In the vertebrate spinal cord, oligodendrocytes arise from the ventral part of the neuroepithelium, a region also known to generate somatic motoneurons. The emergence of oligodendrocytes, like that of motoneurons, depends on an inductive signal mediated by Sonic hedgehog. We have defined the precise timing of oligodendrocyte progenitor specification in the cervico-brachial spinal cord of the chick embryo. We show that ventral neuroepithelial explants, isolated at various development stages, are unable to generate oligodendrocytes in culture until E5 but become able to do so in an autonomous way from E5.5. This indicates that the induction of oligodendrocyte precursors is a late event that occurs between E5 and E5.5, precisely at the time when the ventral neuroepithelium stops producing somatic motoneurons. Analysis of the spatial restriction of oligodendrocyte progenitors, evidenced by their expression of O4 or PDGFRμ, indicate that they always lie within the most ventral Nkx2.2-expressing domain of the neuroepithelium, and not in the adjacent domain characterized by Pax6 expression from which somatic motoneurons emerge. We then confirm that Shh is necessary between E5 and E5.5 to specify oligodendrocyte precursors but is no longer required beyond this stage to maintain ongoing oligodendrocyte production. Furthermore, Shh is sufficient to induce oligodendrocyte formation from ventral neuroepithelial explants dissected at E5. Newly induced oligodendrocytes expressed Nkx2.2 but not Pax6, correlating with the in vivo observation. Altogether, our results show that, in the chick spinal cord, oligodendrocytes originate from Nkx2.2-expressing progenitors.

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.

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 gcmand 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.

In the developing ventral spinal cord, motor neurons (MNs) and oligodendrocyte precursor cells (OPCs) are sequentially generated from a common pool of neural progenitors included in the so-called pMN domain characterized by Olig2 expression. Here, we establish that the secreted Sulfatase 1 (Sulf1) is a major component of the mechanism that causes these progenitors to stop producing MNs and change their fate to generate OPCs. We show that specification of OPCs is severely affected insulf1-deficient mouse embryos. This defect does not rely on abnormal patterning of the spinal cord or failure in maintenance of pMN progenitors at the onset of OPC specification. Instead, the efficiency of OPC induction is reduced, only few Olig2 progenitors are recruited to generate OPCs, meanwhile they continue to produce MNs beyond the normal timing of the neuroglial switch. Using the chicken embryo, we show that Sulf1 activity is required precisely at the stage of the MN-to-OPC fate switch. Finally, we bring arguments supporting the view that Sulf1 controls the level of Sonic Hedgehog (Shh) signaling activity, behaving as an enhancer rather than an obligatory component in the Shh pathway. Our study provides additional insights into the temporal control of Olig2 progenitor cell fate change by the identification of Sulf1 as an extracellular timing signal in the ventral spinal cord.