https://www.rawlins.group.gurdon.cam.ac.uk/index.html

Where is your lab?:

At the Gurdon Institute, which is part of the University of Cambridge. We are located in the city centre of Cambridge, UK.

Research summary:

Emma Rawlins: We ask, how are our lungs built and maintained? We have a particular focus on stem cell biology of the developing and adult lungs and asking how cell-cell interactions regulate growth, patterning and repair. We have also recently started collaborating with human geneticists to explore the genetic contribution to chronic lung diseases in humans, and whether these have their origins in infancy, or the adult, or both.

Lab role call:

• Claire: 1^st year PhD student investigating airway homeostasis in humans.
• Emma: PI – does all the paperwork.
• Hannah: Clinical lecturer – has completed an MD and a PhD and is now balancing postdoctoral work with clinical duties, working on lineage decisions in the developing airway.
• John: Postdoc interested in morphogenesis of the developing human lung.
• Kyungtae: Postdoc interested in cell-cell signalling during lung alveolar development.
• Odara: 1^st year PhD student jointly supervised by Vito Menella who is interested in analysis of volumetric electron microscopy and using human organoids to study toxicology.
• Quitz: Lab manager who keeps us all organised and enjoys thinking about computational biology methods.
• Tessa: PhD student working on epithelial cell fate decisions in the developing mouse lung, and optimal imaging techniques for visualizing lung development.
• Vanesa: PhD student (just submitted her thesis!) jointly supervised by Kristian Franze and works on the role of stiffness in cell fate decision making during human lung development.
• Yihong: Visiting final year PhD student from Zhejiang University who is interested in tuft cells.
• Ziqi: PhD student working on the role of hypoxia in cell fate decision making during normal human lung development.

Favourite technique, and why?

Emma: Clonal lineage-tracing combined with genetic manipulation and microscopy, and now often coupled to single cell transcriptomics. In my opinion, this is the most elegant method to study normal and aberrant cell fate decisions due to the presence of mutant and control cells in the same tissue. One current lab challenge is how to apply this technique to human samples.

Apart from your own research, what are you most excited about in developmental and stem cell biology

Emma: The advances being made in understanding of regeneration from organisms that regenerate on an impressive scale like Axolotls.

How do you approach managing your group and all the different tasks required in your job?

Emma: Most of the time it feels like neither managing my group, or the other admin and teaching tasks, are being done properly. It’s important to recognise that good-enough is sufficient for many tasks we must do, particularly the administrative ones. I try and prioritise my lab as it’s the science that excites me. I block time out in my diary to ensure that I have a detailed science conversation with every lab member every 2 weeks, and other tasks are fitted in around this schedule. I also have annual career planning meetings with everyone in the lab (the University’s appraisal scheme) and try and make sure that I keep up to date with any changing aspirations in addition. That way I/we can be looking out for the best career development opportunities for each person.

What is the best thing about where you work? 

Claire: Working in Cambridge offers so much rich scientific history alongside cutting-edge techniques. It’s incredible to train in a such a collaborative space, and the Rawlins lab has been such a supportive and kind group with lots of advice and guidance as I begin my PhD.

Emma: Cambridge has a really rich scientific environment, it’s very easy to network to find collaborators in pretty much any research area that you are interested in.

Kyungtae: The best place in the world to do science – especially developmental biology. Also, it’s easy to get connected between fields to fields and person to person, leading to fantastic interdisciplinary studies.   

Tessa: The lab environment is very supportive and collaborative, it’s an exciting place to do science and work with like-minded colleagues who are happy to help you troubleshoot or discuss new ideas.

What’s there to do outside of the lab?

Claire: As a PhD student, I’m part of a college here at Cambridge, and you have the opportunity to meet peers across different subject areas over weekly dinners and different social events. My favourite thing to do is explore Cambridge by going on runs around town and trying all the different restaurants!

Kyungtae: There are great walks and places around the Cambridge with beautiful nature which I can feel the universe – that is why it is the University of Cambridge, in Cambridge. Work and walk and feel the Cambridge. 

Tessa: Cambridge has a lot of green space, and (weather permitting!) you can spend lots of time outdoors on the meadows or walking through the backs of the Colleges and admiring the architecture.

(2 votes)

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We are delighted to announce a new series on the Node called ‘Lab meetings’! In these posts, we will be highlighting developmental and stem cell biology labs across the globe. We aim to build up a directory of labs, which will not only showcase the exciting research and researchers in the community, but will also provide a useful resource for anyone looking for their next job. We ask the group leaders about their research and mentoring style, while the group members share what they like about the lab and what they like to do away from work.

Our first ‘Lab meeting’ is with the Rawlins lab, at the Gurdon Institute in Cambridge, which you can read here. You’ll be able to see all our full directory on our ‘Lab meetings‘ page.

Drop us an email if you would like to nominate a lab (including your own), especially if you have (or will soon have) open positions. We look forward to working with you all to build up a useful resource!

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It’s not ridiculous to suggest that the Y chromosome might eventually become so mutation-addled that it disappears entirely. In fact, it’s already happened…in the Amami spiny rat.

Dr Sally Le Page

In the latest episode of the Genetics Unzipped podcast, we’re saying bye-bye to the boys, and exploring whether new gene technologies and climate change will make males extinct.

