Forget about those large amounts of bottles containing thousands of flies, those huge piles of boxes containing different lineages of mice or large tanks filled with happy-hopping frogs. Also, forget about transgenic, mutant, knockout litters… what I am going to tell you is the routine of an emergent lab working (or, better, trying to) with emergent models that you probably haven’t heard about for a while.
My name is Emilio Lanna and I am a young faculty at the Biology Institute in Federal University of Bahia (UFBA) in Salvador, Bahia, Brazil. I coordinate the Embryology and Reproductive Biology lab (LEBR), which currently consists of four grad students and four undergrads; all of them focused in understand the evolution of sponge development and life history.
Sponges (phylum Porifera) are aquatic animals living in the bottom of the oceans (you can find some species in freshwater, too). They are morphologically very simple, having a body adapted to pump as much water as possible to get their aliment, get rid of toxic metabolites, and even reproduce. However, although morphologically simple, their genomes are complex and similar to those of other animals. It is very important to study the development of sponges if we are to understand the evolution of animals as whole, as this group is considered the sister-group of all other metazoans (Figure 1, BUT you may find divergent opinions about that, especially those that put ctenophorans as the sister-group of all metazoans, but this is a history for another time). Their development may also shed light on the ecology and conservation of those animals. And, from an anthropocentric perspective, sponges are a rich source of natural products with a broad range of potential application in pharmaceutics, cosmetics and bioproducts. Last, but not least, the sponges’ cells have the ability to differentiate into other cell types in any stage of their life cycle. Therefore, understanding the mechanisms of cell differentiation in these animals could help us to treat cancer and advance tissue regeneration therapies. Therefore, although they are mainly interesting from an evo-devo perspective, there is a potential for medical application for the knowledge generated studying sponge development.
Figure 1 – Metazoan phylogeny (see Lanna, 2015 for a better discussion about the phylogenetic relationship of the non-bilaterian phyla)
As stated before, we are working with emerging models. We are currently investigating ten different species belonging to two different classes and six orders of Porifera (Figure 2). Before starting our work, we knew nothing about the reproductive biology of these species. Even simple questions such as: “when?”, “how often?”, “how much?” and “how do they reproduce?” were never addressed by other investigators before. So, we are doing a lot of early and basic research here. To do that, the work in our lab always starts with the students heading to a gorgeous beach close to the campus (Figure 3). We usually plan the sampling looking at a tide table, in order to choose the time when the distance of the surface and the bottom is the lowest possible, because we are going to skin dive. No special gear is necessary, as the seawater in Salvador is always around 24-26 ºC (75-79 ºF). Diving is not always necessary, as there are some of the species that hang on nautical ropes in nearby harbour and others that are directly collected in a flat rocky substrate during the low tide. IMPORTANT! Before any sponge collection we have to register the sampling campaign in an online platform of the Brazilian Environmental Ministry.
Figure 2 – In situ photographs of some of the species of sponges investigated by our lab.
Figure 3 – Porto da Barra beach is located at the mouth of the Todos os Santos Bay, the 3rd largest bay in Brazil. It has warm and transparent waters throughout the year (credits: Creative Commons).
The dive can have different objectives and the after-dive work will depend on these. The most usual is to collect pieces of sponges for histological procedures. In this case, the samples are fixed in saline formaline and brought to the lab, where they are processed through standard protocols. The material is then observed with a compound microscope to search for the reproductive elements (eggs, sperm cells, embryos and larvae). Sponges lack any organs, and gonads or reproductive compartments are always absent. Gametes and embryos, when present, are found spread throughout the somatic tissue of the organism and the researcher has to quickly learn how to differentiate a choanocyte (a typical somatic cell) from spermatozoa and to find embryos among the choanocyte chambers (Figure 4). We can count these elements to answer the questions on when, how often and how much the species reproduce, but this procedure tells us little about the gametogenesis and embryogenesis.
Figure 4 – Histological preparations to study the reproduction of sponges. The upper panel shows an egg (arrow) among the choanocyte chambers. The right bottom panel is an embryo in late cleavages and the left bottom panels shows a parenchymella larvae.
To describe gametogenesis and embryogenesis, we are doing electron microscopy and planning three-dimensional reconstruction using a confocal microscope (using DAPI). For the electron microscopy (EM), we take two pieces of the same sponge: one is fixed for EM (with standard fixative cocktails) and other for histology. We first confirm that the reproductive elements are present through the histological slides, because it’s cheaper and routine in our lab. To prepare the samples for EM we have to take them to another lab in another institution (Fiocruz-BA). There, the wonderful crew of technicians do the hard work for us and we have only to check the semithin sections and then go to spend productive hours sitting in front of the screens of the scanning or transmission EM.
In the last few months we started another approach, which has been, at the same time, both challenging and very interesting. We are putting larval traps above the sponges in the field to collect the free-swimming larvae of these animals. We go to this beach few hours before the sunset and set the traps around the sponge. The traps lay there till the next morning when we dive again to collect conical tubes filled with 0.1-0.5 mm long larvae. These tubes are taken to the lab, where we observe the ciliated larvae swimming in circular pattern for hours. Then, with a lot of patience, we observe them to settle, metamorphose and start their development. We do that with direct observation, fixing in different periods and through time-lapse imaging. The larvae lack a functional aquiferous system (the one used to pump water). As the development of this trait is little understood, we are going to investigate the morphogenesis and dynamics of development of this system. We are currently staging the development of three species and are also planning to disturb their development applying drugs and physical disruptors.
As you could see till here, our lab is not what a canonical student of developmental biology might expect to find in a developmental biology lab. We are getting there, though. In a few months, we expect to sequence the transcriptome of different stages and parts of one of the species. After that, we are also planning to do some in situ hybridizations to check which cell type express key developmental genes. Until those issues become routine in our lab, they cannot be described here… so, I may come back in the future.
If you have any question or comment, please send me a message. I´ll be glad to chat with you!
