Genome-wide screens could reveal the liver’s secrets

A new technique for studying liver cells within an organism could shed light on the genes required for regeneration.

Anne Trafton | MIT News Office
November 15, 2022

The liver’s ability to regenerate itself is legendary. Even if more than 70 percent of the organ is removed, the remaining tissue can regrow an entire new liver.

Kristin Knouse, an MIT assistant professor of biology, wants to find out how the liver is able to achieve this kind of regeneration, in hopes of learning how to induce other organs to do the same thing. To that end, her lab has developed a new way to perform genome-wide studies of the liver in mice, using the gene-editing system CRISPR.

With this new technique, researchers can study how each of the genes in the mouse genome affects a particular disease or behavior. In a paper describing the technique, the researchers uncovered several genes important for liver cell survival and proliferation that had not been seen before in studies of cells grown in a lab dish.

“If we really want to understand mammalian physiology and disease, we should study these processes in the living organism wherever possible, as that’s where we can investigate the biology in its most native context,” says Knouse, who is also a member of MIT’s Koch Institute for Integrative Cancer Research.

Knouse is the senior author of the new paper, which appears today in Cell Genomics. Heather Keys, director of the Functional Genomics Platform at the Whitehead Institute, is a co-author on the study.

Extracellular context

As a graduate student at MIT, Knouse used regenerating liver tissue as a model to study an aspect of cell division called chromosome segregation. During this study, she observed that cells dividing in the liver did not behave the same way as liver cells dividing in a lab dish.

“What I internalized from that research was the extent to which something as intrinsic to the cell as cell division, something we have long assumed to be independent of anything beyond the cell, is clearly influenced by the extracellular environment,” she says. “When we study cells in culture, we lose the impact of that extracellular context.”

However, many types of studies, including genome-wide screens that use technologies such as CRISPR, are more difficult to deploy at the scale of an entire organism. The CRISPR gene-editing system consists of an enzyme called Cas9 that cuts DNA in a given location, directed by a strand of RNA called a guide RNA. This allows researchers to knock out one gene per cell, in a huge population of cells.

While this approach can reveal genes and proteins involved in specific cellular processes, it has proven difficult to deliver CRISPR components efficiently to enough cells in the body to make it useful for animal studies. In some studies, researchers have used CRISPR to knock out about 100 genes of interest, which is useful if they know which genes they want to study, but this limited approach doesn’t reveal new genes linked to a particular function or disease.

A few research groups have used CRISPR to do genome-wide screens in the brain and in skin cells, but these studies required large numbers of mice to uncover significant hits.

“For us, and I think many other researchers, the limited experimental tractability of mouse models has long hindered our capacity to dive into questions of mammalian physiology and disease in an unbiased and comprehensive manner,” Knouse says. “That’s what I really wanted to change, to bring the experimental tractability that was once restricted to cell culture into the organism, so that we are no longer limited in our ability to explore fundamental principles of physiology and disease in their native context.”

To get guide RNA strands into hepatocytes, the predominant cell type in the liver, Knouse decided to use lentivirus, an engineered nonpathogenic virus that is commonly used to insert genetic material into the genome of cells. She injected the guide RNAs into newborn mice, such that once the guide RNA was integrated into the genome, it would be passed on to future generations of liver cells as the mice grew. After months of effort in the lab, she was able to get guide RNAs consistently expressed in tens of millions of hepatocytes, which is enough to do a genome-wide screen in just a single animal.

Cellular fitness

To test the system, the researchers decided to look for genes that influence hepatocyte fitness — the ability of hepatocytes to survive and proliferate. To do that, they delivered a library of more than 70,000 guide RNAs, targeting more than 13,000 genes, and then determined the effect of each knockout on cell fitness.

The mice used for the study were engineered so that Cas9 can be turned on at any point in their lifetime. Using a group of four mice — two male and two female — the researchers turned on expression of Cas9 when the mice were five days old. Three weeks later, the researchers screened their liver cells and measured how much of each guide RNA was present. If a particular guide RNA is abundant, that means the gene it targets can be knocked out without fatally damaging the cells. If a guide RNA doesn’t show up in the screen, it means that knocking out that gene was fatal to the cells.

This screen yielded hundreds of genes linked to hepatocyte fitness, and the results were very consistent across the four mice. The researchers also compared the genes they identified to genes that have been linked to human liver disease. They found that genes mutated in neonatal liver failure syndromes also caused hepatocyte death in their screen.

The screen also revealed critical fitness genes that had not been identified in studies of liver cells grown in a lab dish. Many of these genes are involved in interactions with immune cells or with molecules in the extracellular matrix that surrounds cells. These pathways likely did not turn up in screens done in cultured cells because they involve cellular interactions with their external environment, Knouse says.

By comparing the results from the male and female mice, the researchers also identified several genes that had sex-specific effects on fitness, which would not have been possible to pick up by studying cells alone.

Renew and regenerate

Knouse now plans to use this system to identify genes that are critical for liver regeneration.

“Many tissues such as the heart are unable to regenerate because they lack stem cells and the differentiated cells are unable to divide. However, the liver is also a highly differentiated tissue that lacks stem cells, yet it retains this amazing capacity to regenerate itself after injury,” she says. “Importantly, the genome of the liver cells is no different from the genome of the heart cells. All of these cells have the same instruction manual in their nucleus, but the liver cells are clearly reading different sentences in this manual in order to regenerate. What we don’t know is, what are those sentences? What are those genes? If we can identify those genes, perhaps someday we can instruct the heart to regenerate.”

This new screening technique could also be used to study conditions such as fatty liver disease and cirrhosis. Knouse’s lab is also working on expanding this approach to organs other than the liver.

“We need to find ways to get guide RNAs into other tissues at high efficiency,” she says. “In overcoming that technical barrier, then we can establish the same experimental tractability that we now have in the liver in the heart or other issues.”

The research was funded by the National Institutes of Health NIH Director’s Early Independence Award, the Koch Institute Support (core) Grant from the National Cancer Institute, and the Scott Cook and Signe Ostby Fund.

