Research Area: Stem Cell and Developmental Biology

Decoding development

Despite being 2,000 miles apart, two researchers are devising deep learning algorithms to predict embryonic tissue folding.

Raleigh McElvery
May 18, 2020

Since March when MIT’s new COVID-19 policies took effect, the research labs on campus have been vacant, save a skeleton crew of essential workers. Despite being separated from their benches, microscopes, and pipets, biologists have devised creative solutions to continue working remotely. In one lab, a postdoc and an undergraduate are using their time at home to develop a deep learning algorithm to spot hidden clues about embryonic development.

Professor Adam Martin’s lab studies the fruit fly embryo, which consists of a single layer of cells encircling a yolk core about three hours after fertilization. Within the next few minutes, a band of cells on the surface furrows inward, forming a critical fold that helps determine where the cells will go and what roles they will eventually play.

Postdoc Hannah Yevick has spent most of her time in the Martin lab focusing on the protein myosin, which forms a network of connections that links cells together and helps generate the force needed to fold the embryo. With her eye to the microscope, she’s been investigating how this ball of cells compensates for damage and continues to fold correctly despite occasional disruptions to the myosin network. But it remains unclear how cells coordinate to overcome such impediments, and what factors besides myosin aid the process. Yevick began to wonder if there was a way to extract hidden clues from her microscope pictures that would predict which embryos would develop properly and which would not.

Deep learning, a type of machine learning, has become a popular tool to detect and classify visual data. Just like the brain, deep learning algorithms run on sets of interconnecting nodes that can be trained to distinguish features and predict outcomes. (For example, differentiating a cat from a dog, or recognizing a friend in a Facebook picture.) Before an algorithm can complete these tasks on its own, however, researchers must train it using a set of practice images. Some scientists are training algorithms intended for use in clinical settings, from AI-based chatbots to diagnostic assistance that helps predict whether a patient has cancer.

Man in shirt and tie
Prateek Kalakuntla, a third-year Course 20 major and Course 6 minor.

“Deep learning shows great promise in clinical settings,” Yevick says, “and that got me thinking about ways to bring it back into the lab, and dig deeper into fundamental questions about development.”

Although she conducts computational analyses to decipher her microscopy images of fly embryos, Yevick hadn’t considered leveraging deep learning algorithms to predict developmental outcomes until a few months ago. In fact, she’d never tried any machine learning techniques at all. Sitting at home sans microscope during a pandemic seemed like the perfect time to start.

Right before the Martin lab dispersed per MIT’s COVID-19 policies, Yevick gained a collaborator: undergraduate researcher Prateek Kalakuntla, a third-year Course 20 (Biological Engineering) major with a minor in Course 6 (Electrical Engineering and Computer Science). He returned to his home in Dallas, Texas while Yevick remained in Cambridge.

“I was looking for a new project, and this seemed like the perfect one to start from home,” Kalakuntla says. “Our experience of practical machine learning is limited, so we assign ourselves research to do individually, and then check in with each other regularly.”

Despite nearly 2,000 miles separating them, the duo meets via Zoom once or twice a week to discuss their progress. They have been taking online tutorials in deep learning, provided by MIT OpenCourseWare, and gleaning information from scientific papers and colleagues.

“When you’re learning new things, it’s fun to have someone else to bounce ideas off,” Yevick says. “We’re exploring machine learning and gaining basic skills that will help us shape and address important questions moving forward.”

Two people at computer
Adam Martin and Hannah Yevick examine a video of a folding embryo.

At the moment, they’re practicing by constructing codes pulled from online exercises. Eventually, they aim to create and train their own algorithm and feed it images of embryos, taken just a few minutes into the stage of development where the layer of cells begins to furrow inward. The algorithm will then predict whether or not the embryo will develop correctly over the course of the 15-minute folding process.

Yevick and Kalakuntla intend to collect images from the entire lab, gathering as much data as possible to teach the algorithm to discern successful folds from failed ones. But they hope the algorithm will eventually teach them a thing or two as well — namely, where and when critical proteins are working to influence development.

“We’re feeding the algorithm entire images, but it’s pulling out what it deems to be the most interesting parts,” Kalakuntla says. “These could be specific regions of tissue or time periods that provide hints about the necessary proteins and cell shapes, which we can then analyze further.”

Although they’ll train their algorithm on images of fruit fly embryos, Kalakuntla hopes their model could eventually be applied to other organisms like mice or frogs — and even predict outcomes for data sets lacking images of later developmental stages.

