Our 20th instalment of this series comes from South Korea and features an investigation into the molecular basis of how temperature influences developmental transitions in Arabidopsis seedlings, recently published in Developmental Cell. We caught up with joint first authors Jun-Ho Ha and Hyo-Jun Lee, and their supervisor Chung-Mo Park, Professor in the Department of Chemistry, Seoul National University (SNU), to hear the story of the paper.
Jun-Ho, Hyo-Jun and Chung-Mo
Chung-Mo, can you give us your scientific biography – I understand you spent some years in the US before returning to South Korea?
CMP I am currently professor in the Department of Chemistry at SNU. I earned my Bachelor of Science in the Department of Science Education from Seoul National University in 1983 and my PhD in molecular virology from State University of New York at Buffalo in 1993 under the supervision of professor Jeremy Bruenn. The topic of my thesis work was identification of killer toxin genes in a double-stranded virus endogenously residing in Ustilago maydis, a corn smut fungus and functional and structural characterization of the killer toxin proteins. After completing my PhD, I worked as a postdoctoral researcher in the same university and the Hauptman-Woodward Medical Research Institute, Buffalo, until I joined the Kumho Life & Environmental Science Laboratory, Korea, as PI in 1996. In the Kumho Laboratory, I worked on the photochemical and photobiological characterization of phytochrome photoreceptors in higher plants and the cyanobacteria Synechocystis PCC6803 and their associated light signal transduction in photomorphogenic responses.
In 2002, I accepted an associate professor position in the Department of Chemistry, SNU, where I have been since that time. While at SNU, my research team has been working on diverse aspects of plant growth and developmental processes, such as seed germination, phase transition and flowering induction, and leaf senescence. I have also been working on plant responses to environmental stresses with emphasis on temperature extremes and drought stresses. In recent years, my research is focused on plant adaptation to high but nonstressful temperatures (warm temperatures) with emphasis on leaf hyponasty, heat dissipation from leaves, and autotrophic development.
And what is South Korea like as a place to do science?
CMP The Korean government and several biotech companies have been investing a huge amount of research fund during the last 30 years. While industrial research and development has been a priority as a potential driving force of economic growth, the Korean government is also spending heavily on basic research. In plant science, there is a national research supporting program, termed New-Generation Biogreen 21, which is organized and supported by the Korean Rural Development Administration. The Program supports various research on both model plants and crops. It is considered that although not sufficient, enthusiastic plant scientists are able to get enough research funds to perform both basic and applied researches in recent years.
fca mutant seedlings grown at different temperatures, from Figure 1, Ha, et al. 2017
Jun-Ho and Hyo-Jun – how did you come to join Chung-Mo’s lab?
JHH I earned my Bachelor of Science in chemistry. I was also interested in molecular biology with an expectation that combining chemical and biological principles would be exciting in understanding life. While I was looking for an appropriate lab for my graduate study, I met Chung-Mo Park, who is my current thesis advisor. I was greatly impressed by his passion for science and research. It was also impressive that his group is working on plant molecular biology in the Department of Chemistry. I therefore decided to join his laboratory for my graduate study.
HJL Since I was a high school student, I planned to be a scientist with an aim of discovering unknown principles of nature and living organisms. After I entered the Department of Chemistry, Seoul National University, as an undergraduate student, I searched for potential labs in the Department appropriate for my research carrier. I realized that Chung-Mo’ lab is unique among the laboratories in that he is studying plant molecular biology and biochemistry. I thought that understanding molecular biological and biochemical mechanisms underlying plant performance would be helpful for me to find ways to sustain the Earth’s ecosystem. In particular, as a chemist, I thought that applying chemical tools to understanding biological systems would be interesting. I therefore decided to perform my graduate study in his lab.
Before your work, what was known about how plants respond to temperature changes during autotrophic development, and what was the key question you set out to answer?
CMP, JHH & HJL It is well known that extreme temperatures significantly affect plant performance, including autotrophic development. In addition, associated molecular events and signaling schemes are fairly well understood. In nature, the soil temperature is rapidly elevated under warm temperature conditions. Therefore, developing seedlings should cope with high temperatures while they pass through the heat-absorbing soil layer to obtain photosynthetic capacity required for autotrophic growth. However, it is almost unknown how the heat-labile shoot apical meristem tissues of developing seedlings handle the temperature constraints. It has recently been reported that warm temperatures, in a temperature range of 23 – 28oC in Arabidopsis, accelerate cell elongation during early seedling development. Thus, we were curious about whether and how warm temperatures influence chlorophyll biosynthesis during autotrophic development.
TEM images of cotyledons of 3-day-old seedlings, from Ha, et al. 2017.
Can you give us key results of the paper in a paragraph?
CMP, JHH & HJL We demonstrated that developing seedlings are capable of maintaining chlorophyll biosynthesis required for autotrophic development at warm temperature conditions. A group of photooxidoreductase (POR) enzymes is responsible for chlorophyll biosynthesis. Notably, they are susceptible to warm temperatures and thus rapidly inactivated in developing seedlings while they pass through the warm soil layer. We found that an RNA-binding protein FCA maintains the abundance of POR enzymes at warm temperatures in developing seedlings. Without FCA, plants fail to maintain the enzyme abundance, resulting in loss of chlorophyll and thus failure to achieve autotrophic growth. Our work provide a molecular basis for the acquisition of autotrophic growth under fluctuating temperature conditions in plants.
How do have any idea of what is upstream of FCA? How does it sense temperature changes?
