The Marine Biological Laboratory seeks a highly motivated individual to join the laboratory of Dr. Kristin Gribble in the full time position of Research Assistant I, II, or III. The successful applicant will contribute to our projects on the biology of aging, maternal effects on offspring health and lifespan, life history, evolution, and ecology using an aquatic invertebrate model system. The Gribble lab is housed within the Josephine Bay Paul Center, a collaborative research group addressing questions of microbial diversity, molecular evolution, and comparative genomics. Information about our research may be found at: http://mbl.edu/jbpc/gribble
Additional Information: Responsibilities for this position include, but are not limited to, designing and conducting experiments, rotifer and phytoplankton culture, PCR, qPCR, protein extraction and analysis, microscopy, data entry and analysis, and general laboratory maintenance and organization. This position requires occasional work on weekends to accomplish long-term life table experiments. The position will be for 1 year, but may be extended beyond this period contingent upon progress and funding.
Basic Qualifications: Applicants should have a B.A./B.S., or M.A/M.S. in biology, cell/molecular biology, biochemistry, or a related field. This position requires an independent, organized, and self-motivated individual with robust problem-solving skills. Excellent written, verbal, and interpersonal skills; attention to detail; and a strong work ethic are essential. Position level and salary will depend upon education and experience.
Preferred Qualifications: The ideal candidate will have one or more years of experience working in a research laboratory and will be familiar with standard laboratory practices and equipment. Previous experience with DNA, RNA, and protein extractions; next-generation sequencing library construction; PCR and qPCR; protein analysis; RNAi; microscopy; and bioinformatics is preferred. An understanding of basic molecular biology concepts is important.
Instructions: Apply at the MBL website and please provide the following required documents:
Cover letter describing your interests, skills, prior research experience, and motivation for joining the lab;
Curriculum vitae;
The names and contact information for three references (Please do not send letters at this time; we will contact references directly).
The Company of Bioloists’ Workshops provide leading experts and early-career researchers 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.
In November 2019, a Workshop on birth defects is being held with the aim of
dismantling boundaries between developmental biology and clinical birth defects research so that clinical findings can inform our understanding of the processes that construct a human being, which in turn can guide clinicians in order to deliver better care to patients.
There are around 10 funded places for early-career researchers available – a fantastic opportunity to share your research with leading scientists in an intimate setting.
An exciting role is now available with The Company of Biologists to enhance the community content on our journal websites and grow our social media presence in China through our new WeChat channel.
We publish five important journals that serve the biological research community. All have effective marketing and a good social media presence. We now seek to extend our community engagement, increase awareness of our charitable activities and build on our connections with early-career
researchers.
We are looking for an enthusiastic and motivated team player to support us in this initiative, which is initially planned as a one-year position as we determine future directions. Working with experienced marketing and editorial teams, you will be responsible for accurate and engaging short-form
content for the journal websites, generating WeChat content for our growing China-based audience, and writing stories about our grant recipients. We are open to new creative ideas.
Core responsibilities include:
• Engaging the scientific community through the journal websites and social media channels.
• Generating dynamic content ‘snippets’ to draw in readers.
• Developing our new WeChat channel to engage China-based researchers.
• Measuring usage and online behaviours to assess and guide strategies.
• Working with the marketing and editorial teams on other community engagement content such as video interviews and educational materials.
The successful applicant will have:
• A science degree, ideally in a field relevant to one or more of our journals.
• Experience with scientific communications such as social media, marketing or outreach.
• Ability to write accurate and engaging summaries for the non-specialist.
• Experience with a web content management system to create and publish web content.
• Experience with metrics (such as Google Analytics).
• Excellent written and verbal communication skills.
• Confident networking abilities and strong interpersonal skills.
This is an exciting opportunity within a well-established publishing company. The role is based in our attractive modern offices on the outskirts of Cambridge, UK.
The Company of Biologists (biologists.com) exists to support biologists and inspire advances in biology. At the heart of what we do are our five specialist journals – Development, Journal of Cell Science, Journal of Experimental Biology, Disease Models & Mechanisms and Biology Open – two of them fully open access. All are edited by expert researchers in the field, and all articles are subjected to rigorous peer review. We take great pride in the experience of our editorial team and the quality of the work we publish. We believe that the profits from publishing the hard work of biologists should support scientific discovery and help develop future scientists. Our grants help support societies, meetings and individuals. Our workshops and meetings give the opportunity to network and collaborate.
To apply, please send your CV by email to recruitment@biologists.com along with a covering letter that states your current salary, summarises your relevant experience and explains why you are enthusiastic about this opportunity. You must be able to demonstrate your entitlement to work in the UK. Applications should be made as soon as possible and by 21 June (late applicants may be considered).
A postdoc position is available in Uri Frank’s lab in Centre for Chromosome Biology, National University of Ireland, Galway. We study stem cells and regeneration in the cnidarian Hydractinia. This animal can regenerate a whole body from only tiny tissue fragments and is amenable to genome editing, live imaging, cell sorting by FACS, and transplantation. Due to its small size and translucent body, Hydractinia allows performing in vivo experiments that are difficult or impossible to conduct with most other animal models.
The research will be on the molecular mechanisms that drive cellular reprogramming during whole body regeneration in the absence of stem cells. For details, email Uri at <uri.frank@nuigalway.ie>. The position is funded by Wellcome Trust.
Most of what we know about axial patterning in insects comes from decades of careful, beautiful work done in flies. Thanks to the genetic screens of Christiane Nüsslein-Volhard and Eric Wieschaus in the late 1970’s, we learned that distinct classes of genes, many of them transcription factors, act in an elegant procession to achieve the designation of segment identity along the anterior-posterior and dorsal-ventral axes of the insect embryo. This process is robust when nature gets noisy- buffering against noise in RNA gradients to create precise boundaries, detecting coincidence of different proteins and translating their affinities into procedural specificity. Robustness ensures remarkable reproducibility of developmental processes in the face of novelty, since evolution is restless. And it is restless: two fully developed individuals of the same species with the same life history are full of small genetic differences, due to the persistent insinuation of random mutations. When we take a step backwards to look at two closely related species, we see much larger differences. Recognizing the value of this perspective has led to the field of Evo-Devo: that is, using an evolutionary perspective to better understand a developmental process and, in turn, using the knowledge of development to decipher the mechanisms of evolution.
Strength in numbers.
If you have ever looked at insects, you know that they have tremendous variation in morphologies in nature, and it turns out that this diversity arises in part from variation in their embryonic patterning program. Gerhard Krause described variation in developmental styles from his morphological studies of insect embryos in the 1930’s. Later, pioneers in modern Evo-Devo like Michael Akam, Nipam Patel and Diethard Tautz took this further: molecular comparison of evolutionarily distant insects illuminated developmental programs in a way that looking only at one species could not. Discoveries in beetles especially, but also aphids, crickets and others, showed that flies are not typical among insects and therefore are not the only important game in town: a point that insect evo-devo researchers are always quick to point out. And yet, many of us studying less traditional model systems today, like beetles, wasps, or bugs, nevertheless get siloed in our own organism’s biology. One of the greatest strengths of these insect systems is the ability to put things together by looking at where they drifted apart. It is through the diffracting lens of evolutionary differences that we can understand the origins of robustness and of genetic consensus. The ability to move experimentally in a single project between different organisms permits full expression of this colorful perspective.
Collaboration was essential for synthesis of the discoveries of Nüsslein-Vollhard and Wieschaus into the basis of our understanding of axial patterning. And so too is collaboration between developmental biologists in different (insect) systems to make fundamental discoveries about the origins of robustness and functional variation in developmental programs.
Our recent paper in eLife (Ray* and Rosenberg* et al, eLife 2019) describes such a finding about the ancestral function of a highly conserved peptide, which would not have been possible if the study had been limited to any single insect. In this post, we highlight the importance of the collaboration and how it came about, since this ability to move between insect species, and open data sharing, permitted the most profound insights of our work.
Better together.
Ray and Rosenberg et al., is a work that came together from 4 different groups working in different countries, and carried out over almost 10 years. Our story involves a few main molecular characters: Mille-pattes, a regulatory micropeptide; Shavenbaby, a transcription factor previously known for its key role in epidermis development; and Ubr3, an E3 ubiquitin ligase. Our paper’s story was inspired by the publication by Savard et al. (Cell 2006) showing that mille-pattes (mlpt), which they identified in an expression screen in the flour beetle Tribolium castaneum, is essential in Tribolium for abdominal segment formation. This finding was surprising at first because mlpt doesn’t produce a regular protein, but rather is a an apparently long non-coding RNA encoding only four tiny peptides of 11-32 amino acids in length. The suspense grew later, when studies of the same gene in flies, named polished rice (pri) or tarsal less (tal), revealed functions in leg patterning, trachea and epidermis differentiation, but no role in segmentation of the Drosophila embryo (Kondo et al., Nat Cell Biol 2007; Galindo et al., PLoS Biol 2007).
Our collaboration arose from parallel stories, which made possible the insights from comparison. We have decided to tell each of the stories, that became a powerful synergy and ultimately, enabled the most profound results.
1. Ray and Klingler.
Martin Kingler and Suparna Ray
Suparna Ray had joined the lab of Martin Klingler in Erlangen, Germany, towards the end of a collaborative genome-wide screen, iBeetle, whose aim was to uncover all developmental genes in Tribolium. mlpt was the newest kid on the block in the search for a definitive set of gap genes. Of particular interest was its interaction with Notch during Drosophila tarsal leg joint development, since Notch is a missing link long sought between the vertebrate segmentation clock and the segmentation clock of short germ insects, like Tribolium (Dequéant et al., Science 2006; Pueyo and Couso Dev Biol. 2011). Intrigued by the novel regulatory potential of micropeptides encoded from small open reading frames (smORFs) in developmental programs, the Klingler lab began to apply the most cutting edge approaches in Tribolium for functional analysis of gap genes (Schinko et al., BMC Dev Biol 2010 ; Schinko et al., Dev Genes Evol 2012). Shortly after Mlpt was discovered to activate the transcription factor Shavenbaby (Svb) in the fly epidermis (Kondo et al., Science 2010), RNAi knockdown of Triboliumsvb in the Klingler lab revealed its role in abdominal segmentation. Furthermore, the mlpt and svb RNAi phenotypes were similar, suggesting a functional interaction of these two genes in Tribolium embryonic segmentation.
