New DMDD data released on Expression Atlas reveals the effect of single gene knockouts on the expression of all other genes in the mouse genome. The gene expression profiles of 11 knockout lines have been derived from whole embryos harvested at E9.5, and the results can be compared with wild-type controls using an interactive online tool. Users can investigate which genes are differentially expressed as a result of a gene knockout, with the potential to uncover genes with similar roles or compensatory effects when a related gene is knocked out.
Data for additional lines will be released throughout 2017. The ultimate goal is to bring these molecular phenotypes together with the morphological phenotypes that have already been derived by the DMDD programme, to offer new insights about the effects of gene knockout on embryo development.
THE GENOMIC EFFECTS OF Ssr2 KNOCKOUT
The knockout of Ssr2 in the mouse was found to affect the expression level of 325 genes in total, and this is one of the 11 new datasets that can be explored in Expression Atlas.
The differential expression of each gene is described using the log2 fold change – a measure that describes the ratio of gene expression in the knockout to the level of gene expression in a wild-type control. A negative fold change (shown in blue in the image below) means that the gene was expressed at a lower level in the mutant. A positive fold change (shown in red in the image below) means that the gene was expressed at a higher level in the mutant.
Eight genes that are differentially expressed due to a knockout of the gene Ssr2 (above a cut off log2 fold change of 0.4). Six genes are expressed at a higher level, while Mfap2 and Ssr2 are expressed at a lower level.
The interactive tool in Expression Atlas allows different cut-offs to be applied to the fold change, so the genes displayed can be restricted to those with a large differential expression. The image above shows the 8 genes with a fold change greater than 0.4 as a result of knocking out the gene Ssr2.
The tool can also be used to visualise the data in graphical form. The plot below shows the fold change for each gene, allowing the user to quickly ascertain the extent to which a gene knockout caused differential expression of other genes. All 325 genes considered to have a significant change in the level of gene expression are plotted in red, with the rest shown in grey.
A graphical visualisation of the fold change for each gene. The outlier with a fold change of -3.5 is the gene Ssr2, which has a much-reduced expression level in an Ssr2 knockout embryo.
The full list of lines with data currently available is: 1700007K13Rik, 4933434E20Rik, Adamts3, Anks6, Camsap3, Cnot4, Cyp11a1, Mir96, Otud7b, Pdzk1 and Ssr2.
The full dataset for any line can be downloaded for further analysis, while the individual line pages on Expression Atlas integrate the DMDD data with other pre-existing data, in cases where a gene has already been shown to alter expression.
We heard from three Company of Biologists Travelling Fellows – Hanna Hakkinen, Nanami Morookaand Tetsuto Miyashita– who collectively crossed continents to learn new techniques in host labs.
We are reconstructing retinal circuitry with 2.19nm/pixel resolution, to document all of the synapses and gap junctions. #USofSciencepic.twitter.com/OCnAQALbTl
(A/Prof Helena Richardson’s & Prof Patrick Humbert’s laboratories at the Department of Biochemistry & Genetics, La Trobe Institute of Molecular Sciences (LIMS), La Trobe University Melbourne Campus (Bundoora)
We offer a PhD scholarship to an exceptional student (who has achieved a H1 Honours or equivalent) to determine how cell polarity perturbations affect signalling pathways in tissue repair. The project utilizes the model organism, Drosophila, and mammalian epithelial cell culture. This project will have important implications for understanding wound healing as well as cancer.
The Applicant should have Australian citizenship or residency. They should be highly driven and have a high level of achievement, including a first class Honours degree or equivalent in the field of Cell Biology and/or Genetics. Knowledge of Cell Biology theory and techniques is essential, and knowledge of Genetics, Molecular Biology and Biochemistry approaches is desirable. Experience in the Drosophila model organism, although not essential, will be highly beneficial.
