As a first-year graduate student, I had the good fortune of accompanying Dr. Pierre Rasmont (U. Mons, Belgium) and his lab group on an expedition to collect bumble bees in Turkey. At our first stop onto the dry but flower-rich volcanic lands, we each dispersed to collect bees. At the time I was working to reconstruct a phylogeny of world bumble bees and one of each of the endemic species from this region was needed. When we reconvened to share what we found, I, not wanting to sacrifice unnecessary lives, came back with a limited catch. Pierre frowned, so I commented, “I only need one of each” and to that he commented, “They all look the same!”  It was then I learned the extremes to which bumble bees can mimic each other. While in the Eastern United States bumble bees are yellow and black, in Turkey nearly every species is white with black bands and a red tail…only in the details of color and morphology can they be told apart. Across the world there are several dozen bumble bee mimicry patterns each differing by geographic region. These locally shared warning signals enable enhanced predator learning, reducing predation for all – a process known as Mullerian mimicry.

I became interested in deciphering the phylogenetic patterns of this color diversity, noting that color is highly labile, exhibiting so much intraspecific and interspecific diversity that little phylogenetic signal can be recovered. The ~260 species of bumble bees globally can exhibit nearly every combination of red/orange, white, yellow, and black color in their fluffy pile across their segmental sclerites, with some species exhibiting dramatic color variation by geographic region [1].   My PhD advisor, Dr. Sydney Cameron (U. Illinois, U-C), and I were excited about the potential of this system to inform Evo-Devo. How is it that these bumble bees are capable of attaining such color diversity? What mechanisms may be driving the modular coloration we see in these bees? She set out to determine the diversity in patterns and how patterns vary across the bee body, revealing that some segments of the body are more prone to change relative to others [2]. The places on the body where color transitions were most likely to take place appeared to me to be locations where segment fate-determining Hox genes shift.

Examples of typical bumble bee mimicry patterns from different geographic regions. Upper left: Color patterns and their distribution among bumble bee species in subgenus Pyrobombus, demonstrating mimetic convergence (Image credit: H. Hines). Lower left: Bombus melanopygus red and black form (Photo credit: Li Tian, H. Hines), Upper middle: Bombus bimaculatus (Photo credit: H. Hines), Upper right: Bombus haemorrhoidalis (Photo credit: H. Hines), Lower right: Bombus incertus (Photo credit: Pierre Rasmont, available on http://www.atlashymenoptera.net) 

The evolutionary genetics literature however, would not predict that these patterns were driven by direct changes to Hox genes. Hox genes are highly pleiotropic, driving extensive body region-specific morphologies. Thus changes in the amount and locations of these genes should have large scale, often deleterious effects (e.g., antennapedia and bithorax). In Drosophila, nearly every case of color variation involves changes to a cis-regulatory module of a pigment gene in the melanin pathway.  Many pigment enzymes can be altered, but it is nearly always a pigment gene in cis [3]. The upstream regulators, as predicted in evolutionary genetics, stay unchanged.

To understand the genetic basis of color variation in bumble bees, I first set out to determine the pigments of their colored hairs, which yielded its own surprises. We discovered black is eumelanin, yellow is most likely a novel pterin (although melanin likely contributes) and that the red is a pheomelanin, the type of melanin that makes mammal hair and bird feathers red, but which was not thought until recently to be present in insects [4]. Thus color switches such as from black to red melanins lacked a clear enzyme candidate, as phaeomelanins are not part of known insect pigment pathways.  Building on technological advances, I decided to pursue whole genome approaches to discover genes driving color variation, and, the bumble bee Bombus melanopygus was an ideal choice. Bombus melanopygus has two color forms, one with a red abdominal stripe in the Rocky Mountain mimicry complex and another with a black abdominal stripe belonging to the Pacific Coastal mimicry group [5]. Previous work [6] revealed that this phenotype was under simple single-gene Mendelian inheritance, with red dominant to black, and that these color forms meet in a narrow hybrid zone, where they are genetically admixed [7].  These were perfect conditions for obtaining resolution in genome wide association analysis (GWAS).

With a NSF CAREER grant secured to pursue this project [8], graduate student Sarthok Rahman applied GWAS to determine the genetic basis of the color variation in B. melanopygus, identifying a single locus with just a few individuals.  We were excited to discover this locus fell in the cis-regulatory region between the abdominal fate determining Hox genes, abd-A and Abd-B. Is it possible that the pleiotropic Hox genes were changing their expression, counter to evolutionary genetics theory?

