For other posts in this series click here

In my first post a few months back, I talked about the need for working scientists to create new habits of mind by practicing the craft of being a writer.  Since then, I took my own advice.  I abandoned Twitter, helped folks in the lab to get five papers into the BioRxiv, and finally finished an essay I’ve been working on for two years (with luck, you can read it in Development soon…).  Last week, though, I was reading an interview in the New York Times with the newest winner of the Nobel Prize for Literature, the poet Louise Glück, and I was inspired.

She said this: “Though I couldn’t always write, I could always read other people’s writing.”

I love this.  Here’s a Nobel Laureate acknowledging that she couldn’t always write, acknowledging perhaps as well that she can’t always write.  We can all relate.  Any writer faces writer’s block, but in science there’s often a far more tangible reason for not being able to write: Not enough data to write a paper!  So, what do you do?  How do you keep your writing practice moving forward when there’s nothing pressing to write about?  Glück gives us the answer:  You read.

There’s a catch, though; you have to read like a writer.  This is rule #4 of DevBiolWriteClub.  Happily, starting to read like a writer is simple and quick.  The hard part of course is that it only matters if you keep it up, day after day, for a long, long time.  There are no shortcuts.

So, what does it mean to read like a writer?  In my view, there are two components.  The first one is simple.  You just do the work, with intent, every damn day.

Here’s what this looks like for me:  I wake up, get eggs and coffee, and settle into the New York Times for at least 30 minutes, often for an hour.  I do this, seven days a week.  Usually, I read the front section and the columnists.  When the news is too ugly, I skip all that and plunge straight into Arts or Food, sometimes Travel.  I even sometimes read the Style section; I will read any article about Prince Harry and Meghan in its entirety, so long as it’s written well.  Then I’m off to work (just in the next room these days), where of course I read all day.  Emails do not count, but papers certainly do.  At my mid-afternoon coffee break, if I can’t find someone to talk to me, I usually read a book, science history mostly or popular science.  Finally, at the end of the night, I read more.  Now it’s purely for fun, but it still counts.  Novels, short stories, biography, satire, history.  I once read an entire book by Nick Hornby that was just about reading books.  There must be 30 books in various states of read or unread stacked next to my bed.  My wife hates this mess.  Regardless, I read a book in bed before I sleep every single night.

What does your reading schedule look like?   How much did you read yesterday?

Ask yourself:  How does your time spent reading compare with your time on Twitter or Reddit or TikTok?  (Or in my case playing my kids’ Xbox.)  It’s probably obvious that we might carve out at least a little extra time for reading.  Now, do a more challenging exercise:  How did your reading time yesterday compare with your time planning, performing, or interpreting experiments?  Would your career benefit from exchanging 25 minutes a day of lab time for reading time?  Over the next few months, no.  Over the next ten years, though?  The answer is certainly yes.

Next, ask yourself, “what did I read today?”  In fact, ask yourself this question every single day.  It’s a 10 second step that will put you on the right path.  It will instill a habit of noticing what you read; this is the second component of reading like a writer.

As this new habit matures, you’ll find yourself noticing what you read when you read it. Eventually, this will grow into not just noticing what you read but also noticing the writing as you read (that was a really well written sentence; that was not, etc.).   The key to developing this habit is to avoid the normal scientist’s instinct of simply devouring the content of a paper, squeezing it for every possible insight.  Instead, try to carve out a small part of your consciousness and keep it focused on seeing the writing.

This will be an unfamiliar way of reading for many, so some exercises might help develop the practice.  I like to underline sentences I think are particularly well written.  An exclamation mark in the margin is my shorthand to indicate that a sentence was underlined for the writing, not the content.   If you get through a week of papers without ever noticing a great sentence, another exercise may be useful:  Make it a habit to simply ask yourself at the end of each paper, “what was my favorite sentence?” then spend a few minutes answering yourself.  Even when I read for fun, I do something like this: Dog-eared pages in books in my library indicate that hidden on this page, somewhere, is a sentence I really liked.

Ok, so now we know what we need to do.  Read and notice what you’re reading.  The next question, then, is what should you read?  Scientific papers first and foremost.  I tell my PhD students to maintain a steady diet of one paper per day (or seven papers per week).  Now, this does not mean you spend over an hour each day deeply reading, analyzing and annotating each paper (i.e. don’t read each one as if you were preparing it for journal club).  Just read it, start to finish.  Note the parts you like and dislike.  Notice the writing independently from the content.  That 25 minutes I talked about?  Use 22 of them to read the paper and three to think about the writing.  Then you’re done. Until tomorrow.

