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Ttc7a controls HSC functions
in cellular response to stress (Figure 6E). Our transcriptom- ic analysis also highlighted high expression levels of Ttc7a in HSC (Online Supplementary Figure S6). A growing body of evidence suggests that ER stress regulates the function of the HSC pool.21 In particular, a recent study highlighted a link between ER stress perturbation in HSC and an ele- vated reconstitution capacity following BM transplanta- tion.29 Accordingly, we found that several effectors of the ER stress response were significantly downregulated in Ttc7a-deficient HSC, including the UPR master regulator Bip (Hspa5/GRP78), calreticulin, Pdia3, Pdia4, Pdia6, sev-
eral Hsp, as well as Sel1l and Syvn1 which have been shown to regulate an ER-associated protein degradation (ERAD) pathway30,31 (Figure 6F and Online Supplementary Table S1). Overall, our data suggest that Ttc7a loss affects the cellular response to ER stress in HSC.
Ttc7a controls the response to stress in hematopoietic stem cells
ER stress is mainly triggered in response to altered pro- tein homeostasis leading to pro-apoptotic or pro-survival responses. Notably, Ttc7a-deficient HSC had reduced lev-
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Figure 7. Ttc7a controls the response to stress in hematopoietic stem cells. (A) Representative histograms of protein aggregation level of 3-week old ctrl (black line) and fsn (red line) hematopoietic stem cells (HSC). (B) Protein expression of Bip in ctrl and fsn HSC after 3 days of in vitro expansion. (C) Proliferation index (calculated as the ratio between the number of cells at 48 h and 24 h) of HSC after Lin- cells were sorted from ctrl (black bars) and Ttc7a-deficient (fsn – red bars) mice and cultured for 2 days with or without tunicamycin. **P<0.01 (two-tailed t-test). (D-I) Ctrlctrl (black line and bars) and Ctrlfsn (red line and bars) mice were analyzed after they had received a single intraperitoneal injection of 150 mg/Kg of 5-fluorouracil (5-FU), 12 weeks after bone marrow transfer. White blood cell count over time (D). Spleen size (E) and absolute number of lymphoid (F) and myeloid (G) cells in the spleen 15 days after 5-FU injection (n=9). Percentage bromodeoxyuridine (BrdU) incorporation in LSK subpopulations (H) and representative flow cytometry histograms of BrdU incorporation in LK (Lin- Kit+) populations (I) 7 days after 5-FU injection. (n=10 for control- and n=12 for Ttc7a-reconstituted mice) *P<0.05; **P<0.01; ***P<0.001 (two-tailed t-test). AU: arbitrary units; LSK: Lin- Sca1+ cKit+ cells; MPP: multipotent progenitors; HPC: hematopoietic progenitor cells; CMP: common myeloid progenitors; GMP: granulocyte-monocyte progenitors; MEP: megakaryocyte-ery- throid progenitors; CLP: common lymphoid progenitors.
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