Controlling differentiation using biophysical cues from development
Embryonic stem cells have the potential to become any cell type in the adult organism, but coaxing them to a specific fate continues to be a challenge for researchers. While many of the soluble signals involved in patterning the early embryo are well-established, only recently have tools been available to study how biophysical cues synchronize with other signals in the microenvironment to specify cell fate during development. Mechanical signals have been implicated in controlling stem cell fate in a number of contexts, but the molecular mechanisms involved are often not well-defined. By taking cues from embryonic development, we have identified a key role for mechanical signals in driving differentiation of human embryonic stem cells (hESCs) toward mesoderm, work that was recently published in Cell Stem Cell (Przybyla et al., 2016a). We used genetic and pharmacologic manipulation to identify molecular mechanisms whereby tissue tension directs stem cell fate and demonstrated how hESC differentiation protocols can be optimized to direct early progenitor specification.
Substrate compliance can be tuned to alter the ability of hESCs to differentiate toward mesoderm.
Whether scientists are working to regenerate damaged organs or fix genetic defects in existing organs, a major bottleneck in regenerative medicine is generating differentiated cells that exhibit a structure/function that faithfully recapitulates that of the cells within the tissues in healthy adult organs. One approach has been to mimic the soluble cues and transcriptional signaling cells receive and integrate during embryonic development as they transition to tissue-specific differentiation in the adult organism. Yet cells in tissue also receive spatial-mechanical cues that are critical for directing their fate and maintaining the tissue function.
For more than a decade the Weaver laboratoryhas been exploring the role of biophysical cues in adult tissue development and homeostasis and malignancy, in order to clarify how the physical context of a cell in a tissue drives embryogenesis and influences stem cell fate specification. To this end, our experimental efforts have focused on examining how cell-cell and cell-extracellular matrix interactions and tissue geometry regulate early developmental decisions. We have been studying early gastrulation and mesoderm specification using human embryonic stem cells and a simplified polyacrylamide gel system combined with three dimensional cell printers. Our findings revealed that the biophysical microenvironment helps to synchronize embryogenesis to facilitate mesoderm progenitor differentiation.
The complexity of embryonic patterning
The first symmetry-breaking process in embryogenesis is gastrulation, when cells are reorganized and divided up into three germ layers that go on to derive all the structures and organs of the body. This process also marks the first epithelial-mesenchymal transition (EMT), as cells in the epiblast ingress into the newly formed primitive streak and lose their epithelial characteristics to form the mesoderm layer. A major question remains as to how cells know where to go and when, and what to do when they get there. Cells derive this information from the inputs they receive from their cellular and noncellular microenvironment. A major goal of our work is to begin to unravel the molecular mechanisms whereby spatial-mechanical cues regulate gastrulation.
Epithelial versus mesenchymal cell organization can affect the cell’s response to extrinsic cues
When cells switch between epithelial and mesenchymal states, their organization with respect to each other becomes dramatically altered. Cells in an epithelial state have strong cell-cell contacts and exhibit collective cell behaviors such as coordinated migration and mechanical connectivity, whereas cells in a mesenchymal state acquire a motile phenotype that is linked in part to stronger cell-extracellular matrix interactions. After the initial EMT during gastrulation that forms the mesoderm layer, mesoderm cells destined to different downstream fates undergo additional mesenchymal-epithelial transitions (METs) and EMTs as tissues are formed and cells migrate to their appropriate embryonic positions. The objective of our recent studies was to clarify how cell arrangements and rearrangements influenced the cellular response to mesoderm-inducing cues in the context of early embryonic gastrulation. To do this, we first developed a method to control both the hESC plating geometry and density and the mechanical signals presented to hESCs through their extracellular matrix substrate (Lakins et al., 2012; Przybyla et al., 2015). We then used this system to interrogate how cell context influences early mesoderm specification and is able to orchestrate a program of differentiation with striking parallels to embryonic gastrulation.
Mechanical signals can control transcriptional responses
The mechanical stimuli sensed by cells in the developing embryo include the stiffness and topography of their extracellular matrix, the forces exerted between cells, the shear stress applied by surrounding fluid flow, and mechanical strains resulting from interstitial and osmotic pressures. Cells respond to mechanical signals with integrated mechanosensitive machinery, including molecules that hold tension at the cell-cell and cell-substrate interfaces. Cell organization within tissues affects what mechanosensitive machinery is expressed and activated, thereby dictating how cells respond to other environmental cues (Przybyla et al., 2016b). For example, mesenchymal cells may initiate signaling pathways downstream of strong cell-matrix contacts at focal adhesions, whereas strong cell-cell junctions in epithelial cells may allow for efficient transmission of contact-based signals. Mechanical signals can also alter ligand (Giacomini et al., 2012; Wipff et al., 2007) or receptor (Boulant et al., 2011; Gauthier et al., 2011) accessibility, thereby enhancing or inhibiting downstream signaling pathways.
Mechanisms by which mechanical cues can be translated by cells into functional responses.
Tissues and organs all have an intrinsic stiffness that is likely to affect their functionality. When tissue stiffness is altered, as in the case of heart or liver fibrosis, the ability of the organ to function is dramatically compromised. Similarly, developing embryonic tissues have an intrinsic stiffness that rapidly changes during differentiation (Majkut et al., 2013). Though we believe that these intrinsic properties contribute to development as part of the signaling microenvironment that drives cell fate, this has not been demonstrated in a controlled way. Using engineered matrices to control substrate stiffness as a mechanical parameter allowed us to explore this phenomenon during stem cell differentiation in vitro, and relate this to processes that occur in vivo during embryonic development.
Stem cell fate can be controlled by substrate stiffness as a mechanical input
In 2006, Dennis Discher’s group reported that mesenchymal stem cell fate could be controlled by altering substrate stiffness (Engler et al., 2006). Since then, many studies have demonstrated that substrate compliance is able to influence mesenchymal, neuronal, and liver stem cell differentiation (Cozzolino et al., 2016; Gobaa et al., 2015; Saha et al., 2008) as well as stem cell self-renewal (Chowdhury et al., 2010; Gilbert et al., 2010) and they have implicated RhoGTPases and myosins as key regulators of these behaviors. Nevertheless, the relevance of these observations to human embryonic stem cell fate and the molecular mechanisms critical for eliciting these phenotypes remained ill defined.
In our study, we cultured human embryonic stem cells on soft substrates similar in compliance to a gastrulation-stage embryo (400Pa) and substrates that were stiffer by two orders of magnitude. We were able to show that hESCs maintain their ability to grow and self-renew regardless of substrate stiffness, but surprisingly we failed to observe any direct impact of substrate stiffness on hESC differentiation. Nevertheless, we did note a remarkable increase in their propensity to differentiate into mesoderm progenitors when they were stimulated with differentiation-promoting morphogens if grown on the extracellular substrates that mimicked the microenvironment of the embryo. Mesoderm differentiation occurs concomitant with the first EMT of development, so we concluded that it could represent a stage at which cells respond to mechanical inputs by altering their fate.
