Specification, multipotency and plasticity during embryonic and adult neurogenesis
Séminaire FSER organisé par Chaya Kalcheim (IMRIC- Hebrew University, Jerusalem) et Pierre-Marie Lledo (Institut Pasteur, Paris) du 24 au 29 octobre 2011
Nora Abrous, Arturo Alvarez-Buylla, Paola Arlotta, Yves-Alain Barde, James Briscoe, Elisabeth Dupin, Carol Erickson, Patrik Ernfors, Magdalena Götz, Wieland B. Huttner, Jane Johnson, Ryoichiro Kageyama, Chaya Kalcheim, Juergen Knoblich, Arnold Kriegstein, Nicole Le Douarin, Pierre-Marie Lledo, Adi Mizrahi, Amar Sahay, Lukas Sommer, Song Hongjun
In october 24-29, 2011, a group of 21 scientists gathered at the Fondation des Treilles to hold a meeting entitled “Specification, multipotency and plasticity during embryonic and adult neurogenesis”. This meeting was organized by Chaya Kalcheim (Hebrew University of Jerusalem) and Pierre-Marie Lledo (Pasteur Institute).
The field of stem cells and more specifically, neural stem cells, has attracted much attention during the past years. Neural stem cells (NSCs) are the self-renewing, multipotent cells that generate the main phenotypes of the peripheral and central nervous systems. In the embryo, a hierarchy of developmental restrictions generates differentiated cell types from multipotent progenitors. However, the state of specification of progenitor cells at different stages and embryonic sites is not well defined. Elucidation of the above is essential for understanding the mechanisms leading to fate restriction in normal development.
Stem cells are not unique to embryos. In 1992, Reynolds and Weiss were the first to isolate neural progenitors and stem cells from the striatum, including the subventricular zone of adult mouse brain tissue. Since then, neural progenitor and stem cells have been isolated from various areas of the adult spinal cord and brain, and from various species including human. It is hypothesized that neurogenesis in the adult brain originates from NSCs. Yet, the origin, identity and functional properties of NSCs in the adult brain remain to be defined.
The workshop focused on the latest advances in the mechanisms responsible for proliferation, maintenance, differentiation and function of neural stem cell/progenitors. Using an array of model organisms (avians, mice, drosophila, humans, cultured cells), diverse systems were addressed: from Neural Crest progenitors to their peripheral neural vs melanocytic derivatives; several central neuronal types like cortical, hippocampal and olfactory bulb neurons and glia, etc.
The main topics that were discussed included the following:
FROM STEM CELL TO TERMINAL NEUROGENIC DIVISIONS (Huttner, Knoblich, Kriegstein, Kageyama, Song)
During brain development, neurons arise from neural stem and progenitor cells, which initially proliferate by symmetric divisions and later switch to both asymmetric and symmetric neurogenic divisions. Multiple types of progenitors exist with different characteristics and modes of division. To control the number, type, and final location of neurons, the transition from proliferative to neurogenic cell divisions requires a complex network of regulation so that neural specification, cell-cycle exit, cell differentiation and neuronal migration can all occur in concert. These concepts were discussed for bothembryonic and adult processes.
FATE DECISIONS DURING NEURAL DEVELOPMENT (Le Douarin, Kalcheim, Erickson, Ernfors, Briscoe, Sommer, Johnson, Dupin, Barde).
Neuronal fate determination is an essential stage in both embryonic and adult neurogenesis. Embryonic microenvironments within the neural tube/nascent brain, in peripheral ganglia, and neurogenic niches in the adult, provide instructional signals that control proliferation, differentiation and survival of the stem cell pool to ensure that neurogenesis continues throughout life. Current knowledge regarding the mechanisms that control the undifferentiated state and fate determination of embryonic peripheral and central neurons as well as of adult neural stem cells in the hippocampus and olfactory bulb were discussed.
THE COORDINATION BETWEEN SPECIFICATION AND PROGENITOR MIGRATION (Erickson, Kalcheim, Arlotta).
Neural progenitors often migrate through stereotypic pathways until homing to their final destinations. The neural crest offers an excellent example for such a paradigm. Is there a deterministic relationship between the state of specification of a progenitor and the routes of migration it follows?. Alternatively, are these two independently-regulated processes, yet merely coordinated in time to ensure successful morphogenesis? Furthermore, the session dealt with questions regarding the timing of fate decisions and the extent to which these depend upon environmental cues.