CMP, JHH & HJL Our recent findings strongly support that the typical RNA-binding protein FCA plays a critical role through epigenetic control of target genes during high temperature responses and thermomorphogenesis in Arabidopsis. Our data also indicate that FCA sustains the thermos-stable expression of POR enzymes during autotrophic development at warm temperatures. Altogether, these observations suggest that FCA function is thermos-regulated. However, it is current unclear how FCA is activated by ambient temperatures. We found that gene transcription and protein stability of FCA are not altered by temperature changes. Its subcellular localization is also unaltered under fluctuating temperature conditions.
Our preliminary data suggest that warm temperatures activates FCA through post-translational modifications, such as protein phosphorylation. We are currently under way to examine if FCA is differentially phosphorylated or chemically modified in response to temperature changes by employing global-scale proteomics.
Singlet oxygen accumulation, from Figure 4, Ha, et al. 2017.
Do you think your work will have relevance to agriculture in a warming world?
CMP, JHH & HJL Global warming depicts the gradual elevation of the average temperature of the Earth’s climate system. It is widely documented that under high ambient temperature conditions, plants exhibit distinct morphological and developmental traits, such as accelerated hypocotyl growth, leaf hyponasty, reduction of stomatal density, and early flowering, which profoundly influence crop productivity and commercial values. Our findings on plant thermal responses are closely associated with global warming. We propose that the FCA-mediated thermal adaptation of autotrophic development allows developing seedlings to cope with the heat-absorbing soil surface layer under natural conditions. In particular, we found that a single gene mutation causes a total loss of chlorophyll biosynthesis and autotrophic development at warm temperatures, providing a way of enhancing plant adaptation to thermal fluctuations in crop agriculture.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
HJL & JHH In the initial stage of the research, we germinated and grew the FCA-defective mutants at normal temperatures for 3 days before transferred to warm temperatures to see if the fca mutations affect seedling growth. However, we did not observe any phenotypic differences in seedling growth and greening patterns in the mutants. A few months later, we anticipated that the fca mutations might affect the earlier stages of seedling growth. To examine the hypothesis, we germinated and grew the mutant seedlings at 28oC. We were surprised at the albino phenotype of the mutants. This observation triggered the re-examination of the thermal phenotypes of the fca mutants, resulting in the completion of this paper.
At first, we could not figure out why the fca mutants exhibited albinism only when germinated and grown at warm temperatures. As a potential cause of the albino phenotype, we considered several possibilities, such as defects in chloroplast development, chlorophyll biosynthesis, or both. It was found that the expression of POR genes was disrupted in the fca mutants when grown at warm temperatures. Accordingly, the level of chlorophylls was extremely low in the mutants, showing that the thermo-sensitive albino phenotype of the mutants is caused primarily by defects in chlorophyll biosynthesis, consistent with the FCA-mediated stabilization of POR production.
fca seedlings grown in soil, from Figure S6, Ha, et al. 2017
And what about the flipside: any moments of frustration or despair?
HJL & JHH The FCA-defective mutants are well-known late flowering mutants. A set of transgenic fca plants expressing POR genes were required for this study. It needs a lot of time to generate the transgenic plants because it takes 3-4 months to obtain seeds from the transgenic plants. While we were generating transgenic plants, we realized that a wrong expression construct was accidentally used, spending at least 5 additional months to obtain correct transgenic plants.
We also remember the frustrating moment when temperature controllers in the culture room were out of order during last summer, when we experienced a rarely high temperature and thus unstable supply of electricity in Korea. We had to grow a full set of plants again after a period time for fixing the temperature controllers.
What are your career plans following this work?
HJL I am currently a postdoc in Chung-Mo Park’s lab. I will continue studying for a while on molecular and physiological mechanisms underlying plant thermomorphogenesis. I am interested in the as-yet unidentified regulator of POR abundance at warm temperatures. After finishing the experiments, I am planning to find an appropriate postdoc position to extend my research career in environmental control of plant proteomics.
JHH I hope to be able to finish my thesis study in a couple of years, after which I am planning to find postdoc positions in Korea or in USA to extend my research career in the field.
And what next for the Park lab?
CMP We have a well-organized research system with a variety of molecular and biochemical tools, personnel, and facilities. We are specialized in gene regulatory mechanisms with emphasis on induction and activation mechanisms of transcription factors. Using these research tools and system, we will further extend our researches on plant thermomorphogenesis, which is emerging as a hot issue in the field because of the growing concern about global warming. In particular, we are focused on the functional linkage between photomorphogenic responses and growth hormones. We are also preparing a long-term project for engineering crop plants to enhance their adaptation capacity to changing temperature environment.
Location: The Francis Crick Institute, Midland Road, London
Contract: Fixed-term (3 years), Full time
Salary: Competitive with benefits, subject to skills and experience
Vacancy ID: 5003
SHORT INTRODUCTION/SUMMARY
We seek a talented and motivated postdoc to join a Research Group led by Victor Tybulewicz at the Francis Crick Institute. The Group currently consists of 12 scientists, including 6 postdocs and 4 PhD students. One of the two main research interests of the Group is the study of the genetics underlying Down Syndrome. The Group has previously generated a series of mouse models of Down Syndrome that can be used to map the location of dosage-sensitive genes that cause Down Syndrome phenotypes (Lana-Elola et al, eLife 2016).