We next explored the molecular mechanisms underlying the enhanced mesoderm progenitor specification, finding that hESCs on embryo-like substrates coordinated several complementary pathways to initiate and stabilize mesoderm progenitor differentiation. First, β-catenin was not degraded in cells cultured on soft substrates, while rapid proteasomal degradation occurred in cells on stiff substrates due to increased Src and GSK3 activity. Second, cell-cell junctions were stabilized in cells on soft but not stiff substrates, creating colonies with enhanced epithelial characteristics, including circumferential actin and high levels of E-cadherin and β-catenin localized at these junctions. Third, the induction of differentiation caused controlled activation of signals, including P120-catenin-mediated Kaiso sequestration, that coordinately enhanced the ability of β-catenin to enter the nucleus and begin a program of transcription to initiate and reinforce mesoderm differentiation. By upregulating Wnts, this system propagated a feed-forward loop that mitigated expression of secreted Wnt inhibitors to ensure robust, efficient mesoderm differentiation, but only under circumstances where cells were primed on soft, embryo-like substrates. Our findings obtained using this simplified hESC system not only build on prior knowledge of Wnt-dependent mesoderm specification during embryogenesis derived from model organisms, but also definitively link Wnt-stimulated developmental programs to a tightly coordinated cytoskeletal-linked adhesion-directed mechanosignaling pathway.
Mechanism for how cells cultured on soft gels respond to differentiation cues to coordinately initiate mesoderm differentiation
Using an in vivo model of gastrulation, the chick embryo, we also demonstrated that some of the same signaling pathways were upregulated in cells undergoing gastrulation and differentiating toward mesoderm. This study therefore demonstrates a means by which soluble signals synchronize with mechanical cues to drive hESC fate decisions, implying that biophysical parameters sensed by cells in vivo are an important part of the extracellular microenvironment that contributes to cell specification during development.
Understanding the cues required for differentiation during development is a crucial step towards tissue engineering-based cell therapies
Stem cell-based regenerative therapies require generation of specific cell types that perform their physiological function and are arranged properly within tissues. Pluripotent stem cells are particularly important in regenerative medicine because patient-specific induced pluripotent stem cells (iPSCs) can be derived and used to fix genetic defects or regenerate diseased or injured organs without risk of immune rejection. Although protocols exist to differentiate pluripotent cells into specific cell types in vitro, many such protocols generate immature cells in which tissue-specific marker expression is much lower than levels found in corresponding primary adult cell cultures, and these cells often lack the ability to adequately perform the functions required in fully functional tissues.
By demonstrating how one aspect of the biophysical environment, substrate compliance, can be tuned to influence embryonic stem cell fate in a developmentally relevant way, we have taken a step towards recapitulating developmental processes in the dish, and we plan to extend these findings to explore how mesoderm cells incorporate biophysical cues to further differentiate toward a cardiac fate. We hope that recapitulating more aspects of the biophysical microenvironment of the embryo, including shear stresses and mechanical strains, will allow us to develop differentiation protocols that result in functional cells that will be useful in regenerative therapies. To this end, our objective is to further characterize the mechanical properties of the early embryo and develop additional innovative techniques to recapitulate the in vivo embryonic microenvironment in a dish.
References
Boulant, S., Kural, C., Zeeh, J.-C., Ubelmann, F., and Kirchhausen, T. (2011). Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat. Cell Biol. 13, 1124–1131.
Chowdhury, F., Li, Y., Poh, Y.-C., Yokohama-Tamaki, T., Wang, N., and Tanaka, T.S. (2010). Soft Substrates Promote Homogeneous Self-Renewal of Embryonic Stem Cells via Downregulating Cell-Matrix Tractions. PLoS ONE 5, e15655.
Cozzolino, A.M., Noce, V., Battistelli, C., Marchetti, A., Grassi, G., Cicchini, C., Tripodi, M., and Amicone, L. (2016). Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes. Stem Cells Int. 2016, 5481493.
Engler, A.J., Sen, S., Sweeney, H.L., and Discher, D.E. (2006). Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677–689.
Gauthier, N.C., Fardin, M.A., Roca-Cusachs, P., and Sheetz, M.P. (2011). Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading. Proc. Natl. Acad. Sci. U. S. A. 108, 14467–14472.
Giacomini, M.M., Travis, M.A., Kudo, M., and Sheppard, D. (2012). Epithelial cells utilize cortical actin/myosin to activate latent TGF-β through integrin α(v)β(6)-dependent physical force. Exp. Cell Res. 318, 716–722.
Gobaa, S., Hoehnel, S., and Lutolf, M.P. (2015). Substrate elasticity modulates the responsiveness of mesenchymal stem cells to commitment cues. Integr. Biol. Quant. Biosci. Nano Macro 7, 1135–1142.
Lakins, J.N., Chin, A.R., and Weaver, V.M. (2012). Exploring the link between human embryonic stem cell organization and fate using tension-calibrated extracellular matrix functionalized polyacrylamide gels. Methods Mol. Biol. Clifton NJ 916, 317–350.
Majkut, S., Idema, T., Swift, J., Krieger, C., Liu, A., and Discher, D.E. (2013). Heart-specific stiffening in early embryos parallels matrix and myosin expression to optimize beating. Curr. Biol. CB 23, 2434–2439.
Przybyla, L., Lakins, J.N., Sunyer, R., Trepat, X., and Weaver, V.M. (2015). Monitoring developmental force distributions in reconstituted embryonic epithelia. Methods 94, 101-113.
Przybyla, L., Lakins, J.N., and Weaver, V.M. (2016a). Tissue Mechanics Orchestrate Wnt-Dependent Human Embryonic Stem Cell Differentiation. Cell Stem Cell, in press.
Przybyla, L., Muncie, J.M., and Weaver, V.M. (2016b). Mechanical Control of Epithelial-to-Mesenchymal Transitions in Development and Cancer. Annu. Rev. Cell Dev. Biol. 32, in press.
