An emerging trend in developmental biology focuses on the role of cell adhesion in modulating tissue morphogenesis. Spectacular advances have shed light on how modulation of adhesion between equivalent cells orchestrates the acquisition of forms. However, how interactions at complex interfaces between distinct neighboring cell types influence tissue growth remains to be elucidated. In a recent paper, staring mostly at a flat muscle that spreads subcutaneously and its partner motor neurons, I investigated how neuromuscular morphogenesis involves complementary activities of the Fat1 Cadherin in three interdependent cell types – myogenic cells, motor neurons, and the connective tissue through which muscles spreads.

Tissue-specific activities of the Fat1 Cadherin cooperate to control neuromuscular morphogenesis Françoise Helmbacher, PLoS Biology, May 16, 2018 | doi: 10.1371/journal.pbio.2004734 | PMID : 29768404

The story started as I was transitioning from being a post-doc in the lab of Rüdiger Klein in Munich, to establishing my own group in Marseilles. At the time, I was interested in events of communication between motor neuron (MN) populations, whereby a pool of MNs influences the specification of neighbor MNs. I was looking for molecules mediating such function, and Fat1 originally came up as one of my candidate genes. Although Fat1 met many of my criteria, it was initially considered a low priority candidate for the specific question I was asking (which I won’t discuss here). However, there were also good reasons for deciding to work on Fat1, aside from the main project of my freshly established lab: (1) Fat1 was expressed in my favorite MN population (Figure 1), a group of neurons recognizable by the transcription factor they expressed, which innervates a fascinating muscle, the Cutaneous Maximus (CM), (2) there were mouse mutants available (which by chance happened to be already in my former lab), and (3) through pioneer experiments with these mutants, I had gathered preliminary data indicating that Fat1 disruption resulted in defective specification of the CM motor pool and in defective axonal arborization within the target muscle – the phenotype I was expecting.

Whatever the underlying gene function, starting a project with a robust in vivo phenotype guaranteed that an interesting story would emerge from it, even though this was not my main project. For this reason, I kept this as my own side project. I had no hypothesis, but it felt like starting a classical forward genetics approach, with the advantage that I already knew the gene identity. This is also when the field of planar cell polarity (PCP) started blooming, mostly through work in Drosophila. The Drosophila Fat Cadherin, initially regarded as a putative tumor suppressor, had been attributed the mysterious function of propagating information on cell polarity within the plane of epithelia across long distances. I was fascinated by these studies, and curious to ask if MNs could propagate some polarity information to their neighbors too. Well, the story took a completely different path!

The two first key results that I had obtained were that 1) axons of the motor neuron pool innervating my favorite muscle, the CM, were clearly shorter in Fat1-deficient embryos, when all other nerves appeared unaffected; and 2) expression of one of the markers specifically characterizing this MN population was drastically reduced. Given the fact that at that time, I had selected Fat1 because of its selective expression in subsets of MNs encompassing the CM-innervating pool (marked by Etv4 expression, Figure 1B), these data were compatible with the simplistic and straight forward hypothesis of a cell autonomous activity of Fat1 in MNs. Thus, it looked as if this project would be devoted to finding out whether specification or axonal growth was affected first, and to tackle the mechanism, keeping PCP principles in mind, possibly using a combination of genetics, in vitro, and in vitro approaches.

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Prior to jumping to the conclusion that Fat1 indeed controlled MN specification and axon patterning cell autonomously, it was essential to determine whether the target muscle, the CM, had developed normally in Fat1 deficient embryos. Any muscle defect might indeed represent a potential cause – or consequence – of these MN phenotypes, or might even indicate an independent phenotype. With my team, we therefore performed the experiment that changed the course of this story. We visualized muscle development by whole mount in situ hybridization to detect MyoD RNA in myogenic progenitors. To my surprise, this experiment turned out not to yield the anticipated negative result. Instead, Fat1-deficient embryos exhibited a series of very robust muscle defects affecting morphogenesis of subsets of muscles in the face and shoulder, phenotypes that we described in our PLoS Genetics paper in 2013 [1].

When we first saw these muscle phenotypes, there was no reason a priori to dissociate our findings on the altered development of a selective group of muscles and associated defects in the corresponding motor neuron population. However, as I was reflecting on the possible significance of these results in terms of health and biomedical implications, and as, in these early days, I was also in a phase of heavy grant writing, I wondered whether these phenotypes could possibly correspond to symptoms of a rare human pathology that would not have been assigned yet any genetic culprit. I thus started exploring the human genome to see where the human FAT1 gene was located, and discovered the OMIM database (Online Mendelian Inheritance in Man). It didn’t take long to realize that the 4q35 region where FAT1 was localized was also known for being subject to chromosomal abnormalities associated with a human muscular dystrophy called Facioscapulohumeral dystrophy (FSHD) [2]. I was really amazed to learn that the muscle wasting symptoms in this disease affected a highly regionalized group of muscles, with a topography reminiscent of what we had seen in Fat1-deficient embryos. I was also intrigued by the fact that additional non-muscular symptoms of this disease, auditory and vascular retinal abnormalities, were emerging as hallmarks of defective planar cell polarity.

