Osamu Shimomura: Chemistry of Bioluminescence

Bioluminescence is common, especially in marine organisms. Shimomura classified thes into a couple of types: luciferain, photoprotein, and an undefined “other”. Luciferin requires an enzymatic (luciferase) reaction, and luminescence is proportional to the concentration of the substrate. The photoprotein type requires a single molecule — aequorin, symplectin, pholasin, etc.

He summarized how D-Luciferin is converted to Oxyluciferin in the presence of O2 and ATP, catalyzed by luciferase, to produce light, either red light in acidic media or yellow green in alkaline media. There is also a variant of this reaction called coelenterazine-luciferase luminaescence, found in many marine organisms, like Periphylla, a ellyfish, and Chiroteuthis, a squid. Coelenterazine is converted to coelenteramide in the presence of O2 to produce light and CO2.

Another organism is Cyprodana, a crustacean that uses a luciferin variant — produces a very pretty blue glow.

Luminiscent bacteria convert luciferin into a fattye acid, FMN, to produce light, which is also luciferase-catalyzed. Luciferase seems to be popping up all over the place — unfortunately, Shimomura doesn’t talk much at all about variations in the enzyme, but is more focused on giving us the chemical intermediates produced in the reaction. Typical chemist!

This is especially unfortunate since the luciferase reaction in different organisms seems to produce very different reaction products…it’s getting me very curious about how these different forms of the protein must differ to be yielding such different outcomes, all only similar in that they also produce light as a byproduct. And then we see that some very different phyla, such as krill and dinoflagellates, use nearly identical reactions.

Photoproteins: he talks about Aequorea aequorea, which produces green light with a protein called aequorin that binds a complex molecule that resembles coelenterazine, that undergoes a conformational change in the presence of calcium ions to produce green light. If GFP is used, it produces green light.

A squid, Symplectoteuthis, converts dehydrocoelenterazine to symplectin which then produces light.

Then he switches to talking about fungal biolominescence in Mycena and Panellus, mushrooms that glow green. The precursor is decanoylpanal is conferted to luciferin, which produces light in the presence of superoxides, O2, and tetradecanoylcholine. The reaction can take place in the absence of any enzyme.

Shimomura is not exactly a dynamic speaker — he basically just read off his list of reactions — but at least he had lots of pretty pictures of glowing organisms. If only he’d said something about the relationships and evolutionary differences between them all!

Argentina takes over the world!

I am in awe — they did it without anyone noticing. They just infiltrated nations all around the planet, smuggling in individuals to form vast new colonies of billions, all loyal to the overlords back home. Of course, these are very, very short Argentinians, which made them harder to notice: they’re all ants.

In Europe, one vast colony of Argentine ants is thought to stretch for 6,000km (3,700 miles) along the Mediterranean coast, while another in the US, known as the ‘Californian large’, extends over 900km (560 miles) along the coast of California. A third huge colony exists on the west coast of Japan.

While ants are usually highly territorial, those living within each super-colony are tolerant of one another, even if they live tens or hundreds of kilometres apart. Each super-colony, however, was thought to be quite distinct.

But it now appears that billions of Argentine ants around the world all actually belong to one single global mega-colony.

You better start practicing your tango is you hope to get along with our new arthropod overlords.

wednesday morning at Lindau, part 2

This morning was a long session broken into two big chunks, and I’m afraid it was too much for me — my recent weird sleep patterns are catching up with me, which didn’t help at all in staying alert.

Robert Huber: Intracellular protein degradation and its control

This talk was a disaster. Not because it wasn’t good, because it was; lots of fine, detailed science on the regulation of proteases by various mechanisms, with a discussion of the structure and function of proteasomes, accompanied by beautiful mandalas of protein structure. No, the problem was that this listener’s jet lag has been causing some wild precession of my internal clocks, and a quarter of the way through this talk all systems were shutting down while announcing that it was the middle of the night, and I really couldn’t cope. I’m going to have to look up some of his papers when I get home, though.

Walter Kohn: An Earth Powered Predominantly by Solar and Wind Energy

Kohn has made a documentary to illustrate the power of solar energy. It was very basic, a bit silly — John Cleese narrates it — but might be useful in educating the pubic. He showed excerpts from it, and while it was nice, it didn’t fire me up.

