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 to study the dynamic behaviour of cells towards nanoparticles of natural and artificial origins. Why zebrafish? We can manipulate their genome e.g. to label cells of interest by fluorescent proteins, and it allows us to image live embryos in real-time seeing through the tissues non-invasively (click the "Our Imaging Approaches" button below to expand). We are particularly interested in 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 nanoparticles circulating in the blood? What are their roles in inflammation and tissue repair? 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 visualizing 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)
"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.
Yuya Hayashi receives a Hallas-Møller Emerging Investigator grant from the Novo Nordisk Foundation for a project entitled Exosomes: Decrypting the "Blood-Streamed" RNA Communication.
Exosomes: Decrypting "blood-streamed" RNA communication
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 discovery in the past years is the packaged delivery of small RNAs in nano-sized vesicles called exosomes to "stream" the RNA language over a long distance. Much remains unknown, however, about the precise context of such messages that are exchanged between cells of a living organism. This project aims to decipher the secret RNA codes delivered by exosomes using zebrafish embryos as a research model that allows genetic manipulation to capture target exosomes and live imaging of the exosome transport through the bloodstream. The deeper understanding of the exosome-powered RNA communication between distant cells will identify novel targets for nanomedicine.
Our collaboration partners for this project are Prof. Jørgen Kjems (iNANO Interdisciplinary Nanoscience Center, Aarhus University), Dr. Frederik Verweij (Utrecht University, the Netherlands), and Dr. Guillaume van Niel (INSERM, France).
Breaking free from antiviral immunity
"RNA Origami" is a new frontier in self-assembled nanotechnologies exploiting programmable folding of RNA into artificial 3D nanostructures constructed from pre-determined DNA templates. We dream of breakthroughs in nanomedicine by realizing designer machineries that can combine RNA's unique functions in gene regulation and protein-like structural versatility (e.g. aptamers). However, the biomedical application of artificial nucleic acids suffers from the general uncertainty in potential side-effects. It is therefore important that we understand the science behind it already at the time of drug development. Today, however, much still remains unanswered about the complexity of nucleic acid sensing that discriminates self (endogenous RNA) from non-self (virus) – critical knowledge necessary for successful delivery of RNA origami nanostructures injected into a living organism.
The key research strategy in this project is whole-organism imaging of zebrafish embryos for visualization of RNA nanostructures and antiviral immunity in real-time and at ultrastructural resolution. The successful outcome of this project will unravel how life "sees" artificial RNA architectures and thus hold the promise to redefine the RNA nanotechnology field towards a safe-by-design approach.
RNA origami is a nascent technology invented by Dr. Ebbe Sloth Andersen and Prof. Jørgen Kjems (iNANO Interdisciplinary Nanoscience Center, Aarhus University). They are important collaborators on the designing part while our focus is its application as nanomedicine.
Dr. Jean-Pierre Levraud (NeuroPSI & Institut Pasteur, France) and Prof. Søren Riis Paludan (Department of Biomedicine, Aarhus University) are collaboration partners on antiviral immunity in zebrafish.
Biological recognition of the protein corona at nanoparticles
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 the high spatio-temporal resolution.
We work with the collaboration partner Prof. Duncan Sutherland (iNANO Interdisciplinary Nanoscience Center, Aarhus University).
"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.
The zebrafish facility here at MBG has assisted our research since 2014. A number of genetically modified lines generated by us or other zebrafish scientists are available at the facility. Among those, we routinely use those with a fluorescent reporter transgene that labels cells of particular interest for e.g. live imaging. Generation and establishment of a new transgenic line takes min. 7 months but normally longer than that. Enquiries regarding the use of the fish facility should be made to Dr. Kasper Kjær-Sørensen or Prof. Claus Oxvig.
Tumour necrosis factor-alpha (as a signature of macrophage activation)
Tg(tnfa:EGFP-F)ump5 *Membrane-labelling ZFIN
The highly interdisciplinary nature of our research drives close collaborations with experts across different fields. Among them, the mainstream publications listed below are particularly relevant for the group's scientific focus area.
Mohammad-Beigi H, Scavenius C, Jensen PB, Kjaer-Sorensen K, Oxvig C, Boesen T, Enghild JJ, Sutherland DS, Hayashi Y*.
Tracing the in Vivo Fate of Nanoparticles with a ”Non-Self” Biological Identity.
ACS Nano 14. 10666–10679. 2020. DOI
We show how non-self protein coronas can determine the blood clearance kinetics of the nanoparticles leading to vascular damages and inflammation.
Hayashi Y*, Takamiya M, Jensen PB, Ojea-Jiménez I, Claude H, Antony C, Kjaer-Sorensen K, Grabher C, Boesen T, Gilliland D, Oxvig C, Strähle U, Weiss C.
Differential Nanoparticle Sequestration by Macrophages and Scavenger Endothelial Cells Visualized In Vivo in Real-Time and at Ultrastructural Resolution.
ACS Nano 14. 1665-1681. 2020. DOI
This article summarizes our imaging approaches to study the biological fate of nanoparticles with a particular emphasis on macrophage and endothelial biology.
Hayashi Y*, Miclaus T, Murugadoss S, Takamiya M, Scavenius C, Kjaer-Sorensen K, Enghild JJ, Strähle U, Oxvig C, Weiss C, Sutherland DS*.
Female versus male biological identities of nanoparticles determine the interaction with immune cells in fish.
Environmental Science: Nano 4. 895-906. 2017. DOI
Following Yuya's pioneering Ph.D. work on "species differences at nanoparticles", another new concept "sex differences at nanoparticles" was conceived that adds another layer of complexity in the protein corona formation.
We currently have open positions for Ph.D. students to join forces for tackling the new project that starts in Spring 2022. Job advertisements will follow in time, but in general education in any of Nanoscience, Immunology, Cell Biology, Molecular Biology, Genetics and Bioinformatics is advantageous. Previous experience with zebrafish is of course a plus, but we are also happy to make you a new zebrafish scientist ;)
Bachelor's and Master's students are always welcome to take a part in our research. Please send me an e-mail expressing what skills you wish to learn and we can discuss about projects for the mutual benefit!
Please read the application guideline here.
We are grateful for the financial support from various foundations that has given us the wonderful opportunities to promote the zebrafish initiative and excellence of Danish research.