for a two-year fixed term position (possibility for extension) starting as soon as possible. Trial period of six months will be applied.
The position is open in a national research consortium focusing on modeling Finnish disease heritage in mouse. These monogenic diseases are typically rare, but highly enriched in Finland. With CRISPR/Cas9 genome editing we aim at mimicking two rare human fetal syndromes, LCCS1 and LCHAD, and an adult-onset motoneuron disease, the Jokela type of spinal muscular atrophy, in animal models.
Requirements
The successful applicant should have PhD degree and be well acquainted with molecular biology, genome engineering, and most importantly, show self-motivation and enthusiasm towards science, learning new things and technologies. Previous experience in imaging, developmental and cell biology, and tissue processing techniques are acknowledged.
The salary is based on the Finnish university salary (YPJ) system, which take into account previous qualifications, experience and performance.
To apply, please submit a single pdf application including a short cover letter describing your areas of expertise and motivation for the position, CV (max 2 pages), and names and contact information of two to three references through by clicking on “Apply for the position“. Current employees of the University of Helsinki are to submit their applications through the SAP HR portal.
Application deadline is March 29, 2020
For further information, please contact GM-unit Director Satu Kuure, tel. +358 2941 59395, or by mailing
We are pleased to announce the 2020 Santa Cruz Developmental Biology Meeting, which will be held August 8-12 on the beautiful UC Santa Cruz campus. Since 1992, the SCDB meeting has been a premier gathering for developmental biologists from around the world. This year’s meeting is organized around the theme, “Looking to the future: open questions in developmental biology”. Our central goal is to highlight the top challenges facing the field, both conceptual and technical, as we seek fresh insights into the complex mechanisms underlying organismal development, tissue renewal, and the evolution of new forms. The exciting lineup of keynote speakers includes: Philipp Keller, Ruth Lehmann, Roberto Mayor, and Magdalena Zernica-Goetz. There will also be a career-perspective talk from one of the meeting’s founders, Cynthia Kenyon. For the full list of invited speakers, please see the poster.
The SCDB meeting is designed to foster interaction among developmental biologists at all career stages. A sequence of single-platform sessions will be interspersed with career-focused workshops and poster sessions. In addition to invited speakers, at least 16 short talks will be chosen from the submitted abstracts, with preference given to students, postdocs, and junior faculty. Depending on funds, a limited number of travel awards will be available to trainees with financial need.
SCDB Young Investigator Award
If you are a postdoctoral trainee or graduate student, and you would like to be considered for the SCDB Young Investigator Award, we strongly encourage you to submit your CV along with your abstract to scdb2020@gmail.com. The SCDB Young Investigator Awardee will speak in the opening session, receive free registration and housing, a $500 award, and an interview published in Development! This award is sponsored by Development.
Please help us advertise the meeting by printing the attached poster and hanging it in a prominent location, and by forwarding this email to colleagues. If you are a faculty member, please encourage your students and postdocs to submit abstracts and attend the meeting.
This promises to be an exciting meeting and we look forward to seeing you all this summer!
Come for free, participate in our round table discussions, and honor a great scientist and person. Free and open to anyone interested, with an amazing list of speakers and talks, as none of the most renowned scientists in the field that was invited wanted to miss this unique opportunity to honor the amazing Drew Noden.
The following message by Society for Developmental Biology President, Alejandro Sánchez Alvarado (Stowers Institute for Medical Research), was originally posted on the Society for Developmental Biology website February 24, 2020.
Dear Members and Friends of the Society for Developmental Biology,
Alejandro Sánchez Alvarado
We are living in interesting times. Technological advances are moving at neck-breaking speed: artificial intelligence, machine learning, neural networks, quantum computers, advances in optical and electron microscopy, machine miniaturization and gene editing… a list that seems to grow geometrically with every passing month. From the look of it, both lay and scientific publications seem to herald the advent of an age of technological wonders in which previously inaccessible biology is now tangibly within our grasps.
And yet, as a species, we are also facing record challenges. For us biologists at large, and developmental biologists in particular, we are witnessing changes to our planet’s biome that are without precedent in human history. We are witnessing rapid decreases in biodiversity in ecosystems around the world, expansion of species into new territories along with the attendant displacement -even extinction- of endemic organisms that usually follows, and the perturbation of natural processes caused by unnatural warming, acidification and microplastics. The current global environmental deterioration should make us pause, not just as individuals, but particularly as developmental biologists, for where would our field be without the many research organisms we brought from nature into our labs?
Yes, these matters may not appear to be of direct concern to developmental biology. I would argue they should be. For most of the 20th century, modern developmental biology has been limited to the study of a handful of organisms in great part due to the absence of technology that prevented us from taking a more systematic and global approach to understanding fundamental aspects of developmental processes. That limitation is no longer as daunting. Why then should we continue to bring nature into our labs when it is becoming more and more practical to bring our laboratory sophistication to nature and study development there instead?
