By Rachel Bonnington, Carla Lloret Fernández and Laura Molina García

Classically, developmental programmes were believed to be a one-way linear process in which cells progressively acquire their differentiated identities, each with a distinct and highly specialised morphology and function. Since differentiated post-mitotic identities are usually stable, differentiation was first thought to be irreversible. However, there are now many examples that appear to subvert this idea, both in nature and during forced reprogramming experiments, where differentiated cells are able to switch their identity into distinct differentiated cell types (Lambert et al. in press). This process is called transdifferentiation (Eguchi & Kodama, 1993; Selman & Kafatos,1974). In our recent paper (Molina-García et al., 2020), we describe how a glial cell transdifferentiates into a neuron in a sexually dimorphic manner to optimise male mating performance in the nematode worm C. elegans. Our findings demonstrate how genetically-programmed transdifferentiation acts as a developmental mechanism to allow flexibility in innate behaviour.

C. elegans is an excellent model for single cell studies

Two main challenges of studying transdifferentiation are that two distinct, stable identities need to be unambiguously defined, both before and after the proposed cell type conversion, and a direct lineal relationship must be established between them. This remains extremely difficult to determine in most model organisms, in which cellular lineages are highly variable and poorly defined. However, C. elegans conveniently overcomes these barriers, allowing us to study natural cellular reprogramming events in live animals. In fact, the first transdifferentiation in C. elegans was described nearly fifty years ago by Sir John Sulston, one of our scientific heroes, who observed that the rectal-epithelial cell Y converts into the PDA motor neuron (Sulston & Horvitz, 1977). This was possible because development in C. elegans is highly stereotyped and the number of cells in the adult is fixed. This allowed Sulston and colleagues to fully resolve the somatic lineage of the worm, which describes all cellular identities and their positions, for the two sexes: males and hermaphrodites (Sulston & Horvitz, 1977; Sulston et al. 1980; Sulston et al. 1983). C. elegans is also transparent, and transgenics are readily made, so cells can be imaged at a single-cell level, making it possible to unambiguously follow cell-type conversion events. No wonder then, that C.elegans is our model organism of choice in the Barrios and Poole labs at UCL!

PHso1 is a glial cell that changes identity in the worm

Previous work from the Barrios and Poole labs described how a pair of glial cells, called amphid socket (AMso) cells, undergo an identity change into neurons in males, providing the second example of transdifferentiation in the worm. The AMso cells form the cuticular pore of the amphid chemosensory organ in the head of the worm that allows sensory neurons to contact and respond to the external world. In this case, transdifferentiation occurs alongside asymmetric cell division that leads to a self-renewal of the AMso glial cells and the production of a previously unidentified male-specific pair of interneurons, the MCMs (Sammut et al. 2015). The AMso cells retain their structural role in forming the socket of the amphid, while the MCM neurons are involved in male-specific learning.

Following on from our discovery of this glia-to-neuron transdifferentiation, we were interested to examine the sensory pore of the equivalent sensory organ in the tail of worms, the phasmid sensillum. Unlike the amphid, the phasmid is made up of two pairs of socket cells (the sister cells PHso1 and PHso2), which together form a bilayer hollow pore in the cuticle. Again, during his description of the C. elegans lineage, John Sulston noted a difference between the phasmid sockets in males and hermaphrodites, observing that in adult males, PHso1 cells appear to retract from the hypodermis, and that they contain basal bodies (a structural component of cilia – in C. elegans, the only ciliated cells are sensory neurons) (Sulston, 1980). However, no other neuronal characteristics were noted and PHso1 cells continued to be classified as glia.

We were therefore hugely excited when we analysed the lin-48/OVO1 transgenic reporter, known to be expressed in PHso1, and noticed a long, axon-like projection in males, extending towards the pre-anal ganglion, a key neuronal centre in the nematode tail. This strongly suggested a neuronal identity for PHso1!

Could this be the smoking gun we were looking for, proving that in males the anterior-most phasmid socket glial cells (PHso1) were in fact being transformed into neurons? To determine this, we examined these lin-48-expressing cells during different developmental stages in males by time-lapse and compared them to hermaphrodites. We found that during the early stages of larval development, PHso1 cells appear indistinguishable between males and hermaphrodites. During the course of male sexual maturation, PHso1s in males undergo radical remodelling from a socket-glial morphology to a neuron-like morphology. By contrast, PHso1 cells remain as sockets in hermaphrodites (see Figure), thus corroborating John Sulston’s earlier observations of sexual dimorphisms in PHso1 morphology.

