NEUCrest Ph.D Studentships in Developmental Biology, Stem Cell Biology and Cancer Biology
Applications are invited from suitably qualified candidates for full-time fixed term positions as Early Stage Researchers in the lab of Prof Karen Liu, King’s College London (karen.liu@kcl.ac.uk), Centre for Craniofacial and Regenerative Biology, King’s College London.
These positions are funded by the Horizon 2020 programme of the European Union and will be available from 1st January 2020. Appointments will be on a full-time basis for a period of 3 years. Remuneration will be in line with the European Commission rules for Marie Skłodowska-Curie grant holders (Early-Stage Researchers, Initial Training Network).
NEUcrest is a four-year EU Horizon 2020 project funded by Agreement 860635. The neural crest is an essential stem cell population of vertebrate embryos. The project focuses on integrating academic, clinical and industrial research for a better understanding of neural crest development and neural crest related diseases. These pathologies are a major group of congenital diseases in human, and a heavy societal concern. The NEUcrest network comprises 20 partners in academia, industry and hospitals from seven European countries, gathered in a synergistic effort to advance knowledge and outreach of these diseases.
The Ph.D projects are highly multidisciplinary and will develop scientific strategies from experimental embryology, genome editing, imaging and generation of genomic datasets. A full list of projects and partners:
PhD Projects available through the lab of Prof Karen Liu, King’s College London:
PROJECT 1: The Role of ALK and GSK3 in normal and pathogenic neural crest migration. This project led by Prof Karen Liu (King’s College London) and Prof Angela Nieto (UMH, Spain). Applications see: https://tinyurl.com/yycpnlxj
Person Specification: First degree or Masters in Biological Sciences, Cell Biology, Genetics and Molecular Biology. Mobility requirement: EU applicants are eligible to apply. Applicants must not have been based in the country of registration for more than 12 months in the last 3 years.
Start date: Positions are available from 1st January 2020. Informal enquiries to Prof Karen Liu (karen.liu@kcl.ac.uk)
To Apply: Applications, in English, should include a detailed CV, certificates of examination grades (bachelor/master), a letter describing your career goals, skills and experience, as well as two letters of recommendation. Applications: https://tinyurl.com/yycpnlxj. Informal inquiries may be made to karen.liu@kcl.ac.uk. Deadline for applications is 15 November 2019, or until posts are filled.
PROJECT 2: Generation of neural crest cells from patient-derived hiPSCs for disease modelling and therapeutic applications. This industrial project will be led Dr Erin Knock and Dr Wing Chang with student based at STEMCell Technologies, Cambridge UK. Academic registration and supervision by the lab of Prof Karen Liu (King’s College London).
PROJECT 3: Establishment of human iPSCs from syndromic neurocristopathy patients with skeletal dysplasia This project is suitable for a clinical or suitably qualified non-clinical PhD student and is led by Prof Irene Mathijssen (email to i.mathijssen@erasmusmc.nl Erasmus Medical Centre, Netherlands) with secondment to Prof Karen Liu (King’s College London) and STEMCell Tech, Cambridge UK and Phenocell, France.
Cell adhesion molecules (CAMs) of the plasma membrane physically tie cells together into tissues, both via interaction of cells with other cells or with ECM (extracellular matrix); they also constitute hubs of information exchange with neighbouring cells and the environment. CAMs mediate a broad range of biological functions and their aberration contributes to a range of diseases, such as cancer progression or aberrant growth/regeneration of nerves.
On this project, you will study integrins, which are the major adhesion receptor class for extracellular matrices.Integrin signalling is performed by its adhesome, the dynamic population of proteins that link integrins both to the cytoskeleton and to signalling pathways via the cytoplasmic tails of the receptors. The composition and functional state of integrin-associated complexes essentially determines the adhesion state and signalling output.
To understand this, Martin Humphries has long-standing expertise in using proteomics based on mass spectrometry as a means to identify components of the integrin adhesome and study their function in mammalian cell culture [1]. On this project, you will extend such approaches to brain tissues in the living organism and study the function of identified proteins in the context of nerve growth.
For this, you will capitalise on the expertise of Andreas Prokop, who uses neurons of the fruit fly Drosophila to study mechanisms of nerve growth [2]. In this functional context, his team discovered and analysed important roles of integrins and some of their conventional adhesome components (new unpublished results). The small size and genetic tractability of Drosophila makes it feasible to perform proteomics on integrin adhesion complexes extracted from nerve cells of living organisms.
