An important question in developmental biology is how regions with distinct identity are established despite the intermingling of cells that occurs during growth and morphogenesis. Our recent work revisited some old studies of how the vertebrate hindbrain is patterned, and found that sharp and homogeneous segments are formed through a combination of identity switching and border control.
The story started in the late 1980s, in the early days of analysing developmental gene expression using in situ hybridisation. One of the genes we analysed was egr2 (a.k.a. krox20), a transcription factor which had been identified by Patrick Charnay as an early growth response gene in fibroblasts. To our amazement, we found that egr2 is expressed in stripes in the hindbrain, corresponding to two rhombomeres, r3 and r5. We then collaborated with Robb Krumlauf to show that hox genes have segmental expression in the hindbrain. egr2 and hox genes were later found to be components of a network that regulates segmental identity. A striking feature of their segmental expression is that they come to have razor sharp borders, and a clue to how these form came from the work of Scott Fraser and colleagues. They found that once morphological borders are seen in the chick hindbrain, cells do not intermingle between segments.
Sharp and homogeneous segmental expression of egr2 in the hindbrain.
In another collaboration with the Charnay lab, we carried out a screen to identify kinases that are segmentally expressed in the hindbrain. One of these, a receptor tyrosine kinase subsequently named EphA4, is expressed in r3 and r5, and we later found that it is a direct transcriptional target of egr2. We went on to show that Eph receptors and their ephrin ligands underlie cell segregation that sharpens the segment borders. This turned out to be the first example of a general role of Ephs and ephrins in border formation during development.
These findings fit the familiar idea that cell segregation sharpens and stabilises tissue organisation. However, the lineage studies in chick had found that cells marked at early stages can contribute progeny to adjacent hindbrain segments. Furthermore, experiments by Trainor and Krumlauf in mouse, and by Schilling, Prince and Ingham in zebrafish, had shown that cells transplanted between segments change identity to match their new neighbours. Intriguingly, identity switching occurs for single cells but not when groups of cells are transplanted. These findings in the early 2000s suggested that some intermingling occurs, and identity switching ensures that segments nevertheless establish a homogeneous identity. However, this idea languished as intermingling between segments had not been directly visualised, and the mechanism of switching remained a mystery. This was the problem that Megan Addison took on as her PhD project in my lab.
We reasoned that intermingling and identity switching of cells would mainly occur at early stages, when egr2 is first expressed but EphA4 has yet to be upregulated to sufficient levels to drive cell segregation. The key question is whether any egr2-expressing cells intermingle from r3 and r5 into adjacent segments and then downregulate egr2 expression. To address this question, we used the newly-emerging techniques for genome manipulation in zebrafish to create an enhancer trap in which a stable reporter is expressed directly from the egr2 locus. During this work, another lab reported that intermingling does not occur between hindbrain segments in zebrafish, but used reporters expressed one step downstream of egr2, which might miss the time window in which mixing occurs. Indeed, using the early reporter line that we created we found that cell intermingling and identity switching does occur.
Stable reporter for egr2 generated by CRISPR/Cas9 mediated insertion of H2B-Citrine into the egr2 locus. Some cells that have expressed egr2 are found in even-numbered segments. These cells switch identity to match their new neighbours.
We started wondering what the mechanism of switching might be, and here some other old findings came into play. The Charnay lab had reported in 2001 that mosaic ectopic expression of egr2 in the chick hindbrain causes adjacent cells to upregulate egr2. This suggested that egr2 regulates a community effect, which in classical models would involve upregulation of a signal that non-autonomously induces egr2. Such community signaling leads to homogeneous gene expression within a field of cells, and might explain why groups of transplanted cells do not switch identity. However, the puzzle of how egr2 induces egr2 in adjacent cells had also languished in the literature.
Megan analysed whether ectopically expressed egr2 acts non-autonomously in the zebrafish hindbrain. We found that it does when the egr2-expressing cells have a scattered distribution, but not when they later segregate from cells with even-numbered identity. Since our previous work had shown that the segregation is driven by EphA4, we blocked it by simultaneous knockdown of EphA4 and found that non-autonomous induction was restored. Non-autonomous induction thus depends upon how many neighbours of the same or different type you have: egr2 is only induced in cells that are surrounded by egr2-expressing cells.
What might the mechanism of the community regulation of egr2 be, and does it account for identity switching? We started thinking about retinoic acid (RA) signaling as a candidate. An RA gradient establishes segmental identity in the hindbrain, and studies of Tom Schilling in zebrafish had shown that graded expression of an RA-degrading enzyme, cyp26a1, has a key role. The lab of Cecilia Moens found that two other family members, cyp26b1 and cyp26c1, have dynamic segmental expression that also contributes to A-P patterning. We wondered if this segmental expression is under the control of segment identity genes and thus acts in a feedback loop. We found that this is indeed the case: egr2 underlies the lower expression level of cyp26b1 and cyp26c1 in r3 and r5 compared with r2, r4 and r6. Since a high level of cyp26 enzymes can non-autonomously decrease RA levels in neighbouring cells, they could decrease RA signaling in single cells that have intermingled. Indeed, loss of cyp26b1 and cyp26c1 function disrupted the identity switching of egr2-expressing cells that have intermingled into adjacent segments. We showed that in r4 the switching involves upregulation of hoxb1, which in turn represses egr2 expression.
This work has revealed parallel mechanisms of identity switching and border control that establish sharp and homogeneous segments in the hindbrain. At early stages, some cells mix into adjacent segments and switch identity to match their new neighbours. This mediated by a community effect in which there is reciprocal feedback between RA levels and segmental identity. Subsequently, Eph receptors and ephrins are upregulated and they underlie segregation that prevents intermingling and sharpens the border.
These studies of hindbrain patterning raise the question of whether similar mechanisms operate in other tissues. Since mediators of cell segregation are often regulated downstream of regional identity genes, some intermingling between adjacent regions may occur at early stages. Furthermore, plasticity in cell identity is a common feature at early stages of development. Insights will come from the creation of further reporter lines to visualise cell intermingling and cell identity.
In our recent paper published in eLife, we found a novel form of movement-dependent neural feedback that drives early forebrain growth in zebrafish. In this article, I discuss the problems, solutions, and lucky breaks that led to our finding. I also end up giving the mighty zebrafish larvae the credit it so deserves.
A year before the completion of my PhD, I unofficially joined Vince Tropepe’s lab at the University of Toronto as a postdoc over the course of a two hour Skype call. During this call, we slowly came to recognize one another as our academic complements: Vince’s history is firmly rooted in one end of the organismal spectrum, the genetics and cell biology of stem cells, extending up to brief forays into how sensory experiences modulate adult neurogenesis in vertebrates. Conversely, with no history in genetics or cell biology, I had started at the other end of the organismal spectrum, studying the evolution of brain structure and animal behaviour and extending down to how sensory experiences modulate adult neurogenesis in vertebrates. With a common interest in how the environment shapes brain development, together we felt that our collective expertise might offer a novel perspective to studying the importance of sensory experience on the production of new brain cells. Also, Vince promised me that I would become a competent geneticist–a promise we are still both working on today.
A year later, I arrived at the University of Toronto and met both Vince and zebrafish. The fish were much smaller than in the figures. Eager to delve into the behavioural repertoire of zebrafish, I purchased my own set of adults from a local pet store and watched them daily from home. Whereas my mind was dancing among work documenting the acoustic communications of midshipman fish, territoriality in cichlids, and nest construction in sticklebacks, my eyes were settled on a set of four fish that seemed to simply swim and eat and swim again. The fish neither sang nor built a nest, two behaviours I had previously studied in songbirds throughout grad school. Why were people studying zebrafish again?
In countless reviews, I had read that the power of studying zebrafish is in the accessibility of the embryo during development and their genetic tractability. The former of these advantages is evident during the first 24 hours of zebrafish development, over which one can watch a pigment-less embryo develop into a larvae in real time. Now one day old, the genetic tractability of zebrafish became obvious in the variety of transgenic zebrafish strains available in Vince’s lab, starting to glow red or green or red-and-then-green beneath the stereomicroscope. While I may have been taking the nicest micrographs of my career, I soon became worried about my choice in model system if all of the advantages of working with zebrafish–transparent embryos; the ability to introduce mutations or transgenes–only applied to the first couple days of development. At these early stages, larvae had yet to develop the neural connections to process sensory information and by the time they did, pigment would (rudely) form in the skin, blocking my view of the brain. I feared that I was trying to drag zebrafish larvae out of the developmental window for which they were chosen as a model system.