Discovering the expression patterns of these Hox genes was much harder than finding the locus. As a non-model system, the development of bumble bee pupae, the stage when coloration is established, had to be worked out. This was especially needed as bumble bees are highly labile in size, so time-based staging is unreliable. Li Tian, a postdoc at the time, developed a morphological staging system for bumble bee pupae that he used to perform comparative gene expression work [9; see time-lapse of pupal development below or download here]. 

This too took us longer than we would have liked as we originally focused on stages prior to the pigmentation process. Only later did we include the stages where color was being deposited, and discovered that Abd-B was differentially expressed concordant with this stage [10]. By comparing across segments of the body we discovered that Abd-B is upregulated only in the anterior abdominal segments bearing the color differences, a location it normally has little to no expression in, but only in the stages immediately prior to adult emergence. At this stage most abdominal Hox genes have low expression, concordant with the near completion of morphological change aside from color. This late-stage homeotic shift in Hox gene expression enabled segment-specific effects with little pleiotropic consequences, highlighting how upstream selector genes like Hox genes can be excellent evolutionary targets for promoting diversity as long as their shifting expression is compartmentalized.

Going forward we plan to dissect how Hox gene cis-regulatory regions enable these changes with transgenic approaches.  With this hyperdiverse system, our larger goal is to determine the role Hox genes might play in enabling the diversification across more bee species. Perhaps this story is unique to B. melanopygus (our analyses suggests the same variants are not involved in comimics) or perhaps Hox genes are commonly implicated across species but different regulatory targets are involved. With such diversity, our ability to study these mechanisms and fill out this story seems endless.

[1] Hines, H. M., & Williams, P. H. (2012). Mimetic colour pattern evolution in the highly polymorphic Bombus trifasciatus (Hymenoptera: Apidae) species complex and its comimics. Zoological Journal of the Linnean Society, 166(4), 805-826.

[2] Rapti, Z., Duennes, M. A., & Cameron, S. A. (2014). Defining the colour pattern phenotype in bumble bees (Bombus): a new model for evo devo. Biological Journal of the Linnean Society, 113(2), 384-404.

[3] Kronforst, M. R., Barsh, G. S., Kopp, A., Mallet, J., Monteiro, A., Mullen, S. P., … & Hoekstra, H. E. (2012). Unraveling the thread of nature’s tapestry: the genetics of diversity and convergence in animal pigmentation. Pigment cell & melanoma research, 25(4), 411-433.

[4] Hines, H. M., Witkowski, P., Wilson, J. S., & Wakamatsu, K. (2017). Melanic variation underlies aposematic color variation in two hymenopteran mimicry systems. PloS one, 12(7), e0182135.

[5] Ezray, B. D., Wham, D. C., Hill, C., & Hines, H. M. (2019). Müllerian mimicry in bumble bees is a transient continuum. bioRxiv, 513275.

[6] Owen, R. E., & Plowright, R. C. (1980). Abdominal pile color dimorphism in the bumble bee, Bombus melanopygus. Journal of Heredity, 71(4), 241-247.

[7] Owen, R. E., Whidden, T. L., & Plowright, R. C. (2010). Genetic and morphometric evidence for the conspecific status of the bumble bees, Bombus melanopygus and Bombus edwardsii. Journal of Insect Science, 10(1).

[8] National Science Foundation: Division of Environmental Biology: Evolutionary Processes, #1453473. CAREER: The genetics underlying adaptive diversification patterns in bumble bees. H.M. Hines.

[9] Tian, L., & Hines, H. M. (2018). Morphological characterization and staging of bumble bee pupae. PeerJ, 6, e6089.

[10] Tian, L., Rahman, S. R., Ezray, B. D., Franzini, L., Strange, J. P., Lhomme, P., & Hines, H. M. (2019). A homeotic shift late in development drives mimetic color variation in a bumble bee. Proceedings of the National Academy of Sciences, 116(24), 11857-11865.

(2 votes)

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We offer one fully-funded postdoctoral position up to five years in the Laboratory of Genome Integrity located at the National Institutes of Health (NHI/NCI, Bethesda, MD).

Our laboratory uses human and mouse embryonic stem cells (ESCs) as well as mouse embryos to understand the molecular mechanisms underlying the maintenance/exit of pluripotency and self-renewal. Understanding cell plasticity, pluripotency and differentiation to get a better comprehension of embryonic development, cell transformation and cancer are our scientific interests.

The applicant should have or about to have a PhD in Developmental Biology, Genetics or similar, and should have demonstrated expertise on molecular biology/mammalian tissue culture. Knowledge in mouse embryology, computational biology and next generation sequencing technologies will be considered as an advantage.

We seek a highly motivated, creative individual, eager to learn and develop new technologies and complex cell systems based on live cell/embryo imaging, single-cell technologies, 3D modelling and CRISPR-based editing interested in understanding how a single cell can develop into a complex multicellular organism in vitro and in vivo.

Please send a brief cover letter, CV and three reference letters via e-mail to:

sergio.ruizmacias@nih.gov

https://ccr.cancer.gov/Laboratory-of-Genome-Integrity/sergio-ruiz-macias

• Mayor-Ruiz C, et al. ERF deletion rescues RAS deficiency in mouse embryonic stem cells. Genes & Dev. 32: 568-576, 2018.
• Olbrich T, et al. A p53-dependent response limits the viability of mammalian haploid cells. Proc Natl Acad Sci U S A. 114: 9367-9372, 2017.

• Ruiz S, et al. A genome-wide CRISPR screen identifies CDC25A as a determinant of sensitivity to ATR inhibitors. Mol Cell. 62: 307-13, 2016.
• Ruiz S, et al. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells. Nature Commun. 6: 8036, 2015.
• Ruiz S, et al. Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. Proc Natl Acad Sci U S A. 109: 17196-201, 2012

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The Musculoskeletal Extracellular Matrix Laboratory (MEML) in the Department of Mechanical Engineering at the University of Colorado – Boulder is seeking an exceptionally creative postdoctoral researcher to investigate the mechanistic basis for how extracellular matrix (ECM) remodeling directs regeneration in mammals.  The project is a collaborative effort using spiny mice, an emerging model for mammalian musculoskeletal regeneration developed in the Seifert Lab in the Department of Biology at the University of Kentucky.  As such, the selected candidate will work with both groups, but will based at CU-Boulder.  The selected candidate will be trained to leverage cutting edge imaging and proteomic tools employed in the MEML to label, isolate and visualize newly generated ECM.  Using this methodology, in combination with in vitro and in vivo approaches in spiny mice and lab mice, the selected candidate will investigate how ECM production, composition and force generation regulate regenerative healing.

Ideal candidates will have a strong background in at least two of the following areas (and a drive to learn and master the others): biomechanics of soft tissues, developmental biology, 3D/4D visualization of biological tissues, protein engineering and quantitative bioinformatics.  Successful applicants will initially join an NIH funded project.  While this is a funded position, postdocs in the MEML are strongly encouraged to develop their own projects and external funding portfolios as a pathway toward independence.  Salary follows NIH guidelines for postdoctoral researchers.  Informal inquiries by email are strongly encouraged.  For additional information visit: https://www.colorado.edu/mechanical/sarah-calve.

Review of applications will begin on a rolling basis and will continue until the position has been filled.  Ideal start date is Fall 2019/Winter 2020.  Candidates will have completed their Ph.D. prior to starting the position but need not have defended their dissertation prior to applying.  Applicants should send a single pdf document to Sarah Calve (sarah.calve@colorado.edu) that includes their CV, names of three references, and a 1-2-page synopsis of their current research interests and how these complement our overall research program.

The Department of Mechanical Engineering at CU-Boulder houses a strong group of research labs interested in biomedical engineering, materials science, imaging, robotics and micro/nanoscale engineering.  Together, these labs create a vibrant atmosphere to leverage engineering principles and tools to clarify unanswered questions in biology.

The University of Colorado is an equal opportunity and affirmative action employer committed to assembling a diverse, broadly trained faculty and staff. In compliance with applicable laws and in furtherance of its commitment to fostering an environment that welcomes and embraces diversity, the University of Colorado does not discriminate on the basis of race, color, creed, religion, national origin, sex (including pregnancy), disability, age, veteran status, sexual orientation, gender identity or expression, genetic information, political affiliation or political philosophy in its programs or activities, including employment, admissions, and educational programs. Inquiries may be directed to the Boulder Campus Title IX Coordinator by calling 303-492-2127.

