Somites are segmented structures  which give rise to numerous tissues in the vertebrate body. It has long been observed that somites scale in size with the overall size of the embryo, both as development proceeds and between individuals of different sizes, but the molecular underpinnings of this process have remained controversial. A new paper in the current issue of Development uses size-reduced zebrafish embryos to investigate this problem. We caught up with authors Kana Ishimatsu, Tom Hiscock and Sean Megason (Associate Professor of Systems Biology at Harvard Medical School) to find out more about the story.

Sean, can you give us your scientific biography and the questions your lab is trying to answer?

SM I’ve always been interested in how code makes pattern. When I was a kid, my parents bought me a computer (Commodore Vic-20). At some point I got bored with the couple of video games it came with and I taught myself how to program using the built in BASIC interpreter. I ended up making a lot of simulations of patterning based on complex dynamical systems without any training or knowing that it was a field of study. My mom was a high school librarian in the small town I grew up in so she would bring home the old Scientific Americans for me. They used to have a section called “Mathematical Recreations” that inspired a bunch of programming. In college I figured out that how the genome patterns life is the ultimate puzzle and got hooked on developmental biology.

My lab is broadly interested in how groups of cells work together to make things. We are big believers that “mechanism” typically cannot be reduced to a molecule. It emerges as interactions that can span many levels from molecules to cells to tissue mechanics, and that deciphering the mechanisms requires a balanced approach of direct observation, perturbation, and modelling across these scales.

Kana – how did you come to join Sean’s lab and be involved with the project?

KI I came to be interested in somite scaling problem while I was still in Japan, but soon realized I needed more quantitative approaches, especially in imaging. Sean’s lab had an ideal imaging system, a common interest in scaling issue, and good environment to work with people from different backgrounds.

And what about you Tom? You seem to have done a mix of wet and dry science so far in your career?

TH Yes – I started out studying physics and maths… until I joined Sean’s lab for my PhD, and fell in love with embryos and imaging. Now I have returned a little to my physics roots, and am trying to build theories to understand how animals build themselves.

Somites were first shown to scale in 1975, but decades later somite scaling and its relationship to the segmentation clock is still a controversial topic. Why do you think this is?

KI Although there are a number of models explaining somite formation, not only under a normal condition but also experimentally perturbed conditions, we have not been able to distinguish which “class” of model is working in vivo, before going into theoretically and molecularly detailed analyses. This has made more confusion than clarification.

TH Maybe one of the reasons is that there are many models that fit the data, and that it’s been difficult to design experiments to distinguish between the different hypotheses.

SM In our hands distinguishing models required rigorously comparing quantitative data with theoretical predictions, otherwise there’s a tendency to just say “oh it looks close enough”. It also required figuring out new experimental approaches that make different predictions for the different models.

Can you give us the key results of the paper in a paragraph?

KI In order to tackle the long-standing problem of the somite formation mechanism, we started off from simply asking whether somite size scales with PSM size, which was a disputed topic in the field. We discovered that somite size scales with PSM size in a linear manner, only when the time delay between somite boundary specification and formation is taken into consideration. Based on this observation, we hypothesized the key feature of somite formation should scale with PSM size, and found only the Fgf gradient  scales with PSM size, not clock period, axis elongation speed or a spatial wave pattern. Taking the time delay and the gradient scaling in to consideration, we proposed a new model, the “Clock and Scaled Gradient model”, in which scaling of a gradient is responsible for both progression and scaling of the somite formation. This model not only explains all the existing experimental results, but also makes a unique prediction, namely “an echo effect”, which was validated in vivo. Once PSM size is made artificially smaller, the system shows oscillations of somite sizes – becoming smaller and larger over and over again – which cannot be predicted by other classes or version of models.

Do you have any favourite candidates for the molecular players that would control gradient scaling?

KI Though there are several obvious candidates, like Retinoic Acid, it would be interesting if it does not primarily depend on molecules. For example, the scaling might be achieved through a gradient of pH or mechanical force. Anyway, the mechanism has to have fast dynamics because Fgf scaling occurs every somite cycle, implying that mechanisms that require a long time to achieve scaling (such as an expander-repressor mechanism) are not likely to control the gradient scaling in PSM.

