Fig_2.tif (2.57 MB)
Download file

Asymmetric and symmetric divisions revealed by in vivo lineage tracing of progenitors at clonal density.

Download (0 kB)
posted on 18.03.2015, 04:04 by Yung Hae Kim, Hjalte List Larsen, Pau Rué, Laurence A. Lemaire, Jorge Ferrer, Anne Grapin-Botton

(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.