Phantom hourglasses

Holy ribosome, I’ve just written close to two thousand words about a paper. I… think I may have got a bit too excited. Or too bogged down in little technical details. Either way, you got lucky. The two-thousand word monster is not what you’re getting.

The reason I got excited about Piasecka et al. (2013) is that it, er, qualifies some other things I’d previously got excited about. And by “qualifies”, I mean turns inside out and performs a thorough autopsy on.

I previously touched upon the idea of the developmental hourglass – meaning that the embryos of related creatures are most similar to each other somewhere in the middle of development. The great rival of this hypothesis is that of early conservation (or the “funnel”), where embryos diverge from a similar starting point. The latter has been around as long as comparative embryology itself. The hourglass is a pretty intriguing pattern and raises all kinds of questions about what causes it – but of course, to have a cause, it has to exist in the first place.

So my previous excitement had been partly about the observation that the hourglass – originally noted in visible traits of embryos – also exists in the changing sets of genes activated throughout development (the transcriptome). According to various papers, genes expressed in mid-embryogenesis are on average older, slower-evolving and behave more similarly across species than genes active at other stages. If such observations are correct, that would certainly indicate that the hourglass is a real thing and something strange is going on with constraints and evolvability.

But, and here comes the Piasecka paper – is it?

This study is huge. There is (to use a highly technical phrase) a fucking shitload of stuff in it. Instead of looking at some big global property of the transcriptome, these authors went into all kinds of detail about various properties of specific sets of genes. They looked at – well, they say they looked at five different measures of evolutionary constraint, but actually some of those are made up of more than one thing, so really it’s quite a bit more than five.

And when they go down to that level of detail, they find that the hourglass is not a universal property of the developmental genetics of zebrafish embryos (unlike Domazet-Lošo and Tautz [2010] reported). Different measures of evolutionary constraint such as the strength of selection against protein-changing mutations, the age of the genes (which is what the original study focused on), or the conservation of their regulatory elements, show different patterns. There are hourglasses, there are a couple of funnels, and then there are parameters that just don’t exhibit much systematic change at all.

(There’s also a couple of points about potentially dodgy statistical approaches in some of these papers, which may make all the difference between an hourglass and a funnel. That’s a bit scary.)

I can’t say I’ve properly digested this paper. There’s an awful lot in it, and, my head was spinning non-stop when I finished reading. It’s definitely fascinating stuff, though, and once again, the conclusion is that things are More Complicated. (I’m kind of getting used to that at this point…) Before, you could look at a group of creatures, compare their development and ask, funnel or hourglass? Then you could ask why. Now, you can’t just make grand generalisations about anything. Taking Piasecka et al. at face value, “funnel or hourglass” is not even a valid question – it depends on exactly what you’re measuring. So much for “laws” of developmental evolution…

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References:

Domazet-Lošo T & Tautz D (2010) A phylogenetically based transcriptome age index mirrors ontogenetic divergence patterns. Nature 468:815-818

Piasecka B et al. (2013) The hourglass and the early conservation models—co-existing patterns of developmental constraints in vertebrates. PLoS Genetics 9: e1003476

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Shining a light on retinoic acid

I was planning to do more bioinformaticky stuff tonight, but then I saw Shimozono et al. (2013), and… SHINY!

I derive a particular joy from seeing neat methods, and what these guys did is pretty damn neat. They used genetic engineering and a clever trick with fluorescence to (almost) directly study an important but rather elusive molecule in vertebrate development.

Retinoic acid (RA) is related to vitamin A; in fact, it is synthesised from vitamin A by enzymes in our cells. It is what developmental biologists call a morphogen: a molecule that spreads through an embryo by diffusion, and influences development depending on its concentration. Among other things, RA is thought to be responsible for the subdivision of the embryonic body axis by Hox genes, and also the correct formation of somites, the basic repeating units that eventually form our spine.

So RA is pretty darn important, but it’s also a bit difficult to investigate. It’s a relatively small and simple molecule that isn’t encoded in the genome, so some of the popular tools for detecting important molecules don’t work on it. Its activity can be monitored indirectly, though. Retinoic acid works by binding to proteins called retinoic acid receptors (RARs), which then latch onto certain DNA sequences that regulate nearby genes. So you can, for example, construct a piece of DNA that responds to an activated RAR by producing a fluorescent protein. You can also examine the distribution of the enzymes that make and break down RA, the assumption being that this corresponds to the distribution of RA itself.

