Gene regulatory networks and conserved noncoding elements

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We miss something important when we just look at the genome as a string of nucleotides with scattered bits that will get translated into proteins — we miss the fact that the genome is a dynamically modified and expressed sequence, with patterns of activity in the living cell that are not readily discerned in a simple series of As, Ts, Gs, and Cs. What we can’t see very well are gene regulatory networks (GRNs), the interlinked sets of genes that are regulated in a coordinated fashion in cells and tissues.

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The evolution of Hedgehog

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PLoS has recently published a highly speculative but very interesting paper on how a particular signaling pathway, the Hedgehog pathway, might have evolved. It’s at a fairly early stage in hypothesis testing, which is one of the things that makes it interesting — usually all you see published is the product of a great deal of data collection and experiment and testing, which means the scientific literature gives a somewhat skewed view of the process of science, letting the outsider mainly see work that has been hammered and polished, while hiding the rougher drafts that would better allow us to see how the story started and was built. It’s informative in particular for those who follow the creationist “literature”, which often crudely apes the products of actual working science, but lacks the sound methodological underpinnings. In particular, creationism completely misses the process of poking at the real world to develop ideas, since they begin with their conclusion.

So take this description as a work in progress — we’re seeing the dynamic of building up a good working model. As usual, it starts on a sound foundation of confirmed, known evidence, makes a reasonably hypothesis on the basis of the facts, and then proposes a series of research avenues with predicted results that would confirm the idea.

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Brian Goodwin, 1931-2009

It’s sad to see that we’ve lost Brian Goodwin, one of the genuinely original (but not always right!) thinkers of our time. There aren’t many left of the old structuralist tradition in biology, the kind of non-genetic purists who tried to analyze development in terms of the fundamental physical and chemical properties of the organism—they’ve been swallowed up and lost in a triumphal molecular biology research program.

Edge has a nice interview with and essay by Goodwin — they’re good places to start. If that whets your appetite, you should also read his book, How the Leopard Changed Its Spots : The Evolution of Complexity(amzn/b&n/abe/pwll), which is aimed at general audiences and is a good overview of why we should look at more than just genes to explain form.

He was an advocate for one view of nature, and I think he missed the mark by neglecting genes as much as he did; we know now that a lot of details of morphology are directly affected in subtle and not-so-subtle ways by the genetics of the organism. But I think we can also make a case that the modern molecular biological approach is also missing a significant element. Every biologist ought to read a little Goodwin, just to leaven their picture of how biology works with his special perspective.

What caused the Cambrian explosion? MicroRNA!

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No, not really — my title is a bit of a sensationalistic exploitation of the thesis of a paper by Peterson, Dietrich, and McPeek, but I can buy into their idea that microRNAs (miRNAs) may have contributed to the pattern of metazoan phylogenies we see now. It’s actually a thought-provoking concept, especially to someone who favors the evo-devo view of animal evolution. And actually, the question it answers is why we haven’t had thousands of Cambrian explosions.

In case you haven’t been keeping up, miRNAs are a hot topic in molecular genetics: they are short (21-23 nucleotides) pieces of single stranded RNA that are not translated into protein, but have their effect by binding to other strands of messenger RNA (mRNA) to which they complement, effectively down-regulating expression of that messenger. They play an important role in regulating the levels of expression of other genes.

One role for miRNAs seems to be to act as a kind of biological buffer, working to limit the range of effective message that can be operating in the cell at any one time. Some experiments that have knocked out specific miRNAs have had a very interesting effect: the range of expressed phenotypes for the targeted message gene increases. The presence or absence of miRNA doesn’t actually generate a novel phenotype, it simply fine-tunes what other genes do — and without miRNA, some genes become sloppy in their expression.

