Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos.

L Carine Stapel,Dagmar Kainmueller,Nadine L Vastenhouw,Benoit Lombardot,Florian Jug,Coleman Broaddus,Eugene W Myers

doi:10.1242/dev.128918

L Carine Stapel, Dagmar Kainmueller + Show 5 more

Open Access

https://doi.org/10.1242/dev.128918

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Abstract

Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.

Highlights

Analysis of gene expression patterns is an essential tool in many areas of biological research
Sensitive and specific detection and quantification of transcripts To detect mRNA at single-molecule resolution, we developed a protocol for single-molecule fluorescence in situ hybridization (smFISH) on 8 μm cryosections of zebrafish embryos
We performed smFISH for ntla and eif4g2a on sections of zebrafish embryos at 50% epiboly [5.3 hours post fertilization] (Fig. 1B-E, Fig. S1). ntla is involved in mesoderm specification and has been shown to be expressed in the presumptive mesoderm at the margin of the embryo (Harvey et al, 2010; Schier and Talbot, 2005) (Fig. S2A,B)

Summary

Introduction

Analysis of gene expression patterns is an essential tool in many areas of biological research. Broad application of smFISH in complex samples has been hampered by the limited availability of protocols for embryos and by the lack of an automated image analysis pipeline that combines transcript detection with cell segmentation (Bahar Halpern et al, 2015; Itzkovitz et al, 2011; Lyubimova et al, 2013; Oka and Sato, 2015). We present a protocol for smFISH on embryo sections in combination with an analysis pipeline for automated transcript detection and cell segmentation.

Results

Conclusion