One scientific story has dominated the news this week: the first report of CRISPR-edited human babies being born. In an associated Node post, we’ve collected the most useful links we could find surrounding the story, and here we reached out to members of the community for their perspectives.

Some responses are hopefully still coming in so look out for updates, and we’d also love to hear your thoughts – just use the comment box below.

Harry Leitch

MRC London Institute of Medical Sciences, Imperial College London

There is already a prevailing feeling that this work may be false. Certainly it is very difficult to know exactly what has gone on, with the limited information available thus far. I would agree with many other commentators that it is simply too soon to attempt human genome editing in embryos. For one thing there are obvious concerns about off-target effects and overall safety. I am also not convinced of the argument for attempting to make this particular modification. This doesn’t mean such edits are necessarily wrong, but I am not convinced a compelling case has been made especially given the risks.

The story has provoked an interesting debate regarding what type of genome and/or germline modification might be justified. While I agree that in many cases pre-implantation genetic diagnosis (PGD) would be a better and safer option than genome editing, I think many commentators have oversimplified the issue. There are situations in which PGD would be of no use, and genome editing is the only option, and this is not simply restricted to cases in which both parents are homozygous for a recessive disease allele. So to attempt to argue the issue away in this way seems nonsensical. My own group is doing some pre-clinical work to test if a genome editing approach might be curative in just such a scenario. Of course, if such approaches do appear promising, moving forwards to in vivo application will require very careful and meticulous pre-clinical studies to demonstrate safety, as well as  proper ethical debate, public scrutiny and legislation/regulation.

The most worrying aspect of this story is that these critical ethical and safety debates have been skipped. I do hope this does not set the field back, or prevent considerate and nuanced debate going forwards. It would be sad if irresponsible use of the technology in the coming years prevents its judicious application in the future, including potentially curative therapies for patients with no other options.

Insoo Hyun

Case Western Reserve University School of Medicine

The germline editing of human embryos is not new – there have already been a handful of scientific papers published on this type of research. What is new about the He case is that He transferred the edited embryos into women’s wombs, with or without full informed consent – that point is unclear. But aside from concerns about informed consent, the most significant ethical line that he crossed involved his many attempts at uterine transfer. Had He confined his editing work to in vitro activities only, he would have made a very small splash in the scientific community. Current ethical recommendations and guidelines, including the 2016 Guidelines of the International Society for Stem Cell Research, all state that it would be unethical to transfer germline edited human embryos into the womb, although in vitro work alone is permissible. Guidelines such as these should cast a wider net of potential actors.

It is not enough for scientific self-regulation to involve just the usual suspects involved in events like the 1st and 2nd International Summit on Human Genome Editing, where most of the participants are basic scientists and ethics and policy experts. Conspicuously absent from international and national discussions are the fertility clinic physicians and other assisted reproduction medical professionals who would eventually be the ones to provide reproductive gene editing procedures as an option for affected couples seeking to have healthy children. These medical professionals from the world of assisted reproductive technologies need to be involved in the discourse around reproductive germline editing. They need to be brought into the discussion surrounding what it means for scientists (and licensed physicians) to self-regulate on the issue of germline engineering. Fertility clinic doctors need to have a seat at the table.

Janet Rossant

Hospital for Sick Children Toronto, University of Toronto

This announcement from the group in China led by JianKui He is a very unfortunate and unwise development. The consensus from almost every working group internationally, including the National Academies Working Group, of which I was a part, has been that we need to move cautiously on possible germline editing in terms of safety and efficacy and that, even when these barriers are met, this approach would only be used for preventing serious genetic disease, where there is no other option, and where there has been full oversight, ethical approval and societal consensus. None of these applies to this report; ethical review is under question; his own university has disavowed him; the editing, if true, would count as an enhancement and not necessary for the child to be HIV-free; the long-term consequences in terms of susceptibility to other viral diseases could be damaging. The Chinese academies and academics are united in condemning this work and continued work towards international guidelines and regulation is clearly needed.

Paulo Navarro-Costa

The Gulbenkian Institute and at the Institute of Environmental Health in Portugal

As a reproductive biologist I was tremendously relieved by the nearly universal backlash against this purported achievement. The importance of preclinical safety assessment is paramount, particularly when it comes to procedures with a direct impact on our germ cells and resulting embryos. At the moment we still don’t know just how safe human genome editing really is. Another point this controversy makes abundantly clear is the need to ensure a consistent ethical framework across borders. Science and technology are a global enterprise and should be regulated likewise, especially when it comes to the use of human gametes and embryos for research purposes. I’m concerned with the fact that our currently heterogenous regulatory landscape leaves too much room for unethical and exploitative research.

