Watanabe Hiina, Fukuda Ayaka, Ikeda Natsumi, Sato Maoko, Hashimoto Kei, Miyamoto Yasunori
Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan; Institute for Human Life Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan.
Institute for Human Life Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan; Department of Biology, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan.
Brain Res. 2023 May 15;1807:148317. doi: 10.1016/j.brainres.2023.148317. Epub 2023 Mar 8.
To analyze the role of syndecan-3 (SDC3), a heparan sulfate proteoglycan, in cerebellum development, we examined the effect of SDC3 on the transition from cell cycle exit to the initial differentiation stage of cerebellar granule cell precursors (CGCPs). First, we examined SDC3 localization in the developing cerebellum. SDC3 was mainly localized to the inner external granule layer where the transition from the cell cycle exit to the initial differentiation of CGCPs occurs. To examine how SDC3 regulates the cell cycle exit of CGCPs, we performed SDC3-knockdown (SDC3-KD) and -overexpression (Myc-SDC3) assays using primary CGCPs. SDC3-KD significantly increased the ratio of p27 cells to total cells at day 3 in vitro (DIV3) and 4, but Myc-SDC3 reduced that at DIV3. Regarding the cell cycle exit efficiency using 24 h-labelled bromodeoxyuridine (BrdU) and a marker of cell cycling, Ki67, SDC3-KD significantly increased cell cycle exit efficiency (Ki67; BrdU cells/BrdU cells) in primary CGCP at DIV4 and 5, but Myc-SDC3 reduced that at DIV4 and 5. However, SDC3-KD and Myc-SDC3 did not affect the efficiency of the final differentiation from CGCPs to granule cells at DIV3-5. Furthermore, the ratio of CGCPs in the cell cycle exiting stage to total cells, identified by initial differentiation markers TAG1 and Ki67 (TAG1; Ki67 cells), was considerably decreased by SDC3-KD at DIV4, but increased by Myc-SDC3 at DIV4 and 5. Altogether, these results indicate that SDC3 regulates the timing of the transition from the cell cycle exit stage to the initial differentiation stage of CGCP.
为了分析硫酸乙酰肝素蛋白聚糖Syndecan-3（SDC3）在小脑发育中的作用，我们研究了SDC3对小脑颗粒细胞前体（CGCPs）从细胞周期退出到初始分化阶段转变的影响。首先，我们检测了SDC3在发育中小脑的定位。SDC3主要定位于内外部颗粒层，这里发生着CGCPs从细胞周期退出到初始分化的转变。为了研究SDC3如何调节CGCPs的细胞周期退出，我们使用原代CGCPs进行了SDC3敲低（SDC3-KD）和过表达（Myc-SDC3）实验。SDC3-KD显著增加了体外培养第3天（DIV3）和第4天p27阳性细胞与总细胞的比例，但Myc-SDC3在DIV3时降低了该比例。关于使用24小时标记的溴脱氧尿苷（BrdU）和细胞周期标记物Ki67检测细胞周期退出效率，SDC3-KD显著提高了原代CGCP在DIV4和5时的细胞周期退出效率（Ki67；BrdU阳性细胞/BrdU阳性细胞），但Myc-SDC3在DIV4和5时降低了该效率。然而，SDC3-KD和Myc-SDC3在DIV3至5时并不影响CGCPs向颗粒细胞最终分化的效率。此外，在DIV4时，通过初始分化标记物TAG1和Ki67（TAG1；Ki67阳性细胞）鉴定的处于细胞周期退出阶段的CGCPs与总细胞的比例，因SDC3-KD而显著降低，但在DIV4和5时因Myc-SDC3而增加。总之，这些结果表明SDC3调节CGCPs从细胞周期退出阶段到初始分化阶段转变的时间。