Male PHso1 glia transdifferentiate into PHD neurons

At the gene expression and ultrastructural levels, male PHso1 cells also acquire a number of uniquely neuronal characteristics, which we never observed in either the neighbouring PHso2 cells, nor the PHso1&2 cells of hermaphrodites. These include the expression of pan-neuronal genes and genes involved in neuronal transport and communication (assessed by fluorescent reporter expression) as well as the presence of synaptic vesicles, dense-core vesicles and ciliated structures (assessed by electron microscopy). Importantly, at the same stage of sexual maturation, when these neuronal markers are firing up, we see dimming of glial genes, such as the panglial microRNA mir-228 and the AMso and PHso glial subtype marker grl-2, in male PHso1s, before they fully switch off. Together, these results demonstrate that PHso1 glial cells transdifferentiate into a previously undescribed class of neurons that we call PHDs (Phasmid neuron D). This updates the anatomy of the C. elegans male, increasing the number of neurons from 385 to 387 (of which 93 are male-specific) and decreasing the total number of glia from 92 to 90.

The PHso1-to-PHD transdifferentiation seems to occur without a division. It does not require wholesale DNA replication or cell division followed by programmed cell death of one of the daughters, a common strategy used throughout development (reviewed in Conradt et al. 2016). To our surprise, however, we sometimes observed that PHso1 divides in a background-dependent manner to give rise to two apparently equivalent neurons (PHD1 and PHD2). Moreover, when we genetically manipulated the biological sex of the cell (i.e. we masculinised PHso1 in an otherwise hermaphroditic background) we observed a similar transdifferentiation of PHso1 to a PHD-like cell. This suggests that, as we previously published for the AMso, PHso1 is poised to transdifferentiate, awaiting cell-autonomous activation by the sex determination pathway.

We next sought to investigate if the molecular mechanisms known to regulate the Y-to-PDA transdifferentiation mentioned earlier could also control the transdifferentiation of the PHso1 and AMso cells. The Greenwald and Jarriault labs were the first to fully characterise the Y-to-PDA transdifferentiation (Jarriault et al., 2008). They later showed that a complex of conserved NODE-like factors (CEH-6/Oct, SEM-4/Sall and EGL-27/Mta) together with the transcription factor SOX-2, known to regulate mammalian embryonic stem cell pluripotency, are required for the initiation of the process (Kagias et al. 2012). Interestingly, these factors seem to be largely dispensable for the PHso1 and AMso cell identity switches. This could point towards independent mechanisms of transdifferentiation rather than a shared program. It will be interesting to determine if, as for Y-to-PDA, chromatin remodelling (Zuryn et al. 2014) also plays a role in AMso and PHso1 transdifferentiation, or whether transdifferentiation of these cells rely on completely different strategies. The new AMso and PHso1 cellular paradigms provide the perfect scenario for performing forward genetic screens and single-cell sequencing in order to identify, compare and contrast the molecules regulating transdifferentiation in the worm.

What is the function of PHD?

Bearing in mind that the PHD neurons arise during sexual maturation and exclusively in males, we asked ourselves what function these cells might have. First, we tried to identify the sensory stimulus that activates PHDs, by measuring neuronal activity in immobilised animals using a genetically-encoded calcium indicator. Surprisingly, we noticed spontaneous activity of the PHD neurons in the absence of any stimuli. We then realised that, despite immobilisation, some muscles near the PHD neurons were still twitching due to the defecation cycle and spasms of the spicules (the equivalent of the penis in nematodes). Perhaps the PHDs are mechanosensors and they are being activated by the internal deformations caused by those muscle contractions?

To test this idea, we measured PHD activity in worms in which muscle contractions were abolished using an inducible chemogenetic tool (an histamine-gated chloride channel transgene). Indeed, muscle silencing eliminated PHD activity, supporting our hypothesis. Furthermore, it appears that the PHDs sense muscle contractions directly and not through other neurons within the circuit because disrupting their chemical synaptic input (through mutations in genes required for synaptic transmission) did not eliminate activity. PHD neurons are thus likely to have a proprioceptive function – but which process could they be involved in?

