by Amanda Hill, Highlands Ranch, Colorado
When studying human disease and working to develop treatments, researchers typically perform experiments on animal models that represent the disease in a controlled laboratory setting. However, in the case of FSHD, creating an animal model which accurately represents the human disease has proven difficult.
One reason is that FSHD is highly variable from person to person. Another is that the specific form of the DUX4 gene found in humans only exists in other primates, not in common laboratory animals such as mice or fish. So in order to use these common lab animals, scientists must first artificially express the DUX4 gene in them. This can be a difficult and delicate process to get just right.
Now, add to that the fact that scientists are learning about DUX4 every day. They are still trying to understand exactly how, where, and when DUX4 is expressed in FSHD, so artificially re-creating the gene’s behavior in laboratory animals is no easy task. It’s like building a house of cards ─ with a blindfold on.
For a number of years, FSHD scientists have been diligently working to create animal models of FSHD using fruit flies, frogs, zebrafish, and mice. To date, no single model fully recapitulates all the clinical realities of the human disease. However, each model provides unique pros and cons for scientific studies and has taught researchers how to improve their strategies for the next model.
Recently, Louis Kunkel, PhD, and his team in Boston published the results of their work establishing and characterizing a new model of FSHD in zebrafish. Zebrafish are handy creatures for studying muscle diseases because their translucent bodies offer relative ease in seeing and assessing muscle abnormalities. The FSHD zebrafish was engineered so that the researchers can turn on expression of DUX4 in muscle cells by adding a specific chemical to their water. This is a widely accepted strategy in the field and is simple in theory, but not in practice.
You see, the amount of drug that is added to the water affects how much DUX4 will be expressed. And there’s the question of when to express DUX4 ─ when the fish is an adult, or a young hatchling, or immediately after an egg is fertilized? And finally, you have to know for how long to express DUX4. An hour? A day? A week? Answering these questions is where that blindfold comes in. Scientists can’t yet fully answer all these questions about how DUX4 behaves in human FSHD, let alone know how to translate that to zebrafish.
Nevertheless, Dr. Kunkel and his team succeeded in creating a model that has led us one step closer to accurately representing FSHD in the laboratory and better understanding the underlying pathophysiology. The team decided to turn on expression of DUX4 in the zebrafish one day after fertilization of an egg and for only 24 hours, representing a scenario in humans where DUX4 would be expressed only briefly during early development, and its levels would decrease over time to be largely undetectable later in life. Remarkably, this strategy produced zebrafish FSHD that behaved a lot like human FSHD.
In this zebrafish model, not every muscle cell ended up expressing DUX4. Instead, the expression pattern was mosaic, similar to what has been observed in FSHD patient biopsies. At a young age, about 30 percent of zebrafish had abnormal muscle fiber formation and could not swim as far as their non-FSHD counterparts, while the remaining 70 percent appeared unaffected. This is not unlike the percentage of people with FSHD who have earlier onset of symptoms. A bit later in development, all the FSHD zebrafish, on average, generated less force with their muscle contractions than the non-FSHD controls.
As adults, all the FSHD zebrafish exhibited varying levels of abnormal muscle structure, mild inflammation, and asymmetric replacement of muscle with fat or collagen ─ much like in humans. Some of these fish tended to swim more slowly than regular zebrafish, while others were just as fast, again representing the spectrum of disability in human FSHD, even as adults.
Overall, Dr. Kunkel and his team deemed this particular zebrafish model to represent a relatively mild form of FSHD. They went on to compare this artificially expressed DUX4 model to another zebrafish model they had previously established using a different technique. That model had many similarities, but the disease symptoms tended to be more severe. Importantly, the team noted that in both models, temporary expression of DUX4 during early development was sufficient to spawn FSHD later in life.
This observation could be of paramount importance, and suggests that the presence of DUX4 in an adult may not be the sole cause of FSHD. Instead, it could be that DUX4 expression in a fetus or a young child is responsible for setting off a chain of events that eventually leads to the development of FSHD later in life. In that case, one would have some degree of FSHD symptoms regardless of whether DUX4 was still expressed in an adult. Dr. Kunkel and his team are the first to suggest such a theory, and if their hypothesis is correct, it would have important implications for how to treat FSHD.
Much work must still be done to determine whether this observation holds true in humans, and to what extent DUX4 expression during development versus adulthood contributes to disease progression and severity. Moving forward, Dr. Kunkel’s team plans to use these zebrafish models to test potential therapeutic compounds and their ability to correct the FSHD symptoms exhibited by the fish. This will be an important precursor to moving into mouse models, and eventually into humans.
Pakula A, Lek A, Widrick J, Mitsuhashi H, Bugda Gwilt KM, Gupta VA, Rahimov F, Criscione J, Zhang Y, Gibbs D, Murphy Q, Manglik A, Mead L, Kunkel L. Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis. Hum Mol Genet. 2019 Jan 15;28(2):320-331. doi: 10.1093/hmg/ddy348.
Lek A, Rahimov F, Jones PL, Kunkel LM. Emerging preclinical animal models for FSHD. Trends Mol Med. 2015 May;21(5):295-306. doi: 10.1016/j.molmed.2015.02.011. Epub 2015 Mar 20. Review. PubMed PMID: 25801126; PubMed Central PMCID: PMC4424175.