Once candidate components have been identified, you will then be able to capitalise on highly efficient genetic means provided by Drosophila to study their functional contributions during nerve growth [2] – potentially identifying promising drug targets for nerve regeneration therapies. A likely option will be the extension of such functional studies into mammalian models.
Your experimental skill training opportunities will include molecular biology, protein biochemistry, mass spectrometry, genetics, cell biology, a range of imaging techniques, bioinformatics, as well as insights into important concepts of the cell and neurobiology fields. In addition, AP is an expert in science communication providing further training opportunities important for your career development.
Epithelial tubes often have a functional polarity written along their proximo-distal (P-D) axis, with different segments of specialised cell-types carrying out distinct physiological activities. With a handful of notable exceptions, we know very little about how P-D axes and segment-specific differentiation are regulated during organogenesis.
The major objective of this project is to explore the molecular and cellular mechanisms that pattern and maintain functional polarity along the P-D axis in a structurally simple, but functionally sophisticated epithelial tube: the insect renal tubule. Approaches will include: state-of-the-art imaging, single cell RNAseq, genetics, physiological assays and in silico modelling.
The project will be carried out in the laboratory of Barry Denholm (Biomedical Sciences, University of Edinburgh)
A recent example of our work from a related project: Beaven, R. and Denholm, B (2018) Release and spread of Wingless is required to pattern the proximo-distal axis of Drosophila renal tubules eLife 2018;7:e35373 https://elifesciences.org/articles/35373
Miki Ebisuya’s research group at EMBL Barcelona is looking for a postdoctoral fellow who tackles a mystery in developmental biology: why larger animal species tend to show slower time. Why is the gestation period of humans ~9 months while that of mice is only 20 days? What are the molecular mechanisms of such inter-species timing differences?
Thanks to in vitro differentiation of iPS/ES cells, we can now recapitulate several developmental processes of diverse species, including humans. Thus, our plan is to systematically measure biochemical parameters, such as degradation rates, by using proteomics and RNAseq, and to compare them among species.
We are looking for a person who will carry out both cell biology experiments and high throughput data analyses (both can be taught by current lab members and collaborators). For more information, see the job description/application page.
* This is a position supported by EMBL internal grant as well as PGC2018-097872-A-I00 (MCIU/AEI/FEDER, UE) funded by the Spanish Ministry of Science, Innovation and Universities (MCIU) and co-funded by the European Regional Development Fund (ERDF, EU).
A Wellcome Trust/Royal Society funded Research Associate position is available in Dr. Kyra Campbell’s research group. This is a fantastic opportunity to join the Campbell group, who are focused on identifying the molecular mechanisms underlying epithelial cell plasticity during development and disease. We study this during morphogenesis of the Drosophila midgut (Campbell et al, Dev Cell 2011; Campbell and Casanova, Nat Comms 2015), and also in exciting Drosophila cancer models that we have recently generated (Campbell and Casanova, Plos Genetics 2018; Campbell et al, Nat Comms 2019).
We are combining single-cell OMICs approaches and deep-tissue imaging on our own labs dedicated multiphoton confocal microscope, with genetic approaches and CRISPR/Cas9 technologies. We are looking for a motivated and enthusiastic candidate who will play a central role in the lab. You must have a good honours degree and a PhD (or be close to completion) in areas relevant to cell/developmental biology (or have equivalent experience), along with experience in in vivo imaging and image analysis. Applicants are expected to have excellent interpersonal and communication skills, be highly independent and committed to research in a fast-moving and competitive field.
All cells in the body contain the same genetic material. The difference between cells therefore depends solely on which genes are expressed or ‘turned on’. Now, researchers from the University of Copenhagen have gained new insights into how genes are turned on and off and how the cells “forget their past” while developing into a specific cell in the body. This new knowledge is published in Nature and will be crucial for stem cell therapy and potentially treating people with cancer.
Stem cells all share the potential of developing into any specific cell in the body. Many researchers are therefore trying to answer the fundamental questions of what determines the cells’ developmental fate as well as when and why the cells lose the potential of developing into any cell.
Now, researchers from the Novo Nordisk Foundation Center for Stem Cell Biology (DanStem) at University of Copenhagen have discovered how stem cells can lose this potential and thus can be said to “forget their past”. It turns out that the proteins called transcription factors play another role than the scientists thought. For 30 years, the dogma has been that transcription factors are the engines of gene expression, triggering these changes by switching the genes on and off. However, new research results published in Nature reveal something quite different.