“Machine learning can give us a birds-eye view of how cells coordinate collective movements, and show us ‘signatures’ that we might not have otherwise considered,” Yevick says. “Working remotely is certainly not ideal, but it’s given us the chance to gain new skills like this.”

Stretch and relax
Lucy Jakub
April 13, 2020

Consider the fruit fly, Drosophila melanogaster. Though it’s only a couple of millimeters long, its body is intricately complex. But it began, as most animals do, as an amorphous blastula—a hollow ball of dividing cells. During embryonic development, the structures of the body emerge as cells multiply and change shape, sculpting tissues into the mature forms dictated by the genetic code. One of the first structural changes is gastrulation, during which the blastula becomes multilayered with an ectoderm, mesoderm, and endoderm. In the developing fly, this occurs through a tissue folding mechanism. The first fold is the invagination of the mesoderm, when cells fated to become muscles contract and curl inward, leaving the cells fated to become skin on the exterior.

Biologists have traditionally focused on how cells generate force to understand cell and tissue shape change. But researchers at MIT have found that there’s another important, though often overlooked, player in tissue folding: cell division, or mitosis. By combining live-imaging with genetic mutations of developing Drosophila embryos, they observed that cell constriction and division can act together to promote folding, and that mitosis interferes with the accumulation of motor proteins that allows cells to generate force.

“What the results tell us is that the cell cycle and cell division might need to be tightly regulated relative to other shape changes that are happening in the tissue,” says Adam Martin, the senior author of the study published on March 13 in Molecular Biology of the Cell. “They present a new paradigm for thinking about how tissue shape might be regulated during development, and provide insight into what might cause birth defects in humans.” Clint Ko PhD ’20, a former graduate student in the Martin lab, was lead author of the study.

In 2000, three different labs identified a genetic mutation that caused premature cell division in developing Drosophila embryos. They found that the gene tribbles, named for the fuzzy, rapidly-reproducing animals in Star Trek, regulates cell division in the mesoderm of the fly, ensuring that cells only divide at the appropriate time. When that gene is deleted, cell division occurs before the mesoderm can properly internalize. What was notable about this mutant was that the blastula never folded, and remained a ball of cells instead of an envelope of tissue with an inside and an outside. This observation led researchers to believe that cell cycle regulation somehow regulates tissue folding. But, at the time, there was no live-imaging technology to visualize how cells changed in the developing embryo.

By using a fluorescent protein to visualize chromosome condensation, which marks the start of mitosis and the cell’s preparation for division, the researchers were able to use live-cell imaging to see how premature division might be interfering with cell constriction. When a cell prepares to divide, it expands and becomes rounded, before elongating—shape changes that exert force on neighboring cells. But something else was going on, too.Specifically, researchers in the Martin lab wanted to see what was happening to networks of the motor protein myosin, which allows cells to contract, in the tribbles mutant. Myosin is the same protein that allows our muscle tissue to contract when we flex. To facilitate tissue folding in the developing fly, myosin is concentrated at the top of the cells in the mesoderm, where they form the surface of the blastula. As this myosin constricts, the outer surface of the tissue shrinks and contracts inward.

“We noticed that when the cells are dividing, the apical myosin networks that are present disappear,” says Ko. Cells that had already begun to contract relaxed when they entered mitosis, indicating that it’s a loss of contractility in the tribbles mutant that prevents folding. The researchers suspect that this reversal occurs because mitosis disrupts signaling from the gene RhoA, which regulates contractility and cell shape changes during development. An undergraduate researcher in the lab, Prateek Kalakuntla, showed that regulation of RhoA changes at the start of mitosis.

“Initially we were just curious about the tribbles mutant,” says Ko. “But then we started exploring other ways of looking at how cell divisions affect myosin accumulation in cells.” They utilized a mutation in which the gene fog, which is located upstream of myosin activation on the genome, was overexpressed. (Fog is short for “folded gastrulation.”) Cells in the Drosophila ectoderm don’t normally contract, but with ectopic fog overexpression, those cells activated myosin, too. With live-cell imaging, the researchers observed furrows develop across the ectoderm.

“It was a bit unexpected to see these tissues folding when they shouldn’t be folding,” says Ko. Specifically, the folds occurred along the boundaries of mitotic domains, regions of spatiotemporally patterned cell divisions that occur in coordinated pulses. “That led to this sort of novel idea that cell divisions—particularly when they’re in this pattern where they’re interspersed between contractile cells—can actually promote tissue folding.”

