Ercanbrack Wesley S, Dungan Austin, Gaul Ella, Ramirez Mateo, J DelVecchio Alexander, Grass Calvin, Wingert Rebecca A
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, United States.
Front Cell Dev Biol. 2024 Dec 11;12:1496244. doi: 10.3389/fcell.2024.1496244. eCollection 2024.
Friedreich's Ataxia (FRDA) is a genetic disease that affects a variety of different tissues. The disease is caused by a mutation in the gene ( which is important for the synthesis of iron-sulfur clusters. The primary pathologies of FRDA are loss of motor control and cardiomyopathy. These occur due to the accumulation of reactive oxygen species (ROS) in the brain and the heart due to their high metabolic rates. Our research aims to understand how developmental processes and the kidney are impacted by a deficiency of .
We utilized an antisense oligomer, or morpholino, to knockdown the gene () in zebrafish embryos. Knockdown was confirmed via RT-PCR, gel electrophoresis, and Sanger sequencing. To investigate phenotypes, we utilized several staining techniques including whole mount hybridization, Alcian blue, and acridine orange, as well as dextran-FITC clearance assays.
deficient animals displayed otolith malformations, edema, and reduced survival. Alcian blue staining revealed craniofacial defects in deficient animals, and gene expression studies showed that the pronephros, or embryonic kidney, had several morphological defects. We investigated the function of the pronephros through clearance assays and found that the renal function is disrupted in deficient animals in addition to proximal tubule endocytosis. Utilizing acridine orange staining, we found that cell death is a partial contributor to these phenotypes.
This work provides new insights about how deficiency impacts development and kidney morphogenesis. Additionally, this work establishes an additional model system to study FRDA.
弗里德赖希共济失调（FRDA）是一种影响多种不同组织的遗传性疾病。该疾病由基因中的突变引起（该基因对铁硫簇的合成很重要）。FRDA的主要病理学特征是运动控制丧失和心肌病。这些是由于大脑和心脏因代谢率高而积累活性氧（ROS）所致。我们的研究旨在了解缺乏该基因如何影响发育过程和肾脏。
我们利用反义寡聚核苷酸或吗啉代寡核苷酸在斑马鱼胚胎中敲低该基因。通过逆转录聚合酶链反应（RT-PCR）、凝胶电泳和桑格测序确认敲低。为了研究表型，我们利用了几种染色技术，包括整体原位杂交、阿尔辛蓝和吖啶橙，以及葡聚糖异硫氰酸荧光素清除试验。
缺乏该基因的动物表现出耳石畸形、水肿和存活率降低。阿尔辛蓝染色显示缺乏该基因的动物存在颅面缺陷，基因表达研究表明前肾（即胚胎肾脏）有几种形态学缺陷。我们通过清除试验研究了前肾的功能，发现除近端小管内吞作用外，缺乏该基因的动物的肾功能也受到破坏。利用吖啶橙染色，我们发现细胞死亡是这些表型的部分原因。
这项工作为缺乏该基因如何影响发育和肾脏形态发生提供了新的见解。此外，这项工作建立了另一个研究FRDA的模型系统。
