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Ttc7a controls HSC functions
suggested the presence of stress erythropoiesis as a possi- ble attempt to compensate for the peripheral anemia (Online Supplementary Figure S3B).
Thus, our present results show that the absence of Ttc7a in fsn mice is associated with deregulation of the homeostatic balance between hematopoietic lineages, from the HSC stage onwards, and a tendency of all leuko- cyte subsets to expand over time.
Ttc7a has an intrinsic role in the fate of progenitor cells
Since Ttc7a is broadly expressed, it was not possible to distinguish the respective involvements of hematopoietic factors (i.e., HSC) and non-hematopoietic factors (e.g., BM niches and the thymic epithelium) in the generation of the fsn phenotype. In a previous study we found that the skin barrier is impaired in fsn mice;9 this defect may enhance antigen sensitization and thus induce immune system activation. Therefore, in order to determine the Ttc7a- deficient hematopoietic cells’ intrinsic contribution to fsn- associated hematologic manifestations, we generated chimeric mice by reconstituting lethally irradiated control recipients with LSK cells purified from either ctrl or fsn mice. Hereafter, these chimeric mice are respectively referred to as Ctrlctrl and Ctrlfsn. Three-week old mice were chosen as donors so that we could use a similar LSK graft inoculum in both control and fsn samples, and thus mini- mize the potential immune consequences caused by the altered fsn skin barrier. We monitored the hematologic reconstitution over time by collecting blood samples from the recipient mice every 2 weeks. As observed in native
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fsn mice, white blood cell counts were higher in Ctrlfsn mice than in Ctrlctrl mice, and a difference was observed as early as 4 weeks after transplantation (Figure 3A). The Ctrlfsn mice were also anemic (Figure 3B) and developed splenomegaly (Figure 3C), although the latter was less pronounced than in native fsn mice (Figure 1B). The total body weight of Ctrlctrl and Ctrlfsn mice was not different. The distribution of the splenic myeloid, T- and B-cell pop- ulations was the same as in ctrl mice (Figure 3D). As observed in fsn mice, BM cellularity was higher in Ctrlfsn mice than in Ctrlctrl mice (Figure 3E), whereas the LSK counts were slightly increased (Figure 3F). The distribu- tion of the HSC, MPP, HPC-2 and HPC-1 populations was similar in Ctrlfsn and Ctrlctrl mice, suggesting that the low HSC count observed in 12-week old native fsn mice (Figure 2C) is primarily caused by external (i.e., non- hematopoietic) factors. Taken as a whole, these data sug- gest that Ttc7a has an intrinsic role in hematopoietic cells; the absence of Ttc7a in hematopoietic progenitors results in the over-proliferation of the various cell lineages as seen in native fsn mice.
Loss of Ttc7a enhances the reconstitution potential of hematopoietic stem cells
Next, we sought to determine the impact of Ttc7a loss on the reconstitution potential of HSC in a controlled in vivo environment. Using lethally irradiated congenic recip- ients, we transferred equal numbers of LSK cells purified from 3-week old ctrl (LSKctrl)- or fsn (LSKfsn)-(CD45.2+) mice together with competitor wildtype-(CD45.1+) BM cells (i.e., Ctrl-LSKctrl or Ctrl-LSKfsn). We then assessed the
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Figure 3. Transferred Ttc7a-deficient LSK cells reproduce fsn manifestations in a control environment. Control irradiated mice were transferred with LSK cells puri- fied from 3-week old ctrl (Ctrlctrl – black bars) or Ttc7a-deficient mice (Ctrlfsn – red bars). (mean ± standard error of mean) *P<0.05; **P<0.01 (two-tailed t-test). (A, B) Monitoring of the number of leukocytes (A) and red blood cells, hematocrit and hemoglobin (B) during hematopoietic reconstitution. (C) Spleen size determined as percent of body weight 10 weeks after bone marrow (BM) transfer. (D) Relative contribution of myeloid compared to T cells and B cells in the spleen. (E, F) BM cellularity (E) and absolute number of LSK cells (F) 10 weeks after BM transfer. RBC: red blood cells.
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