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Most downloaded bioRxiv papers, all time

in category developmental biology

1,955 results found. For more information, click each entry to expand.

21: Decoding the development of the blood and immune systems during human fetal liver haematopoiesis
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Posted to bioRxiv 31 May 2019

Decoding the development of the blood and immune systems during human fetal liver haematopoiesis
2,400 downloads developmental biology

Dorin-Mirel Popescu, Rachel A. Botting, Emily Stephenson, Kile Green, Laura Jardine, Emily F Calderbank, Mirjana Efremova, Meghan Acres, Daniel Maunder, Peter Vegh, Issac Goh, Yorick Gitton, Jongeun Park, Krzysztof Polanski, Roser Vento-Tormo, Zhichao Miao, Rachel Rowell, David McDonald, James Fletcher, David Dixon, Elizabeth Poyner, Gary Reynolds, Michael Mather, Corina Moldovan, Lira Mamanova, Frankie Greig, Matthew Young, Kerstin Meyer, Steven Lisgo, Jaume Bacardit, Andrew Fuller, Ben Millar, Barbara Innes, Susan Lindsay, Michael J T Stubbington, Monika S. Kowalczyk, Bo Li, Orr Ashenbrg, Marcin Tabaka, Danielle Dionne, Timothy L. Tickle, Michal Slyper, Orit Rozenblatt-Rosen, Andrew Filby, Alexandra-Chloe Villani, Anindita Roy, Aviv Regev, Alain Chedotal, Irene Roberts, Berthold Göttgens, Elisa Laurenti, Sam Behjati, Sarah A Teichmann, Muzlifah Haniffa

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

22: Guided self-organization recapitulates tissue architecture in a bioengineered brain organoid model
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Posted to bioRxiv 19 Apr 2016

Guided self-organization recapitulates tissue architecture in a bioengineered brain organoid model
2,391 downloads developmental biology

Madeline A Lancaster, Nina S Corsini, Thomas R Burkard, Juergen A. Knoblich

Recently emerging methodology for generating human tissues in vitro has the potential to revolutionize drug discovery and disease research. Currently, three-dimensional cell culture models either rely on the pronounced ability of mammalian cells to self organize in vitro1-6, or use bioengineered constructs to arrange cells in an organ-like configuration7,8. While self-organizing organoids can recapitulate developmental events at a remarkable level of detail, bioengineered constructs excel at reproducibly generating tissue of a desired architecture. Here, we combine these two approaches to reproducibly generate micropatterned human forebrain tissue while maintaining its self-organizing capacity. We utilize poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a scaffold to generate elongated embryoid bodies and demonstrate that this influences tissue identity. Micropatterned engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, we reconstitute the basement membrane at later stages leading to characteristic cortical tissue architecture including formation of a polarized cortical plate and radial units. enCORs provide the first in vitro system for modelling the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. We demonstrate their utility by modelling teratogenic effects of ethanol and show that defects in leading process formation may be responsible for the neuronal migration deficits in fetal alcohol syndrome. Our data demonstrate that combining 3D cell culture with bioengineering can significantly enhance tissue identity and architecture, and establish organoid models for teratogenic compounds.

23: Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure
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Posted to bioRxiv 13 Jun 2018

Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure
2,352 downloads developmental biology

Wouter Masselink, Daniel Reumann, Prayag Murawala, Pawel Pasierbek, Yuka Taniguchi, Juergen A. Knoblich, Elly M Tanaka

Turbidity and opaqueness are inherent properties of tissues which limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here we describe 2Eci (2nd generation Ethyl cinnamate based clearing method) which can be used to clear a wide range of tissues, including cerebral organoids, Drosophila melanogaster, zebrafish, axolotl, and Xenopus laevis in as little as 1-5 days while preserving a broad range of fluorescence proteins including GFP, mCherry, Brainbow, and alexa-fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method will open up clearing to a much broader group of researchers, due to its broad applicability, ease of use, and non-toxic nature of Ethyl cinnamate.