As I assume many postdocs do, I started in the lab bursting with creative energy and began experimenting by instead repeating a previous lab member’s work only with a slightly different focus. In my case, I was building off of original work by Ben Lindsey, who had shown in a collection of studies how visual, social, and olfactory experiences modulate the production and survival of new neurons in the adult zebrafish brain (Lindsey and Tropepe, 2014; Lindsey et al., 2014). Ben had integrated zebrafish into the popular field of experience-dependent adult neurogenesis, dominated by rodent models but also involving a menagerie of other vertebrates including amphibians, lizards, fish, and birds. My work differed from Ben’s in that I was manipulating sensory experience in zebrafish larvae only days old, compared to the adult fish in his previous studies. The decision to study postembryonic brain development instead of adult neurogenesis was motivated by both practical advantages and, more importantly, theoretical reasons. Practically, larvae are tiny and easy to manipulate: we could generate hundreds of larvae and even still reap some of the advantages of transgenic strains, such as fluorescent labeling of neuronal populations in histological sections. Theoretically, Vince and I had come to question whether adulthood was the developmental stage best suited to our questions.
Most often, the study of neurogenesis in the vertebrate brain is experimentally bimodal: studies focus on either embryonic neurogenesis, in which neurons are generated to initially produce a central nervous system, or adult neurogenesis, where the brain is functionally mature and neurons continue to be added at the relatively lowest rate in life. We found ourselves interested in the awkward developmental period in between these extremes, broadly referred to here as postembryonic development. Somewhere in postembryonic development, the brain must become sufficiently developed to begin processing incoming sensory input from the environment. During this time, the brain is also still growing, including the incorporation of new neurons. The combination of sensory processing and continued brain growth make postembryonic development the period of brain growth most sensitive to sensory experience. This postembryonic sensitivity is thought to explain why learning new languages or musical instruments is easier earlier than later in life (White et al., 2013). Within the nervous system, these forms of experience-dependent neuroplasticity are traditionally considered to occur through modifications in the connections between pre-existing neurons. We asked whether the continued production of new neurons itself may also mediate the effects of experience on early brain growth.
With a focus on postembryonic neurogenesis, I began a multitude of experiments manipulating the sensory environments of zebrafish larvae. Initially, my work focused on modifying the visual experiences of larvae by rearing them in different lighting conditions and tracking the developmental trajectory of both the retina and its primary target in the midbrain, the optic tectum. This approach benefitted from the extensive published work characterizing 1) neural circuitry in the retinotectal pathway of zebrafish, 2) the switch from spontaneous to visual input-dependent neural activity in the optic tectum upon maturation of visual input by 5 days of age, and 3) Ben’s previous work demonstrating that visual experience would modulate neurogenesis in zebrafish. These experiments culminated in a publication released earlier this year in the Journal of Neuroscience (Hall and Tropepe, 2018a), in which we discovered that visual experience-dependent growth of the zebrafish midbrain is mediated, at least in part, by the modulation of new neuron survival.
Parallel with my work on visual experience, I was also piloting approaches to noninvasively manipulate social and olfactory experience in zebrafish larvae. Across all of these studies, I discovered a novel experimental advantage in using zebrafish that I had never seen reported in a literature review. It turns out that zebrafish larvae are amenable to extreme manipulations in the sensory environment. This advantage is conferred by the combination of the zebrafish larva’s small size, built-in food source (a yolk providing nutrition for the zebrafish for up to 5-6 days of age), and easily met living conditions. Using zebrafish larvae, I was able to isolate, restrict, and enrich distinct sensory modalities noninvasively without grossly altering the larva’s ability to develop. Perhaps the best example of this sensory tractability I’m referring to is in our recently published paper in eLife (Hall and Tropepe, 2018b).
My work on motor experience began after I stumbled upon the ViewPoint Zebrabox, a chamber designed to track zebrafish larval swimming behaviour, sitting idly in one of my testing rooms. Vince explained that he had purchased the system using an equipment grant years ago after a previous postdoc, Bruno Souza, had the unfortunate opportunity to manually code larval swimming from video. Bruno discovered that disruptions to dopamine signaling during postembryonic brain development reduced both the size of an inhibitory neuron population in the zebrafish forebrain and the amount a fish would swim (Souza et al., 2011). One conclusion from this work was that perhaps this inhibitory neuron population is required to support normal motor development. Looking to be a contrarian, I argued the opposite, that the amount a fish swims may guide the development of the neuronal population. Intent on settling the issue, we brainstormed techniques through which we could transform movement, typically recorded as a dependent variable, into a manipulation itself. Since the discovery that aerobic exercise upregulates adult neurogenesis in mammals (van Praag et al., 1999), movement has overwhelmingly been manipulated positively with the use of stationary wheels and wind/water tunnels to encourage running, swimming, and flying. Still a contrarian, I suggested we instead restrict movement, restraining larvae within small cylindrical mesh fences to reduce the available swim space in a similar volume of water compared to controls.
A) Mesh fences we used to restrict the swimming of zebrafish larvae. A single larvae was housed in each fenced well and control larvae were individually housed in unmodified wells. (B-E) Restraining larvae significantly reduced the amount they would swim from 6 days of age onward. Figure originally printed as Figure 1 in Hall and Tropepe (2018b).
One summer semester later, and with the help of one eager undergraduate student, I had my answer as to whether movement drives the development of this inhibitory neuronal population: no. Although our restriction paradigm significantly reduced swimming from both 3-6 and 3-9 days, we could account for every inhibitory neuron produced regardless of swimming experience. Luckily, we noticed something else. When we restrained larval swimming for as long as 6 days, the entire inhibitory neuronal population was there, sure, but the forebrain itself was significantly smaller in our restrained larvae, suggesting that movement could be modulating neurogenesis, albeit it not via the production of the neurons we were focusing on.
Using a variety of histological markers and changing our focus from the neurons being produced to the cells producing them, the neural stem cell and intermediate progenitor populations, we found that restricting swimming led to fewer proliferative cells in the dorsal portion of the forebrain, referred to as the pallium. Subsequent analysis found that this reduction was not attributed to cell death or a change in the population of neural stem cells, but a reduction in the size of the intermediate progenitor populations, which amplify the rate of neurogenesis by adding additional proliferative steps between stem cell and differentiated neuron. Recognizing that reducing swimming by rearing larvae in less physical space may be complicated by reductions in other sensory inputs, such as reduced visual input with less to see, we complemented our approach by rearing larvae against water currents to increase swimming. Using this approach, we conversely found that increasing swimming also increased the population of proliferative cells in the pallium. Together, these experiments formed the basis for our mechanistic work probing how movement itself modulates early brain development.
Thus far, our work seemed to fit nicely among the extensive literature on exercise-dependent increases in vertebrate adult neurogenesis, with the added benefit that we could manipulate movement in both directions. Having learned that working in a cellular and molecular biology department meant you should draw a lot of mechanistic flowcharts, I took a stab at it.
Though wildly underwhelming, the simple framing of movement as affecting neurogenesis led us to discover a notable gap in the literature. Mechanistic work on exercise-dependent adult neurogenesis has implicated genes, neurotransmitters, and growth factors that act within the neurogenic niche. However, these proliferative populations do not often receive direct input from the body, leaving the question as to how the body signals the brain to increase neuron production. Current theories are centred around the concept that muscle engagement could release trophic factors into circulation that are carried to the brain to stimulate growth, though this model is difficult to test. We argued that the best step towards linking bodily movement to brain growth was to ask, “what sort of information does movement provide the brain?” and how each of these modalities may be isolated or removed during swimming. Enter the tolerant zebrafish larvae.
