In spite of our external appearance, our innards are asymmetric. For today’s interview, we feature a paper published recently in Development that provides a cellular and molecular investigation into symmetry breaking in a poorly understood organ, the stomach. We caught up with first authors Adam Davis and Nirav Amin, and their supervisor Nanette Nascone-Yoder, Associate Professor in North Carolina State University, Raleigh, NC.

Nanette, can you give us your scientific biography and the questions your lab is interested in?

NN-Y I have been interested in left-right asymmetry for a long time. I did my graduate work with Mark Mercola at Harvard Medical School, studying the induction and left-right asymmetric patterning of the heart. At NC State University, we’ve been investigating the morphogenesis and evolution of the gastrointestinal tract, specifically, how the embryonic gut tube forms the requisite three-dimensional shape necessary for proper physiological function, including appropriate length and left-right asymmetric curvature.

And Nirav and Adam, how did you come to join Nanette’s lab?

NA During my career, my research interests have focused on the transcriptional control of cell fate specification and organ development. I did my PhD work in the lab of Dr. Jun “Kelly” Liu at Cornell University, where I focused on the transcription factors, and their targets, that controlled muscle and non-muscle fates in the C. elegans mesoderm. From there, I wanted to apply the tools and techniques I used in C. elegans to the classic vertebrate model for developmental biology, Xenopus. I did my postdoctoral work in the lab of Dr. Frank Conlon at the University of North Carolina at Chapel Hill, where my research focused on a transcription factor, Casz1, and its role in cardiac development. As part of my project, I also participated in some RNA-seq and ChIP-seq studies to identify targets of Casz1 in the heart. Eventually, I began exploring methods to determine the functional roles of genes in cardiac development by editing frog genomes via mutagenesis and transgenesis.

Towards the completion of my postdoctoral work, I met Nanette and she told me about a research project in her lab in which she was using a non-model organism, the Budgett’s frog, to investigate how organs establish left-right asymmetry. These frogs have large embryos (three times larger than the Xenopus embryo!), and she was using this to her advantage in some transcriptomic studies. My experience with this type of data and functional studies in the frog made me a perfect fit for the project. I was excited by the possibility of developing this frog as a new model for developmental biology research and I joined the lab three years ago.

AD I’ve always been interested in animals, particularly in how they are shaped. I’ve also always been interested in how our DNA shapes our organs and organ systems. I performed my PhD work in the lab of Dr. Ed Stellwag at East Carolina University. I studied how Hox genes are regulated in the developing brain and pharyngeal arches. I used the Japanese medaka (Oryzias latipes) as a model to study the development and evolution of morphology. I enjoyed learning how transcription factors and their respective cis-regulatory elements regulate gene expression during development. I first met Nanette when I presented a poster of my research at the Southeast regional conference meeting for the Society of Developmental Biology at UNC, Chapel Hill in 2007. I was amazed by her talk and the research her lab was performing at NC State, particularly in how genes influence cellular morphogenesis. Once I finished my Ph.D., I was lucky enough to obtain a postdoc position in her lab under a NIEHS training grant. I was excited to work with Pitx2, as it is a homeodomain-encoding transcription factor. I was also excited to understand how organs are shaped at the cellular level.

You reference some papers from the 1960s hypothesising as to how the stomach gets its curvature. Why do you think it has taken until 2017 for the first experimental investigation into its cellular and molecular basis?

NN-Y The rotation theory was actually put forth in the late 19th century, based on retrospective observations of human embryos. Rotation of the organ nicely explained the final anatomical location of left and right nerves along the front and back of the stomach; hence, the idea of early organ rotation became widely accepted and propagated such that it became dogma in all the textbooks. We were certainly not the first to question this model. Dissenting theories have been proposed by other scientists before now, but these studies were also based largely on retrospective studies in human embryos, and conducted prior to modern molecular developmental biology.

It is important to clarify that the stomach probably does undergo rotation events during later development, in order to refine the final contour and position of the organ as it descends into the abdominal cavity. However, our results indicate that wholesale rotation is not the mechanism of establishing the initial LR asymmetric curvature, as has been assumed for over a century.

What led you to address this question using both mice and frogs?