This talk of buffering expression immediately swivels a developmental biologist’s mind to another term: canalization. Canalization is a process that leads organisms to produce similar phenotypes despite variations in genotype or the environment (within limits, of course). Development is a fairly robust process that overcomes genetic variations and external events to yield a moderately consistent outcome — I can raise fish embryos at 20°C or at 30°C, and despite differences in the overall rate of growth, the resultant adult fish are indistinguishable. This is also true of populations in evolution: stasis is the norm, morphologies don’t swing too widely generation after generation, but still, we can get some rapid (geologically speaking) shifts, as if forms are switching between a couple of stable nodes of attraction.

Where the Cambrian comes into this is that it is the greatest example of a flowering of new forms, which then all began diverging down different evolutionary tracks. The curious thing isn’t their appearance — there is evidence of a diversity of forms before the Cambrian, bacteria had been flourishing for a few billion years, etc., and what happened 500 million years ago is that the forms became visible in the fossil record with the evolution of hard body parts — but that these phyla established body plans that they were then locked into, to varying degrees, right up to the modern day. What the authors are proposing is that miRNAs might be part of the explanation for why these lineages were subsequently channeled into discrete morphological pathways, each distinct from the other as chordates and arthropods and echinoderms and molluscs.

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Martin Chalfie: GFP and After

Chalfie is interested in sensory mechanotransduction—how are mechanical deformations of cells converted into chemical and electrical signals. Examples are touch, hearing, balance, and proprioception, and (hooray!) he references development: sidedness in mammals is defined by mechanical forces in early development. He studies this problem in C. elegans, in which 6 of 302 nerve cells detect touch. It’s easy to screen for mutants in touch pathways just by tickling animals and seeing if they move away. They’ve identified various genes, in particular a protein that’s involved in transducing touch into a cellular signal.

They’ve localized where this gene is expressed. Most of these techniques involved killing, fixing, and staining the animals. He was inspired by work of Shimomura, as described by Paul Brehm that showed that Aequorin + Ca++ + GFP produces light, and got in touch with Douglas Prasher, who was cloning GFP, and got to work making a probe that would allow him to visualize the expression of interesting genes. It was a gamble — no one knew if there were additional proteins required to turn the sequence into a glowing final product…but they discovered that they could get functional product in bacteria within a month.

They published a paper describing GFP as a new marker for gene expression, which Science disliked because of the simple title, and so they had to give it a cumbersome title for the reviewers, which got changed back for publication. They had a beautiful cover photo of a glowing neuron in the living animal.

Advantages of GFP: heritable, relatively non-invasive, small and monomeric, and visible in living tissues. Roger Tsien worked to improve the protein and produce variants that fluroesced at different wavelengths. There are currently at least 30,000 papers published that use fluroescent proteins, in all kinds of organisms, from bunnies to tobacco plants.

He showed some spectacular movies from Silverman-Gavrila of dividing cells with tubulin/GFP, and another of GFP/nuclear localization signal in which nuclei glowed as they condensed after division, and then disappeared during mitosis. Sanes and Lichtman’s brainbow work was shown. Also cute: he showed the opening sequence of the Hulk movie, which is illustrated with jellyfish fluorescence (he does not think the Hulk is a legitimate example of a human transgenic.)

Finally, he returned to his mechanoreceptor work and showed the transducing cells in the worm. One of the possibilities this opened up was visual screening for new mutants: either looking for missing or morphologically aberrant cells, or even more subtle things, like tagging expression of synaptic proteins so you can visually scan for changes in synaptic function or organization.

He had a number of questions he could address: how are mechanotransducers generated, how is touch transduced, what is the role of membrane lipids, can they identify other genes important in touch, and what turns off these genes?

They traced the genes involved in turning on the mec-3 gene; the pathway, it turned out, was also expressed in other cells, but they thought they identified other genes involved in selectively regulating touch sensitivity. One curious thing: the mec genes are transcribed in other cells that aren’t sensitive, but somehow are not translated.