At that time, this was a completely unexpected finding. First, the FAT1 gene was relatively far from the true FSHD locus (where changes in the D4Z4 macrosatellite repeat array were considered to be the primary event linked to FSHD). This distance (~3Mb) was sufficiently large to guarantee unambiguous genetic mapping of a disease locus, but was small enough for a genetic linkage, and for FAT1 regulation to be affected secondarily to chromatin architecture changes occurring as a result of the FSHD abnormalities (although the concept of topologically associated domains (TADs) was not as popular then as it has become). Even though a possible deregulation/contribution of neighboring genes had been considered, FAT1 had largely been ignored or dismissed. Nothing in the literature at that time indicated any likely involvement of Fat1 in muscle biology, whereas Fat1 disruption in mice had instead been reported to result in severe kidney abnormalities [3], the latter not being part of the panoply of FSHD symptoms.

This is when I decided to radically change the topic of the lab. I therefore sought to identify potential collaborators who would help approaching the human disease link. I was lucky to turn to Nicolas Levy, who headed a department of medical genetics at La Timone Hospital in Marseilles, and was really enthusiastic about this new idea. With his help, we were later joined by Marc Bartoli, who started a group on translational myology in Marseilles, and by Julie Dumonceaux (now at UCL, London). To make a long story short, this fruitful collaboration has led to date to 3 papers exploring the possible link between FAT1 dysfunction and FSHD [1, 4, 5]. First, my team provided compelling data indicating that disrupting Fat1 in mice caused muscular and non-muscular phenotypes with an FSHD-like topography [1]. Second, we detected lowered FAT1 RNA and protein levels in affected muscles from FSHD patients with classical diagnosis, at fetal [1] and adult [5] stages. Third, we identified FSHD-like patients carrying pathogenic FAT1 variants, in absence of the traditional FSHD-causal abnormalities [1, 4]. These FAT1 variants included single nucleotide variants affecting RNA splicing or amino-acid structure [4], but also copy number variants deleting a putative cis-regulatory element in the FAT1 locus [1]. Such deletions had the potential to cause tissue-specific depletion of FAT1 expression, thus offering a logical explanation for why some phenotypes resulting from Fat1 deficiency in mice were not part of the clinical picture of FSHD symptoms in human patients.

Because the FAT1/FSHD link was so unanticipated and provocative, the road to our first publication turned into a long and emotionally agitated roller coaster ride. As a result, this paper was submitted multiple times, and went through several rounds of revisions for several journals, until it proudly landed in PLoS Genetics in 2013 [1]. As in the meantime we had produced mice with a conditional Fat1 allele, we were able to show in this first study that Fat1 deletion in the myogenic lineage reproduced in part the aberrant dispersion of myogenic cells in the limb observed in constitutive knockouts [1]. I already knew however that this only gave a partial account of the complexity of Fat1 functions. I just figured that it was unnecessarily challenging to simultaneously report the identification of a potential new genetic actor in a human pathology (even when only considered a modifier gene) and the complexity of its biology. This deserved to be studied as a developmental question per se, independently of its relevance for this disease, which somehow is still a controversial issue, despite our genetic evidence obtained with the Levy/Bartoli groups.

To approach the complexity of the Fat1-driven control of neuromuscular morphogenetic, the new study I have just published [6] gives a central position to one emblematic couple: the Cutaneous Maximus (CM) muscle, and its cognate MN pool. This MN pool can be recognized through its expression of the transcription factor Etv4, which is essential to specify the cell body position and dendritic architecture of these MNs [7]. The CM muscle emerges from the bulk of forelimb levels myogenic progenitors that undergo long range migration. Unlike other limb muscles, the CM muscle progenitors avoid entering the limb bud. Instead, they undergo a sharp caudal turn and start their subcutaneous progression. Throughout this migration phase, CM progenitors secrete Glial-cell-derived-neurotrophic-factor (GDNF), previously known for its neurotrophic activity and for its roles in motor axon guidance and in CM motor pool specification (as it induces Etv4 expression) [8]. Thus, by way of GDNF production, there is an intimate link between muscle progression and partner MN development. Among the muscles vulnerable to Fat1 deficiency, the CM muscle was also the easiest to approach quantitatively, by measuring the orientation of individual progenitor cells in our first study [1], and with simple area measurements in the new study [6]. Such an analysis illustrates how Fat1 disruption robustly interferes with expansion of the area occupied by Gdnf-expressing myogenic progenitors, by differentiating muscle fibers (Figure 2), as well as by axons of the cognate MNs.

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Given my initial selection of Fat1 for its expression in the pool of MNs innervating the CM muscle, this was a region of non-myogenic Fat1 expression of obvious relevance to the phenotype. I was really excited by the possibility that Fat1 expression in the CM pool of MNs might represent a key driver of CM subcutaneous morphogenesis. Quantifications of morphometric parameters confirmed that MN-specific Fat1 ablation interfered with axonal growth and specification of the CM-innervating MNs. In addition, this also non-autonomously slows the subcutaneous progression of CM myogenic progenitors, hence expansion of the GDNF producing domain, without altering the rate of myogenic differentiation. However, this role turned out to be way more modest than what I initially anticipated, with an effect size considerably smaller than the phenotypes of constitutive mutants. This implied that another site of Fat1 activity had to be making a major contribution to neuromuscular morphogenesis.