Understanding the genetic basis for tissue folding, and how our genes control the development of specific bodily features, can help determine how birth defects arise during development. “If cell cycle control is misregulated during development, it could actually alter the shape of that tissue,” says Martin. The study paves the way for further research into how exactly the location of myosin in the cell is regulated, and how it is affected at the molecular level by cell division.

“We observed that when these cells enter mitosis, the localization of myosin activators changes. But we don’t really know how it changes,” says Ko. “That would be a pretty interesting research problem, especially considering that it’s such an integral part of force generation in cells.” Kalakuntla has begun investigating what controls these regulators, which will be an avenue of future research for the lab.

Top image: Myosin networks, in green, contract cell membranes in the mesoderm of a developing Drosophila embryo. Credit: Martin lab.

Citation:
“Apical Constriction Reversal upon Mitotic Entry Underlies Different Morphogenetic Outcomes of Cell Division”
Molecular Biology of the Cell, online March 4, 2020, DOI: 10.1091/mbc.E19-12-0673
Clint S. Ko, Prateek Kalakuntla, and Adam C. Martin

Chimeras offer a new way to study childhood cancer in mice
Eva Frederick | Whitehead Institute
March 5, 2020

In a new paper published March 5 in the journal Cell Stem Cell, researchers in Whitehead Institute Member Rudolf Jaenisch’s lab introduce a new way to model human neuroblastoma tumors in mice using chimeras — in this case, mice that have been modified to have human cells in parts of their nervous systems. “This may serve as a unique model that you can use to study the dynamic of immune cells within human tumors,” says Malkiel Cohen, a postdoc in Jaenisch’s lab and the first author of the paper.

Neuroblastoma is a rare and unpredictable form of childhood cancer that affects around 800 young children in the US each year. Neuroblastoma tumors often occur in parts of the sympathetic nervous system, which includes the nerves that run parallel to the spinal cord and the adrenal medulla, part of the glands that produce hormones such as adrenaline. Neuroblastoma is notoriously hard to study primarily because of its disparate behavior: the tumors often shrink spontaneously in infants, while in toddlers they are highly aggressive and often fatal. “The seeds for the cancer are sown during fetal life,” says Rani George, MD, PhD, an associate professor of pediatrics at Harvard Medical School and a neuroblastoma researcher and physician at Dana-Farber Cancer Institute and Boston Children’s Hospital, and a co-senior author on the paper. “For obvious reasons, you can’t really study the development of these tumors in humans.”

Until now, researchers didn’t have many realistic ways to study these tumors in animal models, either. They could create transgenic mice with cancer-causing genes, but the resulting tumors were mouse tumors, not human ones, and had some key differences. Another method involved taking human tumor cells and implanting them in a mouse — a process called xenotransplantation — but that only worked in mice with compromised immune systems, and didn’t allow researchers to study how the tumors formed in the first place or how they interacted with a fully functioning immune system. “This is where we think the new model is a perfect fit,” said Stefani Spranger, PhD, an assistant professor of Biology at the Massachusetts Institute of Technology (MIT) and the Koch Institute for Integrative Cancer Research at MIT and a co-senior author on the paper.

Human-mouse chimeras have been used in the past to study Alzheimer’s disease and brain development. Jaenisch, who is also a professor of biology at MIT, and his lab had been working for years to create chimeric mice with human cells in the neural crest — the group of developing cells that go on to form parts of the sympathetic nervous system — and published their findings in 2016. “In this study, we hoped to use these mice with human neural crest cells to study how neuroblastoma tumors form and respond to immune system attacks,” Jaenisch says.

To create these chimeric mice, Cohen and coauthors at MIT’s Koch Institute and the Dana-Farber Cancer Institute first engineered human pluripotent stem cells to express two genes known to be abnormal in neuroblastoma, MYCN and mutated ALK, and modified them so they became neural crest cells, from which human neuroblastomas are derived. The genes could be turned on and off with the addition of doxycycline, an antibiotic. They also inserted the gene for eGFP, a brightly glowing fluorescent protein originally isolated from jellyfish. This would allow the team to tell whether the cells were spreading correctly through the bodies of the mice, and would cause any tumors originating from these human cells to be luminous under fluorescent light.

The researchers injected mouse embryos with these cells, and watched over the course of embryonic development as the cells proliferated and human tissues crept into the developing peripheral nervous systems of the tiny mice. To activate the two cancer-causing genes, researchers spiked the pregnant mice’ water with doxycycline, and over the next few days in utero — and in the weeks and months after the pups were born — the researchers inspected the chimeras to see whether tumors would appear.