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In this episode from our centenary series exploring 100 ideas in genetics, we’re telling tales of sex and death, and exploring the very darkest side of genetics. We discover how Francis Galton’s eugenic ideas led to some of the worst atrocities of the 20th century, and ask how his legacy should be honoured today – if at all? Plus, the evolution of sex, and the total eradication of mosquitoes.

Please fill in our short listener survey, and you’ll be entered into a prize draw to win a signed copy of Kat Arney’s book, Herding Hemingway’s Cats.

Listen and download now from GeneticsUnzipped.com, plus full show notes and transcripts.

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Research technician position available for a project focused on functional technique development in annelid worms, with a conceptual focus on evo-devo and regeneration biology. This position is part of an NSF-funded project focused on developing approaches to test gene function in post-embryonic (juvenile and adult) stages of several annelid species. The project is a collaboration between Alexa Bely (U Maryland), Duygu Ozpolat (Marine Biological Laboratory, Woods Hole), and Elaine Seaver (U Florida, Whitney Marine Lab) and there are substantial interactions among the three lab groups.

​This position is in the lab of Elaine Seaver, with primary focus on the annelid Capitella teleta. Position is for 1 year, with possibility of renewal for a second year, and with a start in Fall 2019.

Required qualifications: bachelor’s degree in biological sciences or related field, at least 1 year research experience in molecular or developmental biology, good fine-motor skills, ability to troubleshoot and persevere, effective time management and organizational skills, and team-oriented outlook. Preferred but not required: experience with microinjection, electroporation, and approaches to disrupt gene function.

Through this position, the successful candidate will gain valuable experience in the development of novel methods, will gain experience in both molecular and organismal research, and will be part of a highly interactive and supportive team.

More information about this project and this position is available at: https://wormsontheedge.weebly.com

To apply: send a cover letter, CV, and name and contact information for three references to Elaine Seaver at seaver@whitney.ufl.edu.

Note: There is a similar position at Univ. Maryland – see separate posting at: https://thenode.biologists.com/research-technic…univ-of-maryland/jobs/

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Research technician position available for a project focused on functional technique development in annelid worms, with a conceptual focus on evo-devo and regeneration biology. This position is part of an NSF-funded project focused on developing approaches to test gene function in post-embryonic (juvenile and adult) stages of several annelid species. The project is a collaboration between Alexa Bely (U Maryland), Duygu Ozpolat (Marine Biological Laboratory, Woods Hole), and Elaine Seaver (U Florida, Whitney Marine Lab) and there are substantial interactions among the three lab groups.

​This position is in the lab of Alexa Bely, with primary focus on the annelid Pristina leidyi. Position is for 1 year, with possibility of renewal for a second year, and with a start in Fall 2019.

Required qualifications: bachelor’s degree in biological sciences or related field, at least 1 year research experience in molecular or developmental biology, good fine-motor skills, ability to troubleshoot and persevere, effective time management and organizational skills, and team-oriented outlook. Preferred but not required: experience with microinjection, electroporation, and approaches to disrupt gene function.

Through this position, the successful candidate will gain valuable experience in the development of novel methods, will gain experience in both molecular and organismal research, and will be part of a highly interactive and supportive team.

More information about this project and this position is available at: https://wormsontheedge.weebly.com

To apply: send a cover letter, CV, and name and contact information for three references to Alexa Bely at abely@umd.edu.

Note: There is a similar position at Univ. Florida, Whitney Marine Lab – see separate posting at: https://thenode.biologists.com/research-technic…rine-lab-florida/jobs/

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This interview, the 67th in our series, was recently published in Development

Most bones in the vertebrate skeleton are made in the same way – endochondrial ossification – yet they display a variety of shapes and sizes. The question of how these unique bone morphologies, including the superstructures that protrude from their surfaces, arise during development is still unclear, and the subject of a new paper in Development. We caught up with first author Shai Eyal and his supervisor Elazar Zelzer, Professor in the Department of Molecular Genetics at the Weizmann Institute of Science in Rehovot, Israel, to find out more about the story.

