Research Area: Stem Cell and Developmental Biology

Single-cell database to propel biological studies

Whitehead team analyzes transcriptomes for roughly 70,000 cells in planarians, creates publicly available database to drive further research.

Nicole Davis | Whitehead Institute
April 20, 2018

A team at Whitehead Institute and MIT has harnessed single-cell technologies to analyze over 65,000 cells from the regenerative planarian flatworm, Schmidtea mediterranea, revealing the complete suite of actives genes (or “transcriptome”) for practically every type of cell in a complete organism. This transcriptome atlas represents a treasure trove of biological information on planarians, which is the subject of intense study in part because of its unique ability to regrow lost or damaged body parts. As described in the April 19 advance online issue of the journal Science, this new, publicly available resource has already fueled important discoveries, including the identification of novel planarian cell types, the characterization of key transition states as cells mature from one type to another, and the identity of new genes that could impart positional cues from muscles cells — a critical component of tissue regeneration.

“We’re really at the beginning of an amazing era,” says senior author Peter Reddien, a member of Whitehead Institute, professor of biology at MIT, and investigator with the Howard Hughes Medical Institute. “Just as genome sequences became indispensable resources for studying the biology of countless organisms, analyzing the transcriptomes of every cell type will become another fundamental tool — not just for planarians, but for many different organisms.”

The ability to systematically reveal which genes in the genome are active within an individual cell flows from a critical technology known as single-cell RNA sequencing. Recent advances in the technique have dramatically reduced the per-cell cost, making it feasible for a single laboratory to analyze a suitably large number of cells to capture the cell type diversity present in complex, multi-cellular organisms.

Reddien has maintained a careful eye on the technology from its earliest days because he believed it offered an ideal way to unravel planarian biology. “Planarians are relatively simple, so it would be theoretically possible for us to capture every cell type. Yet they still have a sufficiently large number of cells — including types we know little or even nothing about,” he explains. “And because of the unusual aspects of planarian biology — essentially, adults maintain developmental information and progenitor cells that in other organisms might be present transiently only in embryos — we could capture information about mature cells, progenitor cells, and information guiding cell decisions by sampling just one stage, the adult.”

Two and a half years ago, Reddien and his colleagues — led by first author Christopher Fincher, a graduate student in Reddien’s laboratory — set out to apply single-cell RNA sequencing systematically to planarians. The group isolated individual cells from five regions of the animal and gathered data from a total of 66,783 cells. The results include transcriptomes for rare cell types, such as those that comprise on the order of 10 cells out of an adult animal that consists of roughly 500,000 to 1 million cells.

In addition, the researchers uncovered some cell types that have yet to be described in planarians, as well cell types common to many organisms, making the atlas a valuable tool across the scientific community. “We identified many cells that were present widely throughout the animal, but had not been previously identified. This surprising finding highlights the great value of this approach in identifying new cells, a method that could be applied widely to many understudied organisms,” Fincher says.

“One main important aspect of our transcriptome atlas is its utility for the scientific community,” Reddien says. “Because many of the cell types present in planarians emerged long ago in evolution, similar cells still exist today in various organisms across the planet. That means these cell types and the genes active within them can be studied using this resource.”

The Whitehead team also conducted some preliminary analyses of their atlas, which they’ve dubbed “Planarian Digiworm.” For example, they were able to discern in the transcriptome data a variety of transition states that reflect the progression of stem cells into more specialized, differentiated cell types. Some of these cellular transition states have been previously analyzed in painstaking detail, thereby providing an important validation of the team’s approach.

In addition, Reddien and his colleagues knew from their own prior, extensive research that there is positional information encoded in adult planarian muscle — information that is required not only for the general maintenance of adult tissues but also for the regeneration of lost or damaged tissue. Based on the activity pattern of known genes, they could determine roughly which positions the cells had occupied in the intact animal, and then sort through those cells’ transcriptomes to identify new genes that are candidates for transmitting positional information.

“There are an unlimited number of directions that can now be taken with these data,” Reddien says. “We plan to extend our initial work, using further single-cell analyses, and also to mine the transcriptome atlas for addressing important questions in regenerative biology. We hope many other investigators find this to be a very valuable resource, too.”

This work was supported by the National Institutes of Health, the Howard Hughes Medical Institute, and the Eleanor Schwartz Charitable Foundation.

Study suggests method for boosting growth of blood vessels and muscle

Activating proteins linked to longevity may help to increase endurance and combat frailty in the elderly.

Anne Trafton | MIT News Office
March 22, 2018

As we get older, our endurance declines, in part because our blood vessels lose some of their capacity to deliver oxygen and nutrients to muscle tissue. An MIT-led research team has now found that it can reverse this age-related endurance loss in mice by treating them with a compound that promotes new blood vessel growth.

The study found that the compound, which re-activates longevity-linked proteins called sirtuins, promotes the growth of blood vessels and muscle, boosting the endurance of elderly mice by up to 80 percent.

