Conditional deletion of Jak2 reveals an essential role in hematopoiesis throughout mouse ontogeny: implications for Jak2 inhibition in humans

Abstract

Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5), at postnatal day 4 (PN4), or at ∼2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans.

Figure 1. Deletion of Jak2 during mid/late gestation results in impaired erythropoiesis.

(A) Cartoon illustrating the times at which Jak2 was deleted from mice via Tamoxifen injection and the times at which necropsy was performed. The three selected times for Jak2 deletion are mid/late gestation (E12.5), early prenatal (PN4), and early adult (PN35/PN63). Also shown are the times when necropsy was performed on each group; E16.5–E17.5, PN17–PN20, and PN91, respectively. (B) Representative control and Jak2 cKO E16.5 yolk sacs are shown as well as the PCR analysis that confirmed both the presence of Cre and the null allele in the Jak2 cKO embryos (n = 8), but not the controls (n = 12). Size bar  = 2 mm. (C) E17.5 embryos connected to visceral yolk sacs (top). Blood-filled umbilical vessels were seen in the control embryos, but not the Jak2 cKO embryos (bottom). Size bars  = 2 mm. (D) When compared to controls, spleens from the Jak2 cKO mice were paler (top) with visible histopathological necrosis (bottom, indicated by arrows). Upper panel size bar  = 2 mm while lower panel size bars  = 10 µm. (E) While livers from the control embryos appeared normal, livers from the Jak2 cKO embryos were similarly smaller and paler (top) and histopathology of E17.5 embryonic livers showed marked hypo-cellularity and reduced erythroid and megakaryocytic precursors in the Jak2 cKO mice (bottom). Upper panel size bar  = 2 mm while lower panel size bars  = 10 µm. (F) Determination of Jak2 mRNA levels in the liver from both genotypes (n = 3 for each genotype). **, p<0.01.

Figure 2. Deletion of Jak2 beginning at postnatal day 4 results in profound anemia.

(A) Representative PN17 control (n = 7) and PN17 Jak2 cKO (n = 6) mice and body weights plotted as a function of genotype. Size bar  = 5 mm. (B) Pallor due to poor peripheral perfusion secondary to severe anemia was seen in the tail, forelimb, and mesenteric vasculature of the Jak2 cKO mice, but not the controls. Size bar  = 5 mm. (C) Representative peripheral blood films for both genotypes. Jak2 cKO blood films revealed marked anemia, hypochromasia, and microcytosis. Hematocrits plotted as a function of genotype indicating severe anemia in the Jak2 cKO mice. Size bars  = 10 µm. (D) Representative bone marrow sections from both genotypes and quantification of marrow cellularity plotted as a function of genotype. Size bars  = 500 µm for 4X images and 50 µm for 40X images. (E) Representative spleens from both genotypes and spleen weight to body weight ratios plotted as a function of genotype. Size bar  = 5 mm. (F) Histologic sections through the spleen are shown which indicate a hypoplasia phenotype in the Jak2 cKO mice. Size bars  = 500 µm for 4X images and 50 µm for 40X images. (G) Representative liver sections from both genotypes showing hepatocellular atrophy in the Jak2 cKO tissue, but not in the control tissue. Size bars  = 50 µm for 40X images and 10 µm for 100X images. (H) Determination of Jak2 mRNA levels in the liver from both genotypes. **, p<0.01.

Figure 3. Tamoxifen-inducible deletion of Jak2 during early adulthood results in impaired hematopoiesis and lower mortality rates.

(A) Kaplan-Meier survival curves for control (n = 17) and age-matched Jak2 cKO mice (n = 34). (B) Representative peripheral blood films from both genotypes showing the presence of anemia, thrombocytopenia, and poikilocytosis in the Jak2 cKO mice, but not the controls. Size bars  = 10 µm. (C) Representative spleens from both genotypes and spleen weight to body weight ratios plotted as a function of control (n = 10) and Jak2 cKO (n = 10) genotypes. Histologic sections of spleen found an abnormal splenic architecture in the Jak2 cKO mice characterized by atrophied and disorganized white pulp. Size bars  = 2 mm for top panel, 500 µm for 4X images and 50 µm for 40X images. (D) Representative bone marrow sections from both genotypes indicating increased adipose deposits in the Jak2 cKO mice, but not the controls. Also shown is bone marrow cellularity plotted as a function of control (n = 4) and Jak2 cKO (n = 5) genotypes. Size bars  = 500 µm for 4X images and 50 µm for 40X images. (E) Representative liver sections showing diffuse centrolobular vacuolar degeneration in the Jak2 cKO tissue, but not in the control tissue. Size bars  = 500 µm for 4X images, 50 µm for 40X images, and 10 µm for 100X images. (F) Determination of Jak2 mRNA levels from control (n = 4) and Jak2 cKO (n = 5) livers. **, p<0.01.