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

EZ We walk, run and jump using the complex and ingenious musculoskeletal system. It is therefore puzzling that, although each of its components has been extensively studied, research on the musculoskeleton as an integrated system, and in particular on how it is assembled, has been scarce. My vision as a scientist is to demonstrate the centrality of molecular and biomechanical crossregulation between musculoskeletal tissues in the development and function of this system. In 2004, I established my independent research group at the Weizmann Institute. Over the years, I was joined by a group of extremely talented students. Together, we have made interesting discoveries about various aspects of musculoskeletal biology, including the regulatory interactions between the developing skeleton and its vasculature, the regulation of bone morphogenesis and joint formation by muscle-induced mechanical signals, and the development of secondary structures such as bone eminences and sesamoid bones.

One of the cornerstones of my philosophy as a mentor of young creative people is to allow team members space for individual expression. I believe that this has been key to our ability to develop new avenues of research. Indeed, we have recently opened a new and exciting direction by introducing the significance of proprioceptive signalling in skeletal regulation, specifically in spine alignment, bone regeneration and joint development.

And Shai, how did you come to work in the Zelzer lab, and what drives your research today?

SE I majored in Life Sciences at the Hebrew University of Jerusalem. During that time, I was exposed to various fields of biological research; however, the field that really caught my attention was developmental biology. When I think of how a single cellular unit, a fertilized egg, can develop into endless forms and shapes, it is like biological magic to me. Therefore, when I moved to the Weizmann Institute of Science, I looked for labs in which I could continue studying concepts in developmental biology. Looking into the research that was carried out by the Zelzer lab, I was very intrigued. Skeletogenesis, in particular how the skeletal and muscular systems are integrated, is a very interesting and largely unknown process. With Eli’s superb mentorship and the supportive lab atmosphere, I was able to highlight several key processes taking place during sesamoid bone formation and long bone morphogenesis. Today, my interest has shifted away from skeletogenesis, but I am still as hooked on developmental biology as I was when I began my research more than a decade ago.

Prior to your work, what was known about how each bone gets its distinctive shape?

EZ Although the generic mechanisms of long bone development, in particular elongation, have been extensively studied, far less was known about how each bone gets its distinctive shape. Nevertheless, several intriguing discoveries have been made in recent years. Muscle force was shown to regulate appositional growth, which forms the circumferential shape of the bone (Sharir et al., 2011), and bone superstructures were shown to develop modularly from a distinct population of Sox9 and scleraxis (Scx) double-positive progenitor cells under regulation of TGFβ and BMP signalling (Blitz et al., 2009, 2013). Lastly, the relative positions of superstructures along the bone shaft was found to be determined both by bone modelling and by a unique ratio between growth rates at the two bone ends (Stern et al., 2015).

SE The process of endochondral ossification was described a long time ago. In short, it is a process in which pools of Sox9-expressing chondroprogenitors condense into discrete cartilage templates, which later will ossify into mineralized bones. Until recently, the dogma in the field was that long bones acquire their specific morphologies at the stage of cartilage condensation, with some changes occurring after ossification by bone modelling. However, studies that were done by a former graduate student, Einat Blitz, challenged this dogma and presented evidence of a secondary population of chondroprogenitors that co-express both Sox9 and Scx.These cells are specified after the primary condensation stage and their addition forms superstructures along the bone shaft and thereby contributes to the final bone morphology. This finding served as our starting point and raised the question of how the addition of the Sox9⁺/Scx⁺cells to developing bones at specific sites is controlled.

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

SE & EZ First, we show that the Sox9⁺/Scx⁺ progenitor cells contribute extensively to the formation of bone superstructures in mouse. Importantly, we show that they contribute not only to bone eminences, but also to condyles and sesamoid bones, which are auxiliary bones that are important for proper locomotion. Then, we show that the genetic programme that controls the patterning of these progenitors contains both global and regional regulatory modules, as Gli3 regulated this process globally, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 acted as either proximal or distal regulators. Finally, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in tandem. Together, these results demonstrate genetic regulation of superstructure patterning, thereby supporting the notion that long bone development is a modular process.

How do your findings contribute to the modular model of bone morphogenesis?

EZ Previously, we showed that bone superstructures originate from a dedicated pool of progenitors. Now, we expand the modular model of bone development by identifying part of the genetic programme that controls the patterning of these progenitors at specific locations along the bone. Furthermore, we show that modularity exists within the process of bone superstructure patterning, as some components of this genetic programme regulate all superstructures, whereas others regulate only proximal or distal superstructures. From an evolutionary perspective, this strategy allows changing the position of superstructures and, thereby, the locomotor abilities of the organism without rewriting the entire skeletogenic programme.