Optic flow is the term referring to the visual illusion of movement. As we walk forward, the visual environment appears to move backward. This visual input is processed by the brain to maintain balance during movement and explains why, when stopped in traffic, a neighbouring cars rolling forward creates the illusion that we are rolling backward. In our first experiment, we aimed to divide swimming into a visual component and a physical component. But how could we trick a fish that isn’t swimming into perceiving movement? And conversely, how could we allow a fish to swim without moving throughout the environment? Our solution came from a common treatment in many zebrafish imaging studies–agarose embedding. Although immobilizing larvae in agarose is commonly used in calcium imaging or electrophysiological preparations, these preparations are relatively acute. We found that we could chronically immobilize larvae using a sufficiently low concentration of agarose gel. Embedded larvae are unable to move but remain alive via water flow through agarose and by feeding on the yolk they are born with. Unable to move, we then reintroduced sensory components of movement to our fish. Optic flow was simulated by exposing animals to computerized black and white gratings moving in a given direction. Free-swimming larvae will swim parallel to the direction of movement of a grating (perpendicular to the stripes of the grating). Exposing larvae to a battery of moving gratings through days 3-6 acted as a form of visual movement-dependent input. To reintroduce physical components of movement, we cut larval tails free from the agarose, enabling the bodily movement of swimming without physical displacement in the environment. Using these manipulations, we found that only physical input associated with swimming (tail movement) stimulated increased neurogenesis in the larval forebrain, leading us to cross visual input off the list.
Unlike visual input, which would predictably originate from the retina, physical input during swimming could be sensed by multiple systems in larval zebrafish. Externally, the movement of water against the skin could be detected by the lateral line, a system of cell clusters along the length of teleost fish containing hair cells that extend their cilia outside of the body into the external environment. Water movement causes the cilia in hair cells to bend, which then drives ascending input to the central nervous system. Because of the exposed nature of these hair cells, they can be easily ablated through the treatment of chemicals added to the fish water. We found that ablating lateral line hair cells had no impact on forebrain neurogenesis, suggesting this pathway was not mediating the effects of movement on brain growth observed here.
Internally, bodily movement in zebrafish larvae could be detected via two neuronal populations throughout the trunk of the fish.
Rohon-Beard cell (*) and dorsal root ganglia (arrow) populations visualized in the trunk of a larval zebrafish using the Tg(Isl2b:mgfp) transgenic line. Figure appeared originally as Figure 6A in Hall and Tropepe (2018b).
The first population, Rohon-Beard cells, lie within the spine and extend somatosensory processes throughout the skin. This population of neurons develop early in zebrafish development and appear to die off entirely by adulthood. The second population, dorsal root ganglia, lie on each side of the spine and first appear only by 3 days of age, but will exist for the rest of the fish’s life. Upon identifying these neuronal populations, we at first rejoiced at the opportunity to take advantage of the transgenic tools available in zebrafish, isolating these populations by the genes they uniquely express. While searching for transgenic lines reported to include targeting to either Rohon-Beard cells or dorsal root ganglia, I stumbled upon a bit of luck. One of the transgenic lines isolating Rohon-Beard cells under the expression of the isl2b promoter was already in the lab! Vince had received this line from Chi Bin Chien and I had been using it for the past year as this promoter also isolates the projection from the fish retina to the optic tectum! I reared a clutch of larvae from this transgenic line and inspected their bodies to find bright green Rohon-Beard cells! Unfortunately, I also found bright green dorsal root ganglia beside them. Expression of our fluorescent reporter in both cell populations meant we would be unable to isolate these different sensory inputs genetically.
With a little luck and I lot of reading, I soon found another approach to isolating Rohon-Beard cells and dorsal root ganglia in the work of Judith Eisen’s lab. The Eisen lab had found that precursor cells that would ultimately give rise to dorsal root ganglia must first migrate out of the spine into the body. These precursors require a specific cell signal to tell them to stop in the correct location to generate dorsal root ganglia. If this signal is blocked during this migration time using the drug AG1478, the precursors continue to migrate aberrantly, failing to give rise to any dorsal roots (Honjo et al., 2008). Replicating these results in the lab, we found that acute treatment with low doses of AG1478 blocked the initial formation of dorsal root ganglia, generating larvae lacking one of these two neuronal populations. With a batch of zebrafish larvae lacking a dorsal root ganglia population in the trunk, we repeated our swimming restraint experiments. We found that larvae deficient in dorsal root ganglia exhibit attenuated swimming-dependent forebrain neurogenesis, suggesting dorsal roots mediate sensory feedback during swimming to maintain an expanded pool of proliferating cells in the forebrain.
By treating zebrafish larvae expressing mgfp in Rohon-Beard cell and dorsal root ganglia populations with AG1478, we were able to generate larvae that lacked dorsal root populations in the trunk (A). These larvae deficient in trunk dorsal root ganglia exhibited similar swimming behaviour as controls (B). However, larvae deficient in trunk dorsal root ganglia exhibited reduced forebrain neurogenesis in response to the same amount of swimming (C-G). Figure printed originally as Figure 6 in Hall and Tropepe (2018b).
At this point, we were beginning to feel confident that we may have identified one form of sensory feedback during movement that could drive forebrain growth. And unlike previous models, which suggested these signals would be transported via the circulatory system, our results suggested this input could also come directly from peripheral nervous input. However, like our manipulations of swimming behaviour in the original experiments, we believed that removing dorsal root ganglia and finding a reduction in neurogenesis should be complemented with an experiment in which we drive neurogenesis using peripheral neural input, aimed primarily at dorsal root ganglia. Based on our experiments up to this point, we predicted that activating this peripheral sensory input could drive increased forebrain neurogenesis, even in the complete absence of movement.
After a handful of failed pilots in which I had tested drugs I was hoping may enhance neuronal firing in dorsal root ganglia, I met with Vince, worried our study may have to stop one experiment short of our plans. I was unable to believe there was not some known drug that could be added to the water to activate dorsal root ganglia in zebrafish. Vince was also unable to believe this, particularly after just having sat through a PhD committee meeting earlier that day in which he listened to a graduate student report on Optovin, a drug characterized by Randy Peterson’s lab that can be added to the water to activate peripheral neural feedback, including dorsal root ganglia. Eureka (I should have said). Optovin is a small molecule that interacts with the TRPA1b receptor found in the peripheral nervous system of zebrafish, changing the dynamics of these receptors to open following exposure to purple light (Kokel et al., 2013). With this stroke of luck and a batch of Optovin aliquots generously provided by Jim Dowling, we were able to control peripheral nervous feedback in larval zebrafish embedded in agarose, unable to move. Furthermore, we found that a single session of light pulses in Optovin-treated larvae produced a significant increase in the number of proliferating cells in the zebrafish pallium as early as the next morning. When we repeated these light + Optovin treatments in larvae deficient in dorsal root ganglia, the effect was blocked, consistent with a direct role for dorsal root ganglia in providing neural feedback during swimming to encourage forebrain growth via increased neurogenesis.
Altogether, we believe our study provides a novel perspective on the importance of movement in early brain development both because of our model of sensory feedback, but also for the age of the fish we study. First, our findings suggest a wholly novel form a sensory feedback during movement that may act as a means of coordinating body and brain development. Fish that swam the most also exhibited the highest rates of neurogenesis. Second, although we have borrowed much of our theory from the study of exercise-dependent neurogenesis, we believe our results here may be unique in the examination of postembryonic brain development. Often considered simply an early form of the slower neurogenesis persisting into adulthood, increasing work has suggested that postembryonic neurogenesis in developmentally unique, producing populations of neurons not generated elsewhere in life. Accordingly, we believe our work may specifically highlight the importance of movement-generated sensory feedback during early brain growth in vertebrates. Such a coordination between brain and behaviour may explain the comorbidity of cognitive deficits in diseases predominantly considered to be muscular, such as congenital myopathy.
As a study, we believe the power in our results comes from the collection of experiments we performed as a whole. Whereas we often performed experiments that were met with technical limitations, we could always generate a complementary experiment to overcome these restrictions. And all along the way, from mesh fences to swim tunnels to immobilization and Optovin jolts, we believe no other model organism would’ve kept on developing as well as the zebrafish larvae. So what if they don’t build a nest?