The project will address the role of cell shape (polarity) regulation in epithelial tissue homeostasis, using an in vivo approach utilizing the Drosophila model system, and an in vitro approach with cultured mammalian epithelial cells. Sophisticated genetic techniques will be used to generate mutant patches of cells within an epithelium and the effect on cell morphology, cell extrusion, signalling pathways, cell proliferation, apoptosis and protein-protein interactions will be monitored utilizing sophisticated cell biological approaches involving fixed samples or live cell imaging. The project seeks to reveal novel mechanisms by which mutant cells interact with their microenvironment that can be utilized therapeutically to improve wound repair or to enhance elimination of the mutant cells.
Benefits of the scholarship
Benefits of the scholarship include:
A La Trobe University Research Scholarship for three years, with a value of $26,288 per annum, to support your living costs [2016 rate]
Opportunities to work with outstanding researchers at the Department of Biochemistry & Genetics, LIMS and have access to cutting-edge equipment and professional development programs
Opportunities for authorship on high impact scientific manuscripts.
Opportunities to attend national and international conferences
Contact A/Prof. Helena Richardson by email at h.richardson@latrobe.edu.au, with a full CV, academic transcript, and a cover letter outlining why you would like to be considered for this scholarship.
A/Prof Helena Richardson and Prof Patrick Humbert at the Department of Biochemistry & Genetics, LIMS will carefully review your application and consider you for this Scholarship.
The successful applicant who receives in-principle agreement for supervision, will then submit a complete PhD application to the La Trobe Graduate Research School, attaching a copy of the agreement to admissions.grs@latrobe.edu.au
You will be advised of an outcome by 30th April, 2017.
Closing date
Applications close 1 April 2017, unless filled sooner.
Contact us
If you require further information, please contact: h.richardson@latrobe.edu.au or the La Trobe University Graduate Research School: grs@latrobe.edu.au
A Ph.D. scholarship offered to an exceptional student, to investigate genetic mechanisms which underpin vertebrate birth defects, with a particular focus on craniofacial defects such as cleft palate.
This scholarship will be offered to an independent, proactive, forward thinking and enthusiastic candidate, who wishes to forge an independent career in science.
Applicants should have a high level of achievement, including a first class honours degree or equivalent.
As an applicant you should have an interest in developmental genetics and understanding the processes which govern embryo formation, as well as a keen interest and aptitude in biochemistry and molecular genetics. Your project will address biological and cellular behaviours which regulate how the vertebrate embryos forms, using the mouse, and zebrafish as genetic developmental models.
Benefits of the scholarship
Benefits of the scholarship include:
a La Trobe Research Scholarship for three years, with a value of $26,288 per annum, to support your living costs [2016 rate]
a fee-relief scholarship (LTUFFRS) for four years to undertake a PhD at La Trobe University (international applicants only)
opportunities for authorship on high impact scientific manuscripts.
opportunities to attend national and international conferences
opportunities to work with La Trobe’s outstanding researchers, and have access to our suite of professional development programs
Contact Dr. Seb Dworkin by email at s.dworkin@latrobe.edu.au, with a full CV, academic transcript, and a cover letter outlining why you would like to be considered for this scholarship.
Dr. Dworkin, and the Department of Physiology, Anatomy and Microbiology will carefully review your application and consider you for this Scholarship.
The successful applicant who receives in-principle agreement for supervision, will then submit a complete PhD application to the La Trobe Graduate Research School, attaching a copy of the agreement to admissions.grs@latrobe.edu.au
You will be advised of an outcome by 30th April, 2017.
Closing date
Applications close 1 April 2017, unless filled sooner.
Contact us
If you require further information, please contact: s.dworkin@latrobe.edu.au or the La Trobe University Graduate Research School: grs@latrobe.edu.au
An NIH-funded postdoctoral researcher position is available immediately in Dr. Nadia Dahmane laboratory at Cornell University-Weill Cornell Medicine in the Department of Neurological Surgery to study the transcriptional regulation of normal brain development and brain tumor progression. Our group uses cell biology, mouse genetics, biochemical and genomic approaches to decipher the cellular and molecular mechanisms controlling brain development and brain tumor progression (e.g. Xiang et al. Cell Death and Differentiation 2012; Baubet et al., Development 2012,Tatard et al., Cancer Research 2010; Deng et al. Journal of Cell Science 2012).
