Tamoxifen-inducible Deletion of Jak2 during Mid/late Gestation Results in Hematopoietic Insufficiency and Death
Previous studies demonstrated that germline deletion of Jak2 results in embryonic lethality at E12.5 due to impaired hematopoiesis 
. The floxed Jak2 mice that we obtained for this current study were created independently of those previous two works. Specifically, the floxed Jak2 mice used here had loxP sites introduced around the ATG start codon in exon 2 
. Although it was shown that germline deletion of Jak2 in our mouse 
recapitulates the previous two reports 
, we first wanted to demonstrate that germline deletion of Jak2 in our hands would yield a similar result. We found that germline deletion of Jak2 derived from the Jak2 cKO mice in our hands resulted in a phenotype that was identical to the previous Jak2 conventional knockouts, as all our Jak2 null embryos were dead and partially resorbed at E13.5 and the reason for death was impaired hematopoiesis (Figure S1
). As such, these results demonstrated a proof-of-concept in that they recapitulate the phenotype of the previously reported Jak2 conventional null embryos and therefore allowed for the deletion of Jak2 at later stages of prenatal and postnatal life.
To determine the role of Jak2 beyond germline deletion, we selected three different stages for Jak2 elimination; namely, mid/late gestation (E12.5), early postnatal life (PN4), or early adulthood (~2 months of age). provides a summary of these optimized injection times and the days at which necropsy was performed for each specific cohort.
For the mid/late gestational experiments, timed matings were set with ROSA26CreER/+
males and ROSA26+/+
females. Tamoxifen (TM) was then injected into pregnant dams at 12.5 days post coitum (dpc). No ROSA26CreER/+
newborn pups were found from the first several litters, indicating gestational lethality. To determine why lethality occurred in the Jak2 cKO mice, embryos from pregnant dams injected with TM at 12.5 dpc were examined at E17.5. All ROSA26CreER/+
13) were non-viable and resorbed at E17.5 when compared to controls (n
20). The Jak2 conditional mutants were easily distinguished by pale yolk sacs and undersized embryos (). PCR analysis confirmed that 100% of these atypical yolk sacs were R26CreER/+
13), and the null allele was only detected in these Jak2 mutant embryos (). In contrast, embryos containing the control genotype (ROSA26+/+
) appeared to be phenotypically normal (n
20). The Jak2 mutant embryos themselves exhibited significant erythropoietic deficiency and umbilical vessels lacked red blood cells (). Spleens from the Jak2 cKO embryos were necrotic (). Compared to controls, the fetal livers from the Jak2 cKO embryos were smaller and had hematopoietic insufficiency characterized marked hypo-cellularity, reductions in erythroid and megakaryocytic precursors, and severe anemia (). Lastly, to determine the relative levels of expressed Jak2 in the control and Jak2 cKO embryos, sections of fetal liver were subjected to qRT-PCR mRNA analysis. We found that the Jak2 mRNA levels in the Jak2 cKO livers (n
3) were <1% of that in controls (n
3) (). These results indicate that the severe erythropoietic-deficiency phenotype observed in the Jak2 cKO embryos correlates with the virtual elimination of Jak2 from their livers.
In summary, the data in indicate that timed deletion of Jak2 beginning at mid-gestation (E12.5) results in lethality by E17.5, characterized by necrotic spleens and severely impaired erythropoiesis in the fetal liver.
Tamoxifen-inducible Deletion of Jak2 during Early Postnatal Life Results in Death due to Severe Anemia
To investigate the importance of Jak2 during early postnatal life, TM was administered to ROSA26CreER/+
mice beginning at postnatal day 4 (PN4). We found that all Jak2
cKO mice were dead by PN25 (n
18). Jak2 cKO mice administered TM at PN4 were clearly distinguishable from the control mice due to pallor and a reduced body size at PN17, and quantification of this size difference found it to be significant (). When compared to the controls, the tails, paws, and gastrointestinal system of the Jak2 cKO mice were very pale, suggestive of poor peripheral perfusion (). Analysis of the peripheral blood revealed significantly reduced numbers of platelets and marked microcytic hypochromic anemia and the hematocrits of the Jak2 cKO mice were reduced by ~85%, when compared to controls ().
Deletion of Jak2 beginning at postnatal day 4 results in profound anemia.
