Water is a fascinating substance. Its behavior sets a lot of interesting constraints on both how the surface of our world is shaped geologically and how life on said surface has adapted to optimize its use. Biology and geology, while vastly different in scale, share many commonalities that can we can learn from. Our work found one such connection: we found that the mammalian pancreas is like a river basin.

The mechanics behind the emergence and refinement of the ducts in the pancreas has been a mystery for some decades. First, the ducts form from unconnected microlumen in the pancreatic mass into a meshwork of interconnected tubes, connected seemingly at random (Fig 1). Later, as the pancreas nears maturity, it undergoes a transition from a mesh into a tree-like structure with no redundant ducts. The process does seem to have some stochasticity, as no two individuals has the exact same pattern of tubes when examined.  Yet the function of delivering pancreatic juices to the duodenum is the same across healthy specimens.

In nature, simple rules can often result in very complex behavior. A good example is Turing patterns (1), in which fur and skin coloration can elegantly be explained by reaction-diffusion equations. It is for that reason that physicists often try to use simple models and derive universal laws in order to get some conceptual understanding of the phenomena they are analyzing. And it was with that mindset that we set out to study pancreatic development. Three years later we had made a great discovery by applying network theory and fluid mechanics to describe the developing pancreas.

First, we needed quantifiable measures to describe how the pancreas tubes connect. We sought inspiration in network theory since the pancreas had all we needed – a network. Network (graph) theory is pretty simple at its core. Every network consists of nodes that serve as connection points and edges which are the connections between the points. The idea was to convert the pancreas tubes into edges and the tube intersections into nodes. The first snag we hit was that we found the images we had of the pancreas, while of excellent quality, were hard to automatically convert into a digitized network of nodes and connections between them. In the end we decided to go old school and digitize them by hand….

20000+ nodes later, we had an amazing dataset! We had digitized mouse pancreas networks at three different developmental stages: E12.5, when the network is very interconnected; E14.5, when the network is visually clearly beginning to change into a tree-like structure; and E18.5, when the network has almost fully transformed (Fig 2). What we found was stunning.  It turns out that while the networks are visually very different, most of the quantitative features of the network are strikingly similar. Furthermore some of these quantities change with each developmental stage.  This shows that the pancreas, while seemingly chaotic, must follow a set of rules when developing.  To a physicist like me there is no sweeter thing because then you get try and uncover these rules. In the end, development of the pancreatic ducts can be explained on a macroscopic level by two models. One model creates the mesh, the other matures it into the tree-like structure.

In order to model the creation of the interconnected network that appeared we tried to naively model what we thought we saw: that the microlumen that emerged simply connected to some of the nearest microlumens.  Our model therefore became the following:

1. Create a node close to the bulk of the already existing nodes
2. Form edges to some of the nearest nodes
3. Reiterate 1 and 2 until you have the desired pancreas size

With the addition of some noise, our model makes a very nice approximation to the real pancreas from a network viewpoint.

In order to model the maturation of the pancreatic network from mesh to tree-like structure, we first had to figure out which mechanism could be the driver of such a selection. It is here that we drew inspiration from the flow of rivers. At this time point, we had also noticed that at the end of development the width of the ducts was wider and wider close to the exit, as seen in river basins.

The concept of the path of least resistance is that every entity will select the easiest path from a to b when selecting from multiple potential pathways. Water exhibits such behavior. If you pour water on the top of a hill of sand, all the water will flow on some select trajectories. There is another phenomenon hidden in this analogy: once water has passed through the sand, the water following it will experience less resistance. Therefore water seems to stick with its initial choice. This is why water flowing down hill is confined to specific paths. During a flood the water from a river can branch off in new directions, and if these newly created river branches are more favorable than the original, the river will change shape as the old river branch dries out and the new branch is widened.  The end result is a river that is fairly optimal in delivering water from one or more source points to an end point.

Our idea is that the pancreas may follow similar rules. By doing so, we postulate that the delivery of pancreatic juice from multiple source points (the pancreatic acini) into a single end point (the exit into the duodenum) finds the shortest path. Forming redundant ducts may enable this optimization. When fluid then runs through, the pancreas changes the duct diameter to correspond to the flow (Fig 3a). When the fluid flow is higher than a given threshold, the duct widens. When the flow is lower than the threshold, the duct shrinks in diameter. The ingenious detail in this simple scheme is that if redundant paths exist between the exit and the acini, the suboptimal paths can be shrunk until they no longer exist and the cells lining them may be reused by the surrounding tissue (Fig 3b). If a given path is the only path, it will stabilize into a duct with a diameter perfect for the flow it receives. The end-result is a structure with one optimized path from every acini to the exit that is homogeneous in fluid pressure as every duct has the ideal diameter for the given flow.

The concept was easy enough to test as we had the digitized networks. The model we constructed did the following:

1. Let fluid run through the network either with the acini as the source or every point in the network as the source and wait until approximate steady state.
2. Remove the redundant duct with least flow.
3. Reiterate 1 and 2 until no redundant ducts exist.