The accomplishments of our field thus far have been numerous. Developmental Biologists uncovered the fundamental underpinnings of gastrulation, pattern formation, tissue polarity, organogenesis, sex determination, epigenetics, aging, apoptosis and cellular reprogramming, among others. It is easy to forget, yet important to remember that it was Developmental Biologists who first isolated and cultured stem cells and cloned animals. Morphogens, the genetic unravelling of the major signaling pathways by which cells communicate with each other, RNA-mediated genetic interference (RNAi), microRNAs, the fundamental principles of differential gene regulation, all of them discovered by Developmental Biologists. Importantly, Developmental Biologists not only have introduced technological advances to the study of life such as in situ hybridization, genome manipulation and in vivo imaging, but our discipline has also created the context in which to understand human birth defects and disease.
Now, take a second and imagine the immensity of what we can contribute in this century to address pressing global problems by merely expanding our interrogation of development into unknown and/or understudied organisms. Organisms with which, by the way, we share profound evolutionary ties. Simply put, we have but barely scratched the surface of development: we do not know what is possible. The sheer number of species out there waiting to show us what is indeed biologically possible is staggering. Nature has done many more experiments than any of us can fathom, each extant species a unique interpretation of evolution. Equally remarkable is the fact that our species has the necessary tools to decode and understand them all if we so wished. In fact, merely expanding our knowledge of developmental processes in as many species as possible would stand to provide unimagined knowledge, which would result in–in the words of Abraham Flexner–“undreamed-of utility”.
There is an essential role discovery researchers play in the well-being of science and society. The vitality, longevity and, therefore, relevance of the biomedical sciences is ultimately and intricately dependent on the combined efforts of present and future scientists who are averse to neither risk-taking, nor effectively communicating their work to the general public. More than ever, we in the Society for Developmental Biology need to bring a strong and contemporary approach to meet the challenges facing our current members, and to actively participate in national discussions that affect our research, education and outreach activities. That is why our annual meeting will include in its program scores of early career investigators and a selected number of topics not traditionally considered to be part of our discipline. At the very least, I am quite certain that we will likely leave our annual meeting with a host of new ideas and perhaps new ways of looking at our own science.
I believe that the time is coming when developmental biology will be needed to inform and contribute to the study, and more importantly, to the solutions of some of the major problems affecting the health of our planet and its biome. I am delighted to serve as our Society’s president and to continue to drive forward the major goals of the SDB: to support talented investigators at all stages of their careers, stimulate the exchange of scientific information, and promote a research environment where developmental biologists can achieve their best work.
I am very much looking forward to seeing all of you in Chicago in 2020.
Alejandro Sánchez Alvarado, Ph.D.
President, Society for Developmental Biology
For decades, genetics and biochemistry have formed the bedrock of developmental biology. But it turns out that physical forces — the way cells push, pull and squeeze each other — play a huge role, too.
By David Levin
In every parent’s life there comes a singular moment, a knife edge when the world bisects neatly into two parts. There’s life before, and life after. Mine split irreversibly on a frigid December morning, just past 4 a.m., as my son took his first breath and sent me reeling over the threshold of fatherhood, ecstatic and teary-eyed.
Witnessing the birth of my first child opened up whole new avenues of developmental biology for me, as a science writer, to ponder. How could our little miracle, with his wriggling legs, searching gaze, tiny, grasping hands, emerge from a minuscule ball of cells? The basic concept has been drilled into our brains since we were kids: From the union of sperm and egg comes an embryo. But as that egg divides and grows, how does the structure of a body come to be?
It’s a doozy of a question. For more than half a century, the go-to answer has been DNA and biochemistry: Nearly every aspect of development, researchers thought, could be brought on by triggering the right genes at the right times or adding the right chemical signal, creating a cascade of precise transformations. Over the past few decades, though, a growing body of work has cast a spotlight on another starring role in this show. Physical and mechanical forces — things that pull and stretch and twist and squish cells — act alongside genes, and play a major role in everything from the formation of organs to the structure of our limbs to the onset of disease. Serious conditions like certain types of muscular dystrophy and cancer, which seem entirely disparate on the surface, may result from a breakdown of force-sensing mechanisms in cells.
The idea isn’t exactly new. “If you get books on science from the 1890s to the 1910s, everything’s explained mechanically,” says bioengineer and cell biologist Don Ingber, the founding director of Harvard’s Wyss Institute and coauthor of a 2013 overview of the role of mechanics in cell development in the Annual Review of Cell and Developmental Biology. “Then chemistry came in, genetics came in, and the baby was thrown out with the bathwater.”
Armed with technology that can probe and measure forces at a cellular level, more scientists are starting to return to these century-old concepts — and according to Ingber, it’s becoming increasingly clear that physical forces and mechanics are a crucial part of developmental biology.
A key 2006 study in the journal Cell showed just how dramatically force can change cell function. When adult stem cells grew on a soft surface, the paper reported, they started to form something that looked like developing neurons — but when they grew on a hard substrate, they instead took on the shape of osteoblasts, the precursor cells to bone.