24: Linguistically deprived children: meta-analysis of published research underlines the importance of early syntactic language use for normal brain development
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Posted to bioRxiv 21 Jul 2017

Linguistically deprived children: meta-analysis of published research underlines the importance of early syntactic language use for normal brain development
2,341 downloads developmental biology

Andrey Vyshedskiy, Mahapatra Shreyas, Rita Dunn

We analyzed all published reports of individuals not exposed to syntactic language until puberty: two feral children, who grew up without hearing any language, and eight deaf linguistic isolates, who grew up communicating to their families using homesign or kitchensign, a system of gestures which allows them to communicate simple commands but lacks much in the way of syntax. A common observation in these individuals is the lifelong difficulty understanding syntax and spatial prepositions, even after many years of rehabilitation. This debilitating condition stands in stark contrast to linguistic isolates' performance on memory as well as semantic tests: they could easily remember hundreds of newly learned words and identify previously seen objects by name. The lack of syntactic language comprehension in linguistic isolates may stem from inability to understand words and/or grammar or inability to mentally synthesize known objects into novel configurations. We have previously shown that purposeful construction of novel mental images is the function of the lateral prefrontal cortex (LPFC) ability to dynamically control posterior cortex neurons [1]. Here we have ranked all tests performed on linguistic isolates by their reliance on the LPFC control of the posterior cortex: a) the amount of posterior cortex territory that needs to be recruited by the LPFC and b) the number of disparate objects that have to be combined together by the LPFC in order to answer the test question. According to our analysis, linguistic isolates performed well in all tests that did not involve the LPFC control of the posterior cortex, showed decreasing scores in tests that involved greater recruitment of the posterior cortex by the LPFC, and failed in tests that involved greatest recruitment of posterior cortex necessary for mental synthesis of multiple objects. This pattern is consistent with inadequate frontoposterior connections in linguistic isolates. We discuss implications of these findings for the importance of early syntactic language exposure in formation of frontoposterior connections.

25: Stem cell differentiation trajectories in Hydra resolved at single-cell resolution
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Posted to bioRxiv 03 Nov 2018

Stem cell differentiation trajectories in Hydra resolved at single-cell resolution
2,243 downloads developmental biology

Stefan Siebert, Jeffrey A. Farrell, Jack F. Cazet, Yashodara Abeykoon, Abby S. Primack, Christine E Schnitzler, Celina E. Juliano

The adult Hydra polyp continuously renews all of its cells using three separate stem cell populations, but the genetic pathways enabling homeostatic tissue maintenance are not well understood. We used Drop-seq to sequence transcriptomes of 24,985 single Hydra cells and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified the transcription factors expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. We unexpectedly found that neuron and gland cell differentiation transits through a common progenitor state, suggesting a shared evolutionary history for these secretory cell types. Finally, we have built the first gene expression map of the Hydra nervous system. By producing a comprehensive molecular description of the adult Hydra polyp, we have generated a resource for addressing fundamental questions regarding the evolution of developmental processes and nervous system function.

26: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development
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Posted to bioRxiv 27 Jun 2019

Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development
2,234 downloads developmental biology

Sabina Kanton, Michael James Boyle, Zhisong He, Malgorzata Santel, Anne Weigert, Fatima Sanchis Calleja, Leila Sidow, Jonas Fleck, Patricia Guijarro, Dingding Han, Zhengzong Qian, Michael Heide, Wieland Huttner, Philipp Khaitovich, Svante Pääbo, Barbara Treutlein, J. Gray Camp

The human brain has changed dramatically since humans diverged from our closest living relatives, chimpanzees and the other great apes. However, the genetic and developmental programs underlying this divergence are not fully understood. Here, we have analyzed stem cell-derived cerebral organoids using single-cell transcriptomics (scRNA-seq) and accessible chromatin profiling (scATAC-seq) to explore gene regulatory changes that are specific to humans. We first analyze cell composition and reconstruct differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We find that brain region composition varies in organoids from different iPSC lines, yet regional gene expression patterns are largely reproducible across individuals. We then analyze chimpanzee and macaque cerebral organoids and find that human neuronal development proceeds at a delayed pace relative to the other two primates. Through pseudotemporal alignment of differentiation paths, we identify human-specific gene expression resolved to distinct cell states along progenitor to neuron lineages in the cortex. We find that chromatin accessibility is dynamic during cortex development, and identify instances of accessibility divergence between human and chimpanzee that correlate with human-specific gene expression and genetic change. Finally, we map human-specific expression in adult prefrontal cortex using single-nucleus RNA-seq and find developmental differences that persist into adulthood, as well as cell state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene regulatory features that are unique to humans.

27: A single cell transcriptional roadmap for cardiopharyngeal fate diversification
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Posted to bioRxiv 15 Jun 2017

A single cell transcriptional roadmap for cardiopharyngeal fate diversification
2,206 downloads developmental biology

Wei Wang, Xiang Niu, Tim Stuart, Estelle Jullian, William Mauck, Robert G. Kelly, Rahul Satija, Lionel Christiaen

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programs and mechanisms underlying cardiopharyngeal multipotency and heart vs. head muscle fate choices remain elusive. Here, we used single cell genomics in the simple chordate model Ciona, to reconstruct developmental trajectories forming first and second heart lineages, and pharyngeal muscle precursors, and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signaling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac program, shared by the first and second lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage program, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.