Saha, K., Keung, A.J., Irwin, E.F., Li, Y., Little, L., Schaffer, D.V., and Healy, K.E. (2008). Substrate modulus directs neural stem cell behavior. Biophys. J. 95, 4426–4438.
Wipff, P.-J., Rifkin, D.B., Meister, J.-J., and Hinz, B. (2007). Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J. Cell Biol. 179, 1311–1323.
Mathew Tata was the winner of the BSDB poster competition at the BSDB-BSCB 2016 meeting in Warwick. His prize was a trip to Boston to attend the SDB-ISD meeting in August, where he caught up with the SDB poster prize winner Yusuff Abdu to continue the BSDB-SDB interview chain (have a look at our previous interviews, which started way back in 2012). Yusuff’s prize in turn is to attend the BSDB-BSCB meeting in April, 2017.
The Node listened in as the two prize-winning graduate students chatted over a drink on the final night in Boston.
Yusuff, congratulations on winning the SDB poster prize! Can you tell us a little about the lab you work in?
Thank you very much. I’m inJeremy Nance’s lab, he’s a developmental biologist at NYU, and the lab studies a lot of events in the c. elegans embryo. Some people work on polarity and PAR proteins, but I study the development of the primordial germ cells.
And how long have you been in Jeremy’s lab?
Five years, just starting my sixth.
Can you tell us about your poster?
One common feature in development is this intimate interaction between germ cells and the endoderm, and I feel like we haven’t really fully appreciated it. My work has been looking at a particular form of germ cell remodelling driven by the endoderm: we basically found that parts of the germ cells are eaten by the endoderm, and we’re trying to figure out why that is. It appears to be important in some way for germ cell development.
In essence, the poster is about the active remodelling of germ cells by endodermal cells.
What would say is the single experiment or finding that you are most proud of?
That’s a tough one…This process we study occurs when the embryo is moving rapidly within its eggshell, so the imaging is tough and challenging. We were interested in these huge protrusions the germ cells send out, called lobes, and whether some of them remained attached to the cell body or not. To do this, we basically had to paralyse these embryos and do FRAP on them, photobleaching the lobes and seeing if there is any recovery from the cell body…so I call it imaging acrobatics: you have to keep them alive, but still enough, and do fancy imaging on top of that. I didn’t think it was going to work, but it did!
So what’s next for you?
After I get my doctorate, a postdoc: I think I’ll stay with worms, they’re a great organism to work with and we’re getting new tools everyday. There’s still a lot of weird stuff going on in worms that is yet to be understood: I’d encourage anyone who doesn’t work on them to consider a worm lab in the future.
And are you looking forward to the BSDB next year?
I am, yes: I’ve heard some great things about it.
Well it was a pleasure to meet you, and congratulations again!
We invite applications to this permanent full time appointment in the School of Biological Sciences. The successful candidate will be expected to make a significant contribution to biological research in the School and College, and to develop a research programme in their specialist area that will attract external funding and international recognition. They will also contribute to the teaching of biology and related subjects at undergraduate and postgraduate levels.
The successful candidate will be educated to PhD standard or equivalent and have previous experience across a broad range of sub-disciplines within Biological Sciences. To match our vision for the development of biology in the school we would particularly welcome candidates to carry out research to complement our existing areas of expertise (Animal Physiology; Behaviour and Conservation; Wetlands, Biogeochemistry and Plant Science; Microbiology, Parasitology and Biotechnology). From a teaching perspective, applicants should be prepared to contribute to areas that could include: physiology, immunology, biochemistry, human/primate biology, biotechnology, systems biology, quantitative biology, bioveterinary science or forensic biology.
In addition, the post holder will be expected to make a strong contribution to our existing ethos of inter-disciplinarity and team work in research and teaching, enhancing and complementing our existing area of expertise.
The appointment will be made in the range of Lecturer 1 £31,656 – £37,768 (Grade 7) or Lecturer 2 £38,896 – £46,414 (Grade 8) per annum, depending on previous experience.
Informal enquiries can be made by contacting Prof Chris Freeman (tel: +44 (0) 1248 382353, e-mail: c.freeman@bangor.ac.uk
Closing date for applications: 9th September 2016. Interviews will be scheduled shortly after 21st September 2016.
Today’s paper is from the latest issue of Development and investigates the mechanisms of lizard tail regeneration, revealing distinct cell behaviours in the proximal versus distal regenerating tail. Today’s person is Thomas Lozito, Research Assistant Professor at the Center for Cellular & Molecular Engineering, University of Pittsburgh, and co-author with Rocky Tuan.
So Thomas, tell us a little bit about yourself: how did you come to work in the Center for Cellular & Molecular Engineering (CCME) in Pittsburgh?
I actually came to the CCME with my mentor, Dr. Rocky Tuan, when he moved from the National Institutes of Health (NIH) to the University of Pittsburgh in 2009. I attended the graduate partnership program between the National Institutes of Health and the University of Cambridge. I spent the first part of graduate school at Cambridge, and finished up in Rocky’s lab at the NIH. As I was writing up, Rocky accepted a position as the director of the CCME at the University of Pittsburgh, and asked if I would like to come with him as a Post Doc. So I moved to Pittsburgh, first as a Post Doc, and then as a Research Assistant Professor.
What is the general focus of your work within the CCME?
My work focuses on lizard tail regeneration. I try to understand how lizards regenerate their tails, how the regenerated lizard tail tissues are patterned, as well as the cellular origins of regenerated tail tissues. A large part of my work also involves comparing lizard tails with the tails of other regenerative species, such as salamanders, as well as tails of non-regenerative animals, such as mice, in the hopes of finding that special mix of factors that allow for regeneration.
“Turns out no one knew why lizards regenerate cartilage instead of bone, so that’s where I started.”
How was this particular project conceived?
I’ve always been interested in lizards. In fact, herpetology has been a hobby of mine since I was 4 years old. I started with box turtles, and I maintained a small colony in my back yard. Over the years, I’ve kept and bred many different kinds of reptiles and amphibians, including poison dart frogs, monitor lizards, and geckos. As I was approaching the end of my Post Doc, I started to think about what type of research direction I would like to build a career on. Coming from Rocky’s lab and studying cartilage biology, I immediately thought of lizard tail regeneration because the regenerated lizard tail skeleton is almost completely cartilaginous, not bone like the original tail skeleton. Turns out no one knew why lizards regenerate cartilage instead of bone, so that’s where I started.
