Today’s paper is from the latest issue of Development and  introduces the squid Doryteuthis pealeii as a lophotrochozoan model for eye development. And the people are PI Jeffrey Gross, Director of the Louis J. Fox Center for Vision Restoration and Professor of Ophthalmology and Developmental Biology at the University of Pittsburgh, and lead author Kristen Koenig, who has recently started her own lab at the FAS Centre for Systems Biology at Harvard University.

So Jeffrey, can you give us the brief history of the Gross lab, and what key questions your group is trying to answer?

JG I started the lab 11 years ago at the University of Texas at Austin, and we moved about a year ago to the University of Pittsburgh Medical School.  We’re broadly interested in eye development and diseases.  Major questions in the lab focus on optic cup morphogenesis, the epigenetic regulation of retinal and retinal pigment epithelium development and regeneration.

To be honest, we’re rather unfocused, which I think is great! I encourage the students and postdocs in the lab to come up with their own questions and interests and then to go for them; this is the way I was trained as a Ph.D. student with Dave McClay, and I think it is the best way for Ph.D. students and postdocs to learn how to do science.  As a result, we’ve worked on a fairly diverse set of questions over the time I’ve had the lab and it really makes this is a fun job.

“I encourage the students and postdocs in the lab to come up with their own questions and interests and then to go for them; this is the way I was trained as a Ph.D. student with Dave McClay, and I think it is the best way for Ph.D. students and postdocs to learn how to do science.”

And Kristen: how did you come to join the Gross lab?

KK I decided to go to the University of Texas for graduate school because it seemed like an exciting research environment that had all the scientific resources one could want but was not a competitive or negative place to work. When choosing the Gross lab, I knew I wanted to study evolutionary questions but I was also concerned with generating a positive relationship with my advisor.  The eye is an excellent system to study the evolution and development of complexity and Jeff was amazingly supportive of crazy ideas so it was a good fit.  Also, the developmental biology community at UT was and still is a really exciting and supportive group of researchers.

While your lab usually works on zebrafish, your Development paper focuses on eye development and photoreceptor differentiation in the longfin inshore squid (Doryteuthis paeleii). So why cephalopods, and this squid in particular, for this project?

JG I’ve always been interested in evo-devo work and wanted us to do some work on eye evolution, but this project really didn’t start until Kristen Koenig joined the lab as a Ph.D. student.  The squid project was her idea and I just provided some advice on getting it started – she’s really the brains of the operation!

There is a long history of research using Doryteuthis pealeii at the Marine Biological Laboratory (MBL) in Woods Hole, MA and there is terrific infrastructure for collecting adults and obtaining gametes at the Marine Resources Center at the MBL.  Moreover, Kristen and I have spent a significant amount of time at the MBL, which is truly a magical place, so it made a lot of sense to use the resources there and our connections to build Doryteuthis pealeii as a model.  There are also worse places in the world than Woods Hole to have to spend a summer working in the lab!

“They are definitely charismatic embryos.  It is a pleasure to look at them for as long as I do.”

What’s it like working with squid? They look kind of cute…

KK Doryteuthis pealeii are great to work with.  They are a good size, develop at a reasonable pace, they are abundant and resilient to manipulations. Their eyes are quite large during development so for someone interested in visual systems they are quite exciting organisms. As you mentioned, they are definitely charismatic embryos.  It is a pleasure to look at them for as long as I do.

And you get the embryos from Woods Hole?

KK Yes.  During most of my dissertation work I spent every summer at the Marine Biological Labs in Woods Hole.  As Jeff mentioned, the Marine Resources Center there has an excellent capacity to provide access to adult squid and their eggs.  Without the MRC, none of this work would have been possible.  I only have embryos during the summer months so I have to plan my experiments for the year during that time.

Could you give us the paper’s key results in a short paragraph?

JG, KK Our research interest is to better understand visual system evolution across the Bilateria from a developmental perspective. We established the squid, Doryteuthis pealeii, as a lophotrochozoan model for complex eye development. Utilizing histological, transcriptomic and molecular assays we characterized eye formation in Doryteuthis pealeii. Through lineage tracing and gene expression analyses, we demonstrated that cells expressing Pax and Six genes incorporate into the lens, cornea and iris tissue, suggesting a convergent involvement in lens formation. We identified the sole source of retinal tissue in the squid and functional assays demonstrated that Notch signaling is required for photoreceptor cell differentiation and retina organization. These assays support a conserved role for notch signaling in neurogenesis in the cephalopod eye.

Were there any particularly surprising results that came out of the lineage tracing?

JG, KK Our lineage tracing showed that the cells generating the optic lobe originated from a different place than originally thought.  This ultimately has consequences on how we interpret gene expression at these early stages and also suggests that these cells might be migrating to incorporate into the developing optic lobes.

I’m wondering about the question of conservation or convergence (of photoreceptor cell types and the molecular pathways that generate them, in metazoa). What does your work bring to the debate?

JG, KK The photoreceptor cells in the squid retina are rhabdomeric and express r-opsins. There is still a lot to understand about the molecular pathways that generate these cells to enable gene regulatory network comparisons across organisms.  Our work supports a role for notch signalling during neurogenesis within the squid retina.  This may illuminate a conserved function for notch within pseudostratified neuroepithelia.

“Just seeing the basics of eye morphogenesis continues to blow my mind.”

Was there any part of the work you were most proud of?

KK That’s a tough question because each part of the project presented its own set of unique challenges.  The part I still think about regularly is the staging series of eye formation.  Just seeing the basics of eye morphogenesis continues to blow my mind.  There is so much information in those images and when I look at them it only generates more curiosity and questions.

And any part of the work that was particularly frustrating or intractable?

KK Some parts of the lineage tracing were challenging.  The experiment consisted of hundreds of embryos and had many steps and it was important to keeping track of every individual embryo at each step.  Also, half way through the experiment I had to trust the mail delivery system that my specimens wouldn’t get stalled in transit back to Texas and melt into a sad pool of defeat.  In the end I knew the experiment was possible but it was just very important to stay consistently focused and organized.