REGULATION OF ADULT NEUROGENESIS (Arlotta, Sahay, Abrous, Lledo, Song, Alvarez-Buylla, Götz, Mizrahi)
The molecular pathways important for adult neurogenesis, including transcriptional and epigenetic regulatory factors, and signaling mechanisms during physiological and pathological contexts were discussed.
FUNCTIONAL INTEGRATION OF ADULT-GENERATED NEURONS (Abrous, Song, Sahay, Lledo, Arlotta, Götz, Alvarez-Buylla, Mizrahi)
Newborn neurons in the adult circuits differentiate and integrate within the existing neuronal circuitry. This topic addressed how the impact of adult-born neurons in neuronal networks is dictated by the extent that newborn neurons participate as part of the network and how their intrinsic properties compare with those of existing neurons generated during development.
FUNCTIONAL SIGNIFICANCE OF ADULT NEUROGENESIS (Abrous, Song, Sahay, Lledo, Arlotta, Götz, Alvarez-Buylla, Mizrahi).
Newly formed neurons incorporate into functional networks suggesting important roles for adult neurogenesis in some cognitive functions. This session presented the current evidence regarding the functional roles that newborn neurons play in the adult brain.
NEUROLOGICAL DISORDERS AND REPAIR (Abrous, Song, Sahay, Lledo, Götz, Alvarez-Buylla, Mizrahi).
Recent studies have shown that a wide variety of stimuli, including seizures, stress, stroke, and chronic antidepressant treatment, can profoundly affect adult neurogenesis. Moreover, neurodegenerative disease states are often associated with diminished neurogenesis. This session addressed whether failure of a normal reparative process, i.e., adult neurogenesis, contributes to the development of disease, and/or whether enhancing neurogenesis could be used as a therapeutic strategy in some of these disorders.
Nicole Le Douarin
The Cephalic Neural crest is a powerful regulator of preotic brain development and functions as a third Brain organizer
The role of the neural crest (NC) in the construction of the vertebrate head was demonstrated when cell tracing techniques became available to follow the cells exiting from the cephalic neural folds in embryos of various vertebrate species.
Experiments carried out in the avian embryo, using the quail/chick chimera system, was critical in showing that the entire facial skeleton and most of the skull (except for he occipital region) were derived from the NC domain of the posterior diencephalon, mesencephalon and rhombomeres 1 and 2 (r1, r2). This region of the NC was designated FSNC (for Facial Skeletogenic NC). One characteristic of this part of the head including the neural anlage is that it remains free of expression of the homeotic genes of the Hox-clusters. In an attempt to see whether this rostral Hoxnegative domain of the NC has a specific role in the development of the skeleton, we have surgically removed it in chick embryos at 5-6 somite stage (5-6 ss). The operated embryos showed a complete absence of facial and skull cartilages and bones showing that the Hox expressing domain of the NC caudally located to the excision and expressing Hox genes did not regenerate to replace the anterior NC. In addition to the deficit in skeletal structures, the operated embryos exhibited severe brain defects resulting in exencephaly although the excision of the neural folds corresponding to the FSNC left intact the neural epithelium fated to develop into fore- and mid-brain structures. Experiments that will be described in the presentation have shown that the neural crest cells regulate the amount of Fgf8 produced by the two brain organizers, the Anterior Neural Ridge (ANR) and the isthmus. This regulation is exerted via the secretion of anti-BMP signaling molecules (like Gremlin and Noggin) which decrease BMP production, hence enhancing the amount of Fgf8 synthesized in the ANR (also called “Prosencephalic organizer”) and in the isthmus. Fgf8 turns out to be an essential signal for the growth and patterning of the alar plate territories of the anterior Neural Plate. The role of the NC in the construction of the Vertebrate head was at the origin of the “New Head concept” developed by Gans and Northcutt (1983) who considered that the NC, which in the group of Cordates is an innovation of the Vertebrate phylum, has been an important asset in their evolution since it participated in the development of the head with an increase of the volume and complexity of their brain when compared to that of their ancestors the Protocordates We show here that the NC exerts a direct effect on brain development in promoting the development of the telencephalon, the thalamus and the tectum opticum through its action as a regulator of the production of key signaling molecules for brain neurogenesis.