Isolated lizard tail blastemas, at day 0 and 14 after explant culture. From Fig. S1 in the paper.
People will be familiar with salamanders as models for regeneration, but perhaps not so familiar with their lizard cousins. Why use lizards?
Lizards represent an interesting, yet underused, model organism with potential applications to regenerative medicine. As amniotes, lizards are more closely related to mammals, and yet retain impressive regenerative capabilities. Thus, studies involving lizards may be more relevant to improving human regeneration than work done with urodeles (newts and salamanders), the model organisms traditionally used to study vertebrate regeneration. Indeed, several aspects of the urodelian (salamander) life cycle, particularly those pertaining to the larval stage of metamorphosis, are at odds with those of higher vertebrates. In fact, the urodele species most commonly used in regeneration studies, the axolotl salamander, exhibits neoteny, typically never metamorphosing from its larval form. The absence of a larval form in mammals makes meaningful comparisons with salamanders difficult. Lizards, on the other hand, follow a similar developmental plan as mammals and are able to regenerate their tails as adults, making lizard regeneration particularly attractive from a biological and developmental standpoint.
“Lizards are very alert and surprisingly social. They constantly display to one another for territories and dominance, and they definitely watch you as you work and during maintenance”
And what are lizards like to work with as a model organism?
As a hobbyist, working with lizards is second nature to me, and they are very interesting animals. Most only eat live insect prey, so keeping lizards means also keeping feeder insects like crickets. And the lizards are very alert and surprisingly social. They constantly display to one another for territories and dominance, and they definitely watch you as you work and during maintenance. On the science side, working with lizards comes with challenges, since many primers and antibodies aren’t validated for lizards or reptiles. But the challenges make for an exciting research project, I think.
Could you sum up the key results of the paper in a few sentences?
In investigating regenerated lizard tail cartilage, we noticed that the extreme proximal cartilage in contact with the original tail skeleton ossifies, while the rest of the regenerated cartilaginous skeleton does not. In this paper, we described that these differences in cartilage development between the two areas are due to differences in cell sources and signalling. Proximal cartilage forms from periosteal cells in response to signals from the original tail bone, similar to a cartilage callus during fracture repair, while the distal cartilage forms from blastemal cells in response to signals from the regenerated spinal cord.
Implanting exogenous spinal cords leads to the formation of multiple cartilage tubes, from Fig. 1 in the paper.
When doing this research, was there a particularly exciting result or eureka moment that stuck with you?
We did some experiments with lizard spinal cord implants. Spinal cords pieces were subcutaneously implanted along the tails of lizards. Wherever a spinal cord piece was implanted, an ectopic tail grew. That was fascinating, and really showed that, in terms of lizard tail regeneration, it’s the spinal cord that provides the regeneration spark.
And what about the flipside: any particular moments of frustration?
Isolating primary cells from lizards, which harbour a lot of bacteria and fungi on their scales, always runs of risk of contamination. We try to be as careful as possible, and take the necessary precautions, but there was a period of several months that every lizard cell isolation got contaminated. That was very frustrating.
“I hope that lizards can teach us new ways to improve mammalian regeneration.”
What can lizards teach us about regeneration in general?
I hope that lizards can teach us new ways to improve mammalian regeneration. As I mentioned, in sparking regeneration in lizards, it basically comes down to the spinal cord, which provides the regeneration spark, so to speak. Mammals like mice don’t have spinal cords in their tails as adults, and can’t regenerate their tails. Is getting a mouse to regenerate its tail as simple as making a mouse with a spinal cord in its adult tail? Probably not, but we can start looking to lizards to see what else they have that is lacking in mammals and that is involved in regeneration.
Is there a particular loose end or unexpected result that you’d like to get to the bottom of?
Of course. In this paper, we’ve determined why proximal regenerated lizard tail cartilage ossifies. But we still don’t know why distal regenerated cartilage does not ossify. Yes, distal cartilage is derived from a different cell source than proximal cartilage. But I’d like to know if distal cartilage cells are even capable of ossification. So far our results say no, but I’d like to know why not.
And what are you working on now?
I just got an R01 grant from NIGMS to answer the question “Why don’t lizards regenerate perfect tails like salamanders?”. Regenerated salamander tails are near-perfect copies of their tails, complete with dorsoventral patterning and segmentation. “Imperfect” regenerated lizard tails, on the other hand, lack dorsoventral patterning and do not segment. As part of the grant, I’m trying to identify the molecular and cellular reasons behind these differences, and ultimately use genome-editing technologies like CRIPR/Cas9 to correct some of the “imperfections” in regenerated lizard tails by introducing patterning and segmentation.
What do you like to do when you’re not surrounded by lizards?
Since my hobby is still herpetology, a good portion of my spare time is also spent surrounded by lizards and frogs! I currently keep several types of geckos and frogs as pets. But I am basically interested in all animals. Wherever I travel, I try to visit the local zoo, and most of my vacations are of the “ecotourism” sort where I try to observe some exotic animals in their natural habitats. For example, I went to Costa Rica and Panama a couple years ago to see some of their endangered frogs. And this year I visited Crystal River in Florida to see and swim with the manatees. Closer to home, I enjoy movies, especially super hero movies, and spending time with friends and family, especially when it involves good food.
The joint meeting of the Society for Developmental Biology and the International Society for Differentiation was held in Boston between August 4th and 8th. It was a meeting of firsts for me: first meeting representing the Node, first time at the SDB (having regularly enjoyed the Brit version, I was looking forward to hearing the US angle on what’s exciting at the moment), and first time in Boston. The venue was the gargantuan Copley Place Marriott, which offered a stunning view of the city (from my hotel room at least) as well as a central base to explore. Boston turned out to be beautiful and walkable. Great local beer too.
Boston, looking south from the Marriott
True to our diverse field, the meeting covered a lot of bases, from cell mechanics to genome editing, patterning to computational modelling, as well as life away from the bench (education, publishing, grant-writing) and the planet (designing experiments for NASA’s Genelab). What follows is a selection of my personal highlights from the talks I saw. Let me know if I missed anything or have mangled things beyond comprehension.