SE What allows modularity in long bone morphogenesis is the appearance of the secondary Sox9/Scx progenitors and the fact that they can be regulated independently from the sox9-expressing cells of the bone shaft. Our findings provide further evidence of the abundance and contribution of these progenitors and highlight several genetic components and programmes that regulate their patterning both globally and locally. These are missing from the present dogma of a rigid patterning programme executed by a single condensation step of a single population of progenitors, therefore changing our view of bone morphogenesis.

How do you think that Sox9⁺/Scx⁺ chondroprogenitors get to the right sites to form the superstructures?

SE & EZ Sox9⁺/Scx⁺ chondroprogenitors could get to their locations in one of two ways. The first option is their migration to specific condensation sites, where they will form superstructures. Alternatively, it is possible that the Sox9⁺/Scx⁺ chondroprogenitors are specified from cells that are already present at the designated superstructure formation sites. In the latter scenario, the mechanism would involve activation of a chondrogenic programme in selected cell subpopulations at specific spatiotemporal positions. Either way, it appears that the mechanism that decides where superstructure development should take place involves Gli3 and the Pbx and Hox genes.

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

SE It is hard to choose a specific eureka moment. I think every experiment we performed presented some advancement toward our findings. Perhaps such a moment would be when we first put together the results from our Sox9/Col2A1-CreER;tdTomato 3D reconstructions and the SOX9/ScxGFP-labelled sections. It was then that we saw clearly for the first time how abundant these progenitors were. That was a very reassuring moment that gave me the confidence to move forward with studying the patterning of superstructures.

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

SE I think that working with a model organism, and especially with mutant or transgenic lines, can sometime be a source of frustration. For example, we had to postpone the revision of two papers twice by more than 6 months because we had to revive our double-mutant colonies sufficiently to carry out additional experiments that were requested by the reviewers. Another experiment required us to cross the compound HOX11 mutants to a ScxGFP reporter to follow the Sox9⁺/Scx⁺progenitors. The experiment was straightforward but the genetic ratio was against us and, after two years of trying, we had to submit the manuscript without these results.

So what next for you after this paper – I understand you are now in the US?

SE That is correct. I am now doing my postdoctorate in Professor David Traver’s lab at the University of California, San Diego (UCSD). The main areas researched in the lab are the formation and development of the haematopoietic stem cells and their niche in both juvenile and adult zebrafish. The advantage of working with zebrafish is that their embryonic development is rapid and the developing embryos are transparent. That gives us the opportunity to follow cellular behaviours over long periods of time with high-resolution imaging. Specifically, for my projects I am working on setting up a pipeline that will use cutting-edge labelling and imaging technologies to allow me to label and follow single cells and their cellular fates and lineages. That system will allow me to study heterogeneous populations within the blood system by following individual cells within such populations.

Where will this work take the Zelzer lab?

EZ We are interested in further understanding the process of superstructure development, both upstream and downstream to the

current study. We are investigating the unique identity of the progenitor cells that give rise to superstructures, focusing on their single cell transcriptome and chromatin landscape. At the same time, we are pursuing the molecular and biomechanical signals that regulate superstructure development. These studies will shed light not only on bone morphogenesis, but also on the connection of tendons to bones during musculoskeletal assembly. To achieve these goals, we would welcome new partners. We are always looking for curious students and postdocs on the quest for self-fulfilment, who are interested in musculoskeletal biology, organ shaping or the assembly of complex systems.

In my view, the field of developmental biology is currently at a crossroads. Pushing the field back to centre stage may require the integration of other fields such as physiology, engineering and mechanobiology. The musculoskeleton is the ideal model system for such integration. An example for that is the new direction we have recently taken in studying the regulatory effect of the proprioceptive system on skeletal development and function.

The field of developmental biology is currently at a crossroads

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

EZ I like mountain biking and practicing jujutsu.

SE I have moved to San Diego with my wife and two amazing children. Coming to San Diego opened up many new experiences for all of us. The city is beautiful and there are endless family attractions, especially in the larger area of Southern California. We also like to travel and camp on long weekends. For myself, I started rock climbing during my PhD in Israel and continue to climb in San Diego. In addition, as I am in California, it didn’t take long before I took up surfing, too. It is amazing to have the opportunity to start the day by surfing among sea lions and dolphins.

