Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1.

The Lyn tyrosine kinase plays essential inhibitory signaling roles within hematopoietic cells by recruiting inhibitory phosphatases such as SH2-domain containing phosphatase-1 (SHP-1), SHP-2, and SH2-domain containing 5'-inositol phosphatase (SHIP-1) to the plasma membrane in response to specific stimuli. Lyn-deficient mice display a collection of hematopoietic defects, including autoimmune disease as a result of autoantibody production, and perturbations in myelopoiesis that ultimately lead to splenomegaly and myeloid neoplasia. In this study, we demonstrate that loss of Lyn results in a stem/progenitor cell-intrinsic defect leading to an age-dependent increase in myeloid, erythroid, and primitive hematopoietic progenitor numbers that is independent of autoimmune disease. Despite possessing increased numbers of erythroid progenitors, and a more robust expansion of these cells following phenylhydrazine challenge, Lyn-deficient mice are more severely affected by the chemotherapeutic drug 5-fluorouracil, revealing a greater proportion of cycling progenitors. We also show that mice lacking SHIP-1 have defects in the erythroid and myeloid compartments similar to those in mice lacking Lyn or SHP-1, suggesting an intimate relationship between Lyn, SHP-1, and SHIP-1 in regulating hematopoiesis.


Introduction
The production and lineage commitment of hematopoietic cells is governed by the actions of a multitude of cytokines, hormones, and growth factors that bind to cell surface receptors activating signal transduction cascades that ultimately regulate the expression of genes that control cell fate and effector function. 1Signal propagation in these cells is actively counterbalanced by several families of inhibitory gene products including protein tyrosine phosphatases, 2 phosphatidyl-inositol phosphatases, 3 the suppressors of cytokine signaling (SOCS) proteins, 4 and receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). 5The central role played by tyrosine phosphorylation is exemplified by mutations in particular genes that lead to deregulation of hematopoiesis.For example, mutational activation of either the Abl 6,7 or Janus tyrosine kinases [8][9][10] leads to leukemogenesis.Loss of appropriate negative regulation of signaling may also have catastrophic consequences.5][16] Thus, the appropriate balance of positive and negative elements of signal transduction is essential for maintaining normal hematopoietic cell self-renewal, differentiation, and immune cell function.
5][26][27][28] Indeed, loss of Lyn kinase leads to defects in activation of inhibitory phosphatases that likely underlies the hypersensitivity of deficient cells to immunoreceptor and cytokine stimulation. 20,22,29,3026][27]31 In macrophages, Lyn deficiency leads to enhanced sensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF), diminished phosphorylation of SHIP-1 and SHP-1, enhanced AKT activity, and improved survival in cytokinereduced conditions. 22,23Similarly, loss of either SHIP-1 or SHP-1 leads to enhanced hypersensitivity to growth factors and resistance to cytokine withdrawal-induced cell death. 14,15,32,33e have previously shown that Lyn Ϫ/Ϫ mice exhibit hematopoietic system defects that lead to an age-dependent increase in extramedullary myelopoiesis and widely disseminated myeloid neoplasia.In this study, we have characterized the temporal development of perturbed hematopoiesis, identified an increase in erythroid progenitor numbers in these mice, and conducted a comparative analysis of the Lyn Ϫ/Ϫ phenotype with both Me v /Me v and SHIP-1 Ϫ/Ϫ mice.We show that Lyn Ϫ/Ϫ mice develop a similar, although much less severe, defect in hematopoiesis than Me v /Me v and SHIP-1 Ϫ/Ϫ mice, which is characterized by dramatically increased splenic progenitors and progeny of the myeloid and erythroid lineages.Furthermore, we show that these characteristics of Lyn-deficient mice are transplantable with bone marrow (BM) and independent of B cells and autoimmune disease.

Age-dependent increase of myeloid progenitors in Lyn
We have previously demonstrated that Lyn Ϫ/Ϫ mice develop splenomegaly characterized by a dramatic increase in hematopoietic progenitors responsive to cytokines such as GM-CSF, IL-3, M-CSF, and SCF.Moreover, Lyn Ϫ/Ϫ mice develop a myeloproliferative disease characterized by the accumulation of myelomonocytic cells widely disseminated throughout the animals. 22n order to understand the temporal progression of this phenotype, we investigated hematopoiesis in embryonic day-14 (E14) fetal liver and 4-and 16-week-old mice.This analysis revealed increased numbers of myeloid progenitors in the spleens of Lyn Ϫ/Ϫ mice at both 4 and 16 weeks of age (Figure 1A-B), although, the magnitude of the increase in GM-CSF-and M-CSF-responsive progenitors was enhanced in older mice (GM-CSF/M-CSF progenitors were 13-and 35-fold higher, respectively, in 16-week-old Lyn Ϫ/Ϫ mice compared with Lyn ϩ/ϩ mice, and 4-and 6-fold higher, respectively, in 4-week-old mice).This increase in splenic myeloid progenitors does not appear to be due to an impairment of BM myelopoiesis, as the number of myeloid progenitors in the BM of Lyn Ϫ/Ϫ mice was similar to that in wild-type mice at all ages (Figure 1A-B).While splenic myelopoiesis was enhanced in 4-and 16-week-old mice, analysis of E14 fetal liver revealed no difference in myeloid progenitor numbers between Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice (Figure 1C).