Through reconstruction of serial electron micrographs we identified all the synaptic partners of PHD and we realised that they are not only unique to males but also highly connected to other male-specific neuronal circuits. This was highly suggestive of a role in mating. C. elegans mating behaviour is stereotyped and consists of a sequence of behavioural steps: response to a potential mate, scanning the mate’s body, turning, location of vulva, spicule insertion and ejaculation (reviewed in Barr et al., 2018). We compared the performance of each mating step in control animals and animals in which we removed PHD by laser ablation, and noticed a defect in the scanning behaviour of PHD-ablated males. In a normal mating sequence, the male scans the body of its mate moving backwards (tail-first) in a continuous manner. However, males lacking PHDs could not perform this movement smoothly, and they tended to switch directionality and pause during scanning.

As stated above, C. elegans mating behaviour is sequential and if completion of a step fails, the animal will repeat the previous step and try again. Interestingly, we noticed that wild-type males that were repeatedly unsuccessful at spicule insertion did not always return to scanning backwards in order to relocate the vulva. Instead, they performed a readjustment movement by going forwards (head-first), away from the vulva, and then returning to the vulva backwards to try to insert the spicules again (see Video 1). This was remarkable as all previously described male mating movements involve only backwards locomotion. We called this novel readjustment the ‘Molina manoeuvre’ (MM) after Laura Molina, who first observed it and to acknowledge her good eye for males!

Wildtype (Video 1) and PHD-ablated (Video 2) male worms performing Molina manoeuvres during mating with paralysed hermaphrodites (mutant for unc-51), as shown in our recent eLife paper (Molina-García et al., 2020).

When we looked specifically at MM performance, we found that animals without PHD neurons displayed discontinuous manoeuvres, often stopping at the transition from forward to backward locomotion to return to the vulva (see Video 2). Together, our data show that without intact PHD neurons, backward movement along the mating partner becomes somewhat erratic.

Are PHDs activated during backward locomotion? Consistent with the behavioural defects, we observed higher activity (i.e. rise in Ca²⁺ levels) in PHD during backward locomotion than during forward locomotion while scanning. The same happened during the MM, during which PHD activity also peaked just after the switch to backward locomotion. However, the highest level of PHD activation occurred during intromission. This step involves full insertion of the spicules into the mate’s vulva while sustaining backward locomotion and precedes sperm transfer. Interestingly, PHD-ablated males produced fewer cross-progeny than intact males after a single mating encounter. This suggests that intact PHDs may increase the efficiency of sperm transfer by controlling the male’s posture during intromission, which would be consistent with a putative proprioceptive role for these neurons.

Scientific significance

In summary, the previously undescribed male-specific PHD neurons are born through transdifferentiation during sexual maturation to control backward locomotion during mating. This is of high ethological relevance as the failure to complete mating implies missing a chance to reproduce and therefore failing to pass on one’s genes. Neurogenesis through transdifferentiation could facilitate strict temporal and spatial control of such finely tuned behaviours, repurposing a pre-existing cell for a newly required function, or allowing the generation of a cell only when specific structures are already in place (i.e. to ensure correct neuronal wiring).

Importantly, this is the second example of neurons arising from differentiated glial cells in C. elegans, following our previous work on the AMso. This process resembles neurogenesis in the vertebrate postnatal brain, where radial glial cells produce post-mitotic neurons (reviewed in Kriegstein and Alvarez-Buylla, 2009), raising the intriguing possibility that shared mechanisms may govern glia-to-neuron transdifferentiation in the worm and vertebrate adult neurogenesis. Identifying the mechanisms that regulate these naturally occurring switches in cell identities will improve our understanding of cellular plasticity and will help develop more efficient protocols for reprogramming cells in vitro, which is widely used for cell replacement therapies. Furthermore, a deeper understanding of how locomotion is guided by self-sensory feedback could be applied to improve the execution of behavioural sequences in artificial intelligence and robotics.

References

Barr, MM., García, LR., Portman, DS. (2018) Sexual Dimorphism and Sex Differences in Caenorhabditis elegans Neuronal Development and Behavior. Genetics 208(3): 909-935; https://doi.org/10.1534/genetics.117.300294

Conradt, B., Wu, YC., Xue, D. (2016). Programmed Cell Death During Caenorhabditis elegans Development. Genetics 203(4): 1533-1562; https://doi.org/10.1534/genetics.115.186247

Eguchi, Goro; Kodama, R., 1993. Transdifferentiation. Curr. Opin. Cell Biol. 2, 1023–1028. https://doi.org/10.1016/0955-0674(93)90087-7

Jarriault, S., Schwab, Y., Greenwald, I. (2008). A Caenorhabditis elegans model for epithelial-neuronal transdifferentiation. Proceedings of the National Academy of Sciences of the United States of America, 105(10), 3790–3795. https://doi.org/10.1073/pnas.0712159105

