25 Feb 2015

Should mice be used to study the human gut microbiome?

In recent years, the trillions of bacteria living in our guts have risen from obscurity to stardom. Hyped press releases claim that probiotics and faecal transplants might one day treat almost everything, from bowel inflictions to obesity. These studies often involve mice, but are these rodents really a suitable model for microbiome research?

The gut microbiome has been associated with an ever-growing list of diseases, including obesity, diabetes and even mental disorders such as anxiety and autism. Much like the Human Genome Project around 15 years ago, the booming microbiome research field has promised to deliver new revolutionary treatments, some as simple as eating a yogurt. Perhaps inevitably though, history repeats itself. After a few years of frantic microbiome sequencing and many new biotech start-ups, microbiome researchers are now having to face the hard questions: are the changes in the gut microbiome associated with certain diseases a cause, or a consequence, of the disease? How on earth can bacteria in the gut affect other parts of the body, such as the brain? What are the molecular mechanisms behind all this?

E. coli bacteria thrive in the gut.

Studies in humans can at most reveal correlations between the microbiome composition and a given disease. For example: Bob is obese and happens to have a microbiome with lots of bacteria X, but John, who is slim, doesn’t. This suggests that bacteria X cause obesity, yet, there’s also a good chance that in fact it’s the other way round: obesity might somehow promote growth of bacteria X. Or maybe this type of bacteria thrives on Bob’s diet, or it simply prefers the unique environment of his gut.

It is virtually impossible, and unethical, to perform experiments in humans to explore causal hypotheses (does bacteria X cause obesity?) and control for confounding factors like diet and genetic background. Microbiome researchers have to use the next best thing: mice. There are, however, growing concerns within the scientific community that more often than not, data from mouse can’t be extrapolated to humans for clinical purposes. Or at least, not easily.

In a new study, Jeroen Raes and colleagues at the KULeuven University, in Belgium, carefully compared the human and mouse gut microbiomes to assess the strengths and pitfalls of this model system for studying microbiome-related diseases.

“Microbiome research, notably its association to inflammatory diseases, relies heavily on mouse models […]. It is essential to know the qualities and limitations of each model to choose the correct one to test specific hypotheses”, says Sara Vieira-Silva, one of the authors conducting the study.

Can mice recapitulate the human gut microbiome?
Mice are great for biomedical research. They share most of our genes, and have similar anatomy and physiology. With the many available genetic tools, scientists can easily and quickly discover the function of literally any gene in the mouse genome, and recapitulate human disease in a controlled experimental set up. So where’s the catch? The problem is that although mice and humans share many similarities, there are also many differences.

Rae’s team performed comprehensive statistical analyses for all gut microbiomes from mice and humans published to date. These new data tell us what types of bacteria live in the gut in various scenarios (disease, diet, genetic background…), as well as their relative abundance. The team first compared the gut microbiomes of healthy humans and mice. And the differences start here.


Human and mouse guts have predominantly two ‘families’ of bacteria—Bacteroidetes and Firmicutes—but within these groups, 85% of bacteria species found in mice are not present in humans. And the bacteria found in both? It appears their abundance in the gut also varies between mice and humans; when you’ve got a lot of a certain bacteria in mouse, you may find very little of it in humans, and vice versa. The authors stress that many of these differences could simply be a result of technical limitations, like methodology or interference from external factors (diet, age, etc).



Mouse models of disease
There are over 60 mouse models of Inflammatory Bowel Disease (IBD), but none fully recapitulates the disease. Even so, the changes in the gut microbiome of patients with IBD (when compared to healthy people) are similar to those observed in IBD mouse models. For example, there is a significant reduction in bacterial diversity in both IBD patients and IBD mouse models. However, some specific bacteria species will be more (or less) abundant in mouse but not in IBD patients. The same goes for obesity models. Overall, mice fed on high-fat diet, and also leptin-deficient mice, which cannot control their appetite, recapitulate the microbiome changes observed in obese people. But there are many discrepancies in the data, again likely due to external factors that are difficult to control, at least in human studies.

The conclusion? Well, mice are not people. Raes and colleagues warn microbiome researchers that extreme care should be taken when trying to extrapolate findings in mouse to humans. They should also make bigger efforts to standardise their protocols for animal handling and data analysis, and to share mouse models to eliminate any genetic variability that might skew the data.

