Blood vessels, lymphatic vessels and other tube-like structures
Lymphatic vessels growing wild
Lab insights
Many genes in the zebrafish have the same or similar functions in humans, which is why zebrafish are so suitable for biomedical research.© Uni Münster - Erk Wibberg 
Our team uses microscopy to investigate how genetic modifications in zebrafish affect for example the growth of blood vessels, lymphatic vessels and bones.© Uni Münster - Erk Wibberg 
Zebrafish embryos (seen here through the microscope) develop outside the womb and are transparent for the first five days, allowing us to even observe processes in the organism live – as they unfold – during this period.© Dörte Schulte Ostermann - Uni Münster 
When examining zebrafish embryos under the microscope, we place them in a thermal chamber set to the perfect temperature for them.© Uni Münster - Erk Wibberg 
Around 400 genetically modified zebrafish lines live in small swarms at the Multiscale Imaging Centre. A special filtration and heating system ensures the optimal water quality and temperature.© Uni Münster - Erk Wibberg 
Under these conditions the fish feel comfortable and reproduce quickly. One mating pair can produce hundreds of eggs in a single day.© Uni Münster - Erk Wibberg 
We use CRISPR/Cas9 genome editing (“genetic scissors”) to create specific fish lines. We inject zebrafish embryos with an RNA sequence that binds to the gene we wish to modify, and a Cas9 protein that cuts the DNA at the desired point so that the gene can be modified. © Uni Münster - Erk Wibberg 
Using a fine needle, the components of the CRISPR/Cas9 system are being injected here into a zebrafish embryo (image taken through a microscope). From the fish that grow out of such embryos we breed genetically modified lines.© Dörte Schulte Ostermann - Uni Münster
Lymphatic vessels (green) in a zebrafish that are deformed on account of a mutated gene. The arteries are shown in pink. / Confocal fluorescence microscopy
Lymphatic vessels have an important role to play in the body because they transport fluid and immune cells from the tissue into the blood stream. Defects in these vessels can cause water to accumulate in the tissue and lead to a reduced immune function. Here we can see lymphatic vessels (green) in the head of a zebrafish that have grown too much as the result of a genetic modification. Surprisingly, this deformation only occurs once the zebrafish reaches the age of around three months. By this time the system of lymphatic vessels is normally fully formed. Without the gene we have identified, however, the lymphatic vessel cells will continue to grow unchecked.
We are now studying exactly what the reason for this is and whether this finding has any relevance to the ageing process in humans. In cell culture experiments the gene in question can also be deactivated in human lymphatic vessel cells. We are analysing whether the cells then react differently to proteins that stimulate the growth of lymphatic vessels than normal cells would, and which molecular signal chains regulate vessel growth. In addition, we check whether the same processes can be observed in mice. If a gene has a similar function in both fish and mice, it is highly probable that it will also have evolved in humans. Genetic mutations that lead to uncontrolled growth of lymphatic and indeed blood vessels also occur in humans. Such congenital vascular malformations can be found anywhere in the body and can result in vessels entering tissue and destroying organs. The growth of lymphatic and blood vessels is also altered in people suffering from cancer or inflammation. Understanding the underlying mechanisms is the key to finding ways to prevent or reverse deformations.
Dörte Schulte-Ostermann
Institute for Cardiovascular Organogenesis and Regeneration, University of Münster
Watching blood vessels as they evolve
Red blood cells (yellowish orange) stream through the blood vessels (white) of a three-day-old zebrafish. / Overlaid images of several tissue layers obtained with confocal fluorescence microscopy
Video: Zooming in on the beating heart and on the red blood cells streaming through the blood vessels of the head and torso of a zebrafish / Widefield microscopy
We can watch live how blood vessels grow and other organs develop in the first five days of a zebrafish’s life because they are transparent and grow outside the womb during this initial phase of their development. Blood vessels (white) form within just a few hours. After 26 hours the heart begins to beat, and after 28 hours the first red blood cells (yellowish orange) start moving. An accumulation of red blood cells can be seen in the image – revealing where the heart is. And from there, cells are pumped into the vascular system.