The role of FoxD3 in the specification of premigratory neural crest and the control of neural crest pathfinding.
Trunk neural crest cells migrate from the dorsal neural tube and produce the neurons and glial cells of the peripheral nervous system and the pigment cells of the skin. We and others have discovered that neural crest cells are often lineagerestricted when they leave the neural tube. Aside from determining how the various neural crest lineages will differentiate, this early specification also controls what pathways the neural crest lineages will take. Neural and glial lineages can only migrate ventrally, whereas melanoblasts are uniquely able to take the dorsolateral path between the ectoderm and dermamyotome, where they will differentiate into the pigment cells of the skin. This ability to enter different pathways is dependent upon their response to several signaling molecules in the environment.
We have determined that the lineage switch from neural/glial precursors to melanoblasts is controlled by the transcription factor FoxD3. We have demonstrated that the first neural crest cells to undergo the EMT are specified as neural, glial or bipotent precursors and that FoxD3 is expressed in the dorsal neural epithelium at this time. FoxD3 is downregulated at the time that only melanoblasts are migrating. When FoxD3 is overexpressed either in cultured neural crest cells or in the dorsal neural tube, melanogenesis is repressed and all neural crest cells migrate ventrally. Conversely when FoxD3 is downregulated by siRNA, there is an abundance of pigment cells and they all migrate dorsolaterally. We discovered that FoxD3 represses the expression of the transcription factor Mitf, which regulates melanogenesis. Moreover, we show that FoxD3 does not directly bind the regulatory region of Mitf, but rather prevents the binding of Pax3, which is transcriptional activator required for Mitf expression.
Mitf, in turn, regulates the expression level of EphB2 and EDNRB2 in the neural crest cells that are paused in the migration staging area.When these two receptors are upregulated the neural crest cells are now able to invade the dorsolateral pathway. We know that BMP4 represses melanogenesis and Wnt3 stimulates melanogenesis but at this time it is not known how these two signaling molecules regulate FoxD3 itself.
The dorsal neural tube: a dynamic setting for cell fate decisions
The dorsal neural tube first generates neural crest cells that exit the neural primordium following an epithelial-to-mesenchymal conversion to become sympathetic ganglia, Schwann cells, dorsal root sensory ganglia and melanocytes of the skin. Followingthe end of crest emigration, the dorsal midline of the neural tube becomes the roof plate, a signaling center for the organization of dorsal neuronal cell types. Recent lineage analysis performed before the onset of crest delamination revealed that the dorsal tube ishighly dynamic region sequentially traversed by faterestricted crest progenitors. Furthermore, prospective roof plate cells were shown to originate ventral to presumptive crest and to progressively relocate dorsalward to occupy their definitive midline position following crest delamination. These data raise important questions regarding the mechanisms of cell emigration in relation to fate acquisition, and suggest the possibility that spatial and/or temporal information in the dorsal neural tube determines initial segregation of neural crest cells into their derivatives. In addition, they emphasize the need to address what controls the end of neural crest production and consequent roof plate formation, a fundamental issue for understanding the separation between central and peripheral lineages during development of the nervous system.
The orientation of asymmetric cell divisions directs lineage decisions in the mouse neocortex
Maria Pia Postiglione, Yunli Xie, Christoph Jüschke, Gerald A. Haas, Christoforos Charalambous and Juergen A. Knoblich Institute of Molecular Biotechnology (IMBA), Vienna, Austria
The mammalian brain develops from a layer of progenitor cells located on the apical side of a neuroepithelium. Initially, the progenitor pool expands by symmetric divisions. Later, progenitors divide asymmetrically and generate only one self-renewing daughter cell. The other daughter cell either migrates towards the cortical plate to differentiate into a neuron (direct neurogenesis) or becomes an intermediate progenitor generating two neurons after one additional symmetric division (indirect neurogenesis). In symmetric progenitor cell divisions the mitotic spindle is always parallel to the neuroepithelial surface, but asymmetric divisions can also have vertical or oblique spindles. Whether this reorientation of the mitotic spindle is actually responsible for the establishment of asymmetry during those divisions is highly controversial and unclear. We have addressed this question by generating conditional mutants and overexpression lines for the mouse Inscuteable (mInsc) gene, the homolog of a key regulator of spindle orientation in Drosophila. We find that mutating mInsc almost completely abolishes oblique and vertical mitotic spindles while mInsc overexpression has the opposite effect. Surprisingly, our data indicate that spindle reorientation is not essential for the asymmetric outcome of the progenitor divisions but strongly influences the balance between direct and indirect neurogenesis. Thus, lineage decisions in the vertebrate brain are influenced by the orientation of progenitor divisions. As modified lineages and increased indirect neurogenesis are highly correlated with the expanded cortical size in primates, our data might be relevant for human brain evolution as well.
