Position Description:

We seek highly motivated post-doctoral Fellows interested in cell signaling at the intersection of development, stem cells and cancer. Candidates will join the group of Dr. Andres Lebensohn, an Earl Stadtman Principal Investigator in the Laboratory of Cellular and Molecular Biology (LCMB) at the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), National Institutes of Health (NIH). We combine functional genomics, CRISPR/Cas9-mediated genome editing, cell biology and biochemistry to study how signaling pathways are used, reused and repurposed to drive the myriad different cellular processes that give rise to tissues and organs during embryonic development, and maintain them in adult life. A major focus of the lab is on WNT/R-spondin signaling, a fundamental pathway that orchestrates embryonic patterning and morphogenesis, and promotes stem cell self-renewal and tissue regeneration. We recently discovered a new mode of signaling by R-spondins that does not require LGRs, thought to be the main R-spondin receptors (https://elifesciences.org/articles/33126). We seek a Fellow who will use mouse genetics to elucidate the physiological functions of LGR-independent signaling by R-spondins during embryonic development and in stem cells. Descriptions of this and other research projects in the lab can be found at https://ccr.cancer.gov/Laboratory-of-Cellular-and-Molecular-Biology/andres-m-lebensohn.

About the Center for Cancer Research and the Laboratory of Cellular and Molecular Biology:

The CCR is the largest division of the NCI intramural research program, comprising nearly 250 basic and clinical research groups. In the words of Dr. Tom Misteli, director of the CCR, “Our scientists enjoy complete intellectual freedom and are expected to creatively and innovatively explore the most important questions in the field of cancer research and treatment.” The LCMB (https://ccr.cancer.gov/Laboratory-of-Cellular-and-Molecular-Biology) is located at the NIH Bethesda campus within a vibrant biomedical research community. We have access to state-of-the art research facilities and close links to the NIH Clinical Center, one of the leading clinical research hospitals in the world. The open lab space, collegial atmosphere and joint weekly seminars shared by the seven groups in the LCMB give Fellows a strong support network and plenty of opportunities to present and discuss their research with close peers. A short metro ride to Washington D.C. and the surrounding areas provides Fellows and their families access to numerous free museums (https://washington.org/free-things-to-do), excellent culinary choices and many outdoor activities.

Qualifications:

Candidates must have completed a Ph.D. and should be recent graduates or have less than 3 years of post-doctoral experience by the desired start date. Candidates with backgrounds in developmental, stem cell or cancer biology are encouraged to apply. Experience in mouse genetics is required for this project, and experience with organoid models is welcome but not required. Fellows will have exceptional stipend support for the entire duration of their post-doctorate, funds for travel to scientific conferences, and full healthcare benefits.

How to Apply:

Please send a cover letter describing your research accomplishments and future interests, including your specific interest in my lab, a CV with bibliography, and contact information for three references to andres.lebensohn@nih.gov. Please use the following email subject: “Application for post-doc position in WNT/R-spondin signaling”

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Written by Antoine Fruleux and Arezki Boudaoud

As Lewis Wolpert put it (Wolpert, PLoS Biology 2010), if you extend your two arms, you will likely find that they match in length by better than 0.2%, though they do not seem to directly communicate during their development and growth. Similarly, flowers in an individual plant are likely to be almost identical in size and shape. Such precision, or robustness, in organ size might seem trivial when conceptualizing observations as reflecting a ‘developmental program’ with series of well-defined steps and ‘decisions’, but it is less so when thinking of the myriad of cells that form an organ and that divide and grow asynchronously (Figure 1). This raises the question of how organs and organisms reach well-defined sizes and forms, in the face of considerable spatial and temporal fluctuations of cell growth and deformation during morphogenesis (Hong et al. 2018, Uyttewaal et al. 2012, Kierzkowski et al. 2012, Hipfner et al. 2004).

Figure 1. Cell growth is heterogeneous in space and time. Example of a sepal (green leaf-like organ that protects a flower before it opens) from the model plant Arabidopsis thaliana. The color scale corresponds to growth rates (high in red, low in blue).

This question might find a very simple answer. As an organ grows larger and larger, an individual cell has less and less influence on overall size and shape of the organ; accordingly, precision of organ size would increase with the number of its cells. This is somehow like taking many independent measurements of an experimental parameter, the precision of the mean value of the parameter increases with the number of measurements, or, equivalently, the standard error of the mean (SEM) decreases with the number of measurements according to the law of large numbers. In an organ, if cells expanded randomly and independently from each other, then organ growth rate, which combines growth of all cells, would become increasingly precise as the number of cells increases (Hong et al. 2018). Nevertheless, this scenario cannot be fully true because cells communicate with each other and, therefore, are not necessarily independent. This led us to investigate how growth fluctuates according to the size of the region of interest, from cell to organ, so as to understand how precision of organ size can be achieved (Fruleux & Boudaoud, 2019). Interestingly, an independent and complementary study obtained results that are similar to ours (Damavandi & Lubensky, 2019).