Kagias, K., Ahier, A., Fischer, N., Jarriault, S. (2012). Members of the NODE (Nanog and Oct4-associated deacetylase) complex and SOX-2 promote the initiation of a natural cellular reprogramming event in vivo. Proceedings of the National Academy of Sciences of the United States of America, 109(17), 6596–6601. https://doi.org/10.1073/pnas.1117031109

Kriegstein, A., Alvarez-Buylla, A. (2009). The glial nature of embryonic and adult neural stem cells. Annual review of neuroscience, 32, 149–184. https://doi.org/10.1146/annurev.neuro.051508.135600

Lambert, J., Lloret-Fernández, C., Laplane, L., Poole, R. J. & Jarriault, S. (in press). On the origins and conceptual frameworks of natural plasticity – lessons from single cell models in C. elegans. Current Trends in Developmental Biology.

Molina-García, L., Lloret-Fernández, C., Cook, SJ., Kim, B., Bonnington, RC., Sammut, M., O’Shea, JM., Gilbert, SP., Elliott, DJ., Hall, DH., Emmons, SW., Barrios, A. & Poole, RJ. (2020). Direct glia-to-neuron transdifferentiation gives rise to a pair of male-specific neurons that ensure nimble male mating. Elife 9:e48361. https://doi:10.7554/eLife.48361

Sammut, M., Cook, S. J., Nguyen, K., Felton, T., Hall, D. H., Emmons, S. W., Poole, R. J., & Barrios, A. (2015). Glia-derived neurons are required for sex-specific learning in C. elegans. Nature, 526(7573), 385–390. https://doi.org/10.1038/nature15700

Selman, K., Kafatos, F.C., 1974. Transdifferentiation in the labial gland of silk moths: is DNA required for cellular metamorphosis? Cell Differ. 3, 81–94. https://doi.org/10.1016/0045-6039(74)90030-X

Sulston, J. E. & Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Developmental Biology, 56(1):110-156. https://doi.org/10.1016/0012-1606(77)90158-0

Sulston, J. E., Albertson, D. G., & Thomson, J. N. (1980). The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Developmental biology, 78(2), 542–576. https://doi.org/10.1016/0012-1606(80)90352-8

Sulston, J. E., Schierenberg, E., White, J. G., & Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Developmental Biology, 100(1), 64–119. https://doi.org/10.1016/0012-1606(83)90201-4

Zuryn, S., Ahier, A., Portoso, M., White, E. R., Morin, M. C., Margueron, R., & Jarriault, S. (2014). Transdifferentiation. Sequential histone-modifying activities determine the robustness of transdifferentiation. Science (New York, N.Y.), 345(6198), 826–829. https://doi.org/10.1126/science.1255885

(5 votes)

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In the latest Genetics Unzipped podcast we chat with author Helen Pilcher about how humans have shaped the evolutionary trajectory of species on earth, find out how genetics is used in conservation Alex Ball from the RZSS WildGenes project , and meet Bill Ritchie, the embryologist who cloned Dolly The Sheep at the Roslin Institute in the 1990s.

Genetics Unzipped is the podcast from The Genetics Society. Full transcript, links and references available online at GeneticsUnzipped.com.

Subscribe from Apple podcasts, Spotify, or wherever you get your podcasts.

Head over to GeneticsUnzipped.com to catch up on our extensive back catalogue.

If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip

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I joined the Node and Development out of a postdoc in 2016 and now after five enjoyable years I’m moving on. I’ll write something a bit more reflective closer to my leaving date in June but here I thought I’d let you know what the job entails, since we’re searching for my replacement.

If you’re interested in science communication and helping the developmental biology community in various ways, this job might be ideal for you. And if you know anyone who is thinking of hanging up their pipettes but still wants to stay in touch with science and scientists, please send the job ad their way.

Here’s a collection of the kind of things you’d get up to:

