Supplementary MaterialsS1 Data: Excel spreadsheet containing, in distinct bed linens, the numerical data and statistical for Fig sections 1O, 1P, 2H, 2I, 3N, 3O, 3P, 4A, 4B, 4C, 4D, 5E, 5F, 5G, 6, and Model formulas

Supplementary MaterialsS1 Data: Excel spreadsheet containing, in distinct bed linens, the numerical data and statistical for Fig sections 1O, 1P, 2H, 2I, 3N, 3O, 3P, 4A, 4B, 4C, 4D, 5E, 5F, 5G, 6, and Model formulas. GFP (G) and immunostained for NEUROG3 (H) and SOX9 (I). Inset displays NEUROG3+ MGCD-265 (Glesatinib) cells (H, nuclear sign; arrowheads) in high magnification. Stations in (H) and (I) are masked by indigenous GFP route (G) to exclude nonspecific history in mesenchymal areas. (JCO) Images in 3-D maximum intensity projection of immunostained control explant without imaging and laser exposure. The overall morphology after 48 h of culture, equivalent to 24-h live imaging, reveals that this epithelium branches (J). The white square is an area zoomed in (KCO). Cells in the trunk region are differentiating into MGCD-265 (Glesatinib) NEUROG3+ endocrine progenitors (M, nuclear signal). The epithelium is usually intact as shown by E-CADHERIN staining (M), and branching ducts and acini are apically polarized as revealed by aPKC staining (O). Arrowheads in (K) indicate endocrine cell clusters. The blue channel (M) is usually masked by the native GFP channel (L) to exclude non-specific background in mesenchymal regions. A Z-stack is usually shown in S2 Movie (JCO). Scale bars, 50 m.(TIF) pbio.1002111.s003.tif (6.9M) GUID:?DC6AC6EE-6A55-4ED6-955D-B7603138A8EA S2 Fig: NEUROG3+ endocrine progenitor generation is more efficient in vivo than in vitro. Comparison of two-cell clone frequency distribution from in vivo clonal analysis (left column, = 244), and in vitro clonal analysis (right column, = 96) using E13.5 explants cultured on filters.(TIF) pbio.1002111.s004.tif (133K) GUID:?10891D11-0788-4466-A7CF-972BB6AF4AAB S3 Fig: transgenic line. (A) Construct. A 7.6 kb promoter region is linked to an intron, open reading frame of turboRFP, which contains a nuclear localization signal (NLS) and a Myc-tagC (Myc), and a bGH-PolyA signal (PolyA). The transgenic construct injection resulted in two transgenic mouse lines. (B) Optical section of a pancreatic explant from a embryo, immunostained for NEUROG3 (blue), SOX9 (white) and aPKC (white). The RFP MGCD-265 (Glesatinib) (red) and GFP (green) channels are native signals from each fluorescent protein. (C) Characterization of Neurog3-RFP in the E14.5 pancreas: Proportion of RFP+ (immunostained for Myc) (red) and RFP? (white) in NEUROG3+ cells, and proportion of NEUROG3+, Hormones+ (identified by INSULIN [INS] and GLUCAGON [GCG]), DHX16 and NEUROG3?/Hormones? in RFP+ cells. (D) Optical section of E14.5 pancreas, immunostained for NEUROG3 (cyan), RFP (Orange; immnostained for Myc), and INS and GCG (Magenta). White arrowheads indicate RFP+/NEUROG3+ cells, and yellow arrowheads indicate RFP+/NEUROG3? cells. Scale bars, 20 m. Histograms and error bars represent the mean and standard deviation (= 4).(TIF) pbio.1002111.s005.tif (4.2M) GUID:?5CBD0F64-03FE-491B-8436-865EF15BE690 S4 Fig: Comparison of transgenic line to knock-add-on line. (A) Scheme summarizing the genetic strategy to evaluate fidelity compared to embryos are cultured, and 3-D time-lapse imaging is done for over 48 h. Then, EYFP- and RFP-expressing cells are tracked. (C) Quantification of EYFP and RFP cells at time 0 of time-lapse movies. Note that EYFP+/RFP+ bar (orange) includes cells that are initially RFP? but acquire RFP over time, whereas EYFP+/RFP? bar indicates cells express EYFP only throughout the movie. All RFP+ cells are EYFP+ (brown bar). Histograms and error bars represent the mean and standard deviation (= 3). (D) Lag time of RFP onset after EYFP onset. RFP expression is usually delayed by 4.7 ( 1.1) h in EYFP+ cells. (E) Fluorescence intensity of EYFP and RFP in four cells in time-lapse movies. The green and red lines indicate EYFP and RFP signals, respectively. Note RFP signal is usually delayed by several hours, and both EYFP and RFP signals have comparable pattern of increase and decrease over time. See S5 Table for further data.(TIF) pbio.1002111.s006.tif (859K) GUID:?73E3B555-8DB1-49C2-92FD-BDD5AB267CD2 S5 Fig: Dynamics of during 48-hour time-lapse. (A) Fluorescence intensity of individual Neurog3-RFP cells over time. Different coloured lines indicate individual RFP cells. (B) Normalized fluorescence of all the RFP signals from (A) and aligned by 25% intensity to time 0. Black line indicates average intensity of all RFP signals. (C) Estimation of RFP half-life. Red line indicates the pattern of exponential decay. RFP half-life is usually estimated as 5.3 h.(TIF) pbio.1002111.s007.tif (837K) GUID:?F0E91B98-34D6-49E5-84FA-DB1C4CEB1EEA S6 Fig: Analysis of Neurog3-RFP onset from time-lapse films. (A) RFP starting point period distribution from four time-lapse movie positions (= 56, 89, 54, and 125). Each circle indicates onset time of RFP cell. Red, blue, and yellowish circles indicate RFP cells due to asymmetric, symmetric, and RFP divisions, respectively. (B) Analyses of RFP starting point Coefficient of Variance and slipping home window for oscillatory patterns. The Coefficient of Variance of every time-lapse onset distribution is the same as a homogeneous procedure, which is add up to 1.(TIF) pbio.1002111.s008.tif (773K) GUID:?D869243E-AA2B-485E-8C6A-B36BBD17059E.