Are lemurs the next big thing in genetics?

Onja is struggling tonight – her hands are constantly sliding on a miniature grab bar used to measure her strength. "Come on, you can do better," shouts Zeph Pendleton, who gently supports the mouse lemur as she tries to hold herself firm. Finally, the animal puts his fingers around the bar and pulls it. It records a force of 1 kilogram, impressive for a creature weighing only 41 grams. "Good," says Pendleton, a research assistant who works here in the tropical rainforest of the ValBio Center, a research station at Ranomafana National Park in Madagascar.

Bathed in a dim red light, Pendleton, coming from Stanford University in California, puts Onja to the test. He has her hands placed on a modified iPhone to measure the electrical activity of her heart. It checks its length and weight – it has gained 2 grams in less than a week – then takes a photo and then stores the information in a constantly expanding database on one of the smallest and most abundant primates of the planet.

Finally, Pendleton pushes Onja into a cage and covers her with a black bag to protect the night creature's eyes as he transports her into the bright corridor and returns to the rainforest.

Onja, which translates as an oceanic wave, is one of some 500 animals studied to date in the mouse lemur project, a collaboration that aims to analyze the genetics of this tiny prosimian primate. An original idea of ​​Stanford biochemist Mark Krasnow, the project is studying a large population of gray and brown lemurs – Microcebus murinus and Microcebus rufus, respectively – in nature to determine how their genes are related to differences in biology, health and behavior.

Krasnow believes that mouse lemur could become an important animal for genetic research, potentially competing with the common laboratory mouse Mus musculus, at least for some questions. Genetically, mouse lemurs are more closely related to humans, while retaining many of the advantages of mice in terms of small size, fast reproduction, and relatively large litters. As such, they can shed light on some questions about human biology and disease, which mice simply can not do. "You hear a lot about the success of mice in elucidating the disease. human biology, "says Krasnow. "What you do not hear are aspects of human biology that are not imitated in mice," behaviors to diseases and physical traits. Lemurs could go a long way to solving this problem, he says.

Although it is difficult to establish a new model organism, scientists and funding agencies take into account the mouse lemur. Researchers sequenced his genome (P. A. Larsen et al. BMC Biol. 15110; 2017), and Krasnow and his collaborators Stanford and Biohub Chan Zuckerberg in San Francisco, California, will soon release a single-cell atlas for the creature – a detailed description of the gene's activity in hundreds of thousands of cells. worldwide. body of the animal. This would be the second atlas cell of a mammal, after the mouse. In addition, several laboratories around the world have already established lemur colonies to explore topics as diverse as Alzheimer's disease and its evolution.

A decade after the start of the project, Krasnow says it's already a "huge success". His team has identified dozens of distinctive lemur traits in mice, many of them medically, such as cardiac arrhythmias, movement disorders, and hypercholesterolemia. Researchers are now beginning the difficult task of linking these traits to the mutations found in animal genomes, an approach that could reveal the genetic basis of the complex behaviors of primates and human disease.

"I love the idea of ​​using a new model," says Rochelle Buffenstein, a comparative biologist at Google's anti-aging company, Calico, in South San Francisco. "Mice have made a huge contribution to biology, but they can not go further. The cures for obesity and Alzheimer's disease in mice have never been translated to humans, "says Buffenstein, a pioneer in efforts to use the naked mole rat (Heterocephalus glaber) as a model organization. And although lemurs will probably never replace more established primate models such as the macaque, Buffenstein is quite likely to expand the repertoire. "The more models there are, the better."

An act of faith

The mouse lemur project was started in the hot, dry summer of 2009, when Krasnow's teenage daughter, Maya, and her friends were about to work in her lab at Stanford. "We had asked Mark 1,000 times," says Camille Ezran, a 24-year-old medical student at the University of Rochester in New York. "Finally, he gave in." Ezran thought that she could follow a postdoctoral fellow for a few months. On the first day, Krasnow accused Ezran, Maya and their friend Jason Willick of finding a new genetic model body that is closer to human biology than the mouse (see "All in the family").

