Published online 23 June 2010 | Nature | doi:10.1038/news.2010.315

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Four-legged creatures may have gained a foothold by ditching genes guiding fin development.

Actinodin genes code for rigid fibres in zebrafish embryo fin buds. Neither genes nor fibres are found in mouse embryo limb buds. Jing Zhang

The loss of genes that guide the development of fins may help to explain how fish evolved into four-limbed vertebrates, according to a study.

In the late Devonian period, around 365 million years ago, fish-like creatures started venturing from shallow waters onto land with the help of eight-fingered limbs. The limbs had evolved from fins; during the transition, our back-boned ancestors lost rows of rigid fibres, called actinotrichia, that provide structural support and guide fin development. The number of digits was also later winnowed to a maximum of five on each limb.

Marie-Andrée Akimenko of the University of Ottawa in Canada and her colleagues may now be able to explain how our ancestors lost their fins: they have discovered a family of genes that code for the proteins that make up fins' rigid fibres. The actinodin (and) genes are present in the laboratory model zebrafish and in ancient fish, but not in four-legged vertebrates (tetrapods), the team report today in the journal Nature1. What's more, the researchers found that dampening the expression of and genes in zebrafish also disrupts the expression of genes that regulate the growth of limbs and the number of digits in other animals.

“The real question is: did we lose these genes because we lost the use of fins, or did we lose fins because we lost the genes?” Denis Duboule

Swiss Federal Institute of Technology, Lausanne

These results hint that the loss of and genes is linked to the change from fins to limbs. "It's a very nice example of how changes in one or two genes can be responsible for a huge evolutionary transition," says Axel Meyer, a biologist at the University of Konstanz in Germany who studies gene evolution in fish.

But a causal connection is not certain. "The real question is: did we lose these genes because we lost the use of fins, or did we lose fins because we lost the genes?" says Denis Duboule, an evolutionary developmental biologist at the Swiss Federal Institute of Technology in Lausanne (EPFL). "The problem is that when it's an evolutionary question, you can't do the experiment."

and action

The researchers looked for genes that were most actively expressed in zebrafish fins that were regrowing after amputation, and pinpointed two with previously unknown functions. Both genes code for proteins that make up a complex collagen-like structure called elastoidin, which is found in actinotrichia. Akimenko's team also searched the zebrafish genome database and found two additional genes that they predict produce similar proteins. The expression of all four and genes tracked the appearance of actinotrichia in the zebrafish embryo and in the regenerating fins of adults.

Databases for other bony fish also contain the genes, but they are not found in tetrapods. The gene family may have very ancient roots, as a partial and-like sequence appears in the genome of the elephant shark, which evolved 450 million years ago and is part of the oldest living family of jawed vertebrates.

The team went on to use morpholinos (small molecules that bind to RNA and prevent the manufacture of proteins) to dampen the expression of two and genes in zebrafish embryos. They found no actinotrichia in the embryonic folds that would normally give rise to fins, and the folds were underdeveloped and curled over.

When they disrupted the two genes in the regenerating fins of adults in the same way, they found that the distribution of actinotrichia was affected. Furthermore, zebrafish with reduced and expression showed abnormal expression of genes that regulate the growth of limbs and digits. When similarly abnormal in other animals, these genes can cause the growth of extra digits, like those seen in the early eight-fingered land vertebrates.

You lose some, you win some

"We tend to think that new genes bring new functions, but this study shows that the presence of genes constrains or directs development in certain directions," says Meyer. "Gene loss is actually a creative force in evolution."

The study was limited to only a few days by the short lifespan of the RNA-blocking morpholinos, so it was not possible for Akimenko to determine whether disrupting the and genes also prevents the formation of other parts of the fin skeleton that disappeared during the fin-to-limb transition.

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In the future, she plans to introduce and genes into mice and observe the effects on limb development. She would also like to examine the role of the other two and genes and determine how they are regulated. The genes were not the only factor in the evolution of fins into limbs, however: "this is only one little piece of the puzzle that may help us to understand this transition," Akimenko says.