Wieland B. Huttner
Neural stem and progenitor cells and the evolution of the cerebral cortex
Our group studies the molecular and cellular mechanisms of neurogenesis in the developing neocortex in the context of mammalian brain evolution, specifically the various types of cortical stem and progenitors cells and their modes of division. In terms of their cell biology, two principal classes of cortical stem and progenitors cells can be distinguished. One class comprises stem/progenitor cells exhibiting bipolar morphology and apical-basal cell polarity that divide at the ventricular, i.e. apical, surface of the ventricular zone (VZ). These are the neuroepithelial cells and radial glial cells, which are collectively referred to as apical progenitors (APs). The other class comprises stem/progenitor cells dividing in a more basal, abventricular location, notably the subventricular zone (SVZ). These fall into two subclasses (i) radial glia-related progenitors exhibiting monopolar morphology and basal, but not apical, cell polarity, referred to as basal radial glial (bRG) cells (also called outer SVZ (OSVZ) progenitors, outer radial glia or intermediate radial glia); and (ii) progenitors exhibiting nonpolar morphology and lacking overt apical-basal cell polarity, called basal progenitors (BPs) or intermediate progenitor cells. Recent insights into molecular and subcellular features of cortical stem and progenitors cells will be presented.
The role of Notch signaling in proliferation and differentiation of neural stem cells
Neural stem cells are mostly quiescent in the adult brain, but the precise mechanism of maintenance of quiescent neural stem cells remains to be analyzed. We found that the Notch effector genes Hes1 and Hes5 are highly expressed by adult neural stem cells, and that forced expression of Hes1 is sufficient for maintenance of quiescent neural stem cells. In contrast, when Hes1 and its related genes were deleted in the adult brain, all neural stem cells differentiated into transit-amplifying cells and neurons. As a result, neurogenesis was increased transiently, but three months later, virtually all neural stem cells were depleted and neurogenesis ceased. These results indicate that Notch signaling plays an essential role in maintenance of quiescent neural stem cells in the adult brain. We also examined the mechanism of how the quiescence is maintained by Hes1, and will discuss the significance of Hes1 expression dynamics in the maintenance of quiescent neural stem cells.
Neural stem and progenitor cells in human cortical development
Recent insights gained from studies of the developing cerebral cortex are illuminating potential evolutionary steps that contributed to structural and functional features of the human brain. Radial glial cells (RG), long thought to simply guide embryonic nerve cells during migration, have now been identified as neuronal stem cells in the developing brain. RG cells undergo self-renewing, asymmetric divisions to generate neuronal precursors that can further proliferate in the subventricular zone (SVZ) to increase neuronal number.Unlike the developing rodent cortex, the developing human cortex contains a massively expanded SVZ (OSVZ) that is thought to account for the bulk of cortical neurogenesis. We have begun to characterize the types and locations of progenitor cells responsible for human cortical development.We found that large numbers of radial glia-like cells and intermediate progenitor cells populate the human OSVZ.The OSVZ radial glialike cells have a long basal process but, surprisingly, do not have basolateral polarity and lack contact with the ventricular surface. Using real-time imaging and clonal analysis, we demonstrate that these cells undergo self-renewing asymmetric divisions to generate neuronal progenitor cells that can further proliferate. We have recently found that progenitor cells resembling oRG cells are present in mouse embryonic neocortex, and arise from asymmetric divisions of radial glia.Time-lapse imaging reveals that the cells undergo self-renewing asymmetric divisions to generate neurons.These results suggest that oRG cells are probably present in all mammals and are not a specialization of a larger brain with increased cortical area. Instead, an evolutionary increase in the number of oRG cells and their transit amplifying daughter cells likely amplified neuronal production and contributed to increased cortical size and complexity in the human brain.
Transcription Complexes Couple Neural Differentiation and Neuronal Subtype Specification
The regulation of processes that control the balance of progenitor cell maintenance with neuronal differentiation and specification is essential for generating a functioning nervous system. Members of the bHLH family of transcription factors are central to the regulation of these processes. During development of the dorsal spinal cord, a region critical for processing and relaying somatosensory information, alteration in function and expression of multiple bHLH factors results in disturbances of connectivity, imbalances in excitatory and inhibitory neuron formation and loss of control of neural cell number. Ascl1 (previously Mash1) and Ptf1a are two factors particularly important in these processes. Using mouse genetics and Cre-lox fate mapping strategies, we find that Ascl1 is present in neuronal and glial restricted progenitor cells at separable embryonic stages during neural tube development. It is required in these progenitors, in a balance with Notch signaling, for differentiation into specific subsets of dorsal horn neurons. Indeed, using chromatin immunoprecipitation followed by sequencing, Ascl1 regulates transcription of a diverse set of genes including multiple components of the Notch pathway, and other known regulators of neuronal differentiation. In contrast, Ptf1a is present in neuronal restricted lineages and is essential for generation of inhibitory interneurons in the dorsal spinal cord as well as in cerebellum and retina. Ptf1a is unique for a bHLH factor in that it forms a novel transcription complex that includes Rbpj, the transcriptional effector in Notch signaling. It is notable that Ptf1a dependent neuronal specification is occurring in the neural tube in a similar temporal window as Ascl1 and Notch-dependent neuronal differentiation. Furthermore, a direct downstream target of the Ptf1a-Rbpj transcription complex antagonizes the neuronal specification activity of Ascl1. model is suggested in which distinct transcription complexes utilize shared components to couple decisions in neuronal differentiation and specification. Genome-wide target analysis is being used to define genetic and molecular mechanisms by which neural bHLH factors participate in regulating progenitors as they undergo neuronal differentiation and neuronal sub-type specification.
Sonic Hedgehog Signaling and the control of neural cell behaviour
Secreted signals known as morphogens provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the embryonic neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the proliferation of stem cells and the pattern of neuronal subtype specification. A temporally and spatially changing gradient of Shh signaling determines the pattern of gene expression in neural progenitors. This dynamic signaling gradient is interpreted by the regulatory logic of a downstream transcriptional network that links three transcription factors to Shh signaling. Strikingly, the design of the network, not differential sensitivity of target genes to Shh signaling, is responsible for positioning gene expression boundaries. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis memory of the signal. These data suggest that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.
Molecular development and directed differentiation of projection neuron types in the cerebral cortex
In the developing cerebral cortex, cell-extrinsic and cell-intrinsic signals govern the establishment of neuron subtype-specific identity. Corticospinal Motor Neurons (CSMN) are one subpopulation of cortical projection neurons that reside in layer of the cortex and connect to targets in the spinal cord. We previously identified genes that control the development of CSMN. Among these, the transcription factor Fezf2 is necessary for the fate specification of CSMN and related populations of Corticofugal Projection Neurons (CfuPN) of the cortex. This suggests that Fezf2 alone might be able to instruct projection neuron subtype-specific development from neural progenitors of a different cell fate, within neurogenic niches of other CNS regions. Here, we find that the ectopic expression of Fezf2 in neural progenitors of the striatum is sufficient to instruct the generation of CFuPN, including CSMN, bypassing typical requirements for early cortical progenitor patterning and glutamatergic specification. Fezf2-expressing neurons acquire molecular, morphological and connectivity properties of CFuPN, despite their ectopic location. The work demonstrates that cell-autonomous signals that drive neuron type-specific development in the cortex can override extracellular niche restrictions and instructions over progenitor cell fate, and supports a model in which key transcription factors might be used to direct the differentiation of clinically relevant neuron types of the cortex for therapeutic benefit
Cell type specification in the neural crest
