Seeing is believing. We use zebrafish as a model organism to visualize unsolved mysteries in biology – such as the physiological role of extracellular vesicles – in a way that cannot be achieved by the traditional use of cell cultures or mammalian models. What makes us unique is our interdisciplinary approach, where nanoscience meets zebrafish in search of new inspirations for nanomedicine.
Why zebrafish, not mice? We can manipulate their genome e.g. to label cells of interest by fluorescent (and functional) proteins, and it allows us to image live embryos in real-time seeing through the tissues non-invasively (click the "NanoBiaS – Our Research Strategy" button below to expand). A particularly successful example of our approach is live imaging and electron microscopy of how nanoparticles injected into the bloodstream are captured by macrophages – the cells that eat/clean up foreign materials, pathogens and dead cells just as they do in mice and humans. Therefore by looking at the biology evolutionarily conserved at the cellular and molecular levels, we can study how life behaves at the interface with nanotechnology.
Our current interest centres around bioinspired nanomedicine, for which we have just started a journey to explore novel ideas in extracellular vesicles. We are particularly interested in their mechanisms that drive long-distance cell-to-cell communication, because they can be therapeutic targets for molecular intervention or potentially mimicked as drug delivery vehicles. Much is still not known about these natural nanoparticles that carry biomolecular cargoes. For example, by which mechanisms can extracellular vesicles find the target recipient cells via the bloodstream? What kind of messages are conveyed to regulate/support the recipient cells? With zebrafish as our little partners, we seek answers to these questions by nanoscience approaches, bioinformatics and 4D imaging of live transgenic embryos. The big picture of our research is thus to learn, manipulate and mimic nature's biomolecular architecture to advance the field of nanomedicine.
We do both basic and applied sciences. Apart from the fish, we strongly collaborate with nanoscientists at iNANO and health science researchers at the Department of Biomedicine to make our interdisciplinary research happen!
Transgenic lines with a cell type-specific fluorescent protein reporter allow us to study the dynamic and interacting behaviour of cells such as macrophages and how (injected) nanoparticles are cleared from the bloodstream in a living organism. This power of whole-embryo bioimaging can then be combined with a high-resolution modality, transmission electron microscopy (TEM), by visualizing those biological processes at the nanoscale. Correlative light-electron microscopy (CLEM) is an exciting marriage of the two imaging approaches, by which we can link the fluorescent reporters (i.e. cell-type identity) to ultrastructure observed by TEM. The unrivalled strength of the zebrafish model is also its screening capacity that enables testing of multiple nanoformulations within a short time period. 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)
Extracellular vesicles secreted from the yolk syncytial layer (yellow) circulate in the bloodstream and are captured by endothelial cells (grey) and macrophages (magenta). Eggs obtained from Tg(mpeg1:mCherry) fish were transfected with DNA plasmids by microinjection into the yolk syncytial later that is formed at 3-4 hpf. The caudal vein tissue of the transfected embryos at 3 dpf was imaged every minute for 30 min. (Movie: Yuya Hayashi)
"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.
Our group poster has been updated with new project opportunities in extracellular vesicles and RNA origami. We are looking for students!
We are now also affiliated with Interdisciplinary Nanoscience Center (iNANO) where excellent nanoscience research takes place!
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 main 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.
Bachelor's, Master's, medical and visiting (Erasmus+) students are always welcome to contact Yuya (yuya.hayashi@mbg.au.dk) and he will usually respond very quickly ;) Student projects are designed according to your scientific interests (well, motivation is the key driving factor to complete a project!) but we also help you to define the project based on what we can offer. Once the project starts, you will become a group member and report to weekly group meetings. On the Group Members page, you can have an idea about what kind of projects each member is working on.
Listed below are the scientifc topics that we cover, and techniques that you can learn from us.
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.
Blood vessels (Endothelial cells)
Tg(fli1a:EGFP)y1 ZFIN
Tg(kdrl:Hsa.HRAS-mCherry)s896 *Membrane-labelling ZFIN
Embryonic macrophages
Tg(mpeg1:EGFP)gl22 ZFIN
Tg(mpeg1:mCherry)gl23 ZFIN
Tg(mpeg1:EGFP-Mmu.Rpl10a)uwm16 *TRAP ZFIN
Neutrophils
Tg(lyz:DsRed2)nz50 ZFIN
Tumour necrosis factor-alpha (as a signature of macrophage activation)
Tg(tnfa:EGFP-F)ump5 *Membrane-labelling ZFIN
Currently, we are sharing the lab space with a new group led by Dr. Gilles Vanwalleghem, who is also a team leader at DANDRITE and an expert in bioimaging of zebrafish. We will have regular joint group meetings to exchange relevant skills and knowledge and discuss lab issues. Together with his group, we thrive to promote the zebrafish initiatives here at MBG ;)
Our neighbours are people from Prof. Claus Oxvig's, Dr. Lisbeth Schmidt Laursen's and Prof. Daan van Aaltan's groups who work with cell cultures, zebrafish and mouse models. Students, PhDs and postdoc researchers thus have a vibrant social life in a mixed Danish-international work environment.
We are constantly looking for students who wish to join the group and carry out a project as a part of the educational programme. Please contact Yuya to hear about project opportunities.
We have collaborations with health science researchers, for example, at Department of Biomedicine. Medical students who are interested in our research are advised to contact me to find project opportunities through such collaborations.
ERASMUS students/trainees from abroad 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! There are also possibilities of financial support for foreign students through an internal grant from the Department (read more about the grant possibilities).
We currently have an open position for a PhD student to join forces for tackling the new project that just started in Spring 2022. Job advertisements will follow in time (likely mid/late 2023), but in general education in any of Nanoscience, Immunology, Cell Biology, Molecular Biology, and Bioinformatics is advantageous. Knowledge in RNA origami and/or nucleic acid sensing for antiviral immunity is particularly favoured. Previous experience with zebrafish is of course a plus, but we are also happy to make you a new zebrafish scientist ;)
Please contact Yuya Hayashi (yuya.hayashi@mbg.au.dk) to discuss possibilities.
