Optogenetic manipulation of nuclear Dorsal reveals temporal requirements and consequences for transcription
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1.
Institut de Génétique Moléculaire de Montpellier
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2.
California Institute of Technology
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3.
University of Montpellier
Abstract
Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homologue of NF-κB, which orchestrates dorso-ventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning, and characterized the resulting effect on spatio-temporal transcription of target genes in terms of timing, coordination, and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL is detectable at the level of single cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally-resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.
Copyright and License
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
Acknowledgement
We are grateful to Eileen Furlong and Alessandro Dulja (EMBL, Heidelberg) for help with setting up the optobox, Sarah Bray, Hernan Garcia, and Chris Rushlow for providing fly stocks, Mike Levine for providing plasmid DNA, and Louise Maillard and Leslie Dunipace for comments on the manuscript. We also thank Pedro Prudêncio (Carmo Fonseca lab) and Amal Makrini (IGMM, CNRS) for the analysis of published NET-seq data. We acknowledge the Montpellier Ressources Imagerie facility (France-BioImaging), the Biocampus Drosophila facility of Montpellier, and the Caltech Beckman Institute Imaging Facility. This study was supported by funding from the National Institute of Health grant R35GM118146 to A.S as well as ERC SyncDev and ANR HubDyn to ML. M.D is supported by the CNRS and University of Chicago Joint PhD programme. V.P was initially supported by ERC syncDev and then ANR HubDyn. M.L., O.R., and A.T. are sponsored by CNRS.
Contributions
A.S., M.L, V.P., and J.M. conceived the project and planned the experimental approach. A.S. and M.L. directed the project. J.M. and V.P. performed all experiments, and J.M, VP, and A.T. performed all quantitative analysis of imaging data. A.T implemented the OptoTrack code, suitable for image analysis in the absence of a nuclear marker. M.D and O.R performed mathematical modeling. All authors analyzed and interpreted the data. The manuscript was written by V.P., J.M., A.S., and M.L with edits provided by O.R, M.D and A.T.
Supplemental Material
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Supplemental Figures 1-6[supplements/623729_file03.pdf]
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Movie S1[supplements/623729_file04.avi]
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Movie S2[supplements/623729_file05.avi]
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Movie S3[supplements/623729_file06.avi]
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Movie S4[supplements/623729_file07.mp4]
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Additional details
- National Institutes of Health
- R35GM118146
- Caltech groups
- Division of Biology and Biological Engineering
- Publication Status
- Submitted