Biology subjects

Trapnell, C.

Publications and source records attributed to Trapnell, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Single-cell sequencing of individual retinal organoids reveals determinants of cell fate heterogeneity

With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA-seq multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10x methods produce highly concordant cell class compositions and then expand sci-Plex to analyze the cell class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to six weeks later. Our data show sci-Plexs potential to dramatically scale-up the analysis of treatment conditions on relevant human models.

genomics↗

bioRxiv2023

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

developmental biology↗

bioRxiv2023

Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.

cell biology↗

bioRxiv2023

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways. HighlightsO_LIDe novo designed cyclic oligomers with tunable geometric properties C_LIO_LICyclic, homo-oligomeric FGFR binding modules induce geometry- and valency-dependent activity of isoform-specific FGF signaling C_LIO_LIModulation of FGFR isoform activity controls bifurcation of endothelial and mesenchymal fate during vascular development C_LIO_LIC-isoform activation favors arterial endothelial cell formation while B-isoform induces pericyte differentiation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/532666v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151f6eforg.highwire.dtl.DTLVardef@2ae877org.highwire.dtl.DTLVardef@103e0d1org.highwire.dtl.DTLVardef@19cd695_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy

Chemical genetic screens are a powerful tool for exploring how cancer cells response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-kB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

genomics↗

bioRxiv2023

High-Capacity Sample Multiplexing for Single Cell Chromatin Accessibility Profiling

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

molecular biology↗

bioRxiv2023

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including wholelung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-{kappa}B ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis. One Sentence SummarySilica induces the alveolar epithelium to reprogram recruited and resident pulmonary myeloid cells to become osteoclasts that contribute to pulmonary fibrosis.

pathology↗

bioRxiv2022

Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters

The inability to scalably and precisely measure the activity of developmental enhancers in multicellular systems is a bottleneck in genomics. Here, we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays, resulting in accurate measurement of reporter expression over a >10,000-fold range of activity with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode circularization, these single-cell quantitative expression reporters (scQers) provide high-contrast readouts analogous to classic in situ assays, but entirely from sequencing. Screening >200 enhancers in a multicellular in vitro model of early mammalian development, we identified numerous autonomous and cell-type-specific elements, including constituents of the Sox2 control region exclusively active in pluripotent cells, endoderm-specific enhancers, including near Foxa2 and Gata4, and a compact pleiotropic enhancer at the Lamc1 locus. scQers can be mobilized in developmental systems to quantitatively characterize native, perturbed, and synthetic enhancers at scale, with high sensitivity and at single-cell resolution.

genomics↗

bioRxiv2022

Single-Cell Transcriptomic Profiling of the Zebrafish Inner Ear Reveals Molecularly Distinct Hair Cell and Supporting Cell Subtypes

A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair cells and supporting cell types remains unresolved. Here we perform single-cell RNA sequencing of the zebrafish inner ear at embryonic through adult stages to catalog the diversity of hair cell and non-sensory supporting cells. We identify a putative progenitor population for hair cells and supporting cells, as well as distinct hair cells and supporting cell types in the maculae versus cristae. The hair cell and supporting cell types differ from those described for the lateral line system, a distributed mechanosensory organ in zebrafish in which most studies of hair cell regeneration have been conducted. In the maculae, we identify two subtypes of hair cells that share gene expression with mammalian striolar or extrastriolar hair cells. In situ hybridization reveals that these hair cell subtypes occupy distinct spatial domains within the two major macular organs, the utricle and saccule, consistent with the reported distinct electrophysiological properties of hair cells within these domains. These findings suggest that primitive specialization of spatially distinct striolar and extrastriolar hair cells likely arose in the last common ancestor of fish and mammals. The similarities of inner ear cell type composition between fish and mammals also support using zebrafish as a relevant model for understanding inner ear-specific hair cell function and regeneration.

neuroscience↗

bioRxiv2022

Proteostasis governs differential temperature sensitivity across embryonic cell types

The genetic program of embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. While the stereotyped, consistent phenotypes associated with environmental stress during vertebrate development suggest that some cell types are more sensitive to stress than others, the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single cell RNA-seq to identify cell types and molecular programs within them that drive phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in their development. The notochord is particularly sensitive to temperature stress, which we show is due to a specialized cell type, sheath cells. Further analyses show that sheath cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and irreversible anatomic defects in the embryo. Our study demonstrates that whole-animal single cell RNA-seq can characterize mechanisms important for developmental robustness and pinpoint molecular programs within specific cell types that constitute key failure points.

genomics↗

bioRxiv2022

Deep molecular, cellular and temporal phenotyping of developmental perturbations at whole organism scale

The maturation of single cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of this data, however, has been collected from wild type embryos without an appreciation for latent variation present in development. Here we present single cell transcriptomic data from 1812 individually resolved developing zebrafish embryos, encompassing 19 time points, 23 genetic perturbations, and totaling 3.2M cells. The high degree of replication in our study (8 or more embryos per condition) allows us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about the origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single cell data from large numbers of individual embryos will enable mapping the genetic dependencies of zebrafish cell types, while also addressing long-standing challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

genomics↗

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2022

Single-cell analysis of chromatin and expression reveals age- and sex-associated alterations in the human heart

Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex. Sex dependent alterations include pathways such as TGF{beta} signaling and metabolic shifts by sex, evident in both transcriptional alterations and differing presence of transcription factor (TF) motifs in accessible chromatin. Age was associated with changes such as immune activation-related transcriptional and chromatin accessibility differences, as well as changes in the relative proportion of cardiomyocytes, neurons, and perivascular cells. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs.

