In which a “living fossil’s” genome delights me

I promised myself I wouldn’t go on for thousands and thousands of words about the Lingula genome paper (I’ve got things to do, and there is a LOT of stuff in there), but I had to indulge myself a little bit. Four or five years ago when I was a final year undergrad trying to figure out things about Hox gene evolution, I would have killed for a complete brachiopod genome. Or even a complete brachiopod Hox cluster. A year or two ago, when I was trying to sweat out something resembling a PhD thesis, I would have killed for some information about the genetics of brachiopod shells that amounted to more than tables of amino acid abundances. Too late for my poor dissertations, but a brachiopod genome is finally sequenced! The paper is right here, completely free (Luo et al., 2015). Yay for labs who can afford open-access publishing!

In case you’re not familiar with Lingula, it’s this guy (image from Wikipedia):

In a classic case of looks being deceiving, it’s not a mollusc, although it does look a bit like one except for the weird white stalk sticking out of the back of its shell. Brachiopods, the phylum to which Lingula belongs, are one of those strange groups no one really knows where to place, although nowadays we are pretty sure they are somewhere in the general vicinity of molluscs, annelid worms and their ilk. Unlike bivalve molluscs, whose shell valves are on the left and right sides of the animal, the shells of brachiopods like Lingula have top and bottom valves. Lingula‘s shell is also made of different materials: while bivalve shells contain calcium carbonate deposited into a mesh of chitin and silk-like proteins,* the subgroup of brachiopods Lingula belongs to uses calcium phosphate, the same mineral that dominates our bones, and a lot of collagen (again like bone). But we’ll come back to that in a moment…

One of the reasons the Lingula genome is particularly interesting is that Lingula is a classic “living fossil”. In the Paleobiology Database, there’s even an entry for a Cambrian fossil classified as Lingula, and there are plenty of entries from the next geological period. If the database is to be believed, the genus Lingula has existed for something like 500 million years, which must be some kind of record for an animal.** Is its genome similarly conservative? Or did the DNA hiding under a deceptively conservative shell design evolve as quickly as anyone’s?

In a heroic feat of self-control, I’m not spending all night poring over the paper, but I did give a couple of interesting sections a look. Naturally, the first thing I dug out was the Hox cluster hiding in the rather large supplement. This was the first clue that Lingula‘s genome is definitely “living” and not at all a fossil in any sense of the word. If it were, we’d expect one neat string of Hox genes, all in the order we’re used to from other animals. Instead, what we find is two missing genes, one plucked from the middle of the cluster and tacked onto its “front” end, and two genes totally detached from the rest. It’s not too bad as Hox cluster disintegration goes – six out of nine genes are still neatly ordered – but it certainly doesn’t look like something left over from the dawn of animals.

The bigger clue that caught my eye, though, was this little family tree in Figure 2:

Luo_etal2015-fig2

The red numbers on each branch indicate the number of gene families that expanded or first appeared in that lineage, and the green numbers are the families shrunk or lost. Note that our “living fossil” takes the lead in both. What I find funny is that it’s miles ahead of not only the animals generally considered “conservative” in terms of genome evolution, like the limpet Lottia and the lancelet Branchiostoma, but also the sea squirt (Ciona). Squirts are notorious for having incredibly fast-evolving genomes; then again, most of that notoriety was based on the crazily divergent sequences and often wildly scrambled order of its genes. A genome can be conservative in some ways and highly innovative in others. In fact, many of the genes involved in basic cellular functions are very slow-evolving in Lingula. (Note also: humans are pretty slow-evolving as far as gene content goes. This is not the first study to find that.)

So, Lingula, living fossil? Not so much.

The last bit I looked at was the section about shell genetics. Although it’s generally foolish to expect the shell-forming gene sets of two animals from different phyla to be similar (see my first footnote), if there are similarities, they could potentially go at least two different ways. First, brachiopods might be quite close to molluscs, which is the hypothesis Luo et al.‘s own treebuilding efforts support. Like molluscs, brachiopods also have a specialised mantle that secretes shell material, though having the same name doesn’t mean the two “mantles” actually share a common origin. So who knows, some molluscan shell proteins, or shell regulatory genes, might show up in Lingula, too.

On the other hand, the composition of Lingula’s shell is more similar to our skeletons’. So, since they have to capture the same mineral, could the brachiopods share some of our skeletal proteins? The answer to both questions seems to be “mostly no”.

Molluscan shell matrix proteins, those that are actually built into the structure of the shell, are quite variable even within Mollusca. It’s probably not surprising, then, that most of the relevant genes that are even present in Lingula are not specific to the mantle, and those that are are the kinds of genes that are generally involved in the handling of calcium or the building of the stuff around cells in all kinds of contexts. Some of the regulatory mechanisms might be shared – Luo et al. report that BMP signalling seems to be going on around the edge of the mantle in baby Lingula, and this cellular signalling system is also involved in molluscan shell formation. Then again, a handful of similar signalling systems “are involved” in bloody everything in animal development, so how much we can deduce from this similarity is anyone’s guess.

