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Archive for the ‘Two-Dog-Night Tetrapods’ Category

What a week!

My team had a new technician arrive, Kyle Chadwick from Uni. Virginia, and NSF Postdoctoral Research Fellow, Dr. Ashley Heers (see here for an example  of new stuff she’s starting here at the RVC!), started working with me at the RVC, and then these guys showed up…

Salamanders!

Woo hoo!

First a tiger salamander (Ambystoma) paid a visit, for filming an episode of the Windfall Films/PBS documentary “Your Inner Fish” (a la the famous book):

So cute! Tiger salamander, soon to be a TV celebrity.

So cute! Tiger salamander, soon to be a TV celebrity.

Dr. Stephanie Pierce (who was also a coauthor on a great open access croc paper in Proc Roy Soc B this week) was filmed with Prof. Jenny Clack to recap some of our past work on tetrapod locomotion. Watch out for the 3-part series!

And that gorgeous salamander was a star performer in strutting his stuff for the camera to demonstrate the locomotion of modern tetrapods, including some lovely slo-mo footage from our lab cameras:

(if that’s too slow for you, try the normal-speed footage. I’ll admit, salamanders don’t really need slo-mo video for normal walking, but I like it)

So cool!

But then we got a special package… with three frozen fire salamanders (Salamandra salamandra) from colleagues in Germany!

Three new occupants of the freezers, for planning our studies of salamander locomotion

Three new occupants of the freezers, for planning our studies of salamander locomotion

This marks the start of an exciting new period in my team’s work in the lab. I’ve always liked salamanders and newts, and we’ve scanned and modelled plenty (e.g. this old post), but now we’re going to work with live fire salamanders (a first for me)! We are using the dead ones to plan the new studies with the live ones– these new studies will involve lots of high speed videos and force platform analysis (as shown above), in conjunction with XROMM (biplanar fluoroscopy/3D skeletal motion analysis) and other techniques including computer simulations. We got initial approval this week to work with these salamanders, and found a reputable source this week too, so it was definitely Salamander Week in my group!

This research all will feed into our upcoming studies of extinct tetrapods: we’re using salamanders to figure out how salamanders move and what limits their speed and gait, and then we’re using the same sorts of computer tools to try to estimate how extinct tetrapods may have moved and how locomotion evolved, in much more specific detail than our prior work had done, which was mainly about using 3D reconstructions of anatomy to show what those animals could not do. More about the project here.

Watch this space for more scampering salamanders!

UPDATE: And here’s one! Not quite scampering, but…

Setting up our two fluoroscopes for a test run of our gait studies-- but with one of the deceased salamanders. Gotta get a good image  before any live animal work!

Setting up our two fluoroscopes for a test run of our gait studies– but with one of the deceased salamanders. Gotta get good images before any live animal work begins!

An example of the kind of footage we’re aiming for (single 2D fluoroscope view from Nadja Schilling’s team’s research; see XROMM website for more details on the methodology)

UPDATE 2:

I did a CT scan with a normal medical grade CT scanner at the highest resolution we can manage (0.625 mm slices). Check out the results below, which amuse me:

Looks like a toy; too crude resolution. But we can see major structures, and we can very nicely see the “microchip” (which looks HUGE) that was placed in this animal’s back when in captivity, and then another structure is visible near the pelvis which might be another chip or else remains of some food, pathology, or a really odd pelvis– I am not totally sure!

So this is why we tend to use microCT, which can go down to as low as ~5 micron resolution, to get 3D anatomy of animals this small. It’s no surprise to me, but it is fun to see how far we could push our normal CT machine. The results aren’t horrid but wouldn’t have much scientific value for us. They did confirm for us that this specimen is heavily ossified, so the faint images of bone that we are getting in our x-ray fluoroscopes (above) are due to something going wrong with our camera system, not the animal’s immature skeleton. Stay tuned for more updates as the science happens!

UPDATE 3:

20 wonderful adult Fire Salamanders have joined our team and are relaxing over the coming week before we start taking them for walks. Here is one exploring its new home:

Fire salamander

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…a daily picture of anatomy! And today it is five pictures; zza-zza-zee! ♫

Welcome back againagain, (gasp, pant) and again to Freezermas

I’m letting the dogs out today. Science gone barking mad! Hopefully my puns will not screw the pooch.

Stomach-Churning Rating: 4/10; a dog cadaver’s leg (not messy), then just tame digital images of anatomy.

I am working with Rich Ellis, a former MSc student at Univ. Colorado (see his cool new paper here!), for a fun new collaboration this year. He was awarded a prestigious Whitaker Foundation scholarship to do this research, which focuses on how different animals stand up from a squatting position, with the legs about as bent as they can be.

