Biology subjects

Quake, S. R.

Publications and source records attributed to Quake, S. R..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

A cell atlas of the fly kidney

Like humans, insects rely on precise regulation of their internal environments to survive. The insect renal system consists of Malpighian tubules and nephrocytes that share similarities to the mammalian kidney. Studies of the Drosophila Malpighian tubules and nephrocytes have provided many insights into our understanding of the excretion of waste products, stem cell regeneration, protein reabsorption, and as human kidney disease models. Here, we analyzed single-nucleus RNA sequencing (snRNA-seq) data sets to characterize the cell types of the adult fly kidney. We identified 11 distinct clusters representing renal stem cells (RSCs), stellate cells (SCs), regionally specific principal cells (PCs), garland nephrocyte cells (GCs) and pericardial nephrocytes (PNs). Analyses of these clusters revealed many new interesting features. For example, we found a new, previously unrecognized cell cluster: lower segment PCs that express Esyt2. In addition, we find that the SC marker genes RhoGEF64c, Frq2, Prip and CG10939 regulate their unusual cell shape. Further, we identified transcription factors specific to each cluster and built a network of signaling pathways that are potentially involved in mediating cell-cell communication between Malpighian tubule cell types. Finally, cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia - knowledge that will help the generation of kidney disease models. To visualize this dataset, we provide a web-based resource for gene expression in single cells (https://www.flyrnai.org/scRNA/kidney/). Altogether, our study provides a comprehensive resource for addressing gene function in the fly kidney and future disease studies.

developmental biology↗

bioRxiv2021

Microbial community of recently discovered Auka vent field sheds light on vent biogeography and evolutionary history of thermophily

Hydrothermal vents have been key to our understanding of the limits of life, and the metabolic and phylogenetic diversity of thermophilic organisms. Here we used environmental metagenomics combined with analysis of physico-chemical data and 16S rRNA amplicons to characterize the diversity, temperature optima, and biogeographic distribution of sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California, the deepest known hydrothermal vent field in the Pacific Ocean. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 1/3 of the currently known phylum diversity, showing the microbial community in Auka hydrothermal sediments is highly diverse. Large scale 16S rRNA amplicon screening of 227 sediment samples across the vent field indicates that the MAGs are largely representative of the microbial community. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota (Tharpobacteria) suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison of the average nucleotide identity of the Auka MAGs with MAGs from the Guaymas Basin vent field, found 400 km to the Northwest, revealed a remarkable 20% species-level overlap between vent sites, suggestive of long-distance species transfer and sediment colonization. An adapted version of a recently developed model for predicting optimal growth temperature to the Auka and Guaymas MAGs indicates several of these uncultured microorganisms could grow at temperatures exceeding the currently known upper limit of life. Extending this analysis to reference data shows that thermophily is a trait that has evolved frequently among Bacteria and Archaea. Combined, our results show that Auka vent field offers new perspectives on our understanding of hydrothermal vent microbiology.

genomics↗

bioRxiv2021

The Tabula Sapiens: a single cell transcriptomic atlas of multiple organs from individual human donors

Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual.

cell biology↗

bioRxiv2021

Revealing conserved mechanisms of neurodegeneration in a colonial chordate

Loss of the brains functional ability is a common symptom of aging and neurodegenerative diseases1,2. While the genetic and molecular mechanisms underlying human neurodegeneration are studied in-depth3-6, very little is known about the evolutionary origin of these traits and their involvement in loss of nervous system function in aged invertebrate species. Here we study evolutionarily conserved elements of brain degeneration using the colonial chordate model species Botryllus schlosseri. B. schlosseri reproduces both sexually and asexually7, with adult brains regenerating and degenerating multiple times throughout its adult life. Combining microscopy, transcriptomics and behavioral assays, we characterized adult brains from diverse stages and ages. We found that the number of neurons fluctuates each week, reaching a maximum of [~]1000 cells, and thereafter decreasing while the number of immunocytes increases. Comparing the number of neurons in the adult brains of young and old colonies, we found that older brains are smaller and contain fewer cells. Both during weekly degeneration cycles and overall with age, the decrease in neuron number correlates with reduced response to stimuli and with significant changes in the expression of genes with mammalian homologs associated with neural stem cells and neurodegenerative pathways. These results suggest persistent neural stem cell activity across ages and that cellular and molecular mechanisms of neurodegeneration are evolutionary conserved between tunicates and humans.