Understanding how out of single cells functional tissues and organs develop is a major challenge of biology. Recent progress allows us to grow organ-like cell assemblies (organoids) from stem cells in vitro. Organoids offer great potential for studying diseases and development. However, in many cases we do not yet understand how these complex tissues emerge out of progenitor stem cells. A common feature in the initial growth phase of many organoid systems is the formation of a polarized epithelial cyst with a single or multiple internal apical lumen. This initial transition into an epithelial cyst establishes a tissue template that on the one hand enables maintenance of progenitor/stem cells (niche) and on the other hand guides the patterning of differentiated cells into a functional tissue. Our aim is to understand how the interplay between proliferation (cell divisions), polarization (epithelial transition) and differentiation (patterning) leads to self-organization of this epithelial progenitor template and how this structure facilitates correct patterning into functional organoids. To this end, we will systematically control and characterize the early growth phase of two organoid systems (pancreatic and neural tube) using microfabrication and micro-patterning approaches. We will quantify evolution of cell shapes, adhesion and cortical forces, apical-basal polarization and differentiation as a function of initial cell contact patterns. This approach will provide the means to find rules how local cell interactions (cell-cell, cell-matrix, cell-lumen) are connected to tissue growth and differentiation. We will then test sufficiency of the hypothetical rules to generate the observed organoid structures using an in silico mechano-chemical model. Taken together, by dissecting the early growth phase of two organoid systems, we aim to uncover the common rules on how progenitors establish a polarized epithelial template, and how this template is then differentially used to generate organ specific differentiation patterns.
Candidates will join a team at the Interfaces between Physics and Biology. Applicants with backgrounds in cell and developmental biology, theoretical physics, microfabrication/microfluidics will be considered for interviews.
For more details, candidates should contact the following PIs with CV and motivation letter before June 15th 2018 :
This post highlights the approach and finding of a new research article published by Disease Models and Mechanisms (DMM). This feature is written by Lacey Kennedy as apart of a seminar at The University of Alabama (taught by DMM Editorial Board member, Prof. Guy Caldwell) on current topics related to use of animal and cellular model systems in studies of human disease.
Lacey Kennedy
Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA
Pansarasa O.1, Bordoni M.1,2, Dufruca L.1, Diamanti L.2,3, Sproviero D.1, Trotti R. 4, Bernuzzi S.5, La Salvia S.1, Gagliardi S.1, Ceroni M.2,3, Cereda C.1
Genomic and post-Genomic Center, “C. Mondino” National Neurological Institute, Pavia, Italy.
Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy.
General Neurology Unit, “C. Mondino” National Neurological Institute, Pavia, Italy.
Department of Neurodiagnostics and Services, Laboratory of Clinicals and Chemicals Analysis (SMeL), , “C. Mondino” National Neurological Institute, Pavia, Italy.
Department of “Medicina Diagnostica e dei Servizi”, IRCCS Policlinico San Matteo Foundation, Pavia, Italy.
Pansarasa, O., Bordoni, M., Dufruca, L., Diamanti, L., Sproviero, D., Trotti, R., Bernuzzi, S., La Salvia S., Gagliardi, S., Cereda, C.(2018). ALS lymphoblastoid cell lines as a considerable model to understand disease mechanisms. Disease Models & Mechanisms, (January), dmm.031625. https://doi.org/10.1242/dmm.031625
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons (Hardiman et al. 2017). Although a rare disease, affecting approximately 15,000 Americans between the ages of 55-75 years old, it is one of the many ageing-related diseases growing in prevalence as global lifespan increases (https://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Amyotrophic-Lateral-Sclerosis-ALS-Fact-Sheet). ALS is a multi-factorial disease that causes muscle weakness, spasticity, cognitive impairment, and ultimately ends in a premature death within five years after the diagnosis (http://www.alsa.com/about-als/facts-you-should-know.html). While many causes are still unknown, there are three main genes thought to play a role in the pathogenesis of ALS: Copper zinc superoxide dismutase (SOD1), TAR DNA Binding Protein 43 (TDP-43), and RNA binding protein FUS. SOD1 is a highly conserved enzyme that scavenges for superoxide radicals, but in its mutated form is very unstable and has been found to unfold even at physiological pH and temperature (Lee, S., & Kim, H. 2015). This mutation can lead to an aggressive form of the disease; in fact, ALS can be characterized by an increase of SOD1 expression in peripheral blood mononuclear cells, as well as neurons (Cereda, C. et al., 2013). Furthermore, TDP-43 and FUS, which are both involved with RNA regulation and transcription, appear to be functionally related in ALS pathology (Lee, S., & Kim, H. 2015). FUS mutations appear to play a role in young-onset of the disease and are characterized by predominate degeneration of the lower motor neurons, although a direct mechanism is not well understood (Blokhuis, A. M, etal. 2013).
While serving different functions, when any of these three genes are mutated in ALS patients, mitochondrial function becomes abnormal, which affects neurons, as well as surrounding tissues. Due to the increasing realization that the molecular mechanisms of ALS are not isolated to the nervous system, Pansarasa et al.identified the need for an alternative model for ALS research in a recent Disease Models and Mechanismsarticle. In this study, they sought to identify biologically relevant molecular hallmarks of ALS in lymphoblastic cell lines (LCLs) isolated directly from human ALS patients, with the hope of establishing a new, more effective model for ALS research (Pansarasa, O. et al., 2018). To do this, they looked at the two known pathogenic mechanisms of ALS: protein aggregate accumulation and mitochondrial dysfunction.
Evidence has indicated that mislocalization of SOD1, TDP-43, and FUS could explain many irregularities in ALS signaling(Ido, A., et al,2011). Using western blot,Pansarasa et al. show that SOD1 expression levels decrease in the nucleus of LCLs from patients with sporadic ALS (sALS), SOD1, and TDP-43 mutations as compared to LCLs from healthy patients. They did not see such a change in cytoplasmic SOD1 levels, suggesting that there is a failure to relocate from the cytoplasm to the nucleus. Supporting this idea through immunostaining, the authors found an abnormal presence of protein aggregates in the cytoplasm of SOD1 and TDP-43 mutated LCLs, which further supports the idea of protein mislocalization in ALS patients (Pansarasa, O. et al., 2018). SOD1 and TDP-43 aggregate in certain forms of ALS (Blokhuis, A. M., et al 2013). Therefore, demonstrating the presence of protein aggregates marks an important first step in the establishment of LCLs as a model for ALS as these aggregates are a known hallmark of the disease.
Next, the authors investigated changes in mitochondrial morphology and function. Through TEM microscopy, they confirmed that patients with SOD1, TDP-43, and FUS mutations harbored morphological signs of degeneration in the mitochondria, such as a smaller, rounder size and increased number of vacuoles. After examining the mitochondria, Pansarasa et al. looked at the protein expression levels of proteins regulating the fission and fusion processes. Notably, in patients with a TDP-43 mutation there was a significant increase in expression levels of proteins regulating the fusion process (Pansarasa O. et al, 2018). This increased fusion corresponds to the same increase found in the neurons of ALS patients with TDP-43 mutations.
Finally, the authors investigated the functional changes in mitochondria through the examination of respiration and glycolytic flux. Using the Seahorse Bioanalyzer, Pansarasa et al found that mitochondrial oxygen consumption significantly increased in sALS patients as compared to the controls. Additionally, they found that Spare Respiratory Capacity, the ability of mitochondria to produce energy in conditions of high energy demand, was greatly diminished. The results also suggested a down-regulation of glycolytic flux in SOD1 mutated patients. These changes in mitochondrial activity correspond to a failure of ALS mitochondria to adequately respond to increased energy demands (Pansarasa et al. 2018).
With this, the authors have established a variety of physiological hallmarks of cells affected by ALS. This is of the utmost importance in ALS research as the field changes from the perspective that ALS is just a neurological disease to the view that it is a multifaceted disorder that impacts the entire body. It should be noted, however, that there are some disadvantages to LCLs as a model. Due to the nature of in vitro culturing, LCLs lack environmental factors that affect behavior. For example, Pansarasa et al demonstrated that while some LCL mutations facilitated protein aggregation, there was a lack of aggregation in LCLs from sALS patients, which the authors theorized could be due to a lack of elements regulating nuclear import and export pathways. In addition, there was little significant change in protein aggregation of LCLs with mutated FUS, suggesting that this is not an effective representation of the FUS mutation in ALS patients due to the role of this protein in lymphoblastic cells (Pansarasa O. et al. 2018). Despite this fact, using human LCLs offers a solution to many of the limitations presented with current ALS models.
