Research Area: Stem Cell and Developmental Biology
Graduate student Marlis Denk-Lobnig investigates the biological forces that shape developing tissue to dictate form and function.
Raleigh McElvery
March 22, 2019
A few hours after fertilization, the fruit fly embryo is just a hollow sphere, slightly oblong in shape, until a band of cells on its surface furrows inward to form a new layer. This folding process takes only 15 minutes, but it’s critical for determining where the cells will go and what roles they will eventually play. In humans, errors in tissue folding can result in diseases like spina bifida, where the spine never fully closes.
Fourth-year graduate student Marlis Denk-Lobnig watches this gastrulation process occurring in fly embryos in real time, tagging molecules with fluorescent proteins to probe the forces that eventually shape a fully-formed organism. Every day, she gets to witness new life unfold — literally.
Denk-Lobnig spends most of her time with her eye to a microscope or generating genetic crosses in the “fly room” where she keeps her stocks — rows of tubes containing light brown insect food that emits an unmistakable odor, despite being corked with cotton swabs. Inside each neatly labeled container, scores of tiny flies mill around as they lay eggs and feast.
Given that her mother trained in chemistry and her father in physics, “it didn’t take much creativity to get into science early on.” Denk-Lobnig enjoyed physics throughout high school, but also maintained a keen interest in biology, which became more pronounced after she was diagnosed with an autoimmune disease affecting her thyroid and adrenal glands.
“In some ways, the question of how your own body works is the most tangible question to ask,” she says. “It’s fascinating to connect everyday experiences with mechanisms, and studying biology and medicine seemed like a powerful way to have a direct impact on life.”
She majored in molecular medicine at Georg August University in Göttingen, Germany, located several hours from her childhood home in Heidelberg. Inspired by a summer internship with MIT Biology alum and Rockefeller professor Cori Bargmann PhD ’87, Denk-Lobnig centered her undergraduate thesis on the role glial cells play in disease.
She graduated after only three years, the typical duration in Germany, and spent the next several months traveling and applying to graduate schools. She also visited Nepal, where she taught visual and performing arts — and a bit of gymnastics — at a local boarding school.
When she began at MIT in 2015, Denk-Lobnig took the opportunity to blend her expertise in biology with a renewed enthusiasm for physics. Although she is a full-fledged member of the Department of Biology, she is simultaneously enrolled in the interdepartmental Biophysics Certificate Program.
“Not many people know that MIT has a thriving biophysics community,” she says. “It’s a mix of mechanical engineers, chemists, biologists, and physicists. There are specific course requirements, and we go on retreats and participate in seminars to share our research and discuss collaborations.”
As a member of Adam Martin’s lab, Denk-Lobnig studies the cellular forces that shape tissue form and function. Martin is also affiliated with the certificate program, and was one of the faculty members who initially interviewed Denk-Lobnig for the graduate program.
“Biophysics is all about finding elegant explanations for everyday phenomena, and I really enjoy thinking about physical principles and how they apply to biological problems,” Denk-Lobnig says. “The methods we use in the Martin lab are also incredibly visual. You can literally see a fruit fly embryo fold, and watch as a sheet of cells furrows inside the embryo to form a second layer, which is important for development. It’s both informative and aesthetically pleasing.”
Denk-Lobnig began by focusing on a single molecule called Cumberland-GAP (C-GAP), which regulates one of the many proteins in charge of tissue folding: myosin. Myosin is responsible for muscle contraction, among other duties. With its characteristic forked shape — two “heads” protruding from string-like “tail” domain — myosin can appear to walk along the cell’s scaffolding, sometimes transporting cargo. Denk-Lobnig, though, is most interested in myosin’s ability to pull on developing tissue and create a fold.
Right before graduating, one of Denk-Lobnig’s former labmates noticed that depleting C-GAP seemed to alter the concentration (or “gradient”) of myosin across the tissue. Since this finding pertained to the very regulator she was studying, it piqued Denk-Lobnig’s interest. She wanted to know how molecules like C-GAP might influence myosin and impact folding, and her scope widened from the molecular level to include the entire tissue.
It’s unlikely, she says, that myosin is pulling with equal force across the tissue — “that wouldn’t constrict the sheet of cells very efficiently.” Instead, there’s probably more myosin in middle and less towards the edges, which contracts the cells in the middle of the sheet to a greater degree and creates the curvature that forms the crease of the fold. In the fruit fly, gastrulation occurs just three hours after the eggs are laid. Because the folding happens at the surface of the embryo, there’s no need for dissection to witness the entire event through a microscope.
Denk-Lobnig has begun exploring other regulators besides C-GAP to analyze their effects on the myosin gradient and cell curvature. She was one of the first members of the lab to introduce CRISPR-Cas9 into their testing protocol, and is currently the only one experimenting with optogenetic techniques. She also regularly participates in the lab book club, which features classics like The Bell Jar and One Hundred Years of Solitude.
Outside of lab, Denk-Lobnig serves as the president of MIT’s women’s club gymnastics team, volunteers to help run weekly Gymnastics Special Olympics events, and sings in a graduate student choir. She is also a member of the department’s peer support program, bioREFs.
Long-term, she plans to stay in academia and delve further into physics-based methods, like modeling and coding. If she could find a project that’s just as visual as her current work in the Martin lab, “that would definitely be a plus.”