“We previously thought that transcription factors drive the process that determines whether a gene is expressed and subsequently translated into the corresponding protein. Our new results show that transcription factors may be more analogous to being the memory of the cell. As long as the transcription factors are connected to a gene, the gene can be read (turned on), but the external signals received by the cells seem to determine whether the gene is turned on or off. As soon as the transcription factors are gone, the cells can no longer return to their point of origin,” explains Josh Brickman, Professor and Group Leader, DanStem, University of Copenhagen.
The question of how a cell slowly develops from one state to another is key to understanding cell behavior in multicellular organisms. Stem cell researchers consider this vital, which is why they are constantly trying to refine techniques to develop the human body’s most basic cells into various specific types of cells that can be used, for example, to regenerate damaged tissue. So far, however, investigating the signals required to make cells switch identity has been extremely difficult, since making all the cells in a dish do the same thing at the same time is very difficult.
A protein centered viewpoint
The researchers developed a stem cell model to mimic a cell’s response to signaling and used it to, for first time, precisely determine the sequence of the events involved in a gene being turned on and off in response to a signal in stem cells. The researchers were able to describe how genes are turned on and off and under what circumstances a cell can develop in a certain direction but then elect to return to the starting-point. Part of this work involved measuring how proteins in a cell are modified by phosphorylation using advanced mass spectrometry available through an important collaboration with Jesper Olsen’s Group at the Novo Nordisk Foundation Center for Protein Research. “Combining forces with the Olsen group in the CPR enabled us to provide a unique deep description of how individual proteins in a cell react to signals from the outside,” continues Josh Brickman.
New answers to old scientific questions
These results are surprising. Although the sequence of cell transcription processes could not previously be measured as accurately as in this study, the dogma was that transcription factors comprise the on-off switch that is essential to initiate transcription of the individual gene. This is not so for embryonic stem cells and potentially for other cell types.
“Transcription factors are still a key signal, but they do not drive the process, as previously thought. Once they are there, the gene can be read, and they remain in place for a while after the gene is read. And when they are gone, the window in which the gene can be read can be closed again. You can compare it with the vapour trails you see in the sky when an airplane has passed. They linger for a while but slowly dissipate again,” explains first author, William Hamilton.
This discovery is first and foremost basic knowledge, which changes fundamental assumptions in molecular biology. The new results are especially important for researchers working on stem cells and cancer biology. They provide new insight into how cells develop, how pathways involved in development determine when cells change, and when the point of no return is reached. These pathways are also found frequently mutated in cancer and the findings in this study will be valuable to the study of malignant development.
“In the project, we focused on the fibroblast growth factor (FGF)–extracellular signal–regulated kinase (ERK) signalling pathway, which is a signalling pathway from a receptor on the surface of a cell to DNA inside the cell nucleus. This pathway is dysregulated in many types of cancer, and we therefore hope that many of the data in this study will help to inform aspects of cancer biology by indicating new ways to specifically target this signalling pathway in cancer cells,” concludes Josh Brickman.
They study was funded by the Novo Nordisk Foundation, the Independent Research Fund Denmark, the Danish National Research Foundation, the Human Frontier Science Program and the Lundbeck Foundation. It also involved an important collaboration with the group of Naama Barkai, at the Weizmann Institute for Science, Rehovot, Israel.
Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen | joshua.brickman@sund.ku.dk
Josh Brickman has a background in molecular biology and gene regulation. From a PhD focused on transcriptional regulation he trained in developmental biology as a post-doctoral fellow, working in early mouse, and Xenopus, as well as cultivating embryonic stem cells as a model for developmental biology. He began his own lab with research projects bridging early development in multiple models systems with ES cells in a hybrid approach aimed at understanding conserved mechanisms of lineage specification, pluripotency and self-renewal. He currently seeks to understand how transcription factors regulate cell fate choice in ES cells and early embryos. More specifically, Professor Brickman’s and his group investigate the basis for transcriptional priming and commitment in ES cells and early in the specification of the endoderm lineage. They hope to understand the relevance of these molecular events to cellular decision making, pattern formation, in addition to stem and progenitor cell potency.
Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen | william.hamilton@sund.ku.dk
William obtained his PhD at the Edinburgh University in the labs of Tilo Kunath and Mike Tyers, where he worked on defining factors that regulate MAPK signalling in mouse embryonic stem cells. He then joined the Brickman lab in Copenhagen where he expanded upon this to uncover how MAPK signalling regulates transcription and plasticity during early stem cell differentiation.
Drosophila wing discs are epithelial sac-like organs and a powerful model for investigating the link between proliferation and patterning. Of particular interest is the question of how single cells in the disc integrate information regarding position and growth control, as morphogens that pattern an axis can also regulate cell division. A new Techniques and Resources article in Development reports the application of single cell sequencing technologies to dissociated discs in an effort to understand these problems. We caught up with first author Mingxi Deng and his supervisor Yan Yan, Assistant Professor at The Hong Kong University of Science and Technology (HKUST), to hear more about the story.
Mingxi and Yan (L-R)
Yan, can you give us your scientific biography and the questions your lab is trying to answer?
YY My lab is primarily interested in organ size control, in particular, the roles of cell structural components such as apicobasal polarity proteins and cytoskeletal proteins in this process. When I was a graduate student with Prof. Trudi Schupbach at Princeton University, New Jersey, USA, I performed a genetic screen for mutants affecting Drosophila follicle cell epithelial morphogenesis and proliferation. I then got my postdoc training with Prof. Chris Doe at the University of Oregon, USA, where I learned how Drosophila embryonic neuroblasts lose their apical domains and emerge from neuroepithelia. These experiences were important for me to learn that cell polarity and, more broadly, cell structural proteins, are important for organ size and shape. Another important thing I learned from graduate school is the power of quantification, which influences how we approach questions now in the lab.
Mingxi, how did you come to work in the Yan lab, and what drives your research today?
MD When I was looking for a postgraduate student position at HKUST I found Prof. Yan’s research interesting in combining the power of Drosophila genetics with quantitative biology methods. Her lab also has a good reputation for being supportive to students, so I decided to join. I have always wanted to become a scientist and I get excited when I encounter new problems and need to find a way to solve them.
What was the drive behind doing a single cell analysis of the disc, and how easy was it to set the system up?
MD & YY It started with our study of how scribble (scrib) mutant tumours – which show disrupted tissue architecture – change over time. We found that they showed a high degree of plasticity, and suspected that they might be more heterogeneous than previously assumed. For this we needed to understand how much of the cell heterogeneity in the scrib tumours comes from heterogeneity already existing in wild-type wing discs. That was the starting point of this analysis.
We were lucky to have the help we needed for this study. Prof. Ting Xie from the Stowers Institute for Medical Research, Missouri, USA, happened to visit my university at the time. I went to talk with him and he kindly shared his unpublished fly single cell dissociation protocol. It is also very helpful to have colleagues Jiguang Wang and Hao Ge with whom to discuss methods: their expertise in bioinformatics and mathematics ensures that we are analysing data correctly and robustly. In addition, the single cell community has been very good in providing open-access analytical tools with user-friendly tutorials.
Can you give us the most surprising finding from your paper?
MD & YY The most surprising finding is that pattern formation partially persisted in the scrib mutant tumours. This is surprising because the morphogens important for pattern formation need to properly spread in space, and it suggests that further studies are needed to understand why particular pattern formation processes are robust against loss of tissue architecture.
A combination of different gene expression patterns from the ‘Virtual wing disc in situ’ section of the online database.
What does your scrib tumour model analysis tell you about the link between patterning and growth control in the disc?
MD & YY This is a very good question, but we still do not understand the link between patterning and growth control, although we are able to make more quantitative observations from these data. It was previously shown, with very clear genetic evidence, that patterning factors such as dpp are needed to ensure proper proliferation and growth in the disc. Previous experiments have also shown that cell proliferation and growth are more or less uniform throughout the whole disc. Now, we provide another line of evidence that proliferation and growth states do not appear to be particularly biased in sub-regions marked by any single patterning gene, in both wild-type and scrib mutant discs. Our data also suggest that a well-defined distribution of proliferation and growth states exists in discs and this distribution is severely disrupted in the scrib mutants. Interestingly, the temporal scrib mutant data suggested a positive correlation between formation of correct patterns and a distribution of proliferation and growth states closer to wild type.
Your single cell datasets are available to explore on a database: what questions do you think this database will be particularly useful for addressing?
