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Chronological analysis with fluorescent timer reveals unique features of newly generated β-cells.

ABSTRACT: Although numerous studies have uncovered the molecular mechanisms regulating pancreas development, it remains to be clarified how β-cells arise from progenitors and how recently specified β-cells are different from preexisting β-cells. To address these questions, we developed a mouse model in which the insulin 1 promoter drives DsRed-E5 Timer fluorescence that shifts its spectrum over time. In transgenic embryos, green fluorescent β-cells were readily detected by FACS and could be distinguished from mature β-cells only until postnatal day 0, suggesting that β-cell neogenesis occurs exclusively during embryogenesis. Transcriptome analysis with green fluorescent cells sorted by FACS demonstrated that newly differentiated β-cells highly expressed progenitor markers, such as Sox9, Neurog3, and Pax4, showing the progenitor-like features of newborn β-cells. Flow cytometric analysis of cell cycle dynamics showed that green fluorescent cells were mostly quiescent, and differentiated β-cells were mitotically active. Thus, the precise temporal resolution of this model enables us to dissect the unique features of newly specified insulin-producing cells, which could enhance our understanding of β-cell neogenesis for future cell therapy.

SUBMITTER: Miyatsuka T

PROVIDER: S-EPMC4392905 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Chronological analysis with fluorescent timer reveals unique features of newly generated β-cells.

Miyatsuka Takeshi T Matsuoka Taka-aki TA Sasaki Shugo S Kubo Fumiyo F Shimomura Iichiro I Watada Hirotaka H German Michael S MS Hara Manami M

Diabetes 20140516 10

Although numerous studies have uncovered the molecular mechanisms regulating pancreas development, it remains to be clarified how β-cells arise from progenitors and how recently specified β-cells are different from preexisting β-cells. To address these questions, we developed a mouse model in which the insulin 1 promoter drives DsRed-E5 Timer fluorescence that shifts its spectrum over time. In transgenic embryos, green fluorescent β-cells were readily detected by FACS and could be distinguished ...[more]

PMID: 24834978

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Project description:The molecular mechanisms governing the differentiation of neural stem cells (NSCs) into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg) mice in which production of the fluorescent protein Kusabira-Orange (KOr) is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to green. This two-color initial version of the "Color Timer" mouse system will provide a powerful new tool for neurogenesis research.

Project description:Background & aimsThe lymphatic system plays crucial roles in maintaining fluid balance and immune regulation. Studying the liver lymphatics has been considered challenging, as common lymphatic endothelial cell (LyEC) markers are expressed by other liver cells. Additionally, isolation of sufficient numbers of LyECs has been challenging because of their extremely low abundance (<0.01% of entire liver cell population) in a normal liver.MethodsPotential LyEC markers was identified using our published single-cell RNA sequencing (scRNA-seq) dataset (GSE147581) in mouse livers. Interleukin-7 (IL7) promoter-driven green fluorescent protein knock-in heterozygous mice were used for the validation of IL7 expression in LyECs in the liver, for the development of liver LyEC isolation protocol, and generating liver ischemia/reperfusion (I/R) injury. Scanning electron microscopy was used for the structural analysis of LyECs. Changes in LyEC phenotypes in livers of mice with I/R were determined by RNA-seq analysis.ResultsThrough scRNA-seq analysis, we have identified IL7 as an exclusive marker for liver LyECs, with no overlap with other liver cell types. Based on IL7 expression in liver LyECs, we have established an LyEC isolation method and observed distinct cell surface structures of LyECs with fenestrae and cellular pores (ranging from 100 to 400 nm in diameter). Furthermore, we identified LyEC genes that undergo alterations during I/R liver injuries.ConclusionsThis study not only identified IL7 as an exclusively expressed gene in liver LyECs, but also enhanced our understanding of LyEC structures and demonstrated transcriptomic changes in injured livers.Impact and implicationsUnderstanding the lymphatic system in the liver is challenging because of the absence of specific markers for liver LyEC. This study has identified IL7 as a reliable marker for LyECs, enabling the development of an effective method for their isolation, elucidating their unique cell surface structure, and identifying LyEC genes that undergo changes during liver damage. The development of IL7 antibodies for detecting it in human liver specimens will further advance our understanding of the liver lymphatic system in the future.

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Chronological analysis with fluorescent timer reveals unique features of newly generated β-cells.

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Chronological analysis with fluorescent timer reveals unique features of newly generated β-cells.

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