(1 votes)

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Press release for a new Development paper on reprogramming in the retina.

Scientists at the Federal University of Rio de Janeiro, Brazil, in collaboration with the Max Planck Institute of Molecular Cell Biology and Genetics, Germany, discovered that a single transcription factor drives retinal progenitor cells to reacquire the potency to generate Retinal ganglion cells. The transcription factor is Klf4, which became notorious for its role in reprogramming cells towards pluripotency. The novel findings open a new door to investigate regenerative therapies for retinal diseases that cause irreversible vision loss, such as Glaucoma. The research was published in the scientific journal Development at https://dev.biologists.org/content/early/2019/08/08/dev.176586.

Ganglion cells in the retina are the source of the optic nerve and are vital for seeing as this nerve sends the messages from the retina to the brain. Their degeneration in glaucoma leads millions of people to permanent vision impairment. Current treatments at best control the progression of the disease, but recovery of normal vision require not only the prevention of cell death but also regeneration of cells and their axons.

To address the latter, Mauricio Rocha-Martins, Mariana Silveira and a team of researchers investigated both the role of Klf4 in the normal development of ganglion cells, as well as its potential to coax retinal progenitor cells to generate these neurons.

Although an endogenous role of Klf4 in retinal development could not be confirmed, overexpression of this gene in retinal progenitors of newborn rats led to a switch in cell fate. Instead of giving birth mostly to photoreceptors (which sense light) the progenitors generated retinal ganglion cells, the projection neurons of the retina. Remarkably, less than two days after Klf4 overexpression, a molecular program was activated that is similar to that responsible for the generation of ganglion cells during retinal development.

“We literally cheered the first time we noticed that, instead of positioning on the photoreceptor layer, where most of those cells should be in normal conditions, this transcription factor led the progeny of progenitor cells to move to the region of the retinal ganglion cells, and project axons toward the optic nerve,” the authors said.

Future work will focus both on improving the protocol to guarantee that these induced ganglion cells acquire mature properties, but also to direct this strategy to the glial cells of the retina, of which the ability to regenerate retinal ganglion cells was lost in mammals through evolution, but remains in fish.

“We believe there is a long way towards actual therapy, and plenty to be understood, but our data indicate that the program to generate ganglion cells can be reactivated, which may open new directions for regenerative therapies,” said the authors. “This is a starting point,” they conclude.

(5 votes)

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This post was originally posted on the MRC Weatherall Institute of Molecular Medicine’s blog

Dominic Owens, PhD student in the De Bruijn group at MRC MHU, recounts how puzzling results and a fortuitous lab meeting uncovered unexpected outcomes of CRISPR editing and changed the direction of his research.

It’s May 2018 and I’m feeling nervous. I was presenting the latest results in my PhD research project at a large joint lab meeting involving several research groups. The last few weeks of experiments hadn’t gone the way I expected, and I had gotten some odd results that were puzzling. Unknown to me, from this lab meeting, I would be embarking on a yearlong journey of despair and eventual discovery which became central to my first publication, published in Nucleic Acids Research (Owens et al. 2019, https://doi.org/10.1093/nar/gkz459).

I had been using CRISPR-Cas9 to examine the function of ‘junk DNA’. So-called ‘junk DNA’ contains no genes but instead is increasingly understood to play important roles in regulating gene expression. The enhancers I study are believed to regulate blood stem cell generation, by activating a gene called Runx1 during embryonic development. This gene is required for blood stem cell generation and is frequently mutated in human leukaemia.

In mouse cells, I had been using CRISPR to remove specific enhancers, and wanted to see what the impact on blood cell generation would be. If removing these enhancers made a difference, then I could reveal unknown factors important for blood stem cell generation.

After using CRISPR to cut out this DNA, I needed to sequence short pieces around the cut sites to determine what kind of cut took place.  As is common practice, I used PCR to amplify the DNA and Sanger sequencing to read it, over a region around 200 base pairs on either side of the target sites. After doing this, I often saw cells with what looked like both copies of the region deleted, right where we expected. However, when I tested the function of the cells, I saw inconsistent results. Even though the sequencing told me that the region was missing, some of the cells were behaving like it was still there.

