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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 67,044 bioRxiv papers from 295,122 authors.

Most downloaded bioRxiv papers, all time

in category developmental biology

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

61: Contact-dependent cell communications drive morphological invariance during ascidian embryogenesis
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Posted to bioRxiv 24 Dec 2017

Contact-dependent cell communications drive morphological invariance during ascidian embryogenesis
1,518 downloads developmental biology

Leo Guignard, Ulla-Maj Fiuza, Bruno Leggio, Emmanuel Faure, Julien Laussu, Lars Hufnagel, Gregoire Malandain, Christophe Godin, Patrick Lemaire

Canalization of developmental processes ensures the reproducibility and robustness of embryogenesis within each species. In its extreme form, found in ascidians, early embryonic cell lineages are invariant between embryos within and between species, despite rapid genomic divergence. To resolve this paradox, we used live light-sheet imaging to quantify individual cell behaviors in digitalized embryos and explore the forces that canalize their development. This quantitative approach revealed that individual cell geometries and cell contacts are strongly constrained, and that these constraints are tightly linked to the control of fate specification by local cell inductions. While in vertebrates ligand concentration usually controls cell inductions, we found that this role is fulfilled in ascidians by the area of contacts between signaling and responding cells. We propose that the duality between geometric and genetic control of inductions contributes to the counterintuitive inverse correlation between geometric and genetic variability during embryogenesis.

62: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics
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Posted to bioRxiv 30 Jan 2017

Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics
1,513 downloads developmental biology

Andreas Sagner, Zachary B. Gaber, Julien Delile, Jennifer H. Kong, David L. Rousso, Caroline A. Pearson, Steven E. Weicksel, Manuela Melchionda, Neda S. Mousavy Gharavy, James Briscoe, Bennett Novitch

During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We address the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN) progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we define the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation, and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.

63: Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking
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Posted to bioRxiv 28 Feb 2017

Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking
1,504 downloads developmental biology

Carsten Wolff, Jean-Yves Tinevez, Tobias Pietzsch, Evangelia Stamataki, Benjamin Harich, Stephan Preibisch, Spencer Shorte, Philipp J. Keller, Pavel Tomancak, Anastasios Pavlopoulos

During development coordinated cell behaviors orchestrate tissue and organ morphogenesis to suit the lifestyle of the organism. We have used here the crustacean Parhyale hawaiensis to study the cellular basis of limb development. Transgenic Parhyale embryos with fluorescently labeled nuclei were imaged at high spatiotemporal resolution with multi-view light-sheet fluorescence microscopy over several days of embryogenesis spanning appendage morphogenesis from early specification up to late differentiation stages. Cell tracking with a new tool called Massive Multi-view Tracker (MaMuT) enabled the reconstruction of the complete cell lineage of an outgrowing thoracic limb with single-cell resolution. In silico clonal analyses suggested that the limb primordium becomes subdivided from an early stage first into anterior-posterior and then into dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb bud formation is associated with the spatial modulation of cell proliferation, while limb elongation is also driven by the preferential orientation of division of epidermal cells along the proximal-distal axis of growth. Cellular reconstructions were predictive of the expression patterns of limb development genes including the Decapentaplegic (Dpp) morphogen.

64: Charting the emergent organotypic landscape of the mammalian gut endoderm at single-cell resolution
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Posted to bioRxiv 15 Nov 2018

Charting the emergent organotypic landscape of the mammalian gut endoderm at single-cell resolution
1,502 downloads developmental biology

Sonja Nowotschin, Manu Setty, Ying-Yi Kuo, Vincent Lui, Vidur Garg, Roshan Sharma, Claire S Simon, Nestor Saiz, Rui Gardner, Stéphane C Boutet, Deanna M Church, Pamela A Hoodless, Anna-Katerina Hadjantonakis, Dana Pe’er

To comprehensively delineate the ontogeny of an organ system, we generated 112,217 single-cell transcriptomes representing all endoderm populations within the mouse embryo until midgestation. We employed graph-based approaches to model differentiating cells for spatio-temporal characterization of developmental trajectories. Our analysis reveals the detailed architecture of the emergence of the first (primitive or extra-embryonic) endodermal population and pluripotent epiblast. We uncover an unappreciated relationship between descendants of these lineages, before the onset of gastrulation, suggesting that mixing of extra-embryonic and embryonic endoderm cells occurs more than once during mammalian development. We map the trajectories of endoderm cells as they acquire embryonic versus extra-embryonic fates, and their spatial convergence within the gut endoderm; revealing them to be globally similar but retaining aspects of their lineage history. We observe the regionalized localization of cells along the forming gut tube, reflecting their extra-embryonic or embryonic origin, and their coordinate patterning into organ-specific territories along the anterior-posterior axis.