Richard Behringer

The University of Texas MD Anderson Cancer Center, Houston

Tuesday evening here in Houston, I watched Dr. Jiankui He’s talk live through a video link to the 2nd International Summit on Human Genome Editing in Hong Kong. Dr. He presented a large amount of data about the research that led to the generation of the first humans produced with edited genomes, twin girls. Dr. He said a paper describing the results had been submitted for peer review. The gene that was edited using CRISPR technologies was CCR5. CCR5 encodes a receptor required for HIV infection. There is a relatively common loss-of-function allele called D32 in certain human populations such that there are individuals homozygous for this allele that are apparently normal yet resistant to HIV infection. Dr. He reported his group had generated one infant girl homozygous for CCR5 edited alleles and a twin girl that was heterozygous. He reported that the girls were normal and healthy. He also said that there would be an 18-year follow up on the children.

If the results hold true, then a so-called line has been crossed. In the current situation, normal (wild-type) zygotes were edited to make them resistant to a viral infection. However, in situations to cure a genetic disease, in nearly all cases that I can imagine, there will be carrier embryos and probably wild-type embryos. In these situations, preimplantation genetic diagnosis could identify embryos without the genetic disease for transfer into the womb. Thus, even though human genome editing to generate babies is now apparently possible, I’m not sure how it would be applied for clinical therapies.

Zhao Zhang

Carnegie Institution for Science Department of Embryology

As for the medical reason claimed by He Jiankui on this clinical trial (protecting the babies from HIV), I do not believe it is justified. It appears to me that he is doing an extremely risky, but completely unnecessary, experiment directly on two innocent HUMAN babies. I am therefore totally horrified for what He has done. Meanwhile, I do feel this is an individual case. Although deeply depressing, it is slightly gratifying to see that the whole Chinese Biology Community is unprecedentedly unified to condemn such an irresponsible, unethical, and illegal behavior. Next, I think multiple levels of investigations are needed to first validate the whole case. And we should give the two innocent girls the privacy and a normal life–or at least as close to be normal as possible. As a global community, we should take this case as a hard lesson to find a better and efficient way on implementing the standards and guidelines.

⬇⬇⬇ We’d love to hear your perspectives too⬇⬇⬇

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One scientific story has dominated the news this week: the first report of CRISPR-edited human babies being born. The story’s scientific and ethical aspects stirred up heated debate, as did its means of delivery: rather than a published paper, the story broke with reports of clinical trial documents and then a YouTube video from lead researcher He Jiankui (from the Southern University of Science and Technology in Shenzhen), all on the eve of a conference he was due to speak at  (and whose organisers were seemingly unaware what we was going to speak about).

In an associated Node post, we asked developmental and reproductive biologists to give their reaction to the story (and we’d love to hear yours too), but here we’ve collated a bunch of hopefully helpful links, and some recent Development commentaries on the issues surrounding gene editing in humans.

The story breaks

On 25 November, Antonio Regaldo in MIT Technology Review reported details of the study’s clinical trial data

https://www.technologyreview.com/s/612458/exclusive-chinese-scientists-are-creating-crispr-babies/

The He Lab YouTube channel released this video on the same day (the channel also has four associated videos about the work)

“A surgery that could save a child from a lethal genetic disease like cystic fibrosis or from a life-threatening infection like HIV doesn’t just give that little boy or girl an equal chance at a healthy life. We heal a whole family”

 

He talks to the Associated Press (26/11)

https://www.apnews.com/4997bb7aa36c45449b488e19ac83e86d

Statement from He’s employer, the Southern University of Science and Technology, stating that the university knew nothing about He’s work and plans to set up an independent committee to investigate (in Chinese – translates page reads quite clearly; 26/11)

https://www.sustc.edu.cn/news_events_/5524

Reaction to the initial reports from Nature (26/11)

https://www.nature.com/articles/d41586-018-07545-0

Ewan Birney, Director of EMBL-EBI, gives his thoughts on his blog (26/11)

http://ewanbirney.com/2018/11/crispr-babies-consideration-science-ethics.html

Paul Knoepfler gives his response (26/11)

ipscell.com/2018/11/why-crispr-baby-production-if-it-happened-was-unethical-dangerous/

An OpEd by Eric Topol of the Scripps Research institute (27/11)

nytime.com/2018/11/27/opinion-genetically-engineered-babies-china.html

He talks at the Second International Summit on Human Genome Editing, 28th November

He’s talk begins at 1:18, and a Q&A moderated by Robin Lovell-Badge and Matthew Porteus starts at 1:39.

He’s presentation slides can be seen here:

https://drive.google.com/drive/folders/1T1zLTtHS2z_cgl29fN_7qJg7fLA4qlrd

A transcript of the talk made by Bryan Bishop

http://diyhpl.us/wiki/transcripts/human-genome-editing-summit/2018-hong-kong/jiankui-he-human-genome-editing/

Reaction to the talk

Gaetan Burigo gave a helpful thread particularly regarding the science presented  by He in the summit (28/11)

OK there are a lot to unravel in this He Jankui talk and panel discussion at the #GeneEditSummit on #CRISPR in human embryos leading to the birth of the twins Lulu and Nana. -> Let's analyze the science, the ethics and controversies -> thread pic.twitter.com/3qOCC6IhvJ

— Dr Gaetan Burgio @gaetan_burgio@mstdn.science (@GaetanBurgio) November 28, 2018

