Hox Genes pp 183-195 | Cite as

Tissue Specific RNA Isolation in Drosophila Embryos: A Strategy to Analyze Context Dependent Transcriptome Landscapes Using FACS

  • Arnaud Defaye
  • Laurent PerrinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1196)


The Hox family of transcription factors defines cell identity along the A/P axis of animal body plan by modulating expression of distinct sets of target genes in a tissue specific manner. Identifying such tissue specific target genes is indispensable if one wants to understand how Hox proteins mediate their context dependent function. Genome wide analysis of transcriptional activity in different tissues and contexts regarding Hox genes activity could help in reaching this goal. Such experiments rely on the possibility to selectively purify the cells of interest from developing embryos and to perform a transcriptomic analysis on such purified cell populations. By combining expression of a fluorescent protein and fluorescent activating cell sorting (FACS) technique, it is possible to obtain highly purified specific cell populations. In this chapter we describe the experimental procedure we have established in Drosophila—starting from a genetically marked small cell population (cardiomyocytes, 104 cells)—to dissociate the embryos in order to turn it into a suspension of individual cells, sort cells according to the expression of the introduced genetic marker and purify the total RNA content of the sorted cells. This can be used to analyze the transcriptome landscape of rare cell populations in wild type and mutant contexts. This technique has shown to be useful in the case of cardiac cells but is virtually applicable to any cell type and mutant backgrounds, provided that specific genetic markers are available.

Key words

Cell dissociation of Drosophila embryo Fluorescence activated cell sorting RNA extraction Transcriptomics Cell purification Tissue specific Context-dependent Hox activity 



This work was supported by ANR, partner of the ERASysBio + initiative supported under the EU ERA-NET Plus scheme in FP7.


  1. 1.
    Lohmann I, McGinnis W (2002) Hox genes: it’s all a matter of context. Curr Biol 12:R514–R516PubMedCrossRefGoogle Scholar
  2. 2.
    Ponzielli R, Astier M, Chartier A et al (2002) Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling. Development 129:4509–4521PubMedGoogle Scholar
  3. 3.
    Lo PCH, Skeath JB, Gajewski K et al (2002) Homeotic genes autonomously specify the anteroposterior subdivision of the Drosophila dorsal vessel into aorta and heart. Dev Biol 251:307–319. doi: 10.1006/dbio.2002.0839 PubMedCrossRefGoogle Scholar
  4. 4.
    Lovato TL, Nguyen TP, Molina MR, Cripps RM (2002) The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel. Development 129:5019–5027PubMedGoogle Scholar
  5. 5.
    Perrin L, Monier B, Ponzielli R et al (2004) Drosophila cardiac tube organogenesis requires multiple phases of Hox activity. Dev Biol 272:419–431. doi: 10.1016/j.ydbio.2004.04.036 PubMedCrossRefGoogle Scholar
  6. 6.
    Monier B, Astier M, Sémériva M, Perrin L (2005) Steroid-dependent modification of Hox function drives myocyte reprogramming in the Drosophila heart. Development 132:5283–5293. doi: 10.1242/dev.02091 PubMedCrossRefGoogle Scholar
  7. 7.
    Hueber SD, Bezdan D, Henz SR et al (2007) Comparative analysis of Hox downstream genes in Drosophila. Development 134:381–392. doi: 10.1242/dev.02746 PubMedCrossRefGoogle Scholar
  8. 8.
    Pavlopoulos A, Akam M (2011) Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis. Proc Natl Acad Sci U S A 108:2855–2860. doi: 10.1073/pnas.1015077108 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Thomas A, Lee P-J, Dalton JE et al (2012) A versatile method for cell-specific profiling of translated mRNAs in Drosophila. PLoS One 7:e40276. doi: 10.1371/journal.pone.0040276 PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Miller MR, Robinson KJ, Cleary MD, Doe CQ (2009) TU-tagging: cell type-specific RNA isolation from intact complex tissues. Nat Methods 6:439–441. doi: 10.1038/nmeth.1329 PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Steiner FA, Talbert PB, Kasinathan S et al (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res 22:766–777. doi: 10.1101/gr.131748.111 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Henry GL, Davis FP, Picard S, Eddy SR (2012) Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res 40:9691–9704. doi: 10.1093/nar/gks671 PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Bryantsev AL, Cripps RM (2012) Purification of cardiac cells from Drosophila embryos. Methods 56:44–49. doi: 10.1016/j.ymeth.2011.11.004 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Salmand P-A, Iché-Torres M, Perrin L (2011) Tissue-specific cell sorting from Drosophila embryos: application to gene expression analysis. Fly (Austin) 5:261–265. doi: 10.4161/fly.5.3.16509 CrossRefGoogle Scholar
  15. 15.
    Abreu-Blanco MT, Verboon JM, Parkhurst SM (2011) Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling. J Cell Biol 193:455–464. doi: 10.1083/jcb.201011018 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Technologies Avancées pour le Génome et la Clinique (TAGC), UMR 1090 INSERM, CNRS-FranceAix Marseille UniversitéMarseille cedex 09France

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