Biology subjects

Rajewsky, N.

Publications and source records attributed to Rajewsky, N..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Long-term imaging reveals behavioral plasticity during C. elegans dauer exit

During their lifetime, animals must adapt their behavior to survive in changing environments. This ability requires the nervous system to adjust through dynamic expression of neurotransmitters and receptors but also through growth, spatial reorganization and connectivity while integrating external stimuli. For instance, despite having a fixed neuronal cell lineage, the nematode Caenorhabditis elegans nervous system remains plastic throughout its development. Here, we focus on a specific example of nervous system plasticity, the C. elegans dauer exit decision. Under unfavorable conditions, larvae will enter the non-feeding and non-reproductive dauer stage and adapt their behavior to cope with a new environment. Upon improved conditions, this stress resistant developmental stage is actively reversed to resume reproductive development. However, how different environmental stimuli regulate the exit decision mechanism and thereby drive the larvas behavioral change is unknown. To fill this gap, we developed a new open hardware method for long-term imaging (12h) of C. elegans larvae. We identified dauer-specific behavioral motifs and characterized the behavioral trajectory of dauer exit in different environments to identify key decision points. Combining long-term behavioral imaging with transcriptomics, we find that bacterial ingestion triggers a change in neuropeptide gene expression to establish post-dauer behavior. Taken together, we show how a developing nervous system can robustly integrate environmental changes, activate a developmental switch and adapt the organisms behavior to a new environment.

animal behavior and cognition↗

bioRxiv2022

Urinary single-cell sequencing captures intrarenal injury and repair processes in human acute kidney injury

Acute kidney injury (AKI) is a major health issue, the outcome of which depends primarily on damage and reparative processes of tubular epithelial cells (TEC). Mechanisms underlying AKI remain incompletely understood, specific therapies are lacking and monitoring the course of AKI in clinical routine is confined to measuring urine output and plasma levels of filtration markers. Here we demonstrate feasibility and potential of a novel approach to assess the cellular and molecular dynamics of AKI by establishing a robust urine-to-single cell RNA sequencing (scRNAseq) pipeline for excreted kidney cells via flow cytometry sorting. We analyzed 42,608 single cell transcriptomes of 40 urine samples from 32 AKI patients and compared our data with reference material from human AKI post-mortem biopsies and published mouse data. We demonstrate that TEC transcriptomes mirror intrarenal pathology and reflect distinct injury and repair processes, including oxidative stress, inflammation, and tissue rearrangement. We also describe an AKI-specific abundant urinary excretion of progenitor-like cells. In conclusion, single cell transcriptomics of kidney cells excreted in urine provides non-invasive, unprecedented insight into cellular processes underlying AKI, thereby opening novel opportunities for target identification, AKI sub-categorization and monitoring of natural disease course and interventions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/479234v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@e7175corg.highwire.dtl.DTLVardef@1aed1e6org.highwire.dtl.DTLVardef@5655f4org.highwire.dtl.DTLVardef@2512a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

MicroRNAs are deeply linked to the emergence of the complex octopus brain

Soft-bodied cephalopods such as the octopus are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled mRNAs and small RNAs in 18 tissues of the common octopus. We show that the major RNA innovation of soft-bodied cephalopods is a massive expansion of the miRNA gene repertoire. These novel miRNAs were primarily expressed in neuronal tissues, during development, and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, strikingly, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains. One-Sentence SummarymiRNAs are deeply linked to the emergence of complex brains.

evolutionary biology↗

bioRxiv2022

Optocoder: computational decoding of spatially indexed bead arrays

MotivationAdvancing technologies that quantify gene expression in space are transforming contemporary biology research. A class of spatial transcriptomics methods uses barcoded bead arrays that are optically decoded via microscopy and are later matched to sequenced data from the respective libraries. To obtain a detailed representation of the tissue in space, robust and efficient computational pipelines are required to process microscopy images and accurately basecall the bead barcodes. ResultsOptocoder is a computational framework that processes microscopy images to decode bead barcodes in space. It efficiently aligns images, detects beads, and corrects for confounding factors of the fluorescence signal, such as crosstalk and phasing. Furthermore, Optocoder employs supervised machine learning to strongly increase the number of matches between optically decoded and sequenced barcodes. We benchmark Optocoder using data from an in-house spatial transcriptomics platform, as well as from Slide-Seq(V2), and we show that it efficiently processes all datasets without modification. AvailabilityOptocoder is publicly available, open-source and provided as a stand-alone Python package on GitHub: https://github.com/rajewsky-lab/optocoder

