Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas - PubMed (original) (raw)
Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas
Yung Hae Kim et al. PLoS Biol. 2015.
Abstract
Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo clonal analysis, we reveal the contribution of individual cells to the global behaviour and demonstrate three modes of progenitor divisions: symmetric renewing, symmetric endocrinogenic, and asymmetric generating a progenitor and an endocrine progenitor. Quantitative analysis shows that the endocrine differentiation process is consistent with a simple model of cell cycle-dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Fig 1. Live imaging reveals both asymmetric and symmetric emergence of NEUROG3 cells.
(A) Scheme summarizing the genetic strategy to visualize PDX1+ pancreatic progenitors for live imaging. (B) Scheme of imaging and analysis. Pancreatic explants from E12.5 Pdx1 tTA/+;tetO-H2B-GFP embryos are cultured, and 3-D time-lapse imaging is done for 18–24 h. Then, the explants are immunostained for markers, and endocrine progenitor (NEUROG3) cells are back- and forward-tracked. (C) Model of pancreatic progenitor divisions. A PDX1+ progenitor can produce two PDX1+/SOX9+ progenitor daughters, two NEUROG3+ endocrine progenitor daughters, or one PDX1+/SOX9+ daughter and one NEUROG3+ daughter. (D) Still images of live imaging in 3-D maximum intensity projection from S3 Movie, illustrating a symmetric (P/P) division producing two progenitor daughters (blue spots). White nuclei correspond to H2B-GFP signal in the cells originating from the pancreas epithelium. (E) Still images from S3 Movie, illustrating an asymmetric (P/N) division producing two daughters with different fates (red spots). (F-I) Images of fixed explant with native GFP and nuclear staining DRAQ5 overlay (F) and immunostained for NEUROG3 (G) and SOX9/aPKC (H). Blue spots correspond to cells in (D) and red spots to cells in (E). Inset in (H) shows high magnification image of SOX9 staining. Note both blue spotted cells are SOX9+, but only one red spotted cell is SOX9_low_ (H), and the other red spotted cell is NEUROG3+ (G). (J) Still images from S4 Movie, demonstrating a symmetric (N/N) division producing two daughters with the same fate (pink spots). (K–N) Images of fixed explant with native GFP (K) and immunostained for NEUROG3 (L) and SOX9/aPKC (M). Pink spots correspond to cells in (J), and both are NEUROG3+/SOX9−. Inset in (L) shows NEUROG3 staining (four NEUROG3+ cells in a row) in high magnification. (O) Analysis of progenitor division patterns from live imaging. Total cell divisions are counted from four cropped positions from four live imaging movies, and fraction of NEUROG3-producing cell divisions is calculated from the corresponding positions (yellow bar over grey bar). NEUROG3-producing divisions (pink, blue, and purple bars) are counted from entire position of four movies. (P) Analysis of NEUROG3 emergence from four live imaging movies. 18.4% ± 5.0% cells emerge through P/N divisions, and 29.8% ± 14.2% through N/N divisions. 29.3% ± 5.9% do not exhibit prior division, and 22.4% ± 10.6% were either lost (17.5% ± 7.7%) or dead (8.9% ± 3.4%). Cells lost or gone out of frame were categorized as indeterminable (purple bar). Numbering denotes elapsed time in h:min, and in the cell division diagrams P indicates progenitor and N, NEUROG3 (D,E,J). Scale bars, 20 μm. Histograms and error bars represent the mean and standard deviation (n = 4). See S2 Table for further data.
Fig 2. Asymmetric and symmetric divisions revealed by in vivo lineage tracing of progenitors at clonal density.