Obviously, this regimen will not sustain you over the long term; you have to have variety.  So, find a way to read some non-science writing every single day.  This can be books, magazines, or newspapers.  The key is to read something that is professionally edited, so there is at least some expectation that the writing is good.  (Reddit, Twitter, and internet screeds do not count; good blogs do count.)

A student subscription to the NY Times is $1.88 per week with the LA Times and Washington Post being similar.  Scientific American and National Geographic are 20 bucks a year for students. The Economist is a bit more, but most university libraries and even local libraries provide broad newspaper and magazine access.  Used bookstores provide incredible value.  There’s just no excuse.  You have to read widely, and you have to at least try to notice what you’re reading.  Do this every day, even if only for a few minutes.

Build this habit now, and in a few years, you’ll be a better writer.  Again, I wish I could tell you it’d happen faster, but it won’t.  Just do the work.

I’ll end with something I read in another great piece in the New York Times.  The Pulitzer Prize winner Viet Thahn Nguyen wrote: “… people ask me what it takes to be a writer. The only things you have to do, I tell them, are read constantly; write for thousands of hours; and have the masochistic ability to absorb a great deal of rejection…”  That last bit will sound immediately familiar to scientists.  We’d all do well to make that first part seem familiar as well.

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The Kodjabachian lab at the Institute of Developmental Biology of Marseille (IBDM) is seeking a young and talented postdoctoral scientist with strong background in cell and developmental biology, and a keen interest in integrative quantitative biology and interdisciplinary research. Our lab uses advanced imaging techniques (such as confocal videomicroscopy, super-resolution microscopy and 3D electron microscopy) to study the biology of ciliated epithelia at multiple scales.

In vertebrate ciliated epithelia, flows of biological fluids are powered by the coordinated beating of myriads of ciliaharbored by multiciliated cells (MCC). In recent years, the global MCC transcriptome has been decrypted in Xenopus, mouse and human. Through this project, funded by ANR, we now wish to elucidate the functional MCC proteome. The selected candidate will be in charge of testing the functional importance of candidates selected through proteomic screens currently running in the team. He/she will use Xenopus epidermis, inducible MCC culture, and mouse post-natal brain as models to elucidate the mechanisms underlying vertebrate MCC construction.

IBDM offers a vibrant, international, and interactive environment to study the fundamental principles of cell and developmental biology. IBDM is also part of the Turing Center for Living Systems (CENTURI), a large interdisciplinary program allowing rich collaboration with theoreticians, physicists and computer scientists.

The ideal candidate must hold a PhD for less than two years, and have skills in cell culture, cell imaging, molecular biology, and biochemistry. The position is opened for one year renewable up to 3 years starting as early as January 2021. Applicants must email a CV, a statement of interest and contact details for 2-3 references to Laurent Kodjabachian.

Access to the lab page here

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Job title:          Project Research Scientist

Location:          The Francis Crick Institute, Midland Road, London

Contract:          Full time

Salary:             Competitive with benefits, subject to skills and experience

Vacancy ID:      15088

Short summary

We are seeking a highly motivated and collaborative postdoc in the area of human embryology and stem cell biology to join Professor Kathy Niakan’s laboratory.

The aim of the project is to characterise early lineage specification in early human embryos. We have recently identified several transcription factors and components of key signaling pathways that are highly expressed in epiblast cells of the developing human embryo, which we hypothesize may have an important function for the development of these pluripotent cells. We seek to understand the function requirement of these factors using a range of methods including cutting-edge single cell, imaging and genome editing techniques. Ultimately, this knowledge will provide fundamental insights into human biology and facilitate the development of conditions for the further refinement of implantation models and the establishment of novel human stem cells and stem cell-based models of development.

We seek candidates who are energetic, focused, and productive with a desire to work in a congenial, dynamic, and collaborative research environment. Good organisational, analytical, and communication skills are essential.

Project scope

Dr Niakan’s laboratory focuses on understanding the mechanisms of lineage specification in human embryos and the derivation of novel human stem cells. Details of research projects currently being undertaken can be seen at:  http://www.crick.ac.uk/kathy-niakan

Research techniques used in the laboratory include: molecular biology, advanced microscopy and image quantification, human and mouse preimplantation embryo culture and micromanipulation, genome modification, genome-wide techniques including single-cell RNA-sequencing, multi-omics analysis and human trophoblast, embryonic and induced pluripotent stem cell derivation.

