The Early Postnatal Nonhuman Primate Neocortex Contains Self-Renewing Multipotent Neural Progenitor Cells

The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs) derived from the postnatal day 14 (PD14) marmoset monkey primary visual cortex (V1, striate cortex). While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF-2), dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine

Mapping arealisation of the visual cortex of non-primate species: lessons for development and evolution

In order to integrate and interpret visual stimuli and build a representation of the surrounding environment, the visual cortex is organised in anatomically distinct and functionally unique areas. Each area processes a particular aspect of the visual scene, with the signal flowing from one area to the next in a bottom-up processing sequence. Areal borders can be demarcated both functionally by systematic electrophysiology mapping, and anatomically by sharp changes in cellular distribution and molecular expression profiles. Primates, including humans, are heavily dependent on vision, with approximately 50% of their neocortical surface dedicated to visual processing and possess many more visual areas than any other mammal, making them often the model of choice to study visual arealisation. However, the recent identification of differential gene expression profiles between cortices in a number of species has allowed for the introduction of non-primate animal models in the field to better understand development and evolution. Profiling the mosaic of visual areas in less complex species was pivotal in understanding the mechanisms responsible for patterning the developing neocortex, specifying area identity as well as the evolutionary events that have allowed for primates to develop more areas. In addition, species with fewer areas provide a simpler system in which to study and map cortical connectivity. In this review we focus on non-primate species that have contributed to elucidating the evolution and development of the visual cortex, including small nocturnal species and carnivores. We present the current understanding of the mechanisms supporting the establishment of areal borders during development and the limitations of the predominant mouse model and the need for alternate species

Rôle et expression du facteur lymphangiogénique VEGF-C et de son récepteur VEGFR-3 au cours du développement du cerveau embryonnaire

Le VEGF-C a été caractérisé pour son implication dans le développement des vaisseaux lymphatiques via l activation de son récepteur à activité tyrosine kinase, VEGFR-3. Le VEGF-C liant également les récepteurs Neuropilines exprimés par les cellules neurales, nous avons examiné si les cellules neurales répondaient au VEGF-C et si elles exprimaient le VEGFR-3. J ai d abord montré, in vitro, que le VEGF-C stimule la prolifération des précurseurs neuraux exprimant le VEGFR-3 dans bulbe olfactif embryonnaire ainsi que la prolifération et la migration de précurseurs oligodendrocytaires du chiasma optique chez la souris. J ai localisé par hybridation in situ les sites d expression du Vegf-c et du Vegfr-3 dans les régions septo-hippocampale, pré-thalamique et dorso-latérale du thalamus du cerveau embryonnaire et adulte de poulet. En effet, le modèle de poulet est très utilisé pour ce genre de manipulation du fait de son accessibilité aux stades embryonnaires. J ai ensuite généré et testé la validité d une série d outils moléculaires permettant de bloquer par ARN interférence l expression du Vegfr-3 (siRNAs et shRNAs codé par un plasmide pouvant être électroporé ou dans un lentivirus capable d infecter durablement le cerveau embryonnaire et adulte). J ai mis au point la technique d électroporation et permis d engager les expériences d ARN interférence actuellement en cours. J ai également mis au point les analyses fonctionnelles permettant d évaluer les effets de la perte de fonction du VEGFR-3 sur la prolifération, la survie, la mise en place des populations neuronales exprimant le gène codant le Vegfr-3 dans le septum et ainsi que sur le développement des projections axonales connectant l hippocampe dans lequel le Vegf-c est exprimé.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

V1 derived neurospheres were maintained for at least 6 passages and remained multipotent from passage 0 to 3.

<p>Plot of cell yield at each passage based on a seeding density of 4.8×10⁴ cells per dish (n = 10 independent isolates per condition) (<b><i>A</i></b>). Clonogenicity assays of FGF-2+EGF-generated neurospheres at passages 1–4 (1,000 cell/cm² seeding density) (<b><i>B</i></b>). At passage 3, neurospheres gave rise to MAP2⁺ neurons (<b><i>C</i></b>), O4⁺ oligodendrocytes (<b><i>D</i></b>) and GFAP⁺ astrocytes (<b><i>E</i></b>) after 15 DIV. Data represent mean ± SEM. Scale bar = 50 µm.</p

The neuronal maturation marker NNF is expressed by neurons in marmoset monkey V1 at PD14.