As for “bone genes” – the ones that are most characteristically tied to bone are missing (disappointingly or reassuringly, take your pick). The SCPP protein family is so far known only from vertebrates, and its various members are involved in the mineralisation of bones and teeth. SCPPs originate from an ancient protein called SPARC, which seems to be generally present wherever collagen is (IIRC, it’s thought to help collagen fibres arrange themselves correctly). Lingula has a gene for SPARC all right, but nothing remotely resembling an SCPP gene.

I mentioned that the shell of Lingula is built largely on collagen, but it turns out that it isn’t “our” kind of collagen. “Collagen” is just a protein with a particular kind of repetitive sequence. Three amino acids (glycine-proline-something else, in case you’re interested) are repeated ad nauseam in the collagen chain, and these repetitive regions let the protein twist into characteristic rope-like fibres that make collagen such a wonderfully tough basis for connective tissue. Aside from the repeats they all share, collagens are a large and diverse bunch. The ones that form most of the organic matrix in bone contain a non-repetitive and rather easily recognised domain at one end, but when Luo et al. analysed the genome and the proteins extracted from the Lingula shell, they found that none of the shell collagens possessed this domain. Instead, most of them had EGF domains, which are pretty widespread in all kinds of extracellular proteins. Based on the genome sequence, Lingula has a whole little cluster of these collagens-with-EGF-domains that probably originated from brachiopod-specific gene duplications.

So, to recap: Lingula is not as conservative as its looks would suggest (never judge a living fossil by its cover, right?) We also finally have actual sequences for lots of its shell proteins, which reveal that when it comes to building shells, Lingula does its own thing. Not much of a surprise, but still, knowing is a damn sight better than thinkin’ it’s probably so. We are scientists here, or what.

I am Very Pleased with this genome. (I just wish it was published five years ago 😛 )

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Notes:

*This, interestingly, doesn’t seem to be the general case for all molluscs. Jackson et al. (2010) compared the genes building the pearly layer of snail (abalone, to be precise) and bivalve (pearl oyster) shells, and found that the snail showed no sign of the chitin-making enzymes and silk type proteins that were so abundant in its bivalved cousins. It appears that even within molluscs, different groups have found different ways to make often very similar shell structures. However, all molluscs shells regardless of the underlying genetics are predominantly composed of calcium carbonate.

**You often hear about sharks, or crocodiles, or coelacanths, existing “unchanged” for 100 or 200 or whatever million years, but in reality, 200-million-year-old crocodiles aren’t even classified in the same families, let alone the same genera, as any of the living species. Again, the living coelacanth is distinct enough from its relatives in the Cretaceous, when they were last seen, to warrant its own genus in the eyes of taxonomists. I’ve no time to check up on sharks, but I’m willing to bet the situation is similar. Whether Lingula‘s jaw-dropping 500-million-year tenure on earth is a result of taxonomic lumping or the shells genuinely looking that similar, I don’t know. Anyway, rant over.

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References:

Jackson DJ et al. (2010) Parallel evolution of nacre building gene sets in molluscs. Molecular Biology and Evolution 27:591-608

Luo Y-J et al. (2015) The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization. Nature Communications 6:8301

Putting the cart before the… snake?

Time to reexamine some assumptions (again)! And also, talk about Hox genes, because do I even need a reason?

Hox genes often come up when we look for explanations for various innovations in animal body plans – the digits of land vertebrates, the limbless abdomens of insects, the various feeding and walking and swimming appendages of crustaceans, the strongly differentiated vertebral columns of mammals, and so on.

Speaking of differentiated vertebral columns, here’s one group I’d always thought of as having pretty much the exact opposite of them: snakes. Vertebral columns are patterned, among other things, by Hox genes. Boundaries between different types of vertebrae such as cervical (neck) and thoracic (the ones bearing the ribcage) correspond to boundaries of Hox gene expression in the embryo – e.g. the thoracic region in mammals begins where HoxC6 starts being expressed.

In mammals like us, and also in archosaurs (dinosaurs/birds, crocodiles and extinct relatives thereof), these boundaries can be really obvious and sharply defined – here’s Wikipedia’s crocodile skeleton for an example:

In contrast, the spine of a snake (example from Wikipedia below) just looks like a very long ribcage with a wee tail:

Snakes, of course, are rather weird vertebrates, and weird things make us sciencey types dig for an explanation.

Since Hox genes appear to be responsible for the regionalisation of vertebral columns in mammals and archosaurs, it stands to reason that they’d also have something to do with the comparative lack of regionalisation (and the disappearance of limbs) seen in snakes and similar creatures. In a now classic paper, Cohn and Tickle (1999) observed that unlike in chicks, the Hox genes that normally define the neck and thoracic regions are kind of mashed together in embryonic pythons. Below is a simple schematic from the paper showing where three Hox genes are expressed along the body axis in these two animals. (Green is HoxB5, blue is C8, red is C6.)

