A discussion of “Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signalling,” Nat Cell Biol, 2017. 19(5): p. 433-444.

We have recently revised the model of Follicle Stem Cell (FSC) organization in the Drosophila ovary, showing that there is a much larger population of stem cells than formerly realized, that these FSCs exhibit population asymmetry, and that they give rise to Escort Cells as well as Follicle Cells [1]. Ovarian Germline Stem Cells have long been used as model stem cells, benefiting from easy recognition by virtue of their characteristic location at the anterior of the germarium, morphology, and functional markers that reflect the actions of a key BMP (bone morphogenetic protein) niche signal. FSCs, which are required for the development of germline cells into a mature oocyte, have been more difficult to investigate. No marker specific to these stem cells has been discovered, they are not in an easily recognized location and are not morphologically conspicuous among their neighbors.

In 1995 Margolis and Spradling reported the identification of FSCs (originally named Somatic Stem Cells), which they located midway through the germarium by BrdU labeling and lineage analysis [2]. An FSC is defined by lineage analysis as a cell that can produce Follicle Cells (FCs) but remains in the germarium while germline cysts pass through. FSC derivatives that associate with a germline cyst in the posterior half of the germarium all initially proliferate. A few quickly become specialized post-mitotic polar cells and stalk cells to allow egg chamber budding from the germarium, while the rest form an expanding epithelium around the germline cyst, cease divisions in mid-size egg chambers (stage 6) and adopt position-specific fates about a day later (stage 9). We refer here to all of these FSC derivatives including the earliest precursors (often termed prefollicle cells) associated with a germline cyst as Folicle Cells (FCs). Marked lineages that originate in an FC have a predictable, limited lifespan because all FC progeny move through the germarium and ovariole along with the associated cyst at an established rate. It takes the earliest FC about two days to exit the germarium and five days to exit the ovariole. If a marked cell remains in the germarium or the ovariole for longer than this time then we can deduce that the lineage must have originated in an FC precursor, namely an FSC. Although this definition has been used since 1995, our perception has changed from expecting most FSCs to reside in the germarium much longer than an FC, to realizing that many FSCs are in fact very short-lived before becoming FCs.

History of FSC numbers

The number of stem cells was first calculated as the reciprocal of the fraction of the FC epithelium covered by a marked FSC clone and was measured at 9-11 days after clone induction to be sure that all transient FC clones had exited the ovariole [2]. Care was taken that the FSC clones examined likely originated from a single marked cell by inducing clones at low frequency with a heat-shock inducible recombination event, but an implicit assumption was made that FSCs divide asymmetrically (to produce one stem cell and one non-stem cell at each division) and that there would therefore be one marked FSC throughout the history of generating marked FCs. At the time, this was a common assumption for stem cells, inspired in part by studies of ovarian Germline Stem Cells. We found that FSCs do not repeatedly undergo asymmetric divisions but rather exhibit frequent amplification or loss of individual lineages (see later). An accurate count of FSCs deduced from FC contributions cannot therefore be made at 9 days after clone induction because by this time a single surviving clone will include an unknown number of FSCs. Earlier results therefore reflect the FC contribution of multiple FSCs, leading to a large underestimate of the total number of FSCs.

In 2001, Zhang and Kalderon used 3 genetic lineage labels and detected up to 3 different FSC lineage colors in a single ovariole 8 days after clone induction, concluding that there were likely three FSCs [3].  Identification of an FSC lineage was constrained by the requirement to identify an FC-contributing, surviving FSC in a location roughly consistent with the two FSC positions mapped by Margolis and Spradling; any marked FSCs that were lost prior to 8d, that occupied an unexpected position or did not contribute any FCs over the last five days would not have been scored. In 2007 Nystul and Spradling also used 3-genotype labeling and postulated 2 fixed niches of FSCs on opposite sides of the germarium, stating that the germarium is bilaterally symmetric and lies down on the slide in a particular orientation such that the two FSC niches are on either side of the germarium [4]. They did sometimes see three FC genotypes in an ovariole, but postulated that an FSC daughter could temporarily occupy one of the niches and act as an FSC.

In our more recent experiments we used the same strategies as before (counting the contribution of a single FSC lineage to the FC epithelium or counting the number of distinctly colored FSC lineages that can be generated in a single epithelium) but concluded that there are many more FSCs. The biggest reason for the very different outcome was the consideration that some FSC lineages might be lost very quickly while others amplify, necessitating looking at outcomes at a variety of times after marking. Other important differences included using more colors for lineage marking; consideration of more potential locations for FSCs and the possibility that a marked FSC may only occasionally give rise to an FC; and thorough analysis of all cells in a set of ovarioles selected without bias. We developed a lineage tracing method that yields 6 different combinations of GFP, RFP and β-galactosidase. Our strategy was to induce as many recombinations as possible in dividing stem cells in order to see the maximum number of FSC lineages. Using the enduring definition of a surviving marked FSC (a cell that remains in the 2a/b region and has given rise to at least one FC patch in the same ovariole), we found up to six (the maximum we could score) surviving FSC lineages in a single ovariole and inferred from the number of colors seen in a large sample of ovarioles that there were about five surviving FSC lineages on average.