If the findings translate to humans, this restoration of muscle mass could help to combat some of the effects of age-related frailty, which often lead to osteoporosis and other debilitating conditions.

“We’ll have to see if this plays out in people, but you may actually be able to rescue muscle mass in an aging population by this kind of intervention,” says Leonard Guarente, the Novartis Professor of Biology at MIT and one of the senior authors of the study. “There’s a lot of crosstalk between muscle and bone, so losing muscle mass ultimately can lead to loss of bone, osteoporosis, and frailty, which is a major problem in aging.”

The first author of the paper, which appears in Cell on March 22, is Abhirup Das, a former postdoc in Guarente’s lab who is now at the University of New South Wales in Australia. Other senior authors of the paper are David Sinclair, a professor at Harvard Medical School and the University of New South Wales, and Zolt Arany, a professor at the University of Pennsylvania.

Race against time

In the early 1990s, Guarente discovered that sirtuins, a class of proteins found in nearly all animals, protect against the effects of aging in yeast. Since then, similar effects have been seen in many other organisms.

In their latest study, Guarente and his colleagues decided to explore the role of sirtuins in endothelial cells, which line the inside of blood vessels. To do that, they deleted the gene for SIRT1, which encodes the major mammalian sirtuin, in endothelial cells of mice. They found that at 6 months of age, these mice had reduced capillary density and could run only half as far as normal 6-month-old mice.

The researchers then decided to see what would happen if they boosted sirtuin levels in normal mice as they aged. They treated the mice with a compound called NMN, which is a precursor to NAD, a coenzyme that activates SIRT1. NAD levels normally drop as animals age, which is believed to be caused by a combination of reduced NAD production and faster NAD degradation.

After 18-month-old mice were treated with NMN for two months, their capillary density was restored to levels typically seen in young mice, and they experienced a 56 to 80 percent improvement in endurance. Beneficial effects were also seen in mice up to 32 months of age (comparable to humans in their 80s).

“In normal aging, the number of blood vessels goes down, so you lose the capacity to deliver nutrients and oxygen to tissues like muscle, and that contributes to decline,” Guarente says. “The effect of the precursors that boost NAD is to counteract the decline that occurs with normal aging, to reactivate SIRT1, and to restore function in endothelial cells to give rise to more blood vessels.”

These effects were enhanced when the researchers treated the mice with both NMN and hydrogen sulfide, another sirtuin activator.

Vittorio Sartorelli, a principal investigator at the National Institute of Allergy and Infectious Diseases who was not involved in the research, described the experiments as “elegant and compelling.” He added that “it will be of interest and of clinical relevance to evaluate the effect of NMN and hydrogen sulfide on the vascularization of other organs such as the heart and brain, which are often damaged by acutely or chronically reduced blood flow.”

Benefits of exercise

The researchers also found that SIRT1 activity in endothelial cells is critical for the beneficial effects of exercise in young mice. In mice, exercise generally stimulates growth of new blood vessels and boosts muscle mass. However, when the researchers knocked out SIRT1 in endothelial cells of 10-month-old mice, then put them on a four-week treadmill running program, they found that the exercise did not produce the same gains seen in normal 10-month-old mice on the same training plan.

If validated in humans, the findings would suggest that boosting sirtuin levels may help older people retain their muscle mass with exercise, Guarente says. Studies in humans have shown that age-related muscle loss can be partially staved off with exercise, especially weight training.

“What this paper would suggest is that you may actually be able to rescue muscle mass in an aging population by this kind of intervention with an NAD precursor,” Guarente says.

In 2014, Guarente started a company called Elysium Health, which sells a dietary supplement containing a different precursor of NAD, known as NR, as well as a compound called pterostilbene, which is an activator of SIRT1.

The research was funded by the Glenn Foundation for Medical Research, the Sinclair Gift Fund, a gift from Edward Schulak, and the National Institutes of Health.

A blueprint for regeneration

Whitehead Institute researchers uncover framework for how stem cells determine where to form replacement structures.

Lisa Girard | Whitehead Institute
March 15, 2018

Researchers at Whitehead Institute have uncovered a framework for regeneration that may explain and predict how stem cells in adult, regenerating tissue determine where to form replacement structures.

In a paper that appeared online March 15 in the journal Science, the researchers describe a model for planarian (flatworm) eye regeneration that is governed by three principles acting in concert, which inform how progenitor cells behave in regeneration. The model invokes positional cues that create a scalable map; self-organization that attracts progenitors to existing structures; and progenitor cells that originate in a diffuse spatial zone, rather than a precise location, allowing flexibility in their path. These principles appear to dictate how progenitor cells decide where to go during regeneration to recreate form and function, and they bring us closer to a systems-level understanding of the process.