PROJECT SCOPE/ DESCRIPTION
The postdoc will study the genetics and developmental biology underlying congenital heart defects in Down Syndrome. The overall aim is to understand how increased dosage of genes on human chromosome 21 leads to heart defects. Specifically, the project aims to identify the dosage-sensitive genes that cause heart defects when present in three copies and to elucidate the mechanism by which the genes cause pathology. The work will involve use of genetic, developmental biology and biochemical techniques including microscopy, image analysis, and RNAseq, and will be supported by the excellent core facilities of the Institute. The work is funded by the Wellcome Trust.
The Francis Crick Institute is a biomedical discovery institute dedicated to understanding the fundamental biology underlying health and disease. Its work is helping to understand why disease develops and to translate discoveries into new ways to prevent, diagnose and treat illnesses such as cancer, heart disease, stroke, infections, and neurodegenerative diseases.
An independent organisation, its founding partners are the Medical Research Council (MRC), Cancer Research UK, Wellcome, UCL (University College London), Imperial College London and King’s College London.
The Crick was formed in 2015, and in 2016 it moved into a brand new state-of-the-art building in central London which brings together 1500 scientists and support staff working collaboratively across disciplines, making it the biggest biomedical research facility under a single roof in Europe.
The Francis Crick Institute will be world-class with a strong national role. Its distinctive vision for excellence includes commitments to collaboration; to developing emerging talent and exporting it the rest of the UK; to public engagement; and to helping turn discoveries into treatments as quickly as possible to improve lives and strengthen the economy.
If you are interested in applying for this role, please apply via our website https://goo.gl/IaFC2r
The closing date for applications is 10 June at 23:30 pm.
Please note: all offers of employment are subject to successful security screening and continuous eligibility to work in the United Kingdom.
We are pleased to announce the Center for Dental, Oral, Craniofacial Tissue and Organ Regeneration (C-DOCTOR – www.c-doctor.org) RFP that will award funding to promising dental, oral and craniofacial tissue engineering and regenerative medicine technologies and help them advance toward human clinical trials through customized product development advice and core resources. Please see the full RFP below the cut or here for details – deadline June 9, 2017. We ask that you kindly distribute this RFP widely to investigators who may be interested.
Our latest monthly trawl for developmental biology (and other cool) preprints. See June’s introductory post for background, and let us know if we missed anything
And a good month for bioRxiv was also a good month for developmental biology (and related) preprints. This month we found 115 preprints covering coral regeneration, spider development, a root-on-a-chip, a lot of discussion about publishing in our ‘Research Practise’ section, and a range of stem cell and cell biology, hosted on bioRxiv, F1000Research, PeerJ and arXiv.
Auxin depletion and gene expression, from Caggiano, et al’s preprint
Cell type boundaries organize plant development. Monica Pia Caggiano, Xiulian Yu, Neha Bhatia, André Larsson, Hasthi Ram, Carolyn K Ohno, Pia Sappl, Elliot M Meyerowitz, Henrik Jönsson, Marcus G Heisler
Microtubule structures in tubulin mutant worms from Zheng, et al’s manuscript
Genetical genomics reveals Ras/MAPK modifier loci. Mark G. Sterken, Linda Van Bemmelen van der Plaat, Joost A.G. Riksen, Miriam Rodriguez, Tobias Schmid, Alex Hajnal, Jan E. Kammenga, Basten L. Snoek
Post-transcriptional regulation of adult CNS axonal regeneration by Cpeb1. Wilson Pak-Kin Lou, Alvaros Mateos, Marta Koch, Stefan Klussmann, Chao Yang, Na Lu, Stefanie Limpert, Manuel Göpferich, Marlen Zschaetzsch, Carlos Maillo, Elena Senis, Dirk Grimm, Raúl Méndez, Kai Liu, Bassem A Hassan, Ana Martin-Villalba
Some stressed out coral from Boness, et al’s preprint
Disabling Cas9 by an anti-CRISPR DNA mimic. Jiyung Shing, Fuguo Jiang, Jun-Jie Liu, Nicholas L Bray, Benjamin J Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, Jacob E Corn, Jennifer A Doudna
Nanopore sequencing and assembly of a human genome with ultra-long reads. Miten Jain, Sergey Koren, Josh Quick, Arthur C Rand, Thomas A Sasani, John R Tyson, Andrew D Beggs, Alexander T Dilthey, Ian T Fiddes, Sunir Malla, Hannah Marriott, Karen H Miga, Tom Nieto, Justin O’Grady, Hugh E Olsen, Brent S Pedersen, Arang Rhie, Hollian Richardson, Aaron Quinlan, Terrance P Snutch, Louise Tee, Benedict Paten, Adam M. Phillippy, Jared T Simpson, Nicholas James Loman, Matthew Loose
Improved maize reference genome with single molecule technologies. Yinping Jiao, Paul Peluso, Jinghua Shi, Tiffany Liang, Michelle C Stitzer, Bo Wang, Michael Campbell, Joshua C Stein, Xuehong Wei, Chen-Shan Chin, Katherine Guill, Michael Regulski, Sunita Kumari, Andrew Olson, Jonathan Gent, Kevin L Schneider, Thomas K Wolfgruber, Michael R May, Nathan M Springer, Eric Antoniou, Richard McCombie, Gernot G Presting, Michael McMullen, Jeffrey Ross-Ibarra, R. Kelly Dawe, Alex Hastie, David R Rank, Doreen Ware