Cohn_Tickle1999_hoxRegions

As more studies examined snake embryos, others came up with different ideas about the patterning of serpentine spines. Woltering et al. (2009) had a more in-depth look at Hox gene expression in both snakes and caecilians (limbless amphibians) and saw that there are in fact regions ruled by different Hoxes in these animals, if a little fuzzier than you’d expect in a mammal or bird – but they don’t appear to translate to different anatomical regions. Here’s their summary of their findings, showing the anteriormost limit of the activity of various Hox genes in a corn snake compared to a mouse:

Woltering_etal2009-mouse_vs_snake

Such differences aside, both of the above studies operated on the assumption that the vertebral column of snakes is “deregionalised” – i.e. that it evolved by losing well-defined anatomical regions present in its ancestors. But is that actually correct? Did snakes evolve from more regionalised ancestors, and did they then lose this regionalisation?

Head and Polly (2015) argue that the assumption of deregionalisation is a bit stinky. First, that super-long ribcage of snakes does in fact divide into several regions, and these regions respect the usual boundaries of Hox expression. Second, ordinary lizard-shaped lizards (from which snakes descended back in the days of the dinosaurs) are no more regionalised than snakes.

The study is mostly a statistical analysis of the shapes of vertebrae. Using an approach called geometric morphometrics, it turned these shapes from dozens of squamate (snake and lizard) species into sets of coordinates, which could then be compared to see how much they vary along the spine and whether the variation is smooth and continuous or clustered into different regions. The authors evaluated hypotheses regarding the number of distinct regions to see which one(s) best explained the observed variation. They also compared the squamates to alligators (representing archosaurs).

The results were partly what you’d expect. First, alligators showed much more overall variation in vertebral shape than squamates. Note that that’s all squamates – leggy lizards are nearly (though not quite) as uniform as their snake-like relatives. However, in all squamates, the best-fitting model of regionalisation was still one with either three or four distinct regions in front of the hips/cloaca, and in the majority, it was four, the same number as the alligator had.

Moreover, there appeared to be no strong support for an evolutionary pattern to the number of regions – specifically, none of the scenarios in which the origin of snake-like body plans involved the loss of one or more regions were particularly favoured by the data. There was also no systematic variation in the relative lengths of various regions; the idea that snakes in general have ridiculously long thoraxes is not supported by this analysis.

In summary, snakes might show a little less variation in vertebral shape than their closest relatives, but they certainly didn’t descend from alligator-style sharply regionalised ancestors, and they do still have regionalised spines.

Hox gene expression is not known for most of the creatures for which vertebral shapes were analysed, but such data do exist for mammals (mice, here), alligators, and corn snakes. What is known about different domains of Hox gene activation in these three animals turns out to match the anatomical boundaries defined by the models pretty well. In the mouse and alligator, Hox expression boundaries are sharp, and the borders of regions fall within one vertebra of them.

In the snake, the genetic and morphological boundaries are both gradual, but the boundaries estimated by the best model are always within the fuzzy boundary region of an appropriate Hox gene expression domain. Overall, the relationship between Hox genes and regions of the spine is pretty consistent in all three species.

To finish off, the authors make the important point that once you start turning to the fossil record and examining extinct relatives of mammals, or archosaurs, or squamates, or beasties close to the common ancestor of all three groups (collectively known as amniotes), you tend to find something less obviously regionalised than living mammals or archosaurs – check out this little figure from Head and Polly (2015) to see what they’re talking about:

Head_Polly2015-phylogeny_of_spines

(Moving across the tree, Seymouria is an early relative of amniotes but not quite an amniote; Captorhinus is similarly related to archosaurs and squamates, Uromastyx is the spiny-tailed lizard, Lichanura is a boa, Thrinaxodon is a close relative of mammals from the Triassic, and Mus, of course, is everyone’s favourite rodent. Note how alligators and mice really stand out with their ribless lower backs and suchlike.)

Although they don’t show stats for extinct creatures, Head and Polly argue that mammals and archosaurs, not snakes, are the weird ones when it comes to vertebral regionalisation. For most of amniote evolution, the norm was the more subtle version seen in living squamates. It was only during the origin of mammals and archosaurs that boundaries were sharpened and differences between regions magnified. Nice bit of convergent/parallel evolution there!

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References:

Cohn MJ & Tickle C (1999) Developmental basis of limblessness and axial patterning in snakes. Nature 399:474-479

Head JJ & Polly PD (2015) Evolution of the snake body form reveals homoplasy in amniote Hox gene function. Nature 520:86-89

Woltering JM et al. (2009) Axial patterning in snakes and caecilians: evidence for an alternative interpretation of the Hox code. Developmental Biology 332:82-89

Catching up

So I felt like I couldn’t put off the sixteen hundred articles twiddling their thumbs and tapping their feet in my RSS reader any longer. This is the first part of the crop that has accumulated since late December (yikes!). Legless axolotls, homing starfish, secretly related proteins, and more!

