MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity

We have investigated the genetic circuitry underlying the determination of neuronal identity, using mammalian peripheral autonomic neurons as a model system. Previously, we showed that treatment of neural crest stem cells (NCSCs) with bone morphogenetic protein-2 (BMP-2) leads to an induction of MASH1 expression and consequent autonomic neuronal differentiation. We now show that BMP2 also induces expression of the paired homeodomain transcription factor Phox2a, and the GDNF/NTN signalling receptor tyrosine kinase c-RET. Constitutive expression of MASH1 in NCSCs from a retroviral vector, in the absence of exogenous BMP2, induces expression of both Phox2a and c-RET in a large fraction of infected colonies, and also promotes morphological neuronal differentiation and expression of pan-neuronal markers. In vivo, expression of Phox2a in autonomic ganglia is strongly reduced in Mash1 -/- embryos. These loss- and gain-of-function data suggest that MASH1 positively regulates expression of Phox2a, either directly or indirectly. Constitutive expression of Phox2a, by contrast to MASH1, fails to induce expression of neuronal markers or a neuronal morphology, but does induce expression of c-RET. These data suggest that MASH1 couples expression of pan-neuronal and subtype-specific components of autonomic neuronal identity, and support the general idea that identity is established by combining subprograms involving cascades of transcription factors, which specify distinct components of neuronal phenotype

Neuronal Subtype Generation During Postnatal Olfactory Bulb Neurogenesis

International audienceNew neurons are generated throughout life in two specific regions of the mammalian brain: the hippocampus and the olfactory bulb (OB). In the OB, large numbers of interneurons are permanently issued from stem cells residing in the ven-tricular/subventricular zone (V/SVZ) lining the forebrain ven-tricles. From here, they migrate via the rostral migratory stream (RMS) into the OB where they differentiate as interneurons. These postnatal and adult generated interneurons exhibit considerable phenotypic diversity at several levels. First, they are heterogeneous at the neurotransmitter level. Indeed, it has been shown that neurons using exclusively GABA, neurons that use both GABA and dopamine, and also very few gluta-matergic neurons are generated and integrated. Second, they show varying final locations in the OB. Although most OB interneurons remain in the deep positioned granule cell layer, a substantial fraction integrates in the superficial glomerular layer. Finally, adult-born neurons show a wide spectrum of morphologies, projection patterns, and targets. 1 Lineage studies demonstrated that the diversity of OB interneurons is closely tied to their spatial origin in the stem cell compartment. For example, interneurons generated by progenitors of the dorsal part of the V/SVZ will predominantly integrate in the superficial layers of the OB and express sub-type markers such as calretinin (CR), tyrosine hydroxylase, or the transcription factors (TFs) TBR1/2. In contrast, OB neu-rons produced along the lateral aspect of the ventricle are purely GABAergic and integrate in deeper layers of the OB. The discovery that neuronal heterogeneity is determined by their site of origin led to the notion that the stem cell niche represents a cellular mosaic in which populations of stem cells in defined dorsoventral and anteroposterior positions are predetermined to produce specific neurons for the OB. 2 This, in turn, implies that molecular determinants, for example, differentially expressed TFs, underlie early fate specification and ultimately neuronal function and connectivity. Gene Expression in Space and Time Tiveron et al. 3 set out to systematically identify and functionally characterize such fate determinants based on high-resolution gene expression analyses. Up to now, gene expression analyses performed in the V/SVZ-RMS-OB neurogenic system relied either on microdissection of tissues 4 or on cell sorting based on expression of a limited set of membrane markers 5,6 that define specific differentiation stages. However, in this neurogenic system , such approaches present several limitations. Although the stem cell compartments of the dorsal and lateral lineages are physically separated, these regions harbor progenitors at different differentiation stages (neural stem cells [NSCs], transit amplifying progenitors and migrating neuroblasts). In addition, in the RMS and the OB, both lineages are intermingled and cannot be easily distinguished, let alone isolated. To overcome these limitations, Tiveron et al. performed a lineage tracing approach based on targeted brain electropora-tion. Previous work demonstrated that in vivo brain electropo-ration can be used to transfect DNA-based vectors, 7 or even messenger RNA, 8 into the different stem cell compartments ABSTRACT: In the perinatal and adult forebrain, regionalized neural stem cells lining the ventricular walls produce different types of olfactory bulb interneurons. Although these postnatal stem cells are lineage related to their embryonic counterparts that produce, for example, cortical, septal, and striatal neurons, their output at the level of neuronal phenotype changes dramatically. Tiveron et al. investigated the molecular determinants underlying stem cell regionalization and the gene expression changes inducing the shift from embryonic to adult neuron production. High-resolution gene expression analyses of different lineages revealed that the zinc finger proteins, Zic1 and Zic2, are postnatally induced in the dorsal olfactory bulb neuron lineage. Functional studies demonstrated that these factors confer a GABAergic and calretinin-positive phenotype to neural stem cells while repressing dopaminergic fate. Based on these findings, we discuss the molecular mechanisms that allow acquisition of new traits during the transition from embryonic to adult neurogenesis. We focus on the involvement of epigenetic marks and emphasize why the identification of master transcription factors, that instruct the fate of postnatally generated neurons, can help in deciphering the mechanisms driving fate transition from embryonic to adult neuron production