“Most limitations of murine [mouse] models for fundamental microbiome research can be overcome by methodical study design and statistical testing: either eliminating or keeping track of possible confounders (e.g. diet variation, genetic background) and testing for their influence on the results”, says Vieira-Silva.

Nevertheless, the authors conclude, when it comes to understanding the causes and molecular mechanisms behind human disease, mouse models seem to fit the bill. “Although the mouse microbiota composition is not identical to the human's, most mechanisms of microbiota-host interaction will be shared between mice and humans” concludes Vieira-Silva. “Mice models allow us to study these mechanisms with direct controlled experiments, towards the ultimate aim of providing therapeutic solutions.”

Reference:

Nguyen T.L.A., A. Liston & J. Raes (2015). How informative is the mouse for human gut microbiota research?, Disease Models , 8 (1) 1-16. DOI: http://dx.doi.org/10.1242/dmm.017400

And edited version of this article was published in Lab Times on the 24-02-2015. You can read it here.


5 Feb 2015

'One fossil can overturn anything' Interview with Jenny Clack

Now happily living on land, our Devonian ancestors tried many ways to get out of the murky waters. Jenny Clack has been studying the water-to-land transition of vertebrates for many decades. Her discoveries broke dogmas and rewrote textbooks. 

Jenny Clack's passion for palaeontology began at a young age, but unlike most children, Clack found dinosaurs “rather boring” and was instead fascinated with weird older creatures from the Devonian era, over 360 million years ago. After completing an undergraduate degree in vertebrate palaeontology, Clack worked for about seven years as a display technician at the Birmingham City Museum, until she finally had the opportunity to do a PhD with Alec Panchen at the University of Newcastle upon Tyne (UK). Clack’s talent quickly got noticed, and during her PhD she was offered a position as an assistant curator at the Museum of Zoology of the University of Cambridge (UK). At Cambridge, Clack had an insight that would transform her career and her life. During an arduous field trip to Greenland in 1987, she found spectacular remains of Acanthostega, a tetrapode (four-legged vertebrate) that would overturn decades-old theories. Clack was the first woman in her field to become a fellow of the Royal Society, won numerous distinguished awards and is currently a professor and curator of vertebrate palaeontology at the Museum of Zoology of the University of Cambridge.

When did you know you wanted to be a palaeontologist?
Clack: I was always interested in natural history generally, and as quite a young child, from the age of seven or so, I collected plants and fossils. And certainly by the age of ten I was interested in palaeontology and rocks, and I used to borrow books from the library. I would read geology books and books on fossils and natural history instead of what my teachers would want me to do, which was to read novels, of course. Throughout school, I was always interested in natural history and decided that I wanted to do zoology degree, and went to the University of Newcastle upon Tyne. One of the reasons for choosing Newcastle was because it had a programme in palaeontology as part of the zoology degree. It was just the idea of these ancient creatures... I was always interested in the earliest stuff, rather than dinosaurs. I had a series of volumes of a children encyclopaedia that had sections on various periods from the Palaeozoic, and they were really my inspiration. I wanted to know about the very old fishes and early animals, like the amphibians that were described in those days. When I got the opportunity to study at university then obviously I decided that’s where I wanted to go. But it wasn’t straightforward by any means.

What was it like for a little girl back in the 1960s to pursue an academic career? 
Clack:  It was more that the teachers obviously knew that I was interested in that kind of thing. I remember one of the teachers in junior school identifying me as an “academic type”, even though I had no idea what that meant at the time. Certainly, my parents always encouraged me to do whatever it was I wanted to do. They took me on holidays to places where I mind find fossils and other elements of natural history. […] My career has been a bit of a complicated path because I didn’t go into palaeontology professionally after my degree. I did a Museums Study course, and then worked seven years in the City Museum in Birmingham. And it was only when I had the opportunity to do a PhD that my career really started.

How did you eventually get into academia?
Clack: It was partly encouraged by the museum itself because they allowed people to do three weeks of private studies per year and my mentor-boss at the time was very supportive of this. So, I got back in touch with my old mentor, Alec Panchen, in Newcastle and asked him whether he had any projects I could work on, and in fact he did. He directed me to a specimen in a museum in Bradford that was a Carboniferous tetrapode. To cut the long story short, I took that specimen to his lab and worked on it for the three weeks, during which time I found that there was quite a lot more to the specimen than anybody had realised. And then Panchen said I could probably get a PhD from that material; he applied for grants and got it.

