Effect of depletion of the gne gene in Zebrafish

GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized. In these studies, we aimed to investigate gne functions using zebrafish genetic and transgenic models, and characterized them using macroscopic, microscopic, and molecular approaches. We first established transgenic zebrafish lineages expressing the human GNE cDNA carrying the M743T mutation, driven by the zebrafish gne promoter. These fish developed entirely normally. Then, we generated a gne knock-out (KO) fish using the CRISPR/Cas9 methodology. These fish died 8-10 days post-fertilization (dpf), but a phenotype appeared less than 24 hours before death and included progressive body axis curving, deflation of the swim bladder and decreasing movement and heart rate. However, muscle histology uncovered severe defects, already at 5 dpf, with compromised fiber organization. Sialic acid supplementation did not rescue the larvae from this phenotype nor prolonged their lifespan. To have deeper insights into the potential functions of gne in zebrafish, RNA sequencing was performed at 3 time points (3, 5, and 7 dpf). Genotype clustering was progressive, with only 5 genes differentially expressed in gne KO compared to gne WT siblings at 3 dpf. Enrichment analyses of the primary processes affected by the lack of gne also at 5 and 7 dpf point to the involvement of cell cycle and DNA damage/repair processes in the gne KO zebrafish. Thus, we have established a gne KO zebrafish lineage and obtained new insights into gne functions. This is the only model where GNE can be related to clear muscle defects, thus the only animal model relevant to GNE Myopathy to date. Further elucidation of gne precise mechanism-of-action in these processes could be relevant to GNE Myopathy and allow the identification of novel therapeutic targets. Overall design: Comparative gene expression profiling analysis of RNA-seq data for homozygote gne KO zebrafish, heterozygote gneKO zf and gne WT zebrafish.

GNE肌病（GNE Myopathy）是一种罕见的常染色体隐性遗传性、全球分布的神经肌肉疾病，由人类GNE基因（GNE gene）上的一系列双等位基因突变所致。GNE编码一种双功能酶，负责唾液酸（sialic acid）生物合成通路的限速步骤。然而，GNE突变引发肌肉病理改变的具体机制目前仍不明确。迄今为止已建立的GNE肌病细胞模型与小鼠模型均未提供有效研究线索。此外，学界推测GNE在肌肉组织中还存在其他未被阐明的功能。本研究旨在借助斑马鱼（zebrafish）遗传与转基因模型探究gne的功能，并通过宏观观测、显微分析与分子生物学手段对其进行表征。我们首先构建了以斑马鱼gne启动子驱动、表达携带M743T突变的人源GNE互补DNA（cDNA）的转基因斑马鱼品系，该类转基因鱼发育完全正常。随后，我们利用CRISPR/Cas9基因编辑技术构建了gne敲除（KO）斑马鱼。该类敲除鱼在受精后8~10天（dpf）死亡，且在死亡前24小时内出现特征性表型：躯体轴进行性弯曲、鱼鳔萎陷、运动能力与心率持续下降。肌肉组织学检测显示，早在受精后5天（dpf）即可观察到严重的肌纤维结构紊乱缺陷。外源性补充唾液酸既未能挽救该表型，也未能延长幼鱼的存活时长。为深入探究斑马鱼中gne的潜在功能，我们在3、5、7 dpf三个时间点开展了RNA测序（RNA-seq）。基因型聚类分析呈进行性变化：在3 dpf时，gne KO斑马鱼与野生型（WT）同胞对照相比，仅5个基因存在差异表达。对gne缺失后在5 dpf与7 dpf同样受影响的核心生物学过程进行富集分析，结果显示细胞周期与DNA损伤/修复通路参与了gne KO斑马鱼的病理过程。综上，我们成功构建了gne KO斑马鱼品系，并为gne的功能研究提供了新的见解。这是目前唯一可将GNE与明确肌肉缺陷相关联的动物模型，也是迄今唯一与GNE肌病相关的动物模型。进一步阐明gne在上述过程中的精准作用机制，或可为GNE肌病的研究提供理论参考，并助力新型治疗靶点的鉴定。实验设计概述：对纯合子gne KO斑马鱼、杂合子gne KO斑马鱼与野生型gne斑马鱼的RNA-seq数据开展比较基因表达谱分析。