title: Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos.
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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany.
authors.forename: L Carine
authors.initials: LC
authors.lastname: Stapel
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany.
authors.forename: Benoit
authors.initials: B
authors.lastname: Lombardot
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany.
authors.forename: Coleman
authors.initials: C
authors.lastname: Broaddus
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Kainmueller
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Jug
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany.
authors.forename: Eugene W
authors.initials: EW
authors.lastname: Myers
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden 01307, Germany vastenhouw@mpi-cbg.de.
authors.forename: Nadine L
authors.initials: NL
authors.lastname: Vastenhouw
mesh_terms: D000818:Animals; D001331:Automation; D002462:Cell Membrane / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D012313:RNA
keywords: Cell segmentation; Gene expression; Image analysis pipeline; Zebrafish; smFISH