And you’ve left the Gross lab now? What’s next for you?

KK Yes, I just recently left Texas.  I am now at the FAS Center for Systems Biology at Harvard University.  I am a John Harvard Distinguished Science Fellow and am starting my own lab continuing to study squid eye formation and the evolution and development of visual systems more broadly.

Now you have this squid developmental resource, any future plans to carry on with Doryteuthis? What do you want to know now?

JG I hope to, if Kristen doesn’t mind letting me squat in her lab at Harvard from time to time!  The way the lens develops in the squid is fascinating, and I think the cell biology underlying this will be fun to study.  I hope that we’ll be able to take a look at this in more detail over the next few summers, and use some of the molecular and imaging tools that Kristen has developed to drill down to the mechanism.

(4 votes)

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The story of this paper is also the story of my PhD. It begins as most papers and PhDs do: with a distinct and often unrelated starting project or plan. It is great to have a plan. But time and luck and data bend and twist the plan; until it finally breaks and you end up ditching said plan.

Then the real fun can begin.

I – Enter the plan

Vertebrates are an incredibly diverse group of organisms, as demonstrated by the wide range of body shapes and sizes animals belonging to this group can exhibit. This remarkable flexibility was likely a decisive factor allowing for the amazing adaptation of vertebrates to almost all habitats throughout the globe. The general body plan of a vertebrate is usually determined in embryonic development during the process of axial extension, in which the embryo elongates in a rostral to caudal progression by the activity of a very particular population of cells located in the posterior embryonic end. These are the axial progenitors, and their job is to proliferate and give rise to all post-cranial structures of the embryo (Wilson et al., 2009). Yet, an in-depth study of this important cell population was – and still is – hindered by the lack of specific molecular markers.

My initial project was then, to do a molecular characterization of axial progenitors in the mouse embryo; and my supervisor, Moisés Mallo, and I had a nice, neat strategy designed to fish these markers out. At the same time – like a responsible and efficient (and somewhat neurotic) PhD student – I had compiled quite a list of candidate genes that might be expressed and/or could potentially be important to axial progenitors just by searching for truncation phenotypes in existing databases. This way I could screen those genes while waiting for the other experiment’s results. One of these candidate genes was Oct4.

II – How I met Oct4

Oct4 is the gene equivalent of a rock star. Besides being an integral part of the core pluripotency network, Oct4 is also famously known for being an indispensable part of the reprogramming cocktail able to turn somatic cells back into pluripotent stem cells (Takahashi and Yamanaka, 2006). All vertebrate species studied so far seem to have a functional Oct4 counterpart (Frankenberg et al., 2014). In the mouse embryo, Oct4 is expressed as early as the two-cell stage, becoming gradually down regulated as development progresses until its complete silencing in somatic tissues by mid-gestation stages. However, in 2013, a paper came out by DeVeale and colleagues (DeVeale et al., 2013), which showed that, if Oct4 was inactivated during a very particular window of developmental time, some of the resulting mouse embryos could exhibit significant axial truncations, while others showed dramatically shortened trunks, yet perfectly specified tails. The latter were remarkably similar to a variety of transgenic embryos we had described just a few months before, in which the type I TGF-β receptor Alk5 was constitutively active in axial progenitor-containing areas (Jurberg et al., 2013). This showed that not only Oct4 seemed to be important for trunk development, but also suggested the existence of some sort of inverse functional link between Oct4 expression and TGF-β signalling through Alk5 during mouse axis elongation.

But was that really true? At that point, I and my good friend and former labmate Arnon D. Jurberg had been working with this pathway for quite some time, specifically with the Gdf11 mutant. Gdf11 is the main ligand for the Alk5 receptor in vivo, and mutants for both these proteins have characteristically longer trunks due to a delayed trunk to tail transition (Andersson et al., 2006; Jurberg et al., 2013; McPherron et al., 1999). So we definitely knew that these molecules were important for axial progenitors during the extension process. Thus, we decided to put this putative functional connection to the test using our faithful Gdf11 mutant. We reasoned that if premature activation of Alk5 activity was equivalent to the absence of Oct4 during axis formation, and this excess signalling was able to generate shorter trunks, then perhaps the lack of signalling through Alk5 – by absence of Gdf11, for example – could result in an increased expression of Oct4 and that could maybe account for the longer trunks in these mutants. Admittedly, it was a bit of a stretch. But to our vast surprise and amazement, our preliminary results showed us that we might actually be right. 

III – Oct4 and the “snakefied” mice

Proving beyond any doubt that Oct4 was really and truly ectopically expressed in Gdf11 mutants took sensibly two years, three different methodologies and a healthy dose of stubbornness. However, in the meantime, we still wanted to know if Oct4 missexpression was responsible for the longer trunks these mutants showcased, which required a way to overcome its progressive downregulation throughout development. Therefore, we chose to take advantage of the Cdx2 promoter, which we had previously demonstrated to be active in axial progenitors, and use it to drive Oct4 expression in transgenic mouse embryos. So it was a dark December day – and a national holiday to boot – when Moisés and I were lucky to dissect together a particularly good batch of E18.5 transgenic embryos courtesy of Ana Nóvoa, transgenic wizard extraordinaire. And the results surpassed even our wildest expectations. Not only were we able to obtain embryos that mimicked the Gdf11 mutant axial phenotype, we also got fetuses that yielded up to 20 or 30 trunk segments (Fig.1). Which meant two things: first, that Oct4 missexpression was most likely the main factor responsible by the longer trunks in Gdf11 mutant embryos; and second, that the trunk to tail transition could be delayed almost indefinitely if you just maintained the right level of Oct4 activity during axial extension.