65: In vitro induction and in vivo engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells
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Posted to bioRxiv 15 Feb 2017

In vitro induction and in vivo engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells
1,499 downloads developmental biology

Alyssa J. Miller, David R. Hill, Melinda S. Nagy, Yoshiro Aoki, Briana R. Dye, Alana M Chin, Sha Huang, Felix Zhu, Eric S. White, Vibha Lama, Jason R Spence

The bud tip epithelium of the branching mouse and human lung contains multipotent progenitors that are able to self-renew and give rise to all mature lung epithelial cell types. The current study aimed to understand the developmental signaling cues that regulate bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified that FGF7, CHIR-99021 and RA maintained isolated human fetal lung epithelial bud tip progenitor cells in an undifferentiated state in vitro, and led to the induction of a 3-dimensional lung-like epithelium from hPSCs. 3-dimensional hPSC-derived lung tissue was initially patterned, with airway-like interior domains and bud tip-like progenitor domains at the periphery. Epithelial bud tip-like domains could be isolated, expanded and maintained as a nearly homogeneous population by serial passaging. Comparisons between human fetal lung epithelial bud tip cells and hPSC-derived bud tip-like cells were carried out using immunostaining, in situ hybridization and transcriptome-wide analysis, and revealed that in vitro derived tissue was highly similar to native lung. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed and maintained multi-lineage potential. Furthermore, hPSC-derived epithelial bud tip progenitors successfully engrafted in the proximal airways of injured immunocompromised NSG mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

66: Cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription
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Posted to bioRxiv 07 Dec 2017

Cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription
1,496 downloads developmental biology

Clayton E Friedman, Quan Nguyen, Samuel W Lukowski, Han Sheng Chiu, Abbigail Helfer, Jason Miklas, Shengbao Suo Suo, Jing-Dong Jackie Han, Pierre Osteil, Guangdun Peng, Naihe Jing, Greg J Baillie, Anne Senabouth, Angelika N. Christ, Timothy J Bruxner, Charles E. Murry, Emily S. Wong, Jun Ding, Yuliang Wang, James Hudson, Hannele Ruohola-Baker, Ziv Bar-Joseph, Patrick P L Tam, Joseph E Powell, Nathan J. Palpant

Differentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five-time points over a thirty-day time-course of in vitro cardiac- directed differentiation. Unsupervised clustering and lineage prediction algorithms were used to map fate choices and transcriptional networks underlying cardiac differentiation. We leveraged this resource to identify strategies for controlling in vitro differentiation as it occurs in vivo. HOPX, a non-DNA binding homeodomain protein essential for heart development in vivo was identified as dysregulated in vitro derived cardiomyocytes. Utilizing genetic gain and loss of function approaches, we dissect the transcriptional complexity of the HOPX locus and identify the requirement of hypertrophic signaling for HOPX transcription in hPSC-derived cardiomyocytes. This work provides a single cell dissection of the transcriptional landscape of cardiac differentiation for broad applications of stem cells in cardiovascular biology.

67: Tracking the embryonic stem cell transition from ground state pluripotency
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Posted to bioRxiv 08 Dec 2016

Tracking the embryonic stem cell transition from ground state pluripotency
1,490 downloads developmental biology

Tüzer Kalkan, Nelly Olova, Mila Roode, Carla Mulas, Heather J. Lee, Isabelle Nett, Hendrik Marks, Rachael Walker, Hendrik G. Stunnenberg, Kathryn S Lilley, Jennifer Nichols, Wolf Reik, Paul Bertone, Austin Smith

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition of ES cells. The population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naive pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state exhibit global transcriptome features consistent with features of early post-implantation epiblast and distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a discrete formative phase of pluripotency preparatory to lineage priming.