From previous work, the researchers knew that stem cells are likely reading out instructions from neighboring tissues to guide their path, and it became clear that the process faces some serious challenges in regeneration. “We realized that positional information has to move; it needs to change during regeneration in order to specify the new missing parts to be regenerated. This revised information can then guide progenitor cells that are choosing to make new structures to differentiate into the correct anatomy at the correct locations,” says the paper’s senior author Peter Reddien, a Whitehead Institute researcher, an MIT professor of biology, and a Howard Hughes Medical Institute (HHMI) investigator. “There is a puzzle that emerges, however. Since positional information shifts after injury during regeneration, there is a mismatch between the positional information pattern and the remaining anatomy pattern. Realizing this mismatch exists was a trigger for our study. We wanted to understand how stem cells making particular tissues decide where to go and differentiate. Is it based on anatomy, or is it based on positional information? And when those two things are not aligned, how do they decide?”

Reddien and his lab have spent over a decade unraveling the mysteries of regeneration using a small flatworm, called the planarian. If a planarian’s head is amputated, or its side is removed, each piece will regenerate an entire animal. In order to understand how progenitors decide where to go in the noisy environment of animal regeneration, the researchers used the planarian eye, a visible organ that is small enough to be removed without serious injury and has the added advantage of having defined progenitor cell molecular markers.

The researchers devised a simple experiment to resolve the question of how the progenitors decide where to go: amputate the animal’s head, then after three days remove one of the eyes from the head piece. What they found was that progenitor cells would nucleate a new eye in a position anterior (closer to the tip) to the remaining eye, rather than in the “correct” position specified by the anatomy, symmetrical with the current eye. However, if the same experiment is done, except with one of the eyes removed from the head piece earlier — the same day as the amputation, rather than three days later — there is a different result: The new eye is nucleated at a position symmetrical to the remaining eye, at the “correct” position according to the anatomy, suggesting that when the choices are conflicting, anatomical self-organizing dynamics win.

These simple rules guide the system to successful regeneration and also yield surprising outcomes when the system is pushed to its limits, producing alternative stable anatomical states with three-, four-, and five-eyed animals. Resecting both the side of an animal and amputating the head can place progenitors far enough from existing, self-organizing attractors that they miss them, allowing them to nucleate a new attractor — a third eye — in the head fragments. “If the migrating progenitors are too close to the attractor that is the existing eye, it will suck them in, just like a black hole; if they are far enough, they can escape,” says Kutay Deniz Atabay, first author on the paper and a graduate student in the Reddien lab.

When the researchers did the same surgery on a three-eyed animal, the progenitors miss the attraction of existing eyes and form a five-eyed animal. In each of these cases, all of the eyes are functionally integrated into the brain and exhibit the same light-avoidance response of the planarian eye. The researchers also observed that these alternative anatomical states endure, revealing rules for maintenance of existing and alternative anatomical structures. “This recipe helps provide an explanation for a fundamental problem of animal regeneration, which is how migratory progenitors make choices that lead the system to regenerate and maintain organs,” Atabay says.

“These studies contribute to a proposed framework for regeneration in which competing forces, self-organization, and extrinsic cues, are the guideposts impacting the choice of progenitor targeting in regeneration; and those two forces together determine the outcome,” Reddien says.

This work was supported by the National Institutes of Health (NIH) and the HHMI. Additional funding was provided by the MIT Presidential Fellowship Program and a National Defense Science and Engineering Graduate Fellowship.

Study: Fragile X syndrome neurons can be restored

Whitehead Institute researchers are using a modified CRISPR/Cas9-guided activation strategy to investigate the most frequent cause of intellectual disability in males.

Nicole Giese Rura | Whitehead Institute
February 15, 2018

Fragile X syndrome is the most frequent cause of intellectual disability in males, affecting one out of every 3,600 boys born. The syndrome can also cause autistic traits, such as social and communication deficits, as well as attention problems and hyperactivity. Currently, there is no cure for this disorder.

Fragile X syndrome is caused by mutations in the FMR1 gene on the X chromosome, which prevent the gene’s expression. This absence of the FMR1-encoded protein during brain development has been shown to cause the overexcitability in neurons associated with the syndrome. Now, for the first time, researchers at Whitehead Institute have restored activity to the fragile X syndrome gene in affected neurons using a modified CRISPR/Cas9 system they developed that removes the methylation — the molecular tags that keep the mutant gene shut off — suggesting that this method may prove to be a useful paradigm for targeting diseases caused by abnormal methylation.

Research by the lab of Whitehead Institute for Biomedical Research Founding Member Rudolf Jaenisch, which is described online this week in the journal Cell, is the first direct evidence that removing the methylation from a specific segment within the FMR1 locus can reactivate the gene and rescue fragile X syndrome neurons.

The FMR1 gene sequence includes a series of three nucleotide (CGG) repeats, and the length of these repeats determines whether or not a person will develop fragile X syndrome: A normal version of the gene contains anywhere from 5 to 55 CGG repeats, versions with 56 to 200 repeats are considered to be at a higher risk of generating some of the syndrome’s symptoms, and those versions with more than 200 repeats will produce fragile X syndrome.

Until now, the mechanism linking the excessive repeats in FMR1 to fragile X syndrome was not well-understood. But Shawn Liu, a postdoc in Jaenisch’s lab and first author of the Cell study, and others thought that the methylation blanketing those nucleotide repeats might play an important role in shutting down the gene’s expression.