IV – How the snake got its curves

Needless to say, if something has long, rib-packed and organ-filled trunks… well, it’s snakes. Could Oct4 have a role in the making of their long trunks too? For that we needed to check its expression during snake embryonic development, which was most definitely not our area of expertise. We decided to ask for help from Francisca Leal and Martin J. Cohn from the University of Florida, proud owners of a lovely snake facility and source of infinite wisdom in all snake-related matters. We hit the jackpot: expression studies revealed that Oct4 was still present in snake embryos long after it was gone in equivalently staged mice embryos (Fig.2). That made for a classic case of gene expression heterochrony, in which the timing of a gene’s activation or silencing changes from one species to another.

What could have possibly happened during evolution for Oct4 to be so differently regulated in these two groups of organisms? Thanks to Francisca and her sequence analysis skills, we found out that the genomic landscape upstream of the snake Oct4 locus had diverged dramatically from mice and even lizards. Yet, this entire region was extremely well conserved among snakes. As sequence conservation generally means conservation in function, we wondered if these conserved non-coding sequences 5’ of snake Oct4 had any regulatory potential by testing them in mice. We found that a tiny, little 250bp region – the only region conserved in both lizard and snake species – was extremely active on its own, whereas when embedded within larger snake-specific sequences it was a lot less able to drive reporter gene expression. But these were only scattered, isolated pieces of a bigger puzzle. When we used a snake BAC containing the whole genomic context around Oct4 – a kind gift from Isabel Guerreiro and Denis Duboule – and got no expression in transgenic mice, we realized that this entire region most likely operated as a complex regulatory module, composed by a 250bp core regulatory enhancer under the additional control of neighbouring sequences. In the end, we think that the remarkable conservation in snake non-coding sequences probably resulted from strong selective pressures over Oct4 expression due to the adaptation to a fossorial lifestyle, which favoured long trunks for burrowing and prey constriction.

V – Epilogue

So there you have it: the story of how ditching the initial plan can be a good thing, if you just pay attention and let it happen. Especially when results start acquiring a life, to tell a story of their own. Go with the flow. Get really curious and excited. Talk to people, ask for help, and help out even more. And maybe, just maybe, you might stumble across something that, ultimately, turns out to be your dream Evo-Devo project.

References

Aires R, Jurberg AD, Leal F, Nóvoa A, Cohn MJ, Mallo M. (2016) Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity. Dev Cell. 2016 Aug 8;38(3):262-74

Andersson, O., Reissmann, E. and Ibáñez, C. F. (2006). Growth differentiation factor 11 signals through the transforming growth factor-beta receptor ALK5 to regionalize the anterior-posterior axis. EMBO Rep. 7, 831–7.

DeVeale, B., Brokhman, I., Mohseni, P., Babak, T., Yoon, C., Lin, A., Onishi, K., Tomilin, A., Pevny, L., Zandstra, P. W., et al. (2013). Oct4 Is Required ˜E7.5 for Proliferation in the Primitive Streak. PLoS Genet. 9, e1003957.

Frankenberg, S. R., Frank, D., Harland, R., Johnson, A. D., Nichols, J., Niwa, H., Schöler, H. R., Tanaka, E., Wylie, C. and Brickman, J. M. (2014). The POU-er of gene nomenclature. Development 141, 2921–3.

Jurberg, A. D., Aires, R., Varela-Lasheras, I., Nóvoa, A. and Mallo, M. (2013). Switching Axial Progenitors from Producing Trunk to Tail Tissues in Vertebrate Embryos. Dev. Cell 25, 451–462.

McPherron, A. C., Lawler, A. M. and Lee, S.-J. J. (1999). Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11. Nat. Genet. 22, 260–264.

Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676.

Wilson, V., Olivera-Martínez, I. and Storey, K. G. (2009). Stem cells, signals and vertebrate body axis extension. Development 136, 2133–2133.

(8 votes)

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Who are we?
Hi, my name is Ruairi Kavanagh and I’m a Master’s student at Plymouth University. For my dissertation I am based in The Marine Biological Association (MBA). I am carrying out my research in the recently established Burkhardt Lab. Our lab’s research is focused on tracing the origin and evolution of synaptic proteins, to better understand how synapses and neurons evolved in animals. Strikingly, many of the building blocks of animal synapses originated before the first synapses and neurons evolved. We work on a number of model organisms, including choanoflagellates, sponges, cnidarians, and of course ctenophores (Figure 1). We use choanoflagellates, the closest unicellular relatives of animals, to better understand which synaptic signalling machineries for neuronal functions have been co-opted. Sponges, basal animals with no synapses and neurons, are used to elucidate the origin of synapses and neurons. Ctenophores and cnidarians, basal animals with synapses and neurons, are helping us to answer the question, did synapses and neurons originate once or multiple times independently? Typical analyses are centred on identifying the function of key “neuronal” proteins in these organisms. I work on ctenophores and have identified homologs of synaptic proteins from a transcriptome of the ctenophore Pleurobrachia pileus, a local species that is found in Plymouth sound.

Ctenophore characteristics
Ctenophores or “comb jellies” are fantastic creatures who have recently come into the evolutionary limelight. There are about 150 known species, almost all of which are pelagic marine predators. Comb jellies contain eight longitudinal rows of ciliated “combs” (or ctenes – ctenophore means “comb-bearing”), which are a distinct apomorphy of the phylum (Figure 2). A nerve net envelops the animal in a kind of polygonal mesh, which is most densely concentrated at the apical sense organ (also called the statocyst). The apical sense organ is thought to detect stimuli such as light, gravity and pressure, to coordinate beating of the ciliated combs, and to control the animal’s orientation. At the oral end, the gastro-vascular tract is connected to the mouth, via a pharynx. At the aboral end there are usually two anal pores, on either side of the apical organ. Interior canals distribute nutrients around the body. Ctenophores are generally found with two tentacles, but some species have secondarily lost the need for them. If tentacles are present, they are lined with sticky cells known as colloblasts, which are used in prey capture (and are another apomorphy). The tentacles of the sea gooseberry, Pleurobrachia (pileus), can be 10-15 times the length of its body. Watching them fish for prey really is an amazing sight. They will extend out their tentacles like big nets and sit in the water column waiting for something to swim into their trap. If they feel the slightest contact, they instantly retract the tentacles towards the mouth, which also triggers them to do a sort of death roll, as they suck down whatever it is they have caught. The two other species that occur here in Plymouth are Beroe cucumis and Bolinopsis infundibulum (Figure 2).

