The Company of Biologists’ journals – Development, Journal of Cell Science, Journal of Experimental Biology andDisease Models & Mechanisms – offer Travelling Fellowships of up to £2,500 to graduate students and post-doctoral researchers wishing to make collaborative visits to other laboratories. These are designed to offset the cost of travel and other expenses. There is no restriction on nationality.
They really are an amazing opportunity for ECRs to learn new things, meet new people and travel to new places.
The current round of Travelling Fellowships closes on 31 May (for travel > 15 July 2019)
Butterfly eyespots are striking examples of animal patterning, but their developmental origins are still relatively poorly understood. A new paper in Development– the result of a collaboration between two Singapore-based labs – now combines CRISPR-Cas9 gene targeting with theoretical modelling to address the role of the Distal-less transcription factor in eyespot patterning. We caught up with co-first authors Heidi Connahs and Sham Tlili, and their respective supervisors Timothy Saunders (Assistant Professor at the Mechanobiology Institute, National University of Singapore) and Antónia Monteiro (Associate Professor at the Department of Biological Sciences, National University of Singapore and Yale-NUS College) to find out more about the story.
Antónia, Tim, Heidi and Sham (from left to right)
Tim and Antónia, can you give us your scientific biography and the questions your labs are trying to answer?
AM I was trained in population genetics and developmental biology, and have been trying to figure out how eyespot patterns develop on the wings of butterflies for most of my career, including investigating their origin, their evolution in number and also their ability to change in size with changes in environmental cues such as temperature. We have also been addressing how males and females use these eyespots in sexual signalling and in evading predators.
TS I was trained in theoretical physics but moved to developmental biology in 2007. Initially, I was interested in developing mathematical models, particularly of morphogen gradients, to understand how developing organisms reliably form, despite the presence of temperature variations and other natural fluctuations. I realised that access to experimental data was critical, and so I learnt Drosophila genetics and imaging during a post-doc at the European Molecular Biology Laboratory (Heidelberg, Germany). I have had my own lab since 2013 at the Mechanobiology Institute where we primarily focus on how complex organ shape emerges during development. We use both Drosophila and zebrafish embryogenesis to perform quantitative live imaging of organ formation. The lab also incorporates mathematical modelling and image analysis, to build a deeper understanding of how tissues form complex morphological shapes.
And Heidi and Sham: how did you each come to join your respective labs, and what drives your research?
HC During my PhD, I became fascinated with butterfly wing patterns and their eyespots in particular, so I was already reading a lot of Antonia’s work. And about 18 months before the end of my PhD, I attended a careers seminar where the speaker said that if there was someone we really wanted to post-doc with, we should just go ahead and contact them. So, I emailed Antonia right away and sent her my CV. I got lucky, because a semester before I graduated she contacted me and said she had funding for a postdoc and asked if I was still interested, and of course I said yes! Mostly what drives my research is a desire to understand how butterfly wing patterns develop, and specifically to identify the developmental processes that generate eyespot diversity.
ST I was trained as a physicist and enjoy doing both experimental work and modelling. I am especially interested in biological problems where spatial components play a role, and this necessitates the generation of quantitative maps of quantities such as cellular movements. I completed my PhD in Paris working on the mechanical properties of cell aggregates and cell monolayers. During the last year of my PhD, I had started to think about doing a post-doctoral work on more in vivosystems while keeping a quantitative biophysics approach. I discovered Tim through his website: he had just started his group at the Mechanobiology Institute. I was motivated by the highly interdisciplinary component of his group and by the fact that he was trained as a theoretical physicist developing experimental aspects in the lab. I was finally convinced to join his lab after meeting all the members of the lab during my interview.
How did your two labs come to collaborate on this project?
AM I think we started this collaboration via an undergraduate student, Trisha Loo, who joined Tim’s lab with an interest in modelling and was also doing some wet lab work in my lab. But the collaborative work really got going when we started getting very interesting Distal-less (Dll) crispants, such as butterflies with split eyespot centres, that really begged for modelling of potential morphogenetic processes that were differentiating those centres.
TS Antonia and I first met at a department faculty retreat. On the bus journey to the venue, we discussed patterning and complexity. We realised there was potential synergy between our labs work, and so we jointly took on Trisha to explore mathematical modelling of eyespot centre specification. The project then grew from there, with Heidi and Sham joining, who significantly pushed forward the science. Pleasingly, Tricia is now a PhD student in my lab, doing theory and image analysis – a large change from her undergraduate studies in biology!
The disruption of Distal-less exon 2 via CRISPR-Cas9 led to the differentiation of two eyespot patterns on each sector of the wings of the Squinting Bush Brown butterfly, Bicyclus anynana, instead of the expected single eyespots.
Can you give us the key results of the paper in a paragraph?