Over the course of the next 15 months, 14% of the mice developed tumors — 29 mice out of 198 total. The tumors mostly appeared in the space behind the abdominal cavity close to the nerves along the spinal cord, although one mouse developed a tumor in its adrenal gland. Both locations are common places for human children to develop neuroblastoma. The researchers took samples of the tumors and found that they contained the glowing protein eGFP, which confirmed that they were of human origin.

When the team examined the growth patterns of the cancerous cells, they found that the tumors were remarkably similar to human neuroblastomas: they contained cell markers typical of human tumors, and some grew in characteristic rosette shapes — features that did not often appear in tumors implanted in immunocompromised mice through xenotransplantation.

Having successfully induced neuroblastoma tumors in the chimeric mice, the researchers took the opportunity to examine the communication between immune cells and tumors — and specifically, how the tumors evaded destruction by anti-cancer immune cells called T cells. One factor that makes human neuroblastomas and many other cancers dangerous is their sophisticated strategy for avoiding being destroyed by T cells. “The cancer tricks the immune system,” Cohen says.  By activating chemical signals that exhaust the T cells, the tumors effectively weaken their attack. The tumors in the chimeric mice, Cohen found, use a similar method to human neuroblastomas to evade immune responses.

Cohen and others plan to test the new system’s potential for modeling other cancers such as melanoma, and to use it to investigate potential treatments for neuroblastoma patients. “The obvious next step is to study how treatment of these tumors will allow these chimeric mice to be cured,” he says. “This is a model that will allow us to approach not only how to get rid of the tumor, but also to fix the immune system and recover those exhausted T cells, allowing them to fight back and deplete the tumor.”

This research was funded by the National Institutes of Health, as well as grants from the Emerald Foundation, the LEO Foundation, the Melanoma Research Foundation, and the St. Baldrick’s Foundation.

Citation: Cohen, M., et al. Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras. Cell Stem Cell. March 5, 2020. DOI: https://doi.org/10.1016/j.stem.2020.02.001

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Written by Eva Frederick

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Researchers discover an RNA-related function for a DNA repair enzyme
Raleigh McElvery
February 26, 2020

After decades of speculation, researchers have demonstrated that a classical DNA repair enzyme also binds to RNA, affecting blood cell development.

The DNA-dependent protein kinase, otherwise known as DNA-PK, is one of the most important enzymes that binds DNA and repairs double-stranded breaks. This mode of repair is essential for generating receptors that help the immune system fight off intruders. But DNA-PK doesn’t just bind DNA; it also binds RNA. Although researchers have known this for decades, they didn’t fully understand what kinds of RNAs DNA-PK bound in mammalian cells, or the physiological consequences of this binding.

In a new study published on February 26 in Nature, researchers from MIT and Columbia University have uncovered a mechanism whereby DNA-PK binds to the RNA involved in ribosome assembly. Ribosomes — the cell’s protein synthesis machinery — ensure that stem cells give rise to enough red blood cells. The researchers found that mutating DNA-PK prevents the ribosomes from being built properly, which prevents blood cells from doing their job and leads to blood disorders.

“This is the first biochemical evidence of DNA-PK assembly and activation by RNA inside cells,” says Eliezer Calo, a co-senior author and assistant professor in MIT’s Department of Biology. “We’re still trying to determine the mechanisms that regulate protein synthesis in stem cells, and this study reveals one of them.”

Co-senior author, Shan Zha from Columbia University, had previously studied DNA-PK’s role in DNA repair by generating a mouse model that carried enzymatically-dead versions of DNA-PK. While using this model to investigate tumorigenesis, Zha’s lab found these mutant mice developed a form of blood cancer known as myeloid disease. At the same time, another research group showed that mutations in DNA-PK also led to anemia, which occurs when the body does not have enough healthy red blood cells

Neither myeloid disease nor anemia could be easily explained by DNA repair defects alone. However, the two blood disorders did share some similarities to diseases caused by ribosome defects. Because DNA-PK resides in the same organelle where ribosomes are made, the Zha and Calo labs began to wonder whether DNA-PK could bind to the RNA there and control ribosome biogenesis.

In this new study, the Zha lab found that DNA-PK mutations impaired protein translation in red blood cell progenitors, which might contribute to anemia. In parallel, the Calo lab was investigating ribosomal RNA processing and was surprised to find that DNA-PK seemed to be implicated in ribosome assembly. The Calo lab then mapped all the RNAs in cells that bind DNA-PK. The enzyme unexpectedly attached to U3, a small RNA that helps assemble one of the subunits comprising the ribosome. Once it binds U3, DNA-PK can transfer a phosphate group to several specific sites on one of its own subunits. If DNA-PK is defective and cannot transfer the phosphate group, protein synthesis in blood stem cells is impaired, eventually causing anemia.