neuroscience↗

bioRxiv2021

Progressive Increases in Mesenchymal Cell Diversity Modulate Lung Development and are Attenuated by Hyperoxia

Lung mesenchymal cells play an essential role in development and at birth, as the lung moves from a fluid-filled to an oxygen-rich environment with a stable gas-liquid interface. The molecular details and cellular changes accompanying this highly coordinated process remain incompletely understood. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung in both health and disease to characterize the spectrum of mesenchymal cell states prior to the onset of air-breathing life through late alveolarization to gain insight into their role in orchestrating tissue maturation. We found that cell type diversity in the mesenchymal compartment increases rapidly during normal development but is delayed during neonatal exposure to 80% O2 hyperoxia, a model for bronchopulmonary dysplasia. This study identifies the molecular transitions between populations of mesenchymal cells at discrete developmental time points across fibroblast, smooth muscle, and mural compartments and elucidates the global and cell type-specific effects of neonatal hyperoxia, including the emergence of Acta1+ cells which are absent in normoxic neonatal lungs. These granular insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world.

developmental biology↗

bioRxiv2021

Cell Types of Origin in the Cell Free Transcriptome in Human Health and Disease

Cell-free RNA (cfRNA) can be used to noninvasively measure dynamic and longitudinal physiological changes throughout the body. While there is considerable effort in the liquid biopsy field to determine disease tissue-of-origin, pathophysiology occurs at the cellular level. Here, we describe two approaches to identify cell type contributions to cfRNA. First we used Tabula Sapiens, a transcriptomic cell atlas of the human body to computationally deconvolve the cell-free transcriptome into a sum of cell type specific transcriptomes, thus revealing the spectrum of cell types readily detectable in the blood. Second, we used individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset to create cell type signature scores which can be used to infer the implicated cell types from cfRNA for a variety of diseases. Taken together, these results demonstrate that cfRNA reflects cellular contributions in health and disease from diverse cell types, potentially enabling determination of pathophysiological changes of many cell types from a single blood test.

systems biology↗

bioRxiv2021

RNA splicing programs define tissue compartments and cell types at single cell resolution

More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

bioinformatics↗

bioRxiv2021

Phenotypic diversity and sensitivity to injury of the pulmonary endothelium during a period of rapid postnatal growth

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2), contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury which impairs angiogenesis, induced both common and unique endothelial gene signatures, dysregulated capillary EC cross-talk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

developmental biology↗

bioRxiv2021

Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development

Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells "remember" their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources--the endocardium (Endo) and sinus venosus (SV)--but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary ECs transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well-established but still remodeling, capillary Ecs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.

developmental biology↗

bioRxiv2021

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence background of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating measurement of expression kinetics both in isolated cells and in whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a strong foundation for the development and expansion of transgenic techniques in this unique model system. MotivationThe study of planarians has contributed to advances in our understanding of regeneration, stem cell dynamics, and many other fundamental biological processes. However, the persistent challenge of expressing transgenes in planarians has led to the speculation that they may be resistant to transfection. In this work, we develop methods to express exogenous mRNAs in both isolated planarian cells and whole animals by optimizing delivery techniques, genetic constructs, and detection methods. These methods allow us to study transfection kinetics and post-transcriptional regulation of gene expression in a quantitative manner. Beyond planarian research, this work should also provide a broadly applicable strategy to develop similar tools for animals that are also challenging to modify genetically.

bioengineering↗

bioRxiv2021

Heterogenous impairment of α-cell function in type 2 diabetes is linked to cell maturation state