• Run the Node
  • Commission content – research stories, interviews, meeting reports etc.
  • Edit / give feedback on drafts for authors
  • Write your own content
  • Run competitions and series of posts
  • Maintain the site (including the now 900+ strong Node Network)
  • Develop the site’s future – we’re now 10 years old and well-established, but there are always opportunities for innovation
• Social media
  • For the Node: Twitter (where all the fun stuff happens) and Facebook
  • For Development: Twitter, Facebook, more recently Instagram, and on our YouTube channel you’ll have opportunities for video editing
  • With these accounts you reach thousands of people, specialists and non-specialists, to spread the word about developmental biology
• For Development
  • Write Research Highlights (usually one a week)
  • Conduct ‘The people behind the papers’ interview series (usually one per issue), and longer standalone interviews (could be a Nobel laureate or an up-and-coming star)
  • Write and manage press releases for topical papers
  • Development presents… – help out with our webinar series
  • Attend Editor meetings and strategy sessions – insight into the publishing world
• Conferences
  • In a normal year, go to perhaps six conferences, some abroad, usually focusing on society events. Meet people, hear new science, promote our work, write meeting reports, interview prize winners
  • Help out at Development’s biennial human development meetings and Company of Biologists Workshops
• Working for The Company of Biologists
  • Provide cover for and collaborate with the two other community sites, preLights and FocalPlane
  • Promote our work as a not-for-profit publisher
  • Work with 50 other great colleagues near Cambridge (/home office)

I’d be happy to answer any informal inquiries – just email thenode@biologists.com

Check out the full job ad here:

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The competition is closed and the winner has been announced!

Last month we heard from the students in the 2020 class of the MBL Practical course in Developmental Biology, which is held biennially in Quintay, Chile. It was clearly a transformative experience for the participants, and we hope that the 2022 version can occur as planned (check out the homepage for more information).

During their practical classes the students produced some beautiful images and so, as we did in 2018, we’re going to use them in a competition to find a Development cover. Participating is easy – just vote for your favourite image from the following selection (click to get full size images, voting below the pictures). The winner will then be immortalised in print and on screen in a future issue of the journal – testament to the bright future of developmental biology in Latin America.

One vote per person – voting closes Monday 29 March!

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Last week, we held the sixth webinar in our series, which was chaired by Development editor Thomas Lecuit (IBDM).

Below you’ll find recordings of the talks and live Q&A sessions.

Hongzhe Peng (from Bo Dong’s lab at Ocean University of China)
‘Ciona embryonic tail bending is driven by asymmetrical notochord contractility and coordinated by epithelial proliferation

Read the full article at development here:
https://dev.biologists.org/content/147/24/dev185868

Camille Curantz (from Marie Manceau’s lab at Collège de France)
‘Cell shape anisotropy and motility constrain self-organised feather pattern fidelity in birds’

You can read Camille’s preprint here:
https://www.biorxiv.org/content/10.1101/2021.01.22.427778v1

Chen Luxenburg (Tel Aviv University)
‘Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity’

You can find Chen’s Open Access paper at Development here:
https://dev.biologists.org/content/147/23/dev193425

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The annual Young Embryologist Network Conference is happening this year on May 4^th! Put the date in your calendars now! 

YEN is thrilled to announce that Nobel Laureate Christiane Nüsslein-Volhard from the Max Planck Institute of Developmental Biology in Tübingen, Germany will present this year’s Sammy Lee Memorial Lecture. We are also honoured to host two special speakers: Matthias Lutolf from EPFL in Lausanne, Switzerland and Alexander Aulehla from EMBL in Heidelberg, Germany. Finally, we are delighted that Marianne Bronner from Caltech, USA, Ana Pombo from MDC Berlin, Germany and Patrick P.L. Tam from CMRI, Australia will share invaluable insights from their life as a scientist in our “Scientific Perspectives” session.

YEN 2021 will be held entirely online, which will allow for unprecedented international participation. Spread the word, and let us take the Young Embryologist Network worldwide! 

Talks will be held throughout the day, beginning at 9.15am (BST). We will be using the online conference platform Remo, enabling us to recapitulate that in-person networking feeling of conferences that we are all missing. Interactive poster sessions will also be held via Remo, with further discussions taking place in dedicated Slack channels both during and after the event.

Register here to submit an abstract or to attend as a delegate (and hear what your fellow young embryologists are up to). Registration is free and the deadline for abstract submission is April 4^th, so be sure to sign up as soon as possible!

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In 2020 the Node turned 10 and, along with a virtual networking birthday party and a Development editorial, we ran a community survey for advice on what to improve and where to go next. We gathered some fantastic ideas for content that we’re going to develop soon but also heard many suggestions for things we’d done before. This made us think about how we could better promote historical Node content (going all the way back to 2010), pieces that are currently quite hard to find in the archive. We also felt that the homepage needed a refresh – it hadn’t been updated since 2015 – and identified a few more tweaks we’d like to implement, both from reader and author perspectives. These discussions happened to coincide with a necessary upgrade in our WordPress system to their Gutenberg editor, which gives a lot more freedom in terms of page design, and also changes the user experience for writing posts. And so, towards the end of 2020 we started working on giving the Node a new look: not a full on revolution, more an upgrade, which we’re happy to launch today. Here are the main features we’ve changed: 

Homepage tweaks

One of our first decisions was to refresh our header images. This being the Node, we tapped our greatest resource: the developmental biology community. A competition in February led to over fifty entries which we winnowed down to the final five (you can skip through by refreshing your page). Congratulations to competition winners Markus Schliffka, Rory Cooper, Evan Bardot, Gonzalo Aparicio and Daniel Castranova – you can find out more about their images in our ‘About us’ page.