The model can artificially mature an E14.5 into an E18.5 network, and optimize the average duct distance from every acini to the exit.

So there you have it: what the pancreas needs to optimize delivery of pancreatic juice is flow, a flow sensor, a duct diameter adjustment mechanism and a duct removal mechanism.  Here it should be noted that while the pancreas is the basis of our discovery its task of producing fluid and move it somewhere else is not unique in the body.  Other glands include the salivary (2) and lachrymal gland (3) and the mammary gland (4). It could be immensely interesting for future research to digitize these glands in the same way and see if any follows the same principle.

As a physicist I am happy to end here and say that the pancreas optimizes its own fluid delivering capabilities by locally following the path of least resistance, just like a river basin. My friends and collaborators who are biologists say that while we have seen that the exocrine cells actually secrete fluid as soon as they form a lumen, we have not yet measured flow, that we need to perturb it, that we only have hypotheses regarding the sensor and that we do not know the mechanisms leading to the disappearance of supernumerary loops … it will keep them busy for a while.

References

1 Turing, A. M. (1952). The chemical basis of morphogenesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 237, 37-72.

2 Patel VN, Rebustini IT, Hoffman MP. Salivary gland branching morphogenesis. Differentiation. 2006; 74(7):349–64. https://doi.org/10.1111/j.1432-0436.2006.00088.x PMID: 16916374

3 Gjorevski N, Nelson CM. Integrated morphodynamic signalling of the mammary gland. Nature reviews. Molecular cell biology. 2011; 12(9):581–93. https://doi.org/10.1038/nrm3168 PMID: 21829222

4 Dean C, Ito M, Makarenkova HP, Faber SC, Lang RA. Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation. Development. 2004; 131(17):4155–65. https://doi.org/10.1242/dev.01285 PMID: 15280212

Link to our work

Deconstructing the principles of ductal network formation in the pancreas 
Dahl-Jensen SB, Yennek S, Flasse L, Larsen HL, Sever D, et al. (2018) Deconstructing the principles of ductal network formation in the pancreas. PLOS Biology 16(7): e2002842. https://doi.org/10.1371/journal.pbio.2002842

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Vienna, Austria

July 2018

The use of salamanders in regeneration and developmental research has a long history filled with luminaries of the life sciences. Thomas Hunt Morgan, Hans Spemann and Hilde Mangold, Ross Granville Harrison, Inez Whipple Wilder, and August Weismann all employed salamanders in their work ^1,2. Current experimental research on salamanders largely focuses on studies of regeneration. Their abilities to regenerate limbs, brain, kidney, heart, and tail³ is enviable from the perspective of our limited human regenerative abilities. There is a large and growing toolkit of experimental manipulations, mutant and transgenic lines, and genomic resources that have advanced the use of salamanders to probe critical questions on the evolution of regeneration and its loss. These offer opportunities to discover mechanisms that could lead to new therapies for tissue repair.

The first “Salamander Models in Cross-Disciplinary Biological Research” meeting, organized by Elly Tanaka, Jessica Whited, Karen Echeverri, and Randal Voss, was held this past July (2018) at the Research Institute of Molecular Pathology (IMP) in Vienna, Austria. The meeting was hosted by the intrepid Dr. Tanaka, who is a Senior Scientist at the IMP and a leader in the regenerative biology field. This was a gathering of principal investigators working on various aspects of salamander regeneration, development, genetics, and genomics. The major goals included reviewing recent advances in research tools, along with nuanced tips for employing them, while also establishing a master “to-do” list for the field. Another objective was to lay the groundwork for organizing future salamander meetings intended for the broader community, including postdoctoral and pre-doctoral trainees and potentially representatives from funding agencies.

The Salamanders

Unlike “the” worm or “the” fly, there are multiple salamander models used in molecular studies of development and regeneration. These prominently include the Mexican axolotl (Ambystoma mexicanum), the Iberian ribbed newt (Pleurodeles waltl), the Japanese newt (Cynops pyrrhogaster) and to a lesser extent the North American eastern newt (Notophthalmus viridescens) and several species of lungless salamanders (Plethodontidae). Currently the most commonly used model is the axolotl, which has the longest captive history of any laboratory animal¹. This history includes the importation of a founder population to Paris in 1864, some of which contained the mutant “white” phenotype (an edn3 mutant), and deliberate introgression of an A. tigrinum locus found in 1962, which confers albinism through a tyrosinase mutation^4,5. These salamander species are representatives of three families (Ambystomatidae, Plethodontidae, and Salamandridae – the newts) out of the ten extant salamander families, which likely had extensive limb regenerative abilities at the base of the their clade⁶.