Posted 3.21.19
Greta Friar | Whitehead Institute
February 28, 2019
Cambridge, MA — Red blood cells are the most plentiful cell type in our blood and play a vital role transporting oxygen around our body and waste carbon dioxide to the lungs. Injuries that cause significant blood loss prod the body to secrete a one-two punch of signals – stress steroids and erythropoietin (EPO) – that stimulates red blood cell production in the bone marrow. These signals help immature cells along the path to becoming mature red blood cells. In a healthy individual, as much as half of their blood volume can be replenished within a week. Despite its importance, scientists are still working to unravel many aspects of red blood cell production. In a paper published online February 28 in the journal Developmental Cell, Whitehead Institute researchers describe work that refines our understanding of how stress steroids, in particular glucocorticoids, increase red blood cell production and how early red blood cell progenitors progress to the next stage of maturation toward mature red blood cells.
These findings are especially important for patients with certain types of anemia that do not respond to clinical use of EPO to stimulate the final stages of red cell formation, such as Diamond-Blackfan anemia (DBA). In this rare genetic disorder usually diagnosed in infants and toddlers, the bone marrow does not produce enough of early red blood cell progenitors, called burst forming unit-erythroids (BFU-Es), that respond to glucocorticoids. In both healthy people and DBA patients, these BFU-Es divide several times and mature before developing into colony forming unit-erythroids (CFU-Es) that that, stimulated by EPO, repeatedly divide and produce immature red blood cells that are released from the bone marrow into the blood. But the lack of BFU-Es in DBA patients means that the glucocorticoid signal has a limited target, and the cascade of cell divisions that should result in plentiful red blood cells is contracted and instead produces an insufficient amount.
One of the standard treatments for DBA is boosting red blood cell production with high doses of synthetic glucocorticoids, such as prednisone or prednisolone. But the mechanisms behind these drugs and their normal counterparts are not well understood. By deciphering the mechanisms by which glucocorticoids stimulate red cell formation, scientists may be able identify other ways to stoke CFU-E production – and ultimately red blood cell production – without synthetic glucocorticoids and the harsh side effects that their long-term use can cause, such as poor growth in children, brittle bones, muscle weakness, diabetes, and eye problems.
For more than two decades, Whitehead Institute Founding Member Harvey Lodish, has investigated glucocorticoids’ effects on red blood cell production. In his lab’s most recent paper, co-first authors and postdocs Hojun Li and Anirudh Natarajan, describe their research, which helps decipher how BFU-Es progress through their maturation process.
For more than 30 years, scientists have thought that glucocorticoids bestowed BFU-Es with a stem cell-like ability to divide until an unknown switch flipped and the cells matured to the CFU-E stage. By looking at gene expression in individual BFU-Es from normal mice, Li and Natarajan determined that the developmental progression from BFU-E to CFU-E is instead a smooth continuum. They also found that in mice glucocorticoids exert the greatest effect on the BFU-Es at the beginning of the developmental continuum by slowing their developmental progression without affecting their cell division rate. In other words glucocorticoids are able to effectively compensate for a decreased number of BFU-Es by allowing those that do exist, while still immature, to divide more times, producing in mice up to 14 times more CFU-Es than BFU-Es lacking exposure to glucocorticoids.
Li and Natarajan’s work reveals previously unknown aspects of the mechanism by which glucocorticoids stimulate red blood cell production. With this better understanding, scientists are one step closer toward pinpointing more targeted approaches to treat certain anemias such as DBA.
This work was supported by the National Institutes of Health (NIH grants DK06834813 and HL032262-25) and the American Society of Hematology and was performed with the assistance of Whitehead Institute’s Fluorescence Activated Cell Scanning (FACS) Facility and Genome Technology Core facility. Styliani Markoulaki, head of the Whitehead Genetically Engineered Models Center, and M. Inmaculada Barrasa of Bioinformatics and Research Computing (BaRC) are also co-authors of the paper.
Written by Nicole Giese Rura
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Harvey Lodish’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology and a professor of biological engineering at Massachusetts Institute of Technology (MIT). Lodish serves as a paid consultant and owns equity in Rubius, a biotech company that seeks to exploit the use of modified red blood cells for therapeutic applications.
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Citation:
“Rate of Progression through a Continuum of Transit-Amplifying Progenitor Cell States Regulates Blood Cell Production”
Developmental Cell, online February 28, 2019, https://doi.org/10.1016/j.devcel.2019.01.026
Hojun Li*, Anirudh Natarajan*, Jideofor Ezike, M. Inmaculada Barrasa, Yenthanh Le, Zoë A. Feder, Huan Yang, Clement Ma, Styliani Markoulaki, and Harvey F. Lodish.
*These authors contributed equally
The dynamic process is critical to embryonic development and other cellular phenomena.
Anne Trafton | MIT News Office
February 1, 2019
Embryonic development is tightly regulated by genes that control how body parts form. One of the key responsibilities of these genes is to make sure that tissues fold into the correct shapes, forming structures that will become the spine, brain, and other body parts.
During the 1970s and ’80s, the field of embryonic development focused mainly on identifying the genes that control this process. More recently, many biologists have shifted toward investigating the physics behind the tissue movements that occur during development, and how those movements affect the shape of tissues, says Adam Martin, an MIT associate professor of biology.