A key question is whether there are mechanisms that enhance robustness of organ shape. If growth fluctuates between cells, those growing relatively fast will tend to push their neighbors, generating forces within the tissues. More generally, any tissue with heterogeneous growth builds up internal forces (internal stress) due to conflicts between cells or groups of cells. Such internal forces might be instructive: They could be sensed and used by cells to regulate their growth (Shraiman, 2005). This can be pictured by thinking of a group of blindfolded persons, each holding the hands of two neighbors, all involved in a round dance. Each dancer moves at its own natural pace and so pace fluctuates from dancer to dancer; as a consequence each dancer feels forces from the hands of the neighbors. Can the dancers use information provided by these forces to circle at the same speed?

Cells within a tissue can sense and respond to forces. It was first hypothesized that proliferation is affected by such forces, with cells under mechanical compression or tension reducing or increasing their division rates, respectively (Shraiman, 2005), which has received indirect and direct support in developing fruit fly and zebrafish (LeGoff et al., 2013; Campinho et al. 2013; Mao et al., 2013; Pan et al., 2016). The cytoskeleton may also respond to forces: actomyosin cables are reinforced by mechanical tension in the wing imaginal disk of Drosophila (LeGoff et al., 2013; Duda et al., 2017). The synthesis of the extracellular matrix (ECM) might also be modulated in response to mechanical signals. In plant cells, growth is driven by the cell’s inner pressure and restrained by the cell wall (the equivalent of the ECM) that encloses cells. The deposition of cellulose fibers, the main load-bearing component of the plant cell wall, depends on wall tension (Hamant et al. 2008, Hervieux et al., 2017), likely leading to reinforcing the cell wall in the direction of maximal tension (Sampathkumar et al., 2014). We aimed to incorporate all these responses to mechanical signals in a unifying framework, so as to be able to reach conclusions that may be relevant to robustness of morphogenesis in many developmental systems.

We described a tissue as a two-dimensional (2D) absract continuous medium, so as to make the calculations tractable and the conclusions general; a 2D model captures all main processes at play (notably directional processes such as those related to cytoskeleton), while a continuous model enables to disregard the details of cell arrangements and describe them at a supracellular level. We assumed the medium to be viscous, which accounts for the long-term relaxation and remodeling of cell junctions, cytoskeleton, ECM, or cell wall. We assumed the medium to be anisotropic; anisotropy is quantified with a mathematical object known as nematic tensor, which has been extensively used to measure fibril orientation and degree of orientation in cells and tissues (Boudaoud et al., 2014). We describe in the model the dynamics of density (density of cells, of cytoskeleton, of ECM, or of cell wall) and nematic tensor, that vary in time and space due to tissue expansion and deformation and to synthesis; for instance, ECM density decreases if tissue expands without synthesis,  increases if cells exocytose ECM components, while ECM fibers align passively if the tissue is stretched in one direction.

Up to this point, all assumptions are rather straightforward. Our key additional assumption is that synthesis is modulated according to mechanical stress, reflecting the evidence mentioned above: density is modulated (positively or negatively) in response to level of mechanical stress and anisotropy is modulated (positively or negatively) in response to directional mechanical stress; the corresponding response parameters (which may be positive or negative) are the main parameters of our model. Positive response to stress levels describes putative increase in ECM / cell wall synthesis, and subsequent reduction in expansion, in response to tensional stress; negative response to stress levels describes reduction in proliferation due to compression. Positive response to directional stress corresponds to cytoskeleton / cellulose fibers reinforcement under tension; negative respond to directional stress corresponds to cell intercalation under directional stretching. Accordingly, the sign of the response (positive or negative) allows us to account for biological diversity in mechanical responses.

Figure 2. Response to a localized perturbation.Snapshots of the predicted dynamics following a  perturbation of tissue density in the form of a disk at time t0. The density is color-coded (white corresponds to no deviation from average density and dark red to large deviations). Tissue anisotropy is indicated by the small lines: the stiffest tissue direction corresponds to line orientation and the degree of anisotropy to line length.

We first examined tissue dynamics following a perturbation in tissue density of a small disk-shaped region (Figure 2), which could correspond to a transient depolymerization of cytoskeleton or a transient reduction in ECM / cell wall synthesis The model predicts the expansion of this region due to tissue growth and the relaxation of density to its average value due to tissue response to mechanical stress; anisotropy orients circumferentially around the perturbed region, like in wound healing. We then considered the effects of a stochastic component to synthesis, reflecting temporal fluctuations of cellular dynamics. The model predicts that cells (more rigorously regions as the model is continuous) are correlated to each other at long distances (Figure 3): Cells do not grow independently (although they synthesize materials independently) and growth fluctuations in one cell remain correlated with growth fluctuations in a cell far apart. Long-range correlations are characterized by an exponent indicating how fluctuations of an element of the tissue scale with its size (Figure 3). Thus individual cells may have a stronger impact on organs size than if they were fully independent.

Figure 3. Long-range correlations. Cell-to-cell correlations quantify the degree of spatial coordination between cell fluctuations. The plot shows relative fluctuations in areal expansion of the tissue (growth SD) as a function of size of the region of interest (surface area). If cells wall fully correlated, fluctuations in areal expansion would not depend on size (exponent 0, blue line) and if cells wall fully uncorrelated, fluctuations in areal size would quickly decrease with size (exponent -0.5, green line). In general, the model predicts an intermediate behavior (with exponent ranging between -0.5 and 0, dark line), known as long-range correlations.