Introducing the Amon Award Winners
MIT Koch Institute
October 25, 2022

Cheers to the inaugural winners of the Koch Institute’s Angelika Amon Young Scientist Award, Alejandro Aguilera and Melanie de Almeida. The new award recognizes graduate students in the life sciences or biomedical research from institutions outside the U.S. who embody Dr. Amon’s infectious enthusiasm for discovery science.

Aguilera, a student at the Weizmann Institute of Science in Israel, has developed a platform for studying mammalian embryogenesis. De Almeida, who recently completed her doctoral work at the Research Institute of Molecular Pathology in Austria, develops CRISPR screens to explore cancer vulnerabilities and gene regulatory networks.

Aguilera and de Almeida will visit the Koch Institute in November to deliver scientific presentations to the MIT community and Amon Lab alumni.

Unusual Labmates: How C. elegans Wormed Its Way into Science Stardom
Greta Friar | Whitehead Institute
September 20, 2022

 

Introduction

Michael Stubna, a graduate student in Whitehead Institute Member David Bartel’s lab, peers into his microscope at the Petri dish full of agar gel below. He spots one of his research specimens, a millimeter-long nematode worm known as Caenorhabditis elegans (C. elegans), slithering across the coating of bacteria–the worm’s food source–on the surface of the gel. The worm leaves sinuous tracks in its wake like a skier slaloming down a slope.

 

Michael looks up from the microscope and grabs his worm pick, a metal wire sticking out of a glass tube. He runs the end of the pick through a Bunsen burner flame until the wire glows red, using heat to sanitize the tool. Then he returns his attention to the microscope. He nudges the Petri dish to re-center the worm, and, once the pick has cooled, he coats the tip with some of the sticky bacterial food and uses it to skillfully pluck the worm from the surface of the gel. He puts a fresh dish of agar under the microscope, and presses the pick, with the worm still adhered, to the surface. Almost immediately, the worm sets off, carving fresh tracks into the pristine bacterial lawn.

Michael is cultivating C. elegans in order to use them to study microRNAs, tiny RNA molecules involved in gene regulation. Right now, Michael is the only researcher in the Bartel lab using the worms, but in the wider research world, C. elegans is a popular model organism. At first glance, C. elegans is a rather unassuming animal. Barely large enough to see with the naked eye, in nature the worms reside in soil and decomposing vegetation, feasting on bacteria. Except for their heads, their bodies can only bend up and down, so the animals crawl on their sides. The worms have simple tube bodies and are capable of a limited range of behaviors. Nevertheless, researchers frequently turn to C. elegans to learn about not only their biology, but our own. C. elegans is one of the most intimately understood species in biology—the first animal to have its complete genome sequenced or its neural circuitry completely mapped. How did this simple worm become so well studied and a fixture in laboratories around the world?

Making a model

The species C. elegans was first identified, and used in research, after being found in the soil in Algeria around 1900. However, its popularity as a research model skyrocketed in the 1970s, after biologist Sydney Brenner, then at the Medical Research Council  Laboratory of Molecular Biology and later the founder of the Molecular Sciences Institute, made the case for it as the best new model species for the field of molecular biology. [1], [2]

What makes C. elegans such a good model organism? The worm exists in a “just right” zone of biological complexity: it is complex enough to have many of the features that researchers want to study, but simple enough that those same features can be examined comprehensively. For example, each C. elegans has 302 neurons, which is enough to be a useful model for everything from questions about how brains form, to how they sense and respond to stimuli, to how neuronal pathways give rise to specific behaviors, to how different diseases cause neurodegeneration. At the same time, 302 neurons is a small enough number for researchers to be able to study each individual  neuron and its connections thoroughly. (In comparison, a fruit fly has around 100,000 neurons.)

In the same way, C. elegans has just enough complexity to be used to model other common aspects of animal biology, including muscle function, reproduction, digestion, wound healing, aging, and more. It shares many genes with humans and can even be used to model human disease. For example, researchers have used C. elegans to model neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

Additionally, C. elegans has many advantages as a research subject. The worm’s skin is transparent, so researchers can easily observe and capture images of changes occurring inside of its body down to a cellular or even sub-cellular level.

C. elegans is small, hardy, and easy to rear in the laboratory. Fed a simple diet of bacteria and kept at 20 degrees Celsius, C. elegans will mature from an egg, through four larval stages, to a fertile adult in three days. It can then rapidly reproduce to provide researchers with thousands of specimens. C. elegans live for about three weeks, allowing for quick generational turnover, but if researchers want to keep the worms alive for longer, this is easy to do by putting the worms in stasis.

In nature, it is common to find the worms in a state of suspended animation, in which they can survive for months. [3] During poor environmental conditions, such as when food is scarce, instead of maturing into their usual third larval stage, the worms will enter what is known as a dauer stage, a hardier but inactive larval form. When environmental conditions improve, the worms exit the dauer stage and resume normal development. Researchers can recreate this process in the lab.

If a researcher is going out of town for a few days, they can keep their worms in a refrigerator at 4 degrees Celsius. They also have a simple solution for storing worms long term: freezing them. Worms put in a negative 80-degree Celsius freezer can survive for years and still be recovered. This makes the worms much easier to maintain than other common model organisms, which need constant maintenance to keep them alive, fed, and reproducing.

“With most model organisms, if you go away on vacation you need to find someone to look after your specimens while you are gone,” Stubna says. “That’s not necessary with worms.”