The Japanese team, however, created a modification of retinoic acid receptors that is basically a direct indicator of RA level. Their RARs have been engineered to glow in different ways depending on whether or not RA is bound to them. They were able to zap these miniature RA detectors into zebrafish embryos without affecting the little creatures’ development, creating a gentle way to monitor RA levels in live animals.

They exploited a fascinating phenomenon called fluorescence resonance energy transfer (FRET for short). FRET needs two fluorescent molecules that glow at different wavelengths, such that the wavelength one of them emits is the same that turns the other on. (Wikipedia tells me FRET is actually based on spooky quantum effects involving virtual particles rather than ordinary light travelling from molecule to molecule. Wow, I didn’t know that!)

If the two molecules are very, very close, the emissions of the first one can give the other enough energy to light up. You can detect this by shining the colour of light needed to excite the first molecule on your sample, but then also measuring the fluorescence from the second molecule. The ratio of Molecule 1 to Molecule 2 glow can tell you how much FRETting is going on.

What Shimozono et al. did was to add the code for a FRET-capable pair of fluorescent proteins to various RAR genes. RARs change shape when retinoic acid binds to them, and in these engineered versions this means that they bring their fluorescent tags close enough for FRET to work. (The above figure, from Carr and Hetherington [2000], illustrates the principle – just substitute “Ca2+” with “RA”.) The scientists calibrated their little RA detectors by measuring how much FRET happened at various controlled RA concentrations first; this allowed them to turn FRET intensities into accurate measurements of RA. They then tested whether the detectors were truly RA-specific (and not activated by, say, vitamin A) by using them in fish embryos with their RA-making enzymes crippled.

Of course, they also got round to looking at the behaviour of RA during development, which was, after all, the point of their new toys. They did a basic visualisation of RA concentration throughout developing embryos – and confirmed that the established method of looking for the enzymes involved in RA synthesis and degradation is actually a decent substitute for measuring RA itself.

They then interfered with the production of a protein called FGF8 that is thought to regulate RA synthesis, and found that this altered the RA gradient – as well as the expression of the main enzyme that produces RA. Basically – the technique seems to work, and what it shows agrees with what we’ve thought about RA signalling. Hooray!

And, of course, they got pretty pictures like the ones below, coloured according to the amount of FRET (red = high, green = low) they measured. These two compare a normal embryo (left) and an embryo of the same age whose fgf8 gene has been messed with (right). If you have normal colour vision*, it’s pretty clear how the control embryo has this massive band of redness halfway down its body, and how even nearer the head and tail ends it’s more yellow than the sad green of the treated baby.

(I spliced these together from panels in an overwhelmingly massive figure and labelled them for those of you who don’t look at fish embryos much. No copyright infringement and no financial gain is intended, of course ;))
Shimozono_etal_fgfEdit

I think this whole thing is waaaaay cool. I wish I could come up with something clever like that. Oh well, at least I get to work with fluorescent things and take pretty glowy pictures every now and then. When I’m not neck deep in protein sequences and ‘omics data. 🙂

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*Being a red-green colour blind developmental biologist must be a hell of a lot of fun. I just realised that pretty much everything involving fluorescence in biology is red, green or both – and developmental biologists love sticking fluorescent tags on everything. By the way, this particular figure could have been presented in any old combination of colours – they’re illustrating abstract numbers, not the actual colours of the specimens, which in this case would have been glowing in cyan and yellow. Of course, there’s probably a colour vision deficiency for every combination you can think of, so, uh. I probably overthunk this?

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References:
Carr K & Hetherington A (2000) Calcium dynamics in single plant cells. Genome Biology 1:reports024

Shimozono S et al. (2013) Visualization of an endogenous retinoic acid gradient across embryonic development. Nature 496:363-366

What use is (not even) half a leg?

 

(I’m even further behind on things than usual, so this is not that “hot” off the press, but the walking lungfish can’t not be posted on.)

The evolution of new traits serving new functions is always a bit of a chicken and egg problem. Why would you need wings if you don’t fly, and how could you start flying without them? Why would you need legs if you don’t walk, and how would you walk without legs?

Often, as in the case of wings, the most likely answer is that the trait originally had a different function that didn’t necessitate a “perfect” version of it. Wings that are no good for flying could be anything from egg-warmers/shades through mate attraction devices to balancing organs for prey-wrestling predatory dinosaurs (latter idea from Fowler et al., 2011, which by now has probably gone as viral as scientific papers can).

With legs, though, it seems that the chicken really did come first. We’ve known for a long time that coelacanths (which are somewhat distantly related to vertebrates with legs) sometimes move their pectoral and pelvic fins in an alternating rhythm that resembles walking. (IIRC you can find a fair few YouTube videos in which they are filmed doing that.) Nonetheless, coelacanths use this movement for swimming. They don’t actually get down and plod along the bottom.