Martin, who recently earned tenure, has made key discoveries in how tissue folding is controlled by the movement of cells’ internal scaffolding, known as the cytoskeleton. Such discoveries can not only shed light on how tissues form, including how birth defects such as spina bifida occur, but may also help guide scientists who are working on engineering artificial human tissues.
“We’d like to understand the molecular mechanisms that tune how forces are generated by cells in a tissue, such that the tissue then gets into a proper shape,” Martin says. “It’s important that we understand fundamental mechanisms that are in play when tissues are getting sculpted in development, so that we can then harness that knowledge to engineer tissues outside of the body.”
Cellular forces
Martin grew up in Rochester, New York, where both of his parents were teachers. As a biology major at nearby Cornell University, he became interested in genetics and development. He went on to graduate school at the University of California at Berkeley, thinking he would study the genes that control embryonic development.
However, while in his PhD program, Martin became interested in a different phenomenon — the role of the cytoskeleton in a process called endocytosis. Cells use endocytosis to absorb many different kinds of molecules, such as hormones or growth factors.
“I was interested in what generates the force to promote this internalization,” Martin says.
He discovered that the force is generated by the assembly of arrays of actin filaments. These filaments tug on a section of the cell membrane, pulling it inward so that the membrane encloses the molecule being absorbed. He also found that myosin, a protein that can act as a motor and controls muscle contractions, helps to control the assembly of actin filaments.
After finishing his PhD, Martin hoped to find a way to combine his study of cytoskeleton mechanics with his interest in developmental biology. As a postdoc at Princeton University, he started to study the phenomenon of tissue folding in fruit fly embryonic development, which is now one of the main research areas of his lab at MIT. Tissue folding is a ubiquitous shape change in tissues to convert a planar sheet of cells into a three-dimensional structure, such as a tube.
In developing fruit fly embryos, tissue folding invaginates cells that will form internal structures in the fly. This folding process is similar to tissue folding events in vertebrates, such as neural tube formation. The neural tube, which is the precursor to the vertebrate spinal cord and brain, begins as a sheet of cells that must fold over and “zip” itself up along a seam to form a tube. Problems with this process can lead to spina bifida, a birth defect that results from an incomplete closing of the backbone.
When Martin began working in this area, scientists had already discovered many of the transcription factors (proteins that turn on networks of specific genes) that control the folding of the neural tube. However, little was known about the mechanics of this folding.
“We didn’t know what types of forces those transcription factors generate, or what the mechanisms were that generated the force,” he says.
He discovered that the accumulation of myosin helps cells lined up in a row to become bottle-shaped, causing the top layer of the tissue to pucker inward and create a fold in the tissue. More recently, he found that myosin is turned on and off in these cells in a dynamic way, by a protein called RhoA.
“What we found is there’s essentially an oscillator running in the cells, and you get a cycle of this signaling protein, RhoA, that’s being switched on and off in a cyclical manner,” Martin says. “When you don’t have the dynamics, the tissue still tries to contract, but it falls apart.”
He also found that the dynamics of this myosin activity can be disrupted by depleting genes that have been linked to spina bifida.
Breaking free
Another important cellular process that relies on tissue folding is the epithelial-mesenchymal transition (EMT). This occurs during embryonic development when cells gain the ability to break free and move to a new location. It is also believed to occur when cancer cells metastasize from tumors to seed new tumors in other parts of the body.
During embryonic development, cells lined up in a row need to orient themselves so that when they divide, both daughter cells remain in the row. Martin has shown that when the mechanism that enables the cells to align correctly is disrupted, one of the daughter cells will be squeezed out of the tissue.
“This has been proposed as one way you can get an epithelial-to-mesenchymal transition, where you have cells dissociate from native tissue,” Martin says. He now plans to further study what happens to the cells that get squeezed out during the EMT.
In addition to these projects, he is also collaborating with Jörn Dunkel, an MIT associate professor of mathematics, to map the network connections between the myosin proteins that control tissue folding during development. “That project really highlights the benefits of getting people from diverse backgrounds to analyze a problem,” Martin says.
Findings could help inform new therapies, improve diagnosis.
David Orenstein | Picower Institute for Learning and Memory
January 18, 2019
A new study by researchers at the Picower Institute for Learning and Memory at MIT finds that the protein CPG2 is significantly less abundant in the brains of people with bipolar disorder (BD) and shows how specific mutations in the SYNE1 gene that encodes the protein undermine its expression and its function in neurons.
Led by Elly Nedivi, professor in MIT’s departments of Biology and Brain and Cognitive Sciences, and former postdoc Mette Rathje, the study goes beyond merely reporting associations between genetic variations and psychiatric disease. Instead, the team’s analysis and experiments show how a set of genetic differences in patients with bipolar disorder can lead to specific physiological dysfunction for neural circuit connections, or synapses, in the brain.
The mechanistic detail and specificity of the findings provide new and potentially important information for developing novel treatment strategies and for improving diagnostics, Nedivi says.
“It’s a rare situation where people have been able to link mutations genetically associated with increased risk of a mental health disorder to the underlying cellular dysfunction,” says Nedivi, senior author of the study online in Molecular Psychiatry. “For bipolar disorder this might be the one and only.”
