Antoine de Chevigny

Neuronal fate determinants of adult olfactory bulb neurogenesis.

Adult neurogenesis in mammals is restricted to two small regions, including the olfactory bulb, where GABAergic and dopaminergic interneurons are newly generated throughout the entire lifespan. However, the mechanisms directing them towards a specific neuronal phenotype are not yet understood. Here, we demonstrate the dual role of the transcription factor Pax6 in generating neuronal progenitors and also in directing them towards a dopaminergic periglomerular phenotype in adult mice. We present further evidence that dopaminergic periglomerular neurons originate in a distinct niche, the rostral migratory stream, and are fewer derived from precursors in the zone lining the ventricle. This regionalization of the adult precursor cells is further supported by the restricted expression of the transcription factor Olig2, which specifies transit-amplifying precursor fate and opposes the neurogenic role of Pax6. Together, these data explain both extrinsic and intrinsic mechanisms controlling neuronal identity in adult neurogenesis.

miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons

In the postnatal and adult mouse forebrain, a mosaic of spatially separated neural stem cells along the lateral wall of the ventricles generates defined types of olfactory bulb neurons. To understand the mechanisms underlying the regionalization of the stem cell pool, we focused on the transcription factor Pax6, a determinant of the dopaminergic phenotype in this system. We found that, although Pax6 mRNA was transcribed widely along the ventricular walls, Pax6 protein was restricted to the dorsal aspect. This dorsal restriction was a result of inhibition of protein expression by miR-7a, a microRNA (miRNA) that was expressed in a gradient opposing Pax6. In vivo inhibition of miR-7a in Pax6-negative regions of the lateral wall induced Pax6 protein expression and increased dopaminergic neurons in the olfactory bulb. These findings establish miRNA-mediated fine-tuning of protein expression as a mechanism for controlling neuronal stem cell diversity and, consequently, neuronal phenotype.

NeuroD1 induces terminal neuronal differentiation in olfactory neurogenesis

After their generation and specification in periventricular regions, neuronal precursors maintain an immature and migratory state until their arrival in the respective target structures. Only here are terminal differentiation and synaptic integration induced. Although the molecular control of neuronal specification has started to be elucidated, little is known about the factors that control the latest maturation steps. We aimed at identifying factors that induce terminal differentiation during postnatal and adult neurogenesis, thereby focusing on the generation of periglomerular interneurons in the olfactory bulb. We isolated neuronal precursors and mature neurons from the periglomerular neuron lineage and analyzed their gene expression by microarray. We found that expression of the bHLH transcription factor NeuroD1 strikingly coincides with terminal differentiation. Using brain electroporation, we show that overexpression of NeuroD1 in the periventricular region in vivo leads to the rapid appearance of cells with morphological and molecular characteristics of mature neurons in the subventricular zone and rostral migratory stream. Conversely, shRNA-induced knockdown of NeuroD1 inhibits terminal neuronal differentiation. Thus, expression of a single transcription factor is sufficient to induce neuronal differentiation of neural progenitors in regions that normally do not show addition of new neurons. These results suggest a considerable potential of NeuroD1 for use in cell-therapeutic approaches in the nervous system.

High transfection efficiency of neural stem cells with magnetofection

Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.

Local neurons play key roles in the mammalian olfactory bulb

Over the past few decades, research exploring how the brain perceives, discriminates, and recognizes odorant molecules has received a growing interest. Today, olfaction is no longer considered a matter of poetry. Chemical senses entered the biological era when an increasing number of scientists started to elucidate the early stages of the olfactory pathway. A combination of genetic, biochemical, cellular, electrophysiological and behavioral methods has provided a picture of how odor information is processed in the olfactory system as it moves from the periphery to higher areas of the brain. Our group is exploring the physiology of the main olfactory bulb, the first processing relay in the mammalian brain. From different electrophysiological approaches, we are attempting to understand the cellular rules that contribute to the synaptic transmission and plasticity at this central relay. How olfactory sensory inputs, originating from the olfactory epithelium located in the nasal cavity, are encoded in the main olfactory bulb remains a crucial question for understanding odor processing. More importantly, the persistence of a high level of neurogenesis continuously supplying the adult olfactory bulb with newborn local neurons provides an attractive model to investigate how basic olfactory functions are maintained when a large proportion of local neurons are continuously renewed. For this purpose, we summarize the current ideas concerning the molecular mechanisms and organizational strategies used by the olfactory system to encode and process information in the main olfactory bulb. We discuss the degree of sensitivity of the bulbar neuronal network activity to the persistence of this high level of neurogenesis that is modulated by sensory experience. Finally, it is worth mentioning that analyzing the molecular mechanisms and organizational strategies used by the olfactory system to transduce, encode, and process odorant information in the olfactory bulb should aid in understanding the general neural mechanisms involved in both sensory perception and memory. Due to space constraints, this review focuses exclusively on the olfactory systems of vertebrates and primarily those of mammals.