<p><b><i>A</i></b>, Parasagittal section reveals intense expression in layers 3 and 6 of V1, and the absence of labeling in adjacent area V2. <b><i>B</i></b>, Schematic representation of the marmoset neocortex illustrating posterior visual areas. The vertical dashed line illustrates the plane of surgical excision used to isolate V1 tissue. The lateral ventricle is delineated by the gray shaded region based upon our own observations and the analyses of other groups <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034383#pone.0034383-Palazzi1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034383#pone.0034383-Sawamoto1" target="_blank">[39]</a>. <b><i>C</i></b>, Representative piece of resected V1 tissue subsequently processed for cell culture. Calcarine (Ca), calcarine fissure (CaF), hippocampus (Hip), lateral (L), lateral ventricle (LV), medial (M), operculum (Op), primary visual cortex (V1), secondary visual area (V2), white matter (WM). Scale bar = 2000 µm (<b><i>A</i></b>), 1000 µm (<b><i>C</i></b>).</p

DCX, Sox2, Ki67, NG2 and GFAP are expressed in V1 at PD14.

<p>Hoechst staining demarcates cortical layers (<b><i>A</i></b>). Tbr2 was not expressed in V1 but expressed in the SVZ (<b><i>C</i></b>, LV:lateral ventricle). DCX⁺ cell bodies (<b><i>D</i></b>, *blood vessels) were located at the limit between layers 1 and 2 (<b><i>E</i></b>, arrow), and in white matter (<b><i>F</i></b>, arrow). DCX⁺ cells in the white matter extended long radial processes towards the outer layers (<b><i>F</i></b>; arrowheads). Sox2⁺ cells (<b><i>G</i></b>) were distributed across all cortical layers and white matter, of which a subset co-expressed Ki67 (<b><i>H</i></b>, <i>z</i> = z stack). NG2⁺ cells distributed in the white matter and the cortical layers co-expressed Sox2 (<b><i>I</i></b>, <b><i>J</i></b>) and the proliferation marker PCNA (<b><i>K</i></b>). The majority of Sox2+ cells co-expressed GFAP (<b><i>L</i></b>, <b><i>M</i></b>), a subset of astrocytes expressed Ki67 (<b><i>N</i></b>, *blood cell) however most of the Ki67⁺ cells were GFAP⁻ (<b><i>L</i></b>, arrowhead). Scale bar = 100 µm (<b><i>A, B, C, D, G, I, L</i></b>), 50 µm (<b><i>E</i></b>, <b><i>F</i></b>), 20 µm (<b><i>H, J, K, M, N</i></b>).</p

EGF and/or FGF-2 stimulate the formation of multipotent neurospheres from dissociated V1 tissue.

<p>Plot of primary neurospheres counted in each well of a 96 well plate after 15 DIV. Acutely isolated cells seeded at 500 cells/well (n = 11 independent isolates per condition) (<b><i>A</i></b>). Plot of mean primary neurosphere diameter (n = 6 independent isolates per condition) (<b><i>B</i></b>). After 15 DIV, neurospheres were pulsed with BrdU for 8 hours prior to fixation. BrdU⁺ cells found within the neurospheres (<b><i>C</i></b>, <b><i>D</i></b>, red) were double-labelled with Sox2 (<b><i>C</i></b>, arrowheads), and expressed Nestin (<b><i>D</i></b>). Differentiation of dissociated primary neurospheres for 15 DIV generated MAP2⁺ neurons (<b><i>E</i></b>), O4⁺ oligodendrocytes (<b><i>F</i></b>) and GFAP⁺ astrocytes (<b><i>G</i></b>). Percentages of MAP2⁺ neurons and O4⁺ oligodendrocytes compared to total differentiated cells (<b><i>H</i></b>). Data represent mean ± SEM (*** <i>P</i>&lt;0.0001; **<i>P</i>&lt;0.009, *<i>P</i>&lt;0.03; Mann-Wittney <i>U</i> test <b><i>A,B</i></b>; Kruskal-Wallis test, <b><i>H</i></b>).</p