Similar connectivity profiles in humans and monkeys used to generate a Theory of Mind

The ability to infer emotion or intention in others from their outward appearance and behavior, has been called a “Theory of Mind” (TOM). While cognitive scientists have debated whether animals other than humans possess a TOM, many animals (like monkeys) clearly react to facial expression or body movements. One area of the human brain that has received considerable attention in discussions of TOM, is the temporo-parietal junction (TPJ). If each half of the brain is viewed as a boxing glove, the TPA corresponds to the junction between the “thumb” and body of the glove. To explore whether the TPJ regions of humans and monkeys have similar “functional connectivity” profiles, a group of Oxford researchers turned to high resolution at-rest fMRI. The researchers generated correlation maps between each time series obtained for specific voxel regions of interest. Their results, just published in PNAS, show that the most similar TPJ connectivity profiles correspond to areas that process, among other things, faces and social stimuli within the temporal cortex.

When the brain first begins to develop in the womb, the cortex is basically a smooth sheet. The most noticeable topological feature in the cortex of all higher vertebrates, the lateral or Sylvian fissure, begins to take shape as an invagination in the side that proceeds from front to back. This fold, with the TPA at its apex, remains as the primary feature of the cortex even as it grows increasingly convoluted. It is little wonder that many of the most interesting mental phenomena, and malady, are often attributed to this region. Stimulation of this area has produced effects as widespread as out of body experiences, impostor syndromes, and even phantom body doubles with precise geometrically offsets to the primary body position.

It is a bit of a paradox perhaps, that many studies which look for uniform or predictable features in the brain have instead hit upon the very region where any such pigeonholing is most labile. In other words, when the brain folds, the TPA is precisely the region where the most scrunching happens, with the result the mature structure typically shows the most variance. In animals like cats and many monkeys, the cortical gyri and sulci, have virtually the same pattern in each individual. In humans however, attempts to assign names to specific folds of the TPA region is like playing a game of pin the tail on the donkey. For example, the Angular gyrus, Wernicke’s area, Supramarginal gyrus, and Inferior parietal area, can all be variously designated as part of the TPA.

Recent attempts to define a default mode network (DMN) using fMRI have included this same region. In theory, the DMN can be used to distinguish sleep from arousal. It was noted that neurons which project out of the cortex in this region have, in effect, more options open to them than those virtually anywhere else in the brain. For example, directly under the angular gyrus is the area known as the temporo-parietal fiber association area. It includes at least seven long range white matter superhighways. That is not to say TPA neurons have free reign to board any tract they choose, (especially those like the optic radiations whose foundations are strongly and quickly set by myelin), but certainly the wide variance in behavioral correlates of these cells has an anatomical basis.

The Oxford study used Macaques, a monkey which has been on a separate evolutionary path from humans for around 30 million years. They note that the superior temporal (STS) region of the Macaque contains face cells that have been found to be more responsive to social cues rather than to identity. The researchers included the STS in their MRI meta-analysis, and also incorporated information from the BrainMap database, a large repository of neuroimaging data. While it is encouraging to see big data being put to use, it is often difficult to follow exactly how the data is processed to yield the so-called “activation likelihood estimation maps for activity elicited by theory of mind paradigms and by face discrimination or processing.”

As various federal projects begin to assemble connectomes for the human brain, functional connectivity studies that use highly processed MRI data, will need to be made as simple and straightforward as possible if they are to be put to widespread use. MRI tractography is a related technology that can assign physical connectivity by performing a meta-analysis on diffusion tensor data. Using scans and connectomes to generate theories to explain some of the strange mental phenomena generated secondary to stroke or by various kinds of electromagnetic stimulation are the best approaches we have at the moment. New technologies generated by the BRAIN Initiative will hopefully allow a finer-grained exploration of theory of mind.

Animals in research: zebrafish

Zebrafish are probably not the first creatures that come to mind when it comes to animals that are valuable for medical research.

You might struggle to imagine you have much in common with this small tropical freshwater fish, though you may be inclined to keep a few “zebra danios” in your home aquarium, given they are hardy, undemanding animals that cost only a few dollars each.

