Postdoctoral position in the research group of Dr. Nikolay Ninov at the Center for Regenerative Therapies Dresden and the Paul Langerhans Institute Dresden (PLID) (of Helmholtz Zentrum München and the German Center for Diabetes Research (DZD e.V.).

Our goal is to understand beta-cell regeneration and function in vivo in order to develop innovative cures for diabetes. We use the zebrafish as a model organism.  The zebrafish is ideal to observe the behavior of beta-cells in their endogenous environment using live imaging. To do so, we have developed new tools to visualize beta-cell function and proliferation while performing genetic and lineage-tracing analysis (see Janjuha et al., 2018; Singh et al., 2018, Alfar et al., 2017; Spanjaard et al. 2018).

Currently, we are focusing on the following projects:

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At the last AGM, held at the 2018 Spring Meeting in Warwick, five new BSDB committee members were elected to take term in autumn. They will replace the five leaving members: our Graduate Representative Alexandra Ashcroft, Postdoc Representative Michelle Ware, Secretary Kim Dale, Meetings Officer Josh Brickman, and Communications Officer Andreas Prokop (see a complete list of current committee members here). Please, read below about the new committee members, their careers, research interests and plans for their time on the committee.

Jessica Forsyth – the new Graduate Representative

Charlotte Sophie Louise Bailey – the Postdoc Representative

Tanya T. Whitfield

Shankar Srinivas

Jens Januschke

(1 votes)

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To mark Mental Health Awareness Week, we’re sharing this blog post on the MRC WIMM blog from Gregorio Dias, who describes his experiences with stress and anxiety as a PhD student. If you’ve faced similar issues and want to share them with the community, just get in touch.

Working towards a PhD is an exciting, albeit challenging, narrative in a student’s life. The goals and aspirations that motivate one at the early stages of a PhD project are very likely to change over time.

In my view, this transformative process is much needed, as it builds up personal and scientific maturity. This journey is, nonetheless, accompanied by stress and low points and I discovered that taking time to do other things I enjoy and sharing my experiences with other students was as important as working hard to be successful. That is an accurate summary of my experience as a PhD student and I hope that sharing it here will help others in their own journey.

Aspiring to become an academic professor, I came straight from the Brazilian Amazon to start a DPhil (PhD) at the University of Oxford in the UK. I started an ambitious project aiming at answering a long-standing question in my field of study. In brief, I aimed at understanding how a particular protein of the innate immune system detects the presence of virus RNA in infected cells. Motivated by this exciting question, I immersed myself in the lab to achieve these goals. I had access to all the facilities, reagents and expertise I needed to tackle this question and never felt any pressure from my supervisors or colleagues.

However, the combination of being ambitious and anxious did not mean immediate results, and I soon learnt that top notch science is rather a slow process….

Read the full post over at the  MRC Weatherall Institute of Molecular Medicine Blog

https://www.imm.ox.ac.uk/about/blog/a-bumpy-2013-emotional-2013-ride-towards-a-phd

And in case you missed it, we’ll also direct you to Dave Reay’s piece in last week’s Nature on the his experience of debilitating depression during his PhD

https://www.nature.com/articles/d41586-018-05080-6

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A postdoctoral position is available in the Bush lab bush.ucsf.edu at the University of California, San Francisco to study the cellular basis of morphogenesis using live imaging, mouse genetic, and iPSC and ESC approaches. Our dynamic team focuses on understanding basic mechanisms of signaling control of morphogenesis particularly as related to human structural birth defects. The position is in the collaborative UCSF Department of Cell and Tissue Biology and Program in Craniofacial Biology, located at the UCSF Parnassus Heights campus in the center of San Francisco. UCSF offers an outstanding developmental and stem cell biology community, access to cutting edge technologies, and a supportive working environment. Candidates with a Ph.D. degree in a biological science and research experience in molecular biology, genetics, biochemistry, or live cell or live embryo imaging should submit a C.V. and names of at least 2 references via email to: jeffrey.bush@ucsf.edu

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***DEADLINE IS TODAY – MAY 15!***

In September, Development is hosting the third of its highly successful series of meetings focusing on human developmental biology. Held in the Wotton House estate near Dorking in Surrey and organised by Paola Arlotta, Ali Brivanlou, Olivier Pourquié and Jason Spence, the meeting will cover the latest developments and future prospects for this rapidly evolving field (you can find more information at the meeting homepage).