We seek enthusiastic, highly qualified and motivated individuals to join our research group. The successful candidate should have a Ph.D. degree with a strong background in molecular biology, cell biology, and/or biochemistry. Research experience in developmental neuroscience, cancer biology and animal models of brain diseases would be considered advantageous.
Our laboratory is located on the Weill Cornell Medicine campus in New York City.
Please submit your CV and a cover letter outlining your research interests, career goals and the names of three referees with contact information to Dr. Nadia Dahmane at: nad2639@med.cornell.edu
Here we highlight some developmental biology related content from other journals published by The Company of Biologists.
JCS kicked off 2017 with a Special Issue relevant to many developmental biologists: 3D cell biology. It’s packed full of commentaries, interviews, research articles and techniques, and well worth a browse.
Nicole Gorfinkiel and colleagues showed that α- Catenin stabilises actomyosin foci and E-Cadherin to promote apical contraction in the Drosophila amnioserosa.
A position (#122764) is available immediately for a Research Technician/Faculty Specialist to contribute to our studies in neural crest and placodes. The Technician will conduct research, assist in the training of students, and take part in the management of the laboratory of Dr. Lisa Taneyhill at the University of Maryland. Laboratory skills should include the ability to perform various molecular biology and biochemical assays, such as recombinant DNA/cloning; immunoprecipitation and immunoblotting; and/or immunohistochemistry. Experience with microscopy, chick embryology, and tissue culture is desirable. For more information on the lab, please see http://www.ansc.umd.edu/people/lisa-taneyhill. A Bachelor’s degree (B.A. or B.S.) in a related field and prior laboratory research experience is essential. Fluency in spoken and written English is required. Salaries are highly competitive, negotiable and commensurate with qualifications. Fringe benefits offered. Applicants must apply through eTerp at https://ejobs.umd.edu. Applications will be accepted until a suitable candidate is identified.
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
2017 started where 2016 had left off, with an number of preprints covering most corners of developmental biology, plus more relevant work from related fields. Looking at the list below, it’s clear that a mix of young and established labs are using preprints. Following the trends of last year, they were predominantly found on bioRxiv, with some also on arXiv and PeerJ Preprints.
This month features regeneration in mouse digits and whole ascidians, how body axes form in gastruloids, the link between metabolism and signalling in mice, and developmental plasticity in Arabidopsis. There also were a whole bunch of fly papers, and new tools including a new ImageJ and ‘WikiGenomes’. Happy preprinting!
Molecular and functional variation in iPSC-derived sensory neurons. Jeremy Schwartzentruber, Stefanie Foskolou, Helena Kilpinen, Julia Rodrigues, Kaur Alasoo, Andrew J Knights, Minal Patel, Angela Goncalves, Rita Ferreira, Caroline L Benn, Anna Wilbrey, Magda Bictash, Emma Impey, Lishuang Cao, Sergio Lainez, Alexandre J Loucif, Paul J Whiting, HIPSCI Consortium, Alex Gutteridge,Daniel J Gaffney
Stem cell differentiation is a stochastic process with memory. Patrick S. Stumpf, Rosanna C. G. Smith, Michael Lenz, Andreas Schuppert, Franz-Josef Müller, Ann Babtie, Thalia E. Chan, Michael P. H. Stumpf, Colin P. Please, Sam D. Howison, Fumio Arai, Ben D. MacArthur
Distinguishing Mechanisms Underlying EMT Tristability. Dongya Jia, Mohit Kumar Jolly, Satyendra Chandra Tripathi, Petra Den Hollander, Bin Huang, Mingyang Lu, Muge Celiktas, Esmeralda Ramirez-Pena, Eshel Ben-Jacob, Jose N. Onuchic, Samir M. Hanash, Sendurai A. Mani, Herbert Levine
It′s okay to be green: Draft genome of the North American Bullfrog (Rana [Lithobates] catesbeiana). S Austin Hammond, René L Warren, Benjamin P Vandervalk, Erdi Kucuk, Hamza Khan, Ewan A Gibb, Pawan Pandoh, Heather Kirk, Yongjun Zhao, Martin Jones, Andrew J Mungall, Robin Coope, Stephen Pleasance, Richard A Moore, Robert A Holt, Jessica M Round, Sara Ohora, Nik Veldhoen, Caren C Helbing, Inanc Birol
Progress Towards a Public Chemogenomic Set for Protein Kinases and a Call for Contributions. David H Drewry, Carrow I Wells, David M Andrews, Richard Angell, Hassan Al-Ali, Alison D Axtman, Stephen J Capuzzi, Jonathan M Elkins, Peter Ettmayer, Mathias Frederiksen, Opher Gileadi, Nathanael Gray, Alice Hooper, Stefan Knapp, Stefan Laufer, Ulrich Luecking, Susanne Muller, Eugene Muratov, R. Aldrin Denny, Kumar S Saikatendu, Daniel K Treiber, William J Zuercher, Timothy M Willson
The 4D Nucleome Project. Job Dekker, Andrew S Belmont, Mitchell Guttman, Victor O Leshyk, John T Lis, Stavros Lomvardas, Leonid A Mirny, Clodagh C O’Shea, Peter J Park, Bing Ren, Joan C Ritland, Jay Shendure, Sheng Zhong, The 4D Nucleome Network
Genome Graphs. Adam M Novak, Glenn Hickey, Erik Garrison, Sean Blum, Abram Connelly, Alexander Dilthey, Jordan Eizenga, M. A. Saleh Elmohamed, Sally Guthrie, André Kahles, Stephen Keenan, Jerome Kelleher, Deniz Kural, Heng Li, Michael F Lin, Karen Miga, Nancy Ouyang, Goran Rakocevic, Maciek Smuga-Otto, Alexander Wait Zaranek, Richard Durbin, Gil McVean, David Haussler, Benedict Paten
Biocuration as an undergraduate training experience: Improving the annotation of the insect vector of Citrus greening disease.Surya Saha, Prashant S Hosmani, Krystal Villalobos-Ayala, Sherry Miller, Teresa Shippy, Andrew Rosendale, Chris Cordola, Tracey Bell, Hannah Mann, Gabe DeAvila, Daniel DeAvila, Zachary Moore, Kyle Buller, Kathryn Ciolkevich, Samantha Nandyal, Robert Mahoney, Joshua Von Voorhis, Megan Dunlevy, David Farrow, David Hunter, Taylar Morgan, Kayla Shore, Victoria Guzman, Allison Izsak, Danielle E Dixon, Liliana Cano, Andrew Cridge, Shannon Johnson, Brandi L Cantarel, Stephen Richardson, Adam English, Nan Leng, Xiaolong Cao, Haobo Jiang, Chris Childers, Mei-Ju Chen, Mirella Flores, Wayne Hunter, Michelle Cilia, Lukas A Mueller, Monica Munoz-Torres, David Nelson, Monica F Poelchau, Josh Benoit, Helen Wiersma-Koch, Tom D’elia, Susan J Brown
Recommended by Bob Goldstein, University of North Carolina at Chapel Hill
Sven Hörstadius stands alongside the likes of Boveri, Spemann, Mangold and Driesch as a giant of experimental embryology in the first half of the twentieth century. While his 1950 treatise on the neural crest in vertebrate head development became an early bible for the field, he is probably best known for his work on sea urchins. His 1939 manuscript summarises a whole suite of work from himself and others dealing with various kinds of determination: of the axes of the embryo, of cell fate by instruction or position, and of ‘species character’ by the cytoplasm or the nucleus.
