Ediacaran Underground

As you may have guessed from my blog title, I’m fascinated by the early history of animals. Part of the problem with this early history is that its fossil record is extremely sketchy, extremely difficult to interpret, or both. One of the things that marks the diversification of animals late in the Ediacaran (the last period of the Precambrian) and even more during the Cambrian explosion, is the appearance of burrows and other trace fossils. Marks on the seafloor don’t have to be made by what we usually think of as “complex” animals, though. Burrowing sea anemones are well known, and even single-celled creatures can plough a track in the sediment. That makes things rather complicated when you are looking for the first signs of complex bilaterian animals in the rocks.

Pecoits et al. (2012) are convinced that the trace fossils they found in Uruguay are such a sign, perhaps indeed the oldest. The gently meandering little burrows they report in Science are shaped much like the burrows made by some modern molluscs and annelid worms, possessing features that are hard to explain with a rolling protist or a simple animal. Among other things, they have minute indentations in their walls that may indicate the places where leg-like body parts pushed against the sediment as the animal pulled itself along.

The mysterious burrowers’ path was relatively simple, meandering in broad sine waves that may preserve the unknown creature’s search for food. Burrows often cross, suggesting that their makers made no effort to avoid each other. However, they sometimes disappear and reappear a few millimetres later, as if the animal made a detour upwards or downwards. The few abrupt turns, combined with the width of the burrows, indicate that the creature who left these traces was small, less than a centimetre long.

These burrows – if they are indeed that – are somewhere between 585-600 million years old, at least 30 million years older than the oldest uncontested bilaterians. My question, though, is are they? Bilaterian burrows, I mean. It’s a good thing I double-checked about trace-making protists, since it turns out that the protists in question leave tracks that have supposedly bilaterian characteristics, like consisting of two ruts on either side of a central ridge (Matz et al., 2008). This is one of the similarities the new paper draws between modern bilaterian burrows and their Precambrian precursors! (The weirdest thing is they cite Matz et al. but kind of dismiss the eerily bilaterian character of protist traces…)

Pecoits et al. (2012) do take some time to argue against a protist or non-bilaterian origin of the burrows. There are the little indentations that may have come from something like a worm’s parapodia but are perhaps more difficult to reconcile with a simple rolling ball of cytoplasm. There are also the disappearances and reappearances that indicate the creature moving up and down levels. The authors also argue that the burrows are definitely burrows – i.e. actually under the sediment surface -, though I’m not sure this follows from their evidence.

Then again, they’re the experts here, I’m just a geologically challenged biologist looking at pictures of grooves in rocks… The disappearance-reappearance thing does seem to imply that whatever left these fossils could burrow. (Unless it suddenly hopped off the bottom and landed nearby? And why can’t protists burrow anyway? We didn’t even know they made fake animal traces until a few years ago… I think I’m beginning to sound silly…)

As far as arguments for a bilaterian trace-maker go, a lot is made of the infillings in the burrows, and of the parts that look like the roof collapsed after the animal moved on, but honestly I can’t see what they are talking about in the photos, so I’ll stay on the safe side and reserve judgement there ­čÖé

And here I meander off track (terrible pun fully intended)

Aaaaanyway, let’s assume they are right, and there’s a reasonably complex worm behind these burrows.

That would be awesome.

However, it poses some questions. In fact, it poses the same questions my friend Kimberella does. I’ve been ruminating about this since I wound up explaining what we know of the bilaterian ancestor to some random guy on the internetz. Let me pour out the contents of my brain here, and let’s hope they make sense >_>

Kimberella. This creature is at the very least a bilaterian, but probably not too far off from molluscs. Its amazing fossil record includes incontrovertible evidence that it could move around and graze on whatever it ate (microbes, probably) by scraping it off the seafloor. It was covered in a knobby armour – fairly flexible and probably not made of a single peace, but definitely a shell of sorts. That’s one of the things that suggest ties to molluscs. Either way, something that we can identify as a member of a subgroup of bilaterians must postdate the bilaterian common ancestor by a fair bit – all those lineages needed time to split and evolve their recognisable body plans.

