Quantitative Imaging of Transcription in Living Drosophila Embryos Links Polymerase Activity to Patterning

Spatiotemporal patterns of gene expression are fundamental to every developmental program. The resulting macroscopic domains have been mainly characterized by their levels of gene products. However, the establishment of such patterns results from differences in the dynamics of microscopic events in individual cells such as transcription. It is unclear how these microscopic decisions lead to macroscopic patterns, as measurements in fixed tissue cannot access the underlying transcriptional dynamics. In vivo transcriptional dynamics have long been approached in single-celled organisms, but never in a multicellular developmental context. Here, we directly address how boundaries of gene expression emerge in the Drosophila embryo by measuring the absolute number of actively transcribing polymerases in real time in individual nuclei. Specifically, we show that the formation of a boundary cannot be quantitatively explained by the rate of mRNA production in each cell, but instead requires amplification of the dynamic range of the expression boundary. This amplification is accomplished by nuclei randomly adopting active or inactive states of transcription, leading to a collective effect where the fraction of active nuclei is modulated in space. Thus, developmental patterns are not just the consequence of reproducible transcriptional dynamics in individual nuclei, but are the result of averaging expression over space and time.

[1]  C. Fradin,et al.  The time to measure positional information: maternal Hunchback is required for the synchrony of the Bicoid transcriptional response at the onset of zygotic transcription , 2010, Development.

[2]  John Reinitz,et al.  A database for management of gene expression data in situ , 2004, Bioinform..

[3]  Steven Henikoff,et al.  Modulation of a Transcription Factor Counteracts Heterochromatic Gene Silencing in Drosophila , 2001, Cell.

[4]  P. O’Farrell,et al.  Developmental Control of Late Replication and S Phase Length , 2010, Current Biology.

[5]  Timon Idema.  A new way of tracking motion, shape, and divisions , 2013, European Biophysics Journal.

[6]  Michael Levine,et al.  Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo , 2011, Proceedings of the National Academy of Sciences.

[7]  Ilan Davis,et al.  Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei , 1999, Current Biology.

[8]  Julien O. Dubuis,et al.  Accurate measurements of dynamics and reproducibility in small genetic networks , 2013, Molecular systems biology.

[9]  J. Lis,et al.  RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene , 1991, Molecular and cellular biology.

[10]  P. Swain,et al.  Gene Regulation at the Single-Cell Level , 2005, Science.

[11]  Jun Ma,et al.  Uncovering a Dynamic Feature of the Transcriptional Regulatory Network for Anterior-Posterior Patterning in the Drosophila Embryo , 2013, PloS one.

[12]  F. Cross,et al.  Multiple sequence-specific factors generate the nucleosome-depleted region on CLN2 promoter. , 2011, Molecular cell.

[13]  J. Posakony,et al.  Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element. , 1995, Development.

[14]  Wolfgang Huber,et al.  Genome-wide analysis of mRNA decay patterns during early Drosophila development , 2010, Genome Biology.

[15]  Eric F. Wieschaus,et al.  The Formation of the Bicoid Morphogen Gradient Requires Protein Movement from Anteriorly Localized mRNA , 2011, PLoS biology.

[16]  S. Small,et al.  A System of Repressor Gradients Spatially Organizes the Boundaries of Bicoid-Dependent Target Genes , 2012, Cell.

[17]  Yoav Freund,et al.  Visualization of Individual Scr mRNAs during Drosophila Embryogenesis Yields Evidence for Transcriptional Bursting , 2009, Current Biology.

[18]  Julian Lewis,et al.  From Signals to Patterns: Space, Time, and Mathematics in Developmental Biology , 2008, Science.

[19]  W. Bialek,et al.  Probing the Limits to Positional Information , 2007, Cell.

[20]  Thomas Gregor,et al.  Dynamic interpretation of maternal inputs by the Drosophila segmentation gene network , 2013, Proceedings of the National Academy of Sciences.

[21]  P. O’Farrell,et al.  Progression of the cell cycle through mitosis leads to abortion of nascent transcripts , 1991, Cell.