Why study ctenophores?
For a long time, ctenophores were considered to be closely related to cnidarians, since they look superficially quite similar, are both gelatinous, contain comparable tissue-level organization, and perform similar ecological roles. However on closer inspection it is clear that there are significant differences between the two phyla. The most obvious difference between them has to do with locomotion. If we look at jellyfish, they move by contractions of the “bell”, and can only move in one direction. Whereas I have already mentioned that ctenophores use their ciliated combs. The coordinated beating of these combs enables the animal to swim forwards or backwards, and allows for a very efficient way of rapidly changing their orientation.

The coordinated beating of combs from the ctenophore Beroe cucumis. Video taken by Ruairi Kavanagh and Pawel Burkhardt (MBA).

In addition, recent phylogenetic studies proposed that ctenophores might after all not be as closely related to cnidarians as previously thought. And here is why: the genomes of two different ctenophores have recently been sequenced. The first ctenophore genome sequenced was the one from Mnemiopsis leidyi, and the second from Pleurobrachia bachei. These studies, using whole genome sequences, showed that ctenophores and cnidarians are actually not closely related. But even more strikingly, these studies showed that ctenophores, and not sponges, are the sister-group to the rest of animals. If true, this would have important implications: for example, as all ctenophores possess synapses & neurons, in contrast to sponges, an independent origin of synapses and neurons in this phylum is possible. Indeed, neuronal components that are found widely in other animal groups appear to be missing from the genomes of P. pileus and M. leidyi, and many neuronal components do in fact not localize to neurons, but to other, non-neuronal cells. Other researchers have called for more detailed phylogenetic analyses and the need to sequence more ctenophore genomes before such contrary conclusions can be drawn. Ctenophores have, after all, been in the spotlight for only a short period of time. Our work will provide valuable insights into ctenophore biology, and will hopefully help to resolve the current dispute on the origin of synapses and neurons.

A day in the lab
A typical day for us, as you might have guessed, begins at the crack of dawn. The first job of the day is to go ctenophore hunting. For this we use our research vessel named MBA Sepia (Figure 3). The MBA Sepia can accommodate 12 passengers and offers a spacious laboratory. Ctenophore sampling is done using a standard plankton net, and we do both vertical and horizontal trawls (Figure 3). Ctenophores are made up of 99% water and 0.4% salt so one can imagine how difficult it is to take them from the sea, and get them into culture intact back at the lab. In fact, it is difficult to do anything with these buggers! But we persevere. The MBA houses a state-of-the-art seawater facility for marine biological research with a wide range of tanks and we culture ctenophores in so called pseudo-kreisels (Figure 4A). Basically this is a tank that has fresh sea water pumped in from one end and a filter at the other. A cyclical current naturally occurs, which the ctenophores love. One important lesson (that has been learned the hard way) is that you don’t put Pleurobrachia and Beroe together in the same tank, as Beroe is one of Pleurobrachia’s main predators. Ideally I like to leave Pleurobrachia in the tank for 24 hours or so, as this allows them time to recover from the trauma of being handled. They are then ready to use for my experiments, for example for live imaging microscopy and immunostaining assays (Figure 4B). We have generated a couple of antibodies against ctenophore synaptic proteins and are in the process of studying their intracellular localization. In addition, we are using many different biochemical methods, for example immunoprecipitation techniques, to isolate synaptic proteins from ctenophores to determine their in vivo binding partners (Figure 4B).

Working in the Burkhardt Lab has been a brilliant experience. Probably the most valuable thing I have learned from my time here has been how to properly manage my time in the lab. The MBA molecular lab corridor can be a very busy place. Different lab groups are constantly bustling about, and access to resources has to be negotiated amongst the various research groups. If I want to get everything finished by the end of the day then I have to be very efficient, which means I will usually have three or four things going on at any one time. This takes a lot of concentration, and by the end of the day I am absolutely shattered! My favourite thing about the MBA is that it’s right on the coast, so I like to finish the day with a swim to clear my head. My other favourite thing about the MBA is that there are loads of Italians working here. Tip of the day: in my experience Italian people equals food. After my swim I usually head to one of their houses for some home-made pasta, a glass of vino, and a chat. By the time I get home I am asleep before my head hits the pillow.

The bigger picture
What really attracted me to this project was one simple question. How could such a complex and specialised cell as a neuron possibly have evolved twice in animals? Until very recently, most of us took it for granted that synapses and neurons emerged only once during animal evolution. The two newly available ctenophore genomes have caused us to question our current understanding of the course of neuron evolution. It is exciting to know that the questions surrounding ctenophores and the origin of neurons are only a few years old. As such, these strange and beautiful creatures are likely to remain an important source of information, as we strive to unravel the mysteries of past evolutionary events. For me it will be rewarding to trace the evolutionary origin of synapses and neurons, and find out more about our own evolutionary history. Furthermore, I get to study one of the coolest animals I have ever seen. If this does not excite a biologist, then I don’t know what does!

References

Achim, K. & Arendt, D. Structural evolution of cell types by step-wise assembly of
cellular modules. Curr. Opin. Genet. Dev. 27, 102–108 (2014).

Brusca, R. C., Wendy, M. & Shuster, S. M. Invertebrates. Sinauer Associates, (2016).

Jager, M. & Manuel, M. Ctenophores: an evolutionary-developmental perspective
Curr. Opin. Genet. Dev. 39, 85–92 (2016).

Jekely, G., Paps, J. & Nielsen, C. The phylogenetic position of ctenophores and the
origin(s) of nervous systems. Evodevo 6:1 (2015).

