How Limbs Evolved From Fins

Around 400 million years ago a group of creatures made their way onto land to found the tetrapods, a clade encompassing all four-limbed vertebrates, from frogs to shrews to dinosaurs. Precisely how the transition from fins to limbs was accomplished is still unclear, but a team led by Denis Duboule has shed some light on the regulatory mechanisms involved. In a study published in PLoS Biology, they explored the connection between patterning in fins and limbs, investigating how each gets divided into regions during development.

Animal body plans are laid out by Hox genes, evolutionarily ancient genes which have diverged into different clusters as a result of genome duplications. The HoxA and HoxD clusters are essential for limb development in mammals, and their expression partitions the limb into two regions by the wrist/ankle: a proximal region closer to the body and a distal region which includes the digits. The spatial arrangement of Hox genes plays an important role in their regulation. For example, some Hox genes are arrayed within a cluster in the same order as the body segments. Duboule's team has recently shown that the expression of genes within the HoxD cluster is controlled by 3-dimensional interactions with non-coding DNA elements in the surrounding genomic regions. The cluster is divided into a 'bimodal' regulatory pattern, with genes at each end of the cluster interact with enhancers flanking the cluster on that side. The 5' and 3' genomic regions therefore form distinct regulatory landscapes, with enhancers in the 3' region promoting proximal gene expression and those in the 5' region promoting distal expression; genes located in the middle of the cluster are regulated by enchancers from both sides.

The first thing the team wanted to find out was whether the HoxA cluster was regulated in a similar way. If so, it would mean that this regulatory mechanism probably arose before the duplication at the root of the vertebrate clade that gave rise to HoxA & HoxD. The team found that the same spatial regulatory mechanism controlled HoxA expression, dividing its genes into two expression domains. In other words, this mechanism was present in the genome of the ancestor of modern fish and land animals. "This finding was a great surprise as we expected that this 'bimodal' DNA conformation was exactly what would make all the difference in the genetics for making limbs or making fins," said Joost Woltering, the post-doc who was lead author of the study, in a press release. Rather than providing a mechanism by which fins may have evolved, the spatial regulatory mechanism seems to be an ancestral characteristic of vertebrates.

So do fish have the regulatory elements to pattern a limb into an arm and digits? Yes and no. When the team engineered transgenic mice with the 5' or 3' genomic regions from zebrafsh, the HoxA and HoxD genes were expressed consistently in the arm, even if the transgenic genomic region activated genes in the distal part of the fin in fish. So even though fish have the same spatial regulatory mechanism to divide Hox expression into two regions, it turns out that both regions in the fin correspond to the proximal part of the tetrapod limb. Tetrapod digits evolved by transforming one of the two regulatory landscapes to create a novel domain, the distal limb region. Duboule called this "another surprise", saying that the findings "suggest that our digits evolved during the fin to limb transition by modernization of an already existing regulatory mechanism."

Tetrapod limbs turn out to have evolved by co-opting a spatial regulatory mechanism that dates back to our common ancestor with modern fish and adapting it to new ends. It's a remarkable evolutionary story, and one that raises interesting questions about how we define homology. Fish have the necessary components to make digits, which could then be considered a specialized fin component, but the modification of the regulatory landscape to create a new region for our digits argue against this view. Regardless, the next step will be understanding how these modifications came about. "By now we know a lot of genetic switches from the mouse that drive Hox expression in the digits. It's key to find out exactly how these processes work nowadays to understand what made digits appear and favor the colonization of the terrestrial environment," concluded Duboule.

References
Woltering J.M., et al. Conservation and Divergence of Regulatory Strategies at Hox Loci and the Origin of Tetrapod Digits. PLoS Biology 12(1): e1001773. (2014) doi:10.1371/journal.pbio.1001773
Hoff M. A Footnote to the Evolution of Digits. PLoS Biology 12(1): e1001774. (2014) doi:10.1371/journal.pbio.1001774

Image Credit
The image is from Woltering et al (2014) and is distributed under a CC license.