A postdoctoral position is available in the laboratory of Dr. Sophie Astrof at Rutgers University to study roles of cell-extracellular matrix (ECM) interactions in cardiovascular development and congenital heart disease. We have recently discovered that progenitors within the second heart field (SHF) give rise to endothelial cells composing pharyngeal arch arteries (Dev Biol 421:102–111, 2017). Projects in the lab focus on the role of ECM in regulating the development of SHF-derived progenitors into endothelial cells and their morphogenesis into blood vessels. The successful candidate will combine genetic manipulation, embryology, cell biology, and confocal imaging to study molecular mechanisms by which cell-ECM interactions and tissue microenvironment regulate cardiovascular development. Additional projects focus on the investigation of cell type-specific and cell-autonomous functions of fibronectin in development and signaling (Development 143:88-100, 2016). Interested candidates should send their CV and the names of three references to Sophie.astrof@rutgers.edu
Jeff Rasmussen tells the story behind his recent paper from the Sagasti Lab in Dev Cell.
This project began as an extension of my earlier postdoc work in Alvaro Sagasti’s lab investigating removal of axon debris following skin injuries in the larval zebrafish [1] and led me into scientific territory that I never anticipated. It is a story that would not have happened without open-mindedness, encouragement and—most importantly—help from colleagues in the fish community.
Sensory axon endings profusely innervate the skin, and skin injuries trigger axon degeneration. We previously discovered that keratinocytes are the primary phagocyte for degenerating axons in larval skin. But what cells eat axons that degenerate in the more complex adult skin?
In order to answer this question, I first needed a way to visualize sensory axons in adults. Most work in zebrafish has focused on the larval system, so markers for adults have lagged behind. Luckily, I came across a transgenic line made by a previous graduate student in Alvaro’s lab [2] that showed bright and specific expression of sensory axons in adults. The pattern of adult skin innervation revealed by this line caused this project (and my career) to take a twist.
A Striking Axon Pattern
Axons of Rohon-Beard neurons innervate most of the larval fish skin. Rohon-Beard axons in the skin rarely bundle (fasciculate). By contrast, this transgenic line revealed that axons of dorsal root ganglion (DRG) neurons, which innervate adult skin, were frequently bundled. More surprisingly, these bundles were evenly spaced across the surface of scales, which underlie the adult epidermis (Figure 1). Alvaro whole-heartedly encouraged me to dig deeper into what created these evenly spaced bundles, despite his lab having no experience working with post-embryonic stages.
Figure 1. Anatomy of adult zebrafish skin showing axons, vasculature and scale radii.
Thi’s Developmental Studies
Thi Vo, an undergraduate researcher in Alvaro’s lab, took on the challenge of analyzing post-embryonic development. Because many Rohon-Beard neurons die after only a few days of development, we expected that DRG axons would innervate the epidermis shortly thereafter. However, by staining isolated scales to visualize axons, Thi found that the axon bundles only appeared during late juvenile stages. So Thi next analyzed mutants lacking DRG neurons and found that the bundles never formed, proving that they were indeed DRG axons.
Thi also stained scales with phalloidin to visualize cell morphology and found that a set of elongated cells apparently presaged the path of the axon bundles. What could these cells be?
Lindsey’s Schwann cells
We initially hypothesized that the elongated cells were Schwann cells, the glia of the peripheral nervous system. Schwann cells are neural-crest derived, and Shannon Fisher’s group recently made a neural crest lineage reporter [3], which we learned was growing in Gage Crump’s lab at USC. To determine if the line labeled Schwann cells in adults, I made a trip across town to visit Lindsey Barske, a postdoc in the Crump lab, who was working with the neural crest reporters. One of Lindsey’s lines labeled Schwann cells coating the axon bundles, showing that they are nerves and giving us a key reagent to visualize their development. But when Thi and I analyzed the timing of Schwann cell migration, they appeared too late to pioneer the path of the DRG bundles, ruling out this hypothesis.
Michael’s Vessels
After hearing about our work at a Southern California Zebrafish Meeting, Michael Harrison, a postdoc in Ellen Lien’s lab working on heart and vascular development, suggested we analyze several of his chemokine mutants. Thi and I took a bus trip down Sunset Blvd to CHLA to collect scales from these mutants. Although this effort was ultimately fruitless, as luck would have it, Michael’s mutants expressed multiple transgenic reporters. One of these transgenes was fli1a:EGFP (a vascular reporter made by Brant Weinstein’s group) [4], which revealed that the axon bundles tightly associated with blood vessels (Figure 1). This was unexpected because we had initially ruled out a vascular contribution based on analysis of a vessel reporter not as broadly expressed as fli1a.
Intriguingly, axons and vessels also tightly associate in mammalian skin and axons promote vascular remodeling and arterial differentiation in mouse [5]. Were blood vessels the elongated cells that arose early in scale development? No, since we found that blood vessels only appeared along mature scales, once animals reached adulthood. What about the converse: did axons pattern the vessels? By again analyzing mutants lacking sensory neurons, we found that blood vessels appeared normal. Thus, in contrast to mammals, skin nerves and vasculature are independently patterned in fish. This was an important finding but, once again, the identity of the pioneering cells remained elusive.
Sandeep’s Osteoblasts
The scale surface is made from bone and contains a number of striking patterns. Remarkably, we noticed that the axon bundles and vessels aligned with scale radii, grooves in the bone that radiate from the scale center (Figure 1). Could osteoblasts, bone-forming cells, or osteoclasts, bone-degrading cells, guide axons and vessels? We first examined osteoclasts but found no evidence that they form the radii. Next, I made another crosstown trip to the Crump lab—this time with the help of Sandeep Paul—to look at osteoblast reporters. Sandeep’s lines showed that osteoblasts line mature radii, as suggested by pioneering ultrastructural studies [6, 7]. Imaging osteoblasts early in scale development revealed that they create the radial paths by polarized migration.
To test if osteoblasts promote skin innervation during regeneration, we used an inducible osteoblast ablation line made by Ken Poss’ group [8] and found that blocking scale regeneration by osteoblast ablation resulted in a reduction of axon density. To test if scale development similarly promoted innervation, we analyzed mutants that prevent scale development [9, 10]—provided as part of a “scale care package” by Matt Harris’ lab. These mutants had reduced skin innervation and vascularization, showing that scales are also required during ontogeny.
Figure 2. Intubation allows extended live-cell imaging of scale development and regeneration.
Full Scale Ahead
Although I never planned to work on scales as a model system, I am really excited about the potential for these mini-organs to reveal the cellular and molecular basis for cell type patterning during skin development and repair—questions that I will be pursuing in my newly formed research group at the University of Washington. Scales are evolutionarily related to other types of specialized skin appendages like feathers and hair. Thus, studies of fish scales may reveal general mechanisms for coupling organ maturation and growth to skin patterning. Scales may also yield insights into bone-nerve interactions that occur in diverse tissues, like antlers, teeth and long bones. An increasing number of genetic tools, together with advances in live-cell imaging of post-embryonic stages (Figure 2), suggest the future is bright for a resurgence of scales as a model system.
1 PhD Position : Characterization of Hedgehog morphogens in vitro and in vivo
Hedgehog (Hh) morphogens play important roles in development and cancer, but their mode of extracellular transport to target cells is only poorly understood. Thus, we aim at the characterization of various unusual posttranslational regulatory mechanisms in Hh biology, such as Hh multimerization on the surface of secreting cells via structural and biochemical analysis of Hh clusters, and the unusual mode of Hh transport and gradient formation.
We use a wide range of biochemical methods, such as recombinant protein production and chromatographic/functional characterization of proteins and Heparan sulfate-proteoglycans (HSPGs), determination of Shh/HSPG binding and in vivo testing of any obtained models of protein association and transport (In Drosophila melanogaster).
We invite applications from highly qualified and motivated students of any nationality. The applicant will hold a Masters degree (Biology, Chemistry, Biotechnology, Pharmacy or Biochemistry) and has gained first biochemical research experience. Experience in Drosophila experimentation is highly welcome, but not required. The successful applicant will find strong support within the excellent interdisciplinary environment of the SFB1348 of the University of Muenster.
CENTURI is recruiting up to 10 highly motivated postdoctoral fellows to work in an interdisciplinary life science environment. The available postdoctoral projects will be advertised online, with more projects being added to our website up until November 15.
CENTURI postdocs will have the opportunity to develop their project with more than one research group, bridging biology to other disciplines.