MD & YY Wing discs have been a very good system to study pattern formation, organ size control and regeneration. I hope that our database can provide a good reference point for the community interested in these questions. For example, for researchers interested in how wing disc cells respond to injury during regeneration processes, they would be able to compare the identity of their cells of interest with the wild-type imaginal disc cells in our database.
I hope that our database can provide a good reference point for the community
When doing the research, did you have any particular result or eureka moment that has stuck with you?
MD After I assigned the disc cells correctly to the pouch/hinge region, I was very happy to see that fine patterning processes are well represented in our single cell data. This gave me a sense of connection and also deep respect for the classical works on pattern formation, which I had previously only learned from textbooks.
And what about the flipside: any moments of frustration or despair?
MD I started as the only student working on computation in our lab and needed to learn everything from zero. My lab mates are all excellent experimentalists but they cannot help me with computational problems. It took a while to grow out of the loneliness but I have become more confident now.
So what next for you after this paper?
MD I have just finished my second year as a postgraduate student. I am now pursuing a few quantitative biology projects for which we already have data and a priority for me is to further sharpen my computational and mathematical skills. Hopefully, I can share new exciting stories in a few years when I graduate with my PhD.
Where will this work take the Yan lab?
YY The scrib mutant cells are very interesting, because when they are generated as mosaic clones in the wing discs they behave very differently and undergo cell death through a cell competition process. Building upon this work, we are now trying to better understand the scrib mutant clonal cells, and how different signalling activities contribute to their cell plasticity at the single cell level and eventually alter their growth outcome.
Finally, let’s move outside the lab – what do you like to do in your spare time in Hong Kong?
YY: I have a 4-year-old son and I am expecting another baby in December, so my activities outside the lab revolve around parenting. I find the parenting experience extremely helpful in that I am much more patient with students now than before.
MD: Hong Kong is a surprisingly great place for outdoor activities like hiking and sailing, which I like. I also like to play soccer and computer games.
In the latest Genetics Unzipped podcast we’re reporting back from the Manova Global Health Summit in Minneapolis last month, exploring the latest advances in health technology such as CRISPR-based gene therapies, infection-fighting bacteriophage and the possibility of curing HIV with stem cell transplants.
Plus veteran New York Times columnist Jane Brody’s advice for a healthy life, and reflections on progress in cancer from US journalist and advocate Katie Couric.
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 Follow us on Twitter – @geneticsunzip
We study stem cells, development, and regeneration in the cnidarian Hydractinia. The questions we are interested in are related to how cells make decisions in these contexts. Techniques we use in the lab include random-integration and CRISPR-Cas9 mediated transgenesis/mutagenesis, flow cytometry, cell and tissue transplantation, gene expression analysis, and confocal microscopy for fixed tissues and live imaging experiments.
Cnidarians (sea anemones, corals, and jellies) are emerging model organisms in developmental biology and evolution. Hydractinia symbiolongicarpus, our lab animal, is one of only a few established cnidarian models. The animal grows well in the lab, reproduces sexually every day, and is highly regenerative. A high quality, PacBio based genome sequence is available together with numerous tissue-specific transcriptomes. Hydractinia is small, translucent, and sessile in most stages of its life cycle. This enables in vivo experiments that are very difficult to perform on other animals.
One postdoc will work on characterizing the transcriptomes of all Hydractinia cell lineages at single-cell resolution. The work will also include functional studies on key lineage regulators. The second postdoc will study the transcriptional changes and chromatin landscape that underlie a novel type of regeneration involving natural reprogramming of somatic cells. The positions are funded by NSF and Wellcome, respectively, and are available for three years each.
Candidates must have a PhD in developmental biology, cell biology, or related area. A strong background in molecular biology, experience in working with an animal model, or bioinformatics would be advantageous.
To apply, send a cover letter articulating your interest in one of the projects, your CV, and contact info for at least two references, ideally as a single PDF, to Prof. Uri Frank <uri.frank@nuigalway.ie>. Informal enquiries are welcome.
Stem cells are typically defined by their ability to self-renew and differentiate. These activities are tightly controlled by both intrinsic cues and extrinsic cues from the microenvironment, known as the SC niche. This niche consists of multiple components, among which blood vessels (BVs) are critical as they not only supply oxygen and nutrients to the SCs but also provide molecular signals. BVs form a perivascular-niche for many adult SCs including neural, mesenchymal and hematopoietic SCs. A molecular connection between SCs and vasculature contributes to tissue homeostasis and repair. However, it remains unclear whether this connection also exists in epithelial stem cells, and it’s also unknown whether SCs can conversely promote remodeling of their own environment for proper tissue homeostasis.