We also found that a single FSC lineage contributed to about 15% of the FC epithelium in a highly mosaic fashion with zero contribution to several egg chambers. Most important, counting surviving FSC lineages at different times after marking showed that many more lineages were present at 5d, while there were decreasing numbers at later times. We estimated that there may be about ten surviving FSC lineages at 5d, but we would need more than six color combinations to count more accurately (five or six colors were present in almost all ovarioles). Consequently, our extrapolation that there may have originally been as many as sixteen FSC lineages at day zero is also just an approximation. In these experiments we did not change the basic methodology or logic of deducing FSC numbers: every one of the cells we called an FSC remained in the germarium for longer than an FC can and had produced at least one FC, defining it unequivocally as an FSC.

Even after taking stem cell loss and amplification into account, it turns out that there is a further limitation on what was counted as a functional FSC because we required proof that an FSC produced an FC. It turns out that some of the roughly sixteen FSCs in a germarium, all of which we believe to have the potential to produce FCs, do not produce any FCs in the 4-5 day time span displayed on a fixed ovariole. This stems in essence from the apparently stochastic behavior of FSCs and positional heterogeneity within the FSC population.

FSCs are radially distributed in layers

In order to identify positions of FSCs we had to examine only those FSC clones with a single candidate stem cell (a single FSC of a certain color that matched an FC patch). This approach allowed us to define multiple locations for an FSC anywhere around the circumference of the germarium and in any of three anterior-posterior (A/P) layers adjacent to the Fasciclin 3-expressing FCs.

One of the challenges we faced as we started to realize that there were many more FSCs than expected was how to score them in sufficient detail.  Eventually we developed spreadsheets to record, for each germarium and ovariole, every cell in the 2a/b region by A/P position, and the proportion of each egg chamber occupied by each color. This level of detail was essential to see where FSCs are located and to measure contributions to the FC epithelium. Tabulating the total number of cells in the 2a/b region where FSCs could be found, “the FSC region,” (16 cells on average) provided a second means to deduce the total number of FSCs. The frequency of clones with a single FSC in a given layer was very similar to the distribution of all somatic cells in the FSC region (about 8/16 in layer 1, 6/16 in layer 2 and 2/16 in layer 3, on average), suggesting that all of the roughly 16 cells are FSCs.

At the same time that we started scoring FSCs around the entire circumference, we were surprised to find by live imaging that cells in the FSC region do not stay in fixed locations but undergo back-and-forth radial movements. In addition, the ring of cells around the circumference is indistinguishable when examined by many Gal4 expression patterns and markers [5]. The existence of apparently identical interchangeable cells around the circumference of the germarium at a given A/P position suggests that each ring of FSCs constitutes a pool of equivalent cells.

Population asymmetry

Fundamental to revising our ideas about FSC numbers and behavior is the realization that they are governed by population asymmetry. Population asymmetry is generally understood to mean that the daughters of a stem cell do not necessarily have different fates (one stem and one non-stem cell). Instead, within a homogeneous stem cell population of fixed size, individual stem cells undergo neutral competition during which a stem cell is frequently and stochastically duplicated or lost. Short-lived stem cells are not pre-determined or different from long-lived stem cells—they are selected by chance. If you look at the starting population you can say that only a small subset of those cells will survive as lineages for long time periods but you don’t know which ones. Hence, all of the cells are equivalent and have the potential to survive for a long time and are appropriately given the same name—stem cells. One would have to look very early after marking stem cells to capture them all before any are lost, which is often not practical. Counting the lineages after an arbitrary delay yields the number of stem cell lineages that survived for that time, and eventually only a single lineage will remain. Population asymmetry is by now a well-recognized arrangement that applies to many stem cells, including mammalian gut stem cells[6, 7]. In the mouse small intestine there are around 16 stem cells per crypt but several weeks after labeling individual cells with multiple colors only 1-3 lineages remain in most crypts[7]. We have exactly analogous findings. In both cases researchers first looked long after stem cell marking and, accordingly, thought there were a smaller number of stem cells. The appreciation of the dynamics of population asymmetry resolves these apparent contradictions in both cases.

