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Neurogenesis occurs during embryonic life
ESCs can be isolated from the developing mouse blastocyst around embryonic day (E) 3.5; by E6.5 the ICM comprises three germ layers: the endoderm, the mesoderm and the ectoderm (Ohnuki & Takahashi, 2015; Xu et al., 2017). The endoderm will form the digestive tube with the glands that open into it, the urinary bladder, the epithelial parts of trachea, the lungs, the thyroid, the parathyroids (Kiecker et al., 2016; Solnica-Krezel & Sepich, 2012); the mesoderm will form muscles, bones, cartilage, connective tissue, adipose tissue, the circulatory system, the lymphatic system, dermis, the genitourinary system, serous membranes, and the notochord (Kiecker et al., 2016; Solnica-Krezel & Sepich, 2012; Takemoto, 2013); finally the ectoderm will develop in the surface ectoderm, from which epidermis, hair, nails will derive; the neural crests that will give peripheral nervous system,
melanocytes (Nitzan & Kalcheim, 2013); and the neural tube that gives rise to the brain, spinal cord, posterior pituitary and the retina (Kiecker et al., 2016; Solnica-Krezel & Sepich, 2012; Takemoto, 2013). The process of invagination and closure of the ectoderm, through which the neural tube is formed, is called neurulation; this is a finely regulated process divided into different steps, each of them dependent on the establishment of precise morphogen gradients (Takemoto, 2013). The neural tube will give rise to the central nervous system (CNS) therefore being subjected to a rostro-caudal as well as dorso-ventral differentiation process. In its rostral portion, from which the brain will derive, the neural tube balloons into three primary vesicles: the forebrain (cerebral hemispheres, thalamus, hypothalamus, and retina), the midbrain (tectum and motor pathways of the basal ganglia), and the hindbrain (cerebellum and medulla oblongata) (Darnell & Gilbert, 2017). The inner cavity of these vesicles will later be filled with cerebrospinal fluid (CSF) and will give the four ventricles. Meanwhile the dorsoventral axis of the more caudal portion, which will give rise to the spinal cord, is modified by signals coming from the immediate environment: the dorsal side receives sensory inputs, whereas motor signals emanate from the ventral side (Darnell & Gilbert, 2017). Neurons and macroglia of the brain derive from neuroepithelial cells that can be called embryonic neural stem cells (eNSCs), which line the cerebral ventricles, and undergo different amplification steps giving rise to intermediate progenitor cells (IPCs) (Fig. 1). Cortical neurogenesis begins around E9-10 in mice. By this time, neuroepithelial cells begin to acquire features of glial cells, such as expression of astroglial markers like GLAST (glutamate aspartate transporter), BLBP (brain lipid binding protein), nestin, vimentin and are referred to as radial glia (RG) (Kriegstein & Alvarez-Buylla, 2009; Rowitch & Kriegstein, 2010). These cells also express GFAP (glial fibrillary acidic protein) in some species, such as in primates, and in mice only from E16 (Choi, 1988). RGs stay in contact with the surface of the ventricle and the pia, with their cell bodies found at the level of the ventricular zone (VZ) and connected by adherens junctions (Kriegstein & Alvarez-Buylla, 2009). RG cells proliferate and divide asymmetrically to maintain the eNSC pool and to generate either a neuron or an IPC. These IPCs can be committed to the neuronal lineage (nIPC) or to the oligodendrocyte one (oIPC) (Fig. 1).
NSCs IN THE ADULT BRAIN: THE TWO CANONICAL REGIONS
NCSs can still be found in the adult brain within restricted regions where they can give rise to glial as well as neuronal cells (Alvarez-Buylla & Lim, 2004; Gage, 2000; Ming & Song, 2005; Nottebohm, 2004). The two most studied and characterised regions, where the presence of adult NSCs (aNSCs) has been widely accepted, are the subventricular zone of the lateral ventricles (SVZ), adjacent to the ependymal ciliated single cell layer that lines the wall of the lateral ventricles, and the subgranular zone of the dentate gyrus of the hippocampus (SGZ) (Alvarez-Buylla & Lim, 2004; Gage, 2000; Ming & Song, 2005). However, increasing evidences are pointing towards the hypothesis that more regions retain the ability to produce new neural cells, such as the striatum (Bédard et al., 2006; Luzzati et al., 2007; Nato et al., 2015) and the hypothalamus (Djogo et al., 2016; Kokoeva et al., 2005; Mcnay et al., 2012; Migaud et al., 2010; Pencea et al., 2001; Sharif et al., 2014; Xu et al., 2005) (see chapter 1.7.3).