AM The paper shows that mutations in different exons of the gene Dll affect the morphogenetic process that differentiates the cells at the centre of an eyespot pattern. Close examination of these crispants suggests that Dll is involved in a reaction-diffusion process where continuous variation in Dll levels can lead to eyespots appearing on a wing sector, to eyespots splitting into two, to finally acquiring a tear-drop shape.
TS For me, a key result of the paper is the use of modelling to describe complex phenotypes. The crispants created a diverse range of phenotypes and our model was able to explain all these observations with minimal changes.
ST I would add that modelling the spatial component of the problem was critical in this case to make sense of the complex eyespot shapes obtained.
HC Our work shows that, in Bicyclus anynana, Dll is required for eyespot formation and it also appears to have other roles such as in regulating melanin pigmentation and scale development. The experimental and modelling work suggests that different eyespot phenotypes can be explained by variation in levels of Dll expression. Our findings lead us to conclude that, as Dll expression levels decrease, eyespots become smaller or disappear altogether. However, when Dll levels increase, this leads to the duplication of eyespot centres resulting in extra eyespots developing on the wing.
Your experiments demonstrate exon-skipping/gain-of-function phenotypes from certain CRISPR-induced mutations in Dll. Do you think this is a widespread issue in the field?
AM This exact same Dll exon-skipping phenomenon has recently been shown in sepsid flies, leading to the occurrence of ectopic sternite brushes (Rajaratnam et al., 2018), and it has also been documented in other studies using cell lines (Kapahnke et al., 2016; Lalonde et al., 2017; Mou et al., 2017). What is still unclear to us is the extent that natural variation in exon skipping takes place in natural populations to alter gene function from a reduced or loss-of-function to a gain-of-function outcome.
HC There does seem to be a growing awareness now of the potential for CRISPR to induce exon skipping, and also the importance of sequencing not only the genomic DNA but the mRNA from crispants.
Why might Dll lacking exon 2 induce such weird and wonderful phenotypes?
AM: We still don’t know. The Dll truncated protein, lacking exon 2 but still containing a functioning homeobox, might be more stable and resistant to degradation, mimicking a gain-of-function phenotype.
HC Or perhaps the truncated 5′UTR increases the translation efficiency of the protein. More research is needed to understand the properties of this truncated version of Dll, and this will probably require using Drosophila transgenics to express the truncated protein.
Your theoretical model can replicate eyespot formation and Dll mutant phenotypes: do you think it might be extended to a general mechanism for how organisms make spots?
HC, ST, TS & AM Perhaps, but morphogenetic processes in the insect epidermis are largely considered a bit different from those taking place in the skin of mammals such as leopards or fish. In vertebrates, cells containing pigment molecules actually move about to take their place in a field of cells, whereas in insects, the cells are primarily differentiated in situ via interpretations of local morphogen gradients, etc. We are more excited with the idea that the models developed in this paper could help us understand limb specification in the thorax of insects via similar mechanisms.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
HC When I found my first crispant which had the ectopic eyespots, that was a big eureka moment for me. Also, when I got back some sequencing results and realized that exon 2 had been spliced out, that was very exciting. I had to look at the results multiple times before I could believe it.
ST We first investigated how the classical Gierer-Meinhardt activator-inhibitor model could explain the Dllcrispants. Although this model was giving interesting results, we were struggling to find a unique set of parameters that could explain the ensemble of the crispants phenotypes. Then, Antonia motivated us to look for a model where the morphogens are anti-colocalised instead of colocalised – to be in closer agreement with the experimental data. Shortly after implementing the Gray-Scott model, I found that this model easily generated phenotypes strikingly similar to the crispants phenotypes. This was a very satisfying moment.
And what about the flipside: any moments of frustration or despair?
HC Yes, I definitely had a lot of those moments too. Initially when I started doing the CRISPR experiments, we had a lot of trouble getting it to work and so for several months there was this heart-pounding moment each time I went to check if there were any butterflies with interesting wing phenotypes and it was very disheartening to see normal looking butterflies. Eventually we realized it was the cas9 protein, and once we ordered a new one, the experiments finally worked: that was a huge relief!
ST Maybe after sending the first draft versions, when some readers were not reading the modelling part of the story and thinking the crispants phenotypes were just not making any sense. I think that, in this story, the modelling and experimental aspects are tightly entangled. These criticisms made me realise how important it is to make the model as pedagogical as possible in a highly interdisciplinary context.
I had to look at the results multiple times before I could believe it
So what next for you two after this paper?
HC I am now focusing on targeting the enhancers of Dllusing CRISPR so we can try to understand the origins of butterfly eyespots by identifying pleiotropic enhancers. This has been quite a difficult project to work on as using CRISPR to target enhancers comes with its own unique set of challenges, but we are now starting to get some preliminary results that look quite promising!