They are searching for other touch genes. The touch screen misses some relevant genes because they have redundant alternatives, or are pleiotropic so other phenotypes (like lethality) obscure the effect. One technique is RNAi, and they made an interesting observation. Trying about 17000 RNAis, they discovered that 600 had interesting and specific effects, 1100 were lethal, and about 15,000 had no effect at all. The majority of genes are complete mysteries to us. They’ve developed some techniques to get selective incorporation of RNAis into just neurons of C. elegans, so they’re hoping to uncover more specific neural effects. One focus is on the integrin signaling pathway in the nervous system, which they’ve knocked out and found that it demolishes touch sensitivity — a new target!

They are now using a short-lived form of GFP that shuts down quickly, so they’ve got a sharper picture of temporal patterns of gene activity.

Chalfie’s summary:

  • Scientific progress is cumulative.

  • Students and post-docs are the lab innovators.

  • Basic research is essential. Who would have thought working on jellyfish would lead to such powerful tools?

  • All life should be studied; not just model organisms.

Chalfie is an excellent speaker and combined a lot of data with an engaging presentation.

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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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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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.

How to build a dinosaur

I’ve been reading a new book by Jack Horner and James Gorman, How to Build a Dinosaur: Extinction Doesn’t Have to Be Forever(amzn/b&n/abe/pwll), and I was pleasantly surprised. It’s a book that gives a taste of the joys of geology and paleontology, talks at some length about a recent scientific controversy, acknowledges the importance of evo-devo, and will easily tap into the vast mad scientist market.

It is a little scattered, in that it seems to be the loosely assembled concatenation of a couple of books, but that’s part of the appeal; read the chapters like you would a collection of short stories, and you’ll get into the groove.

The first part is about Horner’s life in Montana, the Hell Creek formation, and dinosaur collecting. Hand this to any kid and get him hooked on paleontology for life; I recall reading every book I could get my hands on that talked about Roy Chapman Andrews as a young’un, and it permanently twisted me…in a good way. This will have the same effect, and many people will think about heading out to Garfield County for a little dusty adventure. I know I am — all that stands in my way is South Dakota.

A good chunk of the book is about molecules and how they show the relatedness of dinosaurs to birds, and to the work of Horner’s former student, Mary Schweitzer, who discovered soft tissue in T. rex bones. Horner presents a good overview of the subject, but is also appropriately cautious. You’ll get a good feel for the difficulty of finding this material, and for interpreting it; he clearly believes that these are scraps of real T. rex tissue, but how intact it is, what kinds of changes have occurred in it, and how much information will be extractable from these rare bits of preserved collagen (or whatever) is left an open question.

Finally, the subject of the title…Horner was an advisor to the Jurassic Park movies, and right away he dismisses the idea of extracting 65 million year old DNA in enough quantity to reconstitute a dinosaur as clearly nothing but a fantasy. That’s simply not how it can be done. But he does have a grand, long-term plan for recreating a dinosaur.

What is it? Why, it’s developmental biology, of course. Development is the answer to everything.

Here’s his vision, and I found it believable and captivating: start with a modern dinosaur, a chicken, figure out the developmental pathways that make it different from an ancient dinosaur, and tweak them back to the ancestral condition. For instance, birds have lost the long bony tail of their ancestors, reducing it to a little stump called a pygostyle. In the embryo, they start to make a long tail, but then developmental switches put a kink in it and reduce it to a stub. If we could only figure out what specific molecules are signaling the tissue to take this modern reducing path and switch them off, then maybe we could produce a generation of chickens with the long noble tails of a velociraptor.

My first thought was skepticism — it can’t be that easy. There may be a simple network of genes that regulate this one early decision to form a pygostyle from a tail, but there have been tens of millions of years of adaptation by other genes to the modern condition; we’re dealing with a large network of interlinked genes here, and unraveling one step in development doesn’t mean that subsequent steps are still competent to respond in the ancient pattern. But then, thinking about it a little more, one of the properties of the genome is its plasticity and ability to respond in a coherent, integrated way to changes in one part of a gene network. That capacity might mean you could reconstitute a tail.

