A discussion of our recent paper: Hicks et al., ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs. Nature Cell Biology, January 1 2018

Approaching the problem of muscle disease with cell replacement therapy

We will all be affected by a muscle wasting disease in our lifetime. The ability to transplant a stem cell into skeletal muscle that can self-renew to make more of itself, and respond to injuries as they arise, could provide a continuous source of cells to replace damaged muscle tissue throughout our life span.

Stem cell replacement is especially imperative to the largest childhood muscle disorder Duchenne Muscular Dystrophy, where a genetic mutation in muscle cells results in muscle death and progressive weakness for about 20,000 new cases each year worldwide. The muscle stem cells of Duchenne patients also contain the genetic mutation rendering them ineffective at repairing the muscle as it dies.

Human pluripotent stem cells (hPSCs) have the potential to differentiate into any cell type making them attractive candidates for drug screening and cell replacement therapies.  Indeed, hPSCs are already being used in the clinical setting and in preclinical models for diseases ranging from macular degeneration to cardiac myopathy, diabetes, and Parkinson’s disease.

Using induced pluripotent stem cells taken from Duchenne patients, our team at UCLA has devised a strategy to permanently correct the genetic mutation in Duchenne cells with CRISPR/Cas9 (Young et al., 2016). Our current challenge is delivering the corrected cells back to the affected patients. Correction of patient-derived induced pluripotent stem cells ex vivo and subsequent differentiation to human muscle stem cells offers one potential route for restoration of dystrophin after transplantation.

Pluripotent stem cells must mature through stages of development similar to natural cells to function like in vivo cell counterparts

When we first started this project, the only way to make skeletal muscle from human pluripotent stem cells was using virus to overexpress muscle transcription factors, which lacks precision in truly recapitulating the cells created from human development.  For example, during regeneration a muscle stem cell must be able to simultaneously make more of itself and differentiate to make new muscle fibers. Forced viral overexpression of genes may limit proper stem cell dynamics.

Directed differentiation is a process that follows developmental cues to allow hPSCs to form any cell lineage. It has been well established that for many cell lineages, including cardiac muscle cells, the progeny made from directed differentiation of hPSCs are immature and do not function as well as adult cells.  While there are a number of recent papers that use directed differentiation to produce muscle cells from hPSCs, no papers have benchmarked the cells being made to those derived from human muscle tissue.  In this study we set out to follow human development of muscle more closely to create a muscle cell similar to those naturally found within the body as a means to obtain a cell for replacement therapy.

We set out to determine the myogenic potential of muscle cells created from hPSCs (termed skeletal muscle progenitor cells or hPSC-SMPCs), compared to the muscle cells from human embryonic, fetal, and adult myogenesis (Figure 1).  Cells were dissociated from human tissues and their ability to fuse to form myotubes were compared to hPSC-SMPCs.  We found that hPSC-SMPCs made from two commonly used directed differentiation protocols created muscle in a dish that looked and fused similarly to the embryonic muscle from primary myogenesis.

When we transplanted hPSC-SMPCs into mice, we found they did not engraft very well. Alternatively, human fetal cells from secondary myogenesis were very capable of forming new muscle in vivo.

Directed differentiation to skeletal muscle produces a number of heterogeneous cell types, and we thought that enrichment for the best-known adult satellite cell marker, known as NCAM or CD56, would improve our ability to engraft cells. However, NCAM enrichment failed, and if anything was slightly worse than unsorted hPSC-SMPCs.  This was an example of the failure of applying adult tissue paradigms to immature hPSC progeny from directed differentiation. It was like comparing apples and oranges. After many months, we were no closer to making an engraftable cell from hPSCs.

Development guides the enrichment of functional muscle progenitors from hPSCs

We thought about how muscle cells created from hPSCs functionally resembled the morphology of muscle from embryonic myogenesis, but how muscle cells from fetal myogenesis were able to engraft very well. We hypothesized rather than trying to immediately create an adult cell from hPSC-SMPCs, we still had to direct their differentiation through more stages of human myogenesis and maturation.

The first thing we needed to do was determine the maturational identity of our cells.  We performed RNA sequencing of NCAM+ hPSC-SMPCs, and compared gene expression to NCAM+ fetal week 17 SMPCs.  As predicted, we found that hPSC-SMPCs were more embryonic than fetal SMPCs, but they were also more differentiated with increased expression of genes such as myogenin. This genetic profile supported the idea that the cells we were making more closely resembled the primary myogenic wave in which founder cells differentiate to establish the first muscle fibers, before a second wave of muscle progenitors arise that go on to make the adult satellite cells. Because NCAM is expressed by many cells in directed differentiation, including immature mesoderm cells, neurons, and more differentiated myoblasts, we hypothesized this was also a reason for the lack of improved enrichment and engraftment in vivo by NCAM+ hPSC-SMPCs.

