfactory nerve layer (ONL), glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL), internal plexiform layer (IPL), and granular cell layer (GCL) (Figure 5D). At the cellular level, it has been shown that projection neurons are mainly generated from olfactory bulb stem cells whereas most of the interneurons originate from the anterior forebrain (Figure 5E; Puche and Shipley, 2001; Lledo et al., 2008). The origin of astrocytes and oligodendrocytes is not as well understood as neurons. However, recent studies have shown that astrocytes and oligodendrocytes may originate partly from intrinsic olfactory bulb progenitors and partly from brain derived progenitors (Ganat et al., 2006; Mendez-Gomez et al., 2011).

Figure 5. Schematic illustration of olfactory bulb development. (A) Olfactory placodes invaginate to form nasal pits which are lined with olfactory epithelium. As development continues nasal pits invaginate further and form a more complex nasal cavity, which is lined with the olfactory epithelium containing OSNs. OSNs send their axons towards anterior forebrain and, (B) as these

15 axons reach the anterior end of the forebrain they are thought to initiate evagination of the olfactory bulb. (C) At the same time, RGCs of the olfactory bulb start to produce projection neurons, which migrate radially into their appropriate positions in the olfactory bulb. In addition, OSN axons cooperate with RGC processes in the olfactory bulb GL to form the first glomeruli. (D) In the six layered mature olfactory bulb, OSN axons form synapses with glomerular, tufted, and mitral cells in the glomeruli, from where odour information is further transmitted into the brain through lateral olfactory tract. (E) Olfactory bulb interneurons are mainly produced outside of the olfactory bulb, where they migrate tangentially through RMS to the olfactory bulb. CX, cortex; EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; LOT, lateral olfactory tract; LV, lateral ventricle; MCL, mitral cell layer; ONL, olfactory nerve layer; NC, nasal cavity; NP, nasal pit; OE, olfactory epithelium; OSN, olfactory sensory neuron; RMS, rostral migratory stream; SVZ, subventricular zone; VZ, ventricular zone.

1.2.1.1 Radial glia of olfactory bulb

Similar to the cortex, the olfactory bulb contains RGCs, which are likely to be important for early embryonic radial migration. It has been suggested that the olfactory bulb contains two types of RGCs, which can be distinguished by their processes (Figure 5C; Puche and Shipley, 2001). Type I RGCs have a process that extends from the epithelium of the olfactory bulb ventricle into the GL, starting at E13.5. These apical processes form the “glial glomeruli” at the same time when OSN axons are forming the “axonal glomeruli” (Figure 5C; Puche and Shipley, 2001). In type II RGCs, the apical process does not enter into the GL, but instead branches within the EPL. It is also likely that olfactory RGCs support the neuronal migration in the olfactory bulb, similarly as in the cortex (Puche and Shipley, 2001). The tight spatiotemporal relationship between the glomerulization of RGCs processes and OSN axons may also have a role in formation and/or stabilization of mammalian glomeruli (Puche and Shipley, 2001). Interestingly, the olfactory RGCs seem to transform into astrocytes after birth, showing resemblance to the lateral ventricle RGCs (Bailey et al., 1999; Alves et al., 2002)

1.2.1.2 Development and origin of olfactory bulb projection neurons and LOT

The neural cell types in the olfactory bulb are generated in a sequential manner during development (Hinds, 1968; Bayer, 1983). The projection neurons, mitral and tufted cells, are generated first and the interneurons later (Hinds, 1968; Bayer, 1983). Mitral cells are the first neurons produced in the olfactory bulb VZ, from where they migrate radially towards the pial surface, reaching their final position in MCL (Hinds, 1968; Bayer 1983; Blanchart et al., 2006). At E11-E13, mitral cells migrate radially from VZ to IZ, there they adopt a tangential orientation and start axonogenesis leading to the formation of the LOT (Hinds, 1968; Blanchart et al., 2006). At E14-E16, mitral cells turn and adopt their final radial orientation together with their dendrites. The transition of mitral cell orientation is associated with a formation of protoglomerulus. Protoglomerulus preceed the mature glomerulus and it start to form at E15, when OSN axons penetrate the olfactory bulb and meet the mitral cell dendrites in the MCL (Hinds and Ruffett, 1973; Blanchart et al., 2006). After E16 to adulthood, mitral cells and their dendrites are refined and matured (Blanchart et al., 2006). The second type of projection neurons, the tufted cells, are born after mitral cells and overlapping times with granule cells at late embryonic and early postnatal stages (Hinds, 1968; Altman, 1969; Bayer, 1983). Tufted cells migrate radially past the mitral cells to the GL, whereas granule cells remain in the GCL. Thus, cells born during the same period of time migrate to different positions in the olfactory bulb (Hinds, 1968; Blanchart et al., 2006). Interestingly, olfactory RGCs consistently extend extensive side branches just below the MCL forming a deep plexus of processes. These deep side branches of RGCs have been proposed to provide structural support for the formation of GCL 16 (Puche and Shipley, 2001). It has been suggested that mitral- and tufted cell axonogenesis and formation of LOT does not depend on innervation by OSNs (Lopez-Mascaraque and de Castro, 2002; Walz et al., 2006a). This finding is strengthened by the presence of mitral and tufted cell projections in Pax6- and Dlx5 mutant mice, where the OSNs fail to innervate the olfactory bulb (Jimenez et al., 2000; Long et al., 2003; Levi et al., 2003). The LOT can be observed first at E12 in mice (Walz et al., 2006b; de Castro, 2009). However, the first extending collateral axons of LOT reach and innervate their primary target areas in the olfactory cortex at E16 (Figure 4; Walz et al., 2006b). The LOT axons extend to olfactory cortex areas in an anterior to posterior order and they innervate these areas in the following sequence: anterior olfactory nucleus, olfactory tubercle, piriform cortex, several amygdaloid nuclei, and entorhinal cortex (Devor, 1976; Derer et al., 1977; Scott et al., 1980; Schwob and Price, 1984; de Castro, 2009). So by birth, LOT has grown in size and most of the olfactory cortex connectivity is established together with synaptic connectivity in the olfactory bulb (Hinds and Hinds, 1976; Schwob and Price, 1984; Waltz et al., 2006b). The myelination of LOT follows the establishment of connectivity and is initiated along the entire length of LOT at P7 in mouse (Foran and Peterson, 1992: Philpot et al., 1995).

1.2.1.3 Development and origin of olfactory bulb interneurons

Interneurons modulate the activity of major projection neurons in the olfactory bulb and play essential roles in olfactory information processing in GCL and GL (Shipley et al., 2004). Most granule and many periglomerular interneurons are GABAergic, but a significant number of periglomerular cells are dopaminergic and a subpopulation of the dopaminergic cells also co- express GABA (Kosaka et al., 1995; Kosaka et al., 1998; Kosaka et al., 2005). Granule cells are the largest population of interneurons in the olfactory bulb, comprising 90% of the total population and out-numbering mitral cells by approximately 100:1 (Lledo et al., 2008; Whitman and Greer, 2009). There are at least three subpopulations of granule cells in the GCL, which are distinguished by their position and cellular contacts in the olfactory bulb (Whitman and Greer, 2009). The GL of the olfactory bulb contains at least three nonoverlapping periglomerular interneuron subtypes: tyrosine hydroxylase (TH), calbindin (CB), and calretinin (CR) producing interneurons (Kosaka et al., 1995; Kosaka et al., 1998; Batista-Brito et al., 2008). The origin of these different interneuron subtypes of the olfactory bulb has been under substantial examination. It has been proposed that the earliest olfactory bulb interneurons are produced from local progenitors (Vicario-Abejon et al., 2003; Lemasson et al., 2005; Vergano-Vera et al., 2006), which are followed by SVZ and RMS derived interneurons (Figure 5E; Lledo et al., 2008; Merkle et al., 2007; Ventura et al., 2007). Apart from the olfactory bulb interneuron origin, it seems that the expression of Dlx1 and/or Dlx2 in interneuron progenitors is an absolute requirement for the proper generation of interneurons into the olfactory bulb (Anderson et al., 1997; Bulfone et al., 1998; Long et al., 2007; Batista-Brito et al., 2008)

1.2.1.3.1 Temporal origin of olfactory interneurons

In mouse, the olfactory interneuron production begins as early as E14.5 (Wichterle et al., 2001; Pencea and Luskin, 2003; Yoshihara et al., 2005), but peaks between E18 and P5 (Hinds, 1968) and continues throughout adulthood (Alvarez-Buylla and Garcia-Verdugo, 2002). The granule cells are mostly generated postnatally, except for the granule cells in the accessory olfactory bulb, which are predominantly produced during the embryonic period (Hinds, 1968; Bayer, 1983). The granule cells generated during the first postnatal week are primarily designated to the superficial part of the olfactory bulb, whereas later born granule cells are more restricted to central areas (Lemasson et al., 2005). It also seems that the subpopulations of granule cells are generated differently, for example, 5T4-antigen containing granule cells are generated at constant rate during embryonic and neonatal 17 periods, whereas the CR producing granule cell population reaches its peak neonatally (Batista Brito et al., 2008). The generation of CR and CB producing periglomerular interneurons occurs mainly postnatally (De Marcis et al. 2007; Batista-Brito et al. 2008), but there seems to be two slightly different views about the temporal generation of TH producing interneurons either during the embryonic period (Batista-Brito et al., 2008) or postnatally (De Marcis et al., 2007).

1.2.1.3.2 Lateral ventricle derived interneurons

Periglomerular interneurons are generated mainly from anterior areas of the SVZ, with TH producing cells rising from the dorsal SVZ areas, CB producing cells from the ventral areas, and CR producing cells from the dorsal and medial SVZ (Lledo et al., 2008; Whitman and Greer, 2009). In turn, superficial granule cells are generated predominantly from dorsal SVZ and deep granule cells from ventral SVZ regions (Lledo et al., 2008; Whitman and Greer, 2009). Genetic fate mapping experiments have revealed, in more detail, that cortical Emx1 expressing SVZ cells generate primarily superficial granule cells, TH producing and few CR producing periglomerular cells, but little or no CB producing periglomerular cells (Kohwi et al., 2007). The interneurons derived from the subpallial Gsx2 or Dlx5/6 expressing progenitor cells appear to generate all periglomerular cell (TH, CR, and CB) subtypes (Kohwi et al., 2007; Merkle et al., 2007; Young et al., 2007). Interestingly, the Nkx2.1 expressing progenitor cells in ventralmost region of the lateral ventricles seem to generate a novel population of olfactory bulb interneurons that are expressing neuronal-spesific proteins HuC/D, but are not producing TH, CB, or CR (Young et al., 2007; Xu et al., 2008).

1.2.1.3.3 RMS derived interneurons

RMS is not only a migratory route for the lateral ventricle VZ/SVZ derived progenitors to the olfactory bulb, but it also contains stem/progenitor cells itself, although, in smaller numbers than SVZ (Gritti et al., 2002; Hack et al., 2005; Alonso et al., 2008). In rodents, the postnatal closure of the olfactory ventricle in the anterior part of the SVZ forms a rostral extension (Peretto et al., 1999) and RGCs in the rostral extension transform into astrocytes to form glial tubes in the mature RMS (Bonfanti and Peretto, 2007; Alonso et al., 2008). These astrocytes are thought to possess similar stem/progenitor cell properties as the radial like astrocytes in the lateral ventricle SVZ and they are found throughout the entire RMS up to the core of the olfactory bulb (Gritti et al., 2002; Alves et al., 2002; Panagiotakos et al., 2007; Merkle et al., 2007; Bonfanti and Peretto, 2007). In addition, RMS expresses the same transcription factors as the lateral ventricle VZ/SVZ during the embryonic and adult period (Parmar et al., 2003; Stenman et al., 2003; Kohwi et al., 2005; Waclaw et al., 2006; Young et al., 2007). Consistent with these observations, the stem/progenitor cells of RMS are able to generate granule and periglomerular interneurons into the olfactory bulb (Merkle et al., 2007; Alonso et al., 2008). In summary, RMS derived interneurons seem to be indistinguishable from lateral ventricle derived interneurons (Young et al., 2007; Alonso et al., 2008), which has led to a concept of neurogenic SVZ-RMS region.

1.2.1.3.4 Origin of adult olfactory bulb interneurons

The olfactory system in the mouse brain displays ongoing neurogenesis in the adult (Figure 3A). In the olfactory system, cells are born in the neurogenic SVZ-RMS area, from where they migrate along the RMS into the olfactory bulb, differentiating into granule or periglomerular interneurons (Alvarez-Buylla and Garcia-Verdugo, 2002; Lledo et al., 2008). The vast majority, about 95%, of the new neurons in the olfactory bulb differentiate into granule cells (Lledo and Saghatelyan, 2005). The minority that differentiate into periglomerular cells, however, form all of the molecular and 18 morphological subtypes of periglomerular cells identified (Bagley et al., 2007; Batista-Brito et al., 2008; Brill et al., 2008). Genetic fate mapping with markers of different embryonic areas has shown that adult SVZ-RMS stem/progenitor cells are largely derived from the MGE, LGE, and cortex (Young et al., 2007). Furthermore, stem/progenitor cells derived from distinct embryonic populations reside in different portions of the SVZ-RMS and give rise to different types of interneurons in the adult olfactory bulb (Young et al., 2007; Batista-Brito et al., 2008).

1.2.1.4 Development and origin of olfactory bulb oligodendrocytes

Mature myelinating oligodendrocytes are generated from proliferative and migratory OPCs during the first few postnatal weeks in rodents (Kessaris et al., 2006; Parras et al., 2007). OPCs express a set of characteristic markers, including platelet-GHULYHGJURZWKIDFWRUDOSKD 3'*)5Į FKRQGURLWLQ sulphate proteoglycan (NG2), SOX10, and OLIG2, which are also co-expressed by many OPCs (Rivers et al., 2008). It has been proposed that at least part of the olfactory bulb oligodendrocytes arise from endogenous OPCs, which are characterized by the expression of proteolipid protein (plp) gene (Spassky et al., 2001). The plp gene encodes two alternative splice variants, PLP and DM-20, which are the major constituents of mature oligodendrocytes and myelin in adults (Spassky et al., 2000). The first plp expressing OPCs are detected in mouse olfactory bulb at E14.5 and they are followed by Olig1/2 and PdgfrĮH[SUHVVLQJ23&VDW( 6SDVVN\HWDO 7KHVHOlig1/2 and PdgfrĮ expressing OPCs are thought to appear from the first wave of MGE and AEP derived oligodendrocytes (see Chapter 1.1.5; Spassky et al., 2001; Kessaris et al., 2006). Therefore, plp expressing OPCs probably belong to a different cell lineage than Olig1/2 and PdgfrĮ expressing OPCs (Spassky et al., 2000; Spassky et al., 2001; Le Bras et al., 2005). Later, at late embryonic and early postnatal stages, OPCs are also derived from the SVZ-RMS area, from where they migrate to the olfactory bulb and differentiate into mature oligodendrocytes or parenchymal proliferative NG2- glia (Nishiyama et al., 1996; Gomes et al., 2003; Parras et al., 2004; Aguirre and Gallo, 2004; Menn et al. 2006; Ganat et al., 2006; Ventura and Goldman, 2007; Maire et al., 2010; Richardson et al., 2011). Interestingly, the parenchymal NG2-glia is thought to persist as local OPCs, which give rise to new mature oligodendrocytes in the early postnatal and adult brain (Zhu et al., 2011; Richardson et al., 2011).

1.2.1.4.1 Origin of adult olfactory bulb oligodendrocytes

It remain somewhat ambiguous, whether olfactory bulb oligodendrocytes are produced in adulthood, but it is known that adult SVZ is able to give rise to oligodendrocytes (Hack et al., 2005; Menn et al 2006; Jackson et al., 2006). These SVZ-produced oligodendrocytes in the adult brain originate from SVZ type-B cells, which express PdgfrĮ (Menn et al. 2006; Jackson et al., 2006) and from transit-amplifying type-C cells that expresses Olig2 (Hack et al., 2005; Menn et al., 2006). In addition, both type-B and -C cells have been shown to generate parenchymal NG2 expressing OPCs (Menn et al., 2006; Gonzalez-Perez and Alvarez-Buylla, 2011). Another unclear issue is the migratory behavior of OPCs originating from the adult SVZ. Menn et al. (2006) has suggested that migration of adult SVZ OPCs is limited to specific locations, excluding the olfactory bulb. However, Suzuki and Goldman (2003) have shown that emerging oligodendrocytes from the neonatal SVZ disperse extensively both radial and tangential planes.

1.2.1.5 Development and origin of olfactory bulb astrocytes and OECs

The olfactory bulb is composed of heterogenous populations of glial cells (Bailey and Shipley, 1993; Goodman et al., 1993; Gonzalez and Silver, 1994). The two outermost layers, ONL and GL are populated first and inner layers later by two types of glial cells, OECs and astroglia (Doucette, 19 1984; Gonzalez et al., 1993). The OECs are derived from the olfactory placode from where they migrate together with OSN axons to the olfactory bulb (Doucette, 1984; Ekberg et al., 2012). The OECs function similarly to the Schwann cells of the peripheral nervous system. They ensheat OSN axons to large bundles, but unlike Schwann cells, OECs do not myelinate OSN axons (Doucette, 1990; Doucette, 1993). The second glial type, astroglia, has been proposed to emerge from early olfactory RGCs, which transform postnatally into GL astrocytes (Gonzalez et al., 1993; Bailey et al., 1999) and from endogenous stem/progenitor cells (Vergano-Vera et al., 2006; Mendez-Gomez et al., 2011). Furthermore, cell lineage studies have shown that GFAP expressing cells can be found throughout the SVZ, RMS, and OB (Gritti et al., 2002; Platel et al., 2009) and some of these astrocytes might migrate postnatally to the olfactory bulb (Ganat et al., 2006).

1.3 Development of forebrain commissures

The mammalian brain hemispheres are connected to each other by three main commissural tracts; the CC, the hippocampal commissure (HC) and the anterior commissure (AC), which transmit information arising from different sense organs (Mihrshahi, 2006). The development of commissures occurs in a sequential manner. The AC forms first, followed by the formation of the HC (Wahlsten, 1981; Katz et al., 1983; Borrell et al., 1999). The CC is thought to form last, in close contact with HC (Wahlsten et al., 2006). Formation of these commissures is controlled by long- and short range guidance molecules, which together with specific cellular populations, guides the axons through the telencephalic midline into the contralateral target area (Richards et al., 2004; Lindwall et al., 2007). Disruptions in axon guidance can cause defective development or complete absence of one or more commissures (Paul et al., 2007; Lindwall et al., 2007). The CC develops exclusively to placental mammals, presenting a true evolutionary novelty (Aboitiz and Montiel, 2003; Mihrshahi, 2006). It has been suggested that CC connects topographic sensory cortices more efficiently than the AC and HC, and provide a faster route for interhemispheric communication (Aboitiz and Montiel, 2003; Mihrshahi, 2006). In mice, development and formation of CC requires first an establishment of a substrate, which is achieved through the fusion of the interhemispheric midline (Silver et al., 1982). The interhemispheric fusion is followed by midline crossing of pioneering axons, which originate from the medial part of the cortex, called cingulated cortex (Figure 6; Koester and O`Leary, 1994). The pioneering axons reach the midline between E14 and E15 in mice, and after crossing the midline they travel to innervate the contralateral cingulate cortex (Koester and O`Leary, 1994; Rash and Richards, 2001). The early pioneering axons originating from cingulated cortex also assist the midline crossing of later callosal axons, which project from neocortical layers 2/3/5 and expand the size of the CC (Polleux et al., 1998; Shu et al., 2003d; Donahoo and Richards, 2009). The callosal axons are also guided towards and cross the interhemispheric midline by specific midline cellular populations: glial wedge (GW), indusium griseum (IG) and subcallosal sling (SCS) (Figure 6; Shu and Richards, 2001). After crossing the midline, callosal axons are further guided towards the homotopic regions of the contralateral cortical layers, where they innervate their target neurons (Donahoo and Richards, 2009). Agenesis of the corpus callosum (ACC) refers to the either partial or complete absence of corpus callosum, which can have genetic and/or environmental background (Paul et al., 2007). In addition, defects in axon guidance and/or in interhemispheric fusion can lead to ACC (Brouns et al., 2000). ACC is also often associated with the formation of apparent longitudinal axon bundles (Probst bundles), which develop ipsilateral to the midline (Donahoo and Richards, 2009). ACC occurs in 1:4000 human individuals (Paul et al., 2007), and is associated with more than 50 different human congenital syndromes (Richards et al., 2004; Ren et al., 2006). Furthermore, over 50 different genes are linked to ACC in humans and mice (Donahoo and Richards, 2009). As CC integrates motor, sensory and cognitive performances between cerebral hemispheres, ACC is also associated with variety of neuropsychiatric and behavioral disorders in humans. The common symptoms of ACC in humans include deficits in 20 speech, verbal intelligence, learning, abstract reasoning, motor coordination, short term memory and sleep (Richards et al., 2004; Paul et al., 2007).

Figure 6. Formation of the CC. Midline glia is thought to facilitate interhemipheric fusion, which takes place prior to the midline crossing of pioneering callosal axons. Pioneering and later arriving callosal axons are guided towards and across the interhemispheric midline by transient cellular structures; glial wedge, induseum griseum and subcallosal sling. IG, induseum griseum; CX, cortex; GW, glial wedge; LV, lateral ventricle; MG, midline glia; SCS, subcallosal sling.

1.3.1 Fusion of forebrain hemispheres

Interhemispheric fusion precedes the formation of forebrain commissures and it progresses in two distinct phases (Silver et al., 1982). First, the rostral regions of the forebrain are fused ventrally, which is followed by a fusion in more dorsal regions, where the callosal axons will cross the midline and form the CC (Silver et al., 1993; Richards et al., 2004). In mice, midline fusion begins from the front of the lamina terminalis at approximately E14-15, and progresses rostrally immediately ahead of the arriving commissural axons (Silver et al., 1982; Silver et al., 1993). Consequently, the midline fusion follows ventral-dorsal and caudal-rostral directions in the developing rodent brain (Silver et al., 1982; Silver et al., 1993). The midline fusion also requires removal of the leptomeninges found between the hemispheres (Donahoo and Richards, 2009). The precise molecular and cellular events related to midline fusion are still poorly understood. However, it has been proposed that a midline zipper glia (MZG), arising in the area, could facilitate the fusion by a yet unknown mechanism (Figure 6; Silver et al., 1982; Silver et al., 1993; Richards et al., 2004). Although, there is a shortage of information related to the midline fusion mechanisms, it is known that the fusion is a critical step for the formation of the CC (Silver et al., 1982; Silver and Ogawa, 1983; Richards et al., 2004; Wahlsten et al., 2006). A defective midline fusion is often associated to the interhemispheric cysts, which are also a common cause of ACC (Richards et al., 2004). For example, the mouse strains BALBc/Wah1 and 129 J (Silver et al., 1982; Wahlsten et al., 2006), as well as mice carrying mutation in hydrocephalous with hop gait (Paez et al., 2007), L1 (Demyanenko et al., 1999), p190-rhoGAP (Brouns et al., 2000) and in myristoylated, alanine-rich C kinase substrate (Stumpo et al., 1995) genes exhibit ACC, which is suggested to be a result of defective midline fusion (Donahoo and Richards, 2009). Thus, Donahoo and Richards (2009) have proposed that in some cases interhemispheric cysts are related to a defective midline fusion.

1.3.2 Role of midline cellular populations in development of CC

The developing CC is surrounded by transient cellular structures, which regulate commissural axon guidance towards the brain midline (Figure 6; Lindwall et al., 2007). These midline structures have been demonstrated to exist in mouse (Shu et al., 2001; Silver et al., 1982), human (Lent et al., 2005; 21 Ren et al., 2006), fly (Hidalgo and Booth, 2000) and zebrafish forebrain (Barresi et al., 2005). As mentioned above, MZG is thought to regulate the fusion of hemispheres at the interhemispheric midline (Silver et al., 1982; Lindwall et al. 2007). The other midline structures, such as GW and IG, guide callosal axons towards the midline by secreting axon guidance molecules (Shu and Richards, 2001). The GW and IG locate to ventral and dorsal side of the developing CC (Silver et al., 1982; Shu and Richards, 2001). The SCS is a U-shaped structure at the cortico-septal boundary (CSB) region between cerebral hemispheres, and is thought to serve as a guidance substratum for the callosal axons (Silver et al., 1982; Shu and Richards, 2001).

1.3.2.1 Glial wedge (GW)

GW is a specialized wedge-shaped glial structure positioned at the medial aspect of the lateral ventricles, from where GW forming cells migrate and send long radial glial-like processes toward the interhemispheric midline (Silver et al., 1982; Shu and Richards, 2001; Shu et al., 2003c). The GW begins to develop at E13.5 and the cells of the GW are amongst the earliest GFAP expressing glial populations to arise in the murine forebrain (Shu and Richards, 2001). New GW cells are generated until Pn2, and GW loses its distinctive shape by Pn10 (Shu et al., 2003c). At E17, GW cells are positive for molecular markers of RGCs, including radial glial cell marker-2 (RC2) and Nestin (Shu et al., 2003c). The GW has been thought to guide callosal axons towards the midline by short range chemorepulsion (Silver et al., 1982; Shu and Richards, 2001; Shu et al., 2003b). It has been shown that this short range chemorepulsion is mediated by SLIT- roundabout (ROBO) signaling (Shu and Richards, 2001; Shu et al., 2003d). In forebrain, the callosal axons express SLIT2 receptors Robo1 and 2, as they grow towards the lateral part of the GW, which expresses chemorepellent Slit2 (Shu et al., 2003d). Consequently, callosal axons turn sharply towards the midline and continue growing along the GW (Shu et al., 2003d). In summary, both the shape and location of the GW direct cortical axons towards the interhemispheric midline and prevent them from entering the underlying septum (Shu and Richards, 2001; Shu et al., 2003c; Shu et al., 2003d)

1.3.2.2 Indusium Griseum (IG)

The IG is composed of neurons and glial cells, which are situated between the pial membrane and dorsal CC (Figure 6; Sturrock, 1978; Smith et al., 2006). The IG has been proposed to act as dorsal repulsive barrier for callosal axons, directing them dorsally towards the midline (Shu and Richards, 2001). Furthermore, IG-induced repulsion is suggested to be mediated by SLIT-ROBO signaling, analogous to GW (Shu and Richards, 2001). The IG is formed by the RGCs of the dorsomedial ventricular wall, which retract their apical endfeet and translocate into the overlaying pial surface, starting at E14.5 in mice (Smith et al., 2006). In the absence of fibroblast growth factor receptor 1(FGFR1), the translocation of these RGCs is disrupted and the IG does not form properly (Smith et al., 2006). Interestingly, FGFR1 deficiency leads to callosal pathfinding defects despite the fact that Slit2 expression is maintained in the area equivalent to IG (Smith et al., 2006). Earlier it was suggested that IG glia and MZG could initially originate from the same population of cells, which are split by the formation of the CSB and the CC (Shu and Richards, 2003b; Moldrich et al., 2010). However, the MZG developed normally in mice where Fgfr1 was inactivated from ventricular RGCs (Smith et al., 2006).

1.3.2.3 Subcallosal sling (SCS)

The SCS is a U-shaped transient structure formed by cells, which are linked together with tight junctions (Silver et al., 1982). The callosal axons cross the interhemispheric midline dorsally to the SCS and GW (Silver et al., 1982; Shu and Richards, 2001). The SCS was originally named as the 22 glial sling, because it was thought to be composed of glial cells (Silver et al., 1982). However, Shu et al. (2003b) showed that SCS cells are not positive for glial marker proteins such as RC2, brain lipid-binding protein (BLBP), glutamate-aspartate transporter (GLAST), or GFAP. Instead, the SCS cells are positive for neuronal markers such as CR, neuronal nuclei (NeuN), and neuron-specific class III beta-tubulin (TUJ1), and demonstrate the electrophysiological properties of neurons (Shu et al., 2003b). The first SCS cells in mice are generated in the medial aspect of the lateral ventricles at E15.5, from where they migrate to the CSB region between cerebral hemispheres, forming a visible “sling” structure by E17.5 (Silver et al., 1982; Hankin and Silver, 1988; Shu et al., 2003b). The SCS cells continue to proliferate inside the SCS at least until Pn2 in mice (Shu et al., 2003b). Despite this ongoing proliferation, the SCS size only increases in proportion to the growth of the whole brain, and actually decreases in size after Pn2, disappearing entirely by Pn10 (Shu et al., 2003b). Two alternative hypotheses have emerged to explain the postnatal disappearance of the SCS. First, Hankin et al. (1988) proposed that macrophage mediated endocytosis leads to a disappearance of the SCS. Later, Shu et al. (2003b) has suggested that SCS cells continue to migrate across the interhemispheric midline to adjacent brain regions, which leads to a gradual disappearance of the SCS. Nevertheless, both ablation and rescue experiments (Silver et al., 1982; Silver and Ogawa, 1983), as well as genetic studies have shown that the SCS is required for the proper development of the CC (Shu et al., 2003b; Ha et al., 2005). Albeit, the question of whether callosal axons direct sling cell migration or whether the sling cells guide callosal axons towards the midline is still unresolved, because the pathfinding of callosal axons coincides with the formation of SCS (Donahoo and Richards, 2009).

1.3.3 Callosal axon guidance during formation of CC

Commissural axon guidance towards the interhemispheric midline is a sequential interplay of axon guidance cues (ligands) and their receptors (Donahoo and Richards, 2009). The midline structures are thought to guide growing commissural axons by producing short- or long distance guidance cues and receptors, which either repulse or attract the commissural axons. The axon guidance cues that have been shown to be involved in guiding callosal axons include Netrins, Slits, Ephrins, Semaphorins and Plexins (Serafini et al., 1996; Shu and Richards, 2001; Mendes et al., 2006; Bush and Soriano, 2009; Piper et al., 2009; Hatanaka et al., 2009), and the receptors include members of the Deleted in colorectal cancer (DCC), Neuropilins (Npns), Robo and Ephrin families (Fazeli et al., 1997; Andrews et al., 2006; Mendes et al., 2006; Bush and Soriano, 2009; Piper et al., 2009; Hatanaka et al., 2009). Futhermore, several intracellular downstream targets of these guidance families have been associated to the proper formation of CC (Lindwall et al., 2007; Donahoo and Richards, 2009). These intracellular signaling cascades downstream of guidance cues can directly modify the morphology of the growth cone and, therefore, influence axon guidance (Donahoo and Richards, 2009). In addition to the guidance cues, morphogens (such as SHH, FGFs, and WNTs) also affect the callosal axon guidance by patterning the forebrain midline and interacting with guidance cues (Lindwall et al., 2007; Donahoo and Richards, 2009). It has been shown that both guidance cues and morphogens interact with extracellular heparan sulfates, chondroitin sulfates and glycoproteins, which may have an impact on the localization of the morphogens and guidance cues in the CSB region (Lindwall et al., 2007). Consequently, molecular defects in this diverse callosal axon guidance network can lead to either partial or complete ACC (Lindwall et al., 2007; Donahoo and Richards, 2009).

23 1.4 The Netrin family of guidance factors

1.4.1 History of Netrins

Cajal (1928) suggested that axons might respond to chemoattractive guidance factors, which led to an extensive search for the first axon guidance molecules in the following decades. The first Netrin and its receptor were found in the early 1990s, which led to a major progression in the conception of axon guidance and cell migration mechanisms (Bradford et al., 2009). First, three uncoordinated (Unc) genes (Unc5, Unc6 and Unc40) were identified in C.elegans and mutations in these genes led to disruptions in axonal pathfinding and cell migration (Hedgecock et al., 1990). Furthermore, Hedgecock et al. (1990) proposed that Unc6 gene encoded a guidance molecule that formed a chemotactic gradient, and UNC5 and UNC40 were sensing this gradient by acting as guidance receptors for UNC6. Later, Serafini et al. (1994) and Kennedy et al. (1994) showed that this guidance system was also relevant in vertebrates, when they identified and purified the chicken orthologues of the Unc6 gene, Netrin1 (Ntn1) and 2. In addition, Keino-Masu et al. (1996) and Leonardo et al. (1997) discovered the first mammalian homologs for UNC40 (DCC) and UNC5 (UNC5A-D), demonstrating the conservation of the Netrin dependent guidance system in vertebrates.

1.4.2 Netrins in different animal species

Netrins are expressed in the midline of all animals with bilateral symmetry. Several C.elegans Unc6 orthologues have been described in invertebrates: a single Netrin has been identified in Ciona intestinalis (GI: 7544126) and in Branchisotoma floridae, named amphinetrin (Shimeld S., 2000), NvNetrin has been identified in Nematostella vectensis (Matus et al., 2006) and Netrin in leech (Gan et al., 1999). Two Netrins, Netrin-A and Netrin-B, have been reported in Drosophila melanogaster (Harris et al., 1996; Mitchell et al., 1996). All invertebrate Netrins identified thus far are secreted proteins (Rajasekharan and Kennedy, 2009). A greater diversity in Netrins is seen, to some extent, in vertebrates, although, only one has been described in Xenopus laveis (de la Torre et al., 1997). In zebrafish and chicken, NTN1 and -2 have been reported (Serafini et al., 1994). In mammals, five Netrins have been identified and cloned, NTN1, -3, -4, -G1, and -G2 (Rajasekharan and Kennedy, 2009). Although mammalian Ntn1 is orthologous to chicken Ntn1, no orthologous gene for zebrafish and chicken Ntn2 has been found in mammals (Wang et al., 1999; Barallobre et al., 2005). The expression pattern of Ntn1 in mouse is significantly similar to the combined expression of Ntn1 and 2 in chicken (Püschel A.W., 1999). This observation has led to a proposal that mammalian Ntn1 may perform the functions that correspond to chicken Ntn1 and 2 (Kennedy et al., 1994; Püschel A.W., 1999; Serafini et al., 1994; Serafini et al., 1996; Barallobre et al., 2005). NTN1, 3, and 4 are secreted molecules, whereas NTNG1 and G2 are anchored to the plasma membrane (Nakashiba et al., 2000; Nakashiba et al., 2002).

1.4.3 Molecular structure of Netrins

Netrins are part of the laminin superfamily (Figure 7A; Rajasekharan and Kennedy, 2009). The amino-terminal (N-terminal) sequences of NTN1, 2, and 3 are closely related to N-terminal sequences found in the laminin-Ȗ1 chain (Serafini et al., 1994; Wang et al., 1999), whereas NTNG1, G2, and NTN4 shares the N-terminal similarity with the laminin-ȕ1 chain (Nakashiba et al., 2000; Nakashiba et al., 2002; Yin et al., 2000; Barallobre et al., 2005; Rajasekharan and Kennedy, 2009). 24 Furthermore, the N-terminus of Netrins can be divided into two domains named VI and V (Barallobre et al., 2005; Rajasekharan and Kennedy, 2009). Domain VI is a homologue of the globular domain VI of laminin and it is followed by three domains called V-1, V-2, and V-3, which are EGF repeats found in domain V (Barallobre et al., 2005; Yurchenco and Wadsworth, 2004; Rajasekharan and Kennedy, 2009). The C-terminal domain varies most between species and it is related to similar domains found in a disparate group of proteins that have diverse biological functions (Rajasekharan and Kennedy, 2009). The C-terminal part of Netrins is also called the netrin-like (NTR) domain and it has been identified in complement proteins C3, C4 and C5, which are members of the thioester-FRQWDLQLQJ Į-macroglobulin protein superfamily and components of the innate immune system (Yurchenco and Wadsworth, 2004; Rajasekharan and Kennedy, 2009). The NTR-domain is also related to a domain found in secreted Frizzled-related proteins that are also involved in axon guidance (Serafini et al., 1994; Barallobre et al., 2005; Rajasekharan and Kennedy, 2009) and the type I procollagen C-proteinase enhancer proteins, which are metalloproteinase inhibitors (Banyai and Patthy, 1999; Rajasekharan and Kennedy, 2009).

Figure 7. Structures of Netrins and NTN1 receptors. (A) Netrins belong to the laminin superfamily. ,QJHQHUDOODPLQLQVDUHKHWHURWULPHULFSURWHLQVFRPSRVHGRIĮ-ȕ- DQGȖ-chains. The N-terminus of 171DQGLVKLJKO\KRPRORJRXVWRODPLQLQȖ-chain, whereas the N-terminus of NTN4, G1 and G2 is closely reODWHGWRODPLQLQȕ-chain. The C-terminal part of secreted NTN1-4 proteins contains C-domain, while NTNG1 and NTNG2 are connected to the plasma membrane by GPI linker. (B) Domain structure of NTN1 receptors. Closely related DCC and Neogenin have four immunoglobulin and six fibronectin type III domains in their extracellular part. The intracellular part of the DCC and Neogenin contains P1, P2 and P3 domains. The UNC5 receptors have two immunoglobulin and two thrombospondin type-I domains in their extracellular part, while intracellular part is composed of ZU-5, DB and death domains. The extracellular part of the DSCAM contains ten immunoglobulin and six fibronectin type III domains, whereas the composition of intracellular part is largely unknown. Integrins arH FRPSRVHG RI WKH Į- DQG ȕ- subunits, which contain many different domains in their extracellular part along with short intracellular tails.

1.4.4 Interactions of Netrins

The N-terminal domains VI and V are known to mediate binding interactions between Netrins and their receptors, however, the exact details are as yet unresolved (Rajasekharan and Kennedy, 2009). Evidence from C. elegans demonstrates that domains V-1, V-2, and V-3 are required for UNC5 25 mediated responses, while domains V-1 and V-3 are required for DCC mediated attraction (Lim and Wadsworth, 2002). Interestingly, NTN4 integrates into basement membranes through interaction of its N-terminal domains with laminins (Schneiders et al., 2007). However, other secreted Netrins do not seem to possess this feature (Rajasekharan and Kennedy, 2009), which could be due to the difference in the N-terminal domain of NTN4 compared to other secreted Netrins (see Chapter above). It is also known that NTN3 binds to the DCC receptor with a lower affinity than to the other receptors, indicating that NTN3 acts mainly through other receptors than DCC (Wang et al., 1999). Anyhow, NTN3 can mimic NTN1 functions in vitro by binding to same receptors as NTN1 (Wang et al., 1999). The C-terminus of Netrins is not needed for receptor binding (Keino-Masu et al., 1996; Leonardo et al., 1997; Qin et al., 2007), nevertheless, deletion of the UNC6 C-terminus in C. elegans generates mild axon guidance defects (Lim et al., 1999). Altogether, the C-terminal sequences of Netrins are rich in basic amino acids, which may act as binding sites for heparin, heparan sulfate proteoglycans, membrane glycolipids and integrins, thereby allowing interaction with components of the ECM or cell surface (Kappler et al., 2000; Cirulli and Yebra, 2007).

1.4.5 Interactions and developmental functions of NTN1 in animals

NTN1 is the best characterized member of the Netrin family. It is expressed in developing and adult nervous system, as well as in various tissues outside of the nervous system (Kennedy et al., 1994; Serafini et al., 1994; Livesey and Hunt, 1999). During development of nervous system, NTN1 participates in axon guidance, branching, and arborisation together with synapse formation (Lai Wing Sun et al., 2011). It also has an important role in neuronal migration, survival, and proliferation during neural development (Barallobre et al., 2005). Outside of the nervous system NTN1 has been linked with the morphogenesis of several tissues and organs (see Chapter 1.4.5.5; Livesey and Hunt, 1999; Barallobre et al., 2005; Lai Wing Sun et al., 2011).

1.4.5.1 NTN1 receptor families

The cellular functions of NTN1 are mainly mediated through transmembrane receptors belonging to the DCC, UNC5, DSCAM, and integrin families (Figure 7B; Barallobre et al., 2005; Hedgecock and Norris, 1997; Yurchenco and Wadsworth, 2004).

1.4.5.1.1 DCC and Neogenin

DCC was first identified as a candidate tumour suppressor gene in human colorectal cancer (Fearon et al., 1990) and Neogenin as a novel neural protein with approximately 50% overall homology to DCC (Figure 7B; Vielmetter et al., 1994). Since then, Neogenin and DCC have been identified in many vertebrates, including zebrafish (Hjorth et al., 2001; Shen et al., 2002), frog (Pierceall et al., 1994; Wilson and Key, 2006), chicken (Vielmetter et al., 1994; Chuong et al., 1994), mouse (Cooper et al., 1995; Keeling et al., 1997), and humans (Fearon et al., 1990; Meyerhardt et al., 1997). However, C. elegans and D. melanogaster have only one Neogenin/DCC-like molecule, named UNC40 in C. elegans and Frazzled in D. melanogaster, suggesting that vertebrate Neogenin and Dcc genes have diverged from a common ancestor (Chan et al, 1996; Kolodziej et al., 1996; Cole et al., 2007). The C. elegans UNC40 and D. melanogaster Frazzled function as guidance receptors for axons and migrating cells, similar to the vertebrate DCC (Barallobre et al., 2005). DCC and Neogenin are single-pass transmembrane proteins containing four immunoglobulin domains and six fibronectin type III (FNIII) repetitions in their extracellular part (Cho et al., 1994; Vielmetter et al., 1994; Barallobre et al., 2005; Rajasekharan and Kennedy, 2009). The fourth and fifth FNIII repeat mediate binding of DCC to NTN1 (Geisbrecht et al., 2003; Kruger et al., 2004; Rajasekharan and Kennedy, 2009; Lai Wing Sun et al., 2011). Neogenin binding to NTN1 is also 26 suggested to occur through FNIII domains, based on the 68% amino acid identity shared by Neogenin and DCC in this area (Cole et al., 2007). The intracellular part of these receptors contains three conserved domains, named P1, P2, and P3 (Kolodziej et al., 1996; Hong et al., 1999; Rajasekharan and Kennedy, 2009). These domains mediate homo- or heterodimerisation of the receptors and interact with cytoplasmic signaling molecules (Kolodziej et al., 1996; Hong et al., 1999; Ren et al., 2004; Li et al., 2004; Cole et al., 2007; Rajasekharan and Kennedy, 2009). Also another ligand family has been identified for Neogenin, the repulsive guidance molecule (RGM) family (Monnier et al., 2002). The RGM binding sites are suggested to overlap with NTN1, but there is no direct evidence whether Neogenin can bind simultaneously to RGMs and NTN1 (Rajagopalan et al. 2004; De Vries and Cooper, 2008).

1.4.5.1.2 UNC5 family

The receptors of the UNC5 family include UNC5 of C.elegans (Leung-Hagesteijn et al., 1992) and its four vertebrate homologues: UNC5A-D (Figure 7B; Hedgecock et al., 1990; Ackerman et al., 1997; Leonardo et al., 1997). The UNC5 receptors are transmembrane proteins with an extracellular part containing two immunoglobulin repeats, which are followed by two thrombospondin type-I modules (Leonardo et al., 1997; Rajasekharan and Kennedy, 2009). The immunoglobulin repeats in the extracellular part mediate NTN1 binding (Geisbrecht et al., 2003). The intracellular part of UNC5 receptors contains a ZU-5 domain of undetermined function (Leonardo et al., 1997), DB (DCC-binding) motif (Hong et al., 1999), and apoptotic signaling related death domain (Hofmann and Tschopp, 1995).

1.4.5.1.3 Down’s syndrome cell adhesion molecule (DSCAM)

The mammalian genome encodes two Dscam genes, Dscam and its paralog, Dscam-like 1 (Yamakawa et al., 1998). DSCAM is a transmembrane protein with an extracellular part containing ten immunoglobulin domains and six FNIII repeats (Figure 7B; Yamakawa et al., 1998). The intracellular structure of DSCAM is largely unknown, although some motifs have been predicted (Yamakawa et al., 1998; Barlow et al., 2001; Ly et al., 2008). The extracellular immunoglobulin domains 7–9 of DSCAM are required for NTN1 binding (Ly et al., 2008). It has been shown that DSCAM forms heterodimer complexes with DCC in the absence of NTN1, while the presence of NTN1 leads to the dissociation of these receptor complexes in cell culture conditions (Ly et al., 2008).

1.4.5.1.4 Integrins

Integrins are a superfamily of transmembrane receptors, which are composed of Į and ȕ subunits. Mammals have at least 18 different Į and 8 ȕ subunits, which can compose 24 different integrin heterodimers (Figure 7B; Takada et al., 2007). Each subunit contains several domains in their extracellular part, while the intracellular part is short and unstructured (Takada et al., 2007; Campbell and Humphries, 2011). The integrin ligands bind to the extracellular domains of the heterodimers and can trigger multiple different cell behaviors such as adhesion, proliferation, survival, apoptosis, polarity, motility, gene expression, and cell differentiation (Takada et al., 2007; Campbell and Humphries, 2011). It is known that soluble NTN1 has a direct physical interaction with ȕ4, Į, DQGĮ6 integrin subunits, but not with a ȕ1 subunit (Yebra et al., 2003; Stanco et al., 2009). However, mouse recombinant or secreted NTN1 has been shown in cell culture conditions to increase the active form of ȕ1 in cells extracted from MGE (Stanco et al., 2009). Furthermore, the integrin KHWHURGLPHUVĮȕDQGĮȕELQG to the C-terminus of NTN1 (Yebra et al., 2003; Cirulli and Yebra, 2007), which raises the possibility that NTN1 may simultaneously bind to other 27 receptors with its N-terminus (Nikolopoulos and Giancotti, 2005). Also NTN4 has been shown to interact with integrins Į2ȕ1, Į3ȕ1, and Į6ȕ1 (Larrieu-Lahargue et al., 2011; Yebra et al., 2011).

1.4.5.2 NTN1 and its receptors in axon guidance

Axon guidance is orchestrated by numerous guidance cues acting through distinct receptors at the tip of the axon, in the growth cone. This guidance cue signaling controls the direction of axonal elongation by affecting filamentous actin and microtubule arrangement in the growth cone (Hall and Lalli, 2010). If guidance cues act as chemoattractants, the cytoskeletal rearrangements lead to growth cone extension and axon outgrowth towards the guidance cue source. In contrast, detection of chemorepellent guidance cues leads to depolymerization of the cytoskeleton and growth cone collapse, which results in axon migration away from the guidance cue source (Hall and Lalli, 2010; Dent et al., 2011). The Rho family of guanosine triphosphates (GTPases), in particular Ras related C3 botulinum toxin substrate 1 (RAC1), cell division cycle42 (CDC42) and Ras homolog gene family-member A (RHOA), are known to be important regulators of growth cone extension and axon outgrowth, acting downstream from most of the guidance cue receptors (Hall, 1998; Hall and Lalli, 2010). RAC1 promotes the formation of actin-based lamellipodia, which provides the tension essential for axon elongation. In turn, CDC42 is responsible for the formation of filopodia, which orients the growth cone by sensing the extracellular environment (Hall and Lalli, 2010). RHOA also affects growth cone morphology by regulating the rearrangement of actin cytoskeleton and growth cone collapsion (Wu et al., 2005). Although, RHOA can either inhibit or promote axon elongation depending on the downstream effector (Hall and Lalli, 2010). The attractive guidance cues are thought to promote axon outgrowth through activation of RAC1 and CDC42, whereas repulsive guidance cues induce growth cone collapse via RHOA activation (Bashaw and Klein, 2010; Hall and Lalli, 2010). Guidance cues can act over a short or long distances depending on their interaction with the molecules in ECM (Barallobre et al., 2005). NTN1 can act as chemoattractant or chemorepellent guidance cue, depending on the receptor combination on target cell plasma membrane (Barallobre et al., 2005; Lai Wing Sun et al., 2011). It has been shown that NTN1 mediates attraction mainly through activation of its receptor DCC (Keino-Masu et al., 1996; Fazeli et al., 1997). NTN1 signaling is converted into repulsive by UNC5 receptors, which make a heterodimeric receptor complex with DCC (Leonardo et al., 1997). UNC5 receptors can also act independently of DCC to repel axons at higher NTN1 concentrations (Hong et al., 1999; Keleman and Dickson, 2001). Also a chemorepulsive receptor, ROBO, can form a heterodimeric receptor complex with DCC and silence the attractive responses towards NTN1. The formation of this complex is dependent on the previous interaction between ROBO and its ligand SLIT (Stein and Tessier-Lavigne, 2001). However, the signaling mechanisms facilitating NTN1 induced attraction are far better known than signaling underlying repulsion (Lai Wing Sun et al., 2011). NTN1 has been suggested to act as short and long distance guidance cue. At short distance, NTN1 acts within one or two cell diameters of its source (Baker et al., 2006; Kennedy et al., 2006). Long distance guidance is thought to be achieved through establishment of an NTN1 gradient within extracellular environment. This gradient is also suggested to be further stabilized by the NTN1 interactions with ECM components such as heparan sulfate proteoglycans (Kennedy et al., 1994; Kennedy et al., 2006; Bradford et al., 2009).

1.4.5.2.1 NTN1 mediated signaling in axon chemoattraction

Neurons responding to NTN1 as an attractant cue through DCC; the intracellular domain of DCC is constitutively bound to non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1) and to focal adhesion kinase (FAK) forming a DCC-NCK1-FAK complex (Figure 8A; Li et al., 2004; Li et al., 2002; Ren et al., 2004; Lai Wing Sun et al., 2011). NTN1 binding to DCC triggers tyrosine 28 phosphorylation of DCC and autophosphorylation of FAK, which facilitates the recruitment of several signaling proteins to the DCC-NCK1-FAK complex (Ren et al., 2004; Ren et al., 2008; Lai Wing Sun et al., 2011). Phosphorylation of DCC is also known to facilitate DCC- Src family kinase Fyn interaction through activated FAK (Li et al., 2004; Liu et al., 2004; Meriane et al., 2004; Ren et al., 2004). The DCC-FYN interaction leads to increased tyrosine phosphorylation of DCC, which in turn facilitates the DCC-FYN interaction, forming a positive feedback loop. This DCC-FAK-FYN protein complex is known to be required for NTN1 stimulated axon outgrowth and attractive growth cone turning (Ren et al., 2008). The tyrosine phosphatase, SHP-2, which also binds to DCC has been suggested to serve as suppressor of the DCC-FYN phosphorylation loop (Ren et al. 2008). FYN is also thought to regulate activity of Rho GTPases; CDC42, and/or RAC1 (Li et al., 2002; Shekarabi et al., 2005; Lai Wing Sun et al., 2011), and inhibit RHOA (Rajasekharan et al., 2009). Similarly, NCK1 has been linked to CDC42 and RAC1 via p21- activated kinase 1 (PAK1), which functions as an adaptor protein that links NCK1 to these Rho GTPases (Bagrodia and Cerione, 1999; Li et al., 2001). As mentioned earlier, the Rho GTPases are known to be critical regulators of axonogenesis by modulating the actin dynamics of the cells (see Chapter 1.4.5.2). They act as binary switches cycling between an inactive GDP-bound form and an active GTP-bound state (Bishop and Hall, 2000). The activity of Rho GTPases can be positively influenced by guanine nucleotide exchange factors (GEFs), which promote the exchange of GDP for GTP (Schmidt and Hall, 2002; Rossman et al., 2005). Two GEFs (TRIO and DOCK180) have been shown to mediate axon guidance by activating RAC1 downstream of DCC (Briancon- Marjollet et al., 2008; Li et al., 2008; Lai Wing Sun et al., 2011). In conclusion, NTN1 signaling through DCC activates CDC42 and RAC1, which in turn recruits downstream effectors including the actin-binding proteins Enabled/vasodilator-stimulated phosphoprotein (ENA/VASP) and Neural Wiskott-Aldrich syndrome protein (N-WASP), which are both modulators of actin polymerisation (Lebrand et al., 2004; Shekarabi et al., 2005). On the other hand, NTN1-DCC signaling inhibits RHOA, which increases DCC numbers in plasma membrane and adhesion towards NTN1 (Moore et al., 2008). Interestingly, protein kinase A (PKA) activation in the presence of NTN1 also regulates DCC numbers in the plasma membrane, and although PKA can directly inhibit RHOA, the PKA induced recruitment of DCC to the plasma membrane seems not to be RHOA mediated (Bouchard et al., 2004; Moore et al., 2008). These findings supports the idea that increased DCC presented by RHOA inhibition and/or PKA activation in growth cone plasma membranes, enhances outgrowth and turning responses of these axons towards NTN1 (Bouchard et al., 2004; Bouchard et al., 2008; Moore et al., 2008). DCC dependent axon attraction also involves activation of the mitogen-activated protein kinase (MAPK) extracellular signal- regulated kinases 1/2 (ERK1/2) signaling pathway, which is required for the stimulation of axon outgrowth and the attractive responses towards NTN1 (Campbell and Holt, 2003; Forcet et al., 2002; Ma et al., 2010). In addition, DSCAM has been shown to be involved in NTN1 dependent axon guidance (Andrews et al., 2008; Ly et al., 2008; Liu et al., 2009). The knowledge of NTN1 signaling through DSCAM remains limited, but in vitro studies have revealed that the intracellular part of human DSCAM interacts with PAK1 (Li and Guan, 2004) and FYN (Liu et al., 2009), which are also downstream targets of DCC (Meriane et al., 2004; Shekarabi et al., 2005).

1.4.5.2.2 NTN1 mediated signalling in axon chemorepulsion

The NTN1 induced chemorepulsion is mainly mediated by UNC5 and UNC5-DCC receptor signaling (Figure 8B; Barallobre et al., 2005; Lai Wing Sun et al., 2011). Deletion analyses of UNC5 have revealed a functional contribution of the cytoplasmic juxtamembrane domain to axon guidance in C. elegans, whereas deletion of the cytoplasmic ZU-5 domain disrupted function in both D. melanogaster and C. elegans (Keleman and Dickson, 2001; Killeen et al., 2002). Most of the UNC5 signaling components are still unidentified, however (Lai Wing Sun et al., 2011). It is 29 known that NTN1 induces phosphorylation of UNC5 in a DCC dependent manner, acting through SRC and FAK (Killeen et al., 2002; Li et al., 2006). Furthermore, the SRC activation leads to the recruitment of SHP2 to UNC5, which potentially modifies UNC5 phosphorylation and function (Tong et al., 2001). One possible modulator of UNC5 mediated axon repulsion is PAK family member MAX-2, which has been identified in C. elegans (Lucanic et al., 2006; Lai Wing Sun et al., 2011).

Figure 8. Schematic model of NTN1 mediated signaling in chemoattraction and chemorepulsion. (A) NTN1 binding initiates homodimerization of DCC via P3 domain, which further leads to the chemoattraction through activation of the intracellular signaling cascades affecting to the rearrangement of actin cytoskeleton. (B) NTN1 signaling is converted into repulsive by UNC5 receptors, which make heterodimeric receptor complex with DCC or act independently. CDC42; cell division cycle42; DD, death domain; ENA/VASP, Enabled/vasodilator-stimulated phosphoprotein; FYN, Src family kinase; FNIII, fibronectin type III ; GEFs, guanine nucleotide exchange factors; Ig, immunoglobulin; MAPK, mitogen-activated protein kinase; NCK1, non- catalytic region of tyrosine kinase adaptor protein 1; NFAT, nuclear factor of activated T cells; N- WASP, Neural Wiskott-Aldrich syndrome protein; PAK1, p21 activating kinase 1; pFAK, phosphorylated focal adhesion kinase; PICK1, C kinase-1; PIP2, phosphatidylinositol 4,5- ELVSKRVSKDWH3,73ĮSKRVSKDWLG\OLQRVLWRO WUDQVIHUSURWHLQĮ3.$protein kinase A; PKC, protein kinase C; PLC, phospholipase C; RAC1, Ras related C3 botulinum toxin substrate 1; RHOA, Ras homolog gene family, member A; SHP2, Src homology region 2 domain-containing phosphatase 2; SRC, Tyrosine kinase sarcoma; TS1, thrombospondin type-I.

1.4.5.2.3 Switches between NTN1 mediated axon attraction and repulsion

NTN1 induced axon turning responses are modulated by the ratio of active cyclic adenosine monophosphate (cAMP) versus cyclic guanosine monophosphate (cGMP) in growth cones (Ming et al., 1997; Nishiyama et al., 2003). At the high ratio of cAMP, NTN1 promotes attractive responses through DCC by triggering phospholipase C (PLC) mediated Ca2+ influx via L-type Ca2+ 30 channels, whereas a lower ratio of cAMP compared to cGMP inhibits Ca2+ influx and induces repulsion (Round and Stein, 2007). DCC may also affect neurite outgrowth and attraction by promoting the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) through binding of phosphatidylinositol WUDQVIHUSURWHLQĮ 3,73Į DQG3/&ZKLFKOHDGVWRWKHUHOHDVHRI&DIURP internal stores (Xie et al., 2005; Xie et al., 2006; De Vries and Cooper, 2008). NTN1 has also been shown to utilise Ca2+ signaling to induce changes in the transcription program of the cell (Graef et al., 2003). NTN1 has been shown to trigger calcineurin-dependent activation of nuclear factor of activated T cells (NFAT), and activation of NFAT transcription complexes is necessary for NTN1 induced axon outgrowth (Graef et al., 2003). Interestingly, NTN1 receptor UNC5A trafficking on the cell surface has been shown to modulate growth cone responses towards NTN1 (Bartoe et al., 2006). UNC5A interacts with protein interacting with C kinase-1 (PICK1) and protein kinase C (PKC) in the growth cone, and activation of PKC leads to the internalisation of UNC5A via endocytosis (Bartoe et al., 2006). This UNC5A decrease in growth cones has been shown to switch chemorepulsion to attraction and inhibit growth cone collapse in hippocampal and cerebellar granule axons (Williams et al., 2003; Bartoe et al., 2006).

1.4.5.2.4 NTN1 assisted axon guidance in mouse CNS

The best known example of NTN1 induced axon outgrowth and guidance comes from chicken and mouse embryonic , where NTN1 is secreted from the ventral floor plate cells (Serafini et al., 1994; Kennedy et al., 1994; Serafini et al., 1996). Furthermore, the secretion of NTN1 from the floor plate cells is thought to form a ventro-dorsal gradient of NTN1 protein (Serafini et al., 1996; Kennedy et al., 2006). This gradient is proposed to guide the axons of dorsal commissural neurons and cerebellofugal axons towards the ventral midline, where they cross the midline (Serafini et al., 1996; Shirasaki et al., 1996). The attractive response of commissural axons towards the NTN1 source is mediated by DCC and DSCAM, which are expressed in commissural axon growth cones (Andrews et al., 2008; Ly et al., 2008; Liu et al., 2009). Once the commissural axons cross the midline, they upregulate surface expression of Robo receptors and acquire SLIT responsiveness (Zou et al., 2000; Stein and Tessier-Lavigne, 2001). SLIT induced repulsion helps commissural axons to exit the midline and prevents them from backcrossing to the “wrong” side of the spinal cord (Zou et al., 2000). In this process, SLIT receptor ROBO antagonizes and silences the sensitivity of commissural axons towards NTN1 by binding directly to DCC, thereby coupling repulsion to the downregulation of attraction (Stein and Tessier-Lavigne, 2001). In Ntn1 deficient mice, some commissural axons project incorrectly away from the floor plate, but many axons still orient properly towards the midline of the spinal cord (Serafini et al., 1996). Interestingly, in C. Elegans and D.Melanogaster, many commissural axons are able to cross the midline even in the total absence of Netrins (Wadsworth et al., 1996; Mitchell et al., 1996). Thus, it has been suggested that Netrins are not the only midline attractant molecules in invertebrate and vertebrate nervous system (Evans and Bashaw, 2010). In addition to its role as an attractant, NTN1 also acts as a repellent for those axons that must not cross the midline in the spinal cord. In this regard, NTN1 repels trochlear (Colamarino and Tessier-Lavigne, 1995), cranial (Guthrie and Pini, 1995; Murray et al., 2010), and spinal accessory motor axons in mice (Dillon et al., 2005). Besides the embryonic spinal cord, NTN1 participates in the development of other midline projections of the nervous system (Barallobre et al., 2005). Ntn1 deficient mice lack the main forebrain commissures: the CC, the HC, and sometimes the AC, although the habenular and posterior commissures are intact in these mice (Serafini et al., 1996; Barallobre et al., 2000). Away from the midline, NTN1 is also involved in guiding many cortical efferents in brain (Barallobre et al., 2005). NTN1 participates in the correct development of the corticofugal-, corticospinal-, thalamocortical-, and circumferential habenular axon projections by attracting and repulsing these axons mainly through UNC5 and DCC receptors (Metin et al., 1997; Richards et al., 1997; Funato et al., 2000; Finger et al., 2002; Braisted 31 et al., 2000; Powell et al., 2008). Furthermore, NTN1 plays a key role during the development of hippocampal projections and its deficiency produces serious alterations in commissural and entorhinohippocampal connections (Barallobre et al., 2000; Pascual et al., 2004). In cerebellum, NTN1 strongly repels the parallel fibers of the cerebellar granule cells (Alcantara et al., 2000), inferior olivary axons (Bloch-Gallego et al., 1999; Kim and Ackerman, 2011), and the internal arcuate fibers of the dorsal column-medial lemniscal system (Kubota et al., 2004). NTN1 is also implicated in the guidance of dopaminergic axons within the ventral midbrain (Lin et al., 2005) and its presence in the head is required for the axons of ganglion cells to enter the optic nerve from the retina (Deiner et al., 1997)

1.4.5.3 NTN1 mediated cell migration in CNS

Along with directing axon guidance, NTN1/UNC6 was shown to guide both dorsal and ventral migration of mesodermal (Hedgecock et al., 1990) and neuronal cells in C. elegans (Chan et al., 1996; Wadsworth et al., 1996). After these early discoveries, NTN1 has been shown to guide cell migration also in many distinct areas of the developing mouse nervous system (Barallobre et al., 2005; Lai Wing Sun et al., 2011). Furthermore, NTN1 dependent cell migration seems mainly to be mediated by the same mechanisms as in axon guidance (Figure 8; Rajasekharan et al., 2009; Lai Wing Sun et al., 2011).

1.4.5.3.1 NTN1 mediated migration of neurons in mouse CNS

In forebrain, NTN1 expression in VZ of ganglionic eminences is thought to guide migrating SVZ cells to the striatal primordium, where they differentiate into striatal neurons (Hamasaki et al., 2001). However, Ntn1 deficient mice exhibit only a minor decrease in the overall striatum size (Serafini et al., 1996; Braisted et al., 2000; Hamasaki et al., 2001). Therefore, Hamasaki et al. (2001) have suggested that the role of NTN1 in striatal neuron migration might be compensated for by other functionally similar proteins. In addition, it has been shown that NTN1 interaction with integrins is required for a proper tangential migration of GABAergic interneuron subpopulations from the ganglionic eminences VZ/SVZ to the cerebral cortex (Stanco et al., 2009). In the olfactory system, it has been suggested that subpopulation of specific early cortical neurons, LOT cells, are guided from the dorsal pallium to the PSB region in a NTN1-DCC dependent way. More precisely, these LOT cells express DCC from E10.5 to E12.5 in the surface layer of the cortex, from where they migrate ventrally towards NTN1 expressing subpallium and align in the future pathway of LOT in the PSB region. In this region, LOT cells participate in the guidance of the olfactory bulb axons to the forebrain (Kawasaki et al., 2006; Rajasekharan et al., 2009). NTN1-DCC signaling also guides the caudal vomeronasal nerve axons, which in turn determine the direction of luteinizing hormone-releasing hormone (LHRH) neurons in the basal forebrain (Schwarting et al., 2001; Schwarting et al., 2004). Thus, in Ntn1 and Dcc mutant mice a reduced number of LHRH neurons are found from their final destinations in basal forebrain hypothalamus (Schwarting et al., 2001; Schwarting et al., 2004). In addition, NTN1 and DCC are required to confine antidiuretic hormone and oxytocin neurons to the supraoptic nucleus and the absence of NTN1 or DCC leads to inappropriate migration of these cells from the supraoptic nucleus to the adjacent hypothalamus (Deiner and Sretavan, 1999). Furthermore, Ntn1 expression in the olfactory bulb MCL has been proposed to attract migrating Dcc and/or Neogenin expressing neural progenitor cells in RMS towards the olfactory bulb (Murase and Horwitch, 2002). NTN1 is also required for the development of the cerebellum. It acts as an attractant for precerebellar neurons, which migrate away from the lower germinative neuroepithelium (lower rhombic lip) to populate and form the pontine nucleus (Yee et al., 1999; Alcantara et al., 2000), inferior olivary nucleus (ION), the external cuneatus nucleus (ECN) and the lateral reticular nucleus 32 (LRN) (Bloch-Gallego et al., 1999; Causeret et al., 2002; Marcos et al., 2009). This NTN1 dependent attraction of precerebellar neurons has been proposed to be mainly mediated by the DCC receptor (Barallobre et al., 2005). Functional disruptions in either NTN1 or DCC signaling results in defects in pontine cell migration towards the midline, which eventually leads to the total absence of the pontine nucleus (Barallobre et al., 2005; Bradford et al., 2009). Similarly, the ION/LRN/ECN cells stop their migration and are found ectopically in the dorso-ventral axis of the hindbrain, with only few cells succeeding to leave the rhombic lip (Bloch-Gallego et al., 1999; Marcos et al., 2009). Interestingly, NTN1 dependent migration and axon guidance of precerebellar neurons requires phosphorylation of the microtubule-associated protein 1 B (MAP1B) (Del Rio et al., 2004; Lai Wing Sun et al., 2011). This association is confirmed by the analogous defects observed in the pontine nucleus and axon tracts of MAP1B deficient mice, when compared to the phenotypes of Ntn1 or Dcc mutant mice (Bloch- Gallego et al., 1999; Del Rio et al., 2004). Furthermore, NTN1 dependent precerebellar migration requires Rho GTPases CDC42/RAC1 for the initial extension of the leading processes (Causeret et al., 2004). Opposite to its role in precerebellar neuron migration, NTN1 does not affect the early migration of cerebellar granule neurons, which originate from the upper rhombic lip (Alcantara et al., 2000; Rajasekharan et al., 2009). Subsequently, NTN1 is expressed by the external granule layer during postnatal cerebellar development, from where it acts as a repellent for the cerebellar granule neuron precursors and cerebellar interneurons (Alcantara et al., 2000; Guijarro et al., 2006). Interestingly, cerebellar interneurons migrate to the opposite direction compared to cerebellar granule neurons precursors. Guijarro et al. (2006) have proposed that the NTN1 gradient generated by the external granule layer might have two actions based on a chemorepulsive mechanism mediated by UNC5 receptors: to prevent interneurons from erroneously entering the external granule layer and to push granule cell precursors out of the external granule layer. In addition, NTN1 receptor DCC is also required for the proper tangential migration of locus coeruleus neurons from dorsal rhombomere 1 to dorsolateral pontine tectum (Shi et al., 2008). In spinal cord, NTN1 attracts subtypes of dorsal commissural interneurons towards the floor plate (Rabe et al., 2009)

1.4.5.3.2 NTN1 mediated migration of glial cells in CNS

Sugimoto et al. (2001) have suggested that NTN1 secretion from the VZ of the third ventricle repels migrating glial precursor cells, which have the capacity to differentiate into oligodendrocytes or astrocytes. However, a direct role of NTN1 in mammalian astrocyte development remains to be proven (Tsai et al., 2006; Rajasekharan et al., 2009). Interestingly, a more recent study has shown that Netrins are required for the proper migration of two distinct glial populations in D. Melanogaster (von Hilchen et al., 2010).

1.4.5.3.3 NTN1 mediated migration of oligodendrocytes in mouse CNS

NTN1 has been shown to direct OPC migration in the embryonic spinal cord and in the optic nerve in mice (Deiner et al., 1997; Jarjour et al., 2003). In the embryonic spinal cord, OPCs arise from the ventral VZ where they migrate towards the axons at the edge of the neural tube (presumptive white matter) (Miller et al., 1996; Jarjour et al., 2003). This migration is thought to be directed by an NTN1 gradient originating from the ventral VZ, by repulsing OPCs through DCC and Unc5 receptors (Jarjour et al., 2003; Tsai et al., 2003). These early studies were conducted mostly in vitro and more recent studies conducted in vivo have shown that Ntn1 deficient mice exhibit a delayed initial dispersal of OPCs from the VZ into the presumptive white matter (Tsai et al., 2006; Tsai et al., 2009). More precisely, OPCs lose their capacity to accomplish directional migration from the VZ towards the white matter in Ntn1 deficient mice, because the orientation or/and polarity of OPCs has altered (Tsai et al., 2006; Tsai et al., 2009). This leads to the accumulation of OPCs in the 33 VZ and the intermediate zone of spinal cord with fewer OPCs reaching the white matter (Jarjour et al., 2003; Tsai et al., 2006; Tsai et al., 2009). Interestingly, Tsai et al. (2009) have shown that the absolute rate of OPC migration from VZ to the presumptive white matter is not altered in Ntn1 deficient mice, which suggests that NTN1 acts as a short distance dispersal cue rather than in a gradient manner in the spinal cord. However, due to the defective migration, fewer OPCs reach the gray-white matter interface in Ntn1 deficient mice (Tsai et al., 2006; Tsai et al., 2009). The OPCs normally proliferate in the gray-white matter interface, propagating the OPC population, and then continuing their migration towards the presumptive white matter (Tsai et al., 2009). In Ntn1 deficient mice, fewer OPCs reach and proliferate in the gray-white matter interface, which later leads to a reduced generation of mature oligodendrocytes (Tsai et al., 2006; Tsai et al., 2009). Normally after OPC proliferation in the grey-white matter interface, postmitotic oligodendrocytes extend and retract their processes over substantial distances, sampling the local environment to locate unmyelinated axons (Kirby et al., 2006; Haber et al., 2009). NTN1 also takes part in this process by promoting extension and branching of oligodendrocyte projections together with inducing formation of myelin-like membrane sheets (Rajasekharan et al., 2009; Lai Wing Sun et al., 2011). Interestingly, NTN1 seems to mediate these effects in an autocrine or/and paracrine manner (Rajasekharan et al., 2009; Lai Wing Sun et al., 2011). The distinct responses of OPCs and postmitotic oligodendrocytes to NTN1 signaling seem to be mediated through Rho GTPase RHOA (Rajasekharan et al., 2010; Lai Wing Sun et al., 2011). The NTN1 dependent OPC repulsion requires DCC and the RHOA effector ROCK together with activation of RHOA (Rajasekharan et al., 2010). On the contrary, NTN1 signaling in postmitotic oligodendrocytes inhibits RHOA, which is a requirement for the process branching (Rajasekharan et al., 2010). NTN1 and DCC mediate axon guidance locally at the optic disc and absence of either NTN1 or DCC leads to optic nerve hypoplasia (Deiner et al., 1997). In addition, optic nerve OPCs express DCC and UNC5 receptors as they migrate from the floor of the third ventricle (optic chiasm) along the optic nerve towards the retina (Ono et al., 1997; Sugimoto et al., 2001; Spassky et al., 2002). To date, two alternative models have been proposed for the role of NTN1 in optic nerve: 1.) The migration of OPCs is guided by a chemorepulsive gradient of NTN1 that emanates from the optic chiasm (Sugimoto et al., 2001; Spassky et al., 2002) and, in addition, NTN1 expression at the optic nerve head (the junction between the retina and the optic nerve), could acts as an stop sign preventing the OPCs entering to the retina (Tsai and Miller, 2002; Baker et al., 2006). 2.) NTN1 expression in the optic nerve head might attract OPCs towards the optic nerve end and the NTN1 source in the chiasmal region might help OPCs to enter the optic nerve from the extramural stream of the ventral diencephalon (Spassky et al., 2002).

1.4.5.4 NTN1 mediated apoptosis through its receptors

In the CNS, NTN1 has been found to be a survival factor for the spinal cord commissural neurons (Furne et al., 2008), inferior olivary neurons (Llambi et al., 2001; Marcos et al., 2009), neural crest cells (Jiang et al., 2003), retinal ganglion cells and displaced amacrine cells (Shi et al., 2010). The survival activity of NTN1 is based on the inhibition of the proapoptotic activity of its receptors DCC, Neogenin, and UNC5A-D, which belong to the family of dependence receptors (Mehlen et al., 1998; Goldschneider and Mehlen, 2010). Dependence receptors are not structurally similar, but they share common functional traits (Goldschneider and Mehlen, 2010). The most significant feature is their ability to activate two alternative signaling pathways, depending on the presence of their ligands. In the presence of a ligand, they activate normal signaling pathways associated with cell migration, differentiation, and survival. On the other hand, the absence of ligand triggers apoptotic signaling in a receptor expressing cell (Thibert and Fombonne, 2010; Goldschneider and Mehlen, 2010). Thus, DCC, Neogenin, and UNC5s are thought to trigger cell death in the absence of NTN1. Furthermore, NTN1 signaling has been shown to be involved in tumorigenesis 34 (Goldschneider and Mehlen, 2010). The loss of function of the dependence receptors DCC and UNC5 leads to reduction in cell death, which gives a selective advantage for the progression and growth of tumours (Mille et al., 2009; Goldschneider and Mehlen, 2010). Tumours may also block DCC and UNC5 mediated cell death by autocrine expression of NTN1 (Mille et al., 2009). Although, many studies have confirmed NTN1 participation in cell survival, the dependence receptor hypothesis is still controversial (Lai Wing Sun et al., 2011). Many studies have also quantified neuronal apoptosis in Ntn1 and Dcc mutant mice without finding any increase in apoptosis (Jarjour et al., 2003; Tsai et al., 2003; Williams et al., 2006; Shi et al., 2010; Xu et al., 2010).

1.4.5.5 NTN1 also have a role in organogenesis outside of the nervous system

NTN1 and its receptors are involved in many developmental processes outside of the nervous system, for example, directing cell migration and mediating cell adhesion during organogenesis of lung, pancreas, mammary gland, vasculature, and muscle (Hinck, 2004; Bradford et al., 2009; Lai Wing Sun et al., 2011). Thus, they also participate in morphogenesis of these different organs and are known to influence branching morphogenesis in lung (Liu et al., 2004), mammary gland (Srinivasan et al., 2003; Hinck, 2004), and the vascular system (Lu et al., 2004). In addition, NTN1 also plays an important role in the morphogenesis of the pancreas (Yebra et al., 2003; Nikolopoulos and Giancotti, 2004) and inner ear, where it was first shown to be involved in cell detachment and basement membrane disruption (Salminen et al., 2000; Matilainen et al., 2007)

1.5 Actin-bundling protein with BAIAP2 homology (ABBA)

ABBA belongs to the Bin–amphiphysin–Rvs167 (BAR) protein superfamily, which represents the largest and most diverse group of membrane deforming proteins (Saarikangas et al., 2009; Zhao et al., 2011). BAR domain proteins are known to deform cell membranes and promote plasma membrane invaginations and protrusions (Saarikangas et al., 2010). This BAR protein mediated regulation of plasma membrane morphology is achieved by directly influencing plasma membrane assembly or through rearrangement of the actin cytoskeleton (Saarikangas et al., 2010; Zhao et al., 2011). The BAR domain containing proteins, based on their structural differences, can be further divided into three diverse subgroups: the BAR, F-BAR, and I-BAR domain proteins (Saarikangas, 2010; Zhao et al., 2011). ABBA is a member of the I-BAR domain proteins, together with insulin receptor tyrosine kinase substrate p53 (IRSp53), missing in metastasis (MIM), insulin receptor tyrosine kinase substrate; also known as (BAIAP2L1) (IRTKS) and FLJ22582 (BAIAP2L2), as the I-BAR domain is found in the N-terminal region of all these five proteins (Zhao et al., 2011). Furthermore, the I-BAR domain containing proteins can be subdivided into two groups: the MIM/ABBA and IRSp53 subfamilies (Yamagishi et al., 2004; Scita et al., 2008). All I-BAR domain proteins promote formation of plasma membrane protrusions, such as filopodia and lamellipodia, through influencing intracellular actin dynamics (Saarikangas et al., 2009; Saarikangas et al., 2010; Zhao et al., 2011). Both filopodia and lamellipodia are implicated in cell motility and migration (Mogilner and Keren, 2009). Interestingly, I-BAR domain proteins do not induce cell invaginations like other BAR and F-BAR domain proteins (Saarikangas et al., 2009). In addition to the I-BAR domain, these proteins have several protein-protein interaction modules (Zhao et al., 2011). The C-terminus of most I-BAR domain proteins contains an actin monomer binding Wiskott– Aldrich syndrome protein (WASP) homology 2 (WH2) domain, which is known to be an actin binding domain, linking I-BAR domain proteins even further to plasma membrane and actin dynamics (Scita et al., 2008; Saarikangas et al., 2010). Additionally, an SH3 domain is present in the IRSp53 subfamily of I-BAR protein, but not in the MIM/ABBA subfamily (Yamagishi et al., 35 2004). The SH3 domain of IRSp53 binds to several regulators of actin filaments, such as N-WASP (Lim et al., 2008), WASP-family verprolin-homologous protein 2 (WAVE2) (Miki et al., 2000), mammalian ena/VASP family member (Mena/VASP) (Krugmann et al., 2001), diaphanous homolog 3 (Diap3/mDIA2) (Fujiwara et al., 2000), and epidermal growth factor receptor pathway substrate 8 (Eps8) (Funato et al., 2004). Furthermore, IRSp53 also interacts with the Rho GTPases CDC42 and RAC1 (Miki et al., 2000; Scita et al., 2008). Consequently, Ahmed et al. (2010) has suggested that the intracellular IRSp53-CDC42 complex links the I-BAR domain driven induction of membrane protrusion to SH3-domain-mediated actin dynamics, leading to filopodia formation. ABBA and MIM, which form a subfamily of I-BAR proteins, share a C-terminal WH2 domain and display a high homology in their C-terminus (~58% identity) (Yamagishi et al., 2004; Saarikangas, 2010). However, they lack the SH3 domain, suggesting that ABBA and MIM regulate actin dynamics in a different way than the IRSp53 subfamily (Ahmed et al., 2010). The I-BAR domain of MIM binds to RAC1 (Bompard et al., 2005), and WH2 domain to actin monomers (Mattila et al., 2003). Furthermore, MIM also interacts with actin regulator protein cortactin through its proline-rich region (Lin et al., 2005). All these protein interactions connect MIM closely to the regulation of actin dynamics. In summary, both the IRSp53- and ABBA/MIM subfamilies seem to generate different cell membrane protrusions by integrating different actin regulators through their I-BAR and protein-protein domains (Zhao et al., 2011). Furthermore, at least MIM and IRSp53 have been associated to cell migration and invasion in mammals (Zhao et al., 2011). However, the cellular functions of ABBA were not known before the publication presented in this thesis.

1.6 Oncogenes affect basic developmental processes of stem/progenitor cells

Oncogenes are, in general, either mutated forms of normal cellular genes (proto-oncogenes) or viral genes, which cause the transformation of normal cells into cancerous tumor cells (Kofman et al., 2011; Vogt, 2012). The viral mechanism includes insertion of additional viral oncogenic genes into the host cell genome and/or enhancement of already existing proto-oncogenes in the host cell genome (Kofman et al., 2011; Vogt, 2012). It is well known that oncogenes have a potential to cause tumours through affecting basic cellular processes, for example, cell proliferation, differentiation, and apoptosis (Hede et al., 2011). These same cellular processes are also a part of normal development of different organs. Hence, viral oncogenic genes and mutations in proto- oncogenes may disturb normal development of organs by disrupting the functionality of signaling pathways related to these basic developmental processes (Westphal and Lamszus, 2011; Henley and Dick, 2012). In the CNS, different neuronal cell types develop in a sequential manner from the stem/progenitor cells to fully differentiated cells (see Chapter 1.1.3). It has been proposed that CNS tumours could be derived either from stem/progenitor cells or fully differentiated cells, which have undergone the process of dedifferentiation (Westphal and Lamszus, 2011; Hede et al., 2011). Alternatively, the stem/progenitor cells or fully differentiated cells could transform into cancer stem cells, which then give rise to different brain tumour types (Visvader, 2011; Westphal and Lamszus, 2011; Lathia et al., 2011). However, it is still unclear whether cancer stem cells originate from normal stem/progenitor cells or from differentiated cells that have acquired the ability to self-renew (see Chapter 1.1.2.1; Hede et al., 2011; Valent et al., 2012). Another, unclear issue is whether brain tumours originate from the neural stem/progenitor cells, differentiated neural cells, or cancer stem cells (Hede et al., 2011).

1.6.1 Human Papilloma Virus (HPV) 16 E6/E7 oncogenes

HPVs are a large group (over 200 different types) of small, non-enveloped, double-stranded DNA tumor viruses (Zheng and Baker, 2006; McLaughlin-Drubin and Münger, 2009a). HPV’s viral DNA integrates into host cell genome leading to a constantly maintained expression of viral E6 and 36 E7 genes, whereas the remaining viral genes are often deleted or their expression is repressed (Münger et al., 2004; McLaughlin-Drubin and Münger, 2009a). The infected cells begin to produce HPV E6 and E7 oncoproteins, which lack DNA binding activity, but can be a part of transcription factor complexes and alter their transcriptional activities (Hwang et al., 2002; McLaughlin-Drubin and Münger, 2009b). For example, HPV E6 and E7 oncoproteins are well known for their association with p53 and E2F transcription factor complexes (Münger et al., 2004; McLaughlin- Drubin and Münger, 2009a). HPV E6 proteins target the p53 for proteasomal degradation (Scheffner et al., 1990), whereas HPV E7 proteins stabilize the p53 and inhibit its transcriptional activity (Eichten et al., 2002; McLaughlin-Drubin and Münger, 2009b). The HPV E7 oncoprotein has been also shown to bind to the retinoblastoma protein (pRb) and inactivate its function by preventing the binding of pRb to E2F transcription factor (Münger et al., 1989). Furthermore, HPV E7 also causes degradation of pRb through the ubiquitin–proteasome mediated pathway (Boyer et al., 1996). In general, the p53 and pRb protein families are thought to be the major targets of HPV E6/E7 oncoproteins. However, E6/E7 proteins also interact with a wide variety of cellular proteins with distinct cellular functions (Münger et al., 2004; McLaughlin-Drubin and Münger, 2009b; Ganguly and Parihar, 2009). Consequently, these protein-protein interactions of E6/E7 oncoproteins modulate the behavior of the host cell by affecting regulation of cell cycle, proliferation, survival, and DNA replication (Ganguly and Parihar, 2009).

1.6.1.1 Tumor suppressor protein p53 p53 is one of the most extensively studied tumor suppressor proteins with many distinct cellular functions (Vousden and Ryan, 2009; Solozobova and Blattner, 2011). As a tumour suppressor protein, p53 is known to arrest cell cycle progress in cases of DNA damage (Solozobova and Blattner, 2011). p53 simply provides the time needed for a DNA to be repaired or, alternatively, it initiates apoptosis to eliminate the damaged cell (Levine, 1997; Solozobova and Blattner, 2011). The lack of p53 seems to raise the probability of tumour development, as over 50% of human tumours contain mutations in the p53 gene and nearly 75% of p53-null mutant mice have developed tumours by the age of 6 months (Donehower et al., 1992; Levine, 1997). p53 is a dispensable protein for development of mice and men (Donehower et al., 1992; Levine, 1997). However, in neural, mammary, hematopoietic, and embryonic stem cells, p53 has a role in regulating self- renewal, symmetric division, quiescence, survival, and proliferation of these cells (Hede et al., 2011; Solozobova and Blattner, 2011). In addition, p53 has an ability to induce differentiation and to suppress dedifferentiation of these stem cells (Hede et al., 2011).

1.6.1.2 Tumor suppressor protein pRb

The Rb family of proteins, pRb, p170, and p130, inhibit the cell-cycle entry from G1 to S phase by interacting with cyclin-dependent kinases (CDKs) and the E2F family of transcription factors (Gordon and Du, 2011; Henley and Dick, 2012). In quiescent cells, Rb proteins bind to the E2F family of transcription factors and facilitate repression of E2F target-genes during G1-phase (Dyson, 1998; Henley and Dick, 2012). The pRb is released from E2Fs by the activity of various mitogen-induced CDKs, which hyperphosphorylate the pRb protein (Gordon and Du, 2011). The disintegration of pRb/E2F complexes leads to the transcription of E2F target-genes, which in turn promotes cell entry to S-phase (Dyson, 1998; McLear et al., 2006; Henley and Dick, 2012). Furthermor, the Rb/E2F complexes can recruit the chromatin remodeling factors in their complexes leading to alterations in chromatin environment and conformation, which can also regulate global gene expression (Gordon and Du, 2011; Henley and Dick, 2012). Interestingly, E2Fs have been shown to activate p53 and this interaction is thought to restrict tumorigenesis by inducing apoptosis (Bates et al., 1998; Polager and Ginsberg, 2009). In embryonic- and adult organs pRb affects 37 proliferation, differentiation, and survival of distinct stem/progenitor cells (Sage, 2012). For example, pRb deficiency in brain leads to increased proliferation and apoptosis in the VZ /SVZ area of the developing forebrain (Clarke et al., 1992; McLear et al., 2006). In summary, pRb deficiency seems to elevate proliferation of stem/progenitor cells by affecting cell cycle entry (Sage, 2012). Furthermore, the increased proliferation is often associated with increased cell-autonomous apoptosis, specifically in the progenitors induced to differentiate (Sage, 2012). The pRb has been thought to favor differentiation and its deficiency often prevents the terminal differentiation of cells (De Falco et al., 2006; Sage, 2012)

38 2. Aims of the study

The main objective of this study was to elucidate the developmental role of NTN1 in the formation of the olfactory bulb and corpus callosum during mouse forebrain morphogenesis. Furthermore, this thesis aimed to gain more information about the regulation of neural stem/progenitor cells derived from mouse forebrain. The more specific aims were:

1. To gain a detailed view of NTN1 influence on neuronal and glial development of the mouse olfactory bulb.

2. To investigate role of NTN1 in interhemispheric fusion and formation of transient cellular structures in the corticoseptal boundary region, and their potential impact on development of corpus callosum.

3. To examine the role of NTN1 in proliferation, differentiation, cell death, and migration processes of forebrain neural stem/progenitor cells.

4. To study the expression of ABBA in developing mouse CNS to begin to understand its cellular and developmental role.

5. To study the effects of HPV16 E6/E7 oncoproteins on differentiation of neural stem/progenitor cells

39 3. Material and methods

Experimental methods, primary antibodies and RNA probes personally applied in this study are listed in tables 1-3. Roman numbers indicate the corresponding article where they are described more in detail.

Table 1: Experimental methods used in this study

Method Publication RNA in situ hybridization I,II,III Immunohistochemistry I,II,III,IV Primary cell culture I Explant cultures I Cell migration assay I ȕ-gal staining I,II Programmed cell death assay I Confocal microscopy I,III,IV Cell transplantation in utero IV Statistical analyses I,IV PCR genotyping of mice I,II

Table 2: Primary antibodies used in immunohistochemical analyses

Primary antibodies Dilution Publication Anti-ABBA 1:1000 III Anti-ȕ-gal 1:1000 I,II Anti-Calbindin 1:500 I Anti-Calretinin 1:200 I,II Anti-CNP 1:100 I Anti-GFAP 1:500-1000 I,II,III,IV Anti-Laminin 1:500 II Anti-L1CAM 1:50 II Anti-NG2 1:1000 I Anti-Nestin 1:500 IV Anti-NeuN 1:150 I Anti-O4 1:80 I,IV Anti-OLIG2 1:800 I Anti-3'*)5Į 1:100 I Anti-PSA-NCAM 1:500 I Anti-RC2 1:500 III Anti-S-100ȕ 1:500 I Anti-TH 1:500 I Anti-TUJ1 1:500 I,III,IV

40 Table 3: RNA probes used for in situ hybridization

RNA probes Publication ABBA III Ntn1 I,II Dcc II Neogenin II Unc5a II Unc5b II Unc5c II Unc5d II

Genetically modified Ntn1 mouse line

The generation of mice carrying a gene trap insertion in Ntn1 locus has been described previously (Salminen et al., 1998; Salminen et al., 2000). In summary, this insertion generated a severely hypomorphic Ntn1 allele, which produces Ntn1 mRNA at very low levels. Furthermore, the gene trap insertion contained IRESȕgeo cassette, which produces ȕ-galactosidase (ȕ-gal) and neomycin phosphotransferase (NeoR) in cells expressing Ntn1. Mice homozygous for the hypomorphic Ntn1 allele show 100% penetrance for the inner ear and corpus callosum phenotypes. Pure C57BL/6 and mixed backround of C57BL/6 and ICR mice were used. All mouse work performed in this study was approved by the University of Helsinki ethical review board.

41 4. Results and discussion

4.1 NTN1 is required for the development of olfactory bulb (I)

Ntn1 deficient mice usually die soon after birth (Serafini et al., 1996; Salminen et al., 2000). Therefore, the significance of NTN1 to postnatal brain morphogenesis and function is unknown. In a mixed backround of C57BL/6 and ICR, Ntn1 deficient mice that survived several days after birth even until Pn16, could be generated. These mice facilitated the study of the role of NTN1 also during postnatal life. Although, the overall body size between Ntn1 deficient mice and control littermates was indistinguishable at birth, postnatal growth of Ntn1 deficient mice was strongly retarded. In addition, postnatal Ntn1 deficient mice had severe balance and movement problems likely due to the inner ear and axonal defects (Serafini et al., 1996; Salminen et al., 2000). Interest here, however, was mainly to study the role of NTN1 in neonatal and postnatal brain development. The postnatal size of Ntn1 deficient mouse brains was clearly smaller compared to the Ntn1 heterozygous and wild-type littermate brains (Fig.4 in I), which could be related to the apparent impairment of overall growth observed in Ntn1 deficient mice (unpublished observations). The size reduction of NTN1 deficient olfactory bulbs, however, was proportionally greater than the remaining brain and specifically obvious in the rostro-caudal direction (Fig.4 in I). At E18.5, Pn1 and Pn16 the length of NTN1 deficient olfactory bulbs in rostro-caudal direction was reduced, respectively, on average 13%, 22%, and 23% compared to Ntn1 heterozygous or wild-type littermates (Fig.4B-C and Fig.7A-D in I). Although, Ntn1 deficient mice had smaller olfactory bulbs, all the distinct neuronal layers were present and appeared relatively normal. For example, no pronounced difference was noticed in the thickness of GCL, EPL, MCL, and IPL. However, GL was clearly reduced in size and the outermost ONL was thinner in Ntn1 deficient mice compared to wild-type littermates (Fig.4D-G in I). These observations suggested that NTN1 might be dispensable for the overall morphological development of olfactory bulbs, but it could have a more specific role in development of GL and ONL.

4.1.1 Ntn1 is expressed in forebrain regions which contribute to the development and renewal of olfactory bulb

To better understand the role of NTN1 in olfactory bulb development, a detailed expression analysis was made for Ntn1 in the developing and postnatal mouse brain. The expression analysis was performed using three different techniques, including RNA hybridization, X-gal staining, and anti- ȕ-gal immunohistochemistry, where the lacZ insertion residing in Ntn1 locus was taken advantage of (Salminen et al., 2000). During brain development at E14.5 and E18.5, Ntn1 mRNA expression was evident and strong in subpallial (ventral telencephalon) areas, especially, in the lateral walls of forebrain ventricles, striatum and septum (Fig 1A, B in I). Furthermore, Ntn1 expression in the lateral ventricle walls was primarily confined to VZ of LGE, MGE, CGE, and telencephalic stalk regions (Fig.1A, B in I). Interestingly, Ntn1 expression was absent from the most dorsal part of LGE at E18.5 (Fig.1B in I), which is thought to produce dopaminergic interneurons into the olfactory bulb (Lledo et al., 2008). At E18.5, Ntn1 expression extended also to the rostral extension of VZ (Fig.1C in I), which forms the most proximal part of the RMS. The postnatal expression pattern of Ntn1 mRNA largely resembled the embryonic distribution of Ntn1 (Fig.1H in I). However, an additional Ntn1 expression was detected along the ventral border of postnatal RMS compared to the embryonic expression (Fig.1H, K in I). In olfactory bulb itself, a very weak Ntn1expression was detected in the GCL at E18.5 and stronger expression at Pn6 (Fig. 1C, H, I in I). Interestingly, no Ntn1 expression could be detected in MCL (Fig.1 in I and unpublished observations), which has previously been proposed by Murase and Horwitz (2002).

42 Ntn1-driven lacZ (Ntn1/lacZ) activity analysis was also performed on developing and postnatal brains of Ntn1 heterozygous mice. In general, Ntn1/lacZ activity corresponded with Ntn1 mRNA distribution during embryonic and postnatal development (Fig.1C, D in I and unpublished observations). However, at E18.5 and Pn6, a strong ȕ-gal activity extended from VZ into RMS (Fig.1D, G, E, J in I), excluding the most dorsal half of the horizontal RMS (Fig.1F in I). Furthermore, the detected ȕ-gal protein distribution in RMS was clearly wider compared to the earlier observed Ntn1 mRNA distribution. More closely, Ntn1 mRNA distribution was confined to the border region of ventral RMS (Fig.1H, K in I), whereas ȕ-gal positive cells could also be found in the middle of RMS throughout its length (Fig.1E, G, J in I). This observed difference between Ntn1 mRNA and ȕ-gal protein distribution suggest that the cells expressing Ntn1 mRNA in the lateral ventricle VZ and in border region of the ventral RMS continue to be positive for ȕ-gal protein (due to its longer stability), as they migrate out from VZ or ventral RMS (Fig.1 G, H, J, K in I). In adult brain, Ntn1/lacZ activity was obvious in the lateral walls of lateral ventricles and in the ventral part of the septal ventricle wall, similar to during development (Fig.1M in I and unpublished observations). The ȕ-gal protein activity in ventricular walls was detected in a rostro-caudal direction from the rostral forebrain to the spinal cord (Fig.1L in I). This suggests that it may conduct similar cell behaviors throughout the of the CNS. Furthermore, ȕ-gal protein activity in the lateral ventricle walls was confined to the continuous layer of cells next to the ventricle lumen, which correspond to the ependymal cell layer (Fig.1M, N, O in I). Taken together, the expression analysis performes here confirmed that Ntn1 is expressed in embryonic and postnatal VZ and its rostral extension. These regions are known to contain neural stem/progenitor cells which produce neural precursor cells that migrate into the olfactory bulb through RMS from early development into adulthood.

4.1.2 Ntn1 expressing cells extracted from embryonic and adult brains possess many characteristics associated with neural stem/progenitor cells

In order to characterize that Ntn1 expressing cells in embryonic VZ and in adult neural stem cell niches included neural stem/progenitor cells, primary cell cultures were made from forebrains of E12.5, E14.5, and E18.5 Ntn1 heterozygous embryos, as well as, from adult spinal cords and lateral walls of the lateral ventricles. In primary cell cultures, neural progenitor/stem cells proliferate and form growing cell aggregates (named neurospheres). Ntn1 expression was detected in all established neurosphere cultures together with many well-known neural stem/progenitor cell markers (Suppl. Fig. 1A, B in I). Furthermore, neurosphere cultures established from adult Ntn1 heterozygous mice revealed that only a subset of cells in a given sphere exhibited Ntn1 activity (Suppl. Fig.1D in I). These results raised a question whether Ntn1 expressing cells are able to proliferate and produce neurospheres by themselves. To answer this question, primary cell cultures were established by using with Ntn1 driven NeoR to select only the cells which expressed or maintained Ntn1 expression during culturing. These results revealed that Ntn1/NeoR selected cells were able to proliferate and produce neurospheres by themselves (Suppl. Fig. 1E in I). In addition, these Ntn1/NeoR selected neurospheres were also expressing common stem/progenitor cell markers, similarly, as did the wild-type spheres (Fig. 3A and Suppl. Fig. 1B in I). Interestingly, the number of neurospheres generated by the Ntn1/NeoR selected cell population was approximately 20% of the sphere numbers generated by the wild-type cell cultures (Fig.3B in I), suggesting that Ntn1 expressing cells extracted from brains constituted a subset of cells that proliferate in vitro. One basic feature of neuronal stem/progenitor cells is their ability to produce distinct postmitotic cell types of CNS. Therefore, neurospheres that contained only Ntn1/NeoR selected cells were induced to differentiate. Ntn1/NeoR selected neurosphere cells differentiated into all three major CNS cell

43 types; neurons, astrocytes, and oligodendrocytes, similar to the non-selected cells (Suppl. Fig 2B-G in I).

4.1.3 NTN1 is not critical factor for proliferation, self-renew or differentiation capacity of neural stem/progenitor cells

Since the above results revealed that Ntn1 was expressed by the cells which possess characteristics of neural stem/progenitor cells, the potential role of NTN1 in these cells was investigated. Cells were extracted to culture from the forebrain of Ntn1 deficient embryos and wild-type littermates at E18.5 and their cellular behavior was compared in vitro. NTN1 deficient cells formed primary spheres as efficiently as wild-type cells (Suppl.Fig.2A in I). In addition, the self-renewal capacity of NTN1 deficient cells was unchanged in comparison to wild-type cells in a clonal analysis, i.e. NTN1 deficient cells disassociated from primary spheres were able to form secondary spheres as efficiently as wild-type cells (Fig. 3C in I). NTN1 deficient cells were able to produce equal proportions of differentiated neurons, astrocytes, and oligodendrocytes compared to the wild-type and Ntn1 heterozygous cells (Suppl.Fig.2B-H in I). These results suggest that NTN1 is not necessary for a proper formation of neurospheres in vitro indicating that proliferation of neurosphere forming neural stem/progenitor cells was not influenced by NTN1 deficiency. Furthermore, the self-renewal and differentiation capacity of NTN1 deficient neural stem/progenitor cells was invariable compared to the wild-type cells. Thus, all in vitro experiments performed with Ntn1 expressing cells or NTN1 deficient cells combined with Ntn1 expression assay, suggest that Ntn1 is expressed in the germinal regions of embryonic and adult mice by a subset of cells which possess characteristics of neural stem/progenitor cells. In addition, NTN1 does not seem to influence proliferation, self-renewal or differentiation capacity of these Ntn1 expressing neural stem/progenitor cells.

4.1.4 Characterization of Ntn1/lacZ positive cells in postnatal RMS

Expression analysis revealed that the postnatal RMS contained Ntn1/lacZ positive cells. Earlier studies have shown that postnatal RMS is composed of many distinct cell populations (Lois and Alvarez-Buylla, 1994; Gritti et al., 2002; Aguirre and Gallo, 2004). These cell populations also include migrating and dividing progenitors, which contribute to both neuronal and glial cell lineages in the olfactory bulb (Lois and Alvarez-Buylla, 1994; Gritti et al., 2002). In order to get a more detailed view of the Ntn1/lacZ positive cells in RMS, double labeling experiments were performed with ȕ-gal antibody together with antibodies recognizing major progenitor/precursor cell types in RMS. The highest proportion of Ntn1/lacZ cells was migrating neural progenitor/precursor cells (Fig.2C, D and Table.1 in I). The second highest proportion of Ntn1/lacZ cells was NG2 positive cells (Fig.2E and Table.1 in I), which is a marker of OPCs and interneuron progenitor/precursor cells migrating towards the olfactory bulb through the RMS (Aguirre and Gallo, 2004). Furthermore, some Ntn1/lacZ cells also co-expressed GFAP antibody (Fig.2B and Table.1), which is a common marker of CNS astrocytes and SVZ progenitor/precursor cells (Doetsch et al., 1999). However, a proportion of Ntn1/lacZ cells did not label with any of the applied antibodies, suggesting that these cells might be more immature cells, such as proliferating stem/progenitors cells, which have also been detected in RMS (Coskun and Luskin, 2002). In summary, the Ntn1/lacZ cells inside RMS appeared to be a heterogeneous population of migratory progenitor/precursor cells, which contribute to both neural- and glial cell lineages in the olfactory bulb.

44 4.1.5 Migration defects in Ntn1 deficient mouse forebrain

Ntn1 expression analysis and characterization of Ntn1/LacZ cells implied that NTN1 may have a role in directing migration of distinct cell types in RMS. Therefore, a more detailed spatiotemporal analysis was performed for Ntn1/lacZ cells in RMS; first comparing Ntn1/lacZ cell distribution between Ntn1 heterozygous and Ntn1 deficient mice at E18.5, Pn1, and Pn6. These results showed that the heterozygous Ntn1/lacZ cells migrated further to the proximal region of horizontal RMS compared to the homozygous Ntn1/lacZ cells (Fig.7A-F in I). Furthermore, at Pn6 heterozygous Ntn1/lacZ cells populated the whole horizontal RMS while homozygous Ntn1/lacZ cells accumulated to the proximal part of RMS (Fig.7E-F in I). Thus, the migration of Ntn1/lacZ cells in RMS appeared to be influenced by the amount of NTN1. To gain more detailed information about the possible role of NTN1 in RMS migration, explant cultures were made from VZ/SVZ-RMS/OB regions of wild-type, Ntn1 heterozygous and Ntn1 deficient embryos. It has been shown earlier that cells which migrate out of these VZ/SVZ-RMS/OB explants in vitro form similar migratory chain structures as in vivo. In these chains, migratory cells move along each other, sliding over their neighboring cells (Wichterle et al., 1997). In these explant cultures, no significant difference could be seen in the overall lengths of the outgrown chains between wild-type, Ntn1 heterozygous, or Ntn1 deficient explants (Fig.7G-I in I). However, a significant 24% reduction was found in outmigration distance of Ntn1/lacZ cells in Ntn1 deficient explants compared to Ntn1 heterozygous explants (Fig.7J-L in I).

4.1.5.1 NTN1 may guide migration of the progenitor/precursor cells in a cell-autonomous or paracrine manner

The results mentioned above suggest that NTN1 does not affect formation of migratory chains or their lengths in vitro. Instead, NTN1 seem to be required for a proper migration of Ntn1/lacZ cells out of the explants in these chains, i.e. NTN1 deficiency reduced the migration speed of cells which had expressed Ntn1 before initiation of migration and/or expressed Ntn1 during migration by 24%. NTN1 has been thought to act as a diffusible long or short-range guidance cue, which influences migrating cells in either a chemoattractive or a chemorepulsive manner. However, an increasing number of studies have proposed that NTN1 has an alternative function as a detachment/release/adhesive factor, which directly influences cell motility through cell-cell and cell-matrix interactions (see Chapter 1.4.5.3.3; Tsai et al., 2006; Tsai et al., 2009; Rajasekharan et al., 2009; Lai Wing Sun et al., 2011). The migration of progenitor/precursor cells to olfactory bulb via RMS has been suggested to be guided by a chemoattractive activity of olfactory bulb (Murase and Horwitz, 2002; Liu and Rao, 2003), chemorepulsive activity of septum (Hu and Rutishauser, 1996; Nguyen-Ba-Charvet et al., 2004) and/or adhesive cell-cell/cell-matrix interactions within RMS (Cayre et al., 2009; Sun et al., 2010). Thus, it is not surprising that overall guidance of RMS migration is orchestrated by multiple molecular families related to cell guidance and adhesion (Cayre et al., 2009). In addition, these molecular families seem to act in a parallel manner affecting only a certain specific aspect of overall migration in RMS. The role of NTN1 in directing RMS migration has been controversial. Murase and Horwitz (2002) suggested that olfactory bulb mitral cells produce NTN1, which establish a long-range chemoattractive gradient affecting speed and directionality of DCC expressing progenitor/precursor cells in RMS. In contrast, Liu and Rao (2003) suggested that olfactory bulb itself has chemoattractive activity, but the NTN1 acts as a chemorepellent to RMS migration. Finally, Hu and Rutishauser (1996) and Mason et al. (2001) have proposed that NTN1 have neither attractive nor repulsive effect to RMS cell migration, although, septum itself has a repulsive effect (Hu and Rutishauser, 1996). Thus, the proposed roles for NTN1 in directing cell migration through RMS 45 seem to cover most of the existing possibilities. So, how do the results presented here fit in this context? No obvious change was detected in overall size of the RMS in Ntn1 deficient mice. In addition, the migratory defects observed in explant cultures together with the spatiotemporal analysis of Ntn1/lacZ cells in RMS suggest that NTN1 does not influence all migrating cells in RMS. Furthermore, Ntn1 expression was detected only in the brain regions where migration of neural progenitor/precursor cells starts towards the olfactory bulb, not in RMS itself, suggesting that NTN1 affects mainly cells which have been expressing Ntn1 at the initiation of migration. In summary, these results suggest that NTN1 acts mainly as a detachment/release/adhesive factor for Ntn1 expressing cells and this function is mediated in an either cell-autonomous and/or paracrine manner. Similar cell-autonomous and/or paracrine function of NTN1 has been proposed to be a part of GABAergic interneuron migration in the cerebellum (Guijarro et al., 2006), OPC migration in spinal cord (Tsai et al., 2009), process branching and elaboration of oligodendrocytes (Rajasekharan et al., 2009), and migration of glioma cells (Rajasekharan et al., 2011). However, the results cannot exclude the possibility that NTN1 might have a subtle simultaneous long-range attractive or repulsive effect on neural progenitor/precursor cells migrating in RMS.

4.1.5.2 Ntn1/lacZ expressing cells accumulate to the proximal RMS in Ntn1 deficient mice

To study the RMS region in more detail, where Ntn1/lacZ cells accumulated in Ntn1 deficient mice (Fig.7A-F in I), a cell quantification assay was done for three different areas of proximal RMS at Pn1 (Fig.8A, B in I). More cells were present in the ventral part of the proximal RMS in Ntn1 deficient samples compared to control samples (Fig.8C in I). In contrast, the cell numbers were significantly reduced in more distal and dorsal areas of the Ntn1 deficient RMS compared to the controls (Fig.8C in I). These results further confirmed earlier results that in Ntn1 deficient mice a reduced number of neural progenitor/precursor cells are able to reach the horizontal RMS. Instead, the migrating neural progenitor/precursor cells are accumulating into the ventral part of proximal RMS area. On the other hand, absence of NTN1 has been thought to trigger apoptosis through its receptors (see Chapter 1.4.5.4; Llambi et al., 2001; Jiang et al., 2003; Furne et al., 2008; Shi et al., 2010). Therefore, the cells that accumulate ectopically in the proximal area of horizontal RMS were assessed for excess programmed cell death. In this analysis, no significant differences were detected in the average numbers of cell death between Ntn1 heterozygous and Ntn1 deficient mice at Pn1 (Suppl.Fig.5A-D in I). Thus, it seemed that cells which accumulate into the proximal area of horizontal RMS in Ntn1 deficient mice do not immediately die through apoptosis. Interestingly, Kirschenbaum et al. (1999) have shown that elimination of entire olfactory bulb leads to slow accumulation of neural progenitor/precursors cells to the SVZ/RMS area without immediate increase in cell death. However, they observed that cell death was increasing in these areas several weeks later (Kirschenbaum et al., 1999). Thus, the accumulation of cells in Ntn1 deficient mice RMS may also lead to the elevated apoptosis in these areas at later stages.

4.1.6 Ntn1 deficient mice have reduced number of granular- and periglomerular cells in the olfactory bulb

To investigate further the consequence of the migration defect observed in Ntn1 deficient mice, the total cell numbers were calculated in GCL and GL of the olfactory bulbs. As mentioned above, the overall thickness of the GCL seemed invariable between postnatal wild-type mice and Ntn1 deficient mice. However, the quantification of total cell numbers revealed that the GCL had a reduced number of cells in Ntn1 deficient mice at Pn6 (11%) and Pn16 (14.9%) compared to control littermates (Table.3 in I). Similarly, in GL the average number of cells/glomerulus had decreased at Pn1 (13%) and Pn16 (15%) in Ntn1 deficient mice compared to control littermates (Table.2 in I). Thus, these results confirmed that Ntn1 deficient mice had a reduced number of cells in their 46 olfactory bulbs. However, olfactory bulbs contain many different cell types originating from several distinct sources and, therefore, the question of whether NTN1 deficiency was affecting any particular cell type specifically was addressed next.

4.1.6.1 Ntn1 deficient mice have reduced number of interneurons in their olfactory bulb

At first, the study was concentrated on two major neural cell types found in the olfactory bulb: projection neurons and interneurons. The projection neurons, mitral and tufted cells, are thought to be descendants of intra-bulbar radial glial cells and generated before interneurons (Hinds, 1968; Bayer, 1983). The numbers of mitral cells was quantified, of which somas are easily detectable in the mitral cell layer (Fig.4F, E in I), and no significant differences were found between wild-type and Ntn1 deficient mice at Pn16 (unpublished observations). In contrast to projection neurons, interneurons migrate to olfactory bulb mainly from outside throughout rodent life (Lledo et al., 2008; Whitman and Greer, 2009). The olfactory bulb interneurons are a heterogeneous population of cells, which can be mainly found from GCL and GL, where they interact with projection neurons to establish olfactory bulb circuits (Lledo et al., 2008). Most of the GCL and many GL interneurons are GABAergic inhibitory interneurons, but a number of GL interneurons are dopaminergic and a subpopulation of these dopaminergic interneurons also co-express GABA (see Chapter 1.2.1.4; Kosaka et al., 1995; Kosaka et al., 1998; Kosaka et al., 2005). At first, GCL interneurons were counted with the help of NeuN antibody that stains nuclei of differentiated interneurons in GCL (Mullen et al., 1992; Richard et al., 2010). NTN1 deficiency led to a significance decrease of NeuN positive interneuron numbers in GCL at Pn6 (Table.3 in I). Secondly, three major non-overlapping interneuron subtypes found in GL were counted and the results compared between Ntn1 deficient and wild-type mice (Table.2 in I). Interestingly, no change was detected in dopaminergic TH producing interneuron numbers between Ntn1 deficient and wild-type mice at Pn1 and Pn16 (Fig.4P-S and Table.2 in I). Instead, a significant decrease in GABAergic CB producing (Pn1; 16% and Pn16; 14%) and CR producing (Pn1; 28% and Pn16; 26%) interneuron numbers was observed in Ntn1 deficient glomeruli (Fig.4H-O and Table.2 in I). Thus, NTN1 deficiency leads to a significant reduction of interneurons without influencing projection neuron numbers in the olfactory bulb. As mentioned above, projection neurons are generated locally in the olfactory bulb itself, as olfactory bulb interneurons mainly migrate from lateral walls of lateral ventricles through RMS to olfactory bulb. Therefore, the observed reduction in interneuron numbers without any effect to projection neuron numbers in Ntn1 deficient mice suggest that NTN1 is not involved in directing the development of olfactory bulb projection neurons. This perspective is further strengthened by Ntn1 expression studies, which showed that Ntn1 is not expressed in the olfactory bulb at the time when projection neurons are generated, but is expressed in the brain regions which generate interneurons to the olfactory bulb.

4.1.6.2 Development of distinct interneuron subpopulations was affected differently in Ntn1 deficient olfactory bulbs

Cell quantification assays indicated that NTN1 deficiency affects the numbers of GABAergic interneurons in GCL and GL, but not dopaminergic interneuron numbers in the GL of olfactory bulb. Earlier studies have shown that different interneuron populations in olfactory bulb have distinct spatiotemporal origins (Lledo et al., 2008). For instance, it has been suggested that TH producing dopaminergic interneurons are generated predominantly at embryonic stages and to a lesser degree during the postnatal period (Batista-Brito et al., 2008; Kosaka and Kosaka, 2009). Furthermore, early embryonic TH producing interneurons have been proposed to originate from the olfactory bulb itself and/or from the dorsal SVZ/VZ region of the lateral ventricle walls (Vergano- Vera et al., 2006; Young et al., 2007; Kohwi et al., 2007; Batista-Brito et al., 2008; Lledo et al., 47 2008). Later, during the postnatal period RMS may also start to generate TH producing interneurons into the olfactory bulb (Hack et al., 2005). However, it has been proposed that GABAergic CB and CR producing interneuron populations of GCL and GL have distinct spatiotemporal origins compared to dopaminergic TH producing interneurons and to each other (Lledo et al., 2008). For instance, CB producing interneurons are suggested to be predominantly generated during pre/neonatal stages from the ventral VZ/SVZ area of lateral ventricle walls and CR producing interneurons during the postnatal period from medial walls of the lateral ventricle VZ/SVZ, RMS, septum, and pallium (Merkle et al., 2007; Batista-Brito et al., 2008; Lledo et al., 2008). Our expression analysis revealed that Ntn1 is expressed in the regions which generate GABAergic interneurons into the olfactory bulb from early development to adulthood. On the contrary, Ntn1 was not expressed in the regions which produce dopaminergic TH producing interneurons into the olfactory bulb. Thus, our results from cell quantification, cell migration, and Ntn1 expression analyses suggest that NTN1 influenced the migration of progenitor/precursor cell subpopulations from brain regions which specifically generates CB and CR producing GABAergic interneurons into the olfactory bulb. Interestingly, NTN1 has also been reported to be involved in GABAergic interneuron migration in other parts of the brain. Stanco et al. (2009) demonstrated that NTN1 interaction with integrin Į3ȕ1 is required for the proper tangential migration of GABAergic interneurons from VZ/SVZ of MGE to upper cortical layers in the cortex. Similarly, Guijarro et al. (2006) has proposed that NTN1 guides migration of GABAergic interneurons in the cerebellum in a cell-autonomous/paracrine manner. Our results strongly suggest that NTN1 functions in a similar manner in the olfactory bulb. However, further investigation is required to explicitly demonstrate that similar mechanisms are operating in the olfactory bulb.