68: A Single-Cell Transcriptome Atlas for Zebrafish Development
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Posted to bioRxiv 19 Aug 2019

A Single-Cell Transcriptome Atlas for Zebrafish Development
1,490 downloads developmental biology

Dylan R Farnsworth, Lauren Saunders, Adam C Miller

The ability to define cell types and how they change during organogenesis is central to our understanding of animal development and human disease. Despite the crucial nature of this knowledge, we have yet to fully characterize all distinct cell types and the gene expression differences that generate cell types during development. To address this knowledge gap, we produced an Atlas using single-cell RNA-sequencing methods to investigate gene expression from the pharyngula to early larval stages in developing zebrafish. Our single-cell transcriptome Atlas encompasses transcriptional profiles from 44,102 cells across four days of development using duplicate experiments that confirmed high reproducibility. We annotated 220 identified clusters and highlighted several strategies for interrogating changes in gene expression associated with the development of zebrafish embryos at single-cell resolution. Furthermore, we highlight the power of this analysis to assign new cell-type or developmental stage-specific expression information to many genes, including those that are currently known only by sequence and/or that lack expression information altogether. The resulting Atlas is a resource of biologists to generate hypotheses for genetic (mutant) or functional analysis, to launch an effort to define the diversity of cell-types during zebrafish organogenesis, and to examine the transcriptional profiles that produce each cell type over developmental time.

69: Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling
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Posted to bioRxiv 31 Jan 2017

Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling
1,476 downloads developmental biology

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

Establishment of the three body axes is a critical step during animal development. In mammals, genetic studies have shown that a combination of precisely deployed signals from extraembryonic tissues position the anteroposterior axis (AP) within the embryo and lead to the emergence of the dorsoventral (DV) and left-right (LR) axes. We have used Gastruloids, embryonic organoids, as a model system to understand this process and find that they are able to develop AP, DV and LR axes as well as to undergo axial elongation in a manner that mirror embryos. The Gastruloids can be grown for 160 hours and form derivatives from ectoderm, mesoderm and endoderm. We focus on the AP axis and show that in the Gastruloids this axis is registered in the expression of T/Bra at one pole that corresponds to the tip of the elongation. We find that localisation of T/Bra expression depends on the combined activities of Wnt/β-Catenin and Nodal/Smad2,3 signalling, and that BMP signalling is dispensable for this process. Furthermore, AP axis specification occurs in the absence of both extraembryonic tissues and of localised sources of signalling. Our experiments show that Nodal, together with Wnt/β-Catenin signalling, is essential for the expression of T/Bra but that Wnt signalling has a separable activity in the elongation of the axis. The results lead us to suggest that, in the embryo, the role of the extraembryonic tissues might not be to induce the axes but to bias an intrinsic ability of the embryo to break its initial symmetry and organise its axes.

70: A high-resolution mRNA expression time course of embryonic development in zebrafish
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Posted to bioRxiv 10 Feb 2017

A high-resolution mRNA expression time course of embryonic development in zebrafish
1,460 downloads developmental biology

Richard J White, John E Collins, Ian M Sealy, Neha Wali, Christopher M Dooley, Zsofia Digby, Derek L Stemple, Daniel N Murphy, Thibaut Hourlier, Anja Füllgrabe, Matthew P Davis, Anton J Enright, Elisabeth M Busch-Nentwich

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1-cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3′ end transcript counting method we characterise the temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5,024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. The data reveal complex and widespread differential use of exons and previously unidentified 3′ ends across development, new primary microRNA transcripts and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data are accessible to browse and download at Expression Atlas and Ensembl.

71: Genome And Epigenome Engineering CRISPR Toolkit For Probing In Vivo cis-Regulatory Interactions In The Chicken Embryo
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Posted to bioRxiv 08 May 2017

Genome And Epigenome Engineering CRISPR Toolkit For Probing In Vivo cis-Regulatory Interactions In The Chicken Embryo
1,458 downloads developmental biology

Ruth M. Williams, Upeka Senanayake, Mara Artibani, Gunes Taylor, Daniel Wells, Ahmed Ashour Ahmed, Tatjana Sauka-Spengler

CRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present a highly efficient toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise efficient guide-RNA expression from novel chick U6-mini-vectors, provide a strategy for rapid somatic gene knockout and establish protocol for evaluation of mutational penetrance by targeted next generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.

