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Me with doctor's cap and polar bear. Photo by Lars Nilse.

People who know me well and in person, friends, family, colleagues, my boss, always looked at me in disbelief. "What? There is no Gene of the Week article on Bsx???" They are astonished because Bsx is the gene that has been with me, or- sometimes even more so – has been following me, for several years. Even if originally this wasn't planned at all, the Bsx gene was holding so many previously unknown functions for me to discover that I ultimately filled most of my doctoral thesis by researching and describing these functions. In other words: I owe this awesome hat (photo) mostly to the Bsx gene. But let’s start at the beginning.

The abbreviation Bsx stands for Brain-specific Homeobox and, as the name describes, it is only active in the brain. But what does "homeobox" mean? Well, this story is so unique that I don't want to miss the opportunity to tell you all about it.

 

Homeotic Mutations and the Legendary Fly Room at Columbia University

Almost 100 years ago, Thomas Hunt Morgan and his colleague Calvin Bridges, two exceptional researchers in an exceptional laboratory, the famous fly room at Columbia University, made an astounding discovery. Among the thousands of fruit flies that they grew with loads of patience and a bit of mashed banana, they found a very strange looking one. Instead of one pair of wings, this fly had two: the third body segment, on which only two short stubs were supposed to sit, was changed in its entire identity so that it corresponded to an additional second, wing-bearing, segment. They described a first homeotic mutation: a natural variation in which one part of the body takes on the identity of another.

Thomas Hunt Morgan (reprinted with kind permission from archives.caltech.edu), Calvin Bridges (reprinted with kind permission from archives.caltech.edu) und Edward Lewis (Caltech yearbook, public domain),as well as a normal fly and an "Ultrabothorx" fly (image from Rachgo20 for wikimedia.org, CC BY-SA 4.0), in which the third body segment took the identity of an additional second (wing-bearing) body segment.

 

It wasn't until fifty years later, in the 1970s, that Edward Lewis described that the very gene that was mutated in this fly was arranged in a special cluster together with other special genes. This gene cluster was characterized by a property that he called colinearity: they get activated along the body axis in precisely the temporal and spatial arrangement in which they are arranged on the chromosome. In the 1980s, Walter Gehring and his colleague Bill McGinnis managed to find out something about the structure of these very special genes: they all contained a very similar section, which the scientists called homeobox. This homeobox codes for the so-called homeodomain, which is chemically designed in such a way that it binds to certain DNA sequence motifs. Homeodomain proteins are transcription factors that can control the activity of many other genes. This homeobox was later found in several other genes that are not hox genes because they are not arranged in these clusters and cannot determine the identity of whole body segments. So all hox genes are homeobox genes, but not all homeobox genes are also hox genes.

The Hox genes, arranged in clusters, encode homeodomain transcription factors, which regulate the identity of certain body segments through regulating several other genes. Hox genes are characterized by colinearity: they are activated both temporally and spatially in exactly the same sequence as they are arranged in the clusters. Image modified from Hueber SD, Weiller GF, Djordjevic MA, Frickey T (2010) Improving Hox Protein Classification across the Major Model Organisms. PLoS ONE 5(5): e10820. CC BY 4.0.

 

After discovering the homeobox, Bill McGinnis soon founded his own laboratory at UCSD in San Diego. In his research, he stuck to homeobox transcription factors and described many more of them. In 1993 he found the gene product of one of them, to be present in only in a few nerve cells in the fly's brain, which is why he called this gene brain-specific homeobox.

Ten years later, in 2004, researchers in Milan described the Bsx gene for the first time in mice. They found it was only active in a few parts of the brain, including the pineal gland and hypothalamus.

 

The Magical Mystical Pineal Gland

The pineal gland is a small structure in the brain that fascinated people a lot for a long time. The famous French philosopher René Descartes (1596 - 1650), for example, saw the pineal gland as the mediator between the body and the mind, two completely different spheres in his thinking. To date, you can easily convince yourself by a google query on the pineal gland, that this structure is in the hands of esotericists of all kinds, who want to "activate" the pineal gland, for example through psychoactive substances or through certain images and sounds. Give it a try, it's worth it (I googling it; not the drugs)!

 

René Descartes imagined the pineal gland as the seat of the soul. Image from Wellcome Library, London. Wellcome Images This email address is being protected from spambots. You need JavaScript enabled to view it. http://wellcomeimages.org via wikimedia.org. CC BY 4.0.

 

The pineal gland not only has an astonishing background in the history of ideas, but also a remarkable evolutionary history. It probably originated as a kind of light-sensitive organ, similar to an eye, in the middle of the head. However, as the mammalian brain continued to fold, it ended up in the middle of the brain, where it was rather dark. Nevertheless, it still responds to light, the information about which comes from the eye through a neural circuit. When there is no light, the pineal gland releases melatonin, a hormone that makes us sleepy. Many of you may know that the model organism I'm working on, is the zebrafish. In fish, the pineal gland is (still) located right under the skull and is directly sensitive to light. 

After preliminary experiments from our laboratory indicated that Bsx might be important for the development of dopamine-producing neurons. Thus we were all very excited when in 2012 a new type of programmable nuclease came into play: TALENs. TALENs work very similar to the CRISPR/Cas9 system, which was developed shortly after that. Both tools can be used to create a double-strand break in the DNA at (almost) any position in the genome. It was really cool to see how I could actually create a mutation in the gene of my choice, Bsx. Unfortunately, some of my euphoria evaporated when I found that the dopamine-producing nerve cells in the manipulated fish still looked fine. So Bsx appears to be not required for the normal development of dopamine-producing neurons.

