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First published online 12 April 2006
doi: 10.1242/dev.02349

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Tracing the first waves of lymphopoiesis in mice

¹ Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
² Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
³ INSERM U506, Hopital Paul Brousse, 94807 Villejuif, France.
⁴ Department of Immunology, University of Toronto, Sunnybrook and Women's Research Institute, Toronto, ON M4N 3M5, Canada.
⁵ University of Pittsburgh and Children's Hospital, Rangos Research Center, Pittsburgh, PA 15213, USA.

View larger version (38K): [in a new window]   Fig. 1. The first lymphoid cells with spontaneous RAG1 expression arise within embryos on day 10.5. (A) Single cell suspensions were made from whole embryos or the indicated tissues (embryo proper, yolk sac, placenta), as described in the Materials and methods. Percentages indicate the frequencies of RAG1/GFP+ cells among viable 7AAD- CD45+ hematopoietic cells. The results are representative of three independent experiments. (B) The bar graphs depict numbers of 7AAD- CD45+ cells (left) or 7AAD- CD45+ RAG1/GFP+ cells (right) in the indicated tissues. The data represent mean values with standard deviations from four embryos and are representative of three independent experiments.

View larger version (41K): [in a new window]   Fig. 2. Notch receptor ligation generates T cells from P-Sp/AGM, but not from YS of early embryos. (A) YS and P-Sp from E9.0-9.5 embryos (somites 15-20) were dissected and cultured on OP9 cells for 14 days without exogenous cytokines. The cultures were then stained for lymphocyte markers including TCRß and TCR. (B) E9.0-9.5 P-Sp were cultured on OP9-DL1 with or without additional cytokines for 14 days in parallel, the cultures were then stained for TCR and TCRß from comparison. (C) E9.0-9.5 P-Sp were cultured on OP9-DL1 with (black bars) or without additional cytokines (white bars) for 14 days in parallel. The total cell number per tissue (left) and T cells produced (right) are shown.

View larger version (38K): [in a new window]   Fig. 3. YS and P-Sp/AGM cells at E9.5 differ with respect to surface markers and lympho-hematopoietic potential. (A) YS and P-Sp/AGM cells from E9.5 embryos (somites 19-25) were stained with FITC-anti-CD41 and APC-anti-Kit or anti-CD45. (B) E9.5 P-Sp/AGM cells were stained with PE-anti-Tie2 and FITC-anti-CD41, FITC-anti-CD34 or APC-Kit. (C) CD41+ cells or Tie2+ cells were sorted from E9.5 (somites 19-25) YS or P-Sp/AGM regions, respectively. The cells were co-cultured with OP9 stromal cells for 2 weeks, and then subjected to flow cytometry analysis after staining with PE-anti-CD19 and APC-anti-CD45. The data are representative of three independent cultures. In all these analyses, dead cells were excluded by staining with 7AAD.

View larger version (68K): [in a new window]   Fig. 4. Characterization of the first wave of T lymphopoiesis in the developing thymus. (A) Thymus rudiment sections from E11.25, E12.5 or E14.5 embryos were stained with an anti-GFP antibody. (B) Flow cytometry analysis showed that 82.3% of total CD45+ mononuclear cells in the E12.5 thymus expressed RAG1/GFP. The data is representative of three independent analyses. (C) GFP+ cells were sorted from the E12.5 thymus, and then stained with anti-PECAM-1/CD31 followed by APC-anti-Kit, biotinylated anti-CD34 and streptavidin-PE-TR. (D) GFP+ cells from E12.5 or E13.5 thymus were stained with PE-anti-CD25 and APC-anti CD44.

View larger version (29K): [in a new window]   Fig. 5. Characteristics of the most primitive lymphoid progenitors and their progeny in the fetal liver. (A) Fetal liver cells from E11.5 embryos were stained with APC-anti-CD45 (upper, left panel) and sorted into CD45- and CD45+ fractions (upper, middle and right panels). The sorted CD45+ cells were then stained with PE-anti-Kit, PE-anti-Mac-1 or biotinylated anti-AA4.1 followed by PE-TR-streptavidin (lower panels). Flow cytometry results are shown illustrating the expression of these markers on RAG-1/GFP+ progenitors. (B) Progenitors from E14.5 fetal liver were sorted on the basis of RAG1/GFP, Kit and Sca1 (Yokota et al., 2003) and then cultured on OP9-control or OP9-DL1 for 14 days. Yields of CD19+ B cells (dotted bars in the top panel), TCR+ (white bars, lower) or TCRß+ (hatched bars, lower) T cells per input progenitors are shown. (C) All lymphoid progenitor containing subsets of fetal liver at the indicated gestational stages were resolved. Percentages of each cell type were multiplied by cell yields to obtain absolute numbers per organ.

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