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WNT signaling suppresses oligodendrogenesis via Ngn2-dependent direct inhibition of Olig2 expression
Molecular Brain volume 13, Article number: 155 (2020)
Olig2 transcription factor is essential for the maintenance of neural progenitor cells (NPCs) in the pMN domain and their sequential specification into motor neurons (MNs) and oligodendrocyte precursor cells (OPCs). The expression of Olig2 rapidly declines in newly generated MNs. However, Olig2 expression persists in later-born OPCs and antagonizes the expression of MN-related genes. The mechanism underlying the differential expression of Olig2 in MNs and oligodendrocytes remains unknown. Here, we report that activation of WNT/β-catenin signaling in pMN lineage cells abolished Olig2 expression coupled with a dramatic increase of Ngn2 expression. Luciferase reporter assay showed that Ngn2 inhibited Olig2 promoter activity. Overexpression of Ngn2-EnR transcription repressor blocked the expression of Olig2 in ovo. Our results suggest that down-regulation of WNT-Ngn2 signaling contributes to oligodendrogenesis from the pMN domain and the persistent Olig2 expression in OPCs.
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 , 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 . It is interesting that endogenous WNT/β-catenin signaling is activated in newly generated MNs . Activation of WNT/β-catenin signaling has been reported to inhibit the specification of OPCs and astrocytes from NPCs during early stages of gliogenesis [9,10,11]. However, the mechanism underlying the inhibition of OPC specification from pMN NPCs by WNT/β-catenin signaling remains to be determined.
Here, we utilized the Olig1Cre/+;Ctnnb1ΔEx3/+ transgenic mice to activate WNT signaling in the pMN domain. At embryonic day 12.5 (E12.5) when oligodendrogenesis commences, expression of Olig1 and Olig2 remains high in the pMN neural progenitor cells from which Pdgfra+ OPCs arise (Fig. 1a). Strikingly, in Olig1Cre/+;Ctnnb1ΔEx3/+ transgenic mice, activation of WNT signaling totally abolished the expression of Olig1, Olig2 and Pdgfra (Fig. 1a), indicating a complete inhibition of oligodendrogenesis. By contrast, the number of ISL1-positive MNs was only decreased slightly in Olig1Cre/+;Ctnnb1ΔEx3/+ mice (Additional file 1: Fig. S1), consistent with the previous finding that Olig1 is intermittently expressed in pMN NPCs and only weakly expressed during neurogenesis stage . Although Olig1Cre was also transcribed in P3 domain at early stages [13, 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 . Together, these results strongly suggest that Ngn2 is the candidate gene that mediates the suppression of oligodendrogenesis from pMN NPCs by WNT signaling.
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).
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This work was supported by the National Natural Science Foundation of China (Grant No. 31871480, 81771028, 31771621); the Natural Science Foundation of Zhejiang Province (Grant No. LY18C090009, LQ15C070001, LQ18C090005).
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The use of animals was approved by the Committee on Laboratory Animals, Hangzhou Normal University.
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Jiang, M., Yu, D., Xie, B. et al. WNT signaling suppresses oligodendrogenesis via Ngn2-dependent direct inhibition of Olig2 expression. Mol Brain 13, 155 (2020). https://doi.org/10.1186/s13041-020-00696-0