Peter Mills, Assistant Director of the Nuffield Council on Bioethics, gives his thoughts (28/11)

nuffieldbioethics.org/blog/what-he-said

Nature piece on reaction to the talk (28/11)

https://www.nature.com/articles/d41586-018-07573-w

The Progress Educational Trust‘s Sarah Norcross gives her reaction (28/11)

George Church speaks to Science (28/11)

https://www.sciencemag.org/news/2018/11/i-feel-obligation-be-balanced-noted-biologist-comes-defense-gene-editing-babies

Statement by the Organizing Committee of the
Second International Summit on Human Genome Editing

http://www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID=11282018b

Akshat Rathi helps make sense of how the story unfolded

https://qz.com/1474814/the-cripsr-baby-news-was-carefully-orchestrated-pr-until-it-all-went-wrong/)

Recent Development content

Here at Development we’ve been thinking about issues of human gene editing for some time, and have commissioned content specifically exploring scientific and ethical aspects. We recently published two Spotlight articles on the theme (published in 2017 and 2018 respectively, before the current story broke).

In Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos, Alvaro Plaza Reyes and Fredrik Lanner  discuss the use of CRISPR-based genome engineering in human embryos and the emerging themes therein.

In Gene editing in human development: ethical concerns and practical applications, Janet Rossant  summarizes some of the ethical considerations associated with the use of gene-editing techniques in human embryos and embryo-like entities, highlighting the need for open and informed debate.

This is obviously a fast moving story so if you have any links you think other readers would find useful, let us know!

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We are rapidly approaching the era for safe mass production of specialized neuronal cell types and insulin-producing beta cells. It will then be possible to test whether transplanting such cells will enable paralysed people to walk again or people with type 1 diabetes to restart their own production of insulin. Until now, the engineering of the specialized cells from pluripotent stem cells has largely been based on empirical knowledge of what works. Results published in the prominent journal Nature by a Danish-led research project represent a major leap forward.

“We have now been able to map the signal that determines whether pancreatic progenitor cells will become endocrine, such as insulin-producing beta cells or duct cells. The cells are analogous to pinballs, whose ultimate score is based on the sum of pin encounters. They are constantly moving around within the developing pancreas, leading to frequent environmental changes. We show that the exposure to specific extracellular matrix components determines the ultimate destiny of the cells,” explains Henrik Semb, Professor and Executive Director, Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen.

The matrix determines the destiny

Progenitor cells are similar to stem cells since they can both self-renew and differentiate into mature cell types. However, their self-renewal capacity is generally limited compared with that of stem cells. The dynamic behaviour of progenitors during organ formation makes them difficult to study. By seeding individual human stem cell–derived progenitors on micropatterned glass slides, the researchers could study how each progenitor, without the influence of neighbouring cells, reacts to its surroundings.

“This enabled us to discover something very surprising. Our investigation revealed that interactions with different extracellular matrix components change the mechanical force state within the progenitor. These forces result from interactions between the extracellular matrix, which is outside the cell, and the actin cytoskeleton, which is within the cell.”

Pancreatic endocrine cells include all hormone-producing cells, such as insulin-producing beta cells and glucagon-producing alpha cells, within the islet of Langerhans, whereas the duct cells are epithelial cells that line the ducts of the pancreas.

“The experiments show that exposure to the extracellular matrix laminin instructs the progenitor cells towards an endocrine fate by reducing mechanical forces within the cells. Whereas exposure to fibronectin results in a duct fate because of increased mechanical forces.”

Mechanism facilitates exploitation

To exploit their discovery, the researchers had to understand the signalling pathway. They showed that components in the extracellular matrix trigger a signal into the cell via an integrin receptor, resulting in changes in mechanical forces transmitted through the actin cytoskeleton. The yes-associated protein (YAP) then senses these forces to turn on and off specific genes.

“This cascade determines the ultimate fate of the progenitor cell. Perhaps the most astonishing achievement is that our data answer an enigma that has puzzled the field for decades. How some progenitors mature into duct cells, whereas others become endocrine cells via Notch signals.”

The researcher show that the seemingly stochastic regulation of Notch function is in fact mediated by the progenitor’s encounters with extracellular matrix interactions via the force-sensing gene regulator protein YAP. They were even able to validate the physiological relevance in vivo during pancreas development.

“We can now replace significant numbers of empirically derived substances, whose mode of action in current state-of-the-art differentiation protocols is largely unknown, with small molecule inhibitors that target specific components of the newly identified mechanosignalling pathway.”

With this new strategy, insulin-producing beta cells can now be more cost-effectively and robustly produced from human pluripotent stem cells for future treatments against diabetes.

“Our discovery breaks new ground because it explains how multipotent progenitor cells mature into different cell types during organ formation. It also gives us the tools to recreate the processes in the laboratory, to more precisely engineer cells that are lost or damaged in severe diseases, such as type 1 diabetes and neurodegenerative diseases, for future cell replacement therapies.”