The researchers are not suggesting that the CPG2-related variations in SYNE1 are “the cause” of bipolar disorder, but rather that they likely contribute significantly to susceptibility to the disease. Notably, they found that sometimes combinations of the variants, rather than single genetic differences, were required for significant dysfunction to become apparent in laboratory models.
“Our data fit a genetic architecture of BD, likely involving clusters of both regulatory and protein-coding variants, whose combined contribution to phenotype is an important piece of a puzzle containing other risk and protective factors influencing BD susceptibility,” the authors wrote.
CPG2 in the bipolar brain
During years of fundamental studies of synapses, Nedivi discovered CPG2, a protein expressed in response to neural activity, that helps regulate the number of receptors for the neurotransmitter glutamate at excitatory synapses. Regulation of glutamate receptor numbers is a key mechanism for modulating the strength of connections in brain circuits. When genetic studies identified SYNE1 as a risk gene specific to bipolar disorder, Nedivi’s team recognized the opportunity to shed light into the cellular mechanisms of this devastating neuropsychiatric disorder typified by recurring episodes of mania and depression.
For the new study, Rathje led the charge to investigate how CPG2 may be different in people with the disease. To do that, she collected samples of postmortem brain tissue from six brain banks. The samples included tissue from people who had been diagnosed with bipolar disorder, people who had neuropsychiatric disorders with comorbid symptoms such as depression or schizophrenia, and people who did not have any of those illnesses. Only in samples from people with bipolar disorder was CPG2 significantly lower. Other key synaptic proteins were not uniquely lower in bipolar patients.
“Our findings show a specific correlation between low CPG2 levels and incidence of BD that is not shared with schizophrenia or major depression patients,” the authors wrote.
From there they used deep-sequencing techniques on the same brain samples to look for genetic variations in the SYNE1 regions of BD patients with reduced CPG2 levels. They specifically looked at ones located in regions of the gene that could regulate expression of CPG2 and therefore its abundance.
Meanwhile, they also combed through genomic databases to identify genetic variants in regions of the gene that code CPG2. Those mutations could adversely affect how the protein is built and functions.
Examining effects
The researchers then conducted a series of experiments to test the physiological consequences of both the regulatory and protein coding variants found in BD patients.
To test effects of non-coding variants on CPG2 expression, they cloned the CPG2 promoter regions from the human SYNE1 gene and attached them to a “reporter” that would measure how effective they were in directing protein expression in cultured neurons. They then compared these to the same regions cloned from BD patients that contained specific variants individually or in combination. Some did not affect the neurons’ ability to express CPG2 but some did profoundly. In two cases, pairs of variants (but neither of them individually), also reduced CPG2 expression.
Previously Nedivi’s lab showed that human CPG2 can be used to replace rat CPG2 in culture neurons, and that it works the same way to regulate glutamate receptor levels. Using this assay they tested which of the coding variants might cause problems with CPG2’s cellular function. They found specific culprits that either reduced the ability of CPG2 to locate in the “spines” that house excitatory synapses or that decreased the proper cycling of glutamate receptors within synapses.
The findings show how genetic variations associated with BD disrupt the levels and function of a protein crucial to synaptic activity and therefore the health of neural connections. It remains to be shown how these cellular deficits manifest as biopolar disorder.
Nedivi’s lab plans further studies including assessing behavioral implications of difference-making variants in lab animals. Another is to take a deeper look at how variants affect glutamate receptor cycling and whether there are ways to fix it. Finally, she said, she wants to continue investigating human samples to gain a more comprehensive view of how specific combinations of CPG2-affecting variants relate to disease risk and manifestation.
In addition to Rathje and Nedivi, the paper’s other authors are Hannah Waxman, Marc Benoit, Prasad Tammineni, Costin Leu, and Sven Loebrich.
The JPB Foundation, the Gail Steel Fund, the Carlsberg Foundation, the Lundbeck Foundation and the Danish Council for Independent Research funded the study.
Nicole Davis | Whitehead Institute
November 27, 2018
Cambridge, MA. – The ability to regrow missing or damaged body parts is one of the great marvels of modern biology. In an effort to lay bare the biological underpinnings of this phenomenon, scientists at Whitehead Institute have begun to define the core features that are required for regeneration in flatworms. Their research, which appears online November 27 in Cell Reports, reveals that a set of cellular and molecular responses — previously thought to be essential for regeneration following amputations and other major injuries — is in fact dispensable.
“This is a real surprise,” said senior author Peter Reddien, a Member of Whitehead Institute, professor of biology at Massachusetts Institute of Technology, and investigator with the Howard Hughes Medical Institute. “These responses are broad, prominent attributes of tissue regeneration and repair and, a reasonable bet was that they function to bring about regeneration.”
About eight years ago, Reddien and his team described a set of biological activities that are triggered by injuries that remove tissue. Whereas a cut or a scrape removes little if any tissue, more damaging injuries, like amputations, cause significant tissue loss. That missing tissue must be regenerated to ensure the organism retains its proper anatomical proportions.