In diabetes, glucagon secretion from pancreatic -cells is dysregulated. We examined -cells from human donors and mice using combined electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses -cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat-feeding of mice, -cells shift towards a {beta}-cell-like electrophysiologic profile in concert with an up-regulation of the {beta}-cell Na+ channel isoform Scn9a and indications of impaired -cell identity. In human -cells we identify links between cell membrane properties and cell surface signalling receptors, mitochondrial respiratory complex assembly, and cell maturation. Cell type classification using machine learning of electrophysiology data demonstrates a heterogenous loss of electrophysiologic identity in -cells from donors with T2D. Indeed, a sub-set of -cells with impaired exocytosis is defined by an enrichment in progenitor markers suggesting important links between -cell maturation state and dysfunction in T2D. Key findingsO_LI-cell exocytosis is suppressed by glucose-dependent inhibition of P/Q-type Ca2+ currents C_LIO_LIDysfunction of -cells in type 2 diabetes is associated with a {beta}-cell-like electrophysiologic signature C_LIO_LIPatch-seq links maturation state, the mitochondrial respiratory chain, and cell surface receptor expression to -cell function C_LIO_LI-cell dysfunction occurs preferentially in cells enriched in endocrine lineage markers C_LI

cell biology↗

bioRxiv2020

Inhibiting USP16 rescues stem cell aging and memory in an Alzheimer's model

Alzheimers disease (AD) is a progressive neurodegenerative disease observed with aging that represents the most common form of dementia. To date, therapies targeting end-stage disease plaques, tangles, or inflammation have limited efficacy. Therefore, we set out to identify an earlier targetable phenotype. Utilizing a mouse model of AD and human fetal cells harboring mutant amyloid precursor protein, we show cell intrinsic neural precursor cell (NPC) dysfunction precedes widespread inflammation and amyloid plaque pathology, making it the earliest defect in the evolution of disease. We demonstrate that reversing impaired NPC self-renewal via genetic reduction of USP16, a histone modifier and critical physiological antagonist of the Polycomb Repressor Complex 1, can prevent downstream cognitive defects and decrease astrogliosis in vivo. Reduction of USP16 led to decreased expression of senescence gene Cdkn2a and mitigated aberrant regulation of the BMP pathway, a previously unknown function of USP16. Thus, we reveal USP16 as a novel target in an AD model that can both ameliorate the NPC defect and rescue memory and learning through its regulation of both Cdkn2a and BMP signaling. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Schematic summarizing therapeutic approaches to mitigate the effects of mutant APP through targeting of Cdkn2a, BMI1, USP16 and BMP.

molecular biology↗

bioRxiv2020

Global Age-Specific Patterns of Cyclic Gene Expression Revealed by Tunicate Transcriptome Atlas

Expression levels of circadian clock genes, which regulate 24-hour rhythms of behavior and physiology, have been shown to change with age. However, a study holistically linking aging and circadian gene expression is missing. Using the colonial chordate Botryllus schlosseri, we combined transcriptome sequencing and stem cell-mediated aging phenomena to test how circadian gene expression changes with age. This revealed that B. schlosseri clock and clock-controlled genes oscillate organism-wide, with daily, age-specific amplitudes and frequencies. These age-related, circadian patterns persist at the tissue level, where dramatic variations in cyclic gene expression of tissue profiles link to morphological and cellular aging phenotypes. Similar cyclical expression differences were found in hundreds of pathways associated with known hallmarks of aging, as well as pathways that were not previously linked to aging. The atlas we developed points to alterations in circadian gene expression as a key regulator of aging. One Sentence SummaryThe Ticking Clock: Systemic changes in circadian gene expression correlates with wide-ranging phenotypes of aging

developmental biology↗

bioRxiv2020

Molecular hallmarks of heterochronic parabiosis at single cell resolution

Slowing or reversing biological ageing would have major implications for mitigating disease risk and maintaining vitality. While an increasing number of interventions show promise for rejuvenation, the effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. We performed single-cell RNA-sequencing on 13 organs to reveal cell type specific responses to young or aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, hematopoietic stem cells, hepatocytes, and endothelial cells from multiple tissues appear especially responsive. On the pathway level, young blood invokes novel gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. Intriguingly, we observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it. Altogether, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

genomics↗

bioRxiv2020

Single-cell transcriptomes of developing and adult olfactory receptor neurons in Drosophila

