Developmental single-cell transcriptomics in the Lytechinus variegatus sea urchin embryo
Ontology highlight
ABSTRACT: Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 hours of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to "stitch" together developmental timepoints. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by sixth cleavage, though a small percentage of ectoderm cells briefly co-expressed endoderm markers indicating an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells originated at 6th cleavage also and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. 79 of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset, and expressed in the correct lineages in which the dGRN circuits operate.
Project description:We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for stitch RNA).
Project description:Single cell-based studies have revealed tremendous cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degree of plasticity during organogenesis. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including liver, pancreas, gallbladder, and extra-hepatic bile ducts. Experimental manipulation of various developmental signals in the mouse embryo underscored important cellular plasticity in this embryonic territory. This is also reflected in the existence of human genetic syndromes as well as congenital or environmentally-caused human malformations featuring multiorgan phenotypes in liver, pancreas and gallbladder. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary, and pancreatic structures are not yet established. Here, we combine computational modelling approaches with genetic lineage tracing to assess the tissue dynamics accompanying the ontogeny of the hepato-pancreato-biliary organ system. We show that a multipotent progenitor domain persists at the border between liver and pancreas, even after pancreatic fate is specified, contributing to the formation of several organ derivatives, including the liver. Moreover, using single-cell RNA sequencing we define a specialized niche that possibly supports such extended cell fate plasticity.
Project description:Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple any developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.
Project description: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.
Project description: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.
Project description:There is substantial interest in the genetic regulatory framework that is established in early human development, and in the evolutionary forces that shaped early developmental processes in humans. Progress in these areas has been slow because it is difficult to obtain relevant biological samples. Recent technological developments in the generation and differentiation of inducible pluripotent stem cells (iPSCs) provide the ability to develop in vitro models of early human and non-human primates developmental stages. We have previously established matched iPSC panels from humans and chimpanzees. Using these panels, we comparatively characterized gene regulatory changes through a four-day timecourse differentiation of iPSCs (day 1) into primary streak (day 2), endoderm progenitors (day 3), and definitive endoderm (day 4). As might be expected, we found that differentiation stage (in effect, cell type) is the major driver of variation in gene expression levels in our study, followed by species. We then identified thousands of differentially expressed genes between humans and chimpanzees in each differentiation stage. Yet, when we considered gene-specific dynamic regulatory trajectories throughout the timecourse, we found that 75% of genes, including nearly all known endoderm developmental markers, have conserved trajectories in the two species. Interestingly, we observed a marked reduction of both intra- and inter-species variation in gene expression levels in primitive streak samples compared to the iPSCs, with a recovery of variation in endoderm progenitors. The reduction in variation in gene expression levels at a specific developmental stage, paired with the high degree of conservation of temporal expression across species, is consistent with the dynamics of developmental canalization. Overall, we conclude that endoderm development in iPSC-based models are highly conserved and canalized between humans and our closest evolutionary relative.
Project description:During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branchpoints contained cells expressing genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework to reconstruct complex developmental trees from single-cell transcriptomes. This SuperSeries is composed of the SubSeries listed below.
Project description:We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2 and R3) and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We inserted STITCH into five different positions of the remaining allele of the locus: \\"STITCH+30kb\\", \\"STITCH+440kb\\", \\"STITCH+1760kb\\" and \\"STITCH+1790kb\\" have the STITCH insertions away from the MYC promoter for the indicated distances to the telomeric side of the p arm of the chromosome. \\"STITCH-30kb\\", at the 30-kb upstream from the MYC. We also made a deletion clone of the enhancer region, termed del(30-440). We made deletion of each CTCF array, L (delL) and R (delR), inversion of R (invR), deletion of the middle five binding sites from L2 to R2 (del(L2-R2)), and deletion of the six sites but for R3 (del(L1-R2)) in STITCH+30kb. We also obtained deletion and inversion of the whole of STITCH (del(L1-R3) and inv(L1-R3)). We integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH+30kb clone (STITCH/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the MYC promoter as a viewpoint to see how STITCH impacts on the chromatin conformation.
Project description:In Xenopus laevis, a number of studies identified vegetal factors that specify the germ line, endoderm and dorsal axis, but there are few studies demonstrating roles for animal-enriched maternal mRNAs. Therefore, we carried out a microarray analysis to identify novel maternal transcripts enriched in animal blastomeres. We sought to maximize differences between animal and vegetal samples. To that end, we dissected 8-cell embryos into animal blastomeres and vegetal blastomeres, and further dissected the vegetal blastomeres into vegetal-most halves (VP) and equatorial regions (discarded).
Project description:Induced pluripotent stem cell (iPSC) derived organoid systems provide models to study human organ development. Single-cell transcriptome sequencing enables highly-resolved descriptions of cell state heterogeneity within these systems and computational methods can reconstruct developmental trajectories. However, new approaches are needed to directly measure lineage relationships in these systems. Here we establish an inducible dual channel lineage recorder, iTracer, that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze state and lineage relationships in iPSC-derived systems. This data set include the iTracer data of 12 cerebral organoids.