Our data counting colored patches in ovarioles with no corresponding FSC at 9d clearly showed that many FSC lineages are lost rapidly. By 30 days the majority of ovarioles are monoclonal (we found an average of 1.5 lineages out of 60 ovarioles scored). There is a corresponding increase in the number of FSCs per lineage over time. This demonstration of rapid FSC lineage loss along with a corresponding increase in the number of 2a/b cells per surviving lineage over time shows that FSCs are maintained by population asymmetry.

Origin of Escort Cells—why had it been difficult to see the relationship to FSCs?

 ECs are also labeled in lineage experiments in adults, showing that they are renewed in adult ovaries. However, Margolis and Spradling concluded that there is no lineage relationship between FSCs and ECs [2]. Kirilly et al. concurred but found that the source of new ECs is region 2a/b, very close to FSCs, postulating that the dividing cells are themselves ECs [8]. In these and other lineage studies, including ours, one could readily see ovarioles with marked FCs but no ECs, or marked ECs but no FCs, but there were also many ovarioles containing marked ECs and FCs (as well as FSCs). It was challenging to unravel the basis of these different outcomes.To prove that an FSC can give rise to both ECs and FCs it is essential to prove that a marked lineage containing all three cell types derived from a single cell.  That is normally accomplished by inducing marked cells at very low frequency. We found it difficult to label single FSCs using the MARCM technique even by using the mildest of heat-shocks. It is understandable that it is quite difficult to target a single FSC for recombination by chance, given that there are about 16, rather than 2-3 per germarium.  We eventually accomplished a satisfactorily low clone induction by additionally taking advantage of multicolor labeling to produce a very low frequency of clones with a specific color combination.

We also became convinced that all adult-born ECs derived from FSCs because the locations of cells in region 2a/b that proliferate, according to EdU labeling, is the same as FSC locations deduced from our multicolor labeling; it was previously thought that most of these cells were ECs. We can now categorize an EC not only by location and morphology but also by the unifying property of exhibiting no proliferation. However, it was puzzling that many lineages contained only ECs or only FCs if all of their progenitors can give rise to both ECs and FCs. We resolved this paradox by a combination of careful analysis of FSC lineages over time and the appreciation that the FSC population is heterogeneous. FSCs can produce only FCs for a few days in succession but a longer time-course shows that virtually all FC-producing lineages eventually produce ECs. Conversely, FSCs can produce only ECs for a few days; ovarioles only reveal FC production over the past 4-5 days, so a constant fraction of FSCs (around 20%) are not associated with marked FCs even though FC production may have been in their past or future. The explanation for periods of producing only ECs or only FCs lies in positional heterogeneity of FSCs.

Heterogeneity of FSCs

We found graded cell division rates and graded marker expression across FSC layers. Most important, we found that recent FC production was tightly correlated with FSC location in the most posterior layer (layer 1), leading us to postulate that FCs come directly only from layer 1 FSCs. The process of associating irreversibly with a germline cyst passing through the FSC region has never been visualized, so there was no clear prior indication of where an FC is first “specified.”  ECs are anterior to FSCs, so it is almost inevitable that they must derive directly from anterior FSCs, and we also observed movement of anterior FSCs to the EC region by live imaging. Thus, the direct precursors of FCs and ECs are in different FSC layers and as long as an FSC lineage is confined to a single layer it will produce only ECs or only FCs, as often observed at short times after FSC marking. At later times, most surviving FSC lineages amplify and occupy more than one layer, followed by production of both ECs and FCs. Those FSC lineages that are then lost will leave a residue of only, generally long-lived, ECs. Movement of FSCs between layers was also inferred more directly by finding the derivatives of single marked cells in more than one layer at a reasonably high frequency even within 3 days.

The heterogeneity of FSCs presents a complication in identifying FSCs by markers because many proteins that are highly expressed in Escort cells are graded in expression across the FSC domain. For example, more anterior FSCs express much higher levels of the Wnt reporter Frizzled3-RFP and the enhancer trap PZ1444 than posterior FSCs. Other markers, such as Castor, are roughly evenly expressed across FSC layers but are also expressed in FCs.  We found that only Fax-GFP stood out as being enriched in FSCs compared with ECs or FCs.

Wnt signaling

We had for many years been puzzled as to why FSCs were lost at an enhanced rate when Wnt pathway activity was either eliminated or increased in FSCs. For Hedgehog and JAK-STAT pathways the results were simpler; more activity caused better survival in competition with wild-type neighbors. With our new understanding of FSC organization and behavior we could look more carefully at the consequences of manipulating Wnt pathway activity, and what we found was straightforward and striking. When Wnt pathway activity is eliminated, FSCs of that lineage become heavily biased towards occupying the most posterior FSC layer and produce FCs, with virtually no EC production. On the other hand, excess Wnt signaling forces FSCs at first into the more anterior FSC layers and eventually to become ECs. These results showed that the strength of Wnt signaling influences the A/P location and fates of FSCs.