bioinformatics↗

bioRxiv2022

Cell type diversity in a developing octopus brain

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The cell types within the octopus brain that control their amazingly rich behavioral repertoire are still unknown. Here we profile cell diversity of the paralarval Octopus vulgaris brain to build a comprehensive cell type atlas that comprises mostly neural cells, as well as multiple glial subtypes, endothelial cells and fibroblasts. Moreover, we spatially map cell types within the octopus brain, including vertical and optic lobe cell types. Investigation of cell type conservation reveals a shared gene signature between glial cells of mice, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila. Taken together, our data sheds light on cell type diversity and evolution of the complex octopus brain. Highlights & Key findingsO_LICharacterization of different cell types present in the early paralarval brain C_LIO_LICross-species comparisons reveal a conserved glial gene expression signature C_LIO_LIVertical lobe amacrine cells in octopus have molecular similarities to fly Kenyon cells C_LIO_LIHomeobox genes are defining transcription factors for cell type identity C_LIO_LIRecently expanded gene families may underlie cellular diversification C_LI

developmental biology↗

bioRxiv2021

Transcriptomic responses of the human kidney to acute injury at single cell resolution

BackgroundAcute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. MethodsWe performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 hours after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. ResultsHigh-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. ConclusionThe study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies.

molecular biology↗

bioRxiv2021

Image-based representation of massive spatial transcriptomics datasets

We present STIM, an imaging-based computational framework focused on visualizing and aligning high-throughput spatial sequencing datasets. STIM is built on the powerful, scalable ImgLib2 and BigDataViewer (BDV) image data frameworks and thus enables novel development or transfer of existing computer vision techniques to the sequencing domain characterized by datasets with irregular measurement-spacing and arbitrary spatial resolution, such as spatial transcriptomics data generated by multiplexed targeted hybridization or spatial sequencing technologies. We illustrate STIMs capabilities by representing, interactively visualizing, 3D rendering, automatically registering and segmenting publicly available spatial sequencing data from 13 serial sections of mouse brain tissue, and from 19 sections of a human metastatic lymph node.

bioinformatics↗

bioRxiv2021

Rapid nuclear deadenylation of mammalian messenger RNA

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition, and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened within minutes after transcription. Our data show that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs escape rapid nuclear deadenylation. Modelling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggest that nuclear deadenylation is a key mechanism for regulating mRNA stability, abundance, and subcellular localization.

systems biology↗

bioRxiv2021

Spacemake: processing and analysis of large-scale spatial transcriptomics data

Spatial sequencing methods increasingly gain popularity within RNA biology studies. State-of-the-art techniques can read mRNA expression levels from tissue sections and at the same time register information about the original locations of the molecules in the tissue. The resulting datasets are processed and analyzed by accompanying software which, however, is incompatible across inputs from different technologies. Here, we present spacemake, a modular, robust and scalable spatial transcriptomics pipeline built in snakemake and python. Spacemake is designed to handle all major spatial transcriptomics datasets and can be readily configured to run on other technologies. It can process and analyze several samples in parallel, even if they stem from different experimental methods. Spacemakes unified framework enables reproducible data processing from raw sequencing data to automatically generated downstream analysis reports. Moreover, spacemake is built with a modular design and offers additional functionality such as sample merging, saturation analysis and analysis of long-reads as separate modules. Moreover, spacemake employs novoSpaRc to integrate spatial and single-cell transcriptomics data, resulting in increased gene counts for the spatial dataset. Spacemake is open-source, extendable and can be readily integrated with existing computational workflows.

