Emilie Pacary

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

Proneural Transcription Factors Regulate Different Steps of Cortical Neuron Migration through Rnd-Mediated Inhibition of RhoA Signaling

Summary Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.

Synergistic effects of CoCl2 and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells

Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl2 on MSCs. CoCl2 treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1α and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl2 effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl2-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl2, an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type

The mechanisms governing the expansion of neuron number in specific brain regions are still poorly understood. Enlarged neuron numbers in different species are often anticipated by increased numbers of progenitors dividing in the subventricular zone. Here we present live imaging analysis of radial glial cells and their progeny in the ventral telencephalon, the region with the largest subventricular zone in the murine brain during neurogenesis. We observe lineage amplification by a new type of progenitor, including bipolar radial glial cells dividing at subapical positions and generating further proliferating progeny. The frequency of this new type of progenitor is increased not only in larger clones of the mouse lateral ganglionic eminence but also in cerebral cortices of gyrated species, and upon inducing gyrification in the murine cerebral cortex. This implies key roles of this new type of radial glia in ontogeny and phylogeny.

An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migration

A transcriptional programme initiated by the proneural factors Neurog2 and Ascl1 controls successive steps of neurogenesis in the embryonic cerebral cortex. Previous work has shown that proneural factors also confer a migratory behaviour to cortical neurons by inducing the expression of the small GTP-binding proteins such as Rnd2 and Rnd3. However, the directionality of radial migration suggests that migrating neurons also respond to extracellular signal-regulated pathways. Here we show that the Plexin B2 receptor interacts physically and functionally with Rnd3 and stimulates RhoA activity in migrating cortical neurons. Plexin B2 competes with p190RhoGAP for binding to Rnd3, thus blocking the Rnd3-mediated inhibition of RhoA and also recruits RhoGEFs to directly stimulate RhoA activity. Thus, an interaction between the cell-extrinsic Plexin signalling pathway and the cell-intrinsic Ascl1-Rnd3 pathway determines the level of RhoA activity appropriate for cortical neuron migration.

Rnd3 coordinates early steps of cortical neurogenesis through actin-dependent and -independent mechanisms

The generation of neurons by neural stem cells is a highly choreographed process that requires extensive and dynamic remodelling of the cytoskeleton at each step of the process. The atypical RhoGTPase Rnd3 is expressed by progenitors in the embryonic brain but its role in early steps of neurogenesis has not been addressed. Here we show that silencing Rnd3 in the embryonic cerebral cortex interferes with the interkinetic nuclear migration of radial glial stem cells, disrupts their apical attachment and modifies the orientation of their cleavage plane. These defects are rescued by co-expression of a constitutively active form of cofilin, demonstrating that Rnd3-mediated disassembly of actin filaments coordinates the cellular behaviour of radial glia. Rnd3 also limits the divisions of basal progenitors via a distinct mechanism involving the suppression of cyclin D1 translation. Interestingly, although Rnd3 expression is controlled transcriptionally by Ascl1, this proneural factor is itself required in radial glial progenitors only for proper orientation of cell divisions.

Angiopoietin-2 Regulates Cortical Neurogenesis in the Developing Telencephalon

Vascular-specific growth factor angiopoietin-2 (Ang2) is mainly involved during vascular network setup. Recently, Ang2 was suggested to play a role in adult neurogenesis, affecting migration and differentiation of adult neuroblasts in vitro. However, to date, no data have reported an effect of Ang2 on neurogenesis during embryonic development. As we detected Ang2 expression in the developing cerebral cortex at embryonic day E14.5 and E16.5, we used in utero electroporation to knock down Ang2 expression in neuronal progenitors located in the cortical ventricular zone (VZ) to examine the role of Ang2 in cortical embryonic neurogenesis. Using this strategy, we showed that radial migration from the VZ toward the cortical plate of Ang2-knocked down neurons is altered as well as their morphology. In parallel, we observed a perturbation of intermediate progenitor population and the surrounding vasculature. Taken together, our results show for the first time that, in addition to its role during brain vasculature setup, Ang2 is also involved in embryonic cortical neurogenesis and especially in the radial migration of projection neurons.

Proneural factor/Rnd pathways control specific migratory properties of neurons via regulation of RhoA in different subcellular compartments

S. Xapelli 1,∗, L. Bernardino 1, L. Cortes 2, C. Schitine 3, E.S. Ferro 4, R.A. De Melo Reis 3, et al 1 Neuroprotection and Neurogenesis in Brain Repair Group, Portugal 2 Microscopy and Flow Citometry Unit, Portugal 3 Instituto de Biofísica Carlos Chagas Filho, Brazil 4 Laboratório de Biologia Celular e do Desenvolvimento, Brazil

20-P008 Regulation of neuronal migration by proneural GTP-binding protein pathways

During development, the pancreatic endocrine cells are specified within the epithelium. They will subsequently delaminate and migrate out of the epithelium in order to form the islets. Neurogenin3 (Ngn3) is a bHLH transcription factor that is responsible for differentiation of all endocrine cell types, but whether or not it has a role in their migration is still an open question. By using the chicken embryo model organism, we found that differentiation and migration programs are two different processes that are induced by Ngn3 and that can be uncoupled. Therefore, we tried to unravel the mechanisms by which Ngn3 can induce endocrine cell migration. We found that, both in chick and mouse models, overexpression of Ngn3 induces a loss of apico-basal polarity, a breakdown of basal lamina, and more importantly, a loss of the epithelial marker E-cadherin (Ecad). We also found that this is not a direct effect mediated by E-boxes in the Ecad promoter. Therefore, we are currently trying to find targets of Ngn3 that could mediate repression of Ecad, focalizing on the zinc-finger transcription factors Snail and Slug. Moreover, we are also using a pancreatic explant culture method, developed in the laboratory of Pr. Jonathan Slack, that allows us to do time-lapse imaging. Taking advantage of our Pdx::Ngn3 transgenic line, we are following Ngn3 overexpressing cells to understand in a more physiological manner how they migrate in the developing pancreas.