Similarity to mammalian epithelial stem cells

With the updated organization of FSCs uncovered, we were amazed to find how similar FSC organization is to mammalian intestinal stem cells. Both FSCs and intestinal stem cells give rise to a constant supply of polarized epithelial cells. Similarities include the number of cells per niche, the organization of stem cells in heterogeneous layers, and use of Wnt as a critical niche factor[9]. Mammalian gut stem cells can produce transit amplifying (TA) or quiescent secretory cells, including Paneth cells, which eventually are retained in the crypt while TA cells migrate away. The initial location of stem cell derivatives that lead to these different outcomes are not clearly defined.  Hence the spatial cues for differences in signaling pathway activities (Notch and Wnt) involved in deciding outcomes are not clear. FSCs appear to present a slightly different and simpler paradigm that may also inspire enquiry into an analogous organizational principle for gut stem cells. Wnt signaling is graded across the A/P axis of the FSC community. The relative levels of Wnt signaling dictate whether FSCs adopt more posterior positions and yield predominantly FCs (low Wnt pathway activity) or adopt more anterior positions and become ECs (high Wnt pathway activity), in accord with the natural gradient of Wnt pathway activity in this region. The paradigm of different direct differentiation outcomes for a stem cell population depending on stem cell location might apply to mammalian stem cell communities as well.

Amy Reilein and Daniel Kalderon

Department of Biological Sciences, Columbia University, New York, NY

References

1. Reilein, A., et al., Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signalling. Nat Cell Biol, 2017. 19(5): p. 433-444.
2. Margolis, J. and A. Spradling, Identification and behavior of epithelial stem cells in the Drosophila ovary. Development, 1995. 121(11): p. 3797-807.
3. Zhang, Y. and D. Kalderon, Hedgehog acts as a somatic stem cell factor in the Drosophila ovary. Nature, 2001. 410(6828): p. 599-604.
4. Nystul, T. and A. Spradling, An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement. Cell Stem Cell, 2007. 1(3): p. 277-85.
5. Hartman, T.R., et al., Novel tools for genetic manipulation of follicle stem cells in the Drosophila ovary reveal an integrin-dependent transition from quiescence to proliferation. Genetics, 2015. 199(4): p. 935-57.
6. Lopez-Garcia, C., et al., Intestinal stem cell replacement follows a pattern of neutral drift. Science, 2010. 330(6005): p. 822-5.
7. Snippert, H.J., et al., Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell, 2010. 143(1): p. 134-44.
8. Kirilly, D., S. Wang, and T. Xie, Self-maintained escort cells form a germline stem cell differentiation niche. Development, 2011. 138(23): p. 5087-97.
9. Beumer, J. and H. Clevers, Regulation and plasticity of intestinal stem cells during homeostasis and regeneration. Development, 2016. 143(20): p. 3639-3649.

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Last year we were approached by Andreas Prokop of The University of Manchester (who is also Communications Officer of the British Society for Developmental Biology), with an offer to write a paper on our experiences of running the Node and using social media to build scientific networks. We – that is Aidan Maartens (the Node’s Community Manager), Catarina Vicente (who previously held the post) and Katherine Brown (Development’s Executive Editor) – accepted, seeing it as a great opportunity to promote our work as well as explore what we have learned after six years. The paper is part of an exciting upcoming Special Issue of Seminars in Cell & Developmental Biology on science communication, and features input from individuals and organisations who use social media 

The following excerpts give a taste of where we came from in the article, and you can find a link to it below.

Launching the Node

Established in 1953 and initially known as the Journal of Embryology and Experimental Morphology, Development (www.dev.biologists.com) is a leading research journal in developmental biology. Run by the not-for-profit publisher The Company of Biologists, whose mission is to support and inspire the biological community, it has a specific remit to support the needs of developmental biologists. In 2009, a survey conducted by the journal highlighted the idea that Development − seen as a community journal − should be doing more for the community. Specifically, the survey identified a desire for an environment where members of this and related fields (most notably stem cell biology, but also other intersecting fields such as cell biology, evolution and genetics) could gather and interact online, bypassing the need for each internet-savvy researcher to build their own network from scratch. Development responded in 2010 by establishing an online hub called the Node. The site’s name reflects its aim: from a technical perspective, a node is simply a connection point, while developmental biologists know the node as an important group of cells that instruct and organise the activity of others in the early embryo. The Node was hence conceived as an online connection point for developmental biologists. It would provide a place where ideas could be discussed and exchanged by the whole community, without the restrictions of more formal publications, and would encourage an informal style and varied content, as well as dedicated pages for job opportunities and events useful for community members. Importantly, the Node would be open to anyone interested in contributing and would be easy to use. Thus, to some extent, the Node could be considered an online (and hence more flexible and accessible) version of a scientific newsletter − an informal form of communication aimed at a defined group of researchers with a remit to facilitate exchange of ideas and provide information on useful resources.