Students scoured the animal world by compiling a spreadsheet of features, including time to sexual maturity, litter size, and conservation status for each candidate. They considered the northern shrew (Tupaia Belangeri), the dwarf marmoset (Callithrix pygmaea) and other prosimian primates, including bush babies (Galagoides Demidoff) and the tarsier (Tarsius Tarsier). But the mouse lemur stood out. It has a gestation period of 2 months, reaches sexual maturity in 6 to 8 months and is one of the most fertile primates, with up to 4 offspring per litter. These factors make it possible to study several generations in just a few years. In contrast, the widely used macaque (Macacca Mulata) takes about 4 years to mature, is 5.5 months pregnant and has only one baby at a time.

Like all lemurs, mouse lemurs only live in Madagascar and some species are critically endangered. But common lemurs are so abundant that they can be studied easily in the wild, explains Ezran. For decades, field biologists implanted them with microchips and followed them throughout their 5 to 10 year life to study everything from voice communication to eating behavior. Large laboratory colonies show that animals can adapt well to life in captivity.

Krasnow has been sold. During the school holidays, with his students, he went on a lemur tour, consulted with primate specialists and visited lemur colonies in North Carolina and Paris before traveling to Madagascar in 2010. "We came back enthusiastic. The researchers welcomed us to their laboratories. Many would become our collaborators and mentors, "says Krasnow.

Krasnow learned that many researchers had never met. Thus, in 2011, with Megan Albertelli, a primate researcher and laboratory veterinarian at Stanford, organized the first-ever mouse lemur conference, held at the Janelia Research campus of the Howard Hughes Medical Institute in Ashburn, in Virginia. They brought together some 40 scientists, representatives of the 20 or so groups around the world who are studying lemurs, as well as human geneticists and model organists. People were enthusiastic, but there was a gap.

"We immediately understood that field biologists consider their research subjects differently than laboratory scientists," says Krasnow. Some of the powerful techniques used by laboratory scientists, such as genetic modification, were unthinkable for field biologists. "They have a passion for their mouse lemur subjects, as do other primatologists, an almost human link."

A scientific strategy has emerged which aims to use powerful genetic tools in a minimally invasive way. Researchers would work with animals in the wild, screen them for distinctive traits, and collect their DNA for further analysis. There would be no genetic modification or repatriation of animals to Stanford or other research institutes abroad. In addition to the periodic catches, the animals would reside in the forest. And the team would train Malagasy researchers to be part of the project. "We wanted to find a respectful way of doing genetics," says Krasnow.

Patricia Wright, a renowned lemur primatologist at Stony Brook University in New York, who founded and runs the ValBio Center Field Station, quickly became a key collaborator. By mid-2012, Krasnow and the students had brought equipment and supplies worth several hundred thousand dollars to equip a molecular biology laboratory. It's now their base.

Catch and release

Late in the afternoon in May, the Krasnow team goes to the rainforest to install traps. The expert tracker Victor Rasendry, who has been working with the lemurs of Ranomafana National Park for more than a decade, is giving advice on setting up the traps; the branches on which they are placed must be inclined to catch a mouse lemur. The researchers started about 30 traps with a banana slice before leaving. Around 9 pm, they come back.

It is very dark and researchers have only headlamps to pierce the darkness. Rasendry delicately manages from tree to tree, checking out the traps with the help of Haja Razafindrakoto and her husband, Mahery Razafindrakoto, two Malagasy biologists working on the project almost from the beginning. One after the other, they go up empty. Then, near the bottom of the hill, Haja enthusiastically calls out, "I have one." A few minutes later, another. One of them is Onja.

The team hoped to have more, but the weather was getting colder and the animals were entering a period of reduced activity called torpor. The team had planned to start field work earlier, but had to delay the trip due to a typhoon and an unprecedented outbreak of bubonic plague in the country.

In the ValBio Center laboratory, Pendleton goes to work. In addition to Onja, they captured a young man, Lahatra, whose name means something like fate. The weaker of the two, Lahatra draws a measly 300 grams on the grip-bar test. He is also a lot more cranky, twisting his head to bite Pendleton's thick gloves. He then lets out a low grunt. "He's restless and lets us know," says Pendleton.