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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Please join us for the International Symposium on Women in Tunicata Biology. The Tunicata are invertebrate chordates, several of which are model organisms in developmental biology (e.g. Ciona intestinalis, Botryllus schlosseri). The symposium will be Tuesday, March 28 and Wednesday, March 29, starting at 6 am Pacific Time/9 am Eastern Time. Speakers will be honoring the work of retired researchers and presenting their own research. All researchers are welcome to attend. Please email Marie Nydam (mnydam@soka.edu) or Anna Di Gregorio (adg13@nyu.edu) for a schedule and Zoom link.

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by Naomi Clements-Brod and Hélène Doerflinger

What is Public Engagement with Research?

Public Engagement with Research (PER) encompasses ways of engaging the public with the design, conduct and dissemination of research. Engagement is, by definition, a two-way process to generate mutual benefit for society and the research community by enhancing research quality and socio-economic impact (See the National Co-ordinating Centre for Public Engagement webpage for more information).

Engaging with the public can take many forms. The format is chosen according to the audience, the aim, the nature of the research and the capacity of the PER team. Examples of public engagement activities are:

• citizen science experiments
• panel discussions
• interactive exhibitions 
• festival participation
• school collaborations 
• co-creation with artists 
• contributions to mainstream and social media

There is no hierarchy of engagement approaches. If meaningful and two-way, they are all valid in their own format, and very often, an event or an activity will contain a blend of these approaches and purposes. 

The mutual benefit for society and the research community is essential to PER. Benefits might include learning, acquiring new skills, gaining new insights or ideas, developing better research, generating a new network, raising aspirations, or being inspired. 

The ways scientists interact with the public across the world differ. Some countries continue with the ‘outreach’ approach, which is mainly a one-way communication to generate attention. In contrast, other countries develop citizen science programmes with multiple educational, social, and economic impacts.

So, we’ve established that public engagement in the UK is more than just educating the public about science – crucially, it is two-way, and involves finding out what the public think about our science too. But how did public engagement come to have this emphasis on two-way communication in the UK?

The story of genetically modified food 

A big part (but not the only part) of this story started in the late 1990’s, when there was widespread scepticism and anxiety about genetically modified (GM) food. (Rowe et al., 2005). This public concern around GM food was not just about the science, either: press coverage at the time reveals it was also a political, environment and consumer issue (Durant & Lindsey, 2000).

In 1998, to ease public scepticism, the UK government decided that GM crops wouldn’t be grown commercially in the UK until a set of experiments on farms had been completed. However, this didn’t dampen public opposition: local communities were angry about not being consulted about these farm experiments in the first place (Mayer, 2003). Then, in 1999, major UK food producers and retailers responded to consumer pressure and removed GM ingredients from products on shelves (Mayer, 2003).

In the midst of the GM food controversy, Parliament also issued a report encouraging scientists to engage in dialogue with the public. They recommended openness and transparency in order to help regain public trust, placed greater emphasis on public attitudes, opinions and values and recognised that without public buy-in, science would struggle to move forward. (Science and Technology Select Committee, 2000).

In 2003, the government ran a series of public debates, in parallel with scientific and economic reviews of the issue of GM food. However, despite asking the public what they wanted, there was still widespread polarisation, opposition and scepticism (Mayer, 2003). And today, more than 20 years later, the legacy of anti-GM sentiment in the UK still lives on.

Lessons Learnt

So why didn’t this dialogue with the public work? One of the big reasons scholars give is that the public were involved much too late and it wasn’t clear how their input would be used in the decision-making process. As a result, engagement professionals today are keenly aware today that public engagement needs to be more than just window dressing: when science will impact people’s lives, scientists can’t just use ‘dialogue’ to try to legitimise a decision that has already been made. You have to ask people what their concerns are early enough so that you can respond to what they tell you in a meaningful way, ensuring that your research. and its potential outcomes. are aligned with societal priorities and expectations. (Mayer, 2003; Rowe et al., 2005; Singh, 2008; Marris, 2015; Morrison & de Saille, 2019; de Saille & Martin, 2018; Gjerris, 2008).

Another big take away from the GM story is that increasing public knowledge about biotechnology won’t necessarily mean that people agree with you about how and whether that biotechnology should be used. Disagreements can be fuelled by different interpretations of the facts, often reflecting individual and cultural values. While science can help us answer ‘can we’ questions, science can’t always directly answer ‘should we’ questions – this is why public input is so vital.

This history informs today’s aim to have two-way conversations with the public, regardless of how fundamental the research is, in order to ensure the ultimate outcomes of science are considered mutually beneficial.

Challenges of engaging with fundamental research

While mutual benefit sounds great, there are certainly some challenges to public engagement with fundamental research, when the research outcomes are often unknown. For example, engaging with the public about implications that are remote and uncertain can encourage ideology-based reactions, leading to polarisation and conflict (Tait, 2009; Tait, 2017).

And while today’s public engagement encourages dialogue over merely informing the public, when speaking with non-scientists about fundamental research, there is often a substantial need to build shared language and understanding of the topic before informed conversations can take place. Meaningful PE with fundamental research can take a lot of time and resources (Clements-Brod et al., 2022). However, it is not impossible!

Case studies 

We’ve put together some examples of how we’re doing public engagement with fundamental research in hope to inspire others to do the same.

