Biology subjects

Gottgens, B.

Publications and source records attributed to Gottgens, B..

52 records · Page 3Linked to original sources

bioRxiv2021

Analysis of phloem trajectory links tissue maturation to cell specialization.

The mechanisms that allow cells in the plant meristem to coordinate tissue-wide maturation gradients with specialized cell networks are critical for indeterminate growth. Here, we reconstructed the protophloem developmental trajectory of 19 cells from cell birth to terminal differentiation at single cell resolution in the Arabidopsis root. We found that cellular specification is mediated near the stem cell niche by PHLOEM EARLY DOF (PEAR) transcription factors. However, the PEAR dependent differentiation program is repressed by a broad gradient of PLETHORA (PLT) transcription factors, which directly inhibit PEARs own direct target ALTERED PHLOEM DEVELOPMENT (APL). The dissipation of PLT gradient around 7 cells from the stem cell activates APL expression, and a subsequent transitional network that results in a "seesaw" pattern of mutual inhibition over developmental time. Together, we provide a mechanistic understanding of how morphogen-like maturation gradients interface with cell-type specific transcriptional regulators to stage cellular differentiation.

plant biology↗

bioRxiv2020

Coordinated Changes in Gene Expression Kinetics Underlie both Mouse and Human Erythroid Maturation

BackgroundSingle cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single cell RNA-Seq permits prediction of future expression states. Here we applied this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis. ResultsIntriguingly, RNA velocity correctly identified epiblast cells as the starting point, but several trajectory predictions at later stages were inconsistent with both real time ordering and existing knowledge. The most striking discrepancy concerned red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes revealed a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be up-regulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1- chimera dataset revealed induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. ConclusionsBy identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.

developmental biology↗

bioRxiv2020

Highly multiplexed spatially resolved gene expression profiling of mouse organogenesis

Transcriptional and epigenetic profiling of single-cells has advanced our knowledge of the molecular bases of gastrulation and early organogenesis. However, current approaches rely on dissociating cells from tissues, thereby losing the crucial spatial context that is necessary for understanding cell and tissue interactions during development. Here, we apply an image-based single-cell transcriptomics method, seqFISH, to simultaneously and precisely detect mRNA molecules for 387 selected target genes in 8-12 somite stage mouse embryo tissue sections. By integrating spatial context and highly multiplexed transcriptional measurements with two single-cell transcriptome atlases we accurately characterize cell types across the embryo and demonstrate how spatially-resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary and the developing gut tube. Our spatial atlas uncovers axes of resolution that are not apparent from single-cell RNA sequencing data - for example, in the gut tube we observe early dorsal-ventral separation of esophageal and tracheal progenitor populations. In sum, by computationally integrating high-resolution spatially-resolved gene expression maps with single-cell genomics data, we provide a powerful new approach for studying how and when cell fate decisions are made during early mammalian development.

developmental biology↗

bioRxiv2020

Disruption of a GATA2, TAL1, ERG regulatory circuit promotes erythroid transition in healthy and leukemic stem cells

Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesised that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, RUNX1) bind key hematopoietic genes in human CD34+ haematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each others, regulatory elements. However, their mutual regulation during normal haematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. Here, we integrated bulk and single cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and in leukemic cells. The heptad factors GATA2, TAL1 and ERG formed an integrated sub-circuit that regulates stem cell to erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits could be harnessed to promote specific cell type transitions and overcome dysregulated haematopoiesis.

cancer biology↗

bioRxiv2020

Quantitative and molecular differences distinguish adult human medullary and extramedullary haematopoietic stem and progenitor cell landscapes