SM This has been a hot topic of debate in the lab! There are some obvious candidates for players in the system (e.g. Retinoic Acid) but my best guess is that this is a case where mechanism cannot be reduced to a molecule. There are multiple signalling gradients and antagonists which all regulate each other, and in the context of lots of dynamic cell rearrangements and tissue movements.

How does somite scaling compare to other mechanisms of scaling in development?

KI Based on what we know thus far, using gradient scaling is a fairly general strategy to achieve scaling in development. When I started this project, I was almost sure that we were going to find something other than gradient scaling that is responsible for somite scaling, such as axis elongation speed or spatial patterns of waves in PSM. However, we ended up finding gradient scaling as the underlying mechanism of somite formation. It would be interesting to ask, at least theoretically, if there is any benefit to employ gradient scaling, rather than other mechanisms. Moreover, it is important to keep looking for other scaling mechanisms underlying embryo patterning.

SM The main reason I was interested in looking at somites in the size-reduced embryos was that I thought it would NOT be based on gradient scaling. In my mind, I reasoned that somite size was the product of clock period and axis elongation (wavefront regression) speed. I thought it was very unlikely that clock period would change since it is set by molecular degradation rates, but it seemed likely that the axis would extend more slowly if there were less cells. So I preferred a mechanism of “intrinsic scaling” where if there is a smaller number of precursors, you naturally get smaller products in a system governed by growth. I liked this hypothesis because it was based on geometrical/mechanical considerations rather than molecules but of course it turned out to be wrong! Gradient scaling is certainly an important way to scale pattern given the central importance of gradients in patterning itself, but I’m hopeful that there are other mechanisms to be discovered.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

KI One was that we discovered that the relationship between PSM size and somite size looks completely different when we take the delay into consideration. Until then, I was a big fan of the idea that travelling waves are playing a central role in determining somite size, but this discovery changed my view 180 degrees. The other was when we came up with echo experiment that can be uniquely predicted by our model. We spent months trying to come up with one experiment that not only existing models but also any modified versions of them cannot predict. It was such an exciting moment when we finally came up with the idea and were able to see the predicted result in vivo!

TM For me, it was when we realized that scaling was a central feature of how somites are made. We’d started by thinking about why smaller embryos have smaller somites – but it got really exciting once we realized that scaling was happening throughout somitogenesis.

And what about the flipside: any moments of frustration or despair?

KI&TM There was a long time where we were really confused by the data – particularly since there is so much known about somites, it’s hard to make sense of it all. But these struggles – particularly afternoons of intense headaches and confusion together – are our fondest memories of this project!

What next for you two after this paper?

KI I would like to expand the strategy we took in this study to a non-repetitive, higher dimensional (2D and 3D) system. The advantages of studying somite formation is that it is simple enough to describe its shape as 1D and that it is a repetitive structure allowing us to easily extract its characteristic feature (length). These advantages allowed us to find its input-output relation (PSM size and somite size) and find the transfer function (gradient scaling). It is exciting if we can use the same strategy more generally to study developmental systems. Of course, I am also excited to study (1) the underlying mechanism of gradient scaling in PSM, and (2) the mechanism that integrates the positional information given by the gradient and temporal information given by the oscillator.

TM I’ve just started a postdoc position with Ben Simons and John Marioni in Cambridge, where I think about how we can use lineage tracing data and single cell sequencing to understanding development.

And where will this work take the Megason lab?

SM The mechanism of PSM gradient scaling is clearly an interesting and open question as is the regulation of the speed and duration of axis extension in general given that somite number and body length vary widely between species but are very constant within a species. The size-reduction technique also opens up the rest of the embryo to scaling questions. I am a big fan of the work of Naama Barkai and colleagues on scaling of dorsal-ventral pattern in the early embryo by Expansion-Repression, but the scaling problem must separately be solved for every subsequent part of the embryo. There’s also the question of why these scaling systems are even there. It’s not so we can chop embryos in a dish and watch them recover. Where we actually take the work just depends on who joins the lab and their interests.

Finally, let’s move outside the lab – what do you like to do in your spare time in Boston?

KI I like to eat a lot of good food, especially Chinese!

TH One of the things I miss a lot about Boston is the lovely cycle ride to Walden Pond, followed by a refreshing swim in the lake, and a big ice-cream in Concord!