Candidates will have a background in any of the following fields: cell or developmental biology, immunology, neurobiology, biophysics, theoretical physics, computer science, bioinformatics, applied mathematics or engineering.
Candidates can either apply to one of the advertised CENTURI projects or submit their own project, providing that they meet the application criteria and that their application is supported by at least one host lab. These projects should be submitted to: postdocproject@centuri-livingsystems.org
Deadline for application and project submission: November 30, 2018
About CENTURI
Mainly located on the Luminy science campus (Marseille France), the Turing Centre for Living Systems is an interdisciplinary project federating a growing community of biologists, physicists, mathematicians, computer scientists and engineers in 15 research institutes.
The project focuses on 3 missions: Research, Education and Engineering
Stem cells are defined by the dual capacity to self-renew and to differentiate. These properties sustain homeostatic cell turnover in adult tissues and enable repair and regeneration throughout the lifetime of the organism. In contrast, pluripotent stem cells are generated in the laboratory from early embryos or by molecular reprogramming. They have the capacity to make any somatic cell type, including tissue stem cells.
Stem cell biology aims to identify and characterise which cells are true stem cells, and to elucidate the physiological, cellular and molecular mechanisms that govern self-renewal, fate specification and differentiation. This research should provide new foundations for biomedical discovery, biotechnological and biopharmaceutical exploitation, and clinical applications in regenerative medicine.
Cambridge Stem Cell Community
The University of Cambridge is exceptional in the depth and diversity of its research in Stem Cell Biology, and has a dynamic and interactive research community that is ranked amongst the foremost in the world. By bringing together members of both the Schools of Biology and Medicine, this four year PhD programme will enable you to take advantage of the strength and breadth of stem cell research available in Cambridge. Choose from over 50 participating host laboratories using a range of experimental approaches and organisms.
Programme Outline
During the first year students will:
Perform laboratory rotations in three different participating groups working on both basic and translational stem cell biology.
Study fundamental aspects of Stem Cell Biology through a series of teaching modules led by leaders in the field.
Learn a variety of techniques, such as advanced imaging, flow cytometry, and management of complex data sets.
Students are expected to choose a laboratory for their thesis research by June 2020, and will then write a research proposal to be assessed for the MRes Degree in Stem Cell Biology. This assessment will also be used to determine whether students continue on to a 3-year PhD.
Physical Biology of Stem Cells
Incorporated into the ‘Stem Cell Biology’ Programme, opportunities are available specifically for candidates with a Physical, Computational or Mathematical Sciences background, wanting to apply their training to aspects of Stem Cell Biology *.
Great inroads have been made towards understanding how stem cells generate tissue and sustain cell turnover, most of which have been made by studying the biochemistry of stem cells. Less is known of their function across scales – from molecules to tissue – or interaction with their physical environment. We aim to identify the importance of physical, chemical, mathematical and engineering considerations in stem cell functionality. This could include mathematical modelling, engineering controlled environments to control stem cell function, single molecule approaches to study molecular interactions, systems biology, or investigating stem cell’s response to forces in its environment.
Eligibility
We welcome applications from those who hold (or expect to receive) a relevant first degree at the highest level (minimum of a UK II.i Honours Degreeor equivalent). You must have a passion for scientific research.
Stem Cell Biology and Medicine Programme (funding by Wellcome)
We welcome applications from EU and non-EU candidates. The Wellcome Trust provide full funding at the ‘Home/EU’ rate. Funding does not include overseas fees, so non-EU applicants will need to find alternative funding sources to cover these.
‘Physical Biology of Stem Cells’ Programme (funding by the MRC)
We welcome applications from UK/EU candidates, with a Physical Sciences, Mathematical or Computational Sciences background. *The Medical Research Council provide full funding for UK applicants only. Applicants from EU countries other than the UK, are generally eligible for a fees-only award. Please check your eligibility status at https://www.mrc.ac.uk/skills-careers/studentships/studentship-guidance/student-eligibility-requirements/ before applying.
There is a vast amount of information known about how some animals pattern their bodies into repeated segments, especially in the fruit-fly Drosophila melanogaster. However, when compared to other arthropods, there are several characteristics that are derived in the fruit fly. It has a very short development time, a syncytium at the blastoderm stage allowing molecules free passage throughout the embryo and the segments are generated almost simultaneously. This is called the “long germ band” type of arthropod development.