In adults, the bone marrow (BM) is the main site of haematopoietic stem and progenitor cells (HSPCs) maintenance and differentiation. It is known that other anatomical sites can contribute significantly to blood production under stress conditions. However limited tissue availability restricts our knowledge on the cellular, molecular and functional composition of extramedullary HSPC pools in humans at steady state or under stress. Here we describe the landscape of human HSPC differentiation across the three major haematopoietic anatomical sites: BM, spleen and peripheral blood (PB), using matched tissues isolated from the same individuals. Single cell RNA-seq of 30,000 HSPCs and 700 phenotypic haematopoietic stem cells and multipotent progenitors (HSC/MPP) demonstrates significantly different dynamics of haematopoiesis between BM and extramedullary tissues. Lineage-committed progenitors of spleen and PB do not actively divide, whereas BM is the primary site of progenitor proliferation. The balance of differentiation in spleen and PB is skewed towards the lymphoid and erythroid lineages, whereas in BM it is tilted towards megakaryocytic and myeloid progenitors. Extramedullary tissues also harbour a molecularly defined subset of HSC/MPP not found in the BM, which is marked by a specific acto-myosin cytoskeletal signature and transcriptional priming for division and lineage differentiation. Collectively, our findings define a unique cellular and molecular structure of the haematopoietic landscape in extramedullary organs, positioned for rapid lineage-primed demand-adapted haematopoiesis. These data also provide a framework for better understanding of human extramedullary haematopoiesis in health and disease.

cell biology↗

bioRxiv2019

Single-cell molecular profiling provides a high-resolution map of basophil and mast cell differentiation

Differentiation of hematopoietic stem and progenitor cells ensure a continuous supply of mature blood cells. Recent models of differentiation are represented as a landscape, in which individual progenitors traverse a continuum of multipotent cell states before reaching an entry point that marks lineage commitment. Basophils and mast cells have received little attention in these models and their differentiation trajectories are yet to be explored. Here, we have performed multicolor flow cytometry and high-coverage single-cell RNA sequencing analyses to chart the differentiation of hematopoietic progenitors into basophils and mast cells in mouse. Analysis of flow cytometry data reconstructed a detailed map of the differentiation, including a bifurcation of progenitors into two specific trajectories. Molecular profiling and pseudotime ordering of the single cells revealed gene expression changes during differentiation, with temporally separated regulation of mast cell protease genes. We validate that basophil and mast cell signature genes increased along the trajectories into their respective lineage, and we demonstrate how genes critical for each respective lineage are upregulated during the formation of the mature cells. Cell fate assays showed that multicolor flow cytometry and transcriptional profiling successfully predict the bipotent phenotype of a previously uncharacterized population of basophil-mast cell progenitor-like cells in mouse peritoneum. Taken together, we provide a detailed roadmap of basophil and mast cell development through a combination of molecular and functional profiling.

immunology↗

bioRxiv2019

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration

Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell-types, during both development and adulthood. Here, we report how runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, but unexpectedly, absence of runx1 results in enhanced regeneration. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes without differentiating into myofibroblasts. Both these populations are absent in runx1 mutants, resulting in a less collagenous and fibrinous scar. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and by upregulation of components of the fibrin degradation pathway, including plasminogen receptor Annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of Plasminogen. In addition, we find enhanced myocardial proliferation as well as increased myocardial survival in the mutant. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell-types and that targeting Runx1 is a novel therapeutic strategy to induce endogenous heart repair.

developmental biology↗

bioRxiv2019

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of mouse hematopoietic cells

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for twenty cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions - along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

genomics↗

bioRxiv2019

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis

Thousands of epigenomic datasets have been generated in the past decade, but it is difficult for researchers to effectively utilize all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for ValIdated Systematic IntegratiON of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By employing IDEAS as our Integrative and Discriminative Epigenome Annotation System, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of over 200,000 candidate cis-regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website (usevision.org) to aid research in genomics and hematopoiesis.

genomics↗

bioRxiv2019

Nanog regulates Pou3f1 expression and represses anterior fate at the exit from pluripotency.

Pluripotency is regulated by a network of transcription factors that maintains early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development its expression is restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses the anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting the neural fate by repressing Pou3f1 at the onset of gastrulation.

