Looking back on the journey: Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development eLife van Lessen et al., 2017

Just over two years ago, while I was a Masters of Neuroscience student at University College London, I became interested in the emerging concepts of brain lymphatics and sleep dependent macromolecule clearance within the brain. Until quite recently, the brain was considered to be without lymphatic vasculature or dedicated mechanisms of clearance of its own. Then in 2013, Lulu Xie of the Nedergaard lab published a blockbuster paper in Science that suggested that sleep facilitates fluid and metabolite clearance from the brain (Xie et al., 2013). Following in 2015, a double header in Nature and The Journal of Experimental Medicine (Louveau et. al. 2015, Aspenlund et al. 2015) demonstrated that lymphatic vessels exist within the dura mater of mouse and human meninges. Together these papers opened an entirely new outlook for how our energy intensive brain might maintain homeostasis, with potential implications for neuropathology and degenerative disease. I was attracted to the interdisciplinary aspects of this budding field, and the topic became a fundamental obsession for me after several involved discussions with Dr. Jeff Illif who had written the original brain paravascular clearance paper (Illif et al., 2012) and moved from the Nedergaard lab to head his own at Oregon Health and Science University in 2013. I was deeply encouraged by the time he took to act as a sounding board and to offer mentorship and direction as I decided how to pursue this field for my PhD.

I had a potential idea for how to tackle this field from a new angle after Dr. Jason Rihel from UCL’s Department of Cell and Developmental Biology gave a lecture for the Master’s program, during which I discovered that his lab uses molecular and genetic techniques to study sleep using zebrafish. I found his approach and style as a creative, big-picture scientist appealing. Zebrafish also made sense to me as a model through which to investigate this complex clearance mechanism, as they are small and optically transparent throughout larval stages, allowing for accessible imaging and high throughput studies. However, when I approached Dr. Rihel (Rihel et al. 2010) with a PhD proposal to investigate these processes in zebrafish, he warned me that not only would this be a new area for his lab, requiring new methods, but we also might find that zebrafish simply lack brain lymphatics/clearance. To finally convince Dr. Rihel that it was worth investigating, I poured over these new papers along with zebrafish papers describing lymphatic development, many of which came from a well-known lymph and vascular lab in Muenster, Germany led by the seasoned scientist, Professor Stefan Schulte-Merker.

About the same time, in Professor Schulte-Merker’s lab, a postdoctoral researcher by the name of Max van Lessen had also caught the brain lymphatics ‘bug’. Digging through the same literature, Max had his own “eureka” moment when he realized that the transgenic vascular and lymphatic lines, including a few created by his colleague Andreas van Impel, might have labelled the brain. Such transgenic tools would allow for dynamic investigation of the origins and molecular properties of brain lymphatics, if they were present and conserved in bony fish.

Serendipitous timing brought Professor Schulte-Merker to our doorstep last March for an unrelated meeting with our colleague and neighbour, Professor Steve Wilson. When I heard from my colleague Dr. Eirinn Mackay that he was down the hall, I jumped up and caught him as he was heading out the door. Taking advantage of the brief moment, I thrust my hand at him for a hearty shake and I began excitedly outlining that I believed brain lymphatics found in mice and humans would be conserved in zebrafish. I told him that in some of the images from papers, I had seen evidence of lymphatic markers present in the head of the zebrafish and wanted to know his opinion. Professor Schulte-Merker smiled and nodded. “Indeed, we have noted this and are looking into it.“ I asked out of curiosity why had everyone in the zebrafish vascular community focused exclusively on the trunk of the fish? Most images of zebrafish lymphatics I found in the literature had frustratingly cropped the top of the head from full view. Professor Schulte-Merker smiled wryly and stated that “everyone was so engrossed with the peripheral system in the past that no one ever bothered to look up.” It struck me that so much of discovery is about simply “looking up”. Not being afraid to challenge long standing norms and established or popular avenues of thought to follow instincts that bring us to look with new eyes upon something we have seen before a billion times.

I also discovered that science is, more often than not, luck, happenstance, and the willingness to cooperate to get more done. We can work alone and hoard, or we can branch out and be, as my friend Seanne in California always likes to put it, “better together”. My PhD supervisor Dr. Rihel cultivates an atmosphere of collaborative research, and his fearlessness in sharing ideas and taking chances paved the way. The Schulte-Merker lab didn’t hesitate to reciprocate in the same spirit, and immediately a cooperation was forged. To begin parallel investigations into brain lymphatics, the Schulte-Merker lab sent us a combined transgenic line with kdr-l expression labelling all vasculature in mCherry (Hogan et al., 2009) and flt4 (Vegfr3), an established venous-lymphatic marker in mCitrine (van Impel et al., 2014).

We at the Rihel Lab have an interest in the conservation of these systems and mechanisms to ultimately elucidate the impact on potential clearance through sleep, and the Schulte-Merker lab thinks about this problem through the angle of vascular and lymphatic origin and development. We have complementary skill sets that allowed the work to begin at an incredible pace, building a whirlwind storyline. My colleagues often remark that this project made them feel like they had been strapped to a rocket ship.

The Mystery Cells

To help with the anatomical search for brain lymphatics, I recruited my remarkably talented second supervisor as a co-investigator (Dr. Tom Hawkins), as he is one of the most highly inquisitive people I have met, with a wide breadth and depth of technical expertise which would clearly be needed. We began by looking at adult transgenic brains, carefully dissecting and removing them using confocal microscopy to examine both the surface of the brain as well as the interior portions of the skull. What we saw initially was somewhat astonishing. We had imagined we might find lumenized vessels; however, along much of the surface of the brain were what looked to be flt4 positive cells of some sort, glowing brightly in mCitrine, clearly differentiated from the kdr-l blood vascular marker in brilliant mcherry. While kdr-l expression labels both arteries and veins, flt4 expression is an established marker of venous and and lympahtic endothelium. Importantly, our cells were entirely negative of kdr-l expression, which in the trunk can only be found in lymphatic vessels.

