A tale of three arms

Once upon a time, deep in the Precambrian, this was the planet of worms. Well, actually, this was, is, and always will be the planet of bacteria, but if you filter your perspective to just organisms above a particular size, and if you're an animal writing about it in the modern day with a chauvinistic attitude that allows you to ignore that it was also a planet of algae, that would become a planet of plants, on a world that also is built of soil formed by lichens and saturated with fungus…if you ignore all that, OK, it was a planet of worms.

Late in the Precambrian, the oceans were full of accumulated muck and it was a good time to be a worm -- a slender, plastic body, able to burrow and plunder the detritus of it's nutrients, digging shelters or writhing in the freshest debris up top. Worms were everywhere, and the family was diverse. There were hairy ones, thick ones, slender ones, spiky ones. There were worms beginning to assemble bits of armor, either extracting calcium from their environment or crosslinking stable sugars to create chitin, because there were also worms that were developing the habit of eating other worms, rather than farming the muck.

Worms were and are diverse. One successful subfamily of worms were segmented -- they had begun to evolve more elaborate mechanisms of specializing body parts, so instead of just a simple tube, they had ways of setting aside regions for essential functions, like a head end for eating, a tail end for excreting and reproducing, and a means of making lots of repetitive copies of subunits of their bodies. So, for instance, when they evolved a way to make a bristle at one place, it would be repeated in a whole chain of bristles down the length of the body.

Now as I said, these worms were diverse -- they'd been proliferating and diverging for hundreds of millions of years, and each new kind of worm created a more complex environment that permitted even more kinds of worms to flourish. Different lineages of worms had different histories and different sets of attributes. At the end of the Precambrian, not only had they fueled their own diversification, but they'd also changed the planet. They'd stirred up long-buried nutrients and brought about a population explosion, and other organisms (you know, those neglected algae) had been changing the atmosphere, and it was a different world, ripe with opportunity for even more novel forms and opening up new environments, and there were worms fortuitously endowed with traits to take advantage of those opportunities.

In this vast family of worms, I'm going to mention just three cousins -- three Precambrian worms that were going to found new dynasties in the Cambrian and on.

One cousin had inherited a couple of extraordinarily useful endowments. She had limbs. Lots of limbs. She was segmented, and initially had figured out how to sprout muscular appendages from each segment, and so she had an extravagant superfluity of limbs, and each could be specialized in novel ways. She was a walking Swiss army knife. She also had acquired bits of chitinous armor, which would also serve her family in good stead -- she was the armored and armed cousin, who would found the great family of arthropods.

If she'd had foresight, she would have smiled wolfishly at the prospects for her descendants, if she'd had the kind of mouth that could smile. Instead, she had a face full of legs, each one already specializing into stabbing needles and gnashing blades and pulverizing clubs, which, come to think of it, is even better than a smile for communicating wolfishness.

The second cousin had not inherited any limbs or armor at all. She was smooth and torpedo-shaped. Her endowment was the beginnings of an internal skeleton, a notochord, which made her a fast and efficient swimmer. She was the mother of chordates, and she was the swift cousin -- what she gave to her descendants was the ability to run away quickly. In contrast to her armed cousin, she also had a purty mouth, a delicate basket of rods and cilia and membranes, a pharynx that was an efficient filter feeding tool. Someday, her descendants would be able to smile, but only after radically rearranging that complex endowment.

The third cousin…well, I'm going to call her the humble cousin. She had no arms, either, just one big muscular foot that she could use to crawl about on the bottom of the ocean. She had a gland to secrete a shell, but it wasn't articulated like that of her armored cousin, so it really was a purely defensive structure -- she couldn't shape it into knives and spears. She also had a talent for mucus, which is not usually the kind of thing to make one popular at parties. She's not so obvious as the founder of a successful family, but she was. She was the mother of molluscs. She didn't have much of a face -- a sheet of muscle with a muscular sphincter -- so she wasn't much for smiling.

Time passes. Arthropods flourish and take over the world -- in the modern day we'd have to say we're living on the Planet of the Insects, except that that external armor turned out to impose some limitations on size so they never got big enough to support a large brain, didn't invent the internet, and aren't writing the story of history. But that doesn't matter much when they're the first to invade the land, first to take to the skies, and are filling practically every ecosystem on the planet. Those limbs turned out to be a really lucky and extremely handy if you wanted to interact with the environment in complex ways. Eating, walking, flying…limbs make those behaviors relatively easy, and enable all kinds of specialization.