ST I just moved back to France after three great years in Tim’s group. I started a new post-doc on embryonic stem cell aggregates mimicking early mammalian development. This project will combine tissue mechanics and potentially morphogen reaction-diffusion again!
Where will this work take the Saunders and Monteiro lab? Any plans for further collaborations?
AM Would love to collaborate further with Tim’s lab. The insights they provided into our crazy looking crispant mutants were amazing. We are now mutating other genes that are also expressed in eyespots and we are observing similar eyespot splits, etc. The reaction-diffusion process seems to involve multiple genes and we might need to model the action of these genes as well.
TS This work has nicely gone alongside our lab’s study on complex shape emergence. Both patterning and mechanics play important roles in organ formation. In the future, we are hoping to integrate reaction-diffusion modelling with mechanobiology. Of course, I’d be delighted to work further with Antonia – the butterfly is an awesome system.
Finally, let’s move outside the lab – what do you like to do in your spare time in Singapore?
AM I love to hike through Singapore’s forested parks on the weekends. My husband and I usually do a 10 km trek that ends in VivoCity – a mall with many lunch options!
TS I climb with my wife regularly and much of my weekend is spent exploring Singapore with our 6-year-old daughter.
HC Usually on the weekends I enjoy relaxing with my boyfriend and visiting different science/art/nature-themed attractions or exhibitions in Singapore. I also enjoy watercolour painting and keeping aquarium fish.
ST In Singapore, I really enjoyed walking in the parks along the harbour which gave a really nice view of the sea, Indonesian islands in the horizon and all these coloured container ships coming from all over the world.
The Clark and Monaghan Laboratory at Northeastern University are seeking a postdoc for an NIH-funded project to image acetylcholine in the mouse peripheral nervous system. This project is an exciting opportunity to use novel fluorescent imaging to image neural communication in vivo to understand nerve-organ interactions. Experience in mouse neurobiology and in vivo imaging is desirable.
Interested candidates should email a cover letter describing their research background and CV to j.monaghan@northeastern.edu.
We are happy to announce the launch of preLists, a new initiative within preLights where early-career researchers curate lists of preprints for the community. These lists follow two main themes: preprints on a specific topic or preprints which have been presented at scientific meetings. preLighters can also add brief one-liner summaries to each preprint, and topic-specific lists continuously get updated as new studies come out. So what was the thinking behind starting this new experiment?
When we launched preLights fifteen months ago, one of our main aims was to facilitate preprint commenting. With four-hundred preLight posts published so far, and over a third of them containing comments from authors, we hope to have played a small role in promoting discussion. We also wanted preLights to become a platform that helps scientists navigate the ever-increasing preprint literature. While preLighters contribute new posts each week to the website, due to the sheer volume of newly posted preprints, they can only cover a fraction of the important new studies deposited on bioRxiv. With preLists, we want to provide an even richer selection of interesting work, grouped into well-defined topics, including technologies (e.g. preprints on CRISPR technology, biomolecular NMR or microscopy) or narrower research areas (e.g. preprints on zebrafish immunology, cellular metabolism and mitochondria or antimicrobials).
In addition, preLists will also feature preprints that were presented at conferences, providing a useful service for both attendees as well as those who were not able to make it to the meeting. An encouraging trend is that researchers are more willing to discuss their non-peer-reviewed work at scientific meetings because they have posted, or are in the process of posting, a preprint on the findings. With well over a hundred preLighters who work in different fields, we hope to be able to cover a good number of conferences. For some first examples, take a look at the preLists from the BSCB/BSDB Meeting 2019, Biophysical Society Annual Meeting 2019, 1st Crick-Beddington Developmental Biology Symposium 2019 and the ASCB/EMBO Meeting 2018.
We believe that preLists will nicely complement preLight posts, and to further support this, the two will cross-link to each other on the website. We hope you will find this new feature on preLights useful, and if you would like to take a part in writing preLights and curating preLists, apply to join our team!
Since the Young Embryologist Network (YEN) was established in 2008, its annual conference grows from strength to strength, and thanks to the introduction of travel grants in 2017, the YEN meeting now welcomes an international delegation of attendees studying a variety of developmental problems. YEN conferences are special because they organised by early-career researchers for early-career researchers, which creates a distinctive atmosphere of support, collaboration and discussion. I attended the YEN meetings many times during my PhD and PostDoc, and last week I was happy to join again on behalf of Development to celebrate the 11th annual conference. It was the second year that the one-day event was hosted at the Francis Crick Institute, where it featured three invited speakers, 12 selected speakers, around 30 poster presentations and a panel discussion on careers.