In order to test this hypothesis, Liu removed the methylation tags from the FMR1 repeats using a CRISPR/Cas9-based technique he recently developed with Hao Wu, a postdoc in the Jaenisch lab. This technique can either add or delete methylation tags from specific stretches of DNA. Removal of the tags revived the FMR1 gene’s expression to the level of the normal gene.

“These results are quite surprising — this work produced almost a full restoration of wild type expression levels of the FMR1 gene,” says Jaenisch, whose primary affiliation is with Whitehead Institute, where his laboratory is located and his research is conducted. He is also a professor of biology at MIT. “Often when scientists test therapeutic interventions, they only achieve partial restoration, so these results are substantial,” he says.

The reactivated FMR1 gene rescues neurons derived from fragile X syndrome induced pluripotent stem (iPS) cells, reversing the abnormal electrical activity associated with the syndrome. When rescued neurons were engrafted into the brains of mice, the FMR1 gene remained active in the neurons for at least three months, suggesting that the corrected methylation may be sustainable in the animal.

“We showed that this disorder is reversible at the neuron level,” says Liu. “When we removed methylation of CGG repeats in the neurons derived from fragile X syndrome iPS cells, we achieved full activation of FMR1.”

The CRISPR/Cas-9-based technique may also prove useful for other diseases caused by abnormal methylation including facioscapulohumeral muscular dystrophy and imprinting diseases.

“This work validates the approach of targeting the methylation on genes, and it will be a paradigm for scientists to follow this approach for other diseases,” says Jaenisch.

This work was supported by the National Institutes of Health, the Damon Runyon Cancer Foundation, the Rett Syndrome Research Trust, the Brain and Behavior Research Foundation, and the Helen Hay Whitney Foundation. Jaenisch is co-founder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.

How some facial malformations arise

Study explains why mutations that would seemingly affect all cells lead to face-specific birth defects.

Anne Trafton | MIT News Office
January 24, 2018

About 1 in 750 babies born in the United States has some kind of craniofacial malformation, accounting for about one-third of all birth defects.

Many of these craniofacial disorders arise from mutations of “housekeeping” genes, so called because they are required for basic functions such as building proteins or copying DNA. All cells in the body require these housekeeping genes, so scientists have long wondered why these mutations would produce defects specifically in facial tissues.

Researchers at MIT and Stanford University have now discovered how one such mutation leads to the facial malformations seen in Treacher-Collins Syndrome, a disorder that affects between 1 in 25,000 and 1 in 50,000 babies and produces underdeveloped facial bones, especially in the jaw and cheek.

The team found that embryonic cells that form the face are more sensitive to the mutation because they more readily activate a pathway that induces cell death in response to stress. This pathway is mediated by a protein called p53. The new findings mark the first time that scientists have determined how mutations in housekeeping genes can have tissue-specific effects during embryonic development.

“We were able to narrow down, at the molecular level, how issues with general regulators that are used to make ribosomes in all cells lead to defects in specific cell types,” says Eliezer Calo, an MIT assistant professor of biology and the lead author of the study.

Joanna Wysocka, a professor of chemical and systems biology at Stanford University, is the senior author of the study, which appears in the Jan. 24 online edition of Nature.

From mutation to disease

Treacher-Collins Syndrome is caused by mutations in genes that code for proteins required for the assembly and function of polymerases. These proteins, known as TCOF1, POLR1C, and POLR1D, are responsible for transcribing genes that make up cell organelles called ribosomes. Ribosomes are critical to all cells.

“The question we were trying to understand is, how is it that when all cells in the body need ribosomes to function, mutations in components that are required for making the ribosomes lead to craniofacial disorders? In these conditions, you would expect that all the cell types of the body would be equally affected, but that’s not the case,” Calo says.

During embryonic development, these mutations specifically affect a type of embryonic cells known as cranial neural crest cells, which form the face. The researchers already knew that the mutations disrupt the formation of ribosomes, but they didn’t know exactly how this happens. To investigate that process, the researchers engineered larvae of zebrafish and of an aquatic frog known as Xenopus to express proteins harboring those mutations.

Their experiments revealed that the mutations lead to impairment in the function of an enzyme called DDX21. When DDX21 dissociates from DNA, the genes that encode ribosomal proteins do not get transcribed, so ribosomes are missing key components and can’t function normally. However, this DDX21 loss only appears to happen in cells that are highly sensitive to p53 activation, including cranial neural crest cells. These cells then undergo programmed cell death, which leads to the facial malformations seen in Treacher-Collins Syndrome, Calo says.

Other embryonic cells, including other types of neural crest cells, which form nerves and other parts of the body such as connective tissue, are not affected by the loss of DDX21.

