Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are highly specialized hematopoietic tissue subcompartments that play a critical role in regulating erythropoiesis. Erythroblastic islands contain a central macrophage surrounded by erythroid cells at different stage of maturation. The central macrophage is different from other resident macrophages and can be distinguished by the surface expression of F4/80 antigen and Forssman glycosphingolipid and by the absence of Mac-I antigen (for a review, see the work of Manwani and Bieker [24
]). The role of the central macrophage in erythroid maturation involves the engulfment of extruded nuclei from erythroblasts and degradation of the nuclear DNA by DNase II-alpha (27
In the fetal liver, nuclei expelled from the developing erythroblast rapidly expose phosphatidylserine on their surface and are phagocytosed by the central fetal liver macrophages (17
). The mechanisms of erythroblastic island formation, macrophage-erythroid cell interaction, and cytokine expression are not fully understood. Cell-cell interactions seem to be mediated by different proteins, but only a few have been characterized (8
One of the main established roles of the central macrophage is to phagocytose and degrade DNA contained in erythrocyte nuclei (27
). The lack or dysfunction of the central macrophage, as in retinoblastoma-KO mice, results in embryonic lethality, anemia, and abnormal maturation of definitive erythroblasts that fail to enucleate (15
). Gene ablation experiments revealed that Dnase2a
is indispensable for definitive erythropoiesis and is responsible for destroying the nuclear DNA expelled from developing erythroid cells. Lack of DNase II-alpha activity is associated with persistence of nucleated definitive erythrocytes in the fetus. Homozygous Dnase2a
-null embryos develop normally up to E12, becoming progressively anemic afterwards and dying around E17 (16
). The defect in definitive erythropoiesis has been established to be non-cell autonomous because liver cells from Dnase2a−/−
mice transplanted into wt mice developed into mature enucleated cells (16
). The central macrophage with no DNase II-alpha is unable to digest nuclear DNA expelled from the developing erythrocyte. Dnase2a−/−
mice suffer from severe anemia due to IFN-β production. Accumulation of DNA directly triggers the production of interferon-β by the central macrophage and induces expression of interferon-responsive genes that cause the embryonic lethality (49
). IFN-β has a cytotoxic effect on erythroid cells and inhibits erythroid cell growth in a dose-dependent manner (49
). Activation of IFN-β is one of the markers of the activation of innate immunity. Innate immunity is activated in Dnase2a−/−
mice in a Toll-like receptor (TLR)-independent mechanism (49
). The signal molecules linking DNase II-alpha deficiency to IFN-β production remain to be determined, but it has recently been shown that IFN-β production can be stimulated by threonine-phosphatase of Eyes absent 4 (EyA4) in response to undigested DNA (30
Here we show that Klf1
is expressed in CMEI and that Dnase2a
is strongly downregulated in the fetal liver of Klf1
KO mice. Dnase2a
downregulation in Klf1
KO mouse fetal liver macrophages has not been previously reported (6
). We have also shown that DNase II enzymatic activity is reduced in Klf1
null mice, fetal liver macrophages are abnormal, their number is decreased, and IFN-β is activated. In addition, we have shown that the promoter of the murine Dnase2a
contains a Klf1
consensus sequence that is bound in vitro
by Klf1 in EMSA experiments and in vivo
in ChIP assay. This sequence is responsible for transactivation of the Dnase2a
promoter by Klf1 in a murine macrophage cell line.
Lentivirus-mediated Klf1-shRNA interference showed a significant downregulation of Klf1 and Dnase2a in transduced CMEIs.
These results show that Dnase2a is a direct target of Klf1 and that Klf1 regulates the expression of Dnase2a in the CMEI. The results further suggest that Klf1 controls the CMEI-specific upregulation of Dnase2a necessary to efficiently degrade the nuclear DNA from developing erythrocytes. A lack of Klf1 lowers DNase II-alpha activity, which slows the capacity of the central macrophage to digest DNA, resulting in activation of IFN-β, which would contribute to the impairment of the definitive erythropoiesis.