Another advantage of C. elegans is how easy it is to generate large numbers of them. Most C. elegans are self-fertilizing hermaphrodites. They cannot mate with each other, but each worm can generate hundreds of offspring on its own. The self-fertilizing nature of C. elegans generally works in researchers’ favor, as along with making it easy to generate lots of new worms, self-fertilization makes genetic inheritance simpler to predict and manipulate in order to maintain a desired mutation throughout the generations. However, sometimes researchers may want to cross-breed their specimens, for example to combine mutations found in separate adults in an offspring.
Fortunately for researchers, the rare C. elegans worm is male—around .1-.2% in the wild [4] — and males can fertilize hermaphrodites’ eggs, enabling cross-breeding. C. elegans males arise in the wild due to a deviation during sex cell division. Hermaphrodite C. elegans have two X chromosomes. When one of these fails to form correctly, the resulting worm with its one X chromosome will be male, having some key anatomical differences. If researchers need lots of males for their work, they can increase the percentage of a hermaphrodite’s offspring that are male by exposing the worm to heat before it reproduces or by using genetic manipulation. Then, researchers can selectively breed male-heavy populations to further increase the ratio over time.
Researchers have developed a variety of tools and approaches over the years with which to manipulate C. elegans genetically. These have enabled researchers to learn a lot about both the worm’s genes, and genes that it shares with humans and other animals. One useful approach is the development of marker strains.

Getting to know C. elegans inside and out

After Brenner landed on C. elegans as an ideal model, his research group began several ambitious projects to comprehensively understand the worm’s biology. In the following decades, the worm’s anatomy and genome would be detailed in unprecedented detail. The more that researchers learned about the worm, the better a model it became.

Creating a complete cell lineage and neural map

C. elegans is remarkable in that every worm has the same exact number of cells: 959 in the adult hermaphrodite (not counting the cells that will become eggs or sperm). 302 of these cells are neurons. Researchers in Brenner’s group created two first-of-their-kind resources documenting the details of this biology. First, they mapped the worms’ complete cell lineage, recording every cell division that occurs during the worms’ development from fertilized egg to adult. This resource makes it easy for researchers to study how different factors contribute to—or can alter—this development.

Then, the researchers created a wiring diagram, or connectome, of the hermaphrodite worm’s 302 neurons and their thousands of synapses—the junction points where neurons interact. Researchers have used this wiring diagram to identify neurons involved in many different behaviors in C. elegans, as well as to understand how brains form and function across animal species. [5,6] C. elegans was the first, and as of 2021 the only, animal to have had its brain completely mapped.

Capturing the complete genome

In 1998, C. elegans made the news as the first animal to have its complete genome sequenced. The completion of the 15-year-long sequencing project, helmed by the C. elegans Sequencing Consortium, was announced in a special issue of Science. Researchers had previously compiled complete genomes for a variety of single-celled species, but as an animal, C. elegans had a significantly larger genome. The complete genome provided many useful insights into individual genes, and the relationships between genes both within C. elegans and between species. The ambitious project also proved instructive for how to sequence large genomes. In fact, the Human Genome Project helped to fund the sequencing of C. elegans as a stepping stone to the ultimate goal of sequencing the human genome, which was achieved in 2003.

With C. elegans’ genetics, anatomy, and other biology so thoroughly documented, the worms became an even more potent model organism. Researchers now had a wealth of foundational knowledge about the worm that they could use to make and test hypotheses about specific questions.

Worm culture

As the use of C. elegans in science grew, a community formed among the worm’s researchers. The C. elegans community was quick to develop and share resources. The Worm Breeder’s Gazette is a semi-annual newsletter first published in 1975, which shares information of interest to the C. elegans community such as experimental techniques and new findings. The Caenorhabditis Genetics Center (CGC), founded in 1979, is a central repository from which researchers can order thousands of different strains of C. elegans for use in their own research.

From the early years, prominent researchers working with C. elegans believed strongly in sharing data both among researchers and with the public. This openness set the tone for the field of molecular biology more broadly; for example, open data sharing policies around the sequencing of the C. elegans genome encouraged the Human Genome Project to follow suit.

The worm community often had to build its own tools in order to share data on the scales its members desired. One big project was the creation of ACeDB (A C. elegans Database), a database management system capable of storing and displaying many different kinds of biological information about C. elegans, including its complete genome, in a user-friendly way. The current iteration of ACeDB, known as WormBase, contains the annotated genomes of C. elegans and related nematodes, information on every known C. elegans gene and its function, genetic maps, the C. elegans cell lineage and connectome, and much more. The ACeDB software was soon used to create similar databases for other model organisms. Such databases now exist for many different species, making detailed biological data widely available to everyone. These databases are also often used to share the latest research, maintain a consensus around scientific terminology and gene annotation, and provide educational resources on the model organism. Anyone looking for general information on C. elegans can also visit WormBook, an open access, online review of C. elegans biology.

With these resources and others, the C. elegans community fostered a culture of sharing and scientific openness that continues to this day.

A few of the many discoveries and further tool development

C. elegans researchwith its wealth of experimental tools and methods, pre-existing data with which to build and test hypotheses, and a worldwide community happy to share resources, has been the source of many important discoveries over the years. Many of these discoveries have also added to researchers’ toolkits, providing new ways to experiment with C. elegans and other research specimens. A few of these myriad impactful discoveries are highlighted below.

Insights into development and programmed cell death

Brenner and two researchers whom he had mentored, John Sulston, then at the The Wellcome Trust Sanger Institute, and Robert Horvitz, then at the Massachusetts Institute of Technology (MIT), were awarded the first Nobel Prize for work done in C. elegans in 2002. When the researchers were creating the C. elegans cell lineage map, they saw that some cells created during development died off at particular moments, and that this programmed destruction of cells, called apoptosis, was an essential part of creating the adult body. They identified key genes that regulate apoptosis, and their work led to insights into the role of apoptosis in human development, as well as in health and disease. For example, cancer cells are able to avoid apoptosis, and many modern cancer therapies work by reenabling apoptosis of cancer cells.

Andrew Fire at the Stanford University School of Medicine and Craig Mello at the University of Massachusetts Medical School used C. elegans to discover RNA interference (RNAi), a process that cells use to stop genes from being expressed. RNAi became an important research tool after researchers figured out how to tailor RNAi to turn off genes that they are interested in studying in different cells and species. Researchers turn off a gene and see what changes, which helps them figure out the gene’s function. People have also found uses for RNAi in medicine and industry. RNAi is easy to use in C. elegans — researchers can apply it to worms by simply feeding them modified bacteria — so this tool made the worms an even better model for genetics research. Fire and Mellow earned a Nobel Prize for their discovery in 2006.