We turned to our colleagues, Drs. Denis Evseenko and Ben Van Handel at the University of Southern California, who were facing a similar challenge with directed differentiation of hPSCs to chondrocytes. To identify unique genes of human chondrocytes, Dennis and Ben had performed a Weighed Gene Analyses (WGCNA) on five types of human fetal tissues including bone, cartilage, tendon, ligament, and skeletal muscle. In the fetal muscle dataset, they identified over 400 cell surface genes and transcription factors unique to skeletal muscle (Furgeson et al., 2018). A fun part of this project was pilfering through the gene data, making candidate lists, performing PubMed searches and refining cell surface genes to about 20 candidates.  We then set up directed differentiations to evaluate new and known cell surface marker expression from the candidates in hPSC-SMPCs. From this screen, we found enrichment for ERBB3 or NGFR produced the most myogenic cell in vitro with robust fusion ability.

Eureka! ERBB3 or NGFR demarcate myogenic cells in human development

We wondered about the relevance of ERBB3 or NGFR in human development and whether these two cell surface markers demarcated the same cell or different cells at stages of development. We decided to analyze and enrich for cells based on ERBB3 or NGFR co-expression in primary and secondary myogenesis. We identified multiple subpopulations based on the expression profiles of these two cell surface receptors.  In all cases, double positive ERBB3+NGFR+ cells resulted the highest PAX7 gene expression. Furthermore, primary and secondary myogenesis could be distinguished based on these markers, where very high expression marked a more immature progenitor (Figure 2). ERBB3 was not expressed by adult satellite cells.

We next applied this same enrichment strategy to hPSC-SMPCs. As expected, in every directed differentiation protocol tested double positive ERBB3+NGFR+ SMPCs could be induced to form homogeneous myotubes. These data demonstrate a striking similarity between the surface marker phenotypes of fetal myogenic progenitors and hPSC-SMPCs undergoing directed differentiation.

Development guides the maturation of muscle progenitors from hPSCs

Despite enabling myogenic enrichment, differentiation of ERBB3+NGFR+ populations isolated from hPSCs yielded myotubes that were thinner and contained fewer nuclei than later stage fetal or adult counterparts. To understand the basis for these differences, we further analyzed our RNA sequencing data comparing hPSC-myotubes and fetal myotubes to their progenitors. These data revealed that activators of TGF-β signaling decreased during the course of fetal myotube differentiation. In contrast, hPSC-SMPCs had higher expression of TGF-β signaling genes and hPSC myotubes failed to down regulate TGF-β signaling, such as myostatin. When we blocked the TGF-β signaling we significantly increased hPSC-myotube fusion and produced morphology similar to late stage fetal myotubes (Figure 3). We performed multiple assays to verify that TGF-β inhibition indeed matured hPSC-myotubes.

ERBB3+ hPSC-SMPCs can generate new muscle fibers upon transplantation in vivo.

The moment of truth had arrived when our enrichment and maturation strategy was applied to hPSC-SMPC engraftment in vivo. After 30 days of waiting, mice injected with ERBB3+ hiPSC-SMPCs and treated with TGF-βi were evaluated for restoration of human dystrophin in a mouse model of DMD. We were gratified to find a greater than 50-fold improvement in number of dystrophin+ fibers using our directed differentiation strategy (Figure 4). This was especially satisfying because these cells were originally taken from boys with Duchenne muscular dystrophy, reprogrammed to induced pluripotent cells, and genetically modified with CRISPR/Cas9 to permanently correct the DMD gene mutation. What’s more, ERBB3+ hiPSC-SMPCs enabled engraftment approaching levels equivalent to directly-isolated human fetal muscle cells. By following human development, our results demonstrated a major advance in the ability to obtain engraftable SMPCs from hPSCs.

The next phase of this research will be to test and develop strategies to mature SMPCs to satellite cells similar to adult, which can reside in the stem cell niche in vivo and continuously repopulate new muscle after damage. We hope to continue to create a more regenerative cell for use in cell replacement therapy for patients with DMD and many other muscle wasting diseases.

References

Chal, J., et al., Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat Biotechnol, 2015. 33(9): p. 962-9.

Furgeson G et al., Transcriptional heterogeneity during lineage specification defines human skeletogenesis.  In Review.

Hicks, M.R., et al., ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs. Nat Cell Biol, 2018. 20(1): p. 46-57.

Shelton M., et al., Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells. Stem Cell Reports, 2014. 3(3): p. 516-29.