Finally, we examined the precision of organ shape and size. Positive response to mechanical stress levels (tissue reinforcement in response to tension) makes organ size less precise than with negative response (tissue softening in response to tension). Interestingly, increasing response to directional stress has a non-intuitive effect on organ precision (Figure 4): first the precision of organ size is enhanced, but then precision reaches a minimum, and then it increases for higher response levels. Thus, the model predicts an optimal response to directional mechanical stress for robustness of organ shape and size (Figure 4). This can be qualitatively understood by getting back to the dance picture. The round dance could be broken if the blindfolded dancers respond too weakly or too strongly to the forces they perceive from their neighbors; accordingly, an optimal response to forces would ensure a harmonious dance.

Figure 4.Precision of size and shape. The predicted ‘error’ on organ size (heterogeneity) is shown as a function of the level of response to directional mechanical stress, and features an optimal response level.

Long-range correlations of growth fluctuations are a crucial prediction of our analysis since it is strongly related to how cellular fluctuations impact robustness of morphogenesis. We therefore believe that correlations need to be measured at many scales in different experimental systems, in various genetic backgrounds, and in absence and presence of transient genetic or chemical perturbations. This would enable to disentangle the contributions of cell proliferation, cytoskeleton, ECM, and cell wall to the control of robustness. In addition, we speculate that long-range correlations could be involved in absolute size control; additional work would be required to investigate how this could occur. Finally, we note that we focused here on precision and robustness of morphogenesis, starting from the examples of bilateral symmetry in animals and flowers in plants; but this does not always need to be the case. For instance, plant leaves and roots are phenotypically highly plastic to adapt to environmental constraints. Tumor expansion and metastasis are likely favored by heterogeneous morphogenesis. Altogether, our work sheds light on the importance of considering fluctuations in understanding developmental processes.

References

Boudaoud, A., Burian, A., Borowska-Wykręt, D., Uyttewaal, M., Wrzalik, R., Kwiatkowska, D., & Hamant, O. (2014). FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nature protocols, 9(2), 457. https://www.ncbi.nlm.nih.gov/pubmed/24481272

Campinho, P., Behrndt, M., Ranft, J., Risler, T., Minc, N., & Heisenberg, C. P. (2013). Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly. Nature cell biology, 15(12), 1405. https://www.ncbi.nlm.nih.gov/pubmed/24212092

Damavandi, O. K., & Lubensky, D. K. (2019). Statistics of noisy growth with mechanical feedback in elastic tissues. Proceedings of the National Academy of Sciences, 116(12), 5350-5355. https://www.ncbi.nlm.nih.gov/pubmed/30819899

Duda, M., Kirkland, N. J., Khalilgharibi, N., Tozluoglu, M., Yuen, A. C., Carpi, N., … & Mao, Y. (2019). Polarization of myosin II refines tissue material properties to buffer mechanical stress. Developmental cell, 48(2), 245-260. https://www.ncbi.nlm.nih.gov/pubmed/30695698

Fruleux, A., & Boudaoud, A. (2019). Modulation of tissue growth heterogeneity by responses to mechanical stress. Proceedings of the National Academy of Sciences, 116(6), 1940-1945. https://www.ncbi.nlm.nih.gov/pubmed/30674660

Hamant, O., Heisler, M. G., Jönsson, H., Krupinski, P., Uyttewaal, M., Bokov, P., … & Couder, Y. (2008). Developmental patterning by mechanical signals in Arabidopsis. science,322(5908), 1650-1655. https://www.ncbi.nlm.nih.gov/pubmed/19074340

Hervieux, N., Tsugawa, S., Fruleux, A., Dumond, M., Routier-Kierzkowska, A. L., Komatsuzaki, T., … & Hamant, O. (2017). Mechanical shielding of rapidly growing cells buffers growth heterogeneity and contributes to organ shape reproducibility. Current Biology, 27(22), 3468-3479. https://www.ncbi.nlm.nih.gov/pubmed/29129534

Hong, L., Dumond, M., Zhu, M., Tsugawa, S., Li, C. B., Boudaoud, A., … & Roeder, A. H. (2018). Heterogeneity and robustness in plant morphogenesis: from cells to organs. Annual review of plant biology, 69, 469-495. https://www.ncbi.nlm.nih.gov/pubmed/29505739

Hipfner, D. R., & Cohen, S. M. (2004). Connecting proliferation and apoptosis in development and disease. Nature Reviews Molecular Cell Biology, 5(10), 805. https://www.ncbi.nlm.nih.gov/pubmed/15459661

Kierzkowski, D., Nakayama, N., Routier-Kierzkowska, A. L., Weber, A., Bayer, E., Schorderet, M., … & Smith, R. S. (2012). Elastic domains regulate growth and organogenesis in the plant shoot apical meristem. Science, 335(6072), 1096-1099. https://www.ncbi.nlm.nih.gov/pubmed/22383847

LeGoff, L., Rouault, H., & Lecuit, T. (2013). A global pattern of mechanical stress polarizes cell divisions and cell shape in the growing Drosophila wing disc. Development,140(19), 4051-4059. https://www.ncbi.nlm.nih.gov/pubmed/24046320