1. Axolotls are good at regenerating – until you make them grow up.

(Portrait of a pale lab/aquarium variety axolotl by Orizatriz, Wiki Commons.)

It’s probably not exactly obvious from my posting record, but a large part of my PhD work is about regeneration. It’s something we humans are pretty shit at, but many other vertebrates aren’t. Axolotls, these adorably dumb-faced salamanders, can easily regrow their legs. However, lab axolotls are kind of permanent babies. Although they can grow up in the sense that they are able to breed, they normally keep larval characteristics like gills throughout their lives. It’s reasonable to suspect that this influences their regenerative ability – after all, tadpoles lose their ability to regrow limbs the moment they turn into frogs.

It’s possible to make axolotls metamorphose, too, if you treat them with thyroxine (the same hormone that induces metamorphosis in “normal” amphibians). And when they turn into proper adult salamanders, they suddenly become much poorer regenerators. They can still replace a limb – kind of. But they take twice as long as non-metamorphosed axolotls of the same age and size, and they invariably wind up with small, malformed limbs, often missing bones. After amputation, new skin is slower to grow over their wounds, and the cells that gather under the new skin are sluggish to divide. Something about metamorphosis – that isn’t simply age – dramatically changes how they respond to amputation.

Reference: Monaghan JR et al. (2014) Experimentally induced metamorphosis in axolotls reduces regeneration rate and fidelity. Regeneration advance online publication, doi: 10.1002/reg2.8

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2. Similar cells repair muscles in crustaceans and vertebrates

“Regeneration” can cover a lot of different processes. For example, depending on the creature and the organ you’ve damaged, regenerated body parts can come from totally different kinds of cells. In planarian flatworms, a single kind of stem cell can replace anything else in the body. In the eyes of newts, mature cells of the iris transform into lens cells to replace a missing lens. In our muscles, there are special cells called satellite cells that are held in reserve specifically to make new muscle cells when needed.

This recent study of a little crustacean called Parhyale hawaiensis suggests that muscle regeneration in the fingernail-sized arthropod works in much the same way. Konstantinidis and Averof shot early embryos of Parhyale with DNA encoding a fluorescent marker, which randomly integrated into the genomes of some of the cells it hit. In a few “lucky” individuals, the marker ended up labelling just one cell lineage, and the pair used these animals to figure out which cells made which tissues in a regenerated limb.

It turned out that cells in Parhyale are limited in their potential. Descendants of the ectodermal lineage could make skin and nerves but not muscle, and the mesodermal lineage built muscle but not skin or nerves. Moreover, labelled cells only contributed to regeneration near their original location – animals with their left sides labelled never regrew glowing limbs on the right side. This is starting to sound a lot like vertebrates, but it’s still a very general observation. However, the similarities don’t end there.

Like vertebrate muscles, the muscles of the little crustaceans contain satellite-like cells derived from the mesodermal lineage that sit beside mature muscle cells and express the Pax3/7 gene. When the researchers transplanted some of these cells from animals with the glowy label into leg stumps of non-glowy animals, there were glowing muscle cells in some of the regenerated limbs. So like satellite cells, these cells can turn into muscle during regeneration. There’s little question that muscle cells have a common origin in vertebrates and arthropods like Parhyale, but it’s really cool to see that their mechanisms of regeneration also might.

Reference: Konstantinidis N & Averof M (2014) A common cellular basis for muscle regeneration in arthropods and vertebrates. Science, published online 02/01/2014, doi: 10.1126/science.1243529

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3. Convergent evolution is a poor explanation of rhodopsins

Proteins can be difficult. I mean, sometimes they do their darnedest to hide their family ties. A protein is a chain of amino acids (on average about 300 of them) often folded into a complex shape. Closely related proteins have obviously similar amino acid sequences. However, more distant relatives can be harder to identify. There are about 20 different kinds of amino acids in proteins, so the number of possible sequences is unimaginably vast. The same function can be carried out by very different sequences, and therefore enough evolution can completely erase sequence similarity.

Protein structures are generally thought to be more conserved than sequences. Like function, structure allows for a huge amount of sequence variation without significantly changing. However, theoretically, it’s possible that two unrelated proteins have similar structures because of their similar functions, not because of common ancestry. Apparently, this has been argued for the two types of rhodopsins – proteins that harvest light in systems as different as a the “solar generator” of a salt-loving microbe and the photoreceptors of our own eyes.

If Type I and Type II rhodopsins are similar despite being unrelated, one would assume that this is because they need to be that way to capture light. There are, after all, astronomical numbers of possible protein structures, and the chances of two protein families accidentally stumbling onto the same one without selection steering are slim to say the least. But, in fact, you can rearrange the structure of a rhodopsin in all kinds of cunning ways without destroying its function. This rather weakens the case for convergent evolution, and suggests that similarity of structure does indicate common ancestry here.