NA One day Nanette pulled me into her office to show me some pictures publicly available on emouseatlas.org. It was clear that a lot of the observations she and Adam had made in Xenopus held true in the mouse stomach. We were fortunate that the Ghashghaei lab here at North Carolina State University had the Foxj1 mouse mutant, enabling us to directly test how curvature is affected when left-right asymmetry is randomized, and to show that the origins of stomach curvature are conserved in mammalian development.

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

NA The J-shaped stomach is characterized by a longer “greater” curvature and a smaller “lesser” curvature. Historically, this shape has been thought (and taught) to be the result of a rotation of the dorsal surface of the gut tube to the left. However, others have suggested that this curvature is not the result of rotation, but an intrinsic asymmetric growth of the stomach to give rise to the greater curvature. In this paper, we use frog and mouse embryos to show that the latter hypothesis is true –differential lengthening of the left wall drives the curvature of the stomach. This lengthening occurs due to radial rearrangement which cause the left stomach wall to thin. Importantly, this process is dependent on Foxj1/Nodal/Pitx2c, key determinants of left-right asymmetries in multiple organisms.

Do you have any idea how Pitx2 is directing stomach morphogenesis?

NA Our working hypothesis for how Pitx2 is directing stomach morphogenesis is through the regulation of cellular effectors that drive radial intercalation. Our current research focus is in the identification of such factors within the left stomach.

Are all asymmetric internal organs made asymmetric in different ways? How does the stomach relate to the rest of the digestive system, for instance?

NN-Y Different types of morphological asymmetries form in different organs. For example, some organs form an acute curvature like the stomach or early heart tube. Other organs adopt left right asymmetric positions, or undergo asymmetric regression or remodeling, such as the spleen and vasculature. In others, grossly different morphologies develop out of their left versus right halves/ counterparts; examples include the cardiac atrial chambers or the contralateral lobes of the liver and lungs. Unfortunately, for the majority of organs, we know surprisingly little about the cellular and molecular events that break symmetry during organogenesis, so it is unclear whether these varied types of morphological asymmetries may form in different ways.

The recent work in intestinal rotation does provide one point of comparison. The appearance of cellular differences between the left and right sides of the dorsal mesentery breaks the symmetry of the structure that suspends the gut tube from the body wall, leading to a leftward shift in gut position, ultimately biasing the direction of intestinal rotation. Interestingly, no developmental asymmetries are thought to exist in the intestine itself. In contrast, we find that the left and right sides of the foregut tube itself undergo distinct morphogenetic processes to drive stomach curvature. At the cellular level, left side cells are more polarized /organized than right side in both stomach and mesentery; however, at the tissue level, the left stomach wall thins and expands, while left mesentery condenses. Only the mesoderm layer of the gut is involved in the mesentery, while both mesoderm and endoderm become asymmetrical in the stomach. So there are both similarities and differences in the development of asymmetry in each context. In the future, we will need to look at a variety of organs in order to determine the degree of universality or divergence in organ-level symmetry breaking.

When doing the research, was there a particularly exciting result or eureka moment that has stayed with you?

NA For me, I was excited by the success of the CRISPR reagents I generated to look at Pitx2c function in the stomach. Though it wasn’t the most crucial data pertaining to this paper, it was ground-breaking for me in that I could use CRISPR/Cas9 to systematically interrogate gene function in the F0 generation for Pitx2 and other genes I identify to be involved in asymmetry. This has proven to be a very time- and money-saving finding.

AD Absolutely! It was when I was examining immunochemically-stained transverse sections of wild-type Xenopus stomachs at several developmental stages. I noticed that several morphometric factors (E-cadherin, aPKC, and γ-tubulin) showed much more robust apical localization in the left endodermal cells of the developing stomach than the right. This was observed in developmental stages prior to gut curvature at the gross anatomical level.

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

NA Having worked with worms and frogs, I have gotten used to being able to follow embryogenesis in real time and in large numbers of animals. When we embarked on the mouse side of this project, the frustration for me came in not being able to 1) get more than 2-3 mutant animals per litter and 2) precisely control the timing of when we get embryos. On the bright side, it made me appreciate the advantages of Xenopus even more!

