In the beginning was the phase separation

The question of the origin of life remains one of the oldest unanswered scientific questions. A team at the Technical University of Munich (TUM) has now shown for the first time that phase separation is an extremely efficient way of controlling the selection of chemical building blocks and providing advantages to certain molecules.

A team at the Technical University of Munich (TUM) has shown for the first time, that phase separation is an extremely
efficient way of controlling the selection of chemical building blocks, providing advantages to certain molecules.
This simple mechanism could have been decisive for the development of life. Left: clear solution; right: inside
 the tiny oil droplets instable molecules survive longer [Credit: Andreas Battenberg/TUM]

Life needs energy. Without energy, cells cannot move or divide, not even basic functions such as the production of simple proteins could be maintained. If energy is lacking, more complex connections disintegrate quickly, early life would die immediately.

Chemist Job Boekhoven and his team at TUM have now succeeded in using phase separation to find a mechanism in simple molecules that enables extremely unstable molecules such as those found in the primordial soup to have a higher degree of stability. They could survive longer, even if they had to survive a period without external energy supply.

The principle of simplicity

Job Boekhoven and his team were looking for a simple mechanism with primitive molecules that could produce life-like properties. "Most likely, molecules were simple in the primordial soup," says Boekhoven. The researchers investigated what happens when they "fed" various carboxylic acid molecules with high-energy carbodiimide condensing agents, thus bringing them out of equilibrium.

The reaction produces unstable anhydrides. In principle, these non-equilibrium products quickly disintegrate into carboxylic acids again. The scientists showed that the anhydrides that survived the longest were those that could form a kind of oil droplet in the aqueous environment.

Single droplets under a fluorescence microscope [Credit: Marta Tena-Solsona/TUM]

Molecules in the garage

The effect can also be seen externally: the initially clear solution becomes milky. The lack of water in the oil droplets is like a protection because anhydrides need water to disintegrate back into carboxylic acids.

Boekhoven explains the principle of phase separation with an analogy: "Imagine an old and rusty car: Leave it outside in the rain, and it continues to rust and decomposes because rusting is accelerated by water. Put it in the garage, and it stops rusting because you separate it from the rain."

In a way, a similar process occurs in the primordial soup experiment: Inside the oil droplet (garage) with the long-chain anhydride molecules there is no water, so its molecules survive longer. If the molecules compete with each other for energy, again those that can protect themselves by forming oil droplets are likelier to survive, while their competitors get hydrolyzed.

Next goal: viable information carriers

Since the mechanism of phase separation is so simple, it can possibly be extended to other types of molecular aggregations with life-like properties such as DNA, RNA or self-dividing vesicles. Studies have shown that these bubbles can divide spontaneously. "Soon we hope to turn primitive chemistry into a self-replicating information carrier that is protected from decay to a certain extent," says Boekhoven.

The study is published in Nature Communications.

Source: Technical University of Munich [May 23, 2018]

Why birds don't have teeth

Why did birds lose their teeth? Was it so they would be lighter in the air? Or are pointy beaks better for worm-eating than the jagged jaws of dinosaur ancestors?

New research suggests that birds gave up teeth to speed up egg hatching
[Credit: Yuri Kadobinov/AFP]

Actually, birds gave up teeth to speed up egg hatching, a research paper published in Biology Letters suggests, challenging long-held scientific views on the evolution of the toothless beak.

Compared to an incubation period of several months for dinosaur eggs, modern birds hatch after just a few days or weeks.

This is because there is no need to wait for the embryo to develop teeth -- a process that can consume 60 percent of egg incubation time, said researchers Tzu-Ruei Yang and Martin Sander from the University of Bonn.

While in the egg, the embryo is vulnerable to predators and natural disasters, and faster hatching boosts survival odds.

This would be a concern for dinos and birds -- all egg layers. In mammals, embryos are protected inside the mother.

"We suggest that (evolutionary) selection for tooth loss (in birds) was a side effect of selection for fast embryo growth and thus shorter incubation," Yang and Sander wrote in the journal Biology Letters.

Previous studies had concluded that birds -- living descendants of avian dinosaurs -- lost their teeth to improve flight.

Oviraptosaurs were omnivores but had a toothless beak [Credit: University of Nagoya,
Japan/Masato Hattori/AFP] 

But this did not explain why some non-avian dinosaurs in the Mesozoic era had independently evolved similar toothless beaks, said the duo.

Other studies had concluded that beaks were better for eating bird food.

But some dinosaurs with a very different, meat-eating diet had also discarded teeth in favour of pointed beaks.

Yang and Sander said their breakthrough came from a study published last year, which found that the eggs of non-flying dinosaurs took longer to hatch than previously thought -- about three to six months.