Young C.S., et al., A single CRISPR-Cas9 deletion strategy that targets the majority of DMD patients restores dystrophin functionin hiPSC-derived muscle cells. Cell Stem Cell, 2016. 18: p. 1-8. 

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An exciting opportunity to be part of a pioneering biomedical research institute, dedicated to innovation and science. A postdoctoral research post in the laboratory of Dr. Alex P. Gould is now available. We are looking for a highly motivated researcher to identify the, as yet unknown, molecular mechanisms by which dietary nutrients and oxidative stress during development can profoundly alter adult metabolism and longevity. The successful applicant will choose which of the Drosophila and/or mouse model(s) established in the lab they wish to focus on (PMID: 21816278, PMID: 26451484, PMID: 29123106 and unpublished). Research techniques used in the lab include genetics, molecular biology, confocal microscopy, biochemistry, metabolomics and mass spectrometry imaging. Details of projects currently being undertaken in the lab can be seen at: THE GOULD LAB. The successful applicant will have good communication and organisational skills and a PhD in a relevant area (or be in the final stages of completion). Informal enquiries to alex.gould@crick.ac.uk.

The Francis Crick Institute is a biomedical discovery institute dedicated to understanding the fundamental biology underlying health and disease. Its work is helping to understand why disease develops and to translate discoveries into new ways to prevent, diagnose and treat illnesses such as cancer, heart disease, stroke, infections, and neurodegenerative diseases. An independent organisation, its founding partners are the Medical Research Council (MRC), Cancer Research UK, Wellcome, UCL (University College London), Imperial College London and King’s College London. The Crick was formed in 2015, and in 2016 it moved into a new state-of-the-art building in central London which brings together 1500 scientists and support staff working collaboratively across disciplines, making it the largest biomedical research facility in one building in Europe. The Francis Crick Institute will be world-class with a strong national role. Its distinctive vision for excellence includes commitments to collaboration; developing emerging talent and exporting it the rest of the UK; public engagement; and helping turn discoveries into treatments as quickly as possible to improve lives and strengthen the economy.

” If you are interested in this role, please apply via our website.
” The closing date for applications is Monday, 19 March 2018 at 23:30.
” All offers of employment are subject to successful security screening and continuous eligibility to work in the United Kingdom.

Key words

” Developmental biology
” Mouse genetics
” Drosophila genetics
” metabolism
” physiology
” developmental origins of health and disease
” mass spectrometry imaging
” metabolomics
” cell biology
” molecular biology

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Charlotte Blackburn

(this blog was first published on the droso4schools website)

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Vertebrate segmentation involves the periodic formation of somites from the presomitic mesoderm, in a manner controlled by oscillating gene expression (the oscillations of the segmentation clock must be one of the marvels of nature!). While in vivo work has provided a framework for studying the process, many aspects of segmentation dynamics are obscured in the embryo. A new Techniques & Resources paper in Development describes a stem cell-derived in vitro system for studying segmentation dynamics. We caught up with first author Marina Matsumiya and her supervisor Ryoichiro Kageyama of Kyoto University to hear more about the story.

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

RK I became Assistant Professor of Kyoto University in 1989 and started a project about transcription factors that regulate mammalian neural development. We reported the basic helix-loop-helix (bHLH)-type repressor genes Hes1, Hes3, and Hes5 in 1992 and the bHLH-type proneural gene Atoh1 (Math-1) in 1995, and since then we have been characterizing the functions of these genes in neural development. We found that the antagonistic regulation between Hes and proneural genes controls neurogenesis and gliogenesis.

In December 1997, I was promoted to full professorship in the same university and continued the bHLH project. In the same month, I met Olivier Pourquié, when he visited my group in Kyoto, and we had discussion about his finding (just published in Cell) that the expression of chick hairy1, a Hes1 homologue, oscillates during somite segmentation. This discussion made me keep pondering on how such oscillatory expression with ultradian rhythms is possible and how such dynamic expression regulates the downstream events. In the 2000s, we reported that Hes1 expression oscillates in many cell types by negative feedback, and that this oscillation is important for cell proliferation. In parallel with this finding, we identified a new member of the Hes family, Hes7, and found that its expression oscillates by negative feedback in the presomitic mesoderm (PSM), thereby regulating the somite segmentation. These findings brought us to the field of the segmentation clock. Hes7 oscillations are synchronized under the control Notch signaling, and the oscillatory expression of the Notch ligand Delta-like1 is involved in the synchronized oscillations. However, the detailed mechanism of the synchronized oscillation with traveling waves in the PSM is still unknown, and we have been trying to answer this key question.

What is the status of developmental biology research in Japan?

