Fig 1.
Tie2Cre-mediated Id1 deletion and Id3 global ablation reduced the lifespan of mice.
A. Genomic PCR detecting Id1 in various tissues of 5–6 month old Id1 flox/ Id1 null mice with Tie2Cre (Tie2Cre+Id1F/-) or without Tie2Cre (Id1F/-). The Id1 null allele served as an internal DNA loading control. Tie2Cre converted the Id1 floxed allele into a Id1 null allele in all hematopoietic tissues without affecting mesenchymal tissues or any other tissues. WAT, white adipose tissue; LN, lymph node; BM, bone marrow. B. Xgal staining to visualize completed flox deletion in all hematopoietic tissues of 5–6 month old Tie2Cre/Rosa26-LacZ reporter mice. The floxed Id1 intermediate without Tie2Cre served as a negative control. (Magnification: 100X) C. Representative images of Id1 immunostaining in the developing embryonic liver (E13.5). Id1 protein was not detected in Id cDKO mice as a result of complete flox deletion of the Id1 gene. (Magnification: 40X) D. Survival curve of Id cDKO mice (n = 59), Id control mice (n = 59) and WT mice (n = 22).
Fig 2.
Id cDKO mice exhibit increased erythropoietin expression in kidneys, abnormal bone structure, and dysregulated bone marrow cells.
5–6 month old WT, Id control, and Id cDKO mice were analyzed unless otherwise noted. A. Representative images of femur and tibia/fibulas. B. Total number of bone marrow cells from femur and tibia of WT (n = 5), Id control (n = 6) and Id cDKO (n = 6) mice. C. RBC parameters from CBC analysis. RBC = red blood cell count, HGB = hemoglobin, HCT = hematocrit, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, PCV = packed cell volume D. Flow cytometric analysis of erythroid cells from early (CD71+TER119-) to late (CD71-TER119+) stages of development expressed as fold difference in percent of erythroid cells in gated bone marrow cells relative to WT levels. WT (n = 3), Id control (n = 4), Id cDKO (n = 4). E. Representative reticulocyte images with quantification (%) F. Colony forming unit assay. Percentage of erythroid colonies formed from different groups. WT (n = 3), Id control (n = 3), Id cDKO (n = 3). G. Erythropoietin (EPO) mRNA expression in kidneys of WT (n = 5), Id control (n = 5) and Id cDKO (n = 5) mice. *: p<0.05 compared to WT; **: p<0.05 compared to Id Control.
Fig 3.
Splenomegaly and dysregulated splenocytes in Id cDKO mice.
A. Gross morphology and total cell counts of representative spleens from 6 month old WT, Id control and Id cDKO mice. B. Spleen-to-body weight ratio (mg/g) represented in a scatter plot from WT (n = 26), Id control (n = 50) and Id cDKO (n = 49) mice at different ages. C. Representative H&E staining of spleens from WT, Id control and Id cDKO mice, WP = white pulp, RP = red pulp. (Magnification: 100X). D. Representative immunofluorescence staining of B220 (green) and CD3 (red) spleen sections from 6 month old WT, Id Control, and Id cDKO mice. E. Flow cytometric analysis of erythroid cells from early (CD71+TER119-) to late (CD71-TER119+) stages of development in 5–6 month old mice expressed as fold difference in percent of erythroid cells in gated splenocytes relative to WT levels. WT (n = 3), Id control (n = 4), Id cDKO (n = 4). F. Representative immunostain showing TER119+ erythroid cells in the spleen. (Magnification: 100X). G. Representative Prussian blue stains demonstrating distribution of released iron (blue color) in the spleen (Magnification: 40X). H. Representative H&E stains of spleens. Arrows indicate megakaryocytes. (Magnification: 100X). **: p<0.05 compared to WT and Id Control.
Fig 4.
Defective hematopoietic microenvironments in Id cDKO mice.
A. Representative H&E stain of longitudinal femur sections from 3 month old WT, Id control and Id cDKO mice. Arrows indicate vessels. (Magnification: 200X) B. TRAP-staining of osteoclasts (red) in femur sections (left) from 3-month-old WT, Id control and Id cDKO mice and quantification of number of TRAP+ osteoclasts per field (right). (Magnification: 200X) C. Representative immunofluorescence showing CD29+ endothelial cells in the spleen. (Magnification: 100X). *: p<0.05 compared to WT; **: p<0.05 compared to Id Control.
Fig 5.
Intrinsic and extrinsic effects on anemia in Id cDKO mice.
A. HUVEC in vitro tubulogenesis assay performed using WT, Id control and Id cDKO serum. Representative images (left, Magnification 200X) and quantification of differences in number of tubes and nodes formed (right). B-D. Comparison of red blood cell counts, hemoglobin and hematocrit between WT (n = 5), Id control (n = 5), Id cDKO (n = 5), WTBMT (n = 2), FBMT (n = 4) and RBMT (n = 4) mice. E. Colony forming unit assay. Percentage of erythroid colonies formed from different groups. Bone marrow cells isolated from 5–6 month old mice. WT (n = 3), Id cDKO (n = 3). WTBMT (n = 2), FBMT (n = 2), RBMT (n = 2). F. Representative flow cytometry maps of erythroid cells from early (CD71+TER119-) to late (CD71-TER119+) stages of development in 5–6 month old bone marrow transplanted mice. G. Representative H&E staining of spleens from bone marrow transplanted mice. WTBMT: WT donor/WT recipient; Id control BMT: Id control donor/WT recipient; FBMT: WT donor/Id cDKO recipient; RBMT: Id cDKO donor/WT recipient (Magnification: 100X)(for WT, Id control and Id cDKO, see Fig 3C). H. Ratio of spleen weight to body weight (mg/g) from 5–6 month old WT (n = 26), Id control (n = 50), Id cDKO (n = 49), WTBMT (n = 5), FBMT (n = 9), RBMT (n = 9). I. Flow cytometric analysis of erythroid cells in spleen from early (CD71+TER119-) to late (CD71-TER119+) stages of development. Fold difference in percent of erythroid cells in gated splenocytes relative to WTBMT levels. *: p<0.05 compared to WT; **: p<0.05 compared to Id Control.
Fig 6.
Bone marrow GATA1 and E47 display changes in occupancy to promoter regions of β-globin and E2A.
A. Western blot measurement of E47 (product of E2A) from whole bone marrow cells (left, top row) and erythroid cells (CD71+TER119+, right, top row) in WT, Id control, and Id cDKO mice at 6 months of age. Protein levels were normalized to GAPDH or β-actin levels (bottom row). B. Schematic of mouse β-globin gene promoter region with location of PCR primers and known E-box and GATA binding sites. Protein binding sites and conserved consensus sequences are underlined. C. Schematic of mouse E2A gene promoter region with location of PCR primers and location of known E-box and GATA binding sites. D. ChIP measurement of fold change in occupancy of GATA1 to the promoter region of β-globin and E2A. E. ChIP measurement of fold change in occupancy of E47 to the promoter region of β-globin and E2A. *: p<0.05 compared to WT.
Table 1.
Dysregulation of various members of the hematopoietic transcriptional network.
The values in the Id control and Id cDKO column reflect fold differences in mRNA expression with respect to WT levels. Values in the FBMT and RBMT columns reflect fold differences in mRNA expression with respect to WTBMT levels.