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Inpp5e Regulated the Cilium-Related Genes Contributing to the Neural Tube Defects Under 5-Fluorouracil Exposure

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Abstract

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.

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Data Availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

Thanks to Dr. Jing Pan for her constructive suggestions during the process.

Funding

This work was supported by the Natural Science Foundation of Beijing Municipal (7222016), Research Foundation of the Capital Institute of Pediatrics (CXYJ-2021-03, JCYJ-2023-05) and National Natural Science Foundation of China (U23A20420).

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  1. Xiuwei Wang and Jialu Yu have contributed equally to this work and share the first authorship.

Authors and Affiliations

  1. Laboratory of Translational Medicine, Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, 100020, China

    Xiuwei Wang, Jialu Yu, Shen Li, Aiyun Yang, Zhiqiang Zhu, Zhen Guan & Jianhua Wang

  2. Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100730, China

    Jialu Yu & Jianhua Wang

  3. Department of Pediatrics, Beijing Chaoyang Hospital of Capital Medical University, Beijing, 100020, China

    Huixuan Yue

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Contributions

JW, ZG, XW and JY contributed to the study conception and design. XW, JY, and HY performed the experiment and drafted the article. SL, AY, ZZ, and ZG analyzed the data. XW and JW made contribution to the funding acquisition. All authors approved the final manuscript.

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Correspondence to Zhen Guan or Jianhua Wang.

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All animal experiments were reviewed and approved by the Ethics Committee of the Capital Institute Pediatrics (DWLL2021013). All procedures are strictly in compliance with the relevant regulations of the Chinese Council on Animal Care.

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Wang, X., Yu, J., Yue, H. et al. Inpp5e Regulated the Cilium-Related Genes Contributing to the Neural Tube Defects Under 5-Fluorouracil Exposure. Mol Neurobiol (2024). https://doi.org/10.1007/s12035-024-03946-7

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