The canonical model

The publication of Marc Tessier-Lavigne’s seminal Cell papers (1, 2) in 1994 describing the identification of netrin1 (from the Sanskrit word, netr, meaning “one who guides”) was a defining moment in my graduate career. My friends and I talked about those papers for weeks, from the incredible technical feat, the biochemical purification of netrin1 from tens of thousands of chicken brains, to the commonality of the neural developmental mechanism, based on the homology of netrin1 with Unc6, a gene previously identified by Ed Hedgecock and Joe Culotti in a C.elegans screen for axon guidance defects (3).

A couple of years later, I interviewed for postdoctoral positions in San Francisco Bay area in neural development laboratories, and had arranged to stay with Marya Postner, a fellow Princeton graduate alumnus. Marya just happened to be married to Tito Serafini, who, with Tim Kennedy, was one of two lead postdocs on the seminal 1994 Cell papers. Tito was still working with Marc at the time. I asked him breathlessly about the status of the netrin1 project – did they have a netrin1 loss of function phenotype yet? Tito looked happy, triumphant even. “Yes!” he said. “We’ve knocked out the mouse homologue. Commissural axons stall before they reach the floor plate.”

This Cell paper came out a couple of months later (4) and cemented the idea of chemotaxis as the prevailing model of axon guidance (5): netrin1 is secreted from floor plate (FP) cells at the ventral midline of the spinal cord, and like a beacon in a harbor guiding ships in the night, orients commissural axons to grow towards it (Fig. 1A). This model fit with insight dating back as far as Cajal (6), including work performed in grasshoppers (7, 8) and then in vertebrates (9, 10). Together, these studies suggested that axons could be guided in a stepwise manner over long distances by chemotropic attractive or repellent cues diffusing from “guidepost” cells that they encountered along the way. The axon guidance field also recognized that axons also grew on local substrates provided by extracellular matrix components, such as laminin. But their contribution was generally considered prosaic, i.e. passive carpets that permitted or prevented axon growth. The more important and interesting contribution to neural circuit formation came from the chemotropic cues, the netrins, semaphorins, the slit/robo pathway and morphogens.

Problems with the chemotropic model for netrin1

I have taught the chemotropic model of axon guidance for years in my undergraduate and graduate lectures, with netrin1 as the centerpiece discovery. It was a beautiful example of scientific daring being rewarded with mechanistic understanding. I was able to trace a path from the first century-old scientific insight to the netrin1 mutant phenotype that strongly supported that hypothesis.

However after starting my own laboratory in 2004, I began to wonder about a reported, but underappreciated, feature of the netrin1 expression pattern. Textbooks often show the distribution of netrin1 in the chicken spinal cord, where netrin1 is expressed at high levels specifically in the FP. However, my first undergraduates – Joe Herrold and Anna Maglunog – found that mouse netrin1 is also expressed in the ventricular zone (VZ), which is the central compartment where the neural progenitor cells reside, oscillating back and forth on radial process as they proliferate. Spinal axons uniformly avoid growing in the VZ, staying rather at the “sides” of the spinal cord in a region that will ultimately segregate into the grey and white matter. Anna found that netrin1 expression extended into the dorsal VZ and appeared to strengthen, rather than diminish, over time (Fig. 1B).

This distribution had been accurately described in Serafini et al (4). But, it remained unresolved why spinal commissural axons first grew around the domain of VZ-derived netrin1 before growing towards FP-derived netrin1 (Fig. 1A). Were commissural axons unresponsive to ventricular netrin1? In that case, how did commissural axons then become responsive to FP-derived netrin1 to grow towards the ventral midline? Was there a molecular switch that controlled this process? Joe also noticed that in netrin1 mutant mice, while the vast majority of commissural axons of the Tag1+ subtype stalled as they entered the ventral spinal cord as published (4), there were always some Tag1+ axons that entered the VZ. Again, this latter phenotype was reported by Serafini et al, but it was not a major focus of the paper. We also wondered why spinal axons usually grew around the VZ. Was there a repellent in the VZ? Could netrin1 be that repellent?

I wrote some of these ideas into an R01 grant application that was eventually funded in 2008, which allowed me to bring a postdoctoral fellow on board to work on these questions. However, the project stalled for two years; the Tag1+ axon mispolarization phenotype was subtle and it remained stubbornly unclear whether these axons originated from within the spinal cord or from the dorsal root ganglia (DRGs) in the adjacent peripheral nervous system. Moreover, my plan to tackle the problem by recapitulating the putative VZ-repellent activity in a tissue co-culture assay proved challenging. No progress was made and alas, the postdoc left my lab. With time running out to fulfill this aim of my grant, I recruited a student, Supraja (Sup) Varadarajan onto the project with the idea that she could complete the characterization of the netrin1 phenotype. It would be a quick paper, I reassured her. One of the first ideas that we had was to characterize the netrin1 mutant using a wider range of axonal markers.

First moment of clarity: many types of spinal axons invade the VZ in netrin1 mutants

Not long afterwards, Sup called me over to her computer very excitedly: “LOOK at the pattern of neurofilament innervation!” she said. Neurofilament (NF) is an intermediate filament present ubiquitously in axons. Sup had made transverse slices of control and netrin1 mutant spinal cords and stained them with antibodies against NF. While control NF+ axons grew their usual orderly way avoiding the VZ, to our amazement we saw that NF+ axons were now profusely growing into the VZ in the netrin1 mutant (Fig. 1D, Fig. 2). Antibodies against another protein, Robo3, which labels all commissural axons, showed a similar phenotype; axons were no longer tightly bundled or fasciculated. Rather, the axons radiated in all directions, including into the VZ (Fig. 2). Thus, the observed stall of Tag1+ commissural axons appeared to be an anomaly: the loss of netrin1 resulted in other spinal axons extending wildly into the VZ. We suddenly had clear evidence that there might be a repellent in the VZ, with netrin1 as a top candidate for that repellent.

Sup then spent considerable time mapping domains of netrin1 expression in the mouse spinal cord at different stages of development. She found two general patterns of behavior: 1) spinal axons rarely grow on netrin1-expressing cells, and 2) spinal commissural axons grow precisely around the boundary of netrin1-expressing cells in the VZ. But were these activities mediated by a long-range activity from the FP or a more local, short-range activity from the VZ? Our genetic manipulations strongly supported the presence of a VZ-derived netrin1 repellent. First, we characterized Gli2 mutants, which have no FP (12) either singly or in combination with a netrin1 mutation. The result was clear-cut – it was not enough to remove the FP; NF+ axons only invaded the VZ in the absence of VZ-derived netrin1. Second, in collaboration with Jennifer Kong and Bennett (Ben) Novitch, we were able to ablate netrin1 expression from a stripe of neural progenitor cells, thereby creating two de novo netrin1(-):netrin1(+) boundaries. To our amazement, axons now detached from their normal trajectories and extended into the VZ to follow along one of the ectopic boundaries of netrin1 expression. Everyone in the Butler/Novitch joint lab meeting applauded when Sup showed these results for the first time.

Revised model: netrin1 provides a growth boundary for axon extension

Our first model was that netrin1, present in the VZ, was repulsive for axon growth. But our results were now suggesting a more complex activity. While spinal axons did generally avoid growing on netrin1-expressing cells, commissural axons appeared to grow preferentially along a netrin1(-): netrin1(+) boundary. Since there wasn’t an obvious term for this phenomenon, which was unwieldy to constantly explain, Sup came up with the concept of a “hederal” boundary. Ivy (genus, hedera) uses a wall (c.f. netrin1) as a necessary scaffold for growth, but it is unable to penetrate this wall as it grows. We wondered whether this hederal activity of netrin1 was more attractive or more repulsive, and tested this idea by examining mice lacking different classes of netrin1 receptors, sent to us by Artur Kania.

In the canonical model, netrin1 results in attractive or repulsive responses in axons by activating different receptor complexes. Thus, Dcc translates the attractive responses of netrin1 (13), whereas the Unc5 family mediates the repulsive responses (14). I was confident we would find that a member of the Unc5 family decoded the netrin1 growth boundary in axons, thus confirming that we were describing a repellent activity. But I was wrong: Sup found only minor axon guidance phenotypes in the Unc5 mutants, many of which had been reported before, and stemmed from the loss of Unc5 expression in the DRGs. However, the Dcc mutants looked just like the netrin1 mutants: Tag1 axons stalled, as already described (13) and NF and Robo3 axons dramatically extended into the VZ (Fig. 1D). Thus, Dcc appears to be the chief receptor that mediated the ability of spinal axons to avoid the VZ, and grow alongside a netrin1(-):netrin1(+) border. Moreover, these findings suggested that netrin1-Dcc might be working through an attractive, rather than repulsive, mechanism.

Second moment of clarity: netrin1 protein is deposited on the pial surface of the spinal cord where it acts as a haptotactic growth substrate

As we started to write the paper, Sup had a thesis committee meeting with Kelsey Martin, Alvaro Sagasti and Larry Zipursky where she was questioned skeptically about the feasibility of our hederal model. Larry, in particular, wanted to know more about the distribution of netrin1 protein. Larry, working with his postdoc Orkun Akin, had just shown that netrin had an adhesive, rather than chemotropic, role in the fly medulla (15). We grudgingly admitted that we had never looked, because we had no expectation that netrin1 protein would be anywhere other than the VZ. A couple of days later, Sup appeared at my door, looking worried. “The staining doesn’t look right,” she said. “There’s a lot of background staining on the pial surface of the spinal cord, and in axons.” I internally cursed the non-specificity of our antibody. “Try antigen retrieval.” I said, “That’s what Tim Kennedy had to do!” (16). Sup came back a day or two later, looking even more worried, and told me that the antigen retrieval protocol made the staining “worse.” Both the pial and axonal staining looked brighter than ever. “It looks real!” she said glumly. This distribution made no sense in our model, and I complained to Larry about his opening up a can of worms, when I saw him at a seminar that afternoon. “It’s a bad molecule,” Larry joked.

Sup was right; the antibody staining did look real. There were very low levels of netrin1 in the VZ as we had predicted, but there were much higher levels on most of the pial surface around the circumference of the spinal cord, and on axons (Fig. 1B). I showed James Briscoe the distribution pattern when he visited UCLA a couple of weeks later. “Of course it’s real,” he said, “The neural progenitor cells are making netrin1, and then depositing it on the pial surface using their radial endfeet.” We corralled Ben in from his office next door to mine to assess what he thought. He thought the staining was real too. The three of us peered excitedly at my computer screen, the realization of how the pattern of the netrin1 transcript related to the distribution of netrin1 protein slowly sinking in. We then discussed nothing else for the rest of the weekend.

Sup went on to show that indeed James’ supposition was correct. Through a trick of their cellular geometry, the neural precursors that make netrin1 then use their radial processes to deposit it as a growth substrate onto the pial surface (Fig. 1B, C). No netrin1 is made by the dorsal-most neural precursors, and indeed there is no netrin1 on the most dorsal pial surface. Sup pointed out that the resulting sharp on:off netrin1 boundary on the dorsal pial surface really suggested that netrin1, a member of a laminin family, could not be highly diffusible. In other exhilarating moments of understanding, we realized that spinal neurons specifically initiate NF+ axonal growth on this netrin1 pial-substrate. And that netrin1 only accumulates on commissural axons as they grow adjacent to the netrin1 pial-substrate, as if netrin1 can transfer from this substrate to axons. In a further remarkable result, Sup found that the putative axonal-transfer of netrin1 requires Dcc.

We finally understood how to model our results: VZ-derived netrin1 acts locally as a substrate to promote fasciculated axonal growth in an oriented manner towards the ventral midline. Netrin1 and Dcc appear to cooperate within axons as part of the mechanism that orients growth (Fig. 1C). The innervation of the VZ that we had observed in both netrin1 and Dcc mutants might in fact be randomized, or defasciculated, growth as a result of losing this adhesive interaction (Fig. 1D). VZ-derived netrin1 thus appears to act by haptotaxis, i.e. by the establishment of a local adhesive surface, the alternative model to chemotaxis. As a mechanistic side note: it  remains unclear how this adhesive surface acts to orient and fasciculate commissural axon growth and whether there is an additional “no go” activity in the VZ. Is this mechanism functioning solely through haptotaxis? Or is there also a “hederal” component? Nonetheless, these activities – the pull of an adhesive substrate that promotes fasciculation, perhaps coupled with the push of a “no go” signal – permit axons to grow along the netrin1(-):netrin1(+) border, i.e. in a circumferential path precisely around the VZ.

Third moment of clarity: FP-derived netrin1 is dispensable for axon guidance

Our model – the ability of neural progenitor cells to deposit a haptotactic substrate of netrin1, which promotes ventrally-directed, fasciculated axon growth – was a notable departure from the textbook view of netrin1. But was this model the only mode of action? Or did netrin1 have long-range or short-range activities depending on the cell type? In his original papers, Marc Tessier-Lavigne argued the case for and against netrin1 acting by chemotaxis or haptotaxis (1), but ultimately settled on the idea, well supported by the data at the time, that netrin1 was presented as a long-range cue from the FP. I discussed this point vigorously and endlessly with Sup, Ben, Artur, Larry and Orkun. We had not doubted this model until now. Could our phenotypes still be explained by a long-range activity from the FP? Were the activities of short-range VZ-derived netrin1 in addition to the long-range activities? Or could FP-derived netrin1 really be dispensable for axon guidance?

In the end, the reviewers decided it. The beauty of the finding that neural progenitor cells lay down a substrate of netrin1 that orients and promotes the axonal trajectories of their own neural progeny was not considered sufficiently novel. It was clear what experiment needed to be done, thus we requested the conditional netrin1 lines from Holger Eltzschig and set about breeding them to Cre recombinase driver lines that would remove netrin1 specifically from either FP cells (ΔFP, Fig. 1E) or the dorsal VZ (ΔVZ, Fig.1F). Sup accomplished her breeding scheme astonishing quickly and the day came when she finally had the answer sitting on a microscope slide. I ran to the confocal room, both anxious and excited, to discover the result. “There’s no effect,” she said. “It doesn’t matter if you remove netrin1 from the FP!” In contrast, removing netrin1 from the VZ had profound effects. Axons grew in all directions, but only locally, specifically in the region where netrin1 had been removed from the VZ. Together with the Gli2-mediated FP ablation data, our studies had found no evidence for long-range chemotaxis in the spinal cord. Who knew? Perhaps Cajal’s model was wrong!

The paper was finally accepted at Neuron (11), and came out at the same time as a paper from Alain Chedotal in Nature (17), with complementary findings in the hindbrain. A few days later, I ran into a colleague on the street onside my lab. “I saw your paper in Neuron!” she laughingly scolded me, “But please don’t tell me I have to change my lecture on netrin1! I liked that lecture!” I liked my lecture on netrin1 too, but now I also have to change it. Netrin1, of course, remains the supreme architect of spinal circuitry, but now acts locally as a directional surface along which axons can extend, akin to the holds used by climbers to pioneer their way to the top of a mountain.

Samantha Butler

Department of Neurobiology,
Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research,
Intellectual and Developmental Disabilities Research Center
University of California, Los Angeles.

References

1. Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell. 1994 Aug 12;78(3):425-35. PubMed PMID: 8062385.
2. Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-Lavigne M. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell. 1994 Aug 12;78(3):409-24. PubMed PMID: 8062384.
3. Hedgecock EM, Culotti JG, Thomson JN, Perkins LA. Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes. Dev Biol. 1985 Sep;111(1):158-70. PubMed PMID: 3928418.
4. Serafini T, Colamarino SA, Leonardo ED, Wang H, Beddington R, Skarnes WC, Tessier-Lavigne M. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell. 1996 Dec 13;87(6):1001-14. PubMed PMID: 8978605.
5. Tessier-Lavigne M, Goodman CS. The molecular biology of axon guidance. Science. 1996 Nov 15;274(5290):1123-33. PubMed PMID: 8895455.
6. Ramón y Cajal S. Histology of the nervous system of man and vertebrates. New York: Oxford University Press; 1995.
7. Bentley D, Caudy M. Pioneer axons lose directed growth after selective killing of guidepost cells. Nature. 1983 Jul 7-13;304(5921):62-5. PubMed PMID: 6866090.
8. Ho RK, Goodman CS. Peripheral pathways are pioneered by an array of central and peripheral neurones in grasshopper embryos. Nature. 1982 Jun 03;297(5865):404-6. PubMed PMID: 6176880.
9. Placzek M, Tessier-Lavigne M, Jessell T, Dodd J. Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant. Development. 1990 Sep;110(1):19-30. PubMed PMID: 2081459.
10. Tessier-Lavigne M, Placzek M, Lumsden AG, Dodd J, Jessell TM. Chemotropic guidance of developing axons in the mammalian central nervous system. Nature. 1988 Dec 22-29;336(6201):775-8. PubMed PMID: 3205306.
11. Varadarajan SG, Kong JH, Phan KD, Kao TJ, Panaitof SC, Cardin J, Eltzschig H, Kania A, Novitch BG, Butler SJ. Netrin1 Produced by Neural Progenitors, Not Floor Plate Cells, Is Required for Axon Guidance in the Spinal Cord. Neuron. 2017 Apr 20. PubMed PMID: 28434801.
12. Matise MP, Epstein DJ, Park HL, Platt KA, Joyner AL. Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development. 1998 Aug;125(15):2759-70. PubMed PMID: 9655799. eng.
13. Fazeli A, Dickinson SL, Hermiston ML, Tighe RV, Steen RG, Small CG, Stoeckli ET, Keino-Masu K, Masu M, Rayburn H, Simons J, Bronson RT, Gordon JI, Tessier-Lavigne M, Weinberg RA. Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene. Nature. 1997 Apr 24;386(6627):796-804. PubMed PMID: 9126737.
14. Leonardo ED, Hinck L, Masu M, Keino-Masu K, Ackerman SL, Tessier-Lavigne M. Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature. 1997 Apr 24;386(6627):833-8. PubMed PMID: 9126742.
15. Akin O, Zipursky SL. Frazzled promotes growth cone attachment at the source of a Netrin gradient in the Drosophila visual system. eLife. 2016 Oct 15;5. PubMed PMID: 27743477. Pubmed Central PMCID: 5108592.
16. Kennedy TE, Wang H, Marshall W, Tessier-Lavigne M. Axon guidance by diffusible chemoattractants: a gradient of netrin protein in the developing spinal cord. J Neurosci. 2006 Aug 23;26(34):8866-74. PubMed PMID: 16928876.
17. Dominici C, Moreno-Bravo JA, Puiggros SR, Rappeneau Q, Rama N, Vieugue P, Bernet A, Mehlen P, Chedotal A. Floor-plate-derived netrin-1 is dispensable for commissural axon guidance. Nature. 2017 Apr 26. PubMed PMID: 28445456.

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http://around.uoregon.edu/content/study-brain-formation-finds-possible-link-human-disease @ http://www.doelab.org/ elife https://elifesciences.org/content/6/e26287

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Here are the highlights from the current issue of Development:

A new model for lineage segregation

Lineage segregation during gastrulation has long been thought to be driven by differential cell adhesion and cortical tension among cells, which would together lead to a differential tissue surface tension (TST) and the spatial segregation of specific cell types. However, this long-standing hypothesis is mainly based on in vitro work, and it is as yet unclear whether it holds true in vivo. Now, on p. 1798 Carl-Philipp Heisenberg and colleagues assess the role of differential TST in lineage segregation and find that, contrary to in vitro work, differential TST is insufficient to explain progenitor cell segregation and germ layer formation within the in vivo gastrulating zebrafish embryo. In the study, the authors describe their unique version of video force microscopy called 3D CellFit, which allows them to analyse surface tensions in 3D within a living organism. Using this method, the authors show that ectoderm and mesoderm tissues do not, in fact, exhibit differential TST in the gastrula. They further present evidence that the apparent discrepancy between the in vitro and in vivo results is due to a difference in osmolarity between the culture medium and the interstitial fluid that surround the cells. Finally, by inhibiting the function of the small GTPase Rac, a key regulator of protrusion-driven cell migration, the authors show that directed cell migration, rather than differential TST, provides the major mechanism that determines the segregation of the germ layer progenitors.

Exciting input for inhibitory neurons

A crucial phase in neuronal development is the integration of newborn neurons into circuits. The right balance must be struck between excitatory and inhibitory neurons; however, the mechanisms that control inhibitory neuron integration and drive the maturation of inhibitory connectivity remain largely uncharacterized. In this issue (p. 1807) Michael Francis and colleagues identify a novel, non-cell-autonomous mechanism that regulates inhibitory neuron synapse formation at the neuromuscular junction (NMJ). The authors examine the electrophysiology and structural organization of GABAergic synapses at the NMJ in a number of different C. elegans mutants with developmental or functional defects in excitatory motor neurons. These analyses reveal that the activity of excitatory cholinergic motor neurons, during a period that coincides with the development of postembryonic GABAergic motor neurons, critically affects the size and distribution of GABAergic pre- and post-synaptic specializations. Furthermore, a severe reduction of cholinergic inputs to newly born GABAergic neurons reduces their synaptic density but increases the synapse size. This study makes an important contribution to our understanding of how neuronal activity impacts synapse development and highlights the functional relationship between excitatory and inhibitory neurons during circuit formation.

Heat shock protein regulates human hepatocyte differentiation

The directed differentiation of human induced pluripotent stem cells (iPSCs) into mature hepatocytes is a major goal of liver research. The approach relies on the recapitulation of developmental processes, and thus a better understanding of what regulates hepatocyte differentiation is essential in order to produce these cells more efficiently and to a greater maturity. In this issue (p. 1764) Stephen Duncan and colleagues identify heat shock protein 90 beta (HSP90β) as a novel regulator of endoderm-to-hepatocyte conversion in differentiating human iPSC cultures. The authors begin the study by conducting a screen for small molecules that modify the activity of master hepatocyte transcription factor HNF4A, identifying 132 candidate ‘hits’. They then focus on the role of molecular chaperone HSP90β and show how it acts at the post-translational level to stabilize HNF4A, thus controlling its half-life and availability. Targeted CRISPR-CAS9 mutations in the gene encoding HSP90 perturbs HSP90β levels, resulting in a dramatic reduction of HNF4A protein levels and reduced expression of HNF4A target genes. Moreover, these experiments reveal that HSP90β is specifically required for endoderm-to-hepatocyte conversion, and not for endoderm commitment generally. This study uncovers a new player in hepatocyte differentiation, and further highlights the utility of an iPSC differentiation platform coupled with chemical screens to uncover novel developmental mechanisms.

PLUS

The developmental biology of genetic Notch disorders

This Review discusses the developmental processes underlying Notch-related congenital disorders in humans, drawing on data from model organisms and genome-sequencing projects, on p. 1743.

An interview with Eric Wieschaus

In our latest interview, Eric Wieschaus tells us about his Nobel Prize-winning fly screens, his interest in the cell biology of development and his love of painting, on p. 1740.

Obituary: Tokindo S. Okada (1927-2017)

A retrospective on the life and work of the pioneering Japanese developmental biologist Tokindo Okada, whose research focussed on cell plasticity and transdifferentiation, on p. 1737.

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The Semrau lab

Our lab is interested in the fundamental molecular mechanisms underlying lineage decision-making in stem cells and in vivo.  We are fascinated by the question how defined and stable cell types are generated by the interplay of signaling inputs and gene regulatory networks. We study this question by precise quantification of the states of single cells in combination with bioinformatics analysis and machine learning.  Based on this quantitative understanding we want to develop new ways to manipulate lineage decisions during in vitro differentiation in precisely controlled ways. Our group is highly interdisciplinary and works at the interface of biology, biophysics, bioinformatics and biomedical sciences.

References:

Semrau, S., van Oudenaarden, A., 2015. Studying Lineage Decision-Making In Vitro: Emerging Concepts and Novel Tools. Annu. Rev. Cell Dev. Biol. 31, 317–345. doi:10.1146/annurev-cellbio-100814-125300

Semrau, S., Goldmann, J., Soumillon, M., Mikkelsen, T.S., Jaenisch, R., van Oudenaarden, A., 2016. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells. bioRxiv 068288. doi:10.1101/068288

Semrau, S., Crosetto, N., Bienko, M., Boni, M., Bernasconi, P., Chiarle, R., van Oudenaarden, A., 2014. FuseFISH: Robust Detection of Transcribed Gene Fusions in Single Cells. Cell Reports 6, 18–23. doi:10.1016/j.celrep.2013.12.002

Project and key responsibilities

The available postdoc project aims to create a single-cell atlas of the human embryonic kidney. Information about the transcriptional profiles and locations of all cell types in the embryonic kidney will improve our understanding of kidney development and will provide an important benchmark for kidney organoids. In this project you will be responsible for performing single-cell RNA-seq and single-molecule FISH measurements of human embryonic kidney samples. The necessary experimental techniques are established in our lab and samples will be provided by our collaborators. In particular, you will dissociate the tissue and prepare single-cell RNA-seq libraries with the drop-seq technique (Macosko et al., Cell, 2015). You will analyze the RNA-seq data (potentially together with a bioinformatics collaborator) and identify cell types using state-of-the-art machine learning tools. Based on these results you will define a set of marker genes that will allow you to locate cell types by single-molecule FISH in intact tissue sections. This comprehensive spatial molecular data set will then allow you, for example, to establish intercellular signaling networks.

Selection criteria

• You hold a PhD degree in one of these disciplines: biology, biochemistry, bioengineering or related disciplines
• You have a strong interest in experimental quantitative biology, in particular related to human development and stem cell differentiation
• You have experience with molecular biology techniques, in particular NGS library preparation
• Experience with programming in R or Matlab and relevant bioinformatics packages is a plus.
• You are proficient in spoken and written English, and have good communication and writing skills
• You are independent, creative and have team spirit

Research at our department

Our lab is part of the Leiden Institute of Physics (http://www.physics.leidenuniv.nl) and situated at the Leiden Cell Observatory (http://cellobservatory.leidenuniv.nl). The Cell Observatory is a highly collaborative community dedicated to the visualization and understanding of the fundamental molecular mechanisms of life, which is part of the core scientific profile of Leiden University. The Cell Observatory houses state-of-the-art bio-imaging facilities shared among the member labs, which actively develop new methods for the quantitative measurement of single-cell properties.

Information

More information about our lab can be found at http://www.semraulab.com/.

Enquiries can be made to Dr. Stefan Semrau (semrau@physics.leidenuniv.nl).

Information about the Faculty of Science can be found at http://www.science.leidenuniv.nl/index.php/english/ and about Leiden University at http://workingat.leiden.edu/.

Applications

To apply for this vacancy, please send an email to Dr. Stefan Semrau (semrau@physics.leidenuniv.nl) until June 18. Please include your curriculum vitae, a letter of motivation and the names of 3 potential references.

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“It finally got accepted!”, fol­­lowed by “It’s finally out!” about a month later. I am certain this ‘finally’ feeling about their paper is very familiar to those well-acquainted with the peer review process, and it was no different for our recently published Resource article. The ‘biotagging paper’, as we call it within the Sauka-Spengler lab, is the culmination of several years’ of hard (and often frustrating) work that eventually paid off in more (unexpected) ways than one. Tatjana spearheaded the initial work for biotagging while still at Caltech, by transferring components and approaches she developed in the chicken system into the zebrafish. She worked together with Le and Tatiana, then postdoctoral fellows at Caltech, before the rest of us joined in for the lengthy optimisation, submission and review stage.

Part I: What is “biotagging”?

Biotagging is essentially an encompassing term for our Do-It-Yourself (DIY) in vivo biotinylation system for zebrafish researchers, which can be utilised in creative ways to suit specific biological needs. In vivo biotinylation was first employed in mouse (de Boer et al., 2003) by John Strouboulis when he was in Frank Grosveld’s lab and then applied for use in nuclei isolation from Arabidopsis thaliana by Roger Deal and Steven Henikoff (Deal and Henikoff, 2010). The technique was also applied to the nematode worm at around the same time (Ooi et al. 2009). The core of the technique lies in the ability of bacterial biotin ligase (BirA) to biotinylate an Avi-tagged protein-of-interest. In our binary biotagging system, the researcher decides where BirA will be expressed, which protein is Avi-tagged, and then generates transgenic lines that express these components. Crossing BirA-driver and Avi-effector heterozygous lines will give rise to ~25% of double-alleled offspring, where biotinylation of the Avi-tagged protein product only occurs in cells that also express BirA. The sky is the limit when it comes to the combinations of BirA/Avi that one can use. In the paper, we present a ‘starter’ toolkit consisting of multiple tissue-(neural crest, heart, blood) and cellular compartment-specific (ribosomes, nuclei) transgenic lines, as well as constructs to make your own lines.

Part II: Trials and Tribulations

The deconstruction (and reconstruction) of biotagging

The elegance of in vivo biotinylation means that we are not the only group to perform this method in vertebrates. For example, Michael Housley from Stainier lab (Housley et al. 2014) utilised in vivo biotinylation in zebrafish to apply the TRAP (Translating Ribosome Affinity Purification) method developed by Myriam Heiman and colleagues (Heiman et al., 2008). In vivo biotinylation experiments are not ‘difficult’ per se, but we found that obtaining a clear difference between nuclear and polyribosomal data required a remarkable amount of troubleshooting and optimisation. Our patience paid off, as this was rewarded by a wealth of information provided by a high resolution view into the migratory neural crest nascent (nuclear) and polyribosomal transcriptomes from ~200k cells.

In fact, the entire optimisation process came about by accident. In the paper, we described our surprising results when comparing the nuclear transcriptome of Sox10-positive cells at 16-18ss (migratory neural crest) to a ubiquitous control. By looking at both non-poly and polyadenylated transcripts (whole nuclear transcriptomes), our data did not yield any statistically significant neural crest-specific signature, which is what one would expect, as the enriched transcripts should be neural crest-specific. On the other hand, analysis of polyadenylated nuclear transcripts at 24hpf yielded a neural crest-specific signature. This led to further pain-staking deconstruction of our technique where, months later, we eventually came to the surprisingly simple but crucial element for the protocol to be as consistent as it is today – ensuring the complete lysis of cells (by using hypotonic buffer in excess) to release subcellular compartments into the lysate and minimise the presence of intact cell surface membranes. It is also worth noting, that a key element to the success of our protocol was the usage of an Avi-tagged chicken nuclear envelope protein, RanGAP, to label nuclei. Weirdly enough, chicken RanGAP expressed in zebrafish localised to the nuclei, but zebrafish RanGAP did not.

Having reconstructed the method, we were now eager to repeat the previous 16-18ss neural crest experiment. Imagine our initial dismay when the results were…strikingly similar. However, this was soon replaced by curiosity that drove us to carefully re-examine our results and try to figure out what IS actually going on…

Biotagging of migratory neural crest nuclei transcriptome reveals…what?

The brainstorming sessions were remarkably memorable. They were always long, often ‘lively’ as we picked at each other’s brains, and at times quite outrageous as frustrations ran high. It didn’t take us very long to notice that bidirectional transcription at non-coding regions was enriched in neural crest nuclei. However, it was a long journey after that, as we tried to quantify the phenomenon genome-wide, reproduce what we saw, believe in what we saw, and build our findings into a coherent story. Ultimately, we needed to drive home our main message – that bidirectional transcription at non-coding regions is tissue-specific, thus introducing a new method to detect active regulatory elements. These elements form the molecular signature of neural crest cells, which is traditionally based on the expression of protein-coding genes that are mainly transcription factors. We were also excited to find developmentally regulated long non-coding RNAs and transposable elements.

In short, we are proud of what we have managed to achieve with biotagging. The journey may have been long and arduous, but we have learned a lot from this project.  We hope that we have provided a cool new system that includes a fully optimised tool (plasmids on Addgene) with clean protocols (available on the Resources page of our lab website), handy transgenic lines to get started with, as well as analysis pipelines tailored to biotagging datasets. Having worked out the technical intricacies of this system, this toolkit allows the zebrafish community (including us!) to study specific cellular populations in vivo on the systems level, tackling biological questions that could be important to development and disease.

Le A. Trinh, Vanessa Chong-Morrison, Daria Gavriouchkina, Tatiana Hochgreb-Hägele, Upeka Senanayake, Scott E. Fraser, Tatjana Sauka-Spengler. 2017. Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome. Cell Reports Volume 19, Issue 2, p425–440

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NEUBIAS, the Network of European
BioImage Analysts (www.neubias.org), is delighted to announce two new Training Schools on BioImage Analysis:

• Early Career Investigators (life scientists)
• Facility staff (imaging & analysis staff)

The focus of training will be on construction and automation of image analysis workflows, using as examples more than one toolbox and different exercises. The schools will be held in Gothenburg 11-14th of September 2017, hosted by the Centre for Cellular Imaging – Sahlgrenska Academy, University of Gothenburg, Sweden.

NEUBIAS schools are an excellent opportunity to learn from many experts in bioimage analysis (we are expecting ~40 specialists at the event) and  “….a great mix of intensive learning and community networking” (former trainee testimonial!).

• applications for Gothenburg are now open (each school has 25 available seats and 10-12 trainers).
• Within the COST framework, a few travel grants are offered to applicants who qualify.
• Registration deadline: 26th of May, 2017 (must submit also “letter of motivation”).
• Selection notification: 1st week of June 2017.

More information about schools (programme & trainers) and venue, travel & lodge available at our website (linked above).

On behalf of all NEUBIAS members,
Julien Colombelli, Chair; Kota Miura, Vice-Chair
Julia Fernandez-Rodriguez, Local organizer

Carolina Wählby, Jan Eglinger, Joakim Lindblad & Nuno P Martins, TS4&5 programme organizers

Gaby G Martins & Fabrice Cordelières, WG2-Training leaders

Perrine Paul-Gilloteaux, WG4-Webtool leader
Sébastien Tosi, WG5-Benchmarking & Sample Datasets leader

NEUBIAS is an European network of currently ~180 members and 35 countries, which aims to promote the communication between Life Scientists, Instrumentalists, Developers and BioImage Analysts and to establish and promote the role of Bioimage Analysts in Life Science. Our mission includes:

• A training programme for 3 different target audiences:Early Career researcher, Facility Staff, Analyst (running until 2020 – expected 400 trainees and 15 training schools).
• Promote different yearly events (NEUNIAS2020 Conference, workshops [training schools], Taggathons)
• Online Resources: Repository of tools and workflows, Benchmarking and Sample datasets, Training material and Open Textbook.
• A Short Term Scientific Mission mobility programme for Scientists to visit Host Labs and get in depth insights into cutting edge Image Analysis technology.
• Outreach material.
  

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Our 20th instalment of this series comes from South Korea and features an investigation into the molecular basis of how temperature influences developmental transitions in Arabidopsis seedlings, recently published in Developmental Cell. We caught up with joint first authors Jun-Ho Ha and Hyo-Jun Lee, and their supervisor Chung-Mo Park, Professor in the Department of Chemistry, Seoul National University (SNU), to hear the story of the paper.

Chung-Mo, can you give us your scientific biography – I understand you spent some years in the US before returning to South Korea?

CMP I am currently professor in the Department of Chemistry at SNU. I earned my Bachelor of Science in the Department of Science Education from Seoul National University in 1983 and my PhD in molecular virology from State University of New York at Buffalo in 1993 under the supervision of professor Jeremy Bruenn. The topic of my thesis work was identification of killer toxin genes in a double-stranded virus endogenously residing in Ustilago maydis, a corn smut fungus and functional and structural characterization of the killer toxin proteins. After completing my PhD, I worked as a postdoctoral researcher in the same university and the Hauptman-Woodward Medical Research Institute, Buffalo, until I joined the Kumho Life & Environmental Science Laboratory, Korea, as PI in 1996. In the Kumho Laboratory, I worked on the photochemical and photobiological characterization of phytochrome photoreceptors in higher plants and the cyanobacteria Synechocystis PCC6803 and their associated light signal transduction in photomorphogenic responses.

In 2002, I accepted an associate professor position in the Department of Chemistry, SNU, where I have been since that time. While at SNU, my research team has been working on diverse aspects of plant growth and developmental processes, such as seed germination, phase transition and flowering induction, and leaf senescence. I have also been working on plant responses to environmental stresses with emphasis on temperature extremes and drought stresses. In recent years, my research is focused on plant adaptation to high but nonstressful temperatures (warm temperatures) with emphasis on leaf hyponasty, heat dissipation from leaves, and autotrophic development.

And what is South Korea like as a place to do science?

CMP The Korean government and several biotech companies have been investing a huge amount of research fund during the last 30 years. While industrial research and development has been a priority as a potential driving force of economic growth, the Korean government is also spending heavily on basic research. In plant science, there is a national research supporting program, termed New-Generation Biogreen 21, which is organized and supported by the Korean Rural Development Administration.  The Program supports various research on both model plants and crops. It is considered that although not sufficient, enthusiastic plant scientists are able to get enough research funds to perform both basic and applied researches in recent years.

Jun-Ho and Hyo-Jun – how did you come to join Chung-Mo’s lab?

JHH I earned my Bachelor of Science in chemistry. I was also interested in molecular biology with an expectation that combining chemical and biological principles would be exciting in understanding life. While I was looking for an appropriate lab for my graduate study, I met Chung-Mo Park, who is my current thesis advisor. I was greatly impressed by his passion for science and research. It was also impressive that his group is working on plant molecular biology in the Department of Chemistry. I therefore decided to join his laboratory for my graduate study.

HJL Since I was a high school student, I planned to be a scientist with an aim of discovering unknown principles of nature and living organisms. After I entered the Department of Chemistry, Seoul National University, as an undergraduate student, I searched for potential labs in the Department appropriate for my research carrier. I realized that Chung-Mo’ lab is unique among the laboratories in that he is studying plant molecular biology and biochemistry. I thought that understanding molecular biological and biochemical mechanisms underlying plant performance would be helpful for me to find ways to sustain the Earth’s ecosystem. In particular, as a chemist, I thought that applying chemical tools to understanding biological systems would be interesting. I therefore decided to perform my graduate study in his lab.

Before your work, what was known about how plants respond to temperature changes during autotrophic development, and what was the key question you set out to answer?

CMP, JHH & HJL It is well known that extreme temperatures significantly affect plant performance, including autotrophic development. In addition, associated molecular events and signaling schemes are fairly well understood. In nature, the soil temperature is rapidly elevated under warm temperature conditions. Therefore, developing seedlings should cope with high temperatures while they pass through the heat-absorbing soil layer to obtain photosynthetic capacity required for autotrophic growth. However, it is almost unknown how the heat-labile shoot apical meristem tissues of developing seedlings handle the temperature constraints. It has recently been reported that warm temperatures, in a temperature range of 23 – 28^oC in Arabidopsis, accelerate cell elongation during early seedling development. Thus, we were curious about whether and how warm temperatures influence chlorophyll biosynthesis during autotrophic development.

Can you give us key results of the paper in a paragraph?