Genomics

Our sessions started with a review of the impressive new work in salamander genomics. Recent published genomes for both the Iberian ribbed newt (Pleurodeles waltl⁷) and Mexican axolotl (Ambystoma mexicanum⁸) provide tremendous new resources for the field. The axolotl genome is roughly ten times the size of the human genome, making it the largest genome to be sequenced and assembled to date (sorry loblolly pine). The publication of both of these genomes promises to help resolve the loci of multiple established mutant lines⁵, and offers the opportunity to establish future forward and reverse genetic screens that will improve our mechanistic understanding of regenerative processes. The community identified additional work needed to make consistent annotations, resolving 5’ ends of genes, and approaching a chromosome scale resolution of contiguous sequence. The latter was recently made available through a bioRxiv preprint from the Smith and Voss labs at University of Kentucky. The lack of whole-genome sequences in salamanders has been a major impediment to the field, and though the assemblies will still require extensive refinements, having these resources should prove to be enormously beneficial to labs currently working with these species as well as those interested in starting salamander research.

Tansgenics and Genome Editing

Genome editing approaches are now available for the axolotl^9,10, the Iberian newt^11,7 and the Japanese fire-bellied newt Cynops pyrrhogaster¹² using TALEN and CRISPR-based approaches. The most recent protocols developed by Ji-Feng Fei¹³ (now at the South China Normal University) for knock-out and knock-in approaches were reviewed. Current best practice techniques focused on improving knock-in strategies without relying on homology-directed repair. These include targeting introns for knock-in’s, screening injected embryo knockouts for efficient guide RNA’s prior to knock-ins, and non-homologous end joining approaches with “ORF Baits.”

The advent of CRISPR and TALEN approaches to genome editing in Pleurodeles was reviewed by Ken-Ichi Suzuki from Hiroshima University. The Suzuki lab is currently working to identify a ROSA-like locus for constitutive expression of knock-in constructs that would also provide a “safe harbor” for exogenous DNA. The intent of this approach is to develop a site that would both be minimally disruptive to normal cellular physiology while experiencing minimal interference from histone modifications in different cell lineages.

A wide range of transgenic and CRISPR edited lines are becoming available, especially in the axolotl. These include constitutive RFP and GFP reporter lines (available from the University of Kentucky’s Ambystoma Stock Center – AGSC), a pax7-mcherry muscle satellite cell marker, a neuronal marker, and a brainbow axolotl¹⁴. Discussions focused on prioritizing existing stocks and facilitating their dissemination through the AGSC (primarily in North America), Medipol University, Max Planck Dresden, and MPI (in Europe). The need to pursue financial resources to enable more extensive repository functions for salamanders, including cryopreservation to biobank lines, was also addressed. A long-term goal of the community is to secure resource funding for these types of valuable operations, which will be necessary to advance discoveries in regenerative biology through this growing research community.

Temporal control of transgene expression

Many genes implicated in regenerative processes have pleiotrophic roles in early development. These make knock out experiments in studying adult regeneration difficult as they can be embryonic lethal. Therefore both temporal and spatial control of gene expression is critical for furthering regeneration research.

There are several creative approaches available to address this potential stumbling block. These include inducible cre-lox systems¹⁵, constitutive cas-9 expression in genomic “safe harbors” with drug inducible guide RNA’s, and viral delivery of foreign transgenes into regenerative blastemas¹⁶¹⁷¹⁸. The latter approach has been championed by both Jessica Whited’s group at Harvard University and the Tanaka lab at MPI. These pseudotyped retroviruses have tremendous potential for further labeling and functional studies of the limb blastema, without raising transgenic embryos or modifying the expression of pleiotrophic genes outside the limb.

Open discussions evaluating approaches and avenues for future research allowed individual labs to share their own research objectives with a collective eye toward developing critical methodologies to advance the community.

Resources needed for the field

The real value of this PI-focused meeting was to allow researchers to define limiting resources they found most pressing for the field. Several additional needs were identified in addition to continued improvements to genome annotations and the temporal control of gene expression. These included detailed histological atlases of regeneration, master lists of validated antibodies, and more stable cell lines for in vitro experiments. One of the most obvious resources needed was the continued communication between labs with subsequent salamander research conferences that should continue to strengthen this growing research community.

Coordination and prioritization of a model system

Several research communities have benefitted from the intentional development and promotion of particular model organisms¹⁹. One-stop repositories of information such as Flybase, Wormbase, ZFinBase, Xenbase, and Sal-Site have all expanded the research capabilities of participating labs. While many of these labs focus on Developmental Biology, the reach of these model systems includes studies of neuroscience, evolutionary biology, physiology, and ecology. Emulating this deliberate approach will provide vital cohesion and an undoubted boon to investigators studying salamander regeneration and development. While salamanders are a remarkable “model” for regenerative research they are also remarkable organisms for their unique evolutionary histories, ecological roles, life history variation, and conservation biology. Development of tools and resources for the molecular biologists and biochemists working on salamanders will undoubtedly have unintentional spillover benefits into a wider range of research fields.

