Brain stem cells: how zebrafish are revolutionizing research

Stem cells, progenitor cells present in our adult organs, combine two properties. The first is to be able to proliferate and generate daughter cells committed to differentiation, that is to say the formation of specialized cells for the construction and functioning of tissues and organs.. The second, called “self-renewal”, is to be maintained in the long term, even after the numerous divisions which give rise to daughter cells.

In humans, stem cells are present in many tissues, such as the intestine and the skin, where their activity ensures the permanent renewal of the epithelium¹a protective barrier against the outside world, or the muscle, where they allow the reconstitution of a fiber damaged by exercise.

However, not all organs have stem cells. In humans, they are notably absent from cardiac tissue, and their presence in the brain until adulthood remains controversial. We can easily imagine the interest in being able to manipulate endogenous stem cells, or to generate and direct them in the laboratory, in vitro, with a view to transplantation to facilitate tissue repair in the event of damage due to trauma, ischemia ² or degeneration.

An imperfect process in humans

Our understanding of the mechanisms of stem cell formation in animals remains incomplete. But great advances have been made possible by research in developmental biology, an absolutely fundamental field of biology which aims to understand the choices of fate and the specialization of cells in the embryo for the genesis of tissues, organs, forms and functions. Thus, the identification of factors responsible for an early progenitor state in the embryo, or for engagement in differentiation, now allows the production of progenitor cells in vitro and their orientation towards a different cell type. A real revolution for modeling, understanding and hoping to treat many pathological conditions.

There is much left to discover. Today, the use of stem cells remains limited to a few tissues and the repair process is often imperfect, particularly in mammals, including humans. How can we better understand the mechanisms controlling the activation of stem cells, particularly when they are in their normal environment, in animals, and not in vitro, cut off from their environment?

This is where the zebra fish comes in, by its Latin name “Danio rerio”, an aquarium fish which belongs to the teleost family, like trout or salmon. Small in size (around 3 cm as an adult) and easy to raise, it was chosen as a model animal for developmental biology in the 1970s by George Streisinger (University of Eugene, United States). Thanks to the rapid development outside the female of its embryos as well as their transparency, we can follow the divisions, movements and fate of each cell in real time under a microscope, which is impossible with a mammal.

Furthermore, it has the same body organization plan as mammals and 70% of genes shared with the human genome. Its spectacular assets and contributions, associated with the implementation of genetic tools, have quickly pushed its use at all scales, including physiology, behavior and pathology. Today, more than 1,200 university and industrial laboratories study it.

On this common basis between teleosts and mammals, the variations are all the more interesting. At the forefront of these are stem cells and their potential. In zebrafish, studies over the last fifteen years show that they are present in abundance in all organs. Better, it is not a late addition, but a maintenance. At the embryonic stage, the number of progenitor cells for each organ is comparable between zebrafish and mice (in proportion to their size). But while they are gradually lost in rodents, they are maintained in this fish to give rise to long-lasting stem cells.

Thus, in the brain of adult zebrafish, up to 16 niches³ Neural stem cells have been described (compared to two or three in mice) which are involved in the permanent formation of neurons. These integrate into existing circuits, increasing their size and making it possible in particular to connect new sensory information from peripheral organs.

A battery of tools for studying zebrafish

What are the mechanisms that allow the maintenance of these stem cells and their long-term activity? A battery of tools is available in zebrafish to study them. For example, we can mark them genetically or chemically to follow them over time, modify the genes that are active there, and even film them within their niche.

This last method, developed in our team in collaboration with that of D^r Emmanuel Beaurepaire at the École Polytechnique, is particularly powerful. Called intravital imaging, it is based on fish spontaneously devoid of pigments and therefore transparent even in adulthood: we can thus, in a completely non-invasive manner, film populations of stem cells, over weeks or even months.

These analyzes highlighted at least three components involved in the maintenance and activity of neural stem cells in the adult zebrafish brain: in the stem cells themselves; at the level of their niche; and during the maturation of their daughter cells into neurons.

Concerning the intrinsic components of stem cells, our recent work on the forebrain and mainly the cortex has revealed the existence of a subpopulation capable of divisions allowing systematic self-renewal at the same time as the production of neurons. In mice, the corresponding cells exist but they have lost this capacity.

With regard to the niche, intravital imaging, which makes it possible to analyze at each moment and over time the comparative behaviors of neighboring stem cells, has provided information that is as unique as it is unexpected. For example, when a stem cell activates to divide, its immediate neighbors generally do not. There is thus a coordination of stem cells in their niche which limits activation events to distribute them homogeneously in this physical space, and protect the stem cells from excessive activation which would lead to their exhaustion. This coordination could be facilitated by the direct juxtaposition of stem cells in their niche, whereas in mice their spatial organization is looser.

Finally, we observed that the maturation of newborn neurons in the adult brain is efficient, leading to the connection and survival of the vast majority of neurons formed, whereas in mice only a minority achieves this. Research is underway to identify the exact mechanisms of these differences and determine whether it is possible to exploit them.

A fish capable of regenerating most of its organs

Another major difference between zebrafish and mammals concerns the capacity for neuronal regeneration. In mice, after injury or degeneration, it is weak, or even absent: the stem cells can show a certain activation but the neural progenitors generated do not mature. In zebrafish, on the contrary, regeneration is rapid and efficient. Thus, a mechanical injury in the cortex region is followed within a few days by a massive activation of local stem cells and the genesis of numerous mature neurons.

Although it has not been possible to reliably assess functional restoration in the cortex, this has been done in other territories responding to sensory information or controlling behaviors that are easy to measure, such as vision or locomotion. . Neuronal regeneration in zebrafish thus appears efficient, rapid and targeted. In most brain areas, it involves increased activation of endogenous stem cells, and is capable of redirecting their destiny towards the specific production of missing neurons.

A major component of the difference between zebrafish and mammals appears to be the inflammatory response, which is massive following injury and allows activation of stem cells. However, it is harmful in the long term in mammals, leading to the formation of a so-called glial scar which prevents the maturation and integration of the neurons generated. In zebrafish, this immune reaction is transient, and its rapid termination allows advanced repair. It remains to understand the mechanisms that stop this reaction, or the differences in inflammatory factors between species.

The examples are not limited to the nervous system: the zebrafish is capable of regenerating most of its organs (heart, liver, fins, etc.), and completely: it produces not only the missing cells but also their organization, thus as the shape and size of the organs.

Endogenous stem cells in adult tissues therefore have a lot to teach us about the factors that maintain them, activate them for tissue renewal, slow them down during aging, and recruit them in the event of repair. As well as on the mechanisms of reparative effectiveness, with the perspective of adapting them to stimulate endogenous neural stem cells in mammals, or to better prepare or direct the stem cells which are used to produce progenitors in vitro for transplantation. Zebrafish are well positioned for important new discoveries in these areas.