Olig2 is the key transcription factor that not only maintains the neural progenitor cells (NPCs) of pMN domain, but also regulates the sequential specification of NPCs into motor neurons (MNs) and OPCs [1,2,3,4,5]. Since persistent expression of Olig2 is inhibitory to post-mitotic MN genes [6], the expression of Olig2 rapidly declines in newly generated MNs, but remains high in later-born cells of oligodendrocyte lineage [2,3,4,5,6]. The mechanism of down-regulation of Olig2 expression in MNs remains elusive. WNT signaling is known to regulate the balance between the proliferation and differentiation of NPCs during neurogenesis [7]. It is interesting that endogenous WNT/β-catenin signaling is activated in newly generated MNs [8]. Activation of WNT/β-catenin signaling has been reported to inhibit the specification of OPCs and astrocytes from NPCs during early stages of gliogenesis [9,10,14], expression of P3 domain marker NKX2-2 was not suppressed in Olig1Cre/+;Ctnnb1ΔEx3/+ mice (Additional file 1: Fig. S1). However, the number of Ngn2-positive cells was dramatically increased within the ventral ventricular region in Olig1Cre/+;Ctnnb1ΔEx3/+ mice (Fig. 1a), demonstrating that WNT activation promotes Ngn2 expression. In support of this notion, overexpression of Ctnnb1ΔEx3 in embryonic chicken spinal cord also caused an increase of Ngn2 expression, coupled with a reduced expression of Olig2 (Fig. 1d). At E18.5, although a few dorsally-derived [15,16,17] OPCs were generated from Olig1Cre/+;Ctnnb1ΔEx3/+ mice, Plp1-positive mature oligodendrocytes were still undetectable (Additional file 1: Fig. S2) since dorsal OPCs differentiate only after birth [14]. Together, these results strongly suggest that Ngn2 is the candidate gene that mediates the suppression of oligodendrogenesis from pMN NPCs by WNT signaling.

Fig. 1
figure 1

WNT signaling inhibit Olig2 expression through upregulation of Ngn2 expression. a Transverse sections of spinal cord at E12.5 from control and WNT signaling activated (Olig1Cre/+;Ctnnb1ΔEx3/+) mice were subjected to IF with anti-OLIG2 antibody or ISH with Olig1, Pdgfrα and Ngn2 riboprobes. The cells positive for OLIG2, Olig1 and Pdgfrα are absent in the spinal cord from Olig1Cre/+;Ctnnb1ΔEx3/+ mice, whereas Ngn2 is upregulated. Inset highlights the expression of OLIG2 in pMN domain, note that vascular development was abnormal in the spinal cord of Olig1Cre/+;Ctnnb1ΔEx3/+ mice. b There are putative Ngn2 binding sequences in the promoter regions of Ngn2 from human, rat and mouse. c Luciferase report assay revealed that Ngn2 but not its DNA binding deficient mutant AQ-Ngn2 inhibit the promoter activity of mouse Olig2. *p < 0.05, t-test. d Over-expression of Ngn2-EnR mimics the phenotype caused by over-expression of Ctnnb1-ΔEx3. Both expression of Ctnnb1-ΔEx3 and Ngn2-EnR suppressed the expression of OLIG2 in ovo. Arrowhead indicates induced expression of chick Ngn2 (cNgn2). Arrows represent reduced expression of endogenous genes. e OLIG2 maintains proliferation of pMN domain neural progenitor cells. High level of WNT signaling upregulates Ngn2 expression, NGN2 in turn coordinate with OLIG2 to promote motor neurons specification and suppress Olig2 expression in newly generated motor neurons. OPCs were specified OLIG2+ cells when WNT signaling is declined at the gliogenesis stage

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In line with this concept, two NGN2 recognition sequences are identified in the upstream promoter of the Olig2 gene in human, rat and mouse (Fig. 1b). Luciferase reporter assay revealed that Ngn2 but not its DNA binding defective mutant AQ-Ngn2 can inhibit the promoter activity of mouse Olig2 (Fig. 1c), demonstrating that Ngn2 can bind to the promoter of Olig2 and repress its expression. To confirm that Ngn2 mediates WNT inhibition of oligodendrogenesis, we overexpressed Ngn2 in embryonic chicken spinal cord by in ovo electroporation and found a significant decrease of Olig2 and Pdgfra expression in the electroporated side at cE7 (Additional file 1: Fig. S3). Since Ngn2 can function either as a transcriptional activator or a repressor, we next investigated whether the inhibition of Olig2 expression is mediated by the transcriptional repressor activity of Ngn2. RCAS-Ngn2-EnR (DNA binding domain of NGN2 fused with EnR transcription repressor) was employed as a repressor-only NGN2 chimeric protein. It was found that overexpression of this chimeric repressor caused a significant reduction of Olig2 expression (Fig. 1d), mimicking the effect of full-length Ngn2 protein. This finding demonstrated that Ngn2 inhibits Olig2 expression by its transcriptional repressor activity.

In conclusion, our results suggest that WNT signaling up-regulates the expression of Ngn2, and Ngn2 in turn inhibits Olig2 expression and oligodendrogenesis during MN specification (Fig. 1e).