”Mechanosignaling via integrins directs pancreatic progenitor fate decisions” has been published in Nature. Henrik Semb, Professor and Executive Director, Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen, and  head of Institute of Translational Stem Cell Research at Helmholtz Zentrum München is last author. Drs. Anant Mamidi, Assistant Professor, DanStem and Christy Prawiro DanStem share first authorship, and the work is the result of a collaboration with Professor Palle Serup’s group, DanStem.. The Novo Nordisk Foundation has awarded grants of almost DKK 700 million (€92 million) to the Center for research between 2010 and 2018.

Read more about:

The Semb group
Professor Henrik Semb
Stem cells

The story was published on sciencenews.dk by Morten Busch

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Here at The Company of Biologists we’ve been debating the Bank of England’s decision to put a scientist on their new £50 note (the highest denomination note in England). The scientist must be deceased (only the Queen can grace notes while still alive) and ‘have shaped thought, innovation, leadership or values in the UK’.

Each of our five journals was asked to come up with their nominations for the face of the fifty. Here’s who they picked and why they picked them:

Development

Anne McLaren

“McLaren was a towering figure in developmental and reproductive biology. She did foundational work in IVF, experimental chimeras and germ cell differentiation, contributed to regulatory policies on human embryo research, and championed pubic engagement”

Journal of Cell Science

Rosalind Franklin

“She studied in Cambridge, and although a chemist, made a crucial, and often unrecognised, contribution to the discovery of the double helix structure of DNA”

Journal of Experimental Biology

Mary Anning

“English fossil collector/palaeontologist. Considered an expert in her field, contributing to important changes in scientific thinking about prehistoric life, at a time when women were mostly excluded from the scientific community”

Disease Models & Mechanisms and Biology Open

Fred Sanger

“Modern biology wouldn’t be what it is without him. Double Nobel winner known for sequencing DNA & pioneering work on the structure of proteins. Declined the offer of a knighthood, as did not wish to be addressed as Sir”

The Company of Biologists Twitter feed has a poll where you can pick your favourite out of the four:

We asked our journal teams for their nominations for the face of the new £50 note. Read the thread below to find out who @Dev_journal @J_Cell_Sci @J_Exp_Biol @DMM_Journal and @BiologyOpen selected and why.
Who out of the four would get your vote? #ThinkScience #50poundnote

— The Company of Biologists (@Co_Biologists) November 28, 2018

What do you think of Development’s choice of Anne McLaren?  Which other developmental biologist do you think could be honoured? Let us know in the comments

(1 votes)

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Regular meetings of scientists such as annual society conferences can create opportunities for scientists to engage the public without extensive effort, making connections between scientists and public audiences. Under the umbrella of a specific topic, events can be created to engage local communities with international researchers and foster forums for discussion of specific areas of research.

With this in mind, we created a space and a time for public engagement and a citizen’s approach to developmental biology in the recent Joint meeting of the Portuguese, Spanish and French Societies for Developmental Biology at Oporto, Portugal (http://devbiomeetingporto2018.pt/).

We invited local citizens through social media, the meeting webpage and local secondary school networks. And at the start of the meeting, which took please at the Almeida Garrett Library in Oporto, we organized an open science event for local Oporto high school students (mainly 16-17 year olds), their teachers and other members of the public.

It began with an informal conversation about what is developmental biology and why do we study it. This was done as a dialogue, with a backup of a few slides showing how embryos develop, some historical background and modern applications of the study of developmental biology. For this first part, we used some of the materials available at the BSDB as well as the Droso4schools and HHMI  websites.

This was followed by an organized speed-dating with scientists with the help of 12 Portuguese researchers working in national institutions as well as abroad. These volunteers were asked in advance what was the main question they were trying to answer with their research, so they could start their informal conversations from this starting point. They were also asked to bring along an object related to their research as a communication “ice-breaker”. The format of the speed dating consisted of groups of three members of the public to one scientist, with seven and a half minute slots of time available. After this time, a new scientist would take the place of the previous one and the cycle would start again. We found this informal set up allowed for fluid dialogue between scientists and the invited citizens. In addition, the speed-dating format allowed for each person to have the opportunity to speak with 5 or 6 different researchers, all in about 1h. When asked for their opinion about the event, one of the teachers told us:

“As far as the activity with the scientists is concerned, the students liked it immensely. They told me that this type of interaction is much more interesting than just a conference.”

Scientific meetings can play a key role in building bridges between scientific research and public audiences. Let’s try to create more of these opportunities in many other scientific conferences.

Participants in the event:

Sofia J. Araújo, Leonor Saúde, Patrick Lemaire, João Amorim, Tomás Azevedo, Gil Carraco, Ana Gali, André Gonçalves, Sofia Moreira, Paulo Navarro-Costa, Pedro Rifes, Lígia Tavares

(9 votes)

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MSc/PhD and postdoc positions available in the Zaidel-Bar Cellular and Tissue Morphogenesis Lab.

We study the regulation of the cytoskeleton from single proteins to the entire organism and system levels, using multiple approaches (including bioinformatics, genetics, biochemistry and live imaging) to understand how cells and tissues change shape, move, sense, and generate forces (for more info: celladhesionlab.com).