About us

The Francis Crick Institute is a biomedical discovery institute dedicated to understanding the fundamental biology underlying health and disease. Its work is helping to understand why disease develops and to translate discoveries into new ways to prevent, diagnose and treat illnesses such as cancer, heart disease, stroke, infections, and neurodegenerative diseases.

An independent organisation, its founding partners are the Medical Research Council (MRC), Cancer Research UK, Wellcome, UCL (University College London), Imperial College London and King’s College London.

The Crick was formed in 2015, and in 2016 it moved into a new state-of-the-art building in central London which brings together 1500 scientists and support staff working collaboratively across disciplines, making it the biggest biomedical research facility under in one building in Europe.

The Francis Crick Institute is world-class with a strong national role. Its distinctive vision for excellence includes commitments to collaboration; developing emerging talent and exporting it the rest of the UK; public engagement; and helping turn discoveries into treatments as quickly as possible to improve lives and strengthen the economy.

• If you are interested in applying for this role, please apply via our website.
• The closing date for applications is 16 November 2020 at 23:45.
• All offers of employment are subject to successful security screening and continuous eligibility to work in the United Kingdom.

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This interview, the 79th in our series, was published in Development earlier this year. 

Cortical development involves a switch from the self-amplification of stem cells to the generation of neuron and glia by progenitors. A new paper in Development investigates the molecular control of mitosis in these two stages, using simultaneous labelling and gene knockout in clones in the developing mouse brain. We caught up the paper’s two authors Caroline Johnson and her supervisor Troy Ghashghaei, Professor of Neurobiology at the College of Veterinary Medicine at North Carolina State University, to find out more.

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

TG I did my graduate work mapping limbic circuits, focusing on connections between the basal forebrain/amygdaloid nuclei and the prefrontal cortices of macaque monkeys. Some of the results raised a number of developmental questions, which led me to pursue a postdoc in developmental neurobiology. Therefore, I did my postdoc work on adult neurogenesis and cortical development, which we have continued in my own lab since 2006. I am now a professor and my lab is currently focused on two main projects: (1) how neural stem cells undergo developmental transitions during distinct time points, and how perturbations of these transitions impact proliferation and differentiation in the forebrain and cortex; (2) how ependymal cells are formed from embryonic neural stem cells and how these fascinating and poorly studied cells regulate forebrain homeostasis during adulthood and aging. We use a genetic approach in understanding the functions of various factors, and employ interdisciplinary tools to identify, characterize and test various phenotypes in the two projects.

Caroline, how did you come to work with Troy and what drives your research today?

CJ When I was an undergraduate at UNC Chapel Hill, I volunteered with the veterinarians at the Duke Lemur Center, and worked at Duke studying the diversity of mouse lemur species. I then worked as a technician at Harvard, which sparked my interest in neuroscience research. When I was looking for graduate programmes, I wanted to move back to North Carolina because there are a lot of great research opportunities here and I could be closer to my family. I was also potentially interested in veterinary medicine research, which led me to look at graduate programmes with the veterinary school at NC State University [NC State]. When I heard about the projects in developmental neuroscience research occurring in Dr Ghashghaei’s lab, I contacted him and enrolled in the Comparative Biomedical Sciences programme. Cortical development is an amazing field. The sensitivity of the cortex to disruptions in cell division and the paradigm of its development permits us to ask questions about basic development, comparative development and cell proliferation, and gives us greater insight into how this fascinating structure has developed and evolved.

How did you come to study Sp2 and what was the main question your current paper was aiming to answer?

TG My colleague Jonathan Horowitz and his lab were researching the function of Sp2 in cancer cells when I first arrived at NC State. Sp family members have a fascinating genomic organization and they are all involved with important aspects of organismal development. When we began this project, Sp2 was the least studied family member. In collaboration with the Horowitz lab we found that Sp2 is highly expressed in neurogenic regions of the postnatal and embryonic mouse forebrain. This led a previous graduate student in the lab, Huixuan Liang, to investigate what happens when Sp2 is deleted during neurogenesis at the embryonic and postnatal stages of forebrain development. She found that Sp2 is required for timely progression through mitosis within neurogenic niches, and the results of that work were published in Development in 2013. That is when Caroline joined the lab and the current paper is a summary of all the work she has done over that last few years. It is important to note that the use of MADM technology was instrumental in this work and our interactions with my friend and colleague Simon Hippenmeyer (a co-author on the 2013 paper) were critical in the initial stages of the project.