Both animals have already been captured and have fleas that allow researchers to identify them immediately. Visitors for the first time would receive a more in-depth review, totaling about 70 scans, during their two hours in the lab. After the microchip of the animals, the team took a small sample of blood in the leg and collected a 2 millimeter ear punch – scientists are culturing mouse lemur skin cells to provide an immediate source of cells and blood. DNA. Then the animals would be subjected to a standard battery of tests.

Scientists check the hearing and sight of lemurs. They measure the base of the tail, where the fat is stored, and the width of the skull, a good indicator of age. They analyze the gait as the animals make their way through a transparent tube called Promenade du Prosimien, modified from a device used to measure children's walking habits. Drosophila fruit flies.

According to Krasnow, with such in-depth studies of natural variations, about one in five animals has an interesting or rather extreme trait: it can be as minor as eye color or as serious as cardiac arrhythmias. Until now, the team has identified more than 20 distinctive features. Relevant biomedical diseases include progressive eye diseases, morbid obesity, early signs of diabetes, and microcephaly, a smaller-than-normal-sized head.

The lemurs' personalities are also very clear, which is often reflected in the names that the researchers gave them. Many are docile and docile, but Feisty will attack. Murderface, another aggressive, lets out a strident, unusual cry. Blinky and Stoic are just as they sound. Pendleton particularly likes Alphy, a male they catch every year except the last. "He is an excellent client. I think he really likes bananas, "says Pendleton, although he's afraid he has not seen so much recently.

Back at Stanford, researchers are mapping the genes that underlie the distinctive traits of animals. Until now, they have identified how certain functionalities are grouped within families and they establish genealogies that will allow them to detect responsible mutations. The researchers focus first on a movement disorder that they call "stuttering," in which animals raise a hand immediately after contact with the ground. They also look at a sometimes fatal human disease, known as sinus syndrome, in which the natural pacemaker, the sinus node of the heart, beats much more slowly or more variably than normal .

Hopi Hoekstra, an evolutionary biologist at Harvard University in Cambridge, Mass., States that this idea of ​​capitalizing on natural variation "has proven incredibly powerful." Hoekstra works with wild mice, which she says are excellent models of human variation because, like humans, they are genetically different. And this variation is subject to natural selection in harsh and wild environments, she says.

The Krasnow team is now entering the second phase, a deep genomic screening designed to identify natural variants that make an inactive gene known as a loss of function mutation. To find the mutations, Krasnow and his group intend to sequence the genomes of each captured animal, not just those with distinctive traits, and compare them to the genome published in 2017. Krasnow hopes to achieve a living library of Animals in lost nonfunctional mutations of almost all 20,000 genes encoding the estimated proteins of the animal. This would be analogous to the International Mouse Phenotyping Consortium, a multicenter collaboration aimed at determining the function of each gene in the mouse genome by intentionally transforming them – by deleting them one by one.

"While others generate mutations, we spot them," says Krasnow. This approach is not only faster and cheaper, he says, but it also avoids the technical and ethical problems of genetic engineering in non-human primates. Researchers interested in the function of a specific gene could look for the mutation in the Krasnow library and determine if there are significant physiological traits in the animal. It is even possible that the Krasnow team could reconquer the animal, or his parents, for a follow-up study.

Buffenstein is everything for. "If Mark can exploit natural variability without doing genetic manipulation, it's fantastic."

High hopes

Krasnow is the first to admit that the lemur of the mouse will never enter the pantheon of established model organisms. But he has high hopes. He does not see the animal as a "boutique" model for studying a particular issue, but what he calls a "canonical" or versatile model, like the mouse, to explore many aspects of biology, diseases and ecology of primates. . Developing a model takes years. And while Krasnow's work is minimally invasive – just a tissue grain of atrial piercing and a blood sample of 200 microliters – any primate research is sensitive.

He thinks he'll know in a year or two if other biologists will accept it. "Once the community exchanges results, techniques and reagents, the more you know about this animal, the more likely it is that someone else will want to use it for the first time. use, "says Krasnow. That is why he is particularly excited about the collaboration of a million US dollars between Stanford and the Biohub Chan Zuckerberg for the creation of a single cell atlas.