SM I chase my three little monsters around and occasionally do some recreational coding and gardening.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation
Kana Ishimatsu, Tom W. Hiscock, Zach M. Collins, Dini Wahyu Kartika Sari, Kenny Lischer, David L. Richmond, Yasumasa Bessho, Takaaki Matsui, Sean G. Megason
Development 2018 145: dev161257 doi: 10.1242/dev.161257

This is #44 in our interview series. Browse the archive here. 

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Hubrecht Institute for developmental biology and stem cell research.
MERLN Institute for technology-driven regenerative medicine.

Laboratory for stem cell-based development.

2 years
The Netherlands

A two-year postdoctoral position is available at the laboratory for stem cell-based development headed by Nicolas Rivron. The laboratory is located at the MERLN institute for technology-driven regenerative medicine (Maastricht) and at the Hubrecht institute for developmental biology and stem cell research (Utrecht). The laboratory is embedded in a highly innovative environment, has access to first-class facilities for microscopy, single cell sequencing and microfabrication, and provides investigators with the opportunity to pursue excellent, multidisciplinary research at the interface between stem cell biology and quantitative science.

For more information, visit https://www.nicolasrivron.org

Our goal is to investigate the flow of information between the blastocyst cells and the impact on post-implantation development, using stem cell-based blastoids [1]. We use a multidisciplinary approach combining genetic engineering, high-content screening of molecules, and single cell sequencing.

You must hold a PhD (or equivalent experience) in a relevant life sciences or biomedical discipline and have a strong interest in stem cells and early development. Extensive experience in embryonic stem cells and molecular biology techniques is essential. A proven publication record, with at least one first author publication in a peer-reviewed international journal is also essential. Expertise in cell signaling and genetic engineering would be an advantage. You must also have the ability to develop and apply new concepts, have a creative approach to problem solving, and be able to write clearly and succinctly for publication.

To apply, send your CV, names and contacts of two scientific references along with a covering letter stating why you are applying for this role (providing evidence against the requirements of the job as per the job description and person specification) via the website of the laboratory. Applications which do not provide a cover letter will not be considered. We only consider people who made a deep thought about joining the lab, are motivated for discoveries, success, and respectful of colleagues.

[1] Rivron NC [corresponding author], Frias-Aldeguer J, Vrij EJ, Boisset JC, Vivie J, Korving J, Truckenmuller RK, van Oudenaarden A, van Blitterswijk CA*, Geijsen N*. In vitro generation of blastocyst-like structures solely from stem cells. *Equal contribution. Nature. volume 557, pages 106–111. 2018.

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An NIH-funded post-doctoral position is available in the Hoffman Lab at Yale University School of Medicine.

Our lab studies the function of genes involved in autism spectrum disorders at the cellular, molecular, and circuit levels. To do this, we use zebrafish as a model system due to their transparent larvae and amenability to high-throughput screens (Hoffman et al. 2016 Neuron). Our lab has generated zebrafish mutants in multiple autism risk genes using CRISPRs.

The post-doctoral associate will be involved in projects investigating how the loss of autism risk genes alters fundamental processes of vertebrate brain development. The post-doctoral associate will perform phenotypic analyses of multiple zebrafish mutants of autism risk genes using a combination of molecular, cellular, and circuit-level approaches.

Job Requirements

1. Recent PhD or MD/PhD with strong experience in molecular biology and microscopy.
2. Strong background in genetics and experience working with genetic models of human disease.
3. Highly motivated, enthusiastic, excellent interpersonal skills, and a strong publication record.
4. Prior experience with in vivo functional imaging and computational skills are preferred but not required.

To apply

Candidates should send the following to Dr. Ellen Hoffman (ellen.hoffman@yale.edu):

1. A cover letter stating a description of your accomplishments and interest in the lab’s research projects.
2. CV.
3. Contact information for three references.

More information about the laboratory can be found at: www.hoffmanlab.net.

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Postdoctoral Fellow positions (can join anytime between 2018 summer to 2019 spring) are available in Lee laboratory at Johns Hopkins University. These positions are not only for stem cell experts, but rather for who has expertise on non-stem cell fields.

Here are the focused research areas for this hiring for 3-4 Postdoctoral Fellows.