CJ My major focus was to dynamically investigate mitosis to understand how its timing was affected by loss of Sp2, as the previous project has identified a defect in M-phase progression. Additionally, I wanted to look at earlier stages of corticogenesis prior to the transition to neurogenesis when the stem cell pool is expanding. A very important finding that emerged from our work has been the revelation of phenotypic differences between bulk and sparse genetic deletion strategies. The complex genetic models we used revealed that bulk deletions can cause major non-autonomous effects that may mask the cell-autonomous role of a protein. By using sparse deletions, we are able to measure in detail genotype-phenotype associations.

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

CJ Essentially, we found that Sp2 is required for neurogenesis during a distinct time point of cortical development. Surprisingly, Sp2 is dispensable for early stages of corticogenesis, when stem cells are amplifying and the switch to neurogenesis occurs, but later, when the production of upper layer neurons begins, loss of Sp2 affects the timing of mitosis and ultimately results in the decreased production of neurons.

Do you think that Sp2 acts primarily in neural progenitor cell or intermediate progenitor division when promoting the production of upper layer neurons?

CJ I think that ultimately disruption of mitosis in the neural progenitor population results in the decreased production of intermediate progenitor cells, but it is also possible that disrupted mitosis in the mother cell has consequences for the proliferative capacity of intermediate progenitor cells. An important next step would be to look at the timing of mitosis in the neural and intermediate progenitor populations at multiple neurogenic stages to see how their populations and mitotic timing are affected. Additional experiments specifically deleting Sp2 from the intermediate progenitor population would also be interesting.

TG While we did not distinguish between the two modes of terminal divisions in our study, we suspect both are affected. However, one has to do the experiment to answer the question directly. The results in our current paper reveal stronger effects on large clones and upper layer-specific sensitivity when Sp2 is deleted at sparse levels, which suggest both types of terminal divisions are likely affected in the absence of Sp2.

Do you have any idea about the transcriptional targets of Sp2 that might ensure timely mitosis?

CJ Recent studies of Sp2-dependent transcription have indicated it is involved in multiple pathways and may require different co-factors. It is unclear what the transcriptional activity of Sp2 is outside of cell lines and in developing tissues, whether or not it interacts with these potential co-factors within the developing cortex, and/or if it is playing a non-transcriptional role at one of these stages. Sp2 is known to interact with the nuclear matrix and may be regulating gene expression through a process independent of transcriptional regulation.

TG Our lab has been pursuing questions related to upstream regulation of Sp2 and downstream targets of this interesting protein, but more work is needed.

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

CJ Realizing that larger clones had a stronger phenotype in response to loss of Sp2 was definitely exciting, as it indicated that the clones that were expanding throughout neurogenesis were most affected (similar to our results in the other experiments). The smaller clones, which likely reflected lineages that immediately entered neurogenesis, were not strongly affected.

Working with animal models is always more complicated than you would hope

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

CJ Of course, working with animal models is always more complicated than you would hope, and the early timepoint experiments were surprising at first because Sp2 has a known role in cell proliferation. However, doing the additional experiments with bulk deletion and an additional Cre driver line helped to reveal that we really weren’t seeing an effect at this early stage of cortical development.

So what next for you after this paper?

CJ I am working with Dr Ghashghaei on projects in the lab related to my previous project. In terms of the future, I’m interested in exploring scientific writing or clinical research careers to help advance biomedical research.

Where will this work take the Ghashghaei lab?

TG We are moving forward with a number of projects that continue to interrogate mechanisms that regulate the transitions that NSCs and NPCs in the forebrain go through as development proceeds.

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

TG I have been playing soccer (football to the rest of the world outside America) for a long time. Playing with old friends as well as coaching and scouting for some of the local clubs forms much of my time outside the lab. In addition, my daughter is a gymnast in college – so I spend quite a bit of time with my wife going to her meets.

CJ Raleigh is a beautiful city and the veterinary school is located next to the art museum and greenway, so I like to run at the trails there. I also enjoy cooking and spending time at my parents’ farm.

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Postdoc position is available at the Max Delbrück Center (MDC, Berlin, Chekulaeva lab). ALS is a neurodegenerative disease affecting motor neurons, i.e. neurons that control skeletal muscle contraction to produce motion. Despite heterogeneous etiology, ALS is characterized by abnormalities in RNA metabolism. The successful candidate will work with the collection of ALS patient-derived hiPSCs and hiPSC-derived motor neurons to investigate how changes in RNA metabolism contribute to the mechanism of neurodegeneration. The project will reply on a combination of omics (transcriptomics, Ribo-seq, CLIP-seq), computational, CRISPR/Cas-mediated gene editing and imaging approaches and will involve a collaboration with international partners, contributing different expertise to the project, including computational analysis, in vivo ALS models and clinical research.

