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

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.