We are located in Tel-Aviv University, which is a top research and teaching institution in the most vibrant and cosmopolitan city in Israel https://english.tau.ac.il/ and https://international.tau.ac.il/

If you are interested in joining us send your CV and a statement of interest to: zaidelbar@tauex.tau.ac.il

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For the last two years, our interview series ‘The people behind the papers‘ has showcased the faces of developmental biology, and we’re excited to announce that the series will now also be printed in Development.

The first ‘paper’ interview is with Chaitanya Dingare and Virginie Lecaudey, first and last authors of a paper reporting a surprising link between the Hippo pathway and zebrafish fertilisation.

Before we put that one up on the Node, we thought we’d look back on the people we’ve met so far….

Martin Beck, Yannick Schwab, Nicole Schrieber & Paolo Ronchi

EMBL, Heidelberg

Thomas Lozito

University of Pittsburgh

Kristen Koenig & Jeffrey Gross

University of Pittsburgh and Harvard

Adam Johnston

Hospital for Sick Children in Toronto and University of Prince Edward Island

Joseph Pickering & Matthew Towers

University of Sheffield

James Nichols

University of Oregon and University of Colorado Denver

Amelia Joy Thompson, Sarah K Foster & Kristian Franze

University of Cambridge

Fernando Ferreira & Min Zhao

University of California Davis

Andrew Schiffmacher & Lisa Taneyhill

University of Maryland

Miguel Brun-Usan & Isaac Salazar-Ciudad

University of Helsinki

Ehsan Pourkarimi & Iestyn Whitehouse

Sloan Kettering Institute, New York

Nicolas Macaisne & J. Mark Cock

Station Biologique de Roscoff, Brittany

Philippe Foerster & Nathalie Spassky

Institut de Biologie de l’Ecole Normale Supérieure, Paris

Rute Tomaz & Véronique Azuara

Imperial College London

Matthias Tisler & Martin Blum

Hohenheim UniversityGermany.

Holly Voges, Enzo Porrello & James Hudson

University of Queensland

Thanh Vuong-Brender & Michel Labouesse

Institut de Biologie Paris-Seine

Dae Seok Eom & David Parichy

University of Virginia

Adam Davis, Nirav Amin & Nanette Nascone-Yoder

North Carolina State University

Jun-Ho Ha, Hyo-Jun Lee and Chung-Mo Park

Seoul National University

Gabriel Krens & Carl-Philipp Heisenberg

Institute of Science and Technology in Klosterneuburg, Austria

Lijun Chi & Paul Delgado-Olguin

Hospital for Sick Children and University of Toronto

Kimberly McArthur & Joseph Fetcho

Cornell University

Ivette Olivares-Castiñeira & Marta Llimargas

Molecular Biology Institute of Barcelona

Giri Dahal, Sarala Pradhan & Emily Bates

University of Colorado Denver

Alaa Hachem & John Parrington

University of Oxford

Diane Shakes, André Pires-daSilva, Gunar Fabig, Thomas Müller-Reichert & Jessica Feldman

The College of William and Mary in Williamsburg, VA, University of Warwick, UK, Technische Universität Dresden, Germany, Stanford University, CA

Dan Dickinson

UNC Chapel Hill and University of Texas

Simon Lane & Keith Jones

University of Southampton

Sabrina Jan, Tinke Vormer, Sjoerd Repping & Ans MM van Pelt

The University of Amsterdam

David Turner & Peter Baillie-Johnson

University of Cambridge

Qiang Shao, Stephanie Herrlinger & Jian-Fu (Jeff) Chen

University of Southern California

Ross Carter, Yara Sánchez-Corrales, Verônica Grieneisen & Athanasius (Stan) Marée

John Innes Centre, UK

Alok Javali, Aritra Misra & Ramkumar Sambasivan

Institute for Stem Cell Biology and Regenerative Medicine in Bengaluru, India

Chloé Dominici & Alain Chédotal

Institut de la Vision in Paris

Marina Matsumiya & Ryoichiro Kageyama

Kyoto University

You Wu & Mineko Kengaku

Kyoto University

Rémi-Xavier Coux & Ruth Lehmann

New York University

Jinjin Zhu & Justin Kumar

Indiana University

Sa Geng & James Umen

Donald Danforth Plant Science Center in St. Louis, Missouri

Samira Benhamouche-Trouillet, Evan O’Loughlin & Andrea McClatchey

Massachusetts General Hospital Cancer Centre

Cathy Pichol-Thievend, Natasha Harvey & Mathias Francois

University of South Australia and University of Queensland

Pauline Anne & Christian Hardtke

University of Lausanne, Switzerland

Kana Ishimatsu, Tom Hiscock & Sean Megason

Harvard Medical School

Martina Nagel & Rudolf Winklbauer

University of Toronto

Ximena Anleu Gil & Dominique Bergmann

Stanford University, CA

Takanori Wakatake & Ken Shirasu

RIKEN Center for Sustainable Resource Science in Yokohama

Anjali Rao & Carole LaBonne

Northwestern University, IL

Jaqueline Kinold & Hermann Aberle

Heinrich Heine University, Düsseldorf

Guillaume Blin, Manuel Thery & Sally Lowell

University of Edinburgh, Université
Grenoble-Alpes and Paris Diderot

Joe Shawky & Lance Davidson

University of Pittsburgh

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The Company of Biologists’ journals – Development, Journal of Cell Science, Journal of Experimental Biology and Disease Models & Mechanisms – offer Travelling Fellowships of up to £2,500 to graduate students and post-doctoral researchers wishing to make collaborative visits to other laboratories. These are designed to offset the cost of travel and other expenses. There is no restriction on nationality.