Role of DNA damage

The researchers also found that mutations of POLR1C and POLR1D also cause damage to stretches of DNA that encode some of the RNA molecules that make up ribosomes. The amount of DNA damage correlated closely with the severity of malformations seen in individual larvae, and mutations in POLR1C led to far more DNA damage than mutations in POLR1D. The researchers believe these differences in DNA damage may explain why the severity of Treacher-Collins Syndrome can vary widely among individuals.

Calo’s lab is now studying why affected cells experience greater levels of DNA damage in those particular sequences. The researchers are also looking for compounds that could potentially prevent craniofacial defects by making the cranial neural crest cells more resistant to p53-induced cell death. Such interventions could have a big impact but would have to be targeted very early in embryonic development, as the cranial neural crest cells begin forming the tissue layers that will become the face at about three weeks of development in human embryos.

The research was funded by the National Institutes of Health, Howard Hughes Medical Institute, and March of Dimes Foundation.

An eye for a mouth: How regenerating flatworms keep track of body parts

Graduate student Lauren Cote identifies genes directing regeneration

Justin Chen
November 16, 2017

An eye for a mouth: How regenerating flatworms keep track of body parts

Person with brown hair in pony tail sits in front of computer and microscope.

Graduate student Lauren Cote identifies genes directing regeneration

Justin Chen

 

Peering down through a microscope at a petri dish, Lauren Cote, a sixth-year graduate student, watches the tip of a worm’s tail. Alone in the petri dish, the brown globule of tissue is regenerating an entirely new digestive system, a brain, and a pair of eye spots. After just a few weeks, the animal — a quarter-inch-long ribbon of flesh capped by a triangular head — is complete again. Swimming through the dish, the worm’s grainy, mahogany body fades to a translucent gray-blue along the edges, stretching and contracting as if hinting at its malleability.

Many animals regenerate. Salamanders replace their tails while zebrafish regrow damaged heart muscle. Even humans can renew large parts of their livers. However, few creatures can regenerate like planarians, a class of flatworms found in fresh and salt water habitats around the world — and in the Reddien lab at the Whitehead Institute.

Because planarians are masters of regeneration, able to replace any body part and even create a new animal from small chunks of tissue, they have become a focus of intense study. By examining the flatworm species Schmidtea mediterranea, Cote and other members of the Reddien lab have uncovered the ways cells communicate after injury to coordinate regeneration. Their work provides insight into how the ability to regenerate evolved, and how the healing process works in a variety of animals, including humans.

– –

Although regeneration seems mysterious, researchers have simplified the feat into two steps. First, planarians create the raw material to make new body parts by stimulating a group of rapidly dividing cells, called stem cells, that are the source of all new tissue in the worm. Second, these new cells need instructions to know what kind of tissue to become. Cote’s goal is to demystify this second step by locating a grid of information, like latitude and longitude lines on a map, that helps planarians keep track of their body parts and sense what is missing.

Hands suctioning small, black dots from petri dish.
Few creatures can regenerate like planarians, a class of flatworms found in fresh and salt water habitats around the world.

“The animal could have lost just the tip of its head or entire left side of its body,” Cote says, “and somehow it regrows the precise anatomy needed to make a complete worm.”

Over the past few years, research in the Reddien lab has demonstrated that a network of muscle cells spread throughout the worm’s body guides regeneration. To accomplish this task, muscle cells rely on a group of genes called position control genes (PCGs) which, based on Cote’s model, are predicted to encode proteins involved in cell communication. Depending on what PCGs are activated or expressed, muscle cells would send out a unique combination of signaling molecules that determine which body parts, such as eyes, stomach, or tail, would form.

“We like to imagine that muscle cells function like satellites and beam down information,” Cote says. “This allows stem cells to know where they are and what new body part to become.”

– –

To systematically identify PCGs from the roughly 20,000 genes expressed in Schmidtea mediterranea, Cote worked in tandem with postdoctoral researcher Lucila Scimone in the Reddien lab to perform a two-part study. First they created maps of gene expression by examining individual muscle cells. After inventorying the genes each individual muscle cell expressed, they aggregated the data into a whole body map, showing gene activity across the entire worm. Some genes were expressed in all muscle cells, implying a general function such as controlling contraction and relaxation. In contrast, other genes were expressed in precise regions of the worm, like the head or midsection, suggesting that they could act as PCGs by defining the identity of each area.

In the second half of the study, Cote and Scimone used molecular techniques to disrupt the activity of potential PCGs. “We hypothesized that if a gene were needed to direct regeneration, the worm would still be able to renew itself without that gene’s activity,” Cote says, “but the animal would end up with an abnormal body.”

Indeed, Cote found that disrupting four genes in particular, encoding signaling molecules and receptor-like proteins, led to defective regeneration; worms either grew extra eyes on their head or grew extra feeding tubes sprouting out of their midsection like elongated suction cups.  Together these four genes, along with a few previously identified genes controlling head and tail regeneration, comprise a short but expanding list of PCGs controlling the location and identity of new tissues. As scientists begin to understand the molecular details of planarian regeneration, they will test whether similar genes are used by other animals and humans.