The Node was conceived as an online connection point for developmental biologists

6 years on

The digital age has opened the doors to a brave new world of communication outside traditional restrictions of geography, funding and editorial control. While the scientific community in general has not necessarily been quick to adopt and exploit all the opportunities now available, these technologies are changing the way we communicate. The Node, born out of a desire among the developmental biology community for an online communication hub, exemplifies both the opportunities and the challenges of community building online. Perhaps the biggest lesson we have learned in the 6 years of operating the Node is that the initial concept of a self-sustaining community site was unrealistic: the Node relies on ongoing financial, technical and strategic support from The Company of Biologists. However, we have also learned that this support is recognised and appreciated: most members of the developmental biology community are now aware of the Node, even if they don’t actively participate in it, and value its utility as an online hub for the field. Social media, particularly Twitter, have helped us reach a wider audience, and better gather and disseminate information. The original vision of the Node – as your online coffee break, a place to catch up on the latest news from the field − has been realised, although we continue to develop and grow. Currently, significant efforts are focussed on improving the resources section of the site, to help with teaching, advocacy and outreach activities, as well as on growing our reader- and authorship in particular geographic and scientific areas where we are less well represented than we would like. We are also exploring formats that can better facilitate online discussion.

The Node exemplifies both the opportunities and the challenges of community building online

You can read the full paper freely here:

The Node and beyond–using social media in cell and developmental biology

Catarina Vicente, Aidan Maartens, Katherine Brown

Seminars in Cell & Developmental Biology

https://doi.org/10.1016/j.semcdb.2017.05.009

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POSTDOCTORAL POSITION is available to study different aspects of lymphatic vasculature development and metabolism in health and disease. Some of the projects include the characterization of the cellular and molecular mechanisms controlling the development of the lymphatic vasculature, endothelial cell plasticity and reprogramming, as well as different aspects related to lymphatic function and metabolism. Highly motivated individuals who recently obtained a PhD. or MD degree and have a strong background in mammalian vascular, molecular and developmental biology are encouraged to apply. Interested individuals should send their curriculum vitae, a brief description of their research interests, and the names of three references to:

Guillermo Oliver, Ph.D

Thomas D Spies Professor of Lymphatic Metabolism

Director Center for Vascular and Developmental Biology

Northwestern University Feinberg School of Medicine

303 East Superior Street, 10-107

Chicago, Illinois 60611

Email: guillermo.oliver@northwestern.edu

http://labs.feinberg.northwestern.edu/oliver/

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A Postdoctoral Research Associate position is available in the Conduit lab to study the role of the nuclear envelope in centrosome assembly. Our lab studies how microtubule formation is regulated in cells, including how microtubule organising centres (MTOCs) are assembled. The post-holder will use Drosophila melanogaster to follow up on our recent discover that the nuclear envelope may help regulate centrosome assembly. They will use CRISPR/HDR to generate a series of new fly strains, and then use advanced live-cell fluorescence imaging, including super-resolution imaging, and biochemistry to establish how the nuclear envelope helps regulate centrosome assembly.

The appointment will be for a period of up to three years starting 2nd October 2017, or as soon as possible thereafter.

Candidates are expected to have (or soon have) a PhD in cell/developmental biology and have experience in fluorescent microscopy. Prior experience in Drosophila, centrosome biology, molecular biology and/or basic biochemistry would be an advantage, but not essential.

Applications should include a C.V. and a brief statement of your scientific background and why you would like to join the lab.

To apply, please follow this link: http://www.jobs.cam.ac.uk/job/13999/

Informal enquiries are welcomed and can be addressed to Dr. Paul Conduit ptc29@cam.ac.uk

Please see the lab website for more information on our research http://conduitlab.zoo.cam.ac.uk

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Job Details

Fully funded postdoctoral positions are presently available in the Conlon Lab whose studies focus on identifying the molecular networks that are essential for early heart development and how alterations in these networks lead to congenital heart disease. For these studies, we use a highly integrated approach that incorporates developmental, genetic, proteomic, biochemical and molecular based studies in mouse, Xenopus and stem cells.

