A PhD position is open in the QARMA team (LIS – Marseille, France). The recruited student will join Paul Villoutreix’s group ( @paulvilloutreix ): Data Science and Developmental Biology. PhD students will also be part of the Turing Centre for Living Systems (CENTURI), an interdisciplinary research centre located in Marseille.
PhD project: The intrinsic geometry of a developing embryo – More info
Deadline: March 01, 2019
PhD duration: 3 years
Expected profile – selection criteria
The team is expecting students with a background in machine learning and a good knowledge of biology as well as a will to open new avenues at the intersection of biology and computer science. Candidates will be evaluated based on the following criteria:
Academic achievements
Past research experience (internships, master thesis)
Interest to work in a multidisciplinary research environment
Enthusiasm and communication skills
How to apply: Students are required to apply on CENTURI’s website. Applications must include the following documents (compiled into a single PDF file):
CV
cover letter
transcript of your MSc’s grades (M1 and M2 if available)
Last year, I started to experiment with signing my reports for peer review of manuscripts, inspired by other people on twitter (@kaymtye, @AndrewPlested who in turn were inspired by Leslie Voshall). This year, the experiment is a bit different. I will only review for journals that allow non-anonymous peer-review.
Why?
That was the question raised by an editor. At first the editor did not want me to sign my review, since that was the default. However, after some back-and-forth over email, permission was granted. My main argument to sign was that I think it makes me a better reviewer (that’s right “I think”, these things are difficult to quantify, you know), since I will be less sloppy more precise, more constructive and more realistic in terms of requesting new experiments. Another advantage is that the authors have a better idea of who they are dealing with. They can better assess the expertise of the referee and respond accordingly. Recently, I received non-anonymous reviews for a submitted manuscript for the first time. This was a very positive experience and it strengthens my opinion that signed reviews make the peer review process more human.
I realize that I can sign my reviews and reveal my identity because I am privileged. I have a permanent position at a well-regarded university in a research group with a solid track record. However, being privileged should not stop me. And I think that the privileged have an important role in improving the peer review system. Signed peer reviews are not necessarily a magic bullet, but a good start would be to move away from the anonymous review as a standard. Journals that allow signed reviews should make that clear to reviewers during the peer review process.
What’s next?
The debate around signed reviews is not new. Similar issues with disclosing one’s identity apply to commenting on preprints. Signing reviews or other type of comments is not without risk for early stage career researchers or other researchers in vulnerable positions. We have discussed this in our preprint journal club and in my opinion early stage career researchers (PhD candidates, post-docs) should not sign public comments by default. If they want to disclose their identity, I’d recommend to directly contact the authors with their feedback by email.
One way to protect young researchers would be to co-review and co-sign with a senior scientist. Another opportunity is the cross-commenting on peer review reports that several journals are implementing. One could imagine that multiple reviewers draft a single review report and sign this together. This generates a review report with an author list, which has the advantage that the comments cannot be traced back to a single person. The downside is that such a collaborative review may require substantially more effort and time.
Finally…
As said in the intro, signing peer review reports is an experiment. So far, I am pleased with the results and I will continue. There may, however, be some unwanted side-effects that will stop my experiment. In the meantime, I hope that reviewers realize that signing review reports is often an option and that they give it some serious thoughts.
We offer one fully-funded postdoctoral position up to five years in the Laboratory of Genome Integrity located at the main campus of the National Institutes of Health (NIH/NCI, Bethesda, MD).Our laboratory uses human and mouse embryonic stem cells (ESCs) as well as mouse embryos to understand the molecular mechanisms underlying the maintenance/exit of pluripotency and self-renewal. Understanding cell plasticity, pluripotency and differentiation to get a better comprehension of embryonic development, cell transformation and cancer are our scientific interests.
The applicant should have or about to have a PhD in Developmental Biology, Genetics or similar, and must have demonstrated expertise in mouse embryology and in vitro embryo manipulation. Knowledge on mammalian tissue culture, molecular biology and/or next generation sequencing technologies and computational biology will be considered as an advantage. The applicant will have the opportunity to develop his/her research program or lead ongoing projects.
We seek a highly motivated, interactive, creative individual, eager to learn and develop new technologies and complex cell systems based on live cell/embryo imaging, 3D modelling and CRISPR-based editing interested in understanding how a single cell can develop into a complex multicellular organism in vitro and in vivo.
Please send a brief cover letter, CV and at least two reference letters via e-mail to:
Welcome to our monthly trawl for developmental biology (and related) preprints.
