The neural crest is a progenitor population with the capacity to contribute to all vertebrate germ layers. The transcription factor and signalling pathway activity underlying this remarkable pluripotency have been well studied, but the role of the epigenetic state is less well understood. A new paper in Development examines the role of histone acetylation in regulating the neural crest’s remarkable pluripotency. We caught up with authors Anjali Rao and Carole LaBonne, Erastus O Haven Professor of Life Sciences at Northwestern University, to find out more about the story.

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

CLB I have always been in love with the beauty of embryonic development, and the elegance of developmental decision-making. I did my PhD work at Harvard studying germ layer formation; specifically the role of FGF signalling in mesendoderm formation. During my graduate work I learned about the neural crest and I knew that was what I wanted to work on as a post-doc. It is an absolutely fascinating cell type, and this year is the 150th year of its discovery by Wilhelm His. Acquisition of the neural crest drove the evolution of vertebrates because these stem cells contribute a myriad of novel structures to the basic chordate body plan. Another interesting feature of these cells is that they are migratory and invasive; they need to actively disperse throughout the embryo to the places where they will form those structures. The mechanisms that they use to do this have been co-opted by cancer cells to mediate metastasis, so we can actually learn important things about tumor progression by studying the neural crest.

Two of Carole’s early papers (in Development!), from her PhD and postdoc

After my post-doc at Caltech I moved to Northwestern, where my lab has continued to study various aspects of neural crest development, including the signalling pathways and GRN components that control the stem cell attributes, behaviour and lineage decisions of these cells. More recently we have also been studying the pluripotent cells of the early blastula embryo. We do most of this work in Xenopus, which is a fantastic system for asking questions about early vertebrate development. Xenopus is closely related to mammals but the embryos develop rapidly and externally in simple saline solution, and the large (~1.2mm) size of the embryos facilitates isolation of explants or single cells. These embryos also provide copious materials for genomic and proteomic studies, which we are doing a lot of these days. Xenopus embryos are ideal for rapid gain/loss of function studies, including CRISPR-mediated genome editing, and the fate of every early embryonic cell has been mapped. We are very fortunate to have a fantastic resource center for this model, the National Xenopus Resource (NXR), that is located at the MBL in Woods Hole MA. The NXR generates and distributes transgenic lines and CRISPR mutants to the community, and also holds training workshops.

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

AR I have always been interested in understanding the regulation of stem cell maintenance. Before starting my PhD, I was working in a lab that studied the regulation of kidney stem cells during kidney fibrosis. Often diseases are caused by the misregulation of developmental pathways, and I hence got interested in studying the regulation of stem cells during embryonic development. The LaBonne lab was the perfect fit. Early during my PhD, our lab proposed a new model for the genesis of the neural crest that suggested that these cells arise due to a retention of stem cell potential, and we became really interested in identifying the mechanism for this. In particular, I was tasked with identifying chromatin remodelers required for neural crest stem cell maintenance, and characterizing the epigenetic changes that take place during the process of neural crest formation.

What led you to become interested in the epigenetic regulation of neural crest and blastocyst pluripotency?

CLB A few years ago we realized that neural crest cells and pluripotent blastula cells share a remarkable number of gene regulatory components. In particular, many of the transcription factors that we had long studied in the neural crest are first expressed in, and play essential roles in, blastula stem cells. That led us to hypothesize that neural crest may have arisen (and thus vertebrates evolved) via retention of features of these early pluripotent cells, even as neighbouring cells became lineage restricted. We began actively studying the shared features of these two cell types at the level of signalling pathways and transcription factors. For example some of our most recent work has focused on FGF-mediated Map Kinase signalling, which is essential for maintaining the developmental potential of both cells types. This was fun for me because it brought me full circle back to my graduate studies.

There is growing literature on the roles of epigenetic readers, writers, and erasers in controlling stem cell attributes and lineage decisions in both cultured ES cells and embryos. Thus, it was clear that we would need to examine conserved and divergent roles for these factors, and epigenetic mechanisms more generally, in neural crest and pluripotent blastula cells. We are finding interesting roles for a number of epigenetic regulatory factors, and so Anjali’s HDAC paper is just the tip of that iceberg.

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

AR & CLB We found that Histone Deacetylase (HDAC) activity is essential for neural crest formation, both in whole embryos and explants of pluripotent cells. HDAC activity is also required for the pluripotency of blastula stem cells. Importantly, both cell types are characterized by low levels of histone acetylation, highlighting another shared feature of these cell populations, and HDACs function to maintain this state. Interestingly, we found that blocking HDAC activity in blastula-derived cells results in aberrant expression of markers of multiple lineages, and prevents the cells from committing to any lineage. Finally, we found that increasing HDAC activity can enhance the reprogramming of cells to a neural crest state. Our data suggests that HDACs play an important role in retaining the cells that will become neural crest cells in a stem cell state through gastrulation and neurulation, even as other cells in the early embryo become lineage restricted.