Outlook

Salamander models continue to be fertile ground for amazing discoveries on regenerative biology, cell differentiation, and development. This conference generated a tremendous amount of motivation for its participants. We have planned a broader 2019 meeting in Massachusetts, which will be a showcase for these recent developments and an iterative checkup on this rapidly growing experimental field.

References

1. Reiß, C., Olsson, L. & Hoßfeld, U. The history of the oldest self-sustaining laboratory animal: 150 years of axolotl research. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 324, 393–404 (2015).
2. Wilder, I. W. The Morphology of Amphibian Metamorphosis. (Smith College, 1925).
3. In Salamanders in Regeneration Research Methods and Protocols (eds. Kumar, A & Andras, S) (Humana Press, 2015).
4. Humphrey, R. R. Albino axolotls from an albino tiger salamander through hybridization. J. Hered. 58, 95–101 (1967).
5. Woodcock, M. R. et al. Identification of mutant genes and introgressed tiger salamander DNA in the laboratory axolotl, Ambystoma mexicanum. Scientific Reports 7, (2017).
6. Fröbisch, N. B., Bickelmann, C. & Witzmann, F. Early evolution of limb regeneration in tetrapods: evidence from a 300-million-year-old amphibian. Proc. Biol. Sci. 281, 20141550 (2014).
7. Elewa, A. et al. Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration. Nat Commun 8, 2286 (2017).
8. Nowoshilow, S. et al. The axolotl genome and the evolution of key tissue formation regulators. Nature 554, 50–55 (2018).
9. Fei, J.-F. et al. CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration. Stem cell reports 3, 444–459 (2014).
10. Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R. & Crews, C. M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development (Cambridge, England) 141, 2165–2171 (2014).
11. Hayashi, T. & Takeuchi, T. Gene manipulation for regenerative studies using the Iberian ribbed newt, Pleurodeles waltl. Methods Mol. Biol. 1290, 297–305 (2015).
12. Nakajima, K., Nakajima, T. & Yaoita, Y. Generation of albino Cynops pyrrhogaster by genomic editing of the tyrosinase Gene. Zool. Sci. 33, 290–294 (2016).
13. Fei, J.-F. et al. Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration. Proc. Natl. Acad. Sci. U.S.A. 114, 12501–12506 (2017).
14. Currie, J. D. et al. Live imaging of axolotl digit regeneration reveals spatiotemporal choreography of diverse connective tissue progenitor pools. Dev. Cell 39, 411–423 (2016).
15. Khattak, S. et al. Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc 9, 529–540 (2014).
16. Kuo, T.-H. & Whited, J. L. Pseudotyped retroviruses for infecting axolotl. Methods Mol. Biol. 1290, 127–140 (2015).
17. Khattak, S. et al. Foamy virus for efficient gene transfer in regeneration studies. BMC Dev. Biol. 13, 17 (2013).
18. Oliveira, C. R. et al. Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration. Dev. Biol. 433, 262–275 (2018).
19. Abzhanov, A. et al. Are we there yet? Tracking the development of new model systems. Trends Genet. 24, 353–360 (2008).

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Position Description
The Boston College Biology Department seeks outstanding candidates for a tenure-track faculty position in the area of Cell and Developmental Biology. Applicants are sought at the Assistant Professor level; however, exceptionally strong candidates will be considered at the Associate Professor level. The university provides competitive startup funds and research space with the expectation that the successful candidate will establish, or bring to the university, a vigorous, funded research program. Special consideration will be given to candidates whose research program synergizes with current faculty interests. We especially encourage applicants whose research utilizes embryonic model systems, those who study developmental neurobiology, and/or those who work at the interface of biophysics and cell biology. The successful candidate will join an active and expanding department with current strengths in cytoskeletal regulation, developmental biology, cancer biology, as well as microbiology, infectious disease, and computational biology.

Visit www.bc.edu/bc-web/schools/mcas/departments/biology/research/faculty-research.html for profiles of the Biology Department’s current research programs.

The university is situated on a beautiful campus dating back to the beginning of the twentieth century and is closely located to downtown Boston and Cambridge. The Biology Department has strong ongoing collaborative efforts with other departments including Chemistry and Physics, surrounding institutes including BU, Harvard, MIT, Northeastern University and Tufts University, and has state of the art (core-) facilities including next-gen sequencing, cleanrooms for nanofabrication, FACS, microscopy (including super-resolution microscopy), microfluidics, robotics, NMR and mass-spec. Moreover, the university has recently announced its decision to establish an interdisciplinary Institute for Integrated Science and Society, and the building is scheduled for construction beginning Spring 2019.

How to Apply
Applicants should submit a cover letter, curriculum vitae, a statement of research plans, a statement of teaching interests, and arrange for three letters of reference. All application materials should be submitted via Interfolio at http://apply.interfolio.com/53713. Review of applications will begin on October 1st and will continue until the position is filled.