Moroz, L. L. et al. The ctenophore genome and the evolutionary origins of neural
systems. Nature 510, 109–14 (2014).

Moroz, L. L. & Kohn, A. B. Unbiased View of Synaptic and Neuronal Gene
Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across
Metazoa? Integrative and Comparative Biology 55, 1028–1049 (2015).

Pang, K. & Martindale, M. Q. Comb jellies (Ctenophora): A model for basal
metazoan evolution and development. Cold Spring Harb. Protoc. 3, (2008).

Philippe, H. et al. Phylogenomics Revives Traditional Views on Deep Animal
Relationships. Curr. Biol. 19, 706–712 (2009).

Pisani, D. et al. Genomic data do not support comb jellies as the sister group to all
other animals. Proc. Natl. Acad. Sci. 112, 201518127 (2015).

Ryan, J. F. et al. The genome of the ctenophore Mnemiopsis leidyi and its
implications for cell type evolution. Science 342, 1336–1344 (2013).

Ryan, J. F. Did the ctenophore nervous system evolve independently? Zoology 117,
225–226 (2014).

Whelan, N. V, Kocot, K. M., Moroz, L. L. & Halanych, K. M. Error, signal, and the
placement of Ctenophora sister to all other animals. Proc. Natl. Acad. Sci. U. S. A.
112, 5773–8 (2015).

(3 votes)

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This Editorial originally appeared in Development, Vol 143, Issue 17.

Olivier Pourquié and Katherine Brown

At Development, we are always trying to improve our processes and service – for authors and readers. In April 2015, we made some changes to our peer review process, which aimed at encouraging a more constructive approach to peer review. As we wrote in the Editorial announcing these changes, ‘there can be a tendency for a review to read like a “shopping list” of potential experiments … Instead, we believe that referees should focus on two key questions: how important is the work for the community, and how well do the data support the conclusions? … In other words, what are the necessary revisions, not the “nice-to-have”s?’ (Pourquié and Brown, 2015). Our new system has been in place for over a year now, and we have seen a change in the tone and nature of referee reports: in general, referees have embraced our new guidelines and we feel that this has had a positive effect – both in helping editors to take a decision, and in providing more streamlined feedback for authors. However, and as we recognised at the time, ‘…these changes are conservative compared with some of the more radical approaches in peer review that have been implemented and trialled elsewhere’ (Pourquié and Brown, 2015). At the beginning of 2016, and while reviewing various aspects of the Company’s journals’ peer review processes and online functionalities, we therefore conducted a community survey to gauge your opinions on how we should further improve. Over 300 of you completed the survey (over 800 across all our journals), and we are very grateful for your detailed responses.

In the first part of the survey, we asked you to rank possible improvements or changes to the way we do peer review in order of importance (see Box 1). Perhaps unsurprisingly, you ranked the two statements that relate to speed of publication as the top two priorities. While our own research and author surveys suggest that our speeds are reasonably competitive compared with other journals, there are always improvements to be made and we are actively looking at ways in which we can accelerate the process. We also recognise that multiple rounds of review and revision can be frustrating for the author, so we do try to avoid these wherever possible – while still ensuring the highest standards of peer review. Moreover, such efforts are rarely made in vain: we make a strong commitment to papers at first decision, accepting over 95% of manuscripts where we invite a revision.

Box 1. Peer review

Our January survey asked:

‘Unbiased, independent peer review is at the heart of our publishing decisions but there is always room for improvement and we are open to experimentation and change based on the needs of our community. Please rank the following in order of importance to you.’

Your responses ranked the statements in the order below – from most to least important.

1. Improve speed from submission to first decision

2. Reduce number of rounds of review and revision

3. Collaborative peer review trial (inter-referee discussion before a decision is made)

4. Name of the Editor who handled the paper published with the article

5. Double-blind peer review trial (author identity withheld from referees)

6. Network for transferring referee reports between journals in related fields

7. Post the peer review reports alongside the published articles

8. ‘Open’ peer review trial (referee identity known to authors but not made public)

9. Post-publication commenting

Speed aside, you ranked ‘collaborative peer review trial’ as the most important potential innovation – in fact, you rated this almost as important as reducing the number of rounds of review. This chimed with regular feedback we receive from the community, who, as authors, have found such models of peer review helpful. The idea is that, once all the reports on a paper have been returned, the editor shares these among the referees, asking for further feedback that might clarify the decision. This can help to resolve differences between referees, identify unnecessary or unreasonable requests, or – conversely – highlight valid concerns raised by one referee but overlooked by the others. Many journals, Development included, have been doing this informally with a subset of difficult cases for many years and it can prove invaluable in helping editors to make the right decision on a paper.

Given your enthusiasm for such a model, we have carefully assessed a number of potential models, including those already in place at other journals. These range from ‘cross-referee commenting’ as implemented by The EMBO Journal (Pulverer, 2010) and others, to the more active discussions embraced by eLife (Schekman et al., 2013). Bearing in mind the need to balance gathering additional feedback against the consequent effects on speed to decision, as well as the fact that some papers will benefit from further input more than others, we believe that a cross-referee commenting model is most appropriate for Development.

For all research papers submitted after mid-September, the full set of referee reports (minus any confidential comments) will be shared among all the referees. These may be accompanied by specific requests for feedback from the Editor. Referees will then be given two working days to respond before the Editor takes a decision – thus minimising any impact on speed (although the Editor may choose to wait for input in cases where the decision is particularly difficult or borderline). We anticipate that in many cases, feedback will either not be received or will not alter the Editor’s decision. For a minority of papers, however, we believe that this process will significantly aid decision-making and help the authors to move forwards with the paper – whether the decision is positive or negative. And, because it is not always possible to predict what will come out of such a process, we think it important to implement this as standard across all papers. We know that our referees already do a great job in helping authors to improve the papers we review, and we are hugely grateful for their efforts. We hope they (you!) will engage in this new development, and that authors will find it helpful. We will also continue to review other possible improvements to how we handle papers – bearing in mind your feedback from the survey.

The second part of the survey focused on how we present the work we publish (see Box 2). Once again, it seems that what matters most to you is not how the paper looks when it comes out, but how quickly it appears. Since mid 2015, we have been posting the author-accepted versions of manuscripts on our Advance Articles page before issue publication, minimising the time between acceptance and appearance of the work online. While there is still some delay (1-2 weeks) before online posting, this is primarily to allow us time to run our standard ethics checks on all papers – helping to ensure the integrity of the work we publish. Articles then typically appear in an issue around 6 weeks after acceptance: this reflects the time required to copy-edit, typeset and prepare the issue, although again we are working on ways of accelerating these processes.

Box 2. Online developments

Our January survey asked:

‘We recently rolled out a new-look website to make it easier for you to find and read content, but new features and functionality are being developed all the time. Which areas do you think should be our focus for 2016 and beyond? Please rank the following in order of importance to you.’

Your responses ranked the statements in the order below – from most to least important.

1. Improve speed from acceptance to online publication

2. Easier viewing of figures alongside the relevant text

3. Easier viewing of supplementary material including movies

4. Graphical abstracts (diagrammatical summaries of papers)

5. Publish final versions of articles one by one, gradually building an issue, rather than waiting for an issue to be complete before publication

6. Easier access to related articles, special issues and subject collections

7. Better text and datamining services

8. Annotation of article PDFs e.g. ReadCube

9. More community web content such as feeds from third-party bloggers

Pourquié, O. and Brown, K. (2015). Developing peer review. Development 142, 1389. doi:10.1242/dev.124206

Pulverer, B. (2010). A transparent black box. EMBO J. 29, 3891-3892. doi:10.1038/emboj.2010.307

Schekman, R., Watt, F. and Weigel, D. (2013). Scientific publishing: the eLife approach to peer review. eLife 2, e00799. doi:10.7554/eLife.00799

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(or: how to avoid misleading representations of statistical data)

Recently, a kickstarter project raised more than 3000€ in one month to campaign for banning the wrong usage of bar plots in scientific journals. This demonstrates two important points: a lot of the plots in scientific journals are quite misleading, and, a growing number of people feel very uneasy about this!

What exactly is wrong about bar plots? Nothing per se, but everything goes wrong if you use a bar plot for statistical data – this kind of plot species is also referred to as the dynamite plot: the bar being the detonator and the error range the firing cable (see figure)! We are talking about the famous vertical or horizontal boxes that often come in a dazzling array of colors or patterns, with big fat black outlines and overly prominent error bars.

                                                 Dynamite Plot                                                                      Data Plot

Are they common? Very much so! My personal survey (footnote 1) of dynamite plots in scientific journals revealed that on average 30-60% of articles use them (see figure). These journals cover a wide range of subjects that include physics, meteorology or psychology where authors typically have rigorous training in applied mathematics. The prevalence of dynamite plots increases as we go towards more life science journals, where 50- 70% of articles are accompanied by a dynamite plot showing a statistical summary (footnote 2).

Most of us are completely accustomed to dynamite plots and happily use them, that is, until we see the light. From then on it is impossible to not hate them! Because it is so obvious they are misleading and they just make it harder than necessary to understand the data! And, as scientists, we thrive for clear and concise information!

The top reasons to avoid dynamite plots

• They hide the real distribution of data. Do all samples cluster closely? Do they form two groups? Or is there one drastic outlier? Generally, we assume a normal distribution of the data around the mean where there might not be one! In my survey of dynamite plots per journal they were more or less normally distributed.
• They hide the sample size. From the bar plot you would not have known that I probed one issue of Nature, two issues of Cell and four issues of Development! But for judging scientific data knowledge of the sample size is essential for a proper evaluation of the data! Too often we have to search for the n in axis labeling, figure text, the results, or the methods section to finally find this information. And sometimes it is omitted entirely. A clear understanding of sample size in my opinion is also critical for the review process of a paper and should be demanded by the reviewers! Not showing data, or only showing summary data, should be treated equally to cropping Western blot bands!
• Many different data distributions lead to the same bar! See also the Anscombe quartet. Bar plots are not intended to show statistic distributions, they are for absolute numbers. And, by plotting the real data we also learn more about the biology!

Not quite convinced? Seeing is believing, check out this figure:

Further information: watch the video of the kickstarter campaign (humor alert!) – ideally with your entire lab! And read this seminal paper on wrong usage of bar charts and this survey of their prevalence in biomedical journals!

Practical advice to avoid dynamite plots

• Plot charts with statistical programing tool R. You have to either learn it, or be really nice to someone who knows it – if your PhD requires 3 boxplots, maybe invest in a friendly relationship with the bioinformatic geek in your department, a couple of coffees go a long way!
• Learn how to make box plots in excel! (Here and here is how, but it’s a bit tedious).
• Can’t be bothered to do either? Use one of the available web tools such as the boxplot maker from the Tyer’s lab or the plot generator from the University of Belgrade.

Footnotes:

1) I probed the top10-articles of Nature in July, the three most recent volumes of Science (August), four issues of Development (Vol 138, 1:3(2011) and Jan 2016), and two issues of Cell journal from 2016 (Jan and August). I was very relaxed in my judgement and gave the benefit of doubt when I wasn’t sure. But I was rigorous when authors mixed right and wrong usage of bar plots. How does this even happen? Mix of co-authors and some know better than others?

2) Disclaimer: this does not mean the other articles have great figure design in any of the journals! I saw multiple uses of 3-dimensional pie charts, rainbow color schemes, other instances of unintentional usage of color, incomprehensible spider graphs and 3-dimensional heat maps! Maybe I will devote another blog post to those.

(11 votes)

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Here are the highlights from the current issue of Development:

Coordinating neuronal specification and differentiation

Neurogenesis – the process of making new neurons – is indispensable for normal development and for adult homeostasis and repair. Many of the signalling and transcriptional events that regulate the specification and differentiation of neural progenitor cells (NPCs) into neurons have been uncovered; however, how these events are coordinated at a post-translational level is not well defined. Now, on p.3085, Miho Matsuda, Ajay Chitnis and colleagues identify the protein Epb41l5 – an adaptor protein that links cytoplasmic proteins to specific membrane compartments – as a new regulator of neuronal differentiation in the developing zebrafish hindbrain. The authors first identify Epb41l5 in a yeast two-hybrid screen against Mib1, a key component of the Notch signalling pathway. Using Epb41l5-deficient embryos, the authors show that loss of Epb41l5 impairs neuronal differentiation, but that this can be partially rescued by knockdown of N-cadherin expression, suggesting a possible role for Epb41l5 in the disassembly of apical adherens junctions. In support of this, the authors further demonstrate that Mib binding to Epb41l5 facilitates its degradation and thus promotes apical adhesions, which may impair proper delamination and differentiation. The authors conclude by proposing a model whereby changes in Notch ligand levels that occur during neuronal differentiation protect Epb41l5 from Mib1-mediated degradation, thereby facilitating neuroepithelial detachment and subsequent differentiation.

Sall4 is dispensable for mouse pluripotency

In order to specify the correct lineage at the correct time, the developing embryo must maintain tight control over the gene regulatory networks that enact these changes. Sall4 has long been associated with the regulation of embryonic stem cell (ESC) self-renewal and differentiation; however, teasing out its precise role has been difficult. Now, on p. 3074, Brian Hendrich and colleagues present a comprehensive analysis of the role of Sall4 in self-renewal and differentiation, and shed light on the nature of its interaction with the NuRD complex during these events. Using a series of phenotypical and transcriptional analyses of double Sall4/1 knockout mouse ESCs (mESCs), the authors show that Sall1 and Sall4 are dispensable for ESC pluripotency but are required to repress neuronal differentiation. Remarkably, the authors observed the spontaneous production of neurons alongside self-renewing mESCs in the double knockout mESCs. Genome-wide analyses demonstrate that, although a small proportion of Sall4 does indeed interact with NuRD, Sall4 neither recruits nor functions through the NuRD complex. Rather, Sall4 is seen to bind to enhancer sequences along with the pluripotency-associated transcription factors Pou5f1, Nanog, Klf4 and Esrrb, which can result in either gene activation or repression. Together, these data shed light on a number of previously unresolved issues with regard to the function of Sall4 in mammalian development.

The asymmetry of asynchrony: new roles for CYB-3 in cell division

Regulation of the cell cycle is a crucial component of development, and has been linked to the execution of cell fate decisions in a wide range of developmental contexts. Although many of the molecular components involved in cell cycle progression have been identified, how these proteins are regulated and how their distribution and abundance can influence cell fate remains unclear. In this issue (p. 3119), Matthew Michael identifies cyclin B3 (CYB-3) as a key regulator of cell cycle timing in the developing C. elegans embryo. Using RNAi to knockdown CYB-3, Michael demonstrates that in the one-cell embryo CYB-3 controls not only mitotic entry but S-phase entry as well – a dual-action that is unique among cyclins. At the two-cell stage, the author shows that CYB-3 is asymmetrically distributed in a par-dependant manner such that somatic precursor cells inherit ∼2.5-fold more CYB-3 than do their germline precursor sister cells. The author uses maternal strains with varying copy numbers of cyb-3 to show how variations in the level of CYB-3 can affect the speed and synchrony of the cell cycle at the two-cell stage, suggesting a novel role for CYB-3 in regulating asynchronous cell cycling in the developing embryo. Together, these data advance our understanding of how the timing of cell division is differentially regulated in the early embryo.

PLUS:

Introducing cross-referee commenting in peer review

Following our recent community survey on priorities in peer review and online publishing, we are making changes to the journal, including some changes to our peer review process. Find out more by reading the Editorial on p. 3035

Slit-Robo signaling

Slits are secreted proteins that bind to Roundabout (Robo) receptors. Slit-Robo signaling is best known for mediating axon repulsion in the developing nervous system. However, in recent years the functional repertoire of Slits and Robo has expanded tremendously and Slit-Robo signaling has been linked to roles in neurogenesis, angiogenesis and cancer progression among other processes. Here, Heike Blockus and Alain Chédotalsummarize new insights into Slit-Robo evolutionary and system-dependent diversity, receptor-ligand interactions, signaling crosstalk and receptor activation. See the Development at a Glance poster article on p. 3037

Metabolism meets development at Wiston House

It is becoming increasingly clear that cellular metabolite levels regulate the activity of signaling pathways, and conversely that signaling pathways affect cellular physiology and growth via metabolic pathways. Thus, metabolism and signaling mutually influence each other. The recent Company of Biologists’ Workshop ‘Metabolism in Development and Disease’ brought together people studying signaling and development with people studying metabolism, particularly in a cancer context. Here, Aurelio Teleman discusses examples of talks that illustrated this principle. See the Meeting Review on p. 3045

Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination

Phosphatidylinositide 3 kinases (PI3Ks) and their downstream mediators AKT and mammalian target of rapamycin (mTOR) constitute the core components of the PI3K/AKT/mTOR signalling cascade, regulating cell proliferation, survival and metabolism. Although these functions are well-defined in the context of tumorigenesis, recent studies – in particular those using pluripotent stem cells – have highlighted the importance of this pathway to development and cellular differentiation. Here, Jason Yu and Wei Cui review the role PI3K/AKT/mTOR signalling plays in the control of pluripotency and differentiation, with a particular focus on the molecular mechanisms underlying these functions. See the Review on p. 3050

The roles of microRNAs and siRNAs in mammalian spermatogenesis

MicroRNAs and siRNAs, both of which are AGO-bound small RNAs, are essential for mammalian spermatogenesis. Although their precise germline roles remain largely uncharacterized, recent discoveries suggest that they function in mechanisms beyond microRNA-mediated post-transcriptional control, playing roles in DNA repair and transcriptional regulation within the nucleus. Here, Andrew Grimson and colleagues discuss the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline. They also evaluate the emerging and differing roles for AGOs and AGO-bound small RNAs in the male and female germlines, suggesting potential reasons for these sexual dimorphisms. See the Review on p. 3061

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This post was originally posted on eurostemcell.org, Europe’s stem cell hub.

by Julia Turan

Part of the fascinating potential of stem cells is their ability to provide replacement cells and tissues to treat diseases. In order to do this most effectively, scientists need to be able to create differentiated cells quickly and accurately. However, making differentiated cells can take weeks to months and the resulting cells might be a mixture of the desired type of cell and others. This is because the steps between pluripotency and the differentiated state are not fully understood.

