Stochastic priming and spatial cues orchestrate heterogeneous clonal contribution to mouse pancreas organogenesis

The pancreas arises from a small population of cells but how individual cells contribute to organ formation is unclear. Here, the authors deconstruct pancreas organogenesis into clonal units, showing that single progenitors give rise to heterogeneous multi-lineage and endocrinogenic single-lineage clones

Poly-I:C Decreases Dendritic Cell Viability Independent of PKR Activation

Vaccination with tumor-antigen pulsed, monocyte-derived dendritic cells (DCs) has emerged as a promising strategy in cancer immunotherapy. The standard DC maturation cocktail consists of a combination of tumor necrosis factor-α (TNF-α)/interleukin (IL)-1β/IL-6 and prostaglandin E2 (PGE2) for generation of standard DCs (sDCs). In order to im-prove IL-12p70 production and cytotoxic T-lymphocyte (CTL) induction, a novel cocktail composed of TNF-α/IL-1β/ interferon (IFN)-α/IFN-γ and polyinosinic:polycytidylic acid (Poly-I:C) has been introduced to generate so-called α-Type-1 polarized DCs (αDC1s). We and others have previously performed a comprehensive comparison of sDCs and αDC1s. Here we demonstrate that the viability of αDC1s is lowered compared to sDCs and that DC apoptosis is medi-ated by Poly-I:C. We speculated that activation of protein kinase R (PKR) could mediate the observed apoptosis, but despite significantly higher PKR expression in αDC1s compared to sDCs and induction of active threonine (Thr)446 autophosphorylation of PKR in αDC1s, Poly-I:C did not influence total PKR expression or autophosporylation, indi-cating PKR-independent Poly-I:C-induced DC apoptosis

Data from: Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas

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

Kim_et_al_model_simulations

Kim_et_al_model_simulation

Proposed model.

<p>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 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.ref011" target="_blank">11</a>], 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 <i>q</i>. Primed cells can either exit the cell cycle and differentiate into NEUROG3 (N) with probability <i>θ</i> 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 (<i>n</i> = 14), whereas “Experiment” bar includes all the asymmetric divisions (refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.g004" target="_blank">Fig. 4C</a>). (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.</p

Live imaging reveals both asymmetric and symmetric emergence of NEUROG3 cells.

<p>(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 <i>Pdx1</i>^<i>tTA/+</i>;<i>tetO-H2B-GFP</i> 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.s016" target="_blank">S3 Movie</a>, 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.s016" target="_blank">S3 Movie</a>, 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^<i>low</i> (H), and the other red spotted cell is NEUROG3⁺ (G). (J) Still images from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.s017" target="_blank">S4 Movie</a>, 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 (<i>n</i> = 4). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.s023" target="_blank">S2 Table</a> for further data.</p

A small number of Neurog3-RFP cells divide into two NEUROG3⁺ cells.

<p>(A) Still images from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002111#pbio.1002111.s021" target="_blank">S8 Movie</a>, demonstrating division of Neurog3-RFP cell (white spots) in RFP channel from a <i>Pdx1</i>^<i>tTA/+</i>;<i>tetO-H2B-GFP;Neurog3-RFP</i> 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.</p