Peter Agre: Canoeing in the Arctic, a Scientist´s Perspective

This was a bit strange. We’ve had all these science talks on global warming, so Agre decided to just show us what we stand to lose, and showed us photos of his vacations on canoeing trips in Canada and Alaska. They were gorgeous photos, but please don’t show me your photo album when I’m crashing hard.

I think my new and revised plan is to take a nap this afternoon and try to recharge a bit. I really must be alert for tomorrow’s session with Shimomura, Chalfie, and Tsien, which are the talks I was most anticipating. There’s also a curious talk by Werner Arber on something called Molecular Darwinism which has my skeptical genes tingling; I’ve got to see what kinds of evidence he provides for that. So brain must not melt down now.

Wednesday morning at Lindau

I’m here for another long session of talks. Unfortunately, this is Big Chemistry day, and I’m struggling to keep up with the unfamiliar. I need more biology for it all to make sense!

Rudolph Marcus: From ‘On Water’ and enzyme caalysis to single molecules and quantum dots. Theory and experiment.

I was afraid of this. This Lindau conference has a primary focus on chemistry, and I am not a chemist…and I just knew there would be a talk or two at which I would be all at sea, and that was the case in Marcus’s talk, which was all hardcore chemistry. I got the general gist — he’s making an argument that you need both a solid grounding in theory in order to carry out computational chemistry, which seemed fairly obvious to me — but I confess that his discussion of the details of on-water catalysis, single molecule enzyme catalysis, and quantum dots lost me, through no fault of his. I don’t have the background to follow the context of the discussion.

Kurt Wüthrich: Structural genomics — exploring the protein universe

This was more of that tricky chemistry stuff, but at least it was related to biology. Wüthrich studies 3D protein structures, specializing in using NMR of proteins in solution. He fave a little background, and talked especially about his particular interest in hemoglobin, an interest that continues — he currently works at catching EPO doping in athletes. The more interesting part of the work is his current contributions to analyzing the structure of proteins in the genome. He made the point that there are currently over 6 million gene sequences tucked away in databases, but we know the the 3D structure of only about 50,000 of them. He’s part of a very large research consortium that is trying to fill in the gaps with high throughput, automated techniques.

Harold Kroto: Science, society and sustainability

If you’ve ever heard a Kroto talk, you know it is pretty much indescribable.

He did present all of chemistry in 30 seconds, but much of it was about about science education, science’s role in society, and how science is going to be necessary to save the world. There was a good strong bit of promotion of atheism (he’s one of us!), and an amusing tour of the Creation “Museum”, which he visited recently. All I can recommend is that you keep an eye on the Lindau site — they will make the lectures available online at some time.

Irwin Neher: Chemistry helps neuroscience: the use of caged compounds and indicator dyes for the study of neurotransmitter release

Ah, a solid science talk. It wasn’t bad, except that it was very basic—maybe if I were a real journalist instead of a fake journalist I would have appreciated it more, but as it was, it was a nice overview of some common ideas in neuroscience, with some discussion of pretty new tools on top.

He started with a little history to outline what we know, with Ramon Y Cajal showing that the brain is made up of network of neurons (which we now know to be approxiamately 1012 neurons large). He also predicted the direction of signal propagation, and was mostly right. Each neuron sends signals outwards through an axon, and receives input from thousands of other cells on its cell body and dendrites.

Signals move between neurons mostly by synaptic transmission, or the exocytosis of transmitter-loaded vesicles induced by changes in calcium concentration. That makes calcium a very interesting ion, and makes calcium concentration an extremely important parameter affecting physiological function, so we want to know more about it. Furthermore, it’s a parameter that is in constant flux, changing second by second in the cell. So how do we see an ion in real time or near real time?

The answer is to use fluorescent indicator dyes which are sensitive to changes in calcium concentration — these molecules fluoresce at different wavelenths or absorb light at different wavelengths depending on whether they are bound or not bound to calcium, making the concentration visible as changes in either the absorbed or emitted wavelength of light. There is a small battery of fluorescent compounds — Fura-2, fluo 3, indo-1 — that allow imaging of localized increases in calcium.