Back to the lab meeting: a colleague who is very experienced with CRISPR from a different lab suggested that I could be seeing larger than expected deletions which meant that one of the PCR primer binding sites I relied on was no longer available. This would in effect prevent one copy of the region from being amplified and sequenced, and thus render PCR blind to such larger deletions. I can still remember that moment—the significance of what she said truly dawning on me. So, after the lab meeting, my lab mates and I discussed what to do next. We decided that I should look for larger deletions in all the cell lines we had made. To do this I used longer range PCR, with primers that bound to the DNA much further away from the CRISPR target sites. This way I would be able to detect any larger deletions, if they were there. And when I looked, I found them. In fact, no matter how far I looked, larger deletions were always there.

This left me desperately wanting answers to three questions: why are these larger deletions happening? Just how large can the deletions be? And can they be predicted? The only problem was: I was working in a lab of exceptional developmental biologists, not genome engineers! So, my supervisor and I reached out to two local experts – Lydia Teboul at MRC Harwell, an expert in CRISPR editing, and Peter McHugh at MRC WIMM, an expert in DNA repair. Both were generous with their time, helping me plan and perform the necessary experiments to answer the questions I was puzzled about.

By sequencing many examples of larger deletions, I found that short regions of homology (microhomologies) were commonly found in places where the cut DNA was repaired back together. This suggested that a DNA repair pathway called microhomology-mediated end-joining was a likely culprit.

Next, I used a technique called droplet digital PCR that is incredibly useful for interpreting the outcomes of CRISPR experiments. The method uses an oil in water emulsion of droplets to perform 15,000 individual PCR reactions in a single tube. This is advantageous because it allows quantification of DNA with much greater accuracy and precision than comparable techniques. Taking advantage of this accuracy, my colleagues and I mapped deletions across thousands of base pairs of DNA in populations of cells targeted with CRISPR, without biases arising from the locations of longer-range PCR primers.

Modelling the effects of various potential predictors on the distribution of deletion sizes revealed the driving forces determining how large a deletion is likely to be. I found that the frequency of larger deletions was highly dependent on CRISPR’s cutting efficiency at a target cut site and the distance from it. This means that the closer to a cut site, the more likely a region is to be lost in a deletion. This makes sense of course, but this distance-dependency shows that the microhomology sequences often associated with the repaired breakpoints themselves cannot be used to predict the size of the deletions. This is in stark contrast to shorter CRISPR deletions, which have recently (Shen et al. 2018, Nature, and Allen et al. 2018, Nature Biotechnology) been shown to be highly predictable based on sequence characteristics such as microhomologies.

The work highlights important caveats about CRISPR that I wished I had known when embarking on this project. Larger deletions occur frequently after CRISPR editing and they will be easily missed if you aren’t looking out for them. Deletions can be any size when looking at individual cells or animals, but in general are much more common within 3 kilobases from a cut site. Even though extremely large deletions are rare, given a large enough population of cells, they should be expected to occur. In the case of gene-edited somatic cells for gene therapy, for example, a single rare oncogenic larger deletion could have disastrous health outcomes for recipient patients. In the future, inhibiting microhomology-mediated end-joining could help reduce this problem. Alternatively, gene-editing approaches that avoid DNA double-strand breaks altogether may be preferable.

I must admit, I was slightly disappointed when the questions I had set out to answer during my PhD didn’t quite work out. But I have also learned valuable lessons. It is often tempting to ignore outliers or repeat experiments that just look weird. But by staying curious and motivated to understand the things that don’t quite add up, you will understand the science more deeply, and maybe find your next big breakthrough!

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We are increasing our investment in paediatric cancer research by launching two new significant funding calls. We would like to encourage proposals to investigate the following concepts amongst others-

• the basis of tumour initiation and progression
• novel models that would enhance pre-clinical research.

Please see the links below for the complete list.

The Cancer Research UK Children and Young People’s Innovation Awards calls on all scientists, from basic biology to clinical research, to propose novel and ambitious approaches to further our understanding of paediatric cancer.

The Stand up to Cancer-Cancer Research UK Paediatric New Discoveries Challenge focuses on multidisciplinary, multi-institutional, transatlantic teams that want to pursue a step change in our understanding of the drivers of paediatric cancers and the development of novel or repurposed medicines, treatment strategies or technologies

If you have any questions then please don’t hesitate to contact Sheona Scales (Cancer Research UK Paediatric Lead) sheona.scales@cancer.org.uk .

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