Klara, a transparent fish for aging research

Characterization of ready. (ONE) Expression for csf1ra was analyzed via RT-PCR using cDNA from FACS-sorted populations of lymphocytes, stem cells and myeloid cells obtained from wild-type whole kidney marrow (WKM) N. furzeri. Csf1ra was detected in all subpopulations, most strongly in myeloid cells. As a negative control, an RT sample (no reverse transcriptase during cDNA synthesis) was used to exclude contamination with genomic DNA. As load control, rpl13a was used. (B) Comparison of cell numbers in the different subpopulations of WKM of fish with the following genotypes: mitfa-/-ltd-/-csf1ra+/+ (n=2), mitfa-/-ltd-/-csf1ra+/- (n=4), and mitfa-/-ltd-/-csf1ra-/- (n=4). One-way ANOVA followed by Tukey’s post hoc test revealed no significant differences. Horizontal line represents median. whiskers show min. to max. value. (C, C’) Man ready fish (C) showed an appearance of melanophores on fin appendages, which was not observed in females (C’). (D,D’) Occurrence of melanophores intensified with age resulting in black fins in male fish (D). In females ready animal (D’) black fins were not observed. Credit: eLife (2023). DOI: 10.7554/eLife.81549

The body’s pigmentation of an organism is caused by pigments and structures found in the cells of skin, hair, feathers or scales, for example. This pigmentation significantly limits investigations of important processes in a living organism (in vivo). To provide a detailed overview of the living body, researchers have used transparent model organisms.

Successfully generated transparent fish models, for example in zebrafish or medaka (Japanese rice fish), are already being used in cancer research. Lifespans of up to five years in the zebrafish or two years in the medaka have largely limited the use of these species in aging research, as they make such investigations too long and expensive.

Researchers at the Leibniz Institute on Aging—Fritz Lipmann Institute (FLI) in Jena have now succeeded in generating a transparent killifish (Nothobranchius furzeri), called klar, using CRISPR/Cas9 technology. With a lifetime of only one year at most, it facilitates clear in vivo studies of age-related processes. This means that new discoveries about the role and function of the cell can be investigated much more extensively and in more detail in the future. The current study has now been published in eLife.

Turquoise killifish – an animal model in aging research

“In recent years, we have successfully established the turquoise killifish – originally from East Africa and known in German as the turquoise great ground carp, due to its beautiful, striking color – as a new model organism in aging research at our institute ,” reports Professor Christoph Englert, research group leader at the Leibniz Institute on Aging—Fritz Lipmann Institute (FLI) in Jena and professor of molecular genetics at FSU Jena.

“With a maximum lifespan of up to one year, this fish is so far the shortest-lived vertebrate that can be kept in the laboratory. Moreover, it is genetically similar to humans, ages extremely quickly and shows typical signs of aging, which makes it very interesting for aging research,” adds Prof. Englert to.

With the complete sequencing of the N. furzeri genome at FLI, an important basis for future analysis has been created: the ability to specifically turn genes on and off and thus learn about the influence of individual genes on aging or in relation to aging-related diseases.

Inactivation of pigmentation using CRISPR/Cas9

“In the turquoise killifish, there are three pigment cell types that are responsible for the fish’s very beautiful color,” explains Dr. Johannes Krug, postdoc in the Englert Research Group.

“The availability of the killifish genome sequence gave us the opportunity to investigate whether sequence-specific genome editing methods, such as CRISPR/Cas9, could be used to inactivate genes responsible for body pigmentation to obtain a transparent fish for use in aging research. These studies were the main focus of my doctoral thesis at FLI.”

‘Klara’ – the transparent killifish

CRISPR/Cas9 is a molecular biology method that can be used to inactivate or modify genes in a sequence-specific manner, as if cutting them with scissors. Using this method, the Jena researchers succeeded in disabling the genes responsible for the fish’s pigmentation, thereby generating a transparent killifish for the first time. The transparent fishing line, which the researchers called klar (“klar” is the German word for “clear”), now gives scientists a clear view of the internal organs and their development inside a living animal.

The transparent fishing line currently includes around 200 animals at FLI, both males and females, which can now be used in a number of aging research projects.

What role do senescent cells play in the aging process?

“Our transparent fish has great potential for aging research and opens up a wide range of completely new areas of application. In my group, the new transparent fish line is already being used for in vivo studies of senescent cells,” reports Prof. Englert. Senescent cells are cells that no longer divide and affect surrounding cells and tissues by constantly releasing pro-inflammatory factors. So far, little is known about the role and function of these cells.

It is known that in humans, but also in mice or killifish, the number of senescent cells increases with age, causing a kind of permanent inflammation in the body during the aging process. Targeted removal of senescent cells can therefore contribute to better health or even slow down the aging process. Research and development of substances known as senolytics, which can effectively remove aging cells from the body, is therefore of great interest not only to the pharmaceutical and antiaging industry, but also to aging research.

“With the klara line, we can now investigate the role of senescent cells in the living organism at a molecular level. By labeling them with fluorophores and then examining them under a fluorescence microscope, we can learn where they appear in the body and whether they are possibly clustered in specific locations, and what effects their removal has on the surrounding cells and tissue,” says Dr. Krug, highlighting the benefits of clara. This will lead to new insights regarding the role and function of this particular cell population during aging.

More information:
Johannes Krug et al, Generation of a transparent killifish line through multiplex CRISPR/Cas9-mediated gene inactivation, eLife (2023). DOI: 10.7554/eLife.81549

Journal information:

Provided by the Fritz Lipmann Institute

Citation: Klara, a transparent fish for aging research (2023, March 17) retrieved March 19, 2023 from https://phys.org/news/2023-03-klara-transparent-fish-aging.html

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