The origin of aNSCs in the SVZ has been identified only recently in a subpopulation of quiescent eNSCs (Fuentealba et al., 2015; Furutachi et al., 2015). It appears that around E13.5 and 15.5, a subset of eNSCs strongly slow down their cell cycle, maintaining their undifferentiated state and in this way persisting until postnatal stages when they become reactivated. The rest of them continue dividing to ensure the development of the CNS and are lost during the process (Furutachi et al., 2015). Moreover, it has been shown that as early as E11.5, these cells have acquired a regional specification, due probably to the expression of distinct combinations of transcription factors (TFs), which they will retain throughout postnatal life and that will determine their progeny (Fuentealba et al., 2015).
NSCs of the DG derive from stem cells located in the subpallium region during embryonic development. However, their exact embryonic origin has not been fully elucidated since the DG is generated by a mosaic of stem cells with different embryonic origins. Embryonic formation of the DG begins around E13.5 in mice. Hippocampal RG cells, which express typical markers such as nestin and GLAST at E15.5, proliferate in a domain of the ventricular zone called the dentate neuroepithelium, which is adjacent to the cortical hem (Li et al., 2013; Rolando & Taylor, 2014). From here, these cells will migrate through the dentate migratory stream and spread, covering the DG. At the end of the first postnatal week, following a process of reorganisation, NSCs are found in the SGZ where they will persist throughout life (Rolando & Taylor, 2014).
PHYSIOLOGICAL RELEVANCE OF ADULT NEUROGENESIS
Neurogenesis increases plasticity on multiple levels; while adding new cells to existing circuits it causes structural remodelling, synaptogenesis, and changes in synaptic strength. Neurogenesis encompasses different stages: from cell birth through fate determination, survival, until integration and acquisition of functional properties. Of particular relevance is the selection occurring through modulation of the survival rate of the newborn neuroblasts both in the SVZ and the DG that ensures at any moment a readily available pool of cells. All these processes are sensitive to environmental signals and are subject to reduction with aging, possibly through the activation of the hypothalamic–pituitary–adrenal axis (Danzer, 2012; Fuchs & Flügge, 2014; Gonçalves et al., 2016; Lledo et al., 2006; van Praag et al., 2002).
Table of contents :
Resumé
Abstract
Résumé substantiel
Poster and oral communications
Abbreviations
INTRODUCTION
Chapter 1 Cell neogenesis in the brain throughout life
1.1 STEM CELLS
1.2 EMBRYONIC AND POSTNATAL DEVELOPMENT
1.2.1 Neurogenesis occurs during embryonic life
1.2.2 Gliogenesis occurs during postnatal life
1.2.2.1 Astrogenesis
1.2.2.2 Oligodendrogenesis
1.3 NSCs IN THE ADULT BRAIN: THE TWO CANONICAL REGIONS
1.3.1 Identity of aNSCs – to be or not to be
1.4 A NICHE OF ONE’S OWN
1.4.1 The SVZ niche
1.4.2 The SGZ niche
1.5 PHYSIOLOGICAL RELEVANCE OF ADULT NEUROGENESIS
1.5.1 A role for adult neurogenesis in the SVZ
1.5.2 A role for adult neurogenesis in the SGZ
1.5.3 The interplay with the reproductive function
1.5.3.1 The SVZ
1.5.3.2 The SGZ
1.6 aNSC AND ADULT NEUROGENESIS IN HUMANS
1.6.1 The human SVZ
1.6.2 The human DG
1.7 CELL NEOGENESIS IN THE HYPOTHALAMUS
1.7.1 Structural features of the hypothalamus
1.7.2 Building the hypothalamus
1.7.2.1 Embryonic life
1.7.2.2 Post-natal life
1.7.3 Cell neogenesis in the adult hypothalamus
1.7.4 Identity of hypothalamic NSCs
1.7.4.1 The parenchymal hypothesis
1.7.4.2 The subependymal astrocytes hypothesis
1.7.4.3 The tanycyte hypothesis
1.7.5 Modulators of hypothalamic neurogenesis
1.7.5.1 Growth factors
1.7.5.2 Diet
1.7.5.3 Other modulators
1.7.6 Functional implications of hypothalamic neurogenesis
1.7.6.1 Beyond energy homeostasis
Chapter 2 The central control of reproduction
2.1 THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS
2.2 THE RAT OVARIAN CYCLE
2.2.1 Patterns of hormonal secretion
2.2.1.1 GnRH
2.2.1.2 Gonadotropins