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The University of Virginia invites applications for a tenure-track Assistant Professor position in the Department of Biology. We seek applicants who have a research vision that addresses new or longstanding fundamental questions in cell biology. Located within the College of Arts and Sciences, the Department of Biology provides an interdisciplinary and collaborative environment for basic research and teaching that spans multiple levels of biological organization, enhanced by close collaborations with colleagues at our Schools of Medicine and Engineering & Applied Sciences. A successful candidate is expected to establish a vigorous, independent, and externally funded research program as well as provide instruction and scientific training at the undergraduate and graduate levels. Applicants with a respect for diversity and a passion for making a positive impact on the world in a collaborative environment are strongly encouraged to apply. The position will begin on August 25, 2019.

Applicants must have a Ph.D. or other doctoral degree and post-doctoral research experience. A successful applicant will also have research accomplishments and plans of outstanding quality and significance as well as a commitment to excellence in teaching and mentoring. Commitment to participate in and further develop a diverse, collegial, interdisciplinary, and collaborative environment is strongly preferred.

To apply, jobs.virginia.edu/applicants/Central?quickFind=85482. Complete a candidate profile online, attach a cover letter that succinctly highlights your most significant research accomplishments, experiences, and qualifications; a curriculum vitae; a research statement that describes your vision for your research program at the university (≤ 3 pages); a statement of teaching goals; and the contact information of three references. The deadline for receipt of applications is October 31, 2018.

For questions regarding the position, please contact search chair Keith Kozminski, Associate Professor of Biology, at biocellsearch@virginia.edu; for questions about the application process, please contact Savanna Galambos, Faculty Search Advisor, at: skh7b@virginia.edu

UVA assists faculty spouses and partners seeking employment in the Charlottesville area. To learn more please visit  http://provost.virginia.edu/dual-career

For more information about UVA and the surrounding area, please visit http://uvacharge.virginia.edu/guide.html

The University of Virginia is an equal opportunity and affirmative action employer. Women, minorities, veterans and persons with disabilities are encouraged to apply.

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The Department of Biological Sciences at the University of South Carolina invites applications for a tenure-track position as an Assistant Professor with a research program in neurobiology. The successful candidate will be expected to establish an independent, extramurally funded research program focusing on cell-cell communication in neural development and/or disease. We are especially interested in applicants studying how interactions among neurons or between neurons and other cell types such as astrocytes, oligodendrocytes, Schwann cells, microglia, macrophages or endothelial cells contribute to nervous system development, disease, or response to injury in any experimental or model system. The successful candidate will have a Ph.D. or an M.D., and will have completed post-doctoral training in a relevant area of neuroscience or biomedical sciences. He or she will interact with research groups in Biological Sciences, including the Center for Childhood Neurotherapeutics that includes neuroscientists focusing on mechanisms of neural development and neural repair. Additionally, the University has a highly interactive neuroscience research community that encourages and precipitates collaborations. The candidate will be responsible for teaching at undergraduate and graduate levels in courses appropriate to his/her expertise.

To ensure full consideration, applications should be received by November 16, 2018. All applicants must fill out an online application at the USC employment website at: http://uscjobs.sc.edu/postings/39584. Qualified candidates should include with their application a curriculum vita, research statement (3 pages) and teaching philosophy (1 page). The names, email addresses and phone numbers of at least three references should also be provided. Additional information on the position and the Department of Biological Sciences can be found at http://www.biol.sc.edu/. For questions or further information, please contact Dr. Fabienne Poulain (fpoulain@mailbox.sc.edu).

The University of South Carolina is an affirmative action, equal opportunity employer. Minorities and women are encouraged to apply. The University of South Carolina does not discriminate in educational or employment opportunities on the basis of race, color, religion, national origin, sex, sexual orientation, gender, age, disability, veteran status or genetics.

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Welcome to our monthly summary of developmental biology (and related) preLights.

preLighters are early-career researchers who select and highlight preprints which they feel are interesting for the life-science community. While writing highlight posts is mostly an individual effort, plenty of interactions between the preLights team members take place on our Slack channel. This is where last month several preLighters decided to respond to a controversial World View article in Nature about the danger of preprints, and they shared their well-argued (and highly read) commentary here on the Node. Apart from that, the month of August was not short of exciting developmental biology (and related) preLights, we hope you enjoy this selection!

Gene expression and cell fate

There was a good mix of preLights dealing with gene expression in various models, such as fish, flies and living neuronal tissue. Idoia Quintana-Urzainqui discussed a preprint showing that in zebrafish embryos, this first wave of transcription is restricted to a special nuclear compartment. Later on in development, transcription factors, which are often tissue-specific, regulate cell fate. Amanda Haage highlighted how the progression from pluripotent blastula cells to neural crest cells is regulated by a transition from SoxB1 to SoxE transcription factors in zebrafish. Moving to flies, Clarice Hong’s and Natalie Dye’s preLight both dealt with transcription factor function. Clarice discussed how binding site orientation and spacing determines the activity and specificity of Hox transcription factors, while Natalie reported on how the transcription factor Doublesex is involved in the decision making of male vs. female gonad stem cell niches. Finally, Theresa Rayon illustrated how cells make transitions during neurogenesis. The study she preLighted imaged the Hes5 transcription factor in real-time and showed that its fluctuating expression in neuronal progenitors turns oscillatory as cells enter differentiation.

Neurodevelopment and morphogenesis

Neurogenesis during development of the cortex was the focus of Boyan Bonev’s preLight. By using a new labelling approach, the study uncovered the temporal order in the production of diverse neuronal subtypes and found that many of them emerge simultaneously. Ashrifia Adomako-Ankomah highlighted the spectacular morphogenetic events occurring during development of the eye. The research identified an important role for the protein nidogen, secreted by the surrounding neurocrest cells, in shaping the optic cup in zebrafish. Nidogen also featured in the preLight of Nargess Khalilgharibi, and it turned out that this protein is not essential for Drosophila morphogenesis, but still has tissue-specific functions. Lastly, Andreas van Impel reviewed how the local translation of a transcript at the leading edge of migrating endothelial cells regulates blood vessel formation.

Tools & Technologies

Undoubtedly, new methodologies are key in paving the way for discoveries in developmental biology. Hannah Brunsdon covered  a technique with enormous potential, in which the authors combined CRISPR lineage tracing with single-cell RNA sequencing to decode early mammalian development. Tools to regulate gene expression are highly desired not only for basic research, but also for potential future gene therapies. Tim Fessenden highlighted a clever technique to control the level of transgene expression by integrating synthetic miRNA target sites into the transgene. Tuning gene expression by changing the regulatory 3D interactions was the focus of Ivan Candido-Ferreira’s highlight, which showcased the use of CRISPR technology in combination with optogenetics. While optogenetics is broadly used in many experimental systems, it seems tricky to get it to work in some organisms, such as the C. elegans embryo. Angika Basant discussed an approach in her preLight that showed how such a limitation (in this case germline silencing) could be overcome. Lastly, Samantha Seah preLighted the development of a microfluidic device that allows imaging of sea anemone larvae, which are models for coral symbiosis. Make sure to read the authors’ comments who tell us about how the project was created.

Do also visit the preLights website to discover further interesting preprint highlights, such as Gautam Dey’s post on a statistical tool that provides an alternative to the p-value , or Nicola Stevenson’s highlight on the horizontal transfer of RNAs in honeybees!

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Last week, Development and our sister journal Journal of Cell Science signed an open letter coordinated by ASAPbio, signalling our intention to publish peer review reports alongside published papers. I’m really delighted to be making this commitment and wanted to take the opportunity to say a few words about our thinking behind this decision.

So why publish peer review reports and why start doing it now? The commentary in Nature that came out to accompany the open letter does, in my opinion, a great job of explaining the both the benefits and the potential pitfalls involved in publishing peer review reports and associated correspondence.  Above all, what we gain is transparency: both in terms of providing the reader additional information about the published paper and in opening up the journal’s decision-making process. Referee reports and author point-by-point responses give valuable insight into why a paper is seen by referees as important for the field, what the caveats with the work might be, and how a paper has evolved through the peer review process. I am proud of the job that Development’s Academic Editors do in helping to select papers to be published in the journal, and I’m happy to showcase the work they do in a more transparent manner.

We still have many details to work out in terms of exactly what information we will be making public, and how we will be doing it, but one thing we are clear on is that referees should still have the right to remain anonymous – both to the authors and the reader. We know from talking to the community that many referees would feel uncomfortable signing their name on a report. While open identities are a nice idea in theory, there is a strong risk that forcing referees to sign their names might compromise the quality and rigour of peer review – would you to be happy to openly criticise a paper written by someone you think might review your next manuscript, or sit on your next grant panel?  If referees want to sign their name, they are more than welcome to do so, but we do not want to make this essential.

With the protection of anonymity, though, we hope that referees will continue to do the excellent job they do in assessing papers for Development. I was part of the team at The EMBO Journal when they initiated their policy of transparent peer review, and we were concerned that referees would refuse to review papers, or would provide only ‘bland’ reports. Neither of these concerns came to pass, and the positive experience I had with transparent peer review there convinced me that it would be a good thing to implement across journals more broadly.

Development has been considering publishing peer review reports for some years. A few of our editors have been strong proponents of the policy for a long time; others have been more cautious – primarily for the reasons detailed above and in the Nature commentary. A few years ago, we conducted a community survey asking about priorities in peer review innovations, and this told us that there were other things our readers cared about more – such as introducing cross-referee commenting (which we implemented a couple of years ago). Now, however, we feel that the time is right to start planning for publishing referee reports – and this is something that our incoming Editor-in-Chief James Briscoe is  keen to implement, with the full support of all our editors. Early reactions to our announcement on social media suggest that this move will be welcomed by the community. We will be working with Journal of Cell Science to implement transparent peer review; the other Company of Biologists journals will be reviewing how things go at Development and Journal of Cell Science and consulting with their communities before deciding on their own plans.