One of the common themes of the day was morphogenesis. Florence Giger presented on the dynamics of forebrain neurulation, using live-imaging to investigate myosin localisation during zebrafish neural tube closure. At the opposite end of the embryo, Lewis Thomson explored the dynamics of the zebrafish presomitic mesoderm, which decreases in every dimension during axis elongation. Similarly, Toby Andrews translated morphology into quantitative data in order to understand axis elongation in amphioxus, and used lineage-tracing experiments to identify axial progenitor cells. Sally Lowell (one of the invited speakers) also quantified morphogenesis and, in her opening talk, revealed new tools to measure epithelial formation in the primitive streak1. Sally explained how morphological changes to tissues can help cells commit to changes in gene expression by manipulating the 3D organisation of epiblast stem cells with micropatterns2. You can find out more about her work in “The people behind the papers” interview with Sally, and her colleagues and collaborators, here on the Node.
In recent years, the YEN conferences have hosted a celebration of the memory of Sammy Lee, a prominent scientist in the field of IVF and a great supporter of early career researchers. This year’s Sammy Lee Memorial Lecture was given by keynote speaker Takashi Hiiragi, who returned to the role of forces during development by demonstrating the role of cortical tension in mouse blastocoel self-organisation and fluid cavities in regulating tissue size3,4. Also working in the pre-implantation embryo, Claudia Gerri showed a conserved molecular cascade during blasocyst formation in the mouse, human and cow. Claudia received the Sammy Lee Award for her talk, which is presented to one of the speakers for an “outstanding piece of research”. The medal was awarded by Sammy’s wife Karen Lee, who was one of the judges along with David Wilkinson and Karen Liu. Also keeping in early mammalian development, Sergio Menchero explained the role of Notch signalling in driving the transition for pre-implantation differentiation5, and you can read the story behind the paper here on the Node. Notch also featured in Valeria Scagliotti‘s talk on the pituitary gland, where she showed that overexpression of Dlk1 increases the proliferation of anterior pituitary gland progenitors.
Karen Lee (left) presented Claudia Gerri (right) with the Sammy Lee Award. The bronze medal shows Sammy’s smile on the front, emphasising the humanity and his ability to communicate. The reverse shows an oocyte with the name of Sammy’s charity REGEN injected into its nucleus. Photo credit: Eva Izmilye.
Many of the speakers are interrogating how cells make decisions. Systems biologist and invited speaker Jordi Garcia-Ojalvo proposed using recurrent neural networks to explain how gene regulatory networks can achieve “memory” of their past multidimensional inputs during differentiation6. Noelia Muñoz-Martín discussed the role of Myc7 and MEIS transcription factors in murine cardiac development, and showed that knockdown of MEIS genes affects calcium signalling. Joana Silva discussed the requirement of the mRNA capping enzyme, CMTR1, during pluripotent cell differentiation in embryoid bodies, and Eleni Chrysostomou presented her research in the hydroid, Hydractinia using fluorescent reporter lines to unpick the roles of SoxB family genes in the differentiation of stem cells to neurons. Sergi Junyent Espinosa revealed the neuronal-like properties of stem cells, which utilise synapse-like interactions to find their niche. Andreas Sagner revealed the dynamics of mouse spinal cord development, using single-cell sequencing data from the developing neural tube8 and won a runner-up prize for his talk. The second runner-up Can Aztekin discussed his work — also involving single-cell sequencing — to identify a new population of cells (ROCs) required for Xenopus tail regeneration9.
The talks concluded with a careers panel discussion featuring Marta Gritti (Senior Editor at EBioMedicine, Elsevier), Andy Powell (Crick, GSK Biomedical LinkLabs), Graham Mills (Co-founder and Managing Director of Techspert), and Silvia Santos (Group Leader at the Crick). The panel talked about their career paths and the responsibilities in their current roles, as well as answering questions from the audience about job security, the necessity of moving abroad, the value of internships, the first steps to starting a new career, and opportunities to return to academia. Overall, the panel agreed that we should do what we like doing, for as long as we want — or are able — to do it!
For the first time, all attendees were invited to vote for their favourite posters. The first place poster prize was awarded to Yan Liang for her poster on appendage repression during the evolution of crustaceans and insects — well done! Congratulations to the runners up: Cato Hastings for her poster on mathematical modelling of the primitive streak and Andrea Szydlo-Shein for her poster on neuronal organisation in the zebrafish visual system.
Participants and award winners. Back row, left to right: Andreas Sagner , Can Aztekin, Nikolaos Angelis, Claudia Gerri, Andy Powell, Karen Lee, Karen Liu, Yan Liang, Sally Lowell and Takashi Hiiragi. Front row, left to right: Cato Hastings, Andrea Szydlo-Shein, Silvia Santos and Marta Gritti. Photo credit: Eva Izmilye.
Throughout the conference I was impressed with the good pace of the talks, the openness of speakers to share unpublished data and new ideas, and their receptiveness for feedback on their research. The conference finished with a drinks reception and the opportunity for the attendees to relax and discuss the topics of the day. Congratulations to the prize winners, thank you to the speakers and, most importantly, a huge thank you to the YEN committee for organising the meeting and all their hard work behind the scenes.