A neurotransmitter system that is very similar to dopamine is the serotonin system, and a marker for serotonin neurons was just one of many markers which I stained in fish with no working Bsx gene. I just wanted to find something useful; this Bsx gene had to do something! I didn't even know, or had forgotten, that serotonin is also a direct precursor to melatonin until I saw a small patch in the brain of my fish where the serotonin marker was staining – but only in normal fish. This staining was completely gone in the fish with no functional Bsx. It appeared that these fish couldn't produce melatonin at all!

 

Bsx in the Pineal Gland

Suddenly my low motivation was history and it followed about a year in which I read as much as I could about the pineal gland and examined this fascinating little structure in my fish with broken Bsx gene in all possible ways. My laboratory colleagues and my boss groaned more and more in meetings. "Oh dear, she's talking about this pineal gland again".

I quickly found out from the literatur that the zebrafish pineal gland is interesting and very well studied in another respect. In embryonic development, a few cells emerge here, which subsequently move to the left; always to the left! There they form a small structure called the parapineal organ. This small organ is necessary for the surrounding brain areas, the habenulae, to develop asymmetrically. Because, like us, the zebrafish naturally has an asymmetrical brain. I was thrilled and couldn't wait to find these cells in my fish and in fact I found them - not! They were simply missing from the bsx mutant fish and as a result their brains developed completely symmetrically.

In zebrafish, the light-sensitive pineal gland sits directly under the skull. It secretes melatonin in the dark. The development of the parapineal organ, to the left of the pineal gland, is important for the two halves of the brain to develop asymmetrically.

 

When I put all of this into a manuscript and submitted it for publication in a well-recognized journal, I was very nervous. I somehow felt like an intruder into a field that I didn't even know about a year ago. When the reports were generally positive, I was all the happier. The description of all these functions of the Bsx transcription factor in the pineal gland became my first paper.

 

Bsx in the Hypothalamus

As for the literature, there is one function of the Bsx gene that has been well described. Studies in mice lacking the Bsx transcription factor found that Bsx regulates the activity of two factors: Agouti-related peptide (Agrp) and Neuropeptide Y (Npy). Agrp and Npy are so-called orexigenic factors; they are upregulated by hormones that are released by the empty stomach and prompt the organism to search for food. In short: Agrp and Npy lead to a feeling of hunger and appetite. The neurons that act as a link between the hormones from the stomach and the so-called "higher order" neurons that lead to foraging and eating, are located in the arcuate nucleus, a small area that belongs to the hypothalamus.

So far, the Bsx gene/protein was mainly known to activate the appetite-stimulating factors Agrp and Npy in a small part of the hypothalamus, the arcuate nucleus.

 

However, Bsx is expressed in a much larger area of ​​the hypothalamus, which extends far beyond the arcuate nucleus. What is Bsx doing there? No one had answered this question before. Over the years, however, I've actually stumbled across a few factors that don't seem develop normally in the hypothalamus of bsx mutant fish. But a new obstacle appeared.

 

Neuroanatomy, who would have thought...

When we describe gene functions in zebrafish, we usually want to make statements that are also valid in mammals and thus in us people. Since the genetic makeup of fish is quite similar to ours, this usually works quite well and many gene functions have remained the same throughout evolution. But this time my problem was not to compare the genes of the fish with those of mice or humans, but rather the brain regions in which I had found something. The hypothalamus of a fish just looks quite different than ours and although the zebrafish has become such a popular model animal, surprisingly nobody seems to have yet thoroughly considered which areas of the fish hypothalamus could relate to which parts of the mammalian hypothalamus. So I started to do a few stainings to identify areas in the fish that are well defined in mammals. Well, this soon enough got completely out of hand. Each staining helped me to create anatomical homologies between the zebrafish and mammals, but always raised new questions which I tried to answer with further staining. After a few months, I probably had over a hundred such stainings, which I tried to put together like a puzzle to form a large picture. When it suddenly made sense and I had a kind of map for the fish hypothalamus in my hand, it suddenly occurred to me: what I did was pure neuroanatomy! This hypothalamus map actually became so extensive that I made it into a paper of its own. This is how the puzzle looked at the end, with all these funny letters standing for even funnier names I am not going to explain in detail:

 

 

Neuroanatomy can really be that colorful. Image from Schredelseker T and Driever W (2020) Conserved Genoarchitecture of the Basal Hypothalamus in Zebrafish Embryos. Front. Neuroanat. 14:3. CC BY 4.0.

 

Equipped with this anatomical reference, I finally had names for all those areas of the fish hypothalamus and was therefore able to describe the regions in which something goes wrong when Bsx is missing. Because in fact, over the years I found the precursor transcripts of several neuropeptides in very different areas of the hypothalamus to be dependent on Bsx. Also monoaminergic neuron populations, for example neurons that release serotonin or histamine, appeared to be affected or were missing in bsx mutant fish. Neurons that use nitric oxide as neurotransmitters were also greatly reduced in number. I then summarized all of this in the third and final paper of my doctoral thesis and earned this very very beautiful hat you saw above.

 

Equipped with my self-made hypothalamic map, I finally had names to describe the areas where I had discovered factors that were missing in fish without Bsx. Image from Schredelseker T, Veit F, Dorsky RI and Driever W (2020) Bsx Is Essential for Differentiation of Multiple Neuromodulatory Cell Populations in the Secondary Prosencephalon. Front. Neurosci. 14:525. CC BY 4.0.

 

And so I've reached the perfect place to say Thank You! Thanks to everyone who supported me on this way! My family and friends, my laboratory colleagues, my boss and of course all readers of this blog! You're awesome!