We’ve also removed the static ‘Featured posts’ bar and replaced it with a moving carousel above the blog posts – we hope this better showcases the diverse range of our recent content. The new, more flexible, homepage will also allow us to better highlight other content and information – you can expect to see the homepage evolving further over the coming months.

Something we discovered in the survey was that many of you still don’t know just how easy it is to contribute to the Node – all you need to do is register for an account, and you’re then free to post without the need for our ‘official’ approval (though we are of course always happy to provide feedback to people interested in writing for us). Hopefully the new ‘welcome’ message at the top of the page reemphasising the fact that the Node is your site will encourage even more community engagement. 

Topic pages

To help readers navigate our extensive archive of content, we are now collating blog posts on particular themes into one place – its own topic page. Here are some examples:

• A day in the life… Our series of posts detailing what it’s like to work with a particular model organism
• Behind the paper stories. We regularly commission scientists to tell us the stories behind their new publications.
• Forgotten classics. A series on unjustly neglected papers in the literature.
• How to. Helpful posts on a wide range of topics
• SciArt Profiles. Profiles of scientists who do art, or artists who dabble with science.

You’ll find links to the topics pages in the ‘Archive’ tab at the top of the page, and we’ll continue adding more pages as they become relevant – if you have an idea for a new collection, just get in touch. 

Jobs page

The jobs page now only shows active job adverts – once a job advert expires, it goes into the archive (all job adverts posted before today can be found in the archive – if you want to see your own advert back on the jobs homepage, simply post it again). We’ll soon make job adverts filterable by categories like location and position – watch this space. 

The author experience

If you’re a returning author, you’ll notice a few changes in how posts are created, as we’ve upgraded to a newer version of WordPress that uses their Gutenberg Editor. This uses a ‘block’ system – blocks can be headers, paragraphs, images, YouTube links, and more, and can be used in any order. We hope that creating a post will still be relatively self-explanatory, but we have a walk through video and a written ‘how to’ over on our FAQ page. If you have any issues, just email us.

Posting a job is now different to posting a blog post – for example, you need to include an expiry date. Just check out our FAQs for more information.

We hope you enjoy our new look and, as ever, would love to hear your ideas for where we can take your community site.

(2 votes)

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Jacqueline Copeland and Marcos Simoes-Costa

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

The neural crest has long been referred to as “the fourth germ layer” for its remarkable ability to give rise to a number of cell types in the vertebrate embryo including neurons, glia, bone, and cartilage. Specified at the border of the neural plate, the neural crest is intimately linked to this adjacent cell population, which will give rise to the central nervous system. Directing the partitioning of the ectoderm into these two territories are inductive interactions driven by signaling systems including Wnt, BMP, and FGF. These signals form overlapping spatial gradients across different ectodermal territories, driving the regulatory programs required to induce the neural crest and the neural plate. Many developmental biologists have studied the timing and levels of signaling required for proper neural crest formation, but fundamental questions remain: How do distinct combinations of signaling molecules give rise to different cell types?  Furthermore, how do fields of cells interpret and respond to these signals?

A conceptual framework for understanding these questions was established by Lewis Wolpert in 1969 with his seminal paper “Positional Information and the Spatial Pattern of Cellular Differentiation”¹. Wolpert’s “French Flag Model” proposed that spatial gradients of signaling molecules known as morphogens drive subdivision of cellular fields based on concentration. Although Wolpert’s model offered foundational insights, today we know these processes are much more complex. For example, there must exist mechanisms by which cells can produce binary responses to distinct ranges of signaling levels. One such mechanism relies on the transcriptional control of signaling system activity through activation of agonists/antagonists. However, transcription can be a leaky process, and may not confer the optimal levels of signaling activity needed for cell fate decisions. Therefore, other modes of regulation may offer robustness in these developmental programs.