Palaeontologists and developmental biologists (Budd and Jensen, 2000; Erwin and Davidson, 2002) made a good case in arguing that the last common ancestor of bilaterians must have been small and simple. We know this creature must have predated Kimberella by some time – and this is where we run into problems. One of the strongest arguments for a small and simple bilaterian ancestor is the paucity of Precambrian trace fossils. Large and complex bilaterians make a big mess of seafloor sediment. They dig into it, they walk over it, they churn it up and eat it and spit it out. Nonetheless, the Precambrian is full of virtually undisturbed microbial mats, an unexploited bonanza for any bilaterian with the means to graze. So the conclusion is that large, complex bilaterians must have been rare or non-existent. But, Kimberella? Burrows of a large-and-complex bilaterian* that predate Kimberella by 30 million years?

Something odd is going on here.

I suppose the question could be put as: if these creatures were around in the late Precambrian, why weren’t there more of them? (And where are the bodies?) Why didn’t they swarm out and eat all the microbial mats that made the preservation of many Ediacaran fossils possible (Narbonne, 2005)? Kimberella was doing its best to graze them off the face of the earth, yet this same type of preservation is common even in the formations everyone’s favourite proto-mollusc comes from.

Too little oxygen? But Kimberella is large, relatively compact and partly covered in armour. It’s the sort of creature that we’d expect to have a specialised respiratory system even in an oxygen-rich modern sea, so at least one animal clearly solved this problem…

I suppose it all comes back to what caused the Cambrian explosion, which is a tough question. (Marshall [2006] is a pretty nice review if memory serves.) If we figure out why molluscs and worms and other bilaterians didn’t take over the oceans long before the Cambrian, we’ll have figured out why they did so in the Cambrian.

I’m not sure that did make sense in the end, but I’m glad I could get it off my chest ­čśÇ

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*A centimetre may not sound very large, but a pretty big percentage of the animal kingdom comes nowhere near it in size.

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References:

Budd GE & Jensen S (2000) A critical reappraisal of the fossil record of the bilaterian phyla. Biological Reviews of the Cambridge Philosophical Society 75:253-295

Erwin DH & Davidson EH (2002) The last common bilaterian ancestor. Development 129:3021-3032

Marshall CR (2006) Explaining the Cambrian “explosion” of animals. Annual Review of Earth and Planetary Sciences 34:355-384

Matz MV et al. (2008) Giant deep-sea protist produces bilaterian-like traces. Current Biology 18:1849-1854

Narbonne GM (2005) The Ediacara biota: Neoproterozoic origin of animals and their ecosystems. Annual Review of  Earth and Planetary Sciences  33:421-442

Pecoits E et al. (2012) Bilaterian burrows and grazing behavior at >585 million years ago. Science 336:1693-1696

Hexapeptide or bust!

I have a big soft spot for Hox genes, or rather, Hox proteins. Thanks to some of my earlier work, I also have a soft spot for all their secret little sequence motifs that help them interact with other proteins and help us classify them (e. g. Balavoine et al., 2002). Probably the best-known such motif is the hexapeptide. (“Hexa-” would kind of imply that it’s made of six amino acids, but people only ever seem to talk about four. Don’t ask me why they call it a hexapeptide…)

This motif is very widespread, occurring not just in Hox proteins but also in many others in the larger class of homeodomain proteins that Hoxes belong to. For many years, the hexapeptide has been regarded as the key to the interaction of Hox proteins with another homeodomain-bearing protein, called Extradenticle in flies and Pbx plus a number (we have 3 of them) in vertebrates*. (Above is a cartoon version of DNA with the homeodomains – the purple curls – of Exd and the fly Hox protein Ubx bound to it, from the Protein Data Bank.) Hox proteins bind DNA to regulate various genes, and are absolutely vital for an embryo to develop the right organs in the right places. This interaction changes their DNA binding behaviour, making the hexapeptide possibly the most important four amino acids in animal development.