We hope to introduce this policy in early 2019. For now, though, we welcome any feedback you may have, and look forward to sharing further details as our plans progress.

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The Developmental Biology Unit seeks to understand the general principles and mechanisms underlying the development of multicellular organisms. Researchers in the unit combine the power of genetic model organisms with quantitative imaging and -omics technologies, synthetic biology, reduced (in vitro) systems and theoretical modelling, to create a cross-cutting approach to modern developmental biology.

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Axon guidance relies on the reception and integration of molecular cues from the environment by growth cones, and defective pathfinding results in misplaced projection patterns in the mature nervous system. A new paper in Development investigates this process in the Drosophila neuromucular system, as well as the consequences of axonal miswiring to locomotion. We caught up with lead author Jaqueline Kinold and her supervisor Hermann Aberle, Professor in the Department of Functional Cell Morphology at Heinrich Heine University, Düsseldorf.

Hermann, can you give us your scientific biography and the questions your lab is trying to answer?

HA In retrospect, I find it quite interesting that my work was all the time somehow associated with cell adhesion and cellular junctions. I started off as a PhD student in Rolf Kemler‘s lab in Freiburg, working on vertebrate Cadherin-Catenin complexes, which connect neighbouring epithelial cells at adherens junctions. As a postdoctoral fellow, I switched topics and changed to Drosophila neuromuscular junctions (NMJs) in Corey Goodman‘s lab at UC Berkeley, where I was involved in a large-scale mutagenesis screen searching for genes that affected the morphology of these terminals. After cloning and functional characterization of the wishful thinking gene, I moved to Christiane Nüsslein-Volhard‘s lab at the MPI for Developmental Biology in Tübingen. There, we cloned several other genes that came out of the screen, including tolloid-related, mical, ankyrin-2 and neuroligin-1. During this time, and inspired by work of Darren Gilmour, we also developed tools and techniques to image migrating motor axons in living Drosophila embryos. Receiving a grant from the German Research Foundation and a generous invitation to join Christian Klämbt‘s department, we moved to the University of Münster, where we not only had fantastic imaging opportunities but also focussed more and more on the function of the axon guidance molecule sidestep (side), which also came out of the screen. In 2010, I received a call from the Heinrich Heine University in Düsseldorf, where we developed the idea to search for behavioural consequences of wiring defects.

Jaqueline, how did you come to join the lab, and what drives your research?

JK I studied biology at the University of Kassel and did my diploma thesis on the topic of spermatogenesis in Drosophila under the supervision of Mireille Schäfer. Afterwards, I wanted to continue my research with Drosophila, but I could imagine a change of topic and laboratory for my PhD very well. Hermann’s job advertisement and also his project description during my interview fascinated me very much. On the one hand, the correct wiring of the muscles is a multifaceted process in which many different components interlock and, on the other hand, the different methods and starting points spoke in favour of joining Hermann’s laboratory.

When did your lab first get interested in the link between neuronal wiring and locomotion in flies?

HA Since the phenotype of side mutants is really strong, I was once asked after a talk if this has any consequences for viability or eclosion. This question stuck in my mind and I started to realize that most papers in the field dealt with the guidance process itself, in embryos, and hardly anybody was exploring postembryonic stages, when the neuromuscular system is actually functional and constantly contracting. Together with the Drosophila community moving more and more into circuit analysis and behavioural studies, I asked myself which behaviour could be affected by our mutations. It took a while until I realized that locomotion and movements are the underlying basis for most, if not all, behaviours. However, it took a while until we had all the necessary technical equipment. In fact, everything truly started as the high-speed video camera arrived.

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

HA We examined the final innervation pattern on all muscles in side mutant third instar larvae. From this survey, we derived three major conclusions. First, innervation defects were permanent, i.e. even if potential rescue mechanisms exist, they failed to restore NMJs at non-innervated muscles. Second, loss of side affected all peripheral motor pathways and thus all body wall regions. Third, innervation errors were non-stereotypical and appeared in each hemisegment differentially. Since Side functions as a substrate-bound attractant, ventral bypass phenotypes or lack of NMJs at distal-most muscles, could be explained by insufficient attraction, which either inhibits defasciculation or slows axonal growth, respectively.

JK Also, overexpression of Side attracted motor axons at the wrong time to the wrong place, resulted in innervation defects. This was particularly evident when we expressed Side in muscle precursors. Attraction was that strong that individual axons travelling in the ISN were stretched into opposite directions leading to split pathways and complete lack of NMJs on dorsal-most muscles. This caused also amazing locomotion phenotypes. During crawling, larvae detached from the substrate and excessively lifted their head and tail segments into the air, despite lack of dorsal innervation. Nobody was expecting such a phenotype. Crawling seems to be much more complex than anticipated.

side mutants show considerably variable phenotypes, even in adjacent hemisegments. What do you think this tells us about how axon guidance works in flies?