While the fruit fly undergoes “long germ band” development, most arthropods belong to the “short germ band” type, in which the head and a small portion of the trunk are generated after gastrulation, but posterior segments are then added one by one from the so called segment addition zone (SAZ). An earlier article on the Node previously outlined the reasons the spider is an excellent study organism (https://thenode.biologists.com/day-life-spider-lab/lablife/) – but we want to highlight a recent discovery that builds to this earlier story: some chelicerates have undergone a whole genome duplication during their evolutionary history (Schwager et al., 2017; Leite et al., 2018). This genome duplication event has allowed some of the conserved sets of genes to retain duplicated copies, potentially having huge implications for the regulation and evolution of developmental processes in these animals – including segmentation.
What do we know about segmentation in the common house spider Parasteatoda tepidariorum? A remarkable discovery in this species was that the Delta-Notch and Wnt signaling pathways were involved in the differentiation of the cells of the SAZ. These findings shifted the view of canonical segmentation in arthropods, since these two pathways are not involved in long germ development of the fruit fly (for further information, see Oda et al., 2007 and Schonauer et al., 2016).
Strikingly, we found in 2017 that another conserved metazoan gene family has retained duplicated copies in Parasteatoda (Paese et al., 2017), the Sox genes (Sry-related HMG Box). In this study, we isolated 14 Sox genes, analysed their expression patterns at different stages of embryonic development (Paese et al., 2017) and studied their functional role with RNA interference (Paese et al., 2018). Interestingly, some of these genes have no detectable expression during embryonic development, and as noted in other studies, Sox genes have a high degree of redundancy (i.e. genes with conserved motifs that therefore can act in the place of the missing gene) and we only observed an obvious phenotype in one out of fourteen genes, a SoxB gene – Sox21b-1. (Expression pattern in Figure 1). This is an example of one of the genes that have undergone a duplication – in which each copy is dispersed in the genome of the spider.
Figure 1
Even though the gene’s name may not be the most memorable when compared to some genes in the fruit-fly, the phenotype when Sox21b-1 is knocked down is very unforgettable. Moreover, this gene has an expression pattern similar to what is seen with a related gene, Dichaete, in other arthropods – which has no detectable expression during spider embryogenesis (for more info, see Paese et al., 2017). For a quick spoiler: we first thought that Sox21b-1 had acquired Dichate function in this spider species. Wouldn’t that be amazing? And how would we be sure of that? Let’s do some functional analysis!
When we injected double-stranded RNA in the opisthosoma of a female spider, we obtained phenotypes of different penetrance. During one rainy night in Oxford, I was analyzing a dish with embryos that I removed from a cocoon that was deposited by a Sox21-b1 dsRNA injected female. Using a stereomicroscope, I was only able to identify the head region and a very strange single pair of appendages growing from where six pairs should be growing. At a first glance, I was very excited about finding a new phenotype, but kept searching for other embryos with the same pattern in different cocoons. Sadly, in this first injection round, only two injected females survived – however, a significant proportion of embryos displayed a phenotype.
I injected another seven spiders and the phenotypes matched the first round of injections (Figure 2 for the phenotypes). In class I phenotypes, most embryos displayed only the first three pairs of appendages (namely chelicerae, pedipalps and leg bearing segment 1 – L1) instead of six pairs, whereas class II lack the L1 segment and class III did not develop further after gastrulation. Much more interesting than lacking all the leg-bearing segments was that this anterior region was able to differentiate even in the absence of a developing SAZ! These findings are further evidence for the hypothesis that the short germ arthropods mechanism for anterior and posterior segmentation are decoupled.
Figure 2
Knowing this, we started analyzing which mechanisms were affected by the knockdown of this Sox gene. First, we assayed the expression of known cumulus formation and migration genes: decapentaplegic and Ets4. The expression of both genes was normal (Figure 3 of the paper – A to D). Then the next targets to be evaluated were genes involved with secondary layer formation (forkhead) and head patterning (hedgehog). Whereas the expression of the latter was normal, forkhead expression was completely lost. This information led us to analyse determinants of mesoderm and endoderm formation in older embryos and we indeed observed an effect on those two layers in the Sox21b-1 phenotype. However, we cannot be sure if this is a direct effect due to the lack of Sox21b-1 or caused by the overall lower number of cells, which has to be investigated further.
Figure 3
As mentioned before, the spider patterns its segments not following the Drosophila canonical mode, but instead through control by Delta-Notch and Wnt signaling, regulating pair-rule gene expression via caudal. Thus, we looked into the effect on expression of the known genes that are related to this mechanism in the spider. Interestingly, the expression of caudal, delta, hairy and wnt8 is lost in the SAZ, but we could still see the expression of the segment markers engrailed and hedgehog in the knockdown embryos. In summary, this means that this Sox gene is upstream of Wnt and Delta/Notch and this is additional evidence for the independence of anterior and posterior segmentation in the spider (Figure 4).
Figure 4
In conclusion, our study adds an important piece towards understanding segmentation in short germ arthropods and the effects of a whole genome duplication on developmental processes (Figure 5).
Figure 5
We might be far from a complete picture of how segmentation is regulated in spiders, but it is indeed exciting that there are many more pieces of the puzzle to be solved!
Supplementary References:
Leite, D. J., Baudouin-Gonzalez, L., Iwasaki-Yokozawa, S., Lozano-Fernandez, J., Turetzek, N., Akiyama-Oda, Y., Prpic, N. M., Pisani, D., Oda, H., Sharma, P. P., McGregor, A. P. (2018). Homeobox gene duplication and divergence in arachnids. Mol Biol Evol.
We invite students to apply for a PhD position in computational morphogenesis at the Gene Expression and Morphogenesis Unit (http://cellcollectives.com/) at the Andalusian Centre for Developmental Biology (http://www.cabd.es), in the charming city of Seville, southern Spain. The candidate will be co-supervised by Dr. Luciano Marcon and Dr. Juan R. Martinez-Moraes and will work in the field of developmental biology and organoid biology.
We are looking for a talented and motivated student with a computational background (computer science, physics, engineering or math) to investigate the morphogenetic movements that shape multicellular organism. The successful candidate will develop software to simulate the gene regulatory networks and cellular movements that underlie morphogenesis of the early embryo. He/she will use the software to fit experimental data and identify the cellular mechanisms that underlie gastrulation and axial elongation in fish and in embrioids derived from mouse embryonic stem cells.
Good programming skills with standard languages (e.g. Java, C++, Python) and fluency in English are required. Experience in finite element simulation (or similar techniques) of gene regulatory networks and tissue mechanical forces is desirable. Knowledge on parallel programming with GPUs (i.e. CUDA and OpenCL) will also be valued positively.
The application deadline is on the 29th October! Interested write as soon as possible to lmarcon@upo.es or jrmarmor@upo.es
The Company of Biologists’ Workshopsprovide leading experts and early career scientists from a diverse range of scientific backgrounds with a stimulating environment for the cross fertilization of interdisciplinary ideas.
The April 2019 Workshop looks a treat for fans of developmental gene regulation. Co-organised by Benoit Bruneau and Joanna Wysocka, ‘Chromatin-based regulation of development‘ will bring together scientists who can or would like to bridge chromatin-level gene regulation, quantitative approaches and developmental biology. It will be held at the beautiful Wiston House, a 16th century Grade I listed building at the foot of the South Downs in West Sussex, England.
The Company of Biologists are offering around 10 funded places for early-career researchers to attend our Workshops along with the 20 speakers. It’s an incredible opportunity for young researchers – find out more here:
Group Leader in Biological Imaging (based at Monash University, Melbourne , Australia)
Expressions of interest are invited from scientists pushing technological boundaries in biological imaging, who seek to make major discoveries running their own independent research program. This EMBL Australia Group Leader position is hosted within the Biomedicine Discovery Institute at Monash University, Australia.
The successful applicant will be housed within the Department of Anatomy and Developmental Biology, one of the five Monash BDI departments. Areas of research focus include renal, lung and reproductive biology, inflammation and cell death, epigenetics, embryology, cancer, stem cells, and cellular reprogramming.
Candidates with a background in developing and/or applying cutting edge imaging technology to address major biological questions are invited to apply. The University particularly welcomes applicants with significant experience and interest in lightsheet microscopy, super-resolution microscopy, and correlative light and electron microscopy.
Welcome to our monthly summary of developmental biology (and related) preLights.