– –

Although a biologist now, Cote began her academic life focusing on mathematics. As an undergraduate math major at the University of Chicago, she studied branches of mathematics such as analysis, algebra, and algebraic topology, a discipline that describes the properties of multidimensional shapes. After a summer project, Cote realized that — while she enjoyed learning mathematics — she found the research far too abstract.

“I was having a mid-college crisis,” she recalls. “I wanted to study something more visual where you could actually see what is going on.” Following this urge, Cote began to work in a lab examining fly development during her junior year. “I remember watching sheets of cells on the outside of a fly embryo folding in on themselves and sliding under the surface away from view. It made me wonder how cells make decisions and choreograph their movements to build a body. That’s how I got interested in developmental biology.”

After graduating from the University of Chicago, Cote worked as a lab technician for two years. During this time, she realized that her background in math and ability to think logically was an asset. “Putting together a mathematical proof is similar to publishing a research paper,” she says. “In both cases you are piecing together smaller bits of evidence into a cohesive argument.”

A series of blobs with white, green, purple and yellow specs inside them.
Gene expression maps from the first half of Cote’s and Scimone’s study. The head of the worm faces the top of the screen while the tail of the worm faces the bottom of the screen. Each worm is marked by purple, yellow, and green dots indicating the expression of three different genes expressed in muscle cells. These colors show how genes are localized to different areas of the worm and could act as PCGs.  In the second half of the study, Cote and Scimone identified PCGs by using molecular techniques to disrupt gene activity and looking for worms that regenerated abnormal bodies.

Encouraged by her successful venture into biological research, Cote decided to pursue a PhD in biology. She learned about the Reddien lab while taking a genetics course during her first year at MIT. Like Cote, many members of this group have backgrounds in other areas of science — including computational biology, development, evolution, biochemistry, and immunology — which helps them examine planarian regeneration from many perspectives.

“They were beginning to put together a story linking muscle cells to regeneration that was really intriguing,” Cote says. “I also liked the challenge of working with planarians because they are a fairly new lab animal. We’re still developing a lot of research tools so there is room to be creative and ask fundamental questions.”

By following an initial strand of curiosity as an undergraduate and identifying PCGs as a graduate student, Cote has begun to decipher the molecular language of regeneration.  As scientists learn more about how planarians replace missing body parts, new areas of exploration open. One pressing question­ is how planarian regeneration compares to that of other animals. To pursue that mystery, Cote plans on studying another animal as a postdoctoral researcher and eventually starting her own laboratory.

“I still haven’t made up my mind, “she says, “but I’m considering a lot of possibilities such as crustaceans, sea squirts, zebrafish, and axolotls.” Regardless of her final choice, Cote will be investigating how cells — essentially fatty membranes encasing a slurry of water and proteins — manage to form complex and intricate structures. She will be pursuing the same questions that first captivated her as an undergraduate in Chicago. “How do cells make decisions? How do they know to become an eye or a stomach or a brain?” she asks. “There is a lot more that I want to understand.”

Photo credit: Raleigh McElvery
Pairing mismatch helps impaired fish RNA cleavage proceed swimmingly
December 21, 2017

Beyond tending to its multitudes of genetic, metabolic, and developmental processes, eukaryotic cells must additionally be vigilant against invasion by parasitic sequences such as viruses and transposons. RNA interference (RNAi) is a defense used by eukaryotic cells to protect themselves from such threats to their genomic harmony. Cellular RNAi components slice and destroy invading double-stranded RNA sequences and also help snip and process microRNAs, RNA sequences encoded by the genome that play key roles in gene regulation. An important process that occurs naturally in our cells, RNAi has also been harnessed by scientists as a tool to study gene function in common models such as worms, fruit flies, and mice. While many researchers have been using RNAi to tease apart gene function for over a decade, those using zebrafish, a powerful vertebrate model, have been forced to use other approaches because RNAi just did not seem to work well in these animals. Now, researchers at Whitehead Institute have uncovered how small changes in the fish Argonaute (Ago) protein, an RNA slicing protein, that happened in its lineage an estimated 300 million years ago greatly diminished the efficiency of RNAi in these animals, while another ancestral feature, in a critical pre-microRNA, was retained that enabled the microRNA to still be produced despite the fish’s impaired Ago protein.

In an article published December 21 in the journal Molecular Cell, graduate student Grace Chen, along with both Whitehead Member David Bartel, also a professor of biology at Massachusetts Institute of Technology (MIT) and investigator with the Howard Hughes Medical Institute, and Whitehead Member and MIT professor of biology Hazel Sive, describe their discovery of a roughly 300 million-year-old, two amino acid substitutions in the fish Ago protein. The substitution is present in the ancestor all teleost fish, the class of fish which includes not only zebrafish but also the vast majority of fish species spanning those populating the ocean, aquarium, and supermarket. These two changes reside in and near the protein’s catalytic site and greatly decrease the ability of the fish Ago to perform its RNA slicing function, offering an explanation for why RNAi has not been a useful tool in zebrafish.

