Project description:As the biology of hematopoietic stem cells (HSCs) has been predominantly studied under transplantation conditions, it has been challenging to study dynamic HSC behaviors in their native niche given under steady state conditions. Here, we describe the generation of a double knock-in fluorescent reporter that restricts the reporter labeling exclusively to the LT-HSC compartment. Single cell RNAseq comparing previously published LSK signatures (GEO: GSE90742) to our sorted fluorescently labeled cells (sorted without the use of any other cell surface markers) demonstrates that the fluorescently marked cells in our unique mouse model are exclusively found in the LT-HSC compartment in terms of their overal RNA content. This confirms that the generated mice label LT-HSCs in a highly specific manner.
Project description:The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.
Project description:Here we directly compare for the first time how the longstanding static model of mouse Dentate Gyrus (DG) development compares with a comprehensive high-resolution live-cell multiphoton (live-MPM) imaging approach. We took advantage of multiple fluorescent protein-based cell-type specific reporters to identify Neural Stem Cells (NSC), Intermediate Neurogenic Progenitors (INPs), and Granule Neurons (GNs) to generate live 4D cellular datasets across embryonic, postnatal and adult ages. Live-MPM revealed that INPs and NSCs migrated long distances along multiple routes to seed the SGZ from multiple directions, and from mosaic progenitor zones along the septo-temporal axis of the hippocampus. We found that dynamic INPs processes and interactions contributed to the architecture of both transient and permanent NSC niches during embryonic development, and that INP cellular plasticity is maintained in the adult SGZ NSC niche. We also used a Molecular Systems (MS) approach to determine the basis for maintained INP cellular plasticity that revealed an overlapping signaling network infrastructure based largely on Rho-family mediated regulation of cytoskeletal dynamics. Our combined strategies revealed that dynamic INPs are a major molecular signaling transition state in the adult SGZ, and that Tbr2 expression defines the initial stage of GN commitment. Our novel findings reveal fundamental new insight into one of the most well studied brain regions key for normal cognitive function, and the importance of analyzing the development of live stem cell niches in vivo. In concert with live-cell imaging, we used microarray analysis to identify genes that may be involved in the development of the Dentate Gyrus NSC niche.
Project description:Quantification of cytokine secretion has facilitated advances in the field of immunology, yet the dynamic and varied secretion profiles of individual cells, particularly those obtained from limited human samples, remain obscure. Herein, we introduce a new technology for quantitative live-cell imaging of secretion activity (qLCI-S) that enables high-throughput and dual-color monitoring of secretion activity at the single-cell level over several days, followed by transcriptome analysis of individual cells based on their phenotype. The efficacy of qLCI-S was demonstrated by visualizing the characteristic temporal pattern of cytokine secretion of group 2 innate lymphoid cells, which constitute less than 0.01% of human peripheral blood mononuclear cells, and by revealing minor subpopulations with enhanced cytokine production. The underlying mechanism of this feature was linked to the gene expression of stimuli receptors. This new technology paves the way for exploring gene expression signatures linked to the spatiotemporal dynamic nature of various secretory functions.
Project description:Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as M-bM-^@M-^XM-bM-^@M-^Xtranscription factories,M-bM-^@M-^YM-bM-^@M-^Y but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9- mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments. Examination of genome occupancy of CDK9 and RNAPII that was performed by ChIP-seq in the MEL cell line as described (Soler et al. 2010, 2011) using CDK9 C20 antibody (Santa Cruz Biotechnology, C20, sc-484) and RNA Pol II antibody (Santa Cruz Biotechnology, N20, sc899),
Project description:To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene containing oleic acid analogue, as a bioorthogonal probe. We here show that this lipid can be readily taken up by dendritic cells without toxic side-effects, and that it can subsequently be visualised in live cells using an inverse electron-demand Diels-Alder (IEDDA) reaction with quenched tetrazine fluorophores. Furthermore, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging. Aside from lipid imaging, the uptake and protein modification of sterculic acid can be studied in live cells via chemical proteomics.
Project description:Single-cell RNA-seq (scRNA-seq) has greatly advanced the characterization of cellular heterogeneity in physiological and pathological conditions. However, since cells are lysed and can thus only be profiled once, it is currently impossible to perform downstream functional assays on the same cells and to obtain a direct scRNA-seq-based read-out of transcriptional dynamics. Here, we present Live-seq, a novel single cell transcriptomic biopsy approach based on fluidic force microscopy that differs from other scRNA-seq methods because the cells can be kept alive for further analysis. We demonstrate that live-seq allows the identification of both cell types and states. In addition, when coupled to an inverted optical microscope, it uniquely enables linking single cell transcriptomes to downstream functional properties through real-time imaging.
Project description:Here we directly compare for the first time how the longstanding static model of mouse Dentate Gyrus (DG) development compares with a comprehensive high-resolution live-cell multiphoton (live-MPM) imaging approach. We took advantage of multiple fluorescent protein-based cell-type specific reporters to identify Neural Stem Cells (NSC), Intermediate Neurogenic Progenitors (INPs), and Granule Neurons (GNs) to generate live 4D cellular datasets across embryonic, postnatal and adult ages. Live-MPM revealed that INPs and NSCs migrated long distances along multiple routes to seed the SGZ from multiple directions, and from mosaic progenitor zones along the septo-temporal axis of the hippocampus. We found that dynamic INPs processes and interactions contributed to the architecture of both transient and permanent NSC niches during embryonic development, and that INP cellular plasticity is maintained in the adult SGZ NSC niche. We also used a Molecular Systems (MS) approach to determine the basis for maintained INP cellular plasticity that revealed an overlapping signaling network infrastructure based largely on Rho-family mediated regulation of cytoskeletal dynamics. Our combined strategies revealed that dynamic INPs are a major molecular signaling transition state in the adult SGZ, and that Tbr2 expression defines the initial stage of GN commitment. Our novel findings reveal fundamental new insight into one of the most well studied brain regions key for normal cognitive function, and the importance of analyzing the development of live stem cell niches in vivo.