This was because of slow dental formation, which researchers analysed by examining growth lines -- almost like tree rings -- in the fossilised teeth of two dinosaur embryos.

Faster incubation would have been aided by early birds and some dinos taking to brooding their eggs in open nests rather than burying them as of old, said the research team.

They conceded their hypothesis was not consistent with toothlessness in turtles, which still have a long incubation period.

Source: AFP [May 23, 2018]

Far from special: Humanity's tiny DNA differences are 'average' in animal kingdom

Researchers report important new insights into evolution following a study of mitochondrial DNA from about 5 million specimens covering about 100,000 animal species.

Today's study, "Why should mitochondria define species?" published as an open-access article in the journal
Human Evolution,builds on earlier work by Drs. Stoeckle and Thayer, including an examination of the
mitochondrial genetic diversity of humans vs. our closest living and extinct relatives. The amount of
color variation within each red box of the Klee diagram illustrates the far greater mitochondrial
diversity among chimpanzees and bonobos than among living humans
[Credit: The Rockefeller University]

Mining "big data" insights from the world's fast-growing genetic databases and reviewing a large literature in evolutionary theory, researchers at The Rockefeller University in New York City and the Biozentrum at the University of Basel in Switzerland, published several conclusions today in the journal Human Evolution. Among them:

• - In genetic diversity terms, Earth's 7.6 billion humans are anything but special in the animal kingdom. The tiny average genetic difference in mitochondrial sequences between any two individual people on the planet is about the same as the average genetic difference between a pair of the world's house sparrows, pigeons or robins. The typical difference within a species, including humans, is 0.1% or 1 in 1,000 of the "letters" that make up a DNA sequence.

• - Genetic variation - the average difference in mitochondria DNA between two individuals of the same species - does not increase with population size. Because evolution is relentless, however, the lack of genetic variation offers insights into the timing of a species' emergence and its maintenance.

• - The mass of evidence supports the hypothesis that most species, be it a bird or a moth or a fish, like modern humans, arose recently and have not had time to develop a lot of genetic diversity. The 0.1% average genetic diversity within humanity today corresponds to the divergence of modern humans as a distinct species about 100,000 - 200,000 years ago - not very long in evolutionary terms. The same is likely true of over 90% of species on Earth today.

• - Genetically the world "is not a blurry place." Each species has its own specific mitochondrial sequence and other members of the same species are identical or tightly similar. The research shows that species are "islands in sequence space" with few intermediate "stepping stones" surviving the evolutionary process.

Among 1st "big data" insights from a growing collection of mitochondrial DNA

"DNA barcoding" is a quick, simple technique to identify species reliably through a short DNA sequence from a particular region of an organism. For animals, the preferred barcode regions are in mitochondria - cellular organelles that power all animal life.

The new study, "Why should mitochondria define species?" relies largely on the accumulation of more than 5 million mitochondrial barcodes from more than 100,000 animal species, assembled by scientists worldwide over the past 15 years in the open access GenBank database maintained by the US National Center for Biotechnology Information.

The researchers have made novel use of the collection to examine the range of genetic differences within animal species ranging from bumblebees to birds and reveal surprisingly minute genetic variation within most animal species, and very clear genetic distinction between a given species and all others.

"If a Martian landed on Earth and met a flock of pigeons and a crowd of humans, one would not seem more diverse than the other according to the basic measure of mitochondrial DNA," says Jesse Ausubel, Director of the Program for the Human Environment at The Rockefeller University, where the research was led by Senior Research Associate Mark Stoeckle and Research Associate David Thaler of the University of Basel, Switzerland.

The study results represent a surprise given predictions found in textbooks, and based on mathematical models of evolution,
 that the bigger the population of a species, the greater the genetic variation one expects to find. In fact, the mitochondrial
diversity within 7.6 billion humans or 500 million house sparrows or 100,000 sandpipers from around the world is about
 the same.The paper notes, however, that evolution is relentless, that species are always changing, and, therefore, the
degree of variation within a given species offers a clue into how long ago it emerged distinctly -- in other words,
 the older the species the greater the average genetic variation between its members
[Credit: The Rockefeller University]

"At a time when humans place so much emphasis on individual and group differences, maybe we should spend more time on the ways in which we resemble one another and the rest of the animal kingdom."

Says Dr. Stoeckle: "Culture, life experience and other things can make people very different but in terms of basic biology, we're like the birds."

"By determining the genetic variety within species of the animal kingdom, made possible only recently by the burgeoning number of DNA sequences, we've documented the absence of human exceptionalism."

Says. Dr. Thaler: "Our approach combines DNA barcodes, which are broad but not deep, from the entire animal kingdom with more detailed sequence information available for the entire mitochondrial genome of modern humans and a few other species. We analyzed DNA barcode sequences from thousands of modern humans in the same way as those from other animal species."