Reference: Mackin KA et al. (2014) An empirical test of convergent evolution in rhodopsins. Molecular Biology and Evolution 31:85-95

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4. Starfish can see their way back home

(Blue starfish, the beast featured in the paper, in its natural habitat. Richard Ling, Wiki Commons.)

Starfish aren’t widely known as visual creatures, but they do have eyes at the tips of their arms. The eyes are a bit… basic – no lenses, just a hundred or two little units filled with photoreceptors. Garm and Nilsson set out to find out how the starfish used their eyes. They measured or calculated the eyes’ visual fields (five arm-eyes together can see pretty much everywhere around the animal), resolution (very coarse), reaction speed (slow), and their sensitivity to various wavelengths (they are colour-blind, most sensitive to ocean blue).

Then they took some poor starfish and dumped them a little way off the coral reefs they like staying on. The creatures could walk home from short distances (about 2 m or less), but if you take them too far away, they just wander around in random directions. Likewise if you take off their eyes (don’t worry, they regenerate) or do the experiment in the dark. In conclusion: starfish eyes aren’t exactly top-end cameras, but they are definitely useful to the animals. And what would a slow, brainless mopper-up of coral reef rubbish do with eagle eyes anyway?

(The paper states the walking speed of these starfish as about 4-5 cm per minute. I have a feeling this wasn’t the most exciting fieldwork these guys have done…)

Reference: Garm A & Nilsson D-E (2014) Visual navigation in starfish: first evidence for the use of vision and eyes in starfish. Proceedings of the Royal Society B 281:20133011

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5. What makes wormies settle

OK, Shikuma et al. (2014) one isn’t so much for its own news value, but I hadn’t known that my favourite worms need bacteria to settle until I saw this paper, so I think it deserves a mention. (Besides, it has beautiful pictures of baby Hydroides in it, which I couldn’t resist posting below. They are So. Cute. Yes, I’m weird.)

Shikuma_etal2014-hydroidesBabies

Tubeworms of the serpulid family have swimming larvae which are in many ways like the acorn worm larvae mentioned in my previous post (except cuter). They are tiny, look nothing like an adult worm, have bands of cilia for swimming and feeding, and live in the plankton until they’re ready to metamorphose. When they find a place they like, they settle and turn into adult worms. And apparently, this particular species (Hydroides elegans) not only needs a specific bacterium to like a place, it needs specific proteins produced by that bacterium.

The proteins in question are the components of a nasty device bacteria probably stole from viruses and then used to poke holes in one another. But to Hydroides larvae, they appear to be necessary for metamorphosis. Put healthy bacteria together with worm babies in a dish, and you’ll get happily settled little worms. Do the same with bacteria with damage to the relevant genes, and nothing happens. Use an extract containing the proteins but not the bacteria, and you still get metamorphosing worms. Use too much, though, and they start dying. Everything in moderation…

(Maybe my dismal failure at raising happy young worms years ago could have been remedied with the right bacteria?)

Reference: Shikuma NJ et al. (2014) Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343:529-533

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6. Relative of animals does strange multicellularity with familiar genetics

Although this idea probably hasn’t reached popular perception, animals are surrounded by other multicellular lineages in the tree of life. Sure, most of them are only part-time multicellular, but that’s beside the point. What’s clear is that multicellularity, at least in its simpler forms, is rampant in our extended family. Slime moulds do it, fungi do it, our closest relatives choanoflagellates do it, and our next closest relatives, filastereans and ichthyosporeans also do it.

These latter two groups are really poorly known (the fact that only a taxonomist could like the latter’s name probably doesn’t help), but the situation is getting better with the attention they are receiving as relatives of animals. There are now genome sequences out, and some people are looking at the life cycles of the little creatures to search for clues to our own origins.

Iñaki Ruiz-Trillo recently published a paper describing an ichthyosporean that can form a weird kind of colony with many nuclei in the same membrane starting from a single cell (Suga and Ruiz-Trillo, 2013). Now his team describe a different kind of multicellularity in a filasterean, Capsaspora owczarzaki. Rather than developing from a single cell, this guy does something more akin to the slime mould way: take a load of individual cells and bring them together. (Below: a clump of Capsaspora cells from Sebé-Pedros et al. [2013]. On the right is a regular photograph of the colony. The two-coloured fluorescence on the left indicates that the colony formed by different cells coming together rather than a single cell dividing.)

Sebé-Pedros_etal2013-F4.capsasporaClump

But, interestingly, some of the genetics involved is similar to what animals use, despite the different ways in which the two groups achieve multicellularity. For example, we’ve known since all those genomes came out that the proteins animals use to glue cells together and make them talk to each other are often older than animals. Well, Ruiz-Trillo’s filasterean appears to ramp up the production of some of these when it goes multicellular. It also uses a gene regulation strategy that animals are really big on: it edits the RNA transcribed from many genes in different ways depending on cell type/life stage before it’s translated into protein.