AD There were a lot more of these than the eureka moments, including antibodies not working properly, needles clogging when trying to inject embryos, frogs not yielding viable eggs, me wanting to pull my hair out, etc., etc.

What next for you following this work?

NA I am very excited to continue this work – we have used transcriptome profiling in the Budgett’s frog to identify some interesting genes that function together with Pitx2 in regulating stomach curvature. Hopefully soon, you’ll be reading more about these!

AD Thanks to my experience with my PhD, and as a postdoc in Nanette’s lab, I am currently a tenure-track assistant professor at Gordon State College. I enjoy teaching Developmental Biology and Human Anatomy and Physiology. I learned a wealth of information regarding molecular and developmental genetics, cellular and gross morphogenesis, anatomy, histology, and microscopy. I used this information to build my developmental biology course. I also enjoy training undergraduate students in research in developmental biology.

And finally – what do you get up to when you are not in the lab?

NA The majority of my non-lab life is dedicated to my wife, 4 year-old daughter, and 2 year-old son. They keep me balanced and make me feel young (and old at the same time!). Now that the weather is getting nicer here in North Carolina, we are constantly outside staying active – we have started our garden and will be camping, biking, and more in the coming months.

AD My non-academic life is dedicated to my wife, Rebecca, and our 7 year-old son, Finn. Like Nirav, they give me balance with my work life. We’re near the Appalachian Mountains, so we love hiking and camping. Finn and I enjoy searching for and photographing salamanders and other wildlife when we hike. Also, last summer, we made our first insect collection together.

Back to you Nanette – where will this paper take your lab?

NN-Y We are currently identifying the cellular morphogenetic events that underlie symmetry-breaking in two other organs. As Nirav mentioned, we have also devised a novel strategy for identifying the molecules involved, using a novel model organism (the Budgett’s frog). The hope is that these genes could be candidates for human organ defects. Variation in these processes may also be involved in generating novel organ anatomy and morphology during evolution.

Adam Davis, Nirav M. Amin, Caroline Johnson, Kristen Bagley, H. Troy Ghashghaei, Nanette Nascone-Yoder. Stomach curvature is generated by left-right asymmetric gut morphogenesis. Development 144: 1477-1483

Browse the People Behind the Papers archive here

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Last Sunday evening found me sitting in the BBC Cambridge radio studio, headphones on and mic in front of me, talking about developmental and stem cell biology with Dr. Chris Smith, better known as the naked scientist. Fortunately, both of us were fully clothed. For those of you who aren’t familiar with The Naked Scientists, it’s an award-winning radio show and podcast that discusses the latest scientific research, answers questions on diverse scientific topics from listeners, and generally aims to make science more accessible to the general public. I’d met Chris a few weeks earlier to talk about a program he was planning that would link developmental and stem cell biology to regenerative medicine, and he asked whether I’d be willing to contribute to the show – providing an introduction and commentary to the interviews he was conducting. Hence the headphones and mic.

Having never been in a radio studio before, let alone appeared on live radio, I found the experience fascinating and daunting in equal measure. How did the whole thing work? What if I said something stupid? Fortunately, Chris and Tom (Crawford – the producer working on this show) were great at putting me at ease and guiding me through the program. And I think (hope!) the end result makes for an interesting listen. In the show, you’ll hear Roger Barker talking about his plans for a clinical trial for Parkinson’s Disease using embryonic stem cell-derived dopaminergic neurons, Hans Clevers discussing how gut organoids can be used for personalised drug testing, and Don Ingber on his amazing organ-on-a-chip technology. Plus Chris and me trying to pull these threads together and provide a perspective on where the field is going.

You can find the full show here; our discussion starts around 25 minutes in, but I’d actually encourage you to listen to the whole thing – which covers topics as diverse as heroin addiction, why airplane travel is likely to get more turbulent, and what determines whether something will ‘go viral’.

I hope you enjoy it!

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Regeneration, the ability to restore lost parts of the body, is a widespread phenomenon in animals. Whilst this ability is somehow limited in classical developmental model organisms, a variety of animals are able to regenerate complex structures, such as limbs or important parts of their body. Regeneration is often based on the presence of populations of stem cells which are either pluripotent and able to regenerate all tissues, or multipotent with a much more restricted potential. Regeneration can also rely on local cell dedifferentiation processes by which differentiated cells are reprogramed into proliferating progenitor or stem cells.