Lungfish, however, do. King et al. (2011) videoed them doing it.

Just to be clear, the animal in question is the West African lungfish (Protopterus annectens). Unlike the respectable paddles of the Australian species, its spindly paired appendages barely even deserve to be called fins, let alone legs. (Drawing below from King et al., 2011)

Yet this creature uses its pelvic fins to propel itself along the bottom in a variety of ways. It can walk with alternating “steps”, it can bound by moving both fins at once, and sometimes it just ambles along in a slightly irregular way (videos here). If there’s no traction on the bottom of the tank, it slips and can’t get anywhere, which indicates that it does indeed propel itself by pushing against the bottom with its hind fins. And sometimes, when the fins push off, you can see part of the body come clear off the ground.

(Interestingly, the lungfish walks and bounds only with its hind fins. Meanwhile, the pectorals flail around doing other things, but they don’t engage with the floor. The diagram above gives a clue why: the animal has huge, air-filled lungs – the grey blob – that help its front half float. It doesn’t need its forefins to stroll around.)

Given how un-leglike the fins of African lungfish are, it is obvious that walking underwater doesn’t require anything as sophisticated as ankles or toes or, heck, even proper fins. Just about any ancient lobe-finned fish we know could have been capable of it. Could this be how our ancestors took their first unknowing steps towards land? Were they bottom-dwelling fish that patrolled their territories in a stately fin-walk? Did increasingly leg-like fins just help them do that better rather than breaking new ground? As the authors remind us, we already know that many of the earliest tetrapods – creatures with true legs – lived in water. If less tetrapod-like creatures could walk, then the picture fits quite nicely together.

And speaking of chickens and eggs, once again nature proves how much human incredulity is worth. Just because you don’t know what to do with half a wing, just because you don’t think X is possible without Y, doesn’t mean solutions don’t exist. Studying nature is a life-long lesson in humility in that way.

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References:

Fowler DW et al. (2011) The predatory ecology of Deinonychus and the origin of flapping in birds. PLoS ONE 6:e28964

King HM et al. (2011) Behavioral evidence for the evolution of walking and bounding before terrestriality in sacropterygian fishes. PNAS 108:21146-21151

The bare bones of fins and limbs

Perhaps the central question in developmental biology is how cells that start out as identical end up making bodies with complex shapes and a multitude of different tissues. And perhaps the central question in evo-devo is how such bodies can change into other bodies during the course of evolution. A really cool paper by Zhu et al. (2010) probes a little bit at both, and shows how relatively simple rules can produce results that are surprisingly similar to what we observe in nature.

The authors modelled the development of limb (or fin) bones in vertebrates. They used a simple model made up of the following:

  1. a virtual limb bud (let me call them “simbuds” hereafter) growing continuously
  2. a signal spreading from the tip of the bud that tells “cells” to keep growing but wanes over time (mimicking the role of the apical ectodermal ridge in real limb buds)
  3. two equations describing the activity of (1) genes that make cells differentiate into bone (“activators”), and (2) genes that prevent cells from doing so (“inhibitors”)

The shape of the simbud could be set at the start, and so could the values of all the parameters in the activator and inhibitor equations.

This is much more simple than real limb develompent. It says nothing about cell movement, and it condenses the effect of genes other than the bone activators and inhibitors into two little parameters in the equations. Yet running it with pretty much any initial settings produces something vaguely limb-like, and some sets of parameters give you simbuds that look eerily like real limbs.

Development of a simbud mimicking a chicken wing, next to drawings of the real thing.

Or fins. Or mutant limbs. Or transitional fossils.

Fully developed simbuds resembling various fossil fins: Brachypterygius was a marine reptile from the Jurassic; the other four are more or less close relatives of tetrapods, among them the famous "fishapod" Tiktaalik.

The similarity is not perfect, of course – but the model is not perfect either. Overall, it’s still pretty amazing what a variety of very realistic limb skeletons you can get out of such a simple setup – and how much you can achieve just by varying small things like how wide the limb bud is to begin with or how strongly two gene networks interact. Evolving fins into limbs should be a piece of cake for a system like that!

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Reference

Zhu J, Zhang Y-T, Alber MS, Newman SA (2010) Bare Bones Pattern Formation: A Core Regulatory Network in Varying Geometries Reproduces Major Features of Vertebrate Limb Development and Evolution. PLoS ONE 5:e10892