Tribolium castaneum (flour beetle), adult
Meanwhile, out of the iBeetle screen (Schmitt-Engel et al., Nature Comm. 2015) came a small group of additional candidate genes showing mlpt-like RNAi phenotype. Intriguingly, these genes did not match any known developmental regulators. Instead, they correspond to enzymes involved in the ubiquitin proteasome system, often seen as a mere degradation factory that removes damaged or misfolded proteins. In particular, two genes located side by side and that give strong mlpt-phenotypes were predicted to encode ubiquitin E3 enzymes. These unexpected results prompted a whole flurry of research questions. Was the proteasome actually involved in Tribolium segmentation? How can a ubiquitous degradation machinery be required for the proper formation of posterior segments? Is the proteasome functionally related to mlpt and svb functions in segmentation, as suggested by strikingly similar phenotypes?
2. Rosenberg and Payre.
Payre and Rosenberg
Miriam Rosenberg was at New York University in the lab of Claude Desplan, studying Nasonia, a wasp whose embryonic development exhibits intermediate character between Tribolium and Drosophila. The Savard paper opened the possibility of a role of mlpt peptide in abdominal segment specification, a function not yet accounted for in control of Nasonia’s posterior development by known fly genes. She began to investigate Nasonia mlpt and found that it exhibits striking expression in the region of the embryo that gives rise, in a delayed fashion, to the most posterior abdominal segments (Rosenberg et al., eLife 2014). Shortly thereafter, François Payre, who has spent many years elucidating the function and evolution of Svb during epidermal differentiation, came to the Desplan lab for a sabbatical. The Payre lab in Toulouse, France, had just shown that the Svb protein is post-translationally processed, from a transcriptional repressor into an activator, in the response to mlpt (pri/tal) peptides in flies (Kondo et al., Science 2010). During his sabbatical in New York City, he began to characterize the expression of svb and of its target genes during Nasonia embryogenesis.
Upon his return to Toulouse, he and Rosenberg continued working together to characterize the expression and function of mlpt and svb in Nasonia. When the Payre lab demonstrated the role of the ubiquitin ligase Ubr3 in fly for Mlpt/Pri-mediated processing of Svb (Zanet et al., Science 2015), ubr3 joined the club in Nasonia, as well. Contrary to what was reported in fly, the long germ embryo of Nasonia showed essential functions for these genes in embryogenesis.
3. Descaras, Toubiana, Khila.
Abderrahman Khila
At the same time, in Lyon, France, Abderrahman Khila and his group were studying development of the water strider, Gerris buenoi. Water striders have the remarkable ability to walk on water, enabled by specialized hairs on the legs which allow it to trap air bubbles between leg and water, as well as differential elongation of the T2 leg (Khila et al., PLoS Genetics 2009; Armisén et al., 2018 BMC Genomics). An RNAi screen, searching for genes involved in these features in Gerris was being conducted by Amelie and William. Since Svb is a key regulator of hair formation in flies, they also assayed a putative role of svb in Gerris legs. When svb function was knocked down, they indeed observed a lack of hairs, but a prevalent phenotype in earlier stages was strong embryonic segmentation defects.
This was the starting point for testing whether the Svb partners were also involved in segment patterning in the water strider.
A collaboration is born!
By this time, Ray and Klingler were deep into the characterization of the E3 ubiquitin ligase when a poster from the Payre lab was presented at the French Fly meeting in Sète in 2014: they reported that the Svb processing triggered by Mlpt/Pri peptides relies on a limited proteasome degradation that critical requires the same E3 ligase, called Ubr3, as deduced from genome-wide functional screening.
François Payre
Excited by these data, Ray and Klingler made contact, and arranged a visit to the Payre lab in Toulouse to discuss each others’ findings. During this meeting, an important common link was made: the complementarity of expression of mlpt and svb that had been observed in Tribolium was also present in Nasonia, and possibly other, more basal insects.
Payre was already in touch with Khila and kept collaborating with Rosenberg, who had moved to Israel and expanded the study of (now) three genes to the milkweed bug, Oncopeltus, working with Tzach Auman, a graduate student in the lab of Ariel Chipman in Jerusalem. Payre suggested to all groups involved that to meet shortly before Christmas of 2017, in Lyon, to discuss interest in joining forces with all insect models into a single story about the functional evolution of this trio of genes.
An eventful meeting.
A consensus was reached, since all data supported the ancient evolutionary origin of mlpt/Svb function in the insect embryo, and in all species with segmentation function, both genes exhibit posterior expression. But in flies, Svb expression is restricted to the head segments. A nice idea was proposed by Amelie Descaras, a student from the Khila lab. Could we use the Drosophila system, with its facile genetic tools, to broadly express Svb in the early fly embryo, to see whether its ability to function in embryonic patterning could be re-awakened? Here, the finesse and fly expertise of Hélène Chanut-Delalande was invaluable in designing and executing experiments that not only established the genetic role for Mlpt in embryo patterning, via Svb, but also highlighted the exquisite specificity of this regulation- using surgical mutations in Svb, required for Ubr3/Mlpt association, that abrogate the segmentation phenotype.