An unanticipated third site of Fat1 expression that turned out to play a major role is the loose connective tissue (CT) that surrounds muscles. During their collective progression, myogenic progenitors and motor axons explore such connective tissues and select their migration trajectory. The CM progenitors in particular encounter an increasing gradient of Fat1 expression in this tissue. Furthermore, Fat1 expression appears fully preserved in mouse mutants lacking migratory muscles (Met or Pax3 mutants, in which myoblast migration is abrogated), indicating that the main site of peripheral Fat1 expression represented non-myogenic cells. To establish the contribution of CT-Fat1 to muscle morphogenesis, Fat1 activity was ablated in the lateral plate derived mesenchyme, in a domain (driven by Prx1-cre) including the limb and flank connective tissues encompassing a large part of the territory through which the CM migrates. This led to severe non-cell autonomous disruption not only of the progression of CM progenitors and subsequent myofiber elongation, but also of motor axon elongation across the same territory, and of acquisition of CM fate characteristics. Similar results were obtained with a Cre line allowing stage-controlled Tamoxifen-inducible Fat1 excision in Pdgfrα-expressing connective tissue at the time of myoblast migration, with a lesser effect size matching the lower excision efficiency. These data identify connective tissue as a tissue type in which Fat1 activity is mandatory for neuromuscular morphogenesis. They imply that Fat1 signaling activity in CT cells is necessary for them to emit proper signals promoting collective myoblast migration, myogenic differentiation, motor axon growth, and MN specification, with the exact mechanisms underlying these cell interactions remaining to be explored.

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In conclusion, looking back on how the story started, the loop has been closed by the identification of gene functions that underlie the phenotype which initially attracted my attention. Although my intuition that this had to be connected to the neuronal expression of this atypical Cadherin was not completely wrong after all, conditional mutagenesis nevertheless revealed that this function was modest in regard of the unexpected predominant activity in a cell type – connective tissue – that I had initially not considered. I have learned many lessons along the way of this unusual story. Above all, I learned not to fall in love with a favorite hypothesis but instead to be open to what the facts tell us: when the results of an experiment are the opposite of what you expect, this pushes you to think outside the box, and challenges you to remain unbiased in conceiving experimental plans and analyzing data. It is now time to switch scale, and start exploring what happens at the connective tissue muscle interface from a cell biologist’s point of view.

References

1. Caruso, N., et al., Deregulation of the protocadherin gene FAT1 alters muscle shapes: implications for the pathogenesis of facioscapulohumeral dystrophy. PLoS Genet, 2013. 9(6): p. e1003550.
2. DeSimone, A.M., et al., Facioscapulohumeral Muscular Dystrophy. Compr Physiol, 2017. 7(4): p. 1229-1279.
3. Ciani, L., et al., Mice lacking the giant protocadherin mFAT1 exhibit renal slit junction abnormalities and a partially penetrant cyclopia and anophthalmia phenotype. Mol Cell Biol, 2003. 23(10): p. 3575-82.
4. Puppo, F., et al., Identification of variants in the 4q35 gene FAT1 in patients with a facioscapulohumeral dystrophy-like phenotype. Hum Mutat, 2015. 36(4): p. 443-53.
5. Mariot, V., et al., Correlation between low FAT1 expression and early affected muscle in facioscapulohumeral muscular dystrophy. Ann Neurol, 2015. 78(3): p. 387-400.
6. Helmbacher, F., Tissue-specific activities of the Fat1 cadherin cooperate to control neuromuscular morphogenesis. PLoS Biol, 2018. 16(5): p. e2004734.
7. Livet, J., et al., ETS gene Pea3 controls the central position and terminal arborization of specific motor neuron pools. Neuron, 2002. 35(5): p. 877-92.
8. Haase, G., et al., GDNF acts through PEA3 to regulate cell body positioning and muscle innervation of specific motor neuron pools. Neuron, 2002. 35(5): p. 893-905.

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We recently published a manuscript in Cell that describes a method to image transcription factor concentration dynamics in real time, in living embryos, using a nanobody-based protein tag that we call the “LlamaTag.” We were particularly excited about these investigations because this new technology overcomes a major technical obstacle to understanding how gene-expression dynamics are regulated in development, which has held back the field for decades.

From Snapshots of Dead Embryos to Movies

Remember when you were first starting to study developmental biology? In one of your lab sections, you probably participated in that classic teaching activity where you watched the cephalic furrow form in Drosophila embryos. Maybe you were one of those students struggling to move embryos with an eyelash glued to a toothpick, and the whole time you were thinking, “I need to hurry up—this thing is gastrulating while I’m messing around here!” There’s a strong contrast between watching development unfold before your eyes and scrutinizing snapshots of FISH data from dead, fixed embryos. This contrast hinders more than budding scientific excitement: how can we truly understand dynamic processes by analyzing static data?

I was particularly struck by this conundrum as a postdoctoral researcher as my mind kept replaying a video of a gastrulating embryo. How could I bridge this gap so that developmental biologists could accurately model—and ultimately construct—dynamic systems as complex as embryos? At the simplest level, we would need to know the input and the output of each genetic circuit as it changes throughout development in order to deduce the logic gates at play. I tackled the output end first: as a postdoc, I developed a technique to quantify transcription in flies at the single-cell level by tagging a gene’s nascent mRNA molecules with fluorescent proteins. How could we accomplish the same feat with the DNA-protein interactions that constitute the input to these genetic circuits? I decided that solving this challenge would be a major driver of the research in my own lab. Jacques Bothma was equally excited about this challenge and decided to join the project as a postdoctoral fellow.

Typically, one would quantify the concentration of a transcription factor by fusing it to a fluorescent protein. However, in flies, worms, fish, and frogs, these fluorescent take more than 40 minutes before they mature and become fluorescent. This delay is actually a major problem: the activators and repressors that drive development often exist for less than 10 minutes before they are degraded. By the time the fluorescent proteins actually became fluorescent, the action they were supposed to report on is already over!