January was notable for the number of preprints on Xenopus development, plus a trio on Piezo channels, two on ctenophores, and a preprint on preprints that has also been preLighted (very meta).
The preprints were hosted on bioRxiv, PeerJ, andarXiv. Let us know if we missed anything, and use these links to get to the section you want:
Hedgehog signaling controls progenitor differentiation timing
Megan Rowton, Andrew D. Hoffmann, Jeffrey D. Steimle, Suzy Hur, Xinan Holly Yang, Alexander Guzzetta, Sonja Lazarevic, Chul Kim, Nikita Deng, Emery Lu, Jessica Jacobs-Li, Shuhan Yu, Mervenaz Koska, Erika Hanson, Carlos Perez-Cervantes, Sunny Sun-Kin Chan, Kohta Ikegami, Daniel J. Garry, Michael Kyba, Ivan P. Moskowitz
A fetus and a placenta from Sandovici, et al.’s preprint
Fetus-derived IGF2 matches placental development to fetal demand
Ionel Sandovici, Aikaterini Georgopoulou, Antonia S Hufnagel, Samira N Schiefer, Fatima Santos, Katharina Hoelle, Brian Y.H. Lam, Giles S.H. Yeo, Keith Burling, Jorge Lopez-Tello, Moritz Reiterer, Abigail L. Fowden, Graham J. Burton, Amanda N. Sferruzzi-Perri, Cristina M. Branco, Miguel Constancia
Piezo1 is required for outflow tract and aortic valve development.
Adele Faucherre, Hamid Moha ou Maati, Nathalie Nasr, Amelie Pinard, Alexis Theron, Gaelle Odelin, Jean Pierre Desvignes, David Salgado, Gwenaelle Collod Beroud, Jean Francois Avierinos, Guillaume Lebon, Stephane Zaffran, Chris Jopling
Preformed Chromatin Topology Assists Transcriptional Robustness of Shh during Limb Development
Christina Paliou, Philine Guckelberger, Robert Schöpflin, Verena Heinrich, Andrea Esposito, Andrea Maria Maria Chiariello, Simona Bianco, Carlo Annunziatella, Johannes Helmuth, Stefan Haas, Ivana Jerković, Norbert Brieske, Lars Wittler, Bernd Timmermann, Mario Nicodemi, Martin Vingron, Stefan Mundlos, Guillaume Andrey
The phylogenetically distinct early human embryo
Manvendra Singh, Thomas J Widmann, Vikas Bansal, Jose L Cortes, Gerald G Schumann, Stephanie Wunderlich, Ulrich Martin, Jose L Garcia-Perez, Laurence D Hurst, Zsuzsanna Izsvak
N6-methyladenosine dynamics during early vertebrate embryogenesis
Havard Aanes, Dominique Engelsen, Adeel Manaf, Endalkachew Ashenafi Alemu, Cathrine Broberg Vagbo, Leonardo Martin, Mads Lerdrup, Klaus Hansen, Sinnakaruppan Mathavan, Cecilia Winata, Robert B. Darnell, Peter Alestrom, Arne Klungland
LINE-1 retrotransposition impacts the genome of human pre implantation embryos and extraembryonic tissues
Martin Munoz-Lopez, Raquel Vilar, Claude Philippe, Raheleh Rahbari, Sandra R. Richardson, Miguel Andres-Anton, Thomas Widmann, David Cano, Jose L. Cortes, Alejandro Rubio-Roldan, Etienne Guichard, Sara R. Heras, Francisco J. Sanchez-Luque, Maria Morell, Elisabet Aguilar, Marta Garcia-Canadas, Laura Sanchez, Angela Macia, Pedro Vilches, Maria Concepcion Nieto-Perez, Antonio Gomez-Martin, Beatriz Gonzalez-Alzaga, Clemente Aguilar- Garduno, Adam D. Ewing, Marina Lacasana, Ignacio S. Alvarez, Richard Badge, Geoffrey J. Faulkner, Gael Cristofari, Jose L. Garcia-Perez
The UTX Tumor Suppressor Directly Senses Oxygen to Control Chromatin and Cell Fate
Abhishek Chakraborty, Tuomas Laukka, Matti Myllykoski, Alison Ringel, Matthew Booker, Michael Tolstorukov, Yuzhong Meng, Sam Meier, Rebecca Jennings, Amanda Creech, Zachary Herbert, Jessica Spinelli, Samuel McBrayer, Benjamin Olenchock, Jacob Jaffe, Marcia Haigis, Rameen Beroukhim, Sabina Signoretti, Peppi Koivunen, William G. Kaelin Jr.