"One might have thought that, due to their high population numbers and wide geographic distribution, humans might have led to greater genetic diversity than other animal species," he adds. "At least for mitochondrial DNA, humans turn out to be low to average in genetic diversity."

"Experts have interpreted low genetic variation among living humans as a result of our recent expansion from a small population in which a sequence from one mother became the ancestor for all modern human mitochondrial sequences," says Dr. Thaler.

"Our paper strengthens the argument that the low variation in the mitochondrial DNA of modern humans also explains the similar low variation found in over 90% of living animal species - we all likely originated by similar processes and most animal species are likely young."

Genetic variation does not increase with population

The study results represent a surprise given predictions found in textbooks, and based on mathematical models of evolution, that the bigger the population of a species, the greater the genetic variation one expects to find.

"Is genetic diversity related to the size of the population?" asks Dr. Stoeckle. "The answer is no. The mitochondrial diversity within 7.6 billion humans or 500 million house sparrows or 100,000 sandpipers from around the world is about the same."

The paper notes, however, that evolution is relentless, that species are always changing, and, therefore, the degree of variation within a given species offers a clue into how long ago it emerged distinctly -- in other words, the older the species the greater the average genetic variation between its members.

Evolutionary bottlenecks: the fresh new beginning of a species

While asteroids and ice ages have played major roles in evolutionary history, scientists speculate that another great driver may have been the microbial world, notably viruses, which periodically cull populations, leaving behind only those able to survive the deadly challenge.

Genetically, 'the world is not a blurry place.' It is hard to find 'intermediates' -- the evolutionary stepping
stones between species. The intermediates disappear. The research is a new way to show that species
are 'islands in sequence space.' Each species has its own narrow, very specific consensus sequence,
just as our phone system has short, unique numeric codes to tell cities and countries apart
[Credit: The Rockefeller University]

"Life is fragile, susceptible to reductions in population from ice ages and other forms of environmental change, infections, predation, competition from other species and for limited resources, and interactions among these forces," says Dr. Thaler. Adds Dr. Thaler, "The similar sequence variation in many species suggests that all of animal life experiences pulses of growth and stasis or near extinction on similar time scales."

"Scholars have previously argued that 99% of all animal species that ever lived are now extinct. Our work suggests that most species of animals alive today are like humans, descendants of ancestors who emerged from small populations possibly with near-extinction events within the last few hundred thousand years."

'Islands in sequence space'

Another intriguing insight from the study, says Mr. Ausubel, is that "genetically, the world is not a blurry place. It is hard to find 'intermediates' - the evolutionary stepping stones between species. The intermediates disappear."

Dr. Thaler notes: "Darwin struggled to understand the absence of intermediates and his questions remain fruitful."

"The research is a new way to show that species are 'islands in sequence space.' Each species has its own narrow, very specific consensus sequence, just as our phone system has short, unique numeric codes to tell cities and countries apart."

Adds Dr. Thaler: "If individuals are stars, then species are galaxies. They are compact clusters in the vastness of empty sequence space."

The researchers say that with the bones or teeth of an ancient hominid, like those found in southern France or northern Spain, scientists might shed further light on the rate of evolution of the human species.

"It would be very exciting if over the next few years physical anthropologists and others were able to compare mitochondrial DNA from hominid species over the last 500,000 years," says Dr. Stoeckle.

Source: The Rockefeller University [May 21, 2018]

Missing ingredient to spark the fireworks of life discovered

Most people can name at least a few bones of the human body, but not many know about the cytoskeleton within our cells, let alone the "microtubules" that give it its shape. Now, a group of Princeton researchers has resolved a long-standing controversy by identifying exactly how the body creates these micron-sized filaments.

Credit: Princeton University

Using a novel imaging technique, Sabine Petry and the researchers in her lab were able to show that a protein called XMAP215, previously known only to help microtubules grow faster and longer, is necessary to creating the nucleus of each microtubule. Their work appears in the May issue of the journal Nature Cell Biology.

"Our study shows that microtubules in the cell are generated through cooperation between two molecules," said Akanksha Thawani, a fourth-year graduate student in Petry's lab who is the first author of the new paper. "XMAP215 functions together with a larger protein complex that forms ring shaped structures, gamma-tubulin ring complex (g-TuRC)."

"Microtubules are like the skeleton of the cell -- they give the cell its architecture," said Petry, an assistant professor of molecular biology and the senior author on the paper. "Beyond that, by positioning organelles, they can also serve as a highway for other components. Motor proteins can actually 'walk' along these microtubules. They really are fundamental to cell biology."

For 30 years, researchers in the field have known that the pillar-like microtubules are built of bricks called "tubulin" that grow from a tiny nucleus, and most agreed that g-tubulin was the only compound that could create that nucleus.

