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 Pdx1tTA/+;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 SOX9low (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.