CMP, JHH & HJL We demonstrated that developing seedlings are capable of maintaining chlorophyll biosynthesis required for autotrophic development at warm temperature conditions. A group of photooxidoreductase (POR) enzymes is responsible for chlorophyll biosynthesis. Notably, they are susceptible to warm temperatures and thus rapidly inactivated in developing seedlings while they pass through the warm soil layer. We found that an RNA-binding protein FCA maintains the abundance of POR enzymes at warm temperatures in developing seedlings. Without FCA, plants fail to maintain the enzyme abundance, resulting in loss of chlorophyll and thus failure to achieve autotrophic growth. Our work provide a molecular basis for the acquisition of autotrophic growth under fluctuating temperature conditions in plants.

How do have any idea of what is upstream of FCA? How does it sense temperature changes?

CMP, JHH & HJL Our recent findings strongly support that the typical RNA-binding protein FCA plays a critical role through epigenetic control of target genes during high temperature responses and thermomorphogenesis in Arabidopsis. Our data also indicate that FCA sustains the thermos-stable expression of POR enzymes during autotrophic development at warm temperatures. Altogether, these observations suggest that FCA function is thermos-regulated. However, it is current unclear how FCA is activated by ambient temperatures. We found that gene transcription and protein stability of FCA are not altered by temperature changes. Its subcellular localization is also unaltered under fluctuating temperature conditions.

Our preliminary data suggest that warm temperatures activates FCA through post-translational modifications, such as protein phosphorylation. We are currently under way to examine if FCA is differentially phosphorylated or chemically modified in response to temperature changes by employing global-scale proteomics.

Do you think your work will have relevance to agriculture in a warming world?

CMP, JHH & HJL Global warming depicts the gradual elevation of the average temperature of the Earth’s climate system. It is widely documented that under high ambient temperature conditions, plants exhibit distinct morphological and developmental traits, such as accelerated hypocotyl growth, leaf hyponasty, reduction of stomatal density, and early flowering, which profoundly influence crop productivity and commercial values. Our findings on plant thermal responses are closely associated with global warming. We propose that the FCA-mediated thermal adaptation of autotrophic development allows developing seedlings to cope with the heat-absorbing soil surface layer under natural conditions. In particular, we found that a single gene mutation causes a total loss of chlorophyll biosynthesis and autotrophic development at warm temperatures, providing a way of enhancing plant adaptation to thermal fluctuations in crop agriculture.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

HJL & JHH In the initial stage of the research, we germinated and grew the FCA-defective mutants at normal temperatures for 3 days before transferred to warm temperatures to see if the fca mutations affect seedling growth. However, we did not observe any phenotypic differences in seedling growth and greening patterns in the mutants. A few months later, we anticipated that the fca mutations might affect the earlier stages of seedling growth. To examine the hypothesis, we germinated and grew the mutant seedlings at 28^oC. We were surprised at the albino phenotype of the mutants. This observation triggered the re-examination of the thermal phenotypes of the fca mutants, resulting in the completion of this paper.

At first, we could not figure out why the fca mutants exhibited albinism only when germinated and grown at warm temperatures. As a potential cause of the albino phenotype, we considered several possibilities, such as defects in chloroplast development, chlorophyll biosynthesis, or both. It was found that the expression of POR genes was disrupted in the fca mutants when grown at warm temperatures. Accordingly, the level of chlorophylls was extremely low in the mutants, showing that the thermo-sensitive albino phenotype of the mutants is caused primarily by defects in chlorophyll biosynthesis, consistent with the FCA-mediated stabilization of POR production.

And what about the flipside: any moments of frustration or despair?

HJL & JHH The FCA-defective mutants are well-known late flowering mutants. A set of transgenic fca plants expressing POR genes were required for this study. It needs a lot of time to generate the transgenic plants because it takes 3-4 months to obtain seeds from the transgenic plants. While we were generating transgenic plants, we realized that a wrong expression construct was accidentally used, spending at least 5 additional months to obtain correct transgenic plants.

We also remember the frustrating moment when temperature controllers in the culture room were out of order during last summer, when we experienced a rarely high temperature and thus unstable supply of electricity in Korea. We had to grow a full set of plants again after a period time for fixing the temperature controllers.

What are your career plans following this work?

HJL I am currently a postdoc in Chung-Mo Park’s lab. I will continue studying for a while on molecular and physiological mechanisms underlying plant thermomorphogenesis. I am interested in the as-yet unidentified regulator of POR abundance at warm temperatures. After finishing the experiments, I am planning to find an appropriate postdoc position to extend my research career in environmental control of plant proteomics.

JHH I hope to be able to finish my thesis study in a couple of years, after which I am planning to find postdoc positions in Korea or in USA to extend my research career in the field.

And what next for the Park lab?

CMP We have a well-organized research system with a variety of molecular and biochemical tools, personnel, and facilities. We are specialized in gene regulatory mechanisms with emphasis on induction and activation mechanisms of transcription factors. Using these research tools and system, we will further extend our researches on plant thermomorphogenesis, which is emerging as a hot issue in the field because of the growing concern about global warming. In particular, we are focused on the functional linkage between photomorphogenic responses and growth hormones. We are also preparing a long-term project for engineering crop plants to enhance their adaptation capacity to changing temperature environment.

Jun-Ho Ha, Hyo-Jun Lee, Jae-Hoon Jung and Chung-Mo Park. 2017. Thermo-Induced Maintenance of Photo-oxidoreductases Underlies Plant Autotrophic Development. Developmental Cell 41(2): 170-179.

Browse the People behind the Papers archive here

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Location:          The Francis Crick Institute, Midland Road, London

Contract:          Fixed-term (3 years), Full time

Salary:             Competitive with benefits, subject to skills and experience

Vacancy ID:      5003

SHORT INTRODUCTION/SUMMARY

We seek a talented and motivated postdoc to join a Research Group led by Victor Tybulewicz at the Francis Crick Institute. The Group currently consists of 12 scientists, including 6 postdocs and 4 PhD students. One of the two main research interests of the Group is the study of the genetics underlying Down Syndrome. The Group has previously generated a series of mouse models of Down Syndrome that can be used to map the location of dosage-sensitive genes that cause Down Syndrome phenotypes (Lana-Elola et al, eLife 2016).

PROJECT SCOPE/ DESCRIPTION

The postdoc will study the genetics and developmental biology underlying congenital heart defects in Down Syndrome. The overall aim is to understand how increased dosage of genes on human chromosome 21 leads to heart defects. Specifically, the project aims to identify the dosage-sensitive genes that cause heart defects when present in three copies and to elucidate the mechanism by which the genes cause pathology. The work will involve use of genetic, developmental biology and biochemical techniques including microscopy, image analysis, and RNAseq, and will be supported by the excellent core facilities of the Institute. The work is funded by the Wellcome Trust.

The Francis Crick Institute is a biomedical discovery institute dedicated to understanding the fundamental biology underlying health and disease. Its work is helping to understand why disease develops and to translate discoveries into new ways to prevent, diagnose and treat illnesses such as cancer, heart disease, stroke, infections, and neurodegenerative diseases.

An independent organisation, its founding partners are the Medical Research Council (MRC), Cancer Research UK, Wellcome, UCL (University College London), Imperial College London and King’s College London.

The Crick was formed in 2015, and in 2016 it moved into a brand new state-of-the-art building in central London which brings together 1500 scientists and support staff working collaboratively across disciplines, making it the biggest biomedical research facility under a single roof in Europe.

The Francis Crick Institute will be world-class with a strong national role. Its distinctive vision for excellence includes commitments to collaboration; to developing emerging talent and exporting it the rest of the UK; to public engagement; and to helping turn discoveries into treatments as quickly as possible to improve lives and strengthen the economy.

If you are interested in applying for this role, please apply via our website https://goo.gl/IaFC2r

The closing date for applications is 10 June at 23:30 pm.

Please note: all offers of employment are subject to successful security screening and continuous eligibility to work in the United Kingdom.

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We are pleased to announce the Center for Dental, Oral, Craniofacial Tissue and Organ Regeneration (C-DOCTOR – www.c-doctor.org) RFP that will award funding to promising dental, oral and craniofacial tissue engineering and regenerative medicine technologies and help them advance toward human clinical trials through customized product development advice and core resources. Please see the full RFP below the cut or here for details – deadline June 9, 2017. We ask that you kindly distribute this RFP widely to investigators who may be interested.

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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

At the end of April, life science preprinting received a boost with the news that the Chan Zuckerberg Initiative will provide funding for the main preprint server, bioRxiv, which had the week before celebrated hosting its 10,000th article.

bioRxiv just passed 10,000 papers! :-)
Huge thanks to all involved https://t.co/xfNeiSBk3v pic.twitter.com/BQhIohafNX

— Richard Sever (@cshperspectives) April 21, 2017

And a good month for bioRxiv was also a good month for developmental biology (and related) preprints. This month we found 115 preprints covering coral regeneration, spider development, a root-on-a-chip, a lot of discussion about publishing in our ‘Research Practise’ section, and a range of stem cell and cell biology, hosted on bioRxiv, F1000Research, PeerJ  and arXiv.