References

Clements-Brod, N., Holmes, L., and Rawlins, E. (2022). Exploring the challenges and opportunities of public engagement with fundamental biology. Development. 149 (18) https://doi.org/10.1242/dev.201170 

de Saille, S. and Martin, P. (2018). Monstrous regiment versus Monsters Inc.: Competing imaginaries of science and social order in responsible (research and) innovation. In Science and the Politics of Openness: Here be Monsters (ed. B. Nerlich, S. Harley, S. Raman and A. Smith), pp. 148-166. Manchester: Manchester University Press. https://library.oapen.org/handle/20.500.12657/30733 [accessed 11 August 2021].

Durant, J. and Lindsey, N. (2000). The ‘great GM food debate’ – a survey of media coverage in the first half of 1999. Parliamentary Office of Science and Technology. Report number: 138 https://www.parliament.uk/globalassets/documents/post/report138.pdf 

Gjerris, M (2008). The three teachings of biotechnology. In David, K.H. & Thompson, P.B. (eds.), What Can Nanotechnology Learn From Biotechnology?: Social and Ethical Lessons for Nanoscience From the Debate Over Agrifood Biotechnology and Gmos. Elsevier/Academic Press., pp. 91-105

Marris, C. (2015). The construction of imaginaries of the public as a threat to synthetic biology. Sci. Culture 24, 83-98. https://doi.org/10.1080/09505431.2014.986320

Mayer, S. (2003) GM Nation? Engaging people in real debate? GeneWatch UK.

http://www.genewatch.org/uploads/f03c6d66a9b354535738483c1c3d49e4/GM_Nation_Report.pdf 

Morrison, M. and de Saille, S. (2019). CRISPR in context: towards a socially responsible debate on embryo editing. Palgrave Communications 5, 110. https://doi.org/10.1057/s41599-019-0319-5

Science and Technology Select Committee. (2000). Science and Society. House of Lords, Parliament. Report number: 3. https://publications.parliament.uk/pa/ld199900/ldselect/ldsctech/38/3802.htm 

Rowe, G., Horlick-Jones, T., Walls, J. and Pidgeon, N. (2005). Difficulties in evaluating public engagement initiatives: reflections on an evaluation of the UK GM Nation? Public debate about transgenic crops. Public Underst. Sci. 14, 331-352. https://doi.org/10.1177/0963662505056611

Singh, J. (2008). The UK Nanojury as ‘upstream’ public engagement. Participatory Learn. Action 58, 27-32. https://www.participatorymethods.org/resource/uk-nanojury-upstream-public-engagement[accessed 29 June 2021].

(1 votes)

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She might not see it the next day. At first glance, the slag in the saltwater aquarium is just a rock covered with purple jasmine polyps and green star polyps. Orange tendrils of finger corals wave with a silent current, an undulating calm that almost seems to slow the heart rate. Suddenly the rock flutters. A part of it shifts, seems to detach and hover in the tank, and the shape of a dwarf cuttlefish emerges as bands of light and dark ripple across its skin.

“They’re like little artists creating impressionist paintings on their skin,” said Tessa Montague, a post-doc in the Axel lab at Columbia University. Her latest scientific mission is to understand how the cuttlefish brain controls dynamic camouflage behavior.

From the moment a cuttlefish is born, its skin already contains thousands of pigment-filled sacs called chromatophores that expand and contract under direct control of the brain. And, compared to more established model organisms like the mouse, the dwarf cuttlefish brain is huge. Nearly 75% of its brain is optic lobe, indicating just how important vision is for cuttlefish to sense the world around them.

“When a cuttlefish sees an object or visual scene,” Tessa explained, “the brain must create an abstract representation of the scene using patterns of neural activity.” But through the secret language of neurons the cuttlefish undergoes a remarkable transformation and recreates an approximation of what it has seen on its skin through the fine control of its chromatophores. By studying this transformation, Tessa and colleagues have an opportunity to learn fundamental principles about how brains process the visual world.

Studying a brain as alien as the cuttlefish requires tools to manipulate the genome and record neuronal activity. The way to to do that is to utilize the cuttlefish’s development to create a transgenic animal. But it’s challenging to design experiments without knowing what’s possible or most likely to succeed. At the start of Tessa’s project, no one had sequenced the dwarf cuttlefish genome, and no one had adapted any of the myriad transgenic tools for cephalopods. How hard could it be?

Tessa and colleagues from UCSD and the Chan-Zuckerberg Initiative sequenced the dwarf cuttlefish genome — a 5.5 GB string of DNA, almost twice the size of the human genome. Then, with colleagues at the Marine Biological Laboratory in Woods Hole, Massachusetts, they sequenced RNA transcripts and assembled the cuttlefish transcriptome. Unfortunately, these were not sufficient to identify candidate transgenic drivers. Unfazed, they teamed up with a group at Columbia to perform ATAC-seq — a method that identifies regions of open chromatin in the genome, revealing hidden stretches of non-coding DNA near a gene that determines when and where that gene will be expressed. Scientists can hijack these promoter regions and repurpose them to drive a transgene of their choosing, usually something that glows. Find the right promoter to drive the right gene, and voila! You have a transgenic construct that will be expressed in a specific population of cells. Tessa’s goal is to drive GCaMP, a fluorescent indicator of neural activity, in the cuttlefish brain.

“After many trials and tribulations, we found a really good promoter within the actin gene,” Tessa told scientists at the Society for Integrative and Comparative Biology (SICB) 2023 Conference in January. “We also identified some promising neuronal promoters, which is very exciting.” But getting a promoter to transiently drive expression of a transgene isn’t good enough. To establish a stable transgenic line, the transgene needs to be integrated directly into the genome so it can be passed on to the offspring. And that requires the right tools.