Figure 4. Histology of non-hematopoietic organs.

Tissues were taken from Control (A-H) or Jak2 cKO (I-P) mice. Shown are representative hearts (A, E, I, and M), kidneys (B, F, J, and N), lungs (C, G, K, and O), and brains (D, H, L, and P). The relative magnifications are 4x (D and L), 10x (A–C and I–K), and 40x (E–H and M–P). For A and I, the yellow arrows indicate equal wall thickness and RV = right ventricle. For C and K, Br = bronchus. Size bars  = 500 µm (D&L), 100 µm (A–C, I–K), and 50 µm (E–H, M–P).

Figure 5. The higher survival rate observed in the adult Jak2 cKO mice is due to re-population of hematopoietic tissues with Jak2 expressing cells.

(A) The levels of hemoglobin (Hb) plotted as a function of genotype and time. Although Hb levels dropped acutely in the Jak2 cKO mice (n = 7) relative to controls (n = 5) after TM injections, they returned to normal levels by day 147. (B) Shown are the relative levels of Jak2 mRNA in control (n = 3) and Jak2 cKO (n = 4) mice plotted as a function of day of collection, genotype, and tissue. (C) The levels of Hb plotted as a function of genotype and time. Shown are the control [Cre(-);f/f], Jak2 cKO [Cre(+);f/f], and the Jak2 compound mutant [Cre(+);f/Δ] genotypes. Conversion of one floxed Jak2 allele to a null allele [Cre(+);Jak2(f/Δ)] resulted in a marked and sustained reduction in Hb levels and ensuing death. *, p<0.05; **, p<0.01.

Figure 6. Tamoxifen-inducible deletion of Jak2 significantly attenuates GBP-2 and IRF-1 expression at all three time points.

Shown are the relative levels of GBP-2 and IRF-1 mRNA in control and Jak2 cKO mouse livers plotted as a function of both time of Jak2 deletion and genotype. A total of 3–4 mice per genotype were used on each day and each sample was measured in triplicate. *, p<0.05; **, p<0.01.

Figure 7. Tamoxifen-inducible deletion of Jak2 during early adulthood decreases stem cell and myeloid progenitors, but not lymphoid progenitors.

(A) Spleens were harvested on the indicated days from the given genotypes and subjected to flow cytometry for analysis of T cell and B cell populations. Shown are the numbers of lymphocytes per 10⁴ total events. 3–5 mice of each genotype were used on each day. (B) Representative flow cytometry plots for Control and Jak2 cKO genotypes indicating the markers and gating that was employed for these studies. (C) The numbers of hematopoietic progenitors plotted as a function of genotype and day. Deletion of Jak2 in young adults diminished the numbers of LT-HSC, ST-HSC, MPP, LSK, CMP, MEP, and GMP, but not CLP. (D) Bone marrow cells were isolated on the indicated days from the given genotypes and cultured in semi-solid medium. The numbers of colonies were then determined and plotted as a function of both genotype and day. Relative to controls, the numbers of CFU-GEMM, CFU-GM, BFU-E, and CFU-MK were all significantly reduced in the Jak2 cKO mice at day 56, but not at days 31 and 120. A total of 3–4 mice per genotype were used on each day and each sample was grown in duplicate. *, p<0.05. **, p<0.01.

Figure 8. Proposed role of Jak2 in adult hematopoiesis.

Hyper-kinetic Jak2 signaling results in various neoplastic disorders in adult animals and in some instances, this can be lethal. On the other hand, the specific loss of Jak2 function in adults results in anemia and thrombocytopenia. The severity of these disorders is directly proportional to the extent to which Jak2 function is lost. For instance, an 85%-95% decrease in Jak2 mRNA levels is coincident with significantly decreased bone marrow cellularity, atrophied spleens, and reduced peripheral blood cell counts. Yet, there is still a potential to recover from this pathogenic state. However, when Jak2 mRNA levels are reduced by >95% (ie, Jak2 is eliminated from >95% of cell), there is concomitant thrombocytopenia, severe anemia, and eventual death.