Before this paper, Peripheral Blood Mononuclear Cells (PBCMs) was the new model growing in popularity for ALS research. While this method shows promise for a potential diagnostic tool, there are major disadvantages to their use in basic research (Nardo, G., et al2011). First, they cannot be maintained long-term because they will lack important environmental stimuli present in the body. Second, although they are part of the immune system, they are not representative of the immune system cells outside of the blood niche. What occurs in PBMCs does not necessarily recapitulate the response of the immune cells from other parts of the body, for example the central nervous system. Finally, researchers are not able to generalize the implications of any one result due to the fact that the immune system health of the donors impacts the response of these cells, resulting in very limited inter-experimental reproducibility (Kleiveland C.R., et al 2015).
The use of LCLs addresses many of these problems, because they can be immortalized and grown in culture. This prospective model represents the potential of a non-nervous system method to study the molecular mechanisms of ALS. Pansarasa et al. have already successfully identified morphological indicators of ALS and have shown that, with the establishment of LCLs as a model, we might be able to one day understand the causes and biological effects of ALS and find ways to do more for patients than simply slow down its progression.
References
Blokhuis, A. M., Groen, E. J. N., Koppers, M., Van Den Berg, L. H., & Pasterkamp, R. J.(2013). Protein aggregation in amyotrophic lateral sclerosis. Acta Neuropathologica, 125(6), 777–794. https://doi.org/10.1007/s00401-013-1125-6
Cereda, C., Leoni, E., Milani, P., Pansarasa, O., Mazzini, G., Guareschi, S., Alivisi, E., Ghiroldi, A., Diamanti, L., Bernuzzi, S., Ceroni, Mauro., Cova, E.(2013). Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis. PLoS ONE, 8(10).https://doi.org/10.1371/journal.pone.0075916
Hardiman, O., Al-chalabi, A., Chio, A., Corr, E. M., Robberecht, W., Shaw, P. J., & Simmons, Z.(n.d.). Amyotrophic lateral sclerosis. https://doi.org/10.1038/nrdp.2017.71
Ido, A., Fukuyama, H. and Urushitani, M.(2011). Protein misdirection inside and outside motor neurons in Amyotrophic Lateral Sclerosis (ALS): a possible clue for therapeutic strategies. Int J Mol Sci. 12(10), 6980- 7003.
Ju Gao1, Luwen Wang1, Mikayla L. Huntley1, G. P. and X. W.(2018). Pathomechanisms of TDP-43 in neurodegeneration Accepted. Journal of Neurochemistry. https://doi.org/10.1111/jnc.14327
Kleiveland C.R.(2015) Peripheral Blood Mononuclear Cells. In: Verhoeckx K. et al. (eds) The Impact of Food Bioactives on Health. Springer International Publishing, https://doi.org/1007/978-3-319-16104-4
Lee, S., & Kim, H. (2015). Prion-like Mechanism in Amyotrophic Lateral Sclerosis: Are Protein Aggregates the Key. Exp Neurobiol., 24(1), 1–7.
Mackenzie, I. R. A., Bigio, E. H., Ince, P. G., Geser, F., Neumann, M., Cairns, N. J., Kwong, L. K., Forman, M. S., Ravits, J., Steward, H., et al (2007). Pathological TDP-43 Distinguishes Sporadic Amyotrophic Lateral Sclerosis from Amyotrophic Lateral Sclerosis with SOD1 Mutations. 4, 427–434. https://doi.org/10.1002/ana.21147
Nardo, G., Pozzi, S., Pignataro, M., Lauranzano, E., Spano, G., Garbelli, S., Mantovani, S., Marinou, K., Papetti, L., Monteforte, M., Torri, V., Paris, L., Bazzoni, G., Lunetta, C., Corbo, M., Mora, G., Bendotti, C. and Bonetto, V. (2011). Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells. PLoS One.6(10),e25545.
Pansarasa, O., Bordoni, M., Dufruca, L., Diamanti, L., Sproviero, D., Trotti, R., Bernuzzi, S., La Salvia S., Gagliardi, S., Cereda, C.(2018). ALS lymphoblastoid cell lines as a considerable model to understand disease mechanisms. Disease Models & Mechanisms, (January), dmm.031625. https://doi.org/10.1242/dmm.031625
This post highlights the approach and findings of a new research article published in Disease Models and Mechanisms (DMM). This feature was written by J. Brucker Nourse Jr. as part of a graduate level seminar at The University of Alabama (taught by DMM Editorial Board member, Prof. Guy Caldwell) on current topics related to use of animal and cellular model systems in studies of human disease.
J. Brucker Nourse Jr.
Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA
1. National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
2. The John Hopkins University/National Institutes of Health Graduate Partnership Program, National Institutes of Health, Bethesda, Maryland
3. National Institute on Aging, National Institutes of Health, Bethesda, Maryland
Developments in modern medicine have allowed humans to reach life expectancies that surpass prior generations (Mills et al., 2016). The tradeoff for increased longevity in most populations has led to greater occurrences of neurodegenerative diseases (NDD), such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). One commonality among NDD is that age is the greatest risk factor (Savica et al., 2013; Nho et al., 2016; Valdez et al., 2012). The bridge between age and NDD incidence remains a mystery, which is why investigation into this link is necessary in order to identify potential therapeutic targets. The majority of NDD cases are associated with individuals over the age of 50. However, there are rare early-onset diagnoses of NDD suggesting that the physiological age of a person may supersede their chronological age, in terms of disease development.
Cyclin-dependent kinase 5 (Cdk5) and its regulation have become a key area of interest for researchers, as the activation of Cdk5 can result in the hyper-phosphorylation of tau and increased amyloid beta aggregation in the brains of AD patients (Wilkanie et al., 2016). Additionally, Cdk5 and one of its activators, p35, were recently found in the Lewy Bodies of the brains of PD patients (Wilkanie et al., 2016). Numerous studies have also demonstrated that abnormalities in Cdk5 expression, both increases and decreases, are associated with multiple types of NDD (Wilkanie et al., 2016). Cdk5 obviously merits researchers’ exploration. A recent article by Spurrier et al. (Spurrier et al., 2018) sought out to observe how manipulating Cdk5α, the Drosophila p35 homologue, would impact NDD and aging in vivo.
Understanding the impacts of alterations to the expression levels of Cdk5αin vivo is a crucial stepping stone into uncovering its role in the aging process. Spurrier and colleagues measured the number of GFP-expressing gamma motor neurons present in the mushroom body (MB) of Drosophila that either had wild-type (WT), overexpression (OE), or knockout (KO) levels of Cdk5α in the motor neurons. Wild-type flies showed a steady, gradual decline of gamma motor neurons over time; however, the Cdk5α-OE and Cdk5α-KO flies had a steep decline with significance appearing at 30- and 45-days old (figure 1B in Spurrier et al., 2018). The associated loss of motor neurons with fluctuations in Cdk5α expression was validated through a motor function assay. Flies with either Cdk5α-OE or Cdk5α-KO demonstrated significant defects in climbing ability from 10- to 45-days old (figure 1D in Spurrier et al., 2018). Flies were also subjected to a lifespan assay that resulted in Cdk5α-OE and Cdk5α-KO flies having a decrease in longevity compared to wild-type animals (figure 1C in Spurrier et al., 2018). It is noteworthy that Cdk5α-OE yielded more severe phenotypes in the neuronal loss and lifespan assays. Collectively, these findings suggest that dysregulation of Cdk5α is involved in accelerating the aging process.