We are excited to announce a competition for a reporter to cover the meeting for the Node. The reporter will provide regular updates of the meeting via Twitter, and write a meeting report of their experience and the sights and sounds of the meeting to be published on the Node.

It’s a fantastic opportunity both to practice your science communication skills and for networking, and of course you’ll also be able to learn about the latest fascinating research.

The prize

The winner will get free registration to the meeting!

How to enter

To find the perfect reporter, we’d like to know why you’re excited about the future of human developmental biology. Send us 300 words answering one of the following questions:

Why is human developmental biology important?

What has been the biggest advance or paper in the field since the last Development meeting in September 2016?

What is the key burning question about human development you want to answer in your research?

The competition is open to PhD students and postdocs.

Please send entries to thenode@biologists.com with “Meeting reporter competition” as the subject.

Deadline = 15^th May (winner announced soon thereafter)

Meeting registration details

If you have registered for the meeting already, you will get a refund on your costs.

If you have not registered but plan to, note that the meeting has limited places and we expect these places to run out. So please do not delay your application for the meeting while you’re writing your piece. The deadline for applying to the meeting is 22^nd June – in your application, include reference to this competition in the box “Please state why this meeting is of particular relevance to your research and your reasons for wishing to attend”; you will not have to pay until the winner of the competition is announced.

Happy writing!

(1 votes)

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The Centre for BioNano Interactions (CBNI), University College Dublin is seeking a highly motivated Post-Doctoral Research Fellow with research background in relevant areas of molecular and cellular biology to join a dynamic team dedicated to advancing the understanding and implementation of targeted medicines at the nanoscale.

The successful candidate will explore novel approaches to re-programme the epigenome in mammalian cells, including gene expression and epigenetic changes, and associated phenotypic outcomes. As such, demonstrated experience in mammalian cell culture, advanced molecular biology and biochemistry techniques such as ChIP-sequencing, CRISPR, flow cytometry and live cell microscopy is highly desirable.

In addition to conducting research, the successful candidate will help supervise and support PhD students working on the same topic, promote publication in high quality peer-reviewed academic publications, assist in the development of funding proposals and the management and reporting of projects and will generally contribute to the professional and smooth running of a highly interdisciplinary team.

About CBNI

Located in Dublin, one of the most vibrant cities in Europe, CBNI is pioneering new techniques and approaches at the research interface between nanoscale science and living systems, with applications in the biomedical arena. The team is highly interdisciplinary and dynamic and would suit young scientists that aspire to be at the leading edge of an emerging field of science and biomedical research.

Key areas of research at CBNI include:

• Fundamental understanding of interactions between nanoparticles and living systems at cellular level and in vivo
• Exosome detection and manipulation
• Novel approaches to vaccine development
• Applications of bio-nanoscale science for therapy and diagnostics

Mandatory Qualifications

• PhD in molecular biology, cell biology or a related discipline
• A strong background and in molecular and cell biology techniques
• Experience in mammalian gene regulations, quantitative proteomics, epigenetics
• Evidence of research activity (publications, conference presentations, awards) and future scholarly output (working papers, research proposals) and ability to outline a research project.
• A demonstrated commitment to research and publications
• An understanding of the operational requirements for a successful research project
• Excellent communication skills (Oral, Written, Presentation etc), the ability to work effectively in a team and be self-motivated
• Excellent organisational and administrative skills including a proven ability to work to deadlines

Salary: €36,854 – €37,383 per annum

Appointment on the above range will be dependent on qualifications and experience.

This is a full-time position for (initially) 1 year.

For further detail and to apply, please refer to the University College Dublin vacancies page available at this link with job Ref number 010298

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The Centre for BioNano Interactions, University College Dublin is seeking a highly motivated Post-Doctoral Research Fellow with research background in relevant areas of molecular and cellular biology to join a dynamic team dedicated to advancing the understanding and implementation of diagnostics and therapeutics at the nanoscale.

The successful candidate will work on detection and manipulation of exosomes as complex nanostructures for use in biomedical applications. As such, demonstrated experience in production and harvesting of exosomes or viruses using bench-scale bioreactors is highly desirable.  Analytical experience of extracellular media, and study of cell culture medium components using chromatographic or other techniques would be advantageous.

In addition to conducting research, the successful candidate will help supervise and support PhD students working on the same topic, promote publication in high quality peer-reviewed academic publications, assist in the development of funding proposals and the management and reporting of projects and will generally contribute to the professional and smooth running of a highly interdisciplinary team.

