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.

Figure 1

Schematic of the MIP-Timer mouse system for sorting early β-cells separately from more-differentiated β-cells. A: In MIP-Timer transgenic mice, DsRed-E5 (pTimer) fluorescent protein is expressed under the control of MIP, shifting its emission spectrum from green to red over time. B: Because newly specified β-cells have only green fluorophores that have recently been translated, they are observed as green fluorescent cells. As the β-cell differentiates, yellow and then red fluorescent molecules appear intracellularly. C: Thus, only β-cells that have recently been generated from their progenitors are observed as green fluorescent cells. When β-cells are supplied by self-renewal, they are never detected as green-dominant cells, having a substantial number of red fluorophores.

Figure 2

Fluorescent patterns in developing pancreata of MIP-Timer mice and mRNA expression profiles for pancreas-specific genes. A: Pancreata from wild-type (top panels) and MIP-Timer mice (bottom panels) were dissociated at E15.5 and E17.5 and at postnatal day 0 (P0) and analyzed using flow cytometry. B: Percentage of green fluorescent cells among total pancreatic cells. C: Relative numbers of green and yellow/red cells. D: MIP-Timer pancreata were dissected at E16.5 and sorted by FACS into three gates (N, non–β-cells; A, early β-cells; B, late β-cells). The sorted cell populations were analyzed by real-time RT-PCR for mRNA encoding endocrine hormones (E–J) and exocrine enzyme (K). All expression levels were normalized to β-glucuronidase. Data in B and E–K are mean ± SE of at least four samples.

Figure 3

Temporal transcriptome analysis for transcriptional regulators in MIP-Timer embryos. The pancreata of MIP-Timer embryos were dissected at E16.5 and sorted by FACS into three gates (N, non–β-cells; A, early β-cells; B, late β-cells). The sorted cell populations were analyzed by real-time RT-PCR for mRNAs encoding transcriptional regulators (A–L). All expression levels were normalized to β-glucuronidase. Expression levels are shown relative to the level in gate N. Data are mean ± SE of four independent experiments. *P < 0.05 vs. population N and B; §P < 0.05 vs. population N and A; #P < 0.05 vs. population A and B.

Figure 4

Proliferation during β-cell neogenesis and maturation. A: Dissociated cells from E17.5 MIP-Timer embryos were stained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry for the three different populations shown in Fig. 2D. B: Percentage of proliferating cells at S/G₂/M phases for each population (N, non–β-cells; A, early β-cells; B, late β-cells). Data are mean ± SE of three samples. C: Differentiated β-cells in gate B were divided equally into three populations according to the intensity of red fluorescence. D: One-third of β-cells with the highest fluorescent intensity (gate B^hi) showed the highest proliferation rate compared with the other two-thirds of β-cells in gate B with lower fluorescence (gate B^lo and B^mid). Data are mean ± SE of three embryos.