Ideal candidate should have experience in cell culture and molecular biology techniques, interest in RNA biology and neurodegeneration. Experience in hiPSC work and RNA methods is an advantage. To apply, please send your motivation letter and CV with contact details of at least two referees to marina.chekulaeva(at)mdc-berlin.de

Recommended reading:

1. von Kuegelgen N and Chekulaeva M^# (2020). Conservation of a core neurite transcriptome across neuronal types and species. WIREs RNA 14:e1590. http://dx.doi.org/10.1002/wrna.1590
2. Ciolli Mattioli C., Rom A., Franke V., Imami K., Arrey G., Terne M., Woehler A., Akalin A., Ulitsky I., and Chekulaeva M. (2018). Alternative 3′ UTRs direct localization of functionally diverse protein isoforms in neuronal compartments. Nucleic Acids Research, https://doi.org/10.1093/nar/gky1270
3. Zappulo, A.*, van den Bruck, D.*, Ciolli Mattioli, C.*, Franke, V.*, Imami, K., McShane, E., Moreno-Estelles, M., Calviello, L., Filiipchyk, A., Peguero-Sanchez, E., Mueller, T., Woehler, A., Birchmeier, C., Merino, E., Rajewsky, N., Ohler. U., Mazzoni, E., Selbach, M., Akalin, A., and Chekulaeva, M. (2017). RNA localization is a key determinant of neurite-enriched proteome. Nature Communications, http://dx.doi.org/10.1038/s41467-017-00690-6

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Research at UM addresses a broad range of exciting developmental biology questions.

• What genes regulate the creation of different neuronal cell-types?
• How do cardiac cells sense and respond to their environment to form a heart?
• What gene-networks underlie the release of leaves, fruits and seeds?
• How do plants respond to changes in light and temperature?

Features of our program include:

• 2 admission paths: a rotation program or direct admission to a laboratory
• Choose the courses that are most appropriate for your research and career
• Teaching assistantships are available
• Additional awards for excellent candidates

Learn more about the department and how to apply at biology.olemiss.edu

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As I’m sure you’ve all heard, last week saw the 2020 Nobel Prize in Chemistry awarded to Emmanuelle Charpentier and Jennifer Doudna for their work on the CRISPR/Cas system. It’s hard to believe that it was only 8 or so years ago that they – along with their colleagues Martin Jinek, Krzysztof Chylinski and others – demonstrated the potential of the Streptococcus CRISPR/Cas9 adaptive defense system to be used for genome editing.

Whether you’re a fan of the Nobel or you think it’s over-rated, it’s hard to argue against the influence that CRISPR technology has had on the biomedical sciences, including developmental biology. Of course, it’s not that we couldn’t edit genomes before CRISPR came along – tools such as zinc-finger nucleases and TALENS were already proving useful to engineer mutations in a range of species – but the relative flexibility and ease of use of the CRISPR system opened up new possibilities for genome engineering both in traditional and non-traditional model systems.

Not only can targeted mutations now be made more quickly, efficiently and cheaply than we might have believed possible just a decade ago, but the precision with which specific edits to the genome can be made has also improved dramatically. Moreover, the CRISPR toolkit has been expanded to a wide range of other technologies – from genetic screening to manipulation of gene expression to barcoded lineage tracing.

We asked some of Development’s editors to sum up how CRISPR has impacted their research. Here are a few of their responses:

“CRISPR-based methods have become so ubiquitous that it is becoming difficult to remember how we were working in the pre-CRISPR era” (François Guillemot)

“Like many developmental biologists, CRISPR/Cas9 has revolutionised our research. Knocking-out a gene, easy-peasy; introducing a point mutation, no problem; deleting a regulatory element, piece of cake. It means we can design more creative and more precise experiments and we’re still only at the beginning of what is possible.” (James Briscoe)

“CRISPR/Cas9 has been a great addition to our tool box and substantially changed our life in the last 5-6 years. Genetic approaches in mice and cultured cells became so easy and became available for even non-standard organisms – the evo-devo field has perhaps been revolutionarily changed…  Not only genetics, but also epigenetics: CRISPR/Cas9 technology is also applied to modify chromatin structures in highly targeted manner.” (Haruhiko Koseki)

“My postdoc knockout mouse took over two years to make. A student can make one in 3 weeks now.” (Benoit Bruneau)

To celebrate the Nobel award, Development has collected together just a few of the CRISPR papers published in the journal over the last 8 years. All these articles are free to read, and we invite you to browse the collection here. In it, you’ll find papers that apply CRISPR technologies in a wide range of model systems, that improve the efficiency with which mutations can be generated, and that use modified Cas9 proteins to manipulate gene expression or enhancer activity in a targeted manner. You’ll also find commentaries that touch on some of the thorny ethical questions that genome editing has thrown our way: how should we consider genome editing in the context of agriculture and genetically modified crops and – perhaps most difficult of all – what about human genome editing? We hope you enjoy this collection of articles.