Despite the zebrafish’s deficiencies in RNAi, it is still able to produce the microRNA miR-451, an important regulator of red blood cell maturation and the only microRNA processed by Ago (the rest are produced with another protein called Dicer). MicroRNAs are short stretches of RNA that can regulate gene expression by inhibiting translation of mRNA into a protein and directing the destruction of mRNA before it can be used to make more protein. Since Chen had discovered that zebrafish lack an efficient Ago protein, it was mysterious as to how are fish were able to produce Ago cleavage-dependent miR-451. The Ago protein must process miR-451 by slicing the sequence out of a longer strand of RNA that has folded up on itself, forming a hairpin structure. What they determined was that in the pre-miR-451 hairpin in zebrafish, at a critical position in the miRNA, they found a “G–G” pairing mismatch that actually appears to facilitate cleavage by the impaired zebrafish Ago. No mismatch, no efficient cleavage.

Exploring the effects of a seed sequence mismatch on Ago-catalyzed cleavage kinetics further, they then tested its ability to slice other bound transcripts. The researchers discovered that while, as might be expected, a G–G mismatch slows Ago binding, it significantly enhances both slicing efficiency as well as the release of the bound product, more than off-setting the slower binding reaction kinetics and suggesting that non- “Watson–Crick” base pairing creates an exceptionally favorable geometry for the cleavage and release parts of the reaction.

These findings offer interesting insights into how animals can survive and thrive without an efficient RNAi system and suggest how the Ago protein could be “repaired” in order to allow zebrafish researchers to use RNAi in their experiments. Restoring a function that a lineage hasn’t had for 300 million years might also fuel additional findings into how the teleost class has diverged over time.

Written by Lisa Girard
***
David Bartel’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute.
***
Paper cited:
Chen GR, Sive H, and Bartel DP. A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially Rescues Severely Impaired Cleavage Found in Fish. Molecular Cell, Dec 21 2017 DOI: 10.1016/j.molcel.2017.11.032.
Small RNA mediates genetic parental conflict in seed endosperm
December 19, 2017

CAMBRIDGE, MA–When it comes to gene expression in the endosperm of seeds, gene provenance matters. In this specialized tissue, plants actively strive to keep the expression of genes inherited from the mother versus the father in balance, according to Whitehead Institute scientists.

The endosperm, the starchy part of a seed that envelopes and nourishes the developing embryo, comprises two-thirds of the calories in a typical human diet. It is the meat of a coconut and the sweet part of the corn on the cob we eat.  In a paper published online December 19 in the journal Cell Reports, Whitehead Member Mary Gehring, first author and former Gehring graduate student Robert Erdmann, and colleagues reveal that the endosperm is also the site where the plant must actively orchestrate a delicate balance between expression of genes inherited from the mother and those of the father.  If this critical balance errs toward one parent or the other, seeds can be too small or even abort.

Unlike most plant cells, which have two copies of the genome, cells within the endosperm have three copies: one inherited from the father, and two inherited from the mother. This ratio is established when a sperm cell in the fertilizing pollen grain fuses with the central cell associated with the egg cell in a flower’s ovule. Unlike most cells, the central cell has two nuclei, so when the sperm’s nucleus merges with the central cell, the resulting endosperm is triploid.

 The 2-to-1 ratio of maternal to paternal gene expression is crucial, and deviation can have dire consequences:  If maternal gene expression is too high, the seeds are too small; if paternal gene expression is too high, the seeds abort. Although plant biologists have known the importance of this ratio for seed viability, the balance was assumed to be passively maintained for the majority of genes.  Previously, Gehring determined that a subset of genes expressed in the endosperm are imprinted—their expression is inherited from their parent. But what about the remaining majority of the genome?

Now Gehring and colleagues have discovered a role for small RNAs—snippets of RNA that interfere with and can reduce gene expression—in actively maintaining this 2-to-1 balance in those genes that are not imprinted.  This the first time scientists have documented small RNAs maintaining such a ratio. Using Arabadopsis thaliana and Arabadopsis lyrata plants, Gehring and her lab determined that these small RNAs tamp down the expression of maternally inherited genes. When the enzyme that creates the small RNAs is mutated, fewer small RNAs are produced, and the plant’s carefully balanced gene expression is thrown off. The resulting seeds have excessive maternal gene expression. To understand the significance of this elevated maternal gene expression, Satyaki Rajavasireddy, a postdoctoral researcher in Gehring’s lab and an author of the Cell Reports paper, turned to plants with seeds that abort  because they have additional copies of paternal genes. When these plants with extra paternal DNA had their small-RNA-producing enzyme mutated, the outcome was striking: The seeds were rescued and developed to maturity.

Although the research analyzed this phenomenon in A. thaliana and A. lyrata, Gehring expects it to be a widespread manifestation of the tug-of-war between maternal and paternal genetic contributions.

“Maintaining this maternal/paternal balance is crucial for seed development, including in crop plants,” says Gehring, who is also an associate professor of biology at Massachusetts Institute of Technology.  “We’ve looked at two species that are separated by 10 million years of evolution, and I anticipate we will find this mechanism in other species as well.”