Collaborators and beer! Hélène Chanut-Delalande, François Payre, Miriam Rosenberg, Martin Klingler, Abdou Khila
All together now…
Together, our research has traced the story of a multi-protein complex whose conserved functional interaction is critical for epidermal differentiation, leg development, and embryo segmentation. While each component of this complex has been strongly conserved through the evolution of insects, the segmentation function was lost in the most derived species, Drosophila. The dynamic expression patterns of mlpt and svb, which we observe in species that have diverged for hundreds of millions of years, underscore the continued importance of their segmentation function throughout the insect phylum. This ancestral role in segmentation calls out for study in additional, independently evolved, long germ insects. But it was the a-ha’s made possible by studies in divergent species with various modes of segmentation that coalesced into our current understanding of phenotypic plasticity and the evolutionary role of multi protein complexes.
When the Node invited us to highlight our recent advances with a post for young scientists about our work, we decided it was an important opportunity to highlight the significance of one central player in our work who does NOT appear in the published manuscript: the collaboration itself.
The authors would like to thank Martin Klingler, Abdou Khila, François Payre, Ariel Chipman and Galit Ophir for helpful comments on this piece.
Welcome to our monthly trawl for developmental biology (and related) preprints.
This month evo-devo is particularly well represented (from choanoflagellates to Portuguese men of war), and there’s a slew of single cell sequencing papers, lots on iPSC differentiation and some rooting around in our plant development section.
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:
EZHIP constrains Polycomb Repressive Complex 2 activity in germ cells
R Ragazzini, R Pérez-Palacios, HI Baymaz, S Diop, K Ancelin, D Zielinski, A Michaud, M Givelet, M Borsos, S Aflaki, P Legoix, PWTC Jansen, N Servant, ME Torres-Padilla, D Bourc’his, P Fouchet, M Vermeulen, R Margueron
Single-cell transcriptional diversity is a hallmark of developmental potential
Gunsagar S. Gulati, Shaheen S. Sikandar, Daniel J. Wesche, Anoop Manjunath, Anjan Bharadwaj, Mark J. Berger, Francisco Ilagan, Angera H. Kuo, Robert W. Hsieh, Shang Cai, Maider Zabala, Ferenc A. Scheeren, Neethan A. Lobo, Dalong Qian, Feiqiao B. Yu, Frederick M. Dirbas, Michael F. Clarke, Aaron M. Newman
Decoding the development of the blood and immune systems during human fetal liver haematopoiesis
Dorin-Mirel Popescu, Rachel A Botting, Emily Stephenson, Kile Green, Laura Jardine, Emily F Calderbank, Mirjana Efremova, Meghan Acres, Daniel Maunder, Peter Vegh, Issac Goh, Yorick Gitton, Jongeun Park, Krzysztof Polanski, Roser Vento-Tormo, Zhichao Miao, Rachel Rowell, David McDonald, James Fletcher, David Dixon, Elizabeth Poyner, Gary Reynolds, Michael Mather, Corina Moldovan, Lira Mamanova, Frankie Greig, Matthew D Young, Kerstin Meyer, Steven Lisgo, Jaume Bacardit, Andrew Fuller, Ben Millar, Barbara Innes, Susan Lindsay, Michael J.T. Stubbington, Monika D Kowalczyk, Bo D Li, Orr Ashenbrg, Marcin D Tabaka, Danielle Dionne, Timothy L. Tickle, Michal Slyper, Orit Rozenblatt-Rosen, Andrew Filby, Alexandra-Chloe Villani, Anindita Roy, Aviv D Regev, Alain Chedotal, Irene Roberts, Berthold D Gottgens, Elisa Laurenti, Sam Behjati, Sarah D Teichmann, Muzlifah Haniffa
Mouse embryos from Lewandowski, et al.’s preprint
The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis
Jordan P. Lewandowski, James C. Lee, Taeyoung Hwang, Hongjae Sunwoo, Jill M. Goldstein, Abigail F. Groff, Nydia Chang, William Mallard, Adam Williams, Jorge Henao-Meija, Richard A. Flavell, Jeannie T. Lee, Chiara Gerhardinger, Amy J. Wagers, John L. Rinn
The maternal-fetal interface of successful pregnancies and impact of fetal sex using single cell sequencing
Tianyanxin Sun, Tania L. Gonzalez, Nan Deng, Rosemarie DiPentino, Ekaterina L. Clark, Bora Lee, Jie Tang, Yizhou Wang, Barry R. Stripp, Changfu Yao, Hsian-Rong Tseng, S. Ananth Karumanchi, Alexander F. Koeppel, Stephen D. Turner, Charles R. Farber, Stephen S. Rich, Erica T. Wang, John Williams III, Margareta D. Pisarska
Contribution of Retrotransposition to Developmental Disorders
Eugene J. Gardner, Elena Prigmore, Giuseppe Gallone, Petr Danecek, Kaitlin E. Samocha, Juliet Handsaker, Sebastian S. Gerety, Holly Ironfield, Patrick J. Short, Alejandro Sifrim, Tarjinder Singh, Kate E. Chandler, Emma Clement, Katherine L. Lachlan, Katrina Prescott, Elisabeth Rosser, David R. FitzPatrick, Helen V. Firth, Matthew E. Hurles, on behalf of the Deciphering Developmental Disorders study
A conserved regulatory program drives emergence of the lateral plate mesoderm