Regenerating parts and patterns
I began the meeting in a satellite symposium, Evolution of Regenerative Capacities: recapitulation of development or novel mechanisms? The symposium set the tone for the rest of the meeting in terms of speaker composition: a mix of postdocs, young PIs and established leaders in the field, plus the odd grad student, and an even gender balance.
Most of the speakers started with the same observation: some animals are great at regeneration, some (like us) not so great, and even within the same animals, some tissues regenerate better than others (though axolotls apparently can regenerate everything!). However, we really lack a mechanistic explanation for this spectrum of regenerative capacity; such an explanation might inform our efforts to coax currently intransigent human tissues to regenerate. The evolution part came from the range of species under investigation, which allowed for a fairly broad phylogenetic sample.
For someone fairly naïve to this field, I was struck by the diversity of mechanisms used in the different contexts, as illustrated by a rough list the proteins discussed: piwi piRNA pathway members, various transcription factors, second messengers, growth factors, cytokines, cell adhesion molecules, glycosylation enzymes, and matrix proteins. (I guess this shouldn’t really have been surprising given the tasks at hand for the wounded tissues.) The extent to which regeneration redeploys developmental pathways also seems to depend on the tissue: for instance, Ken Muneoka described how digit regeneration in the mouse shared some features with normal bone development, but not others.
One of the more remarkable stories involved the moon jellyfish, described by Lea Goentoro. Ephyra (juvenile medusa) stage animals typically have 8 evenly-spaced arms which pulsate synchronously to drive propulsion and feeding. Cut off some of the arms, and the ephyra proceeds not to regenerate them, but to reorganise its remaining limbs to regain radial symmetry, in a manner requiring muscle contractility but not cell division. The ephyra regenerates not body parts but a feature of the body pattern, perhaps because repaired animals have still got a reasonable chance of surviving. Got to love nature sometimes.
Gene expression and chromosome topology
After the regeneration fest, KenZaret kicked off the Presidential Symposium by celebrating developmental biology, and insisting on the continued need for basic research in model systems (his reaction, perhaps, to recent events inthe US and Canada). He then stated that, for him, changes in gene expression are the real heart of development (the symposium did have an epigenetics theme, after all). Ali Shilatifard, in his exploration of histone methylation and childhood leukaemia, agreed. Coming from a bit more of a cell mechanics background, I wanted to come up with a counter argument, but by that point didn’t have the brain power for it.
One of the most remarkable epigenetic feats is the inactivation of an entire X chromosome in female mammals, providing almost complete gene silencing from early development onwards. The long non-coding RNA Xist is key to inactivation, and Jeannie Lee described how RNA proteomics had identified the proteins that interact with Xist (it turns out there are a surprisingly large number of them), andhow super-resolution imaging had visualised individual Xist molecules decorating the inactive X (it turns out there are a surprisingly small (~100) number per X). An analysis of Xist mutants then implicated other silencing mechanisms acting synergistically with Xist, and showed that even modest changes in X gene dosage can have profound effects on viability.
Revelations about the X chromosome resulting from a combination of cutting-edge technology and genetics…history repeated itself the next morning, in Job Dekker’s talk, part of a fascinating Chromosome Topology and Gene Regulation session. Revealing the organisation of the inactive X is complicated by the fact that the two copies of the chromosome are too similar to be distinguishable by HiC. The solution: cross two mice lines, creating a (frankly rather shabby looking) F1 hybrid, which contains enough SNPs to distinguish maternal and paternal chromosomes, and then derive ES cells from these mice and induce X inactivation in them. Dekker showed howthese cells revealed the fine-grained structure of the inactive X, with its two highly compact mega-domains, mysterious boundary region between them, and islands of topologically associating domains around the few genes that remain active.
After a delightful interview with Dave McClay (look out for it in an upcoming Development issue and on the Node), and an hour jaywalking around Boston for lunch, I was back for the Hilde Mangold Postdoctoral Symposium.
From a very strong field (the eight talks were picked from over a hundred applicants), two really stuck with me. Yaniv Elkouby, from Mary Mullins’ lab at UPenn, documented theintricate relationship between the centrosome, telomeres, a chromosomal ‘bouquet’, microtubules, and the Balbiani Body in the polarisation of the fish oocyte. Beautiful in vivo cell biology. Jacqueline Tabler (John Wallingford lab at UTexas), pointed out that given how critical vocalisation is for vertebrates, it’s striking how little we know about the development of the organ that creates vocalisations, the larynx. Aided by her own whistling and beautiful images, she told us how interfering with Hedgehog signalling led to an expansion of neural crest-derived tissues in the larynx, and defective vocalisations in newborn pups. (Her previous work has looked at various aspects of craniofacial development.)
Friday afternoon brought another plenary: Biology at the level of single cells. Single cell sequencing is a remarkable advance, but sequencing for the sake of sequencing does not necessarily advance the field; you need a strong biological question. It was therefore wonderful to hear Mark Krasnow reconstruct the program of alveolar development by sequencing individual cells of various stages from a dissociated lung. With exquisite sensitivity (‘a single RNA in a single cell’), the technique revealed new players in the developmental program by correlating expression with differentiation markers, providing a quick, non-genetic screen.
Tony Hyman then took us back to basics and asked: how does the cell organise chemical reactions without membranes? Part of the answer appears to beliquid-liquid phase separations. Unstructured protein regions (for instance in prion-like domains) promote phase transitions, and changing the sequence of these regions could alter the properties of the resulting agglomeration, from liquids that have fast turnover and promote chemical reactions, to gel-like, stable, dense matrices prohibitive of reactions (such as the Balbiani body we encountered in Yaniv’s talk earlier in the day). The talk really made me think differently about the way cells work.
The Charles River
Fat fish and organoids
I started Saturday in the Emerging model organisms and evo-devo session. The general feeling was that techniques like CRISPR were opening up many organisms to developmental analysis; your question might now dictate your model, rather than the other way round. (Lo and behold:a recent piece in Nature.)
Nicolas Rohner introduced us to Astyanax mexicanus, the blind cave fish, which has emerged as a model for metabolic diseases. These fish are always hungry, get really fat (without any clear health disadvantages), and are remarkably resistant to starvation. They are also an evolutionary windfall: not only do river forms persist (providing an ‘ancestral’ population that blind forms can still breed with), but the cave forms themselves have evolved multiple times independently, so they are a great model for convergent evolution.
