Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.

No abstract

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cellsurface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

Neurons and oligodendrocytes are highly vulnerable to various insults, and their spontaneous replacement occurs to only a limited extent after damage in the adult spinal cord. The environment of injured tissue is thus thought to restrict the regenerative capacity of endogenous neural stem/progenitor cells; strategies for overcoming such restrictions remain to be developed. Here, we combined growth factor treatment and genetic manipulation to stimulate neurogenesis and oligodendrogenesis by endogenous progenitors in vivo. The recombinant retrovirus pMXIG, which was designed to coexpress green fluorescent proteins (GFPs) and a neurogenic/gliogenic transcription factor, was directly injected into the injured spinal cord parenchyma to manipulate proliferative cells in situ. We found that cells expressing Olig2, Nkx2.2, and NG2 were enriched among virus-infected, GFP-positive (GFP ϩ ) cells. Moreover, a fraction of GFP ϩ cells formed neurospheres and differentiated into neurons, astrocytes, and oligodendrocytes in vitro, demonstrating that GFP retroviruses indeed infected endogenous neural progenitors in vivo. Neuronal differentiation of control virus-infected cells did not occur at a detectable level in the injured spinal cord. We found, however, that direct administration of fibroblast growth factor 2 and epidermal growth factor into lesioned tissue could induce a significant fraction of GFP-labeled cells to express immature neuronal markers. Moreover, retrovirus-mediated overexpression of the basic helix-loop-helix transcription factors Neurogenin2 and Mash1, together with growth factor treatment, enhanced the production and maturation of new neurons and oligodendrocytes, respectively. These results demonstrate that endogenous neural progenitors can be manipulated to replace neurons and oligodendrocytes lost to insults in the injured spinal cord.