Our understanding of biological phenomena is often shaped by the technology available to query our system of interest. For years, our understanding of embryonic development has been dictated by static snapshots stemming from technologies that rely on the examination of dead, fixed embryos. In this case, the spatiotemporal evolution of the gene expression programs that shape animal body plans is inferred from static images. The main hypothesis of the proposed work is that the reliance on snapshots rather than movies has hidden deep insights from view relevant to embryonic development. We propose to break free from the reign of static snapshots in the study of developmental biology by introducing new technologies that will make it possible to redo the subject of embryonic development from the standpoint of dynamics through the imaging and quantification of the central dogma in real time at the single cell level within living embryos. We will use the embryonic development of the fruit fly Drosophila melanogaster as a proof of principle in order to (i) develop new technology to quantify fast changing transcription factor concentrations and the resulting transcriptional output in development, (ii) develop new in vivo fluorescent probes to augment cutting-edge lattice light-sheet microscopy to make it possible to directly visualize how single activators and repressors bind to the DNA, and (iii) combine these input-output measurements with our single-molecule measurements in order to directly visualize the molecular mechanisms by which transcription factors perform their regulatory function in real time in living embryos. We argue that only by enabling this real-time description of gene regulatory programs in development can we reach a quantitative understanding that makes it possible to predict how DNA sequence dictates cellular commitment and how, when this regulation goes awry, developmental defects and states of unchecked cellular proliferation ensue.

Public Health Relevance

More than a decade after the release of the human genome sequence, we are discovering that many diseases are linked to DNA regulatory regions, which decide where and when to produce proteins in our body throughout our life. The work proposed here seeks to visualize the mechanisms that single regulatory molecules use to determine animal body plans by binding regulatory DNA. These experiments will lay the foundation for modifying regulatory-molecule action, repairing developmental defects, and halting the states of unchecked cellular growth that underlie cancer.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2HD094655-01
Application #
9350588
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mukhopadhyay, Mahua
Project Start
2017-09-30
Project End
2022-06-30
Budget Start
2017-09-30
Budget End
2022-06-30
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Graduate Schools
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Mir, Mustafa; Reimer, Armando; Stadler, Michael et al. (2018) Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy. Methods Mol Biol 1814:541-559
Bothma, Jacques P; Norstad, Matthew R; Alamos, Simon et al. (2018) LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos. Cell 173:1810-1822.e16