DNA-PK is essential for cellular viability in nearly all human cell lines, including cancer cell lines, while many other proteins involved in same DNA repair pathway are dispensable. Several studies, including one published by the Zha lab, showed that DNA-PK protein levels are 50-fold higher in common human cell lines than in rodent cell lines. The researchers do not yet know why the enzyme is so critical, but they suspect it might have to do with its ability to bind RNA. “We are interested in exploring whether this new role for DNA-PK could provide clues to this puzzle,” Zha says.

Calo says their findings could also have important implications for cancer treatment, because DNA-PK has emerged as a promising target for cancer therapy. Drugs that inhibit DNA-PK could prevent cancer cells from repairing their DNA and replicating successfully, but he warns these same remedies could also impact stem cell function. The next step is to explore DNA-PK’s other RNA binding targets and the related molecular pathways.

“We’ve demonstrated that DNA-PK has an entirely separate role that has nothing to do with DNA repair,” Calo says. “In the future, we’re excited to learn what additional RNA-related duties it may have beyond stem cell maintenance.”

Top Image: Ribosomes are assembled in the nucleoli (shown here in human cells).

Citation:
“DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis”
Nature, online February 26, 2020, DOI: 10.1038/s41586-020-2041-2
Zhengping Shao, Ryan A. Flynn, Jennifer L. Crowe, Yimeng Zhu, Jialiang Liang, Wenxia Jiang, Fardin Aryan, Patrick Aoude, Carolyn R. Bertozzi, Verna M. Estes, Brian J. Lee, Govind Bhagat, Shan Zha, and Eliezer Calo

Mary Gehring: Using flowering plants to explore epigenetic inheritance

Biologist’s studies illuminate a control system that influences how traits are passed along to new generations.

Anne Trafton | MIT News Office
December 16, 2019

Genes passed down from generation to generation play a significant role in determining the traits of every organism. In recent decades, scientists have discovered that another layer of control, known as epigenetics, is also critically important in shaping those characteristics.

Those added controls often work through chemical modifications of genes or other sections of DNA, which influence how easily those genes can be expressed by a cell. Many of those modifications are similar across species, allowing scientists to use plants as an experimental model to uncover how epigenetic processes work.

“Many of the epigenetic phenomena we know about were first discovered in plants, and in terms of understanding the molecular mechanisms, work on plants has also led the way,” says Mary Gehring, an associate professor of biology and a member of MIT’s Whitehead Institute for Biomedical Research.

Gehring’s studies of the small flowering plant Arabidopsis thaliana have revealed many of the mechanisms that underlie epigenetic control, shedding light on how these modifications can be passed from generation to generation.

“We’re trying to understand how epigenetic information is used during plant growth and development, and looking at the dynamics of epigenetic information through development within a single generation, between generations, and on an evolutionary timescale,” she says.

Seeds of discovery

Gehring, who grew up in a rural area of northern Michigan, became interested in plant biology as a student at Williams College, where she had followed her older sister. During her junior year at Williams, she took a class in plant growth and development and ended up working in the lab of the professor who taught the course. There, she studied how development of Arabidopsis is influenced by plant hormones called auxins.

After graduation, Gehring went to work for an environmental consulting company near Washington, but she soon decided that she wanted to go to graduate school to continue studying plant biology. She enrolled at the University of California at Berkeley, where she joined a lab that was studying how different genetic mutations affect the development of seeds.

That lab, led by Robert Fischer, was one of the first to discover an epigenetic phenomenon called gene imprinting in plants. Gene imprinting occurs when an organism expresses only the maternal or paternal version of particular gene. This phenomenon has been seen in flowering plants and mammals.

Gehring’s task was to try to figure out the mechanism behind this phenomenon, focusing on an Arabidopsis imprinted gene called MEDEA. She found that this type of imprinting is achieved by DNA demethylation, a process of removing chemical modifications from the maternal version of the gene, effectively turning it on.

After finishing her PhD in 2005, she worked as a postdoc at the Fred Hutchinson Cancer Research Center, in the lab of Steven Henikoff. There, she began doing larger, genome-scale studies in which she could examine epigenetic markers for many genes at once, instead of one at a time.