HA At the beginning of the screen, in Corey Goodman’s lab, we discussed a lot what kind of phenotypes we might possibly find. One such idea was that we might find genes that affect only a single NMJ on a specific muscle fibre, because we imagined a key-and-lock mechanism for wiring 30 muscle fibres, where unique ligands on motor axons connect to cognate receptors on muscles. Inactivating such a receptor by point mutations should in theory prevent the formation of the associated NMJ. Unfortunately, we did not find this phenotypic class, nor did anybody else. We therefore think that motor axon guidance in Drosophila is not hard-wired by high-affinity ligand-receptor complexes but rather functions via co-operative actions of several guidance molecules. Newer finding in Pablo Labrador‘s laboratory also push this older idea originally developed in the Keshishian and Goodman labs. Guidance decisions therefore seem to be made at several points and sum up along the entire path. If several decisions go wrong in a row, phenotypes become noticeable.

Do the locomotion defects you found in larvae have anything to tell us about movement disorders in mammals?

HA Oh yes, I think this is one of the interesting parts of our story. Similar to Drosophila, there are only a few mammalian studies that correlate axon guidance errors with muscle innervation patterns. But these studies made several interesting observations. First, in mice, if a motor nerve branch fails to develop or is severely stalled, entire muscle fields are not innervated after birth causing muscle atrophy. In limbs, this can lead to paralysis and aberrant locomotion patterns. Second, the phenotypes are not necessarily symmetrical and occasionally affect only one side of the body, i.e. very similar to the unilateral defects observed in side mutants. Third, feet and ankles of affected limbs are occasionally twisted inward in newborn mice, a deformity highly reminiscent to congenital clubfoot in humans. The aetiology of clubfoot is still not fully understood, but since it frequently develops unilaterally, one speculative possibility is that it could develop due to innervation defects.

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

JK There was not one key moment that has stuck with me, but several small highlights, especially at the laser-scanning microscope. I am over and over again enthusiastic about how “beautiful” the axonal pattern in the embryo or larva is.  Further, I had such special moments when larvae showed particularly strong or unusual phenotypes during crawling, which looks sometimes quite funny.

Drosophila larva overexpressing Side in muscles and showing an extreme crawling phenotype.

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

JK Yes, there were some of these frustrating moments, especially handling living larvae for videography. During my research, I have realized that Drosophila larvae have sometimes their own mind, as they decided not to crawl at all or not to crawl in a straight line for the high-speed videography, but rather to crawl away from the nicely prepared agar block.

What next for you after this paper?

JK Since this paper is only a partial project of my PhD thesis, I plan for the next months to press ahead with another project for publication – hopefully we can submit the manuscript successfully at the end of this year.

And where will this work take the Aberle lab?

HA One direction we are heading is how locomotion in animals with hydrostatic skeletons works after all. Crawling behaviour is much more sophisticated and fine-tuned than it appears at first glance. We would like to understand how specific muscle groups contribute to particular movements and how this is antagonized by the liquid-filled body cavity.  Which muscle groups, for example, induce rolling escape behaviours during attacks of predatory wasps or coordinate jumping in some dipteran larvae. Ultimately we would like to find mutations or conditions which activate or inactivate particular movements, in order to better understand the underlying circuitry and machinery. Another line of research will certainly be the functional analysis of the entire Sidestep family. There are 7 Side paralogs in the Drosophila genome and virtually nothing is currently known about their functions.

Finally, let’s move outside the lab – what do you like to do in your spare time in Düsseldorf?

JK As I am a very water-loving person, I like to spend my spare time along the Rhine river – whether for a walk or to make myself comfortable on a blanket on its shores and let my soul dangle watching the ships passing by. I also like to take advantage of the city’s concert events, as two of my favourite bands, “Die Toten Hosen” and “Broilers”, come from Düsseldorf and regularly give concerts in the city. Furthermore, the whole laboratory and I like to celebrate the Düsseldorf Carnival, which takes place every spring.

HA Düsseldorf has a quite lively art scene, and since I enjoyed landscape photography very much during my undergraduate years, I am particularly drawn to the “Düsseldorf School of Photography”, founded by Bernd and Hilla Becher. Whenever there are exhibitions by Andreas Gursky, Axel Hütte, Thomas Ruff, to name a few, I try to not miss the vernissage. I also love to shop for coffee-table books in local stores.

Sidestep-induced neuromuscular miswiring causes severe locomotion defects in Drosophila larvae
Jaqueline C. Kinold, Carsten Pfarr, Hermann Aberle
Development 2018 145: dev163279 doi: 10.1242/dev.163279

This is #49 in our interview series. Browse the archive here.

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