Was it at this time that you decided to focus your career on the fish-to-tetrapode transition?
Clack: I was interested in the same sort of field that Panchen was, which was Carboniferous tetrapodes, so it was a natural expectation that I would study something of that nature. And indeed, the PhD started that ball rolling. While I was still doing my PhD, I applied for a job as an assistant curator at the Museum of Zoology of the University of Cambridge and much to my surprise they offered it to me. This would not happen today. There is no way someone who hasn’t finished their PhD, has got no published papers and has no reputation would get that kind of job. Now, you would have to have a postdoc, at least. I had the museum qualifications and the research background that they were interested in. I fit the bill I guess [laughs]. And it wasn’t until some years later that the opportunity to look at the Devonian material came about. 
After I had finished my PhD in 1984, I wondered what on earth am I going to do next? I didn’t have any very clear ideas. My colleague Andrew Miller said something will come up and indeed it did! It turned up in a drawer in the Earth Science Department across the road. This was a drawer full of Devonian material from Greenland that a former student there had collected without realising what it was, or its potential importance. And from there we got the expedition to go to Greenland in 1987 and collected more of this material, which turned out to be extremely important. A very lucky break indeed.


Fossil remains of Acanthostega.

What exactly did we learn about the water-to-land transition from your discoveries of Acanthostega?
Clack: There were two major discoveries. The first one was about the story we had been told that, as soon as these creatures came onto land, they developed the capacity to hear air-born sound. And it became clear from the work I had done in my PhD, and the work on Acanthostega, that this couldn’t possibly be the case. The story of the origin of terrestrial hearing became much more complicated and it was corroborated by people from other palaeontology groups. But probably the most widely known discovery was that Acanthostega had eight digits in each limb. That was a real surprise. It took a little while for people to believe that this was the case because the dogma was that there were five digits in primitive tetrapods. And here we had an animal with eight digits on each limb! 
We then discovered that a Devonian tetrapod that had been known for decades called Icthyostega had in fact seven digits on its hind limb, and this complemented what we had known about a Russian animal from the Devonian, which has got six digits. All of a sudden it became a pattern of multiple digits in the earliest tetrapods with limbs. This changed the idea of how limbs evolved and what they evolved for. If you look at the old books from the 1940s, for instance, you get an idea of what they thought a proto-tetrapode looked like, and basically it looked like a fish that has got legs with five digits on, and it’s making forays onto the land. But actually our work suggests that the animals already had limbs with digits before they ever came out of the water. So, it kind of turns the story upside down.

Is it the number of digits alone that tells us that, or some other features as well?
Clack: Acanthostega had a number of primitive features. One of those was the proportion of [the bones in] the forearm, of the radius to ulna to each other. In most tetrapods, the ulna is longer than the radius, and that’s true to almost all tetrapods, and most fossil ones as well. But in the fish, from what tetrapods were supposed to evolve, it’s the other way round: the radius is much longer than the ulna. And that was the condition in Acanthostega. It seemed to us that the limb elements of Acanthostega were showing us what the primitive condition was like for limbs in general. Also, the fact that the digits were variable in number through these early tetratpods, suggested that the function of the digits in the limbs was quite different from what we assumed. It’s a paddle basically.

You also discovered new features in Icthyostega
Clack: We discovered that Icthyostega is a really enigmatic animal. We’ve known this more or less since it was discovered, and the more we found out about it, the weirder it looked. It’s got some features in which some limbs elements, like the humerus, are more primitive than that of Acanthostega, and yet other aspects of the anatomy of Icthyosthega suggest it was more terrestrial than Acanthostega. Acanthostega seems to be almost certainly entirely aquatic, but Icthyostega has a really robust front limb that looks as though it could at least raise the front body off the ground, whereas the hind limb is a paddle and points backwards towards the animal’s tail. 
We worked out how this animal could move using information from synchroton CT scans of the limbs and reconstruction software that can help you find out how the limbs actually worked in 3D. It turns out that Icthyostega didn’t walk in a conventional manner. It looks as though one of the possible modes that it used would be a source of crunching motion, with the two front limbs together and the hind limbs acting as breaks or supports, but not actually producing any power on land. They were used to propel the animal in water, so for walking or for moving on land it used its front limbs, sort of pulling it along. And in the water it used its hind limbs as paddles for propulsion.