(A) Scheme summarizing the genetic strategy to label HNF1B+ pancreatic progenitors with membrane-localized GFP reporter (mG) for lineage tracing. Upon CRE recombination, membrane-localized Tomato (mT) is excised, allowing mG expression. (B) Schematic overview of the lineage tracing strategy used to trace the fate of progeny from single progenitor cells labelled at clonal density. E13.5 pregnant mice carrying Hnf1bCreER;mT/mG embryos received a single intraperitoneal injection of 0.175 mg 4-OH tamoxifen. After 24 h, pancreata were subjected to whole-mount immunostaining, imaging, and 3-D reconstruction to detect recombined two-cell clones. (C) Model of two-cell clone lineage tracing. A HNF1B+ progenitor can produce two SOX9+ progenitor clones, two NEUROG3+ endocrine progenitor clones, or one PDX1+/SOX9+ and one NEUROG3+ clones. (D) Maximum intensity projection of 3-D reconstructed E14.5 dorsal pancreas after immunostaining for E-CADHERIN, SOX9, and GFP. Arrowheads indicate clones displaying recombination of the mT/mG reporter, detected by anti-GFP immunostaining, while membrane Tomato signal was diminished during staining process. (E) Optical sections from a whole-mount imaged dorsal pancreas demonstrating symmetric generation of SOX9+ progeny (P/P) from a single dividing progenitor cell. (F) Optical sections demonstrating clonal progeny with asymmetric fates, generating one NEUROG3+ and one SOX9+ daughter (P/N). (G) Optical sections demonstrating clonal progeny with symmetric NEUROG3+ fates (N/N). (H) Quantification of two-cell clone fate patterns after in vivo lineage tracing. 244 two-cell clones derived from 22 dorsal pancreata were scored according to SOX9 and NEUROG3 status. Indeterminable refers to clones that could not be categorized because of one or both daughters being both SOX9- and NEUROG3-negative after immunostaining. (I) Quantification of the number of NEUROG3+ cells generated by the different clone patterns. 84 NEUROG3+ cells were detected in 63 NEUROG3+ two-cell clones. Indeterminable refers to clones in which the second daughter was neither NEUROG3- nor SOX9-positive. Scale bars, 100 μm (D) and 3 μm (E–G). Histograms represent the mean (n = 22). See S3 Table for further data.
Fig 3. Extended live imaging with Neurog3-RFP reporter reveals the dynamics of progenitor cell cycle and differentiation.
(A) Scheme summarizing the genetic strategy to visualize PDX1+ pancreatic progenitors and NEUROG3 + endocrine progenitors for live imaging. (B) Model of pancreatic progenitor divisions with a Neurog3-RFP reporter. After second division of self-renewing progenitors, cell cycle length can be obtained. Using the Neurog3-RFP reporter, endocrine differentiation timing and synchrony can be obtained. (C) Still images from S6 Movie, demonstrating an asymmetric (P/R) division producing one Neurog3-RFP+ daughter and two other granddaughters (white spots) from a Pdx1 tTA/+;tetO-H2B-GFP;Neurog3-RFP explant. After the first division, one daughter turns on RFP (before elapsed time 30:00), and later the other daughter divides, producing two granddaughters (at elapsed time 42:12). (D-G) Images of fixed explant with native GFP (D) and immunostained for SOX9/aPKC (E) and Neurog3-RFP (F, staining for MYC-tag). White spots correspond to cells in (C), and one is RFP+ and two granddaughters are SOX9+ (E,F). Inset in (E) shows high magnification image of SOX9 staining. (H) Still images from S7 Movie, demonstrating a symmetric (R/R) division producing two Neurog3-RFP+ daughters (grey spots). After the division, both daughters turn on RFP. (I–M) Images of fixed explant with native GFP (I) and immunostained for SOX9/aPKC (J), NEUROG3 (K), and Neurog3-RFP (L, staining for MYC-tag). Both daughters are NEUROG3+/RFP+. (N) Fraction of RFP-producing cell divisions. Each category (pink, blue, and purple bars) was counted from three movies. (O) Analysis of RFP emergence from three live imaging movies. In three cases, RFP+ cells divided producing two RFP+ cells each (cyan bar), and the majority of RFP+ cells were either lost or moved out of frame during back-tracking (indeterminable, purple bar). (P) Fraction of asymmetric versus symmetric cell divisions from three different measurements: NEUROG3 tracking, Neurog3-RFP tracking, and in vivo clonal analysis. All three measurements exhibit equivalent rates of divisions. Numbering denotes elapsed time in h:min, and in the cell division diagrams P indicates progenitor and R, Neurog3-RFP (C, H). Scale bars, 20 μm. Histograms and error bars represent the mean and standard deviation (n = 3). See S6 Table for further data.
Fig 4. Analysis of differentiation and cell cycle dynamics from live imaging.