We want to know how animals do this standing up movement, because it is in some ways a very demanding activity. Very flexed/bent limb joints mean that the muscles (and some tendons) are stretched about as far as they ever will be. So this places them at disadvantageous lengths (and leverage, or mechanical advantage) for producing force. We know almost nothing about how any animal, even humans, does this-- how close to their limits of length are their muscles? Which muscles are closest? Does this change in animals with different numbers of legs, postures, anatomy, size, etc? Such fundamental questions are totally unaddressed. It’s an exciting area to blaze a new trail in, as Rich is doing. So far, we’ve worked with quail, humans, and now greyhounds; in the past I did some simple studies with horses and elephants, too. Jeff Rankin from my team and other collaborators have also worked on six species of birds, of varying sizes, to see how their squat-stand mechanics change.  Thus we’ve covered a wide diversity of animals, and now we’re learning from that diversity. “Diversity enables discovery,” one of my former PhD mentors Prof. Bob Full always says. Too true.

Greyhounds are interesting because they are medium-sized, long-legged, quadrupedal, quite erect in posture, and very specialized for fast running. Fast runners tend to have big muscles with fairly short fibres. Short fibres are bad for moving the joints through very large ranges of motion. So how does a greyhound stand up? Obviously they can do it, but they might have some interesting strategies for doing so- the demands for large joint motion may require a compromise with the demands for fast running. Or maybe the two demands actually can both be optimized without conflict. We don’t know. But we’re going to find out, and then we’ll see how greyhounds compare with other animals.

To find out, we first have to measure some dogs standing up. We’ve done that for about 8 greyhounds. Here is an example of a cooperative pooch:

Those harmless experiments, if you follow me on Twitter, were live-tweeted under the hashtag #StandSpotStand… I dropped the ball there and didn’t continue the tweeting long after data collection, but we got the point across– it’s fun science addressing useful questions. Anyway, the experiments went well, thanks to cooperative pooches like the one above, and Rich has analyzed most of the data.

Now the next step involves the cadaver of a dog. We could anaesthetize our subjects and do this next procedure to obtain subject-specific anatomy. But it really wouldn’t be ethically justified (and if I were an owner I wouldn’t allow it either!) and so we don’t. A greyhound is a greyhound as far as we’re concerned; they’ll be more like each other than either is like a quail or a human. Individual variation is a whole other subject, and there are published data on this that we can compare with.

We get a dead dog’s leg — we don’t kill them; we get cadavers and re-use them:

Greyhound hindlimb for CT

We study the hindlimb because birds and humans don’t use their forelimbs much to stand up normally, so this makes comparisons simpler. We’re collecting forelimb data, though, as we work with quadrupeds, for a rainy day.

We then CT scan the leg, getting a stack of slices like this– see what you can identify here:

It’s not so clear in these images, but I was impressed to see that the muscles showed up very clearly with this leg. That was doggone cool! Perhaps some combination of formalin preservation, fresh condition, and freezing made the CT images clearer than I am used to. Anyway, this turned out to be a treat for our research, as follows.

We then use commercial software (we like Mimics; others use Amira or other packages) to “segment” (make digital representations in 3D) the CT scan data into 3D anatomy, partitioning the greyscale CT images into coloured individual objects– two views of one part of the thigh are shown below.

What can you identify as different colours here? There are lots of clues in the images (click to embiggen):

Hindlimb segmentation of greyhound

And here is what the whole thigh looks like when you switch to the 3D imaging view:

Quite fetching image, eh?!

The next steps after we finish the limb segmentation are to apply the experimental data we observed for greyhounds of comparable size by importing the model and those data into biomechanics software (SIMM/OpenSim). We’ve done about 40 models like this for various species. I detailed this procedure for an elephant here.

Then, at long last, science will know how a greyhound stands up! Wahoo! Waise the woof! Stay tuned as we hound you with more progress on this research-as-it-happens. Rich just finished the above thigh model this week, and the rest of the leg will be done soon.

Many thanks to Rich Ellis for providing images used here. And thank you for persevering my puns; they will now be cur tailed.