bioinformatics↗

bioRxiv2021

Optogenetic perturbations of RNA expression in tissue space

Organoids derived from stem cells become increasingly important to study human development and to model disease. However, methods are needed to control and study spatio-temporal patterns of gene expression in organoids. To this aim, we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatio-temporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. High-resolution spatial transcriptomic and single-cell analyses showed that this local induction was sufficient to generate stereotypically patterned organoids in three dimensions and revealed new insights into SHHs contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

bioengineering↗

bioRxiv2021

The neuroinflammatory interleukin-12 signaling pathway drives Alzheimer's disease-like pathology by perturbing oligodendrocyte survival and neuronal homeostasis

Alzheimers disease (AD) is characterized by deposition of pathological amyloid-{beta} (A{beta}) and tau protein aggregates and involves chronic neuroinflammation, ultimately leading to neurodegeneration and cognitive decline. Central in AD-related neuroinflammation is the proinflammatory interleukin-12 (IL-12)/IL-23 signaling pathway whose inhibition has been shown to attenuate pathology and cognitive defects in AD-like mice. In order to explore which cell types are involved in this neuroinflammatory cascade, we used single-nuclei RNA sequencing in AD-like APPPS1 mice lacking or harboring IL-12/IL-23 signaling. We found Il12b transcripts encoding the common p40 subunit of IL-12/IL-23 signaling to be expressed preferentially, but not exclusively, in microglia in an AD-specific manner. In contrast, transcripts for the other subunits of the IL-12 signaling pathway were expressed constitutively in neurons and oligodendrocytes irrespective of AD pathology, while transcripts for IL-23 were almost undetectable. Notably, genetic ablation of IL-12/IL-23 signaling did not affect the inflammatory gene expression profile of the AD-specific disease associated microglia (DAM), but reversed the loss of mature myelin-producing oligodendrocytes and alterations in neuronal homeostasis in APPPS1 mice. Taken together, our results reveal that IL-12, but not IL-23 is the main driver of AD-specific IL-12/IL-23 neuroinflammation, which alters neuronal and oligodendrocyte functions. Given that drugs targeting IL-12 already exist, our data may foster first clinical trials in AD subjects using this novel neuroimmune target.

neuroscience↗

bioRxiv2021

Neurodegeneration in human brain organoids infected with herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) infection of the nervous system may lead to brain damage, including neurodegeneration. However, lack of suitable experimental models hinders understanding molecular mechanisms and cell-type-specific responses triggered by HSV-1. Here, we infected human brain organoids with HSV-1. Known features of HSV-1 infection such as alteration of neuronal electrophysiology and induction of antisense transcription were confirmed. Full-length mRNA-sequencing revealed aberrant 3 end formation and poly(A)-tail lengthening. Single-cell RNA-seq and spatial transcriptomics uncovered changes in the cellular composition of the infected organoids caused by viral replication and dysregulation of molecular pathways in cell-type specific manner. Furthermore, hallmarks of early neurodegeneration were observed, namely extracellular matrix disruption, STMN2 and TARDBP/TDP43 downregulation, and upregulation of the AD-related non-coding RNA BC200/BCYRN1. These hallmarks were weaker/absent when infecting with a mutant HSV-1 control. Together, our data indicate that brain organoids serve as a powerful model to study mechanisms of HSV-1-driven neurodegeneration.

neuroscience↗

bioRxiv2020

Parallel genetics of regulatory sequences in vivo

Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. Traditional reporter assays inform about function of individual regulatory elements, typically in isolation. However, regulatory elements must ultimately be understood by perturbing them within their genomic environment and developmental- or tissue-specific contexts. This is technically challenging; therefore, few regulatory elements have been characterized in vivo. Here, we used inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. To quantify the consequences of mutations on expression, we developed a targeted RNA sequencing strategy across hundreds of mutant animals. We were also able to systematically and quantitatively assign fitness cost to mutations. Finally, we identified and characterized sequence elements that strongly regulate phenotypic traits. Our approach enables highly parallelized, functional analysis of regulatory sequences in vivo.

genetics↗

bioRxiv2020

Bulk and single-cell gene expression profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for therapeutic intervention