Neuronal Subtype Generation During Postnatal Olfactory Bulb Neurogenesis

In the perinatal and adult forebrain, regionalized neural stem cells lining the ventricular walls produce different types of olfactory bulb interneurons. Although these postnatal stem cells are lineage related to their embryonic counterparts that produce, for example, cortical, septal, and striatal neurons, their output at the level of neuronal phenotype changes dramatically. Tiveron et al. investigated the molecular determinants underlying stem cell regionalization and the gene expression changes inducing the shift from embryonic to adult neuron production. High-resolution gene expression analyses of different lineages revealed that the zinc finger proteins, Zic1 and Zic2, are postnatally induced in the dorsal olfactory bulb neuron lineage. Functional studies demonstrated that these factors confer a GABAergic and calretinin-positive phenotype to neural stem cells while repressing dopaminergic fate. Based on these findings, we discuss the molecular mechanisms that allow acquisition of new traits during the transition from embryonic to adult neurogenesis. We focus on the involvement of epigenetic marks and emphasize why the identification of master transcription factors, that instruct the fate of postnatally generated neurons, can help in deciphering the mechanisms driving fate transition from embryonic to adult neuron production

Efficient In Vivo Electroporation of the Postnatal Rodent

Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140) in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general

Effects of sensory deprivation on glomerular interneurons in the mouse olfactory bulb: differences in mortality and phenotypic adjustment of dopaminergic neurons

International audienceNeurogenesis persists in the mammalian subventricular zone after birth, producing various populations of olfactory bulb (OB) interneurons, including GABAergic and mixed dopaminergic/GABAergic (DA) neurons for the glomerular layer. While olfactory sensory activity is a major factor controlling the integration of new neurons, its impact on specific subtypes is not well understood. In this study we used genetic labeling of defined neuron subsets, in combination with reversible unilateral sensory deprivation and longitudinal in vivo imaging, to examine the behavior of postnatally born glomerular neurons. We find that a small fraction of GABAergic and of DA neurons die after 4 weeks of sensory deprivation while surviving DA-neurons exhibit a substantial decrease in tyrosine hydroxylase (TH) expression levels. Importantly, after reopening of the naris, cell death is arrested and TH levels go back to normal levels, indicating a specific adaptation to the level of sensory activity. We conclude that sensory deprivation induces adjustments in the population of glomerular neurons, involving both, cell death and adaptation of neurotransmitter use in specific neuron types. Our study highlights the dynamic nature of glomerular neurons in response to sensory deprivation and provide valuable insights into the plasticity and adaptability of the olfactory system

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors.

International audienceThe cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM(+) enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM(+) enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells

BAD-LAMP defines a subset of early endocytic organelles in subpopulations of cortical projection neurons.

The brain-associated LAMP-like molecule (BAD-LAMP) is a new member of the family of lysosome associated membrane proteins (LAMPs). In contrast to other LAMPs, which show a widespread expression, BAD-LAMP expression in mice is confined to the postnatal brain and therein to neuronal subpopulations in layers II/III and V of the neocortex. Onset of expression strictly parallels cortical synaptogenesis. In cortical neurons, the protein is found in defined clustered vesicles, which accumulate along neurites where it localizes with phosphorylated epitopes of neurofilament H. In primary neurons, BAD-LAMP is endocytosed, but is not found in classical lysosomal/endosomal compartments. Modification of BAD-LAMP by addition of GFP revealed a cryptic lysosomal retention motif, suggesting that the cytoplasmic tail of BAD-LAMP is actively interacting with, or modified by, molecules that promote its sorting away from lysosomes. Analysis of BAD-LAMP endocytosis in transfected HeLa cells provided evidence that the protein recycles to the plasma membrane through a dynamin/AP2-dependent mechanism. Thus, BAD-LAMP is an unconventional LAMP-like molecule and defines a new endocytic compartment in specific subtypes of cortical projection neurons. The striking correlation between the appearance of BAD-LAMP and cortical synatogenesis points towards a physiological role of this vesicular determinant for neuronal function

Agrin-signaling is necessary for the integration of newly generated neurons in the adult olfactory bulb.

In the adult forebrain, new interneurons are continuously generated and integrated into the existing circuitry of the olfactory bulb (OB). In an attempt to identify signals that regulate this synaptic integration process, we found strong expression of agrin in adult generated neuronal precursors that arrive in the olfactory bulb after their generation in the subventricular zone. While the agrin receptor components MuSK and Lrp4 were below detection level in neuron populations that represent synaptic targets for the new interneurons, the alternative receptor α3-Na(+)K(+)-ATPase was strongly expressed in mitral cells. Using a transplantation approach, we demonstrate that agrin-deficient interneuron precursors migrate correctly into the OB. However, in contrast to wild-type neurons, which form synapses and survive for prolonged periods, mutant neurons do not mature and are rapidly eliminated. Using in vivo brain electroporation of the olfactory system, we show that the transmembrane form of agrin alone is sufficient to mediate integration and demonstrate that excess transmembrane agrin increases the number of dendritic spines. Last, we provide in vivo evidence that an interaction between agrin and α3-Na(+)K(+)-ATPase is of functional importance in this system