The focus is on the purple sea urchin, Paracentrotus lividus, which Hörstadius studied during trips to the Stazione Zoologica in Naples, once a haunt of Driesch and Boveri and still an active centre of research. P. lividus was a particularly useful urchin species because the eggs and early embryos have a vegetal pigment band, allowing polarity to be easily traced. In previous decades the basics of itsdevelopment had been sketched out, including where successive cleavage planes lie in the cell, the movements of gastrulation, and fate of some blastomeres. However, while fundamental rules of development could be inferred from observation alone, Hörstadius was an experimentalist, and intervened in development in a variety of creative ways to investigate determination. Like Rosa Beddington, subject of the previous post in this series, he was known as an expert dissector: he could isolate blastomeres with fine glass needles, and create chimeras by fusing part of one animal to part of another and placing a small glass ball on top “to give the necessary pressure”. His 1939 paper is full of this sort of cut and paste embryology, and the outcomes are often unexpected (“The following is a very strange phenomenon…”).
Stages of early Paracentrotus development, from uncleaved egg to 64-cell stage, from Figure 1, Hörstadius, 1939. Reproduced with permission of Wiley†.
The paper also devotes a lot of time accounting for contradictory results from other researchers. In particular, there seems to be a long standing beef with one Leopold von Ubisch, with some wonderfully formal put-downs:
“von Ubisch does not admit the possibility that halves of equatorial and subequatorial eggs are different. This is strange…” (p162)
“von Ubisch concludes that the cytoplasm has no influence on the species character of the skeleton. I do not find this conclusion convincing” (p169)
“The diagram of Fig. 9 has been criticized by von Ubisch. But his objections were, it seems to me, anticipated in the original paper” (p158)
Among the flood of experiments and ideas that Hörstadius describes, some stand out. The first results section addresses the control of spindle position and orientation in the divisions that make the 16 cell embryo with its characteristic micromeres at the vegetal pole. These divisions can be delayed by shaking or adding diluted sea water; once this delay is relieved, the embryo starts dividing again. However, instead of picking up where they left off, the embryos would often skip a cell division such that, for instance, a delayed 4 cell embryo would divide to give micromeres (column B in the figure below).
Cleavage in Paracentrotus in normal (A) and delayed conditions (B-F). From Figure 2, Hörstadius, 1939. Reproduced with permission of Wiley†.
Hörstadius proposed that the micromere division is determined by the ‘activation’ of material in the vegetal cytoplasm, and that this activation occurs independently of how many cell divisions had occurred. After a certain amount of time, the embryo wants to make micromeres, irrespective of the number of divisions completed, and changes spindle position to do so. There is a kind of ‘cleavage clock’ which sets the type of cell division that occurs next.
The next three sections concern the embryonic axes. Hörstadius began his career under the supervision of John Runnström, who proposed that the sea urchin egg held two gradients, one emanating from the animal and one from the vegetal pole. The gradients “interact mutually and are partially hostile to each other”, and a cell’s fate is dependent on the relative strength of each of the gradients in the cytoplasm it inherits. Before the molecular biology revolution, before experimental and what was then called ‘chemical’ embryology were united, the cytoplasm had “qualities” or “forces” that influenced development.
The development of isolated an1 and an2 layers, with or without added micromeres. From Figure 9, Hörstadius, 1939. Reproduced with permission of Wiley†.
One of the ways Hörstadius investigated the double gradient hypothesis was to add micromeres (the inheritors of the most vegetal, and hence most ‘active’ cytoplasm) to layers of animal cells. These layers, if isolated and left alone, go on to form useless ciliated balls. Remarkably, add enough micromeres and you could rescue normal development, and gradually increasing the micromere number led to progressively ‘better’ development. Along with the results of various other experiments involving adding bits of one embryo to bits of another, this implied that the amount of animal and vegetal material you start with is crucial to the outcome. To explain these results, Hörstadius
“…assume[d] an animal and a vegetative gradient, both reaching the opposite pole and progressively diminishing. The animal and the vegetative qualities or forces have to interact in order to bring about normal differentiations, e.g. vegetative influences are necessary for the formation of ciliated band and stomodaeum, animal ones for gastrulation and skeleton formation, and so on. The differentiation depends – within wide limits – upon the relative amounts of animal or vegetative material present.” (p173)
While Lewis Wolpert used these experiments to inform his models of positional information, half a century later the picture had changed as Eric Davidson and others challenged the idea of a double gradient. In the updated model, specification is conditional, and mediated by successive interactions between micromeres and blastomeres, the micromeres having been initially autonomously specified by maternally supplied factors. The model combines modern data on signalling pathways and gene expression with a reinterpretation of Hörstadius’ classic experiments and their insight that cell interactions could direct development.