They possessed large inclusions and spindly processes which appeared to be connecting each to the other in close proximity to surface vasculature. A Sir Henry Welcome Fellow in our lab, Sabine Reichert, took one look at the images and without blinking stated, “they look like some sort of endothelial cell type.” It quickly became clear that she was right.

We dove in with immunohistochemistry using anti GFP and RFP antibodies followed by cryosections in adult brains to investigate carefully where these cells were positioned. Our cryosections revealed that these cells populated the meninges surrounding the entire brain exclusively and were associated with particular vessels diving into the parenchyma from the meningeal layer. This was an exciting moment for us as we recognized that the work done in mice and in humans had placed brain lymphatics in the meninges and our cells were positioned in a similar location. Although much work remained, we in London felt we were on track and may have identified a brain lymphatic system in the fish, suggesting evolutionary conservation within the vertebrate lineage.

Origins

Around the same time Professor Schulte-Merker and I had met, Max and the Schulte-Merker lab in Germany had found beautiful evidence of head lymphatics in the embryonic brain. Max had been capturing live images from the earliest stages of development onwards, patiently waiting to see when, how, and from where expression of these flt4 positive structures would emerge to get a hint of their developmental origin. They reasoned that if peripheral lymphatics sprout from venous structures early in development, perhaps the same would occur in the brain. While training the confocal microscope on the choroidal vascular plexus (CVP) and mesencephalic vein (MsV) which loops around the optic tectum of the zebrafish brain, they saw a clear sprout comprised of flt4 positive cells emerging around 56 hours post fertilization (hpf) from the CVP that then grew in an arc along the MsV at the edges of the tectum in parallel to the vasculature. Residual kdr-l expression in these flt4 positive cells suggested differentiation from venous endothelium.

Over the course of three days, this flt4 positive ring from what we began calling the stereotypical tectal “loop”, a cohort/string of loosely connected single cells which aligns closely to the MsV. In the peripheral nervous system of zebrafish, angiogenesis begins with the sprouting and development of arteries, followed by the concurrent development of veins and lymphatic vessels with both requiring three key genes: ccbe1, vegfc and flt4. Both lymphatic sprouts and venous sprouts initially originate from the posterior cardinal vein and ultimately form their own independent structures as they grow towards the myoseptum (van Impel et al., 2014). Since these novel brain cells sprouted from a vein and expressed flt4, Professor Schulte-Merker remarked that they were most likely lymphatic in origin.

Macromolecule Uptake

If these novel cells were in fact functionally similar to mammalian lymphatic structures, Max hypothesised that these cells may have function in the uptake and clearance of macromolecules, similar to what has been reported for murine brain lympahtic vessels. Incredibly, tracers injected either into the tectal neuropil or into the hindbrain ventricle accumulated at flt4 positive cells forming the tectal loop within minutes of injection. This uptake is so rapid and specific that when I first showed Dr. Rihel a similar dye injection experiments that my colleague Dr. Chintan Trivedi and I had performed on the two-photon microscope in a wild type fish, he thought I was showing him an image of the lymph transgenic line, as the dye took on the precise location and shape of the flt4 positive cells! With so much activity going on in both of our labs, we realized that it was time to have an in-person meeting to facilitate discussion and avoid excessive experimental overlap.

I made a pre-Christmas journey to Muenster to mutually share our findings along with new ideas and techniques. During my time there, I was also introduced to Andreas van Impel, another postdoctoral researcher in the Schulte-Merker lab, with a humble and steadfast demeanour who had created a number of the stunning complex lymphatic lines we were currently using. He brought a great deal of experience and insight to our discussions and grounded us with his thoughtful interjections. When Max presented his work, we both were so excited that we were wildly gesticulating and scribbling notes. At one point, one of us climbed atop of the table to point out a key piece of research from an old paper from the 1930’s, with Andreas calmly leaning in with an amused smile on his face. It was one of the most vibrant scientific visits I have had, and it solidified a strong sense of teamwork. Face to face contact I found is the cornerstone of collaboration. Trust must be earned and built, while looking one another in the eye, so colleagues can both sense and see our intentions, and know we are all labouring for the same goal.

Shortly after my visit, Max shared he had mused that the cells had macrophage or dendritic-like characteristics, even though their lymphatic origin suggested a novel cell type. This had Max hunting after shared receptors or other common characteristics, which may offer further clues about our cells. In this reading, Max discovered a universal pattern recognition receptor called the mannose receptor found in both macrophages and dendritic cells and was a major way in which cargo was endocytosed into these cell types. Thus, he decided to apply both Pyrimidyn-7, a quintessential blocker of dynamin-dependent endocytosis, and mannan, a bacterial polysaccharide that binds to and saturates the mannose receptor in independent experiments to see if he could stop tracer uptake. He shared on a call that he was left deeply puzzled as it looked like the tracers had still localized to the flt4 positive cells anyway, even in the presence of the endocytic blocker.

Fortunately, Max had also injected the pH sensitive dye (pHrodo-advin) which would only fluoresce when it encountered an acidic environment inside of a cell. In previous experiments, he had seen both the tracers and the pHrodo fluoresce at the flt4 positive cells together, demonstrating for the first time lymphatic uptake of macromolecules at the single cell level. Being in the habit of injecting pHrodo along with his usual tracer, he also used this dye cocktail in his search for an effective concentration of mannan or Pyrimidyn-7, which revealed that the usual fluorescent tracer still accumulated at the cells. However the injection of either blocker effectively stopped the internalization of pHrodo. It didn’t make sense! How could the fluorescent tracer be taken up, but the pHrodo not? Suspicious, he took a closer look, and realized that the usual fluorescent tracer had merely localized to the membrane of the cell and was not actually being internalized. 3D reconstructions revealed that the fluorescent tracers were lodged into the membrane. If pHrodo was injected before the blocker it still happily fluoresced in the acidic lysosomal compartments within the cell but never co-localized with the fluorescent tracers lodged in the membrane after endocytic blockage.