During that time, she began studying some of the topics she continues to investigate now, including regulation of the enzymes that control DNA methylation, as well as regulation of “transposable elements.” Also known as “jumping genes,” these sequences of DNA can change their position within the genome, sometimes to promote their own expression at the expense of the organism. Cells often use methylation to silence these genes if they generate harmful mutations.

Patterns of inheritance

After her postdoc, Gehring was drawn to MIT by “how passionate people are about what they’re working on, whether that’s biology or another subject.”

“Boston, especially MIT and Whitehead, is a great environment for science,” she says. “It seemed like there were a lot of opportunities to get really smart and talented students in the lab and have interesting colleagues to talk with.”

When Gehring joined the Whitehead Institute in 2010, she was the only plant biologist on the faculty, but she has since been joined by Associate Professor Jing-Ke Weng.

Her lab now focuses primarily on questions such as how maternal and paternal parents contribute to reproduction, and how their differing interests can lead to genetic conflicts. Gene imprinting is one way that this conflict is played out. Gehring has also discovered that small noncoding RNA molecules play an important role in imprinting and other aspects of inheritance by directing epigenetic modifications such as DNA methylation.

“One thing we’ve found is that this noncoding RNA pathway seems to control the transcriptional dosage of seeds, that is, how many of the transcripts are from the maternally inherited genome and how many from the paternally inherited genome. Not just for imprinted genes, but also more broadly for genes that aren’t imprinted,” Gehring says.

She has also identified a genetic circuit that controls an enzyme that is required to help patterns of DNA methylation get passed from parent to offspring. When this circuit is disrupted, the methylation state changes and unusual traits can appear. In one case, she found that the plants’ leaves become curled after a few generations of disrupted methylation.

“You need this genetic circuit in order to maintain stable methylation patterns. If you don’t, then what you start to see is that the plants develop some phenotypes that get worse over generational time,” she says.

Many of the epigenetic phenomena that Gehring studies in plants are similar to those seen in animals, including humans. Because of those similarities, plant biology has made significant contributions to scientists’ understanding of epigenetics. The phenomenon of epigenomic imprinting was first discovered in plants, in the 1970s, and many other epigenetic phenomena first seen in plants have also been found in mammals, although the molecular details often vary.

“There are a lot of similarities among epigenetic control in flowering plants and mammals, and fungi as well,” Gehring says. “Some of the pathways are plant-specific, like the noncoding RNA pathway that we study, where small noncoding RNAs direct DNA methylation, but small RNAs directing silencing via chromatin is something that happens in many other systems as well.”

Committed to reproduction
Greta Friar | Whitehead Institute
November 21, 2019

Cambridge, MA – Early in mammalian embryonic development, long before the organism’s ultimate form has taken shape, a precious subset of its cells are set aside for future use in creating offspring. This task bestows on that subset of cells a special kind of immortality. While the majority of the embryo’s cells go on to construct the growing body, and their journey begins and ends in that body, the cells that are set aside, called primordial germ cells (PGCs), will eventually produce sperm and eggs, which will in turn produce a new body—and so the circle of life continues.

An embryo’s earliest cells are pluripotent, meaning they have the potential to develop into many different cell types—for example, heart, brain, blood—but the descendants of these cells eventually become committed to a specific identity, after which each can only produce one type of cell. Scientists have long believed that when PGCs are set aside, they are immediately committed to the path of producing egg and sperm cells. However, new research from Whitehead Institute Director David Page, also a professor of biology at the Massachusetts Institute of Technology (MIT) and a Howard Hughes Medical Institute investigator, and postdoctoral researcher Peter Nicholls, suggests that instead, the primordial germ cells’ fate remains flexible for much longer: until much closer to the end of embryonic development. In most species, PGCs are set aside long before the gonads—the testes or ovaries—form, and then later travel to these developing gonads where they will ultimately produce sex cells. Page and Nicholls have found evidence that the fate of these PGCs remains flexible until shortly after they reach the gonads. Their findings, which appear in the journal PNAS on November 21, deepen our understanding of the process of reproduction.

“A fundamental question in biology is how we get from one generation to the next,” Page says. “And the cells that are tasked with producing the next generation are an important part of that story.”

Establishing a new timeline for when PGCs become committed could also shed light on the origins of some reproductive tract cancers, including testicular cancer, the incidence of which is on the rise, and which is already the most commonly diagnosed cancer in young men.