The Drosophila ventral nervous system from Court, et al’s preprint
A Systematic Nomenclature for the Drosophila Ventral Nervous System. Robert Christopher Court, James Douglas Armstrong, Jana Borner, Gwyneth Card, Marta Costa, Michael Dickinson, Carsten Duch, Wyatt Korff, Richard Mann, David Merritt, Rod Murphey, Shigehiro Namiki, Andrew Seeds, David Shepherd, Troy Shirangi, Julie Simpson, James Truman, John Tuthill, Darren Williams
CiliaCarta: An Integrated And Validated Compendium Of Ciliary Genes. Teunis J. P. van Dam, Julie Kennedy, Robin van der Lee, Erik de Vrieze, Kirsten A. Wunderlich, Suzanne Rix, Gerard W. Dougherty, Nils J. Lambacher, Chunmei Li, Victor L. Jensen, Michael R. Leroux, Rim Hjeij, Nicola Horn, Yves Texier, Yasmin Wissinger, Jeroen van Reeuwijk, Gabrielle Wheway, Barbara Knapp, Jan F. Scheel, Brunella Franco, Dorus A. Mans, Erwin van Wijk, François Képès, Gisela G. Slaats, Grischa Toedt, Hannie Kremer, Heymut Omran, Katarzyna Szymanska, Konstantinos Koutroumpas, Marius Ueffing, Thanh-Minh T. Nguyen, Stef J. F. Letteboer, Machteld M. Oud, Sylvia E. C. van Beersum, Miriam Schmidts, Philip L. Beales, Qianhao Lu, Rachel H. Giles, Radek Szklarczyk, Robert B. Russell, Toby J. Gibson, Colin A. Johnson, Oliver E. Blacque, Uwe Wolfrum, Karsten Boldt, Ronald Roepman, Victor Hernandez-Hernandez, Martijn A. Huynen
TOWARDS COORDINATED INTERNATIONAL SUPPORT OF CORE DATA RESOURCES FOR THE LIFE SCIENCES. Warwick Anderson, Rolf Apweiler, Alex Bateman, Guntram A Bauer, Helen Berman, Judith A Blake, Niklas Blomberg, Stephen K Burley, Guy Cochrane, Valentina Di Francesco, Tim Donohue, Christine Durinx, Alfred Game, Eric Green, Takashi Gojobori, Peter Goodhand, Ada Hamosh, Henning Hermjakob, Minoru Kanehisa, Robert Kiley, Johanna McEntyre, Rowan McKibbin, Satoru Miyano, Barbara Pauly, Norbert Perrimon, Mark A Ragan, Geoffrey Richards, Yik-Ying Teo, Monte Westerfield, Eric Westhof, Paul F Lasko
A postdoctoral position is immediately available to pursue cutting-edge musculoskeletal stem cell research in the laboratory of Kathryn Wagner, MD, PhD at the Center for Genetic Muscle Disorders, Kennedy Krieger Institute and Johns Hopkins School of Medicine,
The Wagner laboratory is a moderate-size laboratory that focuses on translational science to develop novel therapies for muscle disorders. Approaches include small molecules, biologics, stem cells and gene therapy. The laboratory is part of a Senator Paul Wellstone Muscular Dystrophy Cooperative Research Center and has extensive collaboration both within Johns Hopkins and across institutions. https://www.thewagnerlab.org.
The ideal applicant will have a PhD in molecular or cell biology. Experience in skeletal muscle is a positive but not a requirement. This is a fully funded position to study transplantation of iPSC-derived myogenic progenitors in models of muscular dystrophy and volumetric muscle loss.
Interested applicants should send a cover letter and CV to Dr. Wagner at wagnerk@kennedykrieger.org
Department of Clinical and Experimental Medicine, Linkoping University, Sweden.
How cells are generated in proper numbers in order to form specific structures in an organism has been a subject of interest for many years. This issue is particularly intriguing in in the central nervous system (CNS), where the number of neurons of each type plays a crucial role in neural circuitry and cognitive capabilities in different species across evolution1. An intriguing feature of the CNS is that different regions have different cell numbers, and the number of neurons along the anterior-posterior (A-P) axis seems to be organized in a wedge-like appearance: the anterior part contains more neurons than the posterior part. This overall feature of CNS organization holds true for many vertebrate and invertebrate species. So how is the generation of proper number of neurons determined in the different regions of the central nervous system? This was, and still is, one of the questions around when I joined Stefan Thor’s lab in Linkoping, and the one which we try to shed light on in this post.
The total number of cells generated in a certain region is determined by several factors: the initial number of progenitors, the number of divisions that those progenitors go through, the proliferative behavior of the daughter cells produced by progenitors, and programmed cell death (PCD) present within the lineage. Drosophila CNS is subdivided into the brain, and the ventral nerve cord (VNC), equivalent to the mammalian and spinal cord respectively. Recent studies in the Drosophila VNC have provided an essential framework for addressing how the proper number of cells are generated in each segment. First, the recent meticulous mapping of the number of neural progenitors or neuroblasts (NBs) in each of the 13 segments of the VNC 2–6; second, our previous discovery of different daughter proliferation modes in NB lineages, where NBs initially bud off daughters that divide once, to generate two neurons/glia, denoted Type I, and subsequently switch to generating daughters that do not divide, instead directly differentiating, denoted Type 0 7; third, the identification of mutants that lack PCD in embryonic stages8. These findings gave us the opportunity to make a global and systematic study of proliferation in the VNC.