There’s no shortage of possibilities. For decades, scientists have been fiddling with different transgenic tools, from transposases to Zinc-fingers to restriction enzymes to CRISPR-Cas9.

Tessa’s research team has tried several of these tools, with limited success. Most recently, while teaching the embryology course at the Marine Biological Laboratory, a student told her about a special protocol using the meganuclease I-SceI that creates transgenic frogs and fish very efficiently. Tessa’s testing it now in her dwarf cuttlefish.

“Every time I describe the method I’m using, someone says, ‘Have you thought about this?’ and they offer me a different approach,” said Tessa. “The community support has been amazing, but I try to avoid the temptation to constantly jump between methods because each one probably requires months of adaptations for the cuttlefish. And I won’t know for sure if any method has worked until months later.” It can be difficult balancing the decision of when to spend time and resources on giving something a fair chance versus when to move on.

Injecting cuttlefish embryos offers its own host of challenges, not least of which is getting the cuttlefish parents to cooperate in the first place. During mating, the male cuttlefish embraces the female with his eight arms and deposits his spermatophores into her cheeks. The female can then store the sperm for weeks, and she decides when to fertilize and lay her eggs at her own leisure.

“They tend to want to do this on weekends and in the middle of the night, which is not very good for us,” Tessa told scientists at SICB. She came up with a simple trick of putting a box with large openings inside the tank. The cuttlefish generally don’t like the box because it’s too exposed. But place a rock on top and it suddenly becomes a safe haven. “Almost like clockwork, within three hours the cuttlefish swim inside and lay a bunch of eggs,” said Tessa.

The eggs themselves are covered in dozens of layers of inky jelly that need to be removed before injecting. If you just tear away the jelly and try to grow the embryos in a dish, they die. The solution? Dunk the embryos in bleach and then gently rinse them off. After this bleach treatment, the ink jelly sloughs off easily and researchers  can plop the embryo into a dish with antibiotics and squash it between two plastic tubes. This creates a positive pressure that, when combined with a quartz needle beveled at 15º to a very sharp 2.5-micron tip, allow researchers to punch through a tough chorion to reach the teensy cell at the top of a glob of yolk.

Left: The so-called “Holy Temple”: an open box with holes and a rock on top with an inky clutch of cuttlefish eggs. Right: Closeup image of a de-jellied cuttlefish egg. (Photo credits: Tessa Montague)

“No matter how delicately we do this, the vast majority of injected embryos die,” said Tessa, “but after years of practice, enough survive that we can do some experiments.” It takes four months, filled with lots of food and care, before the cuttlefish become sexually mature and Tessa can see if her transgenes make it to the germ line and are passed on to another generation of little cuttlefish.

Even without the worry of death by injection, it takes quite a lot of effort to keep cuttlefish alive. Two full-time animal specialists count grass and mysid shrimp and offer them one by one to the 200 dwarf cuttlefish housed in the Axel lab at Columbia University. Because of their fast metabolism, cuttlefish must be fed three times a day, resulting in lots and lots of poop. And they’re extremely sensitive to changes in water quality, meaning the animal tanks must feature myriad filtration systems and be cleaned every day.

“I’m very fortunate to have a team of six or seven people that work with me,” said Tessa. “Creating something out of nothing is very difficult. But every day that goes by, things get a little easier.”

Tessa wants to share her research to inspire other scientists whose burning scientific questions in new model organisms also require technological developments to answer them. To that end, she created Cuttlebase, a website that chronicles the scientific tools her team has developed, including a 3D brain atlas and a staging series of dwarf cuttlefish embryonic development. Her outreach extends beyond scientists to the general public — her personal website hosts a CuttleCam livestream where anyone in the world can watch these alien creatures from the comfort of their own home.

So, if you give a scientist a cuttlefish, know that she may embark on a journey to understand its secret language of colors rippling across its skin. It may be bumpy, but that journey will have at least a few moments that will take her breath away. And if you know where to look — if you can find the fluttering fins of a cuttlefish hidden in the rock — you might find yourself holding your own breath on your own journey. Maybe that just confirms there’s a scientist in all of us.

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“CNKSR2: Identifying and elucidating the role of a novel downstream effector of RA signalling in chick forebrain morphogenesis“

In the February issue of Development, Niveda and her colleagues report on the identification and function of a previously unreported downstream effector of retinoic acid (RA) signalling in the chick forebrain. Niveda shares some insights into the story behind the paper and the science communication outreach initiated by her department at the Indian Institute of Technology Kanpur.

How did you get started on this project? 

The genesis of this project was based on the findings of a study carried out in Prof. Amitabha Bandyopadhyay’s laboratory at the Department of Biological Sciences and Bioengineering (BSBE) at IIT Kanpur, wherein they performed a genome-wide expression screen of Metabolism-Related Genes (MRGs) in the chicken embryo. The result of this experiment was very interesting as the expression of MRGs peaked at the time of differentiation of the various tissues in the embryo. Intrigued by this result, we started to explore the role of Metabolism-Related Genes during the development of the chick brain. 