In order to confirm that Cdk5α-induced neuronal loss occurs in a degenerative manner, Spurrier and colleagues measured neurodegenerative phenotypes in Cdk5α-OE and Cdk5α-KO flies. Dysregulation in autophagy is implicated in the pathogenesis of NDD (Menzies et al., 2015). To follow autophagy in vivo, Spurrier et al. used the two autophagy markers: autophagy-related protein 8 (Atg8), a required component of the autophagosomal membrane, and Ref(2)P, a homologue of nucleoporin p62. Wild-type Drosophila steadily expressed both autophagic proteins, with a slight increase in protein levels as the animals aged (figures 2A-D in Spurrier et al., 2018). Cdk5α-KO flies had a significant increase in autophagic markers; however, Cdk5α-OE flies showed a greater fold change (figures 2A-D in Spurrier et al., 2018). Together, these findings suggest a dysregulation of autophagy. This observation corroborates with previous studies that suggest an increase in autophagy protects against NDD (Menzies et al., 2015). The researchers’ observation of an impairment in autophagy fits the narrative that Cdk5 is involved in the pathogenesis of NDD.
In addition to autophagy, researchers used the flies’ sensitivity to oxidative stress to measure aging (Uttara et al., 2009). Flies of different ages, ranging between 3- and 45-days old, were treated with hydrogen peroxide (H2O2) and paraquat, then tested for viability. Cdk5α-OE and Cdk5α-KO flies were significantly less tolerant for oxidative stress when compared to wild-type for both treatments (figures 6A-B in Spurrier et al., 2018). Young Cdk5α-OE and Cdk5α-KO flies had a stress tolerance that was more similar to older wild-type flies. This further supports the notion that modified activation of Cdk5 fast-tracks the aging process.
A major gap in the field of neuropathology is understanding how aging enhances disease susceptibility. Spurrier and colleagues have approached this question by developing an elegant method to identify the physiological age of flies. They compared the expression levels of the genes typically involved in basic biological processes (e.g. metabolism, mitochondrial homeostasis, immunity) in wild-type flies as they aged to establish a standard curve of gene expression, adjusted over time. They then compared the expression levels of these genes to flies with either Cdk5α-OE or Cdk5α-KO genotypes. Both increasing and abolishing expression of Cdk5α led to accelerated physiological aging (figures 3B, E & 5D-E in Spurrier et al., 2018). Spurrier et al. also revealed an overlap of genes that were affected by Cdk5α levels (figures 4B, D in Spurrier et al., 2018). Even though the expression of Cdk5α is limited to neurons, genes affected by the alteration of its expression showed an accelerated aging trend in both the head and thorax of Drosophila (figures 5D-E in Spurrier et al., 2018). The researchers noted that the results from the thorax were unexpected, but this demonstrates that neuronal Cdk5α can cause systemic changes that alter the lifespan of the organism.
Together, the in vivo assays and bioinformatics analyses in Drosophila suggest that altering the activation of Cdk5 by manipulating Cdk5α expression can lead to accelerated aging and enhanced neurodegenerative phenotypes. While both the overexpression and null mutations were deleterious, the Cdk5α-OE flies presented exaggerated phenotypes in the majority of the assays, which is consistent with previous findings (Wilkanie et al., 2016). Spurrier and colleagues have superbly devised an unbiased way to determine physiological aging through genome-wide expression profiling. Establishing physiological age is a tremendous tool that can be implemented in future NDD studies to validate that physiological aging supersedes chronological aging. The next question to address in this field is identifying potential genetic targets and exogenous factors that manipulate the physiological aging process. The aging-related genes in the present study offer a potential starting point to provide a fuller picture of the aging process and its link to the genesis of diseases.
References
1. Menzies, Fiona M., Angeleen Fleming, and David C. Rubinsztein. “Compromised autophagy and neurodegenerative diseases.” Nature Reviews Neuroscience 16, no. 6 (2015): 345.
2. Mills, Kathryn F., Shohei Yoshida, Liana R. Stein, Alessia Grozio, Shunsuke Kubota, Yo Sasaki, Philip Redpath et al. “Long-term administration of nicotinamide mononucleotide mitigates age-associated physiological decline in mice.” Cell Metabolism 24, no. 6 (2016): 795-806.
3. Nho, Kwangsik, Andrew J. Saykin, and Peter T. Nelson. “Hippocampal sclerosis of aging, a common Alzheimer’s disease ‘Mimic’: risk genotypes are associated with brain atrophy outside the temporal lobe.” Journal of Alzheimer’s Disease 52, no. 1 (2016): 373-383.
4. Savica, Rodolfo, Brandon R. Grossardt, James H. Bower, Bradley F. Boeve, J. Eric Ahlskog, and Walter A. Rocca. “Incidence of dementia with Lewy bodies and Parkinson disease dementia.” JAMA Neurology 70, no. 11 (2013): 1396-1402.
5. Spurrier, Shukla, McLinden, Johnson, Giniger. “Altered expression of the Cdk5 activator-like protein, Cdk5α, causes neurodegeneration in part by accelerating the rate of aging.” Disease Models & Mechanisms. no.11 (2018): 1-14.
6. Uttara, Bayani, Ajay V. Singh, Paolo Zamboni, and R. T. Mahajan. “Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.” Current Neuropharmacology 7, no. 1 (2009): 65-74.
7. Valdez, Gregorio, Juan C. Tapia, Jeff W. Lichtman, Michael A. Fox, and Joshua R. Sanes. “Shared resistance to aging and ALS in neuromuscular junctions of specific muscles.” PloS One 7, no. 4 (2012): e34640.
8. Wilkaniec, Anna, Grzegorz A. Czapski, and Agata Adamczyk. “Cdk5 at crossroads of protein oligomerization in neurodegenerative diseases: facts and hypotheses.” Journal of Neurochemistry 136, no. 2 (2016): 222-233.
An engineer position in molecular and cellular biology is available starting in summer 2018 in the group of Pierre-François Lenne and in collaboration with Rosanna Dono at the Developmental Biology Institute of Marseille (IBDM), France. The initial appointment will be made for 1 year, with a possible extension to up to 2 years.
We are seeking a highly-motivated candidate, who will support an interdisciplinary project on cell dynamics and tissue morphogenesis.
The main tasks of the engineer will be the preparation of cell aggregates from mouse embryonic stem cells (“embryonic organoids”), and the development of cell lines expressing fluorescent reporter proteins to monitor gene expression dynamics through imaging. Specifically, the job requires expert knowledge in cell biology (cell cultures, immuno-staining), imaging (e.g. confocal microscopy) and molecular biology (cloning of large DNA fragments, routine and advanced PCR technology). Expertise in stem-cell biologyis desirable.
The working language in the laboratory is English. Candidates are expected to be fluent in English.
A letter of motivation, a CV and the names of two referees should be sent to Pierre-François Lenne (pierre-francois.lenne@univ-amu.fr)
The Ghavi-Helm lab for Developmental (Epi)Genomics at the Institut de Génomique Fonctionnelle de Lyon (IGFL) in France focuses on the 3D regulatory genomics of early Drosophila embryogenesis.
Our group aims to dissect the functional relevance of chromatin organization by combining state-of-the-art methods in genetics, genomics, and computational biology, including novel single-cell techniques, using Drosophila embryogenesis as a model system. We are looking for a creative and talented postdoctoral fellow to work at the intersection between genomics and developmental biology. The candidate will be expected to plan and carry out research tasks independently and write-up/present findings on a regular basis. The project is funded by an ERC Starting grant “Enhancer3D”.
The IGFL fosters an outstanding international environment at the interfaces of evolution, development, and integrative physiology using functional genomics, bioinformatics, genetics, and comparative approaches. The institute is housed in a new building at the Ecole Nationale Supérieure (ENS) of Lyon within the vibrant research community of the Lyon Gerland campus.
Lyon, the “gastronomic capital” of France, is a modern and dynamic city, with rich cultural and sport offerings. It’s an international transportation hub (< 2h from Paris or Marseille with high-speed train, 1h drive from the Alps).
Requirements for the role:
Ph.D. or MD-Ph.D. degree in biology
Prior research experience in genomics, molecular biology, developmental biology, genetics and/or biochemistry (including a track record of peer-reviewed publications)
Ability to work independently and as part of a team in a dynamic and multidisciplinary environment
Previous experience with single-cell genomics is a plus
Interest and/or experience in bioinformatics is a plus
The working language of the lab is English.
The contract will be initially for one year, renewable.
Start date: summer/fall 2018, negotiable.
Salary: commensurate with experience according to CNRS rules.
To get in touch, please send your CV, a brief description of your present and future research interests, and contact details for 3 references to yad.ghavi-helm@ens-lyon.fr. The deadline for applications is open; applications will be examined until the position is filled.
