Dynamic changes in murine erythropoiesis from birth to adulthood: implications for the study of murine models of anemia

Abstract

Liver, spleen, and bone marrow are 3 key erythropoietic tissues in mammals. In the mouse, the liver is the predominant site of erythropoiesis during fetal development, the spleen responds to stress erythropoiesis, and the bone marrow is involved in maintaining homeostatic erythropoiesis in adults. However, the dynamic changes and respective contributions of the erythropoietic activity of these tissues from birth to adulthood are incompletely defined. Using C57BL/6 mice, we systematically examined the age-dependent changes in liver, spleen, and bone marrow erythropoiesis following birth. In addition to bone marrow, the liver and spleen of newborn mice sustain an active erythropoietic activity that is gradually lost during first few weeks of life. While the erythropoietic activity of the liver is lost 1 week after birth, that of the spleen is maintained for 7 weeks until the erythropoietic activity of the bone marrow is sufficient to sustain steady-state adult erythropoiesis. Measurement of the red cell parameters demonstrates that these postnatal dynamic changes are reflected by varying indices of circulating red cells. While the red cell numbers, hemoglobin concentration, and hematocrit progressively increase after birth and reach steady-state levels by week 7, reticulocyte counts decrease during this time period. Mean cell volume and mean cell hemoglobin progressively decrease and reach steady state by week 3. Our findings provide comprehensive insights into developmental changes of murine erythropoiesis postnatally and have significant implications for the appropriate interpretation of findings from the variety of murine models used in the study of normal and disordered erythropoiesis.

Graphical abstract

Figure 1.

Dynamic changes in erythropoietic activity in the C57BL/6 mouse liver after birth. (A) Terminal erythroid differentiation in the liver for the first 12 days after birth (P1 to P12) was measured by flow cytometry using CD44 vs FSC as markers. (B) The total number of erythroblasts in the liver from P1 to P15 was determined by flow cytometry using Ter119⁺ CD44^hi populations of erythroid cells (red gate is shown in panel A). (C) Absolute number of liver cells from P1 to P15. (D) Evolution of the mouse body weight (blue solid lines) vs liver weight (red solid line) from P1 to P15. (E) Erythropoietic activity in the liver was determined by immunohistochemistry using Ter119 staining at the indicated days (left panels, original magnification ×20; right panels, original magnification ×40). n = 4-13 animals per day; data are presented as mean ± SEM. *P < .05, **P < .01, ***P < .001, ***P < .0001, indicated postnatal day vs P1 (ANOVA with Tukey’s post hoc test with corrections for multiple comparisons).

Figure 2.

The spleen is a transient erythropoietic organ in the neonate C57BL/6 mouse. (A) Terminal erythroid differentiation in the spleen for the first 8 weeks after birth was measured by flow cytometry using CD44 vs FSC as markers. (B) The total number of erythroblasts in the spleen for the first 12 weeks was determined by flow cytometry using Ter119⁺ CD44^hi populations of erythroid cells (red gate is shown in panel A). (C) Absolute number of splenocytes for the first 12 weeks. (D) Evolution of the mouse body weight (blue solid lines) vs spleen weight (red solid line) from week 1 to 12. (E) Erythropoietic activity in the spleen was determined by immunohistochemistry using Ter119 staining 3 weeks (upper panels) and 7 weeks (bottom panels) after birth (left panels, original magnification ×20; right panels, original magnification ×40). n = 4-13 animals per day; data are presented as mean ± SEM. *P < .05, **P < .01, ***P < .001, indicated week vs week 1 (ANOVA with Tukey’s post hoc test with corrections for multiple comparisons).

Figure 3.

Steady-state erythropoiesis in the C57BL/6 mouse bone marrow is established within 7 weeks after birth. (A) Terminal erythroid differentiation in the bone marrow for the first 12 weeks after birth was measured by flow cytometry using CD44 vs FSC as markers. (B) The total number of erythroblasts in the bone marrow for the first 12 weeks was determined by flow cytometry using Ter119⁺ CD44^hi populations of erythroid cells (red gate is shown in panel A). (C) Absolute number of bone marrow cells for the first 12 weeks. (D) Evolution of the mouse body weight (blue solid lines) vs bone marrow weight (red solid line) from week 1 to week 12. n = 4-13 animals per day using 1 femur and 1 tibia for each animal; data are presented as mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, indicated week vs week 1 (ANOVA with Tukey’s post hoc test with corrections for multiple comparisons).

Figure 4.

Red cell parameters in the circulation reflect the dynamic changes from the erythropoietic tissues. Red cell parameters were evaluated in C57BL/6 mice every day from P1 to P14 and then every week until 12 weeks of age using ADVIA 120. Hemoglobin (Hgb) (A), hematocrit (Hct) (B), RBCs (C), MCV (blue solid line) vs RDW (red solid line) (D), MCH (E), and measured mean cell hemoglobin concentration (CHCM) (F). n = 4-13 animals per day; data are presented as mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, indicated postnatal day vs P1 (ANOVA with Tukey’s post hoc test with corrections for multiple comparisons).

Figure 5.

Dynamic changes in the reticulocyte count and EPO levels after birth. (A) Reticulocyte count was evaluated in C57BL/6 mice every day from P1 to P14 and then every week until 12 weeks of age using ADVIA 120. (B) EPO levels in the serum were measured every other day for the first 2 weeks and then every week until 12 weeks of age. n = 4-13 animals per day; data are presented as mean ± SEM. **P < .01, ****P < .0001, indicated postnatal day vs P1 (ANOVA with Tukey’s post hoc test with corrections for multiple comparisons).