Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 89,678 bioRxiv papers from 384,180 authors.
Most downloaded bioRxiv papers, all time
in category developmental biology
2,619 results found. For more information, click each entry to expand.
1,747 downloads developmental biology
Tissue-specific loss-of-function (LOF) analysis is an essential approach for characterizing gene function. Here we describe an efficient CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. To facilitate the construction of these components, we developed convenient tools for generating and evaluating enhancer-driven Cas9 lines, identified a multi-gRNA design that is highly efficient in mutagenizing somatic cells, and established an assay for testing the efficiency of multi-gRNAs in creating double-stranded breaks. We found that excision of genomic DNA induced by two gRNAs is infrequent in somatic cells, while indels more reliably cause tissue-specific LOF. Furthermore, we show that enhancer-driven Cas9 is less cytotoxic yet results in more complete gene removal than Gal4-driven Cas9 in larval neurons. Finally, we demonstrate that CRISPR-TRiM efficiently unmasks redundant gene functions in neuronal morphogenesis. Importantly, two Cas9 transgenes that turn on with different timings in the neuronal lineage revealed the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISRPR tools can be applied to analyze tissue-specific gene function in many biological processes.
1,743 downloads developmental biology
The authors have withdrawn their manuscript given that some conclusions need more research and data support. In addition, they intend to improve the experimental design to make the research more rigorous. If you have any questions, please contact the corresponding author. ### Competing Interest Statement The authors have declared no competing interest.
1,743 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.
1,734 downloads developmental biology
In embryonic development, cells must differentiate through stereotypical sequences of intermediate states to generate mature states of a particular fate. By contrast, direct programming can generate similar fates through alternative routes, by directly expressing terminal transcription factors. Yet the cell state transitions defining these new routes are unclear. We applied single-cell RNA sequencing to compare two mouse motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both undergo similar early neural commitment. Then, rather than transitioning through spinal intermediates like the standard protocol, the direct programming path diverges into a novel transitional state. This state has specific and abnormal gene expression. It opens a ‘loop’ or ‘worm hole’ in gene expression that converges separately onto the final motor neuron state of the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons from embryos.
1,696 downloads developmental biology
Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. In this study we show that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells located superficially and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, we show that these cells express Zfh1- cells and engage in Notch-Delta dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, we consider these novel cells to be the Drosophila equivalent of vertebrate muscle satellite cells.
1,674 downloads developmental biology
Tissues undergoing morphogenesis impose mechanical effects on one another. How developmental programs adapt to or take advantage of these effects remains poorly explored. Here, using a combination of live imaging, modeling, and microsurgical perturbations, we show that the axial and paraxial tissues in the forming avian embryonic body coordinate their rates of elongation through mechanical interactions. First, a cell motility gradient drives paraxial presomitic mesoderm (PSM) expansion, resulting in compression of the axial neural tube and notochord; second, elongation of axial tissues driven by PSM compression and polarized cell intercalation pushes the caudal progenitor domain posteriorly; finally, the axial push drives progenitors to emigrate into the PSM to maintain tissue growth and cell motility. These interactions form an engine-like positive feedback loop, which ensures the tissue-coupling and self-sustaining characteristics of body elongation. Our results suggest a general role of inter-tissue forces in the coordination of complex morphogenesis involving distinct tissues.
1,672 downloads developmental biology
Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to the significant reduction of signal intensities. To overcome this limitation, we enhanced the translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.
