Notch Signaling in Bone Marrow Nestin-Expressing Cells Controls Balance of Erythropoiesis at the Bone Marrow and Spleen

[Background] Nestin-expressing cells (NeC) have been characterized as one of many types of bone marrow (BM) microenvironmental cells, including endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, etc. Recent studies have gradually provided information about anatomy and functions of each of these cells. Nevertheless, subcellular signaling and transcriptional regulations in individual miciroenvironmental cells have poorly been demonstrated. On a different line of studies, it has been suggested that NOTCH signaling in BM microenvironmental cells affects hematopoiesis; despite this, information is limited whether NOTCH signaling plays a role in NeC. It is of note that nestin was originally identified in neural stem cells (NSC), that NOTCH signaling is known to play a pivotal role in the NSC, and that the BM NeC could be derived from neuroectoderm. This potential linkage urged us to investigate whether and how downregulation of NOTCH signaling in BM NeC affects hematopoiesis.

[Method] Mice with an rbpj-flox allele were crossed with those with a CreERT2/GFP (Green Fluorescent Protein) transgene under the nestin promoter. At 8-12 weeks, tamoxifen was intraperitoneally injected for 4-12 weeks to delete the rbpj gene only in NeC (rbpj cKO mice). In this experimental system, GFP is expected to be expressed as a surrogate marker for the rbpj gene deletion, by the deletion of stop codon inserted at 5' to the cDNA of GFP. Then, transplantation assays were performed using rbpj-null BM cells as a donor to reconstitute hematopoiesis in the wild-type mice, or using rbpj-null mice as recipients to see reconstitution of hematopoiesis from wild-type BM cells. The effect of splenectomy was investigated in the untransplanted and transplanted conditions. The littermate rbpj^null/wt orrbpj^wt/wtmice were used as controls.

[Results] GFP was detected in 0.1-0.5% of flow cytometry (FCM)-sorted CD45(-) cells only after tamoxifen injection. Deletion of rbpj was specifically confirmed in GFP-positive BM and spleen cells. Tamoxifen induced mild splenomegaly in rbpj cKO mice compared with littermate control mice. Enlarged spleen showed preserved follicular architecture but increased CD71(+)Ter119(+) mature erythroid cells in the red pulp. In contrast, BM of rbpj cKO mice bearing mild splenomegaly demonstrated marked decrease in the CD71(+)Ter119(+) mature erythroid cells without obvious anemia. There was a substantial animal-to-animal variation in the phenotypes; however, the strength of phenotypes was correlated with the frequency of GFP-positive cells in the BM, suggesting that the phenotypic variation was a result of the efficiency of rbpj gene deletion. We hypothesized that the rbpj cKO mice would develop anemia if spenectomized, because extramedullary erythropoiesis in spleen might compensate the defective BM erythropoiesis. However, tamoxifen did not cause significant anemia in splenectomized rbpj cKO mice. In these mice, reduction of the CD71(+)Ter119(+) mature erythroid cells in BM was significantly milder than nonsplenectomized mice. In transplantation analysis, the recipient rbpj cKO mice transplanted with BM cells from wild-type mice showed a reduction in CD71(+)Ter119(+) mature erythroid cells and mild splenomegaly, as were seen in rbpj cKO mice without transplantation. The phenotypes were again erased by the splenectomy. The recipient wild-type mice transplanted with BM cells from rbpjcKO mice did not show any phenotypes.

[Discussion] Rbpj cKO in NeC induced impaired erythroid differentiation in BM together with mild splenomegaly. We confirmed that these phenotypes were caused by rbpj cKO in BM NeC by transplantation experiments. Surprisingly, such BM erythropoiesis impairment was reversed by splenectomy. This unexpected finding uncovered the presence of a previously unidentified balance controller between BM and spleen erythropoiesis. Hematopoietic stem cell-autonomous NOTCH signaling has been shown to be dispensable for adult murine hematopoiesis; however, NOTCH signaling in BM-NeC is responsible for control of balance of erythropoiesis at the BM and the spleen.