During the coffee break, I interviewed Doug Melton – another wonderful half hour, look out for the write-up in the future – prior to his Jean Brachet lecture, in which he promoted the idea of applied developmental biology in harnessing stem cells to treat diabetes. Applied developmental biology is also being advanced by organoids, which were celebrated in a special plenary session in honour of Yoshiki Sasai. One recurrent problem with organoids is the variation you get between samples, and Jürgen Knoblich described how growing brain organoids on strands of a synthetic polymer reduces variability, and promotes development of a cortical plate-like structure . Hans Clevers discussed modelling cancer with tumour organoids (somewhat counterintuitively, tumour-derived organoids always grow slowly), and promoted the idea that, in many cases, stem cells are not a rigid cell fate, but a labile cell state.
Downtown Boston, looking like a toy town
Circumventing Mendel and celebrating scientists
Sunday: the final day; bleary-eyed and near-saturated, but not dead yet. Before the SDB award lectures, I heard from Ethan Bier on the ‘mutagenic chain reaction’, a CRISPR/Cas9 technique which gives a rapid form of non-Mendelian inheritance. Not only does this drastically cut the time it takes to make mutants (hypothetically, to make a mouse quadruple mutant: 1/64 mice in the 4th generation of a standard cross; 64/64 in the 2nd generation using MCR), and provide a tool for organisms without established genetic techniques, it also allows you to rapidly spread a genetic element through a population (a gene drive). Stunning, revolutionary genetics.
The afternoon Education Symposium was a change of gears, as educators discussed how to provide biological training in institutions with shrinking budgets. The general consensus was that inquiry-driven learning could provide a more engaging route for biology students than your standard rote lab work (this report sums up a lot of the speakers’ ideas). Getting undergrads to feel a sense of ownership over the projects also helps: Sarah Elgin described how hundreds of undergrads had contributed to the annotation of the oft-forgotten dot chromosome of Drosophila melanogaster (leading to a paper with over a thousand authors!).
And finally, the SDB awards. Ida Chow (Viktor Hamburger Outstanding Educator Prize) encouraged all of us to fight for science and science education, and got a raucous standing ovation (from a show of hands, many in the audience had directly benefited from one of her many initiatives). We got to see old pictures of Dave McClay (Lifetime Achievement Award) as a high school football player and an underwater diver, before he described how each of the EMT events in the early sea urchin is driven by a separate gene regulatory network. And finally, we heard from Kathryn Anderson (Edwin G. Conklin medal; I also had the pleasure of interviewing Kathryn, keep an eye out for it) who described the beauty and mystery of gastrulation in the mouse.
The evening ended with drinks, poster awards, dinner (including waiters impatient to get you on to your next course: “coffee with your chicken, Sir?”), drinks, dancing, watching-of-the-dancing, and then final drinks in a dive bar (Cambridge really lacks places like these).
Pre-dinner drinks in the Marriott
I left the meeting with the feeling that developmental biology is in a good state. This was not just down to the general positivity of Americans: new technologies are unlocking doors everywhere, and the increasing applicability of developmental research to human health (with stem cells, organoids) gives a public persona that I hope will draw in more money without side-lining all of the strong non-applied research that sits under the DB umbrella. Coming from England, you also forget how vast the US is and how much wonderful research is being carried out there.
I would also like to commend all of the speakers who suffered technical difficulties and kept their poise to finish their talks, rather than melt down and destroy the equipment (in one of the meeting’s funniest moments, Mina Bissell walked the length of the hall to fix her frozen computer at the back AV desk, all the while giving running commentary).
And finally: the posters. More than 500, each up for one night only, they were excellent too, and included many with added extras (3D printed models, interactive whiteboard sections, and built-in videos!).
Quotes of the meeting (paraphrased, potentially misremembered)
“Three cheers for developmental biology!”
Ken Zaret opening the meeting, cue cheers and applause.
“This is for you, Sarah Palin…”
Ali Shilatifard on how research in flies helped to inform children’s cancer treatment
“Like Republicans, Aedes aegypti ‘repeal and replace’”
Marc Halfon on mosquito GRN evolution (not that I immediately got the reference)
“It is not birth, marriage, or death, or gastrulation, but epithelium formation that is truly the most important time in your life”
Matthew Gibson takes Wolpert back a step
“If your grandfather was a fish, he’d have no problem hearing you”
Shawn Burger on the amazing regenerating ear hair cells of zebrafish
“I justlove the spiny mouse!”
Overheard at the Regeneration symposium (before I found out that they were a species, not a Pokemon)
“Blind cavefish are fat”
Nicolas Rohner pulls no punches in describing his beloved model organism
“We must build the embryo to understand it”
Eric Siggia goes a bit Feynman
“If you’re not arrogant, you can look at your data and see what it’s trying to tell you”
Mina Bissel warning against over-confidence
“There are no ‘higher’ or ‘lower’ organisms”
Richard Berhinger on Twitter
“Other hashtag is … different”
James Gagnon on Twitter, after confusion over #2016SDB (the correct one) versus #SDB2016 (Seventh Day Baptists)
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We provide a dynamic and progressive atmosphere for science-led service delivery, supporting the generation and characterisation of mouse lines for the study of human diseases. With a leading role in the International Mouse Phenotyping Consortium (IMPC) and co-ordinating the UK Genome Editing Mice for Medicine (GEMM) initiative, MRC Harwell is at the forefront of the provision of high-quality mouse lines and phenotyping data to the international genomics community.
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Heart development is a complex process involving numerous cell types and different morphogenetic events to make an organ that begins its vital function long before it reaches its final, intricate shape. That this process is error prone is borne out by the high prevalence of congenital heart defects in humans. For de la Cruz, a Cuban cardiac embryologist who spent most of her career in Mexico City, a precondition for understanding the aetiology of congenital defects was a comprehensive description of normal heart development. She was particularly preoccupied with the question of where the various parts of the final (or ‘definitive’) heart mapped onto earlier developmental stages.
Her 1989 paper begins with a nod to an earlier age: the descriptive work of Carl Davis, who in the 1920s used human embryo samples from the Carnegie Collection to infer the lineage of the compartments of the heart. Davis had described one particular embryo, #3709, the type specimen for stage 9, at which point the heart is a straight, symmetrical tube, yet to undergo looping and formation of the chambers (the same embryohas recently been reconstructed in 3D).