In our research we want to identify the genes that are responsible for healthy vessel formation and whose defects cause disease in humans. Many genes in the zebrafish have the same or similar functions in humans. We can use CRISPR/Cas genome editing to create various fish strains in which we add or deactivate specific genes. When these are compared with non-genetically modified fish (as in the image here), it becomes clear which gene is responsible for which developmental stage.
Sanjay Sunil Kumar and Stefan Schulte-Merker
Institute for Cardiovascular Organogenesis and Regeneration, University of Münster
The “magic” of the liver in 3D
The network of blood vessels in the liver lobe of a mouse. To the trained eye, the central veins, branches of the portal veins, hepatic arteries (smaller structures along the portal veins) and gallbladder (the roundish shape to the left of the image) are visible. / Depth colour-coded projection of an image set taken by light sheet fluorescence microscopy
The liver is the only human organ that regenerates completely if part of it is removed. It can lose more than half of its tissue but still regenerate. The blood vessels play a key role in this “magic”, as many molecular signals that control tissue growth originate in the cells of the blood vessels. Liver cells, lymphatic vessels and bile ducts (not shown) arrange themselves around the blood vessels. The liver’s ability to grow is evident not only when it regenerates but also when tissue structure changes as a result of illness. This can affect the bile ducts, for example – the channels along which bile is transported from the liver to the intestine. When liver samples are examined in the pathology lab, changes in the appearance of the bile ducts are often noticed. This is something we are currently looking into more closely, using tissue samples from mice and humans.
One special feature of our research is that we analyse the liver in 3D. In our opinion this is the only way to create a visual representation of the tiny changes and detect patterns that would otherwise be missed in standard histological tissue sections. At just under three micrometres (0.003 millimetres) thick, these are wafer-thin. Light sheet microscopy, by contrast, allows us to analyse entire blocks of tissue (measuring up to 1.5 x 1.5 x 0.5 centimetres). This special microscopic technique involves taking thousands of digital images of cross-sectional levels and then stitching them back together to create a 3D image on the computer. Unlike with true tissue cross sections, no tissue is lost, nor is any squashed or distorted. We hope that this 3D information about the structure of the liver can ultimately be used to detect the causes of disease even in thin cross sections, enabling more accurate diagnosis.
Stefanie Bobe and Friedemann Kiefer
European Institute for Molecular Imaging, University of Münster
Individual cells of growing tracheae
A section showing the tracheal system as it forms in a fruit fly larva. / 3D reconstruction from confocal fluorescence microscopy
Unlike mammals, insects do not have blood vessels. Their blood – a colourless fluid – flows freely within their bodies, supplying the cells with nutrients. Oxygen is supplied separately via a network of branched tubular structures. Filled with air, this system runs through the insect’s entire body and provides the cells with oxygen. Resembling blood vessels, these tracheae provide a comparatively simple model system that allows us to study how vessels form and how branching patterns develop in a living organism.
Since the fruit fly’s tracheal system is made up of a comparatively small number of cells, its development can be analysed at the level of individual cells. That’s exactly what we did here: we used a genetic tool to mark the cells such that each cell fluoresces in one of three different colours. In each cell, the selection of the colour occurs stochastically. In some cells (on the left-hand side of the image) we rendered the surface using a computer to reveal the shape and orientation of the cells within the tissue. The tracheal system grows because the individual cells stretch along the longitudinal axis of the developing tube. In mammals, including humans, this cellular behaviour plays an important role, for example in the normal development of the kidneys. The image shows tracheal cells at an early stage before they begin to extend along the axis of the tube.
Stefan Luschnig
Institute of Integrative Cell Biology and Physiology, University of Münster
This study was done during my time at the University of Zurich.