Use these links to get to the section you want –

Developmental biology

| Patterning & signalling

| Morphogenesis & mechanics

| Genes & genomes

| Stem cells, regeneration & disease modelling

Cell biology

Modelling

Evo-devo & evo

Tools & resources

Research practice

Why not…

Developmental biology

| Patterning & signalling

An Embryonic System To Assess Wnt Transcriptional Targets. Jahnavi Suresh, Nathan Harmston, Ka Keat Lim, Prameet Kaur, Helen Jingshu Jin, Jay B. Lusk, Enrico Petretto, Nicholas S. Tolwinski

C. elegans Flavin Monooxygenases Regulate C. elegans Axon Guidance and Growth Cone Protrusion with UNC-6/Netrin signaling and Rac GTPases. Mahekta R. Gujar, Aubrie M. Stricker, Erik A. Lundquist

TGF(beta) Mediated Structural Remodeling Facilitates Optic Fissure Fusion And The Necessity Of BMP Antagonism In This Process. Max D. Knickmeyer, Juan L. Mateo, Priska Eckert, Eleni Roussa, Belal Rahhal, Aimee Zuniga, Kerstin Krieglstein, Joachim Wittbrodt, Stephan Heermann

Alternative cleavage of a bone morphogenetic protein (BMP) produces ligands with distinct developmental functions and receptor preference. Edward N Anderson, Kristi A Wharton

MCAM controls cell autonomous polarity in myogenic and chondrogenic differentiation. Artal Moreno-Fortuny, Laricia Bragg, Giulio Cossu, Urmas Roostalu

The Calcineurin-FoxO-MuRF1 Signaling Pathway Regulates Myofibril Integrity in Cardiomyocytes. Jau-Nian Chen, Hirohito Shimizu, Adam D Langenbacher, Jie Huang, Kevin Wang, Georg Otto, Robert Geisler, Yibin Wang

Secretogranin-II Plays A Critical Role In Zebrafish Neurovascular Modeling. Binbin Tao, Hongling Hu, Kimberly Mitchell, Ji Chen, Haibo Jia, Zuoyan Zhu, Vance Trudeau, Wei Hu

Sympathetic Nerve Activity Promotes Cardiomyocyte Cell-Cycle Arrest And Binucleation. Li Chen, Alexander Y Payumo, Kentaro Hirose, Rachel B. Bigley, Jonathan Lovas, Rejji Kuruvilla, Guo N. Huang

| Morphogenesis & mechanics

Par3/Baz levels control epithelial folding at actomyosin-enriched compartmental boundaries. Jose M Urbano, Huw W Naylor, Elena Scarpa, Leila Muresan, Bénédicte Sanson

Large, long range tensile forces drive convergence during Xenopus blastopore closure and body axis elongation. David R Shook, Raymond Keller, Lance Davidson, Eric M. Kasprowicz

Cells From The Same Lineage Switch From Reduction To Enhancement Of Size Variability In Arabidopsis Sepals. Satoru Tsugawa, Nathan Hervieux, Daniel Kierzkowski, Anne-Lise Routier-Kierzkowska, Aleksandra Sapala, Olivier Hamant, Richard S. Smith, Adrienne H. K. Roeder, Arezki Boudaoud, Chun-Biu Li

Cell type boundaries organize plant development. Monica Pia Caggiano, Xiulian Yu, Neha Bhatia, André Larsson, Hasthi Ram, Carolyn K Ohno, Pia Sappl, Elliot M Meyerowitz, Henrik Jönsson, Marcus G Heisler

Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans. Chaogu Zheng, Margarete Diaz-Cuadros, Susan Laura Jao, Ken Nguyen, David H Hall, Martin Chalfie

Myomerger Induces Fusion Of Non-Fusogenic Cells And Is Required For Myoblast Fusion. Malgorzata Quinn, Qingnian Goh, Mitsutoshi Kurosaka, Dilani Gamage, Michael Petrany, Vikram Prasad, Douglas Millay

| Genes & genomes

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos. Shai Joseph, Mate Palfy, Lennart Hilbert, Mukesh Kumar, Jens Karschau, Vasily Zaburdaev, Andrej Shevchenko, Nadine Vastenhouw

Comprehensive Characterization Of The Complex Lola Locus Reveals A Novel Role In The Octopaminergic Pathway Via Tyramine Beta-Hydroxylase Activation. Nadja Dinges, Violeta Morin, Nastasja Kreim, Tony Southall, Jean-Yves Roignant

Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice. Shintaro Yamada, Seoyoung Kim, Sam E Tischfield, Julian Lange, Maria Jasin, Scott Keeney

Germ cell connectivity enhances cell death in response to DNA damage in Drosophila testis. Yukiko M Yamashita, Kevin L Lu

The G-box transcriptional regulatory code in Arabidopsis. Daphne Ezer, Samuel JK Shepherd, Anna Brestovitsky, Patrick Dickinson, Sandra Cortijo, Varodom Charoensawan, Mathew S Box, Surojit Biswas, Philip Wigge

Cross-talk between active DNA demethylation, resetting of cellular metabolism and shoot apical growth in poplar bud break. Daniel Conde, Mariano Perales, Anne-Laure Le Gac, Christopher Dervinis, Matias Kirst, Stephane Maury, Pablo Gonzalez-Melendi, Isabel Allona

A Role For The F-Box Protein HAWAIIAN SKIRT In Plant miRNA Function. Patricia Lang, Michael Christie, Ezgi Dogan, Rebecca Schwab, Joerg Hagmann, Anna-Lena Van de Weyer, Detlef Weigel

Sequence Features Of MADS-Domain Proteins That Act As Hubs In The Protein-Protein Interaction Network Controlling Flower Development. Florian Ruempler, Guenter Theissen, Rainer Melzer

Nuclear Microenvironments Modulate Transcription From Low-Affinity Enhancers. Justin Crocker, Albert Tsai, Anand K Muthusamy, Luke D Lavis, Robert H Singer, David L Stern

Genetical genomics reveals Ras/MAPK modifier loci. Mark G. Sterken, Linda Van Bemmelen van der Plaat, Joost A.G. Riksen, Miriam Rodriguez, Tobias Schmid, Alex Hajnal, Jan E. Kammenga, Basten L. Snoek

5-Hydroxymethylcytosine Is Highly Dynamic Across Human Fetal Brain Development. Helen Spiers, Eilis Hannon, Leonard Schalkwyk, Nicholas Bray, Jonathan Mill

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development. Joel Hrit, Cheng Li, Elizabeth Allene Martin, Mary Goll, Barbara Panning

Transcription Activation Of Early Human Development Suggests DUX4 As An Embryonic Regulator. Virpi Töhönen, Shintaro Katayama, Liselotte Vesterlund, Mona Sheikhi, Liselotte Antonsson, Giuditta Filippini-Cattaneo, Marisa Jaconi, Anna Johnsson, Sten Linnarsson, Outi Hovatta, Juha Kere

Extensive alternative splicing transitions during postnatal skeletal muscle development are required for Ca2+ handling. Tom Cooper

| Stem cells, regeneration & disease modelling

Morphogen And Community Effects Determine Cell Fates In Response To BMP4 Signaling In Human Embryonic Stem Cells. Anastasiia Nemashkalo, Albert Ruzo, Idse Heemskerk, Aryeh Warmflash

A protein phosphatase network controls temporal and spatial dynamics of differentiation commitment in human epidermis. Ajay Mishra, Angela Oliveira Pisco, Benedicte Oules, Tony Ly, Kifayathullah Liakath-Ali, Gernot Walko, Priyalakshmi Viswanathan, Jagdeesh Nijjher, Sara-Jane Dunn, Angus I Lamond, Fiona M Watt

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during trophectoderm and inner cell mass specification. Daosheng Huang, Xiaoping Han, Ping Yuan, Amy Ralston, Lingang Sun, Mikael Huss, Tapan Mistri, Luca Pinello, Huck Hui Ng, Guocheng Yuan, Junfeng Ji, Janet Rossant, Paul Robson, Guoji Guo

A lncRNA/Lin28/Let7 Axis Coupled To DNA Methylation Fine Tunes The Dynamics Of A Cell State Transition. Meng Amy Li, Paulo P. Amaral, Priscilla Cheung, Jan H. Bergmann, Masaki Kinoshita, Tuzer Kalkan, Meryem Ralser, Sam Robson, Ferdinand von Meyenn, Maike Paramor, Fengtang Yang, Caifu Chen, Jennifer Nichols, David L. Spector, Tony Kouzarides, Lin He, Austin Smith

Mouse embryonic stem cells can differentiate via multiple paths to the same state. James Alexander Briggs, Victor C Li, Seungkyu Lee, Clifford J Woolf, Allon Klein, Marc W Kirschner

Pervasive Discordance Between mRNA And Protein Expression During Embryonic Stem Cell Differentiation. Patrick van den Berg, Bogdan Budnik, Nikolai Slavov, Stefan Semrau

Establishment In Culture Of Expanded Potential Stem Cells. Jian Yang, David Ryan, Wei Wang, Cheuk-Ho J Tsang, Guocheng Lan, Xuefei Gao, Liliana Antunes, Adam Clifford Wilkinson, Yong Yu, Aleksandra Kolodziejczyk, Lia Campos, Juexuan Wang, Fengtang Yang, Yosuke Tanaka, Melanie Eckersley-Maslin, Michael Woods, James Bussell, Ramiro Ramirez-Solis, Wolf Reik, Bertie Gottgens, Xiangang Zou, Liming Lu, Cui Wang, Hideki Masaki, Jacqui White, Hiro Nakauchi, Zheng Zhong, Sarah Teichmann, Beiyuan Fu, Zhexin Zhu, Pentao Liu

Fused dorsal-ventral cerebral organoids model human cortical interneuron migration. Joshua A Bagley, Daniel Reumann, Shan Bian, Juergen A. Knoblich

Transcription Factors Orchestrate Dynamic Interplay Between Genome Topology And Gene Regulation During Cell Reprogramming. Ralph Stadhouders, Enrique Vidal, François Serra, Bruno Di Stefano, François Le Dily, Javier Quilez, Antonio Gomez, Samuel Collombet, Clara Berenguer, Yasmina Cuartero, Jochen Hecht, Guillaume Filion, Miguel Beato, Marc A. Marti-Renom, Thomas Graf

Direct Conversion Of Human Fibroblasts Into Osteoblasts And Osteocytes With Small Molecules And A Single Factor, Runx2. Yanjiao Li, YaoLong Wang, Juehua Yu, Zhaoxia Ma, Qiong Bai, Xingfei Wu, Pengfei Bao, Lirong Li, Daiping Ma, Jingxue Liu, Change Liu, Fangyun Chen, Min Hu

RNA helicase, DDX27 regulates proliferation and myogenic commitment of muscle stem cells. Alexis Bennett, Marie Francoise O’Donohue, Stacey Gundry, Aye Chan, Jeffery Widrick, Isabelle Draper, Anirban Chakraborty, Yi Zhou, Leonard Zon, Pierre-Emmanuel Gleizes, Alan Beggs, Vandana Gupta