And now we’re supposed to scrap that?

I’ve just skimmed through Hudry et al. (2012), and died a little inside.

The study claims – on what seems to be good evidence – that the hexapeptide is not all it’s cracked up to be. Out of six fruit fly Hox proteins examined, only two stop interacting with Exd when the hexapeptide is mutated beyond recognition, and even then one of them is kind of half-hearted about it. The team also tested a few mouse Hox genes in cultured cells and – for whatever reason – chick embryos, and got largely the same results.

I rather like their approach, though. I think the method for detecting interaction is incredibly clever, though it’s clearly not something they invented. The idea is based on fluorescent proteins. These are very commonly used to track the levels and whereabouts of other proteins. Since they are pretty small and innocuous, the gene encoding them can be tacked onto the gene of interest, and the resulting protein chimaera will do whatever the target protein would do without its fluorescent companion. The only difference is now it glows wherever it goes. The more protein, the brighter the glow.

The nice thing about fluorescent proteins is that you can cut them in half, and if the two parts get close enough, they’ll still glow. Therefore, if you glue one half of the DNA for a fluorescent protein to gene 1, the other half to gene 2, and let them loose in the same cell, you can tell whether the protein products of gene 1 and gene 2 interact just by looking for the telltale fluorescence. And the tale it’s telling is that all these Hox proteins are getting snug with Exd despite the loss of the motif supposedly necessary for the interaction.

I’ll just go away and quietly get over that now.

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*Vertebrate geneticists have no imagination. Okay, they did come up with lunatic fringe and Sonic hedgehog. After the fly people named fringe and hedgehog.

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References:

Balavoine G et al. (2002) Hox clusters and bilaterian phylogeny. Molecular Phylogenetics and Evolution 24:366-373

Hudry B et al. (2012) Hox proteins display a common and ancestral ability to diversify their interaction mode with the PBC class cofactors. PLoS Biology 10:e1001351

Genes from scratch

When we talk about evolutionary novelty, especially if the talking is to non-specialists, gene duplication is all the rage. From the sophistication of vertebrate blood clotting to the seemingly pointless complexity of a yeast proton pump (Finnigan et al., 2012), accidentally copied genes are undoubtedly an important source of new stuff in evolution. But copying and tweaking is not the only way new genes can arise. Sometimes, new genes really are new.

I admit, I wasn’t nearly excited enough about this possibility until this paper landed in my RSS reader a while back. Toll-Riera et al. (2012) find that the boring repetitive DNA that my gut feeling would’ve dismissed as true “junk” may actually be a great source of new proteins. First, it’s a good theoretical source . Long stretches of repetitive sequence are less likely than random sequence to suddenly and unceremoniously end in a stop codon* and translate to a short and useless amino acid sequence. Second, it appears that younger proteins do contain more repetitive sequence than old ones. What’s more, the repeats are often found within the regions that confer function on proteins. They aren’t just useless filler.

So, okay, a lot of proteins seem come from pieces of “junk” DNA. How?

Maybe they arise from random gene expression noise and turn into proper genes gradually, say Carvunis et al. (2012). It has been known for a while that DNA that doesn’t belong to traditionally recognised genes quite often gets transcribed into RNA in cells. Sometimes, these random bits of RNA may even be translated into an amino acid chain. If some of these accidents are actually useful, the researchers reasoned, they could create a selection pressure to turn the DNA that produced them into a proper gene.

They took this idea and applied it in a study of open reading frames (ORFs) in the yeast genome. An “ORF” is jargon for a stretch of DNA that isn’t interrupted by stop codons. In theory, any ORF could make a “meaningful” piece of protein. Most ORFs that aren’t genes are short, often just a handful of codons; and most ORFs known to be genes are long, with hundreds of codons. The team argued that if random ORFs can give rise to genes, there should be plenty of transitional forms.

