DiSNE Movie Visualization and Assessment of Clonal Kinetics Reveal Multiple Trajectories of Dendritic Cell Development

Abstract

A thorough understanding of cellular development is incumbent on assessing the complexities of fate and kinetics of individual clones within a population. Here, we develop a system for robust periodical assessment of lineage outputs of thousands of transient clones and establishment of bona fide cellular trajectories. We appraise the development of dendritic cells (DCs) in fms-like tyrosine kinase 3 ligand culture from barcode-labeled hematopoietic stem and progenitor cells (HSPCs) by serially measuring barcode signatures and visualize these multidimensional data using developmental interpolated t-distributed stochastic neighborhood embedding (DiSNE) time-lapse movies. We identify multiple cellular trajectories of DC development that are characterized by distinct fate bias and expansion kinetics and determine that these are intrinsically programmed. We demonstrate that conventional DC and plasmacytoid DC trajectories are largely separated already at the HSPC stage. This framework allows systematic evaluation of clonal dynamics and can be applied to other steady-state or perturbed developmental systems.

Lineage tracing on transcriptional landscapes links state to fate during differentiation

“…We modified a classical strategy for clonal labeling by lentiviral delivery of inherited DNA barcodes 15,16 , to allow barcode detection using scSeq 17 . The barcode consists of a random 28-mer in the 3' UTR of an enhanced green fluorescent protein (eGFP) transgene under control of a ubiquitous EF1a promoter (Fig 1b).…”

“…Transcripts of eGFP are captured during scSeq, and the barcode is revealed through analysis of sequencing reads. The theoretical number of barcodes for this construct is 4 28 (or ~10 16 ), though bottlenecks in molecular cloning limited the diversity to ~10 6 barcodes, sufficient to label ~10 4 cells in an experiment with <0.5% barcode overlap between clones. We refer to the barcoding construct as LARRY (Lineage And RNA RecoverY).…”

The clonal and molecular aetiology of emergency dendritic cell development

“…Recent single cell transcriptional profiling and lineage tracing studies have highlighted substantial lineage bias and lineage restriction within HSPCs that had previously been assumed to be multi-or oligo-potent (Dykstra et al, 2007;Lee et al, 2017;Lin et al, 2018a;Naik et al, 2013;Nestorowa et al, 2016;Notta et al, 2016;Velten et al, 2017;Yamamoto et al, 2013).…”

SIS-seq, a molecular ‘time machine’, connects single cell fate with gene programs

“…There could be three possibilities for this: a) there were different molecular routes to the same cell type, and there is precedence for this in DC development 26 ; b) the clusters represent different points of the same developmental trajectory, and the snapshot of our clones captured these different stages; and c) there is heterogeneity within our flow cytometric-sorted populations of DCs and the clusters may represent different clonal routes to these putative subpopulations. Recent evidence suggesting Siglec-H + cells can be further separated by CCR9 expression into cDC progenitors and bona fide pDCs 18,27 , so this might be an example of c) as pDCs were not sorted based on CCR9 in these experiments. That is, Cluster 8 may represent genes that actually correlate with cDC bias, and where cells phenotypically ascribed as pDCs were in fact CCR9 -cDC progenitors.…”

“…Relevant to SIS-seq is that DCs can be generated in vitro 16 for ease of assessment of multiple clones, and be derived from single HSPCs 11 . Moreover, cellular barcoding and clone-splitting experiments in vivo 17 and in vitro 18 demonstrated that DC lineage bias, and also individual DC subtype bias, is heterogeneous amongst HSPCs, but restricted within a given clone. These properties fit the first two conditions for SIS-seq to be feasible.…”