2.2.1.3 Prolactin
2.2.1.4 Ovarian steroids
2.3 REGULATION OF THE GNRH SYSTEM
2.3.1 Neurotransmitters and neuropeptides
2.3.1.1 GABA
2.3.1.2 Glutamate
2.3.1.3 Kisspeptin
2.3.2 Orexigenic and anorexigenic modulators
2.3.2.1 POMC neurons
2.3.2.2 NPY/AgRP neurons
2.3.3 Nitric oxide
2.3.4 Autocrine control of the GnRH neurons
2.3.5 Glial cells
2.3.5.1 Paracrine factors
2.3.5.2 Juxtacrine interactions
2.3.5.3 Structural plasticity
2.3.5.4 Human findings
2.4 EMBRYONIC DEVELOPMENT OF THE GNRH SYSTEM
2.5 POSTNATAL DEVELOPMENT OF THE GNRH SYSTEM
2.5.1 Establishment of the GnRH neural network
2.5.1.1 Inhibitory inputs
2.5.1.2 Excitatory inputs
2.5.1.3 Neuropeptidergic inputs
2.5.1.4 nNOS neurons
2.5.2 Glial cells
2.6 PREGNANCY AND BEYOND
2.6.1 Pregnancy
2.6.2 Lactation
2.6.3 Maternal behaviour
Chapter 3 Aims
Chapter 4 Materials and Methods
4.1 ANIMALS
4.2 BRDU INJECTIONS
4.3 PUBERTY ONSET AND ESTROUS CYCLICITY
4.4 TISSUE PREPARATION
4.5 IMMUNOFLUORESCENCE
4.5.1 Antibodies
4.5.2 Immunohistochemistry
4.5.3 Immunocytochemistry
4.6 MICROSCOPIC ANALYSIS
4.6.1 Postnatal animals
4.6.1.1 Morphological association between GnRH neurons and BrdU+ cells
4.6.1.2 Phenotypic identity of BrdU+ cells
4.6.2 Adult animals
4.6.2.1 Analysis at the level of the POA
4.6.2.2 Analysis at the level of the ME
4.6.2.3 Analysis at the level of the DG
4.7 STATISTICAL ANALYSIS
4.8 HYPOTHALAMIC PROGENITOR CULTURES
4.9 IN VITRO MIGRATION ASSAY
4.10 IN VITRO PROLIFERATION ASSAY
4.11 IMMUNOBLOTTING
4.12 ISOLATION OF HYPOTHALAMIC GNRH NEURONS USING FLUORESCENCE-ACTIVATED CELL SORTING (FACS)
4.13 QUANTITATIVE REAL TIME-PCR ANALYSES
4.14 HIGH-RESOLUTION FLUORESCENT IN SITU HYBRIDISATION (FISH) BY RNASCOPE
4.15 STEREOTACTIC BRAIN INFUSION OF BWA868C
4.15.1 Acute injections
4.15.2 Long-term injections
4.16 HORMONE LEVELS MEASUREMENT
RESULTS
Chapter 5 Cell neogenesis in the postnatal hypothalamus in the female rat: involvement in sexual maturation
5.1 INTRODUCTION
5.2 PREVIOUS RESULTS
5.3 RESULTS
5.3.1 Identification of candidate factors
5.3.2 Development and characterisation of hypothalamic progenitor cultures
5.3.3 PGD2 attracts POA progenitors in vitro via DP1 signalling
5.3.4 The candidate factors do not affect hypothalamic progenitor proliferation in vitro
5.3.5 GnRH neurons recruit newborn cells through the PGD2/DP1 signalling in vivo
5.3.5.1 In vivo expression of PGDS and DP1
5.3.5.2 PGD2/DP1 signalling regulates the recruitment of BrdU+ cells in the vicinity of GnRH neurons in vivo
5.3.6 Impairment of DP1 signalling in the POA during the infantile period perturbs the onset of oestrous cyclicity
Chapter 6 Cell neogenesis in the hypothalamus of the adult female rat
6.1 INTRODUCTION
6.2 RESULTS
6.2.1 Cell proliferation throughout the estrous cycle
6.2.1.1 Cell proliferation at the level of the POA
6.2.1.2 Cell proliferation at the level of the ME
6.2.1.3 Cell proliferation at the level of the DG
6.2.2 Pregnancy increases survival of cells born before mating
Chapter 7 The stem/progenitor cell niche in the adult human hypothalamus
Abstract
7.1 INTRODUCTION
7.2 MATERIALS AND METHODS
7.2.1 Tissue
7.2.2 Fluorescent immunostaining
7.2.3 Antibody characterization
7.2.4 Histological staining
7.2.5 Microscopy
7.2.6 Analysis
7.2.7 Statistics
7.3 RESULT
7.3.1 The human hypothalamus
7.3.2 The rodent and grey mouse lemur hypothalamus
7.3.3 The human SVZ and the dentate gyrus of the hippocampus
7.4 DISCUSSION
References
Chapter 8 Discussion and Conclusions
8.1 DISCUSSION
8.1.1 Cell proliferation in the female rat POA is maintained throughout postnatal development and during adulthood
8.1.2 Newborn cells are associated with GnRH neurons and are necessary for their postnatal development
8.1.3 The recruitment of newborn cells by GnRH neurons is necessary for the correct maturation of the system
8.1.4 Proposed model and future perspectives
8.1.5 Cell neogenesis in the hypothalamus of adult female rats during the estrous cycle
8.1.6 Pregnancy increases cell survival in the MPO
8.1.7 Open questions
8.1.8 The adult human hypothalamus likely contains neural stem/progenitor cells
8.2 CONCLUDING REMARKS
Bibliography