A series of cellular and molecular activities — known collectively as the missing tissue response — were believed to enable this regeneration to occur. They include the sustained action of genes that respond to injury, a period of intense cell division in areas surrounding the wound, and a general increase in cell death throughout the body. “This happens prominently, not only in planarians but also in other organisms capable of regeneration, so we suspected that the missing tissue response must play a very fundamental role in regeneration,” recalled Reddien.
What types of injuries require the missing tissue response for repair, and what is the function of the missing tissue response in regeneration? Graduate student and first author Aneesha Tewari, Reddien and colleagues, including Sarah Stern and Isaac Oderberg, set out to uncover the answers. This work forms the basis of their latest Cell Reports study.
The researchers harnessed an earlier discovery that a gene known as follistatin is required for the missing tissue response in flatworms (known as planarians). By using molecular tools to inhibit this gene, they could block the missing tissue response and observe what happens under various wound conditions, ranging from minimal (the removal of an eye, for example) to moderate (the removal of the pharynx or part of the head) to significant tissue loss (the removal of a complete side of the body). Remarkably, in every case, the missing tissue was regenerated, albeit much more slowly than it would be otherwise.
“These results tell us that what the missing tissue response is really doing is simply pushing the foot down on the gas pedal — basically accelerating the process of regeneration,” explained Reddien. “If you can’t accelerate, you’ll still get there, it just takes longer.”
Tewari, Reddien, and their colleagues also cracked a thorny mystery surrounding the missing tissue response. Although their results show that it is not required across a wide range of injuries, there is one lingering instance in which regeneration failed to occur when they blocked the missing tissue response: head amputation.
“This was a big puzzle,” said Tewari. “It left us wondering whether or not we could generalize our findings to all types of wounds — is there something special about the head that makes it uniquely dependent on the missing tissue response?”
The answer, it turns out, is no. When follistatin is blocked, a key signaling protein, called Wnt1, kicks into overdrive. And when that happens, the tissue destined to form the head does not receive the positional cues it needs to properly regrow, which means regeneration fails to proceed. But, when both the missing tissue response and Wnt1 are blocked, the head does indeed regenerate, the team uncovered.
Taken together, the researchers’ findings begin to clarify what is essential for regeneration to take place and what is not. “Our study greatly simplifies the picture of what it takes to regenerate,” said Reddien. “And that’s an important step along the path towards dissecting the central elements of regeneration in animals that do regenerate well, like flatworms, and then applying that knowledge to understand what the limits might be in those animals that don’t regenerate as well, like humans.”
This research was supported by the NIH (R01GM080639), the National Science Foundation, the Eleanor Schwartz Charitable Foundation, and the Howard Hughes Medical Institute.
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Peter Reddien’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 Massachusetts Institute of Technology.
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Full citation:
Cell Reports, Vol. 25, Is. 9, P2577-2590.E3, November 27, 2018, DOI:https://doi.org/10.1016/j.celrep.2018.11.004
“Cellular and molecular responses unique to major injury are dispensable for planarian regeneration”
Aneesha G. Tewari (1,2), Sarah R. Stern (1,2), Isaac M. Oderberg (1,2,4), and Peter W. Reddien (1,2,3)
1.Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
4. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Greta Friar | Whitehead Institute
November 19, 2018
CAMBRIDGE, Mass. – Different subpopulations of a plant species can have distinct traits, varying in size, seed count, coloration, and so on. The primary source of this variation is genes: different versions of a gene can lead to different traits. However, genes are not the only determinant of such traits, and researchers are learning more about another contributor: epigenetics. Epigenetic factors are things that regulate genes, altering their expression, and like genes they can be inherited from generation to generation, even though they are independent of the actual DNA sequences of the genes.
One epigenetic mechanism is DNA methylation, in which the addition of chemical tags called methyl groups can turn genes on or off. Genes that share the identical DNA sequence but have different patterns of methylation are called epialleles. Several studies have shown that epialleles, like different versions of genes, can cause differences in traits between plant subpopulations, or strains, but whether genetic factors are also at play can be difficult to determine.
The lab of Whitehead Member Mary Gehring, who is also an associate professor at Massachusetts Institute of Technology, has described evidence that epialleles alone can lead to different heritable traits in plants. In research published online November 5 in the journal PLoS Genetics, Gehring, along with co-first authors and former lab members Daniela Pignatta and Katherine Novitzky, showed that altering the methylation state of the gene HDG3 in different strains of the plant Arabidopsis thaliana was enough to cause changes in seed weight and in the timing of certain aspects of seed development.
In plants, methylation states of genes change most frequently during seed development, when genes are switched on or off to progress development of the organism. This period is also when a conflict of interest arises in the genome of each seed between the parts inherited from its mother and father. The mother plants produce seeds fertilized by different fathers at the same time. It’s in the mother’s interest to give an equal share of nutrients to each seed—to have many smaller seeds. But it’s in the father’s interest for its seed to get the most nutrients and grow larger. This conflict plays out through an epigenetic mechanism called imprinting, in which, through differential methylation between the father’s and mother’s copies of a gene, one parent’s copy is silenced in the offspring so that only the other parent’s version of the gene is expressed.
The gene HDG3 is imprinted in one strain of Arabidopsis so that only the father’s copy is expressed. Gehring and her team found that when the strain loses its paternal imprinting, the timing of seed development is affected and the plant ends up with smaller seeds. This is consistent with the theory of imprinting: When the father’s genes have the advantage, the seeds are larger than when both parents’ genes are equally expressed.