genomics↗

bioRxiv2021

Airway epithelial interferon response to SARS-CoV-2 is inferior to rhinovirus and heterologous rhinovirus infection suppresses SARS-CoV-2 replication

IntroductionCommon alphacoronaviruses and human rhinoviruses (HRV) induce type I and III interferon (IFN) responses important to limiting viral replication in the airway epithelium. In contrast, highly pathogenic betacoronaviruses including SARS-CoV-2 may evade or antagonize RNA-induced IFN I/III responses. MethodsIn airway epithelial cells (AECs) from children and older adults we compared IFN I/III responses to SARS-CoV-2 and HRV-16, and assessed whether pre-infection with HRV-16, or pretreatment with recombinant IFN-{beta} or IFN-{lambda}, modified SARS-CoV-2 replication. Bronchial AECs from children (ages 6-18 yrs.) and older adults (ages 60-75 yrs.) were differentiated ex vivo to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 or HRV-16, and RNA and protein was harvested from cell lysates 96 hrs. following infection and supernatant was collected 48 and 96 hrs. following infection. In additional experiments cultures were pre-infected with HRV-16, or pre-treated with recombinant IFN-{beta}1 or IFN-{lambda}2 before SARS-CoV-2 infection. ResultsDespite significant between-donor heterogeneity SARS-CoV-2 replicated 100 times more efficiently than HRV-16. IFNB1, INFL2, and CXCL10 gene expression and protein production following HRV-16 infection was significantly greater than following SARS-CoV-2. IFN gene expression and protein production were inversely correlated with SARS-CoV-2 replication. Treatment of cultures with recombinant IFN{beta}1 or IFN{lambda}2, or pre-infection of cultures with HRV-16, markedly reduced SARS-CoV-2 replication. DiscussionIn addition to marked between-donor heterogeneity in IFN responses and viral replication, SARS-CoV-2 elicits a less robust IFN response in primary AEC cultures than does rhinovirus, and heterologous rhinovirus infection, or treatment with recombinant IFN-{beta}1 or IFN-{lambda}2, markedly reduces SARS-CoV-2 replication.

immunology↗

bioRxiv2021

Systematic reconstruction of the cellular trajectories of mammalian embryogenesis

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single cell zygote gives rise to millions of cells expressing a panoply of molecular programs, including much of the diversity that will subsequently be present in adult tissues. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here we set out to integrate several single cell RNA-seq datasets (scRNA-seq) that collectively span mouse gastrulation and organogenesis. We define cell states at each of 19 successive stages spanning E3.5 to E13.5, heuristically connect them with their pseudo-ancestors and pseudo-descendants, and for a subset of stages, deconvolve their approximate spatial distributions. Despite being constructed through automated procedures, the resulting trajectories of mammalian embryogenesis (TOME) are largely consistent with our contemporary understanding of mammalian development. We leverage TOME to nominate transcription factors (TF) and TF motifs as key regulators of each branch point at which a new cell type emerges. Finally, to facilitate comparisons across vertebrates, we apply the same procedures to single cell datasets of zebrafish and frog embryogenesis, and nominate "cell type homologs" based on shared regulators and transcriptional states.

developmental biology↗

bioRxiv2021

Transcriptomic profiling of tissue environments critical for post-embryonic patterning and morphogenesis of zebrafish skin

Pigment patterns and skin appendages are prominent features of vertebrate skin. In zebrafish, regularly patterned pigment stripes and an array of calcified scales form simultaneously in the skin during post-embryonic development. Understanding mechanisms that regulate stripe patterning and scale morphogenesis may lead to discovery of fundamental mechanisms that govern development of animal form. To learn about cell types and signaling interactions that govern skin patterning and morphogenesis we generated and analyzed single cell transcriptomes of skin from wild-type fish as well as fish having genetic or transgenically induced defects in squamation or pigmentation. These data reveal a previously undescribed population of epidermal cells that express transcripts encoding enamel matrix proteins, suggest hormonal control of epithelial-mesenchymal signaling, clarify the signaling network that governs scale papillae development, and identify a critical role for the hypodermis in supporting pigment cell development. Additionally, these comprehensive single-cell transcriptomic data representing skin phenotypes of biomedical relevance should provide a useful resource for accelerating discovery of mechanisms that govern skin development and homeostasis.

developmental biology↗

bioRxiv2021

Multipotent progenitors and hematopoietic stem cells arise independently during the endothelial to hematopoietic transition in the early mouse embryo

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSC). Although previous studies have shown that yolk sac-derived erythromyeloid progenitors and HSC emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSC originate from common HE. Here we demonstrate by clonal assays and single cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSC. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSC arise independently from HE with distinct phenotypic and transcriptional properties.

developmental biology↗

bioRxiv2021

Engineering a niche supporting haematopoietic stem cell development using integrated single cell transcriptomics

Haematopoietic stem cells (HSCs) develop from haemogenic endothelium (HE) within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, we used single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from HE to HSC, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering revealed dynamics of gene expression during the HE to HSC transition, identifying surface receptors specifically expressed on developing HSCs. Transcriptional profiles of niche endothelial cells enabled identification of corresponding ligands, including those signaling to Notch receptors, VLA-4 integrin, and CXCR4, which, when integrated in an engineered platform, were sufficient to support the generation of engrafting HSCs. These studies provide a transcriptional map of the signaling interactions necessary for the development of HSCs and advance the goal of engineering HSC for therapeutic applications.

developmental biology↗