Although PGCs are precursors of sperm and eggs, they also share many features with pluripotent cells, like embryonic stem cells. If migrating PGCs are isolated and cultured like embryonic stem cells, the PGCs show indicators of pluripotency, and are able to spontaneously form tumors containing multiple cell types—a trademark of pluripotent cells. Page and Nicholls found evidence confirming that shortly after the PGCs reach the gonads, they lose this capacity to produce pluripotent cell lines, and their ability for tumor formation. From that point on, the PGCs can only develop into eggs and sperm, no matter their environment.

The researchers then set out to identify the gene that prompts PGCs to become committed to produce only eggs or sperm. First, Nicholls identified a set of genes that are activated around the time that PGCs enter the gonads in mice and humans, and of those, focused on the genes that appeared to have equivalents involved in sex cell commitment across a variety of animals, not just in mammals. He then narrowed in on one of these genes, Dazl, as the single gene necessary for PGCs to become irrevocably committed to their path as sex cells. Nicholls found that when the Dazl gene is deleted from mice, PGCs travel to the gonads but don’t develop into committed precursors of egg and sperm, suggesting that Dazl is the key ingredient in the recipe for sex cell commitment.

In the absence of Dazl, PGCs remain uncommitted, and in some cases, will form gonadal tumors. The researchers argue, based on their findings, that testicular cancer and other gonadal cancers may develop from PGCs that have travelled to the gonads, but have not properly committed to becoming sex cells and so are prone to forming tumors. In Dazl-deficient mice, which had large amounts of uncommitted PGCs, more than one out of four males developed testicular tumors at a young age. The early onset of the tumors is consistent with that seen in children and men with testicular cancer, most of whom are under 45 years old.

The researchers also found that female Dazl-deficient mice developed gonadal tumors, though at a lower rate than males. Further research demonstrated that the testis environment is particularly favorable for tumor formation from uncommitted PGCs.

“Testicular cancer is on the rise for reasons not yet known, and our findings suggest that the cancer has embryonic origins,” Page says. “Understanding the nature of primordial germ cells will be important for investigating and addressing this disease.”

The researchers hope that, along with providing insights into gonadal cancers, their work could help improve the derivation of eggs and sperm from stem cells in the lab. Figuring out the specifics of the process for sex cell commitment should allow researchers recreate it in a dish. Nicholls is also excited about the evolutionary implications of the work: he found evidence that a similar process of sex cell commitment occurs across a wide variety of species. In particular, research with DAZL-deficient pigs—whose last common ancestor with mice and humans lived 95 million years ago—provides strong evidence that this DAZL-dependent process has been in play since the early days of modern mammals.

“This work completely shifts the timing for when sex cells become committed in mammals,” Nicholls says. “Furthermore, our data suggest that a common set of factors might operate in sex cell commitment not only in mammals, but perhaps across all vertebrates, regardless of how the primordial germ cells are first established.”

This work was supported by the Howard Hughes Medical Institute; a Hope Funds for Cancer Research Fellowship; an Early Career Fellowship; a DFG grant; a research grant from Biogen, Inc.; the National Natural Science Foundation of China; and a National Institutes of Health SBIR award.

Written by Greta Friar

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David Page’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 Howard Hughes Medical Institute Investigator and a Professor of Biology at the Massachusetts Institute of Technology.

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Citation:

“Mammalian germ cells are determined after PGC colonization of the nascent gonad”

PNAS, online, Nov 21, 2019, DOI: 10.1073/pnas.1910733116

Peter K. Nicholls (1), Hubert Schorle (1,2),  Sahin Naqvi (1,3), Yueh-Chiang Hu (1,4), Fan Yuting (1,5), Michelle A. Carmell (1), Ina Dobrinski (6), Adrienne L. Watson (7), Daniel F. Carlson (7), Scott C. Fahrenkrug (7) and David C. Page (1,3,8)

1. Whitehead Institute, Cambridge, MA 02142, USA

2. Department of Developmental Pathology, Institute of Pathology, University of Bonn Medical

School, Bonn 53127, Germany

3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

4. Divisions of Developmental Biology and Reproductive Sciences, Cincinnati Children’s

Hospital Medical Center, Cincinnati, OH 45229, USA

5. Reproductive Medicine Center, Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou,

510655, China

6. Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary

Medicine, University of Calgary, Alberta, T2N 4N1, Canada

7. Recombinetics, Inc., Saint Paul, MN 55104, USA

8. Howard Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142, USA

Study links certain metabolites to stem cell function in the intestine

Molecules called ketone bodies may improve stem cells’ ability to regenerate new intestinal tissue.