The principle of cell fate commitment involves patterning morphogens initiating cascades of combinations of transcription factors that together control cell programs. Diversification of the neural crest to different cell types is under strict control in a temporo-spatial manner by the expression of instructive signals. By changing their competence to respond to an extrinsic signal, cells are able to diversify the possible outcomes of a given signaling pathway. This is likely to occur through changes in the complement of receptors and transcription factors (intrinsic, cell-autonomous components) that are expressed by the cell at any given time and in the way in which these interact with elements from the signaling pathways. The perception of warm, cold, pain, texture, vibration, pressure, shape, surface friction, weight and other somatosensations are mediated by distinct types of neural crestderived sensory neurons displaying unique cellular and functional characteristics that allow them to accurately process the information from the periphery to the spinal cord. will present data on mechanisms participating in the process of diversification of the sensory lineage and in particular, how mechanically sensitive neurons are formed.We are also interested in understanding how gene programs directing neuronal diversification are integrated with those determining the establishment of specific points of axonal projections and contacts in the central nervous system. Here, will present data suggesting a layer of complexity in vertebrates represented by a cell intrinsic mechanism involving transcriptional activities active during cell diversification which also underlie growth differences between positionally different neurons. Thus, such factors act in a concentrationdependent manner to regulate positional differences in axonal extension properties apparently without affecting nerve guidance and branching.
Transcription-specific roles of Wnt/Ⱦ-catenin in the developing nervous system in vivo
Size and complexity of the developing nervous system are determined by the exquisite balance between self-renewal and differentiation of neural stem cells (NSC). Among multiple signaling cascades active in the developing CNS and PNS, the canonical Wnt pathway was shown to regulate NSC proliferation, fate choice and differentiation. The transducer of Wnt signals, beta-catenin, interacts with TCF/LEF family members to induce transcriptional responses, but it also participates in adherens junctions as a bridging component. Thus, experimental overexpression and downregulation of beta-catenin cannot distinguish between its signaling and adhesion functions. Combiningconditional inactivation approach with different beta-catenin mutant alleles, we determined the contribution of beta-catenin transcriptional activity as opposed to adhesion during CNS and PNS development in the mouse. Contrary to previous reports, Wnt/Ⱦ-catenin signaling ablation does not lead to rapid depletion of the NSC pool, but affects timing of fate decisions and the sequential generation of neuronal subtypes by neural progenitors in both the CNS and PNS.
Multipotentiality and lineage plasticity in neural crest cell diversification
A wide range of distinct cell types originate from the neural crest (NC) cells, a transient cell population delaminating from the dorsal neuroepithelium in the vertebrate embryo. Migratory NC cells all along the rostro-caudal axis give rise to neurons and glial cells of the peripheral nervous system and to melanocytes. In addition, the anterior NC yields diverse mesenchymal cell types, which form the facial skeleton and most of the dermis, fat tissue and vascular smooth muscle cells in the head and neck. The identification of progenitor cells and the lineages they produce is key to our understanding of how such enormous diversity of phenotypes is produced by the NC. To address these issues, we undertook in vitro clonal analysis of the developmental repertoire of single NC cells isolated from different axial regions and at various stages of migration in the quail embryo. The results show that the early migratory NC cells display heterogeneous differentiation potentials, comprising multipotent cells as well as more restricted progenitors. The cephalic NC cells include highly multipotent neural-mesenchymal progenitors able to generate neurons, glia, melanocytes, fibroblastes, chondrocytes and osteocytes, indicating that the skeletogenic lineages are not completely segregated at early migratory stages. In the trunk of Amniotes, NC cells do not produce mesenchyme in vivo; however, we show that trunk quail NC cells are capable to differentiate in vitro into chondrocytes, osteocytes and adipocytes. Importantly, these mesenchymal phenotypes arise from multipotent trunk NC cells. Therefore, common progenitors for neural and skeletal phenotypes are present in both cephalic and trunk quail NC cells. These data suggest that, in early vertebrates that bore a dermal skeleton covering their whole body, the primitive NC progenitors were endowed with both neural and mesenchymal fates. During evolution, NC cell ability to yield mesenchymal cell types vanished but was not completely lost in the trunk of Amniotes and it can still be elicited in vitro.