Increased numbers of immature erythroid cells and their progenitors in
Analysis of histologic sections of spleens from Lyn Ϫ/Ϫ mice older than 12 weeks revealed an increase in the number of erythroid cells (not shown).These data, and reports showing increased percentages of Ter119 staining cells in the spleens of Lyn Ϫ/Ϫ mice, 38 prompted us to investigate whether erythroid (RBC) development was also perturbed in Lyn-deficient mice.We used the transferrin receptor monoclonal antibody (mAb) CD71 and the erythroid lineage-restricted marker Ter119, as described in the characterization of STAT5A Ϫ/Ϫ 5B Ϫ/Ϫ mice, [39][40][41] to identify erythroblasts in Lyn Ϫ/Ϫ spleen.Staining with these Abs revealed a large increase in the proportion of CD71 ϩ Ter119 ϩ erythroblasts in the spleens of Lyn Ϫ/Ϫ mice (Figure 2A).To investigate whether there was a corresponding increase in primitive erythroid progenitors in the spleens of Lyn Ϫ/Ϫ mice, we assessed the numbers of colony-and burst-forming unit erythroid progenitors (CFU-e's and BFU-e's, respectively). 42,43While there was a slight increase in CFU-e's in the spleens of 8-week-old Lyn Ϫ/Ϫ mice, a dramatic increase in CFU-e's became evident by 16 weeks of age (Figure 2B).In the BM, we observed an increase in the number of CFU-e progenitors in both 8-and 16-week-old mice (Figure 2C).By contrast, the numbers of BM primitive BFU-e progenitors were similar at both time points.Similarly, enumeration of BM and spleen mixed erythroid/myeloid colonies generated in a cytokine cocktail of EPO, IL-3, and SCF revealed no difference in the number of these progenitors in 8-week-old Lyn ϩ/ϩ and Lyn Ϫ/Ϫ BM (Lyn ϩ/ϩ , 22.3 Ϯ 3.5; Lyn Ϫ/Ϫ , 22.5 Ϯ 2.3; n ϭ 12 mice Ϯ SEM/2 ϫ 10 4 cells) or spleen (Lyn ϩ/ϩ , 2.3 Ϯ 0.6; Lyn Ϫ/Ϫ , 3.1 Ϯ 0.5; n ϭ 12 mice Ϯ SEM/10 5 cells).As was the case with fetal liver myeloid  For personal use only.on April 21, 2017.by guest www.bloodjournal.orgFrom progenitors, no difference in either CFU-e's or BFU-e's was detected in E14 fetal livers (Figure 2D).Additionally, no significant difference in either CD71 ϩ /Ter119 ϩ cell numbers or numbers of apoptotic CD71 ϩ cells were observed in either fetal liver or adult BM (Figure 2E, and not shown).Thus, Lyn Ϫ/Ϫ mice develop a significant age-dependent increase in myeloid and erythroid progenitors with the expansion most apparent in relatively mature lineagerestricted progenitors.