And if you have comments on how CRISPR technologies have impacted your research, we’d love to hear them – please leave a comment below!

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Timothy Fulton, Vikas Trivedi, Andrea Attardi & Benjamin Steventon

As developmental biologists, we often find ourselves carefully looking at developing embryos as they undertake a dramatic and fascinating task: making a complex organism out of a few initial embryonic cells. On the other hand, for engineers, building complex structures out of simple constituent parts under a defined set of rules is what creates excitement every day. Together, the bioengineer and the embryologist wonder how cells behave in a way that shapes the animal, whilst simultaneously deciding on their fate. Clearly, these two processes of morphogenesis and pattern formation must occur in concert.

We know that vertebrate embryos achieve this by integrating cues and signals from the extraembryonic environment. Such cues do not act alone though, as they are coupled to an intrinsic ability of early cells to drive morphogenesis and patterning. This latter feature of embryonic cells, known as self-organisation, is remarkably highlighted when cells are separated from their extra-embryonic constraint. An example of this is given by mouse and human embryonic stem cells (mESCs/iPSCs). Under controlled culturing conditions, these cells aggregate as ‘gastruloids’ (van den Brink et al., 2014; Moris et al., 2020), groups of cells that perform and recapitulate essential aspects of axis specification and pattern formation seen in intact mouse or human embryos.

Such ideas about self-organization of embryonic cells were in the atmosphere during lab meetings and tea time discussions at the Department of Genetics. Vikas, who had recently joined the lab of Alfonso Martinez Arias as a postdoctoral fellow, came with the experience of having worked with zebrafish before and was wondering about a fish equivalent of gastruloids. At the same time, Andrea, a masters student in Ben Steventon’s lab,  while working on a project to lineage trace Neuromesodermal Progenitors during gastrulation, was having similar thoughts (Attardi et al., 2018). The question arose quite spontaneously: could zebrafish embryonic cells tell us something new about morphogenesis and self-organisation, if we cultured them free of their embryonic constraints? That looked like a good opportunity for exploring what the collaboration of bioengineering and embryology could lead us to. We therefore decided to attempt culturing zebrafish embryonic cells at early stages of development, starting by explanting cells from the 256 cell stage embryo, prior to the midblastula transition. Strikingly, we observed that in doing so, these masses of cells elongated over a period of seven to ten hours. We observed this phenomenon in a range of media, with and without serum, including inert Ringer’s Solution. These results nicely echo those of Jane Oppenheimer (1936), who found similar results when explanting blastoderm from another teleost, Fundulus heteeroclitus.

Pescoid Elongating – Wildtype pescoid elongating in normal L15 + 3% FBS Serum imaged on a widefield microscope

After identifying optimal incubation conditions, Tim set about understanding which cell types formed within these elongating explants. After making many hundreds of pescoids, and using multiplex in situ Hybridisation Chain Reaction (Choi et al., 2018), he demonstrated that these explants produce all three germ layers in their elongated form. We observed the mesodermal marker tbxta expressed in the elongating pole, with sox17 marking the endoderm in the core of the explants. In the non-elongating end, we observed sox2 expressed marking the ectoderm. In addition to these marker genes, we also observed expression of germ cell markers nanos and vasa, as well as expression of the hindbrain marker gene krox20 which, as in the embryo, was expressed in two clearly defined stripes of expression in the non-elongating end. Interestingly, we did not observe expression of otx2 positive forebrain cell.

We wanted to test exactly how robust the germ layer formation was to mixing of the cells. In order to test this, we cut and totally dissociated and reaggregated the explants immediately after explanting using two eyelashes glued to capillary tubes. Tim and Ben swept the cells together gently under a microscope in order to break down and reassemble the explant. A very delicate procedure, made harder by Ben’s insistence that we listened to Test Match Special, in order to keep up to date with the Cricket World Cup! When we reaggregated pescoids, we observed that the explants rarely elongated, however the majority still expressed tbxta. We hypothesised that the initial size of the explant is important for elongation potential, as quartered explants rarely elongate, however half sized explants retain this potential.