A heterosperm merogone formed from Paracentrotus lividus cytoplasm and Psammechinus microtuberculatus nucleus, from Figure 12, Hörstadius, 1939. Reproduced with permission of Wiley†.
The paper ends with the question of the role of the nucleus in inheritance. Hörstadius took up work originally started by Boveri half a century earlier, removing the nucleus from eggs of one species and fertilising them with the sperm of another to make ‘heterosperm merogones’. The nucleus coming from one species, and the cytoplasm the other, he could ask which species the larva ended up looking like, and hence where ‘character’ was determined. In one combination, the merogone followed the characteristics of the species from which the nucleus was taken, but due to high variability in other combinations, Hörstadius had to be cautious in his intepretation:
“…the nucleus obviously has a positive effect on the species character. It is to be regretted that the characters in question are not defined sharply enough to permit of a conclusion as to the possible role of the cytoplasm.” (p172)
Even treading this lightly, the results are consistent with the work of Boveri, Spemann, Waddington, Wilson and Stevens, all of which put the nucleus, and the mysterious substance contained within it, in the driving seat of development and heredity.
The paper is testament to the power of cut and paste embryology, and the rich potential of the sea urchin embryo as a model. We can leave it Carl Olaf Jacobsen, one of only two graduate students trained by Hörstadius during his long time as a Professor in Uppsala, to summarise his supervisor’s legacy:
“…his experiments on sea urchin larvae shed light on a couple of the most central findings in developmental biology, namely that the uneven distribution of the egg-cell contents give rise to early embryo cells with shifting qualities, and that communication between these cells has an essential role in the differentiation process.”
This paper’s a fun read because it’s dense with simple and clever experiments. And many of the experiments built a foundation for our current understanding of how animal development works. But I love it most of all because it’s a great example of old literature that includes questions that we, as a field, forgot were fundamental questions. How does Hörstadius’ cleavage clock work? I think we still don’t know.
Hörstadius’ work was the inspiration for much of what I have done over the past 30 years. The following information was circulated over the years first by my mentor who had met Hörstadius, and more stories were circulated years ago when there was a festscrhriften thrown in honor of Hörstadius’ life in Stockholm. Unfortunately it was years after his death. Apparently until after his death the Swedes didn’t realize how well known Hörstadius actually was.
The post mentions that Hörstadius worked under Runnström. That was true and it was the cause for several things in his life. The Swedish system had very few professors so Horstadius worked under Runnström for most, if not his entire career. For that reason the Swedes never considered Hörstadius as particularly famous because he was not in charge. Ruunström was also involved in Hörstadius’ science so I was told. Runnström was a major proponent of morphogenetic gradients, especially double gradients. Consequently, the double gradients that are part of Hörstadius’ sea urchin work fit the ideas of Runnström – or perhaps were strongly suggested by him. Perhaps it was only gossip, but I heard from several senior scientists that Hörstadius never really believed that double gradient nonsense but since he was in Runnström’s lab that was part of the cost. He was later admitted to the Swedish Academy and to the Royal Academy largely for his work on neural crest, but the sea urchin work has stood the test of time with a greater presence than the neural crest work.