RK Developmental biology research is well funded in Japan if a stem cell or regenerative research field is included. However, besides this field, it seems to be more difficult to get sufficient funding. I think that translational research-oriented funding is a general trend in Japan, as observed world-wide. Collaborations are very active and actually essential to many research projects of developmental biology. Grants supporting domestic and international collaborations are also available. In addition, Riken offers a service of generation of transgenic mice as a collaboration basis, and many researchers utilize this service. I do not know the exact number of labs in developmental biology in Japan, but the Japanese Society of Developmental Biologists (JSDB) has about 1200 members. This is a good number, compared to The Society of Developmental Biology (USA), which has nearly 2000 members.

However, I am a bit concerned about the recent trend. There used to be more than 300 poster presentations in the Annual Meeting of the JSDB, but last year, there were only 193, suggesting that the number of young researchers in the field is decreasing over the years. One reason is that there were only a handful of poster presentations of the stem cell or regenerative research field in the Annual Meeting of the JSDB, while many Japanese researchers participated in the ISSCR in Boston last year. I feel that (classic) developmental biology and (fancy) stem cell or regenerative research should go hand-in-hand, but in reality these two fields seem to go separately in Japan. It would be desirable to incorporate more stem cell researchers into the JSDB meetings to advance the field properly.

And Marina, how did you come to be involved with this project?

MM I was interested in the mechanism of somitogenesis, so I joined the Kageyama Lab as a master student. There were no PSM-like cell culture systems, and my colleague, Kumiko Yoshioka-Kobayashi, suggested me that such systems would be important to examine the detailed mechanism of somitogenesis. After discussion with Kumiko and RK, I decided to make a PSM-like cell culture system. At almost the same time, I found that Olivier’s group published a method to induce PSM-like cells from mouse embryonic fibroblasts (MEFs), so we first used MEFs produced from our Hes7 reporter mice. However, we were not able to induce Hes7-expressing cells, and we next tried mouse embryonic stem (ES) cells following the paper from the same group published in 2015, which focused on the induction of muscle fibers from ES cells. We also had a chance to learn an ES cell culture method from Mototsugu Eiraku at Riken CDB, who established a method to induce the eye cup from ES cells. By combining these methods, we succeeded in inducing PSM-like cells, which exhibited Hes7 oscillations.

What are the difficulties of studying the segmentation clock in embryos? And what were the limitations of previous in vitro approaches?

RK Detailed analysis of the segmentation clock has been hampered because it requires the use of live embryos. Analysis involves both genetic and pharmacological approaches: in genetic approaches, mutant animals are generated to activate or inactivate gene functions, while in pharmacological approaches, either embryos or PSM tissues prepared from embryos are used for treatment with chemicals that modulate gene activities. If in vitro cell cultures are available, both genetic and pharmacological analyses would be facilitated, and many attempts have been made to induce PSM-like tissues from ES cells. Although PSM-like tissues have been successfully induced from ES cells, there are no reports of wave-like propagation of oscillatory gene expression in such induced tissues. So, we started the in vitro PSM cell project.

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

MM & RK We established a simple and efficient method to generate mouse ES cell-derived PSM-like tissues, in which Hes7 expression oscillates like traveling waves. In these tissues, Hes7 oscillation is synchronized between neighboring cells, and the posterior-anterior axis is self-organized as the central-peripheral axis, generating somite-like segments at the periphery. This method is applicable to not only chemical library and RNAi screening but also CRISPR-Cas9-mediated gene modifications and will facilitate the analysis of the molecular nature of the segmentation clock.

In your system, Hes7 oscillations did show some variability: do you know what underlies this variability?

MM & RK  There is some variability in the amplitude, period, and patterns of Hes7 oscillations even in the same iPSM colonies, which may hamper to detect effects of chemicals and genetic modifications. This variability may depend on the size and shape of iPSM colonies, and how iPSM colonies expand on dishes. Further improvements will be required to reduce such variability.

Is your system restricted to mouse cells, or could you use it to compare segmentation between species?

MM & RK  We have not examined other species yet, but we are sure that this method may be applicable to ES/iPS cells of other species. We are interested in the mechanism of how species-specific periods of Hes7 oscillations are determined.

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

MM  My eureka moment is when I observed Hes7 oscillation in iPSM colonies for the first time. I still remember the date, and we used the data in Figure 4 of the paper. We did not get nice data for a few years, but after changing the culture conditions, all of sudden, everything went well. We were surprised to see the Hes7 oscillations propagating like waves and leading to somite-like segment formation. When I saw the movie, we celebrated with high five. I could watch it forever. It took 6 months from the first detection of Hes7 oscillation to submit the paper. Every day in this half a year was a series of joy.