4.1.6.3 Development of astroglia was normal in Ntn1 deficient olfactory bulbs

A cell quantification assay was pursued to investigate astroglial development in Ntn1 deficient mice olfactory bulbs. The quantification was conducted with GFAP antibody, which recognizes astrocytes of the CNS. Olfactory bulb has at least two distinct GFAP expressing astroglial cell populations, which differ clearly from each other by their characteristics and origin (see Chapter 1.2.1.4; Doucette, 1984; Bailey and Shipley, 1993; Au and Roskams, 2003). Overall distribution and density of GFAP expressing astrocytes was unaltered in Ntn1 deficient mice olfactory bulbs compared to wild-type and Ntn1 heterozygous olfactory bulbs at Pn6 and Pn16 (Fig 5A, C; Fig 6Q, R and unpublished observations in I). Furthermore, a strong GFAP staining was detected in VZ and GL of wild-type, Ntn1 heterozygous and Ntn1 deficient mice olfactory bulbs at Pn6 and Pn16 (Fig 5A, C; Fig 6Q, R and unpublished observations in I). To verify these preliminary observations, the numbers of GFAP expressing cells in GCL of Ntn1 deficient and Ntn1 heterozygous mice was quantified and no difference was found (Table.3, Fig 6Q, R in I). Thus, it seems that the amount of NTN1 is not affecting the development of GFAP expressing astrocyte populations in the olfactory bulb. These results are also in line with previous findings, which suggest that NTN1 does not influence development of mammalian astroglial cell populations in the CNS (Tsai et al., 2006; Rajasekharan et al., 2009).

4.1.6.4 NTN1 has an impact on olfactory bulb oligodendrogenesis

Previous findings have shown that NTN1 directs OPC migration in embryonic spinal cord and in optic nerve (see Chapter 1.4.5.3.3; Deiner et al., 1997; Jarjour et al., 2003). In addition, NTN1 promotes extension and branching of mature oligodendrocyte projections in the spinal cord (Rajasekharan et al., 2009; Lai Wing Sun et al., 2011). Therefore, the potential involvement of NTN1 in oligodendrogenesis of olfactory bulb was studied. At first, the overall distribution profile of mature oligodendrocytes between wild-type and Ntn1 deficient olfactory bulbs at Pn16 was 48 compared. For this purpose, oligodendroglial anti-ƍƍ-F\FOLF QXFOHRWLGH ƍ-phosphodiesterase (CNP) antibody was employed, which is a marker of mature myelin-forming oligodendrocytes (Gomes et al., 2003; Radtke et al., 2011). Interestingly, the CNP profile was remarkably different between wild-type mice and Ntn1 deficient mice (Fig.5E, F, G, H in I). In wild-type mice, CNP expressing oligodendrocytes populated all olfactory bulb layers, but in Ntn1 deficient mice a strong and significant reduction of CNP expressing oligodendrocytes was evident throughout the olfactory bulb (Fig.5E, F in I). Particularly the outermost layers were lacking CNP-staining in Ntn1 deficient mice olfactory bulbs. However, a strong CNP staining was apparent in both wild-type and in Ntn1 deficient mice LOT area, which contains axons of mitral cells extending to the olfactory cortex (López-Mascaraque and de Castro, 2002). Previous studies have proposed that mature myelinating oligodendrocytes are generated from proliferative and migratory OPCs during the first postnatal weeks in brain (Kessaris et al., 2006; Parras et al., 2007). Furthermore, differentiation of oligodendrocytes from OPCs is suggested to proceed in caudal to rostral and inside-out manner in olfactory bulb (Philpot et al., 1995; Gomes et al., 2003). Consequently, the mature oligodendrocytes residing in the outermost layers of olfactory bulbs differentiate last from the arriving OPCs (Gomes et al., 2003). Thus, these results together with earlier findings imply that the observed decrease of mature oligodendrocytes in the outermost layers of Ntn1 deficient olfactory bulbs could be related to the defective OPC migration. Therefore, a comparative analysis between numbers of OPCs found in wild-type and in Ntn1 deficient mice olfactory bulbs was performed. In this analysis, antibodies for common OPC markers, NG2, 3'*)5Į, and OLIG2, revealed a significant and strong reduction of these markers in Ntn1 deficient olfactory bulbs compared to wild-type (Table.3 and 4, Fig.6, Suppl.Fig.4 in I). More precisely, at all time points studied the observed decrease of OPC numbers varied between the ORZHVW 3'*)5ĮSRVLWLYHFHOOVDW3Q DQGWKHKLJKHVW 1*SRVLWLYHFHlls at Pn6). It has been proposed that olfactory bulb oligodendrocytes emerge from different locations and OPC populations during development (see Chapter 1.2.1.5). The first OPC population is proposed to be endogenous and characterised by their expression of plp and their independence of PDGFRĮ signaling (Spassky et al., 2001). The second OPC population is thought to migrate from MGE and AEP to the olfactory bulb and they are characterized by their expression of Olig1/2 and PdgfrĮ (Spassky et al., 2001; Kessaris et al., 2006). The latest OPCs are proposed to arrive in the olfactory bulb at late embryonic and early postnatal stages from SVZ-RMS areas. Furthermore, these late OPCs are suggested to differentiate into mature oligodendrocytes or parenchymal proliferative NG2-glia in the olfactory bulb (Nishiyama et al., 1996; Gomes et al., 2003; Parras et al., 2004; Aguirre and Gallo, 2004; Menn et al. 2006; Ganat et al., 2006; Ventura and Goldman, 2007; Maire et al., 2010; Richardson et al., 2011). Hence, these results propose that NTN1 is specifically influencing migration of Olig2/PdgfrĮDQG1*-expressing OPCs from MGE/AEP and SVZ-RMS regions to the olfactory bulb. In addition, these two OPC populations emerge from brain regions where Ntn1 is expressed during brain development. Interestingly, the results from migration assays are comparable with previous studies completed with Ntn1 deficient mouse spinal cord (Jarjour et al., 2003; Tsai et al., 2006; Tsai et al., 2009). These previous studies had shown that Ntn1 deficient mice exhibit delayed initial dispersal of OPCs from spinal cord VZ, which leads to accumulation of OPCs into ventricular- and intermediate zones of spinal cord with fewer OPCs reaching their final target area (Jarjour et al., 2003; Tsai et al., 2006; Tsai et al., 2009). Thus, NTN1 deficiency may also lead to defective and/or delayed migration of OPCs from MGE/AEP and SVZ-RMS regions into olfactory bulbs and cause the observed decrease of mature oligodendrocytes in Ntn1 deficient mice. In addition, the observed greater reduction of mature oligodendrocytes in the outermost layers of Ntn1 deficient mice is in line with defective and/or delayed migration hypothesis, as differentiation of oligodendrocytes in the olfactory bulb progress in caudal to rostral and inside-out manner. However, the causality of these different events requires more investigation. 49 4.1.7 A summary of NTN1’s role in the cellular development of the olfactory bulb

Stem/progenitor cells fate and forthcoming location in forebrain is predetermined in the germinal regions of the brain (see chapter 1.1.3). NTN1 is shown here not to influence proliferation, self- renewal, or differentiation capacity of neural stem/progenitor cells extracted from these germinal regions. However, a subset of stem/progenitor cells in the germinal regions of brain does express Ntn1 and this expression is required for the proper migration of these stem/progenitor cells in a cell- autonomous and/or paracrine manner to the olfactory bulb. Furthermore, these Ntn1 expressing progenitor/stem cells were mainly contributing to GABAergic interneuron and oligodendrocyte lineages in the olfactory bulb. GABAergic interneurons and oligodendrocytes originate from distinct domains of germinal regions and they also utilize different migration modes to populate the olfactory bulb (see chapters 1.1.3 and 1.1.5). NTN1 has also been shown to affect migration of GABAergic neurons and oligodendrocytes in other areas of CNS (see chapter 1.4.5.3.1 and 1.4.5.3.3). However, there is no clear consensus of how NTN1 signaling affects migration of GABAergic neurons and oligodendrocytes in the CNS. The migration of Ntn1 expressing stem/progenitor cells was delayed in this study, which led to the accumulation of these cells in RMS and, consequently, to reduction of GABAergic neurons and oligodendrocytes in the olfactory bulb. Interestingly, NTN1 signalling has been shown to affect formation of cell protrusions and polarization of cells, which have a major role in cell migration (Shekarabi and Kennedy, 2002; Chang et al. 2006; Guijarro et al., 2006; Tsai et al., 2009; Ziel et al., 2009; Stavoe et al. 2012; Hagedorn et al., 2013). Therefore, NTN1 signaling may affect stem/progenitor cells polarization and protrusion formation in a cell-autonomous and/or paracrine manner at the beginning of their migration towards the olfactory bulb, influencing mainly the early migration speed and directionality of these cells.

4.2 Role of NTN1 in the development of CC (II)

The CC is the largest axonal pathway in the mammalian brain and it connects the cortical regions of the two cerebral hemispheres with each other. Development of the CC is directed by the cortical network of axon guidance molecules and specific cellular structures emerging to the corticoseptal boundary (CSB) region (see Chapter 1.3; Richards et al., 2004; Lindwall et al., 2007). Formation of the CC begins with fusion of the interhemispheric midline (Silver et al., 1982), which is followed by midline crossing of pioneering callosal axons projecting from the cingulate cortex (Koester and O`Leary, 1994; Rash and Richards, 2001). The CC continues to expand in size as neocortical axons reach the midline and cross it (Polleux et al., 1998; Shu et al., 2003b). Both pioneering and neocortical callosal axons are actively guided towards the interhemispheric midline by specific glial and neuronal cell populations arising to the CSB region (Shu and Richards, 2001; Niquille et al., 2009). In addition, these cell populations form transient midline structures, which direct growing callosal axons towards the midline by secreting repulsive and attractive guidance cues. NTN1 was first found to attract commissural axons from the dorsal spinal cord towards the ventral floor plate in a DCC dependent manner (Serafini et al., 1996; Fazeli et al., 1997). These same studies also observed that Ntn1 and Dcc deficient mice exhibit a complete agenesis of corpus callosum (ACC) (Serafini et al., 1996; Fazeli et al., 1997). Therefore, it was originally suggested that Ntn1 expression in the hemispheric midline is required to attract Dcc expressing callosal axons towards the midline, similarly as in the spinal cord (Serafini et al., 1996; Fazeli et al., 1997). These first studies did not investigate, however, the possibility that NTN1 signalling in the hemispheric midline could also affect interhemispheric fusion and/or formation of transient midline structures in the CSB region. Therefore, NTN1 was assessed as to whether it influences these events and whether it could have an effect on CC development. 50 4.2.1 Overall analysis of Ntn1 deficient CSB region in mice

At first, a hematoxylin-eosin staining was done to obtain a more general view of CC development in Ntn1 deficient and wild-type embryos at E15.5-E17.5 (Fig.1 in II). In control embryos CC started to form at E15.5 and continued to increase in size until E17.5 (Fig.1 in II). Contrary to control embryos, callosal axons did not cross the interhemispheric midline and CC did not form at any stage examined in Ntn1 deficient embryos (Fig.1 in II). The defective formation of CC led to the development of an interhemispheric cyst and formation of longitudinal axon bundles (Probst bundles) in Ntn1 deficient mice (Fig.1 in II). The interhemispheric cyst has been proposed to appear as a consequence of defective midline fusion (Richards et al., 2004; Wahlsten et al., 2006; Paez et al., 2007) and Probst bundles have been suggested to emerge as a result of defective midline crossing of callosal axons, which is caused by disruption of transient midline structures and/or molecules they secrete (Donahoo and Richards, 2009). Therefore, appearance of both interhemispheric cyst and Probst bundles in Ntn1 deficient embryos suggest that NTN1 may influence interhemispheric midline fusion and/or formation of transient midline structures during development of the CC. In addition, there was an absence of bridge shape thickening of cells in medial CSB region and obvious continuous line of cells in the interhemispheric midline of Ntn1 deficient mice compared to control mice (Fig.1E and F in II). These observations further suggested that NTN1 may have an impact on interhemispheric midline fusion and development of transient midline structures in the CSB region. Interestingly, the CC was still absent and presence of interhemispheric cyst was still obvious in Ntn1 deficient mice at Pn6 (Fig.4G and H in II). In summary, the overall analysis of CC development in Ntn1 deficient mice suggested that NTN1 may influence interhemisheric fusion and formation of transient midline structures, which are both required for the proper development of the CC.

4.2.2 Expression of Ntn1 and NTN1 receptors during formation of CC

The expression of Ntn1 and NTN1 receptors DCC, Neogenin, and Unc5a-d during development of the CSB region was verified by using RNA hybridisation and X-gal staining method, where LacZ insertion in Ntn1 locus was utilized (Salminen et al., 2000). This confirmed that Ntn1, DCC, Neogenin, Unc5b, and Unc5d were expressed in the CSB region during development of the CC, but Unc5a and Unc5c were not (Fig.2 in II and unpublished observations). At E15.5, Ntn1, DCC, and Unc5b expression was detected at the interhemispheric midline (Fig.2A, C, G and Fig.6A, B in II), suggesting that NTN1 signalling may participate in the interhemispheric fusion process. More precisely, Ntn1 and Unc5b seemed to be expressed by the leptomeningeal cells in the interhemispheric fusion area and also dorsal to the forming CC next to the cingulate cortex. In contrast, DCC expression was confined to a thin line of cells at the midline, but was not detected in the leptomeningeal area next to the cingulate cortex. Therefore, DCC expression was most likely corresponding to CR-expressing neurons, which has been earlier shown to express DCC in the midline area (Shu et al., 2000; Shu et al., 2003b). Interestingly, Ntn1/lacZ activity at the interhemispheric midline of Ntn1 heterozygous embryos disappeared concurrently with CC formation, while in Ntn1 deficient embryos the interhemispheric midline appeared unfused and Ntn1/lacZ activity remained at the midline (Fig.6C, D in II). Similarly, the NTN1 receptor Unc5b was strongly expressed by the leptomeninges at the interhemispheric midline before fusion and this expression also disappeared concurrently with formation of the CC, but remained in the dorsal to the CC at E17.5 (Fig.2G, H in II). This suggests that NTN1-UNC5B signalling at the midline could be related to interhemispheric fusion. However, 51 meningeal expression of Unc5b in the interhemispheric midline is more likely to be affecting meningeal angiogenesis than interhemispheric fusion, since nervous system specific Unc5b conditional mutant mice have been reported to survive into adulthood with normal brain morphology and Unc5b expression is largely restricted to the developing vascular system (Lu et al., 2004; Larrivee et al., 2007; Navankasattusas et al., 2009; van den Heuvel et al., 2013). Ntn1 expression was also detected in the ventro-medial walls of the lateral ventricles and DCC expression in the dorso-lateral region of septum adjacent to the lateral ventricles walls at E15.5 and E17.5 (Fig.2A, B, C, D in II). This region has been proposed to be birthplace of GW cells and to generate CR producing guidepost neurons, which then migrate towards the interhemispheric midline and form SCS (Shu and Richards, 2001; Shu et al., 2003c). Ntn1, DCC, Neogenin and Unc5d expression was also detected ventrally to the forming CC in septum at E15.5 (Fig.2A, C, E, I in II). At E15.5, overlapping expression of DCC and Neogenin was also detected in the cingulate cortex, which is the birthplace of the first pioneering axons (Koester and O`Leary, 1994). This confirms that NTN1 signalling might influence the guidance of pioneering axons from the cingulate cortex towards the interhemispheric midline. At E17.5, Ntn1 and DCC expression continued to resemble their previous expression patterns in the CSB region (Fig.2B, E in II). However, a strong increase of Ntn1 expression was detected in the cingulate cortex and IG, which was overlapping with DCC and Neogenin expressions (Fig.2B, D, F in II). Furthermore, Ntn1 expression was detected in septum ventral to the CC, similar to E15.5, but expression of DCC, Neogenin, and Unc5d was more restricted to SCS (Fig.2C, F, I in II). This suggests that NTN1 signalling could affect SCS formation. In summary, expression analysis confirmed that Ntn1 and NTN1 receptors DCC, Neogenin, Unc5b,andUnc5d are expressed in CSB regions, which contribute to the formation of the CC.

4.2.3 Development of transient midline glial structures in Ntn1 deficient mice

Histological and expression analysis suggested that NTN1 signalling may influence interhemispheric fusion and formation of transient midline structures. Therefore, development of transient midline glial structures, which has been thought to mediate interhemispheric fusion and guide callosal axons towards the hemispheric midline, was analyzed first (Silver et al., 1982; Silver et al., 1993; Shu and Richards, 2001; Shu et al., 2001; Richards et al., 2004). In order to follow the development of these glial structures, GFAP antibody stainings were performed at E16.5-E17.5 (Fig.3 in II). At E16.5- E17.5, location and positioning of IG, GW, and MZ glia was appropriate in Ntn1 deficient mouse embryos compared to control embryos (Fig.3 in II). Thus, the development of these transient midline glial populations was relatively normal in Ntn1 deficient embryos. However, Ntn1 deficient embryos seemed to have a continuous string of glial cells at the interhemispheric midline, which is in line with a previous study suggesting that MZ and IG glia share a common origin and are separated into two independent structures by the developing CC (Shu et al., 2003c). Thus, the observed absence of CC in Ntn1 deficient embryos may lead to unsuccessful segregation of MZ and IG glia into two clearly independent structures. Another observed difference between Ntn1 deficient embryos and control embryos was morphogenesis of GW (Fig.3 in II). At E16.5, location and morphology of GW was indistinguishable between Ntn1 deficient embryos and control embryos (Fig.3A, B in II). However, Ntn1 deficient embryos GW appeared to lose its uniform shape and intermingle with callosal axons at E17.5 (Fig.3E, F in II). This observed disorganization of GW glia in Ntn1 deficient embryos may also be related to the inability of callosal axons to cross the midline and form CC. In such a case, callosal axons grow backwards and intermingle with GW glia, as they form Probst bundles in the same region (Shen et al., 2002; Donahoo and Richards, 2009). In summary, it appeared that early development of transient midline glial structures was normal in the Ntn1 deficient embryos CSB region and later changes in these structures were most likely secondary 52 defects caused by unsuccessful formation of the CC. In addition, both IG and GW appeared to fulfil their function, as a dorsal and ventral barrier for approaching callosal axons, directing them towards the interhemispheric midline (Shu and Richards, 2001; Shu et al., 2003a; Shu et al., 2003b)

4.2.4 NTN1 deficiency affects the distribution of neuronal guidepost cells in CSB region

Besides midline glial cell structures, callosal axons are also guided towards the interhemispheric midline by transient neuronal guidepost cells (Shu et al., 2003b; Niquille et al., 2009; Niquille et al., 2013). These neuronal guidepost cells include CR-producing interneurons, which have been proposed to emerge from two distinct areas during the development of the CSB region (Shu et al. 2003b; Niquille et al., 2009). At first, CR-expressing guidepost neurons were thought to arise from the medial aspect of the lateral ventricles at E15.5, where they migrate towards the hemispheric midline and form a visible SCS structure by E17 (Silver et al., 1982; Hankin and Silver, 1988; Shu et al., 2003b). However, later studies detected CR-producing guidepost neurons at the interhemispheric midline already at E14.5 (Niguille et al., 2009; Benadiba et al., 2012). Furthermore, it was proposed that these CR-producing guidepost neurons originate from the retrobulbar ventricle and they invade the CC through tangential subpial migration (Niguille et al., 2009; Benadiba et al., 2012). In order to investigate whether NTN1 deficiency affected the development and distribution of CR- producing neurons in the CSB region, a series of stainings using anti-CR antibody was performed at E15.5, E16.5, E17.5, and Pn6. Interestingly, Ntn1 deficient embryos lacked CR-producing neurons in the interhemipheric midline detected in control littermates at E15.5-E17.5 (Fig.4 in II). In addition, Ntn1 deficient embryos also lacked some CR-producing neurons, which formed between IG and the developing CC in control embryos at E15.5 and E16.5 (Fig.4A-D in II). It also appeared that Ntn1 deficient embryos had fewer CR-producing neurons in the prospective area of future SCS in septum (Fig.4A-D in II). At E17.5, the SCS was composed normally in control embryos beneath the CC (Fig.4E). In contrast, no clear SCS was formed in Ntn1 deficient embryos and CR- producing neurons appeared mainly to follow callosal axons into the Probst bundles (Fig.4F). Interestingly, the observed absence of CR-producing neurons in the interhemispheric midline combined with only a partial SCS formation in Ntn1 deficient embryos, suggest that CR-producing guidepost neurons could be derived from two distinct origins during development of the CSB region. Thus, it is possible that CR-producing guidepost neurons could be derived from the retrobulbar ventricle, located ventrally immediately after the olfactory bulb, where they migrate subpially to the marginal zone (Meyer et al., 1998; Meyer et al., 2000; Niquille et al., 2009) and also from the medial aspect of the lateral ventricles, where they migrate towards the interhemispheric midline (Shu et al., 2003b). In addition, we observed less CR-producing neurons in dorsal midline of Ntn1 deficient mice. This defect might be related to the inability of NTN1 to guide callosal axons and/or CR-producing neurons towards the hemispheric midline. Previous studies have shown that NTN1 receptors, DCC and Neogenin, are expressed by callosal axons and CR-producing neurons (Shu et al., 2000; Shu et al., 2003b; van den Heuvel et al., 2013). In addition, guidance of callosal axons and CR-producing neurons has been proposed to progress hand in hand, although, the nature of this relationship is still to be discovered (Donahoo and Richards, 2009). On the other hand, Neogenin also interacts with repulsive guidance molecule (RGM) a and b, which have a repulsive effect to axon guidance, cell adhesion and migration (Conrad et al., 2010; Key and Lah, 2012). These RGMs are also expressed in an overlapping manner with Neogenin in the CSB region (van den Heuvel et al., 2013). Therefore, NTN1 deficiency could convert the hemispheric midline to be more repulsive for approaching Neogenin-expressing callosal axons and CR-expressing cells. We also detected NTN1 receptor Unc5d expression in SCS (Fig.2 in II). NTN1 has been proposed to induce repulsive axon 53 guidance through UNC5 receptors, but UNC5D has also been shown act independently of NTN1 signaling (Takemot et al., 2011; Yamagishi et al., 2011; Miyoshi and Fishell, 2012). Thus, it is difficult to interpret the significance of NTN1-UNC5D signaling in development or function of SCS. In summary, NTN1 affects the distribution of CR-producing neurons in the CSB region and this effect may be mediated through its receptors DCC, Neogenin, and UNC5D, which are expressed either by CR-producing neurons or in the CSB region containing CR-producing neurons. Influence of NTN1 deficiency to the proper distribution of CR-producing neurons has also been shown in the developing cerebral cortex (Stanco et al., 2009) and olfactory bulb (I). Therefore, it seems that NTN1 is preferentially influencing the guidance of either progenitor cells generating CR-producing neurons and/or CR-producing neurons throughout the forebrain. However, NTN1 deficiency does not completely block migration of CR-producing neurons (I, II, Stanco et al., 2009), suggesting that NTN1 signaling is a component of a complex network system responsible for guiding CR- producing neurons to their final destinations. Moreover, postnatal Ntn1 deficient mice appeared to have an increased number of ectopically-distributed CR-producing neurons in the septum compared to control mice (Fig.4G-H in II). This observation suggests that defective formation of CC may delay the disappearance of CR-producing neurons from the CSB region and lead to their ectopic distribution near aberrantly projecting callosal axons in the septum (Shu et al., 2003b).