Recent advances and projects of interest in the Conlon lab include studies that define the cellular and molecular events that lead to cardiac septation, those that explore cardiac interaction networks as determinants of transcriptional specificity, the mechanism and function of cardiac transcriptional repression networks and, the regulatory networks of cardiac morphogenesis.

http://www.unc.edu/~fconlon/

Twitter: @fconlon

Job Requirements

Candidates should have recently obtained or be about to obtain a Ph.D. or M.D. in a field of biological science and should have a strong publication record. Outstanding and highly motivated candidates should apply by email to Dr. Frank L. Conlon and include a CV/resume, three references and description of your specific interest in our research programs.

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Daily life changes when you set foot in Woods Hole. There is a beauty in your surroundings and energy in the air that invigorates you. The days are long (8am to 2 or 3 am most days!) and we have a full schedule but we are all so excited to be here – to learn – to question – to move the field forward.

A typical day in the course starts with morning lecture at 9am. The lecture is broken down into two parts, the first part is general information about the model system we are working with and the second is about the current research project in the speaker’s lab. After lecture we head to the ‘Sweat Box’ where the students ask the speaker questions. This includes grilling them about their research, it was said this session is like a qualifying exam for the professors! We can also use this time to ask them about their career trajectory and for career advice. We then break for lunch; two students take the speaker to lunch where they get to talk more in depth about science and life.

After lunch we head to the lab, we get a ‘cookbook’ for each organism; this includes information on how to take care of them and many protocols. In this ‘cookbook’ there are instructions on how to fertilize the egg, manipulate the embryo and the organism as well as a list of reagents and tools that are available to us. We then brainstorm ideas in groups and have full access to the teaching assistants, professors and course directors to plan and execute our experiments.

We break in the early evening to take a run, shoot some hoops or practice softball for the annual softball match (watch out physio) and eat dinner. Two students also get to take the speaker out to dinner at a local Woods Hole restaurant during this time. Then it’s back to the lab to continue our experiments into the wee hours of the morning!

How do we survive this schedule for six weeks? We drink a lot of coffee!

Follow the course here on the blog and on twitter #embryo2017

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A postdoctoral position is available to study the extrinsic modulation of intestinal proliferation in the research group of Golnar Kolahgar at the Department of Physiology, Development and Neuroscience at the University of Cambridge. The candidate is expected to hold a PhD in cellular, molecular or developmental biology.

Our goal is to identify the components of the extracellular space that contribute to maintaining and remodelling the adult intestine in response to various physiological conditions. We use the genetically tractable Drosophila gut as a paradigm to investigate cell fate decisions in vivo (e.g. Kolahgar et al, Dev Cell, 2015; Suijkerbuijk et al, Curr. Biol, 2016). The aim of this project is to explore how integrin signalling promotes intestinal stem cell proliferation and contributes to gut plasticity, using a combination of Drosophila genetics, lineage tracing and clonal analysis, confocal imaging and whole genome sequencing, thus experience with one or several of these techniques is required.

The post is funded for an initial period of 3 years.

For more information and to apply, follow this link: http://www.jobs.cam.ac.uk/job/13950/

Informal enquiries to Golnar Kolahgar (gk262 [at] cam.ac.uk)

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Development of the placental vasculature – known as the labyrinth – is critical for foetal development. Today’s paper comes from the most recent issue of Development and addresses the signalling events involved in placental vascular maturation. We caught up with lead author Lijun Chi and her PI Paul Delgado-Olguin of the Hospital for Sick Children and University of Toronto.

Can you give us your scientific biography and the main questions your lab is trying to answer?

PD-O As a Ph.D. student at the Universidad Nacional Autónoma de México (UNAM) under supervision of Dr. Ramón Coral Vázquez and Dr. Félix Recillas Targa, I investigated the transcriptional regulation of genes expressed in skeletal muscle and whose mutations cause muscular dystrophies. My reading during my Ph.D. studies led me to numerous landmark papers from Eric Olson’s lab on skeletal muscle gene regulation. I soon discovered Dr. Olson’s and Dr. Deepak Srivastava’s seminal work on the transcriptional pathways controlling heart development, a process that I always found fascinating. Working at Dr. Recilla’s lab, I became very interested in the function of chromatin modifiers in gene control, which led me to mine the scientific literature to learn about the function of chromatin structure regulators in cardiac gene control and development. This made me realize how little was known on the subject at the time, and brought me across a pioneering paper from Dr. Benoit Bruneau’s lab describing an essential function of Baf60c, a subunit the SWI/SNF chromatin remodelling complex, in heart development. This helped me decide what I wanted to do as a postdoc.

