Deletion Of Narfl Leads To Increased Oxidative Stress Mediated Abnormal Angiogenesis And Digestive Organ Defects In Zebrafish

Background Nuclear prelamin A recognition factor-like（NARFL）is a human protein that participates in cytosolic iron-sulfur（Fe–S）protein biogenesis and cellular defense against oxidative stress.In our previous work,NARFL was identified as a causative gene in a family with diffuse pulmonary arteriovenous malformations（PAVMs）,and we constructed a narfl knockout zebrafish model via the CRISPR/Cas9 gene editing technique.This study aims to explore the molecular mechanism of the phenotypes caused by narfl knockout.Methods The spatio-temporal expression pattern of narfl gene in wild zebrafish was detected by reverse transcription PCR（RT-PCR）and whole mount in situ hybridization（WISH）.Sanger sequencing,quantitative real time PCR（q PCR）and Western blot were performed to verify the narfl knockout.Subintestinal vessels were observed by alkaline phosphatase staining and confocal microscopic imaging of vascular transgenic zebrafish,and wild type narfl m RNA was injected into narfl^-/-fish for phenotypic rescue.Transcriptional sequencing and q PCR were performed to analyze the differentially expressed genes and pathways in narfl^+/+and narfl^-/-fish.The digestive organ development was detected by WISH.Biochemical analyses of reactive oxygen species（ROS）and redox substances were conducted to assess the oxidative stress status in narfl^+/+and narfl^-/-fish.The HIF-1αexpression was detected by WISH and Western blot,and 2-methoxyestradiol（2ME2）drug treatment was performed to analyze whether HIF-1αinhibition could rescue the abnormal angiogenesis in narfl^-/-fish.Results The results showed that zebrafish narfl gene was widely expressed in the early stages of embryonic development and adult tissues.Narfl deletion led to larvae lethality,subintestinal vessel malformation and digestive organ defects.Transcriptional sequencing and q PCR results demonstrated that narfl knockout resulted in enhanced oxidative stress and upregulated HIF-1αprotein level,as well as increased expression of genes involved in Bmp pathway and glycolysis signaling pathway.The 2ME2 partially inhibited subintestinal vessel sprouting and activated pathways induced by HIF-1αin mutants.Conclusions Zebrafish narfl knockout leads to increased oxidative stress mediated abnormal angiogenesis and digestive organ defects through a ROS-HIF-1α-Bmp pathway and may contribute to larvae lethality.