On Embryonic Zebrafish Brains
and the Probably Quite Usual Twists and Turns of a PhD Thesis
Theresa actually wanted to investigate a gene candidate for the development of dopamine neurons. Instead, she found strange photoreceptors in the “third eye”, drew colorful maps of the embryonic brain and ultimately could only speculate about the reasons for her beloved zebrafish being so sluggish. Read more about the ups and downs in Theresa’s PhD journey.
Why can you read this text? Isn’t it amazing that your eyes only have to slide over the letters to form images and ideas? Probably most of you came across several impressive facts and figures about the human brain. But very few ask how the brain forms in the first place.
The key principle of embryonic development: Differentiation
How is it possible that all of our cells contain the same genetic code, but not only look very different, but also fulfill completely different functions? The explanation for this can be found in our embryonic development, during which the stem cells, which initially all look the same, increasingly use their genetic code in different ways. For example, almost all cells in the body shut down the insulin gene, only certain cells of the pancreas are constantly reading it and building the hormone of the same name according to the instructions encoded in the insulin gene. We call this process, in which the cells become more and more different, differentiation.
Understanding this miracle of differentiation more precisely is not only based on the deeply human thirst for knowledge. Regenerative approaches are very popular in modern medicine. But in order to understand re-generation, we must first understand generation. For many good reasons, however, human embryos are hardly accessible for research. So a model is needed. Often small rodents such as mice or rats are used for this. The model animal of my choice is less warm and fluffy, more wet and slippery.
Zebrafish are about as long as a little finger, beautifully striped and the dream of every geneticist. For over 80 percent of all human genes that are associated with diseases, there is a corresponding gene in zebrafish. A motivated pair of zebrafish lays 300 eggs in a single morning. These eggs develop outside of the mother, so that you can watch the transparent tiny embryos closely under the microscope. After five days, their organs are developed and matured enough in order for the small larvae to start hunting for food on their own. For this task, above all, the brain already needs to be highly differentiated. The stem cells, which initially all look the same or at least very similar, have already developed into a colorful mixture of nerve cells that use a wide variety of neurotransmitters and are in lively exchange with one another.
Positional information is key behind the differentiation process: depending on the position in which the different body cells arise, different combinations of signal molecules rattle on them. The cells react to this in a variety of ways, but especially in terms of the transcription factors they activate and use. These are proteins that regulate the activity of genes. Different cocktails of transcription factors in the cells therefore means that different genes are switched on or off.
Enough of the forewords; let’s jump right in!
Well, that’s what I thought to myself when I started my dissertation.
A quite promising project that I was allowed to work on was based on the results of a previous doctoral student. She had found indications that a so far hardly described transcription factor called BSX could control the differentiation of dopamine-producing nerve cells.
BSX as a novel genetic factor in dopaminergic neuron development? How would you find out?
These preliminary results had the potential to turn into a hot story: Factors in the differentiation of dopaminergic cells are of particular importance when it comes to regenerative approaches to the treatment of Parkinson’s disease.
The early days of my doctoral thesis also coincided with a gigantic upheaval in the biomedical research field. TALENs and CRISPR were first used for genome editing at this time. So I went to work vigorously and used TALENs to mutate the BSX gene. Just a few years earlier, it was still impossible to just “switch off” a certain selected gene in the zebrafish genome. But it worked right away and I was able to breed a stable line of fish in which the BSX gene is literally knocked out.
It was an exciting time in which I examined the tiny brains of zebrafish larvae that developed without the BSX protein. Using fluorescent staining, I was able to see the multiple branched dopamine-producing nerve cells very nicely.
All
of
them.
They were all there. Even in the mutants. The dopamine system in the brain developed splendidly without any BSX protein. I experienced the stereotype of a doctoral thesis. Our initial hypothesis was shattered and nothing – even parallel projects of mine – seemed to work. So I took a step back. BSX had to be good for something!
Well, at least I was able to show that BSX seemed to be active in the zebrafish brain in very similar regions as it was already shown for mice. So I took a closer look at these areas of the brain.
On remarkable brain regions and how to find them
The pineal gland particularly fascinated the French philosopher René Descartes. He suspected this was the seat of the spirit. Today we know that the pineal gland fulfills a much more mundane but important function. If our eyes perceive darkness, this is communicated to the pineal gland, which then releases melatonin. When we finally put our smartphones aside, this melatonin causes us to get tired and our body can regenerate. In the zebrafish, the pineal gland is located directly under the skullcap and is itself sensitive to light, so it functions a bit like a “third eye”.
It made sense to first check whether the pineal gland can still fulfill its main task when BSX is missing. And it turned out: it can’t! Critical enzymes for the formation of both melatonin and its precursor, serotonin, were missing in the pineal gland of the fish larvae without functional BSX. Suddenly I woke up from my lethargy and was back in the race.
I soon found out that in those fish with the BSX mutation the composition of photoreceptors in this “third eye” was completely mixed up. With this and a few more observations on how the pineal gland developped somehow aberrantly in the BSX mutant larvae, I’ve had enough material for my first scientific article.
BSX is also used in another place in the brain: the hypothalamus, which is probably the most important interface between the brain and the endocrine system. A research group from Heidelberg was able to show in 2007 that mice without functioning BSX lack a certain signaling molecule called AGRP. The activity of these AGRP neurons in living mice can be manipulated in elaborate experiments. It shows: if they are active, the mouse eats. If they are inactive, the mouse stops immediately. Although, contrary to expectations, the mice without BSX and AGRP had a normal body weight, they moved less. And that is exactly what my zebrafish did after I knocked out their BSX gene.
In my search for the causes of this inertia, I came across a new problem. In fact, nobody seemed to have bothered to compare the various core areas of the admittedly frustratingly complex hypothalamus between the two popular model animals mouse and zebrafish. By visualizing numerous signal molecules and transcription factors in the differentiating brain, extensive “maps” have already been created in the mouse; in other words, the “genoarchitecture” of the developing hypothalamus is known very well for mice. I therefore stained teeny-tiny zebrafish brains until my fingers became sore in order to map all the factors that where known in mice to identify certain specific brain regions in the zebrafish larvae. After days, weeks and months of comparing, rotating and superimposing colored surfaces on my monitor, it was then ready: my map of the fish hypothalamus and with it my second scientific article.
Finally being able to identify and define all those different subregions in the embryonic zebrafish hypothalamus, I went through all of them tediously but diligently. Since I knew now from my comparison to the mouse model which hypothalamic regions I delt which, I was able to check if expression of all the various signalling aubstances that are know to develop in those regions, still develop in my BSX knock-out zebrafish embryos. And it turned out, that in addition to AGRP – which was previously described as being dependend on functional BSX in mice – I found over ten other neuronal and hormonal factors that were reduced in the BSX-mutated fish. Thus, for both fish and mouse, we should perhaps reevaluate: if the animals are sluggish, this could be due to the lack of the famous AGRP. Or it could be due to any of the other (approx. 10) signaling substances I described in this third and final article.
These insights have earned me what is undoubtedly the most beautiful doctoral cap that my laboratory colleagues have ever made (I am certainly not biased here and accept challenges any time). For the BSX gene, well, I did shed some light on its functions, but was also raising more new questions than answering old ones. However, I suppose that’s quite common true in science, and probably a good thing. Somehow it is reassuring that nature still has more to discover for us, no matter how close we laready have a look. How your brain helped you today to hopefully generate meaning and perhaps vivid images from this text will remain a mystery for a while.