Happy Freezermas! Sing it: “On the fifth day of Freezermas, this blo-og gave to me: one tibiotarsus, two silly Darwins, three muscle layers, four gory hearts, a-and five stages modelling a doggie!” ♪

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To kick off the New Year just right, our tetrapod team has a new paper in Nature, following up on last year’s Ichthyostega not-so-good-at-walking study (also see here). Yet this paper has a more anatomically descriptive — and also an “evo-devo” — twist to it. For brevity, I’ll let our press release tell the story, since I think it does a good job of it (like I always preach scientists should do, we worked with our PR company to write this together, so we’re happy with how the press release came out). In a nutshell, our study used some very fancy synchotron radiation techniques to image the 3D anatomy of the backbone in early land vertebrates. Our findings surprised even us, and ended up turning around palaeontology/comparative anatomy’s view of how the backbone evolved, giving us a new glimpse into our inner tetrapod.

Stick around for the videos at the end, which are the first four supplementary movies from the paper and are rather pretty (there are two more, for imaging/segmenting afficionados, but they are not as pretty or interesting for most of this blog’s readership). The final figure (Figure 1 from our paper) gives some extra visual context.

The paper is:

Pierce, S.E., Ahlberg, P.E., Hutchinson, J.R., Molnar, J.L., Sanchez, S., Tafforeau, P., Clack, J.A. 2013. Vertebral architecture in the earliest stem tetrapods. Nature, published online [here].

I should note that I’m just 3rd author, so I deserve only modest credit. But I helped. Even though no freezers were involved, or harmed, in the process.

Ichy_vertebrae_final_sm-01

Above image: Julia Molnar‘s illustration of Ichthyostega showing anatomical changes of its spine from front to back, with neural arch/spine in pink, twin pleurocentra in yellow, and intercentrum in green. These four parts, three kinds of bones, made up the backbone of the first land vertebrates. These parts evolved in different ways in later animals, but formed one main bone in all living lineages of vertebrates.

RVC PRESS RELEASE:

Scientists reassemble the backbone of life using a particle accelerator

Research published today (Sunday 13 January 2013) in the journal Nature documents, for the first time, the intricate three-dimensional structure of the backbone in the earliest four-legged animals (tetrapods).

The international team of scientists, led by Dr Stephanie E. Pierce from The Royal Veterinary College and Professor Jennifer A. Clack from the University of Cambridge, bombarded 360 million year old early tetrapod fossils with high energy synchrotron radiation. The resulting high resolution X-ray images allowed the researchers to reconstruct the backbones of the extinct animals in exceptional detail.

The backbone, also known as the spine or vertebral column, is a bony structure found in all tetrapods, along with other vertebrates such as fish. It is formed from many elements or vertebrae all connected in a row – from head to tail. Unlike the backbone of living tetrapods (e.g. humans), in which each vertebra is composed of only one bone, early tetrapods had vertebrae made up of multiple parts.

Lead author Dr Pierce says: “For more than 100 years, early tetrapods were thought to have vertebrae composed of three sets of bones – one bone in front, one on top, and a pair behind. But, by peering inside the fossils using synchrotron X-rays we have discovered that this traditional view literally got it back-to-front.”

For the analysis, the European Synchrotron Radiation Facility (ESRF) in France, where the three fossil fragments were scanned with X-rays, used a new protocol to reveal tiny details of the fossil bones buried deep inside the rock matrix.

Using this new technology, the team of scientists discovered that what was thought to be the first bone – known as the intercentrum – is actually the last in the series. And, although this might seem like a trivial oversight, this re-arrangement in vertebral structure has over-arching ramifications for the functional evolution of the tetrapod backbone. (see here for a now out-of-date image from Wikipedia)

Dr. Pierce explains: “By understanding how each of the bones fit together we can begin to explore the mobility of the spine and test how it may have transferred forces between the limbs during the early stages of land movement”.

But, the findings didn’t end there. One of the animals – known as Ichthyostega – was also found to have an assortment of hitherto unknown skeletal features including a string of bones extending down the middle of its chest.

Professor Clack says: “These chest bones turned out to be the earliest evolutionary attempt to produce a bony sternum.  Such a structure would have strengthened the ribcage of Ichthyostega, permitting it to support its body weight on its chest while moving about on land.”

This unexpected discovery supports recent work done by the same authors that showed Ichthyostega probably moved by dragging itself across flat ground using synchronous ‘crutching’ motions of its front legs – much like that of a mudskipper or seal.

Dr Pierce adds: “The results of this study force us to re-write the textbook on backbone evolution in the earliest limbed animals.”

The next step, the researchers say, is to understand how the backbone aided locomotion in these early tetrapods using sophisticated biomechanical analysis.

The study was funded by the Natural Environment Research Council.

Additional support was provided by the European Research Council and the ESRF, of which the Science and Technology Facilities Council (STFC) is the UK shareholder.

