Cathy Soula

Induction of oligodendrocyte progenitors in the trunk neural tube by ventralizing signals: effects of notochord and floor plate grafts, and of sonic hedgehog.

Recent evidence indicates that oligodendrocytes originate initially from the ventral neural tube. We have documented in chick embryos the effect of early ventralization of the dorsal neural tube on oligodendrocyte differentiation. Notochord or floor plate grafted at stage 10 in dorsal position induced the development of oligodendrocyte precursors in the dorsal spinal cord. In vitro, oligodendrocytes differentiated from medial but not intermediate neural plate explants, suggesting that the ventral restriction of oligodendrogenesis is established early. Furthermore, quail fibroblasts overexpressing the ventralizing signal Sonic Hedgehog induced oligodendrocyte differentiation in both the intermediate neural plate and the E4 dorsal spinal cord. These results strongly suggest that the emergence of the oligodendrocyte lineage is related to the establishment of the dorso-ventral polarity of the neural tube.

Ventral Neural Progenitors Switch toward an Oligodendroglial Fate in Response to Increased Sonic Hedgehog (Shh) Activity: Involvement of Sulfatase 1 in Modulating Shh Signaling in the Ventral Spinal Cord

In the embryonic chick ventral spinal cord, the initial emergence of oligodendrocytes is a relatively late event that depends on prolonged Sonic hedgehog (Shh) signaling. In this report, we show that specification of oligodendrocyte precursors (OLPs) from ventral Nkx2.2-expressing neural progenitors occurs precisely when these progenitors stop generating neurons, indicating that the mechanism of the neuronal/oligodendroglial switch is a common feature of ventral OLP specification. We further show that an experimental early increase in the concentration of Shh is sufficient to induce premature specification of OLPs at the expense of neuronal genesis indicating that the relative doses of Shh received by ventral progenitors determine whether they become neurons or glia. Accordingly, we observe that the Shh protein accumulates at the apical surface of Nkx2.2-expressing cells just before OLP specification, providing direct evidence that these cells are subjected to a higher concentration of the morphogen when they switch to an oligodendroglial fate. Finally, we show that this abrupt change in Shh distribution is most likely attributable to the timely activity of Sulfatase 1 (Sulf1), a secreted enzym that modulates the sulfation state of heparan sulfate proteoglycans. Sulf1 is expressed in the ventral neuroepithelium just before OLP specification, and we show that its experimental overexpression leads to apical concentration of Shh on neuroepithelial cells, a decisive event for the switch of ventral neural progenitors toward an oligodendroglial fate.

Neurogenic role of Gcm transcription factors is conserved in chicken spinal cord.

Although glial cells missing (gcm) genes are known as glial determinants in the fly embryo, the role of vertebrate orthologs in the central nervous system is still under debate. Here we show for the first time that the chicken ortholog of fly gcm (herein referred to as c-Gcm1), is expressed in early neuronal lineages of the developing spinal cord and is required for neural progenitors to differentiate as neurons. Moreover, c-Gcm1 overexpression is sufficient to trigger cell cycle exit and neuronal differentiation in neural progenitors. Thus, c-Gcm1 expression constitutes a crucial step in the developmental cascade that prompts progenitors to generate neurons: c-Gcm1 acts downstream of proneural (neurogenin) and progenitor (Sox1-3) factors and upstream of NeuroM neuronal differentiation factor. Strikingly, this neurogenic role is not specific to the vertebrate gene, as fly gcm and gcm2 are also sufficient to induce the expression of neuronal markers. Interestingly, the neurogenic role is restricted to post-embryonic stages and we identify two novel brain neuronal lineages expressing and requiring gcm genes. Finally, we show that fly gcm and the chick and mouse orthologs induce expression of neural markers in HeLa cells. These data, which demonstrate a conserved neurogenic role for Gcm transcription factors, call for a re-evaluation of the mode of action of these genes during evolution.