With this set up, our first challenge was to establish just how different the anterior segments are from the posterior ones, and that was one of the most exciting parts in the project. The 13 segments of the VNC are generated by a similar number of progenitors, about 64 per segment, in each segment. Our cell number analysis at the end of neurogenesis revealed that these 64 NBs generate different numbers of cells in different segments, with more cells in anterior segments than posterior ones. Based upon this finding we postulated two main processes that could contribute to this wedge-like appearance: removal of excess cells, by PCD, in posterior segments, and/or increased proliferation in anterior segments. Looking at PCD mutants, the A-P differences in numbers were still noticeable, suggesting PCD could not explain the difference. In contrast, we observed increased proliferation, of both NBs and daughters, in anterior segments, which pointed to proliferation as the main contributor to the A-P differences. To further address this notion, we analyzed proliferation in specific NB lineages, serially presented in all VNC segments. Whereas in anterior segments, NBs undergo more rounds of division and display a late or absent Type I->0 daughter proliferation switch, in the posterior segments NBs undergo fewer rounds of divisions and display an earlier Type I->0 switch. The combination of cell number analysis with global and single-lineage proliferation analysis allowed us to extract an average lineage behavior for thoracic segments versus posterior segments.
Anterior-posterior differences in proliferation behavior. Anterior thoracic segment show a higher number of dividing neuroblasts (NBs) and dividing daughter cells than the posterior abdominal ones.
The next goal was to begin identifying some of the players controlling this gradient in proliferation behavior. The A-P axis differences obviously pointed to homeotic domains, and our previous work has identified the Hox gene Antennapedia (Antp) as one of the main triggers of the Type I->0 daughter proliferation switch in the thoracic segments7. Therefore, we analyzed the role of the more posteriorly acting Hox genes of the Bithorax-Complex (BX-C); Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). Strikingly, we indeed observed that BX-C genes were important for the earlier Type I->0 daughter proliferation switch and NB proliferation exit observed in posterior segments. However, the triggering of the Type I->0 switch during later stages of lineage progression implied that the action of BX-C was gated in a temporal manner. Our previous studies of Antp had revealed that its expression in thoracic NBs commences during later stages. Studying this, we found that although Hox genes are indeed expressed in an A-P manner during early embryonic stages, early NBs display little if any BX-C protein expression. As NBs lineage progression proceeds, we observed onset of Hox protein expression, establishing the characteristic overlapping domains along the A-P axis, and this late onset in NBs then promotes the Type I->0 daughter proliferation switch. Based upon these findings we propose a model where at early stages NBs progress in a Hox-free “ground state”, allowing for high proliferative modes, whereas at later stages, the onset of Hox expression in NBs promotes the Type I->0 daughter proliferation switch, and eventually NBs stop dividing. This action of Hox genes establishes a gradient in NB lineage size, and results in the A-P wedge appearance.
Hox-Mediated Proliferation Control along the A-P Axis in the VNC
The hypothesis of the ground state is not new in Drosophila, and has been proposed by Fernando Casares and Richard Mann for appendages9. This idea proposes a “default” program, that we believe applies also to NB proliferation behavior, in thoracic segments, which is modified along the A-P axis by the same cues that determine the body plan. This program allows for homologous NBs in different segments to modify their proliferation behavior to generate the proper number of cells needed for that specific segment.
Since Hox genes are evolutionary conserved, establishing the body plan of invertebrates and vertebrates equally, it is reasonable to envision that they may have a conserved function in proliferation control in the nervous system. Indeed, several studies have identified Hoxc6, Hoxb9 and Hoxb13 related to suppression of proliferation in the vertebrate CNS10,11. Most intriguingly, while our current study was focused on the VNC, the most obvious wedge-like appearance in nervous systems is brain size when compared to nerve cord. Given that we have shown that Hox expression suppresses neural progenitor and daughter proliferation, and that the brain is a Hox free region, it seems reasonable to think that Hox genes could be one of the motors of the evolutionary expansion of the brain.
We are now very enthusiastic about the open possibilities of what is happening in the brain. The work is now focused on addressing how proliferation is controlled in the brain, and to what extent could alternate neural progenitor and daughter cell proliferation modes be a common feature, using similar or homologous tools, to establish A-P differences across different species.
We are looking for a motivated, ambitious and collegial person interested in joining us as a postdoc to pursue projects centered around host-pathogen interaction in human stem cell derived macrophages. Prior exprience with stem cells or high throughput microscopy is required. Please contact Eva directly with your CV or with queries if you are interested.
eva.frickel@crick.ac.uk
frickellab.com
This project will dive into regulation of host immunity to infection or infectious stimuli using macrophages derived from human stem cells. More emphasis will be put on deciphering molecular signaling or cellular response pathways than the actual stimuli/infection under study. This will require efficient gene editing approaches for stem cell derived macrophages (CRISPR, siRNA) and the ability to work with high throughput analysis (image based) as well as handling large datasets (RNASeq, proteomic or metabolomic analysis). Thus I believe a person with an interest in using human stem cells to study intracellular mechanisms is best suited for this post.
Please consider visiting us in Sheffield for this exciting symposium this September (register at lifecourse.group.shef.ac.uk). The symposium is being organised by Marysia Placzek, Stephen Renshaw and David Strutt on behalf of the Bateson Centre at the University of Sheffield (https://www.sheffield.ac.uk/bateson). There is an array of outstanding external speakers already confirmed and the opportunity to present your own work (abstract deadline is 15th June). Sign up by 28th July! We look forward to welcoming you to Sheffield!