How did the first terrestrial animal walked?
Clack: We don’t really have enough information to be sure about that, but people now have been using the same sort of software and techniques to look at Acanthostega in the same way. But being very much aquatic, it’s obviously not going to be comparable in terms of what it was doing. The implication is that there were lots of different experiments going on in locomotion and we have only looked at the tip of the iceberg, in terms of the information that we’ve got, which is so limited. For example, in 2011, scientists published some track ways that were found in Poland that pre-date the Devonian tetrapods we had found by about 15 millions years. We don’t know what made those track ways, but we know it was made by an animal walking supported by water and using its limbs in an alternated fashion […]. So there were some animals around at this early stage that were using this pattern of locomotion, but we don’t know what they looked like because we don’t have any body fossils for them. 

What does it take for a palaeontologist to take on an ambitious expedition like your expedition to Greenland?
Clack: Again it was a series of lucky breaks. The material from Greenland at the time belonged to the Danish government. The material from Icthyostega, for example, was all in Copenhagen. I got in touch with the then curator of the Geological Museum in Copenhagen and told him about the material I had found in the Earth Sciences Department. And the quality and amount of that material convinced him that there was a lot more to be found. So he got in touch with the authorities in Denmark and the Greenland Geological Survey (as it was called then) and they happened that year to be setting up a 3-year project in the very area that we wanted to go. We managed to jump on the bandwagon, their expedition, using their facilities and transport arrangements, to get our expedition together. And the funding came to a large extent from our museum in Cambridge, and a certain amount also from Copenhagen and the Karlsberg Foundation. That’s how it was funded. We did try the Research Council in the UK but they weren’t interested.

Have there been other findings throughout your career that got you as excited as when you found Acanthostega?
Clack: Well actually, the project that I’m working on now which is now half way through. The Tw:eed Project is a consortium looking at what happened at the end of the Devonian. As the story goes… Devonian was the age of fishes, and at the end of the Devonian, quite a lot of them got wiped out, there was a mass extinction. The cause of it isn’t clear, but it seems to have been something climatic. The period after that, for 15 to 20 million years, was an almost complete blank in the fossil record, certainly for tetrapods but also for almost everything else as well. [...] The problem was that after that period of 20 million years, when we begin to pick up fossils of tetrapods again, they were extremely diverse. There was a huge variety of tetrapod forms, from small ones the size of a mouse, to other ones that were three or four meters long. So how did they get there? What happened after the end of the Devonian that allowed them to do that? We knew nothing about how these things became properly terrestrial. And it all happened in that gap. 
This gap was first identified by an American palaeontologist called Al Romer, so it’s called Romer’s gap. There were a few specimens from the period of this gap known from Nova Scotia, although nothing formal had been published on those. And I published a paper in the early 2000s on a complete specimen of a tetrapod from the middle of this gap that had been found in Dumbarton, in Scotland. In subsequent years, some of my colleagues have been looking at the appropriate sorts of sediments in the borders region in Northumberland, in Scotland, for the rocks of this age. They found some material, and it’s that material that we are beginning to work on, and we’re also finding a lot more. We have found numerous fossils of tetrapods, several new sharks, new lungfishes, all sorts of things. We’re beginning to get a handle on how terrestrial features or adaptations in tetrapods could have arisen.

So it is possible to find fossils from the Romer’s gap...
Clack: Yes, that’s right. Our idea is that this particular formation called the Ballagan Formation, which has been known for many years and was described as the Scottish cement stone series, is not commercially viable. There’s no coal and no decent limestone. In the 19th century a lot of the carboniferous fossils were found by miners, and that’s how we knew they were there. But because nobody has been looking for commercially viable rocks, nobody has found anything, and because nobody has found anything, nobody has looked. It’s a sort of self-fulfilling prophecy until you get somebody with the determination to say, well they got to be there. And indeed, it turns out that they were.

Do you think that a multidisciplinary approach is important for palaeontology, or is it just a trend?
Clack: This seems to be increasingly the case, yes. […] In the 1970s and 1980s or earlier, palaeontologists tended to work by themselves, just looking and describing the animals. They were doing some fieldwork to find new stuff too, but definitely that was “one person, one fossil” kind of thing. But now collaboration is the key word because different people have different skills, and with all the new techniques that are coming forward you need collaborations to get all those skills together. And certainly I’ve collaborated with people from the Royal Veterinary College for example, and people from the synchroton facility in Grenoble. You just can’t work by yourself anymore, and this particular project was really perfect for this kind of collaborative effort.