| Stem cells, regeneration & disease modelling
Micropatterned hESC colonies from Britton, et al.’s preprint
Tetraploidy in rodent cardiac stem cells confers enhanced biological properties
Kathleen Broughton, Tiffany Khieu, Nicky Nguyen, Michael Rosa, Sadia Mohsin, Pearl Quijada, Jessica Wang, Oscar Echeagaray, Dieter Kubli, Taeyong Kim, Fareheh Firouzi, Megan Monsanto, Natalie Gude, Robert Adamson, Walter Dembitsky, Michael Davis, Mark Sussman
The Evolution of Placental Invasion and Cancer Metastasis are Causally Linked
Kshitiz Gupta, Junaid Afzal, Jamie D. Maziarz, Archer Hamidzadeh, Cong Liang, Eric M. Erkenbrack, Hong Nam, Jan-Dirk Haeger, Christiane Pfarrer, Thomas Hoang, Troy Ott, Thomas Spencer, Mihaela Pavlicev, Doug Antczak, Andre Levchenko, Gunter P. Wagner
YAP/TAZ as a Novel Regulator of cell volume
Nicolas Andres Perez Gonzalez, Nash Delta Rochman, Kai Yao, Jiaxiang Tao, Mihn-Tam Tran Le, Shannon Flanary, Lucia Sablich, Ben Toler, Eliana Crentsil, Felipe Takaesu, Bram Lambrus, Jessie Huang, Vivian Fu, Andrew Holland, Steven An, Denis Wirtz, Kun-Liang Guan, Sean Sun
Multimodal cell type correspondence by intersectional mFISH in intact tissues
Philip R Nicovich, Michael J Taormina, Christopher A Baker, Thuc Nghi Nguyen, Elliot R Thomsen, Emma Garren, Brian Long, Melissa Gorham, Jeremy Miller, Travis Hage, Alice Bosma-Moody, Gabe J Murphy, Boaz P Levi, Jennie L Close, Bosiljka Tasic, Ed S Lein, Hongkui Zeng
The genome of C57BL/6J “Eve”, the mother of the laboratory mouse genome reference strain
Vishal Kumar Sarsani, Narayanan Raghupathy, Ian T Fiddes, Joel Armstrong, Francoise Thibaud-Nissen, Oraya Zinder, Mohan Bolisetty, Kerstin Howe, Doug Hinerfeld, Xiaoan Ruan, Lucy Rowe, Mary Barter, Guruprasad Ananda, Benedict Paten, George M. Weinstock, Gary A. Churchill, Michael V. Wiles, Valerie A. Schneider, Anuj Srivastava, Laura Reinholdt
Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
Ignacio Garcia-Tunon, Veronica Alonso-Perez, Elena Vuelta, Sandra Perez- Ramos, Maria Herrero, Lucia Mendez, Jesus Maria Hernandez-Sanchez, Marta Martin-Izquierdo, Raquel Saldana, Julian Sevilla, Fermin Sanchez-Guijo, Jesus Maria Hernandez-Rivas, Manuel Adolfo Sanchez-Martin
Perceptions and Prospects in Life Sciences in a Heterogenous Latin American Population
Leonardo M.R. Ferreira, Giovanni A. Carosso, Bruno Lopez-Videla, Gustavo Vaca Diez, Laura Ines Rivera-Betancourt, Yara Rodriguez, Dalila G. Ordonez, Natalia Montellano Duran, Diana K. Alatriste-Gonzalez, Aldo Vacaflores, Soad Bohorquez, Lilian Gonzalez Auza, Christian Schuetz, Carolina Alexander-Savino, Omar Gandarilla Cuellar, Mohammed Andres Mostajo Radji
*all authors contributed equally; cross-posted from here.
The growing adoption of preprints over the last five years in the biological sciences has driven discussion within the academic community about the merits, goals, and potential downsides of disseminating work prior to peer review. However, the community has lacked a systematic bibliometric analysis (Figure 1) of preprints in which to root these discussions. Abdill and Blekhman1 have generated an analysis of the data to identify trends in preprint usage, popularity, and outcomes, and created the website rxivist.org to facilitate future analysis and to provide an alternative platform for discovering actively discussed preprints.
As members of the preLights community who support the increased uptake of – and discussion about – preprints in the life sciences, we have taken this opportunity to reflect on how preprint success can be measured, and what the data provided by Abdill and Blekhman tell us about preprints in the life sciences.