If you’d like to know more about the science during the meeting, check out the Twitter @YEN_community, the hashtags #YENconf2019 & #YEN2019, or see the associated preList!
1Blin et al. NesSys: a novel method for accurate nuclear segmentation in 3D. bioRxiv 2018. doi: https://doi.org/10.1101/502872
2Blin et al. Geometrical confinement controls the asymmetric patterning of brachyury in cultures of pluripotent cells. Development 2018; 145: dev166025. doi: 10.1242/dev.166025
3Chan et al. Hydraulic control of embryo size, tissue shape and cell fate. bioRxiv 2018. doi: https://doi.org/10.1101/389619
5Menchero et al. Transitions in cell potency during early mouse development are driven by Notch. eLife 2019;8:e42930. doi: 10.7554/eLife.42930
6Gabalda-Sagarra et al. Recurrence-based information processing in gene regulatory networks. Chaos 2018; 28(10):106313. doi: 10.1063/1.5039861
7 Noelia Muñoz-Martín et al. Myc is dispensable for cardiomyocyte development but rescues Mycn-deficient hearts through functional replacement and cell competition. Development 2019; 146: dev170753 doi: 10.1242/dev.170753
8Delile et al. Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord. Development 2019; 146: dev173807 doi: 10.1242/dev.173807
9Aztekin et al. Identification of a regeneration-organizing cell in the Xenopus tail. Science 2019; 364(6441), 653-658. doi: 10.1126/science.aav9996
In this episode from our series exploring 100 ideas in genetics, we’re taking a trip to London with William Bateson and discovering that the famous story about him reading Mendel’s paper on the train might not be all that it seems.
Plus, we seek the secrets of snapdragons, and learn how to build an army of MinIONs.
If you enjoy the show, please do rate and review and spread the word. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com
Vascular development critically involves pruning, which helps to remodel an immature network containing excess microvessels into a mature and functioning one. The mechanisms of vascular remodelling and the relationship between the endothelial cells and the other cell types with which they are closely associated are, however, currently poorly understood. A new Development paper now demonstrates a crucial role for oxygen sensing by astrocytes in vascular remodelling of the mouse retina. We caught up with the two-author team behind the paper: research associate Li-Juan Duan and her supervisor Guo-Hua Fong, Professor of Cell Biology at the University of Connecticut Health Center, to hear more about the story.
Li-Juan (L) and Guo-Hua (R)
Guo-Hua, can you give us your scientific biography and the questions your lab is trying to answer?
GHF After graduating from Hangzhou University in 1982, I attended the PhD Program in Biochemistry at the University of Illinois at Urbana-Champaign. My interest in angiogenesis began during my postdoctoral training with Martin Breitman and Janet Rossant in Toronto. After reporting the finding that Flt-1 (VEGFR1) was a negative regulator of vasculogenesis, I set up my first lab at the University of Western Ontario before relocating to where I am now (UConn School of Medicine).
Early in my career as a developmental biologist, I used mouse embryos to study vasculogenesis and angiogenesis, but since about a decade ago I have been primarily focused on mouse retinas. At the molecular level, my current focus is on oxygen sensing and hypoxia signalling because growth of blood vessels is mostly (although not exclusively) an adaptive response to tissue hypoxia. Molecules of interest include hypoxia inducible factor (HIF) 2α (also called EPAS1), a transcription factor stabilized by hypoxia that activates the expression of angiogenic factors, and prolyl hydroxylase domain (PHD) proteins (EGLNs), which sense oxygen and destabilize HIFα proteins by oxygen-dependent prolyl hydroxylation reactions.
Li-Juan: how did you come to join the Fong lab and what drives your research?
LJD With a few exceptions, almost all tissues are vascularized. Mechanisms ensuring that blood vessels are formed at the right place, right time and with right configurations are very fascinating to me. Dr Fong’s lab investigates the molecular and cellular mechanisms regulating blood vessel growth, and has contributed significantly to this rapidly advancing field. So the opportunity to be a part of team and contribute to the exciting discoveries is a major driving force for my research. Also, the potential that knowledge generated from these studies might improve angiogenesis therapies is quite energizing.
What was known about the link between astrocytes, endothelial cells and oxygen in retinal angiogenesis prior to your work?
LJD & GHF The retinal astrocytic network is situated just beneath the vascular network on the inner retinal surface, with foot processes from astrocytes wrapping around vascular endothelial cells (ECs) and supporting their survival. It was thought that the astrocytic network forms before retinal angiogenesis and serves as a template for the subsequent vascular growth. The newly formed vascular network exhibits a honeycomb pattern consisting of almost uniformly sized capillaries, separated by roundish non-vascular tissues. To gain circulatory functions, the nascent vascular plexus undergoes active remodelling to form a tree-like pattern with large trunks and smaller branches. During this process, excess microvessels are pruned away. One widely held but never formally documented view is that oxygen causes pruning by activating EC apoptosis. The other theory is that T cells interact with ECs to cause pruning. In both theories, astrocytes were not suspected to play a role in remodelling.
