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L. Shao et al.
were analyzed by chromatin immunoprecipitation assay. The pro- cedure is described in detail in the Online Supplement. The sequences of primers are provided in Online Supplementary Table S3.
Analysis of hematopoietic stem cell transplantation, apoptosis, luciferase activity, bone micro-computed tomography, sternum vasculature whole-mount imaging and immunostaining
These assays and methods are described in the Online Supplementary Methods.
Statistical analysis
Details of the statistical analysis of the data are provided in the Online Supplementary Methods.
Results
Myelosuppression by 5-fluorouracil or γ-irradiation causes pancytopenia in Notch1+/ΔTAD mice
Myelosuppression by 5-FU is achieved by incorporating an analog of uracil into RNA or DNA of proliferating cells.25 The result of 5-FU treatment during hematopoiesis is apoptosis of proliferating progenitors, followed by acti- vation of HSC and reconstitution of the hematopoietic system.26 To test for a possible hematopoietic defect in Notch1+/ΔTAD mice, 5-week old wildtype (WT) and Notch1+/ΔTAD littermates were treated with two doses of 5- FU, 14 days apart. Terminal hematopoietic analysis was performed 28 days after the first injection (Figure 1A). The condition of the Notch1+/ΔTAD mice deteriorated rapidly after treatment. Analysis of peripheral blood from Notch1+/ΔTAD mice revealed a sharp decrease in red blood cell and platelet counts, as well as hemoglobin concentration (Figure 1D). White blood cell and lymphocyte counts were also reduced in Notch1+/ΔTAD mice (Online Supplementary Figure S1A). During the first 9 days after treatment, all Notch1+/ΔTAD mice exhibited severe pancytopenia. For half of the Notch1+/ΔTAD mice, the body-score condition index and body weight dropped below humane levels (body condi- tion index = 2) (Figure 1B,C), and the animals were sacri- ficed.
Figure S2B). Total body irradiation has a limited effect on enucleated erythroid cells1 (Online Supplementary Figure S2C) but the numbers of white blood cells, neutrophils and lymphocytes were significantly lower in Notch1+/ΔTAD mice than in controls (Online Supplementary Figure S1B). At the 9-day time point, the numbers of donor-derived HSC, ETP, and DN3 cells in the Notch1+/ΔTAD recipient mice were markedly reduced (Online Supplementary Figure S2D), sug- gesting that the Notch1+/ΔTAD BM microenvironment plays a critical role during hematopoietic recovery. However, 90 days after transplantation, the numbers of the donor- derived HSC, ETP, and DN3 populations were comparable between recipients (Online Supplementary Figure S2E) sug- gesting that in the surviving Notch1+/ΔTAD recipients, hematopoietic homeostasis was normalized. Thus, early hematopoietic recovery following γ irradiation also depends on Notch1-TAD signaling.
Transplantation with Notch1+/ΔTAD hematopoietic stem cells reveals a cell-autonomous defect in T-cell development
We next investigated whether there was a pre-existing hematopoietic defect in Notch1+/ΔTAD mice. Neither progen- itors nor HSC were significantly affected in 6-week old Notch1+/ΔTAD animals (Online Supplementary Figure S3A). No effect was seen in marginal zone B cells in the spleen or in B- and T-cell populations in the lymph nodes in Notch1+/ΔTAD mice (Online Supplementary Figure S3B, C). The presence of a single Notch1-ΔTAD allele significantly reduced the fre- quency and number of thymic ETP and DN3 populations (Online Supplementary Figure S3D, E). This T-cell lineage defect could be traced to a reduction in the BM-residing CLP population (Online Supplementary Figure S3F). These findings show an inherent early role for Notch1-TAD sig- naling in the BM lymphoid progenitor population.
To investigate whether the effect of Notch1+/ΔTAD on hematopoietic recovery is cell autonomous, 350 HSC from Notch1+/ΔTAD or WT donors (CD45.2+) were transplant- ed into congenic (CD45.1+/.2+) recipients (Figure 2A). Reconstitution, defined by having 85-90% of blood cells in the recipients derived from Notch1+/ΔTAD or WT donors, and multi-lineage potential was confirmed at 1, 2, and 3 months after transplantation (Online Supplementary Figure S4A). Notch1+/ΔTAD HSC reconstituted recipients similarly to WT HSC. There were equal numbers of donor-derived LSK cells and HSC in recipient BM (Online Supplementary Figure S4B), indicating that Notch1+/ΔTAD HSC were capable of homing and successfully engrafting into the WT recipi- ent niche. In secondary transplants, mice receiving Notch1+/ΔTAD BM cells had no defects in HSC reconstitution (Online Supplementary Figure S4C, D); however, the CLP, ETP, and DN3 populations were significantly reduced (Online Supplementary Figure S4E, F). This finding indicated an inherent T-cell defect resulting from loss of one Notch1 TAD allele but also showed that HSC reconstitution was otherwise unaffected.
To test the effects of chemotherapy on the Notch1+/ΔTAD hematopoietic system, we treated WT recipient mice reconstituted by Notch1+/ΔTAD HSC with two rounds of 5-FU (Figure 2A). Recipients transplanted with Notch1+/ΔTAD HSC exhibited no adverse effects when compared to those that received WT HSC (Figure 2B). Following treatment, com- parable numbers of BM HSC were present in recipient mice (Figure 2C). Both sets of recipients had equivalent platelet, white blood cell, neutrophil, lymphocyte and red
We analyzed bone marrow LSK (Lin-Sca1+cKit+) progen- itors and HSC (LSK+CD48-CD150+) after 5-FU treatment. Notch1+/ΔTAD mice showed significant decreases in the num- bers of BM progenitors and HSC (Figure 1E) 9 days after treatment. The BM-residing common lymphoid progeni- tors (CLP), thymic early T-cell precursors (ETP) and CD4- CD8- double negative 3 (DN3) T-cell progenitors were also significantly reduced in Notch1+/ΔTAD mice after treatment (Figure 1F-H). Thymus size was reduced in 5-FU-treated Notch1+/ΔTAD mice compared to controls (Figure 1I). These findings indicate that TAD-dependent Notch1 signaling plays a critical role in recovery of the hematopoietic sys- tem following myelosuppression.
To determine whether 137Cs γ total body irradiation had a similar effect as 5-FU treatment, WT or Notch1+/ΔTAD recip- ient mice (CD45.2+) were lethally irradiated and subse- quently transplanted with 2.0x106 WT donor (CD45.1+) BM cells. Recipient mice were monitored for 90 days after irradiation/transplantation (Online Supplementary Figure S2A). We observed that the Notch1+/ΔTAD mice suffered severe weight loss (data not shown) and 50% of them had low body-score condition leading to mortality 9 days fol- lowing irradiation/transplantation (Online Supplementary
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