Figure 1, reproduced from Figure 1, Abdill and Blekhman 2019, under a CC-BY-ND-4.0 international license. Total preprints posted to bioRxiv over a 61-month period from November 2013 through November 2018. (a) The number of preprints (y- axis) at each month (x-axis), with each category depicted as a line in a different color. (a, inset) The overall number of preprints on bioRxiv in each month. (b) The number of preprints posted (y-axis) in each month (x-axis) by category. The category color key is provided below the figure.
What makes a preprint successful?
Citation rates have long served as the bibliometric gold standard for measuring the scientific impact of publications. However, follow-up studies and reviews can take years to make their own appearance in the literature, meaning that the impact of any one study based on citations can only be assessed in the long term (read: years to decades).
Standing in sharp contrast to this slow, cumulative view of scientific impact, the core goal of preprints is to accelerate the dissemination of scientific observations – to promote discussion, collaboration, and quick follow-up. In this sense, the ability of a preprint to reach the scientific community quickly and effectively is perhaps the ultimate measure of its success.
This alternate perspective justifies Abdill and Blekhman’s use of preprint downloads, Twitter activity and eventual publication outcomes as key quantitative metrics of preprint success, but these naturally raise some questions as well. Is using Twitter to define “popularity” acceptable? Our experience suggests Twitter is indeed the dominant social media platform for spreading preprints – but this does raise the possibility of excluding communities of scientists, and their opinions, based on a lack of Twitter presence. Our hope is that preprint curation platforms (such as preLights) will play an ever more important role in dissemination.
As with all bibliometric methods, it is important to keep in mind the potential for manipulation or misuse – as summarised in Goodhart’s Law, when a measure becomes a target, it ceases to be a good measure2. While we applaud the introduction of quantitative measures for preprint “success” in the rxivist project and the enablement of detailed analyses conducted by the authors, we are mindful that a small number of metrics should not be used to define a preprint without critical engagement and evaluation.
Social media and the dissemination of science
The use of Twitter activity as a metric for popularity of preprints on the rxivist website helps flag some issues of interest. First, biases in research communication using social media are poorly understood. For example, among scientists active on Twitter, there is widespread variation in the number of their Twitter followers3. As such, the interests of a single popular scientific influencer could potentially drive far greater disparities than are reflected in quality.
Second, preprinting opens up a space – until recently unexplored – for community engagement, spanning the gap between the dissemination of the authors’ unfiltered data and ideas, and the final peer-approved version. The bulk of this engagement takes place on social media platforms like Twitter. While the informal, loose, and relatively egalitarian structure of communicating this way can be immensely liberating, it is also easy to get lost in the noise, or to simply be overwhelmed by information overload. It is possible to go to sleep in the UK and miss a wide-ranging and critical discussion about a preprint taking place in US time zones – by the time you wake up, Twitter has moved on to the next big thing. In a world of science dissemination via social media, preprint curation and journal club initiatives (and the biorXiv comments section) must take on a critical role – that of providing a stable platform for sustained, publicly recorded engagement while remaining responsive to the abbreviated timescales driven by social media.
What happens, then, after the first few weeks, when the tweetstorms have settled, and the commentaries have been posted? We explore this in more detail below.
Time to publication
The eventual publication outcomes of preprints – their appearance in some peer-reviewed journal – feature heavily in the metrics utilised by Abdill and Blekhman. The authors found that two-thirds of bioRxiv preprints posted between 2013 and the end of 2016 were eventually published in peer-reviewed form. This high rate indicates that authors tend to post quality preprints, therefore initial fears that work not meeting certain scientific standards might ‘clog’ bioRxiv appear to be unfounded. The median time from the posting of a preprint on bioRxiv to its final publication in a peer-reviewed journal was just under six months, although this varied substantially, depending on the eventual journal of publication (Figure 2). In terms of accelerating science, pushing forward the public dissemination of new information by an average of six months could be considered a significant success- especially for early-career researchers, for whom six months represents a large fraction of their total career progress. Having their work and ideas publicly available enables them to receive feedback earlier and prepare effectively for career transitions.
Figure 2, reproduced from Figure 6 of Abdill and Blekhman, 2019, under a CC-BY-ND 4.0 international license. The interval between the date a preprint is posted to bioRxiv and the date it is first published elsewhere. (a) A histogram showing the distribution of publication intervals—the x axis indicates the time between preprint posting and journal publication; the y axis indicates how many preprints fall within the limits of each bin. The yellow line indicates the median; the same data is also visualized using a boxplot above the histogram. (b) The publication intervals of preprints, broken down by the journal in which each appeared. The journals in this list are the 30 journals that have published the most total bioRxiv preprints; the plot for each journal indicates the density distribution of the preprints published by that journal, excluding any papers that were posted to bioRxiv after publication. Portions of the distributions beyond 1,000 days are not displayed.