But there was a problem, said Petry. The few researchers who had succeeded in isolating g-TuRC found that when they put it in a test tube, it spectacularly underperformed at creating microtubule nuclei.

"Gamma-TuRC barely does anything," she said. "It nucleates a handful of microtubules, but it should make thousands."

Researchers have been puzzling over this for years, looking for some other factor that could activate or enhance g-TuRC. That search may now be over.

"The microtubule field has known [that g-tubulin] is not sufficient, and that other factors, that were not known, were also needed for full activity," said Eva Nogales, a professor of molecular and cell biology at the University of California-Berkeley who was not involved in this research. "The work by Petry and co-workers now shows that XMAP215, previously considered a microtubule 'polymerase' involved in microtubule elongation, acts synergistically with g-tubulin to promote efficient microtubule nucleation. The work provides an answer to this critical puzzle in our understanding of microtubule regulation in the cell."

These time-lapse videos show microtubules growing after the addition of XMAP215, the protein identified by researchers

 in Sabine Petry’s lab as a necessary component of microtubule nucleation. The different squares show the microtubules 

growing after adding different amounts of XMAP215. The observations are made in the “haystack” of the cell,

 revealing the “fireworks” of the rapidly growing microtubules. Elapsed time is shown in seconds 

and the white scale bar shows 10 μm [Credit: Princeton University]

Complicating their research was the difficulty in seeing the tiny structures at all, explained Thawani, a graduate student in in chemical and biological engineering. "The cell has tens of thousands of these polymers at any time, and our standard ways of observing these under a microscope do not allow for a good resolution," she said.

Petry used the classic metaphor of looking for a needle in a haystack, and she compared looking through a microscope with looking at the whole pile of hay. To solve the problem, she adapted a technique called total internal reflection fluorescence (TIRF) microscopy. Instead of illuminating the whole microscopic sample -- the whole haystack -- she instead imaged only a 100 nanometer-thick sliver. (For reference, a human hair is about 50,000 nanometers wide.)

"With TIRF microscopy, we don't illuminate anything outside that layer," she explained. "We don't see it. That's why we get a much higher signal-to-noise ratio: We don't see the other stuff that otherwise overlaps with the observation. So instead of seeing the haystack, which is still on top of it, we can see the needles against the glass. ... We can actually see the microtubules being born, we can see them grow, we can see what happens to them -- at the resolution of the needle."

A serendipitous find

Thawani hadn't set out to solve the mystery of why g-TuRC underperformed in test tubes. She had wanted to use TIRF microscopy to observe the growth of microtubules whose size she hoped to control with XMAP215, a protein known to encourage microtubule growth.

But instead of just growing longer microtubules, she saw that she was growing many more of them. "We added this protein, and it totally created a blast of microtubules," Thawani said. "That was one of the most mind-blowing bits in the whole process."

"The intention was just to study how these 'fireworks' form," said Petry. "So it was serendipity that Akanksha wanted to make them bigger, but then she saw -- 'Oh my gosh, there are more microtubules!' And the reason why she could see that more microtubules formed was that we have developed this imaging and extract capability. That's why no one else has seen it before."

Their team showed that the same phenomenon was seen in a test tube where g-TuRC generated microtubules together with XMAP215. This was chiefly the contribution of Rachel Kadzik, a postdoctoral researcher in Petry's lab and a co-first-author on the paper who purified the g-TuRC and, with Thawani, purified the XMAP215. G-TuRC is a 44-protein complex that has proven very difficult to purify and study in the 30 years since it was discovered.

Using the pure extractions of each protein, Petry's team was able to see that in a test tube, neither protein can nucleate microtubules without the other. "If we take XMAP215 out, not a single microtubule forms. If you take g-tubulin out, not a single microtubule forms," Petry said.

Their discovery required the combination of Thawani and Kadzik's experiments, viewed with Petry's TIRF microscopy imaging technique. "It was interesting," Petry said. "Rachel came from developmental biology, down to chemistry, and then Akanksha came from engineering up to biochemistry, so together, they were the super-team."

Source: Princeton University [May 16, 2018]

Scientists crack how primordial life on Earth might have replicated itself

Scientists have created a new type of genetic replication system which demonstrates how the first life on Earth - in the form of RNA - could have replicated itself. The scientists from the Medical Research Council (MRC) Laboratory of Molecular Biology say the new RNA utilises a system of genetic replication unlike any known to naturally occur on Earth today.

Liquid brine containing replicating RNA molecules is concentrated in the cracks between ice crystals,
as seen with an electron microscope [Credit: Philipp Holliger, MRC LMB]

A popular theory for the earliest stages of life on Earth is that it was founded on strands of RNA, a chemical cousin of DNA. Like DNA, RNA strands can carry genetic information using a code of four molecular letters (bases), but RNA can be more than a simple 'string' of information. Some RNA strands can also fold up into three-dimensional shapes that can form enzymes, called ribozymes, and carry out chemical reactions.

