Transparent line of turquoise killifish called klara
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Transparent Killifish Enable In Vivo Light Sheet Microscopy with Single Cell Resolution

Successful in vivo imaging of GFP-proteins with single cell resolution inside entire fish achieved with light sheet imaging.

To effectively study aging processes and age-related diseases, researchers have established the turquoise killifish (Nothobranchius furzeri) as a model organism in research on aging. This killifish has a short lifespan of 6-12 months. During this period, it undergoes a full aging process with typical aging symptoms similar to mammals, including spine deformation, decreased locomotor activity, and loss of muscle mass.

One limitation of the killifish for microscopy experiments is its body pigmentation. A research team headed by Prof. Dr. Christoph Englert at the Leibniz Institute on Aging, Fritz Lipmann Institute (FLI), Germany, has published an article that shares their work generating a transparent killifish line. Successful in vivo imaging of a GFP-senescence reporter line with single cell resolution inside entire fish was achieved with light sheet imaging. The transparent fish, which they call klara, will be a critical tool in their research on the genes that regulate organ development and aging.

 

Dr. Johannes Krug with klara killifish

Killifish have been used for behavioral studies, genome editing approaches or experiments including organ removal to perform immunohistological analyses. In vivo studies using fluorescence microscopy have been challenging due to their pigmentation and klara will advance these types of experiments.

Dr. Johannes Krug

Postdoctoral Researcher and Lead Author, Molecular Genetics, Leibniz Institute on Aging, Fritz Lipmann Institute (FLI), Germany

Wild type (pigmented) and klara (non-pigmented) killifish

A wild type killifish in the foreground shows its pigmented patterning compared to a klara killifish in the background.

Generation of klara

A Transparent Killifish Line

In order to generate a transparent killifish line, Dr. Englert's team targeted three genes involved in pigment development for disruption using CRISPR/Cas9: mitfa, ltk, and csf1ra.

They were able to achieve fully transparent animals with homozygous triple mutants showing normal behavior, fertility, and general health. Referring to its "clear" appearance, they named the line klara.

Subsequent work was done to generate a GFP-senescence reporter line based on the senescence marker gene cdkn1a (p21) to test not only if klara could be used as a platform for further genetic manipulation, but also to explore if their work in generating a transparent fish would lead to successful experiments with in vivo fluorescence imaging.

 

 

In Vivo Imaging of Transparent GFP-Senescence Reporter Fish

Imaged using Light Sheet Microscopy

Successful in vivo imaging of GFP positive cells in the dorsal fin area of living klara fish at 17 dph showing cellular senescence with single cell resolution using light sheet imaging.

In Vivo Light Sheet Microscopy of klara Killifish

The GFP reporter line successfully reports cellular senescence

The cdkn1a gene is upregulated in older killifish and is a commonly used marker for senescent cells. As killifish age, they accumulate higher numbers of senescent cells. Dr. Englert's team generated a klara-based senescence reporter line by inserting a GFP reporter cassette into the cdkn1a locus.

Not only did they confirm that this reporter line was functional through initial gamma irradiation tests performed in embryos and fluorescence microscopy, they were successfully able to image living fish with single cell resolution using light sheet microscopy. This microscopic technique enables imaging of anesthetized fish while monitoring their vital functions.

Dr. Johannes Krug with ZEISS microinjection station

The ZEISS light sheet system gives us the resolution we need to identify and analyze individual cells. Our goal is to image the same fish throughout its life cycle, placing it back in the fish tank after imaging. We aim to analyze and understand the accumulation of senescent cells during aging and investigate their impact on life- and health span.

Dr. Johannes Krug

Postdoctoral Researcher and Lead Author, Molecular Genetics, Leibniz Institute on Aging, Fritz Lipmann Institute (FLI), Germany

Research team with pages from their publication in eLIFE
Research team with pages from their publication in eLIFE

Top Right Inset: Research team members, left to right, Dr. Johannes Kurg (lead author), Prof. Dr. Christoph Englert (corresponding author and group leader), Dr. Birgit Perner (second author). Figures are from their article in eLIFE.

Top Right Inset: Research team members, left to right, Dr. Johannes Kurg (lead author), Prof. Dr. Christoph Englert (corresponding author and group leader), Dr. Birgit Perner (second author). Figures are from their article in eLIFE.

Generation of a Transparent Killifish and GFP-Senescence Reporter Line

In their publication, Dr. Englert and colleagues tackled the challenge of generating a killifish line that lacked pigmentation to overcome previous challenges with fluorescence microscopy. Their new transparent line, klara, can serve as a platform for further genetic modifications. This was shown by generation of a GFP-senescence reporter line, which they could successfully image in vivo using light sheet microscopy. 

The full details of their research can be found in their eLIFE article, including contact information for researchers interested in obtaining the klara line for their work.


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