However, as Abdill and Blekhman point out, “time-to-publication” is influenced by a plethora of factors, including journal behavior, when preprints are posted in the publication process, and whether preprints are ever published at all.
It’s worth noting that the two “slowest” and the two “fastest” journals (in terms of time from bioRxiv posting to final publication) both fall within the field of genetics and genomics – Genetics and G3 on the “fast” end and Nature Genetics and Genome Research on the “slow” end. This suggests that field-specific attitudes and norms about preprint usage do not drive the difference in publication times by journal.
Preprints can be posted at many stages of the publication process. More field-specific data on when authors typically post preprints would help us understand how authors are utilising preprints – are they sharing work as close to publication as possible, or aiming to get feedback on their work prior to submitting it to a journal? Although the total number of infractions appears to be small, it’s worth noting that some authors appear to be flaunting the bioRxiv guidelines that state that preprints must be uploaded prior to acceptance in a journal.
Publication as a readout of preprint quality
The authors find a significant correlation between preprint download counts and the impact factor of the eventual journal of publication. This suggests that the popularity of a preprint reflects the eventual perceived impact and quality of the work, and that there is some informal consensus among the scientific community about the scientific quality of both published and unpublished preprints. However, the authors rightly point out that publication, particularly in a high profile journal, may actually drive further downloads. Additionally, both metrics are susceptible to biases that distort the connection between popularity and quality – name recognition of the principal investigator, or “hot” topics within a field, for example. Therefore, further validation will be needed to determine how precisely this measure correlates with the scientific utility of a preprint. For instance, it will be interesting to examine whether the number of downloads a preprint receives in its first month holds predictive value for eventual publication.
On the other end of the scale, what about preprints that are never published in peer-reviewed form? Missing links to final publications in the rxivist website and analysis may be partly a technical issue – changes in authors or preprint title make it difficult for automated formats to match preprints to their final publications – but this is unlikely to account for many preprints. Instead, of greater concern is whether these unpublished preprints are of lower quality and are constructed on the back of poor science that would not pass the stringency of peer review. We argue that a lack of final publication should not be held as an indictment of the quality of a preprint. First, preprints help to communicate results more rapidly, particularly in instances where matching a manuscript to typical journal expectations may be difficult or impossible – for example, following the departure of the primary author from a lab. Second, preprints can serve as useful repositories of negative results, which often remain unpublished – after all, “a negative result is still a result”. Therefore, preprints communicating preliminary or shorter stories can prompt discussion and study in the field and be as successful as those that lead to full publication, especially if they invalidate previous hypotheses or drive changes in research that lead in different directions.
Conclusion
The explosive rise of bioRxiv preprints in the life sciences since 2013 has clearly demonstrated the importance of increased speed and visibility through the publishing process. Leveraging social media has also been key to this process. The rxivist project is a laudable effort to collect and collate data on preprint usage, which can in turn be used to measure the influence and uptake of preprints in the life sciences, while he rxivist website makes this data easily accessible and allows others to easily interact with the metadata. We believe the data indicate that preprints are generally successful and that their increasing adoption is a positive trend for not just the life sciences, but science as a whole. While the metrics for preprints diverge from those used for standard peer-reviewed publications, this appropriately reflects the difference between the standards and goals for preprints and that of peer-reviewed journal articles. We thank the authors of the rxivist project for their work, and are excited to watch as our understanding of preprint publishing continues to grow in the future.
References
Abdill, R. J. & Blekhman, R. Tracking the popularity and outcomes of all bioRxiv preprints. bioRxiv 515643 (2019). doi:10.1101/515643
Biagioli, M. Watch out for cheats in citation game. Nature535, 201–201 (2016).
Côté, I. M. & Darling, E. S. Scientists on Twitter: Preaching to the choir or singing from the rooftops? FACETS3, 682–694 (2018).
Picture: Photographic plate from Raphael Weldon’s 1902 paper in Biometrika.
In the latest episode of Genetics Unzipped, Kat Arney is exploring some more of the leading 100 ideas in genetics. She’s been digging around in the genetic vegetable patch in search of flavourful GM tomatoes, chunky onion genomes and Mendelian peas.