We started the study by first examining the spatiotemporal expression profiling of the MRGs that were reported by the initial genome-wide screen. Our laboratory is interested in understanding the process of midline invagination of the forebrain roof plate, which leads to the formation of the cerebral hemispheres from the single forebrain vesicle. Thus, we were very intrigued when we discovered that one MRG known as Connector Enhancer Kinase Suppressor of Ras 2 (CNKSR2) was expressed very precisely in the middle of the invaginating roof plate of the chick forebrain. We then decided to examine its role in the process of midline invagination.

What was already known about the topic?

Before we started our research, the only information available about the process of separation of the cerebral hemispheres was that certain genes linked to holoprosencephaly, a devastating developmental disorder in humans, may be regulating this process. However, nothing was known about the molecular mechanism involved. In a previous study from our lab, we observed that during the process of midline invagination, the roof plate forms a characteristic W-shaped fold¹. Also, it is through this process that the two hemispheres and the medially derived structures such as the hippocampus and choroid plexus are formed. This W-shaped invagination functions as a secondary signalling centre for pathways such as BMP² and WNT³. In the paper published in 2015, we reported that Retinoic Acid signalling is detected in the middle loop of the W-shaped invagination of the roof plate and its inhibition leads to a flattened forebrain roof plate, a phenotype that resembles the human disorder holoprosencephaly¹, where the hemispheres are improperly separated⁴. In this study, we found that the expression of CNKSR2 exactly coincided with the RA signalling domain in the chick forebrain roof plate, prompting us to investigate the role of this gene in this context.

Can you summarize your findings?

We can summarize our findings as follows: we found the expression of an MRG, CNKSR2, in the middle loop of the invaginating dorsal forebrain roof plate and overlapped with the active RA signalling domain. We manipulated RA signalling in the roof plate and found that the expression of the CNKSR2 transcript changed. This led us to infer that CNKSR2 is a downstream effector of RA signalling in this context.

Further, when we knocked down CNKSR2 from the invaginating roof plate using RNA-interference (RNAi) and obtained roof plate invagination defects which phenocopied loss of RA signalling. We found that the invagination defects upon knockdown of CNKSR2 were related to changes in cell proliferation and patterning of this region. Further, misexpression of mouse CNKSR2 was sufficient to ectopically induce the expression of roof plate midline markers in the lateral forebrain. This led us to conclude that CNKSR2 is necessary and sufficient for roof plate patterning. The final experiment that we performed revealed that CNKSR2 modulates Ras/Raf/MEK signalling to lower levels in the roof plate midline for proper patterning and subsequent chick forebrain morphogenesis.

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

We had three eureka moments during our study. The first was when we found that the knockdown of CNKSR2 in the chick embryo forebrain led to invagination defects with a holoprosencephaly-like phenotype. The second, was when the misexpression of the mouse CNKSR2 in the lateral forebrain was sufficient to induce the roof plate marker genes. And the third, and most unexpected result, was when ectopic downregulation of Ras/Raf/MEK in the lateral forebrain was sufficient to induce the expression of the patterning marker, Bmp7. I believe this third result was the final bit of evidence that helped us piece the story together.

Where will this story take the lab?

This story has helped to identify one important molecule-CNKSR2 in the bigger picture of understanding the process of midline invagination in the chick forebrain. Also, this gene may be used as both a proxy for RA signalling in the chick forebrain, as well as a roof plate midline marker. The process of midline invagination is complex with many aspects such as cell adhesion and cytoskeleton rearrangements also likely to be involved. We are currently investigating the possible role of CNKSR2 in each of these functions to understand the forebrain midline invagination process and the resulting separation of the cerebral hemispheres.

Science outreach and its importance

India is a country with enormous linguistic diversity. Our research group comprises members from across the country who are fluent in many languages. As our research is funded by taxpayers, we as a group believe that the general public in the country should be aware of the kind of research taking place in the lab and the resulting publications.

To fulfil this, the Department of Biological Sciences and Bioengineering (BSBE) at the Indian Institute of Technology Kanpur (IITK) decided to start a new initiative wherein the authors of a publication convey their research in their native language and English, all in layman’s terms. The authors of our study are fluent in Bengali, English, Hindi, Nepali and Tamil. Simple animated videos with narration in each of these languages were made and shared across social media for public awareness. We hope to continue this initiative with future publications, and we anticipate that it will be well-received by the viewers. In the end, we aim to inspire young students to actively consider becoming scientists and join us on this exciting journey!

The links for the videos are attached below:

English: https://youtu.be/ag_Of_dAhDQ

Hindi: https://youtu.be/P__Pw0e-qwc

Tamil: https://youtu.be/MFrL6esJUu4

Nepali: https://youtu.be/DYqUZj_2GWc

Bengali: https://youtu.be/ei7dSGCwRVc

References

1) Gupta S, Sen J. Retinoic acid signalling regulates development of the chick’s dorsal forebrain midline and the choroid plexus. Development. 2015 Apr 1;142(7):1293-8. doi: 10.1242/dev.122390. Epub 2015 Mar 10. PMID: 25758461.

2) Furuta Y, Piston DW, Hogan BL. Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development. 1997 Jun;124(11):2203-12. doi: 10.1242/dev.124.11.2203. PMID: 9187146.

3) Lee SM, Tole S, Grove E, McMahon AP. A local Wnt-3a signal is required for the development of the mammalian hippocampus. Development. 2000 Feb;127(3):457-67. doi: 10.1242/dev.127.3.457. PMID: 10631167.