Other experiments tested the effect of either activating or silencing HDG3 by methylation in a variety of scenarios, both in a separate strain of Arabidopsis in which the gene starts off silenced, as well as in crosses between the two strains. The researchers found that altering the methylation state of the gene was sufficient to affect seed size and the timing of seed development. In the crosses, these traits depended on whether the paternal copy of the gene came from the strain in which HDG3 was normally silenced or the strain in which it was normally activated.
Altogether these experiments demonstrate a link between changes in methylation state and differences in seed development and size. This suggests that epialleles in natural populations function much like variations in genes, creating heritable traits that differ within the larger population.
This work was funded by the National Science Foundation (NSF grant 1453459).
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Mary Gehring’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. She is also an associate professor of biology at Massachusetts Institute of Technology.
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Full citation:
“A variably imprinted epiallele impacts seed development”
PLoS Genetics, online November 5 2018, https://doi.org/10.1371/journal.pgen.1007469
Daniela Pignatta (1,3), Katherine Novitzky (1,3), P. R. V. Satyaki (1), Mary Gehring (1,2)
1. Whitehead Institute for Biomedical Research, Cambridge, MA, United States of America
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States of America
3. These authors contributed equally to this work.
Greta Friar | Whitehead Institute
October 9, 2018
Cambridge, Mass. — Brain development is a delicately choreographed dance in which cell division and differentiation into mature cell types must be performed in the right balance for normal growth. In order to better understand factors affecting brain development, Whitehead Institute researchers investigated a genetic mutation that leads to a brain-specific developmental disorder in spite of the gene’s prevalent expression in other cell types.
Kinetochore null protein 1 (KNL1) acts throughout the body during cell division to help ensure the accurate segregation of chromosomes into each daughter cell. A mutation in the KNL1 gene caused by a single change in its DNA sequence leads to microcephaly, a condition in which the brain fails to properly develop, causing babies to be born with small heads, often accompanied by intellectual disabilities and other health problems. In an article published online October 9 in the journal Cell Reports, Whitehead Institute Founding Member Rudolf Jaenisch and colleagues investigated how this KNL1 mutation can lead to microcephaly without affecting other cell types, providing important insights into the underlying basis of microcephaly and the role that KNL1 normally plays in brain development.
“The key question we were interested in was why, if the gene is ubiquitously expressed, is there a brain-specific phenotype,” says Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology.
Jaenisch lab graduate student Attya Omer Javed, a co-first author on the paper along with past lab members Yun Li and Julien Muffat, used CRISPR-Cas9 to recreate the mutation—a point mutation, or one-letter change in the DNA sequence of the KNL1 gene—in several different cell types derived from human stem cells in the lab. Of the three cell types tested, they found that only the neural progenitor cells, early stage cells that become brain cells, appeared to be affected.
As the brain develops, each neural progenitor can either keep dividing to increase the overall number of cells in the brain, or it can mature into a differentiated brain cell, at which point it is no longer able to divide. For a healthy brain to develop, there needs to be a careful balance between these two processes of proliferation and differentiation. If the progenitors take too long to differentiate, the developing brain won’t have the specific cells it needs to assemble. But if all of the cells differentiate too quickly, before they can divide, there will be a shortage of cells and the brain will be too small.
“Neural progenitors are going through many cell cycles, dividing quickly during brain development. Even a small defect could accumulate to have a huge impact,” Omer Javed says.
The researchers discovered that neural progenitors with the KNL1 mutation differentiated prematurely at the cost of proliferation, resulting in the small brain size that characterizes microcephaly. The brain cells with the mutation also were at a greater risk of cell death, disruption of the cell cycle, ending up with the wrong number of chromosomes, and malfunctions during attempted cell division.
KNL1’s role is in the kinetochore, an assembly of proteins that operate during mitosis to attach chromosomes to the machinery that will pull them apart into the daughter cells. This is why the KNL1 mutation negatively affects cell division. Co-author and Whitehead Member Iain Cheesemanhelped identify KNL1’s role in the kinetochore as a postdoctoral researcher years ago, and his expertise provided an opportunity for collaboration between his lab and Jaenisch’s.
“I have always found it interesting that inherited mutations to the kinetochore seem to lead to microcephaly,” Cheeseman says. “Investigating KNL1 together was an exciting chance to combine our labs’ diverse scientific knowledge.”
In order for the researchers to study the cells in an environment that more closely mimicked a human brain, they used a 3D cell culture technique to grow organoids made up of neural progenitors. Omer Javed found that the neural progenitors were extremely sensitive, as the organoids with the mutation expressed the microcephaly phenotype after as little as two weeks of growth.
Omer Javed then looked for differences between neural progenitors and the other cell types that would explain the brain-specific effects of the mutation. Even with the mutation, the KNL1 gene appeared able to make a functioning protein, explaining its lack of effect on the other cell types. So Omer Javed turned her attention to factors involved in regulating gene expression. For many of our genes to be expressed, first sections called introns must be removed, or spliced out, in order for the correct DNA sequence to then be read into RNA and then translated into a functional protein.