They really are an amazing opportunity for ECRs to learn new things, meet new people and travel to new places.

The current round of Travelling Fellowships closes on 30 November (for travel >14 Jan 2019)

Find out more here:

biologists.com/travelling/fellowships

Also learn more about what the Fellows get up to in their posts for the Node:

thenode.biologists.com/tag/travelling-fellowship/

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We are seeking highly motivated candidates to join us on a project beginning in 2019 on the molecular mechanisms of sensory neurogenesis. We study in particular the nociceptors, the specialized peripheral neurons that detect painful stimuli.

We aim to understand the role and mechanism of action of the Prdm12 gene. Prdm12 encodes an evolutionarily conserved epigenetic regulator of gene expression that has been found mutated in patients that suffer from a rare disease, Congenital Insensitivity to Pain, a dangerous condition that renders individuals completely unable to feel pain since their birth (Chen et al., Nat. Genet., 2015). To understand the molecular mechanisms that cause the painlessness, we are using the frog embryo and have generated Prdm12 null and conditional knock-out mouse models. We expect that the results of our work using these experimental systems as well as the identification of Prdm12 direct targets and interacting partners will help in the development of new strategies for treating pain.

The positions are funded by the Walloon government within the frame of the “Win2wal” program. They are open from 2 to 4 years and starting dates are flexible. The research will be performed in the laboratory of Developmental Genetics (Dr. Eric Bellefroid, http://gendev.ulb.ac.be/bellefroidlab/) that is part of the University of Brussels (ULB) Neuroscience Institute (UNI), the ULB Structural Biology and Biophysic laboratory (Dr. Abel Garcia-Pino, https://www.cm2ulb.be/) and the laboratory of Neuroscience (Dr. Laurence Ris, https://sharepoint1.umons.ac.be/FR/universite/facultes/fmp/services/neurosci/Pages/Equipe.aspx) at the University of Mons.

Preference will be given to applicants with a background in one of the following: mouse genetics, electrophysiology, cell and molecular biology and genome wide approaches (ChIP-seq,…).

Interested candidates should send a letter of motivation (before february 2019) describing past research experiences and full CV to:
Eric Bellefroid (ebellefr@ulb.ac.be), Laurence Ris (Laurence.RIS@umons.ac.be) or Abel Garcia Pino (agarciap@ulb.ac.be) together with the name and e-mail address of 2 references.

Selected related publications:
Thelie et al., (2015). Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus. Development, 142(19), 3416-3428.
Nagy et al., (2015). The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception. Cell Cycle, 14(12), 1799-1808.

(1 votes)

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Written and illustrated by: Bjørt K. Kragesteen, Malte Spielmann, and Guillaume Andrey.

In early development, the forelimb and hindlimb buds of tetrapods are morphologically uniform. However, as limb development proceeds, each individual tissue attains a characteristic morphology that ultimately defines the identity of a forelimb (arm) or a hindlimb (leg). How do undifferentiated limbs bend their morphogenetic trajectories into arm and legs, since they are patterned by similar developmental processes? It is commonly accepted that the differential activities of a handful of genes instruct the formation of either arms or legs; yet the mechanism leading to their differential regulation in fore- and hindlimb buds was unknown. In our recent study, we dissected in detail such regulatory mechanisms and have revealed, once again, how important the function of the non-coding regulatory genome is, representing the overwhelming majority of vertebrate genomes.

In Stefan Mundlos’ research group at the Max Planck Institute for Molecular Genetics in Berlin, the focus is on the lessons that human limb malformations can teach us about gene regulation. Most research projects start with detecting the genomic mutation in human patients with congenital limb malformations at the Charité Berlin university hospital. These limb malformations are frequently caused by large changes in the DNA called structural variants including: deletions (removing information), inversions (displacing information), and duplications (doubling and misplacing information). One can imagine that such mutations might mess up the genetic information, and by detecting them in patient genomes, structural variants can provide a clue as to where important non-coding regulatory elements are located. From this starting point, it is possible to look for neighbouring genes that become misregulated following the mutation, ultimately changing the developmental program and the formation of the embryo.