Mao, Y., Tournier, A. L., Hoppe, A., Kester, L., Thompson, B. J., & Tapon, N. (2013). Differential proliferation rates generate patterns of mechanical tension that orient tissue growth. The EMBO journal, 32(21), 2790-2803. https://www.ncbi.nlm.nih.gov/pubmed/24022370

Pan, Y., Heemskerk, I., Ibar, C., Shraiman, B. I., & Irvine, K. D. (2016). Differential growth triggers mechanical feedback that elevates Hippo signaling. Proceedings of the National Academy of Sciences, 113(45), E6974-E6983. https://www.ncbi.nlm.nih.gov/pubmed/27791172

Sampathkumar, A., Krupinski, P., Wightman, R., Milani, P., Berquand, A., Boudaoud, A., … & Meyerowitz, E. M. (2014). Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells. Elife, 3, e01967. https://www.ncbi.nlm.nih.gov/pubmed/24740969

Shraiman, B. I. (2005). Mechanical feedback as a possible regulator of tissue growth. Proceedings of the National Academy of Sciences, 102(9), 3318-3323. https://www.ncbi.nlm.nih.gov/pubmed/15728365

Uyttewaal, M., Burian, A., Alim, K., Landrein, B., Borowska-Wykręt, D., Dedieu, A., … & Kwiatkowska, D. (2012). Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis. Cell, 149(2), 439-451. https://www.ncbi.nlm.nih.gov/pubmed/22500806

Wolpert, L. (2010). Arms and the man: the problem of symmetric growth. PLoS biology, 8(9), e1000477. https://www.ncbi.nlm.nih.gov/pubmed/20838659

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A postdoctoral position is available in the Laboratory of Gareth Fraser (www.fraser-lab.net) at the Department of Biology, University of Florida. Our lab is focused on a range of research projects based around the central theme of vertebrate evolutionary developmental biology – they include tooth development and regeneration, craniofacial diversity, EvoDevOmics, and skin appendage evolution and development.

We are looking for a motivated and creative scientist to continue to develop the shark and ray as comparative evodevo models for a range of research projects. The primary focus of this position will be stem cell mediated tooth regeneration in sharks and rays, however we do encourage development of the candidates own creative research ideas and interests, so there is potential here to develop a project that suits the candidate in line with the focal themes of the lab. The salary is competitive and commensurate with qualifications and experience, and includes a full benefits package. The applicant selected for this position would begin the beginning of the Fall 2019 term.  The anticipated date would be August 16, 2019.

Qualifications

Candidates should have an interest in evolutionary developmental biology, and vertebrate diversity. Knowledge of developmental biology techniques, RNAseq analyses or stem/regenerative biology is desirable.

Application Instructions

For full consideration, applications must be submitted online at http://apply.interfolio.com/62577 and must include: (1) a letter of application summarizing the applicant’s qualifications and interests, (2) a complete curriculum vitae, (3) a statement on research goals, and (4) ) a list of at least three references. After initial review, applicants who are chosen to receive further consideration will be asked to request confidential letters of recommendation from the references. If you would like further information about the position please contact Gareth Fraser. Email: g.fraser@ufl.edu.

Application deadline is May 29, 2019.

All candidates for employment are subject to a pre-employment screening which includes a review of criminal records, reference checks, and verification of education.

The selected candidate will be required to provide an official transcript to the hiring department upon hire. A transcript will not be considered “official” if a designation of “Issued to Student” is visible. Degrees earned from an educational institution outside of the United States require evaluation by a professional credentialing service provider approved by the National Association of Credential Evaluation Services (NACES), which can be found at http://www.naces.org/.

The University of Florida is an equal opportunity institution dedicated to building a broadly diverse and inclusive faculty and staff. Searches are conducted in accordance with Florida’s Sunshine Law. If an accommodation due to disability is needed in order to apply for this position, please call (352) 392-2477 or the Florida Relay System at (800) 955-8771 (TDD).

The Department particularly welcomes applicants who can contribute to a diverse and inclusive environment through their scholarship, teaching, mentoring, and professional service. The university and greater Gainesville communities enjoy a diversity of cultural events, restaurants, year-round outdoor recreational activities, and social opportunities.

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Applications are invited from highly motivated individuals who are interested in fundamental mechanisms of neurodevelopment and neurogenetic disorders. The focus of the project is to understand neural developmental and neurological phenotypes in mouse models ofTuberous Sclerosis Complex.The fellowship is in the laboratories of Sara Wilson at the Department of Integrative Medical Biology (IMB), and Leif Carlsson at Umeå Center for Molecular Medicine (UCMM) both laboratories at Umeå University, Sweden. The facilities provide an interactive modern environmentwith easy access to good core facilities. The fellowship is administratively placedat IMB, which is an interdisciplinary department focusing on questions in basic and medical sciences. The fellowshipis funded for two years and is available immediately. The working ‘day to day’ language in the laboratories is English.

