Abstract
mRNA synthesis is one of the earliest readouts of the activity of a transcribed gene, which is of particular interest in the context of metazoan cell fate specification. These processes are intrinsically dynamic and stochastic, which makes in vivo single-cell measurements inevitable. Here, we present the application of a technology that has been widely used in single celled organisms to measure transcriptional activity in developing embryos of the fruit fly Drosophila melanogaster. The method allows for quantification of instantaneous polymerase occupancy of active gene loci and thereby enables the development and testing of models of gene regulation in development.
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References
Fusco D, Accornero N, Lavoie B, Shenoy SM, Blanchard JM, Singer RH, Bertrand E (2003) Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr Biol 13(2):161–167
Golding I, Paulsson J, Zawilski SM, Cox EC (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123(6):1025–1036
Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332(6028):475–478. doi:10.1126/science.1202142. 332/6028/475 [pii]
Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445. S1097-2765(00)80143-4 [pii]
Yunger S, Rosenfeld L, Garini Y, Shav-Tal Y (2010) Single-allele analysis of transcription kinetics in living mammalian cells. Nat Methods 7(8):631–633. doi:10.1038/nmeth.1482. nmeth.1482 [pii]
Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods. doi:10.1038/nmeth.1551. nmeth.1551 [pii]
Forrest KM, Gavis ER (2003) Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr Biol 13(14):1159–1168
Garcia HG, Tikhonov M, Lin A, Gregor T (2013) Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Curr Biol 23(21):2140–2145. doi:10.1016/j.cub.2013.08.054
Lucas T, Ferraro T, Roelens B, De Las Heras Chanes J, Walczak AM, Coppey M, Dostatni N (2013) Live imaging of bicoid-dependent transcription in Drosophila embryos. Curr Biol 23(21):2135–2139. doi:10.1016/j.cub.2013.08.053
Bothma JP, Garcia HG, Esposito E, Schlissel G, Gregor T, Levine M (2014) Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos. Proc Natl Acad Sci U S A 111(29):10598–10603. doi:10.1073/pnas.1410022111
Bothma JP, Garcia HG, Ng S, Perry MW, Gregor T, Levine M (2015) Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. Elife 4. doi:10.7554/eLife.07956
Ferraro T, Esposito E, Mancini L, Ng S, Lucas T, Coppey M, Dostatni N, Walczak AM, Levine M, Lagha M (2016) Transcriptional memory in the Drosophila embryo. Curr Biol 26(2):212–218. doi:10.1016/j.cub.2015.11.058
Fukaya T, Lim B, Levine M (2016) Enhancer control of transcriptional bursting. Cell. doi:10.1016/j.cell.2016.05.025
Chen H, Xu Z, Mei C, Yu D, Small S (2012) A system of repressor gradients spatially organizes the boundaries of bicoid-dependent target genes. Cell 149(3):618–629. doi:10.1016/j.cell.2012.03.018
Bateman JR, Lee AM, Wu CT (2006) Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. Genetics 173(2):769–777. doi:10.1534/genetics.106.056945. genetics.106.056945 [pii]
Venken KJ, He Y, Hoskins RA, Bellen HJ (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314(5806):1747–1751. doi:10.1126/science.1134426
Liu F, Morrison AH, Gregor T (2013) Dynamic interpretation of maternal inputs by the Drosophila segmentation gene network. Proc Natl Acad Sci U S A 110(17):6724–6729. doi:10.1073/pnas.1220912110
Figard L, Sokac AM (2011) Imaging cell shape change in living Drosophila embryos. J Vis Exp (49). doi:10.3791/2503
Little SC, Tikhonov M, Gregor T (2013) Precise developmental gene expression arises from globally stochastic transcriptional activity. Cell 154(4):789–800. doi:10.1016/j.cell.2013.07.025
Acknowledgments
The authors are grateful through support provided by the Princeton Dickie Fellowship (H.G.), the Burroughs Wellcome Fund Career Award at the Scientific Interface (H.G.), the Sloan Research Foundation (H.G.), the Searle Scholars Program (T.G. and H.G.), the Shurl & Kay Curci Foundation (H.G.), the Human Frontier Science Program (H.G.), and the National Institutes of Health Grants P50 GM071508 and R01 GM097275 (T.G.).
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Garcia, H.G., Gregor, T. (2018). Live Imaging of mRNA Synthesis in Drosophila . In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_23
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DOI: https://doi.org/10.1007/978-1-4939-7213-5_23
Publisher Name: Humana Press, New York, NY
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