“If the cell is round and floppy, or spread out wide and pulled taut, you get totally different functions,” Ingber says. In other words, the forces that a stem cell “feels” on its surface may determine what sort of tissue it will ultimately become.
It’s still not obvious how those minute forces help to shape the formation of all of a body’s requisite parts, however. What signals do they provide that drive the development of limbs? Of organs? Of a brain that harbors thoughts and feelings and fears?
“Those are the mysteries still being uncovered,” says Celeste Nelson, a developmental bioengineer at Princeton University who studies developmental mechanics and coauthored an article on aspects of the topic in the Annual Review of Biomedical Engineering. “The best guess is that yes, mechanical force is important and mechanical stresses are essential. But what exact signals are being sent, and how those are being translated into alterations in cells at these early stages, is still a bit unclear. This is what half of my lab is working on right now.”
Sensing force
The clues to how this all works, Nelson says, might be revealed by studying how cells sense force in the first place — and there are a few different ways a cell can pull this off.
It can use stretch-sensitive ion channels, tiny valves in the cell membrane that let charged atoms like calcium flow in and out, triggering a variety of biochemical signals. Or it can exploit cell-cell junctions, points where certain types of cells are “spot-welded” to their neighbors by globs of proteins.
It could also be leaning on well-studied structures that sense forces during development: clusters of proteins called focal adhesions.
These adhesions anchor cells to the extracellular matrix, the fibrous scaffold that surrounds them. And as that scaffold pulls or stretches a focal adhesion, two things happen: First, proteins inside the adhesion trigger a flood of biochemical signals that alter structural functions inside the cell. Second, and more profoundly, the focal adhesion tugs on a vast structural web inside of cells, called the cytoskeleton.
This mesh acts like a series of I beams and guy wires, supporting the cell membrane and lending the cell its overall shape and form. Crucially, it also connects the focal adhesions to the cell’s nucleus, where its DNA is stored. And that, says Jan Lammerding, a biomedical engineer at Cornell University, could be one of the keys to how physical force influences a cell’s fate.
Any force on the cell’s surface, he says, may be transmitted through the cytoskeleton and passed on to the nucleus, which itself will be stretched or twisted. As the nucleus is squished around, some genes inside it will be newly exposed, and become more active. Others will be suddenly hidden, making them less active. In effect, it may be possible to modify the genetic function of a cell just by yanking on its nucleus.
This illustration shows how forces on the outside of the cell can be sensed deep within it. Mechanical forces in the extracellular matrix push and pull on the exterior of a cell. The forces are detected by the focal adhesion at the cell membrane. The signals are transmitted via the cytoskeleton to the nucleus. There, the detected forces can turn genes on or off by changing the shape of chromosomes or opening nuclear pores to let signaling molecules in.
Those changes go both ways. When the nucleus senses force, proteins inside it called Lamin A pile together en masse, making the organelle stiffer. As the nucleus hardens, it can alter the activity of genes inside it, and some of these changes can set off a chain of events that makes the extracellular matrix stiffer. The matrix, in turn, exerts even stronger forces back into the cell.
This constant push and pull between a cell and its matrix creates a powerful feedback loop. Any changes in the cell will trigger changes in the matrix; any changes in the matrix will trigger even more changes in the cell, and with each iteration, the cell is steered further and further toward its final incarnation, be it a muscle cell, nerve cell, liver cell or any other cell in the body. Sheets or clusters of those differentiated cells expand and fold and, steered by mechanical forces and other inputs, come to sculpt whole organs.
Matters of the heart
In truly poetic fashion, the first organ to emerge in a human embryo is the heart. It starts as a loosely organized tube, explains Dennis Discher, a bioengineer at the University of Pennsylvania, and as it grows, the cells within it harden gradually more each day. That’s a fact cynics everywhere will relish. But according to a 2016 study by Discher and his lab, this hardening may be responsible for the fact that the heart beats at all.
Inside the nascent organ, molecules of Lamin A collect inside the nucleus of cells called cardiac myocytes, causing them to stiffen and elongate. At the same time, the cells exude an increasing amount of collagen, a tough, stringy protein that hardens the extracellular matrix around them. The forces each exerts on the other progressively increase, like a game of cellular tug-of-war, until they reach a critical threshold, Discher says.
As the forces hit that breaking point, a cell somewhere in the mix will spontaneously contract, yank on its neighbors and cause them to contract as well. The resulting chain reaction ripples out among the cardiac cells, triggering an initial heartbeat. “As soon as you get one contracting a certain amount, if it’s connected to all the other cells around it mechanically, then it’s going to set off a reaction called a peristaltic wave that goes on down the line,” Discher says.
Thanks to the constant feedback between forces inside and outside the cells, every pulse of that wave will increase the stiffness of the cardiac cells and the scaffold they sit in, progressively steering the cells’ development. Gradually, that and other inputs will lead them to morph into the specialized, four-chambered muscle that sustains us throughout our lives.