28: High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney
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Posted to bioRxiv 16 Dec 2017

High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney
2,195 downloads developmental biology

Alexander N Combes, Belinda Phipson, Luke Zappia, Kynan T Lawlor, Pei Xuan Er, Alicia Oshlack, Melissa H. Little

Recent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of off target populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

29: Zika Fetal Neuropathogenesis: Etiology of a Viral Syndrome
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Posted to bioRxiv 29 Apr 2016

Zika Fetal Neuropathogenesis: Etiology of a Viral Syndrome
2,170 downloads developmental biology

Zachary A. Klase, Svetlana Khakhina, Adriano De Bernardi Schneider, Michael V Callahan, Jill Glasspool-Malone, Robert W Malone

The ongoing Zika Virus epidemic in the Americas, and the observed association with both fetal abnormalities (primary microcephaly) and adult autoimmune pathology (Guillain-Barre syndrome) has brought attention to this neglected pathogen. While initial case studies generated significant interest in the Zika virus outbreak, larger prospective epidemiology and basic virology studies examining the mechanisms of Zika viral infection and associated pathophysiology are only now starting to be published. In this review, we analyze Zika fetal neuropathogenesis from a comparative pathology perspective, using the historic metaphor of TORCH viral pathogenesis to provide context. By drawing parallels to other viral infections of the fetus, we identify common themes and mechanisms that may illuminate the observed pathology. The existing data on the susceptibility of various cells to both Zika and other flavivirus infections are summarized. Finally, we highlight relevant aspects of the known molecular mechanisms of flavivirus replication.

30: Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord
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Posted to bioRxiv 16 Nov 2018

Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord
2,144 downloads developmental biology

Julien Delile, Teresa Rayon, Manuela Melchionda, Amelia Edwards, James Briscoe, Andreas Sagner

The coordinated spatial and temporal regulation of gene expression in the vertebrate neural tube determines the identity of neural progenitors and the function and physiology of the neurons they generate. Progress has been made deciphering the gene regulatory programmes responsible for this process, however, the complexity of the tissue has hampered the systematic analysis of the network and the underlying mechanisms. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions of the developing mouse neural tube between embryonic days (e)9.5-e13.5. We confirmed the data accurately recapitulates neural tube development, allowing us to identify new markers for specific progenitor and neuronal populations. In addition, the analysis highlighted a previously underappreciated temporal component to the mechanisms generating neuronal diversity and revealed common features in the sequence of transcriptional events that lead to the differentiation of specific neuronal subtypes. Together the data offer insight into the mechanisms responsible for neuronal specification and provide a compendium of gene expression for classifying spinal cord cell types that will support future studies of neural tube development, function, and disease.

31: Interplay of TGFb superfamily members governs optic fissure closure
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Posted to bioRxiv 24 Oct 2014

Interplay of TGFb superfamily members governs optic fissure closure
2,064 downloads developmental biology

Stephan Heermann, Priska Eckert, Juan L Mateo, Eleni Roussa, Belal Rahhal, Aimee Zuniga, Kerstin Krieglstein, Jochim Wittbrodt

The optic fissure is a gap in the developing vertebrate eye and must be closed as development proceeds. A persisting optic fissure is referred to as coloboma, a major cause for blindness in children. Multiple factors have been linked to coloboma formation, however, the actual process of fissure closure is only poorly understood. Based on our findings we propose an important role of TGFb signaling for optic fissure closure. We show active TGFb signaling in the fissure margins, analyzed by a new TGFb signaling reporter zebrafish. We found BMP antagonists regulated by TGFb. These antagonists we also found expressed in the fissure margins. Finally we show a coloboma phenotype in a TGFb KO mouse. Microarray data analysis indicates intense TGFb dependent remodeling of the extracellular matrix (ECM) during optic fissure closure. We propose that TGFb is driving optic fissure closure by ECM remodeling. As previously shown, inhibition of BMP signaling is important for such TGFb dependent ECM remodeling. We show that this is achieved by the regulation of BMP antagonists, expressed in the optic fissure margins.

32: Single-cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development
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Posted to bioRxiv 07 Aug 2018

Single-cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development
2,049 downloads developmental biology

Thorsten Boroviak, Giuliano Giuseppe G Stirparo, Sabine Dietmann, Irene Hernando-Herraez, Hisham Mohammed, Wolf Reik, Austin Smith, Erika Sasaki, Jennifer Nichols, Paul Bertone

The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single-cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single-cell RNA-seq. Zygotic genome activation correlates with the presence of Polycomb Repressive Complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species-, stage- and lineage-specific. The pluripotency network in the primate epiblast lacks certain regulators operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and nonhuman primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development and underscores the molecular adaptability of early mammalian embryogenesis.

