Article Figures & Data
Figures
- FIGURE 1.
Generation of conditional caspase-8 knockout mice and assessment of Cre-mediated deletion. A, Schematic representation of the targeting strategy. The figure shows the structure of the mouse caspase-8 gene before and after its recombination with the targeting vector and after deletion of the NEOr+TK cassette, as well as after further deletion of its floxed region. The positions of the loxP sites are designated by triangles. The exons, deduced by comparison with the cDNA sequence, are denoted by black boxes. The positions of the 5′ and 3′ external genomic DNA probes used for Southern blot analysis of the recombination are underlined. EcoRV restriction sites (RV) are marked by vertical lines. B, Cre-loxP-mediated deletion of the flanked caspase-8 gene. Southern blot analysis (using the 3′ probe shown in A) of DNA from the littermate mice obtained by crossing Casp8F/+ mice with the Casp8+/−:PGK-Cre transgene. PCR assessment of occurrence of the Cre transgene is shown at the bottom. The 6.8-kb fragment corresponding to the deleted floxed allele (Δ) could be observed only in floxed mice that also expressed Cre. Homozygous deletion and combination of the deleted and knockout (ko) allele were lethal in utero and could be found only in embryos before midgestation. C, Genomic location of the oligonucleotide sequences used to assess the extent of caspase-8 deletion by PCR (A–C) and by real-time PCR (D–E). The figure shows the locations of oligonucleotides used to assess levels of the wild-type (WT), targeted (floxed, F), knockout (KO), and deleted floxed (Δ) caspase-8 alleles. The underlying short bar corresponds to the genomic region whose level was assessed by real-time PCR.
- FIGURE 2.
Deletion of the caspase-8 gene in hepatocytes endows them with resistance to Fas cytotoxicity. A–D, Comparison of Casp8F/+:Alb-Cre and Casp8F/−Alb-Cre mice. A, Western blot analysis of procaspase-8 expression in various tissues. B, Assessment of the levels of the various caspase-8 alleles by PCR (top) and by real-time PCR (numbers at the bottom) in the livers and spleens. C, Evaluation of the extent of caspase-3 processing in the tissues by Western blotting at various times after their injection with anti-Fas Ab. D, Liver histology 6 h after injection of anti-Fas Ab. E, Liver histology of pI-pC-pretreated Casp8F/+:Mx1-Cre and Casp8F/−Mx1-Cre mice 6 h after injection of anti-Fas Ab. Arrows in D and E point to pyknotic nuclei in apoptotic cells.
- FIGURE 3.
Deletion of the caspase-8 gene in endothelial cells results in circulatory failure and cardiovascular and neural tube defects similar to those observed in full caspase-8 knockout. A–J, Pathology of E11.5 Casp8F/−Tie1-Cre embryos at the time of their death (B, D, F, H, J), compared with E11.5 Casp8F/+:Tie1-Cre embryos (A, C, E, G, I). A and B, Whole view, demonstrating congestion and pericardial effusion (arrow) and blood-depleted yolk sacs. C and D, Sagittal section through the whole embryo (H&E staining) demonstrating congestion (circles) as well as an expanded pericardial cavity and thinning of the chest wall (red bars) consistent with pericardial effusion, and rounding of the cardiac apex (A). E and F, Section through the chest demonstrating thinning of the ventricular wall (boxes) and abnormal rounding of the cardiac apex. G and H, Whole-mount anti-PECAM-1 immunostaining of the yolk sacs demonstrating extensive reduction in vascular density. I and J, View of the undulant neural tube (arrows). K, Green fluorescence staining in the head of an embryo of a Casp8F/+:Tie1-Cre mouse crossed with a mouse of the reporter strain (Z/EG) that expresses EGFP upon Cre-mediated excision, manifesting EGFP expression throughout the vasculature. Scale bars: C and D, 250 μm; E and F, 50 μm; G and H, 150 μm.
- FIGURE 4.
Early manifestations of the pathology in Casp8F/−Tie1-Cre embryos before death. A, Normal heart morphology and normal ventricular wall thickness (box) in an E11 embryo. B, Whole-mount anti-PECAM-1 immunostaining of the yolk sac of the same E11 embryo, showing degenerating vasculature (red asterisk) alongside vasculature of normal morphology (green asterisk). C, Whole-mount anti-PECAM-1 immunostaining of an E10.5 yolk sac with normal vasculature. D, Antiactive caspase-3 immunostaining (red fluorescence superimposed on the corresponding differential interference contrast image) of the same yolk sac, demonstrating extravascular location of the apoptotic cells found in increased amounts in the Casp8F/–:Tie1-Cre yolk sacs (e, endothelial cells) at this stage. Scale bars: A and C, 150 μm; B, 250 μm; D, 10 μm.
- FIGURE 5.