How has the development of modern instrumentation (isotope analysis, computer modelling, X-ray computed tomography) changed the field?
Clack: Now we can think of asking and answering questions that would have seemed impossible 10 or 15 years ago. We can ask new questions about how things work, what that might mean, and how the animals developed. And, of course, you’ve got geologists on one side, and then you’ve got technicians, and people doing developmental biology on modern creatures to look at how things could relate to what the fossil record is finding. These collaborations are increasingly common. Developmental biologists and Evo-Devo people are constantly coming to us and asking what we see in the fossil record, and how could this fit with what they’re finding. It’s really encouraging. […] Quite a few people are interested in compiling large databases and then interrogating them; what fossils came from this region, how many species are there in these various time slots and what does the phylogeny tells us. That’s all very well but one fossil can overturn any of that. You still need the data and that’s why it’s so encouraging also that more people are going out and finding new stuff all the time, finding new localities and new areas of the world to explore. And at some of the localities people thought were wiped out, they go back and find new material there, so there’s a wealth of stuff. And of course, communication is so much easier than it used to be.

What is the palaeontology of the future?
Clack: Oh, who knows? If you look at the Society of Vertebrate Palaeontology website, they have their programme for their annual meeting which was in Berlin this year, and the diversity of talks is just stunning, where do we go from here? Well, I think we still need to be fuelled by new material, but that new material can overturn anything that I said! 50 years ago we thought we knew everything about fossils and Palaeozoic vertebrates… no we don’t know, it has been completely overturned since then and there’s no doubt it will be overturned again in the next 50 years.

How can we change the way scientists are perceived by the public? 
Clack: The media like to portrait science as rather esoteric, let's say. BBC tries to do a good job, but I think they have very stereotyped ideas about science and they think the public can’t cope with uncertainties. The message needs to get across that science is about questions and not about answers, and that’s hard to communicate. 

What do you love the most about being a palaeontologist?
Clack: Solving the puzzle, interpreting difficult material, and I think it’s probably one of the things I’m best at. I also quite enjoy writing the papers. I don’t find writing difficult, as I know some people do.

What big exciting questions remain out there for palaeontology, and which ones would you really like to see answered?
Clack: In terms of vertebrates, some of the big questions now are: what’s the origin of vertebrates? How do we get limbs from fins? How do you get fins in the first place? How do you get jaws and teeth, where are they coming form? That’s the sort of thing we can relate to modern developmental genetics as well. Where we can find links with other disciplines it’s really important. If you look at the limb bones of the carboniferous animals, in many cases they’re quite different from those of modern forms. How do we get terrestrially capable limbs? Which bits have to be modified so that you can bear weight? What muscles do you attach and how do they develop?

How would you explain to someone in one sentence that it is important to fund and encourage more palaeontology research?
Clack: It’s a bit like learning History, you know what use is History? What use is the Arts? People don’t seem to ask those questions, but what use is Palaeontology? Oh, that’s no use is it? Well it’s a cultural exercise, it expands the mind, it tells us where we came from and it puts us in our place. It’s all part of the evolutionary story. It’s not like a biomedical science where you want to help people, or invent some kid of drug or something, it’s mind expanding blue skies, learning about the world.

What is the fossil of your dreams?
Clack: I would like a sequence of strata with exceptionally well-preserved soft tissue representations of Devonian forms so that we could find out what sort of reproductive strategy they used. It’s what we call a Lagerst├Ątten, like the Burgess Shale where we can actually see soft tissue preservation of early tetrapods. 


References:

Pierce S.E. & John R. Hutchinson (2012). Three-dimensional limb joint mobility in the early tetrapod Ichthyostega, Nature, DOI: http://dx.doi.org/10.1038/nature11124

Clack J.A. (2002). An early tetrapod from ‘Romer's Gap’, Nature, 418 (6893) 72-76. DOI: http://dx.doi.org/10.1038/nature00824 


Image credits: Museum of Zoology, University of Cambridge. Portrait, Chris Green, Department of Zoology, University of Cambridge.

An edited version of this interview was published in Lab Times in print on the 24-11-2014.