There’s another problem: resolution. Where the concentration of calcium matters most is in a tiny microdomain, a thin rind of the cytoplasm near the cell membrane called the cortex, which is where vesicles are lined up, ready to be triggered to fuse with the cell membrane by calcium, leading to the expulsion of their contents to the exterior. This microdomain is tiny, only 10-50nm thick, and is below the limit of resolution of your typical light microscope. If you’re interested in the calcium concentration at one thin, tiny spot, you’ve got a problem.

Most presynaptic terminals are very small and difficult to study; they can be visualized optically, but it’s hard to do simultaneous electrophysiology. One way Neher gets around this problem is to use unusually large synapses, the calyx of Held synapse, which is part of an auditory brainstem pathway. It’s an important pathway in sound localization, and the signals must be very precise. They have a pecial structure, a cup-like synapse that envelops the post-synaptic cell body — they’re spectacularly large, so large that one can insert recording electrodes both pre- and post-synaptically, and both compartments can be loaded with indicator dyes and caged compounds.

The question being addressed is the concentration of Ca2 at the microdomain of the cytoplasmic cortex, where vesicle fusion occurs. This is below the level of resolution of the light microscope, so just imaging a calcium indicator dye won’t work — they need an alternative solution. The one they came up with was to use caged molecules, in particular a reagent call Ca-DMN.

Caged molecules are cool, with one special property: when you flash UV light of just the right wavelength at them, they fall apart into a collection of inert (you hope) photoproducts, releasing the caged molecule, which is calcium in this case. So you can load up a cell with Ca-DMN, and then with one simple signal, you can trigger it to release all of its calcium, generating a uniform concentration at whatever level you desire across the entire cell. So instead of triggering an electrical potential in the synaptic terminal and asking what concentration of calcium appears at the vesicle fusion zone, they reversed the approach, generating a uniform calcium level and then asking how much transmitter was released, measured electrophysiologically at the post-synaptic cell. When they got a calcium level that produced an electrical signal mimicking the natural degree of transmitter release, they knew they’d found the right concentration.

Caged compounds don’t have to be just calcium ions: other useful probes are caged ATP, caged glutamate (a neurotransmitter), and even caged RNA. The power of the technique is that you can use light to manipulate the chemical composition of the cell at will, and observe how it responds. These are tools that can be used to modify cell states, to characterize excretory properties, or to generate extracellular signals, all with the relatively noninvasive probe of a brief focused light flash.

Christian faith is at odds with science

Yesterday morning, I was in a discussion on UK Christian talk radio on the topic of “Is Christian faith at odds with science?”, with Denis Alexander of the Faraday Institute for Science and Religion. It’s going to be available as a podcast at sometime in the next day, but I may not be able to link to it right away — tomorrow I fly away to Germany for a week, so my schedule is going to be a bit chaotic for a while.

Don’t expect fireworks. It was the usual feeble accommodationist claptrap, but I had my nice man hat on and actually tried to get across some basic ideas. To no avail, of course, but at least I tried.

I have now discovered that I was trying to make the same points Lawrence Krauss is doing in the Wall Street Journal: religion is wrong. It’s a set of answers, and worse, a set of procedures, that don’t work. That’s the root of our argument that religion is incompatible with science.

That word, “incompatibility”, is a problem, though. The uniform response we always get when we say that is “Hey! I’m a Christian, and I’m a scientist, therefore they can’t be incompatible!” Alexander was no exception, and said basically the same thing right away. It’s an irrelevant point; it assumes that a person can’t possibly hold two incompatible ideas at once. We know that is not true. We have complicated and imperfect brains, and even the most brilliant person on earth is not going to be perfectly consistent. When we talk about incompatibility, we have to also specify what purposes are in conflict, and show that the patterns of behavior have different results.

For instance, if you just like to go to church because you enjoy the company, then the purpose of religion to you is to reinforce social bonds — so of course there is no incompatibility between science and religion there. If you go for the choir (as Stephen Jay Gould was known to do), you’re there to enjoy the music, and science does not dictate that human beings are not allowed to enjoy music. For that matter, science doesn’t say that someone is not allowed to enjoy the perverse circumlocutions of theology, so if someone attends for the religion sensu strictu, no problem.

