Yokoyama Kiichi, Hiraoka Yuichi, Abe Yoshifumi, Tanaka Kenji F
Division of Brain Sciences, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.
Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.
J Neurochem. 2025 Jan;169(1):e16218. doi: 10.1111/jnc.16218. Epub 2024 Sep 4.
Oligodendrocyte (OL) differentiation from oligodendrocyte precursor cells (OPCs) is considered to result in two populations: premyelinating and myelinating OLs. Recent single-cell RNA sequence data subdivided these populations into newly formed (NFOLs), myelin-forming (MFOLs), and mature (MOLs) oligodendrocytes. However, which newly proposed population corresponds to premyelinating or myelinating OLs is unknown. We focused on the NFOL-specific long non-coding oligodendrocyte 1 gene (LncOL1) and sought to label NFOLs under the control of the LncOL1 promoter using a tetracycline-controllable gene induction system. We demonstrated that LncOL1 was expressed by premyelinating OLs and that the MFOL-specific gene, Ctps, was not, indicating that NFOLs correspond to premyelinating OLs and that MFOLs and MOLs correspond to myelinating OLs. We then generated a LncOL1-tTA mouse in which a tetracycline transactivator (tTA) cassette was inserted downstream from the LncOL1 transcription initiation site. By crossing the LncOL1-tTA mice with tetO reporter mice, we generated LncOL1-tTA::tetO-yellow fluorescent protein (YFP) double-transgenic (LncOL1-YFP) mice. Although LncOL1 is non-coding, YFP was detected in LncOL1-YFP mice, indicating successful tTA translation. Unexpectedly, we found that the morphology of LncOL1-tTA-driven YFP cells was distinct from that of LncOL1 premyelinating OLs and that the labeled cells instead appeared as myelinating OLs. We demonstrated from their RNA expression that YFP-labeled OLs were MFOLs, but not MOLs. Using the unique property of delayed YFP induction, we sought to determine whether MFOLs are constantly supplied from OPCs and differentiate into MOLs, or whether MFOLs pause their differentiation and sustain this stage in the adult brain. To achieve this objective, we irradiated adult LncOL1-YFP brains with X-rays to deplete dividing OPCs and their progeny. The irradiation extinguished YFP-labeled OLs, indicating that adult OPCs differentiated into MOLs during a single period. We established a new transgenic mouse line that genetically labels MFOLs, providing a reliable tool for investigating the dynamics of adult oligodendrogenesis.
少突胶质细胞（OL）从少突胶质前体细胞（OPC）分化而来，一般认为会产生两类细胞群体：pre - 髓鞘形成型和髓鞘形成型少突胶质细胞。最近的单细胞RNA测序数据将这些群体进一步细分为新形成的（NFOLs）、髓鞘形成的（MFOLs）和成熟的（MOLs）少突胶质细胞。然而，新提出的这些群体中，哪一个对应于pre - 髓鞘形成型或髓鞘形成型少突胶质细胞尚不清楚。我们聚焦于NFOL特异性的长链非编码少突胶质细胞1基因（LncOL1），并试图利用四环素可控基因诱导系统，在LncOL1启动子的控制下标记NFOLs。我们证明LncOL1由pre - 髓鞘形成型少突胶质细胞表达，而MFOL特异性基因Ctps则不表达，这表明NFOLs对应于pre - 髓鞘形成型少突胶质细胞，而MFOLs和MOLs对应于髓鞘形成型少突胶质细胞。然后，我们构建了一种LncOL1 - tTA小鼠，其中四环素反式激活因子（tTA）盒插入到LncOL1转录起始位点的下游。通过将LncOL1 - tTA小鼠与tetO报告基因小鼠杂交，我们获得了LncOL1 - tTA::tetO - 黄色荧光蛋白（YFP）双转基因（LncOL1 - YFP）小鼠。尽管LncOL1是非编码的，但在LncOL1 - YFP小鼠中检测到了YFP，这表明tTA成功翻译。出乎意料的是，我们发现LncOL1 - tTA驱动的YFP细胞的形态与LncOL1 pre - 髓鞘形成型少突胶质细胞不同，标记的细胞反而呈现为髓鞘形成型少突胶质细胞。我们从它们的RNA表达情况证明，YFP标记的少突胶质细胞是MFOLs，而不是MOLs。利用YFP诱导延迟这一独特特性，我们试图确定MFOLs是持续由OPCs供应并分化为MOLs，还是MFOLs会暂停分化并在成年大脑中维持这一阶段。为实现这一目标，我们用X射线照射成年LncOL1 - YFP小鼠的大脑，以耗尽正在分裂的OPCs及其子代。照射使YFP标记少突胶质细胞消失，这表明成年OPCs在单一时期内分化为MOLs。我们建立了一种新的转基因小鼠品系，可对MFOLs进行基因标记，为研究成年少突胶质细胞生成的动力学提供了可靠工具。