I was intrigued by your finding that HDAC inhibited cells co-expressed several lineage markers at once. What do you think explains these cells’ confused identities?

AR That is a great question. It seems that HDAC activity must be critical for keeping the acetylation state of genes controlling a number of different lineage states low in pluripotent stem cells, so that these genes are not expressed before the cells have made a specific lineage decision. Inhibition of HDAC activity releases this control, and therefore causes low-level expression of markers of multiple lineages simultaneously. An apparent consequence of co-expressing genes that promote multiple different lineage states is an inability to commit to any one lineage. During normal development, signals instructive of a specific lineage state likely release HDAC control of gene expression essential to that lineage, while ensuring that other lineage control factors remain unexpressed.

What do you think might be downstream of HDAC in the neural crest, and do you expect these targets to be the same as in the naïve blastula?

AR & CLB We know that global levels of H3K9 and H3K27 acetylation are similar in blastula and neural crest stem cells, and we think that the key targets of HDAC activity are likely to be similar in both of these cell types. We have used RNA-Seq to characterize the changes to the transcriptome that occur in response to HDAC inhibition, and that gives us important clues to what the key targets might be. We are currently examining the deposition of specific acetylation marks genome wide using ChIP-Seq to investigate this further.

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

AR I was really excited when I found that increasing HDAC activity enhanced the ability of blastula cells to be reprogrammed to a neural crest state. I think it definitely was my eureka moment, because it gave us the fundamental insight that HDACs actively promote the retention of pluripotency that leads to formation of the neural crest.

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

AR My most frustrating moments during this project were actually during the revisions for the paper. The reviewers asked us to confirm the phenotypes we observed following chemical inhibition of HDAC activity by using an independent means of down-regulating this activity. This was very challenging because HDAC1 is very highly expressed in the early embryo. However, with Carole’s guidance, I was able to target a morpholino that blocks HDAC1 translation to the neural crest, and show that this phenocopied the effects of the chemical inhibitors.

What next for you after this paper?

AR I am currently diving deeper into understanding the epigenetic regulation of the neural crest using ChIP-Seq and proteomics, and I hope to gain further insights into the role of specific epigenetic marks in stem cell maintenance.

And where will this work take the LaBonne lab?

CLB We are continuing to drill down on the epigenetic control of pluripotency in both naïve blastula cells and neural crest cells, and in developmental decision making more generally. Ultimately we want to understand these processes at single cell resolution using both quantitative imaging and single cell genomics. We are fortunate to be a part of a new NSF-Simons Center for Quantitative Biology focused on understanding emergent properties in developmental biology. Our project is focused on understanding dynamical transitions in cell states, and we have amazing math and modelling collaborators in the Center who are going to help us to take these studies to whole new levels of analysis.

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

AR In my spare time, I enjoy running on the beautiful trail along Lake Michigan and I am currently training to run the New York City marathon in November. I also love reading, particularly historical fiction, and trying out new restaurants around Chicago.

CL I am the mother of two and the Chair of my department, so spare time is a bit of a foreign concept for me! In the time I do have I love to cook, travel and take long walks along the lake with my two standard poodles.

Histone deacetylase activity has an essential role in establishing and maintaining the vertebrate neural crest
Anjali Rao, Carole LaBonne
Development 2018 145: dev163386 doi: 10.1242/dev.163386

This is #48 in our interview series. Browse the archive here.

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PhD position in Nephrology in Freiburg, Germany

Studying mechanisms of genetic renal disease in Drosophila and mouse

The Renal Division of the University Medical Center Freiburg is offering a PhD position (DFG-funded). The position is available for three years and should be filled as soon as possible (starting in fall 2018).

Project:

• The lab is interested in studying mechanisms of genetic renal disease with a focus on monogenic causes of nephrotic syndrome.
• To this end we are utilizing the Drosophila and mouse animal models in conjunction with approaches in vitro.
• The podocyte-like Drosophila nephrocytes will be employed to characterize disease genes functionally and for whole-animal drug screening to develop novel therapeutic strategies.
• The candidate will work in diverse scientific environment with intensive supervision and support.

Requirements:

• We are looking for a highly motivated and ambitious candidate with a strong interest in basic research with clinical relevance.
• The applicant should hold a master’s degree in Biology, Molecular Medicine or an equivalent discipline.
• Experience in cell biology, mice and/or Drosophila is desirable but not mandatory.