33: Interactions between Nodal and Wnt signalling Drive Robust Symmetry Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)
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Posted to bioRxiv 04 May 2016

Interactions between Nodal and Wnt signalling Drive Robust Symmetry Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)
2,016 downloads developmental biology

David Andrew Turner, C.R. Glodowski, L. Alonso-Crisostomo, Peter Baillie-Johnson, P.C. Hayward, J. Collignon, C. Gustavsen, P. Serup, C. Schröter, A. Martinez Arias

Generation of asymmetry within the early embryo is a critical step in the establishment of the three body axes, providing a reference for the patterning of the organism. To study the establishment of asymmetry and the development of the anteroposterior axis (AP) in culture, we utilised our Gastruloid model system. Gastruloids, highly reproducible embryonic organoids formed from aggregates of mouse embryonic stem cells, display symmetry-breaking, polarised gene expression and axial development, mirroring the processes on a time-scale similar to that of the mouse embyro. Using Gastruloids formed from mouse ESCs containing reporters for Wnt, FGF and Nodal signalling, we were able to quantitatively assess the contribution of these signalling pathways to the establishment of asymmetry through single time-point and live-cell fluorescence microscopy. During the first 24-48h of culture, interactions between the Wnt/β-Catenin and Nodal/TGFβ signalling pathways promote the initial symmetry-breaking event, manifested through polarised Brachyury (T/Bra) expression. Neither BMP nor FGF signalling is required for the establishment of asymmetry, however Wnt signalling is essential for the amplification and stability of the initial patterning event. Additionally, low, endogenous levels of FGF (24-48h) has a role in the amplification of the established pattern at later time-points. Our results confirm that Gastruloids behave like epiblast cells in the embryo, leading us to translate the processes and signalling involved in pattern formation of Gastruloids in culture to the development of the embryo, firmly establishing Gastruloids as a highly reproducible, robust model system for studying cell fate decisions and early pattern formation in culture.

34: Developmentally regulated Shh expression is robust to TAD perturbations
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Posted to bioRxiv 15 Apr 2019

Developmentally regulated Shh expression is robust to TAD perturbations
1,985 downloads developmental biology

Iain Williamson, Lauren Kane, Paul S Devenney, Eve Anderson, Fiona Kilanowski, Robert E Hill, Wendy Bickmore, Laura Lettice

Topologically Associating Domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD, creating internal deletions, deleting CTCF sites including those at TAD boundaries, as well as larger deletions and inversions of TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used the investigate changes in chromatin conformation that result from these manipulations. Our data suggest that the substantial alteration of TAD structure has no readily detectable effect on Shh expression patterns during development, except where enhancers are deleted, and results in no detectable phenotypes. Only in the case of a larger deletion of one TAD boundary could some ectopic influence of the Shh limb enhancer be detected on a gene, Mnx1 in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.

35: CytoCensus: mapping cell identity and division in tissues and organs using machine learning
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Posted to bioRxiv 14 May 2017

CytoCensus: mapping cell identity and division in tissues and organs using machine learning
1,978 downloads developmental biology

Martin Hailstone, Dominic Waithe, Tamsin J. Samuels, Lu Yang, Ita Costello, Elizabeth J Robertson, Yoav Arava, Richard M. Parton, Ilan Davis

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on these datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues.

36: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells
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Posted to bioRxiv 18 Nov 2013

An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells
1,926 downloads developmental biology

David A Turner, Jamie Trott, Penelope Hayward, Pau Rué, Alfonso Martinez Arias

Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a ?race for fates? in which the neuroectodermal fate has an advantage.

37: A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species
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Posted to bioRxiv 03 May 2017

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species
1,917 downloads developmental biology

Chiara Sinigaglia, Daniel Thiel, Andreas Hejnol, Evelyn Houliston, Lucas Leclère

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care. We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

38: Unidirectional fork movement coupled with strand-specific histone incorporation ensures asymmetric histone inheritance
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Posted to bioRxiv 04 Jan 2018

Unidirectional fork movement coupled with strand-specific histone incorporation ensures asymmetric histone inheritance
1,906 downloads developmental biology

Matthew Wooten, Jonathan Snedeker, Zehra Nizami, Xinxing Yang, Rajesh Ranja, Elizabeth Urban, Jee Min Kim, Joseph Gall, Jie Xiao, Xin Chen