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

MM  When I used MEFs, I was really nervous. I got some Hes7 signals but not oscillatory expression. I tried many different culture conditions, but none of them worked. It was like a never-ending nightmare, but now I feel that it is a very important step to improve myself mentally and technically.

What next for you Marina?

MM  I have one more year to go before I finish my PhD course. Until then, I will try to reveal new factors or pathways that make Hes7 oscillations synchronized by using the iPSM method. This information will be important for the somitogenesis research. After finishing my PhD course, I would like to do research on refractory diseases of bone formation related to somitogenesis. Now, I will prepare the application of a fellowship to become a postdoc.

Where will this work take the Kageyama lab?

RK By using this system, we now started screening many chemicals to find a new pathway involved in synchronization of Hes7 oscillations. We also plan to use two-color fluorescent reporters (one from Hes7 promoter and the other from another important PSM gene) to examine the oscillation at the single-cell resolution. We are trying to find chemicals that do not repress Hes7 expression but affect synchronization. These chemicals, if found, would help reveal the mechanism of synchronized oscillation besides the Notch pathway. We have recently developed an optogenetic gene induction system and plan to introduce this system to iPSM cells. This system allows precise spatiotemporal control of gene expression, so we would like to see how light-controlled gene expression affects Hes7 oscillations.

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

MM  I would like to sleep, but I also like playing modern ballet. These are very important for me to control my condition.

RK  I like jogging along the Kamo river in Kyoto, which really refreshes me. I also like walking. I used to bike, but after an accident I stopped biking now.

Marina Matsumiya, Takehito Tomita, Kumiko Yoshioka-Kobayashi, Akihiro Isomura, Ryoichiro Kageyama. ES cell-derived presomitic mesoderm-like tissues for analysis of synchronized oscillations in the segmentation clock. Development 2018 145: dev156836

This is #36 in our interview series. Browse the archive here. 

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In spite of the difference between the cell functions responsible for giving rise to a tumour and for the metastasis of this same tumour, studies at IRB Barcelona using the fly Drosophila melanogaster reveal that some genes can drive both phenome

na.

In cancer research, there has been a tendency to differentiate between the genes responsible for tumour development and those that give rise to metastasis and to study these processes separately since the cell functions required for these two mechanisms differ radically. Researchers headed by Jordi Casanova at the Institute for Research in Biomedicine (IRB Barcelona) now demonstrate that genes activated during metastasis are also able to initiate primary tumour development, and they explain the molecular mechanism involved. Made using the fly model Drosophila melanogaster, this finding has been published in PloS Genetics this week.

“The results strengthen the notion that was already being put forward in cancer research,” explains Jordi Casanova, head of the Development and Morphogenesis in Drosophila lab and CSIC professor. He goes on to say “And that is that the same genes can activate tumour growth and metastasis, and therefore some tumours are conferred metastatic capacity from their very outset”.
Two phenomena that share genes

The study is based on the observation that benign human tumours or tumours at stages well before metastasis express some genes associated with this process. “This overlap made us question whether the so-called epithelial-mesenchymal factors—which activate genes associated with cell movement and migration, which are common in embryo development and are also present in metastasis—play a role in tumour initiation,” says Casanova.

In particular, GATA genes are overexpressed in liver metastasis in colon cancer patients, as well as in several human tumours, such as those of the breast, ovary, pancreas and colon. Jordi Casanova’s team examined various genes, among them Serpent (homologue of GATA in Drosophila), and demonstrated that the overexpression of this gene is indeed sufficient to drive tumour growth. In this regard, this gene causes overgrowth of the wing, modifies tissue structure, and activates a series of genetic changes in order to facilitate initial tumour growth.
The fly wing as a model of epithelial tumour

The research was conducted on the fruit fly Drosophila, an established model for studying epithelial tumours. “Once again, studies on Drosophila have contributed to our basic understanding of the behaviour of genes involved in cancer and metastasis, knowledge that will allow cancer researchers to pursue this line further,” says the scientist.

Kyra Campbell, first author of the study, former postdoctoral fellow at IRB Barcelona and currently at the University of Sheffield, in the UK, was supported by Xavier Franch-Marro, at the CSIC’s Institute of Evolutionary Biology.

This work was supported by the Ministry of the Economy and Competitiveness, through ERDFs, and by the Government of Catalonia.