Application: Interested candidates should send an application including a CV, a brief motivation letter and two references to

Dr. Tobias Hermle (tobias.hermle[at]uniklinik-freiburg[dot]de).

Selected References:

1. Hermle, T, Schneider, R, Schapiro, D, Braun, DA, van der Ven, AT, Warejko, JK, et al.: GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome. J Am Soc Nephrol, 2018.

2. Helmstadter, M, Huber, TB, Hermle, T: Using the Drosophila Nephrocyte to Model Podocyte Function and Disease. Front Pediatr, 5: 262, 2017.

3. Hermle, T, Braun, DA, Helmstadter, M, Huber, TB, Hildebrandt, F: Modeling Monogenic Human Nephrotic Syndrome in the Drosophila Garland Cell Nephrocyte. J Am Soc Nephrol,28: 1521-1533, 2017.

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A postdoc position is available in the Heemskerk lab, Department of Cell and Developmental Biology at the University of Michigan Medical School.

We are looking for a postdoc that shares our excitement about using stem cells to discover the developmental mechanisms underlying embryogenesis and organogenesis. The lab is highly interdisciplinary and combines experimental and theoretical methods from biology, physics, and engineering, focusing on quantitative live-cell measurements to study spatial organization and cell fate determination in human pluripotent stem cells.

The broader institutional environment of the University of Michigan – the public university with the highest research spending in the United States – provides exceptional resources for professional development and trailblazing scientific exploration. The university is located in Ann Arbor, which is consistently named among cities with the highest quality of life in the US.

The successful candidate is a rigorous thinker with either a relevant biology background and strong interest in quantitative methods, or a quantitative background (e.g. physics, engineering, math) and strong interest in wet lab biology.

For more information see http://idseheemskerk.com.
To apply, please send a CV with publication list and three references, as well as a cover letter stating your motivation to iheemske@umich.edu.

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

preLights, the preprint highlighting service supported by The Company of Biologists, has now reached over 150 posts since launching in late February. To further promote this platform, we are now featuring preLights content in three of The Company of Biologist’s journals – including Development. In addition, complementing the Node’s monthly trawl for preprints, we are starting a monthly series where we highlight some of the exciting developmental biology (and related) preLights articles. So, on to our first post!

Flies, fish and chicks

preLighters  featured several exciting  preprints using Drosophila. A modern imaging approach to study transcriptional dynamics, combined with mathematical modelling shed new light on the generation of the famous even-skipped stripes, and was covered by Erik Clark. Mathematical modelling was also key in getting at the mechanisms of dorsal closure in a preprint that Yara Sanchez and Arnaud Monnard reviewed  together. Ivana Viktorinova’s preLight discussed how the wing epithelium responds to mechanical stress during morphogenesis, in which the mechanosensitive binding of the endocytic regulator p120 plays a major role. Drosophila embryos also have to tolerate other kind of stresses during their development, such as hypoxia; Sarah Bowling’s preLight featured a study that showed how the fat body of larvae senses low oxygen and modulates TORC1 in response.

There were plenty of exciting preprints coming from the zebrafish community, and the preLighters did their best to cover them. James Gagnon highlighted a fascinating study showing the essential role of a novel small protein for species-specific fertilization. Genes and phenotypes were studied at a much larger scale in a story ­­­­– covered by Daniel Grimes – about an impressive effort to link functions to human schizophrenia-associated loci. The strength of zebrafish in modelling complex human diseases was also nicely featured in preLights posts by Hannah Brunsdon and Giuliana Clemente. Hannah highlighted how a putative human CHD gene variant enhances the phenotype of a known CHD-associated gene, and called attention to the importance of investigating the contribution of risk alleles to existing disease-associated phenotypes. Giuliana wrote about the introduction of elements of the human immune system into zebrafish, which could allow future research to investigate how immune cells contribute to cancer progression and metastasis. It turns out that immune cells, and specifically the spatial-temporal dynamics of cytokine induction, are crucial for spinal cord regeneration, as explored in Shikha Nayar’s preLight.

Wouter Masselink and Ashrifia Adomako-Ankomah both highlighted preprints that combined chick embryology methods with modern molecular approaches to gain new insights into one of developmental biology’s ‘classical’ problems: limb development. Wouter covered a study showing that the collinear activation of Hox genes is important for controlling the position of the forelimb. Ashrifia’s post dealt with the question of how the final size of the limb is determined and a preprint showing BMP signalling-dependent regulation of proliferation rates in the limb bud.

