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.2018 Apr 12;131(15):1712-1719.
doi: 10.1182/blood-2017-06-788505. Epub 2018 Jan 16.

Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis

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Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis

Sara Duarte et al. Blood..

Abstract

Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.

© 2018 by The American Society of Hematology.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Unperturbed myelo-E progenitor hierarchies in steady-state BM ofRbpj-deficient mice. (A-D) Analysis of hematopoietic stem and myelo-E progenitor cells in 6- to 10-week-oldRbpjfl/flVavCre/+ (N = 8) andRbpjfl/flVav+/+ (N = 4) andRbpjfll+VavCre/+ (N = 2) littermate controls. (A) Mean (standard deviation [SD]) BM cellularity per 2 femurs and 2 tibias. (B-D) Representative FACS profiles and mean (SD) frequencies of (B) lineage−Sca-1+c-Kit+CD150+Flt3− HSCs and (C-D) myelo-E progenitor subsets. (E)Rbpj expression in HSCs and myelo-E progenitor subsets purified from 8-week-oldRbpjfl/flVavCre/+ (N = 5) andRbpjfl/flVav+/+ orRbpjfll+VavCre/+ littermate controls (N = 1 and 2, respectively). Mean (standard error of the mean [SEM]) expression normalized to hypoxanthine phosphoribosyltransferase 1 (Hprt). Samples in which the mean value of replicates was ≤0.001 (relative toHprt) were considered below cutoff value (#). (F-G) In vitro colony assays using total BM fromRbpjfl/flMx1Cre/+ (N = 5-6) and age-matched (10-14-week-old)Rbpjfl/flMx1+/+ (N = 2) orRbpjfl/+Mx1Cre/+ (N = 6) control mice treated with Poly(I:C) 4 to 5 weeks before analysis. (F) Mean (SD) in vitro CFU-GM and burst-forming units-E (BFU-E) colonies. (G) Pure (CFU-Mk) and mixed-lineage (Mk-mix) Mk-containing colonies scored after staining with acetylthiocholiniodide. Mean (SD) values from 2 to 3 experiments. (H-I) Mean (SEM [H] or SD [I]) of circulating platelet and red blood cell (RBC) counts in 6- to 10-week-oldRbpjfl/flVavCre/+ (N = 18-12) and age-matched controls (Rbpjfl/flVav+/+ [N = 8-11] orRbpjfl/+VavCre/+ [N = 2-5]). For all data sets (A-I), statistical significance was investigated between homozygouslyRbpj-deleted and control mice. *P < .05; **P < .01; ***P < .001. MegE, Mk-E progenitor; MkP, Mk progenitor; PreGM, pregranulocyte/macrophage progenitor; ProEry, pro-erythroblasts. See also supplemental Figure 1A-G.
Figure 2.
Figure 2.
Canonical Notch signaling is dispensable for stem and myelo-E progenitor cell replenishment in competitive bone marrow chimeras. (A-C) One million BM cells were harvested from 10- to 14-week-oldRbpjfl/flMx1Cre/+ and control (Rbpjfl/flMx1+/+ andRbpjfl/+Mx1Cre/+) mice (CD45.2) 4 to 5 weeks after poly(I:C) treatment and transplanted into lethally irradiated wild-type (WT; CD45.1 or CD45.1/2) recipients together with 1 million competitor WT (CD45.1 or CD45.1/2) adult BM cells. Reconstitution of HSC (N = 2 ofRbpjfl/flMx1Cre/+ and N = 1 and N = 3 ofRbpjfl/flMx1+/+ andRbpjfl/+Mx1Cre/+, respectively) and myeloid progenitor subsets (N = 5 ofRbpjfl/flMx1Cre/+, N = 2 ofRbpjfl/flMx1+/+, N = 3 ofRbpjfl/+Mx1Cre/+) in the BM of engrafted mice was assessed 7 to 9 weeks after transplantation. Percentage donor (CD45.2)-derived reconstitution of (A) HSC (mean ± SD) and (B) myeloid progenitor subsets (mean ±SEM) relative to total BM cells. (C) Mean (SEM) CD45.2 contribution ofRbpjfl/flMx1Cre/+ (N = 6) and control (N = 2 and N = 6 ofRbpjfl/flMx1+/+ andRbpjfl/+Mx1Cre/+, respectively) BM cells to total NK1.1−Mac1+ myeloid, NK1.1−Mac1−CD19+ B and NK1.1−Mac1−CD4/CD8+ T cells. (D) Reconstitution of CD45.2-derived blood platelets (CD41+CD150+eGFP−) of total platelets inVwf-eGFP (CD45.1/2) recipients 4 to 5 weeks after transplantation with 1 million 10- to 11-week-old CD45.2Rbpjfl/flMx1Cre/+ (N = 4) or controlRbpjfl/+Mx1Cre/+ (N = 6) BM cells and 1 millionVwf-eGFP (CD45.1/2) competitor BM cells. (Left) Representative FACS profiles of platelet reconstitution in engrafted mice. (Right) Mean (SEM) percentage of CD150+CD41+ platelets derived from transplanted CD45.2 BM cells. For all data sets (A-D), statistical significance was investigated betweenRbpj-deleted and control mice. ***P < .001.
Figure 3.
Figure 3.
Expression of myelo-E lineage programs and Notch-target genes inRbpj-deficient E and Mk progenitors. (A-D) E and Mk progenitor subsets were purified from individual 10- to 12-week-oldRbpjfl/flMx1Cre/+ (N = 6) and age-matchedRbpjfl/flMx1+/+ (N = 2) andRbpjfl/+Mx1Cre/+ (N = 2) poly(I:C)-treated control mice and subjected to quantitative gene expression analysis for (A,C) erythroid and (B-C) megakaryocytic-related genes and (D)Notch1 andNotch2 receptors. Mean (SEM) values normalized toHprt. No differences betweenRbpjfl/flMx1Cre/+ and control mice reached statistical significance. (E) BM HSCs, Mk, and E progenitor cells (100 cells per replicate) were purified from individual 8-week-oldRbpJfl/flVavCre/+ (N = 5) and age-matchedRbpJfl/flVav+/+ orRbpJfl/+VavCre/+ controls (N = 1 and 2, respectively) and analyzed for expression of the Notch target genesHes1,Hes5, Nrarp, andGata3. Mean (SEM) values. Samples in which the mean value of replicates was ≤0.001 (relative toHprt expression) were considered below cutoff value (#). For all data sets (A-E), statistical significance was investigated betweenRbpj-deleted and control mice. *P < .05; **P < .01. See also supplemental Figure 1H.
Figure 4.
Figure 4.
Canonical Notch signaling is dispensable for phenylhydrazine-induced stress erythropoiesis. Seven- to 8-week-oldRbpJfl/flVavCre/+ (N = 3-6) and age-matchedRbpJfl/flVav+/+ orRbpJfl/+VavCre/+ controls (N = 2-3 and 2-3, respectively) were injected with PHZ to induce acute hemolytic anemia. (A) Circulating red blood cell counts in steady-state (day 0) and 2 and 4 days after last PHZ injection. Mean (SD) values are shown. (B-C) Mean (SEM) absolute numbers of E progenitors (PreCFU-E and CFU-E), 4 days after PHZ-induced anemia in BM (B) and spleen (C). *P < .05.
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