An NIH R01-funded postdoctoral position is available immediately in the laboratory of Dr. Beth Roman in the Department of Human Genetics at the University of Pittsburgh. This position provides an outstanding opportunity for cross-disciplinary training at the interface of physics and human health, requiring close collaboration with the laboratories of Dr. Lance Davidson (Bioengineering) and Dr. Andrew Hinck (Structural Biology). We are looking for a productive, highly motivated scientist to dissect the interaction between bone morphogenetic protein (BMP)/ALK1 signaling and mechanical force in controlling directed endothelial cell migration, using zebrafish and endothelial cell culture models. Our ultimate goal is to understand how ALK1 signaling disruption in the genetic vascular disorder, hereditary hemorrhagic telangiectasia (HHT), causes arteriovenous malformations (AVMs). AVMs are direct connections between arteries and veins that can lead to hemorrhage and stroke.
The University of Pittsburgh is a collaborative, collegial environment for biomedical research. Our laboratory is part of a large, interactive zebrafish community, and we are affiliated with the Heart, Lung, and Blood Vascular Medicine Institute (VMI), which houses numerous laboratories focused on basic and translational cardiovascular research. Our laboratory is also integral to the UPMC/Pitt HHT Center of Excellence, affording the opportunity to translate research findings to a clinical care setting.
Qualified candidates must have a PhD in biology, bioengineering, or other relevant field, a strong publication record, and excellent oral and written English communication skills. Expertise in fluid mechanics and endothelial cell culture is preferred. Previous experience with zebrafish, dynamic live-cell imaging, confocal microscopy, and image analysis is desired. Interested candidates should send a brief cover letter (maximum 1-page) summarizing scientific accomplishments and outlining motivation for this position, a CV, and full contact information for three professional references to Dr. Beth Roman at romanb@pitt.edu.
For more information, please see
On a bright, cold morning at the beginning of March, I went back to the institute I once worked in to interview the man after whom the place was named. Greeting me at the entrance, John Gurdon apologised for being a little late and asked if it was alright to delay the interview for five minutes while he finished up some lab work. I followed him up the stairs to his lab and through the lab to his office, where he took a plate out of an incubator and placed it between old microscopes and pipettes on a bench that ran the length of one side of the room. Sunlight streamed in and the frog embryos bobbed around as John replaced the media – it was something to do with Halo tagging, apparently – and put the plate back in the incubator, before pulling a chair out to start the interview. As anyone who has met him will not be surprised to hear, he was engaging, humble and lucid in his recollection of experiments started half a century ago and which his lab continues today.
John Gurdon is a Distinguished Group Leader in the Wellcome Trust/Cancer Research UK Gurdon Institute and Professor Emeritus in the Department of Zoology at the University of Cambridge. In 2012, he was awarded the Nobel Prize in Physiology or Medicine jointly with Shinya Yamanaka for work on the reprogramming of mature cells to pluripotency, and his lab continues to investigate the molecular mechanisms of nuclear reprogramming by oocytes and eggs. We met John in his Cambridge office to discuss his career and hear his thoughts on the past, present and future of reprogramming.
Your first paper was published in 1954 and concerned not embryology but entomology. How did that come about?
Well, that early paper was published in the Entomologist’s Monthly Magazine. Throughout my early life, I really was interested in insects, and used to collect butterflies and moths. When I was an undergraduate I liked to take time off and go out to Wytham Woods near Oxford to see what I could find. So I went out one cold spring day and there were no butterflies about, nor any moths, but, out of nowhere, there was a fly – I caught it, put it in my bottle, and had a look at it. The first thing that was obvious was that it wasn’t a fly, it was a hymenopteran, but when I tried to identify it I simply could not work out what it was. I don’t like to be defeated, so I went to the Hope Department in Oxford and they didn’t know what it was either, and then to the Natural History Museum, where a curator told me that, amazingly enough, this was a species never recorded in England before! This was intensely irritating to the Entomology Department in Oxford because the professor at the time had a major ecological study of all the insects in those woods, and here was a student who had just caught the first thing he could find, and picked up a new species. So I wrote a couple of paragraphs announcing the discovery, and that’s how I came to have that paper.
And did you keep up your interest in insects?
Not really in a proper scientific way, though I keep thinking I’d like to go back to that, mainly because the colour patterns of lepidopterans are so remarkable. We really know almost nothing about how colour patterns are formed – in any species. You won’t have a gene that puts a spot on one wing, it’s a more complicated process, including diffusion of molecules. I keep thinking that when I actually retire I’ll take that up, but I’ve yet to get to that point!
Half a century ago you started your nuclear transfer experiments, and today your lab is still publishing on it. Why do you think such a conceptually simple experiment has had such a remarkably long shelf life?
When I was doing those early nuclear transfer experiments – and I am permanently grateful to my supervisor, Michael Fischberg, for putting me onto that work – the question at the time was whether all cells in the body have the same genes. One way to determine this was to take a nucleus from one kind of cell, put it into the egg, and see if it can develop. This experiment was conceived as far back as the late 19th century: there’s a paper by a man named Rauber who describes an experiment of putting a toad nucleus into a frog egg, and simply says he didn’t get a result, so it’s not clear whether he did the experiment or not!
Anyway, in the 1950s Briggs and King, two Americans, developed the technique of transplanting the nucleus, and Fischberg decided we should try this in Xenopus. There were several very troublesome technical difficulties which we eventually overcame – as much by luck as skill – and the end result was that you can get essentially normal development by taking the nucleus of a specialised cell, in this case an intestinal cell, and transplanting it to an enucleated egg. That clearly said that the same genes are present in all different kinds of cells.