Yet each year more and more scientists are turning to zebrafish to unravel the mechanisms underlying their favourite genetic or infectious disease, be it muscular dystrophy, schizophrenia, tuberculosis or cancer.

My (conservative) estimate is that zebrafish research is now carried out in at least 600 labs worldwide, including 20 in Australia.

So what is it about zebrafish that has taken them from the freshwater rivers and streams of Southeast Asia, beyond the pet shops and into universities and research institutes the world over?

A short history of zebrafish

A scientist called George Streisinger, working at the University of Oregon in Eugene, USA in the 1970s and 80s, recognised the vast potential of this organism for developmental biology and genetics research.

In contrast to fruit flies and worms, the other simple model organisms established at the time, zebrafish are vertebrates.

They have a backbone, brain and spinal cord as well as several other organs, including a heart, liver and pancreas, kidneys, bones and cartilage, which makes them much more similar to humans than you may have otherwise thought.

But as a vertebrate model, could they be as useful as mice?

Several things captured Streisinger’s imagination.

Most famously, zebrafish embryos, unlike mouse embryos, develop outside the mother’s body and are transparent throughout the first few days of life.

This provides unparallelled opportunities for researchers to scrutinise the fine details of embryonic vertebrate development without first having to resort to invasive procedures or killing the mother.

But this advantage is enhanced by the fact zebrafish reproduce profusely (each pair can produce 200-300 fertilised eggs every week); an ideal attribute for genetic studies. Again, the large, external embryos are a critical part of this success.

When just one or two cells old, zebrafish embryos can be easily microinjected with mRNA or DNA corresponding to genes of interest; undeterred, they then they go on to grow and reproduce, handing down the injected gene to the next generation.

From zebrafish to humans

A paper published last month in Nature unveiled the long-awaited sequence of the zebrafish genome, revealing that zebrafish, mice and human have 12,719 genes in common.

Put another way, 70% of human genes are found in zebrafish.

But even more notable is the finding that 84% of human disease-causing genes are found in zebrafish.

Perhaps not surprisingly then, when these genes are injected into zebrafish embryos, the growing animals are doomed to acquire the same diseases.

And while zebrafish are still used widely to answer fundamental questions of developmental biology, much current research is directed towards combining their many attributes in studies that are designed to improve human health.

This is especially true for cancer research where the expression of cancer-causing genes (oncogenes) can be directed to specific organs, virtually at will.

This process, known as transgenesis, is very straightforward in zebrafish and has allowed researchers to produce zebrafish models of liver, pancreatic, skeletal muscle, blood and skin cancers, to name but a few.

And when the genomic make-up of these zebrafish tumours is deciphered using the latest DNA sequencing technology, the patterns of mutations, or “gene signatures”, are found to overlap substantially with those in the corresponding human tumours.

Trialling cancer drugs

These parallels have encouraged researchers to exploit zebrafish in drug development – in particular for high throughput approaches such as chemical/small molecule screens.

Here, the ability to generate tens of thousands of zebrafish embryos harbouring the same disease-causing mutations is crucial.

Then, as the tumours grow in the synchronously developing larvae, the fish are transferred to small volumes of water containing chemicals that may stop the growth, or better still, kill the cancer cells.

Large collections of drugs can be screened relatively quickly for anti-cancer efficacy in this way.

One drug, Leflunomide, identified in such a screen is now in early phase clinical trials to kill melanoma cells.

The only other drug from a zebrafish chemical screen currently in clinical trials is dimethyl-prostaglandin E2 (dmPGE2).

There, the intent is not to kill cancer cells but rather to make mainstream leukaemia treatment more effective.

Studies of dmPGE2 increased the number of blood stem cells in zebrafish embryos and it is being trialled now as a way to expand the number of stem cells in human cord blood samples.

Human cord blood samples are a valuable commodity to restore bone marrow in leukaemia patients after high dose chemotherapy when a matched bone marrow transplant is unavailable.