Constitutive Immune Activity Promotes Tumorigenesis in Drosophila Intestinal Progenitor Cells. Kristina Petkau, Silvia Guntermann, Edan Foley

Conservation of EMT transcription factor function in controlling pluripotent adult stem cell migration in vivo in planarians. Prasad Abnave, Ellen Aboukhatwa, Nobuyoshi Kosaka, James Thompson, Mark Hill, Aziz Aboobaker

MLL3/4 Prevents Stem Cell Hyperplasia And Controls Differentiation Programs In A Planarian Cancer Stem Cell Model. Yuliana Mihaylova, Damian Kao, Samantha Hughes, Alvina Lai, Farah Jaber-Hijazi, Nobuyoshi Kosaka, Prasad Abnave, Aziz Aboobaker

PHRED-1 Is A Divergent Neurexin-1 Homolog That Organizes Muscle Fibers And Patterns Organs During Regeneration. Carolyn E. Adler, Alejandro Sanchez Alvarado

Post-transcriptional regulation of adult CNS axonal regeneration by Cpeb1. Wilson Pak-Kin Lou, Alvaros Mateos, Marta Koch, Stefan Klussmann, Chao Yang, Na Lu, Stefanie Limpert, Manuel Göpferich, Marlen Zschaetzsch, Carlos Maillo, Elena Senis, Dirk Grimm, Raúl Méndez, Kai Liu, Bassem A Hassan, Ana Martin-Villalba

Exposure to elevated sea-surface temperatures below the bleaching threshold impairs coral recovery and regeneration following injury. Joshua Louis Boness, William Leggat, Tracy Danielle Ainsworth

Necroptosis promotes the Aging of the Male Reproductive System in Mice. Xiaodong Wang, Dianrong Li, Lingjun Meng, Tao Xu, Yaning Su, Xiao Liu, Zhiyuan Zhang

Tissue-specific downregulation of EDTP removes polyglutamine protein aggregates and extends lifespan in Drosophila. Chengfeng Xiao, Shuang Qiu, R Meldrum Robertson, Laurent Seroude

Reversal of cardiac and skeletal manifestations of Duchenne muscular dystrophy by cardiosphere-derived cells and their exosomes in mdx dystrophic mice and in human Duchenne cardiomyocytes. Mark A Aminzadeh, Russell G Rogers, Kenneth Gouin, Mario Fournier, Rachel E Tobin, Xuan Guan, Martin K Childers, Allen M Andres, David J Taylor, Ahmed Ibrahim, Xiang-ming Ding, Angelo Torrente, Joshua I Goldhaber, Ronald A Victor, Roberta A Gottlieb, Michael Lewis, Eduardo Marban

PERTURBATION OF PTEN-PI3K/AKT SIGNALLING IMPAIRED AUTOPHAGY MODULATION IN DYSTROPHIN-DEFICIENT MYOBLASTS. Muhammad Dain Yazid, Janet Smith

Modeling Zika Virus Congenital Eye Disease: Differential Susceptibility of Fetal Retinal Progenitor Cells and iPSC-Derived Retinal Stem Cells to Zika Virus Infection. Deisy Contreras, Melissa Jones, Laura E Martinez, Vineela Gangalapudi, Jie Tang, Ying Wu, Jiagang J. Zhao, Zhaohui Chen, Shaomei Wang, Vaithilingaraja Arumugaswami

Cell biology

Astral microtubule dynamics regulate anaphase oscillation onset and set a robust final position for the Caenorhabditis elegans zygote spindle. Helene Bouvrais, Laurent Chesneau, Sylvain Pastezeur, Marie Delattre, Jacques Pecreaux

Anillin proteins stabilize the cytoplasmic bridge between the two primordial germ cells during C. elegans embryogenesis. Eugenie Goupil, Rana Amini, Jean-Claude Labbe

Local Nucleation Of Microtubule Bundles Through Tubulin Concentration Into A Condensed Tau Phase. Amayra Hernández-Vega, Marcus Braun, Lara Scharrel, Marcus Jahnel, Susanne Wegmann, Bradley T. Hyman, Simon Alberti, Stefan Diez, Anthony A. Hyman

An Arf6- And Caveolae-Dependent Pathway Links Hemidesmosome Remodeling And Mechanoresponse. Naël Osmani, Julien Pontabry, Jordi Comelles, Nina Fekonja, Jacky G Goetz, Daniel Riveline, Elisabeth Georges-Labouesse, Michel Labouesse

Cell size sensing in animal cells coordinates growth rates and cell cycle progression to maintain cell size uniformity. Miriam Bracha Ginzberg, Nancy Chang, Ran Kafri, Marc W Kirschner

Scc2-Mediated Loading Of Cohesin Onto Chromosomes In G1 Yeast Cells Is Insufficient To Build Cohesion During S Phase. Kim Nasmyth

Gradients Of Rac1 Nanoclusters Support Spatial Patterns Of Rac1 Signaling. Amanda Remorino, Simon De Beco, Fanny Cayrac, Fahima Di Federico, Gaetan Cornilleau, Alexis Gautreau, Maria Carla Parrini, Jean-Baptiste Masson, Maxime Dahan, Mathieu Coppey

WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility. Lillian K. Fritz-Laylin, Samuel J. Lord, R. Dyche Mullins

Optogenetic Control of RhoA Reveals Zyxin-mediated Elasticity of Stress Fibers. Patrick W Oakes, Elizabeth Wagner, Christoph A Brand, Dimitri Probst, Marco Linke, Ulrich S Schwarz, Michael Glotzer, Margaret L Gardel

Formin-Dependent Adhesions Are Required For Invasion By Epithelial Tissues. Tim B Fessenden, Yvonne Beckham, Mathew Perez-Neut, Aparajita H Chourasia, Kay F Macleod, Patrick W Oakes, Margaret L Gardel

Fast Activation Cycles Of Rac1 At The Lamellipodium Tip Trigger Membrane Protrusion. Amine Mehidi, Olivier Rossier, Anael Chazeau, Fabien Biname, Amanda Remorino, Mathieu Coppey, Zeynep Karatas, Jean-Baptiste Sibarita, Violaine Moreau, Gregory Giannone

Combinatorial Regulation Of The Balance Between Dynein Microtubule End Accumulation And Initiation Of Directed Motility. Rupam Jha, Johanna Roostalu, Martina Trokter, Thomas Surrey

The Ndc80 complex targets Bod1 to human mitotic kinetochores. Katharina Schleicher, Sara ten Have, Iain M Porter, Jason R Swedlow

Dynactin Binding To Tyrosinated Microtubules Promotes Centrosome Centration In C. Elegans By Enhancing Dynein-Mediated Organelle Transport. Daniel J. Barbosa, Joana Duro, Dhanya K. Cheerambathur, Bram Prevo, Ana X. Carvalho, Reto Gassmann

Long-Term Memory In The Migration Movements Of Enucleated Amoeba proteus. Carlos Bringas, Iker Malaina, Alberto Perez-Samartin, Maria Dolores Boyano, Maria Fedetz, Gorka Perez-Yarza, Jesus Cortes, Ildefonso Martinez de la Fuente

Do gametes woo? Evidence for non-random unions at fertilization. Joseph H Nadeau

Modelling

Pairwise hybrid incompatibilities dominate allopatric speciation for a simple biophysical model of development. Bhavin S Khatri, Richard Goldstein

A model for autonomous and non-autonomous effects of the Hippo pathway in Drosophila. Jia Gou, Lin Lin, Hans G Othmer

Single-Cell Genome Dynamics in Early Embryo Development: A Statistical Thermodynamics Approach. Alessandro Giuliani, Masa Tsuchiya, Kenichi Yoshikawa

On The Principles Of Cell Decision-Making: Intracellular Coupling Improves Cell Responses Fidelity Of Noisy Signals. Andreas Reppas, Eduard Jorswieck, Haralampos Hatzikirou

Correlating Cell Shape and Cellular Stress in Motile Confluent Tissues. Xingbo Yang, Dapeng Bi, Michael Czajkowski, Matthias Merkel, M. Lisa Manning, M. Cristina Marchetti

Evo-devo & evo

apterous A Specifies Dorsal Wing Patterns And Sexual Traits In Butterflies. Anupama Prakash, Antonia Monteiro

Sex Differences In 20-Hydroxyecdysone Hormone Levels Control Sexual Dimorphism In Bicyclus anynana Butterfly Wing Patterns. Shivam Bhardwaj, Kathleen L Prudic, Ashley Bear, Mainak Das Gupta, Bethany R Wasik, Xiaoling Tong, Wei Fun Cheong, Markus R Wenk, Antonia Monteiro

A practical guide to CRISPR/Cas9 genome editing in Lepidoptera. Linlin Zhang, Robert Reed

A novel role for Ets4 in axis specification and cell migration in the spider Parasteatoda tepidariorum. Matthias Pechmann, Matthew Alan Benton, Nathan James Kenny, Nico Posnien, Siegfried Roth

Evolution And Multiple Roles Of The Pancrustacea Specific Transcription Factor zelda In Insects. Lupis Ribeiro, Vitoria Tobias-Santos, Danielle Santos, Felipe Antunes, Georgia Feltran, Jackson de Souza Menezes, L Aravind, Thiago M Venancio, Rodrigo Nunes da Fonseca

Deep experimental profiling of microRNA diversity, deployment, and evolution across the Drosophila genus. Alex S Flynt, Alexandra Panzarino, Md Mosharrof Hossain Mondal, Adam Siepel, Jaaved Mohammed, Eric Lai

Tools & resources

Single Molecule Fluorescence In Situ Hybridisation For Quantitating Post-Transcriptional Regulation In Drosophila Brains. Lu Yang, Josh Titlow, Darragh Ennis, Carlas Smith, Jessica Mitchell, Florence L. Young, Scott Waddell, David Ish-Horowicz, Ilan Davis