Perturbed hematopoiesis is transplanted with Lyn ؊/؊ bone marrow
To investigate whether the hematopoietic compartment perturbation in Lyn Ϫ/Ϫ mice is intrinsic to the stem/progenitor cell, or due to microenvironmental differences, we conducted BM transplantations.Irradiated recipient (Ly5.1)mice received transplants of either Lyn ϩ/ϩ or Lyn Ϫ/Ϫ (Ly5.2) BM.Mice were then analyzed at between 10 to 12 weeks after transplantation for donor cell repopulation and transplantability of perturbed hematopoiesis.Engraftment was assessed by staining peripheral blood, spleen, and BM with the Ly5.2-specific mAb CD45.2 (donor) and Ly5.1 mAb CD45.1 (recipient), which revealed effective reconstitution with donor BM (Figure 3A, and not shown).Analysis of peripheral blood revealed similar percentages of donor-derived CD45.2 ϩ cells (Lyn ϩ/ϩ , 94.7 Ϯ 2.4%; Lyn Ϫ/Ϫ , 89.3 Ϯ 5.3%; n ϭ 4).However, numbers of CD19 ϩ B cells in the peripheral blood (Lyn ϩ/ϩ , 4.2 Ϯ 0.5 ϫ 10 3 /L; Lyn Ϫ/Ϫ , 0.6 Ϯ 0.2 ϫ 10 3 /L) and spleen (Lyn ϩ/ϩ , 13.0 Ϯ 1.8 ϫ 10 7 ; Lyn Ϫ/Ϫ , 2.7 ϫ 10 7 Ϯ 0.4 ϫ 10 7 ; n ϭ 6) were dramatically reduced following transplantation with Lyn Ϫ/Ϫ BM, as previously described. 20Interestingly, analysis of recipients of Lyn Ϫ/Ϫ BM revealed a significant population of cells in the spleen (25% Ϯ 10%) that failed to stain with either CD45.1 or CD45.2 (Figure 3A).Further analysis of this CD45 Ϫ population revealed that they were CD71 ϩ Ter119 ϩ and lacked c-Kit expression, consistent with our previous analysis of the spleens of Lyn Ϫ/Ϫ donors (Figures 2A, 3A, and not shown).This population was increased approximately 5-fold in the spleens of Lyn Ϫ/Ϫ BM recipients compared with Lyn ϩ/ϩ transplants.By contrast, the percentages and absolute numbers of CD71 ϩ Ter119 ϩ cells in the BM of Lyn Ϫ/Ϫ and Lyn ϩ/ϩ BM recipients were similar (Figure 3A,C).Analysis of erythroid and myeloid progenitors in spleen revealed that, like Lyn Ϫ/Ϫ donors, irradiated recipients of Lyn Ϫ/Ϫ BM displayed dramatically increased myeloid and erythroid progenitor numbers (Figure 3B,D).Although there was a modest increase in CFU-e's in the BM of Lyn Ϫ/Ϫ BM recipients, the number of myeloid progenitors and BFU-e's in the BM was similar (Figure 3B,D).Increased numbers of progenitors responsive to M-CSF in peripheral blood were also detected in the transplants, a phenotype we previously observed in 8-week-old Lyn Ϫ/Ϫ mice 22 (Figure 3B,D).Collectively, these results reveal that the agedependent defect observed in Lyn Ϫ/Ϫ donors is recapitulated in Lyn Ϫ/Ϫ BM recipients.The hematopoietic defect in Lyn ؊/؊ mice is not B-cell or autoantibody dependent Lyn Ϫ/Ϫ mice have a B-cell defect that results in hyper-IgM and autoantibody production leading to glomerulonephritis and premature death. 34,44To exclude the possibility that deregulated hematopoiesis is due to underlying autoimmune disease, we eliminated autoantibody production by crossing Lyn Ϫ/Ϫ mice with MT/MT mice, which have a mutation in the B-cell receptor (BCR) -chain leading to impaired B-cell maturation. 35The levels of total immunoglobulin were reduced more than 5000-fold, and antinuclear antibodies were undetectable in Lyn Ϫ/Ϫ ;MT/MT doublemutant mice (Figure 4A-B).However, the number of CD71 ϩ Ter119 ϩ cells previously observed in Lyn Ϫ/Ϫ mice was not altered in the double mutant, with both Lyn Ϫ/Ϫ and Lyn Ϫ/Ϫ ;MT/ MT 9-month-old mice containing approximately 8-to 10-fold more splenic erythroid cells (Figure 4C).The total numbers of CFU-e's and BFU-e's in the spleens of Lyn Ϫ/Ϫ and Lyn Ϫ/Ϫ ;MT/ MT mice were also correspondingly elevated.Likewise, myeloid progenitors were increased in both Lyn Ϫ/Ϫ and Lyn Ϫ/Ϫ ;MT/MT mice (Figure 4D-E).Thus, B-cell-dependent autoimmune disease is not a major contributing factor to this phenotype.

Increased numbers of splenic multipotent progenitors in Lyn
To quantify the number of more primitive progenitor populations, we conducted a day-12 colony-forming unit-spleen assay. 45While the spleens of Lyn Ϫ/Ϫ mice contained approximately 3-fold more CFU-S 12 , the BM contained equivalent numbers of these progenitors (Figure 5A).Thus, in addition to an increase in more mature lineage-restricted progenitors, Lyn Ϫ/Ϫ mice possess increased numbers of primitive progenitors in the spleen without a corresponding alteration in BM progenitors.