Limbs do have a limitation -- they're a real drag for swimming, where simple, streamlined torpedos rule. The swift cousin does well in the open sea, and builds a huge clan of fish. Evolution tinkers with them, too, though. Even a torpedo can benefit from fins, so those fish that can manage a little fin-fold swim straighter, and those that can flex a fin become more maneuverable. The heavy duty modifications occur in the pharynx. The bigger the animal, the less useful a delicate ciliated basket is for feeding, so it's dismantled, rearranged, repurposed. It's used for respiration; parts of it are beefed up and turned into a grasping clamp, the jaws; it becomes a key element of the blood circulation; its membranes are used for ion exchange, and some of the tissue is specialized for managing salt balance; in fact, the whole front end of the chordate -- now vertebrate -- gets massively renovated, not just to recycle elements of the pharyngeal apparatus, but to build a bigger brain and a sensitive sensory apparatus.

But jaws don't quite have the versatility of limbs. It took almost a hundred million years after the beginning of the Cambrian for vertebrates to scrape together a kludge to give some of their members a small set of manipulatory/locomotor appendages. This process involved localizing four patches of epidermal thickening¹, reusing signals to recruit mesodermal tissue into the zones, and then recycling signals used for patterning the longitudinal axis of the animal to pattern the length of the limb. It's an amusing glimpse into the way evolution tinkers to see how vertebrate limbs are such perfect examples of bricolage: a piece here, a piece there, jigger them about and reassemble them to make a protruding limb, with the same Hox genes used to specify the organization of the hindbrain lifted wholesale and used in new ways to organize upper arm, forearm, and digits of the hand.

It's also a testimony to the utility of limbs that they've evolved multiple times. There must be considerable incentive for some lineages to reach out and touch the world somehow, as these projecting bulges keep popping out. But they also reveal the other side of convergent evolution: we can get similar function by very different mechanisms, and internal structure reveals historical differences. Here's a cartoon of cross sections of various limbs to expose those differences.

So arthropods had limbs from the earliest times in their history, and are a defining character of the clade. They also acquired an armored exoskeleton made of chitin. When we look at their limbs in cross-section (top left), what we see is a strong, supportive skeletal tube with muscles (in yellow) on the inside. Those muscles are primarily longitudinal -- that is, they attach to the inner wall of the tube and extend the length of that tube to attach to the inner wall of the next tube in sequence (or to internal protrusions called apodemes), and the tubes are linked by articulating joints.

The subgroup of the vertebrates, the tetrapods, that finally got around to evolving jointed limbs, have a mechanically different solution (top right). Their limbs have a central bony rod to which longitudinal² muscles attach, and extend down to the next rod in sequence. Each bone is articulated with the next with joints made of cartilage. Vertebrates and arthropods have come up with roughly similar solutions to movement, constructing rigid structual elements that can be flexed relative to each other by the actions of muscles, but they're inside out relative to one another. As it turns out, having an internal structural support is arguably better engineering at large sizes, while having an external support that doubles as armor is arguably more efficient at smaller sizes, which is one reason arthropods rule the animal domain below about 10cm in size, while tetrapods are living in lonely majesty above that size.³

But wait! What about that homely cousin, the molluscs? I was wrong to call them homely. They blossomed. The nudibranchs are really the most spectacularly colorful animals in the ocean, and they used a body that, thanks to that muscular foot, is practically a solid wall of intricately controlled muscle fibers in multiple orientations. They have a muscular versatility that puts Terry Crews' pecs to shame, but one thing they lack is a rigid skeletal framework. No bones or exoskeleton to tug on! All they can do is exert force on other muscles and on the flexible dermal connective tissue, which sounds as if it would be a serious limitation, but then you watch a sea slug dance through the water, and you have to rethink that assumption.

This is where convergence gets interesting: some members of the molluscan clade also evolved limbs de novo. Cephalopods are simply slugs with ambition. They also evolved new limbs, which had to be even more difficult than the path tetrapods took -- molluscs lack any kind of articulated skeleton at all, so there aren't many conveniently rigid structural elements to act upon. So they resorted to a different solution. Tentacles.

