Using Zebrafish to Understand How Blood and Lymphatic Vessels Develop

Zebrafish is an excellent animal model for biomedical research as it has a highly conserved genetic control of development and disease.

Dr. Kazuhide Shaun Okuda from the Peter MacCallum Cancer Centre in Victoria, Australia, uses ZEISS confocal microscopes to image zebrafish vessel development in real time. He is the winner of the LMA/VIA 2021 Image Competition of the Australian Microscopy & Microanalysis Society.  

We spoke to Dr. Okuda about his love of fish and latest discoveries:

Zebrafish is an excellent animal model for biomedical research as it has a highly conserved genetic control of development and disease. The most important advantage of the zebrafish model is its optical transparency, allowing researchers like me to image blood and lymphatic vessel development in real time. This is still very challenging in optically dense mammalian models.

One research focus of mine is to generate and characterize genetically modified zebrafish (zebrafish transgenics) that enable visualization of the dynamic activity of key signaling pathways for blood/lymphatic development (recently published here). These zebrafish transgenics could also be utilized to understand molecular mechanisms of blood/lymphatic vessels in various pathological settings in real time. Another focus of my research is to uncover new lymphatic genes that may have functions in both developmental and pathological lymphangiogenesis. These genes could be a therapeutic target for various human diseases associated with excessive lymphangiogenesis, such as cancer and lymphatic malformation.

During my PhD, I contributed to the generation of the most lymphatic-specific fluorescent transgenic lines in zebrafish at the time (see publication). This allowed me to characterize previously hard-to-see lymphatic vessels in zebrafish. I was even given the privilege to name some of these lymphatic vessels! I am proud to say that these transgenic lines are currently one of the most utilized zebrafish models for live-imaging lymphangiogenesis globally.

I still remember the early days in my research career when all my experiments were done on fluorescent microscopes. While this gave us lots of valuable 2D data, the z-resolution information from confocal microscopy images allows us to visualize our data in 3D. This is particularly important for zebrafish research which relies heavily on live-imaging. Taking advantage of the large repertoire of zebrafish transgenics, I have used the confocal microscope to visualize multiple cell types in zebrafish including blood/lymphatic vessels, immune cells and neurons. I have also transplanted fluorescently labelled bacteria/cancer cells into zebrafish to image vascular responses in vivo. The resulting images and movies are not just aesthetically pleasing, but are packed with information that I can spend hours analyzing! It is all worth the effort when it’s published though!

The winning image shows a zebrafish transgenic line we generated for our recent publication in the prestigeous journal Nature Cell Biology. The transgenic line enables real-time visualization of RNA helicase Ddx21 expression in zebrafish. A forward genetic screen conducted by my supervisor Professor Benjamin Hogan during his postdoc days in Utrecht, resulted in the identification of ddx21 zebrafish mutant, with reduced lymphatic vessel development. Surprisingly, blood vessel development was unaltered, showing that lymphatic vessels are selectively sensitive to loss of Ddx21. While Ddx21 is known for its function in ribosomal biogenesis, its role in vascular development had never been explored. Using this transgenic line, we showed that Ddx21 is expressed in zebrafish veins, which give rise to lymphatic vessels. Importantly, we showed that Vegfc/Vegfr3 signaling, essential for lymphangiogenesis, modulates Ddx21 level in endothelial cells. We are now interested in identifying inhibitors for Ddx21, which could be used to treat diseases like cancer and lymphatic malformation.