When I began my first year of graduate school at Cornell University I had never even heard of Lewis Wolpert or the neural crest, for that matter. I was first made aware of this cell population after hearing a rotation talk from my now mentor Marcos Simoes-Costa. Although I had no background in development, I was amazed by the beautiful movies of migrating neural crest cells and the chick model system’s tractability for investigating gene regulatory programs. After Marcos’s talk, I was very excited to rotate in his lab and eager to discuss rotation projects. Prior to my rotation, Marcos had published his first article as a new PI, investigating the role of the Lin28/Let-7 axis in neural crest multipotency². From this work he became interested to look further at the post-transcriptional regulation of this cell type. As an undergraduate, I studied genetic interactions associated with pre-mRNA splicing in Saccharomyces Cerevisiae. Through this work, I developed an immense interest in post-transcriptional gene regulation, which I consider the underdog of the central dogma of molecular biology. As luck would have it, our interests aligned, and we embarked on a new project which culminated in a recent publication in PNAS³.

It has been known for roughly a decade that Dicer, a key enzyme in the miRNA biogenesis pathway, is critical for neural crest development and differentiation. Conditional Dicer knockout mice exhibit severe craniofacial defects due to lack of neural crest-derived structures, highlighting the importance of Dicer, and therefore mature miRNA species, in the formation of this cell type^4-6. However, few studies had investigated the mechanistic roles that miRNAs and their gene targets may play in this cell type. This led us to our major goal of identifying and characterizing miRNA function during early neural crest formation.

It would be quite difficult to assay the phenotypic outcome of inhibiting every individual miRNA expressed in the chicken genome. So, to get an idea of the role miRNAs might be playing during neural crest specification, we first turned to Dicer, examining its expression in relation to that of the neural crest specification marker TFAP2B. What came as quite a surprise was that Dicer, which is thought to be a ubiquitously expressed protein, was enriched in neural crest cells. Furthermore, through interrogating the Dicer locus, we uncovered a neural crest-specific enhancer driving Dicer upregulation in this cell population. This was an exciting finding, as it demonstrated that there may be increased processing and turnover of miRNAs within neural crest cells. This idea made a lot of sense to us, as neural crest cells undergo rapid genomic and morphological changes in their relatively short lifetime, which may be facilitated by different groups of miRNAs.

The chick embryo offers a beautiful system in which we can perform bilateral electroporations to observe control and knockdown phenotypes within the same organism. Using this method, we injected gastrulating chick embryos with a Dicer protein-inhibiting morpholino. Since the phenotypic effects of morpholino based knockdown can be quite broad, we performed a Nanostring analysis in order to more globally assess the effects of Dicer knockdown during neural crest specification. To our surprise, knockdown of Dicer not only resulted in a loss of neural crest markers but was also accompanied by increased expression of several neural plate-specific factors. Observing knockdown embryos, we indeed saw an expansion of the neural plate at the expense of neighboring neural crest cells.

These observations led us to hypothesize that miRNAs (synthesized via Dicer) may play an important role in the cell fate decision between the neural crest and neural plate. To test this hypothesis, we needed to isolate these cell populations in order to identify the abundant and unique miRNAs present in each. For this we turned to a commonly used system in our lab, in which we can inject chick embryos with cell type-specific enhancer reporters and then perform FACS to isolate pure populations of cells. Since we were interested in the neural plate and the neural crest, we isolated cells from each of these populations and performed small RNA-sequencing. Admittedly, getting small RNA-sequencing to work was a feat in itself. Most of my rotation and my first summer in lab were spent optimizing the protocol for small numbers of sorted chick neural crest cells. This involved dissecting hundreds of chick embryos but was accompanied by lots of time with Marcos at the electroporation station discussing what miRNAs could be doing in crest.

Once we finally optimized a protocol for small RNA-sequencing, we were off to the races, identifying miRNAs and determining their modes of action. Through several analyses including miRNA target prediction, we ultimately identified a group of neural crest miRNAs that target components of the FGF signaling pathway. This was an “aha!” moment for us as we knew levels of FGF must be quite precise for neural crest induction: too much and the cells are fated towards the neural plate, but not enough, and neural crest will not form at all. We hypothesized this group of miRNAs was required in crest to keep levels of FGF low, inhibiting a neural plate fate, and confirmed this through several functional approaches. One of my favorite experiments we performed, that truly wrapped up the paper, was the rescue of the Dicer knockdown phenotype by adding back our FGF-targeting neural crest miRNAs. Getting back to post-transcriptional regulation being the underdog of the central dogma, this was amazing to me. miRNAs typically have very modest repressive effects (less than 1.5 fold) on gene expression. Given this, I was not sure if by putting back our FGF-targeting miRNAs we would rescue the Dicer knockdown phenotype. But to my surprise, by working in a concerted fashion to target different FGF pathway components and attenuate levels of FGF, these three miRNAs were quite capable of rescuing the neuralization switch observed upon Dicer knockdown.