The bottom illustration in the diagram above is a cross-section through an octopus arm. There is no skeleton. It's just layers of muscles, some longitudinal, some transverse, some circumferential, all pulling in complex ways to change the shape of the appendage. The closest thing we humans have to something like that is our tongue, which contains intrinsic muscles that do not have a bony attachment, and which can change the organ's shape in interesting ways. But look at that diagram! They have all kinds of muscles in all kinds of orientations, and also unlike the arthropod or tetrapod, a robust central nervous organ with chains of ganglia extending the length of the arm. It takes a lot of local circuitry to control something as complex as a cephalopod arm.

That's three different ways to make a limb right here on our one solitary planet. That's interesting in both a developmental and evolutionary way. Do all three use similar processes? We know quite a bit about tetrapod and arthropod limb development, and they aren't homologous at all. Primitively, every segment in an arthropod has a limb; vertebrates had none. As is typical, limbs had to be built using the common molecular toolkit, and we find that homologous genes are used in similar ways. Insects use a gene called decapentaplegic as a morphogen to organize their limb fields. Vertebrates use the decapentaplegic homolog Bone Morphogenetic Protein to regulate skeletal formation in the embryo. We know that molluscs also have decapentaplegic -- they use it as a morphogen to control shell shape. Does it get recycled into cephalopod arm formation?

We also know how squishy vertebrate limbs grow. There is an epidermal thickening called the apical ectodermal ridge that secretes a morphogen, FGF to induce proliferation and extension, and another part of the limb bud called the zone of polarizing activity that uses another morphogen, Shh, to induce polarity (the dorsal side of your arm is different than the ventral). How do cephalopod arms develop? Is it similar, with the same kinds of patterns of proliferation and extension, and do they also recruit the same or similar molecules to do the job?

People are always asking me why I'm known for my fascination with cephalopods, when I work on a limbless vertebrate, the zebrafish, and this is why. Because asking why things are the way they are requires asking about the way they are not. Understanding how our biology came to take one particular path should involve looking at all the different paths, and thinking about alternatives. So if you want to really understand vertebrate limb development, it's important to compare it with invertebrate limb development.

My introduction has gone on way too long, so I'll post the answers (and mostly, lack of answers) to my questions later. For now, just remember that the issue is how different organisms build a protruding appendage, a very general question, and we want to know more about how a group very different from our own, that has diverged from the vertebrate lineage for at least 600 million years, has constructed their unique version of an arm.

Nödl et al. (2015) The making of an octopus arm. EvoDevo 6:19

¹One of the things that always annoys me in SF movies is the ubiquity of bipeds tottering about. That we only have four limbs is entirely a historical accident of our clade -- and bipeds like us are the result of a clumsy evolutionary effort to free up a pair from locomotory duties to be more manipulatory. We're weird! Why do we keep unthinkingly imposing our historical limitations on every alien species we imagine, especially when our own planet is so rich in species that have no such arbitrary constraint?

And don't use that "movie budget" excuse: we have CGI aliens all over the place, and even there they simply map them onto distorted bipedal forms. We even have movies like Avatar which imagines an entire diverse alien ecosystem with diverse body plans, and then chickens out and turns the intelligent aliens into slender big-eyed anime models⁴.

²Of course we have some greater complexity than just longitudinal muscles -- obliques, for instance, that rotate the radius and ulna, or the layered sheets of muscle that make up the abdominal wall.

³On the topic of imaginary aliens -- use our extant patterns as a guide. Imagine an equivalent Precambrian world elsewhere, where one group gets a mix of traits, a complex oral structure plus an exoskeletal armor, whie another group gets a different mix, multiple segmented limbs plus an internal skeleton. Work forward. Later, you've got small heavily armored snake-like things with intricate mouthparts, and giant fleshy beasts with limbs sprouting all over the place.

And of course, the creatures with all the feet evolve into galactic imperialists, conquering the universe to accumulate enough real estate to house all their shoe stores.

⁴Also chickening out: I liked the diversity of limb structure in the John Carter movie, but once again, the hero species defaulted to fully humanoid, to the point where a human would find them sexually attractive. Which is bizarre -- the Red Martians in Burroughs' stories lay eggs. Somebody needs to write the story of John Carter and Dejah Thoris's wedding night, when the wife disrobes and reveals to her husband…a cloaca. Does the sexual attraction endure?