Characterization of the hematopoietic potential of these animals continued with analysis of the bone marrow, spleen, and liver. The Jak2 cKO marrow was markedly hypocellular and quantification of these data indicated that this difference was significant (). We found that the Jak2 cKO mice displayed an 80% reduction in spleen weight to body weight ratios when compared to controls (). Gross and histologic examination found that the Jak2 cKO spleens were hypoplastic, exhibited disorganized red and white pulp, and displayed erythroid extramedullary hematopoiesis (). Interestingly, analysis of splenic lymphocytes found that the percentage of lymphocytes in the Jak2 cKO mice were significantly elevated, when compared to controls (). Histologic examination of the liver revealed that the Jak2 cKO mice exhibited hepatocellular atrophy and lobular collapse when compared to controls (). Lastly, when compared to the controls, the levels of liver derived Jak2 mRNA were reduced by more than 99% in the Jak2 cKO mice ().
Analysis of splenic lymphocytes at PN15 mice.
Collectively, the data in and indicate that loss of Jak2 beginning at PN4 results in animal death by PN25 via hematopoietic insufficiency and severe anemia.
Tamoxifen-inducible Deletion of Jak2 Beginning in Early Adulthood Results in Impaired Hematopoiesis, but Higher Survival Rates
We next wanted to determine what effect, if any, the loss of functional Jak2 would have on adult animals. To gain some sense of how best to optimize Jak2 deletion in early adults, we used Rosa26-LacZ reporter mice, injected them with TM, and then examined β-gal expression patterns. This was an important issue as the data in and showed that while TM is highly effective at deleting Jak2 (ie, >99%), however, it is not absolute. We found that a total of six TM injections for adult mice, comprised of three consecutive days of TM injections starting at PN35 and three consecutive days of TM booster injections starting at day 63, provided the highest level of β-gal expression when tissues were examined at PN91 (data not shown).
To determine the consequence of Jak2 deletion during early adulthood, control (ROSA26+/+;Jak2f/f) and Jak2 cKO (ROSA26CreER/+;Jak2f/f) mice were injected with TM following the optimized injection paradigm. Overall, we found that adult stage deletion of Jak2 resulted in a gross phenotype that was noticeably milder than that observed with the mid/late gestational and early postnatal Jak2 deletions. This was supported by the fact that death was observed in only 20% of the adult Jak2 cKO mutants, when compare to controls (). To gain a better understanding of the hematopoietic system within these animals, tissues were harvested at PN91 and analyzed. With respect to the peripheral blood, CBC and blood film evaluation indicated a number of abnormalities in the Jak2 cKO mice including significant reductions in red blood cells, hemoglobin, hematocrit, mean corpuscular hemoglobin, platelets, mean platelet volume, white blood cells, lymphocytes, monocytes, eosinophils, and basophils ( and ). The peripheral blood films from Jak2 cKO mice contained poikilocyte types and other morphologic changes including acanthocytes, schistocytes, echinocytes, elliptoechinocytes, spheroechinocytes, stomatocytes, and hypochromic microcytic erythrocytes (). Hemoglobin crystals were also qualitatively increased in Jak2 cKO mice, when compared to controls.
Tamoxifen-inducible deletion of Jak2 during early adulthood results in impaired hematopoiesis and lower mortality rates.
CBCs from adult mice at Day 91.
CBCs from adult mice at Day 91.
The spleen weight to body weight ratios were reduced by an average of 46% in the Jak2 cKO mice when compared to the controls and this was significant (). Histologic examination of this tissue revealed that Jak2 cKO spleens were comprised predominantly of red pulp with extramedullary hematopoiesis and had atrophied and disorganized white pulp (). The bone marrow of the Jak2 cKO mice was significantly less cellular, when compared to controls and this too was significant (). The Jak2 cKO livers exhibited diffuse centrilobular vacuolar degeneration consistent with hydropic change (). Lastly, we found that relative to controls, the levels of Jak2 mRNA in the Jak2 cKO mice at Day 91 were reduced by ~88% ().
Jak2 is expressed in nearly every tissue in the body and it has been implicated in a number of other pathologies including renal injury, hypertension, and heart failure 
. To determine what effect, if any, that deletion of Jak2 had on non-hematopoietic organs, we also examined heart, kidney, lung, and brain sections from these same TM treated animals (). Overall, there was no marked difference in the histological appearance of these tissues between the two genotypes even though their hematopoietic systems were notably different.
Histology of non-hematopoietic organs.
