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Ttc7a controls HSC functions
an additional round of transplantation before dying after the eighth round (Figure 5A, B). Similar results were obtained in experiments with 12-week old donors (Online Supplementary Figure S5A, B). We next determined the abil- ity of ctrl and Ttc7a-deficient BM cells from 3-week old mice to properly reconstitute hematopoiesis over the dif- ferent rounds of transplantation. During the first five rounds, the same phenotype was always observed. Ctrlfsn mice displayed anemia (Figure 5C), together with an ele- vated leukocyte count up until the third round (Figure 5D). The spleen was larger in Ctrlfsn mice than in Ctrlctrl mice until the fifth round (Figure 5E). However, differences between Ctrlfsn and Ctrlctrl mice were no longer observed in the sixth round; this was probably caused by exhausted donor cells that failed to properly reconstitute recipient mice at the end of the reconstitution process (Figure 5C- E). Interestingly, the distribution of splenic leukocyte sub- sets in the Ctrlfsn mice was progressively biased toward myeloid populations at the expense of B cells, and to a lesser extent, T cells, as notably observed for the fifth
AB
C
round (Figure 5F). In Ctrlfsn mice, this bias became detectable in the third round and persisted until the sev- enth round (Online Supplementary Figure S5C). In summary, these data show that Ttc7a-deficient HSC have a greater ability to self-renew and to induce myeloid cell expansion.
Ttc7a-deficiency perturbs the transcriptomic profile of the endoplasmic reticulum stress response in hematopoietic stem cells
In order to gain further mechanistic insight into the Ttc7a-related regulation of HSC homeostasis, we carried out a transcriptomic analysis of HSC isolated from Ttc7a- deficient and control BM (from 4-week old mice). A two- way hierarchical clustering analysis of differentially expressed genes (P value ≤0.05, fold change ≥1.2) revealed a clear-cut separation between Ttc7a-deficient and control HSC samples (Figure 6A). We found that 1,103 genes were significantly upregulated and 928 significantly downregu- lated in Ttc7a-deficient HSC relative to the expression lev- els in control HSC (Figure 6B). Among the differentially
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Figure 5. Ttc7a-deficiency promotes self-renewal ability of hematopoietic stem cells. Lethally irradiated mice were serially transplanted with 3-week old ctrl (Ctrlctrl – black bars, dots and lines) or Ttc7a-deficient (Ctrlfsn – red bars, dots and lines) donor bone marrow (BM) cells (mean ± standard error of mean) *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (two-tailed t-test). These data are representative of four independent experiments with at least four mice in each round of transplan- tation. (A) Percent survival of recipient mice across the seven transplantation cycles. (B) Survival of Ctrlctrl and Ctrlfsn mice during the seventh round and Ctrlfsn mice during the eighth over time (n=4 for control- and n=10 for Ttc7a-reconstituted mice). (C-F) Red blood cell count, hematocrit, and hemoglobin level (C), leukocytes count (D) and spleen weight (E) across the transplantation cycles. (F) Relative contribution of myeloid compared to T- and B-lymphoid cells in the spleen of mice transplanted with BM cells that had undergone five transplantation cycles. RBC: red blood cells; Htc: hematocrit; Hgb: hemoglobin.
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