HEMATOPOIETIC STEM AND PROGENITOR CELLS EXHIBIT CELL TYPE SPECIFIC NF-ΰB SIGNALING DYNAMICS UPON INFLAMMATORY STIMULATION

Abstract

Cells sense external stimuli with receptors and further process these stimuli via a variety of signaling pathways consisting of complex biochemical reaction cascades. For decades, cellular signaling was measured at population average level with snapshot data, without taking into account changing signaling activity over time (e.g. signaling dynamics) and cell-to-cell heterogeneity. Recent studies however show the importance of signaling dynamics for controlling future cell fates. As a master regulator of immune responses, the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-κB) coordinates the expression of hundreds of target genes including its own inhibitors NFKBIA and TNFAIP3 in many cell types. While a link between Nf-κB dynamics and future cell state was shown in cell lines, its role in relevant primary cell types remains elusive. Here, we use continuous single-cell quantitative time-lapse imaging of 10 different hematopoietic cell types, including stem and early progenitor cells, from a GFPp65/H2BmCherry reporter mouse line, in custom microfluidic devices, to observe the dynamics of the Nf-κB signaling pathway activity, and investigate how those dynamics influence their fates. We demonstrate that upon an inflammatory challenge by tumor necrosis factor alpha (TNFα), different HSPCs exhibit different and cell type specific Nf-κB dynamics including sustained, transient and oscillatory behaviors. RNA expression kinetics analyses identify molecular regulators responsible for these different dynamics. Additionally, Nf-κB signaling dynamics can identify novel progenitor cell subpopulations with distinct lineage potential. These data demonstrate that signaling dynamics can identify subpopulations of traditionally described cell populations, and suggest a role of Nf-κB signaling dynamics for lineage choice and cell fate control.