Thus, it seemed that one process “docks” the tracer to the cell membrane while subsequent cargo internalization into acidic compartment is likely mannose receptor dependent. If it hadn’t been for his habit of using the pHrodo he may just have given up thinking the blockers hadn’t worked. I learned a few valuable lessons from Max’s results. First, I learned that experiments don’t have to be complicated and showy. They can often be incredibly straightforward. The other lesson was to always look closer and deeper before walking away from an experiment in frustration. So much of science emerges from happy accidents. We hear it again and again, and that is because it’s just so true.

Max and his colleagues expanded the analysis to additional lymph markers as well as markers for macrophages, etc. For example, they examined transgenic lines labelling classic lymphatic markers such as Lyve1 (Okuda et al., 2012) and Prox1 (van Impel et al., 2014) to further confirm these cell’s lymphatic identity. Much to our combined excitement, each lymphatic line labelled the quintessential tectal loop. Finally, to rule out that these flt4, prox1, lyve1 positive cells were macrophages, Professor Schulte-Merker suggested breeding the lyve1 line in DsRed (Okuda et al., 2012) with the mpeg1 macrophage line in GFP (Ellett et al., 2011). This showed clear differences between the cell types based on location, morphology, and expression. To completely eliminate the possibility, they also examined whether the morpholino knockdown of the macrophage transcription factor pu.1 had any effect on the lymphatic tectal loop. Andreas injected morpholinos, and after a tense few days of waiting for the results, the experiment revealed that our flt4 positive cells remained undisturbed, even as all macrophages were eliminated. We were elated, and we all finally allowed ourselves to believe that we had a novel cell-type on our hands.

Back in London, Tom and I began our hunt for the identity of these cells from a very different angle, using electron microscopy to obtain a definitive view of the location and ultrastructural features of these cells in relation to blood vasculature. Tom has had decades of experience with transmission EM, so he warned me not to get too excited, as disappointment was likely. Using the enormous TEM microscope with the assistance of Mark Turmaine: the quiet EM master, we happily navigated our way over the ultrathin slices in what, to me, was reminiscent of the broadcasts from the first moon landing. To our delight, and despite the forewarning, our first EM run was crystal clear with excellent structural resolution. The cellular landscape was wild and extraordinary, full of intricate interdigitations of membranes and strangeness all around. Guided by venerable EM studies on goldfish meninges, we could clearly identify a distinct meningeal tri-layer, inside which the cells that contained these large inclusions were exclusively found. We were able to immediately identify structures with multiple large inclusions in close proximity to surface brain vasculature with the size and distribution shown in our confocal images. They were clearly separable from perycites, vascular endothelial cells, and their basement membranes.

We combed over mammalian and teleost EM images of the meninges to try to identify these cells. One candidate was the Fluorescent Granular Perithelial Cells (FGP), which were discovered and characterized in rodents by Maso Mato in the 1980’s. Mato cells were known to reside in the meninges and contained numerous, heterogeneous inclusions (Mato et al., 1986, 2002), and we had several hours of heated debates in Dr. Rihel’s office as we stared at side-by-side comparisons of classic EM images and our new data. Ultimately, we decided that our cells were most probably distinct from Mato cells, or at least they represented a specific subtype, as our new cells’ inclusions were less heterogeneous than expected for Mato cells, and they had morphologically distinct shapes.

The ”Competition”

As the first part of our story of these cells was beginning to wrap up, Max and I were made aware that two other labs had come to similar conclusions to us. Suddenly, we were plunged into a race in which we weren’t precisely clear who had what, and when, in entirety.

Moments like these, when you find out other labs are working on similar avenues, can be stressful at best, and to be frank, Max and I had moments of panic together. The prospect of potentially being “scooped” looms heavy above all of our heads at any given moment. Each one of us is deeply connected to our research and have spent countless hours sweating and toiling over our projects. They become a part of us. And hearing that others have found something similar can be particularly disorienting as discovery, though made for the common good, can feel so deeply personal. Professor Schulte-Merker and Dr. Rihel kept insisting that each lab would be contributing to the scientific enterprise and bring novel insights into the nature of these cells.

This also meant that the community would be excited about this discovery. In retrospect, with the three respective papers now published, (Galanternik et al., 2017, Bower et al., 2017, van Lessen et al., 2017), it is clear that each lab approached these cells from unique perspectives. Galanternik et al. discovered these cells using a transgenic mannose receptor line and indicated that they believed these to be fluorescent granular peritheliaI cells (FGP or Mato Cells) (Galanternik et al., 2017). Bower et al. utilized the transgenic lyve1 lymphatic line to reach their discovery, and amongst other interesting findings they ablated these cells to find that the ablations impacted the integrity and development/regeneration of meningeal vasculature (Bower et al., 2017). Both those papers also performed valuable RNAseq analysis using differential gene expression analysis to rule out the cell’s having blood endothelial, smooth muscle, macrophage, or pericyte identity. This exciting combined evidence opens up an entirely new avenue of research into brain lymphatics in zebrafish.