Karin D. Prummel, Christopher Hess, Susan Nieuwenhuize, Hugo J. Parker, Katherine W. Rogers, Iryna Kozmikova, Claudia Racioppi, Eline C. Brombacher, Anna Czarkwiani, Dunja Knapp, Sibylle Burger, Elena Chiavacci, Gopi Shah, Alexa Burger, Jan Huisken, Maximina H. Yun, Lionel Christiaen, Zbynek Kozmik, Patrick Müller, Marianne Bronner, Robb Krumlauf, Christian Mosimann
Cross-species blastocyst chimerism between nonhuman primates using iPSCs
Morteza Roodgar, Fabian P. Suchy, Vivek Bajpai, Jose G. Viches-Moure, Joydeep Bhadury, Angelos Oikonomopoulos, Joseph C. Wu, Joseph L. Mankowski, Kyle M. Loh, Hiromitsu Nakauchi, Catherine VandeVoort, Michael P. Snyder
The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications
Noriko Ichino, MaKayla Serres, Rhianna Urban, Mark Urban, Kyle Schaefbauer, Lauren Greif, Gaurav K. Varshney, Kimberly J. Skuster, Melissa McNulty, Camden Daby, Ying Wang, Hsin-kai Liao, Suzan El-Rass, Yonghe Ding, Weibin Liu, Lisa A. Schimmenti, Sridhar Sivasubbu, Darius Balciunas, Matthias Hammerschmidt, Steven A. Farber, Xiao-Yan Wen, Xiaolei Xu, Maura McGrail, Jeffrey J. Essner, Shawn Burgess, Karl J. Clark, Stephen C. Ekker
Expanding the crispr toolbox with mad7 in zebrafish and human cells
Wesley A. Wierson, Brandon W. Simone, Zachary WareJoncas, Carla Mann, Jordan M. Welker, William A. C. Gendron, Michael A. Barry, Karl J. Clark, Drena Dobbs, Maura McGrail, Stephen C. Ekker, Jeffrey J. Essner
How do genes and their environment interact during development and evolution to generate phenotypic diversity? To answer these questions in the Miura lab, by focusing on diverse animal taxa, we are studying physiological and developmental mechanisms of phenotypic changes in animal life cycles in response to environmental shifts. By the way, I’m Kohei Oguchi, a third year PhD Candidate and a member of Toru Miura’s lab at Tokyo University. Among diverse animal taxa, we have been especially focusing on social insects, “termites”.
Termites
Termites are insects belonging to a major group of social insects with divisions of labors among morphologically and behaviorally differentiated individuals (i.e., castes). In termite colonies, although all individuals share the same genetic background, there are several types of castes such as reproductives, workers and soldiers (Fig. 1A, B). These caste differentiations and caste ratios are regulated by individual interactions among colony members to organize highly sophisticated societies. Therefore, researches on termites help us to understand how genes and environments interact during development, i.e., caste differentiation, and how this interaction generates novel phenotypes, i.e., castes.
Figure 1. Caste system and colony members of the focal termite species Hodotermopsis sjostedti. (A) Inside the colony of H. sjostedti. (B) Caste differentiation pathway of H. sjostedti
Recently, we identified a single gene which is responsible for the mandibular elongation that are seen specifically during soldier differentiation (Fig. 2A). To find genes specifically expressed in mandibles during the soldier differentiation processes, we performed gene expression analysis by RT-qPCR and immunostaining, and finally unraveled that dachshund (dac) was the gene we wanted to know (Fig. 2B). Consistently, RNA interference of dac expression inhibited the soldier-specific mandibular elongation (Fig. 2C).
Figure 2. Soldier mandibles are elongated by dachshund gene under hormonal and Hox-gene controls. (A) Transition of mandibular morphology during the soldier differentiation. (B) Dac protein localizes especially at apical part of soldier mandibles. (C) The mandibular elongation was inhibited by RNAi for dac gene. (D) The relationship among factors that control the mandibular elongation. Dac expression was regulated downstream of the juvenile hormone and insulin and a Hox gene, Deformed (Dfd).
Moreover, we revealed that dac expression was regulated under hormonal factors and Hox genes (Sugime et al., 2019; Fig. 2D). In some previous research, interactions among colony members are known to trigger the physiological changes such as the elevation of juvenile hormone titers leading to soldier differentiation (e.g. Cornette et al., 2008). In addition to JH, insulin signaling is also shown to be responsible for soldier morphogenesis (Hattori et al., 2013). Depletion of juvenile hormone receptor, insulin receptor or particular Hox genes all lead to reduced dac expression, shedding light on the epistatic relationships regulating the dac activity. Therefore, our study strongly suggests that the cross talks between temporal factors, i.e., hormonal pathways, and spatial factors, i.e., Hox genes, lead the caste-specific developmental modifications that are seen during caste differentiation in termites.