Chromatin and genomics

The chromatin biology and genomics fields were well represented on preLights this month. Claire Simon and Sophie Morgani discussed how Sox3 and Pou5f3 act as pioneer transcription factors to open chromatin for genome activation in Xenopus, while the organization of inactive chromatin in senescent cells was the focus of Carmen Adriaens’ post. Lauren Neves’ preLight reported on a remarkable study, which showed that the histone H3-H4 tetramer – a main component of the building blocks of chromatin – also has copper reductase activity. This enzymatic function of histones may have been important for the emergence of eukaryotes. Several preLights featured the ever-increasing power of RNA-seq to answer interesting biological questions. A transcriptomic study in plants, highlighted by Martin Balczerowicz, investigated the level of transcriptional noise among genetically identical species. Rob Hynds’ preLight discussed the use of single-cell transcriptomics combined with bulk proteomics to study healthy ageing in mouse lungs. Finally, an RNA-seq study also gave insight into how a remodelled chloroplast is maintained in a non-photosynthetic alga, preLighted by Ellis O’Neill.

Tools & Technologies

Novel technologies featured heavily this month, for instance Rebekah Tillotson’s preLight on CRISPR gene drives in mouse, which could help scientists create disease models with multiple mutations. Satish Bodakuntla covered  a novel expansion microscopy technique that goes beyond the limits of super-resolution. The most popular type of method to highlight turned out to be optogenetics. Mahesh Karnani reflected on two preprints that made major advances in achieving optogenetic inhibition of neural activity by soma targeting, and the preLight includes insightful comments from both research teams. Patricia’s preLight discussed optogenetic manipulation in locust brains, while moving a bit away from neuronal function, Srivats Venkataramanan preLighted a study that used an optogenetic tool to control the formation of stress granules.

Finally, preLighters’ coverage of reproducibility (see Carmen’s earlier post and Reid Alderson’s preLight from this month) and flaws in experiments (see Fabio Liberante’s post on false detection of circular RNAs from RNA-seq) signalled the importance of preprints in rapid dissemination of studies which are extremely valuable for the community, but often more difficult to get published in journals.

Considering reading something more out of your scope? Then explore the preLights website, where you can also find out about microbes in a Mars-analogue environment, Mycobacteria on your showerheads, or drug repurposing to combat brain-eating amoebae.

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https://www.grimes-lab.com

Postdoctoral positions are available in the laboratory of Daniel T. Grimes, Institute of Molecular Biology and Department of Biology at the University of Oregon. The laboratory focuses on symmetry in the biological world.

We are a new lab opening in January 2019. We want to understand how some features of vertebrate bodies (like limbs and the skeleton) develop with left-right symmetry, while others (like the heart, liver, and gut) develop with striking left-right asymmetries. 

We use zebrafish to address these fundamental questions of developmental biology. Our main techniques include genome editing, confocal and micro-CT imaging, genetics and genomics, single-cell sequencing, and more. Current projects include: understanding how mechanical flow signals are generated and sensed by cilia in left-right patterning, investigating Polycystin signal transduction, elucidating mechanisms by which the spine remains straight during growth. We also model human diseases of aberrant (a)symmetry including heterotaxia, scoliosis, and primary ciliary dyskinesia.

We offer the opportunity to work closely with the PI, and to help build and shape lab culture. Your career and manuscripts are important; we are committed to the mentorship and long-term success of our lab members. We also offer a high quality training in zebrafish development and genetics. Zebrafish research was founded at the University of Oregon. As such, we share a large state-of-the-art aquarium with labs that use the zebrafish for diverse science. There is no better place to work with zebrafish!

The Biology Department at the University of Oregon offers an exceptional environment and a broad range of research, great core facilities and close ties to the Knight Campus, a new $1-billion initiative that brings together engineers, biologists and computational scientists to address pressing biological questions.

We are looking for ambitious candidates who hold (or be due to complete) a relevant PhD and have evidence of excellent scholarship. Experience with confocal imaging is a bonus but previous experience with zebrafish is not essential. Candidates should be able to work independently, to implement new technologies, and be productive and collaborative.

The position is fully funded but the candidate will be expected to seek external funding opportunities during their tenure.

The candidate should email (dtgrimes@princeton.edu) with a cover letter describing their career goals, previous experience, scientific interests and reasons for applying, along with a CV. Informal inquiries are welcome (see grimes-lab.com/join-us-1).

