The vertebrate transition from fin to limb is one of the juiciest mysteries in evolutionary biology, and this week, scientists may have identified another clue to the puzzle. Published online today (June 23) at Nature, a team of researchers describe two previously unknown proteins essential to fin development in bony fishes -- the loss of which may have been a key step in the evolution of fins to limbs during tetrapod development.

"It's a very exciting piece of work," said Paula Mabee, an evolutionary biologist at the University of South Dakota. "Their conclusions are really intriguing and well justified."

Top: Pectoral fin bud of zebrafish embryo showing the array of actinotrichia. Bottom: Limb bud of an mouse embryo. In contrast to the pectoral fin bud of teleost fish, the mouse limb bud does not contain actinotrichia. Jing Zhang

The discovery happened almost by accident during a routine screen for genes involved in fin regeneration in the zebrafish labs of Marie-Andree Akimenko and Marc Ekker at the University of Ottawa in Canada. To identify several unknown genes that popped up during the screen, the team analyzed their expression in the developing zebrafish, and for two of the genes, noted a remarkable pattern: a unique and specific localization along the early median fin fold, which runs along a fish's back, and in the pectoral fin buds, fins that grow on each side of the fish's body behind the gills.

The gene expression pattern suggested the proteins are structural components of actinotrichia -- thin, rigid fibrils that form a scaffold upon which bony fins develop. "This the first time that anyone has really looked at genes underlying actinotrichia," said Mabee.

Akimenko's team named the proteins actinodin 1 and 2 (And1 and And2). A database search identified two other closely related genes, and3 and and4, and determined that none of the four genes are present in tetrapods, four-legged vertebrates.

To determine the function of the two novel proteins, the team knocked down their genes, and1 and and2, using morpholinos, molecules that silence gene expression. When the genes were knocked down individually, fin development proceeded normally. "They probably have a redundant function," Akimenko told The Scientist. But when both were silenced, major changes occurred: First, actinotrichia didn't form. And without actinotrichia, there was no regional recruitment of mesenchymal cells, the precursors to the bones of the fin exoskeleton. It is likely the structural changes would prevent fins from correctly forming, resulting instead in short, truncated fins, the authors suggest. (Morpolinos only knockdown genes transiently, so the team couldn't demonstrate the effects on later stages of development).

But gene expression patterns in the region were also extensively disrupted, especially those of genes important for patterning in both fins and limbs in tetrapods, like fibroblast growth factor and sonic hedgehog. The knockdown gene expression profile is highly similar to an experimental chick and mouse mutant called Gli3 that results in polydactyly, the presence of numerous digits, said Akimenko. Interestingly, one of the earliest aquatic tetrapods, Acanthostega, had eight digits on each hand. "The loss of actinotrichia may have been conducive to ancestral, primitive tetrapods having limbs resembling cases of polydactyly," said Akimenko.

The researchers are now looking for alternative methods to prolong the gene silencing of and1 and and2, to see whether or not the fin ray will develop, and if so, what it will look like. Perhaps like a limb with multiple digits? The team will also try reintroducing the genes in tetrapod species, which normally lack the genes, to see how they affect limb development. "We will continue [investigating] this story," said Akimenko.

The study raises a wealth of evolutionary questions, said Mabee, including when the genes originated and how they might be involved in the evolution of other fins, such as the adipose fin, found on the back of some fish species but not others. "It really made me think," she told The Scientist. "There is a lot [of work] remaining."

J. Zhang et al. "Loss of fish actinotrichia proteins and the fin-to-limb transition," Nature, published online June 23, 2010, doi: 10.1038/nature.09137.


Related stories:

Of mice and paws
[19th February 2009]

Early fish had live birth
[25th February 2009]

The Regeneration Recipe
[1st August 2009]