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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.

Neuronal differentiation has been studied in dissociated cell cultures from early neurulae of Pleurodeles waltl and Ambystoma mexicanum. Cocultures were prepared from the neural primordium and underlying chordamesoderm. NP and NF cultures were prepared from isolated neural plate and neural folds, respectively. Neuronal precursors in NP and NF cultures had distinctive aggregation properties already evident after 1–2 days in culture. After 10–15 days, mature neurones and synapses were observed by electron microscopy in the three culture types. The expression of neurofilament polypeptides and tetanus-toxin-binding sites was also present in these cultures. A small percentage of neurones contained cytochemically detectable catecholamine. Many neurones took up tritiated dopamine with a high affinity. Quantitative measurement of [3H]acetylcholine synthesis and storage from [3H]choline were negative at the early neurula stage and in 5 to 15-day-old NF cultures, and remained low in 5 to 15-day-old NP cultures. Acetylcholine production in cocultures increased linearly with time and was always much higher than in NP cultures. These results suggest that, at the early neurula stage, some neuronal precursors have acquired the capacity to express a high degree of morphological and biochemical differentiation even in the absence of further chordamesoderm influence. However, the chordamesodermal cells in the cultures increased acetylcholine synthesis.