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• Develop tailored bioinformatics approaches and provide advice and assistance to DanStem and CPR researchers in their analyses, focusing in particular on the planning, processing, analysis and publication of transcriptomic and epigenomic next-generation sequencing data.
• Prepare pipelines and scripts for use by DanStem and CPR researchers.
• Provide training and design new approaches to analysis.
• Cooperate and collaborate with DanStem and CPR researchers.
• Cooperate with platform personnel and contribute to platform management.
• Network actively with the global bioinformatics community and stay up-to-date with current trends in the field.

• Fluent programming skills in R/Bioconductor.
• Experience with next generation sequencing data.
• Strong statistics skills.
• Knowledge of UNIX-like operating system.
• Experience in working with biologists and an understanding of molecular biology and genomics.
• Excellent oral and written communication skills and fluency in English.
• Successful experiences in project management, including multi-tasking.
• A service-minded and team player attitude.

• State-of-the-art computational infrastructure for data analysis, including access to cluster computing at the Danish National Supercomputer (Computerome) and excellent IT and systems administration support.
• Stimulating and multifaceted research environment of high scientific and societal impact.
• Possibility for continued education and training, especially to develop further as an expert in single cell bioinformatics.

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When reading about co-evolution of prey and predators, I stumbled across a cute new plot type: a half boxplot, half dot plot to show data distributions.

Wilson used this plot to simultaneously visualize summaries about their data (center, spread) and the actual data points. This allows us, the audience, to learn a lot about their results. That cheetahs are maybe binomially distributed and have outliers, or that zebras show a curious clustering.

Your quick guide to distribution plots:

The half-and-half, aka dox-plot (a friend), led me to explore which visuals are commonly used for showing distributions.

Raw data

To show how the raw data is distributed, we simply use dots or bars (as in barcode plots). When there are overlapping data points, we can use transparency or “jittering”. Jittering is distributing the data points in a given area, for increased clarity: the y-position remains the same, the x- position becomes random.

Summarizing the data: center and spread

Often, we are interested in summarizing statistics to judge and compare data. By convention the center (median) is indicated with a horizontal bar and the spread (variance, standard deviation) with vertical whiskers. Common plot types for this are the “star-wars rebel fighter”, the dot plot and boxplot. Bar plot used to be widely used, but are now banned by most journals for concealing most relevant information, so they are here only for completeness (see previous post). Very often these days I see boxplots that are overlaid with the data points – this works really well for up to 100 data points and is easy to implement with most software.

Show shape and unusual features

For normally distributed data, the center and the spread are highly informative. However, in life science we often have bimodal distributions, clusters, or gaps. Then boxplots become very insufficient and might even conceal interesting aspects (if not outright be misleading).

For faithfully showing distributions, histograms have a long history. Here, one has to be very careful with choosing bin sizes: too large or too small bins can greatly distort the histogram shape and result in a misleading chart. Choosing bin sizes is a science in itself, for details see wikipedia – but basically, it again depends on the data shape and sampling depth.

An alternative to histograms are density plots. Density plots show how data are distributed. They become very useful for large data sets. For large data sets individual points can’t be visualized anymore and the eye can’t anymore judge spread intuitively. A rather recent but so far “happy marriage” is the violin plot. Violin plots are a fusion of the boxplot and its summary statistics, with the density/shape of the data (Hintze and Nelson, 1998 doi: 10.1080/00031305.1998.10480559).

Further reading in a recent preLIGHT by Gautam Dey!

Pro and cons of different plot types:

All figures in one for teaching:

(11 votes)

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In LM Escudero´s group, we like developmental biology, mathematical biology and computational biology. We try to be imaginative and get inspiration from simple things… such as a toilet paper roll. Using this tool (and some computers), we claim that we have described a novel geometrical shape… You will be wondering… how do you do that??? You are going to find here: the story behind the discovery of “scutoids”.

First, you need a multidisciplinary group distributed between two buildings: “the wet lab”, with flies and their epithelial tissues, at Institute of Biomedicine of Seville, IBiS (Fig. 1); and the “computer lab” with… well… with a bit of everything (it does not fit in a simple picture… so, see Video 1) at the Faculty of Biology of University of Seville. It is ideal if the two buildings are separated by 1 km, so you can exercise everyday walking between the two labs to interact with the members of each branch.

Fig. 1. A. Tagua (at right, masterful segmenter and drosophilist) and C. Gordillo looking for more scutoids in the fly lab.

Video 1. The computational lab (that is wet if you open the tap) showing P. Gómez-Gálvez and P. Vicente-Munuera, the two first authors, working hard and listening good music.

Very important! You need also an office where both computational and Drosophila worlds can fit together (Fig. 2).

Fig. 2. Very trendy office. Please note the a/c controller, essential if you are based in Seville.