A similar process may happen as lungs and other organs develop. As cells there mature and tissues stiffen, ones that formerly grew in flat sheets (epithelial cells) change their arrangement. This shuffling exerts new forces that can cause the sheets to buckle and fold, like a pair of hands pushing on a tablecloth. Those folds start to rise out from the sheet, creating bud-like shapes that lend organs their initial three-dimensional structures.
“The way you get the patterns of almost all your tissues is through progressive budding or branching in fractal-like ways. All of these patterns are dependent on branches on top of branches, or buds on top of buds,” Ingber says.
Squeezing out a tooth
Given the number of buds that grow on top of each other in a lung or a pancreas or a liver, breaking down how mechanical forces shape such complex structures is no easy task. “In the pancreas or in blood vessels, there are millions of branches and buds, in all three dimensions,” Ingber says. Instead, the best way to get a sense of how the process occurs is to distill it down to the formation of a single bud.
With that in mind, in an elegant 2011 study, he and his colleagues focused on one of the only organs that emerges naturally from a lone bud — a tooth. Rather than needing millions of branches to form its structure, it requires only one. That branch grows in two sections: a part that bows out to form the exterior, and another that pops inward to form roots. By investigating how and when those sections grew, Ingber’s team hoped to unravel the role of physical force in the process.
Researchers had known for years that proteins called motility factors play a role in this process. Usually, they beckon cells to grow toward them like diehard fans flocking toward a celebrity, which squeezes them together into a dense, taut mass. But what if the growth factors were just a means to an end? What if the key to tooth development wasn’t those proteins themselves, but the physical squeezing that they cause as cells crowd together?
To test this idea, Ingber and his colleagues first removed growth factors from the equation, and found that tooth development ground to a halt. Then they tried growing the developing buds between two sheets of a rubbery polymer. Shockingly, as they squeezed those sheets together, the tooth’s normal formation started once again. The scientists later showed that cells started to mineralize and lay down what looked like dentin, a key toothy building block. The force exerted on the cells by the polymer sheets alone was enough to kick off the entire cascade of development.
A tooth bud — the first step in tooth formation — arises in response to pressure on a flat sheet of cells called the dental epithelium. The pressure comes from the accumulation of mobile mesenchymal cells in the tissue below, which pile up like Black Friday shoppers rushing to enter a store but finding the door is locked. The crush of cells triggers a pressure-sensitive response that causes the tooth bud to form.
When force goes awry
The physical forces in and around early cells may spark the formation of organs — but they can also cause the body to fail in spectacular ways. An entire laundry list of seemingly unrelated diseases can be linked to errors in how cells sense force during and after development.
Spina bifida, a condition where an embryonic structure called the neural tube (which eventually forms the brain and spinal cord) fails to close, is connected to a breakdown in cellular forces, new research suggests. Likewise, cleft palate, which occurs when two sides of a person’s palate are deformed, could be caused by force-sensing gone awry at specific points during embryonic development.
Sometimes, the effects of these force-sensing glitches can lead to problems later in life, notes Lammerding. Some types of muscular dystrophy, a disease that results in muscle atrophy over time, may be linked to a breakdown in force-sensing mechanisms within muscle cells.
“Normally if you go to the gym to exercise, the muscles will adapt to the higher mechanical load,” Lammerding says. When muscle tissue feels the physical force of weight pulling on it, the cells inside it lay down more fibers of proteins called actin and myosin in their cytoskeletons. The more of those fibers the muscle cells contain, the stronger they get.
But in one form of the disease called Emery-Dreifuss muscular dystrophy, the cells have trouble sensing the forces stretching them, so they can’t create more muscle fiber in response to the load. As a result, they don’t grow stronger with exercise, and instead atrophy slowly on the bone. Although the symptoms of the disease set in several years after birth, their root cause is a breakdown in cellular force sensing, Lammerding says.
New treatments
For many of these diseases, there’s currently no way to prevent them before they emerge. But bioengineers are searching for new ways to harness the physical forces that shape cell development, and might one day be able to apply them in treatments.
The dream goal, Nelson says, would be to use mechanical force, along with other inputs, to grow tissues and organs on demand in the lab — a development that could do away with long waiting lists for transplants in patients with life-threatening diseases. “Ultimately, what I want to do is know this well enough that we can take a population of maybe a hundred cells, put them in a dish and provide them with two or three initiating stimuli, and have them build themselves into a lung,” she says. Granted, the idea is far-fetched, but based on how quickly research is progressing, she thinks we may see success in a decade or less.
In the meantime, other researchers are working on more immediate applications. Ingber, for one, is taking what he’s learning about the way cellular scaffolds and mechanical forces affect development to create lifelike organ tissues in the lab.
To accomplish that feat, he’s artificially steering the development of cells by growing them on surfaces that mimic the physical and chemical environment of specific organs. With the right mechanical stimulation, he says, cells placed there will develop into a fully formed layer of organ tissue. The result, called an organ-on-a-chip, provides a thin cross section of working kidney, liver or other organ, complete with its own three-dimensional structure.