Arachnocyte, Kumocyte, BLEC

Finally, as we debated what to name these cells, the other labs disclosed the names with which they were identifying the cells in their papers, with Galanternik et. al. calling them FGPs and Bower et al. settling on perivascular Lymphatic Endothelial Cells (pLECs). We ultimately decided on Brain Lymphatic Endothelial Cells (BLECs) to be precisely descriptive of their properties; however, internally we all enjoyed the name we had additionally selected which was Kumocyte. Kumo means spider in Japanese, describing their spidery appearance, they had previously been colloquialised as arachnocytes for the same reason. I think they will always remain Kumocytes to us somehow, as it speaks to their unique morphology and “personality” and we have taken to fondly calling them that within the Rihel lab.

We are grateful for this opportunity to share our experience as we are so often confined by formality, word limits, and jargon that we miss the opportunity to tell “the story”. The story which discusses the more personal aspects of the craft of discovery, especially the journey behind the scenes that keeps all of us all coming back again and again despite the tough days, which are many. After all, in biology and science, we are all just witnesses to nature, merely reporting what we see with awe and wonder. It’s always been there, but there’s that kid in all of us that gets our mind-blown every time we get to be the ones to see something with human eyes for the “first time.“ I also never want to forget on this journey, not just in science, but in life, that we truly are only as good as those we surround ourselves with. Each and every colleague on my floor has contributed, and given their time and expertise without hesitation. They say it takes a village to raise a child…..and so it also goes with raising a PhD student.

We can’t wait to see what this field of brain lymphatics and clearance holds. Certainly, dogma that the brain is without dedicated lymph structures of its own is being rewritten, even in fishes. As we start at the drawing board and re-examine all of our old assumptions, what else will we discover around the corner?

Shannon Shibata-Germanos and Max van Lessen

(8 votes)

Loading...

Cluster Hire in Quantitative and Computational Developmental Biology, North Carolina State University

As part of the Chancellor’s Faculty Excellence Program, NC State University seeks three outstanding faculty at any rank to expand the new interdisciplinary faculty cluster on Quantitative and Computational Developmental Biology. Together with NC State’s existing strength in quantitative/computational sciences and engineering, this cluster will propel NC State to the forefront of efforts to define new principles in developing biological systems.

Successful candidates will engage in transformative research which integrates expertise in biological, biophysical, computational, and/or mathematical methods to investigate dynamic events in human, animal or plant development on any level, from the nanoscale to the whole organism. The cluster seeks individuals with expertise in using state-of-the-art quantitative methodologies and/or computational modeling to address fundamental aspects of Developmental Biology such as:

• the mechanisms that regulate individual cell behavior, proliferation, differentiation, potency, and reprogramming,
• how material properties and dynamic physical / mechanical forces influence collective embryonic cell behavior and tissue shape,
• the formation, maintenance and appropriate scaling of the geometric configurations of cells and tissues that comprise organs and organisms, and
• the mechanisms that underlie the evolution of new biological forms and facilitate morphological plasticity in response to stress or environmental change.

Although we are interested in individuals who can bridge experiment and theory and have the ability to translate research outcomes to address biomedical or agricultural challenges, applicants whose expertise is mainly in the computational realm are also strongly encouraged to apply. Demonstrated collaboration as a member of a multi-disciplinary team is essential.

Hiring may occur at the level of Assistant, Associate, or Full Professor. The home department is anticipated to be in one of the four participating colleges (Engineering, Sciences, Veterinary Medicine and Agriculture and Life Sciences) and will be determined based on credentials and research fit. Hires will be expected to provide key leadership in quantitative and/or computational biology across the university, teach in existing courses and develop specialized coursework in their area of expertise.

Minimum requirements include a PhD in a relevant field from an accredited institution. Interested candidates should submit: a CV, a 2-3 page research plan, a cover letter describing prior multi-disciplinary research efforts and how their research prospectus addresses the goals of the cluster (see https://facultyclusters.ncsu.edu/clusters/modeling-the-living-embryo/), and
contact information for 3 references. Materials for consideration will be accepted electronically via http://jobs.ncsu.edu/postings/88538. Review of applications will begin immediately and continue until the position is filled. Questions about the position may be directed to Dr. Nanette Nascone-Yoder (nmnascon@ncsu.edu).

NC State University provides a vibrant environment for research, teaching and mentoring across disciplines; ample opportunities will be available for collaborations with existing faculty and other newly hired colleagues in the cluster. Our location in the Research Triangle also facilitates interaction with faculty at Duke University and the University of North Carolina at Chapel Hill, as well as with industry and government agencies.

The Chancellor’s Faculty Excellence Program is bringing some of the best and brightest minds to join NC State University’s interdisciplinary efforts to solve some of the globe’s most significant problems. Guided by a strong strategic plan and an aggressive vision, the cluster hiring program is adding new faculty members in select fields to add more breadth and depth to NC State’s already strong efforts. The Chancellor’s Faculty Excellence Program marks a major initiative of the university’s strategic plan, “The Pathway to the Future”   We invite you to explore more information about the Chancellor’s Faculty Excellence Program and this cluster at http://workthatmatters.ncsu.edu/.

NC State University is an equal opportunity and affirmative action employer.All qualified applicants will receive consideration for employment without regard to race, color, national origin, religion, sex, gender identity, age, sexual orientation, genetic information, status as an individual with a disability, or status as a protected veteran. Persons with disabilities requiring accommodations in the application process please call (919) 515-3148.

(No Ratings Yet)

Loading...

The Mokalled laboratory is seeking applicants for a Research Technician II position in the Department of Developmental Biology at Washington University School of Medicine.  Our lab uses the zebrafish model system to study neural regeneration after spinal cord injury or disease.  We welcome ambitious applicants with enthusiasm for neuroscience, regenerative biology, and zebrafish research. Training will be provided. This position offers opportunities for co-authorships on published manuscripts.