Then, you need a problem to think about while you are walking between the two labs… As animals develop, the initial simple planar epithelia of embryos must be sculpted into complex three-dimensional tissues. These cells pack together tightly. To accommodate the curving that occurs during embryonic development, it has been assumed that epithelial cells adopt either columnar or bottle-like shapes.

We thought that a very logic way to approach the problem of how curved epithelia pack in 3D was to design a computer model with the shape of a toilet paper roll (Fig. 3). The results we saw were weird. Our model predicted that, as the curvature of the tissue increases, columns and bottle-shapes were not the only forms that cells may developed. To our surprise, the discovered geometric solid didn’t even have a name in math! We were happy. Really happy. One does not normally have the opportunity to name a shape. We chose the name scutoid.

Fig. 3. The toilet paper: reality versus model.

The undescribed shape was characterised by having at least a vertex in the lateral surface (Fig. 4). This vertex confers an interesting property to the scutoid: when cells adopt this shape, they can have different neighbours in the upper and bottom surfaces (apical and basal in biology). This is exclusive of scutoids and cannot be done with the “prism”, “trunk” (frusta) or “prismatoid” shapes.

Fig. 4. The brotherhood of the cellular geometry: evolution from prisms to the undescribed scutoid geometrical shapes.

All this was amazing… but we needed help to completely understand the problem. We needed lots of collaborators (we are 16 authors), starting with Dr Grima and Dr Márquez, real mathematicians that helped us with the formal aspects of the toilet paper model.

To verify the model’s predictions, the group investigated the three-dimensional packing of different tissues in different animals. The experimental data, using Drosophila salivary glands, confirmed that epithelial cells adopted shapes and three-dimensional packing motifs similar to the ones predicted by the computational model.  This important validation required more collaborators: Dr Sotillos and Dr Martín-Bermudo groups (CABD, CSIC/JA/UPO, Seville) and Dr Cavodeassi (St. George’s University of London) all with biological background but working in other types of epithelia.

And then, the biophysics. We joined efforts with the lab of Dr Javier Buceta (Lehigh University), to study the role of scutoids in tissue architecture from a mechanical point of view. We argue that the scutoids stabilise the three-dimensional packing and make it energetically efficient. Our conclusion was that we have uncovered nature’s solution to achieving efficient epithelial bending. This was really cool… but not so much as the summary that Dr Buceta has prepared for you if you do not have time to read the whole paper… this is art and science in a single sheet (Fig. 5, click to see at full resolution).

Thanks for reading!!!

If you need more information, you can check our paper: Scutoids are a geometrical solution to three-dimensional packing of epithelia. Gómez-Gálvez P, Vicente-Munuera P, Tagua A, Forja C, Castro AM, Letrán M, Valencia-Expósito A, Grima C, Bermúdez-Gallardo M, Serrano-Pérez-Higueras Ó, Cavodeassi F, Sotillos S, Martín-Bermudo MD, Márquez A, Buceta J, Escudero LM. Nat Commun. 2018 Jul 27;9(1):2960. doi: 10.1038/s41467-018-05376-1.

Here you have the link.

(7 votes)

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An opportunity is available for a Postdoctoral position in the Cox Lab at the Peter MacCallum Cancer Centre in Melbourne, Australia. The position requires a highly motivated and enthusiastic postdoctoral scientist to investigate how metabolic reprogramming contributes to liver regeneration and cancer using zebrafish (Danio rerio) as a model organism. The successful candidate should hold a PhD in biochemistry, molecular biology, developmental biology or a related discipline. The person will be expected to conduct rigorous, valid and ethical research both independently and as part of the research team. The person will be expected to supervise undergraduate and postgraduate students, and technical staff.  For more information on recent publications and projects running in the Cox laboratory refer to: https://www.petermac.org/research/labs/andrew-cox

More information about the details of the application process can be found on the website (https://petermac.mercury.com.au/ViewPosition.aspx?id=/GrfG/WmYM8=&jbc=ere), or contact via email (Andrew.Cox@petermac.org).

(1 votes)

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Alexandra Joyner and Alberto Roselló-Díez tell us the story behind their recent paper in PLoS Biology¹.

Today we have tried a new experiment (we cannot help it). Instead of elaborating too much on the scientific aspect of our recent paper about the control of organ growth in mammals¹, we decided to tell the personal aspect of it. It’s a story about perseverance, collaboration and serendipity, and we hope it will be especially encouraging for young trainees striving to find their niche. Here we go:

Alex: One of the big mysteries in developmental biology is how the robustness of our body plans is achieved, perhaps best exemplified by the near equal lengths of our left and right limbs. Alberto started devising a new experimental approach to this fascinating question with a comprehensive review written by Cliff Tabin’s group in hand², but there appeared to be limited investigations on which to base his work.