72: Morphogen dynamics control patterning in a stem cell model of the human embryo
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Posted to bioRxiv 13 Oct 2017

Morphogen dynamics control patterning in a stem cell model of the human embryo
1,451 downloads developmental biology

Idse Heemskerk, Kari Burt, Matthew Miller, Sapna Chhabra, M. Cecilia Guerra, Aryeh Warmflash

During embryonic development, diffusible signaling molecules called morphogens are thought to determine cell fates in a concentration-dependent manner, and protocols for directed stem cell differentiation are based on this picture. However, in the mammalian embryo, morphogen concentrations change rapidly compared to the time for making cell fate decisions. It is unknown how changing ligand levels are interpreted, and whether the precise timecourse of ligand exposure plays a role in cell fate decisions. Nodal and BMP4 are morphogens crucial for gastrulation in vertebrates. Each pathway has distinct receptor complexes that phosphorylate specific signal transducers, known as receptor-Smads, which then complex with the shared cofactor Smad4 to activate target genes. Here we show in human embryonic stem cells (hESCs) that the response to BMP4 signaling indeed is determined by the ligand concentration, but that unexpectedly, the expression of many mesodermal targets of Activin/Nodal depends on rate of concentration increase. In addition, we use live imaging of hESCs with GFP integrated at the endogenous SMAD4 locus to show that a stem cell model for the human embryo generates a wave of Nodal signaling. Cells experience rapidly increasing Nodal specifically in the region of mesendoderm differentiation. We also demonstrate that pulsatile stimulation with Activin induces repeated strong signaling and enhances mesoderm differentiation. Our results break with the paradigm of concentration-dependent differentiation and demonstrate an important role for morphogen dynamics in the cell fate decisions associated with mammalian gastrulation. They suggest a highly dynamic picture of embryonic patterning where some cell fates depend on rapid concentration increase rather than absolute levels, and point to ligand dynamics as a new dimension to optimize protocols for directed stem cell differentiation.

73: Unlimited genetic switches for cell-type specific manipulation
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Posted to bioRxiv 14 Nov 2018

Unlimited genetic switches for cell-type specific manipulation
1,451 downloads developmental biology

Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, Kent Mok, Minoru Koyama, Tzumin Lee

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions, as well as to deliver precise gene therapy. Transcriptome analyses have allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types have remained inaccessible to genetic manipulation. Here, we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches that each respond to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell type-specific manipulations and combinatorial genetic targeting. CaSSA is thus a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

74: A Transcriptional Lineage of the Early C. elegans Embryo
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Posted to bioRxiv 08 Apr 2016

A Transcriptional Lineage of the Early C. elegans Embryo
1,447 downloads developmental biology

Sophia C Tintori, Erin Osborne Nishimura, Patrick Golden, Jason D Lieb, Bob Goldstein

During embryonic development, cells must establish fates, morphologies and behaviors in coordination with one another to form a functional body. A prevalent hypothesis for how this coordination is achieved is that each cell's fate and behavior is determined by a defined mixture of RNAs. Only recently has it become possible to measure the full suite of transcripts in a single cell. Here we quantify the abundance of every mRNA transcript in each cell of the C. elegans embryo up to the 16-cell stage. We describe spatially dynamic expression, quantify cell-specific differential activation of the zygotic genome, and identify critical developmental genes previously unappreciated because of their partial redundancy. We present an interactive data visualization tool that allows broad access to our dataset. This genome-wide single-cell map of mRNA abundance, alongside the well-studied life history and fates of each cell, describes at a cellular resolution the mRNA landscape that guides development.

75: Establishment In Culture Of Expanded Potential Stem Cells
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Posted to bioRxiv 06 Apr 2017

Establishment In Culture Of Expanded Potential Stem Cells
1,441 downloads developmental biology

Jian Yang, David J. Ryan, Wei Wang, Jason Cheuk-Ho Tsang, Guocheng Lan, Hideki Masaki, Xuefei Gao, Liliana Antunes, Yong Yu, Zhexin Zhu, Juexuan Wang, Aleksandra A. Kolodziejczyk, Lia S Campos, Cui Wang, Fengtang Yang, Zhen Zhong, Beiyuan Fu, Melanie Eckersley-Maslin, Michael Woods, Yosuke Tanaka, Adam C. Wilkinson, James Bussell, Jacqui White, Ramiro Ramirez-Solis, Wolf Reik, Berthold Göttgens, Sarah A Teichmann, Hiromitsu Nakauchi, Xiangang Zou, Liming Lu, Pentao Liu

Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts and contribute to both the somatic lineage and germline when returned to the blastocyst but are normally excluded from the trophoblast lineage and primitive endoderm. Here, we report that cultures of expanded potential stem cells (EPSCs) can be established from individual blastomeres, by direct conversion of mouse embryonic stem cells (ESCs) and by genetically reprogramming somatic cells. Remarkably, a single EPSC contributes to the embryo proper and placenta trophoblasts in chimeras. Critically, culturing EPSCs in a trophoblast stem cell (TSC) culture condition permits direct establishment of TSC lines without genetic modification. Molecular analyses including single cell RNA-seq reveal that EPSCs share cardinal pluripotency features with ESCs but have an enriched blastomere transcriptomic signature and a dynamic DNA methylome. These proof-of-concept results open up the possibility of establishing cultures of similar stem cells in other mammalian species.

76: Self-organization of a functional human organizer by combined WNT and NODAL signalling
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Posted to bioRxiv 15 Dec 2017

Self-organization of a functional human organizer by combined WNT and NODAL signalling
1,433 downloads developmental biology

I. Martyn, T.Y. Kanno, A. Ruzo, E.D. Siggia, A.H. Brivanlou

In amniotes, the development of the primitive streak (PS) and its accompanying 'organizer' define the first stages of gastrulation. Despite detailed characterization in model organisms, the analogous human structures remain a mystery. We have previously shown that when stimulated with BMP4, micropatterned colonies of human embryonic stem cells (hESCs) self-organize to generate early embryonic germ layers1. Here we show that in the same type of colonies WNT signalling is sufficient to induce a PS, and WNT with ACTIVIN is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, epithelial-to-mesenchymal transition (EMT) markers, and expression of the organizer specific transcription factor GSC. Moreover, when grafted into chick embryos, WNT and ACTIVIN treated human cells induce and contribute autonomously to a secondary axis while inducing neural fate in the host. This fulfills the most stringent functional criteria for an organizer, and its discovery represents a major milestone in human embryology.

77: A conserved regulatory program drives emergence of the lateral plate mesoderm
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Posted to bioRxiv 07 Feb 2018

A conserved regulatory program drives emergence of the lateral plate mesoderm
1,432 downloads developmental biology

Karin D. Prummel, Christopher Hess, Susan Nieuwenhuize, Hugo J Parker, Katherine W. Rogers, Iryna Kozmikova, Claudia Racioppi, Eline C. Brombacher, Anna Czarkwiani, Dunja Knapp, Alexa Burger, Elena Chiavacci, Gopi Shah

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolated a pan-LPM enhancer in the zebrafish draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captured the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncovered specific drl reporter activity in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

78: Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis
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Posted to bioRxiv 01 Aug 2017

Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis
1,427 downloads developmental biology

Kenzo Ivanovitch, Susana Temiño, Miguel Torres

During vertebrate heart development two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development.

79: Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona
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Posted to bioRxiv 28 Feb 2016

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona
1,418 downloads developmental biology

Shashank Gandhi, Maximilian Haeussler, Florian Razy-Krajka, Lionel Christiaen, Alberto Stolfi

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

80: Cell type boundaries organize plant development
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Posted to bioRxiv 11 Apr 2017

Cell type boundaries organize plant development
1,408 downloads developmental biology

Monica Pia Caggiano, Xiulian Yu, Neha Bhatia, André Larsson, Hasthi Ram, Carolyn Ohno, Pia Sappl, Elliot M. Meyerowitz, Henrik Jönsson, Marcus G Heisler

In plants the dorsoventral boundary of leaves defines an axis of symmetry through the centre of the organ separating the top (dorsal) and bottom (ventral) tissues. Although the positioning of this boundary is critical for leaf morphogenesis, how the boundary is established and how it influences development remains unclear. Using live-imaging and perturbation experiments we show that leaf orientation, morphology and position are pre-patterned by HD-ZIPIII and KAN gene expression in the shoot, leading to a model in which dorsoventral genes coordinate to regulate plant development by localizing auxin response between their expression domains. However we also find that auxin levels feedback on dorsoventral patterning by spatially organizing HD-ZIPIII and KAN expression in the shoot periphery. By demonstrating that the regulation of these genes by auxin also governs their response to wounds, our results also provide a parsimonious explanation for the influence of wounds on leaf dorsoventrality.

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