Introducing a new visual tag

C. elegans also contributed to the development of another popular and powerful research tool, green fluorescent protein (GFP). GFP is a protein first found in jellyfish. It glows green under certain light waves. Martin Chalfie at Columbia University showed in C. elegans that the genetic code for GFP could be added as a tag to genes of interest, and then the products of those genes would glow, providing researchers with a great visual marker of where and when the genes were expressed. Chalfie shared the 2008 Nobel Prize in Chemistry for this work, and researchers now frequently use GFP and similar molecules as visual markers in experiments across species and cell types.

A model for aging

In 1993, Cynthia Kenyon at the University of California, San Francisco and colleagues discovered that mutations to a single gene, daf-2, along with the normal activity of a second gene, daf-16, could more than double the lifespan of C. elegans. Kenyon and others intrigued by this  discovery would go on to use C. elegans to ask questions about the molecular mechanisms governing aging. Researchers have also studied how equivalent genes affect aging in other animals, including humans.

A model for sex determination, reproduction, and development

C. elegans has been used to explore questions related to sex, reproduction, and development. Barbara Meyer, then at MIT, now at the University of California, Berkeley, discovered the mechanism of sex determination in the worm, and has uncovered mechanisms by which gene expression is regulated to compensate an animal having one or two X chromosomes. Other researchers have used C. elegans to make important discoveries about germ cells, the cells that give rise to eggs and sperm. Judith Kimble and John White, then at the MRC Laboratory of Molecular Biology, now at the University of Wisconsin–Madison discovered the first germline stem cell niche in C. elegans, which is the place where animals maintain a pool of stem cells with which to keep producing new germ cells over time. This finding had implications for fertility and regeneration research. Geraldine Seydoux at Johns Hopkins University has used C. elegans to investigate unique features of germ cells, as well as how sperm and egg interact and how the early embryo prepares to form a complex adult body.

Understanding sense of smell

Thanks in large part to C. elegans having such a well-mapped nervous system, the worm has been a common model for researchers studying how animals sense and respond to stimuli in their environments. Cori Bargmann, an alumna of Whitehead Institute, now at Rockefeller University, studies how C. elegans sense and process outside stimuli, how those stimuli can trigger changes in behavior, and how the brain can be rewired to modify behaviors over time. Bargmann’s research has particularly illuminated the worm’s sense of smell. She found the first evidence of a receptor for a specific smell, and her work more broadly shed light on how animals are able to recognize many different types of smells.

A rich history of discovery

This is just a small sampling of the important discoveries that have been made in C. elegans. WormBook has compiled a list of many such achievements, including the discovery of multiple key molecules and pathways present across animals.

Worms at Whitehead Institute

Michael uses C. elegans to study microRNAs.

C. elegans have long played an important role in microRNA research; in fact, microRNAs were first discovered in C. elegansVictor Ambros and colleagues, and Gary Ruvkun and colleagues, published papers describing the first identified microRNA, lin-4, and its target, in 1993. At first, researchers thought that the small gene-regulating molecule might be an oddity. However, in 2000, Ruvkun discovered a second microRNA, and by the next year researchers—including Whitehead Institute Member David Bartel—had identified many more microRNAs in C. elegans, as well as microRNAs in other species. [12] Collectively, this research implied that microRNAs were a common and important regulator of gene expression across species. The field of microRNA research exploded, and microRNAs became the focus of Bartel’s lab.

In spite of the importance of C. elegans in establishing microRNA research, both in the field at large and in the Bartel lab specifically, no one in the lab was using C. elegans as a research model when Michael joined. However, as Michael–who had previous experience working with C. elegans–began to plan his graduate research, he realized that the worm would be the perfect model in which to explore his topic of interest: how microRNAs are regulated. Bartel agreed, and so C. elegans made their triumphant return to Whitehead Institute.

“It’s great to return to C. elegans,” says Bartel, who is also a professor of biology at MIT and a HHMI investigator.  “Michael is working on sets of microRNAs that we discovered over 20 years ago. Since then, we and others have learned a lot about microRNAs, using a variety of research models. It will be fun to see what new things we will learn with C. elegans.

Michael is using C. elegans to better understand how microRNAs, which degrade messenger RNAs, are themselves degraded. In recent years, researchers discovered a surprising mechanism of microRNA degradation: in some instances, when a microRNA pairs with a messenger RNA, instead of this leading to the destruction of the messenger RNA, it leads to the destruction of the microRNA.

“The normal regulatory logic is completely flipped,” Michael says. “This was discovered in mammalian cells, and our lab and others have been working out the mechanism for how this happens.”

The Bartel lab found that a particular gene is necessary for this process. When Michael joined the lab, he wondered whether that gene’s equivalent in C. elegans serves the same role. He found that it does. However, there are differences between how this process works in C. elegans and mammals, in particular in the way that the RNAs pair to trigger destruction of the microRNA. Those differences, and what they reveal about how microRNAs are regulated, are what Michael is studying now.

“What is the underlying principle of what’s required for microRNAs to be degraded through this pathway in worms? It’s not known, and that’s what I’m trying to find out,” Michael says.

Michael hopes that what he learns from the worms will shed light on the logic for how microRNAs are regulated across animal species. This will in turn give researchers a better understanding of how cells are able to so precisely tailor their gene expression. The prospect of deciphering such a central facet of cell biology is exciting for Michael, but it’s just another day for C. elegans. Decades of results have shown that there’s no limit to what these simple worms can be used to discover.

 

Four from MIT receive NIH New Innovator Awards for 2022

Awards support high-risk, high-impact research from early-career investigators.

Phie Jacobs | School of Science
October 4, 2022

The National Institutes of Health (NIH) has awarded grants to four MIT faculty members as part of its High-Risk, High-Reward Research program.

The program supports unconventional approaches to challenges in biomedical, behavioral, and social sciences. Each year, NIH Director’s Awards are granted to program applicants who propose high-risk, high-impact research in areas relevant to the NIH’s mission. In doing so, the NIH encourages innovative proposals that, due to their inherent risk, might struggle in the traditional peer-review process.