To test this, they first classified all the hundreds of thousands of ORFs in the yeast genome according to their evolutionary age. The ones that were conserved in all of the yeast species they used for comparison were given a score of 10, and ORFs that only brewer’s yeast had were called zeroes. (Most known genes belong to classes 5-10, meaning they evolved quite far back on the yeast family tree.) The next step was to pick the Class Zero ORFs that were actually transcribed and translated, so might be in the pool of potential “proto-genes”. They found this set of “0+” ORFs by analysing RNA sequencing data in both happy yeast cells and yeast deprived of food, just to make sure they caught any sequences that only acted like genes under some circumstances. In addition, they also checked which of those RNAs were associated with ribosomes, the sites of translation. These filtering steps left over a thousand little ORFs that don’t belong to known genes, are completely unique to Saccharomyces cerevisiae, expressed, and probably translated.

Going up the conservation scale, ORFs become increasingly gene-like. The older ones are longer, their RNA copies are more abundant, and more of them appear constrained by natural selection. (Interestingly, when you translate them, the more gene-like ORFs produce less ordered protein structures. Not sure what to make of that.) Proper genes are also better suited to get ribosomes to translate them. Conservation classes 1-4, those ORFs that are shared only by closely related Saccharomyces species, are intermediate in all of these properties (and some more) between the zeroes and the older ORFs.

There is one more thing about this study that definitely bears mentioning When you count how many new gene duplicates this yeast species has versus how many new, potentially functional, random ORFs, the latter come out on top by far. Between them, S. cerevisiae and its closest sister species apparently have somewhere between one and five newly duplicated genes. The same duo also came up with nineteen new ORFs that are under selection and therefore probably functional. Potentially, these random little sequences people might have dismissed as background noise not long ago are more potent sources of new genes than the celebrated gene duplication.

I don’t know about you, but that absolutely fascinates me.

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P.S.: Incidentally, this is all about protein-coding genes. However, thousands of genes in your own genome do NOT encode proteins. They include genes for the good old RNA components of the translation machinery, ribosomal and transfer RNA, but there are also other RNA genes with transcripts involved in everything from keeping parasitic DNA in check to editing the messenger RNAs of other genes. I kind of want to find out how these RNAs form and acquire functions. Also, when we are quite happy to call a piece of DNA that doesn’t have a protein product a “gene”, and cells are swarming with RNA that doesn’t come from things traditionally called “genes”, and some of this RNA actually does encode proteins, what does that do to the definition of a “gene”??

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*Gotta love the mnemonics on that page. I didn’t think three three-letter combinations would be that hard to remember, but I have to admit I chuckled at “U Are Gone”.

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References:

Carvunis A-R et al. (2012) Proto-genes and de novo gene birth. Nature advance online publication, doi: 10.1038/nature11184

Finnigan GC et al. (2012) Evolution of increased complexity in a molecular machine. Nature 481:360-364

Toll-Riera M et al. (2012) Role of low-complexity sequences in the formation of novel protein-coding sequences. Molecular Biology and Evolution 29:883-886

Before they became weird

Echinoderms are weird. They are supposed to be bilaterian animals, but they have abandoned bilateral (mirror image) symmetry for looking like fleshy stars, spiny boobs, strange flowers or funky sausages. When they first appear in the fossil record during the Cambrian period*, they show up with an even weirder menagerie of body plans ranging from almost bilateral through asymmetric to all sorts of variations and twists on the standard five-rayed body plan that we know and love. (Below: a selection of weird and wonderful Cambrian echinoderms from Zamora et al. [2012])

(We only know that some of these creatures were echinoderms or very close relatives thereof because they have skeletons with a unique spongy microstructure (stereom) only seen in echinoderms.)