In the our team (Stem Cells, Development and Evolution team), we use the emerging developmental biology annelid model Platynereis dumerilii to investigate the evolution of stem cells and regeneration. Platynereis worms are able to grow and regenerate their posterior (caudal) part following posterior amputation during most of their life. These two abilities end when the worms become sexually mature and are dependent on the presence of a brain hormone (methylfarnesoate) that blocks sexual maturation. In the frame of a collaborative ANR-FWF funded project with the team of Florian Raible (Origin and Diversification of Hormone Systems, MFPL Vienna, Austria), the postdoctoral fellow will study, using cellular and molecular approaches, how the brain hormone controls the growth and regeneration abilities of Platynereis worms, and participate to the molecular and cellular characterization of the regeneration process.

The Vervoort team belongs to the Institut Jacques Monod (IJM) in Paris (France). The IJM is a leading French biological research institute, comprising about 30 interactive research groups and high-quality technological facilities, including a cutting-edge imaging platform. The working language at the IJM is English, and knowledge of French is therefore not a prerequisite for this position. Successful candidates will collaborate with a dynamic team of developmental, cellular and evolutionary biologists at both the IJM and MFPL.

Starting date is flexible but should be around September 2017 onwards and is funded initially for 14 months. The successful applicant must have, or be in the process of completing, a PhD thesis in a relevant research area and not more than two years of postdoctoral experience. Desirable qualifications include expertise in molecular biology, immunohistochemistry, qPCR, and DNA sequence analysis. Potential candidates should send their application by e-mail to michel.vervoort@ijm.fr with a statement of interests and expertise, a Curriculum Vitae and contact information from two referees. The position will remain open until filled; however, applications received by May 30^th will be given priority. Please contact Michel Vervoort for more information.

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We are seeking an enthusiastic and motivated Postdoctoral Research Scholar to join research projects investigating the molecular basis of neurodevelopmental disorders in the laboratory of Kristen L. Kroll at Washington University School of Medicine. Working in collaboration with Washington University’s Intellectual and Developmental Disabilities Research Center (http://iddrc.wustl.edu/About/MissionOverview), we use directed differentiation of human pluripotent stem cells (embryonic stem cells and patient-derived induced pluripotent stem cells), mouse models, and a wide range of cellular, molecular, biochemical, and genomic approaches, to define gene regulatory networks that control neural cell fate acquisition and the specification and differentiation of specific neuronal cell types, such as cortical interneurons. We are defining roles for epigenetic regulation in controlling these networks and identifying mechanisms by which their dysregulation alters neural development and function and contributes to neurodevelopmental disorders, including inherited epilepsies, autism spectrum disorder, and neural tube defects.

For additional information about our ongoing work and research interests, please see: http://krolllab.wustl.edu/

Setting/Salary/Benefits:

Our laboratory is in an academic setting in the Department of Developmental Biology at Washington University School of Medicine (St. Louis), an internationally recognized research institution with a dynamic research environment and extensive infrastructural and core facility support. Postdoctoral appointees at Washington University receive a starting salary based on the NIH NRSA guidelines and a generous benefit package. Complete information on the benefit package is located on the WUSM Human Resources Benefits Website (http://medschoolhr.wustl.edu). The St. Louis area combines the attractions of a major city with family-friendly and affordable lifestyle opportunities (https://explorestlouis.com/)

Qualifications:

Candidates should hold a recent PhD with less than 2 years of prior post-doctoral experience. Preference will be given to applicants with a strong interest in and research training relevant to the areas of neural development, stem cell biology, and transcriptional or epigenetic regulation. US citizens are preferred. Interested candidates should send a CV/names of references by email to kkroll@wustl.edu or by regular mail to Kristen L. Kroll, Washington University School of Medicine, Campus Box 8103, 660 S. Euclid Ave, St. Louis, MO 63110.

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A  postdoctoral position is available in the laboratory of Dr. Martin Basch to study the regenerative potential of the stria vascularis in cases of congenital deafness. Highly motivated, creative and enthusiastic individuals are particularly invited to apply.