Höstadius was praised for his microsurgical abilities. He was the only one in the world who could do those remarkable dissections of the tiny sea urchin embryos and the cut and paste experiments were considered amazing feats. That was how I was initially taught about his work. He taught himself to dissect on the stage of a compound microscope. That meant he had to teach himself to move his needles in the opposite direction relative to what was seeing through the objective. His dissection tray was a piece of photographic film on the bottom of a shallow petri dish. That was because the photographic film had a coating of gelatin on it which was just thick enough to score a trough in which his embryos would be placed. Then, all of his dissections were done by hand. By contrast, I do all the same dissections but in my case I have major help from micro manipulators, a dissection microscope which shows movements in the same direction as what I can see though the objective, and I can record everything with a camera whereas he had to draw everything. As I indicated, he had great hands in addition to a perceptive brain.
† Further usage of any Wiley content that appears on this website is strictly prohibited without permission from John Wiley & Sons, Inc. Please contact Wiley’s Permissions Department either via email: permissions@wiley.com or use the RightsLink service by clicking on the ‘Request Permission’ link accompanying this article on Wiley Online Library (www.onlinelibrary.wiley.com)”
Aidan Maartens
This post is part of a series on forgotten classics of developmental biology. You can read the introduction to the series here and read other posts in this series here. We also would love to hear suggestions for future Forgotten Classics – let us know in the comments box.
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, Uk
Collective cell migration is involved in many biological processes. In particular it is required to build new tissues during morphogenesis and to repair them during wound healing. Cancer cells however also exploit it during invasion of other tissues. In order for a group of cells to migrate together as a coordinated group they must establish a hierarchy of cellular identities, generally thought of as “leader” and “follower” cells (Friedl & Gilmour 2009). How this hierarchy is established and robustly maintained is key to understanding the process of collective cell migration. To examine this, in our recent study we explored the collective movement of endothelial cells undergoing angiogenesis (the generation of new blood vessels from existing ones) in zebrafish embryos. We were particularly interested in how these collectively migrating cells managed to maintain their organisation while undergoing divisions.
During sprouting angiogenesis the collectively migrating cells take on the roles of either a leading “tip cell” or a following “stalk cell” (Herbert & Stainier, 2011). The tip cell is the first cell to leave the existing vessel and is highly motile. This is then followed by the stalk cells, which are less motile and go on to form the main trunk of the developing vessel. Previous work has established that this hierarchy is driven by differing levels of Vascular endothelial growth factor (VEGF) signalling, with the tip cells having high levels compared to the stalk cells. This is thought to be established via a Notch/Delta controlled lateral inhibition, whereby tip cells induce a reduction in VEGF signalling in the stalk cells (Herbert & Stainier, 2011).
Sprouting angiogenesis in a zebrafish embryo. The “tip cell” (far left) first sprouts from the existing blood vessel and is followed by the “stalk cells”. Blue is DNA and red is the cell membranes
During angiogenic sprouting migrating endothelial cells are required to undergo mitosis (Schoors et al. 2015). This presents the cells with a problem, as the two daughter cells must acquire two different migratory profiles, dependent on their resultant positions. The more distal cell must take on the tip cell identity, while the more proximal cell becomes the trailing stalk cell. However, division will partition the components of the VEGF signalling and Notch pathway components, which if equal will presumably induce competition for tip cell identity between the two daughter cells. This would disrupt the migration of the group of cells and ultimately impede angiogenesis. However, far from this, tip/stalk cell identities are actually established almost instantaneously after division, much faster than notch/delta mediated lateral inhibition is thought to take (upwards of 5 hours) (Matsuda et al. 2015; Bentley et al. 2014).
After division cells immediately display distinct tip and stalk cell behaviours. Costa et al., (2016)
The question – How are the tip/stalk identities of collectively migrating endothelial cells re-established so quickly after mitosis?