– Neurodegeneration (Alzheimer’s disease)

– Optogenetics

– Epigenetics/NGS/single-cell transcriptome analysis

– Biochemistry

The Lee lab has been establishing novel methodologies to specify human induced pluripotent cells (hiPSCs) into multiple lineages and to model human diseases, including induced neural crest (Kim et al., Cell Stem Cell, 2014), peripheral neurons (Oh et al., Cell Stem Cell, 2016; Oh et al., Nature Neuroscience, 2017), Schwann cells (Mukherjee-Clavin et al.,in revision) and skeletal muscle cells (Choi et al.,Cell Reports,2016; Choi et al., submitted) using multiple genetic reporter systems. We continue to study human developmental and degenerative disorders to unravel the underlying cellular/molecular mechanism toward realistic therapeutic approaches.

Compensation is following NIH guideline and JHU is an equal opportunity and affirmative action employer. Applicants can send a CV (with three reference contact info) to the address listed below:

Gabsang Lee, DVM, PhD

Associate Professor, Institute for Cell Engineering, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

leelabjob@gmail.com

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The Collaborative Research Centre 1348 “Dynamic Cellular Interfaces: Formation and Function” at the University of Münster, Germany, invites applications for

10 PhD Positions

(salary level E13 TV-L, 65%)

Projects are available from the earliest possible date for three years. Currently, the regular full employment time is 39 hours and 50 minutes per week.

The Collaborative Research Center 1348 uses an integrated research approach to investigate the formation and function of dynamic cellular interfaces, which are the basis for many biological processes ranging from cellular differentiation to synapse function and maintenance. CRC1348 provides a stimulating, interdisciplinary, international research environment with 20 participating groups from three faculties at the University of Münster and the Max Planck Institute of Molecular Biomedicine. Within CRC 1348, the Integrated Research Training Group (IRTG) offers a structured doctoral programme, including a supervision concept, measures for career development, as well as a tailored training programme with subject-based interdisciplinary research and soft skills courses. Support with administrative matters, accommodation and visas are part of the program.

PhD projects involve state-of-the-art imaging as well as molecular, genetic and biochemical approaches. Projects are available in the areas of cell and developmental biology, neurobiology, vascular biology, virology, biophysics and physical chemistry. For details about the projects, please see http://sfb1348.uni-muenster.de/projects.

We invite applications from highly qualified and motivated students of any nationality with a strong background in life sciences or biomedicine. A master’s or equivalent degree in biology, biochemistry or a related field is required. Applications from candidates interested in quantitative imaging and biophysical approaches are especially welcome. Applicants are expected to show a high level of proficiency in both spoken and written English. German language skills are not required.

The University of Münster is an equal opportunity employer and is committed to increasing the proportion of women academics. Consequently, we actively encourage applications by women. Female candidates with equivalent qualifications and academic achievements will be preferentially considered within the framework of the legal possibilities. We also welcome applications from candidates with severe disabilities. Disabled candidates with equivalent qualifications will be preferentially considered.

Application documents should include a curriculum vitae, a grade transcript and a motivation letter. Applicants should state their scientific interests in one or more of the specific CRC projects. Additionally, applicants should arrange for two letters of recommendation to be submitted directly to applications.crc1348@uni-muenster.de

The application deadline is 15 July 2018. Applications can only be submitted via our online system. For online application and further information please visit http://sfb1348.uni-muenster.de/graduate-school/application.

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Transportation networks play a central role in enabling efficient mass flow over extended domains, where diffusion alone would be too slow. Therefore, transportation networks often play a central role for an organism’s physiology and a high degree of energetic efficiency has been proposed as a guiding principle for the layouts of these networks.

However, biological organisms usually do not construct their networks only after fully developing the body plan, but instead extend them together with the growth of the body, for example in trees, fungi, or myxomycetes. Then it is interesting to ask how they achieve a high degree of efficiency, given that at the time of network construction the final shape of the organism is yet unknown.

Physarum polycephalum: a Crawling Network

The true slime mould Physarum polycephalum (see fig. 1) is a good model for this question, for two reasons. First, it is an easy-to-handle, macroscopic organism that prominently features an adaptive tube network as it extends over surfaces.  More importantly, though, we have an understanding of the rules that shape this network under static conditions: a simple set of equations formulated by Tero et al. (J. Theor. Biol. 2007) known as the ‘current reinforcement rule’.

To learn how network formation occurs during extension, it makes sense to first study the organism’s behaviour under this condition and then find a model that explains this behaviour. A model enables us to make testable predictions that can decide whether we have, in fact, understood the basic characteristics of the organism and to include certain assumptions about the mechanism behind them.