Jacques had a great idea: instead of relying on the synthesis of fluorescent proteins, why not use the localization of already matured fluorescent proteins to report gene expression? Our Cell paper describes the engineering and implementation of this idea with LlamaTags, a technology that enables quantitation of the dynamics of transcription-factor activity without limits from the slow maturation of traditional fluorescent fusion proteins. Instead of these fusions, we employed nanobodies, which are small, highly specific, single-domain antibodies that are raised in llamas (hence LlamaTags). We fused a transcription factor of interest (we started with Hunchback) to a nanobody raised against eGFP, and expressed the construct from the endogenous locus for that transcription factor. Importantly, the embryo was engineered to contain maternally deposited eGFP, which means that eGFP is already mature before the transcription factor of interest is expressed (no waiting around for the fusion to mature!). When the transcription factor-nanobody is translated, it binds cytoplasmic eGFP within seconds, yielding a quantitative increase in nuclear fluorescence when the transcription factor moves to the nucleus to perform its regulatory function. LlamaTags therefore deliver a direct readout of the instantaneous transcription-factor concentration in a given nucleus.

Through a variety of experiments, we showed that LlamaTags serve as specific and faithful reporters of the endogenous concentration dynamics of transcription factors during development, thus capturing the input pertinent to these circuits. So, we finally had the two pieces needed to solve the puzzle of measuring input-output functions. LlamaTags made it possible to measure the input concentration of transcription factors in individual nuclei, while MS2 revealed the transcriptional activity of specific genes to these input levels.

By fusing a LlamaTag to Fushi-Tarazu (Ftz), we obtained measurements of its nuclear concentration during development, from which we extracted the in vivo degradation rate of the construct. We were pleased when our data revealed rapid fluctuations in Ftz concentration—consistent with previous reports of protein bursts that likely arise from stochastic fluctuations in mRNA concentrations. We nailed down this relationship, and uncovered exciting evidence of inter-nuclear communication within the embryo. This coupling could be a major driver of the sharp boundaries that dictate development of the fly embryo. However, our greatest satisfaction came from simultaneously visualizing input transcription-factor activity and output transcription, at the single-cell level, in real time, as stripe 2 of eve was laid down in live embryos:

A New “Microscope” for the Central Dogma in Development

Our recent work establishes a powerful pair of technologies—labeling transcription with MS2 and labeling DNA-protein interactions with LlamaTags—that together constitute a “microscope” for visualizing and interrogating the activity of genetic circuits as they function. We envision that LlamaTags can be applied to quantitatively measure the flow of information along regulatory networks in any multicellular organism that is amenable to transgenic control and live imaging. LlamaTags literally light up the central dogma, in real time, as development unfolds. Importantly, we can do more than map input to output: we can quantitate the dynamics of these connections over time. That’s the difference between trying to create a stop-motion movie where you need a new actor for each frame (a dead embryo), and actually observing development unfold in real time.

Physical Biology of Living Embryos

Like many of you, we believe that in order to construct a system, we should first understand it in quantitative detail. We view this as a call for reaching a “predictive understanding” of development, through which we can calculate developmental outcomes from knowledge of the concentrations of input transcription factors and the DNA regulatory sequence. To reach this predictive understanding, we believe that a powerful dialogue is necessary in which theoretical models make predictions that are subsequently tested experimentally, with measurements fed forward into the model to generate a new cycle of experiments. Our work in Cell is a crucial early step toward enabling the biophysical dissection of developmental programs by making it possible to measure the very same input-output functions predicted by our theoretical models.

(6 votes)

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Applications are now open for this year’s Gene Regulatory Networks for Development which will be at The Marine Biological Laboratory in Woods Hole, USA from October 14- 27.  The application deadline is July 20.  The course is for graduate students, postdoctoral researchers, and faculty members and it focuses on using experimental data and computational modeling to analyze gene regulatory networks that are key to development in animals and plants. 

This unique course is an intense and always interesting experience and has drawn rave reviews in all of its previous incarnations. Students will meet with experts in the field for an in-depth treatment of experimental and computational approaches to GRN science. Through lectures, highly interactive discussions, and group projects we will explore the GRN concept and how it can be applied to solve developmental mechanisms in various systems and contexts. Topics include structural and functional properties of networks, GRN evolution, cis-regulatory logic, experimental analysis of GRNs, examples of solved GRNs in a variety of developmental contexts, and the computational analysis of network behaviour by continuous and discrete modelling approaches. 

Travel fellowships are available.

For more information about the course, go to www.mbl.edu

The 2018 GRN course faculty:

Scott Barolo, University of Michigan

James Briscoe, The Francis Crick Institute, London

Fernando Casares, Andalusian Center for Developmental Biology, Spain

Ken Cho, University of California, Irvine

Doug Erwin, Smithsonian Institution

Robb Krumlauf, Stowers Institute

Bill Longabaugh, Institute for Systems Biology, Seattle

Lee Niswander, University of Colorado, Denver

Isabelle Peter, Caltech

John Reinitz, University of Chicago

Ellen Rothenberg, Caltech

Trevor Siggers, Boston University

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The Karam Teixeira lab (https://karamteixeira.wixsite.com/website) at the University of Cambridge (Department of Genetics) is looking to recruit a Research Assistant to support a range of projects being carried out in our research group. We are primarily interested in germline development (stem cell regulation and genome defense mechanisms) and we use the Drosophila ovary as a model system (see Sanchez et al, Cell Stem Cell 2016; Teixeira et al., Nature 2017).