If a ribozyme could replicate folded RNA, it might be able to copy itself and support a simple living system.

Previously, scientists had developed ribozymes that could replicate straight strands of RNA, but if the RNA was folded it blocked the ribozyme from copying it. Since ribozymes themselves are folded RNAs, their own replication is blocked.

Now, in a paper published in the journal eLife, the scientists have resolved this paradox by engineering the first ribozyme that is able to replicate folded RNAs, including itself.

Normally when copying RNA, an enzyme would add single bases (C, G, A or U) one at a time, but the new ribozyme uses three bases joined together, as a 'triplet' (e.g. GAU). These triplet building blocks enable the ribozyme to copy folded RNA, because the triplets bind to the RNA much more strongly and cause it to unravel - so the new ribozyme can copy its own folded RNA strands.

The scientists say that the 'primordial soup' could have contained a mixture of bases in many lengths - one, two, three, four or more bases joined together - but they found that using strings of bases longer than a triplet made copying the RNA less accurate.

Dr Philipp Holliger, from the MRC Laboratory of Molecular Biology and senior author on the paper, said: "We found a solution to the RNA replication paradox by re-thinking how to approach the problem - we stopped trying to mimic existing biology and designed a completely new synthetic strategy. It is exciting that our RNA can now synthesise itself.

"These triplets of bases seem to represent a sweet spot, where we get a nice opening up of the folded RNA structures, but accuracy is still high. Notably, although triplets are not used in present-day biology for replication, protein synthesis by the ribosome - an ancient RNA machine thought to be a relic of early RNA-based life - proceeds using a triplet code.

"However, this is only a first step because our ribozyme still needs a lot of help from us to do replication. We provided a pure system, so the next step is to integrate this into the more complex substrate mixtures mimicking the primordial soup - this likely was a diverse chemical environment also containing a range of simple peptides and lipids that could have interacted with the RNA."

The experiments were conducted in ice at -7°C, because the researchers had previously discovered that freezing concentrates the RNA molecules in a liquid brine in tiny gaps between the ice crystals. This also is beneficial for the RNA enzymes, which are more stable and function better at cold temperatures.

Dr Holliger added: "This is completely new synthetic biology and there are many aspects of the system that we have not yet explored. We hope in future, it will also have some biotechnology applications, such as adding chemical modifications at specific positions to RNA polymers to study RNA epigenetics or augment the function of RNA."

Dr Nathan Richardson, Head of Molecular and Cellular Medicine at the MRC, said: "This is a really exciting example of blue skies research that has revealed important insights into how the very beginnings of life may have emerged from the 'primordial soup' some 3.7 billion years ago. Not only is this fascinating science, but understanding the minimal requirements for RNA replication and how these systems can be manipulated could offer exciting new strategies for treating human disease."

Source: Medical Research Council [May 15, 2018]

Darwin's finches - where did they actually come from?

In 1835, Charles Darwin visited the Galapagos Islands and discovered a group of birds that would shape his groundbreaking theory of natural selection. Darwin's Finches are now well-known as a textbook example of animal evolution. But just where did a species synonymous with the discovery of evolution come from? A new study from The Auk: Ornithological Advances presents some of the best models to date on where these birds actually originated.

Española cactus finch (Geospiza conirostris) [Credit: S. Taylor]

San Diego State University's Erik Funk and Kevin Burns set out to determine the ancestral biogeography - how a species' distribution varies over space and time - of Coerebinae. Coerebinae is a subfamily of birds called tanagers. This group includes the famous Darwin's Finches and their fourteen closest relatives. Using state-of-the-art statistical software, Funk and Burns modeled two competing hypotheses.

Both hypothesis models contained the same geographic area of the Galapagos, South America, and the Caribbean, but one model divided this area into more subregions than the other. The subregions were based on areas that shared similar plants and animals, such as the the Amazon or the Andes. When eight subregions were included in the model, the results indicated that the Caribbean, not the closer South American mainland, was more likely to be the origin of this bird group. However, the opposing model contains only five regions and indicates that the South American mainland is as likely as the Caribbean to be the home to Darwin's Finches' ancestors. The authors conclude that the current data suggest both potential origin sites are equally likely.

Funk says, "the results...were a bit surprising, because they suggested a dispersal pattern that was not necessarily the most 'straightforward' explanation for how these birds arrived in the Galápagos. I think one of the big take-away messages here is the possibility that biogeographic events, like dispersal, may not necessarily happen like logic tells us they should. Darwin's finches are such a highly studied group, and it is often taken for granted they arrived from mainland South America, but hopefully our results show readers that there is no more support for this hypothesis than there is for a Caribbean origin."