Collectively, the data in and as well as and indicate that deletion of Jak2 in early adulthood results in abnormal hematopoiesis characterized by reduction in marrow cellularity, atrophied spleens, and reduced peripheral blood cell counts. This was coincident with a ~88% reduction in Jak2 mRNA levels in the liver and a 20% death rate. Lastly, there were no marked differences in the histological appearance of the hearts, kidneys, lungs, and brains of these same TM treated animals.
The Higher Survival Rate of the Adult Jak2 cKO Mice is due to Re-population of Hematopoietic Tissues with Jak2 Expressing Cells
As opposed to the 100% lethality observed in the mid/late gestational and early postnatal deletions, deletion of Jak2 from young adult mice resulted in only a 20% death rate. One explanation for this was that TM was limited in its ability to delete Jak2 from the tissues. If this were the case, then over time, surviving Jak2 expressing cells could re-populate the hematopoietic tissues. An alternate explanation could be that in adulthood, there is functional redundancy by other proteins for the loss of Jak2 and this in turn allows for improved survival. To gain a better understanding of this process, mice of both genotypes were again subjected to the TM regimen at days PN35 and PN63. The erythropoietic state of the animals was then monitored longitudinally by measuring hemoglobin levels about once per week. As shown in , TM injection into the control mice was without effect. However, TM injection into the Jak2 cKO mice and subsequent Jak2 deletion resulted in cyclic erythropoiesis with the nadirs occurring around days 56 and 98. Interestingly, the hemoglobin levels returned to baseline levels by day 147. To understand what role Jak2 might play in this process, cohorts of mice were euthanized at days 56 and 147 so that the levels of Jak2 mRNA could be determined. At day 56, the levels of Jak2 mRNA in the Jak2 cKO mice were reduced by ~95% in both the bone marrow and liver, when compared to control animals (). However, by day 147, the levels of Jak2 expression in the surviving Jak2 cKO mice were virtually the same as those of controls in both the liver and bone marrow ().
The higher survival rate observed in the adult Jak2 cKO mice is due to re-population of hematopoietic tissues with Jak2 expressing cells.
The data in show that the levels of hemoglobin in the Jak2 cKO mice correlate positively with Jak2 expression levels in the liver and marrow. As such, these data suggest that the higher survival rate observed in adult animals is more likely due to repopulation of Jak2 expressing cells in hematopoietic tissues, rather than compensation by other proteins functioning in an environment that is devoid of Jak2. To demonstrate this experimentally, we created a compound mutant mouse that was comprised of one floxed and one null Jak2 allele (f/Δ). The advantage of this mouse is that one Jak2 allele in each cell has already been deleted and hence, Cre recombinase only needs to delete the remaining Jak2 allele.
For this experiment, three genotypes were used; control (ROSA26+/+
), Jak2 cKO (ROSA26CreER/+
) and the newly created Jak2 compound mutant (ROSA26CreER/+
). Beginning on day 35, the mice received the TM regimen and hemoglobin levels were again determined weekly. Similar to , injection of TM into the control mice (ROSA26+/+
) had no effect on hemoglobin levels while TM injection into the Jak2 cKO mice (ROSA26CreER/+
) once again caused a significant decrease in the hemoglobin levels with the nadir occurring around day 56 and increasing by day 63 (). However, TM injection into the Jak2 compound mutants (ROSA26CreER/+
), resulted in an even greater and more sustained decrease in the hemoglobin levels and 100% mortality (n
9) by day 72.
When taken together, the data in demonstrate that deletion of Jak2 in young adults temporarily reduces the hemoglobin levels to 6–8 g/dL and this is associated with a 20% death rate. At day 91, these animals have a number of hematopoietic abnormalities including peripheral blood cytopenias with abnormal erythrocyte morphology, decreased marrow cellularity, and splenic atrophy. However, the incomplete deletion of Jak2 by TM leaves some Jak2 expressing cells intact and the subsequent repopulation of the hematopoietic tissues by these cells normalizes the hemoglobin levels by day 147. When the f/Δ mice were injected with TM, there was a pronounced and sustained reduction (<5 g/dL for more than 7 days) of the hemoglobin concentrations and ensuing death. From this, we conclude that innate Jak2 actively restores hematopoietic homeostasis in the adult Jak2 cKO mouse, but that elimination of Jak2 via the combined effect of the null allele and TM-induced deletion of the floxed allele, results in death.