4.2.5 Early callosal axons are guided close to the pial basal lamina in Ntn1 deficient mice

Interestingly, recent results have shown that NTN1-DCC signalling attracts mainly early pioneering axons towards the midline, but later arriving neocortical axons are not attracted nor repulsed by NTN1-DCC signalling (Fothergill et al., 2013). More precisely, NTN1-DCC signalling allows callosal axons to cross the hemispheric midline by interacting and attenuating repulsive SLIT- ROBO signalling at the midline (Fothergill et al., 2013). Early guidance of callosal axons towards the interhemispheric midline was compared by performing double-immunostaining with Laminin and L1 antibodies at E15.5 and E16.5. At E15.5, L1 positive callosal axons in control and Ntn1 deficient embryos were approaching the pial basal lamina, but did not cross the midline (Fig.7A, B, I, J in II). However, in control embryos L1-positive axons already contacted the pial basal lamina (Fig.7 I in II), but in Ntn1 deficient embryos, callosal axons were advancing further away from the pial basal lamina and appeared not to be orientated towards it (Fig.7J in II). At E16.5, L1-positive callosal axons crossed the midline and formed CC in control embryos (Fig.7C in II); whereas, in Ntn1 deficient embryos, many L1-positive axons appeared to avoid midline and turned backwards into adjacent septum, as if they would be repelled by the midline (Fig.7D in II). In conclusion, early callosal axons in Ntn1 deficient embryos are guided close to the midline basal lamina and the majority of these axons seem to be repulsed later by the midline, as they turn backwards into the septum. As mentioned above, NTN1-DCC signaling is proposed to attenuate SLIT-ROBO mediated repulsion at the hemispheric midline (Forhergill et al., 2013). In addition to SLIT-ROBO signaling, guidance ligand Draxin has been shown to mediate repulsion of the callosal axon at the interhemispheric midline through binding DCC (Islam et al., 2009; Ahmed et al., 2011). Thus, NTN1 deficiency may modulate the interhemispheric midline region to be more repulsive for the approaching callosal axons. Furthermore, it has been suggested that interhemispheric leptomeninges act as a repulsive barrier for callosal axons and prevent premature formation of the CC (Choe et al., 2013). For example, Sema3A produced by meningeal cells repulse growing axons in CNS (Niclou et al., 2003). Therefore, formation of the CC requires active guidance of callosal axons towards the interhemispheric midline and the transformation of midline region from a repulsive barrier to a more permissive tissue. In addition to NTN1-DCC signaling, CR-producing neurons in the dorsal midline area express Sema3C, which attracts Npn1-expressing callosal axons towards the hemispheric midline (Niquille et al., 2009). Ntn1 deficient 54 embryos lacked CR-producing neurons from interhemispheric midline, which may also convert the hemispheric midline to be more repulsive for approaching callosal axons. Thus, NTN1 deficiency in the hemispheric midline region may affect to the overall balance of attractive- and repulsive signaling in several ways and convert the hemispheric midline to be more repulsive for approaching callosal axons.

4.2.6 NTN1 deficiency affects interhemispheric midline fusion

Interhemispheric midline fusion is suggested to progress in front of arriving callosal axons in ventral-dorsal and caudal-rostral directions beginning at E14-E15 in mice (Silver et al., 1982; Silver et al., 1993; Livy and Wahlsten, 1997). Therefore, it precedes midline crossing of pioneering callosal axons (Silver et al., 1982). The fusion process includes removal of leptomeninges ( and arachnoid mater) found between the hemispheres (Silver et al., 1982; Silver et al., 1993). Leptomeninges contain pial meningeal cells and blood vessels, which are in close contact with the pial basal lamina (Siegenthaler and Pleasure, 2011). The components of the pial basal lamina such as collagens, fibronectin, laminin, nidogen, perlecan, and heparansulphate proteoglycan are produced by meningeal cells (Sievers et al., 1994; Miner et al., 1997; Halfter et al., 2002). We used anti-Laminin antibody to follow and compare disruption of the pial basal lamina together with progression of interhemispheric fusion between control and Ntn1 deficient embryos at E15.5- E17.5 (Fig.5 and Fig.6 in II). At E15.5, a continuous laminin sheet was detected at the interhemispheric midline of control and Ntn1 deficient embryos, which indicated that disruption of pial basal lamina had not started and interhemispheric fusion had not begun in these embryos (Fig.5A, B and Fig.6G in II). At E16.5, the continuous laminin sheet was absent from interhemispheric midline of control embryos, but still present in Ntn1 deficient embryos (Fig.5C, D in II). This observation suggests that interhemispheric fusion was completed in control embryos, but in Ntn1 deficient embryos disruption of the pial basal lamina had not begun and interhemispheric fusion was not completed at E16.5. Interestingly, the laminin sheet was still obvious in the midline of Ntn1 deficient embryos at E17.5 (Fig.5F in II). Therefore, the results presented here suggest that both removal of leptomeninges and disruption of basal lamina found between hemispheres were defective in Ntn1 deficient mice. In addition, an interhemispheric cyst was detectedin Ntn1 deficient mice midline, which has been proposed to appear as a consequence of defective midline fusion (Richards et al., 2004; Wahlsten et al., 2006; Paez et al., 2007). The cellular and molecular events controlling interhemispheric fusion are still largely unknown, albeit, few mouse strains and genes have been proposed to exhibit defective interhemispheric fusion (Silver et al., 1982; Stumpo et al., 1995; Demyanenko et al., 1999; Brouns et al., 2000; Wahlsten et al., 2006; Paez et al., 2007). Interestingly, MZG which arises and locates conveniently to the fusion area has been proposed to facilitate midline fusion (Silver et al., 1982; Hankin and Silver, 1988; Silver et al., 1993; Richards et al., 2004). However, it is still unrevealed whether the presence of MZG is an absolute requirement for interhemispheric fusion and how MZG is mediating this process. Nfia mutant mice show disrupted formation of the MZG, but no obvious defects in the midline fusion process (Shu et al., 2003a). In addition, MZG was detected spatially and temporally in the correct position at the midline of Ntn1 deficient embryos, but interhemispheric fusion did not take place. Furthermore, Ntn1/lacZ was not expressed in MZG in the interhemispheric midline (Fig.6J in II) and, in general, NTN1 signaling has not been shown to affect the function or development of GFAP-positive glial cells in the mammalian CNS (I; Tsai et al., 2006; Rajasekharan et al., 2009; Lai Wing Sun et al., 2011). Together these observations suggest that MZG may not be the only determinant directing interhemispheric midline fusion. Ntn1 deficient embryos also lacked CR-producing neurons from the interhemispheric midline region. Regulatory Factor X3 (RFX3) and JNK interacting protein (JSAP1) deficient mice present disorganization of CR-producing neurons in the CSB region associated with defects in 55 interhemispheric midline fusion (Ha et al., 2005; Benadiba et al., 2013). Thus, it may be possible that CR-producing neurons at the midline participate in the interhemispheric fusion process. One hypothetical explanation of how CR-producing neurons and NTN1 could facilitate interhemispheric fusion arises from studies conducted with C.Elegans (Ziel et al., 2009; Hagedorn et al., 2013). In C.elegans, NTN1-DCC signaling is required for the removal of basement membrane found between uterine and vulval tissues (Hagedorn et al., 2013). More precisely, NTN1-DCC signaling directs generation and orientation of anchor cell invasive protrusions, which physically penetrate the basement membrane and promote anchor cells invasion from uterine tissue through the basement membrane to vulval tissue. Furthermore, anchor cells lacking DCC or missing vulval side secretion of NTN1 leads to incomplete removal of this basement membrane (Ziel et al., 2009; Hagedorn et al., 2013). Thus, CR-producing neurons expressing DCC at the interhemispheric midline may respond to NTN1 in a similar manner and facilitate the breakdown of laminin-containing pial basal lamina during the interhemispheric fusion process. Ntn1 and Ntn1/lacZ expression was detected in the leptomeningeal cells next to the pial basal lamina prior to interhemispheric fusion. This Ntn1 expression disappeared after interhemispheric fusion in Ntn1 heterozygous mice, but was still obvious in the unfused midline of Ntn1 deficient embryos. Thus, NTN1 might be required for the disruption of pial basal lamina and removal and detachment of the Ntn1 expressing leptomeningeal cells from the interhemispheric midline. It has been shown that leptomeningeal cells and their functionality are required to maintain intact pial basal lamina in mouse brain (Sievers et al., 1994; Beggs et al., 2003). Therefore, the unsuccessful removal of the leptomeningeal cells from the interhemispheric midline in Ntn1 deficient mice may lead to prolonged persistence of pial basal lamina in the midline. NTN1 is required in a similar manner for the detachment of inner ear epithelium from basement membrane and NTN1 deficiency in this area causes inefficient disruption of basement membrane and the epithelium remain intact (Salminen et al., 2000). In addition, NTN1 has been suggested to influence initiation of cell migration either cell-autonomous and/or paracrine manner (I; Jarjour et al., 2003; Tsai et al., 2006). Interestingly, callosal axons did not cross the hemispheric midline and form CC in Ntn1 deficient mice at Pn6. This observation further verifies the previous finding that interhemispheric midline fusion has to be complete before birth to enable callosal axons to cross the hemispheric midline and form CC (Silver et al., 1982).

4.2.7 NTN1 may influence the development of CC in several ways

The results presented here suggest that NTN1 influences, in an either direct or indirect manner, the development of the CC by affecting the formation of transient cellular structures, interhemispheric midline fusion, and guidance of callosal axons. Since these events are partially overlapping and connected to each other, it is very challenging to distinguish and interpret causalities between these events. However, NTN1 is required for the breakdown of pial basal lamina in the interhemispheric midline, which is an essential part of the fusion process and precedes midline crossing of callosal axons. Nevertheless, the question of whether the breakdown of pial basal lamina in the interhemispheric midline is initiated by the CR-producing neurons, MZG, and/or leptomeningeal cells requires further investigation. Interestingly, NTN1 has been shown to be involved in local disruption of basement membranes at the fusion plate area of the developing mouse inner ear (Salminen et al., 2000; Matilainen et al., 2007), and in the uterine wall of C.elegans (Hagedorn et al., 2013). These studies do not reveal any common mechanism for the NTN1 dependent breakdown of basement membranes, however. Thus, these studies imply that NTN1 may influence the disruption of basement membranes in multiple ways and this function could also be environment dependent.

56 4.3 Characterisation of the expression and function of ABBA (III)

Mouse ABBA was identified in a search for novel homologues to I-BAR domain family proteins MIM and IRSp53. To understand the biological role of ABBA, a thorough ABBA expression study was first conducted in mice.

4.3.1 Developmental and adult expression of ABBA in mice CNS

Northern blot analysis showed that ABBA was expressed in several adult mice organs, but the strongest expression was in brain (Fig. 1B in III).Therefore, the main interest of this study was focused to optain more information about the expression and possible function of ABBA in the CNS. In order to gain further information and more detailed view of the developmental distribution of ABBA mRNA in the CNS, a series of RNA hybridization experiments were performed in embryos and also in adult mice. At E9.5-E10.5, ABBA mRNA was detected only in the neural tube floor plate, extending from midbrain to the end of spinal cord. At E12.5, the expression of ABBA was still strong in the floor plate area which contains radial glia-like cells, and in the marginal zone, which contains mainly cellular extensions (Fig. 1C in III). In addition, ABBA expression was detected near the pial surface of the marginal zone, where the radial end-feet attach to the pial membrane (Fig. 1C, F in III). A weaker expression was also detected in the brain and spinal cord parenchyma (Fig. 1C, F in III). At E14.5-E18.5, a strong expression of ABBA was detected in a specific midline glial raphe structure and in the pial surface surrounding the brain tissue (Fig. 1G, I, J, H in III). The midline glial raphe structure has been proposed to emerge from floor plate cells and it forms a continuous wall-like structure separating left and right brainstem in the midbrain, hindbrain, and cervical spinal cord (Van Hartesveldt et al., 1986; Mori et al., 1990; Mckanna, 1993; Shu and Richards, 2001). Furthermore, both floor plate and midline glial raphe structures are suggested to be transient structures formed by radial glia-like cells (Van Hartesveldt et al., 1986; Mori et al., 1990). They also seem to share some functional properties, for example, affecting the formation of midline commissures by guiding midline crossing of approaching commissural axons (Mori et al., 1990; Shu and Richards, 2001). In the adult brain, the strongest ABBA expression was observed in the molecular layer of the cerebellum, and moderate expression was also present throughout the brain (Fig. 1K in III). Taken together, ABBA expression analysis suggested that in the developing CNS, ABBA is strongly expressed by different populations of radial glia-like cells and RGCs. In addition, ABBA is also expressed in the marginal zone, where the end-feet of these cells locate.

4.3.1.1 ABBA protein is specifically present in radial glia-like cells and RGCs of CNS

To confirm protein localisation of ABBA in the mouse CNS, a polyclonal antibody against mouse ABBA protein was raised (Fig.2A in III). Immunohistochemical analysis revealed that ABBA protein was indeed present in the floor plate and in midline glial raphe structures similarly to ABBA mRNA (Fig. 2B, E, I in III). Furthermore, to confirm that ABBA was expressed by RGCs and radial glia-like cells, a series of double labeling experiments with the radial glial marker anti-RC2, neuronal marker TUJ1, and anti-ABBA antibody were pursued. This double labeling assay revealed that ABBA co-localised with RC2 positive radial glia-like cells and RGCs all the way from the spinal cord to the cerebral cortex, but did not co-localise with TUJ1 positive neurons (Fig.2D, L, T in III). Furthermore, co-localisation of ABBA and RC2 seemed to be enriched in radial glia-like cells and RGCs extensions (Fig. 2D, L, T in III). Expression analysis demonstrated that ABBA was expressed during CNS development specifically by glial cells, but not by neuronal cells. In summary, both mRNA and protein analysis suggest a subcellular localization of ABBA in radial glia-like cells and RGCs end-feet. It has been estimated that more than 1000 mRNAs are localized 57 in neuronal protrusions and over 250 different mRNA have been identified in astrocyte protrusions (Thomsen et al., 2013). This subcellular localization of mRNAs includes a variety of classes, for example, structural cytoskeletal proteins and the function of these mRNAs are associated with many cellular processes (Mili and Macara, 2009). Nevertheless, subcellular localization of ABBA mRNA might be required to achieve asymmetric localization and activity of ABBA protein in RGCs (Mili and Macara, 2009). Besides ABBA, for example, Nestin mRNA has been shown to localize predominantly in RGCs end-feet (Dahlstrand et al., 1995).

4.3.1.2 ABBA localizes to the interface between the plasma membrane and the cortical actin cytoskeleton in cultured radial-glial-like cells

Since, ABBA was expressed specifically by the RGCs of the developing CNS, a rat C6-R radial glial cell line, which possesses many characteristics related to RGCs (Friedlander et al., 1998), was used to study the cellular localization of ABBA. Antibody stainings with ABBA, F-actin, and membrane marker CM-Dil in these C6-R cell cultures revealed that ABBA localised to plasma membranes in the front edge of the cortical actin cytoskeleton at perinuclear areas (Fig.3C-H in III). More precisely, ABBA localised into the interface of the cortical actin cytoskeleton and plasma membrane in lamellipodial structures and membrane ruffles (Fig.3I-K in III). Since the majority of cells display different protrusion or extensions in their surfaces and these structures are determined by the dynamic association of cellular actin cytoskeleton with plasma membrane, the observed cellular localisation and genealogy of ABBA proposed that it may have a role in the formation of distinct cell shapes.

4.3.2 ABBA interacts with actin monomers, but lacks actin bundling activity

I-BAR domain proteins are known to promote formation of plasma membrane protrusions by influencing intracellular actin dynamics (Saarikangas et al., 2009; Saarikangas et al., 2010; Zhao et al., 2011). The I-BAR domain of ABBA contains an actin monomer binding domain WH2, which in many ways influences dynamic regulation of actin inside cells (Paunola et al., 2002; Mattila et al., 2003). Therefore, a fluorometric assay was performed to examine whether the WH2 domain of ABBA binds to actin monomers. The results showed that ABBA interacts with actin monomers through its WH2 domain and shows high affinity towards ATP-bound actin monomers and lower affinity towards ADP-bound actin monomers (Fig.4A-D in III). Furthermore, mutated form of WH2 domain did not bind to ATP- or ADP-bound actin monomers, which further confirmed that ABBA interacts with actin monomers through its WH2 domain (Fig.4B, D in III). As mentioned above, regulation of actin dynamics inside a cell has an essential role in the formation of cell membrane protrusions. It is known that actin monomers polymerize to form actin filaments (F-actin) and that ATP-bound actin monomers polymerize easier than ADP–bound. Thus, these results suggest that ABBA may promote the addition of actin monomers into actin filaments. Another feature connecting the I-BAR domain to actin dynamics is its proposed capability to act as actin cross-linking domain, which might organize F-actin into different actin bundles or actin networks inside cells. For example, ABBA was originally named an actin bundling protein based on its sequence similarity to other I-BAR-domain proteins (Yamagishi et al., 2004). Interestingly, when this bundling capability was tested, any ABBA I-BAR domain driven F-actin bundling activity could not be detected (Fig. 4E, F in III). In addition, the I-BAR domain of ABBA localised to plasma membrane, but not to F-actin bundles (Fig.4G in III). These results suggested that ABBA participates in the regulation of actin dynamics by binding a polymerization sensitive form of actin monomers, but does not directly influence F-actin bundling as proposed for the other I-BAR domain proteins. Thus, ABBA may promote actin monomer addition to actin filaments and/or serve as a link between actin filaments and plasma membrane interactions. 58 4.3.3 ABBA binds and deforms plasma membranes in vitro and in vivo

It has been proposed that the formation of cell protrusions requires a dynamic polymerization of actin cytoskeleton, which produces a force needed to push plasma membrane forward (Gov and Gopinathan, 2006). During this process, plasma membranes undergo deformation, which is also suggested to induce formation of protrusions (Mattila and Lappalainen, 2008; Robens et al., 2010; Coutinho-Budd et al., 2012). Therefore, many actin binding proteins contain a membrane deforming domain which allows them to connect cytoskeletal actin dynamics to membrane deformation and vice versa. It has been previously shown that I-BAR domain proteins MIM and IRSp53, which are the closest homologs of ABBA, bind to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-rich membranes and deform them into tubular structures (Mattila et al., 2007; Suetsugu et al., 2006). Thus, the competence of ABBA to bind and deform [PtdIns(4,5)P2]-rich membranes was studied. These results confirmed that ABBA I-BAR domain binds with a high affinity to [PtdIns(4,5)P2]- rich membranes, similarly to MIM and IRSp53 (Fig.5A in III). In addition, these experiments revealed that ABBA I-BAR domain induced clustering and tubulation of the vesicles, whereas the absence of ABBA I-BAR domain led to even distribution of vesicles in a given sample (Fig. 5C in III). Thus, ABBA displayed a strong membrane deforming and tubulation activity, similar to MIM and IRSp53 I-BAR domain proteins (Fig.5 in III). As mentioned above, formation of cell protrusions has been suggested to be driven by actin polymerization and/or membrane deformation. Since, ABBA was displaying activity both in actin polymerization and membrane deformation processes, an assumption that ABBA might participate in the formation of cell protrusions was made. This hypothesis was confirmed as C6-R cells transfected with ABBA I-BAR domain were showing elevated formation of microspikes (Fig.6A and supplementary material Movie 2 in III), which are closely linked to generation and dynamics of lamellipodial/filopodial structures (Svitkina et al., 2003). To gather more information about ABBA I-BAR domain induced formation of microspikes, an assay was conducted, where growth of ABBA I-BAR domain induced microspikes was followed in the presence of an actin polymerization inhibitor. These results showed that extending microspikes were not completely lost in the presence of an actin polymerization inhibitor, although their numbers were severely reduced. Thus, proposing that both the membrane deformation activity and the polymerization of actin filaments are influencing ABBA I-BAR domain induced microspike formation, although, it is not entirely depended on actin polymerization (supplementary material Fig.S5 in III).

4.3.4 ABBA participates in regulation of lamellipodial dynamics and process extension

The expression studies revealed that ABBA localizes to the interface of the actin cytoskeleton and plasma membrane in RGCs extensions. In addition, the ABBA I-BAR domain induced formation of cell protrusion by participating into plasma membrane deformation and actin polymerization processes. To study the influence of ABBA specifically in RGC extensions, ABBA knockdown C6- R cells were produced by using an RNA interference (RNAi) method with short interfering RNAs (siRNA) targeted against ABBA (Fig.6 in III). Interestingly, ABBA knockdown cells did not show any defects in overall cell morphology or formation of filopodial structures. Therefore, a more careful approach was applied to study the dynamics of C6-R cells process extension over time, and a clear reduction in process extensions velocity was observed in ABBA knockdown cells (Fig.6C, D in III). This result was further confirmed by a rescue experiment where an ABBA siRNA resistant construct was able to rescue the observed phenotype (Fig.6F in III). Since endogenous ABBA localised to the leading edge of lamellipodium (Fig.3F-H in III), but did not interfere with filopodial formation, a kymograph analysis, where extension and retraction velocities of lamellipodia can be easily detected was performed with ABBA knockdown cells (Hinz 59 et al., 1999). This analysis revealed that depletion of ABBA leads to a significant decrease in ruffling frequency and growth speed of an individual lamellipodial protrusion compared to control cells (Fig.7B, C in III). Interestingly, ABBA was also found to bind with small GTPase RAC1 (supplementary material Fig.S2A in III), which participates in the generation of cell protrusions and membrane ruffling (Ridley and Hall, 1992; Wu et al., 2009). Taken together, the functional studies with ABBA suggest that it connects plasma membrane deformation to actin dynamics. Thus, it enhances formation of cell protrusions, which are required for regulation of distinct cellular functions, such as cell motility, adhesion, and spreading.