But in a debate about the compatibility of science and religion, we have to put the argument in an appropriate context and define a specific shared purpose for both science and religion — it’s the only legitimate ground for discussion. In this case, what we’re trying to do is address big questions (remember, the Templeton Foundation says they’re all about those “big questions”) about the nature of the universe, about our history, about how we function, and then we encounter a conflict: religion keeps giving us different answers. Very different answers. They can’t all be right, and since no two religions give the same answers, but since science can generally converge on similar and consistent answers, I know which one is right. And that makes religion simply wrong.

We have to look at what they do to see why. In order to probe the nature of the universe around us, science is a process, a body of tools, that has a long history of success in giving us robust, consistent answers. We use observation, experiment, critical analysis, and repeated reevaluation and confirmation of events in the natural world. It works. We use frequent internal cross-checking of results to get an answer, and we never entirely trust our answers, so we keep pushing harder at them. We also evaluate our success by whether the end results work: it’s how we end up with lasers and microwave ovens, and antibiotics and cancer therapies.

Religion, on the other hand, uses a different body of techniques to explain the nature of the universe. It uses tradition and dogma and authority and revelation, and a detailed legalistic analysis of source texts, to dictate what the nature of reality should be. It’s always wrong, from an empirical perspective, although I do have to credit theologians with some of the most amazingly intricate logical exercises as they try to justify their conclusions. The end result of all of this kind of clever wankery, though, is that some people say the world is 6000 years old, that it was inundated with a global flood 4000 years ago, and other people say something completely different, and there is no way within the body of theology to resolve which answers are right. They have to step outside their narrow domain to get an independent confirmation — that is, they rely on science to give them the answers to the Big Questions in which they purport to have expertise.

So what theistic scientists have to do is abandon the operational techniques of religion and use science to address those questions. The “theistic” part of their moniker is nothing but useless baggage which, if they take it at all seriously, would interfere with their understanding of the world. That is what I mean by an incompatibility between the two.

Krauss uses a marvelous and well-known quote from J.B.S. Haldane to make that point more briefly.

My practice as a scientist is atheistic. That is to say, when I set up an experiment I assume that no god, angel or devil is going to interfere with its course; and this assumption has been justified by such success as I have achieved in my professional career. I should therefore be intellectually dishonest if I were not also atheistic in the affairs of the world.

I got Alexander to agree that he does not use religion in the laboratory — I don’t know anyone who would say that they do, other than creationist kooks — but it didn’t seem to sink in that that is an admission of incompatibility. Religion doesn’t work to answer questions in science, which always leaves me wondering…if you accept that, why do you go on thinking it might be giving you correct answers in ordinary daily life? It has an awfully poor track record.

Now one way the defenders of religion like to get around this empirical problem is to change the game in mid-play: one moment we’re talking about tools for understanding the world, where there is a conflict, and then they switch to a completely different purpose, that of establishing a common morality, or appreciating art, or falling in love. I would be the first to admit that science does not and should not dictate morality: the cases in the past where this has happened (eugenics comes to mind right away) have been disastrous. Science is good at explaining what is and how it works, and not so great at telling us how it should work. I also wouldn’t use the scientific method directly to determine whether I like some music or poetry or not.

However, I’m going to have to say that religion doesn’t do a good job at that either. SJ Gould tried to partition the domains of authority for science and religion by explicitly setting a boundary, and saying religion should have the job of defining what is right and good…but I think he failed, because he gave far too much credit to religion for being able to discern and act on a reasonable morality. It’s foundation on authority and its role in defining in and out groups means it is too exclusionary, too narrow and inflexible, and also too willing to ignore empirical evidence. It’s why we have religion behind such immoral acts today as trying to restrict civil rights to people who have only a certain range of sexual behaviors, or facilitating the spread of sexually transmitted disease in Africa by damning sex education and condom use.