Funk and Burns suggested the successful colonization of the Galapagos Islands was a result of two traits. First, the finches' ancestors were more likely to wander than other species and consequently encountered islands more often. Second, these ancestors had a large amount of genetic variation in bill size and shape. This diversity in bill morphology allowed them to establish themselves and exploit their newfound niche. Better understanding the biogeography of Darwin's Finches allows scientists to learn how animals move, and how this affects their subsequent evolution and ability to adapt to new or changing environments.

"In 2018, we still have fundamental things to learn about one of the most studied and celebrated groups of birds, Darwin's Finches. Perhaps we should be calling them Darwin's Tanagers because it is Burns' tree of life for these birds, nesting them firmly in Tanagers, that is enabling new insights into the evolution, morphology, and origins of this remarkable group of birds. Funk and Burns use new biogeographic techniques in conjunction with recent phylogenies to explore the origins of Darwin's Finches," adds Shannon Hackett, Associate Curator in the Department of Zoology, and Head of the Field Museum's Bird Division at the Field Museum, who is an avian diversity and phylogeny expert who was not involved in the research.

Source: American Ornithological Society [May 09, 2018]

Stomata - the plant pores that give us life - arise thanks to a gene called MUTE

Plants know how to do a neat trick. Through photosynthesis, they use sunlight and carbon dioxide to make food, belching out the oxygen that we breathe as a byproduct. This evolutionary innovation is so central to plant identity that nearly all land plants use the same pores -- called stomata -- to take in carbon dioxide and release oxygen.

Close-up images of the epidermis of Arabidopsis. seedlings, taken using a microscope. (A) and (C): Seedlings with typical
arrangement of stomata across the surface. (B) and (D): Seedlings that artificially produce a lot of the MUTE protein,
 and have many stomata as a result. Scale bars are 50 micrometers [Credit: Soon-Ki Han/Xingyun Qi]

Stomata are tiny, microscopic and critical for photosynthesis. Thousands of them dot on the surface of the plants. Understanding how stomata form is critical basic information toward understanding how plants grow and produce the biomass upon which we thrive.

In a paper published in the journal Developmental Cell, a University of Washington-led team describes the delicate cellular symphony that produces tiny, functional stomata. The scientists discovered that a gene in plants known as MUTE orchestrates stomatal development. MUTE directs the activity of other genes that tell cells when to divide and not to divide -- much like how a conductor tells musicians when to play and when to stay silent.

"The MUTE gene acts as a master regulator of stomatal development," said senior author Keiko Torii, a UW professor of biology and investigator at the Howard Hughes Medical Institute. "MUTE exerts precision control over the proper formation of stomata by initiating a single round of cell division -- just one -- in the precursor cell that stomata develop from."

Stomata resemble doughnuts -- a circular pore with a hole in the middle for gas to enter or leave the plant. The pore consists of two cells -- each known as a guard cell. They can swell or shrink to open or close the pore, which is critical for regulating gas exchange for photosynthesis, as well as moisture levels in tissues.

"If plants cannot make stomata, they are not viable -- they cannot 'breathe,'" said Torii, who also is a professor at Nagoya University in Japan.

Without MUTE, Arabidopsis plants cannot produce stomata, and do not develop past the seedling stage
[Credit: Soon-Ki Han/ Xingyun Qi]

Torii and her team investigated which genes governed stomata formation in Arabidopsis thaliana, a small weed that is one of the most widely studied plants on the planet. Past research by Torii's team and other researchers had indicated that, in Arabidopsis, MUTE plays a central role in the formation of stomata. The MUTE gene encodes instructions for a cellular protein that can control the "on" or "off" state of other plant genes.

The researchers created a strain of Arabidopsis that can artificially produce a lot of the MUTE protein, so they could easily identify which genes the MUTE protein turned on or off. They discovered that many of the activated genes control cell division -- a process that is critical for stomatal development.

In Arabidopsis, as in nearly all plants, stomata form from precursor cells known as guard mother cells, or GMCs. To form a working stoma -- singular for stomata -- a GMC divides once to yield to paired guard cells. Since their data showed that MUTE proteins switched on genes that regulated cell division, Torii and her team wondered if MUTE is the gene that activates this single round of cell division. If so, it would have to be a tightly regulated process. The genetic program would have to switch on cell division in the GMC, and then quickly switch it right back off to ensure that only a single round of division occurs.

Torii's team showed that one of the genes activated by the MUTE protein to its DNA is CYCD5;1, a gene that causes the GMC to divide. The researchers also found that MUTE proteins turn on two genes called FAMA and FOUR LIPS. This was an important discovery because, while CYCD5;1 turns on cell division of the GMC, FAMA and FOUR LIPS turn off -- or repress -- the cell division program.