Alberto: Indeed! That’s how this story started. When I was finishing my PhD in Miguel Torres’s lab in Spain, Miguel had a great idea for a departmental retreat. Everyone had to think about a wild project, a particularly difficult question they would like to address if they had the resources to do it. Long story short, I presented my idea that while limb growth was mainly autonomous, perhaps the fine-tuning of limb size involved some kind of limb-limb crosstalk. My proposed approach involved complicated ways of achieving unilateral growth manipulation in a variety of vertebrate and invertebrate models, in order to study the potential recovery of symmetry. Frankly, my approaches were hardly viable, but then I got a suggestion from a brilliant colleague who was also doing his PhD at the time, my friend Juanma González Rosa. He suggested an elegant way of achieving unilateral manipulation: to use mouse Cre lines driven by the regulatory region of some of the known left-specific genes that operate during early development. And that was the seed of the project. We came back to our labs, and while I was writing my thesis, a review article from the great developmental biologist Lewis Wolpert revisited the topic of limb symmetry as one of the remaining mysteries in developmental biology³. This obviously bolstered my interest in the topic, and as soon as I graduated and submitted the revisions of one of our papers, I spent the following 3-4 weeks reading, formulating hypotheses and writing an experimental plan. Finding the left-specific Cre line was no easy task, because the people who had generated them were not interested in limb development, and their papers did not mention the expression in the limbs. Fortunately, they were so kind as to respond to the out-of-the-blue request of a young PhD, and even sent me pictures of staining in the limbs (thank you again, Drs Martin, Shiratori and Hamada). And that’s how I found the Pitx2-Cre driver that would be key for our studies⁴, and that Alex would later import from Japan even before I arrived to the lab. However, the Cre was expressed in the very early embryo, marking the left lateral late mesoderm from that stage onwards, so other genetic components were necessary to enable manipulation specifically at the critical period of limb length establishment. According to Wolpert, the problem had to be addressed at the level of the growth plate of the elongating bones, so I devised an intersectional strategy to restrict any cellular modification of interest to the growth plates of the left long bones. The new transgene I designed depended on the coincidence of two drivers, Cre and (r)tTA, to activate expression of a growth altering protein in a given cell population. The idea became to restrict expression of the protein using Pitx2-Cre and a cartilage specific rtTA in combination with the new transgene. This strategy would provide not only exquisite spatial control, but also inducibility and reversibility, as rtTA requires Doxycycline to be active.

It was March 2011 when I went back to the lab after my post-PhD reflection period. I was full of energy and determined to find a lab in which I could develop my idea. My hypothesis at the time was that the nervous system could be involved in communicating the left with the right limb, and maybe even comparing their lengths, so I ideally had to find a lab with expertise in limb and nervous system development, and the resources and experience to generate the complex genetic models the project required. I thought it would be difficult, but in a curious twist of fate, the opportunity quite literally presented itself when Alex visited our institute to present a fascinating story about nerve-released sonic hedgehog (SHH) having an important role in the fate of the stem cells of the hair bulge⁵. Alex had published several studies on limb development before, was an expert in nervous system development and function (especially the cerebellum), and was developing several models of recovery after organ injury, so Miguel kindly introduced me to her during their allocated meeting time.

Alex: It was unusual to have a recently graduated student on the schedule of meeting Miguel sent me, but then Alberto is an unusual scientist! After telling me about his exciting PhD limb research, Alberto proceeded to tell me about what he wanted to do for his postdoctoral research. Loving a genetic challenging in mice, I was very taken by the elegant approach Alberto had come up with to study one of the most basic and fascinating questions in development – how symmetry is attained. He visited our lab at MSKCC in New York soon after, and there was unanimous excitement to have Alberto join our group and start a new area of research.

Alberto: The excitement was reciprocal! I chose to join the lab because I perceived an atmosphere of constructive feedback and criticism during my interview, and that’s exactly what I needed for this type of project that ventured in uncharted waters. But, to be honest, things were everything but easy when I moved to New York in June 2012. First, generating the mouse lines would be quite resource demanding, so we had to choose our experiments carefully. Second, one of my experiments was more complicated than expected. I decided to try to characterise with high precision the left-right limb asymmetry at several stages of mouse development while I was building the transgene constructs, and also correlate it with the presence or absence of innervation near the growth plate, and determine how it was affected after pharmacological manipulation of candidate neurotransmitter response pathways. The precision required made the process extremely painstaking, and the results of the pharmacological manipulation were inconclusive, so when the first mice of my intersectional model were available, roughly a year after I started in Alex’s lab, we decided I would fully dedicate my time to characterise the brand-new mice. A third challenge then arose, the double-conditional allele (which I eagerly called Dragon, standing for Dox-controlled and Recombinase-Activated Gene-OverexpressiON) was unexpectedly not active in most of the cells that were supposed to express the transgene, seemingly pushing us back to square 1.