Typical day
To achieve these findings, in a typical day, we spend a lot of time on the workbench with several types of termite samples. For gene expression and immunostaining analysis we use several pieces of equipment such as RT-qPCR and a confocal laser microscope (Fig. 3A, B). We also spend on time with live termite individuals for behavioral observations and experimental manipulations such as caste induction and micro injection (Fig. 3C).
Figure 3. Typical day in the laboratory. (A) Observation by confocal laser scanning microscope. (B) Molecular experiments on the working bench. (C) Micro-injection under the microscope. (D) Discussion at seminar.
In our focal termite species damp-wood termite Hodotermopsis sjostedti, several castes can easily be induced. In the reproductive caste induction, we manipulate sex and caste ratio of experimental colonies. On the other hand, soldier differentiation can be easily induced by the application of juvenile hormone. In addition, we often have discussions and seminars to discuss our research progresses and experimental plans (Fig. 3D). In every May, we go for collection trips for termite colonies to the Yakushima Island, which is a subtropical island located south of Kyushu, Japan.
Field day in the forest
Our focal termite species is the damp-wood termite Hodotermopsis sjostedti, that lives in rotten trees as its common name suggests. In Japan, this species is distributed in a subtropical forest of southern islands. My supervisor, Prof. Toru Miura, has been sampling termite colonies at Yakushima island every year for nearly 20 years. Soon after arrival Yakushima island, we discuss about schedules and searching area with a spread map. After discussion, we entry the forest and search the rotten trees. We walk and climb through the forest carefully to search for termite colonies, in addition to avoid any dangerous animals such as Mamushi pit vipers (Fig. 4A). When we break rotten trees with a hammer, we occasionally find termite individuals (Fig. 4B). Then, we cut and split the log to find a whole termite colony, that is then separated into containers, by cutting the wood with a saw and an axe. Sometimes, giant centipedes appear from the log (Fig. 4C). During the field sampling we spend a whole day as if we are lumberjacks. To avoid mixing colonies, we carefully labeled the colony number and identification, and send them to our laboratory. After such physical labour, we have fun by bathing in hot springs and drinking “Mitake”, traditional distilled spirit.
Figure 4. Field day in the forest. (A) Searching termite nests in the forest. (B) Find termite colonies in the rotten trees. (C) Giant centipedes with her babies appear from the log.
Field day in the ocean
Recently, our lab moved to Misaki Marine Biological Station at the University of Tokyo (Fig. 5A, B) from Hokkaido. Two years ago, our environment was largely changed to marine laboratory, so the focus of our studies has expanded to fascinating marine animals which provide us tremendous implications on development and evolution. We are now studying diverse taxa across animal phyla, and trying to establish rearing systems for future analysis to reveal physiological and developmental aspects of flexible phenotypic changes in animal life cycles.
Figure 5. Location and pictures of Misaki Marine Biological Station. (A) Location of Misaki Marine Biological Station, Kanagawa, Japan (https://www.google.co.jp/maps). (B) Research building of the Misaki Marine Biological Station. (C) Memorial building and the ship “Rinkai-maru”.
Misaki Marine Biological Station is one of the oldest marine stations in the world (since 1886). It is located at the tip of the Miura Peninsula in Kanagawa Prefecture, facing the Sagami Bay that shows interesting geological features and a rich diversity of marine fauna (Fig. 5A, C). We are constantly sampling diverse animal species from the ocean throughout a year. We collect a number of marine creatures by walking with waders, snorkeling or scuba diving (Fig. 6A). After sampling, we maintain those creatures in the lab to use for experiments. Now, we are working on ten species of animals belonging to eight phyla (Fig. 6B).
Figure 6. Field day in the ocean. (A) Several types of sampling at the ocean, walking with waders, snorkeling, scuba diving and sorting samples. (B) Marine animals which we are working on. All photos are taken by Lab members and technical staffs at MMBS.
Professor Ferguson-Smith is an authority on genomic imprinting and the epigenetic control of genome function in health and disease, and is recognised for her work on parental-origin effects and epigenetic mechanisms. Her work has uncovered epigenetically regulated processes in development and over the life course, and identified key in vivo mechanisms involved in the maintenance of epigenetic states. She also explores communication between the environment and the genome with implications for health, disease and inheritance.
The DanStem podcast channel aims at providing a voice to the talented people who are behind the microscopes, the equipment or computers; we encourage them to share their passions, hobbies and unique career paths. In each episode, we will bring forward interesting, insightful and inspiring stories, where our guests share their personal experiences and give career-related advice.
The Company of Biologists’ journals – Development, Journal of Cell Science, Journal of Experimental Biology andDisease Models & Mechanisms – offer Travelling Fellowships of up to £2,500 to graduate students and post-doctoral researchers wishing to make collaborative visits to other laboratories. These are designed to offset the cost of travel and other expenses. There is no restriction on nationality.
They really are an amazing opportunity for ECRs to learn new things, meet new people and travel to new places.