Omer Javed found that the KNL1 mutation created a site for splicing inhibitors to bind and silence the KNL1 gene by preventing it from being read into RNA. She also found a disparity in the level of a protein involved in this process between the cell types: the inhibitory splicing protein hnRNPA1 was much more prevalent in neural progenitors than elsewhere. When hnRNPA1 came across the site caused by the mutation, it prevented the gene from being expressed. The high quantity of hnRNPA1 in neural progenitors appears to be the main factor mediating the brain-specific effects of the mutation.
The work complements and extends previous investigations by the researchers into how neural progenitor proliferation may have contributed to the evolution of large human brains, as well as studies investigating why neural progenitors are so vulnerable to the Zika virus, which has been associated with microcephaly. Given their work suggesting that KNL1 could be a regulator of brain size, Omer Javed hopes that future research will reveal its role in the evolution of the human brain.
This research was supported by Boehringer Ingelheim Fonds, the Simons Foundation, the International Rett Syndrome Foundation, Brain & Behavior Research Foundation, the European Leukodystrophy Association, the National Institutes of Health (NIH grants HD 045022, R37-CA084198 and 1U19AI131135-01). Jaenisch is a cofounder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.
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Rudolf Jaenisch’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.
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Full citation:
“Microcephaly modeling of kinetochore mutation reveals a brain-specific phenotype”
Cell Reports, online October 9, 2018
Attya Omer (1,2,8), Yun Li (2,3,4,8), Julien Muffat (2,4,5,8), Kuan-Chung Su (2), Malkiel A. Cohen (2), Tenzin Lungjangwa (2), Patrick Aubourg (1,6), Iain M. Cheeseman (2,7), and Rudolf Jaenisch (2,7).
1. Université Paris-Saclay, ED 569, 5 Rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France
2. Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA
3. Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M4G 0A4, Canada
4. Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
5. Program in Neurosciences and Mental Health, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
6. INSERM U1169, CHU Bicêtre Paris Sud, Le Kremlin-Bicêtre, France.
7. Department of Biology, MIT, 31 Ames Street, Cambridge, MA 02139, USA
8. These authors contributed equally
Nicole Davis
August 2, 2018
The human Y chromosome is, in many ways, a study in contrasts. For decades, scientists have struggled to dissect its evolution in part because it does not have a genetic partner (or homolog), as all of the other human chromosomes do. That solitary existence means the Y chromosome is subject to some unusual evolutionary pressures. For example, it does not swap genetic material with a homologous chromosome — a practice known as recombination that other chromosomes follow — along the lion’s share of its length. However, its lack of recombination presents a unique opportunity: Because so much of its own genetic material stays put, scientists can trace the history of individual human Y chromosomes much further back in time than other chromosomes — in fact, they can go as far back as the data will allow.
That is precisely the approach taken by a team of Whitehead Institute researchers, led by Whitehead Institute Director David Page, who is also a professor of biology at the Massachusetts Institute of Technology and a Howard Hughes Medical Institute investigator. Their work is published in the August 2nd online issue of the American Journal of Human Genetics. Page and his colleagues, including graduate student and first author Levi Teitz, set out to examine a series of regions on the Y chromosome called amplicons — vast stretches of DNA, from tens of thousands to millions of nucleotides in length, which are present in two or more copies per chromosome. While the DNA contained in amplicons is often highly repetitive, it also houses biologically important genes. Although the precise functions of many of these genes remains to be determined, some have been found to play important roles in the development of sperm cells and testicular cancer. However, the amplicons vary drastically among species, so scientists cannot look to other organisms such as mice or chimpanzees to help reconstruct their past.
Page’s team zeroed in on these amplicons. Specifically, they looked at how the number of amplicon copies varies from one person’s Y chromosome to another. The researchers developed sophisticated computational tools to analyze DNA sequencing data collected from more than 1,200 males as part of the 1000 Genomes Project. What they discovered was quite surprising. Although the amplicons are quite variable, they found that overall, the configuration of amplicon copies on the Y chromosome has been painstakingly maintained over the last 300,000 years of human evolution. That means that despite the high level of mutation the chromosome experiences, evolutionary forces work to counteract this change and preserve its ancestral structure.
More work is needed to determine which aspects of the amplicons’ structure are important for chromosome biology, and in turn proper male development and fertility. However, the efforts of Teitz, Page, and their colleagues shed new light on the unusual tricks the solo chromosome uses to maintain its genomic integrity.
This research is supported by the National Institutes of Health and the Howard Hughes Medical Institute.
Written by Nicole Davis
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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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Full citation:
“Selection Has Countered High Mutability to Preserve the Ancestral Copy Number of Y Chromosome Amplicons in Diverse Human Lineages”
American Journal of Human Genetics, online August 2, 2018.
Levi S. Teitz (1,2), Tatyana Pyntikova (1), Helen Skaletsky (1,3), and David C. Page (1,2,3).
1. Whitehead Institute, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
3. Howard Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142, USA
A drug treatment that mimics fasting can also provide the same benefit, study finds.
Anne Trafton | MIT News Office
May 1, 2018
As people age, their intestinal stem cells begin to lose their ability to regenerate. These stem cells are the source for all new intestinal cells, so this decline can make it more difficult to recover from gastrointestinal infections or other conditions that affect the intestine.
