Development of an Automated Imaging Pipeline for the Analysis of the Zebrafish Larval Kidney

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.

针对环境影响下肾发生过程所致肾脏畸形，或遗传性肾病的相关研究，亟需能够实现发育过程活体观测的动物模型。斑马鱼现已成为分析脊椎动物器官发育与功能的优质模型系统，且适用于大规模筛选器官毒性化合物或疾病调节因子。然而，若要充分发挥其在高内涵筛选（high content screening）应用中的潜力，亟需建立专用实验方案，以实现对胚胎肾脏等内脏器官的稳定可视化成像。为此，我们开发了一套适配高内涵筛选的实验流程，用于自动化成像斑马鱼幼虫发育阶段的前肾（pronephros）标准化视图。通过定制化工具，在琼脂糖（agarose）包被的微孔板（microtiter plates）中制备定位凹槽，可实现斑马鱼幼虫的精准定位与定向摆放，进而能够自动获取稳定一致的前肾背侧视图。我们将所建立的流程应用于一项先导筛选，以探究潜在肾毒性药物对Tg(wt1b:EGFP)转基因斑马鱼品系前肾发育的影响——该品系的发育前肾可通过GFP荧光标记得以显现。所获取的一致性图像数据可用于量化大体形态学前肾表型，揭示了多种化合物对肾发生的浓度依赖性效应。此外，我们还在基于吗啉寡核苷酸（morpholino）的、与不同鞭毛内运输（intraflagellar transport）基因相关的人类纤毛遗传性疾病模型中，进一步验证了该成像流程的适用性。本研究开发的工具与实验流程可用于斑马鱼肾脏研究的诸多方向，且可轻松适配其他器官系统的相关分析。