And when it comes to other questions than the cosmic ones about the nature of existence, I prefer that we apply just about any discipline other than religion to the problem: at least they are evidence-based, where religion is not. I’d rather consult a philosopher than a theologian on morality; they’ve been thinking about it with a broader scope than the pious promoters of sectarian belief, anyway, don’t restrict their principles to worshippers of one particular idol, and usually don’t invoke magical rewards and punishments that have never been seen to justify decisions. If I’m in love I’m better off pulling a book of poetry off the shelf than consulting a celibate. I’d rather hear about economics from an economist than from a ouija board or a pulpit, and I like the idea of policy decisions being evaluated for effectiveness, rather than ideological purity. When we’re looking at communities and interactions between individuals, give me a psychologist or a sociologist over a priest any day. The only useful priests in those matters are the ones who understand the principles of psychology and sociology, and apply those, rather than pulling a quote out of their holy book.

Accommodationists are a problem not because accommodation is bad, but because they are pushing for the wrong kind of accommodation. Science doesn’t need to conform, religion does. Religion demands a special kind of privilege in these discussions because if we actually get down to assessing views fairly and objectively, on the basis of what works, it fails. I say, let it.

This is also why so many of us object to the Templeton Foundation. Their agenda consists solely of mixing up science and religion, to the detriment of the former. They just want to compromise…but asking us to compromise science that works with faith that doesn’t is a fool’s bargain. Why should we?

Visiting village dogs

I am horribly envious. I am speaking of the Village Dog Project, some current research going on that looks very cool.

Understanding the evolution and domestication in dogs requires genetic analysis of a global and diverse panel of non-breed-affiliated village dogs. With a network of worldwide and Cornell-affiliated collaborators, we plan to gather dog samples from remote villages, establish a genetic archive containing DNA and phenotypic information from these dogs, carry out genetic analyses on these samples, and develop computational methods for analyzing this dataset. In particular, we are interested in understanding the location, timing, and demographic conditions underlying domestication; the genetic changes involved in the transition of wolf to dog; the relationship between these village dogs and the breed dogs; and the effect that historical forces have shaped village dog diversity.

That looks informative and useful, and I’ll be looking forward to the publication of the research. That’s not what’s got me envious, though: for that, you have to look at their field work. The researchers are spending the summer traveling to exotic, remote locations (admittedly, to the kinds of places rife with scavenging village dogs, but still…) to collect blood samples. They have a travel blog that will be recounting their adventures, and also explains the science a little more.

After initial domestication, dogs probably lived “breed-less” lives as human commensals (hanging around humans, not really helping or harming them but living off their trash) for many thousands of years. During this time, dog populations quickly expanded and spread across the globe. In the last few hundreds of years, several hundred dog breeds were formed from local dogs in many parts of the world; these breed dogs have entirely replaced the non-breed “indigenous” dogs in some parts of the world, notably in Western Europe and the USA. However, most dogs throughout the world still live their lives as non-breed, indigenous, commensal dogs. We refer to these dogs as “pariah” or “village” dogs. They tend to be smallish (25-40 pounds), often tan, short-haired dogs, though the type varies a bit according to the region you’re in. The important point is that these dogs have not undergone the intense genetic bottleneck associated with breed formation. Thus, while breed dogs have only a small subset of the total genetic diversity of all dogs, it is likely that village dogs have a much greater range of the total diversity. Thus, they are very useful for looking at the original domestication event. They are informative of the original genetic bottleneck that led to the formation of domestic dogs many thousands of years ago.

Hmmm. We don’t seem to have many dogs running loose around exotic, remote Morris, Minnesota, but there are a few feral cats living off the dumpsters near the grocery store.

I probably wouldn’t try to read about visiting small midwestern towns to collect cats, though.

Limusaurus inextricabilis

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My previous repost was made to give the background on a recent discovery of Jurassic ceratosaur, Limusaurus inextricabilis, and what it tells us about digit evolution. Here’s Limusaurus—beautiful little beastie, isn’t it?