MOVIES:

These are rotating images of the anatomy, colour-coded, of the four species of early tetrapod that we examined for this study. Each shows the same basic pattern of having a “reverse rhachitomous” (pleurocentra in the front, intercentrum in the back; trying to think of a mullet joke…) anatomy. This is opposite the pattern that essentially all studies since famed evolutionary biologist/palaeontologist Edward Drinker Cope coined the term “rhachitomous” in 1878 have portrayed these and related animals as having. And this realization forces a re-examination of how the backbone structures first evolved in tetrapods and which parts (intercentra? pleurocentra? And where?) formed the spines of later animals.

For once, as authors we all felt that this finding really deserved the painfully hackneyed “rewrite the textbooks” label. It changes a lot of what we thought we knew about this classic evolutionary transition of anatomy. Check a vertebrate palaeontology/comparative anatomy textbook and you’ll likely find rhachitomous vertebrae and/or changes of pleurocentra vs. intercentra told in a way that we now are pretty sure is wrong.

You can also see the “sternebrae” (sternal elements; parts of the sternum that evolved independently in later land animals) in the first movie.  This, to my knowledge, is by far the oldest such evidence. I know of ossified sternal plates in Early Permian mesosaurs like Stereosternum, but nothing earlier although perhaps in some synapsid I don’t know, or a basal diapsid of some kind? Chime in in the comments if you know of something I missed. Regardless, the sternebrae in Ichthyostega have nothing to do directly with those convergently evolved in lissamphibians, lepidosaurs, synapsids and archosaurs, although there may be some parallel developmental mechanisms involved and at least similar dermal tissues recruited into ossification patterns. Even so, these sternebrae are further evidence of how that taxon, at least, was beginning to make forays onto land, as they’d have helped it to support its belly on land and breathe.

The segmented PPC-SRµCT of Ichthyostega stensioi MGUH VP 6115 spinning in yaw and roll.

The segmented PPC-SRµCT of Ichthyostega eigili MGUH VP 29017a spinning in yaw and roll.

The segmented PPC-SRµCT of Acanthostega gunnari MGUH f.n. 1227 spinning in yaw.

The segmented µCT of Pederpes finneyae GLAHMS 100815 spinning in yaw.

FIGURE:

Figure 1_Pierce et al

Above: (a,b) How we used to think the vertebrae were composed in early tetrapods like Ichthyostega. (c) How we found that Ichthyostega‘s posterior thoracic vertebrae actually tend to look. (d) Ichthyostega‘s anterior lumbar vertebral morphology. (e) Acanthostega according to Coates’s important description. (f) Our revision of the anatomy of Acanthostega (anterior dorsal). (g) Our new interpretation of Pederpes‘s morphology, from a posterior dorsal. Focus on the yellow vs. green elements. In a,b and e they are in different positions (reversed) compared with our new versions in c,d,f,g.

To put the above figure and movies into broader context, check this Wikipedia image. We think the red/pink bones (pleurocentra) are in the wrong place relative to the blue ones (intercentrum); the ones currently there in this image actually belong to the vertebral unit behind that one, so the pleurocentra should be moved to the front (left end) of each unit. But also look down toward the bottom of the figure. Some of those vertebrae may need to have their blue/pink bits re-examined and interpreted, too. Is it turtles intercentra all the way down?

There you have it! Welcome to your new, revised, irradiated, reverse-rhachitomous inner tetrapod’s vertebrae. Propagation phase-contrast X-ray synchrotron microtomography FTW!!!!

Science media articles arising from this study–

I like to keep track of media stories covering our research, using this blog, so here are some of the stories about this paper. It’s funny… this was one of the most broadly important papers I’ve ever been on, but the coverage was relatively scant. It was too technical. We knew that would be a problem, and really had a hard time putting into words why the study was so surprising even to us! Most writers wanted the “how did the animals move?” angle, which was not what the study was about. I still feel that this angle was not even needed; the study (and again I take minimal credit for it) is exciting without it. To comparative anatomy and evo-devo specialists, anyway. Well, that’s science for you; sometimes it is just too hard to explain its value to the outside world, even when you feel its importance in your very spine… And the press coverage was not terrible by any means; no sour grapes from me. Regardless, we’re glad it has been well received by specialist researcher colleagues we’ve spoken to, and that matters a lot.

NERC’s Planet Earth (nice story from our funder)- “Scientists had fossil backbone backwards”

BBC online (the only story aside from NERC’s that did more than read the press release) “Tetrapod anatomy: Backbone back-to-front in early animals”

Discovery News online- “First Land Animals Shuffled Like Seals” (good, but is sort of mixing up our this study, our 2012 one and Ahlberg et al’s 2005 seal-analogue study; latter two were more about movement. As often happens, a lot of other media stories basically copied this one’s headline/angle.)