Devices like these, he adds, could provide accurate models of full-sized organs within tiny pieces of translucent material, letting scientists and drug companies test new treatments for disease more rapidly and effectively than ever before.
Other researchers want to manipulate cellular forces to treat severe wounds. Treena Arinzeh, a biomedical engineer at the New Jersey Institute of Technology, is working to develop new types of materials that could help regrow large sections of missing bone, doing away with the need for metal implants or grafts from cadavers. By putting stem cells on a specially engineered scaffold — a sort of spongy structure made in the lab — she provides them with a bed that has just the right amount of stiffness to coax them into becoming bone and cartilage cells.
This, she hopes, can one day help treat injuries from trauma, weakened bones from osteoporosis, and potentially a whole host of other conditions. “Even though the fundamental science is still developing, we are trying to at least take some of that knowledge and develop therapies out of it,” she says. “We’re probably only right at the tip of the iceberg in terms of what we can do.”
As with any cutting-edge science, the knowledge that emerges from research like this will inform the next questions scientists ask to understand how the body takes shape. Eventually, cellular force-sensing might even become the basis for a radical new interpretation of developmental biology. The research is still in its early stages, and there are still many open mysteries about the ways mechanical forces steer the fate of cells. But the answers to those puzzles — and the new medical treatments that they enable — may surface within our lifetime.
Until then, I won’t need to look very far to revel in the unlikely miracle of the human form. Like all new parents, I’m struck each day by the profound wonder of a tiny person developing before my eyes. The growing confidence of little feet plodding across a bare wood floor; the wailing cries that arrive as new teeth sprout from sensitive gums; the tiniest milestones, new abilities and revelatory moments of a new body coming into its own — all of those made possible by the complex ballet of cells and tissue unfolding to create a small, sweet, curious little boy.
10.1146/knowable-012720-2
David Levin is a science writer based in Boston, where he lives with his wife, cat and rapidly developing toddler. He can be reached at david at davidlevineditorial.com.
This article originally appeared in Knowable Magazine, an independent journalistic endeavor from Annual Reviews. Sign up for the newsletter.
The group of Anna Kicheva at IST Austria is looking for outstanding postdoctoral candidates interested in working on tissue growth control during vertebrate development. Candidates with background in developmental, cell or molecular biology are encouraged to apply.
The Kicheva group studies how cell diversification and tissue growth are controlled during development to produce organs with correct final sizes and pattern. Current research topics in the group include: control of tissue growth and morphogenesis in the neural tube and notochord, formation and interpretation of morphogen gradients, coordination between patterning and growth. We work with mouse and chick embryos, as well as with embryonic stem cells and organoids. The group collaborates extensively with biophysicists to interpret data in the context of rigorous mathematical models. We offer students and postdocs opportunities to obtain experience with diverse techniques and work in a vibrant interdisciplinary environment. For more information, visit http://www.kichevalab.com.
Qualified candidates are expected to have completed a PhD in developmental biology or related discipline and have a proven competitive research track record. Self-motivation, excellent communication skills and very good command of English are also required.
Applicants should send their CV, motivation letter and contact details of 3 referees to anna.kicheva@ist.ac.at.
We are looking for a self-motivated, responsible and organized individual who has previous hand-on research experience. This position will be working on research projects focused on enamel matrix protein extraction and analysis, and mouse colony maintenance. The techniques required for these projects include protein extraction, protein and peptide concentration analyses, SDS-PAGE, western blot, HPLC, enzyme activity assay, PCR and gel electrophoresis, and mouse colony maintenance. This individual must keep an organized detailed lab notebook, and will be playing a strong role in preparing the projects for future publication. If you are interested in this position, please submit: curriculum vitae, emails and telephone numbers of two references online at https://apptrkr.com/1835198. Applicant’s materials must list (pending) qualifications upon submission.
Minimum/Basic Qualifications required at the time of application:
• A bachelor’s or master’s degree in Biochemistry, Chemistry, Molecular and Cell Biology, or a related field
• Research experience with basic biochemistry, protein chemistry and molecular biology
• Previous research experience with mouse genetics and mouse tissue collections for lab research
• Ability to conduct troubleshooting based on technical literature reviews and prepares summary reports
This job description is not designed to contain or be interpreted as a comprehensive inventory of all duties, responsibilities and qualifications required of employees assigned to the job.
UC San Francisco seeks candidates whose experience, teaching, research, or community service has prepared them to contribute to our commitment to diversity and excellence.
The University of California is an Equal Opportunity/Affirmative Action Employer. All qualified applicants will receive consideration for employment without regard to race, color, religion, sex, sexual orientation, gender identity, national origin, disability, age or protected veteran status.
The Development and Transcriptional Control group is interested in defining how cellular diversity arises during development, and the roles that regulatory elements play in this process. By developing quantitative methods to probe cell identity, we are defining how diverse cellular outcomes are generated in the head versus the trunk of the embryo. For further details, please visit our lab website.