The Tumbar lab at Cornell University uses the mouse hair follicle as a model system to study SCs. Hair follicles (HFs) are characterized by a cyclic destruction and reconstruction, which consists of three morphologically distinct and synchronous phases (Figure 1) : 1) growth and proliferation which results into the formation of a new hair shaft known as anagen; 2) apoptosis driven regression, or catagen; 3) and the resting phase, or telogen. In these three phases, SCs exhibits distinct behaviors such as proliferation, migration, or quiescence.
Figure 1: Stem cell behavior during hair cycle in adult skin. Hair cycle is divided into morphologically distinct and synchronous phases: 1) growth and proliferation; 2) apoptosis driven regression; 3) and the resting phase. At the end of quiescence phase stem cells (shown as blue circles) migrate out of their niche (shown as purple crescent) and in response to the activation signals and they change their gene expression, these are called early progenitor cells (shown as red circles). Transcription factor Runx1 is highly expressed in these cells. During the growth or proliferation phase stem cells undergo self-renewal to fill the vacant space and they differentiate and proliferate to make hair shaft. Towards the end of the proliferation differentiated cells undergo apoptosis and stem cells return to quiescence.
A decade ago, when Tudorita (Doina) was a postdoc with Elaine Fuchs studied the transcriptional profile of the hair follicle stem cells (HFSCs) by purifying label-retaining cells in the lineage tracing experiment (Tumbar et al., 2004). Interestingly, many differentially expressed genes in this population encode secreted molecules, suggesting that in addition to receiving signals from the niche, HFSCs may also modulate the niche (Fuchs et al., 2004). More recently, the Tumbar lab has identified runt-related transcription factor 1 (Runx1) as a HFSC regulator. Epithelial Runx1 knockout mice have significant delay in hair growth (Hoi et al., 2010; Osorio et al., 2008). Runx1 is highly expressed in activated SCs or early progenitor cells and its expression is lost when these cells are proliferating (Figure 1). Microarray analysis further revealed gene expression changes in response to altered Runx1 level in the epithelium (Lee et al., 2014).
Our journey started when Prachi, Post-doc in the lab, got intrigued by the microarray data: the gene changes include secreted molecules whose functions are implicated in vascular remodeling, indicating a cross talk between vasculature and stem cell activation. We started to look for blood vessel remodeling in response to varying levels of Runx1 using mutants: Runx1 epithelial knockout mice (Runx1 EpiKO) and Runx1 epithelial transgenic overexpression mice (Runx1 EpiTG). Blood vessel connection in the skin has mainly been studied for oxygen and nutrition, and its molecular signaling aspect has been addressed in depth. Quick experiments of immunostaining for CD31, an endothelial marker, showed visual differences that were exciting and prompted us to begin our investigation on the connection between HFSC and the vascular niche. We were interested in testing whether the vasculature signals for HFSC activation and/or if HFSCs themselves send signals to remodel vasculature during normal hair homeostasis.
Our basic idea was to perturb one compartment, either epithelium or endothelium, and see what would happen to the other compartment. Taking lead from our earlier observation that BVs are remodeled in response to varying levels of Runx1, undergrad student Catherine focused on quantifying the direct contact between vasculature and different regions of the hair follicle. By subsequent quantification, we found that Runx1 mutants showed distinct patterns of vasculature contact. However, these patterns were at first confusing, and we were not sure how to interpret them.
We then began our analysis by quantifying the area covered by CD31 immunostained vasculature under the hair germ and above the muscle. The quantification process was not easy. First, the selection of CD31 immunostained vasculature needs to be manual, because software such as ImageJ or ilastik are not yet as smart as humans in accurately picking out the vasculature. Second, some of the images were not good quality – for example, some stainings appeared hazy, or in some slides the hypodermis was washed away. Those images could be used for contact quantification, but would not be good for area quantification. Therefore, a lot of stainings had to be repeated. Another problem we encountered the different way different researchers quantified their data. The first part of quantification was done by Flora Eun, a brilliant undergrad who soon graduated and left the lab, and the second part of the job was then picked up by graduate student Nina Li. However, Nina was able to be more precise than Flora in selecting the vasculature, so it did not make sense to combine their data as it would create large error within the dataset. To avoid this problem, Nina then quantified the entire dataset, and compared her statistical result with Flora’s preliminary result. Since both statistical results show the same trend that Runx1 EpiKO has significantly more vasculature than control mice, we felt comfortable to further pursue the study.