Our story started back in 2010 when Malte Spielmann, a clinical scientist interested in structural variants and congenital disease, tried to work out the genetic cause in three families with a very rare malformation syndrome affecting the arms of the patients, known as  Liebenberg syndrome (named after the doctor that first described it back in 1973). When he looked at the X-rays of the patients upside down, it struck him that the arms looked very much like legs! If we think about our arms and legs, they actually share many similarities: both are formed by one long bone (upper arm/thigh: the humerus/femur), followed by two bones (lower arm/leg: ulna/fibula and radius/tibia), terminating in many small bones (hands/feet: carpals, metacarpals, and phalanges) (see Figure 1).

Nevertheless, they flex in opposite directions, as the arms have a specialised elbow joint, where the tip of the ulna called the olecranon (the pointy elbow) grabs around the distal end of the humerus. The legs have a specialised knee joint with a small bone, the patella (knee cap), ensuring stability and enabling weight bearing. However, the Liebenberg syndrome 3D CT scan showed the following: the fingers were very short (brachydactyly) looking very much like toes; the small bones of the wrist (pisiform, triquetral) were fused together looking like the heel bone (calcaneus), and the olecranon was reduced and no longer grabbing around the end of the humerus. Instead, the end of the humerus was broader as a patella like bone appeared fused to it (Figure 2).

How on earth could this happen? Malte performed DNA analysis (array-CGH) and found a large deletion (107 kb) on chromosome 5 in all affected individuals. The deletions remove the gene H2AFY. However, this gene is a housekeeping gene and in mice in which the gene had been inactivated do not show any limb phenotype. Malte then looked on each side of the gene and found two interesting things:

On the centromeric side lies a gene named PITX1, which is expressed exclusively in the hindlimb during limb development and is known to be the only transcription factor that patterns the tissue. Inactivation of Pitx1 in mice results in reduction of leg morphology, such as loss of the patella and reduction of the calcaneus and the knee joint changing into an elbow-like joint. On the contrary, misexpression of Pitx1 in mouse forelimbs partially transforms the elbow joint into a knee like joint. Thus, Pitx1 misregulation in the forelimb seemed like a good candidate to explain to Liebenberg phenotype. However, Pitx1 regulation in tetrapods was unknown. Why would a deletion 200 kb upstream of the PITX1 promoter cause Liebenberg syndrome?

Interestingly, on the telomeric side of the deletion, 300 kb upstream of PITX1, a non-coding enhancer element can be found, called Pen (pan limb enhancer) that is active in both forelimbs and hindlimbs. Enhancer elements are defined as sequence specific stretches of DNA that are bound by transcription factors that dictate the activity and ensure communication with the correct gene promoters through chromatin folding. Often several enhancer elements regulate a target promoter and the collective activity of the tissue specific enhancer reflects the promoter transcriptional output. Recent development of proximity-ligation chromatin conformation capture technology (e.g. genome wide Hi-C) has demonstrated that the genome is partitioned into topological associating domains (TADs). TADs are scaffolds of preferential interactions between cognate promoters and enhancers and thus protect them from promiscuous activity from neighbouring TADs by boundary elements. These TADs were said to be stable across cell types and evolutionary conserved. With this in mind, the following hypothesis was formulated:

The Liebenberg deletion removes a TAD boundary element that normally separates PITX1 and Pen, resulting in PITX1 adopting a foreign enhancer, i.e. Pen, that is active in both fore- and hindlimbs. This enhancer adoption thus results in abnormal activation of PITX1 in the forelimb, where it should never be expressed, altering the patterning of the forelimb into a hindlimb, and partially transforming the arms into legs (Figure 3).

To test the hypothesis, Bjørt Kragesteen, a PhD student in the lab interested in deciphering non-coding functionality, sought to discern the molecular pathomechanism of Liebenberg syndrome together with Malte. First Bjørt created mouse mutants using CRISPR-Cas9 engineering that was newly established in the lab (back in 2013). She used two gRNAs to generate Liebenberg-like deletions and inversions at the mouse Pitx1 locus. Excitingly, analysis of Pitx1 mRNA expression in the early mutant mouse embryos showed Pitx1 misexpression in forelimbs! Skeletal analysis of adult mice showed a Liebenberg-like phenotype where the olecranon was reduced, an ectopic bone (patella-like) appeared at the humerus and the rotation of the arm was similar to the legs (Figure 4). Amazing. Case solved.

However, many questions remained unanswered. Why is Pen, a strong fore-and hindlimb enhancer, located relatively close to Pitx1? Isn’t that too risky for a key hindlimb patterning gene?

In parallel to Bjørt’s project, Guillaume Andrey, who started his postdoc in the laboratory in early 2014, was investigating the normal regulation of Pitx1, by combining deletions and inversions of its putative regulatory landscape and enhancer assays. He observed that several engineered structural variants, which did not alter the pre-supposed boundary between Pitx1 and Pen, resulted in a mirror image pattern between fore- and hindlimb, identical to the one of the Pen activity and a Liebenberg phenotype. This observation suggested that Pitx1 could be controlled by the Pen element even when the “boundary” was intact. From that point, it became evident that in hindlimbs Pen could play a role in the regulation of Pitx1.