Requirements:Individuals with a background in mouse genetics and handling, developmental biology, neuroscience, molecular and/or cell biology with a keen interest in developmental neuroscienceare encouraged to apply. Experience with rodent motor circuits or rodent motor or seizure behavior analysis is a big advantage.Technical experience with imaging, molecular biology, immunohistochemistry,in situhybridization, vertebrate embryonic model systemsare required. Technical experience with mouse embryo electroporation and /or neurite outgrowth/migration assayswill also be positively evaluated. The successful candidate will have or about to receive a Ph.D.in a relevant discipline, have good communication skills and be proficient in written and spoken English. The most successful candidatewill have ahigh level of motivation, be organized, rigorous and have the ability to work both independently and within a team.

Application:Please submit your application (reference 2019SW7) by 31^stMay 2019 to sara.wilson@umu.seby sending the following documents as a single pdf file:

• A short cover letter (not more than 1 page) to include a description of your research experience and suitability for the position.
• Curriculum Vitaeincluding: publication list, technical expertise, names and contact information for three referees.

Informal enquiries may be directed to Dr. Sara Wilson (sara.wilson@umu.se) or Prof. Leif Carlsson (leif.carlsson@umu.se).

website: https://www.umu.se/en/research/groups/laboratory-of-sara-wilson/

We look forward to your application!

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How can a genome’s cis-regulatory potential drive robust gene expression throughout embryonic development? Do topologically associating domains (TADs) truly contribute to gene expression by spatially constraining the regulatory actions of cis-sequences? And might phase separation by multi-valent proteins also be involved in the regulation of genes and genomic regions? The recent Company of Biologists Workshop on “Chromatin-based regulation of development”, organized by Benoit Bruneau (Gladstone Institutes, UCSF) and Joanna Wysocka (Stanford University), was the perfect playground to discuss important questions like these, and to stimulate interactions between early career and established scientists. Why was this Workshop so great to me (currently a postdoc in the lab of François Spitz, Institut Pasteur, France)? And why should you – a young investigator – apply to attend a future one?
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Have you got a beautiful image of a developing organism you’d like to share with the world? Or a striking computer generated visualisation, or even something you’ve drawn or painted or stitched or crafted? We’re planning a Node calendar for the academic year 2019-20, and want to fill it with your images.

Entering is easy: simply send your image, along with a description of what’s being shown, to thenode@biologists.com, with the subject line ‘Calendar competition’.

Content-wise, the only limitation is that it should have a developmental biology angle to it. We’d love to have as much diversity in organisms and systems as possible. The image dimensions on the calendar are 24 cm L x 19 cm H, and some cropping may occur. One entry per person but individual labs can enter multiple images. 

The deadline for submissions is 13.00 (GMT+1), Friday 10 May

We’ll collate all the entries and then run a public vote the following week. Good luck!

The idea for the calendar didn’t come from nowhere: Development used to print calendars each year and distribute them to researchers until it became economically unfeasible. But people would often ask us about them at conferences (or rather, complain about not having them any more!), and some of us in the Development team remember how nice the calendars looked hanging up in the lab before we left those labs for publishing. We hope our Node calendar will be equally beautiful and useful!

Development’s 2013 calendar

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Feiyue Lu and David Gilmour tell the story behind their recent paper in Molecular Cell

RNA polymerase II (Pol II) is the enzyme responsible for transcribing most genes in eukaryotes. The C-terminal domain (CTD) is a highly repetitive, unstructured domain on the largest Pol II subunit, Rpb1. It consists of numerous repeats of seven amino acids and serves as a binding platform for numerous factors involved in gene regulation, including chromatin modifying enzymes, transcription factors and RNA processing enzymes. Both length and sequence complexity of the CTD greatly vary among species of different lineages. For example, the CTD of S. cerevisiae has 26 repeats, the majority of which are consensus repeats of YSPTSPS. In contrast, mammalian CTDs have 52 repeats, the distal repeats are mostly divergent repeats whose sequence differ from the consensus at one or more positions.

The length and sequence complexity of the CTD were thought to regulate gene expression and be essential for the development of complex organisms. In the past decade, this proposition has been reinforced by several studies in mammalian cells. These studies showed that post-translational modifications occur on certain residues of the divergent repeats, some of which are recognized by factors only present in higher eukaryotes, and that mutations which prevent such modifications lead to misexpression of genes. Yet the significance of the divergent residues has never been systematically examined in the context of development.

Considering the above findings in mammalian cells, one would anticipate that a mouse would die if all of its CTD repeats were replaced with the consensus repeats that predominate the yeast CTD. However, generating transgenic mouse lines is very time and cost consuming. Instead, we decided to perform systematic mutagenesis of the CTD in Drosophila, an organism that is also highly genetically malleable but faster and cheaper to mutate compared to mice. In addition, 40 of the 42 CTD repeats in the fly diverge from the consensus, so it is an ideal model to interrogate the significance of divergent repeats.

The project was started at a time when CRISPR/Cas9 was not yet widely adopted by the fly research community, so initially we relied on RNAi knockdown to deplete the endogenous Rpb1 subunit. We also tested if the co-expression of an RNAi-resistant Rpb1 harboring mutations in the CTD would rescue the RNAi phenotype, making approximately 20 transgenic fly lines expressing various mutant forms of Rpb1 in an attempt to identify essential regions of the fly CTD. To our surprise, despite the high conservation in the amino acid sequence of the CTD within the fruit fly genus, most of our mutant CTD flies were viable, even when internal deletions removed up to 30% of the entire CTD. The only essential region we identified was an 8-repeat region encompassing the only two consensus repeats in the fly CTD (Gibbs et al., 2017). This suggests that the majority of the divergent repeats in the fly CTD are redundant.