It’s been almost eight months since the launch of preLights, and we are very excited about the hugely positive response from the community so far and preLights’ rapid growth – we recently reached two hundred preprint highlights! To further grow this service, we will be announcing a call for recruiting new team members next month, so watch this space if you would like to join us as a preLighter.
The preLights posts from September turned out to be very diverse: from worms and insects to plants and fish, and also plenty of exciting novel techniques. We hope you enjoy this selection!
From Development to Disease
The early C. elegans embryo is a powerful model to tackle questions like how polarity initially emerges in a developing organism. Giuliana Clemente covered two studies that revealed a new role for Aurora A in symmetry breaking and polarity establishment in the worm.
Sarah Bowling discussed why cells die when they are not in the “right place” and highlighted a preprint showing that disrupted EGFR signaling, rather than a fitness recognition event through cell competition, is responsible. The preprint authors gave further insight into the study (check out the author’s response section) and pointed out that the simplest explanation to their question proved to be the correct one.
Erik Clark’s preLight showcased a conserved role for a posttranslational module (involving the micropeptide millepattes) in segmentation across many insect species, but a lack of segmentation phenotypes in Drosophila mutants. While they may not be good models for insect segmentation, Drosophila has provided numerous mechanistic insights into cell and tissue dynamics through the study of its developing egg chamber. Sundar Naganathan highlighted a preprint showing that in this model, 3D tissue elongation occurs through a mechanochemical signaling pathway that translates the external stiffness gradient into altered cadherin trafficking. Amanda Haage’s preLight also featured some fine mechanobiology in cancer cells, showing that actomyosin contractility regulates the activity of PKA, which is important for mechanically guided cell migration (durotaxis).
Figure taken from preprint by Ray et al.; highlighted by Erik Clark
From the plant development field, Martin Balcerowicz wrote about a study showing that two new proteins (Sol1 and Sol2) regulate fate transitions and cell divisions during Arabidopsis stomatal development.
Moving to zebrafish, Alberto Rosello-Diez featured a novel mechanism that initiates regeneration upon injury: in response to ROS signaling, Hegdehog-expressing cells are repositioned to create a new signaling center. Andreas van Impel reviewed zebrafish vasculature development and how the 50:50 balance of intersegmental arteries and veins is regulated by a flow-dependent mechanism. Excessive blood vessel formation, on the other hand, is a hallmark of cancer; Coert Margadant highlighted a promising strategy to inhibit blood vessel formation by blocking recycling of phosphoinositide lipids.
Several preprints harnessed the power of single-cell transcriptomics to gain insight into neural development and neurodegenerative disease. Zheng-Shan Chong covered a study exploring microglial development through scRNA-seq, which discovered a subset of postnatal neural cells resembling degenerative disease-associated microglia (DAM). Yen-Chung Chen preLighted two preprints looking at why not all neuron types are equally vulnerable in neurodegenerative diseases. Applying new transcriptomic techniques, LCM-seq and Axon-seq, potential protective mechanisms utilized by resistant somatic neurons were found. Finally, Carmen Adriaens’ preLight also dealt with high-throughput sequencing (promoter capture Hi-C), and explained the links between SNP-s associated with type 2 diabetes and chromatin architecture of human pancreatic islet cells.
Tools & Technologies
It’s difficult to imagine where the developmental biology field would be today without in situ hybridization techniques that detect mRNA localization. This month, Yen-Chung Chen highlighted work that developed a new fluorescent in situ hybridization method termed SABER, which is more sensitive, cheaper and quicker compared to previous FISH methods. To look at fluctuations in mRNA levels and whether these persist over multiple cell divisions, the preprint covered by Leighton Daigh combined Luria and Delbrück’s fluctuation analysis with population based mRNA sequencing (this new method was termed MemorySeq).
Figures taken from preprint by Kishi et al.; preLighted by Yen-Chung Chen
The number of preprints dealing with CRISPR technology is rapidly growing, but there is still a lot to be understood regarding the targeting and editing accuracy and efficacy in this system. Here, Rob Hynds preLighted a study that uncovered some general principles of CRISPR gene editing by analyzing a large set of indel profiles.
Jacky Goetz wrote about a new method that is able to quantify mechanical stress in vivo by injecting polyacrylamide beads into living embryos and analyzing their deformations. Finally, one of the favorites from this month was Samantha Seah’s preLight that discussed a new way of visualizing data using raincloud plots.
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