Reference article:

Kyra Campbell , Gaëlle Lebreton, Xavier Franch-Marro, Jordi Casanova

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth

PLoS Genetics (2018) https://doi.org/10.1371/journal.pgen.1007167

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The Beddington Medal is the BSDB’s major commendation to promising young biologists, awarded for the best PhD thesis in Developmental Biology defended in the year previous to the award. Rosa Beddington was one of the greatest talents and inspirational leaders in the field of developmental biology. Rosa made an enormous contribution to the field in general and to the BSDB in particular, so it seemed entirely appropriate that the Society should establish a lasting memorial to her. The design of the medal, mice on a stylised DNA helix, is from artwork by Rosa herself. We would like to congratulate the 2018 winner of the Beddington Medal, Emilia Favuzzi, and would like to take this opportunity to give a brief overview of her career and her PhD project that was awarded the Beddington medal.

Emilia’s career and project

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The key results of our recent paper in Nature Cell Biology

Cell polarization defines the spatial biological specificities in a cell. During the first cell cycle of a C. elegans zygote, its symmetry is broken by local remodeling of the cortical actomyosin network. This leads to a segregation of the dedicated polarity regulators, the PAR proteins, into two discrete cortical domains. However, it remains unclear how the mechanical changes driven by actomyosin contractions are transmitted to PAR proteins, and this regulates cellular spatial patterning. In our paper, we revealed that both actomyosin contractions and CDC-42 activity modulate the dynamics of the anterior PAR proteins (aPARs) through stimulation of their clustering at the cell cortex. In the early phase of polarity establishment, contractility of the actomyosin cytoskeleton drives cortical tension, which promotes clustering of PAR-3 (Mov.1, Fig.1). In turn, PAR-3 clusters recruit PKC-3, whereas CDC-42 activity antagonizes PKC-3 clustering during later phase of polarization (Mov.2, Fig.2). The degree of aPARs clustering is significantly associated with the stagnation of aPARs exchange at the cortex, and an effective entrainment with the advective flows of the cortical actomyosin network. This is the first report showing that the cell polarity protein PAR-3 is mechanosensitive, and senses forces to form higher order structures at the cell cortex. These findings depict how actomyosin and PAR proteins interplay and set up asymmetry (Fig.3). This feature may also play a role in other cell patterning events including neuron maturation, wound healing and cancer formation.

Movie 1: Cortical PAR-3::GFP (green) and NMY-2::mKate2 (magenta) during the first cell cycle of a C. elegans embryo.

How I started this project

I had made up my mind to become a researcher during my undergraduate days, after first trying to do a simple plasmid construction in a biological laboratory. Back then I was fascinated by all the equipment and materials in the lab. I went on to obtain my Master’s and Ph.D. degrees in Virology and Neuroscience respectively, before moving to Singapore to commence my first postdoctoral training in Frederic Bard’s lab at the Institute of Molecular and Cell Biology (IMCB), A*STAR. There, I matured as a scientist, and with access to multiple advanced microscopes across the various institutes within the campus, I was able to develop my working skills and knowledge in live-imaging techniques. These abilities, have turned out to be a great help in my career so far.

Before joining Fumio Motegi’s lab at the Temasek Lifesciences Laboratory (TLL), I had worked for over 10 years using cell culture to address different biological questions. So, I had a strong feeling that I must do something different for my next post-doc training. However, it was only after Fumio showed a fantastic movie depicting how anterior and posterior PARs are segregated in worm embryos that I decided to pursue this new area of study. My interests had been captured by how complex, yet exquisite, the underlying regulations can be. After I started working on this project and got some beautiful images of the genome-edited GFP-tagged aPARs, which were generous gifts from Kenneth Kemphus from the Cornell University, I realized that C. elegans is such a powerful tool with which we can address fundamental and important questions. I was also encouraged by the worm society, who kindly shared their materials and information, and welcomed me as a newcomer in their community. I was confident that this was what I wanted to do.

How I found the key results in this paper

The first Eureka moment came when I was inspecting the cortical PAR-3::GFP and PKC-3::GFP in wild-type embryos. They showed beautiful and dynamic puncta structures. This led us to further characterize the implication of those puncta during polarity establishment. We then conducted intensive examinations on the clustering of aPARs in wild-type and polarity-mutant zygotes. Another moment of insight came to me when my colleague Ms. Tricia Low noticed that PAR-3::GFP may exhibit mechanosensitivity. PAR-3::GFP failed to form clusters in the non-muscle myosin-II (nmy-2) mutant embryos, suggesting actomyosin and PAR-3 clustering were somehow connected. The clues became clearer when we observed another two phenomena. Firstly, in wild-type embryos, PAR-3 did not reside with NMY-2, suggesting that PAR-3 clustering is not caused by the direct recruitment of PAR-3 molecules to actomyosin. Secondly, although PAR-3 was unable to form clusters in nmy-2 zygotes, PAR-3 clustering was restored when the embryos underwent shape changes, with cortical ruffling occurring during the later stage of embryogenesis. This suggested that actomyosin contractility itself was not essential for PAR-3 clustering. Instead, physical properties of the cortex appeared to play a crucial role in the control of PAR-3. Hence, we hypothesized that cortical tension driven by actomyosin could promote PAR-3 clustering. We tested and validated this assumption in worm zygotes, as well as mammalian fibroblasts NIH3T3 cells exogenously expressing C. elegans PAR-3, which is achieved with the fantastic help from Dr. Yukako Nishimura from the Mechanobiology Institute (MBI), Singapore. We found that in both cases, PAR-3 clustering was rescued under hypotonic treatment when myosin wasn’t functional. This is the first report stating that PAR-3 reorganizes to higher-order structures in response to tension!