This year, Lindsay Case, Siniša Hrvatin, Deblina Sarkar, and Caroline Uhler have been chosen to receive the New Innovator Award, which funds exceptionally creative research from early-career investigators. The award, which was established in 2007, supports researchers who are within 10 years of their final degree or clinical residency and have not yet received a research project grant or equivalent NIH grant.

Lindsay Case, the Irwin and Helen Sizer Department of Biology Career Development Professor and an extramural member of the Koch Institute for Integrative Cancer Research, uses biochemistry and cell biology to study the spatial organization of signal transduction. Her work focuses on understanding how signaling molecules assemble into compartments with unique biochemical and biophysical properties to enable cells to sense and respond to information in their environment. Earlier this year, Case was one of two MIT assistant professors named as Searle Scholars.

Siniša Hrvatin, who joined the School of Science faculty this past winter, is an assistant professor in the Department of Biology and a core member at the Whitehead Institute for Biomedical Research. He studies how animals and cells enter, regulate, and survive states of dormancy such as torpor and hibernation, aiming to harness the potential of these states therapeutically.

Deblina Sarkar is an assistant professor and AT&T Career Development Chair Professor at the MIT Media Lab​. Her research combines the interdisciplinary fields of nanoelectronics, applied physics, and biology to invent disruptive technologies for energy-efficient nanoelectronics and merge such next-generation technologies with living matter to create a new paradigm for life-machine symbiosis. Her high-risk, high-reward proposal received the rare perfect impact score of 10, which is the highest score awarded by NIH.

Caroline Uhler is a professor in the Department of Electrical Engineering and Computer Science and the Institute for Data, Systems, and Society. In addition, she is a core institute member at the Broad Institute of MIT and Harvard, where she co-directs the Eric and Wendy Schmidt Center. By combining machine learning, statistics, and genomics, she develops representation learning and causal inference methods to elucidate gene regulation in health and disease.

The High-Risk, High-Reward Research program is supported by the NIH Common Fund, which oversees programs that pursue major opportunities and gaps in biomedical research that require collaboration across NIH Institutes and Centers. In addition to the New Innovator Award, the NIH also issues three other awards each year: the Pioneer Award, which supports bold and innovative research projects with unusually broad scientific impact; the Transformative Research Award, which supports risky and untested projects with transformative potential; and the Early Independence Award, which allows especially impressive junior scientists to skip the traditional postdoctoral training program to launch independent research careers.

This year, the High-Risk, High-Reward Research program is awarding 103 awards, including eight Pioneer Awards, 72 New Innovator Awards, nine Transformative Research Awards, and 14 Early Independence Awards. These 103 awards total approximately $285 million in support from the institutes, centers, and offices across NIH over five years. “The science advanced by these researchers is poised to blaze new paths of discovery in human health,” says Lawrence A. Tabak DDS, PhD, who is performing the duties of the director of NIH. “This unique cohort of scientists will transform what is known in the biological and behavioral world. We are privileged to support this innovative science.”

New players in an essential pathway to destroy microRNAs

In a study from the lab of Whitehead Institute Member David Bartel, researchers have identified genetic sequences that can lead to the degradation of cellular regulators called microRNAs in the fruit fly Drosophila melanogaster.

Eva Frederick | Whitehead Institute
September 26, 2022

In a study from the lab of Whitehead Institute Member David Bartel, researchers have identified genetic sequences that can lead to the degradation of cellular regulators called microRNAs in the fruit fly Drosophila melanogaster. The findings were published September 22 in Molecular Cell.

“This is an exciting study that paves the way for a deeper understanding of the microRNA degradation pathway,” says Bartel, who is also a professor of biology at the Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute. “Finding these ‘trigger’ sequences will allow us to more precisely probe the workings of this pathway in the lab, which is likely critical for flies — and possibly other species — to survive to adulthood.”

In order to produce new proteins, cells transcribe their DNA into messenger RNAs (or mRNAs), which provide information required to make the proteins . When a given mRNA has served its purpose, it’s degraded. The process of degradation is often led by tiny RNA sequences called microRNAs.

In previous work, researchers showed that certain mRNA or non coding RNA transcripts, rather than being degraded by microRNAs, can instead turn the tables on the microRNAs and lead to their destruction through a pathway called target-directed microRNA degradation, or TDMD. “This pathway leads to rapid turnover of certain microRNAs within the cell,” says former Bartel Lab graduate student Elena Kingston.

Kingston wanted to further understand the functions of the TDMD pathway in cells. “I wanted to get at the ‘why,’” she said. “Why are microRNAs regulated in this way, and why does it matter in an organism?”

Previous work on the TDMD pathway was primarily conducted in cultured cells. For the new study, the researchers decided to use the fruit fly Drosophila melanogaster.  A fly model could provide more insight into how the pathway worked in a live organism — including whether or not it had an effect on the organism’s fitness or was essential for survival.

The researchers created a model to study TDMD by using flies with mutations in an essential TDMD pathway gene called Dora (the equivalent human gene is called ZSWIM8, as detailed in this paper). Very few flies with mutations in Dora were seen to make it to adulthood. Most died early in development, suggesting the TDMD pathway was likely important for their embryonic viability.

Putting a finger on the triggers of the TDMD pathway

While microRNAs don’t need many complementary base pairs to bind and regulate their mRNA targets, the opposite is true in the TDMD pathway. In order to work properly, the TDMD pathway needs a highly specific trigger, which can either be a mRNA that codes for proteins, or a non-coding RNA. “What’s unique about a trigger is it has a site that the microRNA can bind to that has a lot of complementarity to the microRNA,” Kingston said.

During the isolation of the early Covid-19 pandemic, Kingston set out to write a program that could pick out probable triggers of microRNA degradation in Drosophila based on their sequencesThe program returned thousands of hits, and the researchers set to work narrowing down which sites were the best candidates to test in flies.

“As soon as we were able to get back into lab [after the lockdown], I took our top 10 or so candidates and tried perturbing them in flies,” she said. “Fortunately for me, about half of them ended up working out.”

