New Developments in the Development of Limbs

Humans love to group things. A place for everything, and everything in its place. The science of grouping living things has grown ever more sophisticated as technology has enabled us to characterize organisms at the cellular and even the molecular levels. While some of the fundamental groups proposed by even the earliest naturalists still persist, today they have been refined. Moreover, our understanding of the evolutionary relationships among groups has grown clearer and clearer over time.

Neil Shubin has spent decades piecing together the mechanics of one such transition—the one that links tetrapods (animals that inherited the characteristic of four bony limbs from a common ancestor) to fish. Shubin’s fossil finds, including the celebrated Tiktaalik, and his eagerness for engagement through documentaries, popular writing, and presentations have made this particular evolutionary tale one of the best known and understood by the public. But, it turns out, there were still surprises to be uncovered.

Previous work by Shubin and others has firmly cemented the evolutionary link between so-called lobe-finned fishes (such as the enigmatic coelacanth) and tetrapods. Fossils such as Tiktaalik show a “one-bone, two-bones, lots-of-bones” pattern in their limbs that we recognize in every tetrapod today—at least those that have retained their arms and legs. (Later tetrapod ancestors added on digits, but Tiktaalk lacked them.) The connection with ray-finned fish, however, has been far less clear. Ray-finned fish are the most diverse tetrapod group around today, and includes everything from goldfish and seahorses to swordfish to tuna. We know that we share a 430-million-year-old ancestor with this group, but some of the shared structural links (homologies) between tetrapods and ray-finned fish aren’t as clear-cut or understood.

For example, ray-finned fish—forgive me for stating the obvious—have fins supported by rays. The rays are made up of a type of bone known as dermal bone, which forms directly within the ectoderm (outermost layer) of a developing organism. Lobe-finned fish and tetrapods, however, have fins and limbs supported by robust endochondral bone—bone that forms in the mesoderm (middle layer) of developing organisms and is first laid down in cartilage before being replaced with bone. It’s hard to overstate the fundamental differences between dermal and endochondral bone. A developing animal establishes its three main body layers very early—in the case of humans, about fourteen days after fertilization. As a general rule (but by no means an absolute one), differences established so early in development tend to be pretty darn major.

So how did our fishy ancestors go from dermal-bone-supported fins to fins supported by endochondral bone? Thanks to a couple of students in Shubin’s lab, we now have an idea (free summaries here and here). In 1996, French scientists established that mice limbs relied on the expression of two genes, Hoxa-13 and Hoxd-13. Hox genes are to animal development what a conductor is to an orchestra—responsible for much of the timing, coordination, and patterning. They are ancient genes, which means that every animal has a set that, despite minor differences, aligns pretty well with the set of any other animal. Thus, ray-finned fish have versions of Hoxa-13 and Hoxd-13. Shubin wondered whether these genes could provide insights into the link between the likes of goldfish and humans. The problem was that he didn’t have the molecular tools to do the experiments required.

Then along comes CRISPR—a new crazy-powerful gene editing technique that enables scientists to locate, edit, add, and subtract genes with incredible precision. One of Shubin’s postgraduate students, Tetsuya Nakamura, used CRISPR to prevent the expression of Hoxa-13 and Hoxd-13 in developing zebrafish. The result? Drastically shortened fin rays. But wait—that meant these Hox genes somehow were involved in both endochondral bone development (as in the mice) and in dermal bone development (as in the zebrafish), which was unexpected, to say the least. The expected result would have been malformations up near the base of the fish’s fin, where endochondral bone anchors the fin to the body of the fish. But when it comes to science, unexpected results are often far more exciting than expected ones!

In parallel to the CRISPR work, a graduate student in Shubin’s lab, Andrew Gehrke, ran an experiment of his own. To understand it, you need to know that limbs develop from the body outwards. Previous work had shown that Hoxa-13 and Hoxd-13 genes were expressed in the wrists and ankles of mice, and that the cells that expressed these genes went on to form the feet and hands. Following the previous technique, Gehrke engineered zebrafish embryos so that cells expressing Hoxa-13 and Hoxd-13 would glow red. The expected result? That the base of the fin would turn out to glow. The unexpected and actual result? The fin rays were glowing.

Okay, so what the heck does all this mean? As both types of animals, ray-finned fish and tetrapods, develop, the Hoxa-13 and Hoxd-13 genes seem to direct the development of structures at the most distal (away from the body) parts of the limb. In ray-finned fish, the cells that make up these structures form from dermal bone. In tetrapods, they are made up of endochondral bone. But that major difference aside, there is a obviously deep, evolutionary link—at the molecular level—between the hands and feet of tetrapods and the fin rays of fish.

As Shubin told The New York Times, “Here we’re finding that the digits and the fin rays have some sort of equivalence at the level of the cells that make them.” Was Shubin surprised at the finding? “Honestly,” he said, “you could have knocked me over with a feather—it ran counter to everything that I was expecting after working on this problem for decades.”

But the similarities between fins and limbs can be overstated. These new findings do not suggest that fin rays and fingers are the same thing. Rather, the work suggests that somewhere along our evolutionary path the Hox genes that patterned ray fins got redirected somehow and co-opted to pattern hands and feet. Although a more nuanced bottom line, it’s still a very important result that—well, I’ll let Shubin tell you:

"[This new work] shows us how bodies are built. By understanding the biology of fish, we understand the basic architecture of our bodies, and how genes and cells interact to build us, and how we evolve."

Are you a teacher and want to tell us about an amazing free resource? Do you have an idea for a Misconception Monday or other type of post? Have a fossil to share? See some good or bad examples of science communication lately? Drop me an e-mail or shoot me a Tweet @keeps3.