Qualifications include a PhD in developmental biology, neuroscience, cell biology or related field. Priority will be given to candidates with experience in cell culture and basic molecular biology. Mouse genetics and/or neonate mouse surgery skills are desired but not required.

Our laboratory is part of the Hearing Research Program, at Case Western Reserve University School of Medicine. Currently the program involves five Research Faculty PIs and Physician Scientists with key areas of research, which include molecular otology, otitis media, congenital/acquired hearing loss, inner ear development, and hair cell biology. The strength of our program is enhanced by an excellent interdisciplinary and collaborative intellectual environment at Case.

 Interested candidates should submit their CV and a letter of application (including a brief description of previous research experience and a statement of interests) to:  Martin Basch Ph.D. at mlb202@case.edu.

 For more information on our laboratory, please visit our website at http://www.baschlab.org

 In employment, as in education, Case Western Reserve University is committed to Equal Opportunity and Diversity.  Women, veterans, members of underrepresented minority groups, and individuals with disabilities are encouraged to apply.

Case Western Reserve University provides reasonable accommodations to applicants with disabilities.  Applicants requiring a reasonable accommodation for any part of the application and hiring process should contact the Office of Inclusion, Diversity and Equal Opportunity at 216-368-8877 to request a reasonable accommodation.  Determinations as to granting reasonable accommodations for any applicant will be made on a case-by-case basis

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A large-scale study of DMDD data has shown that inactivating the same gene in mouse embryos that are virtually genetically identical can result in a wide range and severity of physical abnormalities. This suggests that the relationship between gene mutation and embryo development is more complex than previously thought.

The study considered 220 mouse embryos, each with one of 42 different genes inactivated. These genes are part of a set known as ‘embryonic lethal’, because they are so crucial to development that an embryo missing any one of them can’t survive to birth. Studying these genes can help us understand how embryos develop, why some miscarry and why some mutations can lead to abnormalities.

Read more on the DMDD’s blog, Annotations, here:

https://blog.dmdd.org.uk/new-complexities-in-relationship-between-gene-mutation-and-embryo-development/

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Here are the highlights from the current issue of Development:

A new niche for human HSCs

Human haematopoiesis occurs at various anatomical sites throughout development, including the yolk sac, the aorta-gonad-mesonephros region, the liver, the placenta and the bone marrow. Cells marked by high expression of CD34 and low CD45 – suggestive of possible HSCs – have been reported in human fetal membranes; however, their exact niche as well as their functional capacity remain untested. In this issue (p. 1399), Alicia Bárcena and colleagues isolate and interrogate this putative HSC population, and demonstrate for the first time that the human chorion contains transplantable, definitive HSCs. The authors carefully separate the chorion and the amnion, and show via fluorescence-activated cell sorting that only the chorion contains the putative HSCs, and only from 15 weeks of gestation. The cells display markers of HSC and primitive haematopoietic progenitors, such as little CD38 and CD133, low levels of CD117 and CD4, and medium to high levels of HLA-DR, CD31, CD90, CD95, TIE2 and CD71. Cells co-expressing CD34 and CD45 antigens are found either in association with mesenchymal stromal cells or with endothelial cells of chorion vasculature . Using in vivo xenotransplantations, the authors demonstrated that the CD34⁺⁺ CD45^low cells possess multilineage long-term HSC activity specifically between weeks 15 and 24 of gestation. This study reveals novel insight into an unexpected niche for HSCs during human development.