4) Roessler E, Muenke M. The molecular genetics of holoprosencephaly. Am J Med Genet C Semin Med Genet. 2010 Feb 15;154C(1):52-61. doi: 10.1002/ajmg.c.30236. PMID: 20104595; PMCID: PMC2815021.

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Dr. Eun-Deok Kim and Professor Keiko Torii from The University of Texas at Austin, USA, have recently published an article in Nature Plants. The article discusses how heterotypic cis/trans factors drive cell fate commitment during stomatal development. The Node asked us to provide a behind the scenes look at how the story came together.

Nature Plants: Dynamic chromatin accessibly deploys heterotypic cis/trans acting factors driving stomatal cell fate commitment.

How did you get started on this project?

One of the fundamental questions in the development of multicellular organisms is how cells with the same genomic content acquire distinct identities. Recent research has highlighted the importance of chromatin in regulating gene expression ^1-3. Nevertheless, the precise mechanisms by which combinations of cis-regulatory elements (CREs) and trans-acting factors interact to promote cell-fate determination remains unclear. Likewise, we still don’t fully understand the relationship between chromatin architecture and transcription factor binding, leading to cell fate specification during development remains unclear. To address this question, we are using plant stomatal differentiation as a model system.

Can you summarise your findings?

This study is a big step forward in understanding how stomatal cell lineages progress and differentiate, thanks to the use of a multi-omics approach, which includes epigenomics, genetics, and biochemistry. By uncovering the unique combinatorial cis/trans-regulatory codes that drive stomatal cell lineage progression, we’ve made a breakthrough in this field. The early stomatal precursor state is initiated by SPCH and terminated by MUTE, two sister bHLH proteins ^4,5. However, how these proteins switch the cell state from proliferation to differentiation has remained a mystery until now. Using specialized techniques such as stomatal-lineage specific ATAC-seq (INTACT-ATACseq), ChIP-seq, and unbiased transcription factor screens, we identified bHLH (E-box) and BBR/BPC (GAGA-repeat) motifs as unique Co-CREs that signify the early stomatal precursor state ⁶.

Figure 1. adapted from Kim et al. 2022, which is licensed under a Creative Commons Attribution 4.0 International License  

Additionally, we uncovered the exact mechanism by which MUTE drives stomatal cell-fate commitment. MUTE (with its partner bHLH, SCREAM ^4,5,7,8) binds to target DNA (E-box) and can act as a transcriptional activator. At the same time, MUTE directly associates with BPC proteins (binding to the nearby BBR/BPC GAGA repeat DNA motif of the Co-CREs), which recruit Polycomb Repressive Complex2 (PRC2) ^9,10and repress the SPCH locus via deposition of repressive histone marks. Depending on the cell stage, specific heterotypic transcription factor interactions drive the switch from proliferation to the commitment stage. We have shown through genetic, biochemical, biophysical, and in vivo functional evidence that the dysregulation of this mechanism leads to hyper-proliferation or lack of stomatal cell lineages. By integrating chromatin landscape dynamics with molecular mechanistic details, our work has shed light on the novel role of disparate pairs of cis- and trans-acting factors in shaping cell-fate commitment during specialized cell-type differentiation.

Do you think that versatile heterotypic transcription factor interactions are likely to play a key role in regulating differentiation in other systems?

A diverse set of heterotypic TF complexes has been observed during cardiogenesis, hematopoiesis, and myogenesis (the development of muscle tissue)^11-16 For instance, certain TFs like T-box TF TBX5, the homeodomain TF NKX2-5 and the zinc finger TF GATA4 form heterotypic interactions that coordinate cardiac gene expression, differentiation, and morphogenesis. These interactions also limit the potential of these TFs to bind to irrelevant regulatory elements in a given context. Myo-D, a myogenic basic helix-loop-helix (bHLH) protein, and the myocyte enhancer MADS domain TF MEF2 can cooperatively regulate the initiation of myogenesis as another example of a heterotypic complex. I strongly believe that heterotypic interactions not only between transcription factors but also epigenetic regulators play a crucial role in regulating differentiation in many other biological systems.

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

Yes, upon observing the first inducible BPC phenotype and the interaction between MUTE-BPC but not SPCH-BPC. I collaborated with Bridget Fitzgerald, then a research technician, and Hyemin Seo, a graduate student in Keiko Torii lab, and we were all very excited to discover that the inducible overexpression phenotype of BPC suggested the repression of SPCH and a cell-state-specific interaction.

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

During the pandemic, like many others, I didn’t have enough time to work on this project. Additionally, generating multiple transgenics and crosses was time-consuming and demanding.

Where will this story take the lab?

Our next goal is to investigate how the combination of cis- and trans-acting factors, as well as chromatin dynamics, mutually impact gene expression to promote cell fate specification at the single-cell resolution. Furthermore, our study indicates that the timely upregulation of MUTE could play a critical role in driving the switch of the epigenomic landscape towards stomatal differentiation. Previous molecular-genetic research has suggested that the HD-ZIP IV family and other transcription factors may promote MUTE expression, but the direct mechanism of action has not been explored. Nevertheless, the future question to address is how heterotypic TF groups, complexed with epigenetic modifiers, differentially guide the developmental progression at the atomic level.

What next for you after this paper?