Can you give us the key results of the paper in a paragraph?
LJD & GHF We found that retinal astrocytes respond to changes in tissue oxygenation mostly through prolyl hydroxylase domain (PHD) protein 2, an enzyme known to use O2 and oxoglutarate as substrates to hydroxylate transcription factors HIF1α and HIF2α, causing them to degrade. In neonatal mouse retinas, HIF2α is essential for the expansion of the astrocyte population owing to its role in maintaining astrocytes in immature and proliferative states. Angiogenesis imposes a physiological limit to the abundance of retinal astrocytes by causing O2-dependent HIF2α degradation. As such, a proportion of the numerous capillaries generated through robust angiogenesis is unable to acquire astrocytic support and undergoes regression (pruning). In support of this theory, targeted disruption of the Phd2 gene in astrocytes led to accumulation of HIF-2α protein, expansion of retinal astrocyte population, and persistence of extra microvessels. When astrocyte growth was directly stimulated by intraocular injection of PDGFA into the eyes in wild-type mice, vascular pruning was also blocked. Based on these findings, we conclude that oxygen- and PHD2-dependent astrocyte growth arrest is a feedback mechanism to prevent runaway vascularization.
Capillaries are pruned off near arterial branches (green) in wild-type mice (left) but persist in mice deficient for PHD2 in astrocytes (right).
Do you have any ideas about what happens downstream of Phd2/HIFα to induce differentiation in astrocytes?
LJD & GHF We don’t really know yet, but there may be some candidates. HIF2α is known to promote the expression of Oct4, a transcription factor important for the maintenance of stem cell identity. While the astrocyte precursors may not be exactly stem cells, they might share certain features related to stem cells. So it seems to us that HIF2α-dependent expression of Oct4 might be a potential mechanism by which HIF2α helps maintain astrocytes at precursor and immature states. At this point, this idea remains highly speculative but it could be a reasonable starting point for the next step.
Does your work have any relevance to pathological contexts?
LJD & GHF Capillary dropout is a common feature in early stage diabetic retinopathy. The prevailing view at present is that capillary dropout starts with apoptosis of pericytes, a type of supporting cell that adheres to the endothelial cells. Our finding of a crucial role for astrocytes in capillary maintenance raises the question of whether the adverse effects of diabetes on retinal astrocytes might also contribute to capillary dropout? If so, the retinal astrocytes could be a novel therapeutic target for improving retinal vascular stability. In this regard, it may be interesting to note that there’s a publication indicating that retinal astrocyte abundance is decreased in diabetic rats (Ly et al., 2011).
Our findings are also related to retinopathy of prematurity (ROP), a disease associated with premature birth. Whereas pathological mechanisms underlying ROP are complicated and go beyond oxygen alone, sensitivity to oxygen is nonetheless an important component. Thus, targeting astrocytic PHD2 could provide a novel opportunity for minimizing the consequence of ROP.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
LJD I guess the moment was when I found that simply injecting PDGFA into the eyes caused both expanded astrocyte populations and persistence of more microvessels, whereas injection of PBS into the contralateral eyes didn’t. These results indicated that increased astrocyte abundance alone was sufficient to block vascular pruning.
And what about the flipside: any moments of frustration or despair?
LJD Sure, plenty of them. This was actually a lengthy project. While I’ve been working on other projects in parallel, this project was on and off for nearly 6 years. While we observed the overcrowded vascularization at the very start of the project, it was rather challenging trying to explain why. Naturally, our initial attention was on VEGF, and lots of time was wasted in that direction. We couldn’t just publish overcrowded capillaries without being able to explain the underlying mechanism, so the project was really stuck for a long time until one day we realized perhaps it was simply the number of astrocytes itself rather than any specific molecule. We also ran into a lot of technical difficulties. One example was growing primary retinal astrocytes: because they could not be expanded too much, we had to pool the cells from a very large number of retinas. These are just two of the many examples.
Where will this work take the Fong lab?
GHF We will be pursuing several directions. We are interested in identifying the potential targets downstream of PHD2/HIF2α by RNA-seq, and then assessing their functionality in retinal vascular development by CRISPR/Cas9-mediated knockout. We will be also looking into potential interaction between HIF2α with a transcription factor called Nr2e1 or Tlx. We are interested in finding out whether the two transcription factors collaborate in retinal astrocytes in controlling astrocyte differentiation. We are also interested in investigating if PHD2 deficiency in astrocytes also confers protection to capillaries in disease models such as oxygen-induced retinopathy and diabetic retinopathy.