"Our experiments showed that MUTE was turning on both activators of cell division and repressors of cell division, which seemed counterintuitive -- why would it do both?" said Torii. "That made us very interested in understanding the temporal regulation of these genes in the GMC and the stomata."

MUTE is a master regulator of the development of stomata in Arabidopsis
[Credit: Keiko Torii and her daughter Erika]

Through precise experiments, they gathered data on the timing MUTE activation of these cell division activators and repressors. They incorporated this information into a mathematical model, which simulated how MUTE acts to both activate and repress cell division in the GMC. First, MUTE turns on the activator CYCD5;1 -- which triggers one round of cell division. Then, FAMA and FOUR LIPS act to prevent further cell division, yielding one functional stomata consisting of two guard cells.

"Like a conductor at the podium, MUTE appears to signal its target genes -- each of which has specific, and even opposite, parts to play in the ensuing piece," said Torii. "The result is a tightly coupled sequence of activation and repression that gives rise to one of the most ancient structures on land plants."

Author: James Urton | Source: University of Washington [May 07, 2018]

Understanding how DNA is selectively tagged with 'do not use' marks

Not all of your genome needs to be active at any given time. Some regions are prone to hopping around the genome in problematic ways if left unchecked; others code for genes that need to be turned off in certain cells or at certain times. One way that cells keep these genetic elements under control is with the chemical equivalent of a "do not use" sign. This chemical signal, called DNA methylation, is known to vary in different cell types or at different stages of cellular development, but the details of how cells regulate exactly where to put DNA methylation marks have remained unclear.

Salk Assistant Professor Julie Law and Research Associate Ming Zhou, pictured with their
Arabidopsis thaliana plants in a Salk greenhouse [Credit: Salk Institute]

Salk scientists studying plants discovered a small family of proteins that control where in the genome DNA methylation marks are added. Their work on this aspect of genetic regulation is highly relevant for processes that range from normal development to cellular defects and diseases, which can arise due to erroneous DNA methylation patterns in plants and/or humans, respectively. Their paper is published in Nature Genetics.

"If we want to understand how differences in DNA methylation patterns can cause developmental defects in plants, or diseases like cancer in humans, we need to understand how DNA methylation is targeted to specific regions of the genome under normal conditions," says Salk Assistant Professor Julie Law, senior author of the paper. "Until now, factors able to control methylation in such a precise manner have been elusive."

Law studies an easy-to-grow weed, Arabidopsis thaliana, the first plant to have its genome sequenced. In the ensuing years, scientists, including Law, have been working to characterize and understand the plant's DNA methylation patterns, which affect gene activity without changing the DNA code itself. This process is similar in plants and animals, but investigating DNA methylation in Arabidopsis is much easier because plants can tolerate methylation defects better than animals, where global changes in methylation are often lethal.

Law was interested in understanding how the pathways that control DNA methylation are regulated not only to control global patterns of methylation but also to enable the regulation of individual regions--a critical step in generating different patterns of DNA methylation within a given organism.

Previously, it was known that a protein complex called RNA polymerase IV (Pol-IV) played a global role in establishing DNA methylation patterns. This polymerase makes small molecular messages called siRNAs that act like a molecular GPS system, indicating all the locations within the genome where methylation should be targeted. However, how this polymerase might be regulated to control DNA methylation at individual genomic locations was unclear.

To address this question, Law's lab used a combined genetic-genomic approach to investigate the functions of four related proteins, the CLASSY family, that they thought might regulate Pol-IV. It turned out that disruption of each CLASSY gene resulted in different sets of genomic regions--in different locations--losing their siRNA signals, resulting in reduced DNA methylation levels. More dramatically, when all four CLASSY genes were disrupted, the siRNA signals and DNA methylation were lost throughout the entire genome.

"In the CLASSY quadruple mutants, the Pol-IV signal completely disappears--essentially no siRNAs are made," says Ming Zhou, a Salk research associate and the paper's first author. "This is very strong evidence that CLASSYs are required for Pol-IV function."

When Law's team probed further, they discovered that the DNA methylation defects in the CLASSY mutants caused some genes to be erroneously turned on and resulted in global decreases in methylation at mobile DNA elements, increasing their potential to move around and disrupt essential gene activity.

"The CLASSYs are a part of a large superfamily that is common to both plants and animals," adds Law, who holds the Hearst Foundation Development Chair. "We hope that by understanding how specific methylation patterns are generated in plants, we can provide insights into how DNA methylation is regulated in other organisms."

Knowledge of this mechanism for regulating DNA methylation could help scientists develop strategies for correcting epigenetic defects that are associated with reduced yields in crops, or diseases--such as cancer--in humans. In the future, the lab is interested in exploring how DNA methylation patterns are controlled during development and in response to the environment.