Duties:

• Assists with research studies, experiments and assays including collection of data, preparation of solutions and set-up and maintenance of equipment. Main experimental duties include molecular biology, zebrafish husbandry and microinjection.
• Performs data entry and maintains data files on research.
• Complies with established safety procedures and maintains required documentation on laboratory and specimen conditions.
• Ensures lab conditions and equipment are properly cleaned and maintained in accordance with established procedures.
• Assists with general lab maintenance and cleaning.

Required qualifications:

Bachelor’s degree and up to 1 year of experience in lab setting or equivalent combination of education and experience equaling 4 years required.  Please send a cover letter, CV, and list of 3 or more references to mmokalled@wustl.edu.

(No Ratings Yet)

Loading...

Each of our cells has the same genetic information and thus the same potential to become a part of a heart, brain, or a finger. Somehow though, during development our cells manage to figure out exactly which type of cell they should be and which body parts they should help compose. The key to making this work is precise control over gene expression, such that as a single cell divides to make trillions, the correct genes are turned on and off at precisely the right times and places. When gene regulation fails, developmental disorders and diseases like cancer can occur.

Today, transcriptional regulation in animals is understood to involve a complex integration of cis-regulatory elements (enhancers), cell signaling pathways, nucleosome occupancy, and higher order chromatin architecture that all work in concert to direct gene expression. In the McKay Lab we are interested in how spatial and temporal regulatory information is integrated during development, and how that integration produces the distinct cell types and body parts of animals.

Central to the process of differential gene regulation are relatively small genomic regions called enhancers. Enhancers, as their name implies, have long been known to increase gene expression, often over long distances. In extreme cases an enhancer can act on a gene that is over 1 million bases away, such as in the case of the enhancer that regulates the Sonic hedgehog gene¹.

Enhancers mediate gene activation by serving as landing pads for proteins (transcription factors) that recruit the machinery required for gene transcription. Enhancers are remarkable not only for their ability to regulate gene expression across long genomic distances, but also because they exhibit highly flexible sequence characteristics, they work independently of their orientation, and they can be transplanted to different parts of the genome and still retain their ability to activate nearby genes². The role and importance of enhancers has become increasingly apparent, especially as the links between human disease and mutations within enhancers have become much clearer^3-5.
One of the major current question in the field is how networks of enhancers direct the differential gene expression programs during development.

Inside eukaryotic cells, DNA is packaged into chromatin, the basic unit of which is the nucleosome. Wrapping DNA around nucleosomes succeeds in packaging two meters of DNA into a ten-micron nucleus. It also serves as a potential means of controlling access to the DNA sequence because nucleosomes typically block transcription factors from binding their target enhancers. For a transcription factor to bind DNA, its target site must be sufficiently depleted of nucleosomes or “opened.” Consequently, one hypothesis for how transcription factor binding is controlled (and hence for determining which enhancers are active) is through regulation of chromatin accessibility. According to this model, an enhancer with cell-type specific activity would be open in cells in which it is active, but closed in cells in which it is inactive (Figure 1).

During his postdoctoral work, our PI Dan McKay tested this idea by profiling the open chromatin regions during appendage development in the fruit fly Drosophila melanogaster⁶. Using a high-throughput sequencing method that identifies genomic regions of low nucleosome occupancy, called FAIRE-Seq, Dan found that, despite having different morphologies, transcriptional programs, and transcription factors that specify the distinct identity of each appendage (so called master transcription factors), the patterns of open chromatin were surprisingly similar between the cells of wings, legs, and halteres. Even more surprising was the finding that although these patterns were dynamic over time, they remained highly similar between the appendages. In other words, the same regions of the genome appeared to be opening and closing at similar times, even though the cells were in completely different parts of the animal. This indicated that the temporal control of chromatin accessibility might be a more significant driver of differential gene expression during appendage development than spatial control (Figure 2).

Dan’s 2013 paper raised a big question: If the transcription factors that specify the distinct identity of each appendage weren’t primarily responsible for directing chromatin accessibility, what exactly was? And on top of that, how were these changes in open chromatin being coordinated across spatially-separated tissues?

One of the phenomenal things about insects is that the timing of their development is precisely controlled by a steroid hormone called ecdysone. In Drosophila, levels of ecdysone increase at stereotypical developmental timepoints, particularly during major transitions like molting⁷. The work of Michael Ashburner in the 1970s revealed that these ecdysone pulses were responsible for activating a large set of genes, many of which were eventually found to be DNA-binding transcription factors⁸. Because ecdysone acts systemically, and it has a known role in controlling gene expression, we wondered if ecdysone, and in turn ecdysone-induced proteins, contributed to the coordinated changes in chromatin accessibility that Dan had observed.

Focusing our attention on wing development, our first step was to get a better sense of how chromatin accessibility was changing in the wing⁹. Our original FAIRE data had only looked at chromatin accessibility at two time points: a stage right before metamorphosis and a much later stage at the end of metamorphosis. Consequently, it was essential to get FAIRE-Seq data in the pupal wing at finer timepoints. With collaboration from the Buttitta Lab at the University of Michigan, we obtained FAIRE-Seq data that confirmed Dan’s observations; chromatin accessibility was changing significantly, with thousands of regions opening and closing over a relatively brief two-day period. Based on these results we knew that the pupal wing, which undergoes striking morphological changes as it develops, was going to be an excellent model system for us to examine how chromatin access is regulated.

Since ecdysone is a steroid hormone and is essential for even the earliest stages of fly development, we couldn’t remove it completely. Instead, we focused our attention on one of the transcription factors directly induced by ecdysone, Eip93F (E93). To ask whether E93 was involved in changing chromatin accessibility, we repeated our FAIRE-seq experiments in an E93 mutant fly. We found that many regions (~50%) that originally showed dynamic changes in accessibility, failed to change their state in the mutant. So, it appeared that we were on the right track and that E93, and by proxy ecdysone signaling, was required for directing many genome-wide changes in chromatin accessibility. Importantly, our data showed that E93 was required for both opening and closing of chromatin.