The atlas was born from the unfortunate disappearance of Stumpy, one of the few old mouse lemurs who retired from a study conducted elsewhere. Stumpy was about 10 years old, the human equivalent of about 100 years old, when he contracted pneumonia. Aware of Stumpy's failing health, biochemist Stanford and Stanford co-chair Steve Quake organized a team of 52 experts to mobilize on the death of Stumpy. They quickly worked to separate the cells from each organ and perform the sequencing of unicellular RNA. The atlas obtained shows the set of genes expressed in 250,000 cells from 30 major organs. Quake says the researchers will make the data available this year before publishing an article about the project.

The atlas "will really turbocharge the effort of the mouse lemurs," he says. Judging by the atlas of mouse cells released last year, Quake predicts that researchers will use it "for all sorts of things"; for example, examine the relationships between tissues, explore the differences between men and women or find new drug targets.

"It would be an incredible resource," says Anne Yoder, an evolution biologist specializing in mouse lemurs at Duke University in Durham, North Carolina. Since many diseases are tissue-specific, an atlas can provide information on the genetics of disease risk and other aspects of health and biological function, she said. And when the atlas of human cells is complete, researchers will be able to probe the evolving roots of diseases.

Krasnow says the ultimate success will be whether the model can answer important questions that scientists did not think of deepening at the beginning of the project, such as the identification of genes that affect survival ability to deforestation or to climate change.

However, others warn that Krasnow's quest may not match his bold vision. Jeffrey Rogers of the Baylor College of Medicine in Houston, Texas, who, along with his colleagues, has sequenced the mouse lemur genome, describes the plan as "awesome". But he does not see researchers turning away from macaques or baboons, models of primates most used, or marmosets, which progress quickly, because we already know a lot about them. "Sometimes I think Mark is a bit disdainful of other primate models," says Rogers. "The idea that any model can be the model misses the point," he says.

And Daniel MacArthur of Broad Institute of MIT and Harvard in Cambridge, Massachusetts are skeptical of some of Krasnow's ambitions. MacArthur, who studies mutations of natural function loss in humans, "is very doubtful that the study of mouse lemur knockouts gives us a transformative insight into human biology." This is because important mutations leading to loss of function causing illness tend to be very rare, he says. "If you want to study natural knockouts, the best model is the man," he adds, because the sequence data available to more than 2 million people are already available. MacArthur says that although he is optimistic about the possibility of adding a new primate model to researchers' toolkits, he is concerned that even with a very conservative approach, the study of wild lemurs could disrupt these populations. .

And Krasnow is tied to the resistance of his peers. Buffenstein has worked tirelessly to develop the naked mole rat, a long-lived rodent that lives in underground colonies, as a model for research on aging and pain. She predicts that "Mark will have all the hurdles thrown on him. The sections of the study will say, "You do not have the most modern tools for the type of research you do. Why not work with mice?

But Krasnow is already looking for ways to expand his efforts in Madagascar. His goal is to "help transform" the teaching of biology in the country by getting students to explore what he calls the "living lab" right outside. Krasnow is considering a citizen-science project in which Madagascan high school students will select thousands or even hundreds of thousands of mouse lemurs in their own backyard, then sequence their genome, greatly expanding the databases. At first, he teaches mouse lemur genetics to postgraduate students in biology at the University of Antananarivo in Madagascar.

The team is also contacting conservation groups to explain its project and stress the importance of maintaining the lemur mouse habitat in the face of the ongoing destruction of Madagascar's environment. "Anyone working with lemurs is an advocate for the environment," says Yoder.

At the moment, lemurs are in full swing in Ranomafana National Park. At the end of May, at midnight, the rain forest is covered with fog as the team leaves the laboratory and climbs the slippery hill to release Onja and Lahatra. The researchers make a point of placing them on the same branches where they were caught. The animals hesitate before leaving their cages, as to find their way, then climb in the trees. For a moment, scientists can follow the bright eyes of these creatures before the lemurs finally disappear into the rainforest.