Source: Salk Institute [May 07, 2018]

Tracing cerebral cortex evolution

Our cerebral cortex, a sheet of neurons, connections and circuits, comprises “ancient” regions such as the hippocampus and “new” areas such as the six-layered “neocortex”, found only in mammals and most prominently in humans. But when in evolution did the components of cerebral cortex arise and how did they evolve? Scientists at the Max Planck Institute for Brain Research in Frankfurt am Main studied gene expression in the neurons of the cortex of turtles and lizards, and found unexpected similarities and differences with the mammalian cortex. These results are a milestone towards reconstructing the evolution of the vertebrate brain.

Snapshot of the turtle three-layered cortex (left) and distinct types of neurons in the turtle dorsal cortex (right).
The neurons are labeled with fluorescent in situ hybridization for two genes expressed
in the two neuronal types [Credit: MPI f. Brain Research]

We are, in many ways, our cerebral cortex. Its circuits serve to shape our perception of the world, store our memories and plan our behavior. A cerebral cortex, with its typical layered organization, is found only among mammals, including humans, and non-avian reptiles such as lizards and turtles. Mammals, reptiles and birds originate from a common ancestor that lived some 320 million years ago. Neuroscientists believe that this ancestor had a small cortex with three layers, because a similar structure is found today in the hippocampus of mammals and in all cortices of modern reptiles: these three-layered cortices likely correspond to their common ancestral cortex.

By comparing the cortex of today’s reptiles to the old and new cortices of today’s mammals (such as hippocampus and neocortex, respectively), we can search for similarities, potential ancestral traits, and differences – resulting from their independent evolutions – and thus reconstruct the main features of cortical evolution. Comparisons were, until now, based on developmental and anatomical features. This new study, based on the molecular characterization of individual reptilian neurons, provides unprecedented data to help reconstruct cortex evolution.

For decades, the anatomical differences between reptilian and mammalian brains have fueled many disputes about cortical evolution. People argued on whether this part of the reptilian brain corresponds to that part of the mammalian brain, or whether the many layers found in mammalian neocortex actually exist also in reptiles, but in a form that is not detectable with traditional methods. Gilles Laurent and his group at the Max Planck Institute for Brain Research took a different approach and focused on the molecular characterization of the myriad neuronal types that make up cortical circuits.

Transcriptome sequenced

Neuronal “types” differ, among others, by their morphology, neurotransmitters, connections and functional properties. These features all result from the expression of different sets of genes; hence individual neurons can be classified (or typed) by measuring the messenger RNA molecules they contain (their “transcriptome”). Maria Antonietta Tosches, the first author of this study, and her colleagues sequenced the transcriptomes of turtle and lizard cells after capturing them, one by one, in microscopic water droplets using specialized microfluidics platforms.

Using these gene expression profiles, the scientists could categorize thousands of neurons. From each type they could identify diagnostic marker genes, and use them to assess the position of the cell types in the brain. Imagine a picture of the cortex, uniform until then, suddenly transformed into a collage of colored zones, with each zone containing one or several characteristic cell types.

The authors could now compare reptilian molecular maps to those of mammalian brains directly, find one-to-one correspondences and even draw hypotheses about the brain of their common ancestor of 320 million years ago (now extinct).

Forced to fold

“Our results tremendously clarify our understanding of the reptilian brain and thus, of brain evolution”, Tosches says. These new molecular maps show, for example, that reptiles have neuron types that correspond to those found in the mammalian hippocampus, a structure involved in spatial orientation and in the formation of memories. In reptiles, the hippocampus is found towards the center of the brain, but unlike its folded-up mammalian counterpart, looks like a single sheet. “It is as if, in early mammals, the ancestral hippocampus had been pushed by an increasingly dominant neocortex and forced to fold onto itself, to acquire its signature mammalian architecture”, Laurent adds.

The non-hippocampal reptilian cortex, by contrast, revealed the intricate history of mammalian neocortex. Inhibitory neurons, for example, express similar sets of genes in reptiles and mammals, indicating a common ancestry. Excitatory neurons, however, differ substantially across these two groups. “The mammalian six-layered neocortex is a fascinating mosaic of ancient and new neuronal types”, says Tosches. The scientists can now point to the true novelty of the mammalian neocortex, that is, the emergence of new types of excitatory neurons after profound changes of gene expression programs.

This study opens up many new questions. Do ancient neuronal types have the same functions in reptilian and mammalian cortical circuits? And can these molecular similarities and differences inform us on the evolution of brain function and animal behavior? “There is a lot more to explore from these new molecular maps” says Laurent: “this is only the beginning”.

The findings are published in Science.

Source: Max Planck Society [May 04, 2018]