Although these data showed us that E93 was required for changes in chromatin accessibility, it didn’t tell us whether it was directly causing these changes. To help answer this question, we wanted to determine where E93 was physically bound in the genome. These days assaying protein-DNA binding by ChIP-Seq is a standard practice, but the method requires a significant amount of input to get good results. When working with cell culture this isn’t much of a problem, but it presents a serious challenge when working with tissues in tiny animals like Drosophila, because we needed over a thousand wings to have sufficient input. Over the course of a week, the lab worked together to dissect all the wings necessary for the experiment, and in the end the effort paid off. We found that not only was E93 required for many of the changes in chromatin accessibility, it actually bound to about half of these dynamic regions, arguing that in many cases E93 had a direct role in coordinating both opening and closing chromatin.

Next, we had to tackle whether the temporally dynamic chromatin regions we observed in our FAIRE data actually corresponded to functional enhancers. The best method for testing enhancer functionality is still to clone them upstream of a fluorescent reporter gene. This can work great, but makes it much harder to test many candidates with a high degree of throughput, as it can take months to establish and test the transgenic flies. This was right around the time I joined the lab as a first-year graduate student, and I got the chance to work on characterizing the function of several of the putative enhancers we had cloned. We were thrilled to find that the E93 dependent dynamic chromatin regions we had cloned successfully drove GFP expression, and that the timing of their activity correlated with the timing of the accessibility of the native enhancer.

The crux of this project was to finally test if the failures that we saw in enhancers opening or closing in E93 mutants correlated with a functional defect in our reporters. We found that in the absence of E93 there were dramatic changes in enhancer activity, and that these changes followed what we would expect based on the changes we saw in chromatin accessibility. For example, the nub^vein enhancer, which normally opens during pupal wing development, produces striking patterns of fluorescence along the wing veins when it’s cloned upstream of a GFP gene. However, in an E93 mutant fly, this region fails to open to the same degree. When we imaged the nub^vein enhancer reporter in the presence of E93 RNAi we saw complete loss of the normal vein pattern of expression (Figure 3).

What we found most exciting about this project is that it demonstrated that an extrinsic signal, ecdysone, which pulses at specific times in development, alters the accessibility of enhancers to binding by transcription factors. In other words, for a genome that contains far greater regulatory capacity than is used at a given point in time, hormone signaling can help to determine which subsets of the genome are accessible for use at given time in development. Thus, ecdysone-regulated chromatin accessibility provides a temporal-specific input, which is combined with spatial-specific input in the form of tissue-specific transcription factors. Amazingly, both forms of input are integrated by enhancers.

This has left us with a whole new set of questions to go after. What are the mechanisms that drive ecdysone-dependent changes in chromatin accessibility? Does E93 act alone? How is it that E93 is required for both opening and closing chromatin? How does chromatin accessibility fit into other aspects of gene regulation such as long-distance enhancer-promoter interactions? There’s a lot more to figure out, and these questions are actively pushing us to expand our research focus. Fortunately for me, that means there’s a lot of room to explore and opportunity to make some meaningful contributions to the field.

References

1. Lettice LA, Heaney SJ, Purdie LA, Li L, de Beer P, Oostra BA, Goode D, Elgar G, Hill RE, de Graaff E. (2003). A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet. 12(14):1725-35.

2. Shlyueva D, Stampfel G, Stark A. (2014). Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev. 15:272-286.

3. Maurano M, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, et al. (2012). Systematic Localization of Common Disease-Associated Variation in Regulatory DNA. Sci. 337(6099):1190-1195.

4. Visel A, Rubin EM, Pennacchio LA. (2009). Genomic views of distant-acting enhancers. Nat. 461:199-205.

5. Sagai T, Hosoya M, Mizushina Y, Tamura M, Shiroishi T. (2005). Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. Dev. 132:797-803.

6. McKay DJ, Lieb JD. (2013). A common set of DNA regulatory elements shapes Drosophila appendages. Dev Cell. 27:306-318.

7. Richards G. (1981). The radioimmune assay of ecdysteroid titres in Drosophila melanogaster. Mol. and Cell. Endo. 21:181-197.

8. Ashburner M. (1974). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. Dev. Bio. 39:141-157.

9. Christopher M. Uyehara, Spencer  Nystrom, Matthew J. Niederhuber, Mary Leatham-Jensen, Yiqin Ma, Laura A. Buttitta & Daniel J. McKay (2017). Hormone-dependent control of developmental timing through regulation of chromatin accessibility. Genes & Development. 31:862-875.

(2 votes)

Loading...

Deciphering the Mechanisms of Developmental Disorders (DMDD) is a large-scale imaging and phenotyping  programme for genetically modified mouse embryos. For embryos at E14.5, the key imaging technique is High Resolution Episcopic Microscopy (HREM), and the resulting images are used to comprehensively phenotype the embryos using a systematic approach.

With a combination of lectures, demonstrations and hands-on sessions, this three-day workshop (20-22 October, The Medical University of Vienna) will introduce HREM technology and discuss the value of the resulting images when used to score morphological phenotypes. The HREM procedure will be described, while sample preparation and data generation will be demonstrated.

As an introduction to phenotyping, the workshop will cover the normal anatomy of E14.5 mouse embryos and the morphology, topology and tissue architecture of their organs as presented in HREM data. A special focus will be given to developmental peculiarities and norm variations in anatomy. A protocol for scoring abnormalities will be demonstrated, after which hands-on sessions will allow participants to practice scoring both wild-type and mutant embryos whilst receiving feedback.