Heightened sensitivity of Lyn ؊/؊ mice to 5-FU
The increase in hematopoietic progenitors in Lyn Ϫ/Ϫ mice coupled with previous findings illustrating an essential inhibitory signaling role for Lyn within hematopoietic cells led us to speculate that loss of Lyn might enhance the ability of the hematopoietic system to recover from insults that are hematoablative. 46To test this hypothesis, we challenged mice with the chemotherapeutic drug 5-FU, which is cytotoxic to cells in cycle.The cellularity of the indicated compartments was then assessed at 2-day intervals for 16 days following 5-FU challenge.Surprisingly, the total cellularity of each tissue analyzed was similar irrespective of genotype (Figure 5B-D).
However, 5-FU had a more severe effect on the hematocrit of Lyn Ϫ/Ϫ mice than that of control mice (Figure 5E).This, together with the observation that Lyn-deficient mice showed a lag of approximately 2 days in the mobilization/expansion of immature CD71 ϩ Ter119 ϩ erythroblasts in the spleen following 5-FU treatment (Figure 5F), may suggest a greater percentage of progenitor cells in cycle and thus heightened 5-FU sensitivity.Alternatively, an underlying defect in erythropoiesis in Lyn Ϫ/Ϫ mice may lead to an impaired rate of RBC production.To investigate these possibilities we challenged Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice with 5-FU and looked for differential sensitivity of erythroid progenitors to 5-FU.Interestingly, Lyn Ϫ/Ϫ mice, which normally contain between 3-to 5-fold more CFU-e's in BM than Lyn ϩ/ϩ mice (Figure 2C), exhibited approximately 5-fold fewer CFU-e's than Lyn ϩ/ϩ mice following 5-FU challenge (Figure 5G).However, analysis of BrdU uptake into more mature CD71 ϩ erythroblasts or Mac-1 ϩ myeloid cells in untreated mice revealed similar levels of BrdU incorporation (Figure 5H).Thus, Lyn Ϫ/Ϫ mice contain an increase in both the absolute number of erythroid progenitors, and an increase in the cycling status of these progenitors, without a corresponding increase in BrdU incorporation into more mature BM erythroblasts.
Similarities between Lyn ؊/؊ , Me v /Me v , and SHIP-1 ؊/؊ mice We and others have defined a relationship between Lyn's inhibitory signaling role and its ability to recruit and activate SHP-1 and SHIP-1 by membrane localization and/or phosphorylation. 20,22,29,30,47ecent studies have also revealed that Lyn and SHIP-1 act together to negatively regulate M-CSF-dependent AKT activity, 23 a finding supported by our previous results showing that SHIP-1 is a target of Lyn-dependent phosphorylation in primary macrophages and B cells, 22,36 and results illustrating coordinated roles for both Lyn and SHIP-1 in establishing Fc␥ receptor IIb1 (Fc␥RIIb1)-dependent inhibitory signaling in B cells and mast cells. 18,19,24,31To investigate this relationship further, we analyzed both Me v /Me v and SHIP-1deficient mice to see if the phenotype of Lyn Ϫ/Ϫ mice might be explained by deregulation of either SHP-1 or SHIP-1.We have previously highlighted the similarities between Lyn Ϫ/Ϫ , Me v /Me v , and SHIP-1 Ϫ/Ϫ mice in that they develop splenomegaly, increased splenic and peripheral blood myeloid progenitors, and in the case of Lyn Ϫ/Ϫ and Me v /Me v mice, enhanced sensitivity to M-CSF and GM-CSF. 22,32,33o further our characterization of Lyn Ϫ/Ϫ , Me v /Me v , and SHIP-1 Ϫ/Ϫ mice, we conducted a limited analysis of the hematopoietic compartments of the 3 mutants and analyzed erythropoiesis in age-matched 6-to 8-week-old mice.Results presented in Table 1 reveal that both Me v /Me v and SHIP-1 Ϫ/Ϫ mice develop similar degrees of splenomegaly, while Me v /Me v mice exhibit dramatically reduced BM cellularity and more severe anemia than do agematched SHIP-1 Ϫ/Ϫ mice.By contrast, Lyn Ϫ/Ϫ mice do not show evidence of splenomegaly until 10 to 13 weeks of age 22 (Table 1).Interestingly, the spleens of Me v /Me v , like Lyn Ϫ/Ϫ mice, contained a large proportion of CD71 ϩ Ter119 ϩ cells (Figure 6A), which, taking into account the splenomegaly in Me v /Me v mice, corre-sponded to a 30-fold increase in this population (Figure 6B).Indeed, this population is expanded to a similar degree as that of the Mac-1 ϩ myelomonocytic/granulocytic population in the spleens of Me v /Me v mice (not shown).In keeping with the increase in CD71 ϩ Ter119 ϩ cells in the spleen, there was a corresponding increase in splenic CFU-e's, and to a lesser extent, BFU-e's, in Me v /Me v mice (Figure 6C), as previously described. 11Thus, our results showing significant increases in erythroid progenitors in Lyn Ϫ/Ϫ mice highlight another similarity between these 2 mouse strains.However, splenic extramedullary hematopoiesis in Me v / Me v mice is much more severe than that observed in Lyn Ϫ/Ϫ mice of similar age.The BM of Me v /Me v mice also exhibited a more dramatic reduction in the proportion and number of CD71 ϩ Ter119 ϩ cells than that observed in Lyn Ϫ/Ϫ mice, and a decrease in CFU-e's and BFU-e's, not observed in Lyn Ϫ/Ϫ mice (Figure 6A-C).
with the latter 2 mutants both more severely affected than Lyn Ϫ/Ϫ mice.The BM compartments of the 3 mutants, however, are significantly different both in terms of CD71 ϩ Ter119 ϩ erythroid cell numbers and alterations in CFU-e and BFU-e progenitor content.

Lyn ؊/؊ and SHIP-1 ؊/؊ mice exhibit enhanced production of erythroid progenitors but normal recovery of hematocrit following erythropoietic stress
The RBC cytotoxic drug phenylhydrazine induces anemia in mice that is reversed by a wave of erythropoiesis characterized by an accumulation of CD71 ϩ Ter119 ϩ cells in the spleen preceding the restoration of normal RBC numbers and hematocrits.A comparison of the response of Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice to phenylhydrazine revealed a similar rate of hematocrit recovery, reticulocyte production, splenomegaly, and increase in splenic CD71 ϩ Ter119 ϩ cellularity, despite the fact that Lyn Ϫ/Ϫ mice exhibited dramatically increased splenic CFU-e and BFU-e progenitor numbers following phenylhydrazine challenge (Figure 7A-D).Interestingly, comparison of SHIP-1 Ϫ/Ϫ with Lyn Ϫ/Ϫ mice revealed a similar increase in CFU-e's at day 3 after phenylhydrazine challenge in both mutants, although SHIP-1 Ϫ/Ϫ mice also showed increased numbers of reticulocytes at day 3 (Figure 7E-F).However, this increase in splenic erythroid progenitors and peripheral blood reticulocyte numbers (for SHIP-1 Ϫ/Ϫ but not Lyn Ϫ/Ϫ mice) did not result in a statistically significant enhancement in hematocrit recovery from phenylhydrazine challenge in either strain.