How the stomach gets its curve

Left-right asymmetry is a common feature of many organs, and is crucial for their function. The stomach is one such organ, with marked curvature on the left compared with the right, resulting in a distinctive shape that is highly conserved among vertebrates. Although it is well established that activation of Nodal controls left-right asymmetry of visceral organs, the cell- and tissue-level morphogenetic mechanisms that drive this phenomenon are poorly understood, especially in the stomach. Now, on p. 1477, Nanette Nascone-Yoder and colleagues shed light on the mechanisms that drive left-right asymmetric development of the stomach in both mouse and Xenopus embryos. The authors start with a gross examination of stomach curvature during development and compare their findings with two proposed models: a rotation model and an asymmetric growth model. They find no evidence for the former, and therefore suggest that the stomach acquires its asymmetry by an intrinsic mechanism. In support of this, the authors show that there is an asymmetric thickness of the left and right stomach wall, which depends on intact cilia and Nodal signalling as both Foxj1 mutant mouse embryos and Xenopus embryos treated with a Nodal inhibitor show a loss of this asymmetry. The authors show a role for Pitx2 in this process by overexpressing Pitx2 on the right side or knocking down Pitx2, both of which affect stomach curvature in Xenopus. This study demonstrates that asymmetric morphogenesis of the stomach in frogs and mice is driven by FoxJ1-Nodal-Pitx2-dependent asymmetric remodelling of the gastric epithelium on the left side.

Liverworts breathe easy

The vast majority of land plants regulate gas exchange through their stomata – tiny pores usually found on the underside of leaves. The liverwort plant group Marchantiidae is an exception, as it lacks stomata and instead breathes through air pore complexes. This is an important evolutionary adaptation, and yet the mechanisms that regulate air pore complex development in Marchantiidae remain unknown. In this issue (p. 1472), Victor Jones and Liam Dolan identify the zinc finger protein MpWIP as necessary for the morphogenesis of the air pore complex in the epidermis of Marchantia polymorpha. The gene was first identified through a mutagenesis screen, in which overexpression led to the presence of ectopic rhizoids on the dorsal epidermis. Using a construct containing the MpWIP promoter fused to a reporter gene, the authors show that MpWIP is expressed both ventrally and dorsally and that the dorsal expression pattern is within the developing air pore complex cells. To determine whether MpWIP is required for air pore development, the authors use artificial microRNAs to generate plants with reduced expression of MpWIP, which results in defects in air pore complex morphology. Based on chimeric dominant repressor and activator versions of MpWIP expressed separately in transgenic plants, the authors provide some evidence for the possible role of MpWIP as a transcriptional repressor. This study identifies, for the first time, a gene that regulates the development of the air pore complex, which is an important evolutionary innovation in liverworts as an alternative to stomata.

Specialised fibroblasts maintain the nipple epidermis

The skin is the body’s largest organ, and is the first line of defence against the external environment. The epidermis – the outermost layer of the skin – is highly specialised and often exhibits unique characteristics depending on its anatomical location and the function it serves. It has long been known that this specialisation is dependent on inductive signals that originate from underlying fibroblasts; however, the exact nature of the signals and their role in maintaining the epidermis is only just starting to emerge. In this issue (p. 1498), John Foley and colleagues identify an oestrogen-regulated TGFβ signalling pathway that is crucial for the maintenance of the highly specialised nipple epidermis. Using a series of grafting experiments, the authors show that fibroblasts taken from the nipple-like skin of mice can induce reprogramming of trunk keratinocytes into nipple-like epidermis. Transcriptional profiling of the nipple-like fibroblasts identifies oestrogen signalling as a strong candidate factor for the maintenance of the nipple epidermis and, indeed, ablation of oestrogen signalling in ovariectomised mice results in an abnormally thin nipple epidermis. The authors further identify Tgfb1 as a direct target of oestrogen signalling and show how ectopic treatment of TGFβ1 protein into the connective tissue of the nipple causes a decrease in epidermal proliferation and a thinning of the nipple epidermis. Taken together, these data represent an important step forward in understanding the signalling network that maintains the specialisation of the nipple epidermis.

PLUS:

Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells

Retinal degenerative diseases are the leading causes of blindness worldwide. In their Review article, Sally Temple and colleagues review stem cell-based therapies for retinal diseases, describing the challenges involved and discussing how basic developmental studies have contributed to and are needed to advance clinical goals.

Prostate organogenesis: tissue induction, hormonal regulation and cell type specification

Prostate organogenesis is a complex process that is regulated by androgens and subsequent mesenchyme-epithelial interactions. In their Review article, Roxanne Toivanen and Michael Shen provide a comprehensive overview of prostate development, focusing on recent findings regarding sexual dimorphism, bud induction, branching morphogenesis and cellular differentiation.