A lot of the details are going to need further investigation, since this was a global RNA-sequencing study with a bird’s-eye view of what genes are doing. It’s still a nice reminder that, like most other innovations in evolutionary history, the multicellularity of animals didn’t spring fully formed out of nowhere.

References:

Suga H & Ruiz-Trillo I (2013) Development of ichthyosporeans sheds light on the origin of metazoan multicellularity. Development 377:284-292

Sebé-Pedros A et al. (2013) Regulated aggregative multicellularity in a close unicellular relative of metazoa. eLife 2:e01287

Lamprey Hox clusters and genome duplications, oh my!

What the hell is up with lamprey Hox clusters?

Lampreys are among the few living jawless vertebrates, creatures that parted evolutionary ways with our ancestors somewhere on the order of 500 million years ago. If you want to know where things like jaws, paired fins or our badass adaptive immune systems came from, a vertebrate that doesn’t possess some of these things and may have diverged from the rest of the vertebrates soon after others originated is just what you need for comparison.

The vertebrate fossil record is pretty rich thanks to us having hard tissues, so a lot can be inferred about these things from the wealth of extinct fishes we have at our disposal. However, there are times when comparisons of living creatures are just as useful, if not more, than examinations of fossils. (Fossils, for example, tend not to have immune systems. ;))

One of the things you absolutely need a living animal to study is, of course, genome evolution. Vertebrates – well, at least jawed vertebrates – are now generally accepted to have the remnants of four genomes. Our long-gone ancestors underwent two rounds of whole genome duplication. Afterwards, most of the extra genes were lost, but evidence for the duplications can still be found in the structure of our genomes, where entire recognisable gene neighbourhoods of our close invertebrate relatives often still exist in up to four copies (Putnam et al., 2008).

Among these neighbourhoods are the four clusters of Hox genes most groups of jawed vertebrates possess. A “normal” animal like a snail or a centipede only has one of these. Since Hox genes are involved in the making of body plans, you have to wonder how suddenly having four sets of them and other developmental “master genes” might have influenced the evolution of vertebrate bodies.

Of course, to guess that, you need to know precisely when these duplications happened. That’s where lampreys come in: their lineage branched off from our definitely quadruple-genomed one after the next closest, definitely single-genomed group. But was it before, between, or after, the two rounds of duplication?

A few years ago, a phylogenetic analysis of 55 gene families by Kuraku et al. (2009) suggested that the lamprey-jawed vertebrate split happened after the 2R. Just this year, the genome of the sea lamprey Petromyzon marinus was finally published (Smith et al., 2013), and its authors agreed that yes, lampreys probably split off from us post-2R. (I don’t entirely get all the things they did to arrive at this conclusion. Groups of linked genes show up again, among other approaches.)

However, that isn’t the whole story, the latest lamprey genomics paper argues (Mehta et al., 2013). The P. marinus genome assembly couldn’t stitch all the Hox clusters properly together. There were two that sat on nice big scaffolds with the whole row of Hox genes and a few of their neighbours, and then there were a bunch of “loose” Hox genes that they couldn’t link to anything (diagram comparing humans and P. marinus below from Smith et al., 2013; the really pale blue boxes under the numbers represent Hox genes):

Smith_etal2013-F4

Given that Hox9 genes exist in four copies in this species, it seems like there may be four clusters. However, in hagfish, the other kind of living jawless vertebrate, a study found Hox genes that seemed to have as many as seven copies (Stadler et al., 2004). Another round of duplication? It wouldn’t be unheard of. Most teleosts, which include most of the things we call “fish” in everyday parlance, have seven Hox clusters courtesy of an extra genome duplication and loss of one cluster*. Salmon and kin have thirteen, after yet another duplication. Maybe hagfish also had another one – but did lampreys? How many more clusters do those lonely Hox genes belong to?

Mehta et al. hunted down the Hox clusters of Japanese lampreys (Lethenteron japonicum), hoping to pin down exactly how many there were. They used large chunks of DNA derived partly from the testicles, where sperm cells and their precursors keep the full genome throughout the animal’s life (lampreys throw away large chunks of the genome in most non-reproductive cells [Smith et al., 2009]). They probed these for Hox genes and sequenced the ones that tested positive. Plus they also got about two-thirds of the full genome together in fairly big pieces. Together, these data allowed them to get a better idea of the mess that is lamprey Hox cluster genomics.

They assembled four whole clusters, including their neighbouring genes, and a partial fifth cluster. A bunch of other genes sat on smaller sequence fragments containing only a couple of Hoxes, or a Hox and a non-Hox, but they were tentatively assigned to a total of eight clusters, eight being the number of different Hox4 genes in the data (no known vertebrate Hox cluster contains more than one Hox4 gene). The L. japonicum equivalents of the 31 publicly available Hox sequences from P. marinus spread out over six of these, which indicates that both species have at least six clusters. Seems like lampreys had another round of genome duplication after 2R? (Summary of L. japonicum Hox clusters from Mehta et al. below.)