If you enjoy the show, please do rate and review and spread the word. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com
3 year-POSTDOC + Starting grant (OPEN-CALL BEATRIU de PINOS program) to join our lab on EvoDevoGenomics in Barcelona
We are seeking candidates to join our lab to study our favorite chordate model Oikopleura dioica, in which we are currently interested in heart and muscle development, tail elongation and the impact of gene loss on the evolution of gene regulatory signalling networks. Click here for a tour “A day in our lab” posted in The Node
We have also engaged a new EcoEvoDevo line investigating if the developmental mechanisms of marine embryos are ready to respond to climate change, including biotoxins derived from algal blooms. Click here for a tour on this new EcoEvoDevo adventure.
Our approaches include RNAseq, CRISPR, RNAi, Fluorescent-Microscopy
DEADLINE call: March 4th 2019 (contact for enquiries as soon as possible canestro@ub.edu)
REQUIREMENT: to have defended the PhD within the period January 1st 2011 – December 31st 2016
DURATION: 3 years: starting not later than February 2020
FUNDING: 132.300€ total gross salary for 3 years + 12.000€ research funds
CONTACT: Interested candidates, please send an email to Cristian Cañestro (canestro@ub.edu), including a brief letter of interest, a brief CV, including list of publications with their impact, and technical skills for post-doc applications, and official scores for doctoral candidates, all together in ONE single pdf file.
The Company of Biologists, as well as publishing Development and four other journals and offering travel and conference grants, runs a successful series of Workshops. The Workshops provide leading experts and early career scientists from a diverse range of scientific backgrounds with a stimulating environment for the cross-fertilisation of interdisciplinary ideas. The programmes are carefully developed and are intended to champion the novel techniques and innovations that will underpin important scientific advances.
The Workshop Committee are currently seeking proposals for Workshops to be held during 2021.The deadline for applications is 31 May 2019.
For sexually reproducing organisms, the diploid life cycle starts with the fusion of a sperm cell with an egg cell. This process, known as fertilization, results in the formation of a zygote, the first diploid cell from which a multicellular embryo develops. In animals, embryo initiation has been shown to be under maternal control, driven by the gene products stored in the egg cytoplasm. For example, in the absence of zygotic transcription, Caenorhabditis elegans zygotes can progress to the 100 cell stage before arresting1. This raises an important question: if the maternal gene products are able to drive embryo development, why do egg cells depend on sperm cell fertilization for embryo initiation? Why is there a fertilization block in sexual organisms?
This work was started in the Sundaresan lab at UC Davis to understand embryo initiation in rice, particularly the role of BABY BOOM (BBM) genes in it. BBM genes belong to AP2 family of transcription factors. Our previous study showed that the expression of three BBM genes is induced in zygotes after fertilization2. Among these, BBM1 was specifically expressed from the male allele (Fig. 1). Although this was an important observation, it needed further confirmation. We attempted confocal imaging of intact zygotes expressing BBM-GFP, but when BBM-GFP plants were used as either the male or the female parent, it did not work: rice carpels are thick and green, so the GFP signal was impossible to view through this tissue. Isolating zygotes is possible in rice and had been done by our team previously, but it is a tedious process which involves manual dissection. Isolating and imaging a statistically significant number of zygotes for our purpose would have probably taken us a year or more, so we decided to use antibodies against GFP to detect the BBM1-GFP expression in zygotes. However, this created another challenge- is immunohistochemistry sensitive enough to detect the expression in a single nucleus? It did work, and it worked well!
Figure 1. Mechanism of embryo initiation in rice. BBM1, an AP-2 domain transcription factor is expressed in the sperm cell but not in the egg cell. After fertilization, its expression from the male genome activates the embryo initiation program in the zygote. Its expression in the later stages of embryo development is required for embryo organ formation.
We could now confirm that BBM1 is expressed only from the male allele in zygotes, immediately after fertilization (Fig. 1). We also found that ectopic overexpression of BBM1 induces somatic embryogenesis in heterologous tissues like leaves. The latter observation, combined with male-specific expression of BBM1 in the early zygotes, lead us to hypothesize that BBM1 expression probably initiates embryogenesis after fertilization. To test this hypothesis, we drove the expression of BBM1 in egg cells using an Arabidopsis egg cell promoter3. At that time (back in 2014), we were not even sure if this promoter was going to work in rice. Egg cell expression of BBM1 resulted in embryo formation without the need for fertilization, a process known as parthenogenesis. Thus, it turns out that the sperm cell transmitted BBM1 initiates embryogenesis after fertilization (Fig.1). BBM-like genes were first discovered in Brassica microspore cultures4. Although, BBM-like genes from Arabidopsis and Brassica have been shown to induce somatic embryogenesis4, their role is zygotic embryogenesis is not known because their loss-of-function mutants do not show any embryonic phenotypes.