The current round of Travelling Fellowships closes on 31 May (for travel > 15 July 2019)
Butterfly eyespots are striking examples of animal patterning, but their developmental origins are still relatively poorly understood. A new paper in Development– the result of a collaboration between two Singapore-based labs – now combines CRISPR-Cas9 gene targeting with theoretical modelling to address the role of the Distal-less transcription factor in eyespot patterning. We caught up with co-first authors Heidi Connahs and Sham Tlili, and their respective supervisors Timothy Saunders (Assistant Professor at the Mechanobiology Institute, National University of Singapore) and Antónia Monteiro (Associate Professor at the Department of Biological Sciences, National University of Singapore and Yale-NUS College) to find out more about the story.
Antónia, Tim, Heidi and Sham (from left to right)
Tim and Antónia, can you give us your scientific biography and the questions your labs are trying to answer?
AM I was trained in population genetics and developmental biology, and have been trying to figure out how eyespot patterns develop on the wings of butterflies for most of my career, including investigating their origin, their evolution in number and also their ability to change in size with changes in environmental cues such as temperature. We have also been addressing how males and females use these eyespots in sexual signalling and in evading predators.
TS I was trained in theoretical physics but moved to developmental biology in 2007. Initially, I was interested in developing mathematical models, particularly of morphogen gradients, to understand how developing organisms reliably form, despite the presence of temperature variations and other natural fluctuations. I realised that access to experimental data was critical, and so I learnt Drosophila genetics and imaging during a post-doc at the European Molecular Biology Laboratory (Heidelberg, Germany). I have had my own lab since 2013 at the Mechanobiology Institute where we primarily focus on how complex organ shape emerges during development. We use both Drosophila and zebrafish embryogenesis to perform quantitative live imaging of organ formation. The lab also incorporates mathematical modelling and image analysis, to build a deeper understanding of how tissues form complex morphological shapes.
And Heidi and Sham: how did you each come to join your respective labs, and what drives your research?
HC During my PhD, I became fascinated with butterfly wing patterns and their eyespots in particular, so I was already reading a lot of Antonia’s work. And about 18 months before the end of my PhD, I attended a careers seminar where the speaker said that if there was someone we really wanted to post-doc with, we should just go ahead and contact them. So, I emailed Antonia right away and sent her my CV. I got lucky, because a semester before I graduated she contacted me and said she had funding for a postdoc and asked if I was still interested, and of course I said yes! Mostly what drives my research is a desire to understand how butterfly wing patterns develop, and specifically to identify the developmental processes that generate eyespot diversity.
ST I was trained as a physicist and enjoy doing both experimental work and modelling. I am especially interested in biological problems where spatial components play a role, and this necessitates the generation of quantitative maps of quantities such as cellular movements. I completed my PhD in Paris working on the mechanical properties of cell aggregates and cell monolayers. During the last year of my PhD, I had started to think about doing a post-doctoral work on more in vivosystems while keeping a quantitative biophysics approach. I discovered Tim through his website: he had just started his group at the Mechanobiology Institute. I was motivated by the highly interdisciplinary component of his group and by the fact that he was trained as a theoretical physicist developing experimental aspects in the lab. I was finally convinced to join his lab after meeting all the members of the lab during my interview.
How did your two labs come to collaborate on this project?
AM I think we started this collaboration via an undergraduate student, Trisha Loo, who joined Tim’s lab with an interest in modelling and was also doing some wet lab work in my lab. But the collaborative work really got going when we started getting very interesting Distal-less (Dll) crispants, such as butterflies with split eyespot centres, that really begged for modelling of potential morphogenetic processes that were differentiating those centres.
TS Antonia and I first met at a department faculty retreat. On the bus journey to the venue, we discussed patterning and complexity. We realised there was potential synergy between our labs work, and so we jointly took on Trisha to explore mathematical modelling of eyespot centre specification. The project then grew from there, with Heidi and Sham joining, who significantly pushed forward the science. Pleasingly, Tricia is now a PhD student in my lab, doing theory and image analysis – a large change from her undergraduate studies in biology!
The disruption of Distal-less exon 2 via CRISPR-Cas9 led to the differentiation of two eyespot patterns on each sector of the wings of the Squinting Bush Brown butterfly, Bicyclus anynana, instead of the expected single eyespots.
Can you give us the key results of the paper in a paragraph?
AM The paper shows that mutations in different exons of the gene Dll affect the morphogenetic process that differentiates the cells at the centre of an eyespot pattern. Close examination of these crispants suggests that Dll is involved in a reaction-diffusion process where continuous variation in Dll levels can lead to eyespots appearing on a wing sector, to eyespots splitting into two, to finally acquiring a tear-drop shape.
TS For me, a key result of the paper is the use of modelling to describe complex phenotypes. The crispants created a diverse range of phenotypes and our model was able to explain all these observations with minimal changes.
ST I would add that modelling the spatial component of the problem was critical in this case to make sense of the complex eyespot shapes obtained.
HC Our work shows that, in Bicyclus anynana, Dll is required for eyespot formation and it also appears to have other roles such as in regulating melanin pigmentation and scale development. The experimental and modelling work suggests that different eyespot phenotypes can be explained by variation in levels of Dll expression. Our findings lead us to conclude that, as Dll expression levels decrease, eyespots become smaller or disappear altogether. However, when Dll levels increase, this leads to the duplication of eyespot centres resulting in extra eyespots developing on the wing.
Your experiments demonstrate exon-skipping/gain-of-function phenotypes from certain CRISPR-induced mutations in Dll. Do you think this is a widespread issue in the field?