And then, once you’ve got a tailed chicken, you could work on adding teeth to the jaws. And foreclaws. And while you’re at it, find the little genomic slider that controls body size, and turn it up to 11. What he’s proposing is a step-by-step analysis of chicken-vs.-dinosaur decisions in the developmental pathways, and inserting intentional atavisms into them. This is all incredibly ambitious, and it might not work…but the only way to find out is try. I like that in a scientist. Turning a chicken into a T. rex is a true Mad Scientist project, and one that I must applaud.

One reservation I have about this section of the book is that too much time is spent dwelling over ethical concerns. Need I mention that real Mad Scientists do not fret over the footling trivia of the Institutional Review Board? These are chicken embryos, animals that your average member of the taxpaying public finds so inconsequential that they will pay to have them homogenized into spongy-textured slabs of yellow protein to be slapped onto their McMuffin. Please, people, get some perspective.

As for respecting the chickens themselves, what can be grander and more respectful than this project? I would whisper to my chickens, “With these experiments, I will take your children’s children’s children, and give them great ripping claws like scythes, and razor-sharp serrate fangs like daggers, and I will turn them into multi-story towers of muscle and bone that will be able to trample KFC restaurants as if they were matchboxes.” And their eyes would light up with a feral gleam of primeval ambition, and they would offer me their ovaries willingly. I’d be doing the chickens a favor. Maybe some chicken farmers would have cause to be fearful, but I wouldn’t be working on their embryos, so let them tremble.

Oh, all right. Horner is taking the responsible path and putting some serious thought into the ethics of this kind of experiment, which is the right thing to do. It’s also the kind of project that will generate serious and useful information about developmental networks, even if it fails in its ultimate aim.

But I have a dream, too. Of a day when biotechnology is ubiquitous, and middle-class kids everywhere will have a cheap DNA sequencer and synthesizer in their garages, and a freezer with handy vectors and enzymes for directed insertional mutagenesis. And one day, Mom will come home with a box of fresh guaranteed organic free range chicken eggs, and Junior’s eyes will glitter with a germ of a cunning plan, fed by a little book he found in the library…and 30-foot-tall fanged chickens will triumphantly stride the cul-de-sacs of suburbia, and the roar of the dinosaur will be heard once again.

Embryonic similarities in the structure of vertebrate brains

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I’ve been doing it wrong. I was looking over creationist responses to my arguments that Haeckel’s embryos are being misused by the ID cretins, and I realized something: they don’t give a damn about Haeckel. They don’t know a thing about the history of embryology. They are utterly ignorant of modern developmental biology. Let me reduce it down for you, showing you the logic of science and creationism in the order they developed.

Here’s how the scientific and creationist thought about the embryological evidence evolves:

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An observation: vertebrate embryos show striking resemblances to one another.

An explanation: the similarities are a consequence of shared ancestry.

Ongoing confirmation: Examine more embryos and look more deeply at the molecules involved.

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Creationist thinking

A premise: all life was created by a designer.

An implication: vertebrate embryos do not share a common ancestor.

A conclusion: therefore, vertebrate embryos do not show striking resemblances to one another.



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Snails have nodal!

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My first column in the Guardian science blog will be coming out soon, and it’s about a recent discovery that I found very exciting…but that some people may find strange and uninteresting. It’s all about the identification of nodal in snails.

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Why should we care? Well, nodal is a rather important — it’s a gene involved in the specification of left/right asymmetry in us chordates. You’re internally asymmetric in some important ways, with, for instance, a heart that is larger on the left than on the right. This is essential for robust physiological function — you’d be dead if you were internally symmetrical. It’s also consistent, with a few rare exceptions, that everyone has a stronger left ventricle than right. The way this is set up is by the activation of the cell signaling gene nodal on one side, the left. Nodal then activates other genes (like Pitx2) farther downstream, that leads to a bias in how development proceeds on the left vs. the right.

In us mammals, the way this asymmetry in gene expression seems to hinge on the way cilia rotate to set up a net leftward flow of extraembryonic fluids. This flow activates sensors on the left rather than the right, that upregulate nodal expression. So nodal is central to differential gene expression on left vs. right sides.

