Seeing is believing. Starring zebrafish as a model organism, we image life at the nanoscale to "visualize" how innate immunity works at the interface with blood. Central to our approaches is a combination of light and (correlative) electron microscopy. Zebrafish embryos allow us to easily manipulate their genome for labelling cells of interest and thus to image them in real-time by seeing through the tissues non-invasively (see Figure and Movie). Our main focus is on the fundamental biology of macrophages – the cells that eat/clean up foreign materials, pathogens and dead cells to maintain homeostasis of our body. How do they recognize particles circulating in the blood? What are their role in inflammation? With zebrafish, we seek answers to these questions in a manner not possible using cell cultures or mammalian models.
Transgenic lines with a cell type-specific fluorescent protein reporter allow us to study the dynamic behaviour of macrophages and how injected nanomaterials are cleared from the bloodstream in a living organism. Transmission electron microscopy (TEM) can then complement the real-time observations by visualising those processes at the nanoscale. A marriage of the two imaging approaches is correlative light-electron microscopy (CLEM), by which we can link the fluorescent reporters (i.e. cell-type identity) to ultrastructure observed by TEM. dpf, days post-fertilisation. mpi/hpi, minutes/hours post-injection (Image: Yuya Hayashi. Adapted from Hayashi et al. (2020) & Mohammad-Beigi et al. (2020) ACS Nano. Copyright 2020 American Chemical Society)
Macrophages (magenta) with internalized nanoparticles (cyan) crawling along the inner side of blood vessels (yellow). Tg(fli1a:eGFP); Tg(mpeg1:mCherry) embryos at 3 dpf were injected with Pacific Blue-labelled 70 nm SiO2 nanoparticles (2 ng). Time-lapse imaging was performed at the intervals of every 16 s for 15 min at 1-4 hpi. (Reprinted from Hayashi et al. (2020) ACS Nano. Copyright 2020 American Chemical Society)
Tg(tnfa:EGFP-F); Tg(mpeg1:mCherry) embryos at 3 dpf were injected with Pacific Blue-labelled 70 nm SiO2 nanoparticles (10 ng) with protein corona pre-formed of FBS. The caudal vein tissue of the embryos was imaged every 20 min for 1-12 hpi. Left panel shows Cy5-labelled protein coronas (cyan), macrophages (grey), and transcriptional activation of tumour necrosis factor-alpha (yellow). Right panel shows only the latter two after applying a mask created with the macrophage reporter signals. (Movie: Yuya Hayashi. Adapted from Mohammad-Beigi et al. (2020) ACS Nano. Copyright 2020 American Chemical Society)
From an organ to another organ, cells send signals to coordinate the physiology of the entire body. A well-known example is signalling by hormones, but what if cells instead wish to deliver more complex messages than signals? A striking new discovery is the packaged delivery of small RNAs via extracellular vesicles (EVs) that enables cell-to-cell communication over a long distance. The meaning of this biological process is, however, still poorly understood today. This is largely due to technical difficulties in the isolation of EVs (and small RNAs therein) from complex biological fluids. The EVs encompass a wide range of vesicular entities, among which exosomes and microvesicles are two major EV types of particular interest in biomedical applications such as diagnostics and drug delivery. They are nanoscale vesicles nature has created, and thus the approaches we use for bionanoscience have technical overlaps with EV research.
Using zebrafish as an in vivo model, we are developing an affinity purification method to capture EVs tagged by a cell type-specific marker. Our aim is to profile the extracellular RNAs "in transit" from donor cells and mRNAs "in translation" in the recipient cells. Successful development of this method will shed light on the role of extracellular RNAs in cell-to-cell communication.
Our collaboration partners for this project are Prof. Jørgen Kjems (iNANO Interdisciplinary Nanoscience Center, Aarhus University) and Dr. Guillaume van Niel (INSERM, France).
What lies at the interface of biological receptors and nanoparticles? Over the past decade, nanoscientists have studied complex biophysical interactions that take place between biomolecules and nanoparticles. Today, it is widely accepted that cells recognize the biomolecules adsorbed to nanoparticles rather than the bare surface. Proteins are among those biomolecules that form a "corona" around the nanoparticle, and the corona profile is translated into a biological identity that can determine the nanoparticle's fate within a biological milieu.
Our interest is to unravel how innate immunity fights against those nanomaterials through recognition of their biological identities. The zebrafish embryo model now opens up a new opportunity to tackle this scientific question with the power of in vivo imaging at high spatio-temporal resolution.
We work with the collaboration partner Prof. Duncan Sutherland (iNANO Interdisciplinary Nanoscience Center, Aarhus University).
"Differential Nanoparticle Sequestration by Macrophages and Scavenger Endothelial Cells Visualized in Vivo in Real-Time and at Ultrastructural Resolution" by Yuya Hayashi*, Masanari Takamiya, Pia Bomholt Jensen, Isaac Ojea-Jiménez, Hélicia Claude, Claude Antony, Kasper Kjær-Sørensen, Clemens Grabher, Thomas Boesen, Douglas Gilliland, Claus Oxvig, Uwe Strähle, and Carsten Weiss.
ACS Nano 14 (2020) pp. 1665-1681. https://doi.org/10.1021/acsnano.9b07233.
"Tracing the In Vivo Fate of Nanoparticles with a "Non-Self" Biological Identity" by Hossein Mohammad-Beigi, Carsten Scavenius, Pia Bomholt Jensen, Kasper Kjær-Sørensen, Claus Oxvig, Thomas Boesen, Jan J. Enghild, Duncan S. Sutherland, and Yuya Hayashi*.
ACS Nano 14 (2020) pp. 10666–10679. https://doi.org/10.1021/acsnano.0c05178.
Want to inject something in zebrafish embryos?
Please contact Yuya Hayashi (email@example.com) to discuss possibilities.