Our initial systematic mutagenesis of the fly CTD emboldened us to test the idea that all divergent repeats could be replaced with consensus repeats. However, most previous findings in mammalian cells argue that the divergent CTD repeats are essential, so we still expected that flies would die with an all-consensus CTD. The most straightforward way to test this would be to simply replace the fly CTD with 42 consensus repeats. However, since many of the CTD repeats appear redundant, consensus repeats might be able to do what divergent repeats do, but with more or fewer repeats to achieve the same function. Therefore, we tested a series of consensus CTDs ranging from 10 to 52 repeats.

Surprisingly, we obtained normal looking flies with solely consensus repeats. This was achieved with only 20 to 29 repeats, which approximates the length of the yeast CTD. In contrast, flies with 42 consensus repeats, which matches the length of the wild-type fly CTD, barely survived to adulthood. In hindsight, we were fortunate to have tested varying numbers of consensus repeats because had we only tested the 42 consensus repeat CTD, we would have concluded that the divergent motifs are indispensable, which would have driven the project in a different direction. Additionally, flies with 52 consensus repeats died, whereas flies with the human CTD, which is also composed of 52 repeats but contains a mixture of consensus and divergent repeats, were able to survive. Therefore, it seems as though consensus repeats alone are sufficient, yet having too many of them is bad for fly development.

We were shocked by the results of the RNAi rescue experiment but at the same time concerned that residual endogenous Rpb1 might contribute to survival. Therefore, we decided to use CRISPR/Cas9 to mutate the endogenous Rpb1 gene. Remarkably, the CRISPR results agreed with our RNAi experiment. In particular, our flies with 20 to 29 consensus repeats did just as well as flies with the wild-type CTD even under temperature stress.

As flies can survive with solely consensus repeats, consensus and divergent repeats should both be able to interact with the same group of factors. Our data also suggests that interactions provided by the consensus repeats are likely stronger, since in the case of an all-consensus CTD, fewer than half the number of wild-type repeats are needed for such interactions to occur properly. This fits nicely with the phase separation mechanism proposed by Weber and Brangwynne where protein interactions could be mediated by weak, multivalent interactions (Weber and Brangwynne, 2012). In reviews published when our project was being developed, Hnisz et al and Harlen et al proposed that such forces could also drive CTD:factor interactions (Harlen and Churchman, 2017; Hnisz et al., 2017). Also consistent with the phase separation theory was that the deleterious effects of having too many consensus repeats could be reverted either by shortening the CTD (as was the case with our truncated consensus CTDs), or by replacing some of the stronger repeats with weaker ones (as was the case with 52 consensus CTD versus the human CTD), both of which could reduce the overall valency of the CTD.

Coincidentally, our co-author Bede Portz had a chat with Stirling Churchman at the Cold Spring Harbor Mechanisms of Eukaryotic Transcription Meeting about our CTD mutagenesis in flies. Stirling was interested in our findings and was curious to know if the CTD by itself could target transcription sites. Bede thought that the fruit fly salivary glands would be an ideal system to test this. The salivary gland cells undergo rounds of replication without cell division, giving rise to polytene chromosomes where numerous copies of sister chromatids are fused together, which allows for visualization of each transcription site as a distinct band (or ‘puff’). Before us, John Lis’ group had shown that many transcription components such as Pol II are compartmentalized at heat shock puffs in salivary glands (Yao et al., 2006; Yao et al., 2007; Zobeck et al., 2010). Transcription compartments have also been documented recently using live super-resolution microscopy or in other systems where numerous copies of the same DNA sequences were introduced into a genomic location (Cho et al., 2018; Chong et al., 2018). However, the polytene chromosomes provide the power to visually determine a factor’s spatial location with respect to euchromatin, heterochromatin and nucleoplasm without having to rely on super-resolution microscopy or amplifying the number of copies of a candidate gene.

We fused just the CTD to GFP and expressed it in salivary glands. Interestingly, GFP-CTD colocalizes with Pol II on fixed polytene chromosome spreads. One possibility is that the CTD alone dynamically associates with the transcription sites. The alternative is that the GFP-CTD is stably bound by CTD binding partners that are abundant at transcription sites. To distinguish the two possibilities, we turned to live imaging and showed that GFP-CTD on puffs can rapidly recover upon photobleaching, suggesting that the CTD is dynamically recruited to transcription compartments. In addition, phase separation is likely the driving force for the behavior of the CTD, as 1,6-hexanediol, which has been shown to disrupt weak hydrophobic interactions, can disrupt the association of GFP-CTD to chromosomes.