Trophic factors and neural development
Fifty years ago, nerve growth factor (NGF) was identified as a diffusible molecule necessary for the development of the sympathetic nervous system. It was the first growth factor ever to have been identified. NGF was shown thirty years later to bind to the proto-oncogene TrkA, causing phosphorylation of the receptor thereby preventing the death of sympathetic and many sensory neurons. But why should these cells absolutely require NGF to survive, while most others don’t? We found that it is the expression of TrkA that instructively kills neurons when NGF is not present (Nikoletopoulou et al., Nature 2010, 467, 59-63). This observation was made possible by the development of a cell culture system based on embryonic stem cells. These cells can be readily cultured and differentiated into progenitors that have many characteristics in common with Pax6-positive radial glial cells found in the developing cerebral cortex. They can also beengineered to express in a rigorously controlled fashion molecules of interest such as for example the Trk receptors, and subsequently differentiated into what appears to be essentially one type of glutamatergic neurons. As all progenitors drop out of division and begin to differentiate synchronously it was easy to note that both TrkA and TrkC rapidly kill all neurons, while TrkB does not. Death induced by TrkA and TrkC can be prevented by the addition of the corresponding ligands NGF and neurotrophin-3. These findings help explaining previous observations that neurons in the developing brain are much less dependent on growth factor for their survival, compared with those of the peripheral nervous system: most CNS neurons express TrkB at fairly high levels, compared with TrkC for example, with only very few CNS neurons expressing TrkA. TrkA-expressing neurons reside primarily in the PNS. These new findings suggest that with the evolution of the nervous system, new principles were used to regulate cell numbers in the PNS. This seems to parallel the expansion of the Trk receptor family that arose by gene duplication and exon shuffling. In this context it is interesting to note that Branchiostoma floridae only has one Trk receptor that can activated equally well by all mammalian neurotrophins (see Benito-Gutiérez et al. Development 2005, 132, 2191-2202).
Harnessing adult hippocampal neurogenesis to modulate cognition and mood
Understanding how neural circuit-plasticity mechanisms may be harnessed to improve cognition and mood is essential for developing novel treatm nts for psychiatric disorders.Adult hippocampal neurogenesis is a unique form of plasticity that generates new neurons in the dentate gyrus throughout life. Levels of adult hippocampal neurogenesis are elevated by interventions associated with beneficial effects on cognition and mood such as exercise and chronic antidepressant treatment.These properties of adult hippocampal neurogenesis suggest that it may be harnessed to improve hippocampal functions. However, despite a substantial number of loss-of-function studies demonstrating an involvement of adult-born neurons in mediating select cognitive functions and some of the behavioral effects of antidepressants, it is unknown whether stimulation of adult hippocampal neurogenesis is sufficient to improve cognition and mood.Addressing this question is pivotal to determine the potential therapeutic impact of strategies designed to specifically stimulate adult hippocampal neurogenesis. We have found that inducible genetic expansion of the population of adult-born neurons in mice improves performance in a specific cognitive task in which an animal must distinguish between two similar contexts. Mice with increased adult hippocampal neurogenesis show normal object recognition and spatial learning but are more efficient in differentiating between overlapping contextual representations, suggestive of enhanced pattern separation. Moreover, augmenting adult neurogenesis conferred a protective effect in a mouse model of depression and promoted exploratory behaviour when combined with voluntary exercise.Analysis of neuronal ensemble activity using immediate early-gene mapping suggested that new neurons influence the activity of dentate gyrus. Together, these findings suggest that adult neurogenesis is a tractable form of plasticity that maybe targeted to modulate a range of hippocampal functions.
Functional relevance of adult hippocampal neurogenesis
The dentate gyrus (DG) of the hippocampus is one of the few regions of the mammalian brain where new neurons are generated throughout adulthood. This structure is part of an integrated network that plays an important role in memory and emotional processes. Our aim is to reveal the functional significance of adult-born neurons in hippocampal-dependent behaviour. This presentation will focus on memory and give a series of evidence showing that: 1) adult-born neurons are required for navigating through space. Indeed, an alteration of hippocampal neurogenesis plays a pivotal role in the development of pathological aging and genetic ablation of new neurons impairs spatial memory, 2) spatial learning regulates the development of newborn neurons and accelerates their integration within the dentate network. Altogether, these findings highlight the role of adult-born neurons in specific memory functions and emphasize the importance of optimizing neurogenesis for preventing/curing memory pathologies.