The focus of training will be on construction and automation of BioImage Analysis workflows, using as examples more than one toolbox and different exercises. The schools will be held in Edinburgh 16-19th of October 2018, hosted by the MRC Center for Regenerative Medicine/Imaging facility and co-organized by the University of Edinburgh and University of Dundee.
NEUBIAS schools are an excellent opportunity to learn from many experts in Bioimage Analysis (we are expecting >30 specialists at the event) and “….a great mix of intensive learning and community networking” (former trainee testimonial!). The schools will include practical sessions and seminars on ImageJ for analysis and publication, scripting/macros in ImageJ and Matlab, Omero, CellProfiler, QuPath, Ilastik, Ethics in Image Analysis and work on own data.
Applications are now open (each school has 20 available seats and ~7 trainers)
Applicants are highly encouraged to bring their own laptops and data.
Within the COST framework (funders of NEUBIAS), 7 travel grants per school are offered to applicants who qualify.
Registration requires a “letter of motivation” (filled in the application form) and later a confirmation of status.
Application deadline: May 11th, 2018
Selection notification: 1st week of June 2018.
More information about schools (programme & trainers) and venue, travel & lodge available at our website (see the linked pages for each school in above)
We kindly ask that you help us reach all potentially interested applicants.
Stem cells are defined by the dual capacity to self-renew and to differentiate. These properties sustain homeostatic cell turnover in adult tissues and enable repair and regeneration throughout the lifetime of the organism. In contrast, pluripotent stem cells are generated in the laboratory from early embryos or by molecular reprogramming. They have the capacity to make any somatic cell type, including tissue stem cells.
Stem cell biology aims to identify and characterise which cells are true stem cells, and to elucidate the physiological, cellular and molecular mechanisms that govern self-renewal, fate specification and differentiation. This research should provide new foundations for biomedical discovery, biotechnological and biopharmaceutical exploitation, and clinical applications in regenerative medicine.
CAMBRIDGE STEM CELL COMMUNITY
The University of Cambridge is exceptional in the depth and diversity of its research in Stem Cell Biology, and has a dynamic and interactive research community that is ranked amongst the foremost in the world. By bringing together members of both the Schools of Biology and Medicine, this studentship will enable you to take advantage of the strength and breadth of stem cell research available in Cambridge. Choose from over 50 participating host laboratories using a range of experimental approaches and organisms.
PROGRAMME OUTLINE
Students are expected to have chosen a laboratory for their thesis research prior to application, and to have obtained the support of the PI. A list of eligible supervisors can be found here.
Students will have access to our Discussion course during the first year, where they will:
Study fundamental aspects of Stem Cell Biology through a series of teaching modules led by leaders in the field.
Learn a variety of techniques, such as advanced imaging, flow cytometry, and management of complex data sets.
ELIGIBILITY
We welcome applications from those who hold a relevant first degree at the highest level (minimum of a UK II.i Honours Degreeor equivalent) as well as a Master’s Degree in a relevant discipline. You must have a passion for scientific research.
Wellcome provide full funding at the ‘Home/EU’ rate. Funding does not include overseas fees, so non-EU applicants will need to find alternative funding sources to cover these.
Another bumper month for developmental biology and related preprints, this time featuring spiders, lampreys, amphiouxus and plenty of zebrafish, worm and fly research. Also a big month for fans of Sox genes, which turned up in the title of five preprints! My visual highlight is the preprint from David Parichy’s group on zebrafish scale development – well worth downloading the PDF to gawp at the imaging.
The preprints were hosted on bioRxiv, PeerJ, andarXiv. Use these links to get to the section you want:
Discovery of a new path for red blood cell generation in the mouse embryo
Irina Pinheiro, Ozge Vargel Bolukbasi, Kerstin Ganter, Laura A. Sabou, Vick Key Tew, Giulia Bolasco, Maya Shvartsman, Polina V. Pavlovich, Andreas Buness, Christina Nikolakopoulou, Isabelle Bergiers, Valerie Kouskoff, Georges Lacaud, Christophe Lancrin
Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth
Andrew Cox, Allison Tsomides, Dean Yimlamai, Katie Hwang, Joel Miesfeld, Giorgio Galli, Brendan Fowl, Michael Fort, Kimberly Ma, Mark Sullivan, Aaron Hosios, Erin Snay, Min Yuan, Kristin Brown, Evan Lien, Sagar Chhangawala, Matthew Steinhauser, John Asara, Yariv Houvras, Brian Link, Matthew Vander Heiden, Fernando Camargo, Wolfram Goessling
Tracking mandibular arch cell rearrangements in Tao, et al.’s preprint
Oscillatory cortical forces promote three dimensional cell intercalations that shape the mandibular arch
Hirotaka Tao, Min Zhu, Kimberly Lau, Owen Whitley, Mohammad Samani, Xiao Xiao, Xiao Xiao Chen, Noah A. Hahn, Weifan Liu, Megan Valencia, Min Wu, Xian Wang, Kelli D. Fenelon, Clarissa C. Pasiliao, Di Hu, Jinchun Wu, Shoshana Spring, James Ferguson, Edith P. Karuana, R. Mark Henkelman, Alexander Dunn, Huaxiong Huang, Hsin-Yi Henry Ho, Radhika Atit, Sidhartha Goyal, Yu Sun, Sevan Hopyan
Tracking development of the zebrafish retina, from Matejcic, et al.’s preprint
Single-cell analysis identifies EpCAM+/CDH6+/TROP-2- cells as human liver progenitors.
Joe M Segal, Daniel J Wesche, Maria Paola Serra, Bénédicte Oulés, Deniz Kent, Soon Seng Ng, Gozde Kar, Guy Emerton, Samuel Blackford, Spyros Darmanis, Rosa Miquel, Tu Vinh, Ryo Yamamoto, Andrew Bonham, Alessandra Vigilante, Sarah Teichmann, Stephen R Quake, Hiromitsu Nakauchi, S Tamir Rashid
Randal Burns, Eric Perlman, Alex Baden, William Gray Roncal, Ben Falk, Vikram Chandrashekhar, Forrest Collman, Sharmishtaa Seshamani, Jesse Patsolic, Kunal Lillaney, Michael Kazhdan, Robert Hider Jr., Derek Pryor, Jordan Matelsky, Timothy Gion, Priya Manavalan, Brock Wester, Mark Chevillet, Eric T. Trautman, Khaled Khairy, Eric Bridgeford, Dean M. Kleissas, Daniel J. Tward, Ailey K. Crow, Matthew A. Wright, Michael I. Miller, Stephen J Smith, R. Jacob Vogelstein, Karl Deisseroth, Joshua T. Vogelstein
| Genome tools
An optimized toolkit for precision base editing
