PUBLICATION

Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing

Authors
Khuansuwan, S., Gamse, J.T.
ID
ZDB-PUB-140901-3
Date
2014
Source
Developmental Biology   395(1): 144-53 (Journal)
Registered Authors
Gamse, Josh, Khuansuwan, Sataree
Keywords
Bioinformatics, Cell dissociation, Epithalamus, FACS, Parapineal, Pineal, Pineal complex, RNA-seq, Transcriptome analysis, Zebrafish, tbx2b
Datasets
GEO:GSE61202
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • Embryo, Nonmammalian/cytology
  • Embryo, Nonmammalian/embryology
  • Embryo, Nonmammalian/metabolism
  • Flow Cytometry
  • Gene Expression Regulation, Developmental*
  • Gene Knockdown Techniques
  • In Situ Hybridization
  • Pineal Gland/cytology
  • Pineal Gland/embryology
  • Pineal Gland/metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, RNA/methods*
  • T-Box Domain Proteins/genetics
  • Time Factors
  • Transcriptome/genetics*
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics
(all 20)
PubMed
25173875 Full text @ Dev. Biol.
Abstract
We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish.
Genes / Markers
Marker Marker Type Name
gngt1GENEguanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 1
gngt2aGENEguanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 2a
notoGENEnotochord homeobox
otompGENEotolith matrix protein
otx5GENEorthodenticle homolog 5
rdh5GENEretinol dehydrogenase 5 (11-cis/9-cis)
rgraGENEretinal G protein coupled receptor a
tbx2bGENET-box transcription factor 2b
tbxtaGENET-box transcription factor Ta
tdgf1GENEteratocarcinoma-derived growth factor 1
1 - 10 of 10
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Figures
No images available
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Expression
Phenotype
Fish Conditions Stage Phenotype Figure
AB + MO4-tbx2bstandard conditionsPrim-5
AB + MO4-tbx2bstandard conditionsPrim-5
AB + MO4-tbx2bstandard conditionsPrim-5
AB + MO4-tbx2bstandard conditionsPrim-5
AB + MO4-tbx2bstandard conditionsPrim-5
AB + MO4-tbx2bstandard conditionsPrim-5
tbx2bc144/c144standard conditionsPrim-5
tbx2bc144/c144standard conditionsPrim-5
tbx2bc144/c144standard conditionsPrim-5
tbx2bc144/c144standard conditionsPrim-5
1 - 10 of 12
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Mutations / Transgenics
Allele Construct Type Affected Genomic Region
c144
    		Point Mutation
    vu376TgTransgenic Insertion
      1 - 2 of 2
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      Human Disease / Model
      No data available
      Sequence Targeting Reagents
      Target Reagent Reagent Type
      tbx2bMO4-tbx2bMRPHLNO
      1 - 1 of 1
      Show
      Fish
      Fish
      AB
      AB + MO4-tbx2b
      tbx2bc144/c144
      vu376Tg
      1 - 4 of 4
      Show
      Antibodies
      No data available
      Orthology
      No data available
      Engineered Foreign Genes
      Marker Marker Type Name
      KaedeEFGKaede
      1 - 1 of 1
      Show
      Mapping
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      Citation
      Khuansuwan, S., Gamse, J.T. (2014) Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing. Developmental Biology. 395(1):144-53.