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First published online 2 December 2004
doi: 10.1242/dev.01558

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Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing

Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (46K): [in a new window]   Fig. 1. Generation and hematopoietic labeling of the ancestry mice. (A) Structure of the vav-Cre transgenic vector showing the hypersensitivity sites (HS) of the vav elements, the insulator sequences (INS) and the IRES-YFP element, which was not functional in vivo (indicated by the lowercase letters), and of the ROSA26R-YFP locus before and after Cre-mediated excision of the stop cassette. (B) Cross of vav-Cre transgenic mice with ROSA26R-YFP mice. F1 animals with the vav-Cre transgene segregated into two phenotypes, which were distinguished by the expression of YFP in the skin of chimeric control mice (yellow patches) and the absence of such a labeling in vav ancestry mice. The F1 mice without the vav-Cre transgene were used as negative controls. The tubes at the bottom containing grey or yellow `cells' indicate the degree of hematopoietic labeling in the different mice. (C) FACS analysis of mononucleated cells from adult bone marrow for YFP expression (left plot). LSK cells were gated (box, middle plot) and analyzed for YFP expression (right plot). (D) FACS analysis of whole E13.5 fetal liver (left plot). LSK cells were gated (box, middle plot) and analyzed for YFP expression (right plot). Numbers in the top right-hand corner of the plots indicate the percentage of YFP+ cells in the respective cell population. FSC, forward scatter. (E) Comparison of the hematopoietic labeling of vav ancestry and lysozyme ancestry mice. The circles represent cell compartments: L, lymphoid; M, myelomonocytic and HSC, hematopoietic stem cells. Yellow indicates that the cells in a given compartment are YFP labeled, grey that they are unlabeled. The yellow triangles indicate that a subset of cells in the corresponding compartment is YFP+.

View larger version (116K): [in a new window]   Fig. 2. Identification of hepatocytes and endothelial cells in control mice. (A,B) Liver sections from a chimeric control (A) and a negative control mouse (B) showing DAPI stained nuclei (blue) and YFP fluorescence (yellow). Scale bar: 50 µm. (C-H) Frozen liver section from a chimeric control mouse showing hepatic sinusoids: YFP (yellow); CD31 (red); CD45 (purple); DAPI (blue). YFP+ hepatocytes are outlined with a broken line, arrowheads highlight two representative YFP+ endothelial cells; arrow shows the position of one hematopoietic cell close to a vessel. Scale bar: 25 µm. (I-K) Frozen liver sections from a negative control mouse showing part of a larger hepatic vessel: autofluorescence (yellow); CD31 (red); CD45 (purple); DAPI (blue). The position of the autofluorescent basement membrane is indicated by broken lines. Arrows and arrowheads in I-K indicate the positions of hematopoietic cells and the endothelial cell layer, respectively. Scale bar: 25 µm.

View larger version (103K): [in a new window]   Fig. 3. Hepatocyte labeling in vav ancestry and lysozyme ancestry mice. (A,B) A binucleated YFP+ hepatocyte (yellow, outlined) in a liver section of a vav ancestry mouse. Nuclei are stained with DAPI (blue) and hematopoietic cells with F4/80, Mac1 and CD45 (purple). Scale bar: 25 µm. (C) The total number and distribution of hepatocyte clusters in vav ancestry mice. (D-F) Liver section of a vav ancestry mouse showing an YFP+ hepatocyte (yellow, outlined) that was stained for albumin expression (red). Nuclei are shown in blue. Scale bar: 10 µm. (G,H) Liver section of a lysozyme ancestry mouse showing a doublet of YFP+ hepatocytes (yellow, outlined). Nuclei are shown in blue; Kupffer cells, stained with F4/80, are in purple. Scale bar: 25 µm. (I) The frequency of YFP+ hepatocyte clusters per 100,000 cells in vav ancestry (vav), lysozyme ancestry (lys) and lck ancestry (lck) mice. Error bars indicate one s.d.

View larger version (9K): [in a new window]   Fig. 4. Effect of liver injury on hematopoietic to hepatocyte contributions. Distribution of YFP labeled hepatocyte clusters of injured (black bars) and uninjured (gray bars) vav ancestry mice. A total of 995,000 hepatocytes from two vav ancestry mice 3 weeks after CCl4 injection were analyzed. The values for uninjured vav ancestry are the same as in Fig. 3C. Clusters containing three or more YFP+ hepatocytes (the latter were only found in injured mice) were pooled in one group.

View larger version (108K): [in a new window]   Fig. 5. Analysis of hepatic endothelial cells in vav ancestry mice. (A-C) Frozen section showing sinusoidal endothelial cells (CD31, red), YFP fluorescence (yellow) and hematopoietic cells (CD45, purple). Scale bar: 25 µm. (D-G) Magnification of the area boxed in A-C, showing an YFP- endothelial cell (arrowhead) and two YFP+ hematopoietic cells (arrows) that are closely associated with the sinusoids. DAPI (blue), CD31 (red), YFP (yellow) and CD45 (purple). Scale bar: 10 µm. (H-J) Area with a large hepatic vessel visualized by CD31 staining (red). YFP (yellow), CD45 (purple). Hematopoietic cells closely line the vessel. Scale bar: 25 µm. (K-N) Magnification of the area boxed in H-J showing two YFP- endothelial cells (arrowheads) integrated into the vessel wall and one YFP+ hematopoietic cell on the abluminal side of the endothelial lining: DAPI (blue); CD31 (red); CD45 (purple). Scale bar: 10 µm.

View larger version (40K): [in a new window]   Fig. 6. FACS analysis of liver cell suspensions. (A-C) Gating logic for cells in organ cell suspension based on exclusion of dead cells by DAPI staining (A) and doublets by pulse width analysis (B). (C) Endothelial cells (EC) and hematopoietic cells (HC) are defined by marker gene expression. (D) YFP labeling of CD45+ hematopoietic cells isolated from the liver of a vav ancestry mouse. (E-H) YFP labeling of hepatic endothelial cells from a negative control mouse (E), a vav ancestry mouse (F), a chimeric control mouse (G) and a wild-type mouse completely reconstituted (>95%) with bone marrow of a vav ancestry mouse three months after transplantation (H).

View larger version (46K): [in a new window]   Fig. 7. Analysis of renal endothelial cells by flow cytometry. (A-D) YFP labeling of endothelial cells in kidney cell suspensions from a negative control mouse (A), a vav ancestry mouse (B), a chimeric control mouse (C) and a wild-type mouse completely reconstituted with bone marrow of a vav ancestry mouse three months after transplantation (D). The YFP+ cells in B correspond to vasa recta endothelial cells. The data shown in A,B were obtained using the MoFlo cell sorter and the LSRII flow cytometer, respectively; hence, the different representation of the YFP intensity.

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