Maria Paz Zafra, Emma M Schatoff, Alyna Katti, Miguel Foronda, Marco Breinig, Anabel Y Schweitzer, Amber Simon, Teng Han, Sukanya Goswami, Emma Montgomery, Jordana Thibado, Francisco J Sanchez-Rivera, Junwei Shi, Christopher R Vakoc, Scott W Lowe, Darjus F Tschaharganeh, Lukas E Dow
Robust single-cell DNA methylome profiling with snmC-seq2
Chongyuan Luo, Angeline Rivkin, Jingtian Zhou, Justin P Sandoval, Laurie Kurihara, Jacinta Lucero, Rosa Castanon, Joseph R Nery, Antonio Pinto-Duarte, Brian Bui, Conor Fitzpatrick, Carolyn O’Connor, Seth Ruga, Marc E Van Eden, David A Davis, Deborah C Mash, M. Margarita Behrens, Joseph R Ecker
Community-driven data analysis training for biology
Bérénice Batut, Saskia Hiltemann, Andrea Bagnacani, Dannon Baker, Vivek Bhardwaj, Clemens Blank, Anthony Bretaudeau, Loraine Guéguen, Martin Čech, John Chilton, Dave Clements, Olivia Doppelt-Azeroual, Anika Erxleben, Mallory Freeberg, Simon Gladman, Youri Hoogstrate, Hans-Rudolf Hotz, Torsten Houwaart, Pratik Jagtap, Delphine Lariviere, Gildas Le Corguillé, Thomas Manke, Fabien Mareuil, Fidel Ramírez, Devon Ryan, Florian Sigloch, Nicola Soranzo, Joachim Wolff, Pavankumar Videm, Markus Wolfien, Aisanjiang Wubuli, Dilmurat Yusuf, Rolf Backofen, James Taylor, Anton Nekrutenko, Björn Grüning
Seán I O’Donoghue, Benedetta F Baldi2, Susan J Clark, Aaron E Darling, James M Hogan, Sandeep Kaur, Lena Maier-Hein, Davis J McCarthy, William J Moore, Esther Stenau, Jason R Swedlow, Jenny Vuong, James B Procter
David Trafimow, Valentin Amrhein, Corson N. Areshenkoff, Carlos Barrera-Causil, Eric J. Beh, Yusuf Bilgiç, Roser Bono, Michael T. Bradley, William M. Briggs, Héctor A. Cepeda-Freyre, Sergio E. Chaigneau, Daniel R. Ciocca, Juan Carlos Correa, Denis Cousineau, Michiel R. de Boer, Subhra Sankar Dhar, Igor Dolgov, Juana Gómez-Benito, Marian Grendar, James Grice, Martin E. Guerrero-Gimenez, Andrés Gutiérrez, Tania B. Huedo-Medina, Klaus Jaffe, Armina Janyan, Ali Karimnezhad, Fränzi Korner-Nievergelt, Koji Kosugi, Martin Lachmair, Rubén Ledesma, Roberto Limongi, Marco Tullio Liuzza, Rosaria Lombardo, Michael Marks, Gunther Meinlschmidt, Ladislas Nalborczyk, Hung T. Nguyen, Raydonal Ospina, Jose D. Perezgonzalez, Roland Pfister, Juan José Rahona, David A. Rodríguez-Medina, Xavier Romão, Susana Ruiz-Fernández, Isabel Suarez, Marion Tegethoff, Mauricio Tejo, Rens van de Schoot, Ivan Vankov, Santiago Velasco-Forero, Tonghui Wang, Yuki Yamada, Felipe C. Zoppino, Fernando Marmolejo-Ramos
Christos D Arvanitidis, Richard M Warwick, Paul J Somerfield, Christina Pavloudi, Evangelos Pafilis, Anastassis Oulas, Giorgos Chatzigeorgiou, Vasilis Gerovasileiou, Theodoros Patkos, Nicolas Bailly, Francisco Hernandez, Bart Vanhoorne, Leen Vandepitte, Ward Appeltans, Robert Adlard, Peter Adriaens, Ahn Kee-Jeong, Ahyong Shane, Akkari Nesrine, Gary Anderson, Angel Martin, Claudia Arango, Tom Artois, Stephen Atkinson, Ruud Bank, Anthony D Barber, Joao P Barbosa, Ilse Bartsch, Denise Bellan-Santini, Jimmy Bernot, Annalisa Berta, Rüdiger Bieler, Magda Błażewicz, Phil Bock, Ruth Böttger-Schnack, Philippe Bouchet, Nicole Boury-Esnault, Geoff Boxshall, Christopher B Boyko, Simone Nunes Brandão, Rod Bray, Niel L Bruce, Stephen Cairns, Tania N Campinas Bezerra, Paco Cárdenas, Benny KK Chan, Tin-Yam Chan, Lanna Cheng, Morgan Churchill, Laure Corbari, Ralf Cordeiro, Astrid Cornils, Keith A Crandall, Thomas Cribb, Jean-Loup D’hondt, Meg Daly, Mikhail Daneliya, Jean-Claude Dauvin, Peter Davie, Claude De Broyer, Valentin De Mazancourt, Nicole De Voogd, Peter Decker, Danielle Defaye, Henk Dijkstra, Martin Dohrmann, Daryl Domning, Rachel Downey, Inna Drapun, Ursula Eisendle-Flöckner, Christine Ewers-Saucedo, Marien Faber, Diego Figueroa, Julian Finn, Gustavo Fonseca, Ewan Fordyce, William Foster, Hidetaka Furuya, Horia Galea, Oscar Garcia-Alvarez, Rade Garic, Rebeca Gasca, Santiago Gaviria-Melo, Sarah Gerken, David Gibson, João Gil, Arjan Gittenberger, Chris Glasby, Serge Gofas, Samuel E Gómez-Noguera, David González-Solís, Dennis Gordon, Michal Grabowski, Cinzia Gravili, José M Guerra-García, Roberto Guidetti, Katja Guilini, Kerry A Hadfield, Ed Hendrycks, Bachiller Herrera, Ju-Shey Ho, Jens Høeg, Oleksandr Holovachov, Matthew D Hooge, John Hooper, Tammy Horton, Lauren Hughes, Matús Hyžný, Luiz IF Moretti, Tohru Iseto, Viatcheslav N Ivanenko, Gerhard Jarms, Damià Jaume, Krzysztof Jazdzewski, Ivana Karanovic, Young-Hyo Kim, Rachael King, Michelle Klautau, Jürgen Kolb, Alexey Kotov, Traudl Krapp-Schickel, Antonina Kremenetskaia, Reinhardt Kristensen, Andreas Kroh, Sven Kullander, Rafael La Perna, Sara LeCroy, Daniel Leduc, Rafael Lemaitre, Anne-Nina Lörz, Jim Lowry, Enrique Macpherson, Larry Madin, Tomasz Mamos, Renata Manconi, Bruce Marshall, David J Marshall, Patrick Martin, Sandra McInnes, Jan Mees, Tõnu Meidla, Kelly Merrin, Dmitry Miljutin, Claudia Mills, Vadim Mokievsky, Tina Molodtsova, Rich Mooi, André C Morandini, Rosana Moreira Da Rocha, Fabio Moretzsohn, Jonas Mortelmans, Jeanne Mortimer, Luigi Musco, Thomas A Neubauer, Eike Neubert, Peter NG Neuhaus, Anh D Nguyen, Claus Nielsen, Jon Norenburg, Tim O’Hara, Hisayo Okahashi, Dennis Opresko, Masayuki Osawa, Yuzo Ota, Gustav Paulay, Vincent Perrier, William Perrin, Iorgu Petrescu, Bernard Picton, John F Pilger, Andrzej Pisera, Dan Polhemus, Gary Poore, James D Reimer, Hans Reip, Michael Reuscher, Pilar Rios Lopez, Marc Rius, Klaus Rützler, Alexander Rzhavsky, José Saiz-Salinas, André F Sartori, Heinrich Schatz, Bernd Schierwater, Andreas Schmidt-Rhaesa, Simon Schneider, Christine Schönberg, André R Senna, Cristiana Serejo, Shabuddin Shaik, Shokoofeh Shamsi, Jyotsna Sharma, Noa Shenkar, Andrew Shinn, Jacek Sicinski, Volker Siegel, Petra Sierwald, Elizabeth Simmons, Frederic Sinniger, Duncan Sivell, Boris Sket, Harry Smit, Nicole Smol, Jesser F Souza-Filho, Jörg Spelda, Sérgio N Stampar, Eric Stienen, Pavel Stoev, Sabine Stöhr, Malin Strand, Eduardo Suárez-Morales, Mindi Summers, Billie J Swalla, Stefano Taiti, Masaatsu Tanaka, Anne H Tandberg, Danny Tang, Mark Tasker, Harry ten Hove, Jan J ter Poorten, Jim Thomas, Erik V Thuesen, Ben Thuy, Juan T Timi, Antonio Todaro, Xavier Turon, Peter Uetz, Sergiy Utevsky, Jean Vacelet, Risto Väinölä, Sancia ET van der Meij, Ton van Haaren, Virág Venekey, Chris Vos, Genefor Walker-Smith, Chad T Walter, Les Watling, Matthew Wayland, Christopher Whipps, Gary Williams, Robin Wilson, Moriaki Yasuhara, Joana Zanol, Wolfgang Zeidler
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The focus of training will be on construction and automation of BioImage Analysis workflows, using as examples more than one toolbox and different exercises. The schools will be held in Edinburgh 16-19th of October 2018, hosted by the MRC Center for Regenerative Medicine/Imaging facility and co-organized by the University of Edinburgh and University of Dundee.