Alex: Yes, the first year was quite difficult for us but proved to me that Alberto had the resourcefulness to make the project work. Fortunately, in another twist of fate, I got in contact with Dr. Hongkui Zeng whom I had met at the Allen Institute and learned that the Tet-responsive element becomes easily inactivated in the locus we used, but that she had identified an intergenic region after extensive screening that supports (r)tTA-driven gene expression⁶. She had named the locus TIGhtly-REgulated or Tigre, the Spanish word for tiger (quite fitting, as Alberto is from Spain). Intriguingly, Dr. Zeng’s group was also working on a Cre- and (r)tTA-dependent intersectional gain-of-function strategy⁷, so we teamed up with them to modify their system and adapt it to our needs, and they generously offered to perform the embryonic stem cell manipulations.

Alberto: Yes, we quite literally found the crouching Tiger for our hidden Dragon! This collaboration was undoubtedly key to finishing the project in a timely manner, but would still require half a year to make the mice. Therefore, while our Tigre-Dragon mice were being generated, we also developed a fast way of using Pitx2-Cre to induce unilateral cell death in the limb, with which we found that the tissues surrounding the long bones can modulate bone growth⁸. This result made us wonder if there was communication between other tissues and the bones, and if it could work in the opposite direction. Spoiler alert: it does!

Alex: That’s right! When Alberto was finally able to analyse our Tigre-Dragon mice, we were surprised to see that the mice did not show any obvious asymmetry despite having more than 50% of their chondrocytes arrested in the left hindlimb. Long story short, we found that two compensatory mechanisms accounted for symmetry maintenance: local hyper-proliferation of the undamaged cells, almost perfectly tuned to make up for the arrested cells, and more surprisingly, a systemic growth reduction that affected all limbs and the whole embryo in general. Although the systemic effect was subtle, we were quite excited about it, because to our knowledge it was the first time that this phenomenon was shown in an organism other than developing insects.

Alberto: Yes, that was a very positive surprise. I feel lucky that we discovered new fundamental mechanisms of growth regulation right when I was going to start my own lab. We would have liked to uncover more about the molecular mechanisms underlying our observations (and so did the reviewers), but to be fair that took over 30 years in Drosophila, so I guess it will be a long and difficult quest!

Alex: Alberto is starting the next stage of his career as an independent investigator with a wealth of interesting results and transgenic mice as a strong foundation for a lifetime’s worth of exciting projects for his lab. His perseverance has paid off in spades, along with his careful evaluation of every experimental design and result, an openness to collaborate, a thirst for charting new ground and of course a little bit of luck.

Alberto: Don’t forget the mentor that kept me on track!

References

1          Rosello-Diez, A., Madisen, L., Bastide, S., Zeng, H. & Joyner, A. L. Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice. PLoS Biol 16, e2005086, doi:10.1371/journal.pbio.2005086 (2018).

2          Allard, P. & Tabin, C. J. Achieving bilateral symmetry during vertebrate limb development. Semin Cell Dev Biol 20, 479-484, doi:10.1016/j.semcdb.2008.10.011 (2009).

3          Wolpert, L. Arms and the man: the problem of symmetric growth. PLoS Biol 8, doi:10.1371/journal.pbio.1000477 (2010).

4          Shiratori, H., Yashiro, K., Shen, M. M. & Hamada, H. Conserved regulation and role of Pitx2 in situs-specific morphogenesis of visceral organs. Development 133, 3015-3025, doi:10.1242/dev.02470 (2006).

5          Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A. & Joyner, A. L. Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells. Cell Stem Cell 8, 552-565, doi:10.1016/j.stem.2011.02.021 (2011).

6          Zeng, H. et al. An inducible and reversible mouse genetic rescue system. PLoS Genet 4, e1000069, doi:10.1371/journal.pgen.1000069 (2008).

7          Madisen, L. et al. Transgenic Mice for Intersectional Targeting of Neural Sensors and Effectors with High Specificity and Performance. Neuron 85, 942-958, doi:10.1016/j.neuron.2015.02.022 (2015).

8          Rosello-Diez, A., Stephen, D. & Joyner, A. L. Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth. Elife 6, doi:10.7554/eLife.27210 (2017).

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