A subtractive approach to characterize genes with regionalized expression in the gliogenic ventral neuroepithelium: identification of chick sulfatase 1 as a new oligodendrocyte lineage gene.

To address the question of the origin of glial cells and the mechanisms leading to their specification, we have sought to identify novel genes expressed in glial progenitors. We adopted suppression subtractive hybridization (SSH) to establish a chick cDNA library enriched for genes specifically expressed at 6 days of incubation (E6) in the ventral neuroepithelium, a tissue previously shown to contain glial progenitors. Screens were then undertaken to select differentially expressed cDNAs, and out of 82 unique SSH clones, 21 were confirmed to display a regionalized expression along the dorsoventral axis of the E6 ventral neuroepithelium. Among these, we identified a transcript coding for the chick orthologue of Sulf1, a recently identified cell surface sulfatase, as a new, early marker of oligodendrocyte (OL) precursors in the chick embryonic spinal cord. This study provides groundwork for the further identification of genes involved in glial specification.

Role of BMPs in controlling the spatial and temporal origin of GFAP astrocytes in the embryonic spinal cord.

In the vertebrate central nervous system (CNS), astrocytes are the most abundant and functionally diverse glial cell population. However, the mechanisms underlying their specification and differentiation are still poorly understood. In this study, we have defined spatially and temporally the origin of astrocytes and studied the role of BMPs in astrocyte development in the embryonic chick spinal cord. Using explant cultures, we show that astrocyte precursors started migrating out of the neuroepithelium in the mantle layer from E5, and that the dorsal-most level of the neuroepithelium, from the roof plate to the dl3 level, did not generate GFAP-positive astrocytes. Using a variety of early astrocyte markers together with functional analyses, we show that dorsal-most progenitors displayed a potential for astrocyte production but that dorsally-derived BMP signalling, possibly mediated through BMP receptor 1B, promoted neuronal specification instead. BMP treatment completely prevented astrocyte development from intermediate spinal cord explants at E5, whereas it promoted it at E6. Such an abrupt change in the response of this tissue to BMP signalling could be correlated to the onset of new foci of BMP activity and enhanced expression of BMP receptor 1A, suggesting that BMP signalling could promote astrocyte development in this region.

Cells from the early chick optic nerve generate neurons but not oligodendrocytes in vitro

We have recently described neuronal potentialities in neuroepithelial cells of the embryonic chicken optic nerve (Giess et al., Proc. Natl. Acad. Sci. USA, 87 (1990), 1643-1647). To further investigate the developmental repertoire of optic nerve cells, oligodendroglial development was studied in cultures of optic nerve explanted at various developmental stages. Oligodendrocyte differentiation was analyzed using antibodies directed against galactocerebrosides (Gal-C) and against sulfatides. Optic nerves removed at embryonic days 5 and 6 (E5-E6) never gave rise in culture to differentiated oligodendrocytes, even after 3 weeks in vitro. In contrast, in cultures of optic nerves removed from E7 or older embryos, cells expressing both oligodendrocyte markers were rapidly and invariably observed. Absence of oligodendrocytes before E7 was not due to culture conditions being inadequate to support the differentiation of early precursors along this pathway, since neuroepithelial cells from E2 and E4 trunk neural tube cultivated in the same conditions expressed Gal-C after respectively 16 and 10 days. These results demonstrate that the optic nerve territory is initially devoid of oligodendrocyte potentialities. Whether oligodendrocyte precursors that, around E7, populate the optic nerve are induced by a specific developmental signal occurring at this stage or migrate from outside the optic nerve remains to be determined.