Lyn kinase is dispensable for EPO-dependent activation of Jak/STAT/MAPK pathways and SHIP-1 phosphorylation in primary erythroblasts
Given that the loss of Lyn lowers the threshold for B cell, [18][19][20][21] mast cell, [25][26][27][28] and macrophage stimulation, 22,23 and the reported role for Lyn in EPO receptor phosphorylation and STAT5 activation, 48 we investigated whether loss of Lyn might modulate EPO-dependent signaling in primary erythroid cells.We purified CD71 ϩ erythroblasts from spleens of phenylhydrazine-challenged mice and showed that they were predominantly basophilic and early polychromatophilic erythroblasts that expressed both CD71 and Ter119 (Supplemental Figure 1A-B, available by clicking the Supplemental Figure link at the top of the online article on the Blood website), but not c-Kit, or CD45 (not shown).Stimulation of these cells with EPO led to dose-dependent tyrosine phosphorylation of both the EPO receptor and Jak2, and corresponding activation of STAT5 and MAP kinases (Supplemental Figure 1C).However, comparison of EPO-dependent signaling in Lyn ϩ/ϩ and Lyn Ϫ/Ϫ cells revealed no detectable modulation in either the dose response or time course of STAT5 or MAP kinase activation (Supplemental Figure 1D).Moreover, unlike the case in B cells 36 and macrophages, 22 loss of Lyn did not alter the tyrosine phosphorylation of SHIP-1 or SHP-1 in erythroblasts (Supplemental Figure 1E, and not shown).Thus, Lyn is dispensable for EPO-dependent activation of the JAK/STAT and MAP kinase pathways and the tyrosine phosphorylation of SHP-1 and SHIP-1 in erythroblasts.