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As every year, the Spring meeting was the time of awards and medals! This year, we had awardees of three societies who are listed below. For those wanting to have a look at the topics of talks and posters presented at the meeting, please download the abstract book here. Watch the movies of the BSDB award winners below.

Movies of medal lectures

Hooke Medal Lecture: Ewa Paluch

Women in Cell Biology Lecture: Victoria Sanz Moreno

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Macrophages are usually associated with immunity, but have increasingly appreciated functions in development and homeostasis. This week we meet the authors of a recent Science paper that identified a role for macrophages in zebrafish stripe patterning, revealing a remarkable ‘relay’ mechanism whereby macrophages help one type of cell signal to another via cytoplasmic extensions. Postdoc Dae Seok Eom and his supervisor David Parichy, recently appointed Pratt-Ivy Foundation Distinguished Professor of Morphogenesis at the University of Virginia, told us more.

David, can you give us your scientific biography and the questions your lab is interested in?

DP I started out in ecology and evolution as an undergrad at Reed College, studying maternal effects on tadpole growth and survivorship for four years with Bob Kaplan. With that experience I applied to E&E Ph.D. programs and ended up accepting an offer from Population Biology at UC Davis, with Brad Shaffer, a systematist and organismal biologist studying biogeography of salamander populations. But between when I applied and when I got there, I became more and more interested in developmental mechanisms and how they evolve. So I was really lucky that Brad has diverse interests and that Carol Erickson—a developmental biologist working on neural crest—was willing to serve as a co-advisor. For my dissertation I focused on the cellular bases for salamander pigment pattern development and evolution. Those are great animals, but the molecular biology and genetics were difficult at the time. So I switched to zebrafish and its relatives for my postdoc with Steve Johnson at Wash U Medical School in St. Louis. In Steve’s lab, we identified some of the mechanisms underlying the development of stripes and other patterns, and I carried this program into my independent career.

My lab has broad interests but an organizing theme has always been to understand the genes and cell behaviors underlying adult phenotypes, and how changes in developmental genetic mechanisms contribute to variation within and among species. We continue to work on pigment patterns, but we also want to know what regulates the stem cells that give rise to adult pigment cells and how “local” cellular mechanisms intersect with “global” endocrine control during development, homeostasis and regeneration. But our interests are wide-ranging so we’ve also worked on topics including skeletal development, zebrafish natural history, and the behavioural significance and cognitive processing of pigmentation. Right now we are doing a lot of work to understand scale morphogenesis and patterning in fish, and we’ve even started working on salamanders again, both pigment and regeneration.

And Dae Seok, how did you come to join David’s lab?

DSE Actually, when I was at PhD training at the University of Texas at Austin, Dave was a faculty of my department, and I was interested in his work, but by then he was then planning to move to the University of Washington. While I was finishing my PhD training, my wife started her PhD at UW, so I had to find a postdoc position in Seattle. I realised Dave was there, and it was obviously a great second chance to work with him – and he accepted.

Aside from aesthetics, what makes zebrafish stripe development an attractive developmental model?

DP When I was a grad student, I wanted to find a system that could be studied from many different angles—molecular through organismal. And this was why I ended up focusing on pigmentation. For anyone with broad interests it’s just a natural: there’s a deep literature on pigment cells and pattern going back to the turn of the last century, the cells are visible even in the living organism as the phenotype is developing, the patterns themselves often have profound ecological significance, and there’s tremendous diversity of pattern across even closely related species. Of course there are also a variety of pigmentary disorders, including melanoma, and pigment cells develop and regenerate from stem cells—so there is obvious biomedical utility in studying pigment cells, especially with a system like zebrafish in which the genetic and cellular mechanisms are so accessible.

A macrophage drags an airineme to a melanocyte. Eom & Parichy, 2017.

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

DP We showed previously that consolidation of melanophores into adult stripes depends on interactions between these cells and xanthoblasts, the precursors of yellow xanthophores. During a specific stage of stripe development, xanthoblasts that happen to be present in future stripe regions extend very long, thin and meandering filopodia-like processes with membraneous vesicles at their tips. We called these projections “airinemes” after Iris—messenger of the gods—and Sir George Airy, who described limits on optical resolution, because the projections are such a pain to visualize. We saw airinemes “dock” with melanophores, and we found that blocking airineme extension prevented melanophores from consolidating properly into stripes, at least in part because of a defect in Delta-Notch signalling.