Your role and opportunities
You will be eager to work at the intersection of developmental biology and the latest technological advancements in genomics and genome editing. You will have the opportunity to train in a range of techniques including embryonic stem cells, mouse genetics, and computational approaches, with access to state-of-the-art core facilities and infrastructure. In addition, you will enjoy the freedom to develop and carry out your own research within the group’s area of interest.
How to apply
For further details about this role, and to apply, please examine the job description and complete the online application. Application deadline: Sunday, March 1, 2020.
For informal inquiries, please contact Vicki Metzis.
Our understanding of lineage decisions in early human development has been greatly aided by embryonic stem cell lines, which avoid many of the practical and ethical difficulties of in vivo material. A new paper in Development exploits naïve human embryonic stem cells to generate in vitro models for the extra-embryonic endoderm. We caught up with first authors Madeleine Linneberg-Agerholm and Yan Fung Wong, and their supervisor Josh Brickman, Professor of Stem Cell and Developmental Biology at the Novo Nordisk Foundation Center for Stem Cell Biology (DanStem) in Copenhagen, to hear more about the work.
Yan Fung, Madeleine and Josh (L-R)
Josh, can you give us your scientific biography and the questions your lab is trying to answer?
JB Since the beginning of my PhD, I have been focused on the transcriptional basis for cell identity. Following a brief foray into the music industry as both a DJ and journalist, I began a PhD under the guidance of Mark Ptashne at Harvard University, where I worked on general mechanisms of transcriptional synergy and cooperativity. By the end of my PhD, I felt the need to take this work into a more biological context. To this end, I trained as a post-doctoral fellow with Rosa Beddington at the National Institute for Medical Research in London, where I began to work with a combination of early embryos (mouse and frog), and embryonic stem cells (ESCs), to explore the means by which transcription controls anterior specification. In 2001, I started my own group at the Institute for Stem Cell Research (now the MRC Centre for Regenerative Medicine), University of Edinburgh, where I used a combination of ESC models and early embryos to deconstruct the transcriptional basis for lineage specification and potency, focusing on endoderm induction and patterning. In those early years, we used Xenopus embryos with parallel experiments in ESCs as a rapid means to understand conserved mechanisms regulating both pluripotency and differentiation. However, with time, my lab has unfortunately lost touch with its amphibian roots as the lure of stem cells became too much for my students to resist.
One of the most important observations we made in those early years was that ESCs could be used as means to model early development in the primitive endoderm and to trap spontaneously arising transcriptional states in which cells were reversibly and functionally primed for differentiation. This led us to the notion that self-renewing cell culture models could be used to trap intermediate, or uncommitted, transcriptional states in differentiation. We see these states as analogous to transition states for lineage specification, and we have used these models to identify mechanisms governing these reversible transcriptional changes. At the same time, we also began to view karyotypically normal, embryo-derived cell culture as a means to trap decision points in differentiation with a capacity for proliferation. We exploited this idea as a way to isolate and expand lineage-restricted progenitors from differentiating ESCs in both the definitive and later primitive endoderm lineages.
In 2011, my lab relocated to the DanStem at the University of Copenhagen, where we continue to focus on the transcriptional basis for cell fate choice. In particular, we’re interested in the basic mechanisms regulating transcriptional heterogeneities in early embryos and differentiation, how gene regulatory networks can be used to explain the differentiation competence and self-renewal of stem and progenitor cells, and how transient transcriptional states become committed in differentiation. Of course, a number of these questions concern the interface of gene regulatory networks with signalling and this has been a major focus of our recent work, including this new paper.
Before concluding, I think I should tell a short story about the origin of this work. About ten years ago, we had some translational funding to apply our work on mouse endoderm differentiation to human ESCs. I used this money to support a student (Maurice Canham) who was finishing his work in the lab on mouse primitive endoderm priming and wanted to take on this translational project. At the time, all the available human cells were primed pluripotent cells. While he was adapting our culture conditions to human ESCs, he decided to dump human endoderm differentiation media on mouse ESCs and see what happened. He observed this remarkably homogenous differentiation to a cell type he thought resembled a slice of pizza and, therefore, referred to them as pizza cells. At the time we were convinced that ‘pizza cells’ were probably primitive endoderm, but it took another PhD student (Kathryn Anderson) years to prove this was the case, to test the activity of these cytokines side by side on primed and naïve cells, and to work out the conditions for the passaging of naïve extra-embryonic endoderm (nEnd). Years later, naïve human pluripotent cells became available, and we were finally able take this work back to the human cells that it started with.
Madeleine and Yan Fung, how did you come to work with Josh and what drives your research today?