We then noticed that the thickness of the hypodermis was different in each mouse, but were able to confirm that this thickness did not relate to the amount of vasculature. Since we reasoned that Runx1 might modulate the vascular niche via secreted molecules, the vasculature in close vicinity to the hair germ should be the most affected population, and they should also be the most important if they can send signals back to the hair germ. We used a more stringent method to quantify vascular differences: we drew a thin stripe under the hair germ, and only quantified the vasculature in this selected region. As expected, Runx1 EpiKO had significantly higher amount of vasculature than control mice, suggesting that HFSCs may actively modulate their own vascular niche via Runx1 expression.
In the reverse signaling, from endothelium to epithelium, we did an initial screen before pursuing a particular mutant. From Dr. Anne Eichmann, we acquired two vasculature-related mutants: Cdh5-CreERT2 mediated endothelial knockout mice for Neuropilin 1 (Nrp1) and activin-receptor like kinase 1 (Alk1) genes (Nrp1 EndKO and Alk1 EndKO). Both genes are critical for endothelial cell homeostasis, and perturbation of either one can lead to serious vasculature related diseases. Since we wanted to know whether perturbation of the endothelium affects hair follicle homeostasis, the idea was to perturb BV during quiescence stage before beginning of next stages of HFSCs proliferation and differentiation. When Prachi started optimizing time points and dosage for knockouts she wasn’t sure that if Alk1 conditional knockout out would be viable and show phenotypes, as previously reported knockout pups could only survive 48 hours after birth. Another question was whether adult vasculature patterns can be remodeled by introducing genetic mutations.
We first checked the hair cycle progression on a few mutant mice after knockout induction at PD17, and sacrificed around PD35. While Nrp1 EndKO did not exhibit obvious epithelial phenotype, we were excited to find Alk1 EndKO showed a hair cycle delay phenotype. Alk1 EndKO in the quiescence stage of hair cycle seemed to modulate vascular remodeling and result in delayed progression of stem cell activation. Therefore, we decided to make further investigation on Alk1 EndKO. To confirm the hair cycle delay phenotype, we checked more mice and at various time points between PD22 to PD35. To distinguish between quiescence and early proliferation, since overall morphology does not differ much at these stages, we did both immunofluorescence staining for Ki67, a proliferation marker, and H&E staining to carefully determine the hair cycle stages for our samples. After checking more than 20 mice, we concluded that Alk1 EndKO mice have delayed proliferation marked by the lack of Ki67 staining. We also confirmed that observed delay was not due to less number of early progenitor cells in the mutant mice but rather as a result of delay in HFSC proliferation.
We then wondered what changes in the skin vasculature could lead to the Alk1 EndKO hair cycle delay phenotype. Again, we did immunofluorescence staining for CD31 and we found that there are more CD31+ vasculature in vicinity of the hair germ. The similarity between Alk1 EndKO and Runx1 EpiKO led us to wonder what is the importance of the vasculature near the hair germ. To answer this question, we first investigated the nature of this vasculature during hair homeostasis using wildtype mice (Figure 2).
Figure 2: Skin vasculatures during the hair cycle. The hair cycle consists of three stages. In telogen, both bulge cells and primed stem cells (hair follicle stem cells, HFSCs) in the hair germ remain quiescent. When the hair follicle receives signals to enter anagen, bulge cells proliferate and self-renew, and HFSCs in hair germ proliferate and differentiate to give rise to multiple lineages. In catagen, the regression stage, differentiated lineages generated in anagen undergo apoptosis. Eventually, the hair follicle enters telogen again. During the hair cycle, skin vasculatures (shown as red cables) also change in parallel to the hair follicles. From late catagen to telogen, a horizontal plexus under hair germ (HPuHG) assembles. In anagen, endothelial cells proliferate and disperse. In catagen, endothelial cells undergo apoptosis and deposit to form the HPuHG.
