Extended live imaging with Neurog3-RFP reporter reveals the dynamics of progenitor cell cycle and differentiation.
Figures are generally photos, graphs and static images that would be represented in traditional pdf publications.
(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 Pdx1tTA/+;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.