developmental biology↗

bioRxiv2018

Lgr5+ stem/progenitor cells reside at the apex of the embryonic hepatoblast pool

AbstractDuring mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocyte and cholangiocyte cells. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous population of progenitors, which initiate differentiation into hepatocytes and cholangiocytes from E13.5 onwards. Recently, sub-populations of transcriptionally different hepatoblasts have been identified as already present at E11.5 by single cell RNAseq (scRNAseq) analysis. However, whether these transcriptional differences result from functionally heterogeneous hepatoblast populations is unknown. Here we show that the hepatoblast pool is not only transcriptionally but also functionally heterogeneous and that a sub-population of E9.5-E10.0 hepatoblasts exhibits a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a sub-population of bona-fide E9.5 hepatoblasts which express the adult stem cell marker Lgr5 and contribute to liver development by generating both hepatocyte and cholangiocyte progeny that persist for the life-span of the mouse. Using a combination of lineage tracing and scRNAseq, we show that Lgr5 marks E9.5-E10.0 bi-potent liver progenitors residing at the apex of a hierarchy of the hepatoblast population. Notably, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to hepatocyte or cholangiocyte fates dependent upon the culture conditions. Our study represents the first functional demonstration of heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a sub-population of truly bi-potent liver progenitors.\n\nSummary StatementLgr5 positive bi-potential hepatoblasts contribute to liver development and reside at the apex of an embryonic liver progenitor pool.

developmental biology↗

bioRxiv2018

Malaria-induced remodelling of the bone marrow microenvironment mediates loss of haematopoietic stem cell function

Severe infections are a major source of stress on haematopoiesis, where consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow niches, which have been shown to be altered during ageing and haematopoietic malignancies. Whether the bone marrow (BM) microenvironment plays a role in mediating the effects of infection on HSCs remains an open question. Here we used an murine model of malaria coupled with intravital microscopy, single cell RNA-Seq, mathematical modelling and transplantation assays to obtain a quantitative understanding of the proliferation dynamics of haematopoietic stem and progenitor cells (HSPCs) during Plasmodium infection. We uncovered that during Plasmodium infection the HSC compartment turns over significantly faster than in steady state, and that a global interferon response and loss of functional HSCs are linked to alterations in BM endothelium function and osteoblasts number. Interventions that targeted osteoblasts uncoupled HSC proliferation and function, thus opening up new avenues for therapeutic interventions that may improve the health of survivors of severe infections.

cell biology↗

bioRxiv2017

Graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells

Single-cell RNA-seq quantifies biological heterogeneity across both discrete cell types and continuous cell transitions. Partition-based graph abstraction (PAGA) provides an interpretable graph-like map of the arising data manifold, based on estimating connectivity of manifold partitions (https://github.com/theislab/paga). PAGA maps provide interpretable discrete and continuous latent coordinates for both disconnected and continuous structure in data, preserve the global topology of data, allow analyzing data at different resolutions and result in much higher computational efficiency of the typical exploratory data analysis workflow -- one million cells take on the order of a minute, a speedup of 130 times compared to UMAP. We demonstrate the method by inferring structure-rich cell maps with consistent topology across four hematopoietic datasets, confirm the reconstruction of lineage relations of adult planaria and the zebrafish embryo, benchmark computational performance on a neuronal dataset and detect a biological trajectory in one deep-learning processed image dataset.

bioinformatics↗

bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗

bioRxiv2017

Establishment In Culture Of Expanded Potential Stem Cells

Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts1,2 and contribute to both the somatic lineage and germline when returned to the blastocyst3 but are normally excluded from the trophoblast lineage and primitive endoderm4-6. 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.

developmental biology↗

bioRxiv2017

Assessing The Reliability Of Spike-In Normalization For Analyses Of Single-Cell RNA Sequencing Data

By profiling the transcriptomes of individual cells, single-cell RNA sequencing provides unparalleled resolution to study cellular heterogeneity. However, this comes at the cost of high technical noise, including cell-specific biases in capture efficiency and library generation. One strategy for removing these biases is to add a constant amount of spike-in RNA to each cell, and to scale the observed expression values so that the coverage of spike-in RNA is constant across cells. This approach has previously been criticized as its accuracy depends on the precise addition of spike-in RNA to each sample, and on similarities in behaviour (e.g., capture efficiency) between the spike-in and endogenous transcripts. Here, we perform mixture experiments using two different sets of spike-in RNA to quantify the variance in the amount of spike-in RNA added to each well in a plate-based protocol. We also obtain an upper bound on the variance due to differences in behaviour between the two spike-in sets. We demonstrate that both factors are small contributors to the total technical variance and have only minor effects on downstream analyses such as detection of highly variable genes and clustering. Our results suggest that spike-in normalization is reliable enough for routine use in single-cell RNA sequencing data analyses.

bioinformatics↗