But wait, that’s not the end of it.

First of all, although there are undoubtedly four complete Hox clusters in there L. japonicum, the relationships of these clusters to our four are terribly confused. Whether you look at the phylogenetic trees of individual genes, or the arrangement of non-Hox genes on either side of the cluster, only a big pile of what the fuck emerges. Phylogenies are problematic because the unusual composition of lamprey genes and proteins (Smith et al., 2013) could easily throw them off. All the complete lamprey clusters have a patchwork of neighbours that look like a mashup of more than one of our Hox clusters. Might it mean that lampreys’ proliferation of Hox clusters occurred independently of ours? Did we split before 2R after all?

Hox genes are not the only interesting things in a Hox cluster. In the long gaps between them, there are all sorts of little DNA switches that regulate their behaviour. Some of these are conserved across the jawed vertebrates. Mehta et al. aligned complete Hox clusters of humans, elephant sharks and lampreys to look for such sequences – called conserved non-coding elements or CNEs – in the lamprey.

They only found a few, but that’s enough for a bit more head-scratching. Most CNEs in, say, the human HoxA cluster are only found in one elephant shark cluster, and vice versa. Humans have a HoxA cluster, elephant sharks have a HoxA cluster, they’re clearly the same thing, pretty straightforward. Not so for lampreys. Homologues of individual CNEs in the complete lamprey clusters are spread out over all four human/elephant shark clusters. More evidence for independent duplications?

Mehta et al. are cautious – they point out that the silly mix of Hox cluster neighbours in lampreys could just be due to independent post-2R losses, which is plausible if the split between lamprey and jawed vertebrate lineages happened not too long after 2R. There’s also the fact that the weird lamprey sequences are phylogenetic minefields – however, that’s a double-edged sword, since the same caveat applies to analyses that support a post-2R divergence. Then, perhaps the same argument that goes for Hox cluster neighbours could also apply to CNEs. And, of course, this is just Hox clusters. Smith et al.‘s (2013) findings about overall genome structure don’t go away just because lamprey Hox clusters are weird.

So, in summary, thanks, lampreys. Fat lot of help you are! 😛

***

*Actually, two losses of two separate clusters in two different teleost lineages. Because Hox evolution wasn’t already complicated enough.

***

References

Kuraku S et al. (2009) Timing of genome duplications relative to the origin of the vertebrates: did cyclostomes diverge before or after? Molecular Biology and Evolution 26:47-59

Mehta TK et al. (2013) Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). PNAS 110:16044-16049

Putnam NH et al. (2008) The amphioxus genome and the evolution of the chordate karyotype. Nature 453:1064-1071

Smith JJ et al. (2009) Programmed loss of millions of base pairs from a vertebrate genome. PNAS 106:11212-11217

Smith JJ et al. (2013) Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nature Genetics 45:415-421

Stadler PF et al. (2004) Evidence for independent Hox gene duplications in the hagfish lineage: a PCR-based gene inventory of Eptatretus stoutii. Molecular Phylogenetics and Evolution 32:686-694

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…

***

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

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. 🙂

***

*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?

***

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

News bites

Since work is quite frantic lately, and my attention span has gone on holiday, I’ve decided to do something I haven’t done before and just say a few words about papers that caught my interest today without actually reading them. Each of these is probably worth a full-blown meandering of its own, but I know I wouldn’t ever get to them at this rate. Better read their abstracts and give some quick thoughts than let them sink unnoticed into the murk of my “papers” folder!

(1) How many genomes do we (not) have?

Reference: Ali Abbasi A & Hanif H (2012) Phylogenetic history of paralogous gene quartets on  human chromosomes 1, 2, 8 and 20 provides no evidence in favor of the vertebrate octoploidy hypothesis. Molecular Phylogenetics and Evolution, in press (doi: 10.1016/j.ympev.2012.02.028)

(How many papers have authors with alliterating names? :D)

In the circles I move in, it’s pretty much canon that the ancestors of living vertebrates doubled their entire genomes twice. It’s still debated exactly when these duplications occurred, but few people doubt that they did. This so-called 2R hypothesis is supported by things like our possession of several (quite often, four) copies of genes that are singletons in our closest living relatives (read: lancelets*), and more importantly, that whole big chunks of lancelet chromosomes can be matched to chunks of four different vertebrate (mainly, human) chromosomes. Genes that are close to one another in lancelets are often also close together in vertebrates.