Study Approach: Learn, Model, Check

1. Learn

In a study published last year (Schenz et al., J. Phys. D 2017), we first studied how the network of Physarum under extension is shaped. We let the organism extend through a narrow lane that includes some turns (see fig. 2). The organism constructed its main veins at a small distance behind the growth front and did so at a time long before it had fully explored the complete layout of the arena. The main characteristic of the resultant vein trajectory is that it cuts corners at turns but then returns back to the centre line of the arena, even between two turns, where the globally shortest path would dictate to remain on the inside edge. 

Nevertheless, analysis showed that the slime mould’s main vein was only 6% longer than the shortest possible route through the arena. To appreciate this, one has to consider that a naïve strategy of constructing the vein in the centre of the corridor would result in a trajectory 18% longer than the minimum. How can we explain such a high efficiency in the absence of foresight?

2. Model

As a model we considered first the classical current reinforcement model, but it failed to reproduce the characteristic vein pattern. Therefore, current reinforcement dynamics alone are insufficient to explain the organism’s capability. As a consequence we constructed a novel model consisting of three core elements that have each been successfully used in the literature to describe specific aspects of Physarum physiology: wave front dynamics, Calcium-driven oscillation waves, and the current reinforcement tube model. We linked these three elements with appropriate interactions under the assumption that the observed expansion behaviour and network structure are a consequence of their interplay.

The resulting model explained the phenotypical features well both qualitatively and quantitatively, and also contained some physiological assumptions that are testable. We could thus determine that the coupling between growth front extension and tube network evolution has to be of just the right strength to allow the organism some spatial integration necessary to find local optimality of the route trajectory as well as efficient transportation of body mass through the tube network from the rear to the front.

3. Check

The corollary of this finding is that growth front extension history alone should be sufficient to make a prediction on the main vein trajectory (see fig. 3). This hypothesis yielded much better predictions than alternative explanations we tested. This then yields a mechanistic explanation of how the organism can achieve this high degree of optimality in the face of uncertainty and at the same time an interpretation for the biological context for the current reinforcement rule: to enable efficient locomotion of Physarum.

In our ongoing research we develop further the question of what measure of optimality is likely guiding an expanding network. Total network length, as considered above, is not the only dimension along which a network can be evaluated, especially if it is embedded in a foraging organism. We will, therefore, search for such a measure that best predicts the behaviour of the organism given greater degrees of freedom to then evaluate well established concepts such as Optimal Foraging Theory in the context of continuous, network-based systems. This will allow us to consider how efficient networks have to be structured in an unknown environment.

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Summer Meeting of the Anatomical Society

Theme: Human Cerebral Cortex Development

Venue: St. John’s College, University of Oxford, Oxford, UK

Dates: 23-25 July 2018

Many human psychiatric and neurological conditions have developmental origins. Rodent models are extremely valuable for the investigation of brain development, but cannot provide insight into aspects that are specifically human. The human cerebral cortex has some unique genetic, molecular, cellular and anatomical features which need to be further explored. At the winter meeting of the Anatomical Society in 2010 we hosted a symposium focussed on development of the human cerebral cortex cortex. At that time a renaissance in the study of human brain development was getting underway made possible by the availability of new techniques, such as generation of human neural stem cells and organoids ex vivo, in utero MRI, and RNAseq and resources such as the Human Developmental Biology Resource and the Allen Brain Atlas. Eight years later, we feel the time has come to review the spectacular progress made since the last meeting. An international cast of speakers will provide insights into the cellular and molecular features of human cortical expansion and evolution, uniquely human features of cortical circuit formation, the development of the subplate in health and disease, and the origins of human cortical malformations, amongst other topics. We look forward to welcoming you to St John’s College for this exciting event.

Invited Speakers
Andre Goffinet, Bruxelles
Arnold Kriegstein, San Francisco
Bruno Mota, Rio de Janeiro
Charles Newton, Oxford
David Edwards, London
Eleonora Aronica, Amsterdam
Eva Anton, Chapel Hill
Fiona Francis, Paris
Gavin John Clowry, Newcastle
István Adorjan, Budapest
Ivica Kostovic, Zagreb
James Bourne, Melbourne
Kjeld Møllgård, Copenhagen
Mary Rutherford, London
Milos Judas, Zagreb
Nenad Sestan, New Haven
Pasko Rakic, New Haven
Patricia Garcez, Rio de Janeiro
Petra Hüppi, Geneva
Robert Hevner, Seattle
Susan Lindsay, Newcastle
Trygve Bakken, Seattle
Xiaoqun Wang, Beijing
Zoltan Molnar, Oxford