Discussion
Lyn plays a critical role in myelomonocytic cells as a regulator of SHP-1, SHP-2, SHIP-1, and the ITIM-containing inhibitory receptors paired immunoglobulin-like receptor B (PIR-B) and signal regulatory protein ␣ (SIRP␣). 22Failure to appropriately engage inhibitory signaling in the absence of Lyn is thought to underlie the extramedullary hematopoiesis observed in these mice and the hypersensitivity of Lyn-deficient macrophages to proliferation and survival signals provided by GM-CSF and M-CSF.Here, we have further defined the temporal progression of perturbations in hematopoiesis that develop as a consequence of Lyn deficiency.We have For personal use only.on April 21, 2017.by guest www.bloodjournal.orgFrom also identified a novel defect in erythropoiesis that results in a significant increase in erythroid progenitors and erythroblasts in Lyn Ϫ/Ϫ spleen.This characteristic becomes more severe with age and coincides temporally with the development of splenomegaly.By contrast, significant increases in splenic myeloid progenitor numbers were apparent in mice as young as 4 weeks of age and preceded splenomegaly.
In light of the superficial similarities between Lyn Ϫ/Ϫ , SHIP-1 Ϫ/Ϫ , and Me v /Me v mice, and Lyn's critical role in regulating the tyrosine phosphorylation and plasma membrane mobilization of SHIP-1 and SHP-1, we compared hematopoiesis in these mice.Interestingly, the composition of the spleens of both SHP-1-and SHIP-1-deficient mice was remarkably similar in terms of the degree of splenomegaly and erythroblast/erythroid progenitor composition.The BM of both mutants also showed a dramatic reduction in immature erythroid cells and an expansion of myeloid populations.Thus, the phenotypes of SHIP-1 Ϫ/Ϫ and Me v /Me v mice were similar, albeit more severe, than similarly aged Lyn Ϫ/Ϫ mice.However, it should be noted that Lyn Ϫ/Ϫ mice do develop splenomegaly of a similar magnitude to that observed in Me v /Me v and SHIP-1 Ϫ/Ϫ mice as they age. 22Comparison of Lyn Ϫ/Ϫ and SHIP-1 Ϫ/Ϫ splenic myeloid colony numbers also revealed that myeloid progenitors are dramatically increased in SHIP-1 Ϫ/Ϫ mice.This increase in myelopoiesis in SHIP-1 Ϫ/Ϫ mice again is more severe than that observed in Lyn Ϫ/Ϫ mice when the degree of splenomegaly is taken into account.There were also notable differences between Lyn Ϫ/Ϫ , Me v /Me v , and SHIP-1 Ϫ/Ϫ mice including a diminution in CFU-e and BFU-e (Me v /Me v ) numbers in the BM not observed in Lyn Ϫ/Ϫ mice.However, the dramatic expansion of myeloid populations in the BM of Me v /Me v and SHIP-1 Ϫ/Ϫ mice, and thus potential limitations in stromal-cell support, may be a contributing factor to these BM progenitor population perturbations.
Splenomegaly may arise for a variety of reasons, including loss of signal inhibition, such as in SHP-1- 12,13 or SHIP-1-deficient mice, 14,15 or be due to the expression of oncogenes such as BCR/Abl. 6,7Splenomegaly may also reflect an underlying defect resulting in hematologic stress.4][55] Thus, we reasoned that splenomegaly in Lyn Ϫ/Ϫ mice might be due to Lyn's role in signal inhibition, or an as-yetunrecognized role for Lyn in the production, survival, or differentiation of erythroblasts.Severe defects in erythropoiesis in mice are most readily apparent during fetal development, a period of high erythropoietic rate. 39However, analysis of fetal liver hematopoiesis in Lyn Ϫ/Ϫ mice failed to show alterations in either myeloid or erythroid progenitor numbers, and quantitation of neonatal erythroblasts revealed no significant difference between Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice (not shown).This is in keeping with our previous analysis of aged Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice, which failed to show evidence of anemia. 22A thorough analysis of apoptosis in immature erythroblasts in adult BM and embryonic day-14 fetal liver also revealed no differences between Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice, in keeping with our finding that Lyn deficiency does not alter EPO-dependent STAT5 phosphorylation.However, the BM of Lyn Ϫ/Ϫ mice did display a modest decrease in immature erythroid cells and an increase in CFU-e's, suggesting an impairment in the expansion/differentiation of CFU-e progenitors into erythroblasts.Interestingly, Helgason et al 14 have speculated that the diminution of BM CFU-e's and Ter119 ϩ cells in SHIP-1 Ϫ/Ϫ BM may indicate a positive role for SHIP-1 in erythropoiesis.Our data suggesting an underlying defect in erythropoiesis in Lyn Ϫ/Ϫ mice were supported by our finding that 5-FU-dependent anemia 56 was more severe, and the anemia-induced expansion of the splenic CD71 ϩ /Ter119 ϩ erythroblasts was delayed in Lyn Ϫ/Ϫ mice.However, the heightened sensitivity of Lyn-deficient mice to 5-FU was likely due to a greater proportion of cycling Lyn Ϫ/Ϫ progenitor cells, as assessment of CFU-e and BFU-e erythroid progenitor numbers 3 days after 5-FU challenge revealed a more severe diminution of erythroid progenitors in Lyn Ϫ/Ϫ versus Lyn ϩ/ϩ mice.Interestingly, the hematopoietic progenitor cell compartment in SHIP-1 Ϫ/Ϫ mice also exhibits heightened sensitivity to 5-FU and diminished stem cell competitive repopulation ability. 57he similarity between Lyn ϩ/ϩ , Lyn Ϫ/Ϫ , and SHIP-1 Ϫ/Ϫ mice in hematocrit recovery following treatment with the RBC-depleting drug phenylhydrazine argues against a major defect in erythropoiesis in either Lyn Ϫ/Ϫ or SHIP-1 Ϫ/Ϫ mice.Indeed, assessment of erythroid progenitor numbers in the spleens of Lyn ϩ/ϩ , Lyn Ϫ/Ϫ , and SHIP-1 Ϫ/Ϫ mice after phenylhydrazine treatment revealed a more robust expansion of this lineage in both Lyn Ϫ/Ϫ and SHIP-1 Ϫ/Ϫ mice.It is surprising, however, that the large increase in erythroid progenitors in the spleens of both Lyn Ϫ/Ϫ and SHIP-1 Ϫ/Ϫ mice did not ultimately lead to an enhanced recovery from phenylhydrazine.Indeed, this observation raises the possibility that Lyn and SHIP may act as negative regulators during early myelo/erythropoiesis, but may be required for appropriate differentiation of hematopoietic cells at later stages of development.
Given the similarities between Lyn Ϫ/Ϫ , Me v /Me v , and SHIP-1 Ϫ/Ϫ mice, together with studies showing a role for Lyn in EPOdependent signaling, 48 and the reported role of SHP-1 as a direct antagonist of EPO receptor and Jak2 phosphorylation/activation, 58,59 we investigated the role of Lyn in EPO receptor signaling in primary erythroblasts.We found no significant difference in either the dose response or time course of activation of STAT5 or MAP kinase following EPO stimulation.Surprisingly, Lyn was also dispensable for both SHIP-1 and SHP-1 (not shown) tyrosine phosphorylation in these cells.Thus, Lyn does not play a significant role in the EPO-dependent activation of the JAK/STAT or MAP kinase pathways, or in SHP-1 and SHIP-1 tyrosine phosphorylation in erythroblasts at this stage of development.These results suggest that the increased erythropoiesis observed in Lyn Ϫ/Ϫ mice is unlikely to be a simple consequence of heightened EPO sensitivity of Lyn Ϫ/Ϫ erythroblasts.However, Lyn-dependent regulation of other EPO-dependent signaling pathways, or regulation of EPO signaling at other stages of erythroid development, cannot be ruled out.Inhibitory roles for Lyn in signaling from other receptors expressed within this lineage, functions for Lyn in differentiation or mobilization of erythroid cells, [60][61][62] and roles for Lyn in more primitive progenitor populations also remain to be explored.Regardless, it is clear that Lyn plays a critical role in signaling required for appropriate development of the myeloid, erythroid, and B-lymphoid arms of the hematopoietic system.Analysis of combined loss-of-function mutants of Lyn, SHP-1, and/or SHIP-1 will be an important step in assessing the genetic interactions between these signal transduction regulators in hematopoietic stem/progenitor cell biology.