About CBNI

Located in Dublin, one of the most vibrant cities in Europe, CBNI is pioneering new techniques and approaches at the research interface between nanoscale science and living systems, with applications in the biomedical arena. The team is highly interdisciplinary and dynamic and would suit young scientists that aspire to be at the leading edge of an emerging field of science and biomedical research.

Key areas of research at CBNI include:

• Fundamental understanding of interactions between nanoparticles and living systems at cellular level and in vivo
• Exosome detection and manipulation
• Novel approaches to vaccine development
• Applications of bio-nanoscale science for therapy and diagnostics

Mandatory Qualifications

• PhD in relevant disciplines of molecular and cellular biology
• Experience in intercellular communication and cell signalling
• Evidence of research activity (publications, conference presentations, awards) and future scholarly output (working papers, research proposals) and ability to outline a research project.
• A demonstrated commitment to research and publications
• An understanding of the operational requirements for a successful research project
• Excellent communication skills (Oral, Written, Presentation etc), the ability to work effectively in a team and be self-motivated
• Excellent organisational and administrative skills including a proven ability to work to deadlines

Salary: €36,854-€37,383 per annum

Appointment on the above range will be dependent on qualifications and experience.

This is a full-time temporary position for (initially) 1 year.

For further detail and to apply, please refer to the University College Dublin vacancies page available at this link with job ref number 010299

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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.

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.

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.

Cell Identity Switching Regulated by Retinoic Acid Signaling Maintains Homogeneous Segments in the Hindbrain. Megan Addison, Qiling Xu, Jordi Cayuso, David G. Wilkinson. Developmental Cell. DOI: https://doi.org/10.1016/j.devcel.2018.04.003

(2 votes)

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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.

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.

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.

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?

References

• Hall, Z. J., & Tropepe, V. (2018a). Visual experience facilitates BDNF-dependent adaptive recruitment of new neurons in the postembryonic optic tectum. Journal of Neuroscience, 38(8), 2000-2014.
• Hall, Z. J., & Tropepe, V. (2018b). Movement maintains forebrain neurogenesis via peripheral neural feedback in larval zebrafish. eLife, 7, e31045.
• Honjo, Y., Kniss, J., & Eisen, J. S. (2008). Neuregulin-mediated ErbB3 signaling is required for formation of zebrafish dorsal root ganglion neurons. Development, 135(15), 2615-2625.
• Kokel, D., Cheung, C. Y. J., Mills, R., Coutinho-Budd, J., Huang, L., Setola, V., … & Bruni, G. (2013). Photochemical activation of TRPA1 channels in neurons and animals. Nature chemical biology, 9(4), 257.
• Lindsey, B. W., Di Donato, S., Kaslin, J., & Tropepe, V. (2014). Sensory‐specific modulation of adult neurogenesis in sensory structures is associated with the type of stem cell present in the neurogenic niche of the zebrafish brain. European Journal of Neuroscience, 40(11), 3591-3607.
• Lindsey, B. W., & Tropepe, V. (2014). Changes in the social environment induce neurogenic plasticity predominantly in niches residing in sensory structures of the zebrafish brain independently of cortisol levels. Developmental neurobiology, 74(11), 1053-1077.
• Souza, B. R., Romano-Silva, M. A., & Tropepe, V. (2011). Dopamine D2 receptor activity modulates Akt signaling and alters GABAergic neuron development and motor behavior in zebrafish larvae. Journal of Neuroscience, 31(14), 5512-5525.
• Van Praag, H., Kempermann, G., & Gage, F. H. (1999). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature neuroscience, 2(3), 266.
• White, E. J., Hutka, S. A., Williams, L. J., & Moreno, S. (2013). Learning, neural plasticity and sensitive periods: implications for language acquisition, music training and transfer across the lifespan. Frontiers in systems neuroscience, 7, 90.

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HFSP_Call for PhD and Postdoc_May_2018

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 15^th 2018 :

Anne Grapin-Botton : anne.grapin-botton@sund.ku.dk (Copenhagen, Pancreatic organoids)

Alf Honigmann : honigmann@mpi-cbg.de (Dresden, Neural organoids, Imaging)

Daniel Riveline : riveline@unistra.fr (Strasbourg, Cell Physics, Microfabrication)

Masaki Sano : sano@phys.s.u-tokyo.ac.jp (Tokyo, Mathematical modelling)

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