Single molecule biophysics in living organisms
In higher organisms, the early phase of embryonic development is driven by maternally inherited protein and mRNA. After a species-dependent number of cell cycles the zygotic genome is activated and its genes are transcribed (zygotic genome activation, ZGA). We investigate the molecular mechanisms underlying ZGA in live developing Zebrafish embryos at the single molecule level by reflected light-sheet microscopy. Our time and space resolved kinetic measurements allow us to build quantitative models of ZGA.
→ Embryo development
Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model
The influential hindered diffusion model postulates that the global movement of a signaling molecule through an embryo is affected by local tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo for any signaling molecule. Nodal and Lefty are a prime example of an activator- inhibitor signaling pair whose different global diffusivities are thought to arise from differential hindrance. Here, we used single-molecule tracking of Nodal and Lefty to directly probe the tenets of the hindered diffusion model on the nanoscale. We visualized individual fluorescently-tagged Nodal and Lefty molecules in developing zebrafish embryos using reflected light-sheet microscopy. Single-particle tracking revealed molecules in three states: molecules diffusing in extracellular cavities, molecules diffusing within cell-cell interfaces, and molecules bound to cell membranes. While the diffusion coefficients of molecules were high in extracellular cavities, mobility was reduced and bound fractions were higher within cell-cell interfaces; counterintuitively, molecules nevertheless accumulated in cavities. Using agent-based simulations, we identified the geometry of the extracellular space as a key factor influencing the accumulation of molecules in cavities. For Nodal, the fraction of molecules in the bound state was larger than for Lefty, and individual Nodal molecules had binding times of tens of seconds. Together, our single-molecule measurements and simulations provide direct support for the hindered diffusion model in a developing embryo and yield unprecedented insights into the nanometer to micrometer scale transport mechanisms that together lead to macroscopic signal dispersal and gradient formation.
Single-molecule imaging correlates decreasing nuclear volume with increasing TF-chromatin associations during zebrafish development
Zygotic genome activation (ZGA), the onset of transcription after initial quiescence, is a major developmental step in many species, which occurs after ten cell divisions in zebrafish embryos. How transcription factor (TF)-chromatin interactions evolve during early development to support ZGA is largely unknown. We establish single molecule tracking in live developing zebrafish embryos using reflected light-sheet microscopy to visualize two fluorescently labeled TF species, mEos2-TBP and mEos2-Sox19b. We further develop a data acquisition and analysis scheme to extract quantitative information on binding kinetics and bound fractions during fast cell cycles. The chromatin-bound fraction of both TFs increases during early development, as expected from a physical model of TF-chromatin interactions including a decreasing nuclear volume and increasing DNA accessibility. For Sox19b, data suggests the increase is mainly due to the shrinking nucleus. Our single molecule approach provides quantitative insight into changes of TF-chromatin associations during the developmental period embracing ZGA.