Movie 2: GFP tagged PAR-3, PKC-3, and CDC-42 during polarity establishment in one-cell C. elegans embryos.

How we build a team to test our hypothetical model

When we discovered that PAR-3 carries mechanosensitivity and that it could sense and respond to changes in tension in worm zygotes, we wanted to validate and replicate this experiment in cultured cells and monitor PAR-3 clustering using a TIRF microscope. In the meantime, we also needed a proper method to quantify and demonstrate the clustering so that we could report its significance under different conditions. To allow the research to progress without delay, we decided to collaborate with researchers outside of our lab. The MBI and the IMCB instantly came to mind. At MBI, there are dozens of experts in the field of physical biology, as well as a comprehensive collection of advanced microscopes. We therefore felt there was no better choice for collaboration. Moreover, I had established tight connections with people in the IMCB during my post-doctoral training there. In particular, with the Computational Bioimage Analysis (CBA) Unit, which is run by truly reliable and efficient staffs who are doing great jobs in image analysis and quantification. I was fortunate to have the opportunity to establish fruitful collaborations across TLL, MBI, and IMCB. Each institute is highly-respected not only in Singapore, but worldwide, for consistently publishing in high-profile journals, and attracting talented scientists from different countries. Singapore had become one of the most splendid research hubs in the world, which is attributed to the massive investment and strong support from the government into the life science realm.

How we struggled to publish our project

I think the most difficult time for us along the journey was when our manuscript was rejected by the journal in December of 2016. The reviewers appreciated the main point of our story, but they thought the quantification method we had used to measure aPARs clustering was insufficient. Time was a big concern as we considered whether to submit to another journal, which might have been the easier path to publication, or stick with the same journal and strengthen our manuscript. We were in a dilemma. But since we were confident in ourselves, and in our story, we immediately made the decision to amend the quantification method and reanalyze all of our images. Fortunately, the results returned by the modified methods were consistent with our previous conclusions. We quickly revised the manuscript and resubmitted to the same journal and it turned out to be a happy ending. We appreciate Dr. Weimiao Yu and Dr. Laurent Gole from the Computational Bioimage Analysis (CBA) Unit of IMCB for their efforts to come up with the modified code, as well as the detailed descriptions for the quantification method, for the revised manuscript. We are also very grateful to the editor and all our reviewers for their constructive comments and positive encouragements to improve our manuscript. We did enjoy the review process. I hope what we had gone through can be an inspiration to those who might currently be struggling in a difficult time like this. What we need to do is to keep calm and believe in yourself.

Publication of our work along with two other groups

We learned there were several groups working on a similar topic since 2016. Fortunately, we maintained unencumbered communication with these groups, through emails or in person during international conferences, and it is truly exciting to see that we have all successfully published our stories in high-impact journals.

The common conclusion drawn by the three groups is that the degree of PAR-3 oligomerization increases during polarity establishment, and this further promotes the formation of aPAR clusters. The clustering of aPARs supports its segregation along with the actomyosin flow. Moreover, single-molecule pull-down in single embryos, as well as particle tracking, were used by Dickinson et al. to demonstrate that clustered PAR-3 is coupled to the cortical flow and phosphorylation by the mitotic kinase PLK-1 prevents PAR-3 from undergoing oligomerization. Furthermore, through a functional assay, Rodriguez et al. showed that PKC-3 inhibition or activation depends on its interaction with pools of PAR-3 or CDC-42, respectively. We found that PAR-3 carries mechanosensitivity that responds to cortical tension driven by actomyosin contraction. It’s genuinely fantastic that all three papers support each other and yet, by employing distinct approaches, present a unique significance to the questions addressed. I believe this can be a good example to encourage those who may also be facing competition in their research.

