Why Use Zebra sh to Study Human Diseases?

irp.nih.gov

By Elizabeth Burke

Tuesday, August 9, 2016

Scientists use a variety of laboratory techniques to investigate the genetic

cause of human diseases. Research often utilizes patients’ cells or tissue
samples, but to determine if a mutation in a speci c gene can cause a
patient’s symptoms, we often need experimental animal models.

While mice and rats have been common choices for modeling human
diseases in the past, the use of zebra sh is rapidly gaining popularity. Does
this surprise you? Let me explain.

What are zebra sh?

Zebra sh are tropical fresh-water sh in the minnow family. In the wild, they
are found in rivers and ponds of India, however they are now often available
in pet shops. The name “zebra sh” comes from the horizontal blue stripes
on each side of their bodies.

Zebra sh, so named due to their stripes, prefer to live in large groups called
shoals.
How can you model a human disease in sh?

Although humans may appear to be extremely different than zebra sh, we

are actually much more similar to them than you might think. In fact, 70% of
human genes are found in zebra sh.

Moreover, zebra sh have two eyes, a mouth, brain, spinal cord, intestine,
pancreas, liver, bile ducts, kidney, esophagus, heart, ear, nose, muscle, blood,
bone, cartilage, and teeth. Many of the genes and critical pathways that are
required to grow these features are highly conserved between humans and
zebra sh. Thus, any type of disease that causes changes in these body
parts in humans could theoretically be modeled in zebra sh.

Why use zebra sh when you could use mice?

While mice are evolutionarily more similar to humans because they are
mammals, zebra sh have several advantages over their furry competitors.

One important advantage of zebra sh is that the adults are small and prefer
to be housed in large groups, or “shoals”. As a result, they require much less
space and are cheaper to maintain than mice.

The NIH Zebra sh Core houses hundreds of thousands of zebra sh in a

state-of-the-art facility.
Another advantage is that adult zebra sh breed readily (approximately every
10 days) and can produce as many as 50 to 300 eggs at a time. This is quite
different from mice as they generally produce litters of one to 10 pups and
can only bear approximately three litters in their lifetime. Scienti c
experiments are generally repeated multiple times in order to prove that the
results are accurate, so having an animal that can produce a large number
of offspring over and over is helpful.

Zebra sh embryos are also laid and fertilized externally, which allows them
to be easily manipulated in a variety of ways. In vitro fertilization can be
performed if necessary. The one-cell-stage fertilized eggs can be easily
injected with DNA or RNA to permanently modify their genetic makeup in
order to generate transgenic or knock-out zebra sh lines. Working with mice
in this way is much more complicated. Mouse embryos develop inside the
mother, and to access and manipulate them the mother would have to be
sacri ced. To keep the embryos alive after fertilizing or injecting them, they
would need to be transplanted into another female mouse, as well.

Zebra sh larva, the stage of development from between three and thirty
days post-fertilization, grow in length from approximately 3.5 to 8
millimeters.

Furthermore, zebra sh embryos are clear, which allows scientists to watch

the fertilized eggs grow into fully formed baby sh under a microscope.
Their transparency also enables the visualization of uorescently labeled
tissues in transgenic zebra sh embryos. Mouse embryos are not clear and
develop inside the mother, so the observation of live embryo development
like that in zebra sh is not possible.

However, there is a limit on what types of diseases can be studied in

zebra sh. Human diseases caused by genes that do not exist in zebra sh
require a different animal model. Additionally, zebra sh are not useful
models for human diseases that mainly take place in a tissue type or body
part that zebra sh do not have (e.g., prostate, mammary glands, lungs).

How exactly do you use zebra sh to investigate human diseases?

Often a patient’s DNA is sequenced in order to nd a mutation in a gene that

could potentially cause his or her disease symptoms. To determine if loss
of function of that gene could cause the symptoms seen in the patient, the
same gene is mutated or “knocked-out” in zebra sh, and then the sh are
examined for similar symptoms. Although it is much more di cult to do, the
exact mutation that the patient has can be introduced into zebra sh as well
—this is called a “knock-in”.

If one or more of the patient’s symptoms are observed in the zebra sh

knock-out or knock-in model, the zebra sh can be used for further studies
to help determine why the mutation in that gene causes the disease. For
instance, the structure of the muscle bers can be examined for
abnormalities under the microscope if the patient has a muscle disease. Or
if the patient’s disease symptoms began during development in utero,
knock-out or knock-in zebra sh embryos can be examined for gene
expression changes (compared to embryos without the mutation) that
could lead to abnormal development. For a patient with a neurological
disease, the neurons of knock-out embryos can be uorescently labeled to
see if they form incorrectly.
In addition to utilizing zebra sh disease models to characterize human
diseases, researchers can also identify and test new drugs to treat the
diseases being modeled. The ability of zebra sh to generate many embryos
every time they breed makes them especially useful for high throughput
drug screening.

What are some examples of human diseases that have been successfully
modeled in zebra sh?

The generation of a knock-out of the dystrophin gene in zebra sh has been

shown to closely resemble the severity and progression of the human
disease Duchenne muscular dystrophy. Patients with Duchenne muscular
dystrophy have been found to carry mutations in dystrophin and
demonstrate childhood muscle weakness that gets progressively worse. In
both humans and the zebra sh model, the loss of dystrophin gradually
leads to necrotic muscle bers that are replaced by in ammatory cells,
brosis, and abnormally sized muscle bers.

This gure shows visual differences in muscle between wild-type zebra sh

larva (A, B, C) and distrophic larva (A’, B’, C’). Source:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484855/

Human melanoma has also been successfully modeled in zebra sh. The
most commonly identi ed mutation in human melanomas—a single amino
acid change in the gene BRAF—was created in zebra sh to make a knock-in
model. Since cancers are caused by a combination of several genetic
alterations, this knock-in zebra sh line was used to screen other potential
cancer causing mutations. When another commonly observed melanoma
mutation of the gene SETDB1 was added to the BRAF knock-in zebra sh, a
melanoma rapidly developed. These results helped to establish that
SETDB1 is an important gene in melanoma growth.

Images of a knock-in zebra sh that expresses the BRAF mutation alone

(top) and one that was also injected with a transposon-based vector
(miniCoopR) containing a mutant form of the gene SETDB1 (bottom). The
addition of the SETB1 mutation resulted in melanoma (indicated by the
arrow).  Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348545/

Those examples of how humans and zebra sh can manifest the same
disease despite how different we appear make it is easy to understand why
zebra sh are becoming a well-accepted animal model. Here in the NIH
Undiagnosed Diseases Program, we perform studies using zebra sh as one
of several approaches to investigate the potential involvement of altered
genes in our patients’ extremely rare diseases. While mice have been the
predominant animal bridge between the bench and bedside in the past,
recent studies have demonstrated the potential of zebra sh to serve as a
tractable alternative to mice. The timing of the adoption of zebra sh as an
emerging model organism could not be better, as mouse studies often fail
to translate to humans. Although no animal can perfectly model a human
disease, I believe these little striped swimmers have great potential for
advancing medical research in the future.

To learn more about how zebra sh contribute to biomedical science and

human health, visit the websites for the Trans-NIH Zebra sh Initiative
website and the NICHD Zebra sh Core.