Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos

Area of Science:

• Developmental Biology
• Molecular Biology
• Genetics

Background:

• Single-molecule fluorescence in situ hybridization (smFISH) visualizes individual mRNA molecules.
• Subcellular transcript quantification requires integration with cell segmentation.
• Existing smFISH protocols are not optimized for zebrafish, limiting its use in this model organism.

Purpose of the Study:

• To establish a robust smFISH protocol for zebrafish cryosections.
• To develop an automated image analysis pipeline for transcript detection and subcellular localization.
• To enable quantitative analysis of gene expression in zebrafish at single-cell resolution.

Main Methods:

• Optimized smFISH protocol for high-quality zebrafish embryo cryosections.
• Development of a user-friendly, automated software pipeline for cell and nuclear segmentation.
• Implementation of transcript detection algorithms for subcellular localization.

Main Results:

• Successfully adapted smFISH for zebrafish cryosections, yielding high-quality data.
• The automated pipeline accurately segments cells and detects single mRNA molecules.
• Quantitative transcript levels can be determined within specific cellular compartments.

Conclusions:

• This study presents a comprehensive smFISH protocol and analysis pipeline for zebrafish.
• The developed tools facilitate subcellular transcriptomic analysis in a key vertebrate model.
• The freely available software can be applied to other multicellular organisms.