But the success of this approach is currently limited by the scant number of stem cells in individual cord blood samples, requiring the use of two precious samples for each patient.

Tumour growth

As well as the transgenic zebrafish models of cancer described above, researchers are also transplanting cells derived from human tumours into zebrafish embryos and watching them grow and spread.

The creation of a transparent (non-striped) version of adult zebrafish (called casper, after the cartoon ghost) means the behaviour of tumour cells inside these living organisms can be followed for days at a time.

Coupled with the advent of high resolution live-imaging techniques, the birth, growth and spread of tumours can be scrutinised in movies that can be played over and over again.

These experiments are usually conducted in zebrafish that have been genetically modified to express genes that glow in specific body compartments, giving researchers the ability to pinpoint potentially critical connections between “host” cells and tumour cells that may determine whether the latter survive or die.

This type of experiment is revealing a complex interplay of potentially beneficial and detrimental components.

While the proximity of immune cells may instigate mechanisms capable of destroying the tumour, the stimulation of new blood and lymphatic vessel growth towards the tumour is more insidious, since it delivers the tumour with both the nutrients it needs to survive and a network to spread throughout the body.

These processes, once properly understood, are likely to provide opportunities for therapeutic intervention in the future.

The future of zebrafish

Cancer research is just one part of the zebrafish story. In Australia alone, investigators are also using zebrafish to study metabolic disorders such as:

• diabetes
• muscle diseases, including muscular dystrophy
• neurodegenerative disease
• the response of the host innate immune system to bacterial and fungal infections

Excitingly, research is also underway in this country to unravel the genetic mechanisms controlling heart, skeletal muscle and nervous tissue regeneration in zebrafish, in the hope that these processes can be one day recapitulated in humans to address the burgeoning socioeconomic problem of tissue degeneration in our ageing population.

So next time you peer into someone’s home aquarium, imagine the biomedical possibilities inherent in this lively and amiable little fish!

Research update: Imaging fish in 3-D

Zebrafish larvae — tiny, transparent and fast-growing vertebrates — are widely used to study development and disease. However, visually examining the larvae for variations caused by drugs or genetic mutations is an imprecise, painstaking and time-consuming process.

Engineers at MIT have now built an automated system that can rapidly produce 3-D, micron-resolution images of thousands of zebrafish larvae and precisely analyze their physical traits. The system, described in the Feb. 12 edition of Nature Communications, offers a comprehensive view of how potential drugs affect vertebrates, says Mehmet Fatih Yanik, senior author of the paper.

“Complex processes involving organs cannot be accurately recapitulated in cell culture today. Existing 3-D tissue models are still far too simple to model live animals,” says Yanik, an MIT associate professor of electrical engineering and computer science and biological engineering. “In whole animals, the biology is far more complicated.”

Lead authors of the paper are MIT graduate student Carlos Pardo-Martin and Amin Allalou, a visiting student at MIT. Other authors are MIT senior research scientist Peter Eimon, MIT intern Jaime Medina, and Carolina Wahlby of the Broad Institute.

Zebrafish are genetically similar to humans and have many of the same developmental pathways, so scientists often use them to model human diseases including cancer, diabetes, Parkinson’s disease and autism.

Using the new technology, researchers can grow larvae in tiny wells and flow them through a channel to an imaging platform. Once there, the embryos are rotated and 320 images are taken from different angles, allowing 3-D reconstructions to be made using optical projection tomography (OPT). Getting larvae to the platform takes about 15 seconds, and the imaging takes only 2.5 seconds. This allows hundreds or thousands of larvae to be imaged within hours.

In a 2010 paper, Yanik’s team described the system that transports the embryos to the imaging platform, which they combined with high-resolution two-dimensional imaging. In the latest version, they developed a high-speed OPT imaging technique, which takes hundreds of two-dimensional images and subsequently generates a 3-D image, similar to a CT scan.

They also created a computer algorithm that can measure hundreds of traits and use that information to create a comprehensive phenotype map — the overall description of an organism’s characteristics — for each larva. This enables rapid and detailed studies of how different drugs affect those phenotypes.