ML-A Although originally from Denmark, I did my undergraduate degree in the UK. I became really interested in early mammalian development as a result of my bachelor’s thesis project in Ryohei Sekido’s group at the University of Aberdeen, working on Y-linked sex-specific epigenetic modifications in mouse ESCs. After four years abroad, I got homesick, but luckily found Josh’s group in Copenhagen, and was able to return to begin a master’s degree under his supervision. In Josh’s group, I was trained by Fung who became my day-to-day supervisor and taught me hESC culture. I was quite fortunate to join when I did, as it was an exciting time both for the group, as they were about to publish the story of the context dependence in mouse endoderm differentiation (Anderson et al., 2017), and also in the field, as a number of human naïve ESC papers had recently come out. I think what drives my research today is trying to fill in the most fundamental steps in human development and reconcile what we know in other species with ourselves.
YFW I finished my PhD in Hong Kong where I studied gene regulatory networks and organ patterning using C. elegans as a model organism. I then applied to work with Shinichi Nishikawa at the RIKEN Center for Developmental Biology, where I used human cell lines and primary cells as disease models to study epigenetic regulation. During that time, I had the chance to meet stem cell biologists from all over the world, including a former PhD student of Josh’s, Kathryn Anderson (one of the authors in this paper), and she convinced me to think about going to his lab. I then applied to Josh’s lab and met with him in Washington DC, after which he encouraged me to visit the lab in Denmark. In 2013, shortly after the group had relocated from Edinburgh to Copenhagen, I came to visit the new centre, DanStem. Attracted by the passionate people in the group, the newly established research institution, and Josh’s impressive work using stem cell culture systems as models to understand the transcriptional basis for lineage choice, I joined the lab.
Immunofluorescence of human nEnd stained for endoderm and basement membrane markers. Left: E-cadherin (green), vimentin (red), GATA6 (white). Middle: AFP (green), collagen IV (red), GATA6 (white). Right: fibronectin (green), vimentin (red), GATA6 (white).
What makes endoderm induction in the mouse context dependent, and before your study what was known about its conservation in humans?
JB, ML-A & YFW We believe that the context dependence we originally saw in mouse was determined by changes in the enhancer landscape between naïve and primed pluripotency. The interaction of Wnt and Nodal-related TGFβ signalling with the set of enhancers primed in these cell types would determine the trajectory of differentiation. In our mouse nEnd paper (Anderson et al., 2017), we found that there was a correlation between enhancer accessibility and definitive endoderm versus primitive endoderm lineage differentiation. This is a remarkably similar idea to our recent thoughts on specificity of FGF/ERK signalling (Hamilton et al., 2019). Here, we found that ERK directly regulates enhancers, but that the activity of ERK on enhancers is likely to depend on pre-bound transcription factors, that don’t in themselves activate transcription, but prepare the available differentiation trajectories a cell can take when exposed to a signal. We believe a similar mechanism must be at work here with respect to endoderm enhancers that respond to Nodal/Wnt signalling in either naïve or primed pluripotency, with these pathways acting on different pre-wired transcriptional circuits that are stimulated by the same signalling pathways, but in different pluripotent states.
At the time we started, it was known that it was possible to culture human naïve cells and that their culture was usually dependent on FGF/ERK inhibition. However, the role of FGF/ERK described extensively in mouse primitive endoderm and epiblast segregation appeared not to be conserved in human embryos. As we believe that inhibiting primitive endoderm differentiation was a primary function of ERK in naïve ESC culture, we wondered how one could reconcile these observations.
Can you give us the key results of the paper in a paragraph?
JB, ML-A & YFW We found that the context dependence we observed in mouse, in which activation of Wnt/Nodal and LIF signalling could promote lineage-specific endoderm differentiation (i.e. primitive versus definitive) based on the developmentally proximal state of the starting culture, was conserved in human. Thus, human naïve pluripotent cells, which resemble the pre-implantation embryo, differentiated to primitive endoderm in response to these pathways, whereas primed pluripotent cells, which resemble the pre-gastrulation-stage epiblast, gave rise to definitive endoderm. We were then able to use this primitive endoderm differentiation model to show that the role of FGF/ERK in specifying this early lineage, at least in vitro, was conserved. Importantly, we were able to establish conditions for the expansion of these in vitro-derived cells to establish a culture/stem cell system for human hypoblast (as the human primitive endoderm is known). As trophoblast stem cells have recently been produced in human and naïve ESCs are thought to represent epiblast, this new culture system means that there are now human cell lines/in vitro models for all three lineages of the blastocyst.
What changes between the naïve and primed states to direct what kind of endoderm ESCs can give rise to?
JB, ML-A & YFW This was discussed above with respect to mouse. We believe it is the gene regulatory network in these different states that provides that platform on which the signalling pathway acts. The transcription factors expressed in these different stages of pluripotency could be sitting on distinct enhancers preparing cells to adopt different trajectories of differentiation in response to the same signal. It’s as if the transcription factors are laying down a road along which the cells can progress in differentiation in response to these signals. When cells transition from naïve to primed pluripotency the road is diverted and signalling pushes cells in this new direction.
What pressing questions do you think your nEnd cells will be particularly suitable for addressing?