I am currently conducting research on the progression of stomatal lineage cells and the active role of chromatin dynamics in determining stem cell fate specification, differentiation, and maintenance. I am now seeking tenure-track positions to establish my own research group and continue to explore these topics further.

References

1. Cusanovich, D. A. et al. The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature 555, 538-542, doi:10.1038/nature25981 (2018).
2. Cusanovich, D. A. et al. A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility. Cell 174, 1309-1324.e1318, doi:10.1016/j.cell.2018.06.052 (2018).
3.   Marand, A. P., Chen, Z., Gallavotti, A. & Schmitz, R. J. A cis-regulatory atlas in maize at single-cell resolution. Cell 184, 3041-3055.e3021, doi:10.1016/j.cell.2021.04.014 (2021).
4. MacAlister, C. A., Ohashi-Ito, K. & Bergmann, D. C. Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445, 537-540, doi:10.1038/nature05491 (2007).
5. Pillitteri, L. J., Sloan, D. B., Bogenschutz, N. L. & Torii, K. U. Termination of asymmetric cell division and differentiation of stomata. Nature 445, 501-505, doi:10.1038/nature05467 (2007).
6. Kim, E.-D. et al. Dynamic chromatin accessibility deploys heterotypic cis/trans-acting factors driving stomatal cell-fate commitment. Nature Plants, doi:10.1038/s41477-022-01304-w (2022).
7. Kanaoka, M. M. et al. SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation. Plant Cell 20, 1775-1785, doi:10.1105/tpc.108.060848 (2008).
8. Ohashi-Ito, K. & Bergmann, D. C. Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell 18, 2493-2505, doi:10.1105/tpc.106.046136 (2006).
9. Xiao, J. et al. Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis. Nat Genet 49, 1546-1552, doi:10.1038/ng.3937 (2017).
10. Wu, J. et al. Spatiotemporal Restriction of FUSCA3 Expression by Class I BPCs Promotes Ovule Development and Coordinates Embryo and Endosperm Growth. Plant Cell 32, 1886-1904, doi:10.1105/tpc.19.00764 (2020).
11. Cao, J. et al. Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science 361, 1380-1385, doi:10.1126/science.aau0730 (2018).
12. Luna-Zurita, L. et al. Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis. Cell 164, 999-1014, doi:10.1016/j.cell.2016.01.004 (2016).
13. Ogata, K., Sato, K. & Tahirov, T. H. Eukaryotic transcriptional regulatory complexes: cooperativity from near and afar. Curr Opin Struct Biol 13, 40-48, doi:10.1016/s0959-440x(03)00012-5 (2003).
14. Glasmacher, E. et al. A Genomic Regulatory Element That Directs Assembly and Function of Immune-Specific AP-1-IRF Complexes. Science 338, 975-980, doi:10.1126/science.1228309 (2012).
15. Rothenberg, E. V. Encounters across networks: Windows into principles of genomic regulation. Mar Genom 44, 3-12, doi:10.1016/j.margen.2019.01.003 (2019).
16. Rothenberg, E. V. Logic and lineage impacts on functional transcription factor deployment for T-cell fate commitment. Biophys J 120, 4162-4181, doi:10.1016/j.bpj.2021.04.002 (2021).

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Latin-America is well-known as one of the largest hotspots of biodiversity, inspiring a generation of important naturalists during the emergence of comparative developmental biology in the 19^th century. One of these naturalists, the German Johann Friedrich Theodor Müller (Fritz Müller), named the “Prince of Observers” by Charles Darwin, produced seminal work on the evolution of animals and plants in their natural environment¹. The reciprocal admiration of Fritz Müller for Darwin can be seen in the name of the amphipod Chelorchestia darwini (Müller, 1864) (Crustacea: Amphipoda: Talitridae – Figure 1) and in the large number of letters exchanged between them.

Figure 1: The amphipod Chelorchestia darwini (Müller, 1864) (Crustacea: Amphipoda: Talitridae. Left – a male of C. darwini. Right – Fritz Müller showed that adult males from this species display two types of morphology in the gnathopod 2.

In Für Darwin² (1864, For Darwin or Facts and Arguments for Darwin), Fritz Müller chose the crustaceans, a diverse and well-established taxonomic group at that time, to develop a theory of phylogenetic (genealogical) relationships among crustacean families, genera and species, particularly taking into account observations on embryonic and post-embryonic development in the natural environment.

Figure 2: Parhyale hawaiensis in laboratory culture, embryonic observation and manipulation.

Two hundred years after the birth of Fritz Müller³ (1822), I had the great opportunity to join a vibrant laboratory specialized in crustacean regeneration and evo-devo using the emerging model species Parhyale hawaiensis⁴. Interestingly, between 1888-1890 Fritz Müller studied the complete limb regeneration of Ayoida Potimirim (Crustacea; Multicrustacea; Decapoda; Atyidae) in their natural environment and found that several molts are required before the limbs acquire their original size. Müller stated “here [in limb regeneration] ontogeny recapitulates phylogeny” in an allusion to Haeckel´s biogenetic law⁵. The Averof laboratory⁶, at the Institut de Génomique Fonctionnelle de Lyon⁷, in France recently showed that the transcriptional signal from molting makes more difficult to study transcriptional profiles of regeneration⁸, but whether regeneration is dependent or controlled by molting is currently unknown.