“You could probably look at almost any organ or tissue that you’re interested in,” Eimon says. “It gives researchers a way to rapidly measure and quantify and put numbers on the kinds of phenotypes and gene-expression patterns that they’ve been looking at for years and years.”

In this study, the researchers focused on the craniofacial skeleton, which is analogous to the human skull. They measured the length and volume of each of the bones that make up this structure, as well as the angles between the bones.

Each embryo was imaged five days after being treated with one of nine different teratogens — drugs that cause developmental abnormalities. The researchers compared their results with the drugs’ known effects and found that they were very consistent. They also obtained high-resolution, 3-D images of the craniofacial skeletons, which are less than a millimeter long.

“Now that we’re able to load the animals, and we can image them really quickly, and we have a way to start looking at the information, the sky’s the limit,” Pardo-Martin says. “What we have to do now is ask the big questions, because the technology has advanced.”

This kind of analysis could be very valuable for drug developers who need to efficiently screen thousands of drug candidates. It could also be used to study hard-to-detect changes in phenotype caused by genetic mutations, says Joseph Fetcho, a professor of neurobiology and behavior at Cornell University.

“A really high-throughput way to assess phenotype is very important for measuring small effects on the development of an organism,” says Fetcho, who was not part of the research team. “You can see what the phenotype looks like in a large population and quantify it in a very rigorous way.”

Researchers uncover major source of evolutionary differences among species

University of Toronto Faculty of Medicine researchers have uncovered a genetic basis for fundamental differences between humans and other vertebrates that could also help explain why humans are susceptible to diseases not found in other species.

Scientists have wondered why vertebrate species, which look and behave very differently from one another, nevertheless share very similar repertoires of genes. For example, despite obvious physical differences, humans and chimpanzees share a nearly identical set of genes.

The team sequenced and compared the composition of hundreds of thousands of genetic messages in equivalent organs, such as brain, heart and liver, from 10 different vertebrate species, ranging from human to frog. They found that alternative splicing — a process by which a single gene can give rise to multiple proteins — has dramatically changed the structure and complexity of genetic messages during vertebrate evolution.

The results suggest that differences in the ways genetic messages are spliced have played a major role in the evolution of fundamental characteristics of species. However, the same process that makes species look different from one another could also account for differences in their disease susceptibility.

"The same genetic mechanisms responsible for a species’ identity could help scientists understand why humans are prone to certain diseases such as Alzheimer’s and particular types of cancer that are not found in other species," says Nuno Barbosa-Morais, the study’s lead author and a computational biologist in U of T Faculty of Medicine’s Donnelly Centre for Cellular and Biomolecular Research. "Our research may lead to the design of improved approaches to study and treat human diseases."

One of the team’s major findings is that the alternative splicing process is more complex in humans and other primates compared to species such as mouse, chicken and frog.

"Our observations provide new insight into the genetic basis of complexity of organs such as the human brain," says Benjamin Blencowe, Professor in U of T’s Banting and Best Department of Research and the Department of Molecular Genetics, and the study’s senior author.

"The fact that alternative splicing is very different even between closely related vertebrate species could ultimately help explain how we are unique."

Scientists have found that one gene is responsible for variability in locomotion in horses and mice.

Traits such as height are controlled by the interaction of up to 700 genes. So it came as quite a shock to researchers from Uppsala University (UU) and their international collaborators that the mutation of just a single gene is responsible for variability in locomotion in horses and mice. Furthermore, the research team discovered that this gene, DMRT3, is expressed in a previously unknown set of neurons in the spinal cord. These findings provide insight into the neural circuits that coordinate movement in vertebrates.

“The amazing result was that we found one very strong signal on one chromosome which by further work led to the discovery of the DMRT3 mutation,” explains Leif Andersson, co-author of the paper published in Nature, from UU and Swedish University of Agricultural Sciences. “This was unexpected since we had [anticipated] a much more complex genetic background for a trait like this.”