1. by Claudia Orelio, Esther Haak, Marian Peeters, and Elaine Dzierzak
Interleukin-1–mediated hematopoietic cell regulation in the aorta-gonad-mesonephros region of the mouse embryo
by Claudia Orelio, Esther Haak, Marian Peeters, and Elaine Dzierzak
Blood
Volume 112(13):
December 15, 2008
©2008 by American Society of Hematology

2. IL-1R–signaling components are expressed and functional in the midgestation AGM region and liver.
IL-1R–signaling components are expressed and functional in the midgestation AGM region and liver. (A) RT-PCR analysis performed to examine the expression of the IL-1Rs type I (Il1r1), the accessory receptor (Il1rap) and the receptor type II (Il1r2), and several downstream signaling components, including Map3k7ip2 (TAB2), Map3k7 (TAK1), Traf6, and Irak4, and the ligand IL-1β (Il1b) in the E10-E12 AGM region and E11-E12 FL. (B) Overview of the culture method used to study gene induction or IκB degradation in AGM tissues. Single-cell suspensions were made from E11 AGM tissues and cultured in 6-well plates overnight. The next day, cells were treated with IL-1β and harvested for RT-PCR analysis or IκB degradation studies. (C) Representative semiquantitative RT-PCR for the IL-1β target genes Junb and Sod2 (MnSOD) after stimulation of E11 AGM and liver single-cell suspensions with IL-1β (10 ng/mL) for 0, 30, 90, or 120 minutes. (D) Western blot showing rapid IκB degradation after IL-1β stimulation (10 ng/mL) of E12 liver cells (left panel) or 3T3 fibroblasts (right panel).
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology

3. IL-1RI is expressed by cells in the E11 region aortic hematopoietic, endothelial, and mesenchymal cells.
IL-1RI is expressed by cells in the E11 region aortic hematopoietic, endothelial, and mesenchymal cells. Immunostaining performed with (A) a control antibody and (B,C) an IL-1RI–specific antibody on transverse cryosections from E11 embryos. (C) An enlargement of the boxed area in panel B. The dorsal aorta of the AGM region is shown with the ventral side at the bottom of the section. Positive signal is seen as an orange-brown precipitate from the DAB chromogen. The sections were counterstained with hematoxylin. Orange arrowheads indicate single IL-1RI–expressing cells in the circulation and scattered in the tissue. Slides were viewed with an Olympus BX40 research microscope (Olympus Nederland B.V., Zoetewoude, The Netherlands) using an Olympus lens at 20×/0.40 PH. Images were acquired and processed with Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA).
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology

4. IL-1RI is expressed by cells in the E11 region aortic hematopoietic, endothelial, and mesenchymal cells.
IL-1RI is expressed by cells in the E11 region aortic hematopoietic, endothelial, and mesenchymal cells. (A) Representative flow cytometric dot plots showing E11 aorta cells (n = 4) stained with antibodies specific for IL-1RI and CD45 (top panel) or c-kit (bottom panel). Percentages of cells in each quadrant are indicated; 3 to 3.6 × 104 events are shown, and more than 5 × 104 events were analyzed. (B) RT-PCR analysis for IL-1R and ligand expression in sorted HSCs (CD34+c-kit+), endothelial cells (CD45−VE-cadherin+; Endo), and presumed mesenchymal cells (CD45−VE-cadherin−; Mesen) from the E11 aorta. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression serves as the normalization control. −RT indicates no reverse transcriptase control. (C) Flow cytometric analysis of IL-1RI expression on UG26-1B6 stromal cells. X-axis indicates intensity of fluorescent signal from staining with IL-1RI–specific antibody; y-axis indicates the number of cells. (D) Intracellular flow cytometric dot plots showing control antibody (top panel) and anti–IL-1α antibody (bottom panel) staining in E11 AGM cells. Boxed areas indicate percentage positive cells.
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology

5. IL-1β increases hematopoietic cells in the E11 AGM region.
IL-1β increases hematopoietic cells in the E11 AGM region. E11 AGM tissues were cultured for 3 days in the presence or absence of different doses of IL-1β. (A) Flow cytometric analysis showing the absolute number of cells per AGM positive for CD45, c-kit, or Mac1 after AGM explant culture (n = 5). (B) Number of CFU-G, CFU-M, and CFU-GEMM per E11 AGM explant cultured for 3 days in the presence of 0, 1, or 10 ng/mL IL-1β. Colonies were scored in triplicate cultures after 7 days of methylcellulose culture (n = 2). (C) Percentage of adult recipient mice repopulated with donor E11 AGM cells. E11 AGM explants were cultured in 0, 1, or 10 ng/mL IL-1β for 3 days, and cells were injected into irradiated recipients (1 embryo equivalent [ee]). At 4 months after transplantation, recipient peripheral blood DNA was analyzed for donor hematopoietic chimerism by semiquantitative PCR. Only mice with more than 10% donor chimerism were considered repopulated. Each column represents the number of mice repopulated per number of recipients transplanted (13 of 24, 9 of 13, and 4 of 12) with AGM explant cells cultured in 0, 1, and 10 ng/mL IL-1β, respectively). Combined results of 8 separate transplantation experiments. The error bars represent SEM.
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology

6. IL-1R signaling affects AGM HSCs
IL-1R signaling affects AGM HSCs. (A) Flow cytometric analysis of freshly isolated E11 Il1r1+/+ and Il1r1−/− AGM cells showing the absolute number of c-kit+, CD45+, CD34+c-kit+, or CD34−Mac1+ cells per AGM (n = 3).
IL-1R signaling affects AGM HSCs. (A) Flow cytometric analysis of freshly isolated E11 Il1r1+/+ and Il1r1−/− AGM cells showing the absolute number of c-kit+, CD45+, CD34+c-kit+, or CD34−Mac1+ cells per AGM (n = 3). (B) Flow cytometric analysis of E11 Il1r1+/+ and Il1r1−/− AGM explants showing absolute numbers of c-kit+, CD45+, or Mac1+ cells per AGM explant (n = 4-5). (C) Number of CFU total, CFU-M, BFU-E, CFU-G, CFU-GM, and CFU-GEMM per freshly isolated E11 Il1r1+/+ and Il1r1−/− AGM. Colonies from triplicate cultures were scored after 7 days of methylcellulose culture (n = 3, cells from 14 Il1r1+/+ and 15 Il1r1−/− AGMs). (D) Number of CFU total, CFU-M, BFU-E, CFU-G, CFU-GM, and CFU-GEMM per E11 Il1r1+/+ and Il1r1−/− AGM after 3 days of explant culture. Colonies from triplicate cultures were scored after 7 days of methylcellulose culture (n = 3, cells from 8 Il1r1+/+ and 8 Il1r1−/− AGMs). (E) Percentage of adult recipient mice repopulated with HSCs from Il1r1+/+ and Il1r1−/− AGM regions either directly transplanted (direct) or transplanted after 3 days of explant culture (explant). E11 AGM cells (1 and 0.3 or 1 and 0.2 embryo AGM tissue equivalents [ee]) were transplanted into irradiated adult recipients. At 4 months after transplantation, peripheral blood DNA of recipients was analyzed for donor hematopoietic chimerism by semiquantitative PCR. Only mice with more than 10% donor chimerism were considered repopulated. Each column represents the number of mice repopulated per number of recipients transplanted (the numbers for the Il1r1+/+ and Il1r1−/− columns are, respectively, 5 of 7 and 3 of 8 for 1 ee direct transplantation, 1 of 6 and 0 of 8 for 0.3 ee direct transplantation, 2 of 2 and 5 of 5 for 1 ee explant transplantation, and 8 of 10 and 2 of 10 for 0.2 ee explant transplantation). Combined results of 4 separate transplantation experiments. The error bars represent SEM.
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology

7. Expression of IL-1–signaling molecules and hematopoietic regulators in IL-1β–stimulated AGMs. (A) RT-PCR analysis of E11 AGM tissue before and after 3-day explant culture in the presence of 0, 1, or 10 ng/mL IL-1β.
Expression of IL-1–signaling molecules and hematopoietic regulators in IL-1β–stimulated AGMs. (A) RT-PCR analysis of E11 AGM tissue before and after 3-day explant culture in the presence of 0, 1, or 10 ng/mL IL-1β. Changes in gene expression of some of the tested genes (Il1r1, Il1rap, Il1r2, Il1a, Il1b, Il18r1, Il18rap, Il18) are found after explant culture or are induced by the presence of IL-1β. (B) RT-PCR analysis of E11 AGM tissue before and after 3-day explant culture in the presence of 0, 1, or 10 ng/mL IL-1β. Changes in gene expression of some of the tested genes (Cxcl12a and Cxcl12b, Cxcr4, Mmp9) are found after explant culture or are induced by the presence of IL-1β. (C) Flow cytometric analysis of cultured E11 aorta explants for expression of CXCR4. E11 aorta explants were cultured in the presence of 0, 1, or 10 ng/mL IL-1β for 3 days before analysis. The percentage of CXCR4+ cells is indicated in the gated upper section (n = 3); 3 × 104 events were analyzed, and 1.3 to 1.5 × 104 events are shown. (D) RT-PCR analysis of AGM tissue from Il1r1+/+ and Il1r1−/− E11 embryos. AGM tissues before and after 3-day explant culture were used for RNA preparation. Changes in gene expression of some of the tested genes (Il81r1, Il81rap, Il18, Tnfr1, Tnfr2, Tnfa, Cxcl12a, Cxcl12b, Cxcr4, Mmp9) are found in the absence of IL-1RI directly or after explant culture; n = 2-3 for each gene. (E) RT-PCR analysis of UG26-1B6 cells treated with 10 ng/mL IL-1β for various times (2-24 hours) and examined for gene expression of several hematopoietic cytokines (Il6, Kitl) and chemokines (Cxcl12a, Cxcl2b). Representative experiments are shown in panels A, B, C, and E (n = 2). Actin was used as a cDNA normalization control. −RT indicates no reverse transcriptase; +RT, + reverse transcriptase.
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology

8. Model of IL-1–related interactions in the E11 mouse AGM region.
Model of IL-1–related interactions in the E11 mouse AGM region. The IL-1RI is expressed on endothelial, mesenchymal, and at lower levels on some HSCs. Receptor expression appears to be stable. The expression of IL-1 is variable, with high levels being produced by HSCs and other hematopoietic cells. Expression of IL-1 is low or negligible in the endothelial and mesenchymal compartment. IL-1 (from HSCs or other hematopoietic cells) is thought to interact with IL-1RI–expressing endothelial and/or mesenchymal cells on the ventral side of the E11 dorsal aorta. IL-1RI signaling results in the induction of unknown factor(s) (eg, some mobilization factors) indicated with a question mark (?) to modulate HSC maintenance and/or differentiation.
Claudia Orelio et al. Blood 2008;112:
©2008 by American Society of Hematology