Earlybird registration rates (until 22nd June, 2018)

Member* £80
Non – Member £100
Student Member* £50
Student non – member £75

* Member of Anatomical Society, American Association of Anatomists and Sociedad Anatómica Española.

http://www.anatsocmeeting.co.uk/

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Two weeks ago we set up our latest competition to vote for an image to adorn the cover of a future Development issue. The images were taken by students of the International Course on Developmental Biology, an EMBO Practical Course held at the Marine Biology Station of Quintay in Chile.

Voting has just closed, and with precisely 800 votes counted, we can now reveal the top three –

3^rd Place (17% of the votes)

Sea anemone by Maria Belen Palacios

2^nd Place (26% of the votes) 

Drosophila by Eugene Tine

1^st Place (36% of the votes)

Drosophila by Soraya Villaseca

Congratulations to Soraya, and thanks to the other competitors Maria Belen Palacios, Eugene Tine, Luiza Saad and Estefanía Sánchez Vásquez. Look out for Soraya’s image on a Development cover later this year!

(1 votes)

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preLights, The Company of Biologist’s new preprint highlighting service, has now been running for more than three months. At the heart of preLights is the community of early-career researchers who select and highlight interesting preprints in various fields.

We are now ready to grow our team of preLighters and are seeking early-career researchers, who are passionate about preprints and enjoy writing and communicating science. We welcome scientists across the biological sciences and especially those with expertise in Neuroscience, Bioinformatics, Microbiology, Ecology, Biophysics, and Systems Biology.

To join our team of preLighters, please send your application to prelights@biologists.com by the 30^th June, 2018. In your application, please provide:

• A short biography, telling us who you are and what you work on
• A few sentences about why you are interested in joining our community
• A preLight post highlighting a preprint of your choice

We have a flexible format for preLights, but your post should aim to include:

A short ‘tweetable’ summary of the preprint; background of the preprint; key findings of the preprint; what you like about this preprint; future directions and questions for the authors.

The post should reflect your personal opinion on the research in the preprint that you selected. Please also provide the URL link of the preprint. Your post should not exceed 1000 words.

To learn more about the ideas behind preLights, please read this introduction, or check out the interviews with current preLighters on their experience on our News page.

What’s in it for me?

This is a great opportunity for you to gain experience in science writing. You will get editing feedback from us and your peers and we aim to raise your profile as a trusted preprint selector and commentator. You will grow your professional network, and we are happy to support you by offering recommendation letters or in other ways.

But there is also a commitment; we expect you to select and highlight a preprint every one-or-two months.

We might not be able to accept all applicants, but are looking forward to welcoming our new preLighters.

For any enquiries about the process, please email prelights@biologists.com

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The story behind FOXL1+ telocytes

You can find our recently published Nature paper here

Our story began two decades ago when my mentor, Klaus H. Kaestner, identified and cloned the transcription factor FOXL1, as being expressed in the mesenchyme of the mouse fetal gut (Kaestner et al. 1997). The position of FOXL1+ mesenchymal cells in such close proximity to the developing epithelium, as evidenced from In-Situ hybridization studies, suggested that FOXL1-expressing cells might be prime candidates to be key signal givers to the developing and adult gastrointestinal tract.

The early studies focused on the role of FOXL1 itself in regulating the intestinal epithelia by deriving mice homozygous for a FOXL1 null allele (FOXL1 -/-) (Perreault et al. 2001; Katz et al. 2004). FOXL1 null mice exhibit increased epithelial proliferation along with increased activation of the Wnt/β-catenin pathway, linking FOXL1 to Wnt/β-catenin pathway regulation.

The breakthrough came with a change in thinking. Klaus first realized that in order to study the role of FOXL1+ CELLS we should employ FOXL1 as a marker to trace these cells and genetically ablate them. The Kaestner lab derived two mouse models to kill FOXL1+ cells through the use of diphtheria toxin administration; FOXL1hDTR BAC transgenic mice that express the human diphtheria toxin receptor from a 170kb bacterial artificial chromosome that harbors all regulatory elements to direct transgene expression in subepithelial telocytes and Rosa-iDTR mice generated by crossing FOXL1Cre mice to a strain that produces the diphtheria toxin receptor from the ubiquitous Rosa26 locus in a Cre-dependent manner (Buch et al. 2005, Sackett et al. 2007).