Our group in Brazil has been interested in studying the evolution of early embryonic patterning, particularly of non-conventional arthropod models such as the kissing bug and the bovine tick, among others, but we particularly lacked crustacean representatives, thus far. In Lyon, I was exposed to a very friendly and productive group and was able to learn the laboratory routine of this emerging model system for development and regeneration (Figure 2 and 3).

Figure 3: Parhyale hawaiensis two cell-stage white-eggs display more intense auto-fluorescence at the green channel than purple eggs. It is also possible that the purple eggs absorbs the energy at this channel. No auto-fluorescence was observed at the red channel.

During these six months, I also started to investigate the role of the pioneer transcription zelda (zld) factor in this amphipod species. In fruit flies, zld is required for the maternal zygotic transition (MZT) and regulates hundreds of genes during the genome zygotic activation (ZGA) process. Interestingly, zld´s phylogenetic roots dates to the common ancestor of Pancrustacea (the monophyletic group including insects and crustaceans)⁹. Since my host lab had already developed genetic tools for transgenesis and transcriptional profiling in Parhyale (bulk and single-cell datasets for Parhyale leg development and regeneration)¹⁰, I could profit from these resources and start to draw hypotheses on the putative roles of zld in this amphipod. I plan to further develop these ideas now back in Brazil!

 In Brazil, I was very lucky that Professor Gisela Umbuzeiro, a well-established ecotoxicologist, has already established a large colony of Parhyale¹¹, and that Prof. Luciano Fisher from NUPEM-UFRJ and Cristiana Serejo from the National Museum-UFRJ had recently published a guide on naturally occurring amphipod species¹² with the rocky shore fauna of Brazil. Although Parhyale hawainesis was not identified in this survey, other amphipods of the same family (Infraorder Talitridae, Hyalidae) are present and associated with different habitats. The rocky shore fauna of Macaé, Rio de Janeiro is a particularly interesting spot for biodiversity, since it is in the most prominent area of oil exploitation in Brazil and harbors the largest protected sandbank park in the country . Thus, the CNPq/FAPERJ/ERC grant provided an opportunity to start an integrated Eco-Evo-Devo approach, connecting laboratory experiments with field work. If alive, I guess Fritz Müller may have been interested to follow these experiments.

Rodrigo Nunes-da-Fonseca (https://nunesdafonsecalab.com/) is an Associate Professor at the Institute of Biodiversity and Sustainability – NUPEM (https://nupem.ufrj.br/english-version/) of the Federal University of Rio de Janeiro. He is a Scientist of Our State FAPERJ and CNPq researcher. He was awarded with an ERC/FAPERJ/CNPq scholarship to join Averof´s team for six months during the second half of 2022.

Acknowledgments: I would like to thank Michalis Averof and all the members of the laboratory for the remarkable six-months I spent in Lyon, France, during my sabbatical.

References/Further Reading:

1. WEST, David A.., Darwin’s Man in Brazil – The Evolving Science of Fritz Müller. University Press of Florida, Gainesville, FL., 2016316p. ISBN: 9780813062600.

2. Müller F (1864) Für Darwin. Wilhelm Engelmann, Leipzig, 91 p.

3. Leonardo Augusto Luvison Araujo, Fritz Müller: 200 years of a pioneer evolutionist, Biological Journal of the Linnean Society, Volume 137, Issue 4, December 2022, Pages 737–739, https://doi.org/10.1093/biolinnean/blac124

4. Paris M, Wolff C, Patel NH, Averof M. The crustacean model Parhyale hawaiensis. Curr Top Dev Biol. 2022;147:199-230. doi: 10.1016/bs.ctdb.2022.02.001. Epub 2022 Mar 14. PMID: 35337450.

5. Müller, Fr., Haeckel’s biogenetisches Grundgesetz bei der Neubildung verlorener Glieder, in: Kosmos. 8. Bd. p. 3S8. *Note that the the fact that Fritz Müller cited Haeckel here does not assume that Haeckel was correct  in the current knowlegde.

6. https://www.averof-lab.org/

7. https://igfl.ens-lyon.fr/

8. Sinigaglia C, Almazán A, Lebel M, Sémon M, Gillet B, Hughes S, Edsinger E, Averof M, Paris M. Distinct gene expression dynamics in developing and regenerating crustacean limbs. Proc Natl Acad Sci U S A. 2022 Jul 5;119(27):e2119297119. doi: 10.1073/pnas.2119297119. Epub 2022 Jul 1. PMID: 35776546; PMCID: PMC9271199.

9. Ribeiro L, Tobias-Santos V, Santos D, Antunes F, Feltran G, de Souza Menezes J, et al. (2017) Evolution and multiple roles of the Pancrustacea specific transcription factor zelda in insects. PLoS Genet 13(7): e1006868. https://doi.org/10.1371/journal.pgen.1006868

10. https://www.averof-lab.org/pages/2923-resources

11. dos Santos, A. Botelho, M.T et al., The amphipod Parhyale hawaiensis as a promising model in ecotoxicology, Chemosphere, Volume 307, Part 2, 2022, 135959,ISSN 0045-6535, https://doi.org/10.1016/j.chemosphere.2022.135959.

12. Intertidal Rocky Shore Fauna. Vol 1. Crustacea Decapoda and Peracarida, Rio de Janeiro, Brazil. https://www.researchgate.net/publication/369021929_Intertidal_Rocky_Shore_Fauna_Vol_1_Crustacea_Decapoda_and_Peracarida_Rio_de_Janeiro_Brazil

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