We are looking for enthusiastic and proactive candidates with expertise in standard molecular biology techniques (including genotyping, cloning, and RT-qPCR). Prior experience in fly genetics would be an advantage – although training can be provided where necessary. Candidates should have a B.Sc. or M.Sc. degree in a relevant biological subject and should possess good communication skills and the ability to work effectively as part of a team.

• How to apply:

To apply, please follow the link: http://www.jobs.cam.ac.uk/job/17837/

The position start date is flexible. Application deadline: July 13^th, 2018.

For an informal discussion about this position, please contact Dr. Felipe Karam Teixeira (fk319@cam.ac.uk; https://www.gen.cam.ac.uk/research-groups/karam-teixeira/).

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Post-doctoral positions are available in Dr. Samantha Brugmann’s lab to study vertebrate craniofacial development and disease, with an emphasis on the role of the primary cilium in these processes. For information about specific research areas see http://www.cincinnatichildrens.org/research/divisions/p/plastic/labs/brugmann/default/.

Qualifications: Applicants should possess a Ph.D. in a relevant field, such as Biology, Biochemistry, Genetics or another related discipline and be highly motivated, independent and organized. Successful applicants will have a record of communicating research results via publications and/or professional presentations, and be willing and able to participate in collaborative, interdisciplinary research projects. Experience in developmental biology, cell and molecular biology and avian/murine model systems is desirable. Preference will be given to applicants with a proven record in craniofacial research.

Please submit your application to Dr. Brugmann with the following information: A cover letter, a statement of research interests, and a CV with contact details for 3 referees.

Contact: Samantha Brugmann, PhD    Email Address: Samantha.Brugmann@cchmc.org

Cincinnati Children’s Research Foundation

Cincinnati Children’s Hospital Medical Center (CCHMC) is a premier pediatric research institution with over 900 diverse and productive faculty members. Here, researchers work collaboratively across specialties and divisions to address some of the biggest challenges we face today in improving child health. A strong network of research support services and facilities, along with institutional commitment to research, push our team of faculty, postdocs and support staff to explore the boundaries of what is possible, leading to significant breakthroughs. We are driven by our mission to improve child health and transform the delivery of care through fully integrated, globally recognized research, education and innovation.

Post-doctoral research fellows at Cincinnati Children’s are valued for their unique interests and strengths, and are supported by our institution’s strong programming for post-docs through the Office of Postdoctoral Affairs and the Office of Academic Affairs and Career Development. Mentoring, support for international students and an emphasis on crafting high-quality grant proposals are only a few of the features that set our program apart. Cincinnati Children’s is a respected part of the broader, and very vibrant, Cincinnati community. With a thriving arts scene, numerous festivals celebrating music and food, a passionate fan following for our college and professional sports teams, and a variety of opportunities for outdoor activities, our region is truly a great place to work and live.

For further information about working at CCHMC or living in Cincinnati, please contact the CCHMC Postdoc recruiter, Uma Sivaprasad, PhD, at research@cchmc.org

To apply online go to: http://www.cincinnatichildrens.org/careers/apply/default.htm and search for job (requisition) number 95516 or 95517.

Cincinnati Children’s Hospital Medical Center is an Affirmative Action/Equal Opportunity Institution

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Applications are invited from highly motivated individuals dedicated to peruse a PhD and who are interested in nerve/tumour interactions and nervous system development. 

PROJECT DESCRIPTION

During tumour progression nerves and tumours interact resulting in tumour cells using nerves as a metastatic route out of the organ, through a process called perineural invasion (PNI). While this is robustly documented at a histological level, the molecular mechanisms underling this, remain poorly understood. You will join an interdisciplinary collaborative team focusing on the signalling between nerves and tumours, leading to provocation of nerve plasticity (such as growth and remodelling) and tumour metastasis via nerves. You will engage a systematic strategy to identify these mechanisms by (i) examining how nerves form/grow/remodel and migrate normally during embryo development and (ii) pathologically in tumour models with an initial focus on pancreatic ductal adenocarcinoma (PDAC). The formal PhD qualification you will obtain is a PhD in medical science with a focus on cell biology.

LOCATION

The laboratory is located at IMB, Umeå University, Sweden. IMB is an interdisciplinary department, which focuses on questions in basic and medical sciences and provides an interactive modern environment with good core facilities within the wider university. The working ‘day to day’ language in the laboratory is English. The position is for 4 years and is fully funded.

APPLICATION

Apply at the following online system by 24^thAugust 2018 (reference code AN 2.2.1-1186-18): https://umu.mynetworkglobal.com/en/what:job/jobID:215647/

Further application information, including application qualifications, requirements and merits can be found at that webpage.

INFORMAL ENQUIRIES

Informal enquiries may be directed to Dr. S. I. Wilson (sara.wilson@umu.se).

Laboratory webpage: www.imb.umu.se/english/research/research-groups/wilson-laboratory/?languageId=1

We look forward to your application!

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Following our latest data release, the DMDD website (dmdd.org.uk) now contains detailed phenotype data for nearly 700 embryos from 82 different knockout mouse lines. Highlights include the identification of limb defects and cysts in Col4a2 knockouts and replication of the major features of Meckel syndrome in B9d2 knockouts.