Oxygen supply in the brain of a fruit fly
A view of the left half of the brain of a fruit fly larva. The brain is supplied with oxygen by ramified airways (tracheae, shown in white). The degree of oxygen supply (illustrated in false colours on a scale from purple = high oxygen, to red and yellow = low oxygen) depends on the distribution and density of the tracheae. / Computer analysis based on confocal fluorescence microscopy
Video: 3D visualisation of the microscopy data. The signals from two fluorescing proteins (red and green) are visible in unprocessed form.
Cells need oxygen to survive. If too little oxygen is present in the tissue during a fruit fly’s development, the tracheae will form new branches through which the fly’s body will be supplied with oxygen. Similar mechanisms can be observed in mammals, including humans, when a tumour grows and induces the formation of new blood vessels that will supply it with nutrients and oxygen.
Until a few years ago we lacked the tools needed to visualise cellular oxygen supply in the tissue of an animal. We therefore developed such a tool, enabling us to visualise the different degrees of oxygen present in the brain of a fruit fly. We genetically marked the fruit fly’s cells so that each cell produces two different proteins that fluoresce in different colours. One of the two proteins (red) serves as a reference to indicate where cells are located. The other protein (green) is destroyed depending on the amount of oxygen present – the more oxygen there is in the vicinity, the lower will be the intensity of the green-fluorescent signal and vice versa. Using the ratio between the two signals as an indicator, we were able to depict the oxygen supply in each individual cell. We used a colour scale to show the gradations.
Stefan Luschnig
Institute of Integrative Cell Biology and Physiology, University of Münster
The image is the result of a collaboration with Martin Baccino-Calace, Felix Meyenhofer and Boris Egger, University of Fribourg (Switzerland).
Cancer cells in state of oxygen deficiency
Oxygen deficiency (red) and blood vessels (white) in a brain tumour (glioblastoma) of a mouse. Thicker and more irregular vessels grow in the tumour tissue than in the surrounding brain tissue. We used a new method of processing three-dimensional microscopic images to analyse the correlations between abnormal blood vessels and oxygen deficiency in glioblastomas. / Digital reconstruction based on light sheet fluorescence microscopy
Video: Image showing the individual digital levels of the examined tumour tissue and its three-dimensional expansion
When tumours grow, abnormally shaped blood vessels form inside them that do not function in the same way as healthy blood vessels. As a result, the blood supply in the tumour tissue is poor and the tumour does not receive sufficient oxygen (hypoxia). It is known that hypoxic cancer cells are often particularly aggressive and resistant to therapy. To treat glioblastomas – highly malignant brain tumours – drugs are therefore used that normalise the structure of the blood vessels and thus the blood supply in the tumour. This also increases the effectiveness of radio- and chemotherapies, which are frequently combined with these drugs. After initially responding to the therapy, however, glioblastomas usually continue to grow very aggressively.
To better understand why, we studied how exactly such therapies alter the blood supply in glioblastomas. We used a new genetically encoded sensor to mark human cancer cells. The sensor is produced by the tumour cells and causes them to fluoresce under the microscope under hypoxic conditions (red). We implanted these cancer cells into the brain tissue of mice, where they formed tumours. Using light sheet fluorescence microscopy, we took thousands of digital cross-sectional images of these tissues. To process the huge data volumes, we developed a new method based on artificial intelligence. For the first time, this made it possible to reconstruct and analyse a digital three-dimensional image of the size and shape of the blood vessels in relation to the oxygen supply of a tumour in intact tissue (roughly 0.5 x 0.5 x 0.5 cm). Interestingly, we observed that some tumour cells, despite being in the direct proximity of healthy blood vessels and thus receiving a good oxygen supply, continued to behave as if they were in a hypoxic environment. We suspect that these cells, which we will now subject to further study, are particularly aggressive and therapy-resistant.
Nadine Bauer and Friedemann Kiefer
European Institute for Molecular Imaging, University of Münster
Daniel Beckmann and Benjamin Risse
Institute for Geoinformatics, University of Münster