Tamoxifen-inducible Deletion of Jak2 Significantly Attenuates GBP-2 and IRF-1 Expression at all three Deletion Time Points
Conditional deletion of Jak2 beginning at either E12.5, PN4, or PN35 results in marked hematopoietic defects characterized by a lack of definitive hematopoiesis/erythropoiesis (, , ). To demonstrate that these defects were consistent with a loss of signaling that is downstream of Jak2, we conducted gene profile analysis on two genes whose expression is known to be highly Jak2-dependent; namely, GBP-2
. Here, Jak2 was conditionally deleted from mice via TM injection starting at either E12.5, PN4, or PN35 and livers were subsequently harvested at E17.5, PN19, and PN56, respectively. mRNA was extracted from the livers, reversed transcribed, and subjected to quantitative gene expression analysis. Relative to the control samples, we found that both GBP-2
were significantly reduced in Jak2 cKO livers across all three time points, thereby confirming a functional loss of signaling that is known to be downstream of Jak2 (). Furthermore, whereas the magnitude of the decreased expression of IRF-
1 in the Jak2 cKO mice was similar across all three time points, the effect of Jak2 deletion on GBP-2
expression in these same mice was greatest in early post natal life and least during late embryogenesis.
Tamoxifen-inducible deletion of Jak2 significantly attenuates GBP-2 and IRF-1 expression at all three time points.
In summary, the data in indicate that deletion of Jak2 impacts Jak2-dependent downstream signaling as determined by the reduced levels of GBP-2 and IRF-1 mRNA in the livers of Jak2 cKO mice, relative to controls. Furthermore, the effect of Jak2 deletion on IRF-1 expression was similar across the three time points whereas the effect of Jak2 deletion on GBP-2 varied with the age of the animal.
Tamoxifen-inducible Deletion of Jak2 during Early Adulthood Significantly Diminishes Myeloid, but not Lymphoid Progenitors
While the role of Jak2 on hematopoietic progenitor populations has been well defined in early embryogenesis 
, the gestational lethality of Jak2 conventional knockout mice has precluded a similar examination in adult animals. Therefore, to determine the specific hematopoietic lineages that were impacted by Jak2 deletion in the adult mouse, we repeated the longitudinal study shown in whereby mice received the TM dosing regimen starting on day 35 and cohorts of mice were sacrificed either at baseline (Day 31), at the hematopoietic nadir (Day 56), or at recovery (Day 120). Within the spleen, we found no significant differences in the number of T cells and B cells at any of the three adult time points (). We then examined the numbers of hematopoietic progenitors within the bone marrow. displays representative flow cytometry plots at Day 56 while indicates the aggregate numbers of progenitors plotted as a function of both genotype and day. Overall, we found no significant decreases in any hematopoietic progenitors either at baseline (Day 31) or at recovery (Day 120). However, significant decreases were observed in the Jak2 cKO mice at the hematopoietic nadir (Day 56). Specifically, at Day 56, when compared to controls, the Jak2 cKO mice had significantly reduced numbers of LT-HSC, ST-HSC, MPP, LSK, CMP, MEP, and GMP, but not CLP.
Tamoxifen-inducible deletion of Jak2 during early adulthood decreases stem cell and myeloid progenitors, but not lymphoid progenitors.
To determine if the reduced numbers of stem and myeloid progenitors impacted the clonogenic growth potential of these cells, bone marrow cells were also plated in semi-solid media and the number of colony forming units were determined. We observed no significant differences between the two genotypes in the clonogenic growth potential for any of the hematopoietic progenitors collected either on Day 31 or on Day 120 (). However, at Day 56, Jak2 cKO had significantly reduced numbers of CFU-GEMM (3.0±0.59 vs. 1.25±0.2), CFU-GM (41.3±3.0 vs. 24.3±3.2), BFU-E (7.0±1.8 vs. 0.8±0.3), and CFU-MK (13.3±1.6 vs. 2.0±0.4), when compared to controls. Interestingly, although all myeloid lineages were significantly reduced in the Jak2 cKO cells when compared to the controls, there appeared to be a differential effect of Jak2 on myelopoiesis as deletion of Jak2 severely impaired erythropoiesis and thrombopoiesis, but only had a mild to moderate effect on granulopoiesis and monocytopoiesis ().
Collectively, the data in indicate that deletion of Jak2 in the adult mouse diminishes myeloid, but not lymphoid progenitors. Furthermore, within the myeloid compartment, deletion of Jak2 in young adults severely impaired erythropoiesis and thrombopoiesis more so than granulopoiesis and monocytopoiesis. As such, Jak2 appears to have a specific yet critical effect on hematopoiesis in young adult animals.