The partitioning characteristic of a molecule is influenced by its valency. This led us to investigate how the number of consensus repeats impacts the behavior of the CTD. To address this, we fused our series of consensus CTDs to GFP. Phase separation models predict that changes in valency could lead to pathological aggregates (Weber and Brangwynne, 2012). Indeed, our 42 and 52 repeats of consensus CTDs, which do not support normal function in the fly, showed less GFP on puffs and correspondingly more extrachromosomal foci when fused to GFP. Much of the extrachromosomal foci were neither recovered upon photobleaching nor dispersed by 1,6-hexanediol, suggesting that they were stable aggregates. Interestingly, none of our consensus CTDs partition into transcription compartments as readily as the wild-type CTD. These results suggest that the divergent repeats play a role in either preventing aggregation, or facilitating partitioning into transcription compartments, both of which could be forces that constrain the sequence conservation of the CTD in evolution.

At the start of this project, we had anticipated that the divergent repeats would be essential for fly viability since the sequences of the CTD in 12 species of Drosophila are highly conserved. Moreover, the ratio of synonymous to nonsynonymous mutations among the 12 species is high, thus indicating that the fly CTD is under significant purifying selection. Our discovery that the entire fly CTD can be replaced by consensus repeats poses a conundrum since it calls into question the basis for the purifying selection. One possibility is that intra and intermolecular interactions intrinsic to the CTD constrain the sequence. Our SAXs analysis showed that the CTD adopts a compact random coil structure and the compaction implies transient interactions within the CTD (Gibbs et al., 2017). Also, recent results show that CTD molecules self-associate to form droplets (Boehning et al., 2018). Both types of interactions might need to be finely tuned to prevent aggregation and allow for factor binding to the CTD. Despite the sequence differences, the 29 consensus and wild-type Drosophila CTDs appear to meet these constraints.

The theory of constructive neutral evolution provides another answer to the conundrum. This theory was formulated to explain the gap between the simplicity and complexity of molecular machines that serve the same function in different organisms (Gray et al., 2010). In our case, the 29 consensus CTD represents the simple end of the spectrum while the Drosophila CTD represents the complex end. For both to be functionally equivalent, we posit that the essential functions of the CTD are mediated by a limited number of proteins that interact with the consensus repeats. It is notable that while the Drosophila CTD only contains two repeats that exactly match the consensus, these are embedded in a region encompassing seven other near-consensus motifs. Deletion of this region completely eliminated the capacity of Rpb1 to support fly viability. Constructive neutral evolution posits that chance mutations in the consensus sequences are tolerated by the fortuitous binding of other proteins which compensate for the loss of the consensus sequences. This fortuitous interaction might offset the tendency of the mutation to cause the CTD to aggregate or become mistargeted. Alternatively, the loss of consensus repeat could diminish the affinity of an essential protein for the CTD but this could be offset by the fortuitous binding of another protein that simultaneously associates with the mutant repeat and the protein that normally binds directly to the consensus repeat. Occurrence of the fortuitous binding caused by one mutation sets the stage for mutating additional consensus repeats. The evolutionary trajectory is then dictated by chance and could explain why the consensus CTD, the human CTD, and the Drosophila CTD all support Rpb1 function in the fly. If the consensus CTD truly represents the simplest CTD, then there should exist a class of mutations in genes outside of the Rpb1 gene that are deleterious to wild-type flies but not to those carrying the 29 consensus CTD.

Considering the number of CTD mutants that we had to generate to come to our final conclusion, we feel extremely lucky to have started this project in flies: the fruit fly is indeed an amazing model system to study the CTD. In addition to this work, the divergent nature of the fly CTD has allowed our collaborators Dr. Scott Showalter at PSU, Drs. Yan “Jessie” Zhang and Jennifer Brodbelt to map structural changes and post-translational modifications to each individual repeat without having to introduce additional mutations to the CTD (Gibbs et al., 2017; Mayfield et al., 2016). Furthermore, the fly salivary gland present a unique system to characterize the spatial distribution of transcription factors with just a standard confocal microscope. We envision that the currently available gene-editing and optogenetic tools will allow more exciting discoveries of transcription to be made in the salivary glands.

References

Boehning, M., Dugast-Darzacq, C., Rankovic, M., Hansen, A. S., Yu, T., Marie-Nelly, H., McSwiggen, D. T., Kokic, G., Dailey, G. M., Cramer, P., et al. (2018). RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol.

Cho, W.-K., Spille, J.-H., Hecht, M., Lee, C., Li, C., Grube, V. and Cisse, I. I. (2018). Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science.

Chong, S., Dugast-Darzacq, C., Liu, Z., Dong, P., Dailey, G. M., Cattoglio, C., Heckert, A., Banala, S., Lavis, L., Darzacq, X., et al. (2018). Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361,.

Gibbs, E. B., Lu, F., Portz, B., Fisher, M. J., Medellin, B. P., Laremore, T. N., Zhang, Y. J., Gilmour, D. S. and Showalter, S. A. (2017). Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain. Nat. Commun. 8, 15233.

Gray, M. W., Lukes, J., Archibald, J. M., Keeling, P. J. and Doolittle, W. F. (2010). Irremediable Complexity? Science 330, 920–921.

Harlen, K. M. and Churchman, L. S. (2017). The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain. Nat. Rev. Mol. Cell Biol. 18, 263–273.

Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. and Sharp, P. A. (2017). A Phase Separation Model for Transcriptional Control. Cell 169, 13–23.

Mayfield, J. E., Robinson, M. R., Cotham, V. C., Irani, S., Matthews, W. L., Ram, A., Gilmour, D. S., Cannon, J. R., Zhang, Y. J. and Brodbelt, J. S. (2016). Mapping the phosphorylation pattern of drosophila melanogaster RNA polymerase ii carboxyl-terminal domain using ultraviolet photodissociation mass spectrometry. ACS Chem. Biol. 12, 153–162.

Weber, S. C. and Brangwynne, C. P. (2012). Getting RNA and protein in phase. Cell 149, 1188–1191.

Yao, J., Munson, K. M., Webb, W. W. and Lis, J. T. (2006). Dynamics of heat shock factor association with native gene loci in living cells. Nature 442, 1050–1053.

Yao, J., Ardehali, M. B., Fecko, C. J., Webb, W. W. and Lis, J. T. (2007). Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation. Mol. Cell 28, 978–990.

Zobeck, K. L., Buckley, M. S., Zipfel, W. R. and Lis, J. T. (2010). Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells. Mol. Cell 40, 965–975.

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One of the things I learned during my stay in another lab is that you can’t succeed completely by yourself. You can try, sure, but a relevant unanswered question in biology is a hard nut to crack. It usually takes a multidisciplinary approach and great ideas coming from more than one brain. Collaborations are key to overcome this issue.

Most of the times, what keeps you from collaborating with peer scientists is money. Money to travel and money to pay for your accommodation. Renting a room in the US can cost as much as your entire monthly wage in Spain. Time is another big factor. Traveling takes time. As a Ph.D. student though, my time is fully dedicated to research anyway, so that factor is negligible for me.

Collaborate or perish

When Kate McDole and colleagues published their paper (McDole et al., 2018) describing their innovative live imaging microscope, I thought, ‘That is a method I could use for my project’. However, in my research center, I don’t have the facilities nor the expertise to carry it out.

In Miguel Torres Lab, at the Spanish Center for Cardiovascular Research, I am using live imaging to study how the heart establishes its heterogeneous cell types during gastrulation and how mouse embryos manage to form a primitive tube that later on starts beating. However, our methodology does not support imaging live specimens for longer than 20h. Keller’s Lab, at Janelia Reseach Institute, managed to build a fast light-sheet microscope that adapts to the mouse embryo growth and optical properties, allowing its imaging throughout 48h at an unprecedented resolution.

A travel grant to make it happen

The Company of Biologists provided with the funds to cover my expenses, and so I was able to join Keller’s lab for 5 weeks. At Janelia, I found a welcoming and collaborative environment surrounded by cutting-edge facilities. Besides getting some precious datasets from their microscope, I learned how to analyze the vast amount of data generated by time-lapse acquisitions (they run for 48h and take 300 stacks every 4 minutes, do the math…). Learning to program in Python wasn’t in my plans but it ended up being the most valuable thing I learned during the stay. Programming was completely unknown to me before I did the stay, and now I don’t know what I would do without it. To be able to quantify the cell dynamics I am observing in the videos and extract meaningful information becomes way more efficient when one uses programming.

It’s true that a Skype call can help you work together with other scientists, but sometimes you need to be there to learn other techniques, bring up together a project or receive inputs from clear-eyed researchers. That, I think, makes a project more solid and likely to succeed in answering the proposed question. I strongly believe collaborations blossom science, and the role of these traveling fellowships is key to make them happen. At the end of the day, to get to your goal you need a team, and some members of that dream team might be in another part of the world.

If you want to know a bit more about my Ph.D. project, here’s a 4min video explaining it:

(3 votes)

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The Craft lab at Boston Children’s Hospital and Harvard Medical School is looking for an outstanding, highly motivated postdoctoral fellow to join our developmental biology and pluripotent stem cell-based team.

We established directed differentiation protocols to generate distinct articular and growth plate-like cartilage lineages from human and mouse pluripotent stem cells (Craft et al., Development 2013; Craft et al., Nature Biotech 2015). NIH and foundation funded projects in our lab include investigating mechanisms of articular chondrocyte lineage commitment and stability of this fate through deep sequencing and functional assays, specification and characterization of joint progenitor cells using newly generated fluorescent reporter lines, translational/preclinical experiments of cartilage repair in large animals, and developing iPSC models of congenital cartilage disease. Collaborative projects with Harvard faculty include studies of how modifications in regulatory elements of GDF5 impact gene expression and differentiation of joint lineages (with Dr. Terence Capellini, Harvard University), and evaluating the cartilage and tendon-inducing functions of small molecules and their associated signaling pathways (identified by Dr. Jenna Galloway, Massachusetts General Hospital, through zebrafish screens) in embryonic stem cell (ESC) differentiation cultures.

Requirements: The successful candidate will have received a PhD or MD/PhD within the past 2 years, a minimum of 3 years laboratory experience including tissue culture, at least one first author publication, and excellent communication skills in English.  Individuals with experience in one or more of the following are encouraged to apply: limb/joint/cartilage or early embryonic development, signal transduction pathways, ESC/iPSCs, single cell RNAseq/programming.

https://hsci.harvard.edu/craft-lab-postdoctoral-fellow

Apply: Interested applicants should email their CV, a cover letter describing their research background/interests, and contact information for three references to april.craft at childrens.harvard.edu

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