More information (PDF link):

http://www.bioimaging-austria.at/web/media/programs/HREM_DMDDProgram%20JL.pdf

Register here:

http://www.bioimaging-austria.at/web/pages/training/by-cmi-technology-units.php

(No Ratings Yet)

Loading...

I recently saw drawings by Maria Sybilla Merian at Kupferstichkabinett Berlin and the University Library Dresden. Merian, who lived from 1647 to 1717, is renowned for her exceptional illustrations of biological specimens and gained recognition as a scientist for her nature observations, for example, of insect metamorphosis.

Merian evidently was genius in choosing frame and magnification in her drawings, but her pictures lack indications of scale*, which are essential in today’s science images. Scales give the reader the key for aligning the image content with reality. To my knowledge, neither Merian nor her predecessors from Antiquity, Byzantium, or Renaissance included scales in their medical and natural science images*. Even in the beginning of the 20^th century, images were often considered a waste of space and scales unnecessary as scientists were familiar with each other’s apparatuses and objects. Today however we study invisible processes and structures that are unfamiliar to most of our colleagues and therefore have to include scales in our images.

We often include in images a familiar object of a standard size for scale: a penny placed on a rock, a person standing beside a large animal or in a landscape, a measuring tape next to a fossil (or an Earth worm!).

Using familiar objects for scale isn’t possible for tiny things. We don’t have a clear mental image of the size of a salt grain or sesames seed to reliably use them to scale for instance cells**. We therefore include scale bars in microscopy images. With ImageJ/FIJI files from any microscope system can be read in along with their scaling information (shout-out to Curtis and Melissa and the Bio-Formats project!). By using Analyze > Tools > Scale Bar we can add the scale bar with a user-defined length, width, color, position, and label. Now the audience can calculate the actual size of objects and relate image with reality.

Four tips for superb scale bars

• Length: Be kind to your audience and use simple units, such as 100um, 50um, 10 or 2um.
• Color: Scale bars should have a high contrast with the background. Avoid red, green, or blue bars, as these colors might be considered part of the image.
• Position: Lower left corner is a safe place. The upper space should be kept for important information like species, cell type, or gene name.
• Add scale bar last: In the process of writing your manuscript you may re-think the figure size. Also images are re-sized for posters and slides. It is therefore easierst to add only a very fine scale bar with FIJI and then re-draw it in Adobe Illustrator (or PowerPoint, as I I know that about half of you out there use PowerPoint for making figures and posters!).

And finally, do not miss this article by Monica Zoppe with an interesting idea on how to communicate subcellular sclales better!

* I’d be delighted to stand corrected, and if you find old scientific images with scale bars, or interesting scales, send them my way for my collection!

** a great tool to update yourself in comparable scales in biology is here: http://learn.genetics.utah.edu/content/cells/scale/.

I never cease to be amazed at the relative size differences of cells and how they vary over so many magnitudes!

(5 votes)

Loading...

We are seeking outstanding candidates to lead a project studying Notch, ephrinB2, and TGF-b signaling pathways in arterial venous programming/reprogramming during development and disease processes. We take a conditional mouse genetic approach to manipulating gene expression in endothelial cell-specific and temporally controlled fashion. We also use cutting edge in vivo real time imaging technology, including an in-lab constructed two-photon microscope, which provides exceptional access to gene function in vivo at the cellular resolution along with blood flow measurement overtime in live animals. This basic approach is complemented by preclinical studies with our elegant mouse models of diseases, offering outstanding opportunities for translational research. The laboratory is well equipped with state-of-the-art capabilities at the molecular, cellular, and organismic levels. In addition to funding from the PI, we also have an excellent track record in sponsoring postdoc fellowships. We are interested in a well-trained, highly productive recent Ph.D. to continue our innovative breakthroughs in a rewarding training program. This postdoctoral research is an excellent platform for a highly productive Ph.D. with a strong motivation to become a future group leader. Experience with mouse techniques is a plus. UCSF offers outstanding postdoctoral career development opportunities. Please submit your CV, research interests, and the names of three references by email with a subject title “postdoc application” to:

Rong Wang, Ph. D.
Professor
UCSF
rong.wang@ucsf.edu

(No Ratings Yet)

Loading...

Multiple postdoctoral positions are available in the field of quantitative stem cell biology at the Warmflash lab at Rice University. Our lab uses human embryonic stem cells to model early embryonic development with a particular focus on understanding the mechanisms of spatial patterning and morphogen signaling dynamics. Our work combines quantitative experimentation with data analysis and mathematical modeling. For more details, see our lab webpage here and examples of our work can be found in our recent publications: Warmflash et al. Nature Methods 2014, Sorre et al Dev Cell 2014, Nemashkalo et al. Development 2017.

Positions are available for:

1. Theoretically trained scientists interested in working closely with experimentalists. Experience with either mathematical modeling of biological systems or analysis of biological data is preferred but not required.
2. Experimental biologists interested in quantitative approaches and working closely with theorists. Experience with cell culture, microscopy, or molecular biology is preferred but not required.

Interested candidates should email a CV and a brief statement of past research accomplishments and future research interests to aryeh.warmflash@rice.edu.

(No Ratings Yet)

Loading...

A Research Technician position is available in the research group of Mina Gouti at the Max Delbrück Center for Molecular Medicine in Berlin. The group is using human and mouse pluripotent stem cells to study the development and disease of neuromuscular system. Further information about research in the lab can be found at: https://www.goutilab.com

The successful candidate will have a B.Sc. or M.Sc. degree in Biology or other related discipline, extensive experience in molecular biology and cell culture. Excellent communication, organization and prioritization skills are required, as well as flexibility to the work schedule as stem cell maintenance involves weekend attention (on a rotating basis). Proficiency in writing and speaking English is essential. Knowledge of German language will be considered as an additional advantage.