Clonal analysis of neural stem cells in the adult hippocampus
Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question in the neural stem cell field is whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. We have developed genetic marking strategies for lineage-tracing of individual neural precursors of different subtypes in the adult mouse dentate gyrus. Using this clonal approach, we have been examining both the modes of self-renewal by different precursor subtypes and their lineage differentiation potentials in the adult brain in vivo. We are also exploring the underlying molecular mechanisms regulating precursor maintenance, activation, cell division mode and fate specification in vivo.
Parcelation of Stem Cells in the Adult Brain
Large numbers of multiple types of new GABA-ergic interneurons originate from a unique mosaic of stem cells on the walls of the lateral ventricles in the adult rodent brain. These cells migrate tot he olfactory bulb (OB) where they continually replace older cells, a process that decreases with age. Within the OB, neurons integrate into functional circuits and likely play key roles in modulation of plasticity for olfactory perception. will discuss recent data on the role of stem cells’ regional specification in production of multiple neuronal types in the adult brain and the possible functional contribution of this diverse group of neurons. Recent work suggests that Shh signaling is involved in the specification of ventral domains of this adult germinal zone. Further lineage tracing of this domain has revealed additional types of OB specific local-circuit interneurons that are formed during postnatal life in mice. Local parcelation of primary progenitors raises interesting questions about evolution and the organization of the periventricular germinal layer in larger brains. will share recent data on the development of the human periventricular germinal zone that suggest unique features including additional major migratory paths when compared to rodents.The regional parcelation of adult neural stem cells raises very basic questions on how this adult germinal layer is regulated and integrated within the adult brain and its possible contributions to neural repair.
Molecular mechanisms of adult neurogenesis
Glial cells generating neurons: molecular and cellular mechanisms. Our understanding of fate determinants endowing glial cells with the capacity of for neurogenesis is still limited. Towardsbetter understanding of such key regulators will present data of genomewide expression analysis of neurogenic glial cells, such as radial glia and adult neural stem cells, versus non-neurogenic glia, such as reactive astrocytes. An intriguing finding is that known neurogenic fate determinants are already up-regulated at mRNA, but not protein level in adult neural stem cells, while radial glial cells possess much higher mRNA and protein levels of such neurogenic fate determinants thereby resembling more the neuroblasts in the adult neurogenic zones. will then present functional data on such neurogenic fate determinants focussing especially on the interaction of neurogenic transcription factors with the chromatin remodelling machinery.
Activation of adult-born neurons promotes discrimination learning and memory
In mammals, new neurons are recruited into restricted brain areas throughout life. Adult-born neurons produced in the subventricular zone migrate towards the olfactory bulb. Although thousands neurons reach this central structure every day, the functional impact of their integration into mature circuits remains debated. Recent investigations have revealed no striking sensory deficit per se when adult bulbar neurogenesis is challenged. However, cognitive functions, such as perceptual learning or olfactory memory, are clearly impaired. In this presentation, we highlight the role of network activity in shaping ongoing neurogenesis and in turn, how the integration of adult-born neurons refines preexisting network function, and consequently olfactory behaviors.
Imaging sensory response profiles of adult born and local neurons in the mouse olfactory bulb
The mammalian olfactory bulb (OB) receives continuous supply of adult-born interneurons throughout adulthood. Little is known about the sensory response properties of these neurons. will present some of our recent efforts to study the sensory physiology of adult born neurons using in vivo two photon calcium imaging. Using viruses we induced expression of the genetically encoded calcium indicator GCamp3.0 either in newborn neurons or in local populations in the OB. We then combined viral injections with chronic window preparation in order to follow the response profiles of single neurons over time. Basal odor responses were imaged repeatedly from distinct local populations of neurons, revealing unique response signatures for different neuronal subtypes. Repeated imaging of the same local neurons over several weeks revealed remarkable stability in the response profiles of neurons to a small set of odors and concentration. This preparation will allow us test how the response profiles of newborn neurons change as they mature in the intact OB.