We were also interested to discover how much cell mixing was occurring in these explants naturally. Using two different methods, Chaitanya labelled either the edge of the explant, or the marginal cells of the embryo prior to explanting, and then tracked these cells post explant. In all situations, we observed that the labelled cells mix significantly with the non-labelled cells prior to elongation. Schauer et al. (2020) recently conducted very similar experiments to us, and demonstrated that the prepatterns provided to the embryonic cells by the yolk prior to the stage at which we take explants are essential for the formation of the elongation. We also found that the whole process is dependent on early Nodal activity, a result that fitted nicely with the work of (Williams and Solnica-Krezel, 2020).

Nodal in Explants – Activin Response Element::GFP reporter for Nodal activity demonstrating correlation of Nodal signalling with the elongation

We had some excellent discussions with Alexandra Schauer at the EDBC meeting in Alicante in 2019 and really enjoyed being able to share and discuss our findings with one another. However, the extensive mixing we observe here demonstrates that whatever the mechanism between prepattern and pattern formation is, it must also be highly robust to cell mixing over long timescales of 3 to 5 hours. Overall the individual cells regularly swap positions with one another, however, at the organism level, the explants form reproducible domains of stable gene expression.

How these different domains of gene expression came about remained unclear, and to investigate this, we looked at the signalling events taking place within the explant. We observed that initially the activity of Wnt and BMP signalling completely overlapped. During elongation however, these two activity domains separate, with Wnt activity being restricted to the elongation, and BMP to the non-elongating end. We hypothesized that the elongation may be the driving force behind the separation of these two domains, and by inhibiting morphogenesis both genetically and pharmacologically, we demonstrated that this is indeed the case; without morphogenesis, the two domains remain overlapping and two krox20 stripes were never observed. The explants however are unable to produce an environment with very low levels of both Wnt and BMP, and therefore this may explain why Oxt2 positive cells are not produced.

Pescoids Elongating with TCF::GFP and BRE::RFP. TCF::GFP reporter for Wnt activity, demonstrating Wnt signalling occurs across the entire explant their localises to just the elongation (Green to White). BMP Response Element fusion of RFP demonstrates BMP signalling is active across the entire explant then localises to just the non-elongating end.

We therefore had a situation where our explants are displaying a very high level of cell mixing, with cells regularly changing positions with one another, whilst also maintaining distinct regions of gene expression. Whilst our work here begins to answer how these domains form, it does not answer how these domains are then maintained. This opens an intriguing question: how do cells, via signalling and gene regulatory networks, update the cell’s positional information whilst constantly moving?

Traditionally, this type of question would invoke ideas of the French Flag Model, which describes a situation where morphogen gradients inform cells of their position and, therefore, of their fate decision. However, as Wolpert described in the French Flag Problem (Sharpe, 2019; Wolpert, 1969), this idea becomes difficult to apply when cells also regularly change positions with one another and therefore scramble the pattern as it is being produced. This is something which Tim is continuing to investigate as part of his PhD. Using live imaging data and tracking of individual cells, we are overlaying dynamical systems models of a GRN onto in vivo tracks  in order to model cell fate decisions in a tissue undergoing active morphogenesis. The Steventon Lab is also interested in investigating how pattern formation may be an emergent property of gene regulatory networks and morphogenesis working together. Using the idea of tectonic plates, the term tissue tectonics (Busby and Steventon, 2020) describes how morphogenetic movements regulate the timings of interactions between different tissues as they slide together or apart, similar to as we see in these pescoid explants, and other embryonic systems. The Trivedi lab is pursuing studies on understanding the biophysics of self-organization that leads to patterning in early embryonic systems including pescoids.

Overall, this work, and the ideas of tissue tectonics propose a model where pattern formation is as much the output of genes and local cell rearrangements, as it is the more global tissue movements which shift cells into and out of range of one another. By considering patterns as an emergent property of morphogenesis and GRNs, we can begin to ask questions about how these components are brought together, and what role cell-cell signalling plays in this.

Read the full paper in Current Biology 

Axis Specification in Zebrafish Is Robust to Cell Mixing and Reveals a Regulation of Pattern Formation by Morphogenesis. Timothy Fulton, Vikas Trivedi, Andrea Attardi, Kerim Anlas, Chaitanya Dingare, Alfonso Martinez Arias, Benjamin Steventon. 