Key responsibilities will include:

• Performing research projects involving molecular biology techniques, pluripotent stem cell culture and differentiation as well as mouse and chick embryological techniques.
• Maintenance of mouse and human pluripotent stem cell lines for the lab stock.
• Generation of new pluripotent stem cell lines using the Crispr/Cas9 system.
• Oversee the maintenance of mouse colony, monitoring and submission of animal protocols.
• Provide technical support to other lab members when required.
• Establish, maintain and improve research protocols and maintain lab records.
• Oversee the effective running of the lab by monitoring stock levels, ordering consumables and reagents, maintaining equipment and updating lab databases.
• Attending safety courses in order to support and maintain good laboratory practice and safety procedures.

Minimum Experience:

Two years of lab experience working with molecular biology techniques. Previous experience with maintenance of a mouse colony and/or pluripotent stem cell culture will be an additional advantage.

The salary will be according to the TV-L9a scale and the contract will be initially for two years with the possibility of renewal. Applicants should send their CVs along with names and emails of at least two referees to Dr Mina Gouti (mina.gouti@mdc-berlin.de).

Deadline : 31st of August 2017

(No Ratings Yet)

Loading...

Blog post written by Isabelle Vea – 2017 Embryology Student

All 24 of the 2017 Embryology students came to Woods Hole to learn from the best scientists in the developmental biology field. We were immersed in a unique setting to interact with established and promising investigators. In general, each invited lecturer came and spent from a few days to a couple of weeks at the Marine Biological Laboratories and was dedicated to interacting with us. Through our interactions with the faculty we quickly learned that it was more than just learning knowledge and techniques. For instance, the course directors carefully planned lunches and dinners with one faculty and two of the students allowing us to discuss informally about all sorts of topics.

In this post, I would like to share some of the interactions I had with the course faculty that are not related to embryos, yet were extremely meaningful.

Science and art

I always thought that science required creativity and so for me, art/crafts and science is a natural combination. But is it possible to combine both in our daily lives? Isn’t science already a lot? One of my most memorable discussions was with Bob Goldstein from UNC Chapel Hill, who managed to integrate his artistic views into the academic world.

Bob creates posters for scientific seminars in his department using screen printing. It was refreshing to be able to discuss screen printing techniques, inks etc. with Bob. We also discussed using arts and craft as a medium for talking about science at outreach events.

I do think it is possible to combine research with other hobbies or non-scientific activities. The bonus (and actually what Bob taught me through meeting him) is to be able to find other researchers with the same artistic interests.

If you are an artistic scientist, give a shout out in the comments! And if you are interested in discovering scientists with an artistic mind, check out #sciart on social media platforms.

Leadership

As graduate students and postdocs, our primary concern is to find the next research position. As a postdoc myself, I am still struggling to figure out what type of researcher I want to become in the long term. I have always wanted to know how one scientist decides on becoming a leader in his department or institution. Unfortunately, I have never had the opportunity to ask such questions in my home institutions. Here at the course, asking these questions seemed natural and many discussions took place informally in a pub or the hallway.

I was particularly interested to hear from Claudio Stern about his leadership experiences. Claudio Stern from UCL is involved in service to the scientific community, he is part of the scientific council at the Institut Pasteur in Paris and the previous president of the International Society of Developmental Biology. We discussed how you decide to become a leader in academia, and what opportunities may lie beyond your own lab. I learned that at some point in our science careers, we may ask ourselves whether we would like to help improve our colleagues’ work environment and to do so, we need to be able inspire them.

Enthusiasm in research

Despite my undivided love for invertebrates and the excellence of every module, my favorite week was the zebrafish/frog one. Into the second week of the course, I had been overwhelmed by C. elegans powerful tools to examine cellular mechanisms and going into vertebrate species was quite intimidating. I initially thought that my lack of knowledge in vertebrate anatomy would be detrimental to learn the techniques suggested during the module but something special happened.

The zebrafish lecturers (Elke Ober from University of Copenhagen and Tatjana Piotrowski from Stowers Institute) were very present during our lab time. They were not only physically here but kept checking on our experimental progress throughout the week and transferred a lot of their enthusiasm to us.

I had decided to perform a simple experiment that would back up my very risky one. As expected my risky experiment failed but I had managed to obtain time lapse images of the simple one. The night before show and tell, I asked one of the lecturers if there were still time to set up a new experiment: transferring cells of an embryo that I would have injected with fluorescent dye, into another embryo and see where the cells develop. I had never transferred any cells from one organism to another and the task seemed impossible to do with less than 24 hours left. But Elke just looked at me with excited eyes: “Yes! You should try! I will help you.” I could not not try.

Next thing I knew, I was injecting embryos with fluorescent dyes and transferring cells at 1 am in the very last hours of the module and both Elke and Tatjana were there as moral supports! The next day we checked the embryos, some of them survived and it worked! It was the most rewarding result of the course, not only because it worked but because I was inspired by their enthusiasm.

For sure, on a daily basis, experiments are not always successful (a big proportion of mine actually failed during the course), but just having one experiment work and a supportive and passionate community makes your day.

Overall, each faculty member came to Woods Hole with something to share with us. They taught us what they know about embryos but to me, they also conveyed their passion, shared their life experience and how this can be relatable to us, as budding independent investigators. And for this, the course was invaluable.

Isabelle Vea

Isabelle Vea is a Marie Skłodowska-Curie Fellow at the University of Edinburgh interested in the evolution of scale insects.

Twitter: @thecochenille

(13 votes)

Loading...