Discover 80beats- “Paleontologists Use 3-D Models to Rewrite Evolution” (also in “top stories”)

Popsci- “Particle Accelerator Reveals That First Land Animals Walked Like Seals”

Daily FMail (nice pics)- “Astonishing 3D images reveal the first four-legged land animals in amazing detail – and overturn a century of research” (wins longest headline award)

Red Orbit- “Study Reveals First Ever Images Of Early Tetrapod Backbone And How It Helped In Land Evolution”

Examiner.com- “X-ray study rewrites tetrapod backbone evolution (Photos)”

Everything Dinosaur- “Ichthyostega Gets a Re-Think”

Business Standard- “Scientists recreate earliest quadraped’s backbone” (Proofread, editors! Quadruped.)

Geekosystem- “Early Land-Dwelling Animals Moved About Like Seals, Probably Didn’t Balance Balls on Their Noses” (scores some pts for humour)

…and the PR-copying, non-spellchecking fail of the week award goes to… Physorg! “Scientists reassemble the backbone of life with a particle acceleratorynchrotron [sic] X-rays”

Warming up the acceleratorynchrotron for our next study… :)

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Party time! Let the media onslaught begin! We’ve published a paper in Nature on the limb motions of Ichthyostega (and by implication, some other stem tetrapods). Since we did use some crocodile specimens from Freezersaurus (see below) in this study, I figured WIJF could cover it to help celebrate this auspicious event. Briefly. Particularly since we already did a quasi-blog on it, which is here:

http://www.rvc.ac.uk/SML/Research/Stories/TetrapodLimbMotion.cfm

and some juicy fossily images at:

http://www.rvc.ac.uk/SML/Research/Stories/TetrapodImages.cfm

However I want to feature our rockin’ cool animations we did for the paper, to squeeze every last possible drop of science communicationy goodness out of them. So here they are in all their digital glory. Huge credit to Dr. Stephanie Pierce, the brilliant, hardworking postdoc who spearheaded the work including these videos! Dr. Jenny Clack is our coauthor on this study and the sage of Ichthyostega and its relatives- her website is here. Also, a big hurrah for our goddess of artsy science, Julia Molnar, who helped with the videos and other images. Enjoy!

The computer model

The forelimb model

The hindlimb model

We used some of my Nile crocodile collection to do a validation analysis of our joint range of motion (ROM) methods, detailed in the Supplementary info of the paper, which I encourage anyone interested to read since it has loads more interesting stuff and cool pics. We found that a bone-based ROM will always give you a greater ROM than an intact fleshy limb-based ROM. In other words, muscles and ligaments (and articular cartilage, etc.). have a net effect of reducing how far a joint can move. This is not shocking but few studies have ever truly quantitatively checked this with empirical data from whole animals. It is an important consideration for all vert paleo types. Here is a pic of one of the crocodiles from the study, with (A) and without muscles (B; ligaments only):

I’ll close with Julia Molnar’s jaw-droppingly awesome flesh reconstruction from our model. Why Nature wouldn’t use this as a cover pic, I’ll never understand, but I LOVE it! When I first saw it enter my email inbox and then opened it to behold its glory, my squeal of geeky joy was deafening.

(edit: Aha! Fellow Berkeley alum Nick Pyenson’s group made the Nature cover, for their kickass study of rorqual whale anatomy, including a “new” organ! Well, we don’t feel so bad then. Great science– and a win for anatomy!!!)

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I’m gearing up for a major post with lots of striking pictures from The Freezers, but to tide you over, here’s a simple movie from one of my CT scans of a Hellbender salamander (Cryptobranchus alleganiensis; with a supportive rod down its GI tract; this was a museum specimen that apparently needed the rod to keep its body straight). We’ve scanned loads of salamanders and other cool critters for our NERC-funded project on the evolution of terrestrial locomotion in the earliest tetrapods (more about that coming up in a future post, as we have some Big News from that study!); this is just one of them.

The huge gaps between limb joints indicate extensive articular cartilage (typical of many aquatic animals, especially some amphibians) and would make a sauropod jealous. The relatively homogenous vertebral column, without much differentiation from head to tail, is also striking, contrasting even with that observed in animals with vaguely similar locomotor styles such as lizards; not to mention mammals, which take that regionalization to an extreme. Also, like most (all?) salamanders, this one has almost no ribs — this is a secondary reduction during their evolution; early fossil tetrapods had nice big ribs. But those ribs aren’t useless (they play a role in moving and breathing), and at least one caudate (member of the broader salamander group) has evolved a very cool use for them!

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