JB, ML-A & YFW We think these cells will be particularly useful for the study of human primitive endoderm patterning and differentiation. They will be an excellent tool for studying how regulatory networks become stabilised in self-renewal in the endoderm and how these can then initiate patterning. As nEnd represents the third cell type from the blastocyst, they will also be very useful in experiments designed to determine the self-organising properties of early embryonic cells in order to generate embryoids. Finally, they provide a system in which to study the differentiation of the primitive endoderm and understand how it compares to the definitive endoderm.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
ML-A For me, there were three moments that really stood out for different reasons. The first one was when I saw the first naïve colony after chemical resetting from primed hESCs. It was the first ‘big’ experiment that I did, both on this project and also in my time in the group, so that was a big moment of success for me. The second was when I saw the first patch of primitive endoderm after my first ever differentiation from naïve hESCs. That was when we knew the project was going somewhere, and it was likely that endoderm specification between mouse and human was conserved. The third was when we figured out the expansion conditions for primitive endoderm to make nEnd, which I was stuck on for easily half a year. When the expansion worked, I started to see the future potential of what I was doing beyond this paper and all the exciting experiments that it could lead to.
YFW Expansion, and the excitement of getting expansion working!
When the expansion worked, I started to see the future potential of what I was doing beyond this paper
And what about the flipside: any moments of frustration or despair?
ML-A A lot! It was really challenging having to learn the most basic aspects of doing research at same time as having such an ambitious project, from cloning and doing my first RT-qPCR to learning R (thank you, Stack Overflow). But I think that just made it all the more rewarding, or at least that’s how I feel now.
YFW When I found out that I could not detect HHEX expression in differentiating primitive endoderm from human naïve ESCs, I thought we had a problem with the cells. However, based on the single cell transcriptome data on the human blastocyst it turned out to be true.
So what next for you two after this paper?
ML-A I graduated with my master’s degree this summer and now I’m taking a year ‘off’ working as a research assistant in the lab. My plan is to start my PhD with Josh next year. I am continuing with human nEnd projects, but I’ve started working with mouse endoderm as well, as it offers a whole new world of experimental possibilities.
YFW Besides this work, I am finishing other projects related to foregut endoderm expansion and differentiation to visceral organs, including pancreas and liver. The main focus is to understand how extrinsic signals influence transcriptional networks or chromatin accessibility. I am interested in how these networks impact the choice these progenitor cells make between self-renewal and lineage specification. I hope this work will bring us one step closer to understanding human embryonic development and perhaps translating this knowledge into strategies for regenerative medicine.
Where will this work take the Brickman lab?
JB As a lab we are very excited about these cell lines. We are excited by the potential of exploiting nEnd to explore the self-organising properties of human primitive endoderm, both on its own and when recombined with other cell types. We are also excited about using nEnd as a model to understand human visceral endoderm patterning.
Since I first started my lab, I have worked on gastrulation-stage endoderm patterning and using ESC differentiation as a model for this. While we have just begun this sort of work in human models, nEnd will complement them nicely. We are looking forward to using these cells to explore ‘extra-embryonic’ endoderm differentiation in human.
Finally, the in vitro model we describe here for human primitive endoderm differentiation will provide us with an excellent platform to collect evidence for our ideas about signalling context. How does the enhancer state or gene regulatory network in naïve and primed pluripotency determine signalling response?
Finally, let’s move outside the lab – what do you like to do in your spare time in Copenhagen?
ML-A Just like in the UK, it definitely depends on the weather. If it’s nice, I like going for walks with my dog in a forest north of Copenhagen called Dyrehaven, which is actually a UNESCO World Heritage Site. On rainy days, I like to try and find the best ramen place in Copenhagen (currently Ramen To Bíiru) or stay at home watching ‘90s rom-coms and playing video games.
YFW Hygge with family and friends, discussing the big and small things in life.
We are pleased to announce a call for applications for a post-doctoral researcher position in the lab of Professor Jonathan Chubb in the MRC Laboratory for Molecular Cell Biology (http://www.ucl.ac.uk/lmcb/research-group/jonathan-chubb-research-group). To understand how cells decide their fates, during development and reprogramming, the lab develops and implements powerful new technologies to directly monitor gene activity in single cells (eLife e13051, Curr Biol 27:1811-17, PNAS 115:8364-8369). This position is an ideal platform for developing an independent research career in a rapidly expanding sector of the life sciences.
Candidates are expected to be exceptional, highly motivated scientists with a strong track record of research in a relevant area of the life sciences. We will also consider applicants with a PhD in physics, engineering, mathematics or computer science with a strong interest in biology.
Work will be carried out at the MRC Laboratory for Molecular Cell Biology http://www.ucl.ac.uk/LMCB/. The LMCB is a focal point for molecular, cell and tissue biology in the UK and is situated in the main UCL campus, in the heart of central London.
Please contact j.chubb@ucl.ac.uk for informal enquiries. Deadline 28th February. Apply using this link:
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