These six new triggers more than double the list of known RNA sequences that can direct degradation of microRNAs. To take this finding a step further, the researchers conducted an analysis of what happened to the flies when a trigger was disrupted.

The researchers found that one of the triggers — a long non-coding RNA — plays a role in proper development of the cuticle, or the waterproof outer shell of a fly embryo. “We noticed that when we perturbed this trigger, the cuticles of fly embryos had altered elasticity,” Kingston said. “When we popped the embryos out of their egg shells, we could see these cuticles expand up and bloat.”

Because of the bloated phenotype, Kingston decided to name the long non-coding RNA marge after Aunt Marge, a character in the Harry Potter series. In “Harry Potter and the Prisoner of Azkaban”, Aunt Marge’s taunts lead Harry to accidentally perform magic on her, causing her to inflate and float away.

In the future, Kingston, who has since graduated and begun a career in the biotech industry, hopes researchers will pick up the torch on learning the roles of other TDMD triggers. “We still have several other triggers [from this paper] where there’s no known biological role for them in the fly,” she said. “I think this opens up the field for others to go in and to ask the questions, ‘Where are these triggers acting? What are they doing? And what’s the phenotype when you lose them?’”

Notes

Elena Kingston, Lianne Blodgett and David Bartel. “Endogenous transcripts direct microRNA degradation in Drosophila, and this targeted degradation is required for proper embryonic development.” Molecular Cell, September 22, 2022. DOI: https://doi.org/10.1016/j.molcel.2022.08.029

Germ cells move like tiny bulldozers
Eva Frederick | Whitehead Institute
September 15, 2022

During fruit fly embryo formation, primordial germ cells — the stem cells that will later form eggs and sperm — must travel from the far end of the embryo to their final location in the gonads. Part of the primordial germ cell migration is passive; the cells are simply pushed into place by the movements of other cells. But at a certain point in development, the primordial germ cells must move on their own.

“A lot of the background in this field has been established by studying  how cells move in culture, and there’s this model that they move by using their cytoskeleton to push out their membranes to crawl,” said Benjamin Lin, a postdoctoral researcher in the lab of Whitehead Institute Director Ruth Lehmann. “We weren’t so sure they were actually moving that way in vivo.”

Now, in a new paper published September 14 in Science Advances, Lehmann who is also a professor of biology at the Massachusetts Institute of Technology, and researchers at Whitehead Institute and the Skirball Institute at New York University School of Medicine show that germ cells in growing fly embryos are in fact using a different method of movement which depends on a process called cortical flow, similar to the way bulldozers move on rotating treads. The research also reveals a new player in the pathway that governs this germ cell movement. “This work brings us one step closer to understanding the regulatory network that guides the germ cells on their long and complex journey across an ever-changing cellular landscape,” Lehmann said.

The research could also provide researchers with a new model for studying this type of cell movement in other situations — for example, cancer cells have been shown to move via cortical flow under certain conditions. ”We think there are more general implications for this mode of migratory behavior that go beyond primordial germ cells and apply to other migratory cells as well,” said Lin.

Balloon-shaped cells 

The first clue that Lehmann and Lin found that germ cells might not move the way scientists thought came from a simple observation. “When we began to study how these primordial germ cells move in the embryo, we saw that the cells actually remain shaped like a balloon while they’re moving and they don’t actually change their shape at all,” said Lin. “It’s really different from the crawling model.”

But if the cells weren’t moving by crawling, how were they moving through the embryo? To find out more, the researchers developed new techniques to image the germ cells in live fly embryos, and were able to watch clusters of a protein called actin moving backwards in each cell, as the cell itself was moving forward.

“There’s this thin layer of actin cytoskeleton just under the membrane of cells called the cortex, and they actually moved by making that cortex ‘flow,’” said Lin. “It’s like if you think of the tread of a bulldozer that’s moving backwards as the bulldozer is moving forward. The cells move that cortex backwards to generate friction to move the cell forward.”

Lin hypothesizes that this method of movement is especially well-suited to germ cells moving through a crowded embryo with many different cell types because instead of depending on recognizing specific proteins to “grab” in order to pull themselves through the embryo, it allows the germ cells to move independently. “Everything is pretty individualistic for primordial germ cells,” he said. “They don’t actually signal to each other at all, all the signaling is within each cell… And germ cells have to move through so many different tissues that they need a universal method of movement.”

A new role for a known protein

The researchers also found new information about how the cells control this form of motility. “We found that a protein called AMPK can control this pathway, which was really unexpected,” Lin said. “ Most people know it as a protein that senses energy. We found that this protein was important for helping these cells navigate. It’s one of these upstream players that can control how fast the cell goes, and in which direction.”

In the future, the researchers hope to map the entire pathway that allows germ cells to get to the right place at the right time in development. They also hope to learn more about the mechanisms behind cortical flow. “We want to figure out what is important for establishing these flows,” Lin said. “Our findings here could have implications not just for germ cells, but for other migrating cells as well.”

Notes

Benjamin Lin, Jonathan Luo, Ruth Lehmann. “An AMPK phosphoregulated RhoGEF feedback loop tunes cortical flow–driven amoeboid migration in vivo.” Science Advances, September 14, 2022. DOI: 10.1126/sciadv.abo0323

Brandon (Brady) Weissbourd

Education

  • Graduate: PhD, 2016, Stanford University
  • Undergraduate: BA, 2009, Human Evolutionary Biology, Harvard University

Research Summary

We use the tiny, transparent jellyfish, Clytia hemisphaerica, to ask questions at the interface of nervous system evolution, development, regeneration, and function. Our foundation is in systems neuroscience, where we use genetic and optical techniques to examine how behavior arises from the activity of networks of neurons. Building from this work, we investigate how the Clytia nervous system is so robust, both to the constant integration of newborn neurons and following large-scale injury. Lastly, we use Clytia’s evolutionary position to study principles of nervous system evolution and make inferences about the ultimate origins of nervous systems.