Trace amounts of mouse β-globin
expression have been detected in isolated primary fetal liver macrophages. Even though expression of globins in macrophages has been reported before (21
), this result could also be explained by the presence of residual erythroid cells. However, the expression of Klf1
in CMEI has been confirmed here by immunohistochemical analysis.
The decreased number of macrophages in Klf1 KO fetal liver may represent a secondary effect of DNase II-alpha shortage and is only marginally responsible for the 5-fold DNase II-alpha downregulation observed in Klf1 KO fetal liver, taking into account the 9-fold upregulation of DNase II-alpha in the CMEI.
Taken together, these data indicate a non-cell-autonomous role of Klf1 in definitive erythropoiesis in addition to its cell-autonomous role. A non-cell-autonomous role of Klf1 has been previously suggested by Perkins et al. (33
) to explain the incongruity between the full Klf1
KO mice and Klf1
KO/wt chimeras. Lim et al. (20
) first showed that Klf1
-null embryonic stem (ES) cells injected into wt blastocysts were able to undergo erythropoiesis and produce enucleated erythrocytes with an extended life span when a γ-globin transgene was present. Subsequently, Perkins et al. (33
) attempted to rescue the Klf1−/−
phenotype by mating Klf1+/−
mice with a transgenic line expressing a high level of the γ-globin
gene (γ+). Surprisingly, Klf1
-null γ+ expressing mice did not have any survival advantage compared to the Klf1
-null γ− mouse littermates, even though the globin chain balance was restored.
In one report, the authors mainly employed the in vitro
culture of primary erythroid progenitors, thus lacking macrophages (6
). In another report, an erythroblast immortalized cell line (cell line B1.6) and E14 fetal liver were analyzed, and only those genes that were up- or downregulated in both biological samples were taken into consideration, therefore excluding the genes expressed in macrophages (14
). In data obtained by subtractive hybridization, only a few downregulated genes were identified, overlooking most of the genes found to be downregulated by microarray expression profile experiments (36
). In addition, microarrays used by the authors were not representative of the full genome and in some cases were enriched for erythroid-specific genes (6
Enucleated definitive red cells are a feature of mammals. In fish, amphibian, reptiles and birds, definitive erythrocytes are nucleated. The lack of enucleated red cells correlates with the absence of erythroblastic islands in definitive erythropoietic tissue in birds (2
) and seems to correlate to the absence of DNase II-alpha in nonmammalian vertebrates. Only in mammals (http://www.ncbi.nlm.nih.gov/gene
), two members of the DNase II enzyme family have been identified: DNase II-alpha, involved in definitive erythropoiesis, and DNase II-beta, involved in lens cells differentiation. The only other Dnase2a
present in the public databases (http://www.ncbi.nlm.nih.gov/gene
) is the single Dnase2
of Danio rerio
, in which no Klf1 binding site is present in the proximal promoter region, in agreement with the status of nucleated definitive red cells in fish.
Our observations, along with those previously reported by others, highlight Klf1 as a key factor of definitive erythropoiesis contributing to the regulation of both cell-autonomous and non-cell-autonomous mechanisms. A non-cell-autonomous role of Klf1 in erythropoiesis is strongly suggested by the presence of mature enucleated erythrocytes in Klf1
chimeric mice (20
). This predicts that in a CMEI conditional Klf1
KO mouse model, nucleated definitive red cells with normal erythropoiesis would be present. Klf1 may regulate additional genes in fetal liver macrophages. Its full role in the CMEI, and therefore its role in the fetal liver blood island homeostasis, needs further investigation.
In summary, we have shown that Klf1 has a non-cell-autonomous role in definitive erythropoiesis through the regulation of DNase II-alpha activity in the central macrophages of erythroblastic islands. The downregulation of DNase II-alpha activates the production of IFN-β in macrophages, which, via an as-yet-to-be-elucidated mechanism, contributes to the block in erythroid differentiation in Klf1-null mice.