Figure 1 .
Figure 1.Normal fetal and bone marrow myelopoiesis but enhanced splenic myelopoiesis in 4-and 16-week-old Lyn ؊/؊ mice.Progenitors responsive to GM-CSF, IL-3, or M-CSF in the spleen and BM of (A) 16-week-old or (B) 4-week-old sex-matched Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice were assessed by in vitro semisolid agar assays.Data presented in panels A-B correspond to the mean (Ϯ SEM) for 2 to 4 experiments using 2 to 3 mice per experiment.(C) Day-14 myeloid fetal liver progenitor populations in Lyn ϩ/ϩ (n ϭ 9), Lyn ϩ/Ϫ (n ϭ 17), or Lyn Ϫ/Ϫ (n ϭ 8) mice were assessed in the presence of the indicated cytokines (mean Ϯ SEM).Fetal livers were derived from day-14 embryos obtained from time-mated Lyn ϩ/Ϫ mice.The splenic myeloid data presented in panel A (left panel) have been reported previously 22 (reprinted from Immunity, Vol 15, Harder KW, Parsons LM, Armes J, et al, "Gain-and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage," pages 603-615, copyright (2001), with permission from Elsevier).

Figure 2 .
Figure 2. Expansion of immature erythroid cells in Lyn ؊/؊ spleen and increased erythroid CFU-e numbers in BM and spleen of Lyn ؊/؊ mice.(A) A 3-color flow cytometric analysis of 8-week-old Lyn ϩ/ϩ and Lyn Ϫ/Ϫ spleen populations stained with CD71 and Ter119.Propidium iodide (PI)-positive and mature RBCs were excluded from the analysis.Relative percentages (Ϯ SD) of immature erythroid (upper right quadrant) and more mature erythroid (lower right) cells of 3 mice of each genotype are indicated.Erythropoiesis in (B) spleen, (C) BM of 8-and 16-week-old Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice, and (D) embryonic day-14 fetal liver was assessed by methylcellulose culture.Mature erythroid (CFU-e) and immature erythroid (BFU-e) progenitors were determined at day 2 and day 10, respectively.(E) Relative percentages (Ϯ SD) of annexin-FITC-positive erythroid cells in fetal liver (n ϭ 7-8/genotype) and adult bone marrow (n ϭ 5/ genotype) are indicated.

Figure 3 .
Figure 3. Perturbed myelo/erythropoiesis is transplantable with Lyn ؊/؊ BM. (A) BM from C57BL/6 Ly5.2 background donor Lyn ϩ/ϩ or Lyn Ϫ/Ϫ mice was transplanted into irradiated C57BL/6 Ly5.1 mice.Engraftment was assessed by identifying donor and recipient cells by FACS analysis with CD45.2 (donor) and CD45.1 (recipient) mAbs.Alternatively, immature erythroid lineage cells in spleen and BM were assessed by staining with CD71 and Ter119 mAbs.Both PI-positive and mature RBCs were excluded from the analysis.The relative percentages of each population (Ϯ SD) in 4 recipient mice of each genotype are presented.(B) CFU-e and BFU-e content in the spleen and BM of recipients of either Lyn ϩ/ϩ or Lyn Ϫ/Ϫ BM are shown.(C) The relative number of CD71/Ter119 double-positive erythroid cells in spleen and BM assessed 10 to 12 weeks after transplantation.(D) Myeloid progenitors in spleen, BM, and peripheral blood responsive to the indicated cytokines were determined in mice reconstituted with either Lyn ϩ/ϩ or Lyn Ϫ/Ϫ BM.Progenitors were scored following plating of 10 5 spleen cells, 2.5 ϫ 10 5 BM cells, or 1 to 2 L peripheral blood.Data presented in panels B and D correspond to the mean (Ϯ SEM) for 3 experiments using 2 mice/ experiment (*P Ͻ .01,Student t test).Data presented in panel C were obtained from the analysis of 4 mice in 2 experiments (mean Ϯ SEM).

Figure 4 .
Figure 4. B-cell developmental defects and autoimmune disease are not linked to the hematopoietic changes observed in Lyn ؊/؊ mice.(A) Lyn ϩ/ϩ , Lyn Ϫ/Ϫ , Lyn Ϫ/Ϫ ;MT/MT, and MT/MT control mice were assessed for total immunoglobulin levels by ELISA, or (B) antinuclear antibody titers using DNA and nuclear antigen-coated ELISA plates incubated with a 1:500 dilution of sera from mice of the indicated genotypes.Relative optical densities are shown.(C) Total numbers of CD71 ϩ Ter119 ϩ cells were enumerated following FACS analysis of spleen cell preparations from mice of the indicated genotypes.(D) Splenic CFU-e's and BFU-e's in mice of the indicated genotypes.Data in panels C-D are means (Ϯ SD) for 2 mice per genotype.Mean spleen weights were Lyn ϩ/ϩ , 0.11 Ϯ 0.01 g; Lyn Ϫ/Ϫ , 0.31 Ϯ 0.08 g; and Lyn Ϫ/Ϫ MT, 0.14 Ϯ 0.05 g. (E) Splenic myeloid progenitor numbers responsive to the indicated cytokines in mice of the indicated genotypes.Data represent the mean (Ϯ SEM) for 2 mice per genotype per experiment in 2 separate experiments.

