Page 63 - 2019_11 Resto del Mondo-web

Loss of Notch1 TAD interferes with niche recovery
Transcriptional defect in Notch1+/ΔTAD impairs T-cell development
Notch function in T-cell development has been well documented,27,28 so to determine how lack of a single TAD allele affects T cells, we measured target expression in sorted WT and Notch1+/ΔTAD DN3 cells before and after 5-FU treatment. During resting conditions, no significant change was observed in the expression of Hes1 or Myc, but a decrease in the levels of Hey1 and Dtx1 was seen in Notch1+/ΔTAD DN3 cells (Figure 2H). However, the expres- sion of Hes1, Hey1, and Dtx1 was severely reduced after 5- FU treatment, and expression of pre-T-cell receptor alpha (pTα) and Myc was moderately reduced (Figure 2I), sug- gesting that Notch1ΔTAD strongly impairs target gene expression in developing thymocytes during regeneration.
To determine whether production of active Notch1 was equal among our different model systems, CD25+ thymo- cytes were isolated from WT, Notch1+/-, and Notch1+/ΔTAD mice. The abundance of cleaved, full-length intracellular domains of Notch (ICN) was comparable between Notch1+/- and Notch1+/ΔTAD samples; however, there was an accumulation and a nearly 3-fold increase of the levels of cleaved ICN ΔTAD protein (Figure 2J).
Next, we employed an in vitro U2OS-based Notch luciferase reporter assay to assess the effects of accumulat- ed ICN ΔTAD on transcription.29 We chose U2OS cells, a human osteosarcoma cell line, because they express very low levels of endogenous Notch signaling and are highly transfectable. Constructs expressing Notch1 WT or ΔTAD mutant were transfected into U2OS cells at various ratios of ICN to ICNΔTAD (Figure 2K, L). ICNΔTAD had a lower capacity of transcriptional activation compared to ICN (Figure 2M, lanes 3 and 4). Dose-dependent transcrip- tional inhibition was observed when ICNΔTAD was co- transfected with ICN. The presence of a 1:4 ratio of ICN:ICNΔTAD caused a 4-fold decrease in luciferase activity (Figure 2M, lane 6). This TAD-dependent regi- ment was rescued by inverting the ratio of ICN to ICNΔ TAD (Figure 2M, lane 7). Our findings uncovered a role for the ΔTAD-mediated suppression of WT Notch signaling which we termed TAD transcriptional interference. These findings showed that the presence of a Notch1ΔTAD pro- tein impaired Notch target expression. Since Notch signal- ing is crucial for T-lineage development, we conclude that this transcriptional interference negatively affected in vivo development of thymocytes.
Chemotherapy severely damages bone marrow microvasculature and induces endothelial cell apoptosis in Notch1+/ΔTAD mice
Our findings suggest that the cell-intrinsic hematopoiet- ic defect in Notch1+/ΔTAD is limited to the T-cell lineage, which cannot account for the pancytopenia, morbidity, and the eventual mortality observed in the Notch1+/ΔTAD mice (Figures 1 and 2). Thus, our attention turned to the BM microenvironment. First, to elucidate whether loss of one copy of TAD in the Notch1 locus affects bone and mesenchymal cells, we performed micro-computed tomography analysis of femoral bones. This assay indicat- ed comparable bone length, bone volume fraction (BV/TV) and cortical thickness in WT and Notch1+/ΔTAD mice (Online Supplementary Figure S6A-C). Furthermore, CD51 and Sca1 were used to identify osteoblasts and mesenchy- mal stem/precursor cells.30 Flow cytometry analysis showed that there were similar numbers of osteoblasts
and mesenchymal stem/precursor cells in digested bones from WT and Notch1+/ΔTAD mice (Online Supplementary Figure S6D, E). These data indicate that loss of one copy of Notch1 TAD does not significantly affect bone develop- ment in Notch1+/ΔTAD mice.
To test whether the presence of one Notch1ΔTAD allele affects formation of the bone vasculature network, whole- mount sternal imaging was conducted. Imaging of the vas- culature labeled by CD31 and VE-cadherin showed com- parable vessel number and vessel length between WT and Notch1+/ΔTAD mice under resting conditions (Figure 3A). Next, we wanted to determine the effect of chemotherapy on the recovery and regeneration of BM EC and specifical- ly the sinusoidal EC, which have been shown to be crucial components of the adult hematopoietic niche.31-33 Primary bone CD31+ EC (pBEC) were harvested from WT mice 3 days after 5-FU treatment. Expression of the Notch1 recep- tor and several Notch canonical gene targets were upregu- lated after the chemotherapy (Figure 3B). Target gene upregulation corresponded with Notch1 receptor cleavage (Figure 3B, inset), showing an increase in Notch activation which occurred in pBEC as early as 3 days after 5-FU treat- ment.
To determine the condition of the BM EC niche at the time point when the Notch1ΔTAD-expressing heterozy- gous hypomorphs succumb to myelosuppressive treat- ment, pBEC in WT or Notch1+/ΔTAD littermates were ana- lyzed 9 days after 5-FU treatment (Figure 3C). Annexin V staining showed that 20% of the pBEC in Notch1+/ΔTAD mice were apoptotic compared to <7% of WT pBEC (Figure 3D). This sorted pBEC population from digested bones, over 90% of which expressed CD31+ and less than 5% expressed CD45+ (Figure 3E, left panel), was further ana- lyzed for expression of pro-apoptotic genes. A correspon- ding 2-fold increase in the expression of Puma and Bax34 was observed in sorted Notch1+/ΔTAD pBEC (Figure 3E, right panel). Absolute numbers of Notch1+/ΔTAD pBEC were markedly decreased, reflecting apoptosis (Figure 3F).
To visualize the BM microenvironment,35 sections of the tibia of WT and Notch1+/ΔTAD littermates were analyzed before and after chemotherapy. Under resting conditions, as suggested by the whole mount imaging of the sternum (Figure 3A), there was no difference in the abundance of hematopoietic cells in the BM, and similar CD31+ and endomucin+ sinusoidal endothelial structures were present in WT and Notch1+/ΔTAD littermates (Figure 3G-I, top panels). After 5-FU treatment, retention of hematopoietic cells was markedly reduced in Notch1+/ΔTAD BM (Figure 3G, bottom panel). Severe damage to the BM niche following 5-FU treatment was evident by the lack of CD31+ cells and irregular endomucin+ endothelial staining in the Notch1+/ΔTAD mice following this treatment (Figure 3H-I, bottom panel). Overall, our findings indicate that the hematopoietic defect observed in Notch1+/ΔTAD mice can be attributed to the failure of endothelial regeneration after chemotherapy.
Endothelial cell-specific deletion of Notch1 receptor confirms the requirement for endothelial Notch signaling during niche recovery
To specify a tissue-specific role for Notch signaling in the recovery and regeneration of BM EC, Notch1f/fVE-cadherin- CreERT2+ mice were treated with tamoxifen 1 week prior to administration of 5-FU (Figure 4A). The tamoxifen regi- ment led to loss of Notch1 protein (Figure 4B) and a
haematologica | 2019; 104(11)
2169