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What about snails? Snails are cool because their asymmetries are just hanging out there visibly, easy to see without taking a scalpel to their torsos (there are also internal asymmetries that we’d need to do a dissection to see, but the external markers are easier). The assymetries also appear very early in the embryo, in a process called spiral cleavage, and in the adult, they are obvious in the handedness of shell coiling. We can see shells with either a left-handed or right-handed spiral.

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Chirality in snails. a, Species with different chirality: sinistral
Busycon pulleyi (left) and dextral Fusinus salisbury (right). b, Sinistral (left)
and dextral (right) shells of Amphidromus perversus, a species with chiral
dimorphism. c, Early cleavage in dextral and sinistral species (based on ref.
27). In sinistral species, the third cleavage is in a counterclockwise direction,
but is clockwise in dextral species. In the next divisions the four quadrants
(A, B, C and D) are oriented as indicated. Cells coloured in yellow have an
endodermal fate and those in red have an endomesodermal fate in P. vulgata
(dextral)15 and B. glabrata (sinistral)28. L and R indicate left and right sides,
respectively. d, B. glabrata possesses a sinistral shell and sinistral cleavage
and internal organ organization. e, L. gigantea displays a dextral cleavage
pattern and internal organ organization, and a relatively flat shell
characteristic of limpets. Scale bars: a, 2.0 cm; b, 1.0 cm; d, 0.5 cm; e, 1.0 cm.

Until now, the only organisms thought to use nodal in setting up left/right asymmetries were us deuterostomes — chordates and echinoderms. In the other big (all right, bigger) branch of the animals, the protostomes, nodal seemed to be lacking. Little jellies, the cnidaria, didn’t have it, and one could argue that with radial symmetry it isn’t useful. The ecdysozoans, animals like insects and crustaceans and nematodes, which do show asymmetries, don’t use nodal for that function. This suggests that maybe nodal was a deuterostome innovation, something that was not used in setting up left and right in the last common ancestor of us animals.

That’s why this is interesting news. If a major protostome group, the lophotrochozoa (which includes the snails) use nodal to set up left and right, that implies that the ecdysozoans are the odd group — they secondarily lost nodal function. That would suggest then that our last common ancestor, a distant pre-Cambrian worm, used this molecule in the same way.

Look in the very early mollusc embryo, and there’s nodal (in red, below) switched on in one or a few cells on one side of the embryo, the right. It’s asymmetrical gene expression!

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(Click for larger image)
Early expression of nodal and Pitx in snails. a, 32-cell stage L.
gigantea expressing nodal in a single cell. b, Group of cells expressing Pitx in
L. gigantea. c, Onset of nodal expression in B. glabrata. d, A group of cells
expressing Pitx in B. glabrata. e, 32-cell L. gigantea expressing nodal (red) in
a single cell (2c) and brachyury (black) in two cells (3D and 3c).
f-h, brachyury (black) is expressed in a symmetrical manner in progeny of 3c
and 3d blastomeres (blue triangles in g), thus marking the bilateral axis, and
nodal (red) is expressed on the right side of L. gigantea in the progeny of 2c
and 1c blastomeres, as seen from the lateral (f) and posterior (g, h) views of
the same embryo. i, A group of cells expressing nodal (red) in the C quadrant
and Pitx (black) in the D quadrant of the 120-cell-stage embryo of L.
gigantea. j, nodal (red) and Pitx (black) expression in adjacent areas of the
right lateral ectoderm in L. gigantea. L and R indicate the left and right sides
of the embryo, respectively. The black triangle in b and i, the green, yellow
and pink arrows in f and i, and the black and pink arrows in f and h point to
the equivalent cells. Scale bars: 50µm.

