Project description:Lineage tracing of individual cells during directed differentiation human iPSC into alveolospheres was performed using a lentiviral barcode labeling system (Weinreb et al., 2020) as described in Hurley et al., 2020.
Project description:Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-Seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-Seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ propDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs, and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s
Project description:Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell-type in terms of their origins and functional effects in vivo. Methods: Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen inducible Cre for cellular lineage tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Results: Lineage tracing with 4 additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin+ myofibroblasts reduces collagen production and scar formation after myocardial infarction. Periostin-traced myofibroblasts also revert back to a less activated state upon injury resolution. Conclusions: Our results define the myofibroblast as a periostin-expressing cell-type necessary for adaptive healing and fibrosis in the heart, which arises from Tcf21+ tissue-resident fibroblasts. Fluidigm C1 whole genome transcriptome analysis of lineage mapped cardiac myofibroblasts
Project description:We investigate non-genomic mechanisms determining cellular response to EGFR inhibitors in triple negative breast cancer (TNBC). We integrate methods for cellular barcoding and single-cell transcriptomics to enable cell lineage tracing and explore the subclonal evolution of adaptation in an established preclinical model of TNBC in response to incremental concentrations of Afatinib, a second generation EGFR-TKI that irreversibly inhibits both EGFR and HER2. Retrospective lineage tracing data analysis uncovered a pre-existing subpopulation of rare Afatinib-tolerant cells displaying distinct biological features, such as elevated mRNA levels of the IGFBP2 gene. Furthermore, we investigated temporal coordination of transcriptional programs in drug resistant clones with high replication fitness by reordering cells along a pseudotime trajectory. Interestingly, it revealed the activation of biological processes, such as fatty acid metabolism, which have previously been linked to EGFR-TKIs resistance mechanisms.
Project description:Multicellular systems develop from single cells through a lineage, but current lineage tracing approaches scale poorly to whole organisms. Here we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, records lineage relationships in the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable, and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult zebrafish organs derive from relatively few embryonic progenitors. Genome editing of synthetic target arrays for lineage tracing (GESTALT) will help generate large-scale maps of cell lineage in multicellular systems.
Project description: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 scar-trace, a strategy for massively parallel whole-organism lineage tracing based on Cas9 induced genetic scars in the zebrafish.
Project description:Cell plasticity is a core biological process underlying a myriad of molecular and cellular events taking place throughout organismal development and evolution. It has been postulated that cellular systems thrive to balance the organization of meta-stable states underlying this phenomenon, thus maintaining a degree of populational homeostasis compatible with an ever-changing environment and, thus, life. Notably, albeit circumstantial evidence has been gathered in favour of the latter conceptual framework, a direct observation of meta-state dynamics and the biological consequences of such a process in generating non-genetic clonal diversity and divergent phenotypic output remains largely unexplored. To fill this void, we developed a lineage-tracing technology termed Barcode-decay Lineage Tracing-Seq. BdLT-Seq is based on episome-encoded molecular identifiers that, supported by the dynamic decay of the tracing information upon cell division, ascribe directionality to a cell lineage tree in a time-resolved manner whilst directly coupling non-genetic molecular features to phenotypes in comparable genomic landscapes. Herein we show that cell transcriptome states are both inherited and dynamically reshaped following constrained rules encoded within the cell lineage, leading to intra-clonal non-genetic diversity in basal growth conditions and while adjusting populational phenotypic output upon oncogene activation and throughout the process of reversible resistance to therapeutic cues.
Project description:Cell plasticity is a core biological process underlying a myriad of molecular and cellular events taking place throughout organismal development and evolution. It has been postulated that cellular systems thrive to balance the organization of meta-stable states underlying this phenomenon, thus maintaining a degree of populational homeostasis compatible with an ever-changing environment and, thus, life. Notably, albeit circumstantial evidence has been gathered in favour of the latter conceptual framework, a direct observation of meta-state dynamics and the biological consequences of such a process in generating non-genetic clonal diversity and divergent phenotypic output remains largely unexplored. To fill this void, we developed a lineage-tracing technology termed Barcode-decay Lineage Tracing-Seq. BdLT-Seq is based on episome-encoded molecular identifiers that, supported by the dynamic decay of the tracing information upon cell division, ascribe directionality to a cell lineage tree in a time-resolved manner whilst directly coupling non-genetic molecular features to phenotypes in comparable genomic landscapes. Herein we show that cell transcriptome states are both inherited and dynamically reshaped following constrained rules encoded within the cell lineage, leading to intra-clonal non-genetic diversity in basal growth conditions and while adjusting populational phenotypic output upon oncogene activation and throughout the process of reversible resistance to therapeutic cues.
Project description:Cell plasticity is a core biological process underlying a myriad of molecular and cellular events taking place throughout organismal development and evolution. It has been postulated that cellular systems thrive to balance the organization of meta-stable states underlying this phenomenon, thus maintaining a degree of populational homeostasis compatible with an ever-changing environment and, thus, life. Notably, albeit circumstantial evidence has been gathered in favour of the latter conceptual framework, a direct observation of meta-state dynamics and the biological consequences of such a process in generating non-genetic clonal diversity and divergent phenotypic output remains largely unexplored. To fill this void, we developed a lineage-tracing technology termed Barcode-decay Lineage Tracing-Seq. BdLT-Seq is based on episome-encoded molecular identifiers that, supported by the dynamic decay of the tracing information upon cell division, ascribe directionality to a cell lineage tree in a time-resolved manner whilst directly coupling non-genetic molecular features to phenotypes in comparable genomic landscapes. Herein we show that cell transcriptome states are both inherited and dynamically reshaped following constrained rules encoded within the cell lineage, leading to intra-clonal non-genetic diversity in basal growth conditions and while adjusting populational phenotypic output upon oncogene activation and throughout the process of reversible resistance to therapeutic cues.
Project description:Cell plasticity is a core biological process underlying a myriad of molecular and cellular events taking place throughout organismal development and evolution. It has been postulated that cellular systems thrive to balance the organization of meta-stable states underlying this phenomenon, thus maintaining a degree of populational homeostasis compatible with an ever-changing environment and, thus, life. Notably, albeit circumstantial evidence has been gathered in favour of the latter conceptual framework, a direct observation of meta-state dynamics and the biological consequences of such a process in generating non-genetic clonal diversity and divergent phenotypic output remains largely unexplored. To fill this void, we developed a lineage-tracing technology termed Barcode-decay Lineage Tracing-Seq. BdLT-Seq is based on episome-encoded molecular identifiers that, supported by the dynamic decay of the tracing information upon cell division, ascribe directionality to a cell lineage tree in a time-resolved manner whilst directly coupling non-genetic molecular features to phenotypes in comparable genomic landscapes. Herein we show that cell transcriptome states are both inherited and dynamically reshaped following constrained rules encoded within the cell lineage, leading to intra-clonal non-genetic diversity in basal growth conditions and while adjusting populational phenotypic output upon oncogene activation and throughout the process of reversible resistance to therapeutic cues.