ABSTRACT
The molecular program underlying infrequent replication of pancreatic ß-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating ß-cells and determined their transcriptome. Replicating ß-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in ß-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating ß-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in ß-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are "left behind" and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating ß-cells in vivo.
Subject(s)
Cell Division/genetics , Cell Proliferation/genetics , Insulin-Secreting Cells/metabolism , Transcriptome , Animals , Flow Cytometry , Gene Expression Regulation , Insulin-Secreting Cells/cytology , Membrane Potential, Mitochondrial/physiology , Mice , Mice, TransgenicABSTRACT
Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of replicating cells in their in vivo niche.