I am quite proud of this paper, not only because it is my first publication in graduate school, but because it tells a story of how small RNAs can work together to drastically influence the cell fate decisions of neighboring cell populations in the ectoderm. I also appreciate how it unifies two opposite ends of the of neural crest regulatory spectra: signaling system inputs that jumpstart the neural crest GRN and post-transcriptional regulation of these signaling systems to ensure the proper thresholds of activity are met. Moving forward, I would like to explore regulation of signaling systems via miRNAs at a broader level, considering their contribution to patterning events related to other tissue types, as well as the regulation of the miRNAs that reside in those tissues. I also hope that this work can inform upon other developmental scenarios where tuning of signaling systems via miRNAs is critical for cell fate commitment.

1. Wolpert, L., Positional information and the spatial pattern of cellular differentiation. J Theor Biol, 1969. 25(1): p. 1-47.
2. Bhattacharya, D., et al., Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis. Elife, 2018. 7.
3. Copeland, J. and M. Simoes-Costa, Post-transcriptional tuning of FGF signaling mediates neural crest induction. Proc Natl Acad Sci U S A, 2020. 117(52): p. 33305-33316.
4. Zehir, A., et al., Dicer is required for survival of differentiating neural crest cells. Dev Biol, 2010. 340(2): p. 459-67.
5. Huang, Z.P., et al., Loss of microRNAs in neural crest leads to cardiovascular syndromes resembling human congenital heart defects. Arterioscler Thromb Vasc Biol, 2010. 30(12): p. 2575-86.
6. Huang, T., et al., Wnt1-cre-mediated conditional loss of Dicer results in malformation of the midbrain and cerebellum and failure of neural crest and dopaminergic differentiation in mice. J Mol Cell Biol, 2010. 2(3): p. 152-63.

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Two Postdoctoral Research Associate positions (PDRA) are available in the Spagnoli lab. in the Centre for Stem Cell & Regenerative Medicine at King’s College London. Our team uses interdisciplinary approaches to study pancreatic development and stem cells. The candidates will join a Wellcome Trust-funded research programme aiming at studying the pancreatic tissue microenvironment in all its complexity using cutting-edge models.

Single-cell sequencing has unveiled a high degree of cellular heterogeneity within the pancreatic microenvironment and has opened the way for a systematic study of intercellular interactions. We seek to spatially reconstruct the organisation of functional niches in the pancreas and study how they induce distinct pancreatic differentiation programmes using mouse models and human pluripotent stem cells. This will set the stage for manipulating combinatorial 3D organ niches towards engineering pancreatic cells for regenerative medicine.

The research programme will require expertise in transcriptomics, high-resolution imaging, stem cell culture, human tissue, mouse genetics, computational analytical methods. Applicants should have a recent Ph.D. degree or have submitted his/her Ph.D. thesis. We wish to appoint one PDRA with background in development and stem cell biology and one PDRA with computational background interested in single-cell omics, spatial transcriptomics and image analysis.

The Spagnoli lab. is a young dynamic team, member of the outstanding Centre for Stem Cell & Regenerative Medicine at King’s College London. This is a world-class research environment with all facilities essential for this ambitious research programme.

Interested candidates should get in touch with Francesca Spagnoli (francesca.spagnoli@kcl.ac.uk) for further information. Please send your CV, research interests, and names and contact information of three references.

Positions will be available starting in October 2021.

More information on the group, publications and research topics in the group can be found in the laboratory website: https://www.spagnolilab.org/

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In the latest episode of Genetics Unzipped we’re delving into the science behind so-called ‘genetic superheroes’, and explaining why you might have hidden powers within your genes. Despite the name, these superheroes don’t have the ability to shoot webs from their fingers or save the universe, but something with a lot more real world relevance to human health.

Instead, these people have a much more down to earth ability: carrying genetic alterations that should make them seriously ill, yet they are apparently healthy.

We take a closer look at the search for genetic superheroes, the science behind their secret powers, and what their existence means for our understanding of genetics.

Genetics Unzipped is the podcast from The Genetics Society. Full transcript, links and references available online at GeneticsUnzipped.com.

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