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(Click for larger image)
Photograph (a) and line drawing (b) of IVPP V 15923. Arrows in a point to a nearly complete and fully articulated basal crocodyliform skeleton preserved next to IVPP V 15923 (scale bar, 5 cm). c, Histological section from the fibular shaft of Limusaurus inextricabilis (IVPP V 15924) under polarized light. Arrows denote growth lines used to age the specimen; HC refers to round haversian canals and EB to layers of endosteal bone. The specimen is inferred to represent a five-year-old individual and to be at a young adult ontogenetic stage, based on a combination of histological features including narrower outermost zones, dense haversian bone, extensive and multiple endosteal bone depositional events and absence of an external fundamental system. d, Close up of the gastroliths (scale bar, 2 cm). Abbreviations: cav, caudal vertebrae; cv, cervical vertebrae; dr, dorsal ribs; ga, gastroliths; lf, left femur; lfl, left forelimb; li, left ilium; lis, left ischium; lp, left pes; lpu, left pubis; lsc, left scapulocoracoid; lt, left tibiotarsus; md, mandible; rfl, right forelimb; ri, right ilium; rp, right pes; sk, skull.

What’s especially interesting about it is that it catches an evolutionary hypothesis in the act, and is another genuine transitional fossil. The hypothesis is about how fingers were modified over time to produce the patterns we see in dinosaurs and birds.

Birds have greatly reduced digits, but when we examine them embryologically, we can see precisely what has happened: they’ve lost the outermost digits, the thumb (I) and pinky (V), and retain the forefinger, middle finger, and ring finger (II-IV), which have been reduced and fused together. This is called Bilateral Digit Reduction, BDR, because they’ve lost digits from the medial and lateral sides, leaving the middle set intact.

Dinosaurs, when examined anatomically, seem to have a different pattern: they have a thumb (I), forefinger (II) and middle finger (III), and have lost the lateral two digits, the ring and pinky finger (IV-V). This arrangement has been advanced as evidence that birds did not evolve from dinosaurs, since they have different bones in their hands, and getting from one pattern to the other is complicated and difficult and very unlikely.

The alternative hypothesis is that there is no conflict, and that dinosaurs actually underwent BDR and their digits are II-III-IV…but that what has also happened is a frame shift in digit identities. So dinosaurs actually have three digits, which are the index, middle, and ring finger, but they’ve undergone a subtle shift in morphology so that their forefinger develops as a thumb, and so forth.

Now we could resolve all this easily if only the physicists would get to work and build that time machine so we could go back to the Mesozoic and study dinosaur embryology, but they’re too busy playing with strings and quanta and dark matter to do the important experiments, so we’ve got to settle for another plan: find intermediate forms in the fossil record. That’s where Limusaurus steps in.

Limusaurus has a thumb, a tiny vestigial nubbin, and has lost its pinky completely. This is a (I)-II-III-IV pattern, and is evidence of bilateral digit reduction in a basal ceratosaur. In addition, the forefinger has become very robust, and while still distinctly a digit II, has been caught in the early stages of a transformation into a saurian first digit. It’s evidence in support of the dinosaurian II-III-IV hypothesis and the frameshift in digit identity! It’s almost as good as having a time machine.

Want to learn more? Carl Zimmer has a summary of the digit changes, while one of the authors of the paper, David Hone, also discusses the digits (the story is a little more complicated than I’ve laid out), and also has more on the rest of the animal—it’s a herbivorous ceratosaur, which is interesting in itself.

Xu X, Clark JM, Mo J, Choiniere J, Forster CA, Erickson GM, Hone DWE, Sullivan C, Eberth DA, Nesbitt S, Zhao Q, Hernandez R, Jia C-k, Han F-l, Guo Y (2009) A Jurassic ceratosaur from China helps clarify avian digit homologies. Nature 459(18):940-944.

Digit numbering and limb development

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Answers in Genesis has evolutionary biology on the run now. In an article from 2002, Ostrich eggs break dino-to-bird theory, they explain that development shows that evolution is all wrong, since developmental pathways in different animals are completely different, and can’t possibly be the result of gradual transformations.

The first piece of evidence against evolution is the old avian digit problem. Birds couldn’t have evolved from dinosaurs, because they have the wrong finger order!

The research conclusively showed that only digits two, three and four (corresponding to our index, middle and ring fingers) develop in birds. This contrasts with dinosaur hands that developed from digits one, two and three. Feduccia pointed out:

‘This creates a new problem for those who insist that dinosaurs were ancestors of modern birds. How can a bird hand, for example, with digits two, three and four evolve from a dinosaur hand that has only digits one, two and three? That would be almost impossible.’