4.3.5 ABBA may participate in maintenance of radial migration in brain

The results of this study indicate that ABBA participates in the regulation of lamellipodial protrusions by enhancing actin dynamics and connecting plasma membrane deformation to actin cytoskeleton. Interestingly, ABBA was especially expressed by RGC extensions during CNS development, particularly, in glial end-feet near meningeal pial basement membrane. During early development of brain, interaction between radial glial end-feet and pial basement membrane is required to maintain radial migration and integrity of pial basement membrane (Nakano et al., 1996; Weimer et al., 2009; Myshrall et al., 2012; see chapter 1.1.3.1.1). Thus, ABBA may have an important role in the maintenance of radial migration and interactions of radial glial end-feet with pial basement membrane. Interestingly, disruptions in radial glial end-feet linkage with meningeal basement membrane leads to retraction of these extensions (Sievers et al., 1985; Graus-Porta et al., 2001), and subsequent apoptosis of these RGCs, which leads to reduced production of neuronal cells and, consequently, reduced size of cortex (Radakovits et al., 2009; Siegenthaler and Pleasure, 2011). Furthermore, disruptions in radial glia end-feet interactions with meningeal basement membrane are associated with breaches in pial basement membrane and ectopic migration of neurons beyond the pial surface (Saito et al., 1999; Myshrall et al., 2012). This overmigration of neuronal cells to meningeal arachnoid space causes many cortical and cerebellar malformations, which are jointly called cobblestone lissencephaly type II syndromes (Haubst et al., 2006; Devisme et al., 2012; Barkovich et al., 2012). Therefore, it would be beneficial to further assess the role of ABBA in genetically modified mice.

4.4 Impact of HPV16 E6/E7 oncoproteins on neural stem/progenitor cells (IV)

E6/E7 oncoproteins can modulate behavior of the host cell by altering regulation of cell cycle, proliferation, survival, and DNA replication (Ganguly and Parihar, 2009). It is known that E6/E7 oncoproteins degrade and interact with a wide variety of cellular proteins (Münger et al., 2004; McLaughlin-Drubin and Münger, 2009b). This degradation effect of E6/E7 oncoproteins was verified with western blotting, which showed a clear reduction in protein levels of p53, pRb, p130, and p170, in neural stem/progenitor cells transduced with E6/E7 oncogene construct (Fig.1B in IV). In addition, neural stem/progenitor cells isolated from p53 deficient mice were used for the experiments. Therefore, the results obtained by this study are also related to cellular functions of p53 protein and pRb protein family.

4.4.1 HPV16 E6/E7 oncogenes increase cell proliferation and self-renewal of neural stem/progenitor cells

The proliferative effects of E6/E7 oncoproteins and p53 deficiency on neural stem/progenitor cells were investigated by using bromodeoxyuridine (BrdU) assay and colorimetric MTT assay, which measures metabolic activity of cells. These assays revealed a significant increase in proportional numbers of proliferative cells and decrease in time required for doubling the original cell population 60 of E7- and E6/E7-transduced or p53 deficient neural progenitor/stem cells compared to control cells (Fig.1C in IV). Thus, E6/E7 oncoproteins or p53 deficiency was expanding the population of proliferative cells in vitro. To identify whether E7- and E6/E7-transduction or p53 deficiency was expanding the proliferative cell pool of neural stem/progenitor cell by affecting their self-renewal capacity, a clonal analysis, where primary neurospheres were disassociated to single cell suspension and their capacity to form secondary spheres was measured. The clonal analysis revealed that E6/E7 oncogenes and p53 deficiency increased the self-renewal capacity of neural stem/progenitor cells, although, the enhanced self-renewal capacity of E6-expressing cells was not statistically significant (Fig.1D in IV). These results propose that E6/E7 oncoproteins and p53 tumor suppressor protein have an important role in regulation of neural stem/progenitor cell proliferation and self-renewal. Consequently, E6/E7 and p53 oncoproteins may cause a defective maintenance and/or expansion of neural stem/progenitor pool during brain development (see chapter 1.1.2). On the other hand, these results also raised a question as to whether E6/E7 and p53 oncoproteins could directly or indirectly influence the normal differentiation process of neural stem/progenitor cells. Since, it has been assumed that cells must exit from cell cycle before entering the differentiation process, however, the mechanisms coordinating and connecting these two events are still elusive (Hindley and Philpot, 2012).

4.4.2 Neural stem/progenitor cells expressing E6/E7 oncogenes maintain their capacity to proliferate after initiation of differentiation

As mentioned above, onset of differentiation is normally related to terminal mitosis (cell cycle exit) and that has been thought to be an irreversible process (Lipinski and Jacks, 1999; Blais et al., 2007). Furthermore, the time interval between terminal mitosis and neuronal differentiation has been proposed to be around 24h in vitro (Rohrer and Thoenen, 1987). To investigate whether E6/E7 oncogenes or p53 deficiency affected the initiation of the differentiation process in neural stem/progenitor cells, E6/E7 transduced or p53 deficient neural stem/progenitor cells were introduced to a growth factor free environment, which induces and promotes initiation of cell differentiation. In these cell culture conditions, virtually all the cells, including E6/E7 transduced cells or p53 deficient cells, attached to the bottom of cell culture wells and presented cell morphology associated with differentiation. Interestingly, when growth factors were re-introduced to these differentiation cultures, E6/E7 transduced and p53 deficient cells showed substantial reformation of neurospheres compared to the control cells, suggesting that they had retained their capacity to proliferate (Fig.2A-C in IV). To understand how E6/E7 oncogenes or p53 deficiency was leading to reformation of neurospheres after initiation of differentiation, a sequential BrdU incorporation analysis was performed. These results showed that almost all control neural stem/progenitor cells did exit the cell cycle within the first 24-72h after initiation of differentiation (Fig.3A-B in IV). On the contrary to control cells, a large proportion of E6/E7 expressing neural stem/progenitor cells did not exit the cell cycle and continued entering to S-phase even after 192h after onset of differentiation (Fig.3A in IV). Also, p53 deficient and only E7 oncogene expressing neural stem/progenitor cells displayed delayed cell cycle exit, although the effect was much milder compared to E6/E7 expressing neural stem/progenitor cells (Fig.3A-B in IV). These results suggest that E6/E7 oncogenes and/or p53 deficiency inhibited or delayed the cell cycle exit during in vitro differentiation of neural stem/progenitor cells.

4.4.3 E6/E7 oncoproteins do not affect cell differentiation

The results mentioned above proposed that E6/E7 oncogenes enhance proliferation and self-renewal of neural stem/progenitor cells. To understand better if enhanced proliferation and self-renewal 61 affected cell differentiation, the differentiation process of E6/E7 expressing cells was followed with both in vitro and in vivo experiments. The reverse-transcriptase PCR results showed that expression patterns of neural bHLH transcription factor genes, Ascl1, Hes1, Hes5, and Id2 was similar between undifferentiated E6/E7 expressing and control neural stem/progenitor cells in vitro (Fig.4D in IV). Furthermore, E6/E7 expressing and control neural stem/precursor cells differentiated into neurons (TUJ1 positive) and glia (GFAP positive) after seven days of differentiation (Fig.4E-H in IV). The bHLH transcription factors are known to affect neural progenitor/stem cells proliferation and commitment to a specific cell fate (Kageyama et al., 2005; Powell and Jarman, 2008). Thus, these results suggest that E6/E7 oncogenes fail to influence the proliferation of neural stem/progenitors in a bHLH dependent way and E6/E7 expressing neural stem/progenitor cells are able to differentiate into two major cell types found in the brain, similar to normal neural stem/progenitor cells in vitro. Interestingly, both E6/E7 expressing and control cells were positive for neural stem/progenitor marker nestin after 92h of differentiation, indicating that the differentiation process was not fully completed even after the control cells were withdrawn from the cell cycle. To characterize differentiation and proliferation capacity of E6/E7 expressing neural stem/progenitor cells in a more natural environment, EGFP-E6/E7 expressing and control neural stem/progenitor cells were transplanted into the lateral ventricles of E14.5 mouse embryos in utero. These results were analyzed 92h after transplantation and compared to the results obtained from previous in vitro experiments. Immunohistochemistry showed that transplanted EGFP-E6/E7 cells co-expressed TUJ1, GFAP, or O4 antibodies, demonstrating that transplanted EGFP-E6/E7 cells were able to differentiate into the three major cell types found in brain, neurons, astrocytes, and oligodendrocytes (Fig.5A-I in IV). Thus, the differentiation capacity of E6/E7 expressing neural stem/progenitor cells appeared to be similar both in vitro and in vivo. To analyze whether transplanted EGFP-E6/E7 and control cells were still in cell cycle after 92h of transplantation, a cell cycle analysis from frozen sections was performed with a widely used cell cycle marker: Ki-67 antibody (Gerdes et al., 1984). Interestingly, none of the transplanted cells was observed to co- express Ki-67. Hence, the transplanted EGFP-E6/E7 expressing cells did not exhibit similar inhibited or delayed cell cycle exit as in vitro. This observation suggests that the in vivo environment might be more potent in promoting cell differentiation and cell cycle exit than the in vitro environment. Furthermore, similar results have also been obtained in other studies, where in vitro propagated neural/stem progenitor cells have lost their stem/progenitor abilities, as they have been transplanted to the in vivo environment (Marshall et al., 2006; Darsalia et al., 2007).

4.4.4 Proliferation and self-renewal capacity of neural progenitor/stem cells may not be coupled

When pRb expression was restored in E6/E7 expressing neural stem/progenitor cells, the observed increase in proliferation rate decreased back into normal levels in these cells (Fig.6A, B in IV). However, the observed elevation in self-renewal capacity of E6/E7 expressing neural stem/progenitor cells did not decrease with restored pRb expression (Fig.6C in IV). Thus, the cellular amount of pRb was mainly affecting the cell proliferation rate without influencing the self- renewal capacity of neural stem/progenitor cells. Furthermore, these results suggest that proliferation rate and self-renewal capacity may not be coupled in neural stem/progenitor cells. Therefore, the search was extended to signaling pathways related to E6/E7 oncogenes and influence on cell proliferation, differentiation, and self-renewal. One of the best characterised pathways affecting cell proliferation is mitogen activated protein kinases (MAPK) pathways, which has been also shown to be a part of E6/E7 oncogenes mediated signaling (Antinore et al., 1996; Chakrabarti et al., 2004). MAPK pathways can be classified into distinct groups, which form signaling cascades/networks connecting extracellular signals into their intracellular targets (Seger and Krebs, 1995; Zassadowski et al., 2012). The most interesting result was obtained when the 62 relationship of cell proliferation and self-renewal was studied by using MEK/ERK pathway inhibitor U0126 (Favata et al., 1998) and cyclin dependent kinase (CDK) -1,-2,-5,-7 and -9 inhibitor R-roscovitine (Meijer et al., 1997; Bach et al., 2005) in neural stem/progenitor cultures (Fig.7 in IV). MEK/ERK pathway is known to integrate into a wide variety of intracellular networks related to cell proliferation, differentiation, survival, and apoptosis (Shaul and Seger, 2007). Also, CDKs are known from their ability to regulate cell cycle progression and at least CDK-2,-4,-5 and -6 are expressed by neural stem/progenitor cells (Ferguson et al., 2000). In addition, the MEK/ERK pathway and CDKs interact with each other to some extent (Shaul and Seger, 2007). The results showed that inhibition of MEK/ERK pathway and CDKs had a similar effect on cell proliferation rate in control cells and in E6/E7 expressing neural stem/progenitor cell cultures by decreasing proliferation more than 50% (Fig.7B, D in IV). However, the inhibition of the MEK/ERK pathway and CDKs had a distinct effect on the self-renewal capacity of neural stem/progenitor cells. MEK/ERK inhibition almost abolished formation of secondary spheres having a much greater effect on the self-renewal capacity compared to proliferation (Fig.7C in IV). In contrast to inhibition of the MEK/ERK pathway, inhibition of CDKs reduced formation of secondary spheres only to a level comparable with proliferation (Fig.7E in IV). Thus, inhibition of the MEK/ERK pathway had a much stronger impact on self-renewal capacity of neural stem/progenitor cells than inhibition of CDKs, which further suggest that self-renewal capacity and proliferation may not be entirely under the same regulatory network. Later studies have also shown that inhibition of the MEK/ERK pathway reduces self-renewal capacity of stem cells (Li et al., 2007; Sunayama et al., 2010; Singh et al., 2013). Furthermore, it has been proposed that the MEK/ERK pathway promotes differentiation of stem cells and/or enhances maintenance of stem cells in the pluripotent state (Li et al., 2007; Sunayama et al., 2010; Wray et al., 2010; Singh et al., 2013) as a functional part of PI3K/Akt - RAF/MEK/ERK - canonical Wnt signaling network (Singh et al., 2013). Therefore, the MEK/ERK pathway also seems to have an important role in maintaining balance between self-renewal capacity and differentiation process in stem/progenitor cells (Sunayama et al., 2010; Singh et al., 2013).

4.4.5 E6/E7 oncoprotein mediated degradation of p53 and pRb protein family may facilitate brain tumor formation

The E6/E7 oncogenes did not influence the differentiation potential of neural progenitor/stem cells in vitro or in vivo. However, they had a significant impact on proliferation and self-renewal capacity of neural progenitor/stem cells, which could be linked to the degradation of p53 protein and pRb protein family (Münger et al., 2004; Solozobova and Blattner, 2011; Sage, 2012). Similarly, the observed inhibition and/or delayed cell cycle exit of E6/E7 oncogenes expressing neural stem/progenitor cells might be related to degradation of p53 protein and pRb protein family in these cells. It has been proposed that p53 has an ability to promote differentiation and cell cycle exit of stem/progenitor cells (Spike and Wahl, 2011; Hede et al., 2011). Furthermore, the pRb protein family has been proposed to couple cell cycle exit and initiation of differentiation by interacting with cyclin-dependent kinases (CDKs) and E2F family of transcription factors (Lipinski and Jacks, 1999; Gordon and Du, 2011; Henley and Dick, 2012). Thus, disruptions in p53 and pRb family mediated signaling may lead to delayed cell cycle exit combined with a normal differentiation process. In addition, the observed capacity of differentiated E6/E7 expressing cells to maintain their ability to proliferate may be a result of pRb inability to couple irreversible cell cycle exit to differentiation and/or inability of p53 to suppress dedifferentiation of these cells (Lipinski and Jacks, 1999; Hede et al., 2011). However, this phenomenon was environment dependent, as we were unable to find any proliferative E6/E7 expressing cells after transplantation. Interestingly, observed increase in cell proliferation and self-renewal capacity combined with delayed cell cycle exit in E6/E7 63 expressing cells are also features associated with brain tumor formation, and mice transgenic for human E6/E7 oncogenes exhibit elevated numbers of brain tumors (Arbeit et al., 1993). Therefore, it is possible that E6/E7 viral oncogenes by blocking p53- and pRb tumor suppressor pathways serve as a model for tumor formation in brain. It has been proposed that brain tumors could arise either from neural stem/progenitor cells or more differentiated cells (Westphal and Lamszus, 2011; Hede et al., 2011; Visvader, 2011; Lathia et al., 2011). Arbeit et al. (1993) noticed that most of the tumors in E6/E7 transgenic mice were found from the ventricular zone of brain, where neural stem/progenitors reside. However, they also detected that some tumors appeared to arise from more differentiated cells (Arbeit et al., 1993). Taken together, HPV16 E6/E7 oncoprotein mediated degradation of p53 and pRb family seems to facilitate a defective coupling of proliferation, self-renewal, and differentiation processes inside a cell, which can lead to an imbalance in normal tissue homeostasis and formation of brain tumors from both neural stem/progenitor cells and more differentiated cells in favorable environment.

64 5. Conclusions

The role and importance of NTN1 for the development of mouse olfactory bulb was shown for the first time in the studies included in this thesis. Furthermore, more information of its participation in the fusion process of brain hemispheres related to the CC formation was provided. In addition, neural stem/progenitor cells derived from mouse forebrain were investigated to gain novel information about the potential roles of NTN1, ABBA, and HPV E6/E7 in these cells. The main results of this thesis demonstrate that:

1. NTN1 has a significant impact on the proper migration of stem/progenitor cells from the forebrain germinal zones to the olfactory bulb. Furthermore, these Ntn1 expressing progenitor/stem cells were mainly contributing to the GABAergic interneuron and oligodendrocyte lineages in the olfactory bulb. In addition, the results suggest that NTN1 acts mainly as a detachment/release factor for Ntn1 expressing cells and this function is mediated in either cell-autonomous or paracrine manner.

2. NTN1 is required for the fusion of the two cerebral hemispheres, which precedes and is the prerequisite for the formation of the CC.More precisely, NTN1 seems to be required for the removal of leptomeningeal cells and disruption of pial basal lamina found between the hemispheres, which in turn enable the midline crossing of callosal axons.

3. ABBA participates in the regulation of lamellipodial protrusions by enhancing actin dynamics and connecting plasma membrane deformation to actin cytoskeleton. ABBA mRNA and protein was detected in radial glial cell extensions during CNS development, particularly, in glial end-feet near meningeal pial basal lamina. During early development of brain, interaction between radial glial end-feet and pial basement membrane is required to maintain radial migration and integrity of pial basal lamina. Thus, ABBA may have an important role during development of the forebrain.

4. HPV E6/E7 oncoproteins mediated degradation of tumor suppressor proteins p53 and pRb in neural stem/progenitor cells had an impact on proliferation, maintenance, and self-renewal capacity of these cells. In addition, the results suggested that proliferation, self-renewal, and differentiation capabilities of neural stem/progenitor cells are largely influenced by the environment of these cells.

65 6. Concluding remarks

Regulation of stem/progenitor cellular behavior has an important role during brain development, since virtually all the cells in brain originate from stem/progenitor cells. The cellular processes affecting brain development include cell proliferation, migration, differentiation, and apoptosis, which are regulated in a spatiotemporal manner by developmental genetic programs and interactive proteins networks in a given environment. In this thesis, the influence of HPV E6/E7 mediated degradation of tumor suppressor proteins, NTN1, and ABBA to the neural stem/progenitor cellular processes were studied and assessed. HPV E6/E7 mediated disruption of pathways regulating different proliferation processes suggested that neural stem/progenitor cell proliferation, self- renewal, and differentiation capacities are connected, but may not be entirely under the same regulatory network. Furthermore, the results suggest that neural stem/progenitor cell proliferation, self-renewal, and differentiation processes are greatly influenced by the actual environment. NTN1 affected the migration of neural stem/progenitor cells, but not their proliferation, self-renewal, or differentiation. Furthermore, the migratory effect of NTN1 mainly influenced the subpopulation of neural stem/progenitor cells, which utilizes tangential migration and contribute to the interneuron and oligodendrocyte lineages in the olfactory bulb. Thus, the results suggest that tangential migration of precursor cells to their final target areas from forebrain VZ/SVZ is regulated in overlapping manner by various distinct guidance cue families. Interestingly, ABBA was expressed by radial glia-like cells and RGCs, which function as primary neural stem/progenitor cells during the early brain development and may affect radial migration of neural cells by participating in formation and maintenance of radial glia-like cells and RGCs glial end-feet connection to pial basement membrane. Since the development of forebrain is more than a progressive unfolding of genetic programs in distinct developmental stages, it can also be perceived as a dynamic and interactive network of actively modulated cellular behaviors, which are controlled by various extrinsic and intrinsic signaling cascades. Furthermore, forebrain areas differ by the cell type they contain, the input they receive, and types of connection they make with other brain areas. This structural divergence of forebrain areas also affects the functional differences observed between distinct forebrain areas (Stiles and Jernigan, 2010). In other words, cellular and functional development of forebrain areas proceed in time from more simple conformation towards complex forms of networks and interactions, which is combined with an increasing specificity of different forebrain areas. Thus, even though NTN1, HPV E6/E7 mediated degradation, and ABBA proteins were affecting different cellular processes of neural stem/progenitor cells, in summary, all these cellular processes participate in morphogenetic events shaping the forebrain and, therefore, the future functionality of it. These studies also strengthen the view that defective regulation of distinct neural stem/progenitor cellular processes may lead to different congenital diseases. Thus, identification and functional studies of the proteins that participate in the regulation of neural stem/progenitor cells cellular processes, eventually, leads to a more comprehensive knowledge of the developmental mechanisms behind these diseases. In addition to cellular development, a proper wiring ensures the functionality of forebrain. These results confirmed that NTN1 is required for the development of the largest axon tract in brain, the CC. The development of CC is directed by a chronological chain of developmental events. These results suggest that NTN1 is primarily influencing, in either a direct or indirect manner, the interhemispheric fusion, which precedes and is an absolute requirement for the midline crossing of callosal axons. However, additional investigations are required to unfold the mechanisms leading to this observed phenotype. Finally, axon guidance and cell migration shares many common molecular mechanisms, which control cell shape changes and chemotactic modifications required for the correct guidance of axons and cells to their target regions (Marin et al., 2010). Many axon guidance

66 cue families are known to affect both axon guidance and cell migration. Thus, it is not surprising that NTN1 has been shown to affect both axon guidance and cell migration. Furthermore, NTN1 has been proposed to accomplish these tasks either by influencing the formation of different cell shapes and/or by establishing an external long or short range chemotactic gradient (Lai Wing Sun et al., 2011). The results in this thesis further confirmed that NTN1 is involved in migration of many cell-types during forebrain development. In addition, results in this thesis strengthen the idea that NTN1 acts in many cases in a cell-autonomous and/or paracrine manner as a short range factor.

67 7. Acknowledgements

This study was carried out at the Institute of Biotechnology and the Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki.

I want to thank my supervisor Marjo Salminen for all the support and guidance that she gave me during this thesis project. It has been the most essential and indispensable part of this thesis. I am also grateful to supervising professor Jyrki Kukkonen for the support and help in practical matters. Pre-reviewers Juha Partanen and Claudio Rivera are sincerely acknowledged for their valuable remarks and advices, which improved the quality of this thesis.

I had a great privilege to be a part of a small commune, which shared daily life experiences in lab, office and outside world. In other words, I was able to share my thoughts and interact with magnificent individuals of our research group; Tanja Torttila, Antti Oinas, Maarja Haugas, Kersti Lilleväli and Sebastien Duprat. Thank you! I want also to thank Raija Savolainen, Eija Koivunen, Christel Pussinen, Luydmula Rasskazova, Virpi Syvälahti, Suzan Cinqi and personnel in Laboratory Animal Center of Viikki for helping and sharing their technical knowledge with me.

I appreciate the great opportunity to be involved in excellent collaboration projects, and I am very grateful to all the collaborators and co-authors of the publications, especially, Katja Piltti, Kirmo Wartiovaara, Juha Saarikangas, Pieta Mattila and Pekka Lappalainen. These collaboration projects gave me a lot of valuable knowledge and perspectives to the ways to conduct research and think over science, thanks!

During these years I have found many friends from Viikki and their contribution to this thesis, in form of having good times (beer) and discussing life (and science), has been invaluable! Thus, my sincere gratitude goes to Mark Tümmers, Isaac Salazar-Ciudad, Heikki Helanterä, Tomi Jukkola, Kaia Achim, Janne Tornberg and Pauli Turunen.

Integrative life science doctoral program (ILS) is appreciated for their support in studies and Finnish Cultural Foundation, Oskar Öflund Stiftelse, Ella and Georg Ehrnrooth Foundation and University of Helsinki for providing financial support.

I sincerely thank my parents for the encouragement and support. Finally, I owe incredible thanks to Päivi for all the support, advices, understanding, sharing and joy that she has provided me during this time. You gave me a massive amount of strength and renewable energy!

Janne

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