1,652 downloads developmental biology
Is the human early embryo unique in lacking an inner cell mass (ICM) and having parallel rather than step-wise development? Here we reanalyse single-cell transcriptomic data and stain human embryos in situ to reveal both classical step-wise development and the missing ICM, a transcriptomic homolog of macaque ICM, that differentiates to epiblast and primitive endoderm. This apparent classicism obscures numerous features that render our blastocyst phylogenetically distinct: unlike mice, human epiblast has hallmarks of self-renewal and we have abundant, previously unrecognized, blastocyst non-committed cells (NCCs), part of an apoptosis-mediated quality control/purging process. Comparative transcriptomics further reveals the transcriptomes of the pluripotent cells to be especially fast evolving, rendering all primate embryos unique. Rapid transcriptome turnover is in large part owing to endogenous retrovirus H (ERVH) activity, ERVH being associated with recent major gene expression gain and loss events of pluripotency-associated genes. Each species is characterised by the ERVHs that are active and the neighbour genes whose expression are in turn modulated. The current portfolio of naive cultures, putative in vitro mimics of pluripotent cells, are both developmentally and phylogenetically "confused" in part owing to a lack of HERVH expression.
1,650 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.
1,643 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.
1,635 downloads developmental biology
The neural crest is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional profiles and chromatin accessibility of neural crest cells in the basal sea lamprey, in order to gain insight into the ancestral state of the neural crest gene regulatory network (GRN) at the dawn of vertebrates. Transcriptome analyses reveal clusters of co-regulated genes during neural crest specification and migration that show high conservation across vertebrates for dynamic programmes like Wnt modulation during the epithelial to mesenchymal transition, but also reveal novel transcription factors and cell-adhesion molecules not previously implicated in neural crest migration. ATAC-seq analysis refines the location of known cis-regulatory elements at the Hox-α2 locus and uncovers novel cis-regulatory elements for Tfap2B and SoxE1. Moreover, cross-species deployment of lamprey elements in zebrafish reveals that the lamprey SoxE1 enhancer activity is deeply conserved, mediating homologous expression in jawed vertebrates. Together, our data provide new insight into the core elements of the GRN that are conserved to the base of the vertebrates, as well as expose elements that are unique to lampreys.
1,630 downloads developmental biology
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.
1,627 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, Alexa Burger, Jan Huisken, Maximina H. Yun, Lionel Christiaen, Lionel Christiaen, Patrick Müller, Marianne Bronner, Robb Krumlauf, Christian Mosimann
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.
1,617 downloads developmental biology
During development, gene regulatory networks allocate cell fates by partitioning tissues into spatially organised domains of gene expression. How the sharp boundaries that delineate these gene expression patterns arise, despite the stochasticity associated with gene regulation, is poorly understood. We show, in the vertebrate neural tube, using perturbations of coding and regulatory regions, that the structure of the regulatory network contributes to boundary precision. This is achieved, not by reducing noise in individual genes, but by the configuration of the network modulating the ability of stochastic fluctuations to initiate gene expression changes. We use a computational screen to identify the properties of a network that influence boundary precision, revealing two dynamical mechanisms by which small gene circuits attenuate the effect of noise to increase patterning precision. These results establish design principles of gene regulatory networks that produce precise patterns of gene expression.
1,601 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 G. 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.
1,595 downloads developmental biology
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.
1,592 downloads developmental biology
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.
1,590 downloads developmental biology
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.
1,588 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.
1,586 downloads developmental biology
Classical mouse embryology has established a sequence of lineage bifurcations underpinning early mammalian development. Consistent with this paradigm, mouse embryonic stem cells have lost the capacity to generate extraembryonic trophectoderm. We show here, however, that human naive epiblast stem cells readily produce this lineage. Inhibition of ERK signalling, fundamental to naive cell propagation, is unexpectedly instrumental in trophectoderm induction. Transcriptome analyses authenticate trophoblast fate and expose a trajectory via reversion to inner cell mass (ICM). Nodal inhibition enhances differentiation and BMP signalling is not engaged. Strikingly, after formative transition primed stem cells cannot make trophectoderm but respond to BMP and form amnion. Gene perturbations in naive cells reveal that YAP and TFAP2C promote trophectoderm as in mouse, while NANOG suppresses distinctively in human. Finally, ICMs from expanded human blastocysts efficiently regenerate trophectoderm. Thus in human, retained trophectoderm potential is an integral feature of emergent pluripotency that confers higher regulative plasticity.
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