Computer modelling suggested that the answer to this question could be that these cells undergo a form of asymmetric cell division and produce daughters of different sizes. The model predicted that a larger daughter cell would inherit more of the VEGF signalling machinery, giving it higher levels of VEGF signalling and thereby establishing it as the tip cell. Live imaging of zebrafish angiogenic endothelial cells revealed that indeed the most distal daughter cell (the tip cell position) is on average 1.8-1.9 times larger than the more proximal daughter cell (the stalk cell position). Furthermore, the size of each daughter cell was shown to be proportional to its migratory speed, meaning that sister cells that had the biggest difference in their size also have the biggest difference in their speeds. Furthermore, in vitro work also demonstrated that larger cells inherit a larger proportion of VEGF receptor mRNA, as well as having higher levels of VEGF signalling. In order to demonstrate that these differing levels of VEGF signalling were necessary to define post-mitotic tip/stalk cell identities, zebrafish embryos were treated with low levels of a drug that blocks VEGF signalling. This low dose didn’t inhibit signalling altogether but prevented any cell from signalling at the levels necessary to be a tip cell. Under these conditions both daughter cells assumed stalk-like identities after mitosis.
A) The divisions of zebrafish angiogenic endothelial cells are asymmetric; one cell (the tip cell, cell 1.1) is larger than the other (the stalk cell, cell 1.2) B) The size of a cell is proportional to its speed of migration. Costa et al., (2016)
How might the asymmetry in daughter cell size be achieved?
Preliminary data suggests that the size asymmetry seen in angiogenic endothelial cells is (at least in part) generated by the positioning of the mitotic spindle towards the proximal pole of the cell (Costa et al. 2016). Thereby shifting the division plane away from the volumetric centre of the cell. Other classical asymmetric cell divisions also result in cells of different sizes, for example Drosophila neuroblasts and one-cell C. elegans embryos, though the role of this asymmetry has not been extensively explored (Cabernard et al. 2010; McNally 2013). However some clues may be gleaned as to how angiogenic endothelial cells manage to position their mitotic spindles such that two differently sized daughters are produced. A canonical set of proteins is known to generate the membrane associated pulling force that acts upon mitotic spindles. Partner of Inscuteable (Pins) (LGN in Drosophila and GPR1/2 in C.elegans), anchored at the membrane by Gαi (GOA-1 and GPA-16 in C.elegans), binds to Nuclear mitotic apparatus (NuMA) (Mud in Drosophila and LIN-5 in C.elegans), which in turn binds to the dynein/dynactin complex, this then pulls on the plus ends of astral microtubules (Bergstralh et al. 2013). Asymmetric enrichment of this complex can cause the spindle to be pulled towards one side of the cell, such as in the one-cell C.elegans embryo (Kiyomitsu, 2015). However, it remains to be seen whether the spindle orienting machinery is involved in positioning the mitotic spindle of angiogenic endothelial cells.
Downward movement of the mitotic spindle positions the plane of division such that 2 differently sized daughter cells are produced. Costa et al., (2016)
Asymmetric cell division is a well-described phenomenon traditionally thought of as a process employed by cells to enable them to generate cells different from themselves. An asymmetric inheritance of fate determinants or a position dependent asymmetry in external cues normally results in daughter cells becoming two different cells types. Size asymmetry (and a resultant asymmetry in signalling strengths) between daughter cells offers a simple way of introducing subtle heterogeneity into a population of a single cell type. Furthermore, if it is controlled so that the larger and smaller cells are positioned specifically, patterns (such as leading and following cells) can be produced. Further work is needed to elucidate the mechanism behind this new form of asymmetric division and it will be interesting to see whether other collectively migrating systems, or indeed any other cell types, undergo similar divisions.
References
Bentley, K., Harrington, K.I. & Regan, E.R., Can active perception generate bistability? Heterogeneous collective dynamics and vascular patterning. ALIFEhttp://dx.doi.org/10.7551/978-0-262-32621-6-ch053 (2014)
New DMDD data released on Expression Atlas reveals the effect of single gene knockouts on the expression of all other genes in the mouse genome. The gene expression profiles of 11 knockout lines have been derived from whole embryos harvested at E9.5, and the results can be compared with wild-type controls using an interactive online tool. Users can investigate which genes are differentially expressed as a result of a gene knockout, with the potential to uncover genes with similar roles or compensatory effects when a related gene is knocked out.
(No Ratings Yet)
Loading...
JCS 
(7 votes)
(2 votes)