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health threat with more than two million infected people since its emergence in late 2019. Detailed knowledge of the molecular biology of the infection is indispensable for understanding of the viral replication, host responses, and disease progression. We provide gene expression profiles of SARS-CoV and SARS-CoV-2 infections in three human cell lines (H1299, Caco-2 and Calu-3 cells), using bulk and single-cell transcriptomics. Small RNA profiling showed strong expression of the immunity and inflammation-associated microRNA miRNA-155 upon infection with both viruses. SARS-CoV-2 elicited approximately two-fold higher stimulation of the interferon response compared to SARS-CoV in the permissive human epithelial cell line Calu-3, and induction of cytokines such as CXCL10 or IL6. Single cell RNA sequencing data showed that canonical interferon stimulated genes such as IFIT2 or OAS2 were broadly induced, whereas interferon beta (IFNB1) and lambda (IFNL1-4) were expressed only in a subset of infected cells. In addition, temporal resolution of transcriptional responses suggested interferon regulatory factors (IRFs) activities precede that of nuclear factor-{kappa}B (NF-{kappa}B). Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 charperone activity by Tanespimycin/17-N-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in a reduction of viral replication, and of TNF and IL1B mRNA levels. In summary, our study established in vitro cell culture models to study SARS-CoV-2 infection and identified HSP90 protein as potential drug target for therapeutic intervention of SARS-CoV-2 infection.

systems biology↗

bioRxiv2019

The transcriptome dynamics of single cells during the cell cycle

Despite advances in single-cell data analysis, the dynamics and topology of the cell cycle in high-dimensional gene expression space remains largely unknown. Here, we use a linear analysis of transcriptome data to reveal that cells move along a circular trajectory in transcriptome space during the cell cycle. This movement occurs largely independently from other cellular processes. Non-cycling gene expression (changes in environment or epigenetic state) adds a third dimension and causes helical motion on a hollow cylinder. The circular trajectory shape indicates minimal acceleration of transcription, i.e. the cell cycle has evolved to minimize changes of transcriptional activity and its entailing regulatory effort. Thus, we uncover a general design principle of the cell cycle that may be of relevance to many other cellular differentiation processes. One Sentence SummaryCells traverse high-dimensional gene expression space in a 2D circular motion, thus minimizing changes of expression changes ("Acceleration").

systems biology↗

bioRxiv2019

Predicting cellular position in the Drosophila embryo from Single-Cell Transcriptomics data

Single-cell RNA-seq technologies are rapidly evolving but while very informative, in standard scRNAseq experiments the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to keep the localization of the cells have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To bridge the gap, we organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as gold standard genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize rare subpopulations of cells. Selection of predictor genes was essential for this task and such genes showed a relatively high expression entropy, high spatial clustering and the presence of prominent developmental genes such as gap and pair-ruled genes and tissue defining markers.

systems biology↗

bioRxiv2019

Single-cell RNA-sequencing of Herpes simplex virus 1-infected cells identifies NRF2 activation as an antiviral program

Herpesvirus infection initiates a range of perturbations in the host cell, which remain poorly understood at the level of individual cells. Here, we quantified the transcrips of single human primary fibroblasts during the first hours of lytic infection with HSV-1. By applying a generalizable analysis scheme, we defined a precise temporal order of early viral gene expression and found unexpected bifurcations and bottlenecks. We identified individual host cell genes and pathways relevant in early infection by combining three different computational approaches: gene and pathway overdispersion analysis, prediction of cell-state transition probabilities as well as future cell states. One transcriptional program, which was turned on in infected cells and correlated with increased resistance to infection, implicated the transcription factor NRF2. Consequently, Bardoxolone methyl, a known NRF2 agonist, impaired virus production, suggesting that NRF2 activation restricts the progression of viral infection. Our study provides novel insights into early stages of HSV-1 infection and serves as a general blueprint for the investigation of heterogenous cell states in virus infection.

systems biology↗

bioRxiv2019

SURF1 mutations causative of Leigh syndrome impair human neurogenesis

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

neuroscience↗