PUBLICATION

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

Authors
Stapel, L.C., Lombardot, B., Broaddus, C., Kainmueller, D., Jug, F., Myers, E.W., Vastenhouw, N.L.
ID
ZDB-PUB-151225-6
Date
2016
Source
Development (Cambridge, England)   143(3): 540-6 (Journal)
Registered Authors
Stapel, Carine, Vastenhouw, Nadine
Keywords
Cell segmentation, Gene expression, Image analysis pipeline, smFISH, Zebrafish
MeSH Terms
  • Transcription, Genetic
  • Zebrafish/embryology*
  • Zebrafish/genetics*
  • Embryo, Nonmammalian/metabolism*
  • RNA, Messenger/genetics
  • RNA, Messenger/metabolism
  • In Situ Hybridization, Fluorescence/methods
  • Cell Membrane/metabolism
  • RNA/genetics
  • RNA/metabolism*
  • Animals
  • Automation
  • Subcellular Fractions/metabolism
  • Gene Expression Regulation, Developmental
(all 14)
PubMed
26700682 Full text @ Development
Abstract
Analysis of differential gene expression is critical 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 like 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.
Genes / Markers
Marker Marker Type Name
akap12bGENEA kinase (PRKA) anchor protein 12b
eif4g2aGENEeukaryotic translation initiation factor 4, gamma 2a
mex3bGENEmex-3 RNA binding family member B
slc7a8aGENEsolute carrier family 7 member 8a
sox19aGENESRY-box transcription factor 19a
tbx16GENET-box transcription factor 16
tbxtaGENET-box transcription factor Ta
zic2bGENEzic family member 2 (odd-paired homolog, Drosophila) b
1 - 8 of 8
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Figures
Figure Gallery (10 images)
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Expression
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay Figure
akap12bAB/TLstandard conditionsSphereISH
akap12bAB/TLstandard conditionsSphereISH
akap12bAB/TLstandard conditionsDomeISH
eif4g2aAB/TLstandard conditionsDomeISH
eif4g2aAB/TLstandard conditionsDomeISH
eif4g2aAB/TLstandard conditionsSphereISH
eif4g2aAB/TLstandard conditionsSphereISH
eif4g2aAB/TLstandard conditions50%-epibolyISH
mex3bAB/TLstandard conditionsDomeISH
mex3bAB/TLstandard conditionsDomeISH
1 - 10 of 18
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Phenotype
No data available
Mutations / Transgenics
No data available
Human Disease / Model
No data available
Sequence Targeting Reagents
No data available
Fish
Fish
AB/TL
1 - 1 of 1
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Antibodies
No data available
Orthology
No data available
Engineered Foreign Genes
No data available
Mapping
No data available
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Citation
Stapel, L.C., Lombardot, B., Broaddus, C., Kainmueller, D., Jug, F., Myers, E.W., Vastenhouw, N.L. (2016) Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos. Development (Cambridge, England). 143(3):540-6.