Anne Trafton | MIT News Office
August 22, 2019

MIT biologists have discovered an unexpected effect of a ketogenic, or fat-rich, diet: They showed that high levels of ketone bodies, molecules produced by the breakdown of fat, help the intestine to maintain a large pool of adult stem cells, which are crucial for keeping the intestinal lining healthy.

The researchers also found that intestinal stem cells produce unusually high levels of ketone bodies even in the absence of a high-fat diet. These ketone bodies activate a well-known signaling pathway called Notch, which has previously been shown to help regulate stem cell differentiation.

“Ketone bodies are one of the first examples of how a metabolite instructs stem cell fate in the intestine,” says Omer Yilmaz, the Eisen and Chang Career Development Associate Professor of Biology and a member of MIT’s Koch Institute for Integrative Cancer Research. “These ketone bodies, which are normally thought to play a critical role in energy maintenance during times of nutritional stress, engage the Notch pathway to enhance stem cell function. Changes in ketone body levels in different nutritional states or diets enable stem cells to adapt to different physiologies.”

In a study of mice, the researchers found that a ketogenic diet gave intestinal stem cells a regenerative boost that made them better able to recover from damage to the intestinal lining, compared to the stem cells of mice on a regular diet.

Yilmaz is the senior author of the study, which appears in the Aug. 22 issue of Cell. MIT postdoc Chia-Wei Cheng is the paper’s lead author.

An unexpected role

Adult stem cells, which can differentiate into many different cell types, are found in tissues throughout the body. These stem cells are particularly important in the intestine because the intestinal lining is replaced every few days. Yilmaz’ lab has previously shown that fasting enhances stem cell function in aged mice, and that a high-fat diet can stimulate rapid growth of stem cell populations in the intestine.

In this study, the research team wanted to study the possible role of metabolism in the function of intestinal stem cells. By analyzing gene expression data, Cheng discovered that several enzymes involved in the production of ketone bodies are more abundant in intestinal stem cells than in other types of cells.

When a very high-fat diet is consumed, cells use these enzymes to break down fat into ketone bodies, which the body can use for fuel in the absence of carbohydrates. However, because these enzymes are so active in intestinal stem cells, these cells have unusually high ketone body levels even when a normal diet is consumed.

To their surprise, the researchers found that the ketones stimulate the Notch signaling pathway, which is known to be critical for regulating stem cell functions such as regenerating damaged tissue.

“Intestinal stem cells can generate ketone bodies by themselves, and use them to sustain their own stemness through fine-tuning a hardwired developmental pathway that controls cell lineage and fate,” Cheng says.

In mice, the researchers showed that a ketogenic diet enhanced this effect, and mice on such a diet were better able to regenerate new intestinal tissue. When the researchers fed the mice a high-sugar diet, they saw the opposite effect: Ketone production and stem cell function both declined.

Stem cell function

The study helps to answer some questions raised by Yilmaz’ previous work showing that both fasting and high-fat diets enhance intestinal stem cell function. The new findings suggest that stimulating ketogenesis through any kind of diet that limits carbohydrate intake helps promote stem cell proliferation.

“Ketone bodies become highly induced in the intestine during periods of food deprivation and play an important role in the process of preserving and enhancing stem cell activity,” Yilmaz says. “When food isn’t readily available, it might be that the intestine needs to preserve stem cell function so that when nutrients become replete, you have a pool of very active stem cells that can go on to repopulate the cells of the intestine.”

The findings suggest that a ketogenic diet, which would drive ketone body production in the intestine, might be helpful for repairing damage to the intestinal lining, which can occur in cancer patients receiving radiation or chemotherapy treatments, Yilmaz says.

The researchers now plan to study whether adult stem cells in other types of tissue use ketone bodies to regulate their function. Another key question is whether ketone-induced stem cell activity could be linked to cancer development, because there is evidence that some tumors in the intestines and other tissues arise from stem cells.

“If an intervention drives stem cell proliferation, a population of cells that serve as the origin of some tumors, could such an intervention possibly elevate cancer risk? That’s something we want to understand,” Yilmaz says. “What role do these ketone bodies play in the early steps of tumor formation, and can driving this pathway too much, either through diet or small molecule mimetics, impact cancer formation? We just don’t know the answer to those questions.”

The research was funded by the National Institutes of Health, a V Foundation V Scholar Award, a Sidney Kimmel Scholar Award, a Pew-Stewart Trust Scholar Award, the MIT Stem Cell Initiative, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the Koch Institute Dana Farber/Harvard Cancer Center Bridge Project, and the American Federation of Aging Research.