“Weird biology”

In Dae Seok’s new paper, we show that airineme extension and vesicle delivery depend on macrophages that are cruising around the local tissue environment. The macrophages recognize surface blebs on xanthoblasts and try to engulf them but continue to wander, pulling a membrane filament from the xanthoblast as they go. Eventually they wander across a melanophore and the vesicle and filament are deposited on the melanophore surface. If we get rid of macrophages, we prevent airineme extension and melanophores remain dispersed. Weird biology.

What convinced you to deplete macrophages in the first place? It might not seem like the most obvious place to look for patterning regulators…

DSE The idea initially came from the question of what would happen to unbound airineme vesicles. When xanthoblasts don’t meet the target melanophores, airineme vesicles detach from the airineme filaments and wander away. We knew the vesicles carry DeltaC and possibly other signalling molecules. Thus, these unbound vesicles should be somehow eliminated. The question then was what cell types can do that? One of the answers was the macrophage.

DP Plus we could constantly see macrophages wandering around in Dae Seok’s movies. How could we not try getting rid of them?

Is there anything in the macrophage-depleted fish to suggest that other patterning or morphogenetic events may be affected, or is this just tissue specific?

DP We’re only starting to investigate roles for macrophages as well as airinemes in other tissues. Because macrophages wander all over, you could imagine a whole variety of possibilities for diffusion-like dissemination—or regulated attenuation—of signals in other contexts. The more people look, the more interesting things macrophages seem to do.

Can you hazard a guess as to whether you think the macrophages actively select signalling targets or just wander around randomly?

DSE We have no idea at this time but my guess is macrophages constantly probe the environment, and signals from the airineme vesicles instruct macrophages to detect the targets and drop the vesicles while they are randomly wandering around.

DP Yes, because Dae Seok showed that targeting is specific to a subset of melanophores, there has to be some sort of airineme vesicle–melanophore recognition system. It will be interesting to see whether this is somehow instructive to the macrophage itself or whether the macrophage is simply trying to eat the vesicle and can’t manage to do so, and therefore the vesicle gets displayed at the cell surface often enough that it can stick to a melanophore as its carrier macrophage wanders along.

When doing the research, was there a particularly exciting result or eureka moment that has stayed with you?

DSE I do not have good explanation for it, but I had very strong gut feeling that there are something going on between macrophages and airinemes. So of course, my most exciting moment was when I saw the macrophages interacting with extending airinemes.

Xanthoblast airinemes. Eom and Parichy, 2017.

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

DSE Our original plan was to add additional mechanisms of airineme vesicle-macrophage interactions beside phosphatidylserine, as we have several additional candidates. We ran into technical problems for this first paper but are now working on it again.

What next for you following this work?

DSE We have many questions about airineme-macrophage interactions, for example, what other signalling molecules carried in the airineme vesicles and how macrophages know what the targets are. I’m very excited about using our new super-resolution microscopes – they will open up many new possibilities.

“Our new super-resolution microscopes will open up many new possibilities.”

And where will this discovery take the Parichy lab?

DP Of course we’ll continue to pursue the mechanisms underlying this signalling relay in zebrafish pigment pattern formation. And we’d really like to know how these and other interactions have evolved to generate the very different patterns we see in Danio and beyond. But you know, a lot depends on the interests of the grads and postdocs who come to my lab. I just try to foster an intellectual environment and provide the resources to let people explore and go where the science leads. When we decided eight years ago to invest in live imaging we didn’t know what we’d find and we certainly didn’t expect this. So I’d be hesitant to make firm predictions.

Finally – what do you get up to outside of the lab when you are not playing with fish?

DSE One of my favourite things to do is visiting local breweries. My wife and I have a Saturday routine of having a lunch at a brewery, and then heading to the lab.

DP Legos. I like to spend as much time as I can with my four year old and my wife. So we do a lot of legos, trains and construction. Helicopters are big, too.

Dae Seok Eom & David M. Parichy. A macrophage relay for long-distance signaling during postembryonic tissue remodeling. Science, 355(6331): 1317-1320

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