Inducible ablation of FOXL1+ cells in adult mice caused a dramatic disruption to the intestinal epithelium, loss of stem and progenitor cell proliferation and the experimental mice died a few days after loss of telocytes had been initiated, demonstrating that FOXL1+ cells play a major role in stem cell function (Aoki et al. 2016).

I joined the Kaestner lab for my post-doctoral training during the time when these cell ablation models where being characterized in details. I am trained as a developmental biologist and therefore studying the potential cross talk between epithelia and mesenchyme was of great interest to me, even though at the time  very little was known about the nature and function of FOXL1+ cells. In fact, we had no way to detect FOXL1 protein in tissue sections or biochemically, as multiple attempts at obtaining anti-FOXL1 antibodies by commercial outfits had been unsuccessful.

Thankfully, Chris V. E. Wright’s lab at Vanderbilt University came to our aid and generated multiple monospecific anti-FOXL1 antibodies for us. Antibody staining of mouse fetal gut showed a protein expression pattern for FOXL1 that was very similar to the one seen two decades earlier using radioactive In-situ hybridization to detect FOXL1-mRNA (Kaestner et al. 1997)  (Figure 1 A-B).

However, the immunostaining for FOXL1 protein in the adult mouse intestine was disappointing at first. FOXL1 protein was present in the nuclei of selected mesenchymal cells; However, the abundance was very low (Figure 1C-D). On average, there were two to three FOXL1+ cells per crypt and their location was at mid-crypt region and along the villi core, in-addition to the crypt base where the stem cells reside.

It is well known that the driving force for intestinal stem cell function is the Wnt/β-catenin pathway, which acts as a short range signaling-system. Potential cell types that might provide Wnt ligands should be in close contact with the stem cells. Do FOXL1+ cells touch stem cells? Do FOXL1+ cells express Wnts? Do they provide the essential Wnts that maintain stem cell identity? These were the key questions that I set out to answer.

With these questions in mind, I met Chris Wright for the first time in person, at the Gastrointestinal tract FASEB meeting in August 2015. During our little discussion, Chris mentioned “cytonemes”, cellular projections that are specialized for exchange of signaling molecules, and suggested that I investigate FOXL1+ cell structure. If FOXL1+ cells have long extensions and/or posses a unique cell structure, this might allow them to contact all epithelial cells.

Back in the lab, I had a clearer understanding as to which steps I should take –

1. I planned to inhibit all Wnt secretion from FOXL1+ cells and ask: Are stem cells affected?
2. I needed to sort GFP-labeled FOXL1+ cells (using a FOXL1-Cre ;Rosa-YFP mice) and prepare RNA seq libraries. Determine their gene expression profile: Do FOXL1+ express Wnts, and if so which ones?
3.  I had to label FOXL1+ cells with a membrane reporter so that I could determine the extent of FOXL1+ cell structure

Targeting secretion of all 19 mammalial Wnt proteins can be done by deleting either Wntless or Porcupine, two essential Wnt processing enzymes, for which floxed mutant mice were already  available. In order to inhibit Wnt secretion from adult and not fetal FOXL1+ cells, I need an inducible-FOXL1 driven Cre.  With the help of fellow postdoc Kirk Wangensteen, I built a FOXL1-CreERT2 BAC and generated a new transgenic mouse line.

The next challenge arose when I tried to sort GFP-labeled FOXL1+ cells. To be able to sort FOXL1+ cells I had to selectively digest the mesenchyme from the intestinal epithelium in a single cell suspension. It was difficult to determine the optimal conditions to digest the mesenchyme as harsh digestion killed FOXL1+ cells, while mild digestion did not liberate any GFP-positive cells. Together with fellow postdoc Yue Wang, we devised a strategy to enable the selection to be successful. We isolated FOXL1+ cells, made RNA seq libraries and submitted them for sequencing.

The last piece of the puzzle was determinning FOXL1+ cell structure. To achieve this goal, I crossed our FOXL1 Cre mice to the Rosa-mTmG reporter in which FOXL1 promoter driven Cre activity lead to the expression of a membrane-bound version of GFP, which labeled the plasma membrane and allowed me to see the full extent of FOXL1+ cell.

