Biology subjects

Horns, F.

Publications and source records attributed to Horns, F..

11 recordsLinked to original sources

bioRxiv2026

Regenerative base editing enables deep lineage recording

Reconstructing the lineage histories of individual cells can reveal the dynamics of developmental and disease processes. In engineered recording systems, cells stochastically edit synthetic barcode sequences as they proliferate, creating distinct, heritable edit patterns that can be used to reconstruct the lineage trees relating individual cells in a manner analogous to phylogenetic reconstruction. However, recording depth is often limited by the kinetics of the editing process: the rate of editing declines exponentially over time for an array of independently editable targets, leading to most edits occurring in early generations. Here, we introduce the hypercascade, a regenerative molecular recording system that takes advantage of the predictability of A-to-G base editing to progressively create new target sites over time. The hypercascade packs 4 editable target sites in every 20 bp of sequence, enabling high density information storage. More importantly, the hypercascades regenerative logic leads to an approximately constant rate of mutation accumulation over time. This in turn facilitates reconstruction of deep lineage relationships. We demonstrate this by reconstructing trees spanning 23 days of editing and approximately 17 generations after a single polyclonal engineering step. Finally, simulations show that the hypercascade has the potential to record chromatin state transition dynamics across multiple genomic loci in parallel. The hypercascade thus provides a flexible and broadly useful tool for molecular recording.

bioengineering↗

bioRxiv2024

Simultaneous protein and RNA analysis in single extracellular vesicles, including viruses: SPIRFISH

Interest in using nanoparticles for delivery of therapeutic RNA has been steadily growing, provoking a need to precisely understand their structure and contents. Single-particle and single-molecule analysis techniques provide snapshots of single biological nanoparticles, including viruses, liposomes, and extracellular vesicles (EVs). While existing methods primarily focus on protein detection, RNA delivery is becoming increasingly prevalent. A method to simultaneously detect protein and internal RNA in the same particle would reveal variability in size, structure, and RNA packaging efficiency, enabling optimization of nanoparticle delivery. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor (SP-IRIS) with single-molecule fluorescence in-situ hybridization (smFISH). Using SPIRFISH, we detect HIV-1 envelope protein and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNA within EVs. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles. Teaser: A new single particle analysis technique simultaneously detects specific RNA and protein in biological nanoparticles.

molecular biology↗

bioRxiv2022

Fate Bias and Transcriptional Memory of human B cells

Lineage tracking offers a direct approach to study cell fate determination. In this work we combined single cell transcriptomics and lineage tracing to better understand fate-choice in human B cells. Using the antibody sequence to trace cell lineage during in vitro differentiation, we identified intrinsic proliferative and cell fate biases of B cell subtypes. Clonal analysis revealed that IgM memory B cells were more proliferative than any other B cell subtype, and that cells from the same clone had highly concordant fates. We found that transcriptional memory within clones varies across genes, with strongest persistence in genes related to cell fate determination. Similar persistent transcriptional programs were observed in human plasma cells from bone marrow, suggesting that these programs maintain long-term cell fate in vivo. These results show that cell-intrinsic fate bias influences human B cell differentiation and reveal molecular programs underpinning cell fate determination in B cells.

immunology↗

bioRxiv2021

Lineage Recording Reveals the Phylodynamics, Plasticity and Paths of Tumor Evolution

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth, expansion to neighboring and distal tissues, and therapeutic resistance. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma which enabled us to track tumor evolution from single transformed cells to metastatic tumors at unprecedented resolution. We found that loss of the initial, stable alveolar-type2-like state was accompanied by transient increase in plasticity. This was followed by adoption of distinct fitness-associated transcriptional programs which enable rapid expansion and ultimately clonal sweep of rare, stable subclones capable of metastasizing to distant sites. Finally, we showed that tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates tumor progression by creating novel evolutionary paths. Overall, our study elucidates the hierarchical nature of tumor evolution, and more broadly enables the in-depth study of tumor progression.

cancer biology↗

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

Temporal evolution of single-cell transcriptomes of Drosophila olfactory projection neurons