We have begun to add immunohistochemistry image data for the brain and spinal cord of some embryos at E18.5. These images give further information about lines in which the embryos appeared morphologically normal at E14.5, but were still not viable. We have also added viability data for every line at both E9.5 and E14.5.

Together with the placental phenotype data that we hold for more than 100 knockout lines, the DMDD website is a rich resource for those investigating the effect of gene mutations on embryo development, and may provide clues about the genetic basis of rare diseases.

LIMB DEFECTS SEEN IN Col4a2 KNOCKOUTS

In humans, COL4A2 mutations have been linked to porencephaly, a rare disorder with phenotypes that include the development of intracranial cysts. In the latest DMDD data, Col4a2 knockouts have a variety of nervous system disorders in line with porencephaly. However, all four embryos also show abnormal autopod morphology and cysts between the nasal septum and the oral cavity, as well as other morphological defects.

B9d2 KNOCKOUTS MODEL MECKEL SYNDROME

In humans, mutations of the gene B9D2 have been linked to Meckel syndrome, a severe disorder caused by dysfunction of the primary cilia during the early stages of embryogenesis. Meckel syndrome is characterised by multiple kidney cysts, occipital encephalocele (where a portion of the brain protrudes through an opening in the skull) and polydactyly, but it also commonly affects the brain and spinal cord, eyes, heart, lungs and bones.

B9d2 knockout mouse embryos included in our latest data release show the major features of Meckel syndrome, including polydactyly and defects in the brain, peripheral nervous system, heart and vascular system. They also display situs defects, where the left-right asymmetry of the body did not develop as expected. The image below shows a B9d2 knockout embryo with left pulmonary isomerism and symmetric branching of the principle bronchi from the trachea.

NEURAL IMAGE DATA NOW AVAILABLE

In around 20% of embryonic lethal lines, embryos appear morphologically normal at E14.5 but still go on to die before or shortly after birth. To understand more about why these embryos were not viable, DMDD colleagues Professor Corinne Houart and Dr Ihssane Bouybayoune at Kings College London analysed the lines at E18.5 – when embryo development is almost complete. They used immunohistochemistry to identify abnormalities in the brain and spinal cord that could not be picked up in our standard, whole-embryo morphological analyses. This data is now available for the line Trappc9, and additional lines will be added in future data releases.

Neural images are available as 20-micron sections through the brain and spinal cord, and the images from different embryos can be compared side by side using the stack viewer. A separate Nissl stain was used to highlight neural death and these images can also be explored online.

A FULL LIST OF NEW DATA IN THE LATEST RELEASE

• HREM image data added for Auts2, Col4a2, Ehbp1l1, Frrs1, Gtpbp3, Nr0b1, Pitx1, Pthlh
• HREM phenotype data added for Auts2, B9d2, Col4a2, Morc2a, Zmynd11

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Development is pleased to welcome submissions for an upcoming special issue on ‘Development at the single cell level’. This special issue aims to showcase the best research in stem cell and developmental biology, building on the rapidly evolving tools of single cell analysis. The issue will be guest-edited by Allon Klein and Barbara Treutlein, working alongside our team of academic editors.

Embryonic development and stem cell differentiation are inherently multicellular phenomena, and every cell can have an important story to tell. The analysis of developmental and stem cell biology at single cell resolution has a long and distinguished history, for example in tracing the lineage of tissues, defining concepts such as commitment, and revealing the existence of adult stem cells. More recently, innovations in single cell transcriptomic, genomic, live imaging and quantitative approaches have opened up new questions about the nature of developmental and stem cell hierarchies, the repertoire of differentiating cell types in tissues and the functional and mechanistic basis of cell types. These new analytical tools are rapidly evolving. They include not just descriptive methods but also fate-tracing and perturbative approaches. They hold promise not just in providing a deeper understanding of cell fate specification and commitment in a native context, but also for understanding and controlling differentiation, de-differentiation and trans-differentiation in vitro and in disease contexts.

Development is the natural home for papers applying single cell analytical and perturbative methods to gain insights into developmental biology and stem cells. We invite you to showcase your breakthrough research that has either been driven by the creative application of single cell approaches; or has created a novel single cell methodology for developmental and stem cell systems.

We also welcome proposals for review-type articles for this special issue. Please send us a short synopsis detailing the scope and structure of the proposed article, and including key references. The deadline for submission of proposals is August 15th and reviews must be submitted by October 31st.

The issue will be published in mid-2019 (note that, in our new ‘continuous publication‘ model, we will be able to publish your article as soon as it is accepted; you will not have to wait for the rest of the issue to be ready) and the deadline for submission of articles is October 31st*. This special issue will be widely promoted online and at key global conferences – guaranteeing maximum exposure for your work. Please refer to our author guidelines for information on preparing your manuscript for Development, and submit via our online submission system. Please highlight in your cover letter that the submission is to be considered for this special issue. Prospective authors are welcome to send presubmission enquiries, or direct any queries, to dev.specialissue@biologists.com

*Please note that not all articles accepted for publication will be included in the special issue; they may instead by published in earlier or later issues of the journal.

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The story behind our recent paper: Counter-rotational cell flows drive morphological and cell fate asymmetries in mammalian hair follicles. Maureen Cetera, Liliya Leybova, Bradley Joyce & Danelle Devenport, Nature Cell Biology. 

