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L. Shao et al.
While further work is required to determine whether spe- cific subsets of BM EC are more susceptible to loss of Notch signaling during niche regeneration, our work suggests that a majority of EC, which express CD31, VE-cadherin and endomucin, require robust Notch1 signaling to recovery after myelosuppressive injury.
Mediators of BM niche recovery include VEGF-A, Tie2, and the Tie2 agonist Ang1.2,53,54 Initiation of regenerative signaling is believed to hinge upon VEGF-A produced by osteoblasts and osteocytes55,56 and Ang1 produced by osteoprogenitors and hematopoietic precursors.10,57 We observed that there was a short pulse of Tie2 phosphory- lation after chemotherapeutic damage while Notch signal- ing remained at elevated levels for at least 7 days after chemotherapy. Tie2 activation increased Notch receptor cleavage and the Tie2 agonist, Ang1 stimulated Notch sig- naling and promoted the expression of two canonical Notch ligands: Dll4 and Jag1. However, Tie2 stimulation, after 5-FU treatment failed to rescue the survival of Notch- deficient EC which expressed the hypomorphic Notch1ΔTAD allele. These results indicate that Notch sig- naling functions downstream of Tie2 and that Notch1 sig- naling is ultimately responsible for the regeneration of the BM endothelium.
The Notch1+/ΔTAD mice exhibited no inherent HSC defect in the absence of BM injury, corroborating reports that indi- cate a lack of involvement of Notch signaling in adult HSC expansion.44,45,47 However, chemotherapy severely impaired the generation of HSC in Notch1+/ΔTAD mice suggesting that Notch signaling is required for HSC self-renewal. Transplantation of Notch1+/ΔTAD HSC reconstituted irradiated WT hosts which showed no pancytopenic symptoms after chemotherapy, indicating that there is no cell-intrinsic defect in HSC generation due to the presence of the Notch1ΔTAD protein. In fact, the reverse experiment, in which WT HSC were transplanted into Notch1+/ΔTAD recipi- ents had the same severe phenotype as the constitutive Notch1+/ΔTAD or Notch1f/fVE-cadherin-CreERT2+ models, further supporting a specific role for Notch signaling in the regen- eration of the BM EC niche. Thus, we attribute the loss of HSC and progenitor cells observed after 5-FU treatment to the destruction of the BM EC niche, a process which requires robust Notch activity for its recovery.
Previous work has established that successful T-cell development requires Notch signaling up until β-selection of αβ T cells in the thymus.27,58,59 Notch1+/ΔTAD mice exhibited a moderate decrease in developing T cells and had no defect in mature single-positive T cells (data not shown). This inherent defect was exacerbated by 5-FU treatment and extended to the BM-residing CLP population. These results suggest that the Notch-driven commitment of the T-cell lineage begins at an early stage in the BM. A recent study, in which genetic deletion of Dll4 in the bone pro- ducing mesenchymal Ocn+ cells decreased CLP numbers and limited the production of T cells in the thymus,60 sup- ports our observations. Thus, the present findings suggest an important role for Notch1 signaling in mediating the differentiation of early lymphoid hematopoietic progeni- tors and the existence of a specific Notch-signal-promot- ing niche in the BM for T-cell commitment prior to thymic migration.
In summary, we employed a novel strategy for physi- ological inhibition of Notch signaling in adult tissues using a hypomorphic in vivo model system. We demon- strated that high levels of Notch1 signaling are required for the regeneration of BM EC after myelosuppressive chemotherapy and radiotherapy. Optimal recuperation of the EC niche relies on several signaling pathways, but it is ultimately a Notch-dependent process that supports regeneration of the BM niche and hematopoietic recov- ery.
Acknowledgments
We thank Dr. Warren Pear for invaluable advice and for shar- ing the Notch1+/ΔTAD murine model system. We also thank Dr. Kishore Wary for sharing the Cdh5-CreERT2 mouse model. Drs. Jon Aster and Stephen Blacklow for advice and thoughtful discus- sion, Dr. Dawson Gerhardt for her help in generating the Notch1- ΔTAD plasmids and vector constructs, Dr. Jan Kitejewski for helpful advice on Notch mutant mice and Drs. Fotini Gounari and Linda Dagenstein of the University of Chicago transgenic mouse facility for help in maintaining the transgenic mouse colonies. The following cores at the University of Illinois at Chicago contributed to this study: RRC Histology Core and RRC Flow Cytometry Core. This study was funded by NIH grants 1R01HL134971 to KVP and 1R01HL136529 to DL.
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