Several open questions about cell polarisation

The evolutionarily conserved PAR machinery is predominantly localized at the actomyosin cortex, which lies adjacent to the cell membrane. It is likely that the principle we uncovered is applicable to other types of tissue, and other organisms. For instance, it has been shown that self-assembly of the PAR-3 N’-terminal domain is critical for axon specification of neurons. In this case, the direct binding of the oligomerized domain to the microtubule promotes microtubule bundling and stabilization. It would be interesting to test if this formation of higher-order PAR-3 can be promoted by the activation of non-muscle myosin, which in turn changes local cortical tension. Another example is during Drosophila embryogenesis. Here, during the process of dorsal closure, actomyosin repeatedly assembles and disassembles in the epithelial amnioserosa cells. In this case, PAR-3 forms patches and overlaps with myosin at the apicomedial surface to promote actomyosin contraction. It is intriguing to explore whether myosin assembly induces structural changes in PAR-3, and whether this contributes to developmental morphogenesis. Finally, some follow-up questions remain elusive and need to be further addressed. What are the molecular mechanisms by which PAR-3 clustering or CDC-42 activity apply to compete for the PKC-3/PAR-6? How do aPARs clustering and actomyosin activity regulate and feedback to each other with a temporal-spatial precision during cell-cycle progression? It would be exciting to see emerging studies resolving more of the puzzles surrounding cell patterning in response to extrinsic and intrinsic stimulation in the near future.

References

Wang, S.C., Low, T.Y.F., Nishimura, Y., Gole, L., Yu, W. & Motegi, F. Cortical Forces and CDC-42 Control Clustering of PAR proteins for C. elegans Embyonic Polarization. Nat Cell Biol 19, 988–995 (2017).

Dickinson, D. J., Schwager, F., Pintard, L., Gotta, M. & Goldstein, B. A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization. Developmental Cell 42, 416–434.e11 (2017).

Rodriguez, J. et al. aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity. Developmental Cell 42, 1–35 (2017).

Chen, S. et al. Regulation of Microtubule Stability and Organization by Mammalian Par3 in Specifying Neuronal Polarity. Developmental Cell 24, 26–40 (2013).

David, D. J. V., Tishkina, A. & Harris, T. J. C. The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila. Development 137, 1645–1655 (2010).

(7 votes)

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The Turing Centre for Living Systems aims at attracting students willing to work in an interdisciplinary life-science environment and with backgrounds in cell or developmental biology, immunology, neurosciences, theoretical physics, biophysics, computer science, bioinformatics, applied mathematics, engineering.

Candidates are asked to select the projects in order of interest (up to 3) and rank them in the application form.

Link to the PDF file.

Deadline for application: 15^th April

Informal enqueries: info@centuri-livingsystems.org

PHD2018-01 – Using single cell transcriptomics to classify neuron types in two brain regions relevant to social behavior: the olfactory bulb and the hippocampal CA2

PHD2018-02 – Visualizing deep hippocampus neuronal functional activity in vivo using ultra-thin 2-photon endoscopes

PHD2018-03 – Understanding uORF functions through dendritic cells biology and computational approaches

PHD2018-04 – Using higher-order biological networks to explore temporal patterns in cell cycle control

PHD2018-05 – 3D optical microscopy for quantifying T lymphocyte activation

PHD2018-06 – Life and death of Salmonella-containing vacuoles

PHD2018-07 – Dynamics of a self-organized multicellular system

PHD2018-08 – Bacterial adaptation by OXPHOS dynamics: from single cell biology to gut microbiota

PHD2018-09 – Emerging near-infrared chromophores for photoacoustic bio-imaging

PHD2018-10 – Fluid-induced self-organization of ciliated-cell activity in human lungs

PHD2018-11 – Muscle building: bridging molecular order to macroscopic morphogenesis

PHD2018-12 – Deciphering the activation states of plasmacytoid dendritic cells, their dynamical relationships and their molecular regulation

PHD2018-13 – Mathematic modelling of cell migration in confined domains

PHD2018-15 – A versatile virtual reality system to understand animal navigation

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The DRSC/TRiP-Functional Genomics Resources is a Drosophila community resource with three main focus areas: (1) cell-based Drosophila cell screening, (2) fly stock production, and (3) bioinformatics. Our TRiP fly stock production platform is taking nominations for production of CRISPR sgRNA fly stocks. Researchers can nominate genes for knockout (TRiP CRIPSR-KO) or activation (TRiP CRISPR-OE), or both. To search existing and in-production stocks, and to nominate genes for production of new stocks, please visit the gRNA Tracker website. In keeping with our long-time policies, all fly stocks are deposited to the Bloomington Drosophila Stock Center. We look forward to continuing to serve the needs of Drosophila developmental biologists and others through this effort!

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