Inpp5e Regulated the Cilium-Related Genes Contributing to the Neural Tube Defects Under 5-Fluorouracil Exposure
Xiuwei Wang1, Jialu Yu1,2, Huixuan Yue3
1Laboratory of Translational Medicine, Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, 100020, China.
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.
Insights
Maternal 5-fluorouracil exposure disrupts neural tube closure by inhibiting Inositol polyphosphate-5-phosphatase (Inpp5e) expression. This leads to reduced cilium-related gene expression and impaired primary cilia development, causing neural tube defects (NTDs).
Area of Science:
- Developmental biology
- Genetics
Background:
- Primary cilia are essential for neurogenesis and neural tube closure.
- Inositol polyphosphate-5-phosphatase (Inpp5e) is linked to neural tube defects (NTDs) but its specific role is unclear.
Purpose of the Study:
- To investigate the association between Inpp5e and neural tube closure.
- To determine the impact of Inpp5e on cilium-related gene expression and primary cilia development.
Main Methods:
- Established a mouse model of NTDs using 5-fluorouracil (5-FU) exposure.
- Generated Inpp5e knockdown mouse embryonic stem cells (mESCs) via CRISPR/Cas9.
- Quantified gene and protein expression of Inpp5e and cilium-related genes (Ift80, Mkks, Kras) using qRT-PCR, western blot, and immunofluorescence.
Main Results:
- 5-FU exposure significantly increased NTD incidence (37.10%) with neuroepithelial cell disarray.
- Inpp5e, Ift80, Mkks, and Kras mRNA and protein levels were significantly decreased in NTD embryos.
- Inpp5e knockdown in mESCs reduced Ift80, Mkks, and Kras expression and α-tubulin levels.
Conclusions:
- Maternal 5-FU exposure inhibits Inpp5e expression, downregulating cilium-related genes.
- This disruption impairs primary cilia development, leading to neural tube closure defects.
- Inpp5e is crucial for maintaining neural tube integrity and primary cilia function.