I obtained my Ph.D. degree in June 2005, and by August I was in Dr. Bruneau’s laboratory ready to start my postdoctoral research. In Dr. Bruneau’s lab I investigated the function of the histone methyltransferase Ezh2 in the heart. This enzyme tri-methylates the lysine 27 of histone H3 to repress gene expression, and its global deletion causes lethality early in mouse development. Surprisingly, we found that deletion of Ezh2 in cardiac progenitor cells, despite altering embryonic gene expression, did not alter heart development, and mutant mice had normally structured hearts. However, adult mutants developed heart disease. This raised the possibility that epigenetic alterations in differentiating cardiovascular progenitor cells early in development might program adult heart disease susceptibility. To address this possibility, my lab at The Hospital for Sick Children (SickKids) has been studying the function of several histone modifiers in cardiac and vascular development, and in regulating adult cardiovascular system function since 2012. Because the placental vasculature is required for proper embryo development, and its malfunction can affect heart development and program adult disease in the offspring, my lab is also interested in uncovering the mechanisms controlling its development. My lab will continue to focus on uncovering the mechanisms controlling cardiovascular development and programming postnatal disease during embryogenesis.

And what is Toronto like to do science in?

PD-O Toronto is a great place to do science. It is home for numerous world-class scientists, research institutes, numerous hospitals with very strong research programs, and the University of Toronto, which has an outstanding research curriculum. These attributes make Toronto and ideal place to develop multidisciplinary research of the highest quality. For example, my lab investigates fundamental biological processes, and being at SickKids and in Toronto’s rich scientific environment allowed establishment of key collaboration with clinician scientists in neighbouring institutions, which has facilitated me to explore the translation potential of my lab’s research.

Lijun, how did you come to join the Delgado-Olguin lab?

I obtained my PhD. at the University of Oulu, Finland under the supervision of Professor Seppo Vainio. My PhD thesis, published in 2007, describes the role of Sprouty2 in development of the urogenital system. I then pursued a postdoctoral fellowship in Dr. Norman Rosenblum’s laboratory at SickKids in Toronto, Canada. During my fellowship I investigated the function of the cilia protein Kif3a, whose deficiency causes polycystic kidney in human and mouse models. After finishing my fellowship in 2011, I wanted to learn about cardiovascular development, and I knew Paul was just about to Join SickKids. Working at the Delgado-Olguin lab has given me the opportunity to work in exciting projects to understand the basis of cardiovascular development and disease.

What was known about signalling pathways controlling placental vascular maturation before you started this work?

PD-O Because of its relevance in embryogenesis and in postnatal health, I was surprised to find out how little we knew about pathways controlling placental vascular maturation before we started our work. Perhaps the most informative report on the subject is from Knox & Barker (2008), who performed global gene expression analyses on the embryonic portion of mice placental vasculature, known as the labyrinth, at consecutive days of development. This analysis revealed a sharp molecular transition defining the developmental and the maturation phases of the placenta. In this transition, over 700 genes change their expression from embryonic day 12.0 to E13.5, and functions associated with these genes provided a general idea of some of the processes occurring in each phase. For instance, genes that are expressed in the developmental phase are involved in growth, metabolic processes, DNA and RNA processing, and cell cycle regulation. While genes active in the maturation phase are involved in pregnancy and reproduction. However, these studies were done on whole labyrinth, and thus tell us little on the pathways controlling this transition in specific cell types. To the best of my knowledge, our work is the first one to address the regulation of the transition from development to maturation in placental vascular maturation, and to define pathways active in endothelial cells regulating this process.

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

PD-O We found that the histone methyl transferase G9a activates the Notch pathway in endothelial cells to promote maturation of the placental vasculature. We inactivated the histone methyltransferase G9a in endothelial progenitors and their derivatives, and found that mutant embryos died with placental defects. Closer examination revealed that the gross morphology of the placentae from mutant embryos appeared normal at E12.5, but had a smaller vascularized area at E13.5. Because the transition from the developmental to the maturation phase of the placenta occurs precisely between these stages, this raised a possible function for G9a as a regulator placental vascular maturation. Analysis of cell proliferation revealed that growth of the labyrinth is coordinated with decreased growth of the spongiotrophoblast during the development to maturation transition, and that this balance is lost in G9a mutant placentae. To uncover regulatory pathways we performed global gene expression analysis, which revealed that effectors of the Notch pathway were downregulated in G9a mutant placental endothelial cells. We then introduced a transgene to activate the Notch pathway in G9a mutants, and we found that the placental morphology was rescued! This opened the possibility that a G9a-Notch axis might be disrupted in placental diseases with vascular maturation defects. Indeed, we found that G9a, and Notch regulators were downregulated in human placentae from pregnancies affected with intra uterine growth restriction.

How might your work inform efforts to diagnose or even treat placental defects during pregnancy?