Awards

  • Pathway to Independence Award (K99/R00), National Institute of Neurological Disorders and Stroke, 2020
  • Life Sciences Research Foundation Fellow, 2017
The blueprint of a body
Eva Frederick | Whitehead Institute
July 20, 2022

Multicellular organisms evolved over millennia into a dazzling array of differently adapted creatures. With each generation, tiny worms, lavishly plumed birds, and even humans must create themselves anew from a single cell. To do so, they require a plan.

“How that multifunctional body plan is created is one of the deepest questions in developmental biology,” said Zak Swartz, until recently a postdoctoral researcher in the lab of Whitehead Institute Member Iain Cheeseman. “How do you take a single cell and pattern into a body that has different functions and features along it?”

Whitehead Institute researchers are tackling this question through a variety of different lenses. Researchers in Iain Cheeseman’s lab, including Swartz, have delved into the mysterious forces that underlie the polarity of an organism’s first cell. For the lab led by Pulin Li, research comes in at a later stage of development, when multiple cells combine to form a tissue and must communicate with each other to become an organized whole. Work on regeneration in Peter Reddien’s lab shows how some creatures can access their body blueprint throughout their lives to repair nearly any injury, and Yukiko Yamashita’s group studies how organisms pass on their body blueprints to their offspring through germ cells. Jonathan Weissman and his lab have created a “map” which researchers can use to find the function of a given gene, allowing them access to an organism’s most fundamental plans. Read on to learn about these scientists’ work, and more.

Laying out the plan

All multicellular organisms begin with a single cell, the fertilized egg. This cell has an essential role in setting out the body plan for the rest of an organism. It all starts with establishing polarity — in other words, figuring out which side of the cell is the top, and which is the bottom. This polarity establishes an axis of symmetry for the growing organism, and sets the stage for other developmental processes to come.

In a 2021 study, Cheeseman and postdoctoral researcher Zak Swartz investigated how one protein in specific, called Disheveled, localizes in a cell to help create this polarity in sea star embryos. Swartz found that Disheveled started out uniformly distributed in small aggregations throughout the egg cell, or oocyte. As the cell prepared to divide, Disheveled aggregations dissolved and then reformed at what would become the “bottom” of the oocyte.

Once the initial polarity is established, the oocyte can divide, creating a bilaterally symmetric sea star larvae. The burgeoning cluster of cells must then undergo other processes to define the several axes of symmetry that adult sea stars are known for.

Talking through it 

If an organism’s developmental blueprints are to be followed as development progresses, cells must be able to effectively communicate with each other. That cell to cell communication is the area of expertise of Whitehead Institute Member Pulin Li.

During her postdoctoral fellowship at the California Institute of Technology, Li studied tissue patterning — the mechanisms by which an organism’s newly forming tissues are laid out. Specifically, she investigated a developmental mechanism called morphogen gradient formation.

These gradients, composed of chemicals present in developing embryos, function as spatial coordinate systems and help determine how various cell types will be arranged in the organism — for example which groups of cells will form the liver, or the bones, or the brain, and where they will be within the body.

Li was able to recreate these gradients in the lab, in a Petri dish, and then interpret their signals using time lapse imaging and mathematical modeling. Here at Whitehead Institute, she follows a “bottom-up” approach to studying these complex systems. The best way to understand how something works, she says, is to build it yourself.

A key process in asymmetric cell division preserves the immortality of the germline
Eva Frederick | Whitehead Institute
July 27, 2022

During cell division, chromosomes are replicated into two copies — one for each daughter cell. These copies, called sister chromatids, are usually considered identical. In fact, it’s the two pairs of sister chromatids that make up the symmetrical X shape usually shown when visualizing chromosomes.

A 2013 paper from the lab of Whitehead Institute Member Yukiko Yamashita showed that in the case of asymmetric cell division — such as when a stem cell is dividing into two different kinds of daughter cells (i.e. a stem cell and a differentiating daughter)  — sister chromatids of sex chromosomes actually may carry distinct information, and the dividing cell “chooses” which of the daughters receive a specific copy.

What that “choice” means, and how it’s executed, has been a mystery — until now. A new paper from Yamashita, who is also a professor of biology at the Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute, published in Science Advances on July 27, illuminates the mechanisms that underlie nonrandom sister chromatid segregation, and suggests that the whole process may serve as a way to maintain the amount of ribosomal DNA (or rDNA) that is passed on to subsequent generations. “Tying together these two processes — rDNA copy number maintenance and nonrandom chromatid segregation — is an unexpected and exciting advance in our understanding of how germ cells are able to maintain their immortality,” said Yamashita.

George Watase, a postdoctoral scholar in the Yamashita Lab, led the study. Watase began his research intent on discovering the genetic underpinnings of nonrandom segregation of X and Y chromosomes in the fruit fly Drosophila melanogaster. As he surveyed the genome for genes that were essential to nonrandom segregation, it became apparent that ribosomal DNA was key for the process.

When rDNA was left intact, the sister chromatid  with more rDNA was preferentially chosen by the daughter stem cell instead of the differentiating daughter cell. When rDNA was removed from X and Y chromosomes, however, Watase found that the sister chromatids segregated randomly to the daughter cells.

Ribosomal DNA, or rDNA, is composed of a long stretch of repeats of certain base pairs. The rDNA provides the instructions and material to make ribosomes, which are essential for cells to create proteins. “Most genes exist only as a single copy, but in the case of rDNA we have hundreds of copies in our genome,” Watase said. “The reason for this is that we need a massive amount of ribosomes to synthesize proteins to maintain our cells’ viability.”

As organisms age, most of their cells naturally lose some of those rDNA repeats, including germline stem cells.  However, germline cells are sometimes called “immortal” — while all other cells in the body are made anew with each generation and die when an organism dies, germline cells such as sperm and eggs must carry DNA between generations. THerefore, the stem cells that produce sperm and eggs thus cannot keep losing rDNA repeats, and must bypass the mortality of other cells, by maintaining  a high number of rDNA repeats over time.

By isolating proteins that bind to rDNA, Watase discovered one specific gene, the protein product of which bound to rDNA and somehow assigned the sister chromatid with more rDNA repeats to the daughter cell that was destined to remain a germline stem cell.