References

Attardi, A., Fulton, T., Florescu, M., Shah, G., Muresan, L., Lenz, M.O., Lancaster, C., Huisken, J., van Oudenaarden, A., and Steventon, B. (2018). Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics. Development dev.166728.

van den Brink, S.C., Baillie-Johnson, P., Balayo, T., Hadjantonakis, A.-K., Nowotschin, S., Turner, D.A., and Martinez Arias, A. (2014). Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141, 4231–4242.

Busby, L., and Steventon, B. (2020). Tissue Tectonics and the Multi-Scale Regulation of Developmental Timing.

Choi, H.M.T., Schwarzkopf, M., Fornace, M.E., Acharya, A., Artavanis, G., Stegmaier, J., Cunha, A., and Pierce, N.A. (2018). Third-generation in situ hybridization chain reaction: Multiplexed, quantitative, sensitive, versatile, robust. Dev. 145.

Moris, N., Anlas, K., van den Brink, S.C., Alemany, A., Schröder, J., Ghimire, S., Balayo, T., van Oudenaarden, A., and Martinez Arias, A. (2020). An in vitro model of early anteroposterior organization during human development. Nature 1–6.

Oppenheimer, J. (1936). The Development of Isolated Blastoferms of Fundulus Heteroclitus. 72, 247–269.

Schauer, A., Pinheiro, D., Hauschild, R., and Heisenberg, C.P. (2020). Zebrafish embryonic explants undergo genetically encoded self-assembly. Elife 9.

Sharpe, J. (2019). Wolpert’s French Flag: what’s the problem? Development 146.

Williams, M.L.K., and Solnica-Krezel, L. (2020). Nodal and planar cell polarity signaling cooperate to regulate zebrafish convergence & extension gastrulation movements. Elife 9.

Wolpert, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1–47.

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Tenure-track Assistant or Associate Professor of Genetics or Genomics

In anticipation of substantial growth over the next five years, the Department of Human Genetics at the University of Utah School of Medicine (www.genetics.utah.edu) seeks outstanding applicants for one or more tenure-track positions at the level of Assistant or Associate Professor. We seek highly creative scientists who use genetics to investigate fundamental biological problems. We encourage applicants whose research focuses on evolutionary and functional genetics and genomics; human and medical genetics; computational genomics; and research programs using established model or unconventional organisms. As part of a vibrant community of faculty with a strong track record of collaborative mentorship, research, and funding, the Department of Human Genetics lies at the interface between basic and clinical sciences.  This creates ample opportunities for interdisciplinary research (e.g., our Center for Genetic Discovery, Transformative Excellence Program in Evolutionary Genetics and Genomics, and Center for Genomic Medicine).  As a department, we value diversity and equity, and believe that the best science is done when researchers of diverse backgrounds are integrated and supported in an inclusive manner. We seek faculty who share these values. Our institution is set in a unique geographical landscape that attracts a heterogeneous and productive scientific community.  Successful candidates will receive a generous startup package and enjoy a stimulating research environment that places a strong emphasis on innovation and interaction.

Apply here: http://utah.peopleadmin.com/postings/108145

Applicants are asked to submit:

Curriculum Vitae – CV and 3 most relevant reprints or preprints

Research statement – Describe your most significant scientific accomplishments, your goals for research as a faculty member, and the qualifications and experience that have prepared you to achieve these goals.

Teaching statement – Describe your commitment to education, your teaching philosophy, and the courses you might potentially teach.

Diversity, equity, and inclusion statement — Describe your past and future contributions to diversity, equity, and inclusion through research, teaching, and service.

The University of Utah is an Affirmative Action/Equal Opportunity employer and does not discriminate based upon race, national origin, color, religion, sex, age, sexual orientation, gender identity/expression, status as a person with a disability, genetic information, or Protected Veteran status. Individuals from historically underrepresented groups, such as minorities, women, qualified persons with disabilities and protected veterans are encouraged to apply. Veterans’ preference is extended to qualified applicants, upon request and consistent with University policy and Utah state law. Upon request, reasonable accommodations in the application process will be provided to individuals with disabilities. To inquire about the University’s nondiscrimination or affirmative action policies or to request disability accommodation, please contact: Director, Office of Equal Opportunity and Affirmative Action, 201 S. Presidents Circle, Rm 135, (801) 581-8365.

The University of Utah values candidates who have experience working in settings with students from diverse backgrounds, and possess a strong commitment to improving access to higher education for historically underrepresented students.

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