One of the most fundamental questions in developmental biology concerns how cells with identical genomes differentiate into distinct cell types. One important context for understanding cell fate specification is asymmetric cell division, where the two daughter cells establish different cell fates following a single division. Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell1-5. Here we show that histone H4 is inherited asymmetrically in asymmetrically dividing Drosophila male germline stem cells, similar to H36. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy with the chromatin fiber method, we are able to study histone inheritance patterns on newly replicated chromatin fibers. Using this technique, we find asymmetric inheritance patterns for old and new H3, but symmetric inheritance patterns for old and new H2A on replicating sister chromatids. Furthermore, co-localization studies on isolated chromatin fibers and proximity ligation assays on intact nuclei reveal that old H3 are preferentially incorporated by the leading strand while newly synthesized H3 are enriched on the lagging strand. Finally, using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional DNA replication on germline-derived chromatin fibers and DNA fibers. The unidirectional fork movement coupled with the strand preference of histone incorporation could explain how old and new H3 are asymmetrically incorporated by replicating sister chromatids. In summary, our work demonstrates that the intrinsic asymmetries in DNA replication may help construct sister chromatids enriched with distinct populations of histones. Therefore, these results suggest unappreciated roles for DNA replication in asymmetrically dividing cells in multicellular organisms.

39: Mbd3/NuRD is a Key Inhibitory Module During the Induction and Maintenance of Naïve Pluripotency
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Posted to bioRxiv 06 Mar 2015

Mbd3/NuRD is a Key Inhibitory Module During the Induction and Maintenance of Naïve Pluripotency
1,884 downloads developmental biology

Asaf Zviran, Yoach Rais, Nofar Mor, Noa Novershtern, Jacob H Hanna

Our group has published a study on induced pluripotent stem cell (iPSC) reprogramming (Rais et al. Nature 20131) that reached the following conclusions: a) Mbd3/NuRD is a repressor of inducing naïve pluripotency from mouse Epiblast stem cells (EpiSCs), primordial germ cells (PGCs), murine somatic cells and human secondary fibroblasts; b) Up to 100% iPSC formation efficiency can be achieved via optimized Mbd3/NuRD depletion, in concert with optimized OKSM delivery and naïve pluripotency conditions (2i supplement applied only after 48 hours, human LIF, hypoxia and Vitamin C containing Knockout serum replacement)1. This represented the first proof for deterministic/near-deterministic iPSC reprogramming, and highlighted a previously unappreciated role for Mbd3/NuRD in hampering the re-establishment of pluripotency. Recent reports have seemingly provided contradictory results and attempted to dispute our iPSC efficiency quantifications and/or the role of Mbd3/NuRD in blocking reprogramming2,3. Here we provide a detailed response to these reports based on extended discussions and providing new data. The synthesis presented herein disagrees with claims made by Silva, Hendrich, Bertone and colleagues2,3, and reconfirms that Mbd3/NuRD is a major pathway that inhibits the maintenance and induction of pluripotency1. Further, we foresee that its controlled manipulation is likely to become an integral pathway for inducing and maintaining naïve pluripotency in a variety of species.

40: Brd4 and P300 regulate zygotic genome activation through histone acetylation
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Posted to bioRxiv 13 Jul 2018

Brd4 and P300 regulate zygotic genome activation through histone acetylation
1,853 downloads developmental biology

Shun Hang Chan, Yin Tang, Liyun Miao, Hiba Darwich-Codore, Charles E Vejnar, Jean-Denis Beaudoin, Damir Musaev, Juan P. Fernandez, Miguel A. Moreno-Mateos, Antonio Giraldez

The awakening of the zygote genome, signaling the transition from maternal transcriptional control to zygotic control, is a watershed in embryonic development, but the factors and mechanisms controlling this transition are still poorly understood. By combining CRISPR-Cas9-mediated live imaging of the first transcribed genes (miR-430), chromatin and transcription analysis during zebrafish embryogenesis, we observed that genome activation is gradual and stochastic, and the active state is inherited in daughter cells. We discovered that genome activation is regulated through both translation of maternal mRNAs and the effects of these factors on the chromatin. We show that chemical inhibition of H3K27Ac writer (P300) and reader (Brd4) block genome activation, while induction of a histone acetylation prematurely activates transcription, and restore genome activation in embryos where translation of maternal mRNAs is impaired, demonstrating that they are limiting factors for the activation of the genome. In contrast to current models, we do not observe triggering of genome activation by a reduction of the nuclear-cytoplasmic(N/C) ratio or slower cell division. We conclude that genome activation is controlled by a time-dependent mechanism involving the translation of maternal mRNAs and the regulation of histone acetylation through P300 and Brd4. This mechanism is critical to initiating zygotic development and developmental reprogramming.

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