A Versatile Compressed Sensing Scheme For Faster And Less Phototoxic 3D Fluorescence Microscopy. Maxime Woringer, Xavier Darzacq, Christophe Zimmer, Mustafa Mir

Three-Dimensional Two-Photon Optogenetics And Imaging Of Neural Circuits In Vivo. Weijian Yang, Luis Carrillo-Reid, Yuki Bando, Darcy S. Peterka, Rafael Yuste

Covalent Protein Labeling By SpyTag-SpyCatcher In Fixed Cells For Super-Resolution Microscopy. Veronica Pessino, Y. Rose Citron, Siyu Feng, Bo Huang

Photoacoustic molecular rulers based on DNA nanostructures. James Joseph, Philipp Koehler, Tim J. Zuehlsdorff, Daniel J Cole, Kevin N. Baumann, Judith Weber, Sarah E. Bohndiek, Silvia Hernandez-Ainsa

Rapid Whole Brain Imaging Of Neural Activities In Freely Behaving Larval Zebrafish. Lin Cong, Zeguan Wang, Yuming Chai, Wei Hang, Chunfeng Shang, Wenbin Yang, Lu Bai, Jiulin Du, Kai Wang, Quan Wen

A general method to fine-tune fluorophores for live-cell and in vivo imaging. Jonathan B. Grimm, Anand K. Muthusamy, Yajie Liang, Timothy A. Brown, William C. Lemon, Ronak Patel, Rongwen Lu, John J. Macklin, Phillip J. Keller, Na Ji,Luke D. Lavis

Real-Time Observation Of Light-Controlled Transcription In Living Cells. Anne Rademacher, Fabian Erdel, Jorge Trojanowski, Karsten Rippe

FiloQuant reveals increased filopodia density during DCIS progression. Guillaume Jacquemet, Ilkka Paatero, Alexandre Carisey, Artur Padzik, Jordan Orange, Hellyeh Hamidi, Johanna Ivaska

An organ-on-a-chip approach for investigating root-environment interactions in heterogeneous conditions. Claire E. Stanley, Jagriti Shrivastava, Rik Brugman, Dirk van Swaay, Guido Grossmann

Disabling Cas9 by an anti-CRISPR DNA mimic. Jiyung Shing, Fuguo Jiang, Jun-Jie Liu, Nicholas L Bray, Benjamin J Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, Jacob E Corn, Jennifer A Doudna

Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression. Yuping Huang, Caitlin McCann, Andrey Samsonov, Dmitry Malkov, Greg D Davis, Qingzhou Ji

The WPRE Improves Genetic Engineering With Site-Specific Nucleases. Jessica M. Ong, Christopher R Brown, Matthew C. Mendel, Gregory J Cost

A Versatile Genetic Tool For Post-Translational Control Of Gene Expression With A Small Molecule In Drosophila melanogaster. Sachin Sethi, Jing W. Wang

Rapid DNA Re-Identification for Cell Line Authentication and Forensics. Sophie Zaaijer, Yaniv Erlich, Daniel Speyer, Robert Piccone, Assaf Gordon

Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing. Andrew M Smith, Miten Jain, Logan Mulroney, Daniel R Garalde, Mark Akeson

Single-cell analysis of clonal dynamics in direct lineage reprogramming: a combinatorial indexing method for lineage tracing. Brent A Biddy, Sarah E Waye, Tao Sun, Samantha A Morris

Nanopore sequencing and assembly of a human genome with ultra-long reads. Miten Jain, Sergey Koren, Josh Quick, Arthur C Rand, Thomas A Sasani, John R Tyson, Andrew D Beggs, Alexander T Dilthey, Ian T Fiddes, Sunir Malla, Hannah Marriott, Karen H Miga, Tom Nieto, Justin O’Grady, Hugh E Olsen, Brent S Pedersen, Arang Rhie, Hollian Richardson, Aaron Quinlan, Terrance P Snutch, Louise Tee, Benedict Paten, Adam M. Phillippy, Jared T Simpson, Nicholas James Loman, Matthew Loose

Beyond The Linear Genome: Comprehensive Determination Of The Endogenous Circular Elements In C. elegans And Human Genomes Via An Unbiased Genomic-Biophysical Method. Massa Shoura, Idan Gabdank, Loren Hansen, Jason Merker, Jason Gotlib, Stephen Levene, Andrew Fire

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes. James M Burke, Rodney P. Kincaid, Francesca Aloisio, Nicole Welch, Christopher S. Sullivan

Improved maize reference genome with single molecule technologies. Yinping Jiao, Paul Peluso, Jinghua Shi, Tiffany Liang, Michelle C Stitzer, Bo Wang, Michael Campbell, Joshua C Stein, Xuehong Wei, Chen-Shan Chin, Katherine Guill, Michael Regulski, Sunita Kumari, Andrew Olson, Jonathan Gent, Kevin L Schneider, Thomas K Wolfgruber, Michael R May, Nathan M Springer, Eric Antoniou, Richard McCombie, Gernot G Presting, Michael McMullen, Jeffrey Ross-Ibarra, R. Kelly Dawe, Alex Hastie, David R Rank, Doreen Ware

A Systematic Nomenclature for the Drosophila Ventral Nervous System. Robert Christopher Court, James Douglas Armstrong, Jana Borner, Gwyneth Card, Marta Costa, Michael Dickinson, Carsten Duch, Wyatt Korff, Richard Mann, David Merritt, Rod Murphey, Shigehiro Namiki, Andrew Seeds, David Shepherd, Troy Shirangi, Julie Simpson, James Truman, John Tuthill, Darren Williams

MAPseq: Improved Speed, Accuracy And Consistency In Ribosomal RNA Sequence Analysis. Joao F Matias Rodrigues, Thomas SB Schmidt, Janko Tackmann, Christian von Mering

High Accuracy Base Calls in Nanopore Sequencing. Philippe Christophe Faucon, Robert Trevino, Parithi Balachandran, Kylie Standage-Beier, Xiao Wang

BasePlayer: Versatile Analysis Software For Large-Scale Genomic Variant Discovery. Riku Katainen, Iikki Donner, Tatiana Cajuso, Eevi Kaasinen, Kimmo Palin, Veli Mäkinen, Lauri A Aaltonen, Esa Pitkänen

CiliaCarta: An Integrated And Validated Compendium Of Ciliary Genes. Teunis J. P. van Dam, Julie Kennedy, Robin van der Lee, Erik de Vrieze, Kirsten A. Wunderlich, Suzanne Rix, Gerard W. Dougherty, Nils J. Lambacher, Chunmei Li, Victor L. Jensen, Michael R. Leroux, Rim Hjeij, Nicola Horn, Yves Texier, Yasmin Wissinger, Jeroen van Reeuwijk, Gabrielle Wheway, Barbara Knapp, Jan F. Scheel, Brunella Franco, Dorus A. Mans, Erwin van Wijk, François Képès, Gisela G. Slaats, Grischa Toedt, Hannie Kremer, Heymut Omran, Katarzyna Szymanska, Konstantinos Koutroumpas, Marius Ueffing, Thanh-Minh T. Nguyen, Stef J. F. Letteboer, Machteld M. Oud, Sylvia E. C. van Beersum, Miriam Schmidts, Philip L. Beales, Qianhao Lu, Rachel H. Giles, Radek Szklarczyk, Robert B. Russell, Toby J. Gibson, Colin A. Johnson, Oliver E. Blacque, Uwe Wolfrum, Karsten Boldt, Ronald Roepman, Victor Hernandez-Hernandez, Martijn A. Huynen

Research practice

Looking Into Pandora’s Box: The Content Of Sci-Hub And Its Usage. Bastian Greshake

Anticipated effects of an open access policy at a private foundation. Eesha Khare, Carly Strasser

Gender disparity in computational biology research publications. Kevin S. Bonham, Melanie I. Stefan

Why Do Scientists Fabricate And Falsify Data? A Matched-Control Analysis Of Papers Containing Problematic Image Duplications. Daniele Fanelli, Rodrigo Costas, Ferric C Fang, Arturo Casadevall, Elisabeth M Bik

Addressing the digital divide in contemporary biology: Lessons from teaching UNIX. Serghei Mangul, Lana Martin, Alexander Hoffmann, Matteo Pellegrini, Eleazar Eskin

The appropriation of GitHub for curation. Yu Wu​, Na Wang, Jessica Kropczynski, John M Carroll

The earth is flat (p>0.05): Significance thresholds and the crisis of unreplicable research. Valentin Amrhein​, Fränzi Korner-Nievergelt, Tobias Roth

What is open peer review? A systematic review. Tony Ross-Hellauer

Standardising and harmonising research data policy in scholarly publishing. Iain Hrynaszkiewicz,Aliaksandr Birukou, Mathias Astell, Sowmya Swaminathan, Amye Kenall, Varsha Khodiyar

TOWARDS COORDINATED INTERNATIONAL SUPPORT OF CORE DATA RESOURCES FOR THE LIFE SCIENCES. Warwick Anderson, Rolf Apweiler, Alex Bateman, Guntram A Bauer, Helen Berman, Judith A Blake, Niklas Blomberg, Stephen K Burley, Guy Cochrane, Valentina Di Francesco, Tim Donohue, Christine Durinx, Alfred Game, Eric Green, Takashi Gojobori, Peter Goodhand, Ada Hamosh, Henning Hermjakob, Minoru Kanehisa, Robert Kiley, Johanna McEntyre, Rowan McKibbin, Satoru Miyano, Barbara Pauly, Norbert Perrimon, Mark A Ragan, Geoffrey Richards, Yik-Ying Teo, Monte Westerfield, Eric Westhof, Paul F Lasko

Why not…

Caterpillars lack a resident gut microbiome. Tobin J Hammer, Daniel H Janzen, Winnifred Hallwachs, Samuel L Jaffe, Noah Fierer

Estimating The Extinction Date Of The Thylacine Accounting For Unconfirmed Sightings. Colin J Carlson, Alexander L Bond, Kevin R Burgio

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