For whatever reasons, the vascular biology community has paid very little attention to astrocytes. The major focus is still on endothelial cells, and their interaction with pericytes, vascular smooth muscle cells and leukocytes. In fact, we also stumbled into this subject accidentally, but we increasingly appreciated the opportunity that fell upon us as we began to realize the importance of this cell type to vascular growth, stability and function. Because so little is known about astrocytes in the context of vascular biology, there are plenty of exciting findings to be made. In the coming years, we will be devoting much of our efforts trying to understand how astrocytes communicate with endothelial cells to regulate various aspects of vascular homeostasis.
Because so little is known about astrocytes in the context of vascular biology, there are plenty of exciting findings to be made
Finally, let’s move outside the lab – what do you like to do in your spare time in Connecticut?
LJD Spring is beautiful in Connecticut. I enjoy growing flowers, watching them blossom in the morning. There are also plenty of water reservoirs with hiking trails open to the public. I enjoy taking long walks around the lakes on weekends, especially in the spring and fall.
GHF Connecticut may not be a frequently talked-about place but it is not exactly in the middle of nowhere either – in fact the UConn School of Medicine campus is just about 2 h from Boston and New York, respectively. So it’s relatively convenient to spend a day in the weekend visiting museums or friends there, or even shopping. Also Connecticut is full of very long hiking trails at different levels of difficulty, so one could spend hours (or days if they like) hiking to fairly distant locations, with some leading all the way into neighbouring states.
The Poché Lab is seeking a highly motivated postdoctoral research associate/fellow with experience in mouse retinal developmental biology, regeneration, and transcriptome analysis. This position is focused on the study of the molecular mechanisms blocking mammalian Müller glial cell (MG)-mediated retinal regeneration. Our long-term goal is to determine whether the mouse retina retains latent regenerative potential, akin to other vertebrates species, and whether we can genetically “awaken” that potential to restore sight.
Special emphasis will be placed on the investigation of MG transcriptional reprogramming to a progenitor-like state. Preference will be given to candidates with a strong background in mouse genetics and in techniques to probe the retinal transcriptome and epigenome. This expertise should ideally include next gen sequencing (RNA-seq, ChIP-seq, ATAC-seq, single cell-seq, etc.) data analysis.
The Poché lab employs a multi-disciplinary approach utilizing genetic loss- and gain-of-function experiments, fate mapping, gene therapy, molecular biology, and live retinal confocal microscopy. We are housed in the Department of Molecular Physiology and Biophysics at Baylor College of Medicine (BCM). Located in the Texas Medical Center, the largest medical center on the world, BCM postdocs have a tremendous amount of technical and intellectual resources at their disposal.
In your application, please include a cover letter, current CV, and contact information for three references. Application review will begin immediately and will continue until the position is filled. Please contact Dr. Poché at poche@bcm.edu.
Two BBSRC-funded post-doctoral positions will become available as part of a collaboration between the Universities of St Andrews and Aberdeen:
evolutionary developmental biologist (three years, currently proposed start date 1 January 2020, mostly based in St Andrews)
molecular developmental biologist/RNA molecular biologist (three years, currently proposed start date 1 January 2020, mostly based in Aberdeen)
The collaboration will study evolution of Wnt signalling and alternative transcript expression, splicing and function.
A BHF-funded position will become available in Aberdeen:
theoretical biologist/bioinformatician/mathematician/physicist (three years, flexible start date, e.g. 1 August 2019)
This project will model and simulate Gene-Regulatory Networks controlling heart muscle (cardiomyocyte) cell differentiation as part of a BHF Programme Grant in collaboration with two laboratory developmental biologist already working with stem cells and embryos, respectively.
These positions have not yet been officially advertised. At this early stage, informal enquiries are encouraged to Dave Ferrier <dekf@st-andrews.ac.uk> and Stefan Hoppler <s.p.hoppler@abdn.ac.uk>. Official announcements with more details will follow as soon as possible.
With over 10,000 votes cast, almost 6,000 people viewing the galleries and a new record for daily page views on the Node, we can now announce the winners of our inaugural calendar competition. We were blown away by the quality of the entries – 62 images of all kinds of cells, tissues and embryos. Check out the original postto see all the entries – as you’ll see, so many beautiful images missed out, and we’d like to thank everyone who took part.
So, category by category, here are the 12 winners who will make the final print calendar, and below them a full vote rundown (there were quite a few close calls!). We’re aiming to take the calendars with us to two upcoming meetings: SDB in Boston in July and the European Developmental Biology Congress in Alicante in October. Come grab one if you’re going.