The second problem is that frogs and people develop hands in completely different ways, ways that are even more different than the order of the digits.

This is not the only example where superficially homologous structures actually develop in totally different ways. One of the most commonly argued proofs of evolution is the pentadactyl limb pattern, i.e. the five-digit limbs found in amphibians, reptiles, birds and mammals. However, they develop in a completely different manner in amphibians and the other groups. To illustrate, the human embryo develops a thickening on the limb tip called the AER (apical ectodermal ridge), then programmed cell death (apoptosis) divides the AER into five regions that then develop into digits (fingers and toes). By contrast, in frogs, the digits grow outwards from buds as cells divide (see diagram, right).

Dang. I might as well hang it up right now. There is no possible way around these intractable differences. Take me, Jesus, I have seen the ligh…oh, wait a minute. That isn’t right. It looks to me like Jonathan Sarfati is just hopelessly confused on the first problem (I can’t really blame him, though—it is a complicated issue that has been the subject of scientific arguments for two centuries), and is simply completely wrong on the second (and that one I do blame him for. Tsk, tsk.)

So first, let’s tackle the tricky problem, digit identity in evolution. Extend your right hand out in front of you, palm down. Your thumb should be sticking out towards the left, and by convention, that’s Digit I. Counting from left to right, your index finger is Digit II, middle finger is Digit III, ring finger is digit IV, and your pinky is Digit V. We have the primitive pentadactyl (five-fingered) hand, so figuring out who is who is fairly easy. The difficulties arise in species that have reduced the number of their digits—when they extend their three-fingered hand, we have to figure out which digits are missing before we assign numbers to the remaining fingers.

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One way is by looking at the adult anatomy. Looking at your hand, you probably notice that your thumb is quantitatively different from the other fingers: it only has two joints, instead of three. This is common, that Digit I has fewer phalanges, or segments, than the others, and this is the kind of property that allows anatomists to figure out whether Digit I is present or not. To the right, for instance, is the hand of the raptor Deinonychus (the left hand, sorry to confuse you) with its digit numbering, from DI to DII to DIII, an assignment that was made on the basis of the anatomy. You can see that the ‘thumb’, DI, has fewer phalanges than the others.

You can try to do the same thing with the digits of birds, but it’s harder. Avian digits are reduced and fused into that pointy thing you find at the end of a chicken wing, and it takes an expert to sort out what bones are blended together in there. Anatomists tried, though, and initially and long ago (Meckel came to this conclusion in 1825), decided the bones were numbered DI, DII, and DIII, just like the ones we see in three-fingered dinosaurs…so no dilemma, right?

Wrong. There’s another way of looking at the identity of these bones, and that is by watching them develop. What some birds do is start to make five fingers—they form four or five little nubbins of cartilage, called condensations, and then shut down the development of some of them. What another old time anatomist noticed (Owen, in 1836) was that one of the condensations that got thrown away was the first one—which means that the bird digits are actually derived from Condensation II, Condensation III, and Condensation IV. The data is even stronger in this day of molecular markers: bird digits arise embryonically from the second, third, and fourth cartilaginous condensations.

Now this is a complication for evolution. We have three-fingered dinosaurs, and three-fingered birds, but it looks like they aren’t the same fingers. Bird ancestors would have had to resurrect their discarded Digit IV, then eliminate Digit I, all before fusing the whole assemblage into a bony gemisch anyway. It’s not parsimonious at all. (Of course, it’s even less parsimonious to throw away more than a century of data supporting evolution, as Jonathan Sarfati would like us to do.)

There is another, better explanation that Wagner and Gauthier have made that clarifies everything to me, at least.

Note that anatomists initially assigned digit numbers I, II, and III to bird limbs on the basis of their form, but later had to revise that to II, III, and IV on the basis of embryology. Dinosaur digits are assigned numbers I, II, and III on the basis of their adult form (which is admittedly much less ambiguous than adult bird digits!)…but what about their embryology? If we had access to information about expression of molecular markers and early condensations in the dinosaur limb, would we have to revise their digit numbers?

We don’t have fetal dinosaur hands to experiment on, but our growing knowledge about how limbs develop suggests that that might just be the case. This diagram illustrates the sequence of development in the hand of an alligator (a) and an ostrich (b).

