Project description:Single-cell lineage tracing based on CRISPR/Cas9 gene editing tracks both cell states and genetic lineages at a single-cell resolution. We developed DuTracer, a single-cell lineage tracing method, to avoid rapid target exhaustion and barcode information dropout. Firstly, we introduced this system into HEK293T cells and initiated DuTracer for tracing. We collected single-cell transcriptome and barcode amplicon, demonstrating that this system could record more hierarchical information with minimal inter-site deletions. Subsequently, we applied DuTracer to a mouse embryoid body differentiation model to track lineage transitions of different cell types. We revealed cell potency hierarchy during the stochastic differentiation process of mES cells.
Project description:To obtain new insight into the mammalian kidney, anatomy-guided, single cell RNA sequencing was performed on the adult male and female mouse kidney. Key observations were followed up through a variety of secondary studies including genetic lineage tracing. The data document novel cell diversity, unexpected origins to key cell types, and significant sex differences centered on proximal tubules segments of the nephron and principal cells of the collecting system. A searchable database integrating these data will facilitate an understanding of gene to cell relationships in the normal and diseased mammalian kidney.
Project description:Single cell-based studies have revealed tremendous cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degree of plasticity during organogenesis. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including liver, pancreas, gallbladder, and extra-hepatic bile ducts. Experimental manipulation of various developmental signals in the mouse embryo underscored important cellular plasticity in this embryonic territory. This is also reflected in the existence of human genetic syndromes as well as congenital or environmentally-caused human malformations featuring multiorgan phenotypes in liver, pancreas and gallbladder. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary, and pancreatic structures are not yet established. Here, we combine computational modelling approaches with genetic lineage tracing to assess the tissue dynamics accompanying the ontogeny of the hepato-pancreato-biliary organ system. We show that a multipotent progenitor domain persists at the border between liver and pancreas, even after pancreatic fate is specified, contributing to the formation of several organ derivatives, including the liver. Moreover, using single-cell RNA sequencing we define a specialized niche that possibly supports such extended cell fate plasticity.
Project description:We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multigenerationa lineage tracking under controlled culture conditions. Examination of lineage and cell cycle dependent transcriptional profiles in two cell types
Project description:Plant roots can regenerate after complete excision of their tip, including the stem cell niche, but it is not clear what developmental program mediates such repair. Here, we use a combination of lineage tracing, single-cell RNA-seq, and marker analysis to test different models of tissue reassembly. We show that rapid cell-identity transitions lead to the formation of a new stem cell niche from multiple remnant tissues. The transcriptome of regenerating cells prior to stem cell activation resembled that of the embryonic root progenitor, and regeneration defects were more severe in embryonic versus adult root mutants. Furthermore, the signaling domains of the hormones auxin and cytokinin mirrored their embryonic dynamics, and manipulation of both hormones altered the position of new tissues and stem cell niche markers. Our findings suggest that plant organ regeneration resembles the developmental stages of embryonic patterning and is guided by spatial information laid down by complementary hormone domains. 215 single cells isolated from marked stele tissue (either using WOL or AHP6 promoters), before, at 3h, 16h and 46h post root tip decapitation
Project description:Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a new mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We have developed the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We used the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which revealed that the stem cell gene, Sox2, is not essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.
Project description:Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics enabling high-resolution analysis of cell states while preserving lineage relationships. However, reliance on transcriptional profiling limits their adaptation to an ever-expanding tool kit of multiomic single-cell assays. With CellTag-multi, we present a novel approach for profiling lineage barcodes with single-cell chromatin accessibility without relying on co-assay of transcriptional state, paving the way for truly multiomic lineage tracing. We validate CellTag-multi in mouse hematopoiesis, characterizing transcriptional and epigenomic lineage priming across progenitor cell populations. In direct reprogramming of fibroblasts to endoderm progenitors, we use CellTag-multi to comprehensively link early cell state with reprogramming outcomes, identifying core regulatory programs underlying on-target and off-target reprogramming. Further, we reveal the Transcription Factor (TF) Zfp281 as a novel regulator of reprogramming outcome, biasing cells towards an off-target mesenchymal fate via its regulation of TGF-β signaling. Together, these results establish CellTag-multi as a novel lineage tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming.