In 1999, a clownfish (Amphiprion ocellaris) hatched in the aquarium of a tropical fish hobbyist in the UK.
These clownfish are prized by aquarists for their unique pattern of three straight white bars bordered by a thin black line. But this UK fish was special: instead of the usual straight bars, it had wavy, corrugated patterns, symmetrical on both sides. The patterns were inherited across generations, leading to a lineage named "Snowflake," but the mechanism causing this irregular patterning remained a mystery.
Two decades later, researchers from the Okinawa Institute of Science and Technology (OIST), Academia Sinica in Taiwan, Kyoto University, and the University of Virginia have finally revealed the exact gene responsible for the change, and in the process, have uncovered clues towards the overarching mystery of how nature creates regular patterns. Their findings were published in Nature Communications .
"Conceptually, it should be simple," says Professor Vincent Laudet from the Marine Eco-Evo-Devo Unit at OIST . "But in practice, it's mysterious. How does a cell know whether to be black, white, or orange, in a way that consistently creates clearly organized patterns at a macroscale? Snowflake has now not only pointed us to a genetic mechanism, but also to a universal framework for studying pattern formation across species."
From a single amino acid change to universal models
Snowflake fish are like other clownfish in all respects except for their unusual patterning, making them the perfect model organisms for investigating the genetics of pigmentation. To understand why they look the way they do, the researchers turned to another fish. Zebrafish are well-studied for their horizontal lines, which stay proportional in size and spacing as the fish grows. Thanks to another mutant, the "Leopard" zebrafish, which develops spots instead of stripes, researchers have previously identified one of the genes implicated in pattern formation. It encodes a specific gap junction protein that essentially serves as a telephone wire between cells, allowing them to exchange information in the form of electric current and small molecules.
When the team compared the genomes of Snowflake to wild-type clownfish, they discovered a striking similarity. Laudet says: "We saw it straight away — Snowflake had a mutation in exactly the same gap junction gene as Leopard!"
However, this eureka led to more questions than answers. In zebrafish, the gap junction protein was thought to be responsible for directing so-called Turing pattern growth. Named after the famous British mathematician Alan Turing, this model describes how the protein mediates stripe formation by inhibiting short-range contact while promoting long-range interactions between pigmentation cells, leading to the development of uniform patterns in zebrafish.
But the Turing model couldn't account for clownfish patterning, as their stripes are fixed in sequence and position throughout their lives, meaning that information is instead exchanged to specify where and when a bar should form. The study's first author, Dr. Marleen Klann of the Marine Eco-Evo-Devo Unit, continues: "We found that the gap junction protein is not specific to Turing patterning in zebrafish, and showed that it ensures clear cell-to-cell communication more generally. It is evidently also much older than we thought: freshwater zebrafish and saltwater anemonefish diverged more than 200 million years ago."
This key piece of evidence points researchers toward a universal biological mechanism for organizing bordering cells. The team turned to another domain to better understand the principles governing the organization of pigmentation cells in clownfish: membrane physics. Here, they found that the so-called Edwards-Wilkinson model is the simplest possible model that accurately accounts for the formation and disruption of the clean border between pigmentation cells in clownfish and beyond.
"The model describes two forces," explains study co-author Professor Simone Pigolotti from the Biocomplexity Unit at OIST . "One is surface tension, which favors a smooth membrane. The other is noise, which has the opposite effect. The balance between those two forces determines the degree to which the membrane — and the pigmentation pattern — is corrugated."
"The model is a general tool that allows us to understand our observations as well as provide clues about where to look next, including across species, which in turn can help elucidate general principles. It's a positive spiral between theory and experimentation," says Pigolotti.
Laudet concludes: "It's thanks to Snowflake and our transgenic anemonefish, which we've successfully engineered with Professor Masato Kinoshita at Kyoto University, that we're now a step closer to understanding the amazingly complex mechanisms behind the conceptually mundane task of cellular organization."