The variety of rice that we use for our experiments, Kitaake, has one of the shortest generation times among different experimental varieties of rice (still about four months!). However, generating new transgenic lines from tissue culture can take up to 8 months. So, using rice for studying genetics can be a lengthy affair. However, we decided to take this long path and study the genetics of BBM genes in rice. The vectors for CRISPR-Cas9 gene editing were provided by Dr. Bing Yang from Iowa State University. We created a single knockout mutant of BBM1, but it did not show any phenotype (Fig. 2a). The double mutant of BBM1 and BBM3 (bbm1 bbm3) did not show any embryonic phenotype either (Fig 2a). An attempt to create a triple mutant using a single CRISPR-Cas9 construct was unsuccessful as the triple knockout construct would not regenerate plants in the tissue culture. This was a setback; however, it also meant that triple mutant is probably embryo lethal. To work around this, we created another double mutant of BBM2 and BBM3 (bbm2 bbm3), crossed it with bbm1bbm3 double mutant and selfed the progeny for the next two generation (Fig. 2b). The triple mutant was indeed embryo lethal (Fig. 2b). But instead of observing a typical 25% lethality (expected from Mendelian genetics), because the mother plant was segregating only for BBM1 (BBM1/bbm1 bbm2/bbm2 bbm3/bbm3), there was a 36% lethality. This was found to be linked to the male transmission of BBM1 from the sperm cell (Fig. 1). This proves that a functional copy of BBM1 from the sperm cell is essential for embryo initiation in rice. This is a novel mechanism that explains why the egg cell (at least in rice) depends on sperm cell fertilization to initiate embryo development.
Figure 2. Schematic showing constructs for mutants and redundancy in BBM1, BBM2 and BBM3 genes. (a) bbm1 mutant alone, or in combination with bbm3 have no embryo phenotypes. Also, loss-of-function bbm2 bbm3 double mutant shows no aberrant embryonic phenotype. (b) bbm1 bbm3 and bbm2 bbm3 double mutants were crossed to create bbm1 bbm2 bbm3 triple knockout mutant, which was embryo lethal.
This work was started purely as a development biology project, but we soon realized this understanding of the basic mechanism of embryo initiation can have agricultural applications. The first application is the conversion of egg cell directly into an embryo without fertilization: it meant we could generate haploid plants. Haploids have only one set of chromosomes, in this case maternal. This makes them efficient agricultural breeding tools as homozygous lines can be produced in one generation after chromosome doubling, bypassing the several generations it takes by inbreeding procedures. The technique we developed does not involve the tedious and laborious tissue culture techniques used in microspore cultures or rescuing and culturing haploid embryos in subsequent generations as seen in wide crossing procedures. The haploids can be grown simply from functional seeds (Fig. 3a).
Figure 3. Haploids and synthetic apomicts in rice. (a) Two segregating sibling plants from BBM1 egg cell expressing mother. The haploid plant is a result of parthenogenesis and diploid plant of sexual reproduction. (b) Progenies from a diploid synthetic apomixis mother. Diploid plant develops from the parthenogenesis of diploid egg cell created by MiMe and hence maternal clone. Tetraploid sibling develops sexually from the fusion of diploid gametes.
The second application is synthetic apomixis by which an unreduced diploid egg cell is converted into a maternal clone, allowing for the maintenance of hybrid vigor. Hybrid vigor, or heterosis refers to the increase in yield, growth or other quantitative characteristics in F1 hybrids, compared to parental inbred lines. However, the genetic combinations that lead to this vigor in F1 hybrids, segregate in the F2 generation due to sexual reproduction and thus resulting in loss of vigor. For this reason, farmers need to buy hybrid seeds, every sowing season. Therefore, a hybrid crop that could self-reproduce through seeds while maintaining the parental heterozygosity would solve this problem.