Planar cell polarity (PCP) is a fascinating biological problem because it spans such vast biological scales, from nanometers to meters of spatial organization. When we think of PCP – the coordinated polarization of cellular structures across a tissue plane – we often imagine the Drosophila wing, where individual cells produce actin-rich protrusions that point in a uniform orientation. But PCP is also observed in much more complex epithelia like the vertebrate epidermis, where multicellular structures, which produce scales, feathers, or fur, collectively align across the entire skin surface. Although we showed nearly 10 years ago that mammalian hair follicles polarize through the same conserved core PCP pathway that orients Drosophila wing hairs (Devenport and Fuchs, 2008), how these multicellular structures polarize was an intractable question because we lacked the long-term live imaging methods to follow their morphogenesis at cellular resolution.

Movie 1

Enter Team Hair Follicle – a collaboration between two postdocs, Maureen Cetera and Brad Joyce, and a graduate student Liliya Leybova. Through trial and error they figured out that by culturing the embryonic epidermis at an air liquid interface, using a transgenic line expressing bright fluorescent membrane labels (mT/mG) and imaging with a spinning disc confocal to minimize phototoxicity, they could acquire time-lapse movies spanning 24 hours of epidermal development with cellular resolution. The first batch of movies took our breaths away. Never had I dreamed the developing skin would be so dynamic. We never imagined that skin epithelial cells, which we often think of a rigidly adherent with adherens junctions and desmosomes, would behave as if they were fluid and extensively rearrange (Movie 1).

Focusing on a 10-12 hour window of time during which hair placodes polarize, and using automated segmentation and cell tracking, we discovered that during polarization the placode epithelium undergoes extensive rearrangements organized in a counter-rotating pattern (Movie 2-3).

Movie 2

Movie 3

Cells that are positioned centrally move anteriorly to occupy the growing tip of the follicle while more peripheral cells are swept posteriorly and incorporate into the trailing rear (Figure 1).

Figure 1

These movements were abolished in the absence of PCP, non-muscle myosin and Rho kinase activities, with the resulting follicles growing vertically rather than anteriorly (Figure 2, Movie 4).

Figure 2

Movie 4

The counter-rotational movements we observed were striking because 1) they explained how the placode gains its morphological asymmetry, by completely remodeling the placode epithelium from a radial to planar polarized organization; and 2) the pattern of movements closely resemble the elaborate ‘polonaise’ movements of gastrulating chick embryos, a process previously linked to the PCP pathway (Voiculescu et al, 2007). During formation of the chick primitive streak, convergent extension in the posterior hemisphere of the embryo displaces cells in the anterior into two counter-rotating flows (Chuai and Weijer, 2008; Rozbicki et al, 2015). It is remarkable that the same pattern of cell movements involving thousands of cells in the chick embryo is made by just a few dozen cells confined within and scattered in a periodic pattern across the skin epithelium.

We were then confronted with the difficult task of connecting PCP to the counter-rotating rearrangements we observed. A key feature of the PCP pathway is that its core components, as well as downstream cytoskeletal factors, are asymmetrically localized to intercellular junctions. What was difficult to explain, and will require much more work to fully understand, was how the asymmetry of the core PCP components, which align along a common axis amongst all cells across the skin epithelium, could generate cell movements in opposite directions. So Maureen broke down the collective movement into its local component parts and asked how the asymmetry of PCP components correlated to those behaviors. First, she determined that in the posterior half of the placode, cells intercalate towards the placode midline via polarized shrinking and growth of intercellular junctions (Figure 3). Junctions that were lost tended to be vertically oriented where new junctions formed in a horizontal orientation. In the anterior half, cells also underwent neighbor exchanges but with the opposite polarity so they moved away from the midline and posteriorly (Figure 3).

Figure 3

PCP protein localization correlated with junction shrinkage, even in the anterior half of the placode, where PCP junctions were rotated relative to the AP axis (Figure 4). This reminded us of a result from a previous study where we showed that when cells exchange neighbors through junction remodeling, the local axis of PCP asymmetry rotates relative to its original orientation (Aw et al, 2016). We hypothesized that early remodeling events in the posterior of the placode could cause PCP-enriched junctions to rotate, thereby generating a new PCP axis for junction shrinkage in the anterior.

Figure 4

The discovery of counter-rotational movements in the placode also explained how PCP generates cell fate asymmetry. In our initial 2008 study we reported that the earliest two hair follicle lineages were distributed in a planar polarized organization (Devenport and Fuchs, 2008), with hair matrix precursors at the anterior and stem cell precursors at the posterior. But the relationship between morphological and cell fate asymmetry was unclear in part because the two events couldn’t be temporally separated. By live imaging we could now show that cell fate asymmetry arises from counter-rotating cell rearrangements. Initially, the two progenitor populations are specified in a bullseye pattern, with a central cluster of matrix progenitors surrounded by a halo of stem cell precursors. Counter-rotating cell rearrangements moved the two cell populations into their respective, anterior-posterior positions and shifted the placode from vertical to anterior-directed growth (Figure 5).

Figure 5

From these studies we learned that a just a single PCP-dependent process – counter rotating cell rearrangements – could generate both morphological and cell fate asymmetry. Additionally, we learned that PCP-dependent cell intercalation is a deeply conserved morphogenetic tool, used to shape tissues in ways other than convergent extension. We learned, of course, that live imaging reveals unexpected and delightful new insights beyond any hypothesis I was ever able to muster. This study was also an really successful experiment in team research. Whereas most projects in the lab and in my previous training had centered mostly around individuals, this was a true collaborative effort.  Perhaps it is appropriate that the insights we gained about how PCP polarizes cell collectives would finally come, not from the efforts of just one individual, but from the collective.