Figure 5 .
Figure 5. Lyn ؊/؊ mice have elevated numbers of CFU-S12 in the spleen but are more severely affected by the cytotoxic drug 5-FU.(A) Numbers of primitive hematopoietic progenitors in the spleen and BM of Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice capable of forming macroscopic colonies in the spleens of irradiated recipient mice 12 days after injection of BM or spleen cell populations (*P Ͻ .01,Student t test).Total cellularity of the (B) BM, (C) spleen, (D) peripheral blood, and (E) hematocrits of Lyn ϩ/ϩ and Lyn Ϫ/Ϫ was determined at 2-day intervals following intravenous injection of the cytotoxic drug 5-FU.Results for spleen and blood are shown as percent of untreated, since young C57BL/6 background Lyn Ϫ/Ϫ mice have diminished splenic and white blood cell (WBC) counts compared with control mice.(F) The total cell number of CD71 ϩ Ter119 ϩ double-positive cells in the spleen of 5-FU-treated Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice determined by FACS analysis at 2-day intervals over 16 days.In experiments depicted in panels B-F, 5 age-and sex-matched mice per genotype were analyzed at each time point.Data presented are the mean (Ϯ SEM).(G) Numbers of BM CFU-e's and BFU-e's were assessed 3 days after challenge with 5-FU.Data represent the mean (Ϯ SD) of 3 to 4 mice/genotype.(H) Percentage BrdU pos erythroid (CD71 ϩ ) and myeloid (Mac-1 ϩ ) cells in BM 2.5 hours after injection of BrdU (40 mg/kg).Data presented are the mean (Ϯ SD) of 3 mice per genotype with similar results observed in 2 independent experiments.

Figure 6 .
Figure 6.Motheaten viable and SHIP-1 ؊/؊ mice exhibit a similar but more severe hematopoietic phenotype than Lyn ؊/؊ mice.(A) FACS profiles of 6-to 8-week-old wild-type (ϩ/ϩ), Me v /Me v , and SHIP-1 Ϫ/Ϫ spleen and BM populations stained with CD71 and Ter119 mAbs.PI-positive and mature RBCs were excluded from the analysis.Relative percentages of immature erythroid (upper right quadrant) and more mature erythroid (lower right) cells are indicated for 4 mice of each genotype (Ϯ SD).(B) Total numbers of CD71 ϩ Ter119 ϩ cells in the spleen and BM of ϩ/ϩ, Me v /Me v , SHIP-1 Ϫ/Ϫ , and Lyn Ϫ/Ϫ mice are shown.Values represent the mean (Ϯ SD) of 5 C57BL/6 background mice per group, except Me v /Me v , for which 2 mice per group are shown.(C) Mature erythroid (CFU-e) and immature erythroid (BFU-e) progenitors in ϩ/ϩ, Me v /Me v , and SHIP-1 Ϫ/Ϫ spleen and BM were determined at day 2 and day 10, respectively.Data represent the mean (Ϯ SEM) for 2 Me v /Me v mice and 4 ϩ/ϩ and SHIP-1 Ϫ/Ϫ mice.(D) Myeloid progenitor content in the spleens of 8-week-old SHIP-1 Ϫ/Ϫ mice was assessed as in Figure 1 in the presence of the indicated cytokines.Values represent the mean (Ϯ SEM) for 4 animals in 2 experiments.

Figure 7 .
Figure 7. Lyn ؊/؊ mice exhibit normal hematocrit recovery following phenylhydrazine challenge but show enhanced erythroid progenitor expansion.(A) Changes in hematocrit and percentages of reticulocytes over a 17-day period were assessed in Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice after 2 consecutive intraperitoneal injections of phenylhydrazine (60 mg/kg) on days 1 and 2. Mean hematocrit (squares) and reticulocyte (circles) values for 3 to 8 mice per time point (Ϯ SD) are shown.(B) Changes in spleen weights in Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice were recorded following phenylhydrazine challenge.Values represent the mean (Ϯ SD) for 3 to 5 mice per time point, excepting days 5 to 7, where 6 to 19 mice per point are shown.(C) Representative 2-color FACS profiles of spleen cell populations from phenylhydrazinetreated Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice stained with the mAbs CD71 and Ter119.Mature RBCs and PI-positive cells were excluded from the analysis.(D) Numbers of splenic CFU-e's and BFU-e's in Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice were determined 3, 6, and 9 days after phenylhydrazine challenge.Values are the mean (Ϯ SD) of 5 mice per genotype.(E) The response of SHIP-1 ϩ/ϩ and SHIP-1 Ϫ/Ϫ mice to phenylhydrazine challenge was determined by assessing reticulocyte and hematocrit levels at days 0, 3, and 6 after challenge.Values are the mean (Ϯ SD) of 4 mice per genotype.(F) Numbers of splenic CFU-e's in SHIP-1 ϩ/ϩ (f) and SHIP-1 Ϫ/Ϫ mice (Ⅺ) were determined 3 and 6 days after phenylhydrazine challenge.Values are the mean (Ϯ SD) of 4 mice/genotype.