We checked the vasculature arrangement at different stages of the hair cycle. Interestingly, we found that at quiescence stage, a horizontal vascular plexus is formed under the hair germ, which we named it “Horizontal Plexus under Hair Germ (HPuHG)”. This vascular plexus quickly disperses as the hair follicle starts proliferating. Then after apoptosis, the skin vasculature deposits to form the HPuHG again. Since we looked at the quiescence stage in Runx1 EpiKO and in Alk1 EndKO, a stage when HPuHG exists, we believed that the vascular phenotype in the two mutants is in fact an increase in the HPuHG vasculature. These observations strengthened our idea that BVs have distinct role in molecular signaling in addition to nutrition and oxygen, contrary to our belief that increased vasculature causes delay in hair growth. Moreover, distinct pattern of BVs during hair growth supports the idea that BVs are important part of HFSCs niche.
To understand whether molecular signal derived from endothelial cells could cause the HFSC activation defect, we checked if a well-established HFSC quiescence factor BMP4 is also expressed in skin endothelial cells. Previous study indicated endothelial cells express BMP4 in the lung (Frank David et al., 2005). We did immunofluorescence staining for CD31 and BMP4, and found colocalization between the two signals, indicating skin endothelial cells are a source of BMP4. While the expression level of BMP4 in endothelial cells and the average BMP4 intensity in interfollicular epidermis are not altered by the mutation of Alk1 or Runx1, the average intensity of BMP4 in the selected HPuHG region is higher in mutants than in control mice. Western blots also showed elevated BMP4 level in Alk1 EndKO. Though more solid evidence such as RNA-Seq of hair follicle cells from the mutant skin is needed to conclude that endothelial BMP4 is the cause of the HFSC activation delay, our data showed a correlation between BMP4 level, amount of vasculature, and HFSC activation defect. Specifically, our data suggested that excessive HPuHG vasculature may lead to excessive BMP4 in the vicinity of the hair germ, thus delaying the HFSC activation.
There have been previous investigations into the association between the skin vasculature and the hair follicle, but these have focused mainly on hair growth related to vasculature and failed to provide molecular evidence on how the vasculature regulates hair homeostasis (Ellis and Moretti, 1959; Mecklenburg et al., 2000). Our current research focuses on the quiescence stage that has been overlooked for many years, and how cross talk between HFSC and their environment influences their activity. Our data support a model where HFSCs are capable of sending signals to their vascular niche, and the vascular niche can reciprocally control HFSC activation (model summarized in Figure 3).
Figure 3: Hypothetical model of the cross-talk between HFSCs and their vascular niche. Our work suggested that a cross-talk exists between HFSCs and their vascular niche. In our hypothetical model, HPuHG (shown as red cables) starts to form from late catagen to telogen. Endothelial cells near the HPuHG secrete quiescence factors such as BMP4 to maintain the quiescence of HFSCs. In the reverse signaling, HFSCs in the hair germ (shown in green) use Runx1 as a master regulator to modulate their vascular niche. Specifically, many secreted molecules encoded by vasculature-related genes are downstream of Runx1. In response to Runx1 expression, HFSCs remodel the vasculature around them. Proper dispersal of the vasculature leads to the activation of HFSCs.
To our knowledge, this is the first publication suggesting a niche modulation role of HFSCs, and also a first documentation of the non-cell autonomous role of Runx1. Though more molecular details underlying the cross-talk between HFSCs and their vascular niche are to be elucidated, our research has opened a new window for future investigation. The Alk1 mutation has been studied in hereditary hemorrhagic telangiectasia, a disease marked by the fusion between arteries and veins. Our current study may further the understanding of its prevalence in skin, and may also provide new thoughts for future therapy. It also has implications for tissue regeneration and in clinical settings where targeting cancer SC niche through anti-angiogenic targets can be promising therapeutics.
Mecklenburg, L., Tobin, D.J., Müller-Röver, S., Handjiski, B., Wendt, G., Peters, E.M.J., Pohl, S., Moll, I., and Paus, R. (2000). Active Hair Growth (Anagen) is Associated with Angiogenesis. Journal of Investigative Dermatology 114, 909-916.
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Cell adhesion molecules (CAMs) of the plasma membrane physically tie cells together into tissues, both via interaction of cells with other cells or with ECM (extracellular matrix); they also constitute hubs of information exchange with neighbouring cells and the environment. CAMs mediate a broad range of biological functions and their aberration contributes to a range of diseases, such as cancer progression or aberrant growth/regeneration of nerves.
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