Induced ubiquitous deletion of the caspase-8 gene compromises hemopoietic progenitor function: CFU-C and CFU-S tests. Assessments of the functionality of the hemopoietic progenitors in the BM of pI-pC-injected (A, B, D–F) and IFN-injected (C) Casp8F/+:Mx1-Cre and Casp8F/−Mx1-Cre mice. A–C, In vitro progenitor assay. A, Gross appearance of in vitro myeloid clonogenic progenitor (CFU-C) tests, and B, yield of colonies (Total; erythroid burst-forming unit (E); CFU-granulo-macrophagic cells (GM); CFU-granulocytic-erythroid-megacaryocytic-macrophagic cells (Mix); and CFU B cells (CFU-B) in the BM of pI-pC-injected mice (mean ± SD, n = 8, where n stands for the number of tests done, each with a single mouse). C, Yield of myeloid colonies in the BM of IFN-injected mice (n = 4). D—F, In vivo CFU-S assay. D, Appearance of spleens; E, number of colonies in the spleens (n = 5); and F, spleen weights in irradiated mice that were reconstituted with BM cells (n = 5). No colonies were detectable in the spleens of irradiated mice that were not reconstituted. Average weights of these spleens (16.7 mg on day 8 and 23.7 mg on day 13) were subtracted from the weights of the spleens of reconstituted mice. Unless otherwise indicated, solid (▪) and open columns (□) in all figures correspond, respectively, to Casp8F/+ and Casp8F/− cells.
- FIGURE 6.
Induced ubiquitous deletion of the caspase-8 gene compromises hemopoietic progenitor function: study of radiation chimera. A and B, Analysis of the T cell and B cell compartments in BM and lymphoid organs of irradiated RAG-1−/− mice 7 wk after their reconstitution with the BM cells of pI-pC-injected Casp8F/+:Mx1-Cre and Casp8F/−Mx1-Cre mice (or, as a control, irradiated RAG-1−/− mice that were not reconstituted). The cell numbers in these organs are given at the top left corners of the panels. Cell numbers in the inguinal lymph nodes were also determined, and found to be 8–14 × 104 in the nonreconstituted RAG-1−/− mice, 15–15.2 × 106 in the mice reconstituted with BM cells of the Casp8F/+:Mx1-Cre mice, and 6–10 × 104 in the mice reconstituted with BM cells of the Casp8F/−Mx1-Cre mice. Also shown are the percentages of living cells identified as B lymphocytes (in the BM subdivided into mature and immature) and T lymphocytes (CD4, CD8, and, in the thymus, double positive). Shown are representative data from the analysis of three transplanted mice in each of the three presented groups. Similar cell numbers were found when data from the mice were analyzed 6 mo after reconstitution (data not shown), suggesting that caspase-8 deletion compromises the function of both committed and uncommitted hemopoietic progenitors. C and D, In vitro progenitor assay (CFU-C) of the hemopoietic progenitor levels in BM of pI-pC-injected radiation chimera. C, Irradiated C57BL/6 (Ly-5.1) mice reconstituted with BM cells of Casp8F/+:Mx1-Cre or Casp8F/−Mx1-Cre mice. D, Irradiated Casp8F/+:Mx1-Cre (▪) and Casp8F/−Mx1-Cre mice (□) reconstituted with BM cells of C57BL/6 (Ly-5.1) mice (n = 3).
- FIGURE 7.
Deletion of the caspase-8 gene in macrophage precursors compromises their differentiation. A, Yield of colonies in in vitro progenitor tests (CFU-C) performed with Casp8F/+:LysM-Cre and Casp8F/−LysM-Cre mouse BM cells (n = 3). B–J, Features of leukocytes generated by in vitro culturing of BM cells (B) of pI-pC-treated Casp8F/+:Mx1-Cre and Casp8F/−Mx1-Cr mice, and (C–J) of Casp8F/+:LysM-Cre and Casp8F/−LysM-Cre mice. C–F, BM-derived macrophages generated by culturing with M-CSF. B and C, Appearance of adherent cells after culturing of BM cells for 7 days, and D, their yield after culturing for the indicated time periods (n = 3). E, Comparison of the extent of deletion of the floxed caspase-8 allele in the adherent cells on their 7th day of culture (n = 6). F, Cell viability after 5 days in culture: FACS analysis of the nonadherent cells for expression of CD11b, a macrophage marker, and staining with annexin V, a cell death marker. G, Yield of granulocytes (Ly6G+ cells) generated by culturing of the BM cells for 7 days with mrG-CSF. H–J, Dendritic cells generated by culturing of the BM cells for 7 days with mrGM-CSF: H, yield of CD11c+ cells; I, extent of deletion of caspase-8 in the immature CD11c+ MHC-IIlow; and J, in the mature CD11c+, MHC-IIhigh dendritic cells. K and L, Extent of caspase-8 deletion in vivo: K, in the peritoneal macrophages; and L, in the peritoneal neutrophils. Deletion was assessed by PCR (top) and real-time PCR (numbers at the bottom). WT, Wild-type caspase-8 allele; F, floxed allele; ko, knockout allele; Δ, deleted floxed allele (n = 4).
In this issue