To tackle these obstacles, Irving L. Weissman and his team based at Stanford University studied how the cells of the middle layer of the embryo – the mesoderm – turn into 12 differentiated cell types. They mapped out 1) the intermediate cell types formed as a cell becomes more and more differentiated, 2) how these intermediates are formed via alternate paths at each branching point and 3) the signals that encourage and discourage cells to follow a path at each branching point.  

Identifying the signals that cells receive to determine which path they follow allowed the researchers to make differentiated cells that were 80-99% the desired cell type in days rather than weeks or months.

To show that these human cells behave as expected in a living model organism (‘in vivo’), they transplanted the cells that can produce bone, cartilage or smooth muscle (‘ventral somite progenitors’), into mice without an immune system.

The cells shown in the image are human cells inside of a mouse. The blue indicates cartilage and the light pink indicates bone. The process of cartilage turning into bone occurs as the blue cells secrete a scaffold of cartilage (‘proliferative cartilage’). The blue cells then enlarge and die (‘hypertrophic cartilage’), and the cartilage they leave behind turns into bone (‘ossification’). The light pink ossified cells are lined in diagonal rows, showing the structure of bone on a microscopic level.

The process developed by Weissman and his team mimics natural bone development and shows that the cells produced via differentiation can integrate into a living system properly. This suggests that, with further research, these cells could be transplanted into humans. With this faster, more efficient technique, therapies utilizing such differentiated cells could be significantly improved.

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We are seeking outstanding postdoctoral candidates to join the Campas lab (Morphogenesis and Self-organization of Living Matter lab) at the University of California, Santa Barbara (UCSB). Our group combines theoretical and experimental approaches to study the spatiotemporal control of tissue mechanics during morphogenesis, using zebrafish as model organism. We have recently developed two microdroplet-based techniques that enable direct measurements of forces and mechanical properties (such as stiffness and fluidity) within developing 3D tissues, as well as allowing the application of controlled forces. Using these techniques, we are studying the molecular control of spatiotemporal variations in tissue mechanics and the role of mechanical feedback on cell behavior, all within developing embryos.

We are specifically seeking independent, passionate, and motivated applicants for a postdoctoral position to work on the mechanics of embryonic development in zebrafish. The candidate will be able to work in a collaborative manner with a highly interdisciplinary group of researchers, including theoretical physicists and engineers. A Ph.D. in the biological sciences, biophysics or related fields and at least 3 years of laboratory research experience in zebrafish development are required. Applicants with quantitative biology or biophysics backgrounds, in addition to experience in zebrafish development, will be considered positively.

This is a renewable, two-year position with full benefits, reappointed annually according to the performance of the candidate. Salary will be competitive and dependent on the level of experience of the candidate. Applicants should email a CV and a description of research interests to campas@engineering.ucsb.edu, and should also arrange for at least two references to submit letters of recommendation of their behalf. Applications submitted by October 31st 2016 will receive priority consideration, but the position will remain open until filled. Start date is flexible and could be as early as November 2016.

The University of California, Santa Barbara (UCSB) provides an exceptional, interdisciplinary and collaborative environment for scientists interested in quantitative biology and systems biology. Researchers at UCSB enjoy regular visits from world-leading scientists and workshops on quantitative biology and biophysics through the Kavli Institute for Theoretical Physics, in addition to exposure to the Summer School on Quantitative Biology.

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The Campàs Lab (“Morphogenesis and Self-Organization of Living Matter” lab) at the University of California, Santa Barbara, works on tissue and organ morphogenesis in zebrafish. The lab focuses on understanding the role of mechanical cues in the development of embryonic structures using novel in vivo force transducers and actuators.

Our lab is seeking independent, passionate, and motivated applicants for a Research Specialist position. This position will assist with experiments, training, and general lab duties, as well as participate in multiple research projects. The candidate will be able to work in a collaborative manner with a highly interdisciplinary group of researchers, and will have many opportunities for co-authorships on published manuscripts. A master’s degree in the biological sciences or related field, and at least 3 years of laboratory research experience in zebrafish are required (lab experience can include graduate training). Knowledge of zebrafish molecular biology, imaging, microinjection, genetics and husbandry are necessary. Applicants with a Ph.D. will be considered positively.

This is a long-term, renewable, full-time position with benefits, reappointed annually. Anticipated begin date may be as soon as November 2016, but the start date is flexible. Salary will be competitive and dependent on the level of experience of the candidate. Applications must be submitted electronically at https://recruit.ap.ucsb.edu/apply/JPF00759 and must include a CV and research statement. Applicants will also need to arrange for at least 3 references to submit letters of recommendation of their behalf via the recruitment website. Applications submitted by October 31st 2016 will receive priority consideration, but the position will remain open until filled.

The department is especially interested in candidates who can contribute to the diversity and excellence of the academic community through research, teaching, and service. The University of California is an Equal Opportunity/Affirmative Action Employer and all qualified applicants will receive consideration for employment without regard to race, color, religion, sex, sexual orientation, gender identity, national origin, disability status, protected veteran status, or any other characteristic protected by law.

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