Modulating Glypican4 Suppresses Tumorigenicity of Embryonic Stem Cells While Preserving Self-Renewal and Pluripotency

Self‐renewal and differentiation of stem cell depend on a dynamic interplay of cell‐extrinsic and ‐intrinsic regulators. However, how stem cells perceive the right amount of signal and at the right time to undergo a precise developmental program remains poorly understood. The cell surface proteins Glypicans act as gatekeepers of environmental signals to modulate their perception by target cells. Here, we show that one of these, Glypican4 (Gpc4), is specifically required to maintain the self‐renewal potential of mouse embryonic stem cells (ESCs) and to fine tune cell lineage commitment. Notably, Gpc4‐mutant ESCs contribute to all embryonic cell lineages when injected in blastocyts but lose their intrinsic tumorigenic properties after implantation into nude mice. Therefore, our molecular and functional studies reveal that Gpc4 maintains distinct stemness features. Moreover, we provide evidence that self‐renewal and lineage commitment of different stem cell types is fine tuned by Gpc4 activity by showing that Gpc4 is required for the maintenance of adult neural stem cell fate in vivo. Mechanistically, Gpc4 regulates self‐renewal of ESCs by modulating Wnt/β‐catenin signaling activities. Thus, our findings establish that Gpc4 acts at the interface of extrinsic and intrinsic signal regulation to fine tune stem cell fate. Moreover, the ability to uncouple pluripotent stem cell differentiation from tumorigenic potential makes Gpc4 as a promising target for cell‐based regenerative therapies. Stem Cells2012;30:1863–1874

Dynamic expression of the pro-dopaminergic transcription factors Pax6 and Dlx2 during postnatal olfactory bulb neurogenesis

Olfactory bulb (OB) neurogenesis generates neurons that use GABA or dopamine as their neurotransmitters throughout life. Regionalized stem cell populations in the periventricular zone (PVZ) of the lateral ventricles (LVs) have been shown to be at the basis of neuronal diversity in the system. For example dopaminergic neurons arise predominantly from neural stem cells (NSCs) residing in the dorsal PVZ and depend on the expression of the transcription factors Pax6 and Dlx2 for their specification. In addition, Dlx2 is required for neurogenesis in general. Using targeted in vivo electroporation combined with immuno-fluorescence imaging and microarray analysis, we provide here detailed spatial and temporal expression data with cellular resolution in this system. We find that all along the neurogenic process Pax6 expression remains restricted to the dorsal PVZ, whereas nearly all neuroblasts express Dlx2, including those of the dorsal lineage, which are switched on for Dlx2 when they enter the rostral migratory stream (RMS). These data allow to explain and precise the functions of these two genes in postnatal OB neurogenesis.

MiR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed for this process. Insight into the role of this regulatory pathway in the brain is still limited. We performed a sequential experimental approach using postnatal olfactory bulb neurogenesis in mice, starting from global expression analyses to the investigation of functional interactions between defined microRNAs and their targets. Deep sequencing of small RNAs extracted from defined compartments of the postnatal neurogenic system demonstrated that the miR-200 family is specifically induced during late neuronal differentiation stages. Using in vivo strategies we interfered with the entire miR-200 family in loss- and gain-of-function settings, showing a role of miR-200 in neuronal maturation. This function is mediated by targeting the transcription factor Zeb2. Interestingly, so far functional interaction between miR-200 and Zeb2 has been exclusively reported in cancer or cultured stem cells. Our data demonstrate that this regulatory interaction is also active during normal neurogenesis.

Reducing Glypican-4 in ES Cells Improves Recovery in a Rat Model of Parkinson's Disease by Increasing the Production of Dopaminergic Neurons and Decreasing Teratoma Formation

The heparan sulfate proteoglycan Glypican 4 (Gpc4) is strongly expressed in mouse embryonic stem (ES) cells where it controls the maintenance of self-renewal by modulating Wnt/β-catenin signaling activities. Here we show that mouse ES cells carrying a hypomorphic Gpc4 allele, in a single-step neuronal differentiation protocol, show increased differentiation into dopaminergic neurons expressing tyrosine hydroxylase (TH) and nuclear receptor related-1 protein (Nurr1) 1. In contrast to wild-type cells, these differentiating Gpc4-mutant cells expressed high levels of DOPA decarboxylase and the dopamine transporter, two markers expressed by fully mature dopaminergic neurons. Intrastriatal transplantation of Gpc4 hypomorphic cells into a 6-OHDA rat model for Parkinsons disease improved motor behavior in the cylinder test and amphetamine-induced rotations at a higher level than transplanted wild-type cells. Importantly, Gpc4 hypomorphic cell grafts, in contrast to wild-type cells, did not generate teratomas in the host brains, leading to strongly enhanced animal survival. Therefore, control of Gpc4 activity level represents a new potential strategy to reduce ES cell tumorigenic features while at the same time increasing neuronal differentiation and integration.

Zic-proteins are repressors of dopaminergic forebrain fate in mice and C. elegans

In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegans. SIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species.