(A) Analysis of Neurog3-RFP onset in four locations from three different time-lapse movies (n = 56, 89, 54, and 125, respectively). Each vertical bar symbol indicates an onset event, and the yellow area displays the probability, obtained by kernel density estimation, of an event occurring over time. These suggest that cell differentiation might not be a homogeneous process; however, further statistical analysis does not rule out this possibility (S1.4 Text). (B) Lag time of Neurog3-RFP onset between daughters derived from symmetric (R/R) divisions (n = 19). Symmetrically fated daughters exhibited synchronized expression of Neurog3-RFP, as pointed out by the highly correlated lag time between division and RFP onset (inset). (C) Lag time between division and Neurog3-RFP onset in asymmetric (P/R, n = 27) versus symmetric (R/R, n = 38) divisions. Note the data are pooled from three live imaging movies. RFP cells from P/R divisions took a significantly longer time to turn on RFP than cells from R/R divisions. Black-outlined red circles from P/R division (n = 14) indicate P/R divisions producing grand-daughters through progenitor daughter division. (D) Doubling time of progenitors originating from either symmetric (P/P, black circle) or asymmetric (P/N and P/R, pink circle) divisions. Doubling time of asymmetrically generated progenitors took longer than symmetrically generated progenitors. Statistical analyses were done using two-tailed Mann-Whitney test. *** p < 0.0001 and * p = 0.04 (C,D).
Fig 5. Proposed model.
The observations from 3-D live imaging suggest that a distinct temporal induction of endocrine progenitor fate during the cell cycle may result in different fates of progenitors. As the majority of NEUROG3 cells are known to be largely post-mitotic [11], we propose models for three modes of cell division, resulting in NEUROG3 daughter differentiation according to a priming time point. (A) Asymmetrically fated daughter differentiation after progenitor cell division (P→P/N). Only one daughter may be induced (arrow) after the mother division, resulting in exit of cell cycle and its differentiation to NEUROG3, while the other daughter is fated as a progenitor, resulting in self-renewal. (B) Symmetrically fated daughter differentiation after progenitor cell division (P→N/N). Before mitosis, the progenitor may be induced to differentiate into an endocrine progenitor, complete the cell cycle, and divide, resulting in both daughters differentiating into NEUROG3. (C) Symmetrically fated daughters through symmetric division of endocrine progenitors (N→N/N). Considering endocrine progenitors post-mitotic, the progenitor may be induced to differentiate into endocrine progenitor, but has not yet finished the cell cycle before the cell actually differentiates into an endocrine progenitor. Therefore, to complete the cell cycle, a recently differentiated NEUROG3 cell may divide and give rise to two NEUROG3 daughters. (D–F) We have developed a mathematical model of cell cycle–dependent stochastic priming of progenitors to endocrine fate. (D) Schematic of the model in which pancreatic progenitors (P, green circles) stochastically are primed for differentiation with probability q. Primed cells can either exit the cell cycle and differentiate into NEUROG3 (N) with probability θ or conclude the cycle (L) and give rise to two NEUROG3 cells. (E) The proposed model accounts for the observed frequencies of each division mode and predicts differential RFP onset dynamics in asymmetric and symmetric divisions. “Experiment*” bar in Asymmetric category denotes asymmetric divisions accounting for a RFP daughter and a self-renewing RFP− daughter (n = 14), whereas “Experiment” bar includes all the asymmetric divisions (refer to Fig. 4C). (F) The model with experiment-matching number of clones (out of 10,000 simulated clones) predicts a larger lag time between division and RFP onset in cells stemming from asymmetric divisions versus symmetric divisions. (G) Correlation of RFP lag times between sibling cells predicted by the model also matches that which was experimentally measured.
Fig 6. A small number of Neurog3-RFP cells divide into two NEUROG3+ cells.
(A) Still images from S8 Movie, demonstrating division of Neurog3-RFP cell (white spots) in RFP channel from a Pdx1 tTA/+;tetO-H2B-GFP;Neurog3-RFP explant. (B–F) Images of fixed explant with native GFP (B) and immunostained for Neurog3-RFP (C, staining for Myc-tag), NEUROG3 (D) and Sox9/aPKC (E). Both daughters are NEUROG3+/RFP+. Numbering denotes elapsed time in h:min (A). Scale bars, 20 μm.
References
- Jorgensen MC, Ahnfelt-Ronne J, Hald J, Madsen OD, Serup P, et al. (2007) An illustrated review of early pancreas development in the mouse. Endocr Rev 28: 685–705. - PubMed
- Gu G, Dubauskaite J, Melton DA (2002) Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129: 2447–2457. - PubMed
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