I don’t know nearly enough about echinoderms to properly discuss the latest addition to the march of the weirdos, but damn me if I don’t at least give a proper fangirlish SQUEEE! to a new Cambrian echinoderm – with bilateral symmetry! Zamora et al. (2012) actually describe two fossil finds, but one of them is new specimens of a previously known animal. However, the other is brand new, and what a pretty thing, too! Behold Ctenoimbricata spinosa, straight out of science fiction – or a nightmare :-P! (OK, don’t start having Ctenoimbricata nightmares just yet. The whole animal was less than an inch long.)

The creature was reconstructed from fossils found in Middle Cambrian (about 510 million years old) rocks in northern Spain. The shape of its body and the arrangement of its many armour plates most closely resemble an obscure group of ancient echinoderms called ctenocystoids (represented by fossil A in the first picture). Typical ctenocystoids have slight asymmetries manifested as different arrangements of armour plates on their left and right sides. However, some are well-behaved bilaterians. That’s the other point of the paper: new fossils belonging to a previously known ctenocystoid demonstrate its symmetry. The authors think that the similarly symmetrical Ctenoimbricata was an even more primitive relative of ctenocystoids. In their view, echinoderms started out with mirror image symmetry, then became asymmetric, and only then did they evolve the radial symmetry starfish exemplify.

Ctenoimbricata, according to Zamora et al., is the most primitive echinoderm ever found. The fact that it doesn’t have a stalk or arms suggests that it wasn’t a filter feeder. Instead, it probably operated flat on the seafloor, gulping sediment and sifting out the edible bits. Since ctenocystoids are also stalk- and armless, this might mean that the last common ancestor of all echinoderms lived in a similar way, which has apparently been a matter of some debate. Yay!

Incidentally, I had no idea that Europe had such awesome Cambrian fossils. I thought the best sites were all at a minimum of a half-day plane ride away. So: double squee for our tiny spiny sandmower!

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*People have argued that a Precambrian fossil called Arkarua may be an echinoderm ancestor, but I wouldn’t bet on that. Just about the only thing those tiny imprints can be shown to share with echinoderms is the five-part symmetry, and it’s not like unusual body symmetries were… unusual for Precambrian animals.

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Reference:

Zamora S et al. (2012) Plated Cambrian bilaterians reveal the earliest stages of echinoderm evolution. PLoS ONE 7: e38296

Damn those ugly facts!

Aw, I’m gutted.

In an undergrad developmental biology course that was otherwise not nearly as interesting as I’d expected to be, there were a few bits neat enough to make me (figuratively ;)) bounce up and down with joy. The differential cell adhesion hypothesis was one of these. It’s the idea that different types of cells in an embryo can sort themselves simply by basic physical forces depending on how sticky they are. If you mix cells with different stickiness, the ones that adhere more strongly to one another will clump on the inside. The great thing about this idea is that it’s a ridiculously simple explanation for the intricate cellular dance of gastrulation, which starts a homogeneous-looking ball of cells on its way to becoming a complex, multi-layered animal body.

If there’s one thing science should’ve taught me, it’s that simple ideas are usually too good to be the (complete) truth. And indeed, when Ninomiya et al. (2012) manipulated the stickiness of cells in frog embryos by changing the level of the “glue” protein C-cadherin, they were in for a little surprise. If differential cell adhesion was indeed responsible for cells organising into layers during gastrulation, then manipulating adhesion in the embryo should produce a visible gastrulation defect. But the only way this happened in this study was when the cells became so un-sticky that the tissue physically disintegrated. Altering general cadherin levels any less than that didn’t change a thing, and changing cadherin expression in just part of a single cell layer didn’t cause that layer to separate into two.

Interestingly, a soup of individual cells does sort itself into layers based on different stickiness. It just doesn’t happen in an intact embryo. So obviously, differential cell adhesion could do what it’s been theorised to do – but it’s not the (only) mechanism real live embryos use to organise themselves. The authors propose that cells in the embryo are (1) using adhesion/repulsion mechanisms other than cadherin to regulate their behaviour, (2) concentrating however much cadherin they possess where it’s most needed, compensating for an overall scarcity by changing the distribution of the protein. And probably cackling evilly to themselves. You thought you were clever, scientist? Well, screw you.

Reference:

Ninomiya H et al. (2012) Cadherin-dependent differential cell adhesion in Xenopus causes cell sorting in vitro but not in the embryo. Journal of Cell Science 125:1877-1883

Did early tetrapods walk on land?

“Fishapods” like Tiktaalik and Ichthyostega are among the iconic transitional fossils that mark part of our own evolutionary journey. Understanding how these intriguing animals lived is key to understanding why they ended up out of water, and how evolution took them there. Of course, one of the most important changes that happened to our fishy forebears is gaining the ability to walk. While “walking” may be less of a challenge to fish than we thought, just how good “classical” proto-tetrapods like Ichthyostega were at it still isn’t entirely clear. (Below: model of Ichthyostega emphasising its aquatic capabilities; photo by Dr G├╝nther Bechly, Wikimedia Commons.)

The recent discovery of tetrapod-like trackways in Mid-Devonian rocks in Poland (Nied┼║wiedzki et al., 2010) added to the pile of evidence on the “pretty good” side. The footprints appeared to come from feet that looked like those of Ichthyostega, and the trackways betrayed an animal that walked essentially like a modern tetrapod, lifting its body clear off the ground. (The fact that these trackways were left in a different environment and 18 million years earlier than any previously known tetrapod is just the icing.)

As is usually the case in science, the paper wasn’t allowed to be simply right. Using a computerised Ichthyostega skeleton and comparisons to modern tetrapods, Pierce et al. (2012) argue that whatever made the Polish tracks and others like them, it couldn’t have been much like Ichthyostega.

The problem is that Ichthyostega had pretty stiff hip and shoulder joints. The joint surfaces are kind of elongated (and, in the case of the shoulder, also twisted), allowing the bones to hinge in various directions but not to rotate around their long axes. The unrotating hip and rigid knee of the hindlimb made it impossible for the animal to put its soles on the ground. The hindlimb looks very paddle-like with its broad, flattened bones – now it appears that paddling is all it was good for. (Below: a decade of Ichthyostega from Dennis Murphy’s wonderful Devonian Times. See his caption for the sources of the reconstructions.)

By all appearances, Ichthyostega couldn’t rotate its arms and legs enough to walk properly, and couldn’t have left even one of the presumed Polish hindlimb prints without dislocating several joints. The most it could have done is haul itself along like a seal or a mudskipper, which, considering its powerful arms and mobile elbows,might have been exactly what it did. Pierce et al. also argue that other known proto-tetrapods were unlikely to be the trackmakers; while they didn’t examine their limb joints in detail, the skeletons share many of the same features that limit the mobility of Ichthyostega‘s limbs.

That raises plenty of questions. If it wasn’t like any of the known early tetrapods, what sort of creature made those footprints? Were there other ancient tetrapod groups with more limber joints that didn’t leave body fossils because of where they lived? Did the known proto-tetrapods go back to a more aquatic existence and more rigid limbs? (Pierce et al. say that Ichthyostega‘s joints were even stiffer than some of its fishier cousins’!) Which kind begat the lineage that gave rise to living tetrapods? What does all of this say about the significance of walking-like movements we observe in living lobe-finned fish?

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References:

Nied┼║wiedzki G et al. (2010) Tetrapod trackways from the early Middle Devonian period of Poland. Nature 463:43-48

Pierce SE et al. (2012) Three-dimensional limb joint mobility in the early tetrapod Ichthyostega. Nature advance online publication, 23 May 2012. doi: 10.1038/nature11124