Laboratory of Translational Medicine, Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, 100020, China.
Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100730, China.
Mol Neurobiol. 2024 Sep;61(9):6189-6199. doi: 10.1007/s12035-024-03946-7. Epub 2024 Jan 29.
Primary cilia are crucial for neurogenesis, and cilium-related genes are involved in the closure of neural tubes. Inositol polyphosphate-5-phosphatase (Inpp5e) was enriched in primary cilia and closely related to the occurrence of neural tube defects (NTDs). However, the role of Inpp5e in the development of NTDs is not well-known. To investigate whether Inpp5e gene is associated with the neural tube closure, we established a mouse model of NTDs by 5-fluorouracil (5-FU) exposure at gestational day 7.5 (GD7.5). The Inpp5e knockdown (Inpp5e) mouse embryonic stem cells (mESCs) were produced by CRISPR/Cas9 system. The expressions of Inpp5e and other cilium-related genes including intraflagellar transport 80 (Ift80), McKusick-Kaufman syndrome (Mkks), and Kirsten rat sarcoma viral oncogene homolog (Kras) were determined, utilizing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blot, PCR array, and immunofluorescence staining. The result showed that the incidence of NTDs was 37.10% (23 NTDs/62 total embryos) and significantly higher than that in the control group (P < 0.001). The neuroepithelial cells of neural tubes were obviously disarranged in NTD embryos. The mRNA and protein levels of Inpp5e, Ift80, Mkks, and Kras were significantly decreased in NTD embryonic brain tissues, compared to the control (P < 0.05). Knockdown of the Inpp5e (Inpp5e) reduced the expressions of Ift80, Mkks, and Kras in mESCs. Furthermore, the levels of α-tubulin were significantly reduced in NTD embryonic neural tissue and Inpp5e mESCs. These results suggested that maternal 5-FU exposure inhibited the expression of Inpp5e, which resulted in the downregulation of cilium-related genes (Ift80, Mkks, and Kras), leading to the impairment of primary cilium development, and ultimately disrupted the neural tube closure.
初级纤毛对神经发生至关重要，纤毛相关基因参与神经管的闭合。肌醇多磷酸-5-磷酸酶（Inpp5e）在初级纤毛中富集，与神经管缺陷（NTDs）的发生密切相关。然而，Inpp5e 基因在 NTDs 发育中的作用尚不清楚。为了研究 Inpp5e 基因是否与神经管闭合有关，我们通过在妊娠第 7.5 天（GD7.5）用 5-氟尿嘧啶（5-FU）暴露建立了 NTD 小鼠模型。通过 CRISPR/Cas9 系统产生了 Inpp5e 基因敲低（Inpp5e）的小鼠胚胎干细胞（mESCs）。利用定量实时逆转录聚合酶链反应（qRT-PCR）、western blot、PCR 阵列和免疫荧光染色，测定了 Inpp5e 及其他纤毛相关基因（包括鞭毛内运输 80（Ift80）、Mkks 和 Kirsten 大鼠肉瘤病毒癌基因同源物（Kras））的表达。结果显示，NTD 的发生率为 37.10%（23 个 NTD/62 个总胚胎），明显高于对照组（P<0.001）。NTD 胚胎神经管的神经上皮细胞明显排列紊乱。与对照组相比，NTD 胚胎脑组织中 Inpp5e、Ift80、Mkks 和 Kras 的 mRNA 和蛋白水平均显著降低（P<0.05）。Inpp5e 基因敲低（Inpp5e）降低了 mESCs 中 Ift80、Mkks 和 Kras 的表达。此外，NTD 胚胎神经组织和 Inpp5e mESCs 中α-微管蛋白的水平也明显降低。这些结果表明，母体 5-FU 暴露抑制了 Inpp5e 的表达，导致纤毛相关基因（Ift80、Mkks 和 Kras）下调，初级纤毛发育受损，最终破坏神经管闭合。