Recognition of environmental cues is essential for the survival of all organisms. Precise transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of Drosophila olfactory receptor neurons (ORNs), thermosensory and hygrosensory neurons from the third antennal segment at an early developmental and adult stage. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using these receptors and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuronal types across multiple developmental stages. Cell-type-specific transcriptomes, in part, reflected axon trajectory choices in early development and sensory modality in adults. Our analysis also uncovered type-specific and broadly expressed genes that could modulate adult sensory responses. Collectively, our data reveal important transcriptomic features of sensory neuron biology and provides a resource for future studies of their development and physiology.

neuroscience↗

bioRxiv2020

Mapping single-cell atlases throughout Metazoa unravels cell type evolution

Comparing single-cell transcriptomic atlases from diverse organisms can elucidate the origins of cellular diversity and assist the annotation of new cell atlases. Yet, comparison between distant relatives is hindered by complex gene histories and diversifications in expression programs. Previously, we introduced the self-assembling manifold (SAM) algorithm to robustly reconstruct manifolds from single-cell data (Tarashansky et al., 2019). Here, we build on SAM to map cell atlas manifolds across species. This new method, SAMap, identifies homologous cell types with shared expression programs across distant species within phyla, even in complex examples where homologous tissues emerge from distinct germ layers. SAMap also finds many genes with more similar expression to their paralogs than their orthologs, suggesting paralog substitution may be more common in evolution than previously appreciated. Lastly, comparing species across animal phyla, spanning mouse to sponge, reveals ancient contractile and stem cell families, which may have arisen early in animal evolution.

bioinformatics↗

bioRxiv2020

An inexpensive semi-automated sample processing pipeline for cell-free RNA

Despite advances in automated liquid handling and microfluidics, preparing samples for RNA sequencing at scale generally requires expensive equipment, which is beyond the reach of many academic labs. Manual sample preparation remains a slow, expensive, and error-prone process. Here, we describe a low-cost, semi-automated pipeline to extract cell-free RNA (cfRNA) that like many RNA isolation protocols, can be decomposed into three subparts - RNA extraction, DNA digestion, and RNA cleaning and concentration. RT-qPCR data using a synthetic spike-in confirms comparable RNA quality as compared to manual sample processing, the gold-standard used in our prior work. The semi-automated pipeline also shows striking improvement in sample throughput (+12x), time spent (-11x), cost (-3x), and biohazardous waste produced (-4x) as compared to its manual counterpart. In total, this protocol enables cfRNA extraction from 96 samples simultaneously in 4.5 hours; in practice, this dramatically improves time to results as demonstrated in our recent work [1] where it was used to process 404 samples in 27 hours. Importantly, any lab already has most of the parts required (manual pipette, corresponding tips and kits) to build a semi-automated sample processing pipeline of their own and would only need to purchase or 3D-print a few extra parts ($5.5K total). This pipeline is also generalizable for many nucleic acid extraction applications, thereby increasing the scale of studies, which can be performed in small research labs.

bioengineering↗

bioRxiv2020

Kinetics and Fidelity of Psychrophilic DNA Polymerases

The discovery of extremophiles helped enable the development of groundbreaking technology such as polymerase chain reaction. Temperature variation is often an essential step of these technology platforms, but the effect of temperature on the error rate of polymerases from different origins is under-explored. Here, we applied high-throughput sequencing to profile the error rates of DNA polymerases from psychrophilic, mesophilic, and thermophilic origins with single-molecule resolution. We found that reaction temperature substantially increases substitution and deletion error rates of psychrophilic and mesophilic DNA polymerases. Our motif analysis shows that the substitution error profiles cluster according to phylogenetic similarity of polymerases, not reaction temperature, thus suggesting that reaction temperature increases global error rate of polymerases independent of sequence context. Intriguingly, we also found that the DNA polymerase I of a psychrophilic bacteria exhibits higher polymerization activity than its mesophilic ortholog across all temperature ranges, including down to -19{degrees}C which is well below the freezing temperature of water. Our results provide a useful reference for how reaction temperature, a crucial parameter of biochemistry, can affect DNA polymerase fidelity in organisms adapted to a wide range of thermal environments.

biochemistry↗