This particular gene had not been described before, and Watase and Yamashita were now tasked with naming it. Fruit fly genes are named after what happens to the animal when the gene is removed. When this new gene was knocked down, the germ cells of subsequent generations gradually lost the immortality that separates germline stem cells from their differentiated counterparts.

Watase wasn’t sure how to convey the intricacies of this outcome. In the end, it was Watase’s wife who came up with the perfect name: Indra. In Hindu scriptures, Indra, the lord of all deities, was given a garland of fragrant flowers by a sage called Durvasa. Indra placed the garland on the trunk of his elephant, but the animal was irritated by the smell of the flowers and threw the garland down, trampling it underfoot. When Durvasa saw this, he became enraged and cursed Indra, taking away his immortality.

The name also opened up a world of possibilities for naming future genes that are important in nonrandom sister chromatid segregation. “People sometimes pull from Roman or Greek myths when naming genes, but not as many people use names from Hindu myths,” he said. “And since this is new biology, if we identify additional related genes in the future, we can use names from Hindu myths again.”

Watase and Yamashita’s study opens new avenues for future research. For example, the paper focused primarily on male fruit flies and the production of sperm via asymmetric division. Indra is expressed in the female germline as well, and when the gene is knocked down in females, the resulting phenotype is much more severe. “There must be some mechanism in female germ cells to avoid rDNA copy number reduction,” said Watase. “We just don’t know what that mechanism is.”

In the future, Watase and Yamashita also hope to elucidate how exactly Indra is interacting with cell division machinery to influence which chromatid ends up in the stem cell and which in the differentiating cell, and beyond this mechanism, how the stem cell “selects” the longer chromatid.

“Many biologists study germ cells, but few specifically study how they maintain their immortality,” said Yamashita. “This study is a step towards understanding this fascinating property of germ cells. It’s a really fascinating area and we really have to keep digging deeper into this phenomenon.”

Investigating how cell orientation drives tissue growth during development

In a new study published July 7, 2022 in eLife, Adam Martin’s lab at the MIT Department of Biology identified the mechanical forces and molecular cues that help the spindles in cells located in the portion of the embryo destined to become the fly’s head to assume the same orientation.

Raleigh McElvery
July 7, 2022

Raleigh McElvery

During development, virtually all multicellular creatures must build themselves up from a ball of cells to a multilayered, fully-functioning organism. In the case of a fruit fly embryo, in order go from blob to organism, the cells must coordinate their divisions along the same axis to drive tissue growth in a specific direction — eventually going on to form the three germ layers known as the endoderm, ectoderm, and mesoderm.

Scientists have long studied how cells orient themselves to divide in a specific direction. It’s clear that this orientation is determined by a bundle of tiny fibers inside the cell called the spindle, which helps segregate the chromosomes so they can be distributed between the two daughter cells as the parent cell splits. When the spindles in neighboring cells are parallel with one another, then the cells will divide along the same axis.

In a new study published in eLife on July 7, 2022, Adam Martin’s lab at the MIT Department of Biology identified the mechanical forces and molecular cues that help the spindles in cells located in the portion of the embryo destined to become the fly’s head to assume the same orientation.

According to Martin, an associate professor of biology and the study’s senior author, his lab was among a handful of labs to take interest in this coordinated spindle orientation in the early fruit fly embryo — and identified the molecular, mechanical, and molecular cues that orient the spindle in a living organism.

“Cell divisions in the fly embryo was thought to be regulated independently of cell shape changes and morphogenetic movements,” Martin says. “However, we found that forces associated with cell invagination actually oriented cell divisions through a novel mechanism that we describe.” First author Jaclyn Camuglia, he explains, spearheaded the project from start to finish.

Prior to Camuglia’s work, researchers knew that spindle orientation was controlled by a complex of multiple proteins, including one protein in fruit flies called Pins (known as LGN in vertebrates). The entire protein complex, including Pins, is coupled to motor proteins that help to rotate the spindle. However, it was still unclear what mechanical forces were orienting Pins to dictate spindle rotation.

“It’s a very striking phenomenon when you look into the microscope at a fly embryo and see all the spindles orienting together,” Camuglia says. “We wanted to know: How are these spindles oriented and why is that directionality important?”

The fly is an ideal organism for probing cell division, because it has a very consistent and predictable cell cycle. During development, cells divide a set number of times, pause briefly, and then start up again in very discrete pockets across the embryo. The researchers wanted to know how the cells in a subset of those pockets in the fly’s head were coordinating their divisions.

Camuglia found that, in healthy embryos, Pins was always recruited to the end of the cell along the anterior-posterior axis (from the fly’s head to rear). By cutting the embryo with a laser, treating it with chemical inhibitors, disrupting the connections between cells, and depleting certain transcription factor proteins, she was able to characterize the mechanical forces that directed Pins to the anterior-posterior axis and thus rotated the spindle in the proper direction. As it turns out, these forces stem from other large-scale tissue movements that occur at the same time to force the embryo to fold in on itself and eventually form the fly’s muscles.

The researchers don’t yet know the role that these coordinated divisions along the anterior-posterior axis play in the developing fruit fly head — or what might happen during development if the divisions were to go awry. But Martin and Camuglia suspect this process facilitates tissue elongation, and helps compensate for any cells that are lost from the tissue during the division process as the embryo folds in on itself.

“This study is unique because it connects mechanical forces to the specific molecular cues like Pins that coordinate cell division,” Camuglia explains. “The factors that shape a developing embryo are critical to understand, because all organisms — from fruit flies to humans — experience these driving forces.” This intersection between physics and biology, she says, is what makes the study so exciting.”

Video: Groups of cells divide in a coordinated and oriented manner in the fruit fly embryo.
Top Image: Enrichment of cortical cues at one end of the cell coordinates the orientation of the mitotic spindle. Credit: Jaclyn Camuglia

Citation:
“Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation”
eLife, online 07/07/2022, DOI: https://doi.org/10.7554/eLife.78779
Jaclyn Camuglia, Soline Chanet, and Adam C Martin