And then there was this gap of 50 years before Yamanaka developed the induced pluripotent stem cell technique, which really opened the field to useful clinical potential. The frog experiments (and much subsequent work, including the generation of Dolly the sheep in the 1990s) said you can reverse or rejuvenate a specialised nucleus right back to the beginning again, but clinical translation became a realistic possibility in humans only when Yamanaka showed you did not need to obtain human eggs or embryos to make stem cells. This idea that you could derive new cells of one kind starting with adult cells of a completely different kind obviously chimed with our work from half a century beforehand but, interestingly enough, this was absolutely not evident when these early experiments were done. ‘Reprogramming’ wasn’t even the aim of the experiments. I imagine I would not get support for carrying on these nuclear transfer experiments today were it not for their relevance to reprogramming in humans.
So then the question is how does this process work? What underlies the egg’s ability to rejuvenate a nucleus? We were always interested in that question, but it became increasingly interesting with Yamanaka’s experiments. And I would point out that people still don’t really know why the Yamanaka procedure works – even after ten years, they don’t really understand the mechanism. So we take the view, and it is true, that the egg does a rather better job of reversing differentiation compared with overexpressing transcription factors, and therefore think that if you knew what all the egg components are, and knew how to make them exchange with the somatic ones, you wouldn’t need the Yamanaka factors. That is why we are actively pursuing the mechanism of reprogramming by the egg, using the same procedure aswas done 60 years ago, but with awhole lot of new ways of investigating it. To me, this exemplifies the interesting principle that work which was done at one time can have a subsequent, much greater relevance in the light of later advances.
“We are actively pursuing the mechanism of reprogramming by the egg, using the same procedure as was done 60 years ago, but with a whole lot of new ways of investigating it”
And what is your current understanding of the molecular mechanisms of reprogramming by the egg?
It’s almost certainly due to a high concentration in the egg of chromatin components, particularly histones. There are numerous variants of histones, in terms of how they are modified, and quite a lot of our recent work has been describing the histone changes that are imposed by the egg on an incoming nucleus. This chromatin change is perhaps the first key stage – there’s a particular histone variant present in eggs which is very important, and it’s likely that the replacement of adult chromatin components by ones present in the egg is ultimately what helps to cause the change.
There are two aspects to this problem. One is how does the egg use its components to replace those of the somatic nucleus, and so rejuvenate it? The second is why doesn’t reprogramming work perfectly? I like to illustrate it like this: there’s a battle between the egg, trying to turn everything back into an embryonic state, and the somatic nucleus, which is designed to be exactly the opposite – it’s meant not to change. Most of our cells don’t change, and it’s quite important that cells are extraordinarily stable. So the egg tries to overrule the nucleus, and the nucleus tries to resist it; those are the two complementary parts of our research project at the moment.
To complement this, we’re also looking at the changes that occur to a sperm nucleus that make it so remarkably receptive to reprogramming; ultimately, we’d like to convert the somatic nucleus into the same condition as the sperm, and then reprogramming should work very well.
While I think most readers will be familiar with your reprogramming experiments, I’d like to discuss some of your other work. In a series of papers in the 1970s you studied the translation of injected RNA in frog oocytes: can you tell us a bit about this work?
The experiment that appealed to me enormously at the time, and still does, is to inject messenger RNA (mRNA) into eggs. I was doing this work when people, notably Hubert Chantrenne in Belgium, had first isolated mRNA. I was a good friend of a wonderful man named Jean Brachet, and told him that what I’d really like to do is to transplant not nuclei but mRNA into eggs.
Jean Brachet and Hubert Chantrenne, from Jean Brachet and his school by H Alexendre, Int. J. Dev. Biol, 1992.
He gave me an introduction to Chantrenne, who was making rabbit globin RNA and gave us some, thanks to Brachet. The stuff was known to be extremely RNase sensitive, so you almost had to take a bath in chromic acid before you touched anything! Now had I proposed that experiment as a grant it would have been rejected because the egg was known to be full of ribonucleases: to put sensitive mRNA into a ribonucleic environment would make no sense. Nevertheless, it worked, and astonishingly well – the globin message went into eggs, and by the time the eggs had turned into tadpoles there was still rabbit globin being made. Almost certainly the reason for the success is that microinjection doesn’t open up the lysosomes, where the ribonucleases are partitioned. So there’s another interesting principle: when someone tells you something won’t work, it’s much better to try it than to take their word for it. And mRNA injection has turned out to be a very useful approach for all sorts of questions. These RNA experiments were really a derivative of the technological results of nuclear transfer – if it works for nuclei, what else can you transfer? Eddy de Robertis and I even had a paper calling the Xenopus egg a living test tube.
You were also interested in the process of induction, and identified a ‘community effect’ in the induction of the Xenopus mesoderm. What is the basis of this effect?
For many decades people had been transplanting tissue – take a piece of tissue and graft it onto another host. But the tissue is obviously composed of many cells, which may not all be the same, and for me it was always desirable to do a single-cell transplant. And so I did a lot of those, moving single progenitor cells from one part of the embryo to another, but I could never get it to work – the cells always died. There must have been some reason why you can successfully transplant multiple cells but not single cells. That led me to perform injections of smaller and smaller numbers of cells. It turned out that transplanted cells release secreted molecules – signalling proteins for instance – that are necessary for them to do anything in the host. A single cell has difficulty doing much with what it secretes – the concentration is too low – but multiple cells will build up a high enough concentration to actually work. This ‘community effect’ is somewhat analogous to the quorum sensing identified in bacteria.
What is your perspective on where developmental biology as a field is today? What are the gaps in our understanding, and what do we need to do to fill the gaps?