[22]  E. R. Gavis,et al.  Live Imaging of Endogenous RNA Reveals a Diffusion and Entrapment Mechanism for nanos mRNA Localization in Drosophila , 2003, Current Biology.

[23]  M. Ardehali,et al.  Tracking rates of transcription and splicing in vivo , 2009, Nature Structural &Molecular Biology.

[24]  L. Mirny,et al.  Nucleosome-mediated cooperativity between transcription factors , 2009, Proceedings of the National Academy of Sciences.

[25]  Eric Mjolsness,et al.  Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy , 2011, Nature Protocols.

[26]  A. Lander,et al.  How Cells Know Where They Are , 2013, Science.

[27]  R. Singer,et al.  Localization of ASH1 mRNA particles in living yeast. , 1998, Molecular cell.

[28]  David H. Sharp,et al.  Dynamic control of positional information in the early Drosophila embryo , 2004, Nature.

[29]  Hans J. Tanke,et al.  In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains , 2009, PloS one.

[30]  Terence Hwa,et al.  Combinatorial transcriptional control of the lactose operon of Escherichia coli , 2007, Proceedings of the National Academy of Sciences.

[31]  E. Davidson.  The Regulatory Genome: Gene Regulatory Networks In Development And Evolution , 2006 .

[32]  Wael Tadros,et al.  The maternal-to-zygotic transition: a play in two acts , 2009, Development.

[33]  Danyang Yu,et al.  Precise Registration of Gene Expression Boundaries by a Repressive Morphogen in Drosophila , 2008, Current Biology.

[34]  C.-ting Wu,et al.  Site-Specific Transformation of Drosophila via ϕC31 Integrase-Mediated Cassette Exchange , 2006, Genetics.

[35]  Michael W. Perry,et al.  Precision of Hunchback Expression in the Drosophila Embryo , 2012, Current Biology.

[36]  T. Hwa,et al.  Gene length may contribute to graded transcriptional responses in the Drosophila embryo. , 2011, Developmental biology.

[37]  Mike Rothe,et al.  Loss of gene function through rapid mitotic cycles in the Drosophila embryo , 1992, Nature.

[38]  E. Wieschaus,et al.  Short-term integration of Cdc25 dynamics controls mitotic entry during Drosophila gastrulation. , 2012, Developmental cell.

[39]  Johannes Jaeger,et al.  Cellular and Molecular Life Sciences REVIEW The gap gene network , 2022 .

[40]  W. Bialek,et al.  Stability and Nuclear Dynamics of the Bicoid Morphogen Gradient , 2007, Cell.

[41]  Hye Yoon Park,et al.  A transgenic mouse for in vivo detection of endogenous labeled mRNA , 2010, Nature Methods.

[42]  E. O’Shea,et al.  Chromatin decouples promoter threshold from dynamic range , 2008, Nature.

[43]  Hernan G. Garcia,et al.  Transcriptional Regulation by the Numbers 2: Applications , 2004, q-bio/0412011.

[44]  Julia Zeitlinger,et al.  Paused Pol II Coordinates Tissue Morphogenesis in the Drosophila Embryo , 2013, Cell.

[45]  N. Dostatni,et al.  Bicoid Determines Sharp and Precise Target Gene Expression in the Drosophila Embryo , 2005, Current Biology.

[46]  E. Cox,et al.  Real-Time Kinetics of Gene Activity in Individual Bacteria , 2005, Cell.

[47]  Liat Rosenfeld,et al.  Single-allele analysis of transcription kinetics in living mammalian cells , 2010, Nature Methods.

[48]  Shawn C. Little,et al.  Precise Developmental Gene Expression Arises from Globally Stochastic Transcriptional Activity , 2013, Cell.

[49]  Bin Wu,et al.  Real-Time Observation of Transcription Initiation and Elongation on an Endogenous Yeast Gene , 2011, Science.

[50]  Wolfgang Driever,et al.  The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo , 1989, Nature.