PD-O These are very exciting possibilities. Intra uterine growth abnormalities are diagnosed only when the foetus is smaller than expected for the gestational age or when the placenta is already malfunctioning. The ability to identify preclinical placental insufficiency is limited because we know very little about the regulation of placental vascular development, and on the events that precede placental malfunction. We found that imbalanced growth of the labyrinth vs the spongiotrophoblast precedes the appearance of gross morphological abnormalities in G9a mutant placentae. Based on these results, we think that being able to define the growth ratio of these placental cell types, and detect imbalances might offer a way of identifying foetuses at risk of defective intrauterine growth. In terms of prevention or treatment, our findings open the possibility that activating the Notch pathway might be investigated as a means to promote placental vascular maturation. Our mouse model, combined with availability of pharmacologic compounds that activate the Notch pathway, will allow us to further investigate these possibilities.

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

LC In the initial stage of the research, we were mainly focused on identifying cardiac defects in G9a mutants. However, I noticed that mutants had placentae with reduced vascularization. I decided to analyze embryos at consecutive developmental stages and I found that the vascular defect was obvious only at E13.5 and onwards. When I realized that the transition from the developmental to the maturation phase occurs precisely from E12.5 to E13.5, I hypothesized that G9a might regulate this transition. Given that the regulation of placental vascular maturation is very poorly understood, we decided to investigate further and test the hypothesis.

What about the flipside: any moments of frustration or despair?

LC As often happens with genome wide gene expression data, it was difficult to identify potentially relevant targets downstream of G9a from our RNAseq results. Fortunately, we found published reports demonstrating the involvement of the Notch signalling pathway in vascular maturation in the retina. We also found a report showing downregulation of some Notch regulators in placentae from pregnancies affected by intrauterine growth restriction. These reports encouraged me to confirm downregulation of Notch effectors in G9a mutant placental endothelial cells, and later on to test the effect of activating the Notch pathway in G9a mutant endothelial cells. These experiments were nerve wracking, because if the vascular phenotype were not to be corrected at least partially, I would have had to keep trying to identify functionally relevant G9a targets.

What are your career plans following this work?

LC With the completion of this current study, I am planning to test whether pharmacologically activating the Notch pathway in the G9a mutant placental vasculature promotes the maturation process and ameliorates or prevents placental defects. This might open the door to new experiments to try to promote vascular maturation in other models of intrauterine growth restriction.

And what next for the Delgado-Olguin lab?

PD-O We will delve deeper into the mechanisms by which G9a controls placental vascular maturation and the effects of activating Notch signalling in the placental vasculature. There are outstanding questions from our work. Particularly intriguing is how G9a activates the Notch pathway, as it is predominantly known as a transcriptional repressor, while its function as a transcriptional activator is less understood. Dissecting the mechanisms of action of G9a in placental endothelium will likely reveal additional regulatory pathways and potential approaches to promote vascular maturation. More broadly, our lab will continue to investigate the mechanisms by which postnatal cardiovascular disease is programmed during embryogenesis.

Finally, what do you two like to do in Toronto when you are not in the lab?

PD-O I enjoy hiking the many trails and parks in the city with my family and dogs, visiting museums, and fishing. Also, being an avid foodie, living in a city where there is food from all over the world available close by is a great bonus!

LC During my spare time, I am a passionate reader who enjoys a diversity of novels. I am also a skilful cook who loves exploring different recipes and trying out new styles. To stay physically well rounded, swimming is one of my weekly activities.

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Today marks Day 3 of the Embryology: Concepts & Techniques in Modern Developmental Biology course (http://www.mbl.edu/education/courses/embryology/) at the Marine Biological Laboratories in Woods Hole, MA (http://www.mbl.edu/).

24 students along with faculty, teaching assistants and course assistants arrived over the weekend to embark on a life changing summer experience. Since my days in undergraduate research in cell biology, I always heard fascinating stories about the science and life at Woods Hole. I knew it was an experience that I wanted to take part in during my training. The MBL at Woods Hole is a mecca of research, creativity, ingenuity and curiosity it gives everyone who sets foot on its campus the opportunity to learn new concepts and techniques. This course and the community here allow you to test your cool and creative hypotheses and push you out of your comfort zone. It’s an experience that empowers scientific adventure and will leave you wanting to come back year after year.

In the first days here as a student in one of the longest standing classes at the MBL I have been welcomed into an amazing group of scientists. My fellow students and I had the opportunity to share our research with our peers and some of the course faculty during an informal poster session over pizza, listened to amazing lectures on echinoderm development, and started to learn how to make the tools and run the microscopes that will enable us to conduct our experiments.

I can’t wait to see what we learn and discover this summer!

Follow the course here on the blog and on twitter #embryo2017.

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