AM This exact same Dll exon-skipping phenomenon has recently been shown in sepsid flies, leading to the occurrence of ectopic sternite brushes (Rajaratnam et al., 2018), and it has also been documented in other studies using cell lines (Kapahnke et al., 2016; Lalonde et al., 2017; Mou et al., 2017). What is still unclear to us is the extent that natural variation in exon skipping takes place in natural populations to alter gene function from a reduced or loss-of-function to a gain-of-function outcome.
HC There does seem to be a growing awareness now of the potential for CRISPR to induce exon skipping, and also the importance of sequencing not only the genomic DNA but the mRNA from crispants.
Why might Dll lacking exon 2 induce such weird and wonderful phenotypes?
AM: We still don’t know. The Dll truncated protein, lacking exon 2 but still containing a functioning homeobox, might be more stable and resistant to degradation, mimicking a gain-of-function phenotype.
HC Or perhaps the truncated 5′UTR increases the translation efficiency of the protein. More research is needed to understand the properties of this truncated version of Dll, and this will probably require using Drosophila transgenics to express the truncated protein.
Your theoretical model can replicate eyespot formation and Dll mutant phenotypes: do you think it might be extended to a general mechanism for how organisms make spots?
HC, ST, TS & AM Perhaps, but morphogenetic processes in the insect epidermis are largely considered a bit different from those taking place in the skin of mammals such as leopards or fish. In vertebrates, cells containing pigment molecules actually move about to take their place in a field of cells, whereas in insects, the cells are primarily differentiated in situ via interpretations of local morphogen gradients, etc. We are more excited with the idea that the models developed in this paper could help us understand limb specification in the thorax of insects via similar mechanisms.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
HC When I found my first crispant which had the ectopic eyespots, that was a big eureka moment for me. Also, when I got back some sequencing results and realized that exon 2 had been spliced out, that was very exciting. I had to look at the results multiple times before I could believe it.
ST We first investigated how the classical Gierer-Meinhardt activator-inhibitor model could explain the Dllcrispants. Although this model was giving interesting results, we were struggling to find a unique set of parameters that could explain the ensemble of the crispants phenotypes. Then, Antonia motivated us to look for a model where the morphogens are anti-colocalised instead of colocalised – to be in closer agreement with the experimental data. Shortly after implementing the Gray-Scott model, I found that this model easily generated phenotypes strikingly similar to the crispants phenotypes. This was a very satisfying moment.
And what about the flipside: any moments of frustration or despair?
HC Yes, I definitely had a lot of those moments too. Initially when I started doing the CRISPR experiments, we had a lot of trouble getting it to work and so for several months there was this heart-pounding moment each time I went to check if there were any butterflies with interesting wing phenotypes and it was very disheartening to see normal looking butterflies. Eventually we realized it was the cas9 protein, and once we ordered a new one, the experiments finally worked: that was a huge relief!
ST Maybe after sending the first draft versions, when some readers were not reading the modelling part of the story and thinking the crispants phenotypes were just not making any sense. I think that, in this story, the modelling and experimental aspects are tightly entangled. These criticisms made me realise how important it is to make the model as pedagogical as possible in a highly interdisciplinary context.
I had to look at the results multiple times before I could believe it
So what next for you two after this paper?
HC I am now focusing on targeting the enhancers of Dllusing CRISPR so we can try to understand the origins of butterfly eyespots by identifying pleiotropic enhancers. This has been quite a difficult project to work on as using CRISPR to target enhancers comes with its own unique set of challenges, but we are now starting to get some preliminary results that look quite promising!
ST I just moved back to France after three great years in Tim’s group. I started a new post-doc on embryonic stem cell aggregates mimicking early mammalian development. This project will combine tissue mechanics and potentially morphogen reaction-diffusion again!
Where will this work take the Saunders and Monteiro lab? Any plans for further collaborations?
AM Would love to collaborate further with Tim’s lab. The insights they provided into our crazy looking crispant mutants were amazing. We are now mutating other genes that are also expressed in eyespots and we are observing similar eyespot splits, etc. The reaction-diffusion process seems to involve multiple genes and we might need to model the action of these genes as well.
TS This work has nicely gone alongside our lab’s study on complex shape emergence. Both patterning and mechanics play important roles in organ formation. In the future, we are hoping to integrate reaction-diffusion modelling with mechanobiology. Of course, I’d be delighted to work further with Antonia – the butterfly is an awesome system.
Finally, let’s move outside the lab – what do you like to do in your spare time in Singapore?
AM I love to hike through Singapore’s forested parks on the weekends. My husband and I usually do a 10 km trek that ends in VivoCity – a mall with many lunch options!
TS I climb with my wife regularly and much of my weekend is spent exploring Singapore with our 6-year-old daughter.
HC Usually on the weekends I enjoy relaxing with my boyfriend and visiting different science/art/nature-themed attractions or exhibitions in Singapore. I also enjoy watercolour painting and keeping aquarium fish.
ST In Singapore, I really enjoyed walking in the parks along the harbour which gave a really nice view of the sea, Indonesian islands in the horizon and all these coloured container ships coming from all over the world.
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