References

Devenport, D. & Fuchs, E. Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles. Nat Cell Biol 10, 1257-1268, doi:10.1038/ncb1784 (2008).

Voiculescu, O., Bertocchini, F., Wolpert, L., Keller, R. E. & Stern, C. D. The amniote primitive streak is defined by epithelial cell intercalation before gastrulation. Nature 449, 1049-1052, doi:10.1038/nature06211 (2007).

Chuai, M. & Weijer, C. J. The mechanisms underlying primitive streak formation in the chick embryo. Curr Top Dev Biol 81, 135-156, doi:10.1016/S0070-2153(07)81004-0 (2008).

Rozbicki, E. et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation. Nat Cell Biol 17, 397-408, doi:10.1038/ncb3138 (2015).

Aw, W. Y., Heck, B. W., Joyce, B. & Devenport, D. Transient Tissue-Scale Deformation Coordinates Alignment of Planar Cell Polarity Junctions in the Mammalian Skin. Curr Biol 26, 2090-2100, doi:10.1016/j.cub.2016.06.030 (2016).

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For a Ph.D. holder, the idea that any career can be classified as “alternative” is obsolete. With a smaller percentage of postdocs going on to tenure-track positions every year, universities and research institutions are stepping up efforts to help recent graduates figure out how to put to good use all the specialized training that they have received. The National Institutes of Health (NIH), for example, has for the past decade hosted a free career symposium open to young scientists from any institution. This annual one-day event, organized by the Office of Intramural Training and Education (OITE) of the NIH and held at its Bethesda, Maryland campus, aims to expose early-career scientists to the various paths available to them professionally, and to help trainees identify the steps that they can take in their current positions to set themselves up for future success.

This year’s symposium, held on May 18th 2018, saw a record attendance of over 1000 participants, mostly Ph.D. students and postdoctoral fellows split evenly from within the NIH and from outside institutions. After a warm welcome address by Dr. Lori Conlan, Director of the OITE Career Services Center and of the Office of Postdoctoral Services, participants attended one of four concurrent panels for three different sessions. Each panel consisted of about four professionals, several of whom were NIH alumni, who had each made the transition from graduate student or postdoc to their current roles and were therefore credible experts. There was a deliberate effort to include panels from a wide range of career options, and in addition to several industry and academic panels, there were panels on careers in the federal government, science administration, science writing, outreach and policy, among others. The day ended with “Skill Blitz” sessions, which gave practical advice on specific skills critical to the job hunting process, such as writing a great CV, interviewing, and negotiating tactics after getting a job offer (The very helpful resources used for these sessions are available online on the OITE website).

The most important lessons from this event transcended any specific panel or career path and were reiterated continuously throughout the day. These general sentiments were:

1) NETWORK! – Networking was mentioned by almost every panelist as key to a successful job search. Having inside information on a job description or hearing about an impending job post before it goes public could be just what you need to stand out in a pool of applicants. Networking does not have to be intimidating. You could start small by making personal connections with colleagues in your department or purposefully arriving at seminar venues early to interact with participants before the talk begins.

2) Get Experience– With so many applicants from a Ph.D. background, having some experience in the field to which you plan to transition is critical. For tenure track jobs, experience with grant writing (especially grants that get funded) is an important way to distinguish one’s self, and for a teaching-intensive faculty position, teaching experience is essential. Volunteering is a great way to get experience in almost any field. For instance, for a career in technology transfer and patents, volunteering at a university tech transfer office would be a valuable addition to your CV. Likewise, working with a start-up company would be useful for a future career in investment. Volunteering to judge school science fairs shows an interest in outreach, as does writing for your department newsletter for a career in science writing. Volunteering can also help you test-run several career options to confirm the right path for you. Step out of your comfort zone, use your weekends (instead of spending them all in the lab) and diversify your experience. Even little things you do could have big payback.

3) Conduct Informational Interviews– Speaking with professionals in the field to which you plan to transition can help you get a clearer idea of what exactly the job entails and what essential skills are involved, as some of this may not be obvious when you’re looking at a job from the outside. Informational interviews can strengthen networking efforts, giving you valuable job leads and inside contacts. These interviews are also great for research into the prospects for growth and advancement in a specific field, and to find out if there are any nuances in applying for jobs, such as in academia where jobs are announced seasonally. By speaking with enough people, you get a well-rounded image of what your future holds with any chosen path, giving you valuable information on how truly suited you are for that career.

4) Publish– Especially for an academic career, publications show productivity. Both first author and middle author papers are important, as first author papers showcase the ability to lead a project and to see it through, and middle author publications show the ability to collaborate with others and work with a team.

5) Be kind– Perhaps the most unexpected but insightful piece of advice came at the start of the day when Dr. Sharon Milgram, Director of OITE, encouraged participants to be kind, both to themselves and to others. The requirements for careers in science can be tough on a person mentally and physically. Don’t be too hard on yourself for apparent shortcomings, and don’t take out your frustrations on others, turning mentoring into “tormentoring”, as she put it. Taking care of yourself boosts resilience, and looking out for the people you work with, especially those you mentor, helps change the culture that extreme stress and anxiety are inseparable from research.

The take home message from this symposium was simple: Opportunities abound. With the right fit, any career can lead to a full, fulfilling professional life. To distinguish yourself in your job applications down the line, though, be aggressive in finding opportunities to network and to gain experience in your chosen field. In all, this was a very productive way to spend the day.

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