We thus next deleted the Pen enhancer to see what would happen. A visit to the animal facility some weeks later revealed that some of the adult homozygous Pen deletion mice developed club feet, dragging their hindlimbs behind. Both mouse and human patients haploinsufficient for Pitx1 develop clubfeet. Moreover, the mutants showed reduced Pitx1 expression and skeletal abnormalities whereby the patella was missing (Figure 5). This was solid evidence that Pen indeed is a Pitx1 enhancer!

With this exciting finding we identified something not hereto described: A gene can be regulated by an unspecific long range enhancer; however, changing its location in the 2D regulatory landscape results in misregulation of the target gene and can transform tissue morphologies. We continued the search for a hindlimb specific Pitx1 enhancer, but no matter how much cloning and testing of enhancer elements we did, no hindlimb specific enhancer could be found. We thus decided to join forces, merging the projects about the normal and Liebenberg regulation of Pitx1, in order to understand how a gene that is solely expressed in the hindlimb can be controlled by unspecific enhancer elements active in both forelimbs and hindlimbs.

The research question thus became: what prevents Pitx1 expression in forelimbs in wildtype animals if its enhancer is active in both pairs of limbs?

The next obvious layer of gene regulation to scrutinise was the chromatin folding at the Pitx1 locus in limbs.  We first employed circular chromatin conformation capture (4C) technology in forelimbs vs hindlimbs to detect which regions surrounding Pitx1 were in close proximity to the gene. But, using the Pitx1 promoter region as a viewpoint, no differences were observed. However, Guillaume was using a slightly alternative 4C viewpoint in the gene body, and using the same library could see very strong differences between the tissues and clear Pitx1-Pen interactions in hindlimbs, indicating that the locus structure was highly defined. This was further confirmed using capture-C variation of the method using a larger fragment as a “bait” to see the interactions with the Pitx1 promoter region. Excitingly, differences between forelimbs vs hindlimbs emerged whereby Pitx1 showed differential interactions with Pen: in hindlimbs, Pen and Pitx1 come into close proximity enabling its tissue specific activation, while in forelimbs they are kept separated! Yet, this did not give the complete picture. At that point in time (2016) a new C-method, capture Hi-C, was developed and we established it in the lab. Here RNA probes are used to pull down a region of interest, and we enriched 3 megabases surrounding the Pitx1 locus. This provides a more complete picture of the interactions over the whole locus. Contrasting forelimbs and hindlimbs interaction heat-maps showed a clear difference: in hindlimbs Pitx1 forms loops with several regions (RA1, RA3 and Pen), which were almost completely diminished in forelimbs, but with a forelimb specific loop with the repressed gene Neurog1 occurs.

Still, this type of analysis gave us a 2D image of what was going on at the locus and we had a hard time imagining what this looks like in 3D, despite many hand drawn depictions. We thus initiated a collaboration with physicists in Italy in Mario Nicodemi’s research group. They used our capture Hi-C data and ran computational simulations using a so-called strings and binders model. They sent us the modelling results and 3D videos of forelimbs vs hindlimbs as well as an inversion mutant that shows the Liebenberg phenotype. The result was jaw-dropping: in wildtype forelimbs, the locus forms an inactive conformation whereby Pitx1 and Pen are kept a part, and where the repressed Pitx1 is embedded within its own domain next with repressed Neurog1 (neither of the genes are active in forelimbs and thus are covered with repressive epigenetic marks and hang out together). In wildtype hindlimbs, the locus folds in three domains and now Pitx1 is sitting at the surface of its domain directly facing Pen, thus enabling communication and robust transcription. Finally, in mutant forelimbs, bringing Pen closer to Pitx1 results in active folding of the whole locus, such as that in hindlimbs. Thus, not only do Pen and Pitx1 interact ectopically in mutant forelimbs, but the 3D-conformation becomes hindlimb-like, resulting in ectopic Pitx1 expression and arm-to-leg transformation.

With all these data in hand and the genetic evidence that Pen was responsible when misplaced in the nuclear 3D space for the ectopic expression of Pitx1 in the “Liebenberg” forelimb, we could determine that the Pitx1 locus dynamic structure modulates the Pen activity in normal embryos. Specifically, in forelimb it represses the enhancer by keeping it away from Pitx1 promoter and that upon activation in hindlimb, it can participate in Pitx1 robust expression. Finally, it also showed that ectopic interaction between a gene and its own enhancer, following structural variant, in a process called gene endo-activation can cause gene misexpression and disease.

Read the full story:

Kragesteen B.K.*, Spielmann M.*, Paliou C., Heinrich V., Schöpflin R., Esposito A., Annunziatella C., Bianco S., Chiariello A.M., Jerković I., Harabula I., Guckelberger P., Pechstein M., Wittler L., Chan W.L., Franke M., Lupiáñez D.G., Kraft K., Timmermann B., Vingron M., Visel A., Nicodemi M., Mundlos S. and Andrey G.

Dynamic 3D Chromatin Architecture Contributes to Enhancer Specificity and Limb Morphogenesis.

Nat Genet. 2018 Doi: 10.1038/s41588-018-0221-x

*These authors contributed equally

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