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological features. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage--neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

neuroscience↗

bioRxiv2019

Transcriptional program of memory B cell activation, broadly binding anti-influenza antibodies, and bystander activation after vaccination revealed by single-cell transcriptomics

Antibody memory protects humans from many diseases. Protective antibody memory responses require activation of transcriptional programs, cell proliferation, and production of antigen-specific antibodies, but how these aspects of the response are coordinated is poorly understood. We profiled the molecular and cellular features of the antibody response to influenza vaccination by integrating single-cell transcriptomics, longitudinal antibody repertoire sequencing, and antibody binding measurements. Single-cell transcriptional profiling revealed a program of memory B cell activation characterized by CD11c and T-bet expression associated with clonal expansion and differentiation toward effector function. Vaccination elicited an antibody clone which rapidly acquired broad high-affinity hemagglutinin binding during affinity maturation. Unexpectedly, many antibody clones elicited by vaccination do not bind vaccine, demonstrating non-specific activation of bystander antibodies by influenza vaccination. These results offer insight into how molecular recognition, transcriptional programs, and clonal proliferation are coordinated in the human B cell repertoire during memory recall.

immunology↗

bioRxiv2019

Coordinating Receptor Expression and Wiring Specificity in Olfactory Receptor Neurons

The ultimate function of a neuron is determined by both its physiology and connectivity, but the transcriptional regulatory mechanisms that coordinate these two features are not well understood1-4. The Drosophila Olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. As in mammals5, each Drosophila ORN class is defined by the expression of a single olfactory receptor or a unique combination thereof, which determines their odor responses, and by the single glomerulus to which their axons target, which determines how sensory signals are represented in the brain6-10. In mammals, the coordination of olfactory receptor expression and wiring specificity is accomplished in part by olfactory receptors themselves regulating ORN wiring specificity11-13. However, Drosophila olfactory receptors do not instruct axon targeting6, 14, raising the question as to how receptor expression and wiring specificity are coordinated. Using single-cell RNA-sequencing and genetic analysis, we identified 33 transcriptomic clusters for fly ORNs. We unambiguously mapped 17 to glomerular classes, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN classes. We found that each ORN expresses ~150 transcription factors (TFs), and identified a master TF that regulates both olfactory receptor expression and wiring specificity. A second TF plays distinct roles, regulating only receptor expression in one class and only wiring in another. Thus, fly ORNs utilize diverse transcriptional strategies to coordinate physiology and connectivity.

neuroscience↗

bioRxiv2018

Activation of polarized cell growth by inhibition of cell polarity

A key feature of cells is the capacity to activate new functional polarized domains contemporaneously to pre-existing ones. How cells accomplish this is not clear. Here, we show that in fission yeast inhibition of cell polarity at pre-existing domains of polarized cell growth is required to activate new growth. This inhibition is mediated by the ERM-related polarity factor Tea3, which antagonizes the activation of the Rho-GTPase Cdc42 by its co-factor Scd2. We demonstrate that Tea3 acts in a phosphorylation-dependent manner controlled by the PAK kinase Shk1 and that, like Scd2, Tea3 is direct substrate of Shk1. Importantly, we show that Tea3 and Scd2 compete for their binding to Shk1, indicating that their biochemical competition for Shk1 underpins their antagonistic roles in controlling polarity. Thus, by preventing pre-existing growth domains from becoming overpowering, Tea3 allows cells to redistribute their polarity-activating machinery to prospective sites and control their timing of activation.

cell biology↗

bioRxiv2017

Signatures of Selection in the Human Antibody Repertoire: Selective Sweeps, Competing Subclones, and Neutral Drift

Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the molecular signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses of five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the linkage between B cell fitness and antibody binding affinity, we demonstrated the potential for using signatures of selection to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering.\n\nOne Sentence SummaryMolecular signatures of somatic evolution reveal that diverse evolutionary processes ranging from strong positive selection to neutral drift sculpt human antibodies.

immunology↗

bioRxiv2017

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-cell RNA Sequencing

How a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely-related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.

genomics↗