During sexual reproduction, meiosis results in recombination and segregation of genetic traits, and fertilization creates new genetic combinations. MiMe (mitosis instead meiosis), is a genetic approach that skips meiosis and converts meiotic cell division into a mitotic like division5. This approach was developed by our collaborator, Raphael Mercier’s group at INRA France. We combined MiMe with our BBM1 induced parthenogenesis system. The two approaches together produced progenies which have the same genetic constitution as that of the mother plant. In other words, the progenies are genetic clones of the mother plant (Fig. 3b). The MiMe produces diploid egg cells which parthenogenetically develop into embryos due to BBM1 expression. The clonal nature of progeny and mother plant was confirmed by whole genome sequencing (Dr. Debra Skinner analyzed the sequences). The endosperm that developed was, however, sexual. Since the gametes are diploid, the endosperm in progenies is hexaploid instead of the usual triploid. This 6X endosperm increases the seed size (Fig. 4a). Thus, this synthetic apomixis approach not only results in clonal propagation but also increases the seed size and hence the yield. This mode of apomixis is seen in some naturally apomictic plants like Boechera6 and others. Engineering apomixis in crop plants ensures fixation of hybrid vigor and stabilization of superior heterozygous genotypes. Also, the yield, quality and exchange of vegetatively propagated true seed crops can been improved by introgression of apomixis (hence seed propagation) as the accumulation of somatic mutations, viruses, and other pathogens over successive generations can be avoided. The successful engineering of apomixis in crop plants is a significant step towards achieving the food security for growing world population.
Figure 4. Seed size in synthetic apomixis plants. (a) Seeds from different genotypes having different ploidy for embryo (em) and endosperm (en). The seed size increases in synthetic apomictic pants due to increase in endosperm ploidy (6X). 1, wild-type; 2, haploid synthetic apomictic seed; 3, control MiMe; 4, diploid apomictic seed; and 5, tetraploid apomictic seed. (b) Genotyping team at Sundar lab, UC Davis.
A major challenge we faced during this study was to be able to genotype the thousands of rice plants used in this study (Fig. 4b). The plants needed to be genotyped for T-DNA insertions, copy number determination and CRISPR-Cas9 mutation analysis (quite often for three genes in the same plant). We highly appreciate the helping hand from Bao Nguyen (now at UC Santa Cruz) and Alina Yalda. Preparing samples for flow cytometry for ploidy determination was another time-consuming procedure. However, the final outcome of being able to decipher the mechanism of sexual reproduction in rice and utilization of this knowledge to make it reproduce asexually, made it all worth the effort!
Full Time one year employment contract (renewable for three years)
Location: University of Cyprus
The University of Cyprus (www.ucy.ac.cy) invites applications for one full time postdoctoral associate to work in the Laboratory of Cell and Developmental Biology (http://xeno.biol.ucy.ac.cy).
DUTIES AND RESPONSIBILITIES
The researcher will be part of a team working on a highly ambitious Research Promotion Foundation Strategic Infrastructure grant that aims to establish super-resolution microscopy in Cyprus and explore questions related to how cells sense and respond to mechanical stimuli. These will be addressed using cutting edge instrumentation and methodologies both in vitro and in vivo. The team will go on to employ the principles revealed by these studies for the development of novel therapeutic approaches for the treatment of cancer and metastatic disease, biomedical applications and in regenerative medicine. The post-doctoral associate will also be responsible for supervision of PhD and MSc candidates participating in the project, together with the group leader.
REQUIRED QUALIFICATIONS
PhD Degree in Cell or Molecular Biology or related area
An excellent research and academic record
Previous laboratory experience and knowledge of basic molecular and cellular biology techniques
Research experience in the areas of cell biology and/or mechanobiology
Ambition, enthusiasm and motivation, with an interest in a long-term career in research
Good publication record
APPLICATION PROCEDURE
All applications must include:
A cover letter, that describes how the applicant meets both the selection criteria and their motivation
Curriculum Vitae, including description of past research experience
Name and contact details of at least two referees
Qualified applicants will be invited for a personal interview
TERMS OF EMPLOYMENT
The position will be funded through the grant INFRASTRUCTURES/1216/0060 andinitiated on a year-long contract, renewable annually for 3 years. It will include a competitive compensation package, depending on the candidate’s qualifications and experience. The monthly salary is set between €2200- €2700, depending on the candidate’s qualifications and experience. Employee and employer contributions will be deducted from the above amount. 13th Salary bonus or medical insurance coverage are not provided, but the candidate can be included in the UCY health plan if they choose to do so.
Contact information
Applications must be emailed to skourip@ucy.ac.cy by 5th February 2019, with the title “Postdoc position-MRU-2019”.
For more information please contact the coordinator of the project Dr. Paris A. Skourides tel.: +357-22892895 or email: skourip@ucy.ac.cy.
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