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C. Leveau et al.
points analyzed (Online Supplementary Figure S2A). The proportion of HSC (Lin- Sca1+ cKit+ CD150+ CD48-)25 was decreased in fsn mice, whereas the proportion of more mature hematopoietic progenitor cells (HPC-1: Lin- Sca1+ cKit+ CD150- CD48+) was increased, compared to the pro- portions in ctrl mice (Figure 2B and Online Supplementary Figure S2B). At 12 weeks of age, the HSC progenitor count was significantly lower in fsn mice than in ctrl mice, while the numbers of multipotent progenitors (MPP: Lin- Sca1+ cKit+ CD150- CD48-) were unchanged and those of HPC-2 (Lin- Sca1+ cKit+ CD150+ CD48+) and HPC-1 were slightly higher (Figure 2C). Within the committed progen- itor compartment, the numbers of common myeloid pro- genitors (CMP: Lin- Sca1- cKit+ CD34+ CD16/32low) and granulocyte-monocyte progenitors (GMP: Lin- Sca1- cKit+ CD34+ CD16/32+) were lower than ctrl values at 3 weeks of age, although the differences disappeared with time (Figure 2D, E). There were no significant differences in fsn
AB
vs. ctrl values in the numbers of common lymphoid pro- genitors (CLP: Lin- Sca1- cKitint) or megakaryocyte-ery- throcyte progenitors (MEP: Lin- Sca1- cKit+ CD34- CD16/32-) (Figure 2D, E). We confirmed previous reports that the profound anemia observed in fsn mice (Online Supplementary Figure S3A) is peripheral in nature and does not result from a decreased number of early erythroid progenitors but rather from a defect in the last step of ery- thropoiesis (Online Supplementary Figure S3B, C).7 Erythropoiesis and enucleation processes have been shown to involve chromatin compaction26 and actin cytoskeleton dynamics.27 Interestingly, we previously showed that Ttc7a plays a role in actin dynamics15,16 as well as in chromatin compaction and genomic stability.28 Hence, it is tempting to speculate that altered actin dynamics and chromatin organization, as a consequence of Ttc7a-deficiency, contribute to defective erythrocyte generation in fsn mice. A high splenic erythroblast count
C
DE
Figure 2. Ttc7a-deficiency alters the hematopoietic stem and progenitor cell compartment. The hematopoietic stem and progenitor cell compartment was analyzed in the bone marrow (BM) of 3-, 6-, and 12-week old control (ctrl - black bars) and Ttc7a-deficient (fsn - red bars) mice (mean ± standard error of mean) *P<0.05; **P<0.01; ****P<0.0001 (two-tailed t-test). (A) Quantification of total femoral BM cells (n≥6). (B) Representative flow cytometry at 12 weeks (left panel) and per- centage of hematopoietic stem cells (HSC: Lin- Sca1+ cKit+ CD150+ CD48-) among LSK (Lin- Sca1+ cKit+) cells (right panel) (n≥7). (C) Quantification of LSK cell popu- lations, HSC, multipotent progenitors (MPP: Lin- Sca1+ cKit+ CD150- CD48-), HPC-2 (Lin- Sca1+ cKit+ CD150+ CD48+) and HPC-1 (Lin- Sca1+ cKit+ CD150- CD48+). (D) Representative flow cytometry at 12 weeks and (E) quantification of common lymphoid progenitors (CLP: Lin- Sca1- cKitint CD127+), common myeloid progenitors (CMP: Lin- Sca1- cKit+ CD34+ CD16/32-), granulocyte-monocyte progenitors (GMP: Lin- Sca1- cKit+ CD34+ CD16/32+) and megakaryocyte-erythroid progenitors (MEP: Lin- Sca1- cKit+ CD34- CD16/32-) (n≥7). (B-D) Numbers adjacent to outlined areas indicate percent cells in the parent gate (mean). Numbers in parentheses indicate per- centage among leukocytes in the BM (mean).
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haematologica | 2020; 105(1)