Davis inferred that each of the regions of the straight tube heart is the primordium of a definitive cardiac cavity; that is, by stage 9, the atria and ventricles of the heart are mapped out onto the straight tube heart, and subsequent development involves morphogenesis of these pre-patterned regions.
For de la Cruz, decades later, purely descriptive embryology on fixed and sectioned samples was inadequate to really test lineage, as there was no means of following regions in the same heart. She thus turned to in vivo labelling, which allowed the “study of the cardiac zones up to their anatomical expression in the mature heart in a continuous and uninterrupted sequence”. Of course she required a non-human model, and used the easily accessible eggs of leghorn chickens (like mammals, birds have a four-chambered heart). Regions of the heart could be injected with iron oxide particles, and the embryo left to develop either in vitro or in ovo until the desired stage. Over the decade preceding the paper, she and others had gathered evidence which questioned the existence of Davis’ primitive cardiac cavities; for instance, the primordial atria were not apparent in the heart tube tissue, but only later in the loop stage heart.
Excerpt from Figure 2 showing labelled regions in St. 9 hearts, and where they ended up in St. 12. de la Cruz, et al. 1989. J Anat. 165:121-131. Reproduced with permission of Wiley.
The 1989 paper was her latest attempt to map out where the regions of the heart tube ended up, and would she hoped “allow us finally to discard the term ‘primitive cardiac cavities’.” To start with, the straight tube heart was labelled in two caudal regions (a and b in the figure excerpt), and then the label was observed in the loop heart stage. Label a, at the left border of the heart tube, ended up in the left border of the loop stage heart, consistent with previous descriptive embryology. But label b showed something different: cells at the tube’s midline ended up at the right border of the loop, which dismisses a simple ‘one to one’ correlation between the regions of the tube and the loop. A final caudal label, at venous edge of the tube, ended up in the border between the left ventricle and left aorta in the mature heart. These labelling experiments were complemented by SEM and histology to compare the morphologies of the straight tube heart with later stages.
This paper, along with de la Cruz’s previous and later work, demonstrated that the straight tube heart does not contain single primordia for each of the definitive cavities. As the initial heart tube only contributes to a subset of the final heart (mainly the left ventricle), the rest of the heart must be added during later development. This work predated the molecular definition of additional regions of the mesoderm that give rise to the heart (the so-called second heart field) by more than a decade, but appears not to have been widely appreciated, perhaps due to the methods employed. It could however be argued that the power of the method is its simplicity: label a cell or set of cells and see where they end up, and deduce from there how the final organ is built. Indeed lineage tracing is still being used to great effect to this day.
Thoughts from the field:
Benoit Bruneau (Gladstone Institute for Cardiovascular Disease)
“Victoria de la Cruz used somewhat crude methods to map out where segments of the early heart tube end up in the more developed heart, and the results were not what people might have expected based on preconceived notions. Her work went largely ignored as people examined gene expression patterns and made erroneous conclusions about the chambers of the heart being already patterned and present as primordia in the linear heart tube. The discovery of the second heart field brought back to the forefront her work, which suggested that there might be an additional source of heart cells.”
“The chick embryo is easily accessible and has been used extensively for fate mapping studies. This paper by Maria Victoria de la Cruz is a prime example of classic mapping experiments that contributed to changing the thinking in the field. Her work using labeling of the linear heart tube with iron oxide particles, indicated that new heart segments are added successively, in particular to generate outflow myocardium. She also concluded that precursors for the right and left primitive atria are not yet present in the early straight heart tube but become incorporated later during loop stages. This was not fully appreciated until the origins of secondarily added cell populations were discovered; in the chick using essentially similar approaches, and in mouse using genetic labelling.”
“This 1989 paper was an important step in a series of cell labeling studies from Maria Victoria de la Cruz and colleagues that demonstrated the dynamic nature of early heart development. Her purely embryological approach was initially underappreciated yet set the scene for many of the molecular and genetic studies underway today. MV de la Cruz was one of the first to realise that there was a myocardium-forming region outside the linear heart tube, a critical advance in emergence of the second heart field model of vertebrate heart development. Indeed, the concepts that certain parts of the heart are late added components and that primitive cardiac regions only contribute to parts of the definitive cardiac chambers stem from this work and have had significant impact on our understanding of congenital heart defects. Her book Living Morphogenesis of the Heart is highly recommended further reading for those interested in the major embryological questions, including heart tube growth, septation, coronary development and the mechanisms underlying anomalous cardiac development, that continue to drive the field today”
A little more reading
I learned a little about Maria de la Cruz’s life from two obituaries (1, 2)
This post is part of a series on forgotten classics of developmental biology. You can read the introduction to the series here and read other posts in this series here.
We are seeking an enthusiastic and outstanding postdoctoral researcher to join a multidisciplinary team led by Prof. Chris Thompson at the University of Manchester. You will use single cell RNA sequencing to identify groups of heterogeneously expressed genes within normal populations of cells, and study the role of these genes in cell fate choice.
Cell fate choice and proportioning are typically considered to be ordered, robust and reproducible. However, noise and stochasticity can lead to heterogeneous gene network activity. Consequently, it has been proposed that gene networks may be ‘wired’ to buffer these fluctuations. Alternatively, heterogeneity may be functionally important to prime cells or increase the spectrum of differentiation capabilities. Addressing these questions represents one of the greatest challenges in Developmental and Stem Cell Biology. However, to date it has been impossible to follow entire gene network behaviour in individual cells, or to follow their temporal changes in activity in individual cells as cells commit to differentiation along different lineages. Single cell gene expression analysis, together with novel computational reconstruction of gene network dynamics provides this opportunity.
This work builds upon our recent finding (Chattwood et al, eLife 2014) that the interplay between dynamic heterogeneity in Ras-GTPase activity and nutritional status is required for normal lineage priming and robust running of an ultradian cell fate oscillator. Computational approaches will be used to identify putative genes involved in lineage priming and cell fate choice. In addition, the role played by these genes will be tested in the lab through the analysis of gene knockout strains, live cell imaging and molecular genetics.
Candidates with extensive experience of using either computational approaches or wet lab approaches to understand the molecular basis and gene networks will be considered.
You should currently hold or be about to obtain a PhD in a relevant field.
The post funded by the Wellcome Trust and is available for up to 3 years.
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