This work was supported by the National Science Foundation (NSF CAREER grant 1453459).

Written by Nicole Giese Rura
* * *
Mary Gehring’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. She is also an associate professor of biology at Massachusetts Institute of Technology.
* * *
Full Citation:
“A small RNA pathway mediates allelic dosage in endosperm”
Cell Reports, online December 19, 2017.
Robert M. Erdmann (1,2), P.R. V. Satyaki (1), Maja Klosinska (1), Mary Gehring (1,2).
1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142 USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA
Jacqueline Lees

Education

  • PhD, 1990, University of London
  • BSc, 1986, Biochemistry, University of York

Research Summary

We identify the proteins and pathways involved in tumorigenicity — establishing their mechanism of action in both normal and tumor cells. To do so, we use a combination of molecular and cellular analyses, mutant mouse models and genetic screens in zebrafish.

Rethinking transcription factors and gene expression

Study shows that, like proteins, genomes must fold appropriately to function properly and that some transcription factors provide the structural support.

Nicole Giese Rura | Whitehead Institute
December 7, 2017

Transcription — the reading of a segment of DNA into an RNA template for protein synthesis — is fundamental for nearly all cellular processes, including growth, responding to stimuli, and reproduction. Now, Whitehead Institute researchers have upended our understanding of how transcription is controlled and the role of transcription factors in the process.

The paradigm shift, described in an article online on Dec. 7 in the journal Cell, hinges on a small protein that plays a key role in genome structure and gives us new insights into how changes in the control of transcription and gene expression can lead to disease.

Transcription has several important players that must all be in the right place at the right time: the transcription machinery, transcription factors, promoters, and enhancers.  According to the existing model, transcription factors are proteins that bind to enhancer regions of the genome and recruit the transcription machinery to the promoter DNA regions, which then initiate the genes’ transcription.

“We’ve always assumed that the role of transcription factors was to recruit the transcription machinery to genes to turn them on or turn them off,” says Richard Young, a Whitehead Insistute member and professor of biology at MIT. “But we never imagined that the transcription factors we’ve studied for three decades actually contribute to the genome’s structure. And as a consequence, they regulate genes. So we now look at genomes like proteins: They have to fold up appropriately in order to control genes.”

Scientists have known that the genome’s structure — how it bends and folds — is essential for efficiently compressing two meters of DNA into each human cell, which is the equivalent of packing a strand ten football fields long into a space the size of a marble. Yet until recently, researchers have not had the tools necessary to appreciate this architecture’s importance in fine control of gene expression or study the genome’s structure at sites ready for transcription.

In 2014, Young and his lab determined that portions of the genome reside in loop-based structures, creating insulated neighborhoods that bring enhancers, promoters, and genes into close proximity. Each loop is tied at the top by a pair of molecules, called CTCF, that are bound together. This structure is essential for proper gene control: If the loop structure is broken, gene expression is altered, and cells can become diseased or die.

In the current research, Young along with co-first authors Abraham Weintraub and Charles Li took a closer look at a protein that is well known but not well understood: Yin Yang 1 (YY1). Hundreds of scientific papers have linked YY1 dysfunction to diseases such as viral infections, cancer, and arthritis, and yet the studies produced seemingly contradictory observations of YY1’s function.

According to Young and colleagues, YY1 is a unique transcription factor that occupies both enhancers and promoters, is essential for cell survival, and is found in almost every cell type in humans and mice. Like CTCF, YY1 can also pair with itself and bind to DNA to form loops that enhance DNA transcription.

“YY1 is expressed broadly, and it is necessary for establishing enhancer-promoter loops in multiple cell types,” says Weintraub. “That’s its job, not recruiting the transcription apparatus. When the structure created by YY1 is removed, the genome is no longer folded properly, gene control is lost and transcription of the affected genes is significantly diminished, which can cause dysfunction.”

This model of YY1’s function could account for its association with a number of disparate diseases. Earlier this year, scientists reported YY1 syndrome — a genetic syndrome causing cognitive disabilities in people with mutations in their YY1 gene.

According to Young, YY1 is probably not the only transcription factor with this loop-forming role, and his lab will be searching for additional factors with similar functions.

“YY1 is most likely just the first one, and there are probably a bunch of collaborators that have similar roles,” says Young. “Instead of the classic function that we thought these transcription factors had — interacting with the transcription apparatus and giving instructions on how much or how little of a gene’s transcript to produce — they are bringing together regulatory elements with the gene. The whole job of these transcription factors is just making structure. We are realizing that the things that form physical structures are much more important than we had appreciated.”

The researchers’ work was supported by the National Institutes of Health, the Ludwig Graduate Fellowship funds, the National Science Foundation, the American Cancer Society, a Margaret and Herman Sokol Postdoctoral Award, the Damon Runyon Cancer Research Foundation, and the Cancer Research Institute. The Whitehead Institute has filed a patent application based on this study.