The relationship is not perfect – in well over 500 million years of evolution, genes inevitably get lost and bits of chromosome scrambled. And, thus, there is always room to question the 2R scenario, which is what this paper clearly does. They propose that those four-gene families originated at all sorts of different times, from small local duplications and rearrangements. If they are right, this is a very important result. It basically uproots every bit of speculation ever proposed on how the genome duplications contributed to the evolution of vertebrates, which, far as I can tell, is a hell of a lot of speculation. Not having read the whole paper, I would still put my money on 2R, but who knows what the future holds? Maybe we are facing a minor paradigm shift?

(2) The segmentation clock also ticks in insects!

Reference: Sarrazin AF et al. (2012) A segmentation clock with two-segment periodicity in insects. Science, advance online publication (doi: 10.1126/science.1218256)

The evolutionary history of segmentation is one of my random interests, and from my point of view, the above is a good reason to squee in a most fangirlish way. Segmentation is the construction of a body from repeating units. In its purest form, which isn’t that common in modern animals, the animal is essentially made up of identical repeated blocks containing a copy of each key organ like kidneys, nerve centres, limbs and muscles. (Even in the most perfectly segmented creatures, head and tail ends form something of an exception. Ragworms make a nice example.) More commonly, only some components are repeated, and they are repeated with slight differences along the body. Vertebrates’ spine and associated muscles are a good example, and so are the defining traits of arthropods, their jointed exoskeletons equipped with repeated pairs of appendages.

Although traditionally it has been thought that arthropod and vertebrate segmentation have independent origins, parts of the genetic machinery are shared between both groups (as well as segmented worms). Various “segmentation genes” are active in distinct stripes in our embryos, marking out future segments even before we can see the segments themselves. In vertebrates, cells periodically switch “segmentation genes” on and off, and this combined with the growth of the embryo produces a dynamic stripey pattern of gene expression. While segments and stripes of gene expression are darn obvious in arthropods, this is the first time anyone has confirmed that some arthropod segmentation genes actually oscillate like their vertebrate counterparts do, as opposed to, say, the cells expressing them moving about. Whether this is a spectacular example of convergent evolution or evidence of a shared ancestral heritage, I couldn’t say, but it’s really cool either way.

(3) Old genes are entrenched, new genes are redundant after all?

Reference: Chen WH et al.(2012) Younger genes are less likely to be essential than older genes, and duplicates are less likely to be essential than singletons of the same age. Molecular Biology and Evolution advance online publication (doi: 10.1093/molbev/mss014)

So, this claims to resolve a conundrum I wasn’t even aware of before. Gene duplication is thought to be important for the evolution of new functions because two copies of a gene mean there is a backup if one of them fails at its original function. Hence, theory goes, duplicate genes are much less restricted in the evolutionary paths they can take. Apparently, studies in mice have contradicted this common wisdom by claiming that duplicate genes are just as likely to be indispensable as genes without backup copies. However, Chen et al. are saying that this is wrong, confounded by gene age. Since new genes are less likely to be essential than old genes (which had more time to evolve interactions with the rest of the genome), and mouse duplicates are on average older than mouse singletons, the two effects end up cancelling out. When they factor in gene age, duplicates are indeed less essential than loners. One of the central tenets of current thinking about (genetic) novelty stays in the ring for another round…

(4) Is reducing complexity easier than increasing it?

Reference: Harjunmaa E et al. (2012) On the difficulty of increasing dental complexity. Nature advance online publication (doi: 10.1038/nature10876)

How complexity increases in evolution is more than a breeding ground for creationist incredulity, it’s also quite interesting for bona fide evolutionary biologists. Looking at the development of mouse teeth, Harjunmaa et al. notice that increases and decreases in the complexity of tooth shapes require different sorts of mess-ups. Simpler-than-normal teeth are common in mutants and easy to make in experiments. More complex teeth – i.e. those with more cusps – are rarely if ever seen in natural mutants. Turns out they are perfectly possible – you just need to manipulate several genetic pathways at the same time to produce a clear result.

Can this be generalised? Is greater complexity usually harder to achieve? When does this apply and when does it not? I’ve recently read papers that explore how complexity increases easily and completely by chance (I have a half-written post about them languishing on my hard drive, FWIW). Are the rules different for different levels of organisation? The aforementioned complexity-by-chance papers analyse the molecular level: one is about the architecture of gene switches, the other about a protein machine. Teeth are pretty large pieces of life with thousands upon thousands of such machines participating in their production. Does that make a real difference, or is what I’m seeing just coincidence? Dunno, but it’s fascinating to think about!

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Heh, it looks like I took rather bigger “bites” of these news than I planned to. I kind of managed to write the equivalent of a full-blown meandering anyway. The only difference is that I didn’t painstakingly reference this one. I hope that doesn’t mean that half of what I wrote off the top of my head is wrong 😀

***

Note:

*Lancelets are now not considered our closest relatives. Unbelievable as that may seem, that honour goes to sea squirts and their ilk. However, the sea squirt bunch are ridiculously weird in all sorts of respects, and their genomes are jumbled beyond recognition. So… not so great if you want to learn anything about our ancestors.