Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney

PLoS One. 2013 Dec 4;8(12):e82137. doi: 10.1371/journal.pone.0082137. eCollection 2013.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Automation
  • Disease Models, Animal
  • Embryo, Nonmammalian / anatomy & histology
  • Embryo, Nonmammalian / drug effects
  • Gene Knockdown Techniques
  • Humans
  • Imaging, Three-Dimensional*
  • Indomethacin / pharmacology
  • Kidney / anatomy & histology*
  • Kidney / drug effects
  • Kidney / embryology
  • Kidney / growth & development*
  • Kidney Diseases / pathology
  • Larva / anatomy & histology
  • Phenotype
  • Pilot Projects
  • Pronephros / anatomy & histology
  • Pronephros / drug effects
  • Pronephros / embryology
  • Zebrafish / embryology
  • Zebrafish / genetics
  • Zebrafish / growth & development*

Substances

  • Indomethacin

Grant support

This project was supported by the European Commission Seventh Framework Programme funded projects DOPAMINET (http://www.dopaminet.eu/; grant no. 223744) and Eurenomics (http://www.eurenomics.eu/; grant no. 305608) to UL, and SYSCILIA (http://www.syscilia.org/; grant no. 241955) to PLB, the Stiftung Landesbank Baden-Württemberg to JHW, the Dutch Kidney foundation (CP11.18) to PLB and MS and institutional funding from Innovation Department, Karlsruhe Institute of Technology (KIT), Germany. MS was funded by an Action Medical Research UK clinical training fellowship (RTF-1411) and PLB is a Wellcome Trust Senior Research Fellow. We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Karlsruhe Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.