Mammals
1st place: Light up
By Paul Gerald Layague Sanchez
(EMBL Heidelberg)
E14.5 mouse embryo labeled for cartilage (Sox9-GFP, in biop-SpringGreen) and vasculature (highlighter ink circulated by injection in a blood vessel, in mpl-magma). Vasculature “lights up” the embryo, including within the developing bones of the limbs. Image taken using a microscope kindly sponsored by Zeiss during the 2018 Embryology Course at the Marine Biological Laboratory in Woods Hole, MA.
2nd place: Human neuron
By Nicholas Gatford
(Institute of Psychiatry, Psychology and Neuroscience, King’s College London)
Super-resolution image acquired via structured illumination microscopy of a single developing human neuron ectopically expressing the autism risk gene neuroligin-4X with enlarged growth cones. F-actin is in green, HA-tagged neuroligin-4X is in magenta, doublecortin is in cyan confirming its neuronal identity, and the nuclear marker DAPI is in grey.
Zebrafish
1st place: Zebrafish head
By Oscar Ruiz
(Department of Genetics MD Anderson Cancer Center)
Transgenic zebrafish (Danio rerio) larva expressing red fluorescent protein in the developing mouth and olfactory epithelium. A subset of cells also express a construct that labels actin filament with green fluorescent protein. DAPI (blue) is used to label DNA in the nuclei of all cells.
2nd place: Zebrafish gills
By Philippa Carr
(Bateson Centre, University of Sheffield)
Developing gill vasculature in a 120h old zebrafish and also features the heart. This image was taken using lightsheet microscopy in two transgenic lines, one that marks the endothelial actin and the other marks the endothelial nuclei. After acquisition it was processed as a colour coded depth projection.
Vertebrate variety show
1st place: Alligator
By Daniel Smith Paredes
(Department of Geology and Geophysics, Yale University)
Alligator mississipiensis embryo at stage 13-14 immunostained against Myosin heavy chain showing the developing muscles and (red) and neurofilament labeling axons of nerves.
2nd place: Chicken embryo
By Laurel Yohe
(Department of Geology and Geophysics, Yale University)
Stage 35 chicken embryo, cleared and immunostained for DAPI (orange) and Pax3 (cyan) demonstrating the developing neural crest and spinal cord. Image was taken on the Nikon AZ-C2 macro-confocal with image analysis performed in Imaris. Image was taken in collaboration with Andrea Attardi at the Max Planck Institute of Molecular Cell Biology and Genetics during the Woods Hole 2018 Embryology course.
Drosophila
1st place: Drosophila ovary
By Yujun Chen
(Kansas State University Division of Biology).
*Yujun also wins the ‘Star of Instagram’ award for most-liked post (we posted all 62 individually from Development’s account!), and the image is the new profile pic*
Drosophila whole ovary stained for f-actin (Red), nuclei (Cyan) and actin (Green).
2nd place: Metallic flight
By Marisa Merino
(Department of Biochemistry, University of Geneva)
Drosophila mutant showing a decreased eye size compared to wild type. This line is not able to generate descendants with wild type flies.
Invertebrate variety show
1st place: Bobtail squid
By Martyna Lukoseviciute
(Weatherall Institute of Molecular Medicine, University of Oxford)
Live Hawaiian Bobtail Squid (Euprymna scolopes), stained with vital dyes (CellMask, LysoTracker and Hoechst) to understand its cellular and sub-cellular organisation during development. Blue is labelling cellular nuclei, green – cell plasma membranes and red – lysosomes that are important for cellular waste removal. This species is a candidate model organism that yet holds many answers to developmental biology questions, such as nervous system and eye development. The image was taken during the MBL 2018 Embryology Course with the confocal microscope provided by Zeiss. Animals were supplied by the cephalopod researcher Carrie Albertin.
2nd place: Hydractinia
By Indu Patwal
(Centre for Chromosome Biology, National University of Ireland Galway)
DIC and fluorescence image of Hydractinia male sexual (left) and feeding polyps (right) on a chitin bed. Chitin is shown in green. Noncycling cells probed with cyclin-dependent kinase inhibitor (CDKI) are shown in yellow, which are mainly in nematocytes, male gonophore, and gastrodermis.
Plants, Fungi and Choanoflagellates
1st place: Arabidopsis lateral root
By Robertas Ursache
(University of Lausanne, Switzerland)
The development of a lateral root in Arabidopsis thaliana. The sample has been cleared and stained with Calcofluor White to outline the cell walls and the green fluorescent nuclei represent a protein expressed specifically in the outer cell layer of developing lateral root.
Art and illustration
1st place: The yin and yang of developmental patterning
By Beata Edyta Mierzwa
(Ludwig Institute for Cancer Research and the University of California, San Diego, and www.beatascienceart.com)
This drawing illustrates the remarkable self-organization capacity of cerebral organoids that allows them to recapitulate human brain development in vitro. Each color represents a different type of cell, and the dorsal and ventral areas are separated by a defined boundary – like a yin and yang symbolizing the balance between distinct but complementary entities.
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