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(Click for larger image)

What you’re seeing is the pattern of early condensations in the limb. We tetrapods have a standard pattern: the very first digit to develop as an extension of the limb is Condensation IV, your ring finger, forming what is called the metapterygial axis. Next, the pinky (CV) forms as a little afterthought along one side of the metapterygial axis, and a new axis of condensation hooks over the palm, with the middle finger (CIII) forming next, then the index finger (CII), and lastly the thumb (CI). From a developmental standpoint, the easiest digits to lose are that odd little CV, and the thumb, CI. CI is the very last to form, so you can stop its formation by changing the timing of development in a process called heterochrony, and just halting the development of that axis hooking across the palm early. You can see that in the ostrich, which just stops making fingers after CII, so CI doesn’t form. The hardest digit to lose is CIV, because it’s kind of the lynchpin of the process—all the other digits follow after IV, so it would be difficult to suppress IV without losing all of the other digits. (Who would have thought that the ring finger was so central and important to hand development?)

The numbering of the dinosaur limb is a problem then…it suggests that they don’t have a Digit IV, which looks like a complicated and unlikely thing to do. But they do have a ‘thumb’, or Digit I. How do we resolve this seeming contradiction?

The answer is that there are two developmental processes going on. The first is the formation of the condensations, CI through CV. This process partitions the terminal region into an appropriate number of chunks, but doesn’t actually specify the identity of the digits. The second process takes each of those chunks and assigns a digit identity to them, and this process is to some degree independent of the first and uses a different set of signals. Wolpert et al. have noticed this in modern embryos:

For example, digit identity is specified at a surprisingly late stage in limb development, and identity remains labile even when the digit primordia have formed. It now appears that digit identity is specified by the interdigital mesenchyme and requires BMP signaling. There is also evidence that mechanisms other than a diffusible morphogen operate to lay down the initial pattern of cartilage, which is then modified by a signal from the polarizing region…

What Wagner and Gauthier propose is that three-fingered dinosaurs accomplished that reduction by shedding the two easiest digits to lose, CI and CV, so that if we enumerated them by the same criteria we use in modern birds, they possess Condensations II, III, and IV. What also happened, though, was that there was a frame shift in the mechanism that assigns digit identity, so CII develops as DI, CIII as DII, and CIV as DIII.

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The timing of this shift can be mapped onto saurian phylogeny, and it all makes sense and is consistent. And it doesn’t involve taking seriously the silly sequence of the biblical account, which has birds appearing before all of the land animals.

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(Click for larger image)

What about Sarfati’s second line of evidence against evolution, that frogs and humans use completely different mechanisms to build their limbs?

Simple answer: it’s all bullshit. It’s a blatant denial of basic information you’ll find in any developmental biology textbook.

We’ve got a pretty good handle on the outline of limb development in multiple tetrapod lineages now, and they all use the same tools. Contrary to Sarfati’s implication, they all have apical ectodermal ridges (with some rare exceptions in a few highly derived, direct-developing frogs) and zones of polarizing activity, they all use the same set of molecules, including FGF-4 and FGF-8 and the same Hox genes and retinoic acid and BMPs. If there’s one thing we know, it’s that limb development is dazzlingly well conserved.

It is true that frogs have less apoptosis between their digits than we do, but that’s because they have webbed feet. Suppress apoptosis in other vertebrates, and you get the same phenomenon, retention of membranous webs between the digits. There is a simple functional reason why they differ in this regard, and it takes advantage of a common property of limb development in all tetrapods.

I can sympathize with Sarfati having difficulty sorting out digit numbering—it’s subtle and sneaky and has puzzled smarter people than either of us. But the uninformed rejection of some of the most straightforward, clearest examples of common mechanisms in development, something that you can find described in the most introductory biology textbook…that’s hard to forgive.

Wagner GP, Gauthier JA (1999) 1,2,3=2,3,4: A solution to the problem of the homology of the digits in the avian hand. Proc. Natl. Acad. Sci. 96:5111-5116.

Wolpert L, Beddington R, Jessel T, Lawrence P, Meyerowitz E, Smith J (2002) Principles of Development. Oxford University Press.