Determination of Glial Lineages During Early Central Nervous System Development

The rapidly cycling neuroepithelial cells, located in the ventricular and subventricular zones of the neural tube, give rise to most of the neurons and macroglial cells, astrocytes and oligodendrocytes, in the vertebrate central nervous system (CNS). In most CNS areas, neurons are the first to develop, followed by astrocytes, and at later stages by oligodendrocytes. An important issue in the study of early CNS development is to understand the lineage relationships of the various CNS cell types and the mechanisms by which these lineages segregate and differentiate. One aspect of this question is to define when and how precursor cells become committed to a specific differentiation pathway. For example, neuroepithelial cells could initially all be endowed with equivalent differentiation capabilities. The specification of these multipotential precursor cells towards a defined phenotype could be controlled by instructive and selective cues arising progressively from their immediate environment. Alternatively, neuroepithelial progenitors could be, from early stages in nervous system ontogeny, already segregated into subpopulations with differing potentialities. In this case, environmental cues would be less critical than intrinsic developmental programs in regulating phenotypic choices.

Moving the Shh Source over Time: What Impact on Neural Cell Diversification in the Developing Spinal Cord?

A substantial amount of data has highlighted the crucial influence of Shh signalling on the generation of diverse classes of neurons and glial cells throughout the developing central nervous system. A critical step leading to this diversity is the establishment of distinct neural progenitor cell domains during the process of pattern formation. The forming spinal cord, in particular, has served as an excellent model to unravel how progenitor cells respond to Shh to produce the appropriate pattern. In recent years, considerable advances have been made in our understanding of important parameters that control the temporal and spatial interpretation of the morphogen signal at the level of Shh-receiving progenitor cells. Although less studied, the identity and position of Shh source cells also undergo significant changes over time, raising the question of how moving the Shh source contributes to cell diversification in response to the morphogen. Here, we focus on the dynamics of Shh-producing cells and discuss specific roles for these time-variant Shh sources with regard to the temporal events occurring in the receiving field.

Identification of a Sulf2-dependant astrocyte subtype that stands out through the expression of Olig2 in the ventral spinal cord

During spinal cord development, both spatial and temporal mechanisms operate to generate glial cell diversity. Here, we addressed the role of the Heparan Sulfate-editing enzyme Sulf2 in the control of gliogenesis in the mouse developing spinal cord and found an unanticipated function for this enzyme. Sulf2 is expressed in ventral spinal progenitors at initiation of gliogenesis, including in Olig2-expressing cells of the pMN domain known to generate most spinal cord oligodendrocyte precursor cells (OPCs). We found that Sulf2 is dispensable for OPC development but required for proper generation of an as-yet-unidentified astrocyte precursor cell (AP) subtype. These cells, like OPCs, express Olig2 while populating the spinal parenchyma at embryonic stages but also retain Olig2 expression as they differentiate into mature astrocytes. We therefore identify a spinal Olig2-expressing AP subtype that segregates early under the influence of the extracellular enzyme Sulf2.

Neuron/Glia Lineages During Early Nervous System Development in Amphibian and Chicken Embryos

Neurons and macroglial cells of the vertebrate central nervous system (CNS) arise from neuroepithelial precursor cells that proliferate rapidly in the ventricular and subventricular zones and present similar morphological characteristics. In most CNS regions, neurons develop first, together with a subpopulation of immature glial cells, the radial glia. The other glial cell types, astrocytes and oligodendrocytes, develop later on according to spatio-temporal schemes specific for each central structure. This sequence of differentiation events raises several key questions regarding the lineage relationships of the various CNS cell types and the mechanisms by which these lineages segregate and differentiate. One important aspect of these questions is to define when and how precursor cells become irreversibly committed to a specific differentiation pathway. Neuroepithelial cells initially could be all endowed with equivalent differentiation capabilities. The specification of these multipotential precursor cells toward a defined phenotype could occur progressively, resulting, as development proceeds, in the formation of discrete families of determined progenitors. Alternatively, they could be restricted in their developmental fate only when they undergo their final round of division, just before differentiating. In either of these two alternatives, the environment should play an important role in specifying the ultimate cell phenotype. In another possible scheme, neuroepithelial progenitors could be already segregated into subpopuladons with differing potentialities at early stages in nervous system ontogeny. In this case, environmental cues would be less critical than intrinsic developmental programs in regulating phenotypic choices.