This age-related loss of stem cell function can be reversed by a 24-hour fast, according to a new study from MIT biologists. The researchers found that fasting dramatically improves stem cells’ ability to regenerate, in both aged and young mice.
In fasting mice, cells begin breaking down fatty acids instead of glucose, a change that stimulates the stem cells to become more regenerative. The researchers found that they could also boost regeneration with a molecule that activates the same metabolic switch. Such an intervention could potentially help older people recovering from GI infections or cancer patients undergoing chemotherapy, the researchers say.
“Fasting has many effects in the intestine, which include boosting regeneration as well as potential uses in any type of ailment that impinges on the intestine, such as infections or cancers,” says Omer Yilmaz, an MIT assistant professor of biology, a member of the Koch Institute for Integrative Cancer Research, and one of the senior authors of the study. “Understanding how fasting improves overall health, including the role of adult stem cells in intestinal regeneration, in repair, and in aging, is a fundamental interest of my laboratory.”
David Sabatini, an MIT professor of biology and member of the Whitehead Institute for Biomedical Research and the Koch Institute, is also a senior author of the paper, which appears in the May 3 issue of Cell Stem Cell.
“This study provided evidence that fasting induces a metabolic switch in the intestinal stem cells, from utilizing carbohydrates to burning fat,” Sabatini says. “Interestingly, switching these cells to fatty acid oxidation enhanced their function significantly. Pharmacological targeting of this pathway may provide a therapeutic opportunity to improve tissue homeostasis in age-associated pathologies.”
The paper’s lead authors are Whitehead Institute postdoc Maria Mihaylova and Koch Institute postdoc Chia-Wei Cheng.
Boosting regeneration
For many decades, scientists have known that low caloric intake is linked with enhanced longevity in humans and other organisms. Yilmaz and his colleagues were interested in exploring how fasting exerts its effects at the molecular level, specifically in the intestine.
Intestinal stem cells are responsible for maintaining the lining of the intestine, which typically renews itself every five days. When an injury or infection occurs, stem cells are key to repairing any damage. As people age, the regenerative abilities of these intestinal stem cells decline, so it takes longer for the intestine to recover.
“Intestinal stem cells are the workhorses of the intestine that give rise to more stem cells and to all of the various differentiated cell types of the intestine. Notably, during aging, intestinal stem function declines, which impairs the ability of the intestine to repair itself after damage,” Yilmaz says. “In this line of investigation, we focused on understanding how a 24-hour fast enhances the function of young and old intestinal stem cells.”
After mice fasted for 24 hours, the researchers removed intestinal stem cells and grew them in a culture dish, allowing them to determine whether the cells can give rise to “mini-intestines” known as organoids.
The researchers found that stem cells from the fasting mice doubled their regenerative capacity.
“It was very obvious that fasting had this really immense effect on the ability of intestinal crypts to form more organoids, which is stem-cell-driven,” Mihaylova says. “This was something that we saw in both the young mice and the aged mice, and we really wanted to understand the molecular mechanisms driving this.”
Metabolic switch
Further studies, including sequencing the messenger RNA of stem cells from the mice that fasted, revealed that fasting induces cells to switch from their usual metabolism, which burns carbohydrates such as sugars, to metabolizing fatty acids. This switch occurs through the activation of transcription factors called PPARs, which turn on many genes that are involved in metabolizing fatty acids.
The researchers found that if they turned off this pathway, fasting could no longer boost regeneration. They now plan to study how this metabolic switch provokes stem cells to enhance their regenerative abilities.
They also found that they could reproduce the beneficial effects of fasting by treating mice with a molecule that mimics the effects of PPARs. “That was also very surprising,” Cheng says. “Just activating one metabolic pathway is sufficient to reverse certain age phenotypes.”
Jared Rutter, a professor of biochemistry at the University of Utah School of Medicine, described the findings as “interesting and important.”
“This paper shows that fasting causes a metabolic change in the stem cells that reside in this organ and thereby changes their behavior to promote more cell division. In a beautiful set of experiments, the authors subvert the system by causing those metabolic changes without fasting and see similar effects,” says Rutter, who was not involved in the research. “This work fits into a rapidly growing field that is demonstrating that nutrition and metabolism has profound effects on the behavior of cells and this can predispose for human disease.”
The findings suggest that drug treatment could stimulate regeneration without requiring patients to fast, which is difficult for most people. One group that could benefit from such treatment is cancer patients who are receiving chemotherapy, which often harms intestinal cells. It could also benefit older people who experience intestinal infections or other gastrointestinal disorders that can damage the lining of the intestine.
The researchers plan to explore the potential effectiveness of such treatments, and they also hope to study whether fasting affects regenerative abilities in stem cells in other types of tissue.
The research was funded by the National Institutes of Health, the V Foundation, a Sidney Kimmel Scholar Award, a Pew-Stewart Trust Scholar Award, the Kathy and Curt Marble Cancer Research Fund, the MIT Stem Cell Initiative through Fondation MIT, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the American Federation of Aging Research, the Damon Runyon Cancer Research Foundation, the Robert Black Charitable Foundation, a Koch Institute Ludwig Postdoctoral Fellowship, a Glenn/AFAR Breakthroughs in Gerontology Award, and the Howard Hughes Medical Institute.