The results were impressive; FOXL1+ GFP labeled cells were very large in extent and thus in contact with the entire epithelium from crypt base to the tip of the villi, with each and every single epithelial cell being touched by FOXL1+ mesenchymal cells!

During the same week I determined FOXL1+ cell structure, the RNAseq data was returned from sequencing. FOXL1+ cells indeed express a specific subset of Wnts and also the Wnt pathway inducers, R-Spondins. However, FOXL1+ cells also made high levels of Wnt inhibitors as well as BMPs, which are known to oppose Wnt signaling. How could this be, as we had shown through cell ablation that critical Wnt signals emanate from FOXL1+ cells?

I reasoned that since I had sorted FOXL1+ cells from anywhere along the crypt-villus axis for my RNAseq study, FOXL1+ cells might compartmentalize expression of signaling molecules based on their specific position along the crypt-villus axis.

To test this hypothesis, I contacted Shalev Itzkovitz from the Weizmann Institute, who had optimized a single molecule mRNA-Fluorescence In-Situ Hybridization (smFISH) technique for the mouse intestine. My goal was to focus on mRNA localization of different signaling molecules in FOXL1+ cell projections along the crypt-villus.

For this, I had to devise a way to label the full extent of FOXL1 cells, not just their nuclei. Unfortunately, I could not employ my Foxl1Cre Rosa-mTmG mice, as the reporter has a global tomato fluorescent protein expression, which bleeds through all analysis channels, thereby interfering with the smFISH signal. FOXL1 antibody staining would also not work, as it would label only the nuclei. What I needed was to identify a surface marker that could be specifically used to label the cells.

The RNAseq data revealed high expression of platelet derived growth factor receptor α  (PDGFRα), member of the “villus cluster genes” characterized previously during gut development (Walton et al., 2012, Shyer et al., 2013, Shyer et al., 2015, Walton et al., 2016). Does PDGFRα label FOXL1+ cells? And if so, would it be possible to use it as a marker to label FOXL1+ cell extensions? YES! We demonstrated that all FOXL1+ cells are PDGFRα+.

In Shalev’s lab, Beáta Tóth combined PDGFRα immunostaining with smFISH and we were able to show regional differentiation in mRNA localization of different signaling molecules along FOXL1+ projections, with FOXL1+ cells near the crypt bottom producing abundant Wnt2b, a canonical Wnt pathway activator, while those further up the crypt-villus axis expressed high levels of Wnt pathway inhibitors.

I was puzzled by the unusual structure of FOXL1+ cells, which I also confirmed by electron microscopy. These cells are extremely thin but very large, with diameters in excess of 200 micrometer (for comparison, an intestinal epithelial cell is only about 10 micrometer in size). I was wondering if such unique stromal cells had been described in the literature, based on histological technologies.

My literature search came up with several reports by Popescu (the late eminent Romanian pathologist) and Faussone-Pellegrini (Popescu et al., 2005, Popescu and Pellegrini 2010, Cantarero et al., 2011, Cretoiu et al. 2012, Vannucchi et al., 2013) describing primarily through the use of electron microscopy, the existence of a new stromal cell type that is present in many organs.

Popescu named these cells “Telocytes” from the Greek words “telos” meaning end, “cytes” meaning cells. Telocytes are cells characterized by extremely long and thin projections called telopodes that may reach millimeters long and express PDGFRα in both human and mouse gut.

Apparently, neurons are not unique; telocytes also have long extensions that make direct contact with each other. Would it be possible to use the neuroscientists’ technology to study the 3D network of these cells? X-CLARITY is a method designed by neuroscientists for clearing tissues in order to visualize neurons in their 3D structure within the brain without the need for sectioning (Chung and Deisseroth 2013). Could we apply this technique to clear the intestine and visualize telocytes in their 3D structure?

In fact, clearing whole intestine and immunostaining for PDGFRα in green and EpCAM to label epithelial cells in red, allowed me to visualize the comprehensive stromal network of telocytes that form a plexus that supports the entire epithelium (Figure 2).

My journey has just begun, and many exciting questions remain: Does FOXL1 label all telocytes? What is the origin of this remarkable cell? How and when do cells acquire telocyte characteristics? How do these cells compartmentalize signaling? What are the mechanisms by which telocytes signal to the epithelium.

The next decade of research will be a lot of fun!

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