Seeing it expressed is tantalizing, but the next question is whether it actually does anything in these embryos. The test is to interfere with the nodal-Pitx2 pathway and see if the asymmetry goes away…and it does, in a dramatic way. There is a chemical inhibitor called SB-431542 that disrupts this pathway, and exposing embryos to it does interesting things to the formation of the shell. In the photos below, the animal on the left is a control, and what you’re seeing is a coiled shell (opening to the right). The other two views are of an animal treated with SB-431542…and look! Its shell doesn’t have either a left- or right-handed twist, and instead extends as a straight tube.

i-055b648d0eaf32f767e5cc31f9f9a19b-nodal_shell.jpeg
(Click for larger image)
Wild-type coiled and drug-treated non-coiled shells of B.
glabrata. Control animals
(e) display the normal sinistral shell morphology. Drug-treated animals
(f, g, exposed to SB-431542 from the 2-cell stage onwards) have straight
shells. f and g show an
individual, ethanol-fixed, and shown from the side (f) and slightly rotated
(g).

What this all means is that we’ve got a slightly better picture of what genes were present in the ancestral bilaterian animal. It probably had both nodal and Pitx2, and used them to build up handedness specializations. Grande and Patel spell this out:

Although Pitx orthologues have also been identified in non-deuterostomes such as Drosophila melanogaster and
Caenorhabditis elegans, in these species Pitx has not been reported in
asymmetrical expression patterns. Our results suggest that asymmetrical expression of Pitx might be an ancestral feature of the bilaterians.
Furthermore, our data suggest that nodal was present in the common
ancestor of all bilaterians and that it too may have been expressed
asymmetrically. Various lines of evidence indicate that the last common ancestor of all snails had a dextral body. If this is true, then our
data would suggest that this animal expressed both nodal and Pitx on
the right side. Combined with the fact that nodal and Pitx are also
expressed on the right side in sea urchins, this raises the possibility
that the bilaterian ancestor had left-right asymmetry controlled by
nodal and Pitx expressed on the right side of the body. Although
independent co-option is always a possibility, the hypotheses we present can be tested by examining nodal and Pitx expression and function in a variety of additional invertebrates.

It’s also, of course, more evidence for the unity of life. We are related to molluscs, and share key genes between us.

Grande C, Patel NH (2009) Nodal signalling is involved in left-right asymmetry in snails. Nature 457(7232):1007-11.

Man-hating Chinese doctors murder baby!

God blessed a Chinese woman with twin baby boys, each one ensouled at the instant of fertilization with personhood and a personal divine fate. At their birth, though, the doctors callously ended one proud male life…and they’ve probably got the poor fellow pickled in a jar somewhere. Here’s a photo of the pair.

i-1bebb470f7aee1ac66b17c45c220bc00-Baby_with_spare.jpeg

That is a baby. The odd blob on his back? That’s his brother, a nicely formed penis growing and thriving there. This is a case of fetus in fetu, in which a mass of cells, either an absorbed twin or a large teratoma (a surprisingly well differentiated, but abnormal, fragment of embryonic tissue), form a partial individual fused to a larger fetus. In this case, all that made it was a penis and what looks like some other adjoining tissues.

Please note that Little Wang (the kids were unnamed, but I have to call him something) is made up of entirely human tissue, and is genuinely alive — blood flows through him, he was probably innervated with sensory and motor nerves, he is obviously sufficiently differentiated that we have no problem calling him male, and he is a miracle of creation — yet what did the doctors do? In a cruel three hour operation, they cut him away from his brother and let him bleed to death.

Yeah, I know what you’re going to say: he was a parasite, entirely dependent on his brother for survival, and could not live on his own—but we all know that argument is totally bogus, because…because…well, because it just is! Life is precious, and you must choose life!

(OK, seriously, this was an awkward developmental difficulty for the child and his parents, but it’s also fascinating. Not enough detail is given in the articles; I’d like to know about deeper levels of organization. Were the various ducts present? Was there any recognizable pelvic tissue? Three hours of surgery implies there may have been some internal entanglements? And most importantly, that location implies that there might have been some spinal involvement — I hope the kid is entirely all right now, and that there won’t be any long term problems.)

(And again seriously, it does raise issues of individuality and human identity. I have no problem saying that the extra penis is an unwanted growth that can be eliminated with no ethical difficulties…but people who claim personhood for a tiny ball of cells ought to have greater problems with this case.)