A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ1. However, organizing the resulting taxonomy of cell types into lineage trees to understand developmental origin of cells remains challenging. Here we present LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences)—a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.
Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B‐cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB‐exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti‐MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies.
The adult zebrafish heart has a high capacity for regeneration following injury. However, the composition of the regenerative niche has remained largely elusive. Here, we dissected the diversity of activated cell states in the regenerating zebrafish heart based on single-cell transcriptomics and spatiotemporal analysis. We observed the emergence of several transient cell states with fibroblast characteristics following injury, and we outlined the proregenerative function of collagen-12-expressing fibroblasts. To understand the cascade of events leading to heart regeneration, we determined the origin of these cell states by high-throughput lineage tracing. We found that activated fibroblasts were derived from two separate sources: the epicardium and the endocardium. Mechanistically, we determined Wnt signalling as a regulator of the endocardial fibroblast response. In summary, our work identifies specialized activated fibroblast cell states that contribute to heart regeneration, thereby opening up possible approaches to modulating the regenerative capacity of the vertebrate heart.
3 equal contribution 4 correspondence: janphilipp.junker@mdc-berlin.de (J.P.J.), a.vanoudenaarden@hubrecht.eu (A.v.O.) A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scartrace, a strategy for massively parallel whole-organism lineage tracing based on Cas9 induced genetic scars in the zebrafish.The timing of each cell division and the fate of the progeny define the lineage of an organism. Analysis of the lineage history of cell populations can reveal the developmental origin and the clonality of cell populations 1 . Genetically encoded fluorescent proteins are widely used as lineage markers 2,3 , but due to limited spectral resolution this approach has mostly been restricted to tracking the lineage of a relatively small subset of cells. Recent progress in live imaging has allowed for following many individual cells over time in optically transparent samples such as early fly and zebrafish embryos 4,5 . Nevertheless, direct observation of all cell divisions is generally only possible at the earliest developmental stages. RNA sequencing has emerged as a powerful method for systematic expression profiling of single cells and for computational inference of differentiation dynamics 6-8 . However, our ability to harness the enormous multiplexing capacity of high-throughput sequencing for lineage tracing has so far been lagging behind, despite pioneering studies in the hematopoietic system based on viral barcoding 9,10 or transposon
2 equal contributions 3 correspondence: janphilipp.junker@mdc-berlin. de (JPJ) A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many different cell types. Single-cell RNAsequencing (scRNA-seq) has become a widely-used method due to its ability to identify all cell types in a tissue or organ in a systematic manner 1-3 . However, a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here, we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNAseq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses, LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences) can be used as a systematic approach for identifying the lineage origin of novel cell types, or of known cell types under different conditions. Measuring lineage relationships between cell types is crucial for understanding fundamental mechanisms of cell differentiation in development and disease 4,5 . In early development and in adult systems with a constant turnover of cells, short-term lineage predictions can be computed directly on scRNA-seq data by ordering cells along pseudo-temporal trajectories according to transcriptome similarity 6-8 . However, for identifying the developmental origin of cells in the adult body, additional information is required. Genetically encoded fluorescent proteins are widely used as lineage markers 9,10 , but due to limited spectral resolution, optical lineage tracing methods have mostly been restricted to relatively small numbers of cells. Pioneering studies based on viral barcoding 11,12 , transposon integration sites 13 , microsatellite repeats 14 , somatic Author contributions JPJ, BS and BH conceived and designed the project. BH developed the experimental protocol, and BH and NM performed experiments. BS developed computational methods and analyzed the data. JPJ guided experiments and analysis. JPJ and BS wrote the manuscript, with input from all other authors. All authors discussed and interpreted results.
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