My own view of development is that one has to try to narrow things down to single entities, whether it’s a cell or a nucleus or a molecule, and I’m often ridiculed because I always ask people what concentration their molecule is at, and they’ll say that it doesn’t matter.
I’d say that concentration and time are the two critical things in development. You need to know the concentration, and you need to know how long it has to be there to make a difference – because for cells, a particular concentration of a molecule for a few seconds may not be the same as that concentration for 10 minutes. So I would take the view that what we really lack in developmental biology at the moment is any ability to determine the concentration of proteins, analogous to the measurement of nucleic acids using PCR.
In my own experience, I got involved in experiments on a protein called Activin, a TGF-β molecule. Rather amazingly – and I still like this experiment – you can take blastula cells, completely dissociate them in suspension, and then add Activin at a known concentration for a known time. Then you wash the cells and let them reaggregate and ask how they differentiate. It turned out that the outcome – whether these cells made ectoderm, mesoderm or endoderm – depended not only on the amount of Activin but also on the time you bathed the cells in it. It was an interesting principle that concentration and time can have completely different effects depending on which one you alter, and by how much.
But to really understand amazing phenomena like this in vivo, knowing the concentration of proteins is really going to be important, and I think we completely lack that at the moment. In the future we will gradually be working with single cells, known concentrations, known amounts of time, and then we can get to an understanding of what’s going on in these differentiation events.
“Concentration and time are the two critical things in development”
Your work will probably be most clinically influential in the field of cell replacement – what do you think of the current challenges and prospects?
I think the prospects for cell replacement are very good, but scientific progress might be hindered by other things. The example I often use is of age-related macular degeneration, where the photoreceptors die and so you go blind. These photoreceptors are supported by retinal pigmented epithelial cells, and researchers in London and elsewhere can use the Yamanaka procedure to make thin layers of the epithelial cells, and then insert them into the eye by a process that is no more complicated than lens replacement. Whenever I talk about this, people come up to me and ask when they can get it done. The answer is that they are not allowed to, and the reason in my opinion goes back ultimately to legal issues. If something goes wrong, the lawyers will fight for huge amounts of compensation. If you do the procedure one hundred times, and it goes wrong once – ninety-nine people will gain tremendously in not going blind, but one will get such a massive financial award that the medical profession will shy away from it. I think this is a real challenge to the field – the resistance of the medical profession because of potential legal and financial consequences.
You’ve previously talked about the importance of the guidance your PhD supervisor, Michael Fischberg, and many of your mentees have talked of you as a great mentor. What is the Gurdon style of leadership?
Well, I would be highly self-critical here – I don’t sit down with everyone for an hour a week to go through their results, I just wait until I see them over coffee and ask how things are going. So I must be a terribly bad mentor in the sense of not really doing a regular and methodical check of things. But I do like to think that people will get something just from ordinary conversation. Someone like Doug Melton was a really fantastic colleague, but that was all through his own ability – I can’t think what he got from me! I simply try to persuade people coming in to my lab to work on a worthwhile project, and then let them enjoy it.
I should just comment that Michael Fischberg really was a remarkable and generous mentor. He put me on to this nuclear transfer work, telling me that I should try anything I wanted to, and was extremely supportive. The very first paper on nuclear transfer – he didn’t do the experiments, but he was an author on it, and quite rightly so. But after that, almost to my embarrassment, he said ‘you take the endoderm cells, I will take the rest’. And so he wasn’t an author on the further papers – it was remarkably generous, really.
I had planned to ask if you are still connected to the lab bench, but I got my answer when I arrived to your office today as you were changing the media for a batch of Xenopus eggs. Is it important to you to maintain this connection?
I’ve always maintained my lab work, even when I was doing other things as well, and still teach nuclear transfer to my colleagues. This connection to the bench of course is not realistic for everyone, but I like to think that by doing it you sometimes discover things that might not be obvious. There’s no point in me using PCR machines or that sort of thing, and one of my colleagues at the moment is running a western blot for me. But the lab work I am doing now is more dependent on trying to find ways of getting these cells to do what I want them to do – and this is something I know well.
Has the Nobel Prize changed your life appreciably?
Well yes, in the sense that I get a ludicrous amount of invitations, which is running now at about 200 per year. You can’t begin to handle that – I travel less than I used to, and I am rather selective about what I accept. I get a lot of invitations not for my scientific contribution but rather for my school report, in which my biology master wrote that I would have no chance of succeeding as a scientist, and which is framed above my desk. That story obviously made a big impression too.
There’s also the recognition of the public. Very soon after the Nobel award was made known I happened to be in South Korea. Walking along the street, someone stopped me and asked if I was Dr Gurdon, and told me my photograph was in the paper. It was quite remarkable really, the coverage that the award got. It’s also obviously nice for people to appreciate my work, and Yamanaka’s, and that people were talking about reprogramming.
Is there anything that Development readers would be surprised to find out about you?
I take the view that it is important to keep reasonably fit and healthy. I’ve always kept an interest in various sporting activities, most notably skiing, skating and squash, which were my major activities, though I have turned in recent years to tennis from squash.
But I suppose what might surprise readers is that I am a complete non-intellectual. I just don’t read books, I hate reading, and I don’t go to the theatre either. If I’m